scholarly journals A segregating human allele of SPO11 modeled in mice disrupts timing and amounts of meiotic recombination, causing oligospermia and a decreased ovarian reserve†

2019 ◽  
Vol 101 (2) ◽  
pp. 347-359 ◽  
Author(s):  
Tina N Tran ◽  
John C Schimenti
Keyword(s):  
Variants Of Unknown Significance ◽  
Y Chromosomes ◽  
Sperm Counts ◽  
Meiotic Dsbs ◽  
Unknown Significance ◽  
Causes Of Disease ◽  
Underlying Causes ◽  
Timely Fashion ◽  
Difficult Challenge ◽  
The Impact

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.

scholarly journals A segregating human allele ofSPO11modeled in mice disrupts timing and amounts of meiotic recombination, causing oligospermia and a decreased ovarian reserve

2019 ◽  
Author(s):  
Tina N. Tran ◽  
John C. Schimenti
Keyword(s):  
Variants Of Unknown Significance ◽  
Y Chromosomes ◽  
Sperm Counts ◽  
Meiotic Dsbs ◽  
Unknown Significance ◽  
Causes Of Disease ◽  
Underlying Causes ◽  
Timely Fashion ◽  
Difficult Challenge ◽  
The Impact

ABSTRACTA major challenge in medical genetics is to characterize variants of unknown significance (VUS), so as to better understand underlying causes of disease and design 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 nonsynonymous (proline-to-threonine at position 306) change inSpo11, 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 femaleSpo11P306T/P306Tmice 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 thanSpo11+/−animals, suggesting that theSpo11P306Tallele 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.

scholarly journals Case Report: Infantile Urticaria as a Herald of Neonatal Onset Multisystem Inflammatory Disease With a Novel Mutation in NLRP3

2021 ◽  
Vol 12 ◽  
Author(s):  
Anna E. Patrick ◽  
Eden M. Lyons ◽  
Lisa Ishii ◽  
Alan S. Boyd ◽  
Joseph M. Choi ◽  
...  
Keyword(s):  
Clinical Symptoms ◽  
Early Recognition ◽  
Novel Mutation ◽  
Genetic Diagnosis ◽  
Inflammatory Disorder ◽  
Laboratory Findings ◽  
Genetic Sequencing ◽  
Variants Of Unknown Significance ◽  
Unknown Significance ◽  
The Impact

Neonatal multisystem onset inflammatory disorder (NOMID) is a severe autoinflammatory syndrome that can have an initial presentation as infantile urticaria. Thus, an immediate recognition of the clinical symptoms is essential for obtaining a genetic diagnosis and initiation of early therapies to prevent morbidity and mortality. Herein, we describe a neonate presenting with urticaria and systemic inflammation within hours after birth who developed arthropathy and neurologic findings. Pathologic evaluation of the skin revealed an infiltration of lymphocytes, eosinophils, and scattered neutrophils. Genetic analysis identified a novel heterozygous germline variant of unknown significance in the NLRP3 gene, causing the missense mutation M408T. Variants of unknown significance are common in genetic sequencing studies and are diagnostically challenging. Functional studies of the M408T variant demonstrated enhanced formation and activity of the NLRP3 inflammasome, with increased cleavage of the inflammatory cytokine IL-1β. Upon initiation of IL-1 pathway blockade, the infant had a robust response and improvement in clinical and laboratory findings. Our experimental data support that this novel variant in NLRP3 is causal for this infant’s diagnosis of NOMID. Rapid assessment of infantile urticaria with biopsy and genetic diagnosis led to early recognition and targeted anti-cytokine therapy. This observation expands the NOMID-causing variants in NLRP3 and underscores the role of genetic sequencing in rapidly identifying and treating autoinflammatory disease in infants. In addition, these findings highlight the importance of establishing the functional impact of variants of unknown significance, and the impact this knowledge may have on therapeutic decision making.

scholarly journals Novel frameshift variant in MYL2 reveals molecular differences between dominant and recessive forms of hypertrophic cardiomyopathy

2019 ◽  
Author(s):  
Sathiya N. Manivannan ◽  
Sihem Darouich ◽  
Aida Masmoudi ◽  
David Gordon ◽  
Gloria Zender ◽  
...  
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Exome Sequencing ◽  
Rapid Screening ◽  
Functional Study ◽  
Ventricular Muscle ◽  
Loss Of Function ◽  
Variants Of Unknown Significance ◽  
Unknown Significance ◽  
The Impact

AbstractHypertrophic cardiomyopathy (HCM) is characterized by enlargement of the ventricular muscle without dilation and is often associated with dominant pathogenic variants in cardiac sarcomeric protein genes. Here, we report a family with two infants diagnosed with infantile-onset HCM and mitral valve dysplasia that led to death before one year of age. Using exome sequencing, we discovered that one of the affected children had a homozygous frameshift variant in Myosin light chain 2 (MYL2:NM_000432.3:c.431_432delCT: p.Pro144Argfs*57;MYL2-fs), which alters the last 20 amino acids of the protein and is predicted to impact the C-terminal EF-hand (CEF) domain. The parents are unaffected heterozygous carriers of the variant and the variant is absent in control cohorts from gnomAD. The absence of the phenotype in carriers and infantile presentation of severe HCM is in contrast to HCM associated with dominant MYL2 variants. Immunohistochemical analysis of the ventricular muscle of the deceased patient with the MYL2-fs variant showed marked reduction of MYL2 expression compared to an unaffected control. In vitro overexpression studies further indicate that the MYL2-fs variant is actively degraded. In contrast, an HCM-associated missense variant (MYL2:p.Gly162Arg) and three other MYL2 stopgain variants that lead to loss of the CEF domain are stably expressed. However, stopgain variants show impaired localization suggesting a functional role for the CEF domain. The degradation of the MYL2-fs can be rescued by inhibiting the cell’s proteasome function supporting a post-translational effect of the variant. In vivo rescue experiments with a Drosophila MYL2-homolog (Mlc2) knockdown model indicate that neither MYL2-fs nor MYL2:p.Gly162Arg supports regular cardiac function. The tools that we have generated provide a rapid screening platform for functional assessment of variants of unknown significance in MYL2. Our study supports an autosomal recessive model of inheritance for MYL2 loss-of-function variants and highlights the variant-specific molecular differences found in MYL2-associated cardiomyopathies.Author SummaryWe report a novel frameshift variant in MYL2 that is associated with a severe form of infantile-onset hypertrophic cardiomyopathy. The impact of the variant is only observed in the recessive form of the disease in the proband and not in the parents who are carriers of the variant. This is in contrast to other dominant variants in MYL2 that are associated with cardiomyopathies. We compared the stability of this variant to that of other cardiomyopathy associated MYL2 variants and found molecular differences in the disease pathology. We also show different protein domain requirement for stability and localization of MYL2 in cardiomyocytes. Further, we used a fly model to demonstrate functional deficits due to the variant in the developing heart. Overall, our study shows a molecular mechanism by which loss-of-function variants in MYL2 are recessive while missense variants are dominant. We highlight the use of exome sequencing and functional testing to assist in the diagnosis of rare forms of diseases where pathogenicity of the variant is not obvious. The new tools we developed for in vitro functional study and the fly fluorescent reporter analysis will permit rapid analysis of MYL2 variants of unknown significance.

Genotype-first in a cohort of 95 fetuses with multiple congenital abnormalities: when exome sequencing reveals unexpected fetal phenotype-genotype correlations

2020 ◽  
pp. jmedgenet-2020-106867
Author(s):  
Mathilde Lefebvre ◽  
Ange-Line Bruel ◽  
Emilie Tisserant ◽  
Nicolas Bourgon ◽  
Yannis Duffourd ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Congenital Abnormalities ◽  
Diagnostic Yield ◽  
Causal Variant ◽  
Developmental Abnormalities ◽  
Variants Of Unknown Significance ◽  
Unknown Significance ◽  
Bibliographic Search ◽  
The Impact ◽  
Multiple Congenital Abnormalities

PurposeMolecular diagnosis based on singleton exome sequencing (sES) is particularly challenging in fetuses with multiple congenital abnormalities (MCA). Indeed, some studies reveal a diagnostic yield of about 20%, far lower than in live birth individuals showing developmental abnormalities (30%), suggesting that standard analyses, based on the correlation between clinical hallmarks described in postnatal syndromic presentations and genotype, may underestimate the impact of the genetic variants identified in fetal analyses.MethodsWe performed sES in 95 fetuses with MCA. Blind to phenotype, we applied a genotype-first approach consisting of combined analyses based on variants annotation and bioinformatics predictions followed by reverse phenotyping. Initially applied to OMIM-morbid genes, analyses were then extended to all genes. We complemented our approach by using reverse phenotyping, variant segregation analysis, bibliographic search and data sharing in order to establish the clinical significance of the prioritised variants.ResultssES rapidly identified causal variant in 24/95 fetuses (25%), variants of unknown significance in OMIM genes in 8/95 fetuses (8%) and six novel candidate genes in 6/95 fetuses (6%).ConclusionsThis method, based on a genotype-first approach followed by reverse phenotyping, shed light on unexpected fetal phenotype-genotype correlations, emphasising the relevance of prenatal studies to reveal extreme clinical presentations associated with well-known Mendelian disorders.

Web-Based Bioinformatics Predictors: Recommendations to Assess Lysosomal Cholesterol Trafficking Diseases-Related Genes

2019 ◽  
Vol 58 (01) ◽  
pp. 050-059 ◽  
Author(s):  
Laura López de Frutos ◽  
Jorge J. Cebolla ◽  
Pilar Irún ◽  
Ralf Köhler ◽  
Pilar Giraldo
Keyword(s):  
Genetic Variants ◽  
Lysosomal Storage ◽  
Cholesterol Trafficking ◽  
Variants Of Unknown Significance ◽  
Unknown Significance ◽  
Deleterious Gene ◽  
The One ◽  
Matthew’S Correlation Coefficient ◽  
Sensitivity Specificity ◽  
The Impact

Introduction The growing number of genetic variants of unknown significance (VUS) and availability of several in silico prediction tools make the evaluation of potentially deleterious gene variants challenging. Materials and Methods We evaluated several programs and software to determine the one that can predict the impact of genetic variants found in lysosomal storage disorders (LSDs) caused by defects in cholesterol trafficking best. We evaluated the sensitivity, specificity, accuracy, precision, and Matthew's correlation coefficient of the most common software. Results Our findings showed that for exonic variants, only MutPred1 reached 100% accuracy and generated the best predictions (sensitivity and accuracy = 1.00), whereas intronic variants, SROOGLE or Human Splicing Finder (HSF) generated the best predictions (sensitivity = 1.00, and accuracy = 1.00). Discussion Next-generation sequencing substantially increased the number of detected genetic variants, most of which were considered to be VUS, creating a need for accurate pathogenicity prediction. The focus of the present study is the importance of accurately predicting LSDs, with majority of previously unreported specific mutations. Conclusion We found that the best prediction tool for the NPC1, NPC2, and LIPA variants was MutPred1 for exonic regions and HSF and SROOGLE for intronic regions and splice sites.

scholarly journals Cell Painting predicts impact of lung cancer variants

2021 ◽  
Author(s):  
Juan C Caicedo ◽  
John Arevalo ◽  
Federica Piccioni ◽  
Mark-Anthony Bray ◽  
Cathy L Hartland ◽  
...  
Keyword(s):  
Single Cell ◽  
Low Cost ◽  
Transcriptional Profiling ◽  
Patient Specific ◽  
Cell Level ◽  
Functional Impact ◽  
Variants Of Unknown Significance ◽  
Online Portal ◽  
Unknown Significance ◽  
The Impact

Most variants in most genes across most organisms have an unknown impact on the function of the corresponding gene. This gap in knowledge is especially acute in cancer, where clinical sequencing of tumors now routinely reveals patient-specific variants whose functional impact on the corresponding gene is unknown, impeding clinical utility. Transcriptional profiling was able to systematically distinguish these variants of unknown significance (VUS) as impactful vs. neutral in an approach called expression-based variant-impact phenotyping (eVIP). We profiled a set of lung adenocarcinoma-associated somatic variants using Cell Painting, a morphological profiling assay that captures features of cells based on microscopy using six stains of cell and organelle components. Using deep-learning-extracted features from each cell's image, we found that cell morphological profiling (cmVIP) can predict variants' functional impact and, particularly at the single-cell level, reveals biological insights into variants which can be explored in our public online portal. Given its low cost, convenient implementation, and single-cell resolution, cmVIP profiling therefore seems promising as an avenue for using non-gene-specific assays to systematically assess the impact of variants, including disease-associated alleles, on gene function.

scholarly journals A predicted deleterious allele of the essential meiosis gene MND1, present in ~ 3% of East Asians, does not disrupt reproduction in mice

2019 ◽  
Vol 25 (10) ◽  
pp. 668-673 ◽  
Author(s):  
Tina N Tran ◽  
Julianna Martinez ◽  
John C Schimenti
Keyword(s):  
Protein Function ◽  
Genetic Defect ◽  
Nucleotide Polymorphisms ◽  
East Asians ◽  
Major Health Problem ◽  
Variants Of Unknown Significance ◽  
Unknown Significance ◽  
Clinical Action ◽  
Infertility Patients ◽  
The Impact

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.

The landscape of RET alterations from 56,970 adult patients with cancer: Clinical implications.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. 3106-3106
Author(s):  
Alexander Andreev-Drakhlin ◽  
Jason Roszik ◽  
Vivek Subbiah
Keyword(s):  
Mapk Pathway ◽  
Functional Characterization ◽  
Great Promise ◽  
Precision Oncology ◽  
Missense Mutations ◽  
Variants Of Unknown Significance ◽  
Patients With Cancer ◽  
Unknown Significance ◽  
Cancer Types ◽  
The Impact

3106 Background: Activating receptor-tyrosine kinase rearranged during transfection ( RET) mutations and fusions have been recognized as potent drivers of oncogenesis. Recent identification of highly potent and selective RET inhibitors holds great promise in the management of RET-dependent tumors. Here we present a comprehensive analysis of RET alterations in pan-cancer adult malignancies. Methods: We analyzed 59,347 samples from 56,970 patients available from AACR Project GENIE (Cancer Discov. 2017) database for the prevalence of RET fusions, mutations, and copy number alterations in diverse cancer types. Results: A total of 1414 RET alterations were detected, including 91 fusions (6.4%), 1166 missense mutations (82.5%), 136 truncating mutations (9.6%), and 21 in-frame mutations (1.5%). RET fusions were observed in 0.15% of tumor samples and were most commonly identified in non-small cell lung cancer, thyroid cancer, colorectal cancer, prostate cancer, and gastric cancer (62.6%, 18.6%, 5.5%, 4.4%, 3.3% of identified RET fusions, respectively). RET fusions were significantly co-altered with MAPK3/ERK1 (p=0.045), SETD2 (p=1.36E-07 ), and EIF4E (p=0.045), while there was a negative association between RET fusions and EGFR (p=0.009634) , TP53 (p=0.02267), and KRAS (p=2.53E-05) alterations. Most common RET gene upstream partners were KIF5B, CCDC6, and NCOA4 (42.9%, 24.2%, 7.7% of identified RET fusions, respectively). RET missense mutations were found in 2.0% of tumor samples; 136 (11.7%) of identified missense mutations, including 8 RET gatekeeper V804M/L mutations, were characterized as likely oncogenic, 12 (1.0%) as likely benign, and 1018 (87.3%) as variants of unknown significance using OncoKB database. RET amplifications occurred in 1.5% of tested samples. Conclusions: While RET fusions represent extremely rare events in multiple cancers, RET missense mutations occur in 2% of malignancies. Most RET missense variants are described as variants of unknown significance, limiting the impact of precision oncology for the majority of patients with RET alterations. Further functional characterization of RET variants is warranted. MAPK pathway co-alterations in patents with RET fusions may present a strategy for future therapeutic combinations.

scholarly journals Genomic imbalances in the placenta are associated with poor fetal growth

2021 ◽  
Vol 27 (1) ◽  
Author(s):  
Giulia F. Del Gobbo ◽  
Yue Yin ◽  
Sanaa Choufani ◽  
Emma A. Butcher ◽  
John Wei ◽  
...  
Keyword(s):  
Fetal Growth ◽  
Sex Chromosome ◽  
Copy Number Variants ◽  
Placental Insufficiency ◽  
Potential Contribution ◽  
Genomic Imbalances ◽  
Significant Difference ◽  
Underlying Causes ◽  
Risk Of Disease ◽  
The Impact

Abstract Background Fetal growth restriction (FGR) is associated with increased risks for complications before, during, and after birth, in addition to risk of disease through to adulthood. Although placental insufficiency, failure to supply the fetus with adequate nutrients, underlies most cases of FGR, its causes are diverse and not fully understood. One of the few diagnosable causes of placental insufficiency in ongoing pregnancies is the presence of large chromosomal imbalances such as trisomy confined to the placenta; however, the impact of smaller copy number variants (CNVs) has not yet been adequately addressed. In this study, we confirm the importance of placental aneuploidy, and assess the potential contribution of CNVs to fetal growth. Methods We used molecular-cytogenetic approaches to identify aneuploidy in placentas from 101 infants born small-for-gestational age (SGA), typically used as a surrogate for FGR, and from 173 non-SGA controls from uncomplicated pregnancies. We confirmed aneuploidies and assessed mosaicism by microsatellite genotyping. We then profiled CNVs using high-resolution microarrays in a subset of 53 SGA and 61 control euploid placentas, and compared the load, impact, gene enrichment and clinical relevance of CNVs between groups. Candidate CNVs were confirmed using quantitative PCR. Results Aneuploidy was over tenfold more frequent in SGA-associated placentas compared to controls (11.9% vs. 1.1%; p = 0.0002, OR = 11.4, 95% CI 2.5–107.4), was confined to the placenta, and typically involved autosomes, whereas only sex chromosome abnormalities were observed in controls. We found no significant difference in CNV load or number of placental-expressed or imprinted genes in CNVs between SGA and controls, however, a rare and likely clinically-relevant germline CNV was identified in 5.7% of SGA cases. These CNVs involved candidate genes INHBB, HSD11B2, CTCF, and CSMD3. Conclusions We conclude that placental genomic imbalances at the cytogenetic and submicroscopic level may underlie up to ~ 18% of SGA cases in our population. This work contributes to the understanding of the underlying causes of placental insufficiency and FGR, which is important for counselling and prediction of long term outcomes for affected cases.

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