Intronic Variant Research Articles

CSF ADAM10 levels in AD patients in relationship to ADAM10 gene variants

AbstractBackgroundThe disintegrin metalloproteinase 10 (ADAM10) is the main α‐secretase acting in the non‐amyloidogenic processing of APP. Some ADAM10 gene variants (Q170H, R181G) have been associated with higher susceptibility to develop late‐onset AD and have been shown to promote AD‐type changes in mice models. We tested the hypothesis that some ADAM10 rare variants could increase the risk for AD through conferring an earlier age‐related downregulation of a‐secretase activity.Method ADAM10 gene was sequenced in 103 AD cases (78% with family history) with positive CSF biomarkers, and 96 cognitively preserved nonagenarians. ADAM10 in CSF of the AD cases was analyzed by means of ELISA (total levels) and western blot (segregated by isoforms of 50 kDa, 55 kDa, and 80 kDa) in comparison with controls and non‐AD dementia cases. We examined whether there was an increase of rare gene variants (MAF < 0.01) in the AD group and their potential association with lowers CSF protein levels.ResultRare variants were found in 15,5% of AD cases (in 23% of early‐onset versus 7,8% in late‐onset, p = 0.058) and in 12,5% of nonagenarians, and several of them were exclusive of the group. However, all were intronic variants except the Q170H change found in three AD cases and one nonagenarian. The SNP rs74016945 (3’UTR), with MAF close to 0.01, was found in 6% of the nonagenarians (OR 0.146, Fisher exact test p = 0.057). ELISA ADAM10 levels were not significantly different comparing AD cases with controls, carriers of rare variants versus non‐carriers, and did not differ consistently according to age at onset or APOE genotype. The three ADAM10 species were also similar in AD cases and controls, and did not differ according to genetic variants or age at onset.ConclusionRare ADAM10 exonic variants are uncommon in AD cases and the presence of rare intronic variants (more frequent in early versus late‐onset cases) is not associated with decreased ADAM10 levels in CSF.

A novel intronic variant causing osteogenesis imperfecta – A rare case study

A novel intronic variant causing osteogenesis imperfecta – A rare case study

Association Study Between Kynurenine 3-Monooxygenase (KMO) Gene and Parkinson's Disease Patients.

The influence of various risk factors such as aging, intricate cellular molecular processes, and lifestyle factors like smoking, alcohol consumption, caffeine intake, and occupational factors has received increased focus in relation to the risk and development of Parkinson's disease (PD). Limited research has been conducted on the assessment of lifestyle impact on kynurenine 3-monooxygenase (KMO) gene in PD. A total of 164 subjects, including 82 PD cases and 82 healthy individuals, were recruited based on specific inclusion and exclusion criteria. The severity of PD and clinical assessment were evaluated using the Unified Parkinson's Disease Rating Scale (UPDRS) and Hoehn and Yahr (HY) scaling. Sanger sequencing was performed to analyse the KMO gene in the recruited subjects, and case-control studies were conducted. The UPDRS assessment revealed significant impairments in smell, tremors, walking, and posture instability in the late-onset PD cohorts. The HY scaling indicated a higher proportion of late-onset cohorts in stage 2. Moreover, both alcoholic and non-alcoholic groups showed significantly increased levels of 3-HK in late-onset PD. Gene analysis identified missense variants at position g.241593373T > A (rs752312199) and intronic variants at positions g.241592623A > G (rs640718), g.241592800C > A (rs990388262), g.241592802A > C (rs1350160268), g.241592808T > C (rs1478255936), and g.241592812G > T (rs948928931). The alterations in the KMO gene were found to influence the levels of kynurenic acid (KYNA) and 3-hydroxykynurenine (3-HK). Genomic analysis revealed a high prevalence of missense mutations in the late-onset PD groups, leading to a decline in 3-HK levels in patients. This leads to the reduction of the progression of disease in late-onset groups which shows that this mutation may lead to the protective effect on the PD subjects. This study suggests the use of KYNA and 3-HK as potential biomarkers in analysing the progression of disease. This study is limited by its small sample size. To overcome this limitation, a larger study involving in greater number of participants is needed to thoroughly investigate the KMO gene and KP metabolites, to enhance our understanding of Parkinson's disease progression, and to enhance diagnostic capabilities.

Relationships between polymorphisms in keratin genes and Alzheimer’s disease phenotypes.

AbstractBackgroundKeratins are genetically polymorphic and are the largest subset of intermediate filaments. Keratins are also implicated in signaling pathways, inflammation, and disease states. Keratin 9 (KRT9) protein has been identified to have high diagnostic accuracy for Alzheimer’s disease (AD) (PMID:22045497); it was detected in cerebrospinal fluid (CSF) of AD patients but not healthy controls (PMID:24959311). Plasma keratin 9 concentrations also positively correlated with AD‐associated proteins (e.g., apolipoprotein E and tau) in AD patients (PMID:26973255). Post‐translational modification of keratin 83 (KRT83) protein has been associated with serum asymmetric dimethylarginine levels (PMID:34181092), a vascular risk factor implicated in AD. However, our understanding of the role of genetic variation in AD risk is incomplete. Here, we investigate associations between single nucleotide polymorphisms (SNPs) in KRT9, KRT83, and KRT2 (Keratin 2, couples with keratin 9) and AD biomarker phenotypes.MethodIndividuals with mild cognitive impairment (MCI) or AD, and clinically normal controls were selected among ADNI participants. Participants underwent genome‐wide genotyping. Keratin gene variants were identified from the genome‐wide data using dbSNP variant IDs and genotypes were coded additively. Linkage‐disequilibrium‐based clumping on minor allele frequency (MAF) was applied to prioritize more common SNPs. CSF‐Aβ42, ‐tau, and phosphorylated‐tau (p‐tau) levels were quantified via Roche Elecsys immunoassays. Brain‐Aβ deposition was evaluated via normalized PET‐18F‐AV‐45 cortical summary measures (SUVR). White matter hyperintensities (WMH) were obtained from MRI 3D‐T1 and FLAIR sequences via an automated atlas‐based segmentation pipeline. Linear regression models controlling for age, sex, Mini‐Mental State Exam, APOE‐ε4 status, and head size were used to assess association and interaction effects between SNPs and the AD phenotypes in R.ResultAmong included participants (n = 907, 73±7.2 years, 44% female), SNPs in KRT2 (e.g. 3’UTR‐variant rs117041267‐G/A; MAF = 1.4%) and KRT83 (e.g. upstream‐variant KRT83‐rs17119838‐C/T; MAF = 15.6%) were associated with higher CSF‐Aβ42 concentrations (F(2,905) = 4.26,p = 0.014; and F(2,905) = 6.61,p = 0.0014, respectively). Upstream‐variant KRT83‐rs182354391‐C/G (MAF = 1%) showed an association with lower CSF‐tau (F(1,904) = 5.44,p = 0.020). Multiple KRT9 and KRT2 SNPs also showed association with WMH, while KRT2 and KRT83 intron variants showed associations with PET‐Aβ.ConclusionThese candidate gene analyses suggest involvement of multiple keratin family genes in AD, as indicated by differences in amyloid and tau biomarkers, and white matter disease.

Genetic Variants in Canonical Wnt Signaling Pathway Associated with Pediatric ITP

Introduction : Pediatric immune thrombocytopenia (ITP) is thought to develop when a triggering event inappropriately activates an autoimmune anti-platelet response in the context of predisposing genetic factors. Inborn errors of immunity are increasingly being identified in cases of chronic, refractory autoimmune cytopenias. We sought to identify genetic variants associated with predisposition to 1) primary or secondary pediatric ITP of any disease duration versus healthy controls and 2) chronic ITP (cITP) versus disease that spontaneously resolves in < 1 year (sr-ITP). Methods : DNA and clinical data were derived from 8 sites from the Pediatric ITP Consortium of North America's (ICON) biorepository. Samples were genotyped using the Illumina Global Screening Array BeadChip. Genotypes were imputed using the Michigan Imputation Server HRC reference panel. Genetic variants that passed standard quality metrics and had a MAF ≥1% were retained. Control samples (n=8,372) were obtained from the National Longitudinal Study of Adolescent to Adult Health (Add Health) cohort. Logistic regression models were used to estimate adjusted odds ratios (aORs) comparing (1) ITP and control cohorts and (2) cITP and sr-ITP cohorts. Models assumed additive allelic effects and accounted for the top 3 principal components. Genome-wide significance was defined as P <5 × 10 -8 and suggestive of genome-wide significance, P <1 x 10 -5. Results : Six-hundred ten unique patient samples were genotyped. Nineteen (3.1%) did not pass quality control, leaving 591 for analysis. This cohort included 307 (52.0%) cITP patients, 209 (35.4%) sr-ITP patients, and 75 (12.7%) individuals without complete follow-up data to allow for classification of cITP or sr-ITP. The ITP cohort was 50.1% male, 50.1% non-Hispanic (NH) white, 29.4% Hispanic, 6.1% NH Black, 3.1% NH Asian, and 11.3% Other/Unknown. Compared with healthy controls, we identified 6 loci associated with ITP at genome-wide significance (Figure 1). Located within the intronic region of SLC24A4 rs75650997 was associated with an increased risk of ITP compared with controls (aOR: 2.38, P=3.9x10 -8). An intronic variant, rs2165798, in NAV2 was inversely associated with ITP (aOR: 0.52, P=3.2x10 -11). A variant in NKD1 rs4785216, was inversely associated with ITP (aOR: 0.43, P=8.86x10 -15).Another intergenic variant, rs4785426, in close proximity to NKD1 was also inversely associated with ITP (aOR: 0.48, P=1.84x10 -16) . An additional 20 variants met suggestive of genome-wide significance. When comparing sr-ITP and cITP patients, no SNVs (single nucleotide variant) reached genome-wide significance, but one variant, rs117609649, in ABCC4 met suggestive of genome-wide significance (aOR: 0.26, P=4.49x10 -7). Discussion : This is the largest, unbiased genetic study of pediatric ITP patients. The analysis comparing ITP versus healthy controls identified 20 suggestive SNVs and 6 which met genome-wide significance. Several of the top SNVs identified in this study affect Wnt/β-catenin signaling. Wnt-signaling impacts the development and regulation of immune cell populations including dendritic cells, macrophages, NK cells, B cells, and T cells. NAV2 activates Wnt/β-catenin signaling. NAV2 variants have been implicated in autoimmune conditions including Celiac disease and rheumatoid arthritis. NKD1 inhibits the canonical Wnt/β-catenin signaling pathway. The cumulative impact of these variants on Wnt activity in ITP should be evaluated in the future. No genetic variants met genome-wide significance in a comparison of sr-ITP and cITP patients. Collectively, these findings suggest there may be genetic variants implicated in ITP risk, but those genetic features are not specific to sr-ITP or cITP. A focused analysis of 632 genes that cause inborn errors of immunity (IEI) is ongoing. This includes gene burden testing and analysis of pathogenic variants within this gene subset. Variants in IEI genes will also be evaluated in a sub-population of patients with Evans syndrome. Conclusion : ITP is highly heterogenous in its presentation, disease course, and response to therapy. Mirroring its clinical behavior, the genetic contribution to ITP development is complex. This study identified potential SNVs associated with ITP, but not specific to sr-ITP or cITP. Variants in genes that affect the canonical Wnt-signaling pathway may play a role in ITP risk.

Aquaporin splice variation differentially modulates channel function during marine teleost egg hydration.

Aquaporin-mediated oocyte hydration is a developmentally regulated adaptive mechanism that co-occurs with meiosis resumption in marine teleosts. It provides the early embryos with vital water until osmoregulatory systems develop, and in the majority of marine teleosts causes their eggs to float. Recent studies have shown that the subdomains of two water channels (Aqp1ab1 and Aqp1ab2) encoded in a teleost-specific aquaporin-1 cluster (TSA1C) co-evolved with duplicated Ywhaz-like (14-3-3ζ-like) binding proteins to differentially control their membrane trafficking for maximal egg hydration. Here, we report that in species that encode the full TSA1C, in-frame intronic splice variants of Aqp1ab1 result in truncated proteins that cause dominant-negative inhibition of the canonical channel trafficking to the plasma membrane. The inhibition likely occurs through hetero-oligomerization and retention in the endoplasmic reticulum (ER) and ultimate degradation. Conversely, in species that only encode the Aqp1ab2 channel we found an in-frame intronic splice variant that results in an intact protein with an extended extracellular loop E, and an out-of frame intronic splice variant with exon readthrough that results in a truncated protein. Both isoforms cause dominant-negative enhancement of the degradation pathway. However, the extended and truncated Aqp1ab2-type variants can also partially escape from the ER to reach the oocyte plasma membrane, where they dominantly-negatively inhibit water flux. The ovarian follicular expression ratios of the Aqp1ab2 isoforms in relation to the canonical channel are lowest during oocyte hydration, but subsequently highest when the canonical channel is recycled, thus leaving the eggs endowed with >90% water. These findings suggest that the expression of inhibitory isoforms of Aqp1ab1 and Aqp1ab2 may represent a new regulatory mechanism through which the cell-surface expression and the activity of the canonical channels can be physiologically modulated during oocyte hydration in marine teleosts.

Characterization of pre-mRNA Splicing Defects Caused by CLCN5 and OCRL Mutations and Identification of Novel Variants Associated with Dent Disease.

Dent disease (DD) is an X-linked renal tubulopathy characterized by low-molecular-weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis and progressive renal failure. Two-thirds of cases are associated with inactivating variants in the CLCN5 gene (Dent disease 1, DD1) and a few present variants in the OCRL gene (Dent disease 2, DD2). The aim of the present study was to test the effect on the pre-mRNA splicing process of DD variants, described here or in the literature, and describe the clinical and genotypic features of thirteen unrelated patients with suspected DD. All patients presented tubular proteinuria, ten presented hypercalciuria and five had nephrolithiasis or nephrocalcinosis. CLCN5 and OCRL genes were analyzed by Sanger sequencing. Nine patients showed variants in CLCN5 and four in OCRL; eight of these were new. Bioinformatics tools were used to select fifteen variants with a potential effect on pre-mRNA splicing from our patients' group and from the literature, and were experimentally tested using minigene assays. Results showed that three exonic missense mutations and two intronic variants affect the mRNA splicing process. Our findings widen the genotypic spectrum of DD and provide insight into the impact of variants causing DD.

Compound Heterozygosity for Late-Onset Cardiomyopathy-Causative ALPK3 Coding Variant and Novel Intronic Variant Cause Infantile Hypertrophic Cardiomyopathy.

Hypertrophic and dilated cardiomyopathy (HCM, DCM) are leading causes of cardiovascular morbidity and mortality in children. The pseudokinase alpha-protein kinase 3 (ALPK3) plays an essential role in sarcomere organization and cardiomyocyte differentiation. ALPK3 coding mutations are causative of recessively inherited pediatric-onset DCM and HCM with variable expression of facial dysmorphism and skeletal abnormalities and implicated in dominantly inherited adult-onset cardiomyopathy. We now report two variants in ALPK3-a coding variant and a novel intronic variant affecting splicing. We demonstrate that compound heterozygosity for both variants is highly suggestive to be causative of infantile-onset HCM with webbed neck, and heterozygosity for the coding variant presents with adult-onset HCM. Our data validate partial penetrance of heterozygous loss-of-function ALPK3 mutations in late-onset hypertrophic cardiomyopathy and expand the genotypic spectrum of autosomal recessive ALPK3-related cardiac disease with Noonan-like features.

Molecular characterization of G6PD mutations identifies new mutations and a high frequency of intronic variants in Thai females.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked enzymopathy caused by mutations in the G6PD gene. A medical concern associated with G6PD deficiency is acute hemolytic anemia induced by certain foods, drugs, and infections. Although phenotypic tests can correctly identify hemizygous males, as well as homozygous and compound heterozygous females, heterozygous females with a wide range of G6PD activity may be misclassified as normal. This study aimed to develop multiplex high-resolution melting (HRM) analyses to enable the accurate detection of G6PD mutations, especially among females with heterozygous deficiency. Multiplex HRM assays were developed to detect six G6PD variants, i.e., G6PD Gaohe (c.95A>G), G6PD Chinese-4 (c.392G>T), G6PD Mahidol (c.487G>A), G6PD Viangchan (c.871G>A), G6PD Chinese-5 (c.1024C>T), and G6PD Union (c.1360C>T) in two reactions. The assays were validated and then applied to genotype G6PD mutations in 248 Thai females. The sensitivity of the HRM assays developed was 100% [95% confidence interval (CI): 94.40%-100%] with a specificity of 100% (95% CI: 88.78%-100%) for detecting these six mutations. The prevalence of G6PD deficiency was estimated as 3.63% (9/248) for G6PD deficiency and 31.05% (77/248) for intermediate deficiency by phenotypic assay. The developed HRM assays identified three participants with normal enzyme activity as heterozygous for G6PD Viangchan. Interestingly, a deletion in intron 5 nucleotide position 637/638 (c.486-34delT) was also detected by the developed HRM assays. G6PD genotyping revealed a total of 12 G6PD genotypes, with a high prevalence of intronic variants. Our results suggested that HRM analysis-based genotyping is a simple and reliable approach for detecting G6PD mutations, and could be used to prevent the misdiagnosis of heterozygous females by phenotypic assay. This study also sheds light on the possibility of overlooking intronic variants, which could affect G6PD expression and contribute to enzyme deficiency.

A Novel Splice Site Mutation in the FBN2 Gene in a Chinese Family with Congenital Contractural Arachnodactyly

Congenital contractural arachnodactyly (CCA) is a rare connective tissue disorder characterized by arachnodactyly, multiple joint contractures, progressive kyphoscoliosis, pectus deformity and abnormal crumpled ears. FBN2 is the only gene currently known to be associated with CCA. In this study, we report on a prenatal case presented with skeletal, cardiac and spinal malformations. And his father had elongated limbs, contractures of the proximal interphalangeal joints, high myopia and scoliosis. We conducted whole exome sequencing (WES) on the fetus-parental trio and a heterozygous variant (hg19 chr5:127,673,685, c.3598 + 4A > G, NM_001999.4) in intron 27 of the FBN2 gene was successfully identified, inherited from the father. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to evaluate the potential splicing effect of this variant, which confirmed that the variant caused a deletion of exon 27 (126 bp) by disrupting the splice-donor site and destroyed the 17th calcium-binding epidermal growth factor-like (cbEGF) domain. Our research not only finds the etiology of the disease in affected individuals and expands the mutation spectrum of FBN2 gene, but also provides genetic counseling and fertility guidance for this family.

Deciphering the impact of coding and non-coding SCN1A gene variants on RNA splicing.

Variants that disrupt normal pre-mRNA splicing are increasingly being recognized as a major cause of monogenic disorders. The SCN1A gene, a key epilepsy gene that is linked to various epilepsy phenotypes, is no exception. Approximately 10% of all reported variants in the SCN1A gene are designated as splicing variants, with many located outside of the canonical donor and acceptor splice sites, and most have not been functionally investigated. However, given its restricted expression pattern, functional analysis of splicing variants in the SCN1A gene could not be routinely performed. In this study, we conducted a comprehensive analysis of all reported SCN1A variants and their potential to impact SCN1A splicing and conclude that splicing variants are substantially misannotated and under-represented. We created a splicing reporter system consisting of 18 splicing vectors covering all 26 protein-coding exons with different genomic contexts and several promoters of varying strengths in order to reproduce the wild-type splicing pattern of the SCN1A gene, revealing cis-regulatory elements essential for proper recognition of SCN1A exons. Functional analysis of 95 SCN1A variants was carried out, including all 68 intronic variants reported in the literature, located outside of the splice sites canonical dinucleotides; 21 exonic variants of different classes (synonymous, missense, nonsense and in-frame deletion) and six variants observed in patients with epilepsy. Interestingly, almost 20% of tested intronic variants had no influence on SCN1A splicing, despite being reported as causative in the literature. Moreover, we confirmed that the majority of predicted exonic variants affect splicing unravelling their true molecular mechanism. We used functional data to perform genotype-phenotype correlation, revealing distinct distribution patterns for missense and splice-affecting 'missense' variants and observed no difference in the phenotype severity of variants leading to in-frame and out-of-frame isoforms, indicating that the Nav1.1 protein is highly intolerant to structural variations. Our work demonstrates the importance of functional analysis in proper variant annotation and provides a tool for high-throughput delineation of splice-affecting variants in SCN1A in a whole-gene manner.

Generation of an iPSC line (RMCGENi020-A) from a patient with Stargardt disease harboring the recurrent intronic ABCA4 variant c.4253+43G>A

Pathogenic variants in ABCA4 are associated with Stargardt disease (STGD1), an autosomal recessive macular dystrophy characterized by bilateral central vision loss due to a progressive degeneration of retinal cells. An induced pluripotent stem cell (iPSC) line was generated from late-onset STGD1 patient-derived fibroblasts harboring bi-allelic ABCA4 variants by lentivirus-induced reprogramming. The obtained iPSC line (RMCGENi020-A) showed pluripotent features after the reprogramming process. The generation of this iPSC line facilitates its use to differentiate it into relevant retinal-like cell models, with the aim to adequately evaluate the effects of the ABCA4 variants.

HSP47 levels determine the degree of body adiposity

Adiposity varies among individuals with the influence of diverse physiological, pathological, environmental, hormonal, and genetic factors, but a unified molecular basis remains elusive. Here, we identify HSP47, a collagen-specific chaperone, as a key determinant of body adiposity. HSP47 expression is abundant in adipose tissue; increased with feeding, overeating, and obesity; decreased with fasting, exercise, calorie restriction, bariatric surgery, and cachexia; and correlated with fat mass, BMI, waist, and hip circumferences. Insulin and glucocorticoids, respectively, up- and down-regulate HSP47 expression. In humans, the increase of HSP47 gene expression by its intron or synonymous variants is associated with higher body adiposity traits. In mice, the adipose-specific knockout or pharmacological inhibition of HSP47 leads to lower body adiposity compared to the control. Mechanistically, HSP47 promotes collagen dynamics in the folding, secretion, and interaction with integrin, which activates FAK signaling and preserves PPARγ protein from proteasomal degradation, partly related to MDM2. The study highlights the significance of HSP47 in determining the amount of body fat individually and under various circumstances.

Structural and non-coding variants increase the diagnostic yield of clinical whole genome sequencing for rare diseases

BackgroundWhole genome sequencing is increasingly being used for the diagnosis of patients with rare diseases. However, the diagnostic yields of many studies, particularly those conducted in a healthcare setting, are often disappointingly low, at 25–30%. This is in part because although entire genomes are sequenced, analysis is often confined to in silico gene panels or coding regions of the genome.MethodsWe undertook WGS on a cohort of 122 unrelated rare disease patients and their relatives (300 genomes) who had been pre-screened by gene panels or arrays. Patients were recruited from a broad spectrum of clinical specialties. We applied a bioinformatics pipeline that would allow comprehensive analysis of all variant types. We combined established bioinformatics tools for phenotypic and genomic analysis with our novel algorithms (SVRare, ALTSPLICE and GREEN-DB) to detect and annotate structural, splice site and non-coding variants.ResultsOur diagnostic yield was 43/122 cases (35%), although 47/122 cases (39%) were considered solved when considering novel candidate genes with supporting functional data into account. Structural, splice site and deep intronic variants contributed to 20/47 (43%) of our solved cases. Five genes that are novel, or were novel at the time of discovery, were identified, whilst a further three genes are putative novel disease genes with evidence of causality. We identified variants of uncertain significance in a further fourteen candidate genes. The phenotypic spectrum associated with RMND1 was expanded to include polymicrogyria. Two patients with secondary findings in FBN1 and KCNQ1 were confirmed to have previously unidentified Marfan and long QT syndromes, respectively, and were referred for further clinical interventions. Clinical diagnoses were changed in six patients and treatment adjustments made for eight individuals, which for five patients was considered life-saving.ConclusionsGenome sequencing is increasingly being considered as a first-line genetic test in routine clinical settings and can make a substantial contribution to rapidly identifying a causal aetiology for many patients, shortening their diagnostic odyssey. We have demonstrated that structural, splice site and intronic variants make a significant contribution to diagnostic yield and that comprehensive analysis of the entire genome is essential to maximise the value of clinical genome sequencing.

In search of genetic modifiers that explain the phenotypic variability in SMAD3-related aortopathy

Abstract Introduction Loeys-Dietz Syndrome (LDS) is an autosomal dominant connective tissue disorder presenting with thoracic aortic aneurysm and dissection (TAAD). Remarkably, some LDS patients remain cardiovascularly unaffected throughout life, while others carrying the exact same genetic variant die early because of an aortic dissection. We hypothesize that genetic modifiers are the basis of this observation. Objectives Identify genetic modifiers that explain the variability in LDS-related aortopathy. Generate a patient-specific iPSC-vascular smooth muscle cell (VSMC) model to allow functional validation of genetic modifiers. Material and Methods We have access to a large LDS family segregating a pathogenic SMAD3 (p.Arg287Gln) variant. Identification of candidate modifiers in this family encompasses genome-wide SNP-based linkage analysis (n=19, available mutation carriers) and WGS (3 affected (AMC) and 4 unaffected mutation carriers (UMC)). Subsequent functional validation involves CRISPR/Cas-based modifier correction in iPSC-VSMCs of an affected variant carrier, which are created using the CytoTune iPS 2.0 Kit and the Granata et al. VSMC differentiation protocols. Results Linkage analysis suggests the presence of an aggravating modifier at chr2 (LOD: 2.68). The region of 6 Mb contains 19 protein coding genes of which the TNFAIP6 gene was ToppGene prioritized. There were no exonic variants in any of the genes that segregate with affection status at MAF 0.01%. However, we are currently adapting the the filtering strategy and will look into more common variants, MAF 10% for exonic and MAF 5% for intronic variants. iPSCs of an AMC and its isogenic control were created. They express core pluripotency markers and possess trilineage differentiation potential in the absence of the Sendai vectors. SNP array confirmed the genomic stability and identity of the cells. Conclusion and Future Work The obtained data suggest the presence of an aggravating modifier at chr2. Further analyses are ongoing to pinpoint the exact modifier variant explaining the linkage signal. Subsequently, the identified modifier(s) will be modelled in an in-vitro created iPSC-VSMC model.

Genome-wide association meta-analysis of 5,864 individuals highlights 25 new loci implicated in the pathogenesis of cancer therapy-related cardiac dysfunction

Abstract Background Cancer therapy-related cardiac dysfunction, which commonly includes varying degrees of asymptomatic ventricular dysfunction and/or heart failure, is the main adverse effect of anticancer therapy. The mechanism by which it develops is still quite uncertain. In recent years several genetic association studies have identified common genetic variants associated with cardiotoxicity in genes related to DNA damage, drug transportation and metabolism, antioxidant mechanism, sarcomere dysfunction and impaired iron metabolism, but the evidence is still limited. Rare variants in familial cardiomyopathies genes have also been identified. Purpose This uncertainly requires larger studies to identify genetic variants allowing to predict the risk of cardiotoxicity. In this sense, we carry out a genome-wide association meta-analysis to identify genetic variants associated with cancer therapy-related cardiac dysfunction. The development of a risk predictor model combining clinical and genetic factors would make it possible to anticipate the adverse effects of this therapy. Methods Genome wide meta-analysis was performed with a total of 594 patients from the CARDIOTOX registry and 3545 healthy individuals from the National DNA Bank, together with three previously published independent cohorts with 1446, 227 and 52 individuals receiving cancer therapy. Patients were classified with different cardiotoxicity degrees based on the CARDIOTOX registry and exhaustive clinical information was obtained from medical records. The study was ethically approved by local institutional review boards and all participants provided written informed consent. DNA samples were genotyped and imputed to perform a GWAS meta-analysis for case-control (n=5864) and extreme phenotypes (n=5317) looking for genetic variants that predispose to developing cancer therapy-related cardiac dysfunction. A functional characterization of the significant loci was performed by colocalization with eQTLs. Results The case-control analysis showed 17 loci that achieved suggestive significance (< P=1 x10-5) (Figure 1). The rs6765460, an intergenic variant between TPRG1 and TP63 genes, had the strongest relationship with the development of cardiotoxicity (P=1.89x10-7). The extreme phenotypes analysis showed additional 8 loci that achieved suggestive significance. The rs12904266, an intron variant of SLCO3A1, had the strongest relationship with severe cardiotoxicity (P=3.20E-07). eQTLs colocalization of the significant loci revealed association with genes involved in the mitochondria, inflammation, immune system, and fibrosis. Conclusions Our GWAS meta-analysis identified 25 novel genetic variants that may be associated with cancer therapy-related cardiac dysfunction. Genotyping of these markers could be considered in a genetic risk score which, together with the already known clinical risk factors, would improve the cardiotoxicity prediction in cardio-oncology units.Figure 1.Miami plot with P values

Genetic polymorphisms in vitamin E transport genes as determinants for risk of equine neuroaxonal dystrophy.

Equine neuroaxonal dystrophy/equine degenerative myeloencephalopathy (eNAD/EDM) is an inherited neurodegenerative disorder associated with vitamin E deficiency. In humans, polymorphisms in genes involved in vitamin E uptake and distribution determines individual vitamin E requirements. Genetic polymorphisms in genes involved in vitamin E metabolism would be associated with an increased risk of eNAD/EDM in Quarter Horses (QHs). Whole-genome sequencing: eNAD/EDM affected (n = 9, postmortem [PM]-confirmed) and control (n = 32) QHs. eNAD/EDM affected (n = 39, 23-PM confirmed) and control (n = 68, 7-PM confirmed) QHs. Allele frequency (AF): Publicly available data from 504 horses across 47 breeds. Retrospective, case control study. Whole-genome sequencing was performed and genetic variants identified within 28 vitamin E candidate genes. These variants were subsequently genotyped in the validation cohort. Thirty-nine confirmed variants in 15 vitamin E candidate genes were significantly associated with eNAD/EDM (P < .01). In the validation cohort, 2 intronic CD36 variants (chr4:726485 and chr4:731082) were significantly associated with eNAD/EDM in clinical (P = 2.78 × 10-4 and P = 4 × 10-4 , respectively) and PM-confirmed cases (P = 6.32 × 10-6 and 1.04 × 10-5 , respectively). Despite the significant association, variant AFs were low in the postmortem-confirmed eNAD/EDM cases (0.22-0.26). In publicly available equine genomes, AFs ranged from 0.06 to 0.1. Many PM-confirmed cases of eNAD/EDM were wild-type for the 2 intronic CD36 SNPs, suggesting either a false positive association or genetic heterogeneity of eNAD/EDM within the QH breed.

Abstract 15609: ParSE-seq: A Calibrated Multiplexed Assay for Clinical Interpretation of Putative Splice-Altering Variants

Background: Up to 10% of disease-associated variants are predicted to affect RNA-splicing. Interpreting the clinical significance of putative splice-altering variants outside the 2-base pair canonical splice sites remains difficult without functional studies. Goal: To develop a calibrated, high-throughput assay to adjudicate variant effects on RNA splicing. Methods: We established Parallel Splice Effect Sequencing (ParSE-seq), a multiplexed minigene assay, to test variant effects on RNA splicing quantified by high-throughput sequencing. We used the tool SpliceAI to prioritize exonic and intronic candidate splice variants, and ClinVar to select curated benign and pathogenic controls in SCN5A , which encodes the major voltage-gated cardiac sodium channel. We transfected 271 barcoded minigene constructs into HEK cells or induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), and calibrated to the American College of Medical Genetics and Genomics scheme. Variants were interpreted using the calibrated functional data, and experimental data correlated to SpliceAI predictions. We studied selected variants by automated patch clamp (APC) in HEK cells and by assessing splicing and channel function directly in CRISPR-edited iPSC-CMs. Results: ParSE-seq revealed the splicing effect of 245 SCN5A variants in iPSC-CMs. The assay had concordant scores for 43/45 benign and pathogenic control variants, achieving both PS3- and BS3-strong levels of evidence after calibration. 41/114 exonic variants and 30/66 intronic variants with determinate scores disrupted splicing. 9 of 42 Variants of Uncertain Significance were reclassified, and 30 of 34 variants with conflicting interpretations were reinterpreted using the functional data. Predictions from SpliceAI performed well on experimental data (AUC = 0.96). We identified 3 clinically relevant splice-altering missense variants that had normal function when tested with a cDNA-based APC assay. A splice-altering intronic variant, c.1891-5C&gt;G also disrupted splicing and sodium channel electrophysiology when introduced into iPSC-CMs by CRISPR-editing. Conclusions: ParSE-seq is an efficient, calibrated, high-throughput assay to facilitate splice-altering variant interpretation.

Pathogenicity evaluation of variants of uncertain significance at exon-intron junction by splicing assay in patients with Mowat–Wilson syndrome

High-throughput sequencing has identified vast numbers of variants in genetic disorders. However, the significance of variants at the exon-intron junction remains controversial. Even though most cases of Mowat–Wilson syndrome (MOWS) are caused by heterozygous loss-of-function variants in ZEB2, the pathogenicity of variants at exon-intron junction is often indeterminable. We identified four intronic variants in 5/173 patients with clinical suspicion for MOWS, and evaluated their pathogenicity by in vitro analyses. The minigene analysis showed that c.73+2T>G caused most of the transcripts skipping exon 2, while c.916+6T>G led to partial skipping of exon 7. No splicing abnormalities were detected in both c.917-21T>C and c.3067+6A>T. The minigene analysis reproduced the splicing observed in the blood cells of the patient with c.73+2T>G. The degree of the exon skipping was concordant with the severity of MOWS; while the patient with c.73+2T>G was typical MOWS, the patient with c.916+6T>G showed milder phenotype which has been seldom reported. Our results demonstrate that mRNA splicing assays using the minigenes are valuable for determining the clinical significance of intronic variants in patients with not only MOWS but also other genetic diseases with splicing aberrations and may explain atypical or milder cases, such as the current patient.

Abstract 14479: Compound Heterozygosity With Two Novel Mutations in MYBPC3 as a Cause of Severe and Early-Onset Hypertrophic Cardiomyopathy

Case presentation: A 8-year-old male was diagnosed with severe hypertrophic cardiomyopathy (HCM) after a cardiac arrest from which he was successfully defibrillated. Maximal wall thickness was 23 mm (z-score 6.36) on echocardiography. An implantable cardioverter defibrillator (ICD) was implanted and atenolol was started. After 4 ventricular fibrillation-terminating ICD shocks in the following 2 months, further treatment is under consideration. A genetic test identified the likely pathogenic intronic variant (c.1927+337G&gt;T) in the MYBPC3 gene. An additional variant in the same gene (p.Arg817Gly), of uncertain significance but potentially relevant for the phenotype, was also found. His 10-year-old sister was also diagnosed with severe HCM, genetic test identified the two previously referred variants and ICD was also implanted. In the 4-year-old one sister, who is apparently healthy, only the likely pathogenic intronic variant (c.1927+337G&gt;T) was found. Regarding their parents, both with no evidence of cardiomyopathy, c.1927+337G&gt;T variant was identified in the father, while p.Arg817Gly was present in the mother. Discussion: This clinical case is noteworthy as an example of compound heterozygosity in which each parent donates one alternate allele, being these alleles located at different loci within the same gen, which emphasizes the importance of family genetic testing. The variant of uncertain significance p.Arg817Gly appears to be a disease modifier, causing the phenotype to be expressed early and severely in the presence of the probably pathogenic intronic variant c.1927+337G&gt;T in MYBPC3. This deep intronic variant has not been described previously but was found in thirteen index cases referred for HCM and a history of familial sudden death at an early age, being overrepresented in the cohort of HCM in the north of Spain. Being a carrier of both these two novel variants appears to cause an extremely aggressive childhood-onset phenotype.