O-115 Parental whole-exome sequencing allows the discovery of genetic causes of extreme IVF phenotypes such as oocyte/embryo developmental arrest and recurrent low fertilization

Abstract Study question Do whole-exome sequencing (WES) data from infertile women provide valuable information for the discovery of genes/pathways involved in extreme IVF phenotypes, i.e. oocyte/embryo developmental arrest? Summary answer The development of a specific bioinformatic WES pipeline revealed known and new candidate genes/pathways for isolated oocyte/embryo developmental failure,providing the foundation to scale up research. What is known already The use of IVF has made it possible to identify extreme and isolated infertility phenotypes such as recurrent low oocytes maturity (LMR), recurrent low fertilization rate (LFR), or preimplantation developmental arrest (PDA) that would remain concealed in natural conception attempts. Recent applications of WES in families with such extreme adverse IVF phenotypes have led to the discovery of new genes and pathways affecting unique functions of gametes and exclusive mechanisms necessary for early embryo development. Here, we apply a tailored bioinformatic approach to WES from women displaying extreme IVF phenotypes to discover new causative genes/pathways involved in unexplained infertility. Study design, size, duration Twenty-two infertile consanguineous women(December 2018-September 2020) suffering from long-term unexplained infertility. Eight cases were classified as PDA (<20% normally developed embryos in > 2 IVF cycles), 8 as LMR (<20% mature oocytes in > 2 IVF cycles), 4 as LFR (<20% of normally fertilized oocytes in > 2 IVF cycles). Two women with recurrent IVF failure (>10 IVF cycles) were also included. A control set of 1660 WES from oocyte donors was used to control for false-positive discoveries. Participants/materials, setting, methods WES at 30X was performed on enrolled women’s gDNA using Illumina short-reads technology. Following annotation, variants were filtered to prioritize putative detrimental variants in genes relevant for oocyte/embryonic development using a previously developed and validated pipeline that minimizes false-positive discoveries. Runs of homozygosity (ROH) within each sample were identified using Refined IBD software. Individual-level single-cell RNAseq (scRNAseq) dataset from 18 human oocytes was used to verify the expression of the identified target genes. Main results and the role of chance The variant prioritization pipeline employed identified 1,160 unique variants in 1,017 genes (average per sample 59.9 sd 8.5). 453 variants were private to this study compared to the 1000 Genomes and gnomAD databases, 3% affecting splicing and/or the gene product length. Significant 5-fold enrichment of 41 genes involved in DNA-damage and repair pathways commonly associated with ovarian function/oocyte quality was observed (p < 0.001). TP53/AKT pathway also showed significant 5-fold enrichment for 45 genes (p < 0.001). This finding is consistent with the known relationship between infertility and cell-cycle/cancer genes. Overall, 66.4% (675/1,017; 95%CI:63.4-69.3) of the targeted genes were expressed in MII human oocytes. Two women (9%) were homozygous carriers of missense pathogenic variants in known candidate genes previously associated to oocyte/embryo developmental arrest (TRIP13, chr5_901344_C/T, CADD percentile 0.999; PADI6, chr1_17394384_C/G, CADD percentile 0.999). Remarkably, four additional women were carriers of high-impact variants in JAKMIP1, a member of a recently characterized family of proteins involved in various cellular processes, including cytoskeleton rearrangement, cell polarization, and intracellular transport. High-impact JAKMP1 variants were never observed in the oocyte donor control dataset. JAKMIP1 mRNA was detected in each individual biological replicate of scRNAseq analysis of MII oocytes with a mean of 6 transcripts per million. Limitations, reasons for caution Functional analysis is ongoing to validate the newly identified genes, data need to be verified in different ethnicities. Nevertheless, this study demonstrates the establishment of a specific and scalable analytical framework that can be employed for the identification of genetic causes in unexplained infertility cases characterized by defective developmental patterns. Wider implications of the findings Scaling up this investigative approach would provide an effective strategy for discovering new genes/pathways in what is considered idiopathic infertility, further defining precision reproductive medicine interventions. Importantly, this study revealed lesions in genetic patterns involved in chronic diseases providing a molecular footprint of the well-established link between infertility and comorbidities. Trial registration number none

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Genome sequencing of ion-beam-induced mutants facilitates detection of candidate genes responsible for phenotypes of mutants in rice

10.1101/666677 ◽

2019 ◽

Author(s):

Yutaka Oono ◽

Hiroyuki Ichida ◽

Ryouhei Morita ◽

Shigeki Nozawa ◽

Katsuya Satoh ◽

...

Keyword(s):

Exome Sequencing ◽

Candidate Genes ◽

Whole Exome Sequencing ◽

Dna Sequences ◽

Ion Beam ◽

Heading Date ◽

High Impact ◽

Induced Mutations ◽

Whole Exome ◽

A Genome

AbstractIon beams are physical mutagens used for plant and microbe breeding that cause mutations via a distinct mechanism from those of chemical mutagens or gamma rays. We utilized whole-exome sequencing of rice DNA in order to understand the properties of ion beam-induced mutations in a genome-wide manner. DNA libraries were constructed from selected carbon-ion-beam-induced rice mutants by capturing with a custom probes covering 66.3 M bases of nearly all exons and miRNAs predicted in the genome. A total of 56 mutations, including 24 single nucleotide variations, 23 deletions, and 5 insertions, were detected in five mutant rice lines (two dwarf and three early-heading-date mutants). The mutations were distributed among all 12 chromosomes, and the average mutation frequency in the M1 generation was estimated to be 2.7 × 10-7 per base. Many single base insertions and deletions were associated with homopolymeric repeats, whereas larger deletions up to seven base pairs were observed at polynucleotide repeats in the DNA sequences of the mutation sites. Of the 56 mutations, six were classified as high-impact mutations that caused a frame shift or loss of exons. A gene that was functionally related to the phenotype of the mutant was disrupted by a high-impact mutation in four of the five lines tested, suggesting that whole-exome sequencing of ion-beam-irradiated mutants could facilitate the detection of candidate genes responsible for the mutant phenotypes.

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P–541 Identification of novel variants and candidate genes in women with familial idiopathic premature ovarian failure using whole-exome sequencing

Human Reproduction ◽

10.1093/humrep/deab130.540 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

R Morale. Sabater ◽

B Lledo ◽

J A Ortiz ◽

F Lozano ◽

A Bernabeu ◽

...

Keyword(s):

Exome Sequencing ◽

Candidate Genes ◽

Allele Frequency ◽

Whole Exome Sequencing ◽

Minor Allele Frequency ◽

Premature Ovarian Failure ◽

Ovarian Function ◽

Ovarian Failure ◽

Minor Allele ◽

Whole Exome

Abstract Study question Is it possible to identify a genetic cause of familial premature ovarian failure (POF) with whole-exome sequencing (WES)? Summary answer Whole-exome sequencing is the most efficient strategy to identify probably pathogenic mutations in different genes in pathologies of polygenic etiology such as premature ovarian failure. What is known already Premature ovarian failure is the loss of ovarian function before the age of 40, and it is a common cause of infertility in women. This pathology has a heterogeneous etiology. Some chromosomal and genetic alterations have been described, and could explain approximately 20% of cases. However, in most patients the origin remains unknown. Recent studies with next-generation sequencing (NGS) have identified new variants in candidate genes related with premature ovarian insufficiency (POI) or premature ovarian failure (POF). These genes are not only involved in processes such as folliculogenesis, but also with DNA damage repair, homologous recombination, and meiosis. Study design, size, duration Fourteen women, from 7 families, affected by idiopathic POF were included in the study from October 2019 to September 2020. Seven POF patients were recruited when they came to our clinic to undergo assisted reproductive treatment. In the anamnesis, it was found that they had relatives with a diagnosis of POF, who were also recruited for the study. The inclusion criteria were amenorrhea before 38 years old and analytical and ultrasound signs of ovarian failure. Participants/materials, setting, methods WES was performed using TrusightOne (Illumina®). Sequenced data were aligned through BWA tool and GATK algorithm was used for SNVs/InDel identification. VCF files were annotated using Variant Interpreter software. Only the variants shared by each family were extracted for analysis and these criteria were followed: (1) Exonic/splicing variants in genes related with POF or involved in biological ovarian functions (2) Variants with minor allele frequency (MAF) ≤0.05 and (3) having potentially moderate/strong functional effects. Main results and the role of chance Seventy-nine variants possibly related with the POF phenotype were identified in the seven families. All these variants had a minor allele frequency (MAF) ≤0.05 in the gnomAD database and 1000 genomes project. Among these candidate variants, two were nonsense, six splice region, one frameshift, two inframe deletion and 68 missense. Thirty-two of the missense variants were predicted to have deleterious effects by minimum two of the four in silico algorithms used (SIFT, PolyPhen–2, MutationTaster and PROVEAN). All variants were heterozygous, and all the families carried three or more candidate variants. Altogether, 43 probably damaging genetic variants were identified in 39 genes expressed in the ovary and related with POF/POI or linked to ovarian physiology. We have described genes that have never been associated to POF pathology, however they may be involved in key biological processes for ovarian function. Moreover, some of these genes were found in two families, for example DDX11, VWF, PIWIL3 and HSD3B1. DDX11 may function at the interface of replication-coupled DNA repair and sister chromatid cohesion. VWF gene is suggested to be associated with follicular atresia in previous studies. PIWIL3 functions in development and maintenance of germline stem cells, and HSD3B1 is implicated in ovarian steroidogenesis. Limitations, reasons for caution Whole-exome sequencing has some limitations: does not cover noncoding regions of the genome, it also cannot detect large rearrangements, copy-number variants (large deletions/duplications), mosaic mutations, mutations in repetitive or high GC rich regions and mutations in genes with corresponding pseudogenes or other highly homologous sequences. Wider implications of the findings: WES has previously shown to be an efficient tool to identify genes as cause of POF, and has demonstrated the polygenic etiology. Although some studies have focused on it, and many genes are identified, this study proposes new candidate genes and variants, having potentially moderate/strong functional effects, associated with POF. Trial registration number Not applicable

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Whole exome sequencing identifies FOXL2, FOXA2 and FOXA3 as candidate genes for monogenic congenital anomalies of the kidneys and urinary tract

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfab253 ◽

2021 ◽

Author(s):

Bixia Zheng ◽

Steve Seltzsam ◽

Chunyan Wang ◽

Luca Schierbaum ◽

Sophia Schneider ◽

...

Keyword(s):

Urinary Tract ◽

Exome Sequencing ◽

Candidate Genes ◽

Whole Exome Sequencing ◽

Congenital Anomalies ◽

De Novo ◽

Horseshoe Kidney ◽

Missense Variant ◽

Whole Exome ◽

Fox Genes

Abstract Background Congenital anomalies of the kidneys and urinary tract (CAKUT) constitute the most common cause of chronic kidney disease in the first three decades of life. Variants in four Forkhead box (FOX) transcription factors have been associated with CAKUT. We hypothesized that other FOX genes, if highly expressed in developing kidney, may also represent monogenic causes of CAKUT. Methods We here performed whole exome sequencing (WES) in 541 families with CAKUT and generated 4 lists of CAKUT candidate genes: A) 36 FOX genes showing high expression during renal development, B) 4 FOX genes known to cause CAKUT to validate list A; C) 80 genes that we identified as unique potential novel CAKUT candidate genes when performing WES in 541 CAKUT families, and D) 175 genes identified from WES as multiple potential novel CAKUT candidate genes. Results To prioritize potential novel CAKUT candidates in FOX gene family, we overlapped 36 FOX genes (list A) with list C and D of WES-derived CAKUT candidates. Intersection with list C, identified a de novo FOXL2 in-frame deletion in a patient with eyelid abnormalities and ureteropelvic junction obstruction, and a homozygous FOXA2 missense variant in a patient with horseshoe kidney. Intersection with list D, identified a heterozygous FOXA3 missense variant in a CAKUT family with multiple affected individuals. Conclusion We hereby identified FOXL2, FOXA2 and FOXA3 as novel monogenic candidate genes of CAKUT, supporting the utility of a paralog-based approach to discover mutated genes associated with human disease.

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Searching for the missing genetic determinants of hereditary breast cancer risk by whole-exome sequencing of BRCA-negative patients: new candidate genes USP39, SLIT3, CREB3

Problems in oncology ◽

10.37469/0507-3758-2021-67-1-111-116 ◽

2021 ◽

Vol 67 (1) ◽

pp. 111-116

Author(s):

Kirill Zagorodnev ◽

Aleksandr Romanko ◽

Uliy Gorgul ◽

Aleksandr Ivantsov ◽

Anna Sokolenko ◽

...

Keyword(s):

Breast Cancer ◽

Exome Sequencing ◽

Candidate Genes ◽

Whole Exome Sequencing ◽

Hereditary Breast Cancer ◽

Germ Line ◽

Genetic Diagnosis ◽

Clinical Signs ◽

Causative Mutation ◽

Whole Exome

The search for the new hereditary mutations and a precise molecular genetic diagnosis that determines the causative mutation in each specific case of hereditary breast cancer (BC) is a clinically important task since it helps to define the personal therapeutic approach and increase the effectiveness of preventive measures. Using whole-exome sequencing (WES) we analyzed the full spectrum of hereditary variations in 49 Russian patients with clinical signs of a hereditary disease which allowed us to compile a list of 229 candidate probably pathogenic germ-line variants. Then, the selected candidate mutations were validated by Sanger sequencing and molecular-epidemiological studies, the predisposing roles of three oncologically relevant mutations (USP39 c.*208G>C, SLIT3 p.Arg154Cys, and CREB3 p.Lys157Glu) were confirmed. Our candidate genes are first mentioned in connection with the hereditary risk of BC. The final proofs of the causative roles of these variants could be obtained through functional tests as well as via the analysis of the mutations segregation in BC families.

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Whole-Exome Sequencing Analysis of Human Semen Quality in Russian Multiethnic Population

Frontiers in Genetics ◽

10.3389/fgene.2021.662846 ◽

2021 ◽

Vol 12 ◽

Author(s):

Semyon Kolmykov ◽

Gennady Vasiliev ◽

Ludmila Osadchuk ◽

Maxim Kleschev ◽

Alexander Osadchuk

Keyword(s):

Exome Sequencing ◽

Ethnic Groups ◽

Whole Exome Sequencing ◽

Complex Traits ◽

Semen Quality ◽

Population Sample ◽

Sperm Concentration ◽

Sequencing Analysis ◽

New Genes ◽

Whole Exome

The global trend toward the reduction of human spermatogenic function observed in many countries, including Russia, raised the problem of extensive screening and monitoring of male fertility and elucidation of its genetic and ethnic mechanisms. Recently, whole-exome sequencing (WES) was developed as a powerful tool for genetic analysis of complex traits. We present here the first Russian WES study for identification of new genes associated with semen quality. The experimental 3 × 2 design of the WES study was based on the analysis of 157 samples including three ethnic groups—Slavs (59), Buryats (n = 49), and Yakuts (n = 49), and two different semen quality groups—pathozoospermia (n = 95) and normospermia (n = 62). Additionally, our WES study group was negative for complete AZF microdeletions of the Y-chromosome. The normospermia group included men with normal sperm parameters in accordance with the WHO-recommended reference limit. The pathozoospermia group included men with impaired semen quality, namely, with any combined parameters of sperm concentration <15 × 106/ml, and/or progressive motility <32%, and/or normal morphology <4%. The WES was performed for all 157 samples. Subsequent calling and filtering of variants were carried out according to the GATK Best Practices recommendations. On the genotyping stage, the samples were combined into four cohorts: three sets corresponded to three ethnic groups, and the fourth set contained all the 157 whole-exome samples. Association of the obtained polymorphisms with semen quality parameters was investigated using the χ2 test. To prioritize the obtained variants associated with pathozoospermia, their effects were determined using Ensembl Variant Effect Predictor. Moreover, polymorphisms located in genes expressed in the testis were revealed based on the genomic annotation. As a result, the nine potential SNP markers rs6971091, rs557806, rs610308, rs556052, rs1289658, rs278981, rs1129172, rs12268007, and rs17228441 were selected for subsequent verification on our previously collected population sample (about 1,500 males). The selected variants located in seven genes FAM71F1, PPP1R15A, TRIM45, PRAME, RBM47, WDFY4, and FSIP2 that are expressed in the testis and play an important role in cell proliferation, meiosis, and apoptosis.

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