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1.
Proc Natl Acad Sci U S A ; 120(30): e2219925120, 2023 07 25.
Article in English | MEDLINE | ID: mdl-37459509

ABSTRACT

Infertility is a heterogeneous condition, with genetic causes thought to underlie a substantial fraction of cases. Genome sequencing is becoming increasingly important for genetic diagnosis of diseases including idiopathic infertility; however, most rare or minor alleles identified in patients are variants of uncertain significance (VUS). Interpreting the functional impacts of VUS is challenging but profoundly important for clinical management and genetic counseling. To determine the consequences of these variants in key fertility genes, we functionally evaluated 11 missense variants in the genes ANKRD31, BRDT, DMC1, EXO1, FKBP6, MCM9, M1AP, MEI1, MSH4 and SEPT12 by generating genome-edited mouse models. Nine variants were classified as deleterious by most functional prediction algorithms, and two disrupted a protein-protein interaction (PPI) in the yeast two hybrid (Y2H) assay. Though these genes are essential for normal meiosis or spermiogenesis in mice, only one variant, observed in the MCM9 gene of a male infertility patient, compromised fertility or gametogenesis in the mouse models. To explore the disconnect between predictions and outcomes, we compared pathogenicity calls of missense variants made by ten widely used algorithms to 1) those annotated in ClinVar and 2) those evaluated in mice. All the algorithms performed poorly in terms of predicting the effects of human missense variants modeled in mice. These studies emphasize caution in the genetic diagnoses of infertile patients based primarily on pathogenicity prediction algorithms and emphasize the need for alternative and efficient in vitro or in vivo functional validation models for more effective and accurate VUS description to either pathogenic or benign categories.


Subject(s)
Infertility, Male , Mutation, Missense , Humans , Male , Mice , Animals , Reproduction , Alleles , Infertility, Male/genetics , Disease Models, Animal , Septins/genetics
2.
Nat Commun ; 13(1): 6384, 2022 10 26.
Article in English | MEDLINE | ID: mdl-36289231

ABSTRACT

With an incidence of ~1 in 800 births, Down syndrome (DS) is the most common chromosomal condition linked to intellectual disability worldwide. While the genetic basis of DS has been identified as a triplication of chromosome 21 (HSA21), the genes encoded from HSA21 that directly contribute to cognitive deficits remain incompletely understood. Here, we found that the HSA21-encoded chromatin effector, BRWD1, was upregulated in neurons derived from iPS cells from an individual with Down syndrome and brain of trisomic mice. We showed that selective copy number restoration of Brwd1 in trisomic animals rescued deficits in hippocampal LTP, cognition and gene expression. We demonstrated that Brwd1 tightly binds the BAF chromatin remodeling complex, and that increased Brwd1 expression promotes BAF genomic mistargeting. Importantly, Brwd1 renormalization rescued aberrant BAF localization, along with associated changes in chromatin accessibility and gene expression. These findings establish BRWD1 as a key epigenomic mediator of normal neurodevelopment and an important contributor to DS-related phenotypes.


Subject(s)
Cognition Disorders , Down Syndrome , Mice , Animals , Down Syndrome/genetics , Down Syndrome/metabolism , DNA Copy Number Variations/genetics , Disease Models, Animal , Cognition Disorders/genetics , Chromatin/genetics , Mice, Transgenic
3.
Nat Commun ; 12(1): 5005, 2021 08 18.
Article in English | MEDLINE | ID: mdl-34408140

ABSTRACT

Embryonic aneuploidy from mis-segregation of chromosomes during meiosis causes pregnancy loss. Proper disjunction of homologous chromosomes requires the mismatch repair (MMR) genes MLH1 and MLH3, essential in mice for fertility. Variants in these genes can increase colorectal cancer risk, yet the reproductive impacts are unclear. To determine if MLH1/3 single nucleotide polymorphisms (SNPs) in human populations could cause reproductive abnormalities, we use computational predictions, yeast two-hybrid assays, and MMR and recombination assays in yeast, selecting nine MLH1 and MLH3 variants to model in mice via genome editing. We identify seven alleles causing reproductive defects in mice including female subfertility and male infertility. Remarkably, in females these alleles cause age-dependent decreases in litter size and increased embryo resorption, likely a consequence of fewer chiasmata that increase univalents at meiotic metaphase I. Our data suggest that hypomorphic alleles of meiotic recombination genes can predispose females to increased incidence of pregnancy loss from gamete aneuploidy.


Subject(s)
Abortion, Spontaneous/genetics , Aneuploidy , Embryo Loss/genetics , MutL Protein Homolog 1/genetics , MutL Proteins/genetics , Abortion, Spontaneous/metabolism , Abortion, Spontaneous/physiopathology , Alleles , Animals , Crossing Over, Genetic , DNA Mismatch Repair , Embryo Loss/physiopathology , Female , Homologous Recombination , Humans , Litter Size , Male , Meiosis , Mice , MutL Protein Homolog 1/metabolism , MutL Proteins/metabolism , Pregnancy , Reproduction , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism
4.
Nat Commun ; 10(1): 4141, 2019 09 12.
Article in English | MEDLINE | ID: mdl-31515488

ABSTRACT

Each human genome carries tens of thousands of coding variants. The extent to which this variation is functional and the mechanisms by which they exert their influence remains largely unexplored. To address this gap, we leverage the ExAC database of 60,706 human exomes to investigate experimentally the impact of 2009 missense single nucleotide variants (SNVs) across 2185 protein-protein interactions, generating interaction profiles for 4797 SNV-interaction pairs, of which 421 SNVs segregate at > 1% allele frequency in human populations. We find that interaction-disruptive SNVs are prevalent at both rare and common allele frequencies. Furthermore, these results suggest that 10.5% of missense variants carried per individual are disruptive, a higher proportion than previously reported; this indicates that each individual's genetic makeup may be significantly more complex than expected. Finally, we demonstrate that candidate disease-associated mutations can be identified through shared interaction perturbations between variants of interest and known disease mutations.


Subject(s)
Gene Frequency/genetics , Genetic Variation , Genetics, Population , Alleles , Animals , Base Sequence , Disease/genetics , Genetic Predisposition to Disease , Genome, Human , HEK293 Cells , Humans , Mice , Mutation, Missense/genetics , Phenotype , Polymorphism, Single Nucleotide/genetics , Protein Binding/genetics
5.
Mol Hum Reprod ; 25(10): 668-673, 2019 10 28.
Article in English | MEDLINE | ID: mdl-31393579

ABSTRACT

Infertility is a major health problem affecting ~15% of couples worldwide. Except for cases involving readily detectable chromosome aberrations, confident identification of a causative genetic defect is problematic. Despite the advent of genome sequencing for diagnostic purposes, the preponderance of segregating genetic variants complicates identification of culprit genetic alleles or mutations. Many algorithms have been developed to predict the effects of 'variants of unknown significance', typically single nucleotide polymorphisms (SNPs), but these predictions are not sufficiently accurate for clinical action. As part of a project to identify population variants that impact fertility, we have been generating clustered regularly interspaced short palindromic repeats-Cas9 edited mouse models of suspect SNPs in genes that are known to be required for fertility in mice. Here, we present data on a non-synonymous (amino acid altering) SNP (rs140107488) in the meiosis gene Mnd1, which is predicted bioinformatically to be deleterious to protein function. We report that when modeled in mice, this allele (MND1K85M), which is present at an allele frequency of ~ 3% in East Asians, has no discernable effect upon fertility, fecundity or gametogenesis, although it may cause sex skewing of progeny from homozygous males. In sum, assuming the mouse model accurately reflects the impact of this variant in humans, rs140107488 appears to be a benign allele that can be eliminated or de-prioritized in clinical genomic analyses of infertility patients.


Subject(s)
Asian People/genetics , Cell Cycle Proteins/genetics , Infertility/genetics , Meiosis/genetics , Polymorphism, Single Nucleotide , Reproduction/genetics , Alleles , Animals , Asia/epidemiology , Asian People/statistics & numerical data , Female , Gene Frequency , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Models, Animal , Pregnancy , Species Specificity
6.
Biol Reprod ; 101(2): 347-359, 2019 08 01.
Article in English | MEDLINE | ID: mdl-31074776

ABSTRACT

A major challenge in medical genetics is to characterize variants of unknown significance (VUS). Doing so would help delineate underlying causes of disease and the design of customized treatments. Infertility has presented an especially difficult challenge with respect to not only determining if a given patient has a genetic basis, but also to identify the causative genetic factor(s). Though genome sequencing can identify candidate variants, in silico predictions of causation are not always sufficiently reliable so as to be actionable. Thus, experimental validation is crucial. Here, we describe the phenotype of mice containing a non-synonymous (proline-to-threonine at position 306) change in Spo11, corresponding to human SNP rs185545661. SPO11 is a topoisomerase-like protein that is essential for meiosis because it induces DNA double stranded breaks (DSBs) that stimulate pairing and recombination of homologous chromosomes. Although both male and female Spo11P306T/P306T mice were fertile, they had reduced sperm and oocytes, respectively. Spermatocyte chromosomes exhibited synapsis defects (especially between the X and Y chromosomes), elevated apoptotic cells, persistent markers of DSBs, and most importantly, fewer Type 1 crossovers that causes some chromosomes to have none. Spo11P306T/- mice were sterile and made fewer meiotic DSBs than Spo11+/- animals, suggesting that the Spo11P306T allele is a hypomorph and likely is delayed in making sufficient DSBs in a timely fashion. If the consequences are recapitulated in humans, it would predict phenotypes of premature ovarian failure, reduced sperm counts, and possible increased number of aneuploid gametes. These results emphasize the importance of deep phenotyping in order to accurately assess the impact of VUSs in reproduction genes.


Subject(s)
Endodeoxyribonucleases/metabolism , Meiosis/physiology , Oligospermia/genetics , Ovarian Reserve/genetics , Recombination, Genetic/physiology , Alleles , Animals , Crossing Over, Genetic , Endodeoxyribonucleases/genetics , Female , Male , Mice , Polymorphism, Single Nucleotide
7.
Hum Mol Genet ; 27(22): 3911-3918, 2018 11 15.
Article in English | MEDLINE | ID: mdl-30085085

ABSTRACT

Whole-exome or whole-genome sequencing is becoming routine in clinical situations for identifying mutations underlying presumed genetic causes of disease including infertility. While this is a powerful approach for implicating polymorphisms or de novo mutations in genes plausibly related to the phenotype, a greater challenge is to definitively prove causality. This is a crucial requisite for treatment, especially for infertility, in which validation options are limited. In this study, we created a mouse model of a putative infertility allele, DMC1M200V. DMC1 encodes a RecA homolog essential for meiotic recombination and fertility in mice. This allele was originally implicated as being responsible for the sterility of a homozygous African woman, a conclusion supported by subsequent biochemical analyses of the mutant protein and by studies of yeast with the orthologous amino acid change. Here, we found that Dmc1M200V/M200V male and female mice are fully fertile and do not exhibit any gonadal abnormalities. Detailed immunocytological analysis of meiosis revealed no defects suggestive of compromised fertility. This study serves as a cautionary tale for making conclusions about consequences of genetic variants, especially with respect to infertility, and emphasizes the importance of conducting relevant biological assays for making accurate diagnoses in the era of genomic medicine.


Subject(s)
Cell Cycle Proteins/genetics , DNA-Binding Proteins/genetics , Infertility/genetics , Meiosis/genetics , Nuclear Proteins/genetics , Alleles , Animals , Disease Models, Animal , Female , Gonads/growth & development , Gonads/pathology , Humans , Infertility/physiopathology , Male , Mice , Mutation , Phosphate-Binding Proteins , Recombinases , Recombination, Genetic
9.
Exp Eye Res ; 83(3): 638-50, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16682026

ABSTRACT

Loss of retinal pigment epithelial (RPE) cells via apoptosis plays a prominent role in several retinal degenerative diseases, such as age-related macular degeneration, and with light damage. Strategies for preservation of vision that would interrupt the apoptotic cascade require understanding the molecular events associated with apoptosis. This study investigated the susceptibility of RPE to caspase-dependent and -independent apoptotic pathways when challenged with different stimuli, including oxidants, anti-Fas antibody, and activated cytotoxic T lymphocytes (CTLs). These experiments used novel RPE cell lines developed from wildtype and heterozygous mice with reduced levels of either Mn superoxide dismutatse (SOD) or CuZnSOD. Peroxide and 4-hydroxynonenal induced apoptosis through both caspase-independent and -dependent pathways, respectively. With both oxidants, translocation of apoptosis inducing factor into the nucleus was observed. Cells containing reduced levels of CuZnSOD were the most susceptible to oxidant-induced cell death. Targeted killing by CTLs and activation of the Fas death receptor induced caspase-dependent apoptosis. These results show stimulus-specific activation of either the caspase-dependent or -independent pathway. Since cultured RPE express the protein components required for different apoptotic pathways, they provide a good model system for studying molecular events associated with multiple signals that lead to cell death.


Subject(s)
Macular Degeneration/pathology , Pigment Epithelium of Eye/pathology , Animals , Apoptosis , Blotting, Western , Cell Line , Cell Survival , Fas Ligand Protein , Flow Cytometry , Humans , Immunohistochemistry/methods , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Oxidation-Reduction , Phagocytosis , Reverse Transcriptase Polymerase Chain Reaction , Superoxide Dismutase/analysis , T-Lymphocytes, Cytotoxic/immunology , Tumor Necrosis Factors/metabolism
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