Candidate Pathogenic Variants Research Articles

In vitro human cell culture models in a bench-to-bedside approach to epilepsy.

Epilepsy is the most common chronic neurological disease, affecting nearly 1%-2% of the world's population. Current pharmacological treatment and regimen adjustments are aimed at controlling seizures; however, they are ineffective in one-third of the patients. Although neuronal hyperexcitability was previously thought to be mainly due to ion channel alterations, current research has revealed other contributing molecular pathways, including processes involved in cellular signaling, energy metabolism, protein synthesis, axon guidance, inflammation, and others. Some forms of drug-resistant epilepsy are caused by genetic defects that constitute potential targets for precision therapy. Although such approaches are increasingly important, they are still in the early stages of development. This review aims to provide a summary of practical aspects of the employment of invitro human cell culture models in epilepsy diagnosis, treatment, and research. First, we briefly summarize the genetic testing that may result in the detection of candidate pathogenic variants in genes involved in epilepsy pathogenesis. Consequently, we review existing invitro cell models, including induced pluripotent stem cells and differentiated neuronal cells, providing their specific properties, validity, and employment in research pipelines. We cover two methodological approaches. The first approach involves the utilization of somatic cells directly obtained from individual patients, while the second approach entails the utilization of characterized cell lines. The models are evaluated in terms of their research and clinical benefits, relevance to the invivo conditions, legal and ethical aspects, time and cost demands, and available published data. Despite the methodological, temporal, and financial demands of the reviewed models they possess high potential to be used as robust systems in routine testing of pathogenicity of detected variants in the near future and provide a solid experimental background for personalized therapy of genetic epilepsies. PLAIN LANGUAGE SUMMARY: Epilepsy affects millions worldwide, but current treatments fail for many patients. Beyond traditional ion channel alterations, various genetic factors contribute to the disorder's complexity. This review explores how invitro human cell models, either from patients or from cell lines, can aid in understanding epilepsy's genetic roots and developing personalized therapies. While these models require further investigation, they offer hope for improved diagnosis and treatment of genetic forms of epilepsy.

Novel likely pathogenic variant in the EYA1 gene causing Branchio oto renal syndrome and the exploration of pathogenic mechanisms

ObjectiveBranchio-oto-renal syndrome (BOR, OMIM#113,650) is a rare autosomal dominant disorder that presents with a variety of symptoms, including hearing loss (sensorineural, conductive, or mixed), structural abnormalities affecting the outer, middle, and inner ear, branchial fistulas or cysts, as well as renal abnormalities.This study aims to identify the pathogenic variants by performing genetic testing on a family with Branchio-oto-renal /Branchio-otic (BO, OMIM#602,588) syndrome using whole-exome sequencing, and to explore possible pathogenic mechanisms.MethodsThe family spans 4 generations and consists of 9 individuals, including 4 affected by the BOR/BO syndrome. Phenotypic information, including ear malformation and branchial cleft, was collected from family members. Audiological, temporal bone imaging, and renal ultrasound examinations were also performed. Whole-exome sequencing was conducted to identify candidate pathogenic variants and explore the underlying molecular etiology of BOR/BO syndrome by minigene experiments.ResultsIntra-familial variability was observed in the clinical phenotypes of BOR/BO syndrome in this family. The severity and nature of hearing loss varied in family members, with mixed or sensorineural hearing loss. The proband, in particular, had profound sensorineural hearing loss on the left and moderate conductive hearing loss on the right. Additionally, the proband exhibited developmental delay, and her mother experienced renal failure during pregnancy and terminated the pregnancy prematurely. Genetic testing revealed a novel heterozygous variant NM_000503.6: c.639 + 3 A > C in the EYA1 gene in affected family members. In vitro minigene experiments demonstrated its effect on splicing. According to the American College of Medical Genetics (ACMG) guidelines, this variant was classified as likely pathogenic.ConclusionThis study highlights the phenotypic heterogeneity within the same family, reports the occurrence of renal failure and adverse pregnancy outcomes in a female patient at reproductive age with BOR syndrome, and enriches the mutational spectrum of pathogenic variants in the EYA1 gene.

The diagnostic yield of exome sequencing in liver diseases from a curated gene panel

Exome sequencing (ES) has been used in a variety of clinical settings but there are limited data on its utility for diagnosis and/or prediction of monogenic liver diseases. We developed a curated list of 502 genes for monogenic disorders associated with liver phenotypes and analyzed ES data for these genes in 758 patients with chronic liver diseases (CLD). For comparison, we examined ES data in 7856 self-declared healthy controls (HC), and 2187 patients with chronic kidney disease (CKD). Candidate pathogenic (P) or likely pathogenic (LP) variants were initially identified in 19.9% of participants, most of which were attributable to previously reported pathogenic variants with implausibly high allele frequencies. After variant annotation and filtering based on population minor allele frequency (MAF ≤ 10–4 for dominant disorders and MAF ≤ 10–3 for recessive disorders), we detected a significant enrichment of P/LP variants in the CLD cohort compared to the HC cohort (X2 test OR 5.00, 95% CI 3.06–8.18, p value = 4.5e−12). A second-level manual annotation was necessary to capture true pathogenic variants that were removed by stringent allele frequency and quality filters. After these sequential steps, the diagnostic rate of monogenic disorders was 5.7% in the CLD cohort, attributable to P/LP variants in 25 genes. We also identified concordant liver disease phenotypes for 15/22 kidney disease patients with P/LP variants in liver genes, mostly associated with cystic liver disease phenotypes. Sequencing results had many implications for clinical management, including familial testing for early diagnosis and management, preventative screening for associated comorbidities, and in some cases for therapy. Exome sequencing provided a 5.7% diagnostic rate in CLD patients and required multiple rounds of review to reduce both false positive and false negative findings. The identification of concordant phenotypes in many patients with P/LP variants and no known liver disease also indicates a potential for predictive testing for selected monogenic liver disorders.

A missense GDF5 variant causes brachydactyly type A1 and multiple-synostoses syndrome 2.

This study aimed to identify the molecular defects and clinical manifestations in a Chinese family with brachydactyly (BD) type A1 (BDA1) and multiple-synostoses syndrome 2 (SYNS2). A Chinese family with BDA1 and SYNS2 was enrolled in this study. Whole-exome sequencing was used to analyze the gene variants in the proband. The sequences of the candidate pathogenic variant in GDF5 was validated via Sanger sequencing. I-TASSER and PyMOL were used to analyze the functional domains of the corresponding mutant proteins. The family was found to have an autosomal-dominantly inherited combination of BDA1 and SYNS2 caused by the S475N variant in the GDF5 gene. The variant was located within the functional region, and the mutated residue was found to be highly conserved among species. Via bioinformatic analyses, we predicted this variant to be deleterious, which perturb the protein function. The substitution of the negatively charged amino acid S475 with the neutral N475 was predicted to disrupt the formation of salt bridges with Y487 and impair the structure, stability, and function of the protein, consequently, the abnormalities in cartilage and bone development ensue. A single genetic variant (S475N) which disrupt the formation of salt bridges with Y487, in the interface of the antagonist- and receptor-binding sites of GDF5 concurrently causes two pathological mechanisms. This is the first report of this variant, identified in a Chinese family with BDA1 and SYNS2.

Abstract 18674: Deep Phenotyping Arrhythmogenic MVP With Imaging and Genotyping Reveals Important Shared Genetic Etiology With Arrhythmia and Cardiomyopathy Genes

Introduction: Mitral valve prolapse (MVP) has a high population prevalence 2-4% with an arrhythmogenic subtype presenting with premature ventricular complexes (PVC) ventricular tachycardia (VT) and sudden death. There is a continued search for markers of MVP to help risk stratify patients. Hypothesis: Arrhythmogenic MVP shares genetic etiology with Mendelian causes of arrhythmia and cardiomyopathy Methods: Consecutive adult patients with arrhythmogenic MVP patients prospectively evaluated Results: A total of 33 cases (age 55±15 years, female 51.5%) presented with history of hypertension (33%), diabetes (3%), coronary disease (12%), atrial fibrillation (45%), bileaflet MVP (58%) and syncope (18%). ECG findings include inverted T-waves in inferior leads in 6%, pleomorphic PVC in 6%, Ventricular couplets in 51.5%, non-sustained VT in 73% and sustained VT in 33%; High PVC burden was seen in 55% of cases with burden 15±14%. Echocardiography noted LV ejection fraction of 55±8%, mitral regurgitations in 70% (severe 18%, moderate 27%, mild 24%), mitral annular disjunction in 39%, and Pickelhaube sign in 9% of cases. Cardiac magnetic resonance showed LVESV 113±119mm, LVEDV 117±70mm, and late Gadolinium enhancement in 48% with highest prevalence in basal inferolateral segment. PET-CT performed in 17 cases and showed fluorodeoxyglucose uptake in 24% cases, mostly in basal inferolateral segment as well. Genetic testing was performed in 23 cases and reported a putative pathogenic variant in 21.7%: FLNC (Exemplar case, Figure 1 ) cause of dilated cardiomyopathy, TBX5 cause of Holt-Oram syndrome, DSP cause of arrhythmogenic cardiomyopathy , TTN causes of dilated cardiomyopathy, MTO1 cause of mitochondrial hypertrophic cardiomyopathy. Conclusion: Full evaluation of arrhythmogenic MVP with multimodality imaging and genetic testing reveals high burden of fibrosis, inflammation, and candidate pathogenic variants.

Identification of potential susceptibility genes in patients with primary Sjögren’s syndrome-associated pulmonary arterial hypertension through whole exome sequencing

BackgroundPulmonary arterial hypertension (PAH) is a rare complication of primary Sjögren’s syndrome (pSS). Several genes have proven to be associated with pSS and PAH. However, there is no study specifically addressing the genetic susceptibility in pSS combined with PAH.MethodsThirty-four unrelated patients with pSS-PAH were recruited from April 2019 to July 2021 at Peking Union Medical College Hospital. Demographic and clinical data were recorded in detail, and peripheral blood samples were collected for whole-exome sequencing (WES). Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to predict the functional effect of mutant genes. Genetic variants identified by WES were confirmed by polymerase chain reaction (PCR)-Sanger sequencing.ResultsWe totally identified 141 pathogenic variant loci of 129 genes in these 34 pSS-PAH patients, using WES analysis. Patients with a family history of rheumatic diseases are more likely to carry FLG mutations or carry gene variations related to the biosynthesis of the amino acids pathway (p < 0.05). According to Sanger sequencing confirmation and pathogenicity validation, we totally identified five candidate pathogenic variants including FLG c.12064A > T, BCR c.3275_3278dupCCGG, GIGYF2 c.3463C > A, ITK c.1741C > T, and SLC26A4 c.919-2A > G.ConclusionOur findings provide preliminary data of exome sequencing to identify susceptibility loci for pSS-PAH and enriched our understanding of the genetic etiology for pSS-PAH. The candidate pathogenic genes may be the potential genetic markers for early warning of this disease.

A de novo mutation of ADAMTS8 in a patient with Wiedemann–Steiner syndrome

BackgroundWiedemann–Steiner syndrome (WDSTS) is a rare autosomal dominant disorder caused by mutations in the KMT2A gene and is usually characterized by hairy elbows, short stature, developmental delay, intellectual disability and obvious facial dysmorphism.Case presentationHere, we report a 5-year-old girl with clinical features similar to WDSTS, including postnatal growth delay, retarded intellectual development, and ocular hypertelorism. Through whole-exome sequencing (WES), a frameshift variant of KMT2A was found in the patient but not in her parents’ genomic DNA. By bioinformatics analysis, the KMT2A variant was demonstrated to be the top candidate pathogenic variant for the clinical phenotype consistent with WDSTS. Moreover, a duplication of exon 1 in ADAMTS8 (belonging to the zinc metalloproteinase family) was found in the genomic DNA of this patient, which may be responsible for the characteristics that are different from those of WDSTS, including early teething, rapid tooth replacement, and dysplastic enamel.ConclusionsFrom the above results, we propose that in our patient, the frameshift variant in KMT2A is the main reason for the WDSTS phenotype, and the unreported mutation in ADAMTS8 may be the candidate reason for other characteristics that are different from those of WDSTS. Therefore, this study not only provides a new KMT2A variant associated with WDSTS but is also a reminder that combined mutations may be present in a case with more characteristics than those seen in WDSTS.

Identification of compound heterozygous variants in MSH4 as a novel genetic cause of diminished ovarian reserve

BackgroundDiminished ovarian reserve (DOR) is a common cause of female infertility, with genetic factors being a significant contributor. However, due to high genetic heterogeneity, the etiology of DOR in many cases remains unknown. In this study, we analyzed the phenotype of a young woman with primary infertility and performed molecular genetic analysis to identify the genetic cause of her condition, thus providing important insights for genetic counseling and reproductive guidance.MethodsWe collected the patient’s basic information, clinical data, as well as diagnostic and therapeutic history and performed whole-exome sequencing on her peripheral blood. Candidate pathogenic variants were validated by Sanger sequencing in family members, and the pathogenicity of variants was analyzed using ACMG guidelines. We used bioinformatics tools to predict variant effects on splicing and protein function, and performed in vitro experiments including minigene assay and expression analysis to evaluate their functional effects on HEK293T.ResultsWe identified biallelic MSH4 variants, c.2374 A > G (p.Thr792Ala) and c.2222_2225delAAGA (p.Lys741Argfs*2) in the DOR patient. According to ACMG guidelines, the former was classified as likely pathogenic, while the latter was classified as pathogenic. The patient presented with poor oocyte quantity and quality, resulting in unsuccessful in vitro fertilization cycles. Bioinformatics and in vitro functional analysis showed that the c.2374 A > G variant altered the local conformation of the MutS_V domain without decreasing MSH4 protein expression, while the c.2222_2225delAAGA variant led to a reduction in MSH4 protein expression without impacting splicing.ConclusionsIn this study, we present evidence of biallelic variants in MSH4 as a potential cause of DOR. Our findings indicate a correlation between MSH4 variants and reduced oocyte quality, as well as abnormal morphology of the first polar body, thereby expanding the phenotypic spectrum associated with MSH4 variants. Furthermore, Our study emphasizes the importance of utilizing whole-exome sequencing and functional analysis in diagnosing genetic causes, as well as providing effective genetic counseling and reproductive guidance for DOR patients.

Prenatal diagnosis to identify compound heterozygous variants in PKDCC that causes rhizomelic limb shortening with dysmorphic features in a fetus from China

BackgroundRhizomelic limb shortening with dysmorphic features (RLSDF) has already been a disorder of the rare autosomal recessive skeletal dysplasia, just having a few reported cases. RLSDF is caused by protein kinase domain containing, cytoplasmic(PKDCC)gene variants. In this study, we describe the clinical features and potential RLSDF molecular etiology in a fetus from China.MethodsGenomic DNA (gDNA) extracted from the fetal muscle tissue and parents’ peripheral blood was subjected to chromosomal microarray analysis (CMA) and trio-based whole exome sequencing (Trio-WES). The candidate pathogenic variants were verified by using Sanger sequencing.ResultsTrio-WES identified two compound heterozygous variants in PKDCC, c.346delC (p.Pro117Argfs*113) and c.994G > T (p.Glu332Ter), inherited from the father and mother, respectively. Both variants are classified as pathogenic according to American College of Medical Genetics and Genomics guidelines.ConclusionsWe reported the first prenatal case of RLSDF caused by PKDCC in the Chinese population. Our findings extended the variation spectrum of PKDCC and emphasized the necessity of WES for the early diagnosis of skeletal dysplasia and other ultrasound structural abnormalities in fetuses.

A Novel Pair of Compound Heterozygous Mutation of EYS in a Han Chinese Family with Retinitis Pigmentosa.

Background: Retinitis pigmentosa (RP) is a complex inherited and progressive degenerative retinal disease. The eyes shut homolog (EYS) is frequently associated with RP is surprisingly high. Exploring the function of EYS is quite difficult due to the unique gene size and species specificity. Gene therapy may provide a breakthrough to treat this disease. Therefore, exploring and clarifying pathogenic mutations of EYS-associated RP has important guiding significance for clinical treatment. Methods: Clinical and molecular genetic data for EYS-associated RP were retrospectively analyzed. Sanger sequencing was applied to identify novel mutations in these patients. Candidate pathogenic variants were subsequently evaluated using bioinformatic tools. Results: A novel pair of compound heterozygous mutations was identified: a novel stop-gain mutation c.2439C>A (p.C813fsX) and a frameshift deletion mutation c.6714delT (p. P2238fsX) of the EYS gene in the RP family. Both of these mutations were rare or absent in the 1000 Genomes Project, dbSNP, and Genome Aggregation Database (gnomAD). These two mutations would result in a lack of multiple functionally important epidermal growth factor-like and Laminin G-like coding regions in EYS. Conclusions: A novel compound heterozygote of the EYS gene in a Chinese family with an autosomal inheritance pattern of RP was identified. Identifying more pathogenic mutations and expanding the mutation spectrum of the EYS gene will contribute to a more comprehensive understanding of the molecular pathogenesis of RP disease that could be gained in the future. It also could provide an important basis for the diagnosis, clinical management, and genetic counseling of the disease.

The novel compound heterozygous variants identified in a Chinese family with glucose phosphate isomerase deficiency and pathogenicity analysis

Background and Aims:Glucose phosphate isomerase (GPI) deficiency is an extremely rare autosomal recessive disorder caused by mutations in the GPI gene. In this research, the proband displaying typical manifestations of haemolytic anaemia and his family members were recruited to analyse the pathogenicity of the detected variants.Methods:Peripheral blood samples were collected from the family members and genomic DNA was extracted and targeted for capture and sequencing. The effect of the candidate pathogenic variants on splicing was further investigated using the minigene splicing system. The computer simulation was also used for further analysis of the detected data.Results:The proband carried the compound heterozygous variants c.633 + 3 A > G and c.295G > T in the GPI gene, which have never been reported before. In the genealogy, co-segregation of the mutant genotype with the phenotype was established. The minigene study showed that intronic mutations resulted in abnormal pre-mRNA splicing. Specifically, the two aberrant transcripts: r.546_633del and r.633 + 1_633 + 2insGT were transcribed by the minigene plasmid expressing the c.633 + 3 A > G variant. The missense mutation c.295G > T in exon 3 resulted in altering glycine at codon 87 to cysteine which was predicted to be pathogenic in an in silico analysis. Deeper analyses revealed that the Gly87Cys missense mutation led to steric hindrance. Compared to the wild-type, the mutation G87C led to a significant increase in intermolecular forces.Conclusion:Overall, the novel compound heterozygous variants in the GPI gene contributed to the etiology of the disease. Genetic testing can assist in the diagnosis. The novel gene variants identified in the present study has further expanded the mutational spectrum of GPI deficiency, which can better guide family counselling.

Contribution of Somatic Ras/Raf/Mitogen-Activated Protein Kinase Variants in the Hippocampus in Drug-Resistant Mesial Temporal Lobe Epilepsy

Mesial temporal lobe epilepsy (MTLE) is the most common focal epilepsy subtype and is often refractory to antiseizure medications. While most patients with MTLE do not have pathogenic germline genetic variants, the contribution of postzygotic (ie, somatic) variants in the brain is unknown. To test the association between pathogenic somatic variants in the hippocampus and MTLE. This case-control genetic association study analyzed the DNA derived from hippocampal tissue of neurosurgically treated patients with MTLE and age-matched and sex-matched neurotypical controls. Participants treated at level 4 epilepsy centers were enrolled from 1988 through 2019, and clinical data were collected retrospectively. Whole-exome and gene-panel sequencing (each genomic region sequenced more than 500 times on average) were used to identify candidate pathogenic somatic variants. A subset of novel variants was functionally evaluated using cellular and molecular assays. Patients with nonlesional and lesional (mesial temporal sclerosis, focal cortical dysplasia, and low-grade epilepsy-associated tumors) drug-resistant MTLE who underwent anterior medial temporal lobectomy were eligible. All patients with available frozen tissue and appropriate consents were included. Control brain tissue was obtained from neurotypical donors at brain banks. Data were analyzed from June 2020 to August 2022. Drug-resistant MTLE. Presence and abundance of pathogenic somatic variants in the hippocampus vs the unaffected temporal neocortex. Of 105 included patients with MTLE, 53 (50.5%) were female, and the median (IQR) age was 32 (26-44) years; of 30 neurotypical controls, 11 (36.7%) were female, and the median (IQR) age was 37 (18-53) years. Eleven pathogenic somatic variants enriched in the hippocampus relative to the unaffected temporal neocortex (median [IQR] variant allele frequency, 1.92 [1.5-2.7] vs 0.3 [0-0.9]; P = .01) were detected in patients with MTLE but not in controls. Ten of these variants were in PTPN11, SOS1, KRAS, BRAF, and NF1, all predicted to constitutively activate Ras/Raf/mitogen-activated protein kinase (MAPK) signaling. Immunohistochemical studies of variant-positive hippocampal tissue demonstrated increased Erk1/2 phosphorylation, indicative of Ras/Raf/MAPK activation, predominantly in glial cells. Molecular assays showed abnormal liquid-liquid phase separation for the PTPN11 variants as a possible dominant gain-of-function mechanism. Hippocampal somatic variants, particularly those activating Ras/Raf/MAPK signaling, may contribute to the pathogenesis of sporadic, drug-resistant MTLE. These findings may provide a novel genetic mechanism and highlight new therapeutic targets for this common indication for epilepsy surgery.

Candidate variants in DNA replication and repair genes in early-onset renal cell carcinoma patients referred for germline testing

BackgroundEarly-onset renal cell carcinoma (eoRCC) is typically associated with pathogenic germline variants (PGVs) in RCC familial syndrome genes. However, most eoRCC patients lack PGVs in familial RCC genes and their genetic risk remains undefined.MethodsHere, we analyzed biospecimens from 22 eoRCC patients that were seen at our institution for genetic counseling and tested negative for PGVs in RCC familial syndrome genes.ResultsAnalysis of whole-exome sequencing (WES) data found enrichment of candidate pathogenic germline variants in DNA repair and replication genes, including multiple DNA polymerases. Induction of DNA damage in peripheral blood monocytes (PBMCs) significantly elevated numbers of gammaH2AX foci, a marker of double-stranded breaks, in PBMCs from eoRCC patients versus PBMCs from matched cancer-free controls. Knockdown of candidate variant genes in Caki RCC cells increased gammaH2AX foci. Immortalized patient-derived B cell lines bearing the candidate variants in DNA polymerase genes (POLD1, POLH, POLE, POLK) had DNA replication defects compared to control cells. Renal tumors carrying these DNA polymerase variants were microsatellite stable but had a high mutational burden. Direct biochemical analysis of the variant Pol δ and Pol η polymerases revealed defective enzymatic activities.ConclusionsTogether, these results suggest that constitutional defects in DNA repair underlie a subset of eoRCC cases. Screening patient lymphocytes to identify these defects may provide insight into mechanisms of carcinogenesis in a subset of genetically undefined eoRCCs. Evaluation of DNA repair defects may also provide insight into the cancer initiation mechanisms for subsets of eoRCCs and lay the foundation for targeting DNA repair vulnerabilities in eoRCC.

Novel Candidate loci and Pathogenic Germline Variants Involved in Familial Hematological Malignancies Revealed by Whole-Exome Sequencing.

The familial occurrence of hematological malignancies has been underappreciated. Recent studies suggest that up to 15% of adults with myeloid neoplasms carry germline pathogenic variants in cancer-predisposing genes. This study aimed to identify the underlying germline predisposition variant in patients with a strong family or personal onco-hematological history using whole exome sequencing on sixteen uncharacterized individuals. It was carried out in two groups of patients, one with samples available from two affected relatives (Cohort A) and one with available samples from the index case (Cohort B). In Cohort A, six families were characterized. Two families shared variants in genes associated with DNA damage response and involved in cancer development (CHEK2 and RAD54L). Pathogenic or likely pathogenic germline variants were also found in novel candidate genes (NFATC2 and TC2N). In two families, any relevant pathogenic or likely pathogenic genomic variants were identified. In Cohort B, four additional index cases were analyzed. Three of them harbor clinically relevant variants in genes with a probable role in the development of inherited forms of hematological malignancies (GATA1, MSH4 and PRF1). Overall, whole exome sequencing is a useful approach to achieve a further characterization of these patients and their mutational spectra. Moreover, further investigations may help improve optimization for disease management of affected patients and their families.

A novel homozygous TUB mutation associated with autosomal recessive retinitis pigmentosa in a consanguineous Chinese family

BackgroundRetinitis pigmentosa (RP) is the most common type of inherited retinopathy. At least 69 genes for RP have been identified. A significant proportion of RP, however, remains genetically unsolved. In this study, the genetic basis of a Chinese consanguineous family with presumed autosomal recessive retinitis pigmentosa (arRP) was investigated.MethodsOverall ophthalmic examinations, including funduscopy, decimal best-corrected visual acuity, axial length and electroretinography (ERG) were performed for the family. Genomic DNA from peripheral blood of the proband was subjected to whole exome sequencing. In silico predictions, structural modelling, and minigene assays were conducted to evaluate the pathogenicity of the variant.ResultsA novel homozygous variant (NM_003320.4: c.1379A > G) in the TUB gene was identified as a candidate pathogenic variant in this parental consanguineous pedigree. This variant co-segregated with the disease in this pedigree and was absent in 118 ethnically matched healthy controls. It’s an extremely rare variant that is neither deposited in population databases (1000 Genomes, ExAC, GnomAD, or Exome Variant Server) nor reported in the literature. Phylogenetic analysis indicated that the Asn residue at codon 460 of TUB is highly conserved across diverse species from tropicalis to humans. It was also completely conserved among the TUB, TULP1, TULP2, and TULP3 family proteins. Multiple bioinformatic algorithms predicted that this variant was deleterious.ConclusionsA novel missense variant in TUB was identified, which was probably the pathogenic basis for arRP in this consanguineous family. This is the first report of a homozygous missense variant in TUB for RP.

The novel compound heterozygous rare variants may impact positively selected regions of TUBGCP6, a microcephaly associated gene

IntroductionThe microcephaly is a rare and severe disease probably under purifying selection due to the reduction of human brain-size. In contrast, the brain-size enlargement is most probably driven by positive selection, in light of this critical phenotypical innovation during primates and human evolution. Thus, microcephaly-related genes were extensively studied for signals of positive selection. However, whether the pathogenic variants of microcephaly-related genes could affect the regions of positive selection is still unclear.MethodsHere, we conducted whole genome sequencing (WGS) and positive selection analysis.ResultsWe identified novel compound heterozygous variants, p.Y613* and p.E1368K in TUBGCP6, related to microcephaly in a Chinese family. The genotyping and the sanger sequencing revealed the maternal and the paternal origin for the first and second variant, respectively. The p.Y613* occurred before the second and third domain of TUBGCP6 protein, while p.E1368K located within the linker region of the second and third domain. Interestingly, using multiple positive selection analyses, we revealed the potential impacts of these variants on the regions of positive selection of TUBGCP6. The truncating variant p.Y613* could lead to the deletions of two positively selected domains DUF5401 and Spc97_Spc98, while p.E1368K could impose a rare mutation burden on the linker region between these two domains.DiscussionOur investigation expands the list of candidate pathogenic variants of TUBGCP6 that may cause microcephaly. Moreover, the study provides insights into the potential pathogenic effects of variants that truncate or distribute within the positively selected regions.

In silico interpretation of variants identifies novel causes of inherited eye disease in the 100 000 genomes project

AbstractInherited retinal dystrophy (IRD) describes a widely heterogeneous spectrum of disorders with over 270 genes implicated to date across all inheritance patterns and an estimated prevalence of around 1 in 2000 people. Genetic testing for IRD had evolved dramatically since the discovery of the first pathogenic variant in 1990: the advent of massively parallel sequencing means that the more than half of patients are now able to receive a molecular diagnosis although there is still significant missing heritability (up to ~40% of patients). Novel genes, non‐coding variants, structural variants/copy number variants, novel gene/disease associations, non‐Mendelian inheritance and non‐genetic disease are some of the mechanisms thought to account for the missing heritability of rare disease. This presentation will focus on non‐coding variant analysis.Whole genome sequencing (WGS) enables the capture and interrogation of all variants covering up to approximately 95% of the genome, and advances in genomic analysis technology, genome and variant databases, bioinformatics and in silico tools mean that nowadays variant prioritization pipelines are able to filter the huge variant numbers leaving only a handful of candidate pathogenic coding variants per patient.For the missing heritability in patients whose phenotype cannot be fully explained by this approach, however, non‐coding variant analysis is more challenging. Many more variants exist outside of coding exons and our understanding of their effect on transcripts and proteins is limited. Functional assays, especially for retina specific genes may be complex and difficult to establish, so accurate variant effect predictions are critical to enable molecular geneticists and clinical laboratories to maximize their ability to provide a definitive molecular diagnosis.This presentation will focus on the analysis of non‐coding variants in IRD genes in a cohort of patients who underwent WGS in the UK's 100 000 genomes project. Key to this study, patients were identified for study following multidisciplinary team discussion to highlight the most likely gene to be implicated in their disease. Identification of candidate non‐coding variants, filtering and prioritization were performed to identify the most compelling variant, followed by molecular investigation and confirmation of the predicted effect. Exemplar case studies will demonstrate the power of this analysis pipeline and accuracy of in silico predictions.

Myosin Mutations and Sudden Sensorineural Hearing Loss: Results of Whole Exome Sequencing.

Idiopathic sudden sensorineural hearing loss (ISSNHL) affects 66,000 patients per year in the United States. Genetic mutations have been associated with progressive hearing loss; however, genetic mutations associated with ISSNHL have not been identified. A prospective cohort study of adults older than 18 years presenting with ISSNHL at a tertiary academic medical center. Whole exome sequencing (WES) was conducted using Genome Analysis Toolkit best practices. An automated diagnostic screen employing a variety of models for pathogenicity was conducted across all genes with no specific targets. Candidate pathogenic variants were reviewed by a team of geneticists and clinicians. Variants were crossed-referenced with 92 known hearing loss associated genes. Twenty-nine patients with SSNHL were screened using WES. The average age of patients was 53 ± 17.1 years, and most patients were White (62%) and men (55%). The mean pure tone average was 64.8 ± 31.3 dB for the affected ear. Using a 0.1% allele frequency screen, 12 (41%) cases had a mutation in any of the nine selected myosin genes. When we restrict to singletons (allele frequency = 0%), 21% (n = 6) of cases have qualifying variants, whereas only 3.8% (n = 481) of 12,577 healthy controls carry qualifying variants (p < 0.01). Most mutations (80%) were missense mutations. Of the novel mutations, one was a frameshift mutation, and two were a stop-gained function. Three were missense mutations. Myosin mutations may be associated with ISSNHL. However, larger population screening is needed to confirm the association of myosin mutation with ISSNHL and steroid responsiveness.

Long-Read Sequencing Resolves Complex Structural Variants and Identifies Missing Disease-Causing Variants in Unsolved Cases of Hemophilia

Introduction: Despite recent advances in genetic testing, many individuals with inherited bleeding disorders remain unsolved after a complete clinical evaluation. In some cases, no variant is found in a particular gene or genes of interest, while in other cases a large structural variant (SV) is found or suspected that cannot be resolved using existing methods. In the large U.S. hemophilia genotyping initiative MyLifeOurFuture, ~2% of participants with hemophilia A had no F8 variant identified despite exhaustive investigation. Additionally, large SVs were common in severe hemophilia and associated with inhibitor risk but were incompletely characterized in nearly all cases. We hypothesized that long-read sequencing (LRS) would identify missing disease-causing variants and fully characterize SVs associated with hemophilia in these individuals. Methods: We performed targeted or whole-genome LRS on the Oxford Nanopore platform of 25 hemophilia cases with FVIII levels <14% in which prior testing identified no pathogenic variant or identified a suspected SV impacting F8. After basecalling with guppy and alignment to GRCh38 using minimap2, single nucleotide and insertion or deletion variants were identified with Clair3. All variant calls were refined using population frequency and pathogenicity predictions. SVs were called with Sniffles2, SVIM, and CuteSV. We performed de novo assembly of SVs and called methylation in all samples. Methylation differences were identified using an in-house developed pipeline. Results: We identified candidate pathogenic variants and previously undetected SVs in 10/10 cases with no variant found on clinical testing. Candidate intronic variants predicted to affect splicing were validated by exon trap assays. SVs identified included deletions and large inversions. Among 11 cases with suspected SVs, LRS resolved the candidate SV providing detailed genomic structure, orientation, and predicted impact of the variant. Whole genome LRS resolved SVs involving the large segmental duplication within F8 intron 22 (Int22h1), revealing variants that have not previously been observed including unsuspected orientations in the F8 intron 22 positive control sample. We analyzed four females with unexplained low FVIII levels and identified skewed methylation profiles that may help explain their phenotype. Conclusion: In a single genetic test LRS can identify disease-causing variants missed by prior genetic testing, resolve complex SVs, and identify methylation differences that explain hemophilia phenotypes. These comprehensive methods are necessary to fully understand causes of hemophilia and complications such as inhibitor development. We expect this approach will similarly resolve "missing” genetic variation in other inherited disorders. Supported and funded by the BBI at UW, WACBD, HTRS MCRA (Genentech), and NHLBI TOPMed.

Genotype and phenotype spectrum of 10 children with STXBP1 gene-related encephalopathy and epilepsy.

STXBP1 mutations are associated with early onset epileptic encephalopathy (EOEE). Our aim was to explore the phenotype spectrum, clinical treatment and prognosis of STXBP1-related encephalopathy (STXBP1-E). Clinical and genetic data were collected from 10 patients with STXBP1 mutations. These patients were examined and diagnosed from 2015 to 2021 at the Pediatric Department of Qilu Hospital. Blood samples were collected and sequenced by next generation sequencing and Candidate pathogenic variants were identified using Sanger sequencing in all family members. All of the patients showed severe epilepsy, varying degrees of intellectual disability and delayed motor. The patients developed multiple seizure types and abnormal electroencephalography (EEG) results at onset, and focal seizures were the most frequent seizure type. Among the patients, 2 were diagnosed with Ohtahara syndrome, 2 patient was diagnosed with West syndrome. The other 6 patients could not be diagnosed with any specifically recognized epilepsy syndrome. Five of the 10 patients had a history of fever with seizures, 4 of whom had eliminated intracranial infection according to the results of cerebrospinal fluid (CSF) examinations, and the other patient was diagnosed with anti-myelin oligodendrocyte glycoprotein (MOG) -associated encephalitis. We identified one patient with a complete deletion of STXBP1 and 9 patients with de novo heterozygous mutations of STXBP1. Among those mutations, 4 were novel (c.56°C > T, c.1315A > T, c.751G > C, and c.554_559del), and 5 had been previously reported [c.364C > T, c.569G > A (2 cases), c.748C > T, and c.1651C > T]. For 8 of our patients, different combinations of anti-seizure medications (ASMs) led to seizure freedom. One patient with MOG antibodies in his serum obtained a poor therapeutic effect from the traditional ASMs treatment, so he had to achieve seizure-free status through vagus nerve stimulation (VNS), which had little effect on his psychomotor ability. Fortunately, in one case, patient psychomotor ability was improved through VNS. Our study shows that STXBP1 screening should be considered in patients with neonatal seizures with intellectual disability, and frequent seizures with fever should also be considered with the STXBP1 mutation when intracranial infection is eliminated. VNS has expanded outcome measures to include behavioral and developmental function as well as seizure control.