Analysis Of Whole Exome Sequencing Data Research Articles

Exome-Wide Search for Genes Associated With Central Nervous System Inflammatory Demyelinating Diseases Following CHIKV Infection: The Tip of the Iceberg.

Chikungunya virus (CHIKV) is a re-emergent arbovirus that causes a disease characterized primarily by fever, rash and severe persistent polyarthralgia, although <1% of cases develop severe neurological manifestations such as inflammatory demyelinating diseases (IDD) of the central nervous system (CNS) like acute disseminated encephalomyelitis (ADEM) and extensive transverse myelitis. Genetic factors associated with host response and disease severity are still poorly understood. In this study, we performed whole-exome sequencing (WES) to identify HLA alleles, genes and cellular pathways associated with CNS IDD clinical phenotype outcomes following CHIKV infection. The cohort includes 345 patients of which 160 were confirmed for CHIKV. Six cases presented neurological manifestation mimetizing CNS IDD. WES data analysis was performed for 12 patients, including the CNS IDD cases and 6 CHIKV patients without any neurological manifestation. We identified 29 candidate genes harboring rare, pathogenic, or probably pathogenic variants in all exomes analyzed. HLA alleles were also determined and patients who developed CNS IDD shared a common signature with diseases such as Multiple sclerosis (MS) and Neuromyelitis Optica Spectrum Disorders (NMOSD). When these genes were included in Gene Ontology analyses, pathways associated with CNS IDD syndromes were retrieved, suggesting that CHIKV-induced CNS outcomesmay share a genetic background with other neurological disorders. To our knowledge, this study was the first genome-wide investigation of genetic risk factors for CNS phenotypes in CHIKV infection. Our data suggest that HLA-DRB1 alleles associated with demyelinating diseases may also confer risk of CNS IDD outcomes in patients with CHIKV infection.

Whole-Genome Sequencing in Diagnostics of Selected Slovenian Undiagnosed Patients with Rare Disorders.

Several patients with rare genetic disorders remain undiagnosed following comprehensive diagnostic testing using whole-exome sequencing (WES). In these patients, pathogenic genetic variants may reside in intronic or regulatory regions or they may emerge through mutational mechanisms not detected by WES. For this reason, we implemented whole-genome sequencing (WGS) in routine clinical diagnostics of patients with undiagnosed genetic disorders and report on the outcome in 30 patients. Criteria for consideration included (1) negative WES, (2) a high likelihood of a genetic cause for the disorders, (3) positive family history, (4) detection of large blocks of homozygosity or (5) detection of a single pathogenic variant in a gene associated with recessive conditions. We successfully discovered a causative genetic variant in 6 cases, a retrotranspositional event in the APC gene, non-coding variants in the intronic region of the OTC gene and the promotor region of the UFM1 gene, repeat expansion in the RFC1 gene and a single exon duplication in the CNGB3 gene. We also discovered one coding variant, an indel, which was missed by variant caller during WES data analysis. Our study demonstrates the impact of WGS in the group of patients with undiagnosed genetic diseases after WES in the clinical setting and the diversity of mutational mechanisms discovered, which would remain undetected using other methods.

Large-scale Identification of Clonal Hematopoiesis and Mutations Recurrent in Blood Cancers

Clonal hematopoiesis of indeterminate potential (CHIP) is characterized by detectable hematopoietic-associated gene mutations in a person without evidence of hematologic malignancy. We sought to identify additional cancer-presenting mutations useable for CHIP detection by performing a data mining analysis of 48 somatic mutation studies reporting mutations at diagnoses of 7,430 adult and pediatric patients with hematologic malignancies. Following extraction of 20,141 protein-altering mutations, we identified 434 significantly recurrent mutation hotspots, 364 of which occurred at loci confidently assessable for CHIP. We then performed an additional large-scale analysis of whole exome sequencing data from 4,538 persons belonging to three non-cancer cohorts for clonal mutations. We found the combined cohort prevalence of CHIP with mutations identical to those reported at blood cancer mutation hotspots to be 1.8%, and that some of these CHIP mutations occurred in children. Our findings may help to improve CHIP detection and pre-cancer surveillance for both children and adults.

Pathogenic Mutations and Putative Phenotype-Affecting Variants in Polish Myofibrillar Myopathy Patients.

Myofibrillar myopathies (MFM) are heterogeneous hereditary muscle diseases with characteristic myopathological features of Z-disk dissolution and aggregates of its degradation products. The onset and progression of the disease are variable, with an elusive genetic background, and around half of the cases lacking molecular diagnosis. Here, we attempted to establish possible genetic foundations of MFM by performing whole exome sequencing (WES) in eleven unrelated families of 13 patients clinically diagnosed as MFM spectrum. A filtering strategy aimed at identification of variants related to the disease was used and included integrative analysis of WES data and human phenotype ontology (HPO) terms, analysis of muscle-expressed genes, and analysis of the disease-associated interactome. Genetic diagnosis was possible in eight out of eleven cases. Putative causative mutations were found in the DES (two cases), CRYAB, TPM3, and SELENON (four cases) genes, the latter typically presenting with a rigid spine syndrome. Moreover, a variety of additional, possibly phenotype-affecting variants were found. These findings indicate a markedly heterogeneous genetic background of MFM and show the usefulness of next generation sequencing in the identification of disease-associated mutations. Finally, we discuss the emerging concept of variant load as the basis of phenotypic heterogeneity.

Exome sequencing reveled a compound heterozygous mutations in RTTN gene causing developmental delay and primary microcephaly.

RTTN (Rotatin) (OMIM 614833) is a large centrosomal protein coding gene. RTTN mutations are responsible for syndromic forms of malformation of brain development, leading to polymicrogyria, microcephaly, primordial dwarfism, seizure along with many other malformations. In this study we have identified a compound heterozygous mutation in RTTN gene having NM_173630 c.5225A > G p.His1742Arg in exon 39 and NM_173630 c.6038G > T p.Cys2013Phe in exon 45 of a consanguineous Saudi family leading to brain malformation, seizure, developmental delay, dysmorphic feature and microcephaly. Whole exome sequencing (WES) techniques was used to identify the causative mutation in the affected members of the family. WES data analysis was done and obtained data were further confirmed by using Sanger sequencing analysis. Moreover, the mutation was ruled out in 100 healthy control from normal population. To the best of our knowledge the novel compound heterozygous mutation observed in this study is the first report from Saudi Arabia. The identified compound heterozygous mutation will further explain the role of RTTN gene in development of microcephaly and neurodevelopmental disorders.

Novel MYO15A variants are associated with hearing loss in the two Iranian pedigrees

BackgroundClinical genetic diagnosis of non-syndromic hearing loss (NSHL) is quite challenging. With regard to its high heterogeneity as well as large size of some genes, it is also really difficult to detect causative mutations using traditional approaches. One of the recent technologies called whole-exome sequencing (WES) has been thus developed in this domain to remove the limitations of conventional methods.MethodsThis study was a report on a research study of two unrelated pedigrees with multiple affected cases of hearing loss (HL). Accordingly, clinical evaluations and genetic analysis were performed in both families.ResultsThe results of WES data analysis to uncover autosomal recessive non-syndromic hearing loss (ARNSHL) disease-causing variants was reported in the present study. Initial analysis identified two novel variants of MYO15A i.e. c.T6442A:p.W2148R and c.10504dupT:p.C3502Lfs*15 correspondingly which were later confirmed by Sanger validations and segregation analyses. According to online prediction tools, both identified variants seemed to have damaging effects.ConclusionIn this study, whole exome sequencing were used as a first approach strategy to identify the two novel variants in MYO15A in two Iranian families with ARNSHL.

The pleiotropic effect of a deleterious DES mutation in familial atrial fibrillation and the role of PDE4DIP as a genetic modifier for heart block

Abstract Background Atrial fibrillation (AF) is a common cardiac arrhythmia and is a major cause of ischemic stroke. It is a heritable disease, which is occasionally transmitted with an autosomal dominant pattern of inheritance. Methods We recruited a three-generation Lebanese kindred with five living family members affected by early onset familial AF (in early 30 years of age) with and without heart block and cardiomyopathy. Affected and unaffected family members underwent whole exome sequencing (WES). Two additional members of the family with undefined arrhythmias, heart block and pacemaker implantation had died from non-ischemic cardiomyopathy (NICM). Results A pathogenic mutation in the DES gene resulting in a serine to phenylalanine substitution at codon 13 in the Desmin protein segregated with early-onset AF. Four of the five patients had heart block and only two developed NICM later in life. Desmin, an intermediate filament protein, plays a pivotal role in the electromechanical functioning of cardiomyocytes and mutations in the DES gene have been implicated in skeletal myopathies, dilated cardiomyopathies and cardiac conduction disease. These mutations exhibit often incomplete penetrance and pleiotropic effects, but modifier genes that explain this behavior have not been identified. Analysis of WES data identified twelve shared variants predicted as damaging by both PolyPhen2 or SIFT software segregated with both early onset AF and heart block among DES mutation carriers. After filtering for known disease genes and genes not expressed in the heart, a rare novel non-conservative mutation in phosphodiesterase 4 D interacting protein (PDE4DIP) gene was identified that perfectly segregated with both heart block and AF. The mutation results in a non-conservative substitution of alanine at codon 123 for threonine. In-silico analysis predicted creation of a putative MAPK phosphorylation site. Functional in vitro characterization of the PDE4DIP p.A123T substitution showed increased cAMP levels by FRET sensor imaging in response to isoproterenol stimulation compared to wildtype transfected cells. cAMP mediates the catecholaminergic control of cardiac conduction and contractility. The PDE4DIP protein anchors PDE4D to the Golgi/centrosomal area in the sarcomeres of the myocardium and skeletal muscle and plays a critical role in subcellular partitioning of the cAMP signal and its dependent pathways. Surprisingly, there was decreased beta-adrenergic receptor phosphorylation in the mutant cells, suggesting reduced membrane PKA activity, possibly due to mislocalization of PDE4D. Inactivation of PDE4D has been linked to cardiomyopathy and arrhythmias including AF. Interestingly, genome-wide association studies have linked both PDE4D and PDE4DIP to ischemic stroke. Conclusion Our findings establish early onset AF as a presenting phenotype for DES mutations and identifies PDE4DIP as a modifier gene for its role in the development of conduction disease. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): National Institutes of Health, USA

Whole-Exome Sequencing of Discordant Monozygotic Twin Families for Identification of Candidate Genes for Microtia-Atresia.

ObjectiveWe used data from twins and their families to probe the genetic factors contributing to microtia-atresia, in particular, early post-twinning variations that potentially account for the discordant phenotypes of monozygotic twin pairs.MethodsSix families of monozygotic twins discordant for congenital microtia-atresia were recruited for study. The six patients shared a consistent clinical phenotype of unilateral microtia-atresia. Whole-exome sequencing (WES) was performed for all six twin pairs and their parents. Family segregation and multiple bioinformatics methods were applied to identify suspicious mutations in all families. Recurring mutations commonly detected in at least two families were highlighted. All variants were validated via Sanger sequencing. Gene Ontology (GO) analysis was performed to identify candidate gene sets and related pathways. Copy number variation (CNV), linkage analysis, association analysis and machine learning methods were additionally applied to isolate candidate mutations, and comparative genomics and structural modeling tools used to evaluate their potential roles in onset of microtia-atresia.ResultsOur analyses revealed 61 genes with suspected mutations associated with microtia-atresia. Five (HOXA4, MUC6, CHST15, TBX10, and AMER1) contained 7 de novo mutations that appeared in at least two families, which have been previously reported as pathogenic for other diseases. Among these, HOXA4 (c.920A>C, p.H307P) was determined as the most likely pathogenic variant for microtia-atresia. GO analysis revealed four gene sets involving 11 pathways potentially related to underlying pathogenesis of the disease. CNVs in three genes (UGT2B17, OVOS, and KATNAL2) were detected in at least two families. Linkage analysis disclosed 13 extra markers for the disease, of which two (FGFR1 and EYA1) were validated via machine learning analysis as plausible candidate genes for the disease.ConclusionBased on comprehensive genetic and bioinformatic analyses of WES data from six families of discordant monozygotic twins with microtia-atresia, we identified multiple candidate genes that may function in post-twinning onset of the disease. The collective findings provide novel insights into the pathogenesis of congenital microtia-atresia.

Comprehensive Exonic Sequencing of Known Ataxia Genes in Episodic Ataxia.

Episodic Ataxias (EAs) are a small group (EA1–EA8) of complex neurological conditions that manifest as incidents of poor balance and coordination. Diagnostic testing cannot always find causative variants for the phenotype, however, and this along with the recently proposed EA type 9 (EA9), suggest that more EA genes are yet to be discovered. We previously identified disease-causing mutations in the CACNA1A gene in 48% (n = 15) of 31 patients with a suspected clinical diagnosis of EA2, and referred to our laboratory for CACNA1A gene testing, leaving 52% of these cases (n = 16) with no molecular diagnosis. In this study, whole exome sequencing (WES) was performed on 16 patients who tested negative for CACNA1A mutations. Tiered analysis of WES data was performed to first explore (Tier-1) the ataxia and ataxia-associated genes (n = 170) available in the literature and databases for comprehensive EA molecular genetic testing; we then investigated 353 ion channel genes (Tier-2). Known and potential causal variants were identified in n = 8/16 (50%) patients in 8 genes (SCN2A, p.Val1325Phe; ATP1A3, p.Arg756His; PEX7, p.Tyr40Ter; and KCNA1, p.Arg167Met; CLCN1, p.Gly945ArgfsX39; CACNA1E, p.Ile614Val; SCN1B, p.Cys121Trp; and SCN9A, p.Tyr1217Ter). These results suggest that mutations in these genes might cause an ataxia phenotype or that combinations of more than one mutation contribute to ataxia disorders.

Tumour immune characterization in adenoid cystic carcinoma identifies prognostic and immunotherapeutically relevant messenger RNA signatures.

e15191 Background: Here, we profiled genetic hallmarks of Chinese adenoid cystic carcinoma (ACC) patients and immune cells infiltration levels by gene expression-based computational methods to investigate the correlation with immunotherapy. Methods: 54 patients with pathologically confirmed ACC admitted from January 2016 to October 2019 were included. Mutation and expression profiles were obtained by whole-exome sequencing (WES) and RNA-seq, respectively. A nomogram that integrated clinicopathological features with the immune signature to predict recurrence or distant metastasis probability was constructed by multivariate Cox regression. Results: As expected, we observed MYB fusion and somatic mutations in our cohort (57%, 31/54), strengthening the role of these aberrations as critical events in ACC. Mutations in NOTCH1 were also found in our cohort (19%, 10/54). Median tumour mutational burden (TMB) of these patients is 0.85 Muts/Mbp (0-11 Muts/Mbp). Analysis of WES data showed that all patients were microsatellite instability (MSI)-negative, and 17 of them were confirmed by conventional MSI-PCR testing. Lack of PD-L1 expression is 96.0% (n = 46/48) at a tumour proportion score 0% and 22.9% (11/48) of cases showed more than 10% CD8+ prevalence. We compared cancer types using an immune infiltration score (IIS) and an antigen presenting machinery expression (APM) score and find that ACC is among the lowest for both scores. Statistically significant correlations were found between IIS and distant metastasis-free survival (univariate analysis, HR = 2.64, 95% confidence interval [CI], 1.134 to 6.145, p = 0.023). Patients with higher IIS had shorter distant metastasis-free survival time (log-rank p = 0.020), and patients with higher T cell infiltration score (TIS) had shorter locoregional recurrence-free survival time (log-rank p = 0.050). In addition, we investigated the pretreatment immune profile of five ACC patients treated with PD-1 checkpoint inhibitors, and found that both TIS and APM were elevated in responding patient (with a partial response to camrelizumab) whereas they were in the lowest for patients with progressive disease on camrelizumab or pembrolizumab. Conclusions: Our study highlights the immune infiltration patterns and unravels mRNA signatures with prognostic utility and immunotherapeutic biomarker potential in ACC.

Circulating tumor DNA dynamics as prognostic and predictive biomarkers of response to pembrolizumab in patients with virally-related tumors (VRT) treated within the INSPIRE study.

3068 Background: We previously showed a correlation between circulating tumor DNA (ctDNA) dynamics and response to pembrolizumab in a cohort of mixed tumors treated in the INSPIRE study (Yang et al, ESMO 2019). We investigated the prognostic and predictive value of ctDNA dynamics in patients (pts) with VRT. Methods: Pts with VRT (HPV+ squamous cell carcinoma (SCC), EBER+ nasopharyngeal carcinoma (NPC) and MCPyV+ Merkel carcinoma (MC)) and a control cohort of non-VRT (HPV- head and neck SCC) treated with single-agent pembrolizumab were selected for the analysis. ctDNA was assayed at baseline and start of cycle 3 using a pt-specific amplicon-based NGS assay (Signatera). Samples were considered ctDNA positive if ≥2 of 16 pt-specific targets met the qualifying confidence score threshold. Whole exome sequencing (WES) performed in baseline tumor tissue; presence of HPV, EBV and MCPyV in tumor determined through bioinformatic analysis of WES data (VirusFinder, PMID23717618). Changes in tumor size (mm) and response data using RECIST 1.1 were collected. Progression-free survival (PFS) and overall survival (OS) were estimated by Kaplan-Meier method. Results: Twenty pts with VRT (HPV+ head and neck = 8, cervical = 2 and anal = 2 SCC; EBER+NPC = 2; MC = 6) and 11 pts with non-VRT were included. Median follow-up: 11 months (0,5-11). Treatment response: VRT 6 responders (CR + PR + SD &gt; 18weeks) and 14 non-responders (SD &lt; 18 weeks + PD); non-VRT 3 responders and 8 non-responders. Median OS and PFS for all pts were 10.61 and 3.2 months, respectively. No differences in PFS (p = 0.60) nor OS (p = 0.66) were observed among responders between VRT and non-VRT. Among non-responders, VRT had significantly higher OS but not PFS when compared to non-VRT (HR 0.30, p = 0.01 and HR 0.82 p = 0.62, respectively). VRT had quantitatively higher ctDNA at baseline vs non-VRT (Mean 7.9 vs 0.4 ng, p &lt; 0.001). ΔctDNA (Change in ctDNA between baseline and cycle 3) strongly correlated with changes in tumor measurements and response by RECIST 1.1 (Spearman Rho = 0.75) and was associated with survival regardless of viral status (Table). Conclusions: ΔctDNA strongly correlated with changes in tumor response and survival in both VRT and non-VRT. Higher baseline ctDNA was found in VRT. Correlation with circulating viral DNA and radiomics analyses is on-going. [Table: see text]

Multi-cancer analysis of clonality and the timing of systemic spread in paired primary tumors and metastases.

Metastasis is the primary cause of cancer-related deaths, but the natural history, clonal evolution and impact of treatment are poorly understood. We analyzed whole-exome sequencing data from 457 paired primary tumor and metastatic samples from 136 breast, colorectal and lung cancer patients, including untreated (n=99) and treated (n=100) metastases. Treated metastases often harbored private ‘driver’ mutations whereas untreated metastases did not, suggesting that treatment promotes clonal evolution. Polyclonal seeding was common in untreated lymph node metastases (n=17/29, 59%) and distant metastases (n=20/70, 29%), but less frequent in treated distant metastases (n=9/94, 10%). The low number of metastasis-private clonal mutations is consistent with early metastatic seeding, which we estimated occurred 2–4 years prior to diagnosis across these cancers. Further, these data suggest that the natural course of metastasis is selectively relaxed relative to early tumorigenesis and that metastasis-private mutations are not drivers of cancer spread but instead associated with drug resistance.

Homozygosity mapping and whole exome sequencing provide exact diagnosis of Cohen syndrome in a Saudi family

BackgroundCohen syndrome (CS) is a rare multi-system autosomal recessive disorder with a high prevalence in the Finnish population. Clinical features of Finnish-type CS are homogeneous, however, in non-Finnish populations, CS diagnosis is challenging due to broad phenotypic variability. MethodsWe studied a consanguineous family having three affected individuals with clinical features of severe intellectual disability and global developmental delay. Clinical diagnosis of the phenotype could not be established based on the features. Therefore, whole genome SNP genotyping and whole exome sequencing (WES) were performed on DNA samples from affected and unaffected family members. ResultsHomozygosity mapping identified a shared loss of heterozygosity region on chromosome 8q22.1-q22.3 and WES data analysis revealed an insertion-deletion (indel) mutation (c.11519_11521delCAAinsT) in the VPS13B gene. The indel is predicted to cause a frameshift resulting in a premature termination of the VPS13B protein (NP_060360.3:p.Pro3840Leufs*2). ConclusionVPS13B encodes a giant transmembrane protein called vacuolar protein sorting 13 homolog B. VPS13B is known to play a role in the glycosylation of Golgi proteins and in endosomal-lysosomal trafficking. Moreover, it is thought to function in vesicle mediated transport and sorting of proteins within the cell. The mechanism by which abnormalities of the VPS13B protein lead to the phenotype of CS is currently unknown. Here, in this study, we successfully established a clinical diagnosis of CS cases from a family using genomic analyses. Clinical re-examination of the patients revealed characteristic ocular abnormalities.

Whole-exome Sequencing for the Identification of Rare Variants in Primary Immunodeficiency Genes in Children With Sepsis: A Prospective, Population-based Cohort Study.

BackgroundThe role of primary immunodeficiencies (PID) in susceptibility to sepsis remains unknown. It is unclear whether children with sepsis benefit from genetic investigations. We hypothesized that sepsis may represent the first manifestation of underlying PID. We applied whole-exome sequencing (WES) to a national cohort of children with sepsis to identify rare, predicted pathogenic variants in PID genes.MethodsWe conducted a multicenter, population-based, prospective study including previously healthy children aged ≥28 days and <17 years admitted with blood culture-proven sepsis. Using a stringent variant filtering procedure, analysis of WES data was restricted to rare, predicted pathogenic variants in 240 PID genes for which increased susceptibility to bacterial infection has been reported.ResultsThere were 176 children presenting with 185 sepsis episodes who underwent WES (median age, 52 months; interquartile range, 15.4–126.4). There were 41 unique predicted pathogenic PID variants (1 homozygous, 5 hemizygous, and 35 heterozygous) found in 35/176 (20%) patients, including 3/176 (2%) patients carrying variants that were previously reported to lead to PID. The variants occurred in PID genes across all 8 PID categories, as defined by the International Union of Immunological Societies. We did not observe a significant correlation between clinical or laboratory characteristics of patients and the presence or absence of PID variants.ConclusionsApplying WES to a population-based cohort of previously healthy children with bacterial sepsis detected variants of uncertain significance in PID genes in 1 out of 5 children. Future studies need to investigate the functional relevance of these variants to determine whether variants in PID genes contribute to pediatric sepsis susceptibility.

Новая гомозиготная мутация в гене ARL6IP1 - второй случай редкой спастической параплегии

Актуальность. Наследственные спастические параплегии (НСП) - обширная, высоко гетерогенная группа нейродегенеративных заболеваний, характеризующихся прогрессирующим нижним спастическим парапарезом, вызванным поражением кортико-спинального тракта. Постоянно растущее число генов (картировано более 80 локусов, известно 60 генов), ассоциированных с НСП, осложняет постановку точного диагноза. Это особенно актуально для форм НСП, где описаны единичные случаи заболевания, как, например, для аутосомно-рецессивной спастической параплегии типа 61 (SPG61, OMIM: 615685). Введение в практику новых технологий секвенирования позволяет сократить время исследования и выявить молекулярно-генетическую причину заболевания в большинстве случаев, особенно в семьях с редкими НСП. Цель - описать клиническую картину редкой осложненной НСП с ранним началом (SPG61) в семье даргинцев, состоящих в близкородственном браке, и установить ее молекулярно-генетическую причину. Материалы и методы: семейный анамнез, неврологическое обследование, электроэнцефалография, МРТ головного мозга, выделение ДНК, секвенирование полного экзома, анализ данных полноэкзомного секвенирования, секвенирование по Сэнгеру. Результаты. В результате секвенирования полного экзома с последующим анализом полученных данных был обнаружен не описанный ранее гомозиготный вариант нуклеотидной последовательности c.[92T&gt;C];[92T&gt;C] (p.[(Leu31Pro)];[(Leu31Pro)], NM_015161.1) в экзоне 2 гена ARL6IP1 - второй вариант, найденный в этом гене в мире и первый в России. Наличие выявленного варианта было подтверждено методом прямого автоматического секвенирования по Сэнгеру. Вариант c.92T&gt;C был зарегистрирован в гомозиготном состоянии у обоих пациентов и в гетерозиготном состоянии у родителей, тем самым была показана его сегрегация с заболеванием в данной семье. В статье приведено подробное описание клинических проявлений заболевания в данной семье и сравнение клинических проявлений у больных в двух семьях с выявленными изменениями в гене ARL6IP1 (описанной ранее и изученной нами). Выводы. Проведенное исследование дополняет характеристику клинических проявлений, связанных с изменениями в гене ARL6IP1, приводящих к осложненным НСП с ранним началом. Background. Hereditary spastic paraplegias (HSPs) are a large group of neurodegenerative disorders characterized by progressive lower limbs spasticity and weakness caused by a retrograde axonal degeneration of the corticospinal tracts. The considerable and constantly increasing number of HSP-associated genes (more than 80 different loci with 60 corresponding spastic paraplegia genes) complicates the diagnosis in every particular case, especially with a single reported occurrence like the autosomal recessive spastic paraplegia 61 (SPG61, OMIM: 615685). However, new sequencing methods allow to accelerate the process and find the molecular cause of the disease much more reliably, especially in families with rare HSPs. Aims. To describe a rare complicated early-onset HSP (SPG61) in a Dargin consanguineous family and find out its molecular genetical cause. Materials and methods: personal and family history analysis, neurological examination, electroencephalography, brain MRI, blood DNA extraction, whole exome sequencing (WES), WES data analysis, Sanger sequencing. Results. During a session of whole-exome sequencing and analysis, a new homozygous variant c.[92T&gt;C];[92T&gt;C] (p.[(Leu31Pro)];[(Leu31Pro)], NM_015161.1) has been discovered in exon 2 of the ARL6IP1 gene, which makes it the second variant found in this gene worldwide and the first one in Russia. Sanger sequencing of the patients’ and parents’ DNA confirmed the p.(Leu31Pro) variant status (homozygous in both patients and heterozygous in both parents) and its segregation with the disease status. Here we describe the clinical findings of the disease in this family and a clinical data comparison for two families with variants in the ARL6IP1 gene (described previously and studied in our laboratory). Conclusions. Our research broadens the diversity of symptoms associated with ARL6IP1 gene mutations. The discovered variant expands the causative mutation spectrum of complicated early-onset HSPs.

Characterization of a novel pathogenic variation c.1237T>G in the FZD4 gene presenting new inheritance from an Iranian individual suffering vitreoretinopathy.

Whole Exome Sequencing (WES) has been used increasingly in genetic determination of various known and unknown genetic disorders. Various genes are involved in the development of the vascular network of retina. Assessment of a collection of these genes could be provided by WES. Here we used WES for a patient suffering vitreoretinopathy to detect the disease causing variant. Sanger sequencing has been applied for variant verification and allelic segregation. After analysis of WES data we found a new variant c.1237T>G in the FZD4 locus which causes retinopathy of prematurity and exudative vitreoretinopathy (MIM number: 133780). Sanger sequencing showed this single nucleotide variation inherited as homozygous in the patient and heterozygous in her unaffected parents. Notably, bioinformatics analysis predicted the variant as disease causing and it has not been described yet in home datasets and public SNP databases. FZD4 mutations are mostly inherited as autosomal dominant traits. Our findings showed the first autosomal recessive inheritance of the FZD4 gene related retinopathy. On the other hand, our data shed light on the significance of an Exome sequencing application as a genetic test to identify and characterize the comprehensive spectrum of genetic variation and classification for patients with retinopathies.

Genetic diagnoses in epilepsy: The impact of dynamic exome analysis in a pediatric cohort.

We evaluated the yield of systematic analysis and/or reanalysis of whole exome sequencing (WES) data from a cohort of well-phenotyped pediatric patients with epilepsy and suspected but previously undetermined genetic etiology. We identified and phenotyped 125 participants with pediatric epilepsy. Etiology was unexplained at the time of enrollment despite clinical testing, which included chromosomal microarray (57 patients), epilepsy gene panel (n = 48), both (n = 28), or WES (n = 8). Clinical epilepsy diagnoses included developmental and epileptic encephalopathy (DEE), febrile infection-related epilepsy syndrome, Rasmussen encephalitis, and other focal and generalized epilepsies. We analyzed WES data and compared the yield in participants with and without prior clinical genetic testing. Overall, we identified pathogenic or likely pathogenic variants in 40% (50/125) of our study participants. Nine patients with DEE had genetic variants in recently published genes that had not been recognized as epilepsy-related at the time of clinical testing (FGF12, GABBR1, GABBR2, ITPA, KAT6A, PTPN23, RHOBTB2, SATB2), and eight patients had genetic variants in candidate epilepsy genes (CAMTA1, FAT3, GABRA6, HUWE1, PTCHD1). Ninety participants had concomitant or subsequent clinical genetic testing, which was ultimately explanatory for 26% (23/90). Of the 67 participants whose molecular diagnoses were "unsolved" through clinical genetic testing, we identified pathogenic or likely pathogenic variants in 17 (25%). Our data argue for early consideration of WES with iterative reanalysis for patients with epilepsy, particularly those with DEE or epilepsy with intellectual disability. Rigorous analysis of WES data of well-phenotyped patients with epilepsy leads to a broader understanding of gene-specific phenotypic spectra as well as candidate disease gene identification. We illustrate the dynamic nature of genetic diagnosis over time, with analysis and in some cases reanalysis of exome data leading to the identification of disease-associated variants among participants with previously nondiagnostic results from a variety of clinical testing strategies.

Genomic landscape of a case of multiple myeloma by whole exome sequencing

Multiple Myeloma (MM) is the malignancy of plasma cells that results in organ damage. These subjects diagnosed with MM are in need of new treatment strategies to improve better outcome. Whole exome sequencing (WES) was performed on the blood sample of a 47 years old male subject, diagnosed with MM in 2010. Analysis of WES data to identifying driver and passenger mutations was performed using various tools and algorithms. Analysis revealed that translocations in IGH and IGK loci found to be primary aberrations in this case. Prioritizing the screening for those genes that are known to be secondary mutations led to identification of critical tumor suppressor genes, KMT2C (MLL3), BCL2, NOTCH2 and DIS3 as driver mutations. We also identified list of mutations that indicted secondary abrasions are continually happen during clonal expansion in MM. Myeloma is individual specific and each patient is unique. Therefore, the genomic data of each case and relevant clinical feature would be important guidelines for treatment strategies.

Exome sequencing study of Russian breast cancer patients suggests a predisposing role for USP39.

Germline variants in known breast cancer (BC) predisposing genes explain less than half of hereditary BC cases. This study aimed to identify missing genetic determinants of BC. Whole exome sequencing (WES) of lymphocyte DNA was performed for 49 Russian patients with clinical signs of genetic BC predisposition, who lacked Slavic founder mutations in BRCA1, BRCA2, CHEK2, and NBS1 genes. Bioinformatic analysis of WES data was allowed to compile a list of 229 candidate mutations. 79 of these mutations were subjected to a three-stage case-control analysis. The initial two stages, which involved up to 797 high-risk BC patients, 1504 consecutive BC cases, and 1081 healthy women, indicated a potentially BC-predisposing role for 6 candidates, i.e., USP39 c.*208G > C, PZP p.Arg680Ter, LEPREL1 p.Pro636Ser, SLIT3 p.Arg154Cys, CREB3 p.Lys157Glu, and ING1 p.Pro319Leu. USP39 c.*208G > C was strongly associated with triple-negative breast tumors (p = 0.0001). In the third replication stage, we genotyped the truncating variant of PZP (rs145240281) and the potential splice variant of USP39 (rs112653307) in three independent cohorts of Russian, Byelorussian, and German ancestry, comprising a total of 3216 cases and 2525 controls. The data obtained for USP39 rs112653307 supported the association identified in the initial stages (the combined OR 1.72, p = 0.035). This study suggests the role of a rare splicing variant in BC susceptibility. USP39 encodes an ubiquitin-specific peptidase that regulates cancer-relevant tumor suppressors including CHEK2. Further epidemiological and functional studies involving these gene variants are warranted.

Exploring the molecular basis of hereditary spinocerebellar degeneration in a large Sudanese family

Spinocerebellar neurodegeneration (SCD) is phenotypically and genetically complex forming a heterogeneous spectrum of disorders with hereditary spastic paraplegias on one end and hereditary ataxias on the other. Genetics of SCD has been a target for extensive researches worldwide, yet little is known about SCD genetics in Sub-Saharan African population. We recruited a large consanguineous Sudanese family with five affected siblings. Extracted genomic DNA was screened for genetic variations using whole exome sequencing (WES). Analysis was done to identify the culprit variations using bioinformatics tools. Clinical results showed a complex phenotype of progressive spastic-ataxia, deafness and microcephaly. Analysis of WES data suggested two homozygous missense variants in two candidate genes (MYO15A and SEMA5D) that were not reported to be linked to similar disease before. The first variant in MYO15A gene (NM_016239.3: c.1634C>T) was reported to cause autosomal recessive hearing loss but was not reported in similar neurological disease. The second variant in SEMA4D gene (NM_006378.3:c.1588G>A) which involved in brain development but not reported to be associated with inherited neurological conditions before. Both variants were extremely rare and highly conserved. They were predicted to be highly pathogenic using bioinformatics tools. The scarcity of genetic data in the highly consanguineous Sudanese population makes whole exome sequencing a powerful and cost effective strategy to identify both known and new pathogenic variations and genes. Sanger sequencing and further functional studies are recommended to prove the association of MYO15A gene and SEMA4D gene with the complex clinical phenotype of deafness, spasticity and ataxia.