Human Genetic Variation Database Research Articles

P-716 Genetic mutations in women with recurrent assisted reproductive technology failure

Abstract Study question Are there genetic mutations which may lead to recurrent failures of assisted reproductive technology? Summary answer In this study, some genes were found to be candidates for causing repeated ART failures. What is known already Although reproductive aging is one of the greatest hindrances to successful pregnancy, many patients remain infertile, enduring recurrent ART failures, and approximately 20% of infertility is idiopathic. At present, the mutation of 8 genes, PADI6, NLRP2, TLE6, NLRP5, BTG4, KHDC3L, REC114 and OOEP has been reported to cause early embryonic arrest, while the mutation of PATL2 with GV stage arrest; TUBB8 and TRIP13 with failure of polar body extrusion; PANX1 with oocyte death; ZP1, ZP2, ZP3 with empty follicle syndrome; WEE2, TLE6 and CDC20 with fertilization failure have been found. Study design, size, duration Between January and October 2021, at Hanabusa Women’s Clinic, 25 infertile women, who experienced IVF / ICSI failure more than 5 times, with blastocyst formation rates of less than 10%, were recruited, after informed consent was obtained. 10 staff members of Hanabusa Women’s Clinic who conceived and delivered naturally were compared as normal controls. Participants/materials, setting, methods Genomic DNA was extracted from whole blood and genomic DNA samples were subjected to whole exome sequencing. High impact variants with allele frequencies of ≦0.20 in the 1000 Genome EAS, which were not found in the control subjects, were selected. Data from the Human Genetic Variation Database (HGVD) was used for the Hardy-Weinberg equation (HWE) to evaluate possible candidates for recurrent ART failures caused by genetic mutations. Main results and the role of chance High impact homozygous variants were detected in 57 genes among the study subjects of infertile women, but not among the control subjects. Among these 57 genes, significant differences in the allele frequencies between 8.3KJPN and the patients were detected in ADAM33 (p = 0.009), CEP89 (p = 0.012), CRIPAK (p < 0.001), LGALS9B (p < 0.001), PDZRN3 (p = 0.001), RAET1E (p = 0.007) and SPATA31A3 (p = 0.045). The HWE of each gene was calculated based on the data from HGVD. Among the 57 genes mentioned above, significantly lower HWE was detected in patients as compared to the HGVD in ADAM33 (p < 0.001), CEP89 (p = 0.033), MICA (p < 0.001), OR2T29 (p = 0.003), OR52J3 (p = 0.0497), RABL2A (p < 0.001), RNF17 (p = 0.0499), SPATA31C1 (p = 0.030) and WWTR1 (p < 0.001). Having identified one of the piRNA pathway genes, RNF17, which had significantly reduced HWE, the localization of RNF17 in the primordial follicle was examined. Immunofluorescent staining showed that RNF17 was sporadically localized in the cytoplasm of primordial follicles. Limitations, reasons for caution The sample size of this study was relatively small, and precise information such as age, gender in the database used in this study did not match that of the study patients, thus, the interpretation of the results is limited and further research is required to confirm our findings. Wider implications of the findings Among abovementioned genes, one of the piRNA pathway genes, RNF17, and one of the Hippo signal pathway genes, WWER1, were the most likely causes of repeated ART failures. The findings may provide potential diagnostic markers for patients with recurrent ART failures, helping us understand the genetic basis of female infertility. Trial registration number Not applicable

Rare variants and HLA haplotypes associated in patients with neuromyelitis optica spectrum disorders.

Neuromyelitis optica spectrum disorders (NMOSD) are rare, debilitating autoimmune diseases of the central nervous system. Many NMOSD patients have antibodies to Aquaporin-4 (AQP4). Prior studies show associations of NMOSD with individual Human Leukocyte Antigen (HLA) alleles and with mutations in the complement pathway and potassium channels. HLA allele associations with NMOSD are inconsistent between populations, suggesting complex relationships between the identified alleles and risk of disease. We used a retrospective case-control approach to identify contributing genetic variants in patients who met the diagnostic criteria for NMOSD and their unaffected family members. Potentially deleterious variants identified in NMOSD patients were compared to members of their families who do not have the disease and to existing databases of human genetic variation. HLA sequences from patients from Belgrade, Serbia, were compared to the frequency of HLA haplotypes in the general population in Belgrade. We analyzed exome sequencing on 40 NMOSD patients and identified rare inherited variants in the complement pathway and potassium channel genes. Haplotype analysis further detected two haplotypes, HLA-A*01, B*08, DRB1*03 and HLA-A*01, B*08, C*07, DRB1*03, DQB1*02, which were more prevalent in NMOSD patients than in unaffected individuals. In silico modeling indicates that HLA molecules within these haplotypes are predicted to bind AQP4 at several sites, potentially contributing to the development of autoimmunity. Our results point to possible autoimmune and neurodegenerative mechanisms that cause NMOSD, and can be used to investigate potential NMOSD drug targets.

Brain region-specific effects of nearly fixed sapiens-derived alleles

The availability of high-coverage genomes of our extinct relatives, the Neanderthals and Denisovans, and the emergence of large, tissue-specific databases of modern human genetic variation, offer the possibility of probing the effects of modern-derived alleles in specific tissues, such as the brain, and its specific regions. While previous research has explored the effects of introgressed variants in gene expression, the effects of Homo sapiens-specific gene expression variability are still understudied. Here we identify derived, Homo sapiens-specific high-frequency (≥90%) alleles that are associated with differential gene expression across 15 brain structures derived from the GTEx database. We show that regulation by these derived variants targets regions under positive selection more often than expected by chance, and that high-frequency derived alleles lie in functional categories related to transcriptional regulation. Our results highlight the role of these variants in gene regulation in specific regions like the cerebellum and pituitary.

Four Variations Detected in 3 Genes Related to Thrombophilia by Multiplex ARMS – PCR

Thrombophilia is a condition that may cause venous obstruction, leading to serious illnesses such as stroke, respiratory failure, and even death. This condition is hereditary or related to the immune system. Inherited thrombophilia is screened by analysing genetic variants (allele). In this study, we analyse 4 variations in 3 genes, which occur at high variable frequency, namely Methytetrahydrofolate Reductase (MTHFR C677T, MTHFR A1298C), Factor V Leiden (FVL C1691T) and Plasminogen Activator Inhibitor 1 (PAI-1 5G/4G), using allele-specific polymerase chain reaction (ARMS-PCR). The primers were designed using FastPCR program and PCR conditions were optimized to identify 8 alleles of 4 variants through 3 multiplex ARMS - PCR reactions. The accuracy of the ARMS - PCR results was confirmed by Sanger sequencing and the real time PCR commercial kit (SNP). Mastering the allele specific PCR technique contributes to the survey of genetic variations in the Vietnamese population in order to screen and treat thrombophilia-related diseases.
 Keywords
 Thrombophilia, allele-specific polymerase chain reaction (ARMS-PCR), variants MTHFR C677T, MTHFR A1298C, FVL C1691T, PAI-1 4G/5G.
 References
 [1] A. Dautaj, G. Krasi, V. Bushati et al., Hereditary Thrombophilia, Acta Biomedica, Vol. 90, No. 10S, 2019, pp. 44-46, https://doi.org/10.23750/abm.v90i10-S.8758.[2] M. G. Beckman, W. C. Hooper, S. E. Critchley, T. L. Ortel, Venous Thromboembolism: A Public Health Concern, American Journal of Preventive Medicine, Vol. 38, No. 4S, 2010, pp. S495-S501, https://doi.org/10.1016/j.amepre.2009.12.017.[3] G. E. Raskob, R. Silverstein, D. W. Bratzler et al., Surveillance for Deep Vein Thrombosis and Pulmonary Embolism: Recommendations from A National Workshop, American Journal Preventive Medicine, Vol. 38, No. 4S, 2010, pp. S502-S509, https://doi.org/10.1016/j.amepre.2010.01.010.[4] J. A. Heit, M. D. Silverstein, D. N. Mohr et al., The Epidemiology of Venous Thromboembolism in the Community, Thrombosis and Haemostasis, Vol. 86, No. 1, 2001, pp. 452-463, https://doi.org/10.1055/s-0037-1616243.[5] M. Cushman, A. W. Tsai, R. H. White et al., Deep Vein Thrombosis and Pulmonary Embolism in Two Cohorts: The Longitudinal Investigation of Thromboembolism Etiology, The American Journal of Medicine, Vol. 117, No. 1, 2004, pp. 19-25, https://doi.org/10.1016/j.amjmed.2004.01.018.[6] J. A. Heit, The Epidemiology of Venous Thromboembolism in the Community: Implications for Prevention and Management, Journal of Thrombosis and Thrombolysis, Vol. 21, No. 1, 2006, pp. 23-29, https://doi.org/10.1007/s11239-006-5572-y.[7] R. H. White, The Epidemiology of Venous Thromboembolism, Circulation, Vol. 107, No. 23 S1, 2003, pp. I4-I8, https://doi.org/10.1161/01.cir. 0000078468.11849.66.[8] A. Wahed, A. Dasgupta, Thrombophilias and Their Detection, Hematology and Coagulation: A Comprehensive Review for Board Preparation, Certification and Clinical Practice, Elsevier, United States of America, 2015, pp. 263-275.[9] SNP Biotechnology, SNP Detection Real-Time PCR Kits, http://www.snp.com.tr/EN,37/snp-detection-real-time-pcr-kits.html/, 2021 (accessed on: May 1st, 2021).[10] V. S. Le, K. T. Tran, H. T. P. Bui et al., A Vietnamese Human Genetic Variation Database, Human Mutation, Vol. 40, No. 10, 2019,pp. 1664-1675, https://doi.org/10.1002/humu.23835.[11] dbSNP – NCBI, Reference SNP (rs) Report (rs6025, rs1799762), https://www.ncbi.nlm.nih.gov/snp/,02021 2021 (accessed on: May 1st, 2021).[12] D. C. Rees, M. Cox, J. B. Clegg, World Distribution of Factor V Leiden, The Lancet, Vol. 346, No. 8983, 1995, pp. 1133-1134, https://doi.org/10.1016/s0140-6736(95)91803-5. A. Fateh, M. Aghasadeghi, S. D. Siadat et al., Comparison of Three Different Methods for Detection of IL28 rs12979860 Polymorphisms as A Predictor of Treatment Outcome in Patients with Hepatitis C Virus, Osong Public Health and Research Perspectives, Vol. 7, No. 2, 2016, pp. 83-89, https://doi.org/10.1016/j.phrp.2015.11.004. [13] T. Huang, J. Zhuge, W. W. Zhang, Sensitive Detection of BRAF V600E Mutation by Amplification Refractory Mutation System (ARMS)-PCR, Biomarker Research, Vol. 1, No. 3, 2013, https://doi.org/10.1186/2050-7771-1-3.[14] R. F. V. Medrano, C. A. D. Oliveira, Guidelines for the Tetra-primer ARMS-PCR Technique Development, Molecular Biotechnology, Vol. 56, 2014, pp. 599-608, https://doi.org/10.1007/s12033-014-9734-4.[15] C. Runcharoen, K. Fukunaga, I. Sensorn et al., Prevalence of Pharmacogenomic Variants in 100 Pharmacogenes Among Southeast Asian Populations under Tarmacogenomics Research Network (SEAPharm), Human Genome Variation, Vol. 8, No. 7, 2021, https://doi.org/10.1038/s41439-021-00135-z.

Prevalence of Wilson disease based on genome databases in Japan.

Wilson disease (WD) is an autosomal recessive disorder caused by mutations in the ATP7B gene. In 1984, Scheinberg and Sternlieb estimated the prevalence of WD to be 1:30000. However, recent epidemiological studies have reported increasing prevalence rates in different populations. The carrier frequency of ATP7B variants and the prevalence of WD in the Japanese population have not been reported using multiple databases. Multiple public databases were used. First, we included mutations in the ATP7B gene that were registered in the Human Gene Mutation Database (HGMD) Professional, where 885 ATP7B variants were identified as pathogenic. Next, we investigated the allele frequencies of these 885 variants in Japanese individuals using the Human Genetic Variation Database (HGVD) and the Japanese Multi Omics Reference Panel (jMorp). Of the 885 variants of ATP7B, 7 and 12 missense and nonsense variants, zero and three splicing variants, and zero and two small deletions were found in the HGVD and in jMorp, respectively. The total allele frequencies of the ATP7B mutations were 0.011 in the HGVD and 0.014 in the jMorp. According to these data, the carrier frequencies were 0.022 (2.2%) and 0.028 (2.8%), respectively, and patient frequencies were 0.000121 (1.21/10000 individuals) and 0.000196 (1.96/10000 individuals), respectively. This is the first study to report the carrier frequency of ATP7B variants and the prevalence of WD in Japan using multiple databases. The calculated prevalence of WD was comparatively higher than that of previous reports, indicating previous underdiagnosis or the existence of less severe phenotypes.

Allele frequency of variants reported to cause adenine phosphoribosyltransferase deficiency

Adenine phosphoribosyltransferase deficiency is a rare, autosomal recessive disorder of purine metabolism that causes nephrolithiasis and progressive chronic kidney disease. The small number of reported cases indicates an extremely low prevalence, although it has been suggested that missed diagnoses may play a role. We assessed the prevalence of APRT deficiency based on the frequency of causally-related APRT sequence variants in a diverse set of large genomic databases. A thorough search was carried out for all APRT variants that have been confirmed as pathogenic under recessive mode of inheritance, and the frequency of the identified variants examined in six population genomic databases: the deCODE genetics database, the UK Biobank, the 100,000 Genomes Project, the Genome Aggregation Database, the Human Genetic Variation Database and the Korean Variant Archive. The estimated frequency of homozygous genotypes was calculated using the Hardy-Weinberg equation. Sixty-two pathogenic APRT variants were identified, including six novel variants. Most common were the missense variants c.407T>C (p.(Met136Thr)) in Japan and c.194A>T (p.(Asp65Val)) in Iceland, as well as the splice-site variant c.400 + 2dup (p.(Ala108Glufs*3)) in the European population. Twenty-nine variants were detected in at least one of the six genomic databases. The highest cumulative minor allele frequency (cMAF) of pathogenic variants outside of Japan and Iceland was observed in the Irish population (0.2%), though no APRT deficiency cases have been reported in Ireland. The large number of cases in Japan and Iceland is consistent with a founder effect in these populations. There is no evidence for widespread underdiagnosis based on the current analysis.

AScan: A Novel Method for the Study of Allele Specific Expression in Single Individuals

In diploid organisms, two copies of each allele are normally inherited from parents. Paternal and maternal alleles can be regulated and expressed unequally, which is referred to as allele-specific expression (ASE). In this work, we present aScan, a novel method for the identification of ASE from the analysis of matched individual genomic and RNA sequencing data. By performing extensive analyses of both real and simulated data, we demonstrate that aScan can correctly identify ASE with high accuracy and sensitivity in different experimental settings. Additionally, by applying our method to a small cohort of individuals that are not included in publicly available databases of human genetic variation, we outline the value of possible applications of ASE analysis in single individuals for deriving a more accurate annotation of “private” low-frequency genetic variants associated with regulatory effects on transcription. All in all, we believe that aScan will represent a beneficial addition to the set of bioinformatics tools for the analysis of ASE. Finally, while our method was initially conceived for the analysis of RNA-seq data, it can in principle be applied to any quantitative NGS assay for which matched genotypic and expression data are available. AvailabilityaScan is currently available in the form of an open source standalone software package at: https://github.com/Federico77z/aScan/. aScan version 1.0.3, available at https://github.com/Federico77z/aScan/releases/tag/1.0.3, has been used for all the analyses included in this manuscript. A Docker image of the tool has also been made available at https://github.com/pmandreoli/aScanDocker.

Splice site mutation of LMNA causes severe dilated cardiomyopathy via strong dominant reduction of total lamin expression

Abstract Objective LMNA is a known causative gene of dilated cardiomyopathy (DCM) and familial cardiac conduction disturbance (CCD). Genetic variants affecting the pre-mRNA splicing process often lead to premature stop codons and result in nonsense-mediated mRNA decay (NMD), followed by degradation of mutated alleles. The misssense variant LMNA c. 936G>C was previously reported in a French family affected by muscular dystrophy, CCD, and DCM, but no detailed analysis has been performed. We so far identified the same variant in two Japanese families affected by CCD and DCM. In this study, we investigated the molecular consequences of the variant located at the last codon of LMNA exon5 to demonstrate its pathogenicity. Methods Genomic DNA and total RNA were isolated from patients' peripheral blood lymphocytes or cardiac tissue. LMNA-coding exons were screened by direct sequencing. Complementary DNAs (cDNAs) were generated by reverse transcription PCR from RNA. Quantitative PCR (qPCR) was performed to quantify the LMNA cDNA amount by using specific primers for lamins A and C. The protein expressions of both isoforms were analyzed by western blotting. Results We detected the heterozygous LMNA c.936 G>C (p. Q312H) variant at the end of exon 5 by genomic DNA sequencing in two unrelated Japanese families (figure. pedigree) affected by DCM and CCD. In a genomic database survey, we did not find the variant in either gnomAD, TogoVar, or the Human Genetic Variation Database. The two commonly used splice site predictor tools, NetGene2 and FSPLICE, estimated that this site was a splice donor site. Sequencing of cDNA demonstrated that the mutated allele was absent. By qPCR assay, we confirmed a 90% reduction in LMNA cDNA. Western blot analysis revealed that lamin A and C expression was reduced far more than 50% (figure. western blot). Conclusions We report a LMNA missense mutation found in two families, which disrupts a normal splicing site, leads to NMD, and resulted in severe cardiac laminopathy. The drastic reductions of lamin expression at the cDNA and protein levels suggested that other co-existing mechanisms may also have suppressed the expression of the healthy wild type allele. Pedigree and western blot assay Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Japan Society for the Promotion of Science KAKENHI

Mesothelioma developing in carriers of inherited genetic mutations.

Malignant mesothelioma is associated with the exposure to asbestos fibers. Recent discovery of the BAP1 cancer syndrome, a Mendelian disorder with high-penetrance autosomal dominant inheritance fostered the genotyping for nucleotide-level or larger structural alteration of germline DNA. Inherited heterozygous mutations of the BAP1 gene increase the susceptibility to carcinogenic fibers, leading to a concept of gene x environment interaction (GxE) as a pathogenetic mechanism of mesothelioma. Several studies on cohorts of unselected patients with mesothelioma or on familial/early-onset cohorts of mesothelioma cases converged on BAP1 as the more frequent germline mutated gene, followed by other genes involved in DNA repair and homologous recombination. Evidence has been emerging that patients with mesothelioma carrying germline mutations of BAP1 and of other genes, such as those involved in DNA repair and tumor suppressor genes, have better prognosis and higher chemosensitivity when compared with patients with germline wildtype Bap1. We report here a germline genomic analysis targeted 22 genes in a cohort of 101 Japanese patients irrespective of asbestos exposure, age at diagnosis, or personal or family history of cancer. By comparing the results with the Human Genetic Variation Database (HGVD) and the Genome Aggregation Database (gnomAD) we selected rare germline variants with a Combined Annotation Dependent Depletion (CADD) >20. We show here that 31 of 101 subjects were carrying 25 rare variants in 14 genes, neither reported in the HGVD nor in the gnomAD database for 14/25 variants. Besides pathogenic variants of BAP1, rare missense variants were found in genes encoding lysine-specific histone methyltransferase SETD2 and SETDB1 and genes encoding subunits of the mSWI/SNF chromatin remodeling complex. The complete scenario of the genetic background consisting of pathogenic germline variants required for the predisposition and GxE for pathogenesis of mesothelioma appears complex, and further large-scale studies are warranted.

Association of Germline Variants of TCF3 and PAX5 with Pediatric Acute Lymphoblastic Leukemia Development

Background: The development of B-lymphocyte is controlled by the coordinated action of transcription factors networks, such as IKZF1, TCF3, EBF1 or PAX5. It is increasingly recognized that germline variants of those genes which disrupt their function are responsible for inborn errors of humoral immunity. Meanwhile, it is well understood genetic alterations of those molecules are frequently altered in B-lineage acute lymphoblastic leukemia (ALL) and are associated with leukemogenesis. However, those involvements are usually attributed to somatic mutation. Germline variations may also trigger cancer development in some cases. For instance, genome-wide association studies (GWAS) have identified B-cell precursor (BCP-) ALL susceptibility variants within IKZF1. Furthermore, germline variants of IKZF1, TCF3, and PAX5 are reported to be associated with B-ALL development. Therefore, we hypothesize that attenuated function due to germline variation in those genes may predispose to ALL development in Japanese population to some extent. Method: Saliva DNA from 583 pediatric ALL patients in remission registered in Tokyo Children's Cancer Study Group (TCCSG) were analyzed in this study. Targeted sequencing for exons and exon-intron boundaries of IKZF1, TCF3, and PAX5 was performed using next-generation sequencer. Frequency of the detected variant alleles was compared to data from the Exome Aggregation Consortium (ExAC) East Asian Cohort (n=4327), Tohoku Medical Megabank Organization (ToMMo) cohort (n=3554), and the Human Genetic Variation Database (HGVD) cohort (n=1208) by chi-square test or Fisher's exact test. Results: In total, 113 variants (28 nonsynonymous, 19 synonymous, 56 intronic, 8 untranslated regions, and 2 deletions) were detected. We could not detect any nonsynonymous variations, insertion or deletion in IKZF1. On the other hand, we detected 8 rare variations in TCF3 and 7 of them are found to be deleterious by in silico analysis. Among them, 1 variant, TCF3 p.S514L, was significantly more common in ALL patients (0.26% in patients vs 0% in ExAC, 0.01% in ToMMO and 0% in HGVD, p=0.0017, 0.0115 and 0.0343 respectively). In PAX5, we found 6 rare variations and 3 of them are found to be deleterious by in silico analysis, However, we could not identify a statistically significant difference in frequency among these variants between TCCSG and normal cohorts. On the other hand, a patient who harbored heterozygous PAX5 p.V26G, which had been reported as a somatic mutation in B-ALL, was diagnosed with primary antibody deficiency after treatment of B-ALL. He is receiving immunoglobulin replacement therapy without relapse of ALL for 15 years. Discussion: Our study implied TCF3 p.S514L is associated with ALL development. Besides, we identified a case of heterozygous PAX5 p.V26G variant who exhibited antibody deficiency and B-ALL. These findings suggest those rare variants impair the normal B-cell development and may predispose to ALL development. GWAS is effective in evaluating common genetic variants. However, it is hard to identify the genes involved Mendelian inheritance or functionally defective rare variants associated with disease susceptibility. Although it is rare, our study suggests that rare variants of genes involved in B cell development could be associated with ALL susceptibility and immunodeficiency, and we revealed the frequency of functionally defective rare variants in Japanese childhood ALL cohort. Disclosures No relevant conflicts of interest to declare.

A Vietnamese human genetic variation database.

Large scale human genome projects have created tremendous human genome databases for some well-studied populations. Vietnam has about 95 million people (the 14th largest country by population in the world) of which more than 86% are Kinh people. To date, genetic studies for Vietnamese people mostly rely on genetic information from other populations. Building a Vietnamese human genetic variation database is a must for properly interpreting Vietnamese genetic variants. To this end, we sequenced 105 whole genomes and 200 whole exomes of 305 unrelated Kinh Vietnamese (KHV) people. We also included 101 other previously published KHV genomes to build a Vietnamese human genetic variation database of 406 KHV people. The KHV database contains 24.81 million variants (22.47 million single nucleotide polymorphisms (SNPs) and 2.34 million indels) of which 0.71 million variants are novel. It includes more than 99.3% of variants with a frequency of >1% in the KHV population. Noticeably, the KHV database revealed 107 variants reported in the human genome mutation database as pathological mutations with a frequency above 1% in the KHV population. The KHV database (available at https://genomes.vn) would be beneficial for genetic studies and medical applications not only for the Vietnamese population but also for other closely related populations.

Biomolecular Databases and Subnetwork Identification Approaches of Interest to Big Data Community: An Expert Review.

Next-generation sequencing approaches and genome-wide studies have become essential for characterizing the mechanisms of human diseases. Consequently, many researchers have applied these approaches to discover the genetic/genomic causes of common complex and rare human diseases, generating multiomics big data that span the continuum of genomics, proteomics, metabolomics, and many other system science fields. Therefore, there is a significant and unmet need for biological databases and tools that enable and empower the researchers to analyze, integrate, and make sense of big data. There are currently large number of databases that offer different types of biological information. In particular, the integration of gene expression profiles and protein-protein interaction networks provides a deeper understanding of the complex multilayered molecular architecture of human diseases. Therefore, there has been a growing interest in developing methodologies that integrate and contextualize big data from molecular interaction networks to identify biomarkers of human diseases at a subnetwork resolution as well. In this expert review, we provide a comprehensive summary of most popular biomolecular databases for molecular interactions (e.g., Biological General Repository for Interaction Datasets, Kyoto Encyclopedia of Genes and Genomes and Search Tool for The Retrieval of Interacting Genes/Proteins), gene-disease associations (e.g., Online Mendelian Inheritance in Man, Disease-Gene Network, MalaCards), and population-specific databases (e.g., Human Genetic Variation Database), and describe some examples of their usage and potential applications. We also present the most recent subnetwork identification approaches and discuss their main advantages and limitations. As the field of data science continues to emerge, the present analysis offers a deeper and contextualized understanding of the available databases in molecular biomedicine.

Impact of cardiac myosin light chain kinase gene mutation on development of dilated cardiomyopathy.

AimsCardiac myosin light chain kinase (cMLCK) phosphorylates ventricular myosin regulatory light chain 2 (MLC2v) and regulates sarcomere and cardiomyocyte organization. However, few data exist regarding the relationship between cMLCK mutations and MLC2v phosphorylation, particularly in terms of developing familial dilated cardiomyopathy (DCM) in whom cMLCK gene mutations were identified. The purpose of the present study was to investigate functional consequences of cMLCK mutations in DCM patients.Methods and resultsThe diagnosis of DCM was based on the patients' history and on echocardiography. We screened cMLCK gene mutations in DCM probands with high resolution melting analysis. Known DCM‐causing genes mutations were excluded by exome sequencing of family members. MLC2v phosphorylation was analysed by Phos‐tag sodium dodecyl sulfate–polyacrylamide gel electrophoresis assays. We also performed ADP‐Glo assays for determining the total amount of adenosine triphosphate used in the kinase reaction. Unrelated DCM probands (109 males and 40 females) were enrolled in this study, of which 16 were familial and 133 sporadic. By mutation screening, a truncation variant of c1915‐1 g>t (p.Pro639Valfs*15) was identified, which was not detected in 400 chromosomes of 200 healthy volunteers; it is listed in the Human Genetic Variation Database with an allele frequency < 0.001. In the proband, the presence of mutations in known DCM‐causing genes was excluded with exome analysis. Familial analysis identified a 19‐year‐old male carrier who manifested slight left ventricular dilation with preserved systolic function. Phosphorylation assays analysed by Phos‐tag SDS‐PAGE revealed that the identified p.Pro639Valfs*15 mutation results in a complete lack of kinase activity, although it did not affect wild‐type cMLCK activity. ADP‐Glo assays confirmed that the mutant cMLCK had no kinase activity, whereas wild‐type cMLCK had a Km value of 5.93 ± 1.47 μM and a V max of 1.28 ± 0.03 mol/min/mol kinase.ConclusionsThese results demonstrate that a truncation mutation in the cMLCK gene p.Pro639Valfs*15 can be associated with significant impairment of MLC2v phosphorylation and possibly with development of DCM, although a larger study of DCM patients is required to determine the prevalence of this mutation and further strengthen its association with disease development.

Genotypic and Phenotypic Analysis of Adolescents with Heavy Menstrual Bleeding and Low Von Willebrand Activity - Interim Report of a Multi-Center Study

Genotypic and Phenotypic Analysis of Adolescents with Heavy Menstrual Bleeding and Low Von Willebrand Activity - Interim Report of a Multi-Center Study

Abstract 3887: Whole exome sequencing to identify genetic markers for trastuzumab-induced cardiotoxicity

Abstract Although trastuzumab-induced cardiotoxicity is an important determinant to limit the use of this drug, the molecular mechanism of risk for this toxicity is not well understood. To identify a genetic marker(s) determining the risk of trastuzumab-induced cardiotoxicity, we performed whole exome sequencing of germline DNA samples from 9 patients with trastuzumab-induced cardiotoxicity, and conducted a case-control association study of 2,258 genetic variants between 9 cases (with trastuzumab-induced cardiotoxicity) and general Japanese population controls registered in Human Genetic Variation Database (HGVD). To further validate the result of the screening study, we carried out a replication study of 10 variants showing Pmin &amp;lt; 0.001 in the screening study using 234 independent patients treated with trastuzumab, including 10 cases and 224 controls (without trastuzumab-induced cardiotoxicity). In the replication study, we observed that three variants had effect in the same direction as in the screening study (SNV1 in exon 2 of HERADR1, SNV2 in exon 2 of HERADR2 and SNV3 in exon 44 of HERADR3). A combined result of the screening and the replication studies suggested an association of a locus on chromosome 6q12 with trastuzumab-induced cardiotoxicity (SNV3 in HERADR3, combined-Pmin = 0.00056, OR = 13.73). This finding provides new insights into personalized trastuzumab therapy for the patients with HER2 positive cancer. Citation Format: Chihiro Udagawa, Hiromi Nakamura, Hiroshi Ohnishi, Kenji Tamura, Tatsunori Shimoi, Masayuki Yoshida, Teruhiko Yoshida, Hiroshi Okamura, Yasushi Totoki, Tatsuhiro Shibata, Hitoshi Zembutsu. Whole exome sequencing to identify genetic markers for trastuzumab-induced cardiotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3887.

Analysis of All 34 Exons of the SPINK5 Gene in Japanese Atopic Dermatitis Patients.

Lympho-epithelial Kazal-type-related inhibitor (LEKTI) is a large multidomain serine protease inhibitor that is expressed in epidermal keratinocytes. Nonsense mutations of the SPINK5 gene, which codes for LEKTI, cause Netherton syndrome, which is characterized by hair abnormality, ichthyosis, and atopy. A single nucleotide polymorphism (SNP) of SPINK5, p.K420E, is reported to be associated with the pathogenesis of atopic dermatitis (AD). We studied all 34 exons of the SPINK5 gene in Japanese 57 AD patients and 50 normal healthy controls. We detected nine nonsynonymous variants, including p.K420E; these variants had already been registered in the SNP database. Among them, p.R654H (n=1) was found as a heterozygous mutation in the AD patients, but not in the control. No new mutation was detected. We next compared the data of the AD patients with data from the Human Genetic Variation Database provided by Kyoto University; a significant difference was found in the frequency of the p.S368N genotype distribution. PolyPhen-2 and SIFT, two algorithms for predicting the functional effects of amino acid substitutions, showed significant scores for p.R654H. Therefore, R654H might be a risk factor for epidermal barrier dysfunction in some Japanese AD patients.

Whole exome sequencing to identify genetic markers for trastuzumab-induced cardiotoxicity.

Although trastuzumab‐induced cardiotoxicity is an important determinant to limit the use of this drug, the molecular mechanism of risk for this toxicity is not well understood. To identify genetic variants determining the risk of trastuzumab‐induced cardiotoxicity, we carried out whole exome sequencing of germline DNA samples from 9 patients with trastuzumab‐induced cardiotoxicity, and conducted a case‐control association study of 2258 genetic variants between 9 cases (with trastuzumab‐induced cardiotoxicity) and general Japanese population controls registered in the Human Genetic Variation Database (HGVD). The top variant which showed the lowest P‐value in the screening study was rs139503277 in PHD Finger Protein 3 (P min = .00012, odds ratio [OR] = 51.23). To further validate the result of screening study, we carried out a replication study of 10 variants showing P min < .001 in the screening study using 234 independent patients treated with trastuzumab, including 10 cases and 224 controls (without trastuzumab‐induced cardiotoxicity). In the replication study, we observed that three variants had an effect in the same direction as in the screening study (rs78272919 in exon 2 of Keratin 15, rs5762940 in exon 2 of zinc and ring finger 3, and rs139944387 in exon 44 of Eyes shut homologs [EYS]). A combined result of the screening and the replication studies suggested an association of a locus on chromosome 6q12 with trastuzumab‐induced cardiotoxicity (rs139944387 in EYS, combined P min = .00056, OR = 13.73). This finding provides new insights into personalized trastuzumab therapy for patients with human epidermal growth factor receptor 2 (HER2)‐positive cancer.

Whole-exome sequencing in a Japanese family with highly aggregated diabetes identifies a candidate susceptibility mutation in ADAMTSL3

AimsThe aim of this study was to clarify the genetic background of a family with multiple cases of diabetes accompanied by absolute insulin deficiency using whole-exome sequencing (WES). MethodsIn a Japanese family, WES was performed in four affected members with absolute insulin deficiency and two unaffected members. We focused on variants that were predicted to be disease-causing by bioinformatics and were shared by all of the four affected members but were not present in the two unaffected members. We assumed that the familial clustering of diabetes was caused by rare variants excluding those with allele frequency of more than 0.01 in the 1000 Genomes Project, the Human Genetic Variation Database, or a cohort of 105 normoglycemic controls in Japan. The rare variants were then genotyped in 2102 Japanese without diabetes and 119 Japanese with diabetes. ResultsAmong the variants detected by WES and predicted to be disease-causing, 16 variants shared by all of the four of the affected members and not present in the two unaffected members were confirmed to be rare. Genotyping of the 16 rare variants revealed that only A137T in ADAMTSL3 (rs181914721) was observed more frequently in the 119 subjects with diabetes than in the 105 normoglycemic controls, and the allele frequency of the variant was significantly higher in the 119 subjects with diabetes than in another cohort of 2102 Japanese without diabetes. ConclusionsWe propose that A137T in ADAMTSL3 is a candidate mutation for susceptibility to diabetes in this family and in the Japanese population.

Association between SCO2 mutation and extreme myopia in Japanese patients.

To investigate the role of SCO2 in extreme myopia of Japanese patients. In total, 101 Japanese patients with extreme myopia (axial length of≥30mm) OU at the Kyoto University Hospital were included in this study. Exon 2 of SCO2 was sequenced by conventional Sanger sequencing. The detected variants were assessed using in silico prediction programs: SIFT, PolyPhen-2 and MutationTaster. To determine the frequency of the mutations in normal subjects, we referred to the 1000 Genomes Project data and the Human Genetic Variation Database (HGVD) in the Human Genetic Variation Browser. The average age of the participants was 62.9±12.7years. There were 31 males (30.7%) and 70 females. Axial lengths were 31.76±1.17mm OD and 31.40±1.07mm OS, and 176 eyes (87.6%) out of 201 eyes had myopic maculopathy of grade 2 or more. Among the 101 extremely myopic patients, one mutation (c.290 C>T;p.Ala97Val) in SCO2 was detected. This mutation was not found in the 1000 Genomes Project data or HGVD data. Variant type of the mutation was nonsynonymous. Although the SIFT prediction score was 0.350, the PolyPhen-2 probability was 0.846, thus predicting its pathogenicity to be possibly damaging. MutationTaster PhyloP was 1.268, suggesting that the mutation is conserved. We identified one novel possibility of an extreme myopia-causing mutation in SCO2. No other disease-causing mutation was found in 101 extremely myopic Japanese patients, suggesting that SCO2 plays a limited role in Japanese extreme myopia. Further investigation is required for better understanding of extreme myopia.

Human genetic variation database, a reference database of genetic variations in the Japanese population.

Whole-genome and -exome resequencing using next-generation sequencers is a powerful approach for identifying genomic variations that are associated with diseases. However, systematic strategies for prioritizing causative variants from many candidates to explain the disease phenotype are still far from being established, because the population-specific frequency spectrum of genetic variation has not been characterized. Here, we have collected exomic genetic variation from 1208 Japanese individuals through a collaborative effort, and aggregated the data into a prevailing catalog. In total, we identified 156 622 previously unreported variants. The allele frequencies for the majority (88.8%) were lower than 0.5% in allele frequency and predicted to be functionally deleterious. In addition, we have constructed a Japanese-specific major allele reference genome by which the number of unique mapping of the short reads in our data has increased 0.045% on average. Our results illustrate the importance of constructing an ethnicity-specific reference genome for identifying rare variants. All the collected data were centralized to a newly developed database to serve as useful resources for exploring pathogenic variations. Public access to the database is available at http://www.genome.med.kyoto-u.ac.jp/SnpDB/.