presentation on dna replication

DNA Replication (Advanced Detail)

• DNA & RNA

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Description.

This animation shows the process of DNA replication, including details about how the mechanism differs between the leading and lagging strand.

DNA replication starts with the separation of the two DNA strands by the enzyme helicase. The two strands are referred to as the 3' and 5' strands based on the direction by which the component nucleotides are joined. The 3' DNA strand is also known as the leading strand; DNA polymerase copies the leading strand to produce a complementary strand. The 5' strand is also known as the lagging strand. DNA polymerase copies the lagging strand in sections. These two different mechanisms of copying DNA occur because DNA polymerase can only synthesize DNA in the 5' to 3' direction.

This animation brings the DNA replication process to life, showing three-dimensional representations of the molecules involved. Depending on students’ backgrounds, it may be helpful to pause the animation at various points to identify the molecules and describe their interactions.

base, helicase, lagging strand, leading strand, nucleic acid, nucleotide, Okazaki fragment

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In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. This process occurs in all living organisms and is the basis for biological inheritance. DNA is made up of a double helix of two complementary strands. During replication, these strands are separated. Each strand of the original DNA molecule then serves as a template for the production of its counterpart, a process referred to as semiconservative replication. Cellular proofreading and error-checking mechanisms ensure near perfect fidelity for DNA replication. [1][2] In a cell, DNA replication begins at specific locations, or origins of replication, in the genome. [3] Unwinding of DNA at the origin and synthesis of new strands results in replication forks growing bi-directionally from the origin. A number of proteins are associated with the replication fork to help in the initiation and continuation of DNA synthesis. Most prominently, DNA polymerase synthesizes the new strands by adding nucleotides that complement each (template) strand. DNA replication occurs during the S-stage of interphase. DNA replication can also be performed in vitro (artificially, outside a cell). DNA polymerases isolated from cells and artificial DNA primers can be used to initiate DNA synthesis at known sequences in a template DNA molecule. The polymerase chain reaction (PCR), a common laboratory technique, cyclically applies such artificial synthesis to amplify a specific target DNA fragment from a pool of DNA.

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DNA replication - PowerPoint PPT Presentation

DNA replication

Dna replication, meselson and stahl experiment, bacterial- theta replication, viral- rolling circle replication, eukaryotic replication, telomere replication. 3'-5' and 5' to 3' exonuclease activity – powerpoint ppt presentation.

• By Pallavi Bohra
• Process of producing two identical replicas from one original DNA molecule
• During preparation for cell division (S phase)
• Each strand of the original DNA molecule serves as a template 
•  Semiconservative replication concept
• Helicase separates the DNA to form a replication fork at the origin of replication where DNA replication begins.
• Replication forks extend bi-directionally as replication continues.
• Okazaki fragments are formed on the lagging strand, while the leading strand is replicated continuously.
• DNA ligase seals the gaps between the Okazaki fragments.
• Primase synthesizes an RNA primer with a free 3'-OH, which DNA polymerase III uses to synthesize the daughter strands.
• Inside cytoplasm
• Only one origin of replication 1000-2000nt
• Rich in AT sequences
• Two replication fork form in each DNA-bidirection
• Only one replicon
• Nick by rep protein (replication initiator protein) within ori.
• Rep protein binds to the 5 end of the nicked strand while the free 3-OH end is used as a primer for DNA synthesis (by host machinery, poly III)
• Other enzymes- helicase called PcrA
• Multiple copies of linear single stranded
•  DNA polymerase I removes the primer, replacing it with DNA, and DNA ligase

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Introduction

Overview of rrd syndromes, updates on cmmrd and surveillance recommendations, updates on other rrd cancer syndromes and associated surveillance recommendations, current therapeutic landscape for rrd cancers, genetic counseling for rrd syndromes, future directions, authors’ disclosures, clinical updates and surveillance recommendations for dna replication repair deficiency syndromes in children and young adults.

Clin Cancer Res 2024;30:3378–87

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• Version of Record August 15 2024
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• Accepted Manuscript June 11 2024

Anirban Das , Suzanne P. MacFarland , Julia Meade , Jordan R. Hansford , Kami W. Schneider , Roland P. Kuiper , Marjolijn C.J. Jongmans , Harry Lesmana , Kris Ann P. Schultz , Kim E. Nichols , Carol Durno , Kristin Zelley , Christopher C. Porter , Lisa J. States , Shay Ben-Shachar , Sharon A. Savage , Jennifer M. Kalish , Michael F. Walsh , Hamish S. Scott , Sharon E. Plon , Uri Tabori; Clinical Updates and Surveillance Recommendations for DNA Replication Repair Deficiency Syndromes in Children and Young Adults. Clin Cancer Res 15 August 2024; 30 (16): 3378–3387. https://doi.org/10.1158/1078-0432.CCR-23-3994

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Replication repair deficiency (RRD) is a pan-cancer mechanism characterized by abnormalities in the DNA mismatch repair (MMR) system due to pathogenic variants in the PMS2 , MSH6 , MSH2 , or MLH1 genes, and/or in the polymerase-proofreading genes POLE and POLD1 . RRD predisposition syndromes (constitutional MMR deficiency, Lynch, and polymerase proofreading–associated polyposis) share overlapping phenotypic and biological characteristics. Moreover, cancers stemming from germline defects of one mechanism can acquire somatic defects in another, leading to complete RRD. Here we describe the recent advances in the diagnostics, surveillance, and clinical management for children with RRD syndromes. For patients with constitutional MMR deficiency, new data combining clinical insights and cancer genomics have revealed genotype–phenotype associations and helped in the development of novel functional assays, diagnostic guidelines, and surveillance recommendations. Recognition of non-gastrointestinal/genitourinary malignancies, particularly aggressive brain tumors, in select children with Lynch and polymerase proofreading–associated polyposis syndromes harboring an RRD biology have led to new management considerations. Additionally, universal hypermutation and microsatellite instability have allowed immunotherapy to be a paradigm shift in the treatment of RRD cancers independent of their germline etiology. These advances have also stimulated a need for expert recommendations about genetic counseling for these patients and their families. Future collaborative work will focus on newer technologies such as quantitative measurement of circulating tumor DNA and functional genomics to tailor surveillance and clinical care, improving immune surveillance; develop prevention strategies; and deliver these novel discoveries to resource-limited settings to maximize benefits for patients globally.

DNA replication during S-phase of the cell cycle is performed by polymerase enzymes. This is a highly conserved yet error-prone process ( 1 ). Errors in base incorporation lead to single-nucleotide variations (SNV), whereas slippages result in insertions and deletions (indel), with the latter being frequent in repetitive genomic segments called microsatellites ( 2 , 3 ). As replication fidelity is vital for genomic integrity, this process is tightly controlled by the exonuclease proofreading domains of the DNA polymerases encoded by the genes POLD1 and POLE , and the mismatch repair (MMR) proteins encoded by PMS2 , MSH6 , MLH1 , and MSH2 ( 4 ). Therefore, it is not surprising that aberrations in MMR and polymerase-proofreading genes lead to uncontrolled mutagenesis and genomic instability resulting in cancers characterized by a high burden of SNVs [(or point mutations) conventionally referred to as a high tumor mutation burden [TMB]), as well as high microsatellite instability (MSI). High TMB, MSI, and specific mutational and microsatellite signatures caused by MMR and/or polymerase proofreading–deficiencies (PPD) therefore form the hallmarks of DNA replication repair deficiency (RRD; refs. 2 , 5 , 6 ) in cancer ( Fig. 1A ).

RRD cancer and related cancer predisposition syndromes. A, Replication fidelity is maintained by the MMR system and the polymerase-proofreading genes. Aberrations lead to single-base mismatches and insertions/deletions, leading to RRD cancers with high SNV and MSI burden. B, Germline defects in RRD predisposition syndromes. Of note, due to increased somatic mutagenesis, cancers in all such individuals can develop both MMR and polymerase-proofreading deficiencies, leading to complete RRD in their cancers. (Adapted from an image created with BioRender.com and Adobe Illustrator v.27.8.).

RRD is widely recognized across multiple cancer types. Patients with RRD tumors have a specific clinical behavior, characterized by resistance to chemotherapeutic agents, and a higher probability of response to immune checkpoint inhibition (ICI; refs. 7 , 8 ). Furthermore, germline RRD stemming from pathogenic variants in MMR or replication polymerase-proofreading genes lead to cancer predisposition syndromes (CPS) with overlapping characteristics ( Fig. 1B ). Therefore, the focus of this article is to summarize together the different syndromes predisposing to RRD cancers and their surveillance in childhood and adolescence.

Since the last guideline published by this AACR Working Group in 2017 ( 9 ), there have been significant developments in the field ( 7 , 10 , 11 ). These include development of novel assays ( 12 ), deeper insights into the biology ( 6 , 13 ), expansion of the syndromes and their cancer spectra ( 14 ), and appreciation of genotype–phenotype associations ( 15 ). These discoveries prompted collaborations to develop new diagnostic criteria ( 16 ), investigate the impact of the previous surveillance guidelines ( 7 , 8 , 10 , 17 ), and development of newer management strategies ( 7 , 8 , 10 ). The current review summarizes these developments related to each syndrome and provide consensus recommendations generated during the AACR’s Childhood Cancer Predisposition Workshop (CPWG) held in July 2023.

Germline pathogenic variants (PV) in both alleles of any MMR gene result in the autosomal recessive constitutional MMR deficiency syndrome (CMMRD; ref. 9 ). Most individuals will be affected by multiple cancers during childhood ( Fig. 1B ; refs. 10, 15). Germline heterozygous PV in any one of the four MMR genes in the germline give rise to Lynch syndrome (LS; ref. 18 ). Cancers in LS may be replication-repair proficient or deficient ( Fig. 1B ), but the absolute risks of cancer development in childhood for a patient with LS is currently considered small ( 19 ). Germline 3′ deletions of the EPCAM gene, located upstream of MSH2 , result in hypermethylation of the MSH2 promoter in epithelial tissues, with tissue-specific MSH2 deficiency, and cancers similar to patients with LS ( 20 ). In contrast, MSH3 encodes another member of the MMR pathway but does not lead to classic LS, and patients with biallelic germline PV in this gene present with a polyposis disorder in adulthood ( 21 ). Similarly, biallelic truncating PV in MLH3 (c.3563C>G; p.Ser1188 ∗ ), reported as a founder variant in the Finnish population, causes adult-onset polyposis and other adult-onset cancers in that population ( 22 ). Germline heterozygous PV in the POLE and POLD1 genes cause cancer predisposition with tumors characterized by hypermutation in both adults and children ( Fig. 1B ; refs. 23 , 24 ). Although the absolute risks of cancer in childhood are small, determinants of such aggressive childhood phenotypes are currently unknown. Digenic syndromes with heterozygous germline PV in both polymerase and MMR genes have also been recently reported to present with cancers in childhood ( 25 ). Overall, although most of the pediatric data in the last 6 years relates to CMMRD, these studies also provide insights into the other closely related pediatric RRD syndromes. As such, here we discuss the data not only for CMMRD but also consider their impact on related syndromes.

Clinical and cancer phenotype

CMMRD is frequent in countries with high consanguinity, where most patients are homozygous for an identical PV in one of the four MMR genes ( 26 ). However, in countries with low-population prevalence of consanguinity, compound heterozygosity of variants in the same MMR gene is more frequently observed. Importantly, homozygosity may still be more prevalent in specific subpopulations like minorities, indigenous communities, and immigrants ( 15 ). This emphasizes the importance of considering the diagnosis of CMMRD independent of a history of consanguinity in the family. It is also important to recognize that although a history of cancer is frequent in the extended family, parents and other adult relatives who are obligate heterozygotes with LS rarely develop cancers at young enough ages to be diagnosed with LS, when their children present to the oncologist ( 15 ). This can be related to the enrichment for heterozygous PMS2 and MSH6 carriers among parents of patients with CMMRD who can have a milder LS phenotype, as well as the relatively young ages of the parents when their children present with a cancer ( 27 ).

Noncancerous clinical features overlapping with neurofibromatosis type-1 (NF1) can complicate the clinical diagnosis in up to a third of patients ( 15 ). Patients with CMMRD can harbor not only café-au-lait macules but also axillary freckling, neurofibromas, Lisch nodules, and other NF1 stigmata ( 15 ). A higher frequency of skin hypopigmentation, pilomatrixoma, and multiple developmental venous anomalies ( 28 ) in CMMRD can distinguish it from NF1 ( 15 , 29 ). In addition, cognitive and learning disabilities are less frequent in CMMRD than NF1 ( 15 , 30 ). Co-occurrence of NF1 mosaicism in patients with CMMRD has been reported ( 31 ). Clinically significant immunodeficiency and autoimmunity that was reported in select PMS2 cases previously ( 32 ) were recently found not to be higher in CMMRD than in the general population ( 15 ).

Early onset of cancers is still the hallmark of CMMRD. The risk for cancer is reported to be higher than that reported in most other CPS, affecting 90% of patients by 18 years of age and almost all by 40 years of age in a recent study ( 15 ). Moreover, most survivors of an initial cancer will develop a second one within 2 years ( 15 ). In addition, there is a 25% risk of presentation with synchronous cancers ( 10 ). These data have a major impact on surveillance guidelines and the clinical evaluation for a child with CMMRD presenting with their first cancer. Hence, at presentation, these children should be screened for additional synchronous primaries and surveillance should continue during their active cancer treatment.

Tumors initially recognized in CMMRD, including high-grade gliomas, T-cell lymphomas, and gastrointestinal tumors, are still considered the most frequent cancer types, but the tumor spectrum in CMMRD is expanding ( Fig. 2A ). Central nervous system (CNS) tumors also include medulloblastoma ( 33 ), ependymoma ( 34 ), and other gliomas ( 35 ). Hematologic malignancies include B-cell, mixed lineage, and myeloid leukemia, as well as Hodgkin lymphoma. Importantly, adult-type cancers like breast, prostate, genitourinary, pancreatic cancers, and melanoma are being observed in young adults with CMMRD at earlier than the usual age of presentation of such cancers ( 15 ). Overall, although the age range for cancer onset varies, hematological and CNS cancers develop earlier (median age: <10 years) and are the major cause of death early in childhood. Non-embryonal solid tumors (median age: >15 years) and gastrointestinal cancers occur relatively later in adolescence and young adulthood ( 15 ). Polyposis is almost universal and multiple, discrete polyps in the upper and lower intestine can be detected as early as 6 years of age. Adult-type cancers occur from the third decade of life, suggesting the need for modified surveillance for cancer survivors with CMMRD beyond childhood ( Fig. 2B ; ref. 15 ).

Updates for CMMRD. A, Expanded cancer spectrum in CMMRD and ( B ) prevalence of major cancer types by age. Adapted from recently published IRRDC data on 201 patients with CMMRD who developed 339 cancers ( 15 ). C, Clinical phenotypes and survival in CMMRD patients by affected gene type. Adapted from recently published IRRDC data on 201 patients with CMMRD ( 15 ). D, Performance of different assays used to detect RRD uniformly suggests highest sensitivity for the published functional genomic assays ( 12 , 36 , 37 , 68 ) in comparison with commercial MSI panels, TMB, and IHC. E, Improvement in survival from birth for CMMRD patients who were on the surveillance protocol vs. those not on surveillance. Adapted from recently published IRRDC data on 85 children with CMMRD who were followed up prospectively ( 15 ). Comp het, compound heterozygosity; Hem, hematologic; Hom, homozygosity; GI, gastrointestinal. (Adapted from an image created with BioRender.com and Adobe Illustrator v.27.8.).

Cancer biology and genotype–phenotype associations

Cancers in CMMRD frequently exhibit hypermutation ( 5 ). Extreme or ultra-hypermutation (TMB ≥100 mutations/megabase) is also common, especially in MMR-deficient cancers that acquire an additional somatic POLE/POLD1 mutation ( Fig. 1B ; ref. 5 ). Similarly, high MSI and corresponding signatures are universal in cancers from patients with CMMRD on both genome and exome analyses ( 2 , 12 , 36 , 37 ).

Recent genetic and genomic analyses in CMMRD have provided important insights for RRD genotype to phenotype associations ( 12 , 15 ). Disease penetrance as measured by cancer risk in adult patients with LS due to PMS2 and MSH6 PV is substantially lower than MSH2 and MLH1 ( 27 ). In CMMRD, PMS2 and MSH6 are the most frequent genes with biallelic PV and patients present with cancers in early childhood. However, patients with CMMRD due to the less-frequent MLH1 and MSH2 PV have more aggressive disease phenotype, including an earlier age of cancer onset and inferior survival post cancer treatment, leading to poor overall outcomes ( Fig. 2C ). Furthermore, within each gene, missense variants, both in individuals who are homozygous or compound heterozygous, have a less-severe phenotype when compared with those with a truncating/nonsense variants ( 15 ).

Diagnostic assays

Immunohistochemistry for the four MMR proteins remains a universal and feasible screening test for RRD ( 38 ). An absence of protein expression in tumor cells alone is a useful marker of somatic MMR deficiency and warrants follow up with genetic testing for LS. Importantly, the added loss of expression in noncancerous cells is the hallmark of CMMRD ( 16 , 38 , 39 ). Although IHC is a relatively specific assay, interpretation can be challenging and requires expertise, as some nontruncating or mosaic variants cause only partial loss of staining due to the expression of some nonfunctional proteins ( 40 ). Negative results in an appropriate clinical context should therefore be interpreted cautiously ( 12 ).

RRD can be detected from tumor sequencing data, which can then prompt germline testing. High TMB and the presence of specific mutational signatures can diagnose RRD in cancers ( 6 ). Methylation arrays can identify specific tumor subgroups enriched for RRD ( 35 , 41 ). However, these assays are neither sensitive nor specific, cannot distinguish somatic from germline findings, and are also expensive with limited availability.

MSI is a hallmark of RRD and can be used for diagnostic purposes ( 2 , 12 ). However, conventional MSI panel testing from several different commercial sources were not found to be reliable for detecting MSI in tumors and germline samples of children with CMMRD ( 2 ). The large number of MSI loci in the genome (>23,000,000) enabled the use of multiple loci or low-pass genome to quantify MSI in cancer and normal cells. These newer functional assays provide robust measures of global MSI status in RRD tumors, surpassing the yields of IHC, TMB, and conventional MSI panels across tumor types ( Fig. 2D ; refs. 12 , 36 , 37 ). These quantifiable assays are also robust for the detection of germline MSI and hence, distinguish CMMRD from other cancer syndromes and controls, using blood, saliva, and other nonmalignant tissues ( 12 ). As these assays are relatively inexpensive (<USD 150/sample), they can be useful to resolve diagnostic ambiguities even in less-resourced settings ( 12 , 40 ), especially for patients reported to have VUS, or where IHC is challenging to interpret. One such functional assay, developed and currently accessible through the International Replication Repair Deficiency Consortium (IRRDC), is in the process of accreditation for wider use as a clinical tool.

New diagnostic criteria for CMMRD

Recognition of the challenges in diagnosing CMMRD led to consensus recommendations by an international working group including the two largest interest groups [IRRDC ( https://replicationrepair.ca ) and European Consortium Care for CMMRD (C4CMMRD)] in 2021 ( 16 ). We recommend that these guidelines should be used by all clinicians for the diagnosis of CMMRD. The new criteria include clinical manifestations based on the previously published C4CMMRD scoring system as an entry point for evaluation ( 39 ), rigorous germline analyses using multigene panels that include all relevant genes as well as close mimickers, and importantly, recommendations for ancillary/functional investigations ( Table 1 ). Panel-sequencing performed in isolation can be falsely erroneous due to an overlap of pseudogenes with PMS2 and frequent reports of variants of uncertain significance (VUS) in all four genes, in addition to the challenges with the limitations of the technology, high cost and lack of global availability. Specifically, accurate testing for PMS2 gene variants may need to include long-range polymerase chain reaction, which is only available in select specialized laboratories ( 42 ). The new functional assays can resolve some of these ambiguities in a more rapid and accessible fashion, allow clinical diagnosis, and reclassification of pathogenicity for previous VUS and are therefore included as important ancillary tests in the updated diagnostic algorithm ( 12 , 16 , 36 , 37 ).

Updated diagnostic criteria for CMMRD.

Reproduced from Journal of Medical Genetics , Diagnostic criteria for constitutional mismatch repair deficiency (CMMRD): recommendations from the international consensus working group, Aronson et al., 59, 318‐327, copyright 2021, with permission from BMJ Publishing Group Ltd. ( 16 ). Readers are encouraged to review this for additional details.

Biallelic, impacts same gene on both parental alleles (i.e., PMS2/PMS2 ); P, pathogenic (ACMG C5); LP, likely pathogenic (ACMG C4); VUS (ACMG C3). Multigene panel testing is recommended to investigate overlapping conditions. Consider phenotype of individual to rule out overlapping syndromes. All families should be assessed in a specialized center for diagnosis.

Ancillary testing is described in further detail in this article and in Aronson and colleagues ( 16 ). Does not include tumor mutation burden and signature currently. Functional testing should be published with proven high sensitivity and specificity performed in an accredited (e.g., CAP inspected) laboratory authorized to give a clinically usable report. If discrepancy occurs among tests, multiple ancillary tests should be used to reach more conclusive decision.

In trans variants, can be proven by testing parents, offspring, or other relatives and rarely through direct testing methods. If unavailable to confirm variants in trans, individual should fulfill criterion 3.

Hallmark CMMRD cancer: glioma or CNS embryonal tumors <25 years, hematological cancer (excluding Hodgkin lymphoma) <18 years, GI adenocarcinoma <25 years, or >10 adenomatous GI polyps <18 years (after ruling out polyposis conditions).

C4CMMRD criteria as previously published ( 39 ).

If unavailable to confirm variants in trans , individual should fulfill criterion 6.

A history of consanguinity further supports a diagnosis of CMMRD due to a homozygous MMR gene mutation that is unidentifiable.

Individuals with two positive ancillary tests for CMMRD in the absence of the described phenotype can be assessed on a case-by-case basis, but these are atypical CMMRD cases and additional assessment is required to determine surveillance.

Surveillance recommendations for patients with CMMRD

The committee recommends following cancer surveillance guidelines for both definitive and likely CMMRD patients ( 16 ), even in the absence of a confirmed genetic diagnosis. The cancer surveillance protocols previously established have now been prospectively evaluated ( 14 , 17 ). Surveillance was found to be excellent for the early detection of extracranial solid and brain tumors but suboptimal for preclinical detection for hematologic malignancies ( 14 ). Furthermore, the biological finding of a high rate of benign to malignant transformation in CMMRD brain (low-grade gliomas) and gastrointestinal lesions (polyps) illustrates the importance of early detection through surveillance that contributes to improved overall survival for patients on surveillance ( Fig. 2E ; ref. 14 ).

Based on these observations, brain MRI and ultrasound of the abdomen are recommended every 6 months, starting at diagnosis, along with whole-body MRI and upper and lower gastrointestinal endoscopy annually, starting at 6 years ( Table 2 ; refs. 9 , 14 ). The increased recognition of the risk of malignant transformation of benign skin neoplasms including pilomatrixoma, and the detection of other skin cancers like melanoma in emerging young adult patients with CMMRD led to a consensus for inclusion of an annual skin examination, starting at diagnosis, with early referral to a dermatologist in case of any concern ( 14 ). We also recommend that screening for genitourinary cancers using dedicated transabdominal pelvic ultrasound starts at the age 12 (instead of 20 years in the previous guideline), as recent data from the IRRDC suggested that adolescents developed these malignancies as early as at 14 years of age ( 15 ). As regular blood counts have failed to detect presymptomatic hematological malignancies, we recommend detailed clinical examination at the time of each imaging visit in lieu of regular blood tests, with the rationale that detection of the signs and symptoms of a hematologic malignancy (pallor, lethargy, tachycardia, organomegaly, etc.) will trigger further investigation by the clinician. There was discussion on testing blood lactate dehydrogenase levels to improve yield for lymphoma detection ( 15 ). In the absence of robust data, the decision to do such regular blood tests is left to the individual physician and family, after a thorough discussion about the pros and cons of undergoing tests on a regular basis.

Updated surveillance recommendations for patients with CMMRD.

In case any abnormality is detected on surveillance, further imaging frequency needs to be more frequent depending on the diagnosis at the clinician’s discretion.

Abbreviations: GI, gastrointestinal; US, ultrasound; VCE, video capsule endoscopy; WB-MRI, whole-body MRI.

MRI brain should not be replaced by WB-MRI. The first MRI brain is recommended with contrast, and subsequent surveillance without, until any abnormality is detected, which would then need to be appropriately evaluated.

For hematologic cancers, a thorough physical examination at surveillance visits is recommended. Complete blood count and lactate dehydrogenase may be considered every 3–4 months.

Urine cytology has not found to be useful in LS and other surveillance studies for urological cancer diagnosis, and there are no data to suggest that it aids early cancer diagnosis in CMMRD.

A general principle for performing surveillance and early tumor detection in CMMRD is to resect, if feasible, all lesions detected presymptomatically (including currently benign lesions such as low-grade gliomas, polyps, pilomatrixoma, and bone tumors), before they accumulate further deleterious mutations and transform to aggressive malignancies ( 14 ). Individual polypectomies should be routinely performed. It is currently debatable whether total colectomy with a pouch procedure, that can dramatically impact a child’s quality of life, should be still advised for patients with numerous presymptomatic polyps in whom individual polypectomies are not feasible. This is because ICI has emerged as an extremely effective treatment for MMR-deficient colon carcinoma ( 43 ) should any of the multiple polyps transform to malignancy. The current consensus guidelines described here for cancer surveillance in CMMRD are summarized in Table 2 .

With the improved awareness and diagnostic assays, data on other germline RRD syndromes, in which children, adolescents, and young adults develop cancers with very similar and overlapping immunobiology, have emerged over the past few years.

Children and young adults with LS

Established surveillance guidelines for LS focus on gastrointestinal and genitourinary cancers, starting at ages 20 to 25 years and 30 to 35 years, respectively ( 44 – 47 ). However, there have been increased reports of aggressive CNS tumors ( 8 , 48 – 51 ) in a subset of patients with LS at relatively younger ages. A recent study revealed that the prevalence of RRD in gliomas occurring in individuals less than 40 years of age exceeds 5% ( 52 ), with almost all stemming from the germline, and LS being more frequent than CMMRD ( 52 ). Other studies have also suggested that pediatric patients with solid tumors can have an LS diagnosis ( 50 , 51 , 53 – 55 ). Some but not all such tumors can have a RRD biology. Importantly, MMR gene PV are frequently reported for children having exome or genome sequencing ( 51 , 53 ). The detection of heterozygous MMR gene PV in the germline, as a secondary finding, can lead to an early diagnosis of LS in children without a typical LS spectrum cancer. It is important to consider the need for testing for RRD in the tumors of such patients using appropriate assays (please see above; Fig. 1B ). As recent studies are reporting significantly improved outcomes for otherwise aggressive RRD tumors using checkpoint inhibition ( 7 , 8 , 56 ), it is vital that the presence of RRD is not missed ( 51 ).

As the absolute risk of tumor development in children with heterozygous PV in genes associated with LS is not currently recognized to be significantly high, our recommendation is to initiate routine cancer screening at adulthood. However, as the determinants of early cancer onset in a subset of LS patients is not well-understood at present, it is important to consider the role of a family history and possible genotype–phenotype associations. In this context, it is important to underscore that the existing guidelines do recommend starting gastrointestinal surveillance at least 2 to 5 years earlier than the youngest member developing colon cancer in an LS family. Practically, this can result in the need for surveillance in adolescence in families with a colon cancer diagnosed in their teens or early twenties ( 44 , 45 , 47 , 57 ). Similarly, the risks and determinants of aggressive CNS cancers in young LS patients and their families are also unknown. Hence, our added recommendation is for increased awareness among both physicians and families about the common signs and symptoms of brain tumors in children and young adults with LS and at-risk family members. It is also important to underscore that if a diagnosis of an RRD malignant glioma is established in a patient with LS, similar principles for therapy as in patients with CMMRD, including need for aggressive resection and immunotherapy, should be implemented.

Germline POLE/POLD1 patients

Individuals with germline, heterozygous, exonuclease domain PV in POLE and POLD1 historically presented in adulthood with colorectal, endometrial, and other cancers and were diagnosed with polymerase proofreading–associated polyposis syndrome ( 58 ). However, a recent pooled analysis suggested that polyposis was overall common, and both colorectal carcinomas and duodenal polyps could present in adolescents ( 58 ). In addition, aggressive CNS malignancies including gliomas and medulloblastoma have been described in children and young adults ( 24 , 25 , 58 – 60 ). However, robust genotype–phenotype correlations are yet to be unearthed ( 58 ). Akin to LS, without awareness, the germline diagnosis and its implication can be missed for these patients. Importantly, some children with germline heterozygous POLE PV can present with phenotypic overlap with CMMRD, including presence of café-au-lait macules ( 22 , 60 ). In addition, there are reports of patients with germline compound heterozygous PVs in POLE and MMR genes who have phenotypic mimicry of CMMRD and develop similar cancers ( 25 ). On the other hand, it is important to recognize that with increased availability of sequencing, variants can be reported in both patients’ tumor and their germline. In such situations, the true functional impact of the reported exonuclease domain variant needs to be evaluated comprehensively using existing data and appropriate tests, and thereafter its possible germline etiology needs to be investigated if indicated ( 2 , 6 , 61 ).

For patients with germline POLE/POLD1 PV, we recommend following the recently published guidelines ( 58 ), which include surveillance colonoscopies every other year starting from age 14 years, upper gastrointestinal surveillance from age 25 years ( 58 ), and gynecological evaluations/surveillance later in life ( 58 , 62 ). In addition, based on the experience of our group as well as previously published experience ( 58 ), we recommend that families and physicians maintain a heightened awareness for other cancers, especially malignant brain tumors. At present, there is insufficient evidence for offering a more intensive CMMRD-like surveillance for select patients deemed at higher risk based on their genotype and family history ( 6 , 63 ). It was agreed that more data needs to be collected in the future for these patients.

Surveillance plans for other rarer digenic patients may need to be individualized by discussing with an expert consortium (e.g., IRRDC/C4CMMRD). Treatment of cancers for all such individuals should follow the same principles as for all RRD cancers.

Other RRD syndromes

Data from patients with germline biallelic EPCAM 3′ deletions reveal that the tissue-(epithelium)-specific nature of the MSH2 deficiency results in multiple gastrointestinal cancers without involvement of other organs ( 64 ). Due to the aggressive gastrointestinal phenotype, the committee recommends offering annual upper and lower gastrointestinal surveillance for patents with this rare disorder from the time of diagnosis. Of note, patients with germline heterozygous 3′ deletions in EPCAM follow the same surveillance as those with MSH2 LS ( 47 , 58 ).

Biallelic MSH3 patients presenting with gastrointestinal polyposis and CNS tumors as young adults have been reported ( 21 ), but current evidence is insufficient to recommend CMMRD-like surveillance in childhood for patients with these rare genotypes.

ICI improves survival for children, adolescents, and young adults with aggressive and chemo-radiation refractory RRD cancers, regardless of their predisposition being from CMMRD, LS, germline POLE or digenic syndromes ( 7 , 8 , 65 ). Data demonstrate ICI efficacy in intra and extracranial solid tumors but not in T-cell malignancies ( 7 ). For lymphoma in patients with CMMRD, standard multiagent chemotherapy is effective for most patients ( 66 ). However, RRD solid cancers have poor survival with conventional therapies ( 67 ), possibly due to inherent resistance to specific chemotherapeutic agents related to MMR deficiency ( 9 ). ICI is increasingly being considered as a first-line therapy for many of these malignancies. High TMB and MSI are independent biomarkers for ICI response, driving high neoantigen expression and contributing to an immune-rich tumor microenvironment ( 7 ). For patients with synchronous and metachronous RRD cancers, ICI can be used as tumor-agnostic treatment for both cancers, ( 10 ) sparing the patient from reaching the maximal threshold of specific chemotherapy agents and radiation, which may be needed to treat future malignancies. It is important to emphasize that in contrast to other DNA damage repair syndromes, chemotherapy and radiotherapy can result in unacceptable toxicity, mismatch repair deficiency primarily affects DNA replication. Therefore in RRD, the normal tissue response to external genotoxic agents and radiotherapy is preserved and thus there is no indication to reduce the standard treatment doses for these children. In fact, re-irradiation has been reported to be safe and effective therapeutic strategy for RRD gliomas and adds to immune synergism with ICI ( 56 ). Lastly, it has been recently shown that regimens combining ICI agents allow continued immune surveillance and confers beneficial responses in patients progressing on ICI monotherapy ( 56 ).

The genetic testing and counseling article in this AACR CPWG series addresses many nuances and updates to testing children for CPS. Given specific considerations in patients and families with RRD syndromes, it is our recommendation that all families should receive genetic counseling. International consortia like the IRRDC and C4CMMRD also provide vital information and support, and these are resources of which health professionals, patients, and their families should be made aware. Cascade testing for RRD syndromes may be complicated when the proband’s germline multigene panel testing is non-diagnostic. In such cases, the options for ancillary functional analyses in the proband, and subsequently, cascade testing of parents, siblings, and other relatives should be discussed. Preconception/prenatal testing options should be discussed with patients with RRD and their families. Options may be limited or complicated if germline testing for the proband was non-diagnostic. If testing prior to or during pregnancy is not to (or cannot) be performed, testing after birth should be pursued through functional and other assays, to avoid increased surveillance in children who did not inherit the CPS. Testing siblings of a child with CMMRD may reveal an LS diagnosis. The diagnosis of pediatric cancer in a child with LS may also raise questions about testing siblings for LS. Genetic counseling may be helpful for these families in navigating these difficult situations. Usually for adult-onset CPS-like LS and polymerase proofreading–associated polyposis, genetic testing should generally be deferred for asymptomatic siblings until adulthood or until management could be impacted ( 19 ). However, if testing is pursued, comprehensive ongoing genetic counseling, pretest, post-test, and as the child develops, are important. Testing is recommended for those presenting with a cancer diagnosis after proper counseling.

Improvements in outcomes for RRD cancers and related predisposition syndromes require multidisciplinary and global collaboration. This is especially true for the rarer childhood phenotypes of LS and POLE variants, for which current data are limited to case reports or small series . Even for CMMRD, for which such collaborative efforts have been immensely fruitful, the surveillance guidelines recommended here will continue to be updated in the future. More data will accumulate on specific cancer risks in the emerging young adult population with CMMRD. The recently unearthed genotype–phenotype associations may allow for more personalization of management strategies, including the intensity and timing of tissue-specific surveillance strategies. As the genomic MSI-based functional assays become clinically validated mainstream tools, these will not only allow accurate diagnosis for more patients globally but may also allow personalization of surveillance by quantifying the genotype effects on cancer risk. Similarly, quantifying circulating tumor DNA in patients can help improve early detection of cancer and can potentially be incorporated into future surveillance strategies in addition to current imaging-based modalities. As children with CMMRD remain at risk of multiple cancers at an alarming rate of a new cancer every 2 years, prevention strategies are also being explored by the interest groups and if successful, will change our approach to overall management. Finally, despite the high burden of the disease, access to many such recent advancements unfortunately remains out-of-bounds for most low- and middle-income countries. Multipronged approaches, including broader availability of inexpensive and accurate diagnostic assays, tailoring of surveillance regimens, and equitable access to immunotherapy, need to be systematically explored to truly maximize patient benefit from such emerging discoveries on a global level.

J. Meade reports personal fees from Alexion Pharmaceuticals and American Society of Pediatric Hematology/Oncology and nonfinancial support from Children’s Oncology Group outside the submitted work. J.R. Hansford reports personal fees from Bayer Pharmaceuticals and Alexion Pharmaceuticals and other support from Servier International outside the submitted work. K.W. Schneider reports other support from AACR during the conduct of the study; personal fees from the University of Pennsylvania and ACMG outside the submitted work; and employment in a position to provide genetic counseling to children and their families with, or at-risk for, DNA replication-repair deficiency syndromes. H. Lesmana reports personal fees from Pharming and grants from VeloSano outside the submitted work. H.S. Scott reports other support from Medicare Benefits Schedule and grants and other support from Omico during the conduct of the study; other support from Children’s Cancer Institute outside the submitted work; and providing presentations for Roche Australia for fees. S.E. Plon reports grants from St. Baldrick’s Foundation during the conduct of the study, as well as being a member (unpaid) scientific advisor panel in Baylor Genetics. No disclosures were reported by the other authors.

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Nuclear Deformation Causes DNA Damage by Increasing Replication Stress

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• Other Affiliation: Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA.
• Affiliation: College of Arts and Sciences, Department of Physics and Astronomy
• Other Affiliation: Robert Frederick Smith School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY, USA
• Affiliation: College of Arts and Sciences, Department of Applied Physical Sciences
• Other Affiliation: Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA
• Cancer metastasis, i.e., the spreading of tumor cells from the primary tumor to distant organs, is responsible for the vast majority of cancer deaths. In the process, cancer cells migrate through narrow interstitial spaces substantially smaller in cross-section than the cell. During such confined migration, cancer cells experience extensive nuclear deformation, nuclear envelope rupture, and DNA damage. The molecular mechanisms responsible for the confined migration-induced DNA damage remain incompletely understood. Although in some cell lines, DNA damage is closely associated with nuclear envelope rupture, we show that, in others, mechanical deformation of the nucleus is sufficient to cause DNA damage, even in the absence of nuclear envelope rupture. This deformation-induced DNA damage, unlike nuclear-envelope-rupture-induced DNA damage, occurs primarily in S/G2 phase of the cell cycle and is associated with replication forks. Nuclear deformation, resulting from either confined migration or external cell compression, increases replication stress, possibly by increasing replication fork stalling, providing a molecular mechanism for the deformation-induced DNA damage. Thus, we have uncovered a new mechanism for mechanically induced DNA damage, linking mechanical deformation of the nucleus to DNA replication stress. This mechanically induced DNA damage could not only increase genomic instability in metastasizing cancer cells but could also cause DNA damage in non-migrating cells and tissues that experience mechanical compression during development, thereby contributing to tumorigenesis and DNA damage response activation.
• Genomic Instability
• Stress, Physiological
• Cell Nucleus
• Cell Movement
• DNA Replication
• Nuclear Envelope
• Carcinogenesis
• https://doi.org/10.17615/pafm-bw69
• https://doi.org/10.1016/j.cub.2020.11.037
• In Copyright
• Current Biology
• National Institute of Biomedical Imaging and Bioengineering
• Office of the Director
• National Institute of General Medical Sciences
• Volkswagen Foundation
• Howard Hughes Medical Institute
• Gordon and Betty Moore Foundation
• New York Stem Cell Foundation
• Directorate for Engineering
• Directorate for Mathematical & Physical Sciences
• National Heart Lung and Blood Institute
• Congressionally Directed Medical Research Programs
• Directorate for Biological Sciences
• National Cancer Institute
• Directorate for STEM Education

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DNA Replication and Repair

Oct 29, 2019

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DNA Replication and Repair. Learning Objectives. Student will be able to Discuss DNA Replication Explain DNA Repair Solve clinical problem. DNA Replication. The process of making an identical new DNA copy of a duplex ( double-stranded ) DNA , using existing DNA as a template.

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• dna replication
• dna polymerase
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Learning Objectives • Student will be able to • Discuss DNA Replication • Explain DNA Repair • Solve clinical problem

DNA Replication • The process of making an identical new DNA copy of a duplex (double-stranded) DNA, using existing DNA as a template. • In humans and other eukaryotes, replication occurs in the cell nucleus.   

DNA Replication • The genetic information found in DNA is copied and transmitted to daughter cells through DNA replication.

Do you have any Idea aboutprocess of DNA Replication ?

The flow of information from DNA to RNA to protein is termed as “central dogma” of molecular biology

Secret behind Central Dogma • The Central Dogma. This states that once ‘information’ has passed into protein it cannot get out again. In more detail, the transfer of information from nucleic acid to nucleic acid, or from nucleic acid to protein may be possible, but transfer from protein to protein, or from protein to nucleic acid is impossible. Information means here the precise determination of sequence, either of bases in the nucleic acid or of amino acid residues in the protein.

DNA Replication • DNA replication takes place by separation of the strands of the double helix, and synthesis of two daughter strands complementary to the two parental templates.

DNA Replication • DNA replication is called semiconservative because half of the parent structure is retained in each of the daughter duplexes.

Separation of the two DNA Strands • For the initiation of Replication Process • The two strands of the parental double stranded DNA dsDNA must first separate (or melt) over a small region. • Because polymerase use only ssDNA as a template.

In prokaryotes • DNA replication begins at a single unique nucleotide sequence, a site called the origin of the replication , or ori. • The ori includes short , AT-rich segments that facilitate melting.

In eukaryotes • Replication begins at multiple sites along the DNA helix. Having multiple origins of replication • Which provides the mechanism for rapidly replicating the great length of eukaryotic DNA molecule.

Proteins Required for DNA strand Separation • Initiation of replication requires the recognition of the origin by a group of the protein that forms a prepriming complex. • These proteins are • DNA A protein • DNA helicases • Single stranded DNA –binding protein

DNA A Protein • This protein binds to specific nucleotide sequences (DnaA boxes) within the origin of replication, causing tandamly arranged (one after the other) AT-rich regions in the origin to melt. • Melting is adenosine triphosphate (ATP) dependent and results in strand separation with the formation of localized region of ssDNA.

DNA helicases • These enzymes bind to ssDNA near the replication fork and then move into the neighboring double stranded region, forcing the strands apart (in effect , unwinding the double helix). • Helicases require energy provided by ATP. • Unwinding at the replication fork causes supercoiling in other regions of the DNA molecule.

Single stranded DNA-binding protein • This protein binds to the ssDNA generated by helicases. • The SSB protein are not enzymes, but rather serve to shift the equilibrium between dsDNA and ssDNA in the direction of a single stranded forms. • These proteins not only keep the strands of DNA separated in the area of the replication origin, but also protects the Dna from nucleases that degrade ssDNA

Supercoiling • As the two strands of double helix are separated a problem is encountered namely, the appearance of positive supercoils in the region of DNA ahead of the replication fork as a result of over winding. • And negative supercoils in the region behind the fork. • The accumulating positive supercoils interfere with further unwinding of the double helix.

To solve this problem there is a group of enzymes called Dnatopoisomerases responsible for removing supercoils in the helix by transiently cleaving one or both of the DNA strand.

Type I DNA topoisomerases • These enzyme reversibly cleaves one strand of the double helix. • They have both strand cutting and strand resealing activities. • They do not require ATP but store energy from phosphodiester bond they cleave. • Reuse the energy to reseal the strand

Each time a transient “nick” is created in one DNA strand, the intact DNA strand is passed through the break before it is resealed, thus relieving (“relaxing”) accumulated supercoils.

Type II DNA topoisomerases • These enzymes bind tightly to the DNA double helix and make transient breaks in both strands. • The enzyme then causes a second stretch of the DNA double helix to pass through the break and, finally, reseals the break (Figure 29.13). As a result, both negative and positive supercoils can be relieved by this ATP-requiring process.

Direction of DNA replication • The DNA polymerases responsible for copying the DNA templates are only able to “read” the parental nucleotide sequences in the 3'→5' direction, and they synthesize the new DNA strands only in the 5'→3' (antiparallel) direction. • Therefore, beginning with one parental double helix, the two newly synthesized stretches of nucleotide chains must grow in opposite directions— • one in the 5'→3' direction toward the replication fork and one in the 5'→3‘ direction away from the replication fork.

Leading strand: The strand that is being copied in the direction of the advancing replication fork is called the leading strand and is synthesized continuously. • Lagging strand: The strand that is being copied in the direction away from the replication fork is synthesized discontinuously, with small fragments of DNA being copied near the replication fork. • These short stretches of discontinuous DNA, termed Okazaki fragments, are eventually joined (ligated) to become a single, continuous strand. The new strand of DNA produced by this mechanism is termed the lagging strand

RNA primer • DNA polymerases cannot initiate synthesis of a complementary strand of DNA on a totally single-stranded template. Rather, they require an RNA primer—that is, a short, double-stranded region consisting of RNA base-paired to the DNA template, with a free hydroxyl group on the 3'-end of the RNA strand (Figure 29.15). • This hydroxyl group serves as the first acceptor of a deoxynucleotide by action of DNA polymerase.

Primase • A specific RNA polymerase, called primase (DnaG), synthesizes the short stretches of RNA (approximately ten nucleotides long) that are complementary and antiparallel to the DNA template. In the resulting hybrid duplex, the U in RNA pairs with A in DNA. As shown in Figure, these short RNA sequences are constantly being synthesized at the replication fork on the lagging strand, but only one RNA sequence at the origin of replication is required on the leading strand.

The substrates for this process are 5'-ribonucleoside triphosphates, and pyrophosphate is released as each ribonucleosidemonophosphate is added through formation of a 3'→5‘ phosphodiester bond. • [Note:The RNA primer is later removed]

Primosome • The addition of primase converts the prepriming complex of proteins required for DNA strand separation to a primosome. The primosome makes the RNA primer required for leading strand synthesis, and initiates Okazaki fragment formation in lagging strand synthesis. As with DNA synthesis, the direction of synthesis of the primer is 5'→3'.

Chain elongation • Prokaryotic (and eukaryotic) DNA polymerases elongate a new DNA strand by adding deoxyribonucleotides, one at a time, to the 3'- end of the growing chain. • The sequence of nucleotides that are added is dictated by the base sequence of the template strand with which the incoming nucleotides are paired.

DNA polymerase III • DNA chain elongation is catalyzed by DNA • polymerase III. Using the 3'-hydroxyl group of the RNA primer as the acceptor of the first deoxyribonucleotide, DNA polymerase III begins to add nucleotides along the single-stranded template that specifies the sequence of bases in the newly synthesized chain.

DNA polymerase III is a highly processive” enzyme—that is, it remains bound to the template strand as it moves along, and does not diffuse away and then rebind before adding each new nucleotide. The processivity of DNA polymerase III is the result of its β subunit forming a ring that encircles and moves along the • template strand of the DNA, thus serving as a sliding DNA clamp.

The new strand grows in the 5 – 3 direction , antiparallel to the parental strand. • All four substrates deoxyadenosinetriphosphate, deoxythymidinetriphosphate , deoxycytidinetriphosphate and deoxyguanosinetriphosphate must be present for DNA elongation to occur. If any one in short supply DNA synthesis will stop.

DNA Replication • Exonuclease Activities of DNA Polymerases • DNA polymerase I is involved in DNA repair and also removes RNA primers and replaces them with DNA. • Exonucleases degrade nucleic acids by removing 5’ or 3’ terminal nucleotides.

The exonuclease activities ofDNA polymerase I

DNA Replication (18) • Initiation of Replication in Eukaryotic Cells • Eukaryotes replicate their genome in small portions (replicons). • Initiation of DNA synthesis in a replicon is regulated.

DNA Replication • The Eukaryotic Replication Fork • Replication activities are similar in eukaryotes and prokaryotes. • There are several DNA polymerases in eukaryotes. • Eukaryotic DNA polymerases elongate in the 5’-to-3’ direction and require a primer; some have 3’-to-5’ exonuclease activity.

Some Proteins Required for EukaryoticDNA Replication

DNA Repair • DNA repair is essential for cell survival. • DNA is the cell molecule most susceptible to environmental damage. • Ionizing radiation, common chemicals, UV radiation and thermal energy create spontaneous alteration (lesions) in DNA. • Cells have a number of mechanisms to repair genetic damage.

A pyrimidine dimer that has formed within a DNA duplex following UV irradiation

Nucleotide excision repair (NER) removes bulky lesions, such as pyrimidinedimers and chemically altered nucleotides. • It consists of two pathways: • A transcription-coupled pathway which is the preferential pathway and selectively repairs genes of greatest importance to the cell. • A global genomic pathway which is less efficient and corrects DNA strands in the remainder of the genome.

DNA Repair • Nucleotide excision repair (continued) • TFIIH is a key component of the repair machinery and is also involved in the initiation for transcription. It links transcription and DNA repair. • A pair of endonucleases cut on both sides of the lesion, and the damaged strand is removed by helicase. • The gap is filled by a DNA polymerase and sealed by DNA ligase.

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• Open access
• Published: 14 August 2024

Molecular regulation of DNA damage and repair in female infertility: a systematic review

• Xiuhua Xu 1 , 2   na1 ,
• Ziwei Wang 1   na1 ,
• Luyi Lv 1 ,
• Lili Wang 1 ,
• Ya-nan Sun 1 ,
• Zhiming Zhao 1 ,
• Baojun Shi 1 ,
• Qian Li 2 &
• Gui-min Hao 1  

Reproductive Biology and Endocrinology volume  22 , Article number:  103 ( 2024 ) Cite this article

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DNA damage is a key factor affecting gametogenesis and embryo development. The integrity and stability of DNA are fundamental to a woman’s successful conception, embryonic development, pregnancy and the production of healthy offspring. Aging, reactive oxygen species, radiation therapy, and chemotherapy often induce oocyte DNA damage, diminished ovarian reserve, and infertility in women. With the increase of infertility population, there is an increasing need to study the relationship between infertility related diseases and DNA damage and repair. Researchers have tried various methods to reduce DNA damage in oocytes and enhance their DNA repair capabilities in an attempt to protect oocytes. In this review, we summarize recent advances in the DNA damage response mechanisms in infertility diseases such as PCOS, endometriosis, diminished ovarian reserve and hydrosalpinx, which has important implications for fertility preservation.

Introduction

DNA damage is a form of cellular stress, defined as any type of alteration in the DNA that disrupts its primary functions (replication and transcription) [ 1 ]. Cells possess DNA damage response (DDR) mechanisms to counter DNA damage. The role of the DDR is to detect damaged DNA and signal repair. The DNA damage response is composed of a variety of proteins, which can be divided into sensors, mediators, transducers and effectors. Cell dysfunction and death, as well as carcinogenesis and the aging process, are all linked to DNA damage. Human genome faces about one million lesions per day, such as adducts, modifications, or fragmentation of the sugar phosphate backbone of DNA [ 2 ].Without repaired, mutations such as base substitutions and chromosomal translocations may occur, and interfering normal gene expression and producing abnormal protein molecules. To cope with such damage, cells have a variety of enzymes and mechanisms for DNA-repair. Defects in one or more of the key components of these pathways can lead to unstable genome spread. This instability can lead to the death of germ cells (a way of eliminating abnormal cells) or cellular changes (a key step in cancer development). Exogenous and endogenous are the two forms of DNA damage.

Exogenous factors of DNA damage

Damage induced by environmental forces is referred to as exogenous damage. Exogenous factors are mainly divided into two categories, namely physical impacts and chemical impacts. The most common ambient physical agents that damage DNA include ionizing and non-ionizing radiation, both natural and manufactured. The majority of ionizing radiation (IR)-induced DNA damage is caused by interactions with hydroxyl radicals produced by radiolysis of water. And the most common DNA damage caused by IR includes ‘simple’ oxidative damage, for instance, modified bases, DNA single-strand breaks (SSB), or abasic sites, as well as more “complex” clustered lesions, or double-strand breaks (DSB) [ 3 ] Ultraviolet (UV) light from the sun is the main source of non-IR-induced DNA damage. Nearby pyrimidines dimerize to create helix-distorting photoproducts, which are the most critical UV targets. In addition, a range of genotoxic compounds present in the environment such as air, soil, water and food. They generate ROS that produce base modifications or SSBs. Occupational exposure to DNA damaging chemicals may occur in particular industrial, laboratory, and clinical settings, in addition to unintentional environmental exposure.

Endogenous factors of DNA damage

The excess of reactive oxygen species (ROS) is one of the most important endogenous factors causing DNA damage. Hydroxyl radicals, oxygen atoms, hydrogen peroxide, and superoxide radicals all belong to ROS. Cellular defense systems, such as ROS-scavenging enzymes, antioxidant enzymes, and vitamins can usually neutralize these reactive chemicals. Free radicals are extremely active and unstable. Oxidative stress ensues when the production of free radicals exceeds the amount of the body’s natural antioxidant defenses. And they become stable by absorbing electrons from nucleic acids, lipids, proteins, carbohydrates, or any other adjacent molecule, culminating in a chain reaction that damages DNA and cells. Next, ROS bind to macromolecules such as lipids, proteins, DNA and damage them. Abasic sites and DNA strand breaks are two of the more common lesions caused by ROS action on the glycophosphate skeleton. In the repair process, such lesions are also the result of enzymatic processing of oxidized bases [ 4 ].

In the reproductive system, DNA damage and repair are important. Infertile men have abnormal sperm. ROS causes strand breaks in sperm DNA, resulting in base loss or base modifications such as 8-oxy-G. Sperm DNA damage has been positively correlated with lower fertilization rates in IVF, impaired implantation rates, increased incidence of miscarriage and disease in the offspring, including childhood cancer [ 5 ]. However, less attention has been paid to oxidative stress and infertility in women, but there is a negative correlation between the ability to fertilize oocytes and elevated DNA damage in cumulus cells [ 6 , 7 ]. A study performed chromosomal analysis on patients who had two or more spontaneous miscarriages or who had not been pregnant for more than 2 years, in which all known causes of miscarriage were excluded to classify true idiopathic infertility. DNA damage (MN frequency) increased in infertile or aborted couples compared to fertile couples with no history of miscarriage and children younger than 2 years old [ 8 ]. In addition, increased MN frequency has been shown to be associated with recurrent miscarriage (at least three consecutive miscarriages) [ 9 ]. DNA integrity and stability are important factors for cell survival. This is especially true for female germ cells. DNA integrity is fundamental to successful conception, pregnancy, embryo development and healthy offspring. About 15% of infertility problems in men and 10% in women can be attributed to genetic abnormalities [ 10 ]. In this review, we summarize recent advances in the DNA damage response mechanisms in infertility diseases such as PCOS, endometriosis, diminished ovarian reserve and hydrosalpinx, which has important implications for fertility preservation. This article describes the current research status of DNA damage and repair mechanisms in female infertility, emphasizes the important role of oocyte DNA damage in aging and fertility decline. This area of research will potentially lead to new ideas for the prevention of female infertility, early detection of female infertility and treatment of female infertility at the genetic level, further contributing to the knowledge and understanding of female reproductive health.

Classic DDR and DNA repair mechanisms in somatic cells

DDR involves many cellular responses, including cell cycle arrest, chromatin remodeling, damage repair, and apoptosis, which are one of the more comprehensive cellular responses to stimuli [ 11 ]. The DDR mechanism is mainly activated by cells in the G1/S and G2/M phases. Ataxia telangiectasia mutated (ATM) and ATM and Rad3 related (ATR) proteins are important DNA damage checkpoint kinases. In the G1 phase, ATM and ATR kinases are recruited to DNA damage sites and undergo phosphorylation, immediately activating downstream checkpoint kinase1 (CHK1) and checkpoint kinase2 (CHK2) [ 12 , 13 ]. The CHK1 and CHK2 kinases continue to activate downstream effecter p53. Under the mediation of p53, p21 binds and inhibits the activity of cyclin-dependent kinase (CDK), thereby causing cell cycle arrest [ 14 , 15 ]. In the G2 phase, DNA damage sites also recruit and activate ATM/ATR kinases and CHK1/CHK2 kinases. The difference is that the CHK1/CHK2 kinase inhibits the activation of CDK1 by inhibiting cell division cyclin25 (CDC25) phosphatase, leading to cell cycle arrest [ 15 ]. During cell cycle arrest, cells repair damaged DNA through complex mechanisms. After DNA repair is completed, the DNA damage checkpoint kinase undergoes dephosphorylation and the cell cycle resumes [ 15 ]. When DNA damage cannot be fully repaired, p53 activates the transcription of pro apoptotic genes such as p53 upregulated modulator of apoptosis (Puma) and phorbol-12-myristate-13-acetate induced protein 1 (Pmaip1/Noxa), thereby inducing cell apoptosis [ 16 , 17 ].

For different types of DNA damage, cells can be repaired by appropriate DNA repair mechanisms. Base mismatches that occur during DNA replication can be corrected by mismatch repair (MMR) mechanisms; Bases that undergo minor chemical changes can be removed by the base excision repair (BER) mechanism; Larger DNA lesions can be removed through the nucleotide excision repair (NER) mechanism; The repair process of DNA single strand breaks involves a series of enzyme cascade reactions; Homologous recombination (HR) and non homologous end joining (NHEJ) are two mechanisms for repairing DSBs; The mechanism for elimination of ICLs involves a complex set of reactions related to Fanconi anemia proteins [ 18 ]. It is generally believed that DSBs are the most severe types of DNA damage, which can lead to genome rearrangement and structural changes, such as deletions, translocations, fusion, etc. [ 11 , 18 ]. The repair mechanisms of DSBs include HR and NHEJ. In the HR repair process, MRN complexes are first recruited to the ends of DSBs and the DNA ends are processed and cleaved to produce single stranded DNA [ 19 ]. Afterwards, replication protein A (RPA) wraps single-stranded DNA to protect it from nuclease action and remove its secondary structure. Under the mediation of breast cancer protein 2 (BRCA2), RPA is replaced by DNA repair protein RAD51 (DNA repair protein RAD51). Subsequently, RAD51 mediated the invasion of single strand DNA into the uninjured sisters’ chromosome [ 20 , 21 ]. Finally, under the action of polymerase, nuclease, helicase, and other molecules, DNA is extended and repaired [ 22 , 23 ]. In contrast to HR, NHEJ directly connects the ends of DSBs through DNA ligases. First, Ku70/Ku80 proteins recognize and bind to the ends of DSBs, followed by recruitment and activation of DNA dependent protein kinase catalytic subunits (DNA-PKcs) [ 24 ]. Afterwards, DNA-PKcs recruits the recombinant enzyme Artemis to process the DNA ends, while also recruiting a protein complex composed of X-ray repair cross complementing protein4 (XRCC4) and DNA ligase 4 (DNAligase4, LIG4) to connect the DNA ends [ 24 , 25 ]. Finally, under the action of polymerase, nuclease, helicase, and other molecules, DNA is extended and repaired [ 22 , 23 ]. Unlike HR, NHEJ directly connects the ends of DSBs through DNA ligases. Firstly, Ku70/Ku80 proteins recognize and bind to the ends of DSBs, followed by recruitment and activation of DNA dependent protein kinase catalytic subunits (DNA-PKcs) [ 24 ]. Afterwards, DNA-PKcs recruited the recombinant enzyme Artemis to process the DNA ends, while also recruiting a protein complex composed of X-ray repair cross complementing protein4 (XRCC4) and DNA ligase 4 (DNAligase4, LIG4) to connect the DNA ends [ 24 , 25 ]. HR occurs in the S and G2 phases of the cell cycle and is repaired with undamaged sister chromosomes, making it more precise. NHEJ directly connects the two ends of DSBs together, although imprecisely, it can operate throughout the entire cell cycle [ 23 , 26 ]. It is widely believed that the accumulation of DNA damage and incorrect DNA repair can easily cause gene mutations and chromosomal aberrations, leading to the decline and loss of cellular function, which may promote aging and the occurrence of diseases [ 11 , 27 ]. Therefore, it is essential that cells maintain the stability and integrity of their genome.

DNA damage and oxidative stress in female reproductive system

Researchers believe that 40% of infertility is caused by male factors with abnormal sperm. Low sperm count, poor motility, and aberrant morphology are all examples of abnormal sperm. 40% of infertility is caused by female factors. The reason for the last 20% is uncertain. The main contributors to the genetic reasons of infertility and recurrent miscarriage are chromosomal abnormalities.

DNA damage in oocyte

The sensitivity of the oocyte to DNA damage is less well documented than that of the spermatozoon, possibly due to the difficulty of acquiring oocytes for research. However, oocytes are known to be more susceptible to external stimulation at certain times. Oocytes are more susceptible to DNA damage during division.

Oogenesis is the process of producing mature oocytes after mitosis and meiosis. During prenatal development, oogenesis begins in the ovary and then stops. Rapid cell division creates 7 million oogonia during the second and seventh month of pregnancy, which are either destined to develop and form germ cell cysts, or disappear through natural atresia. Breakdown of the germ cell cysts is accompanied by the transition of precursor oocytes, enveloped by a single layer of flattened follicular epithelial cells, into primordial follicles [ 28 ]. Immediately after birth, the first phase of meiosis begins. The primordial follicle is particularly susceptible to DNA damage due to its extreme longevity and the unique design of the primordial nucleus [ 29 ].

As mentioned above, DNA damage and apoptosis may be related to fertility and posed a certain threat to fertility. Currently, it is more common in DNA damage caused by anticancer therapy. In detail, the major molecular mechanism of the depletion of ovarian reserve due to exposure to genotoxic stressors is apoptosis mediated by transactivation of p63 (TAp63) [ 30 ]. The effect of TAp63 on the apoptotic pathway is mainly achieved through several processes. Ataxia telangiectasia (mutated) (ATM) kinase and checkpoint kinase 2 (CHK2) trigger the phosphorylation process required for TAP63 activation. This process promotes the induced transcription of BH3-only pro-apoptotic factors, PUMA, and NOXA. The upregulation of these pro-apoptotic factors promotes their interaction with the pro-apoptotic BCL2 family members BAX and BAK. As a result, mitochondrial apoptotic proteins are released, and the crucial proteolytic enzyme caspase-9 for apoptosis and death is activated [ 31 ]. Under the premise of evolution, removing damaged oocytes avoids the risk of passing the mutation on to offspring. This will undoubtedly affect reproductive function and lead to infertility. It is expected to be able to cope with damage or repair through this period of its developmental arrest.

Primordial follicles are released from arrest after birth and continue to develop into primary oocytes. TAP63 levels remain high in primary oocytes, which are also dependent on TAp63-mediated DNA damage responses, but not found in advanced follicles after ovulation [ 32 ]. Taken together, these findings suggest that TAp63 has a conserved function in the removal of defective oocytes from the germline in both primary and primordial follicles.

There may be a way of genetic protection of the female reproductive system without TAp63. Through experiments with rats, researchers found that when the ovaries were exposed to bisphenol A (BPA), a group of DNA repair genes involved in the classic double-strand break (DSB) DNA repair pathway were considerably up-regulated in just 24 hours. However, it was also found that there is a certain threshold of exposure source, beyond which the repair effect fails and apoptosis begins. This can lead to impaired ovarian function [ 33 ].

In contrast to the growing follicle, oocyte at the germinal vesicle stage is transcriptionally dormant. Studies in mice have shown that oocytes in preovulatory follicles have the ability to recognize DNA damage. This can be seen from the development of ɣH2AX foci after exogenous DSB induction. The phosphorylation of H2AX requires both MRN complex and ATM activation. ATM kinase, the master regulator of DNA damage response pathway, cannot be effectively activated in mature GV oocytes, so oocytes carrying DSB DNA have the opportunity to enter the first meiotic M-phase (MI), unless the degrees of damage are quite high [ 34 ]. This explains why oocytes are particularly vulnerable to the accumulation of DNA damage, because of their prolonged pause in meiotic prophase.

In addition, during the G2/M stage of meiosis, the spindle assembly checkpoint (SAC) protects the integrity of the female germline, by examining the state of kinetochore–microtubule attachment and inhibiting the activity of the anaphase-promoting complex (APC) before chromosomes are ready for full division to prevent the occurrence of aneuploidy [ 35 ]. The SAC acts as a gatekeeper. Ovotoxins, UV-B, and ionizing radiation can activate the SAC, which in turn leads to the failure of oocytes from MI to MII. In mice, researchers discovered that roughly 53 proteins are involved in DNA repair, replication, and recombination in oocytes. Including double-strand break (DSB) DNA repair, base excision repair (BER), single-strand break (SSB) repair and any other proteins associated with similar pathways [ 36 ]. There is a basal BER activity in MII oocytes, and this pathway repairs oxidation-induced DNA damage. MII oocytes may have proteins necessary to carry out this repair. Thus, this pathway reduces the burden of oocytes carrying oxidative DNA damage. Taken together, this is the susceptibility of oocytes to DNA damage, and the mechanisms of their defense.

Oxidative stress in oocyte

In the ovary, oocytes are exposed to a variety of reactive oxygen species. This is necessary for normal reproductive activity. Oocyte maturation and ovulation can be compared with an inflammatory response, resulting in the production of large amounts of ROS [ 37 ]. Oocytes have a certain resistance to it, so as we can see the high levels of antioxidants in follicular fluids may be related to that. The biomarkers of the oxidative stress (OS) have already been found nowadays. The expression of antioxidants such as Cu-Zn superoxide dismutase (SOD), Mn-SOD and glutathione peroxidase(GSH-Px) in the follicular microenvironment, which may be related to folliculogenesis, maturation and luteal function, and they are well expressed in MII period [ 38 ]. Reduced glutathione (GSH) is found in abundance in all mammalian cells and acts as a potent antioxidant. Decreased GSH levels have been linked to increased oxidative stress. Low fertilization was associated with downregulation of GSH-Px. Nitric oxide (NO) is also an unstable free radical that can participate in ROS reactions. It also directly acts on DNA to deaminate it. Low levels of NO in the follicular fluid are associated with the eventual fertilization success of the oocytes. And it is negatively correlated with embryo quality and cleavage rate [ 39 ]. H 2 O 2 is a very weak oxidant. DNA damage caused by H 2 O 2 cannot be mediated by H 2 O 2 alone. H 2 O 2 can penetrate cell membranes quickly, and once inside, it binds to iron and copper ions to produce more harmful substances, such as hydroxyl radicals. Hydroxyl radicals effectively interact with DNA, resulting in single- and double- strand breaks. Certain substances and ionizing radiation induce DNA strand breaks by producing hydroxyl radicals.

In some cases, such as certain diseases or ovarian lesions, elevated levels of oxidants in the oocyte or disrupted of the balance between oxidative stress and antioxidants resulting in higher ROS than normal levels, which can affect the oocyte quality, altering its cytoskeleton and microtubules, lead to chromosomal abnormalities that ultimately affect fertilization. Guanine is the most easily to oxidation of the four bases. 8-oxoG is the most often oxidized form of guanine, which is the causal molecule for spontaneous and inheritable germ lineage cell mutations. And it is endogenously produced by ROS [ 40 ]. In addition, increased ROS levels in the tubal and peritoneal environment may affect gametes in the fallopian tube, and their ability to interact and syngamy.

Infertility and DNA damage

Polycystic ovary syndrome and dna damage.

Polycystic ovary syndrome (PCOS) is one of the most common endocrine diseases in women of reproductive age. It is a heterogeneous disease with different clinical and endocrine manifestations. It is a disorder with a wide range of phenotypes such as obesity, hyperandrogenism, irregular menstrual cycles, anovulation, ovarian cysts, and low-grade chronic inflammation-related stress indicators.

Testosterone(T)

Plasma lipid peroxides are increased in PCOS patients, and testosterone has an inductive influence on lipid peroxidation. Hyperandrogenism combined with reduced catalase activity causes free O 2 and peroxynitrite accumulation in PCOS women. These endogenous free radicals can cause lipid peroxidation. In turn, faster lipid metabolism leads to increased oxidative damage. The results showed that H 2 O 2 , hydroxyl radical production and lipid peroxidation were enhanced and GSH levels decreased after treatment of androgen-responsive prostate cancer cell lines with physiological levels of androgens [ 41 ]. In animal experiments, it was found that after long-term combined use of testosterone and estrogen, the DNA strand of the dorsolateral prostate in rats was broken and lipid peroxidation was increased [ 42 ]. In humans, free testosterone is positively correlated with DNA strand breaks, as is H 2 O 2 -induced DNA damage. Therefore, it is believed that women with PCOS may have more DNA strand breaks as a result of increased androgen production.

Estrogen(E2)

Estrogen levels are elevated in PCOS patients. Although estrogen is considered a kind of antioxidant, some of its compounds also metabolize to produce ROS [ 43 ]. Estrogen can interconvert between reduced and oxidized states, potentially generating ROS that can damage DNA [ 44 ]. Studies of estrogen-induced development of DNA adjuncts in Syrian hamster kidneys suggested possible mechanisms of estrogen-induced DNA damage. It was found that estrogen could be metabolized to catechol estrogens, which then undergo an oxidative cycle to produce hemiquinones, reactive oxygen species that are extremely destructive to DNA. Other oxidation-generating processes, such as prostaglandin synthesis or the phage/leukocyte infiltration pathway, may be activated in response to the combined effects of testosterone and estrogen, leading to DNA damage and lipid peroxidation [ 42 ].

Glutathione (GSH)

In PCOS patients, ROS is induced by increased androgen production, which may lead to depletion of GSH. GSH can protect cells by antagonizing and react with ROS through its thiol group in the reduced state. Glutathione plays an important role in cell metabolism. Activation of transcription factors that affect gene expression has been shown to depend on intracellular GSH levels [ 45 ]. In addition, decreased GSH levels may enhance the oxidative stress vulnerability of biological components such as DNA.

Body mass index(BMI)

In addition to effects of hormones on PCOS patients, obesity is also related to the development of PCOS. The average frequency of DNA damage is positively correlated with waist circumference (WC) [ 46 ]. Obesity may be linked to certain oxidants [ 47 ]. Obesity is known to induce an increase in free radicals, which can contribute to cellular DNA damage. The free radicals may be the link between DNA damage and obesity status. Some researchers have found that high BMI leads to increased frequency of micronuclei, a form of chromosomal aberration in interphase cells that may be associated with loss of centromeres during anaphase and DNA damage. Meanwhile, there may also be a correlation between oxidative stress and inflammation in PCOS patients, which are also factors inducing DNA damage. Among them, low-grade inflammation with obesity is most likely to contribute to the pathogenesis of PCOS [ 48 ].

In addition, PCOS patients are often accompanied by metabolic syndrome (MS), and there is also a strong link between obesity and metabolic syndrome, each of them makes patients suffer from genetic damage. Obese women with MS have significantly higher levels of DNA damage compared to healthy non-obese people [ 46 ], and with the progression of MS disease severity, antioxidant capacity decreases and DNA damage increases. Meanwhile, studies have shown that MS patients have lower levels of total antioxidant capacity (TAC) and higher levels of DNA damage and oxidative stress index (OSI). Insulin resistance exists is a characteristic of PCOS and PCOS patients with MS. Insulin resistance causes oxidative stress by activating NADPH oxidase, which response to free radicals or extremely reactive oxygen species. Elevated blood glucose caused by abnormal glucose metabolism may exacerbate oxidative stress by promoting the generation of reactive oxygen species and diminishing the body’s natural antioxidant defenses [ 47 ].

Animal studies have also found that a high-fat diet can cause weight increase in mice and DNA damage in many organs [ 49 ]. There is a correlation between DNA damage and overweight. Weight gain impacts the DNA repair system through many molecular pathways. The brain and ovary are the organs with the lowest DNA repair activity. In addition, increased body weight can lead to DNA damage through decreased telomere length, inflammation, hormonal effects and reactive oxygen species formation [ 50 ]. It is now known that nucleotide excision repair (NER) proteins play an important role in cellular regulation of oxidative DNA damage. In peripheral blood lymphocytes, a negative correlation was found between NER levels and BMI in humans. Therefore, the lower activity of this repair pathway in obese animals may be related to the oxidation of DNA bases. Impaired repair processes lead to genomic instability in obese animals. Weight loss reduces the oxidative stress associated with obesity, and after weight loss, an increase in NER activity has been found in multiple organs such as liver, colon, testes, and ovaries.

Inflammation

Low-grade chronic inflammation is closely associated with PCOS. Increased inflammation can lead to insulin resistance, leading to a “vicious cycle” of metabolic disorders in PCOS patients [ 51 ]. Inflammatory markers and genetic markers were higher in PCOS patients. Women with PCOS had elevated CRP, interleukin 18(IL-18), interleukin 6(IL-6), and tumor necrosis factor (TNF-α) and white blood cells (WBC) compared with age- and BMI-matched controls. Its hyperinsulinemia, obesity, hyperandrogenism, and inflammatory states are interrelated. Inflammatory factors can also cause endothelial cell dysfunction, which in turn leads to the risk of cardiovascular disease. In addition, Iron overload exists in PCOS patients, and high levels of ferritin and transferrin can lead to decreased levels of anti-inflammatory factors and antioxidant molecules [ 52 ].

These all prove that the inflammatory response is higher in PCOS patients. Some studies suggest that this is inseparable from the disease characteristics of PCOS. If the WBC of PCOS patient is higher than that of the normal population, it may be positively correlated with androgens, insulin resistance and BMI. Meanwhile, with a multiple regression analysis, the researchers found that testosterone was one of the predictors of WBC [ 53 ]. Thus, androgens play an important role in the development and activation of WBCs, as well as in low-grade inflammation. In addition to androgens, as we mentioned above, obesity is also one of the triggers of inflammation. As obesity increases, so does its inflammatory state. In obese women, an imbalance between classically activated macrophage (MI) and alternatively activated macrophage (M2) is observed. The higher concentration of MI-type macrophages, which are associated with pro-inflammatory factors, suggests that pro-inflammatory processes dominate, leading to low-grade chronic inflammation throughout the body [ 54 ]. In addition, elevated levels of leptin may also be associated with obesity status, and leptin also has pro-inflammatory effects. Hyperleptinemia further promotes the production of pro-inflammatory cytokines [ 55 ]. Insulin resistance and hyperglycemia also have similar effects in PCOS patients.

Inflammation can lead to accelerated mutagenesis and gene instability [ 56 ]. DNA damage caused by inflammation is mostly related to reactive oxygen and nitrogen species (RONS). RONS secreted by immune cells destroy pathogens, but can also damage neighboring human cells. Most importantly, RONS can cause efficient mutagenesis in DNA. Inflammatory responses may exacerbate their production or effect. For example, NO is a pleiotropic RONS that is an indispensable signaling molecule at concentrations below 400nM. However, during inflammation, immune cells produce high levels of NO, which can induce apoptosis through protein nitrosation, nitration, and alkylation when NO concentrations approach or exceed 1µM [ 57 ]. In addition, macrophages and neutrophils can produce superoxide and participate in enzymatic reactions, producing a series of RONS [ 58 ]. Apart from that, proinflammatory cytokines can also lead to the production of intracellular RONS. RONS can cause DNA damage in multiple ways as RONS are strong oxidants. As we mentioned above, strong oxidants can cause DNA damage. Nitrosated RONS can cause deamination of DNA bases. Deamination products are especially mutagenic, and the corresponding chemical reactions can occur on hydrogen bonds, eventually leading to base mismatches. Inflammatory cells, such as neutrophils, can also produce hypohalous acid [ 59 ]. These hypohalous acids easily react with DNA to form adducts during inflammation, which is even higher than the accumulation of DNA damage caused by oxidation, deamination, or lipid peroxidation.

In conclusion, the DNA damage in PCOS patients is higher than that in the normal population. This may be associated with increased levels of estrogen, androgen, obesity, decreased glutathione levels, and high levels of inflammation.

Endometriosis and DNA damage

Endometriosis (EMS) is a common benign and estrogen dependent disease in fertile women. The incidence among women in fertility is about 10–15%, which is often accompanied by chronic pelvic pain and infertility. Its main characteristic is the growth of endometrioid tissue outside the uterus.

Estrogen and its metabolites have been recognized as genotoxic mutagens. Evidence from animal studies suggests a causal relationship between exposure to environmental factors, such as dioxins, and endometriosis. Although the serum estrogen in patients with endometriosis is not high, the local estrogen concentration in ectopic lesions is significantly increased. Estrogen receptors α and β are encoded by the ESR1 and ESR2 genes located on non-homologous chromosomes. The expression of ERα is higher than that of ERβ in normal endometrium, but in endometriosis, the opposite is true [ 60 , 61 ]. This may be related to the hypermethylation of ESR1 promoter. In endometriosis, the main manifestation is the high expression of local estrogen mainly mediated by ERβ. In addition, the expression of enzymes that mediate estrogen synthesis also plays a crucial role. The expression level of steroidogenic acute regulatory protein(StAR), aromatase, 17β-hydroxysteroid dehydrogenase(17β-HSD) in ectopic lesions of patients with endometriosis is significantly higher than that in normal endometrial tissue [ 62 ].

Natural estrogen plays an important role in regulation. Physiological concentrations of estrogen are essential for maintaining cell growth and several other biological activities. In addition, elevated estrogen levels are known to have adverse effects such as embryotoxicity, teratogenicity, and carcinogenicity. Estrogens are genotoxic and reactive estrogen metabolites may act at the genetic and chromosomal levels. Some researchers found that hamsters exposed to E 2 also occurred in vivo-induced DNA single-strand breaks [ 63 ]. Although it is known that estrogen and its metabolites can cause a lot of DNA damage, whether estrogen in patients with endometriosis can cause corresponding damage still lacks relevant evidence-based medicine evidence, and further research is needed.

Iron overload

Iron is essential for cell growth and metabolism. Low molecular weight iron pools are a major source of toxic iron, which can be reduced by binding to corresponding proteins. This is also one of the ways in which iron regulates the production of reactive oxygen species. Reflux of menstrual blood leads to rapid increase of iron and heme content in EM lesions in a short period of time. Compared with women without endometriosis, patients with endometriosis had significantly higher levels of iron and ferritin in the peritoneal fluid, so as the lipid peroxidation in low-density lipoprotein (LDL) [ 64 ]. Hemorrhage and iron overload are common in the tissues of endometriotic lesions, and the expression level of transferrin gene receptor is higher. Besides, extensive ferritin staining was also observed in macrophages [ 64 , 65 ]. From this perspective, there is a certain imbalance of iron homeostasis in patients with endometriosis. As mentioned above, this can directly induce the occurrence of oxidative stress, as well as the generation of ROS, which may be one of the reasons of iron-induced DNA damage. In addition, iron can directly act as a catalyst to indirectly promote the generation of a series of free radicals through the fenton reaction, which further promoting the generation of oxidative stress [ 66 ]. At the same time, iron further activates intracellular tyrosine kinase or casein kinase II, and activates the p50/p65 NF-kappaB dimer, which can bind to DNA in the nucleus and participate in transcription, thereby mediating the production of a series of cytokines and involving in the development of EMS disease. From this perspective, EMS and oxidative stress are mutually reinforcing [ 67 ]. Direct contact of iron with DNA causes toxic lesions in a dose- and time-dependent manner. In the process of iron-induced DNA oxidative damage, it may lead to the transversions of G to T, transversions of G: C to A: T, and transitions of G: C to C: G in DNA. It also causes coding errors and reading errors in DNA polymerases [ 68 ]. This may also cause errors in base pairing. 8-Oxoguanine is the most common DNA oxidation marker, and Fe-NTA, an iron chelate, generates hydroxyl radicals through the Fenton reaction, which greatly induces the hydroxylation of guanine and pyrimidine and simultaneously produces 8-oxoguanine. In addition, iron can form 8-hydroxyguanine adducts, which can lead to point mutations and DNA strand breaks, as well as induce DNA hypermethylation and shorten telomere length. Furthermore, hemoglobin, heme, and iron derivatives helped to fine-tune the expression of several genes associated with oxidants and antioxidants, resulting in high levels of nuclear factor erythroid 2(Nrf2) and heme oxygenase-1(HO-1) expression. These two counteract inflammation and oxidative stress, which in turn induces malignant transformation of endometriotic cells with persistent DNA damage [ 69 ]. In summary, in the development of endometriosis, severe hemolysis occurs that results in the production of free hemoglobin–haptoglobin(Hb), heme, and iron, which are toxic and act as inflammatory molecules, oxidatively modifying lipids and proteins, and ultimately causing cellular and DNA damage.

Changes in metabolism

Studies have found that the concentration of lipid peroxides in the peritoneal fluid of women with pelvic endometriosis is significantly increased [ 70 ]. The product of unsaturated fatty acid peroxidation promoted by free radicals in cell membrane is lipid peroxide. For example, malondialdehyde (MDA), because it is relatively stable, can be used as a cumulative measure for this process. Vitamin E is one of the non-enzymatic antioxidants that prevent lipid peroxidation or its spread. Some studies have found vitamin E levels are reduced after pituitary blockade with GnRH-a [ 71 ]. One of the antioxidant activities of glutathione is the elimination of oxidized tocopherols, which is important for recycling and maintaining physiological levels of vitamin E, which is essential for fighting oxidative stress [ 72 ]. Vitamin E and glutathione levels were significantly lower in women with moderate and severe endometriosis compared with women with mild disease, speculated that the reduction in antioxidants may be due to their ability to protect against endogenous oxidative stress and therefore consume more in the endometrium [ 73 ]. Women with endometriosis had lower glucose levels and up-regulated pyruvate, both indicative of enhanced glycolysis in these women. Consequently, two TCA cycle intermediates, citrate and succinate, were also elevated which may be related to impaired mitochondrial respiration, and the possibility of ROS generation by the mitochondrial electron transport chain is also increased. This may be another important reason for the increase of ROS in the lesions of patients with endometriosis, leading to oxidative stress [ 74 ].

Inflammatory

Endometriosis is essentially an inflammatory response. Endometriosis is often accompanied by changes in the quantity and function of inflammatory factors, including TNFα, IL-1, IL-6, IL-8, macrophage migration inhibitory factor(MIF), CC chemokine monocyte chemoattractant protein-1(MCP-1) and serum amyloid A(SAA). Among several inflammatory factors, researchers have found that TNF-α plays a role in endometriosis. The expression of TNF-α is increased in the tissues of patients with endometriosis. It is regulated by urocortin 2 and urocortin 3 secreted by the endometrium, thus further illustrating that TNF-α may be a key cytokine in the inflammatory aspects of endometriosis [ 75 ]. At the same time, neutrophils and macrophages were determined to have higher chemotaxis in endometrial proliferative and luteal phase biopsies compared with normal endometrium. However, neutrophil activation is associated with disease severity in endometriosis, and only in patients with stage III and IV disease does the activation signal show a relevant response. It further illustrated that endometriotic tissue has a pro-inflammatory effect[ 76 ]. For macrophages, it may be regulated by estrogen. The expression of the estrogen receptor ER-a is positively correlated with the expression of inflammatory factors in macrophages of endometriosis [ 77 ]. RANTES, a chemotactic protein, is a chemotactic cytokine for a variety of inflammatory cells and plays an active role in recruiting leukocytes to sites of inflammation. However, increased RANTES was found in both the peritoneal fluid and endometrial tissue of patients with endometriosis. The expression of RANTES is also induced by TNF-α, which in turn promotes the recruitment of macrophages to endometriotic tissues. In addition, increased expression of T cells was found in patients with endometriosis. In animal models of endometriosis, there is a decrease in peripheral regulatory T cells and an increase in peritoneal fluid, which may lead to endometriosis associated infertility [ 78 ]. In macrophage MI and MII patterns, MI is characterized by a pro-inflammatory phenotype, while MII is an anti-inflammatory phenotype. We have found that polarization in favor of MII macrophages is observed in endometriosis [ 79 ]. Iron-induced ROS mentioned above can induce cellular and DNA damage by activating KB and increase the expression of pro-inflammatory genes NFκB. Various indications show that there is an inflammatory response in patients with endometriosis, including initial infection and subsequent sterile inflammation. Abnormal increasing in inflammation leads to some degree of genetic damage.

Diminished ovarian reserve and DNA damage

DNA damage is especially problematic for cells that do not divide or divide slowly, such as oocytes. DNA damaged cells undergo complex reactions such as cell cycle arrest, DNA repair and apoptosis. DNA damage accumulates over time. During the formation of the ovarian reserve, germ cells are in an active phase of DNA replication, proliferation and meiotic recombination. This stage is prone to DNA damage, which may lead to the loss of primordial follicles. This in turn affects ovarian reserve. From this perspective, diminished ovarian reserve (DOR) and even premature ovarian failure (POF) are one of the outcomes of DNA damage. DNA damage repair is crucial. The accumulation of DNA damage, and the impair function of DNA damage repair, ultimately lead to a decrease in ovarian reserve.

There are a variety of factors that can lead to DNA damage and thus affect ovarian function. 8-oxoguanine DNA glycosylase(OGG1) is an important component of the DNA damage repair process. OGG1 plays an important role in OS. Elevated serum OGG1 levels in DOR patients may suggest elevated levels of OS and severe DNA damage in DOR patients. Oxidative stress should be associated with diminished ovarian reserve (DOR) [ 80 ]. Excess free radicals can increase DNA damage and cause cellular decline with age [ 81 ]. ROS-induced DNA damage may lead to replication errors, base modifications, genomic instability, mutations and cell apoptosis. Among them, 8’OHdG is its marker. This eventually leads to premature ovarian insufficiency (POI), which may also be associated with mitochondrial dysfunction (MD) [ 82 ]. A variety of DNA damage can cause impairment of ovarian function and even lead to POI.

Double-strand break(DSBs)

DSBs can significantly alter the genetic integrity, which are difficult to repair and extremely toxic. Therefore, DSBs are the most harmful of all types of DNA damages. DSBs may be caused by genotoxic stress or replication fork defects [ 83 ].

Obviously, impaired DSB repair also affects ovarian reserve function. Repair efficiency is the key factor for oocyte loss. In a study of FVB mice, researchers found that the percentage of γH2AX-positive primordial follicles was significantly higher in 11- to 12-month-old compared with 3- to 4-week-old FVB mice, as was the percentage of γH2AX-positive GV-phase oocytes. In contrast, the expression of DNA DSB repair genes was reduced in old mice. The expression of BRCA1, MRE11, Rad51 and ATM was significantly decreased in the aged mice by qRT-PCR. All of these genes are involved in DNA DSB repair. This demonstrates that the reduction of DSB repair in old mouse oocytes results in the accumulation of DSB with age. In addition, the same pattern as in mice was found by studying the expression of key DNA DSBs repair genes in 24 individual human oocytes. Further studies in BRCA1 mutant mice revealed that BRCA1 mutant mice produced fewer oocytes after ovarian stimulation and had fewer litters after mating compared to wild-type mice, and the proportion of γH2AX cells was increased in 4-month-old BRCA1 mutant mice. This indicated inadequate DSB repair in BRCA1 mutant mice and significantly increased accumulation of DNA damage. In humans, people with the BRCA1 mutation have lower AMH levels than people without the BRCA1 mutation. The damage of DSB repair mechanism is associated with the accelerated loss of ovarian reserve, which is related to the accumulation of DSB in oocytes [ 84 ]. In addition to BRCA1, Rad51 and MRE11 are also critical in the process of ATM-mediated DNA DSB repair [ 85 , 86 ]. Therefore, knockdown any of these essential genes reduces the efficiency of DSB repair, which leads to a severe accumulation of DNA damage that triggers the cell death mechanism that explains the diminished function of ovarian reserve. Maintenance complex component (MCM) is also a family of proteins involved in important physiological processes such as DNA replication, meiosis and homologous recombination repair. Especially, both MCM8 and MCM9 promote the aggregation of RAD51 at the site of DNA damage. MCM8 is an important protein involved in the DNA homologous recombination repair mechanism. It is important for the homologous recombination process during the early meiotic prophase of oocytes and DSBs repair during the later developmental stages [ 87 ]. MCM8 mutation leads to impaired repair of DSBs, which can lead to excessive regulation of oocyte death. A study found that MCM8 gene polymorphisms were closely associated with early menopause in women, suggesting that MCM8 may play an important role in the maintenance of ovarian function in humans [ 88 ]. Female mice with MCM8 gene knockout are infertile and prone to ovarian tumors, as are female mice with MCM9 gene knockout [ 87 ]. Several studies have reported that homozygous mutations of MCM8/9 gene have been detected in families of primary amenorrhea. The patients were accompanied by hypergonadotropin, small ovaries and infantile uterus, delayed development of secondary sexual characteristics, short stature and other symptoms, while heterozygous mutation carriers had no phenotype [ 89 ]. MutS homologue 5(MSH5) is a member of the MutS protein family. It is mainly involved in the homologous recombination repair mechanism of DSBs mainly through interaction with Holliday Junction. MSH5 knockout female rats develop progressive oocyte loss, ovarian atrophy and infertility after birth, which is very similar to the clinical presentation of human premature ovarian failure (POF) [ 90 ]. In addition, meiosis specific with OB-fold (MEIOB) protein stabilizes the binding of the recombinant enzyme to the DSB site. Both female and male mice with MEIOB gene knockout were infertile and sterile. Deletion of this gene has been found in patients with POI [ 91 , 92 ]. These genes play an indispensable role in the process of DSB repair, and deletion of the corresponding genes will result in ineffective DSB repair, leading to its accumulation in the cells and eventually initiating the program of apoptosis, leading to the loss of oocytes. This process results in diminished ovarian reserve function and even the development of POI.

Base mismatches

DNA damage involves base mismatches. Abnormal repair can also lead to apoptosis. Diminished DNA repair responses were observed in mice with knockout of the homologous recombination (HR) pathway gene Brca2. There was significant DSB accumulation and chromosome mismatch in their germ cells. The number of follicles was reduced by approximately half [ 93 ].

The mismatch repair (MMR) pathway can repair base mismatch. The relationship between the MMR pathway and follicle development needs to be further explored. In addition, the MMR pathway can repair some DNA damage such as base mismatches and may be beneficial in maintaining integrity of germ cell genome. Whole-exome sequencing of family members of a POI patient identified MSH4-pure mutations. This suggests that defects in the MMR pathway lead to germ cell development arrest and genomic instability, which may be associated with POI [ 94 ]. The MSH5 protein mentioned above is a member of the MutSγ protein family and is involved in a variety of DNA damage repair processes, especially in correcting base mismatches during DNA replication. Msh5 knockout female mice showed impaired chromosome pairing, meiotic prophase I arrest, and attenuated oocyte numbers. These mice exhibited sterility. MSH5 not only plays an important role in homologous chromosome pairing in oocyte meiosis, but may also accelerate follicle failure by affecting the process of homologous recombination repair during mitosis in granulosa cells [ 95 ]. This evidence suggests that base mismatches may affect ovarian function. However, further experimental confirmation is still needed.

Interstrand cross-linking

DNA interstrand cross-linking (ICL) is a highly toxic form of DNA damage that results from the covalent bonding of two bases on complementary DNA strands. The main features of Fanconi anemia are the occurrence of spontaneous DNA breaks and the ICL that can occur after cross-linking agent induction. Therefore, the classical ICL repair pathway was named the Fanconi anemia (FA) pathway accordingly. It can regulate ICL repair during DNA replication to maintain genome integrity. Both endogenous metabolites and exogenous inducers can lead to the development of ICLs, thereby, impeding DNA transcription and replication [ 96 ]. ICLs have been reported to be particularly harmful to rapidly dividing cells, and the genomes of germ cells are particularly vulnerable to widespread DNA damage.

The FA pathway mainly targets the repair of ICLs, including ICL recognition, lesion bypass, DNA excision and DSB repair [ 97 ]. Cells with impaired FA pathways are highly sensitive to DNA cross-linking agents and exhibit increased chromosome breaks, decreased cell viability, and cell cycle arrest [ 98 , 99 ]. Mutations in the biallelic sites of the FANCL gene have now been found to result in the typical FA phenotype. However, FANCL knockout mice exhibited only reproductive defects, and other body systems were not significantly affected. It was also found that FANCL deficiency causes premature ovarian insufficiency in mice [ 100 ]. One study confirmed that the level of γH2AX was increased in FANCL knocked out cells, indicating that the deletion of FANCL impaired DNA repair [ 101 ]. In addition to the FANCL gene, three FA-related gene mutations have been identified in POI patients, including FANCD1/BRCA2, FANCM, and FANW/XECCR. In summary, the decrease in the number of germ cells may be related to the failure of repair of ICLs during the rapid division phase, leading to reduced proliferation and/or increased mediation.

Replication forks stalling

Replication stress can cause replication fork advancement to slow down or even stall, affecting DNA synthesis. Sustained replication stress can lead to the collapse of replication forks, resulting in double-strand breaks, which can be very damaging and difficult to repair for cells and living organisms. Replication fork stalling due to replication stress is also a major source of genomic rearrangements and mutations in cancer cells. Minichromosome maintenance complex (MCM) 2–7 is an important factor required for DNA replication, and a decrease of MCMs leads to an increase in replication stress, which may be associated with a decrease in the dormant origins. Mice carrying the CHAOS3 allele of MCM4 (Mcm c3 ) have depleted MCM, had an increased numbers of micronucleated cells and were highly susceptible to cancer. FANCM is involved in DNA replication fork repair and mice lacking FANCM have impaired replication forks and consequently lead to genomic instability in embryos, as confirmed by an increased number of micronucleated cells [ 102 ]. The FA pathway we mentioned above also protects the nascent DNA strands of stalled replication forks from lysogenic degradation. There are two ways to restart a stalled replication fork, the 53BP1-dependent cleavage-free pathway and the BRCA1-dependent break-induced replication- (BIR-) like pathway.

Hydrosalpinx and DNA damage

It is well known that hydrosalpinx (HSF) is believed to affect the success rate of IVF. Mechanical scour of the endometrium, endometrial receptivity damage and embryotoxic effects of hydrosalpinx were considered to be the main reasons for the decreased success rate.

Oxidative stress may also play a role in the pathophysiology of HSF. One study excised the affected side of the fallopian tube in patients with HSF, and extracted the fluid. This confirmed the presence of ROS, lipid peroxidation (LPO) and total antioxidant capacity (TAC) in the fluid. It has also been suggested that low concentrations of ROS may be an indicator of normal fallopian tube secretory function, which may have trophic effects on the embryo. ROS in HSF may originate from immune cells associated with chronic salpingitis. LPO, the product of OS, was detected in all HSFs, indicating that OS occurred in the acute phase of the disease. The embryotoxic effect of HSF was concentration-dependent when the concentration of HSF was greater than 50%. The blastocyst formation rate decreased with the increase of HSF concentration. Although the specific mechanism has not been formalized, DNA damage caused by ROS-induced oxidative stress couldn’t be excluded [ 103 ].

Conclusions

Increased DNA damage can have an impact on oocytes, spermatozoas, and the developing embryos, leading to infertility, miscarriages, and birth defects. Maintenance of gamete quality is a prerequisite for successful conception, embryo development and pregnancy. Therefore, it is essential to understand whether oocytes respond to DNA damage and which mechanisms of DDR are active to prevent the transfer of genomic damage to the embryo. In this review, we have explored the relationship between infertility related diseases and DNA damage and repair. Researchers have tried a variety of approaches to study the presence and functional mechanism of DDR in mammals, reducing DNA damage and enhancing DNA repair in an attempt to protect female fertility. However, much work remains to be done to elucidate DNA repair pathways in mammalian oocytes and infertility related diseases.

Data availability

No datasets were generated or analysed during the current study.

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Acknowledgements

Not applicable.

This study was supported by Hebei Natural Science Foundation (H2022206019). S&T Program of Hebei (21377721D, 21377720D, 22377795D). Medical Science Research Project of Hebei Province (20211494, 20240414). National Key R&D Program of China(2021YFC2700605). China Health Promotion Foundation. Hebei Provincial Government Funded Clinical Medicine Excellent Talent Program (2021).

Author information

Xiuhua Xu and Ziwei Wang are the first authors who contributed equally to this work.

Authors and Affiliations

Hebei Key Laboratory of Infertility and Genetics, Hebei Clinical Research Center for Birth Defects, Hebei Medical Key discipline of Reproductive Medicine, Hebei Collaborative Innovation Center of Integrated Traditional and Western Medicine on Reproductive Disease, Department of Reproductive Medicine, The Second Hospital of Hebei Medical University, Shijiazhuang, 050000, China

Xiuhua Xu, Ziwei Wang, Luyi Lv, Ci Liu, Lili Wang, Ya-nan Sun, Zhiming Zhao, Baojun Shi & Gui-min Hao

Cardiovascular platform, Institute of Health and Disease, Hebei Medical University, Shijiazhuang, 050000, China

Xiuhua Xu & Qian Li

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Contributions

Conceptualization, XH.X. and GM.H.; validation, XH.X., ZW.W., and LY.L.; formal analysis and investigation, C.L. LL.W. and YN.S.; resources, GM.H. and Q.L.; writing—original draft preparation, XH.X. and ZW.W.; writing—review and editing, Q.L. and GM.H.; visualization, XH.X. and ZW.W.; supervision, GM.H. and ZM. Z.; project administration, BJ.S.; funding acquisition, XH.X.,GM.H.,BJ.S. All authors have read and agreed to the published version of the manuscript.

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Correspondence to Qian Li or Gui-min Hao .

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Xu, X., Wang, Z., Lv, L. et al. Molecular regulation of DNA damage and repair in female infertility: a systematic review. Reprod Biol Endocrinol 22 , 103 (2024). https://doi.org/10.1186/s12958-024-01273-z

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Received : 02 January 2024

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Published : 14 August 2024

DOI : https://doi.org/10.1186/s12958-024-01273-z

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• DNA damage response
• Infertility

Reproductive Biology and Endocrinology

ISSN: 1477-7827

• General enquiries: [email protected]

Xiao P. Peng , MD , PhD

Pediatric genetics, medical genetics.

• Johns Hopkins School of Medicine Faculty

14 Insurances Accepted

Physician’s office phone.

443-287-9494

Professional Titles

• Director, Genetics of Blood and Immunity Clinic
• Clinical Advisor, Johns Hopkins Genomics DNA Diagnostics Lab

Primary Academic Title

Assistant Professor of Genetic Medicine

Xiao Peng, M.D., Ph.D., is currently Assistant Professor and Director of the Genetics of Blood and Immunity Clinic in the Johns Hopkins Department of Genetic Medicine. At Johns Hopkins, Dr. Peng also completed a combined residency in pediatrics and medical genetics, followed by a year as Genetics Chief Resident and research fellow supported by a National Institutes of Health T32 grant.

Dr. Peng’s clinical focus is on patients with suspected inborn errors of blood and immunity. She is co-author of multiple book chapters, several key reviews and scientific articles of the condition, and is a founding member of the Center for Immune-Related Disorders, a multi-disciplinary consortium of providers developed to help establish better resources for diagnosis and treatment of patients with suspected immune-related disorders.

Dr. Peng graduated in 2005 with a B.S. in chemistry with honors from Caltech and then worked as a research assistant, sponsored by the Howard Hughes Medical Institute, in the Cancer Genomics program at the Broad Institute of Harvard/MIT. During this time, she participated in many published basic science and translational medicine projects. At the Broad Institute, Dr. Peng also gained extensive experience in genomics and other cutting-edge technologies.

She graduated with an M.D., Ph.D. from the Weill Cornell/Rockefeller/Memorial Sloan-Kettering Tri-Institutional M.D.-Ph.D. Program in 2017, where she continued to pursue her interest in fundamental biological processes by studying the interface between DNA replication/repair, transcriptional regulation and post-translational protein modification pathways. She also published additional research in immunology and developmental gene regulation.

Since being at Johns Hopkins, she has adapted her basic science background into an innovative and mechanistic approach to the diagnosis and treatment of human disease. Her teaching and research interests in have synergistically converged with her passion for the care of patients with disorders of immunity and hematopoiesis. She continues to teach courses on immunity, genetics/genomics and their interface to trainees at many levels.

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• Ataxia Telangiectasia Clinical Center
• Immune and Blood Related Disorders, Center for

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600 N. Wolfe Street Blalock 1008 Baltimore, MD 21287

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Selected Publications

Peng XP , Caballero-Oteyza A, Grimbacher B. Common Variable Immunodeficiency: More Pathways than Roads to Rome. Annu Rev Pathol . 2022 Oct 20. doi:10.1146/annurev-pathmechdis-031521-024229. Epub ahead of print

Peng XP , Lim S, Li S, Marjavaara L, Chabes A, Zhao XL. Acute Smc5/6 depletion reveals its primary role in rDNA replication by restraining recombination at fork pausing sites. PLoS Genet . 2018 Jan 23; 14(1): e1007129

Peng XP , Schnappauf O, de Jesus AA, Aksentijevich I. Chapter: Autoinflammatory Disorders. Section Editor: Abraham R. Section: Inborn Errors of Immunity. Manual of Molecular and Clinical Laboratory Immunology , 9th Edition. Washington, D.C., ASM/Wiley. In press

Meng X, Wei L, Peng XP , Zhao X. Sumoylation of the DNA polymerase ε by the Smc5/6 complex contributes to DNA replication. PLoS Genet . 2019 Nov 25;15(11):e1008426

Zheng Y, Josefowicz S, Chaudhry A, Peng XP, Forbush K, Rudensky AY. Role of conserved non-coding DNA elements in the Foxp3 gene in regulatory T cell fate. Nature . 2010 Feb 11;463(7282):808-12

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• Systems, Genes, Mechanisms of Disease - Immunity, JHU SOM Dept of Genetic Medicine
• Transition to the Wards - "Genetics in Action"
• TIME Course: Genomics 101
• Translational Medicine - Immunology
• PRECEDE Session for Pediatric Clerkship - Genetics Evaluation in Pediatrics
• Current Topics in Clinical Genetics - Inborn Errors of Immunity
• Physician Scientist Training Program Research Microgrant Award, JHU School of Medicine, 1/24/22
• Primary Immunodeficiency Summer School Participant, Clinical Immunological Society, 10/27/21
• Margaret Nielsen Fellowship in Genetic Medicine, Johns Hopkins Dept of Genetic Medicine, 6/12/20
• Jay Lawrence Award for Clinical Proficiency in Infectious Diseases, Weill Cornell Medicine, 6/1/17
• Weill Cornell International Health Grant in Infectious Disease/Tropical and Travel Medicine, Weill Cornell Medicine/Ludwig-Maximilians-Universit√§t, 4/17/17
• Paul & Daisy Soros Fellowship for New Americans, Paul & Daisy Soros Foundation, 7/1/11
• Axline Merit Scholarship, California Institute of Technology, 9/1/01

Lectures & Presentations

Inborn Errors of Immunity - Genetic Pitfalls and Paradigms, Lecture/Seminar, Quarterly Meeting, Baltimore Washington Genetics Group, 4/21/21

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• Clinical Immunological Society

Professional Activities

ClinGen, Antibody Deficiencies Variant Curation Expert Panel

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