Exonic Variants Research Articles

Bidirectional Risk Modulator and Modifier Variant of Dilated and Hypertrophic Cardiomyopathy in BAG3.

The genetic factors that modulate the reduced penetrance and variable expressivity of heritable dilated cardiomyopathy (DCM) are largely unknown. BAG3 genetic variants have been implicated in both DCM and hypertrophic cardiomyopathy (HCM), nominating BAG3 as a gene that harbors potential modifier variants in DCM. To interrogate the clinical traits and diseases associated with BAG3 coding variation. This was a cross-sectional study in the Penn Medicine BioBank (PMBB) enrolling patients of the University of Pennsylvania Health System's clinical practice sites from 2014 to 2023. Whole-exome sequencing (WES) was linked to electronic health record (EHR) data to associate BAG3 coding variants with EHR phenotypes. This was a health care population-based study including individuals of European and African genetic ancestry in the PMBB with WES linked to EHR phenotypes, with replication studies in BioVU, UK Biobank, MyCode, and DCM Precision Medicine Study. Carrier status for BAG3 coding variants. Association of BAG3 coding variation with clinical diagnoses, echocardiographic traits, and longitudinal outcomes. In PMBB (n = 43 731; median [IQR] age, 65 [50-76] years; 21 907 female [50.1%]), among 30 324 European and 11 198 African individuals, the common C151R variant was associated with decreased risk for DCM (odds ratio [OR], 0.85; 95% CI, 0.78-0.92) and simultaneous increased risk for HCM (OR, 1.59; 95% CI, 1.25-2.02), which was confirmed in the replication cohorts. C151R carriers exhibited improved longitudinal outcomes compared with noncarriers as assessed by age at death (hazard ratio [HR], 0.85; 95% CI, 0.74-0.96; median [IQR] age, 71.8 [63.1-80.7] in carriers and 70.3 [61.6-79.2] in noncarriers) and heart transplant (HR, 0.81; 95% CI, 0.66-0.99; median [IQR] age, 56.7 [46.1-63.1] in carriers and 55.6 [45.2-62.9] in noncarriers). C151R was associated with reduced risk of DCM (OR, 0.42; 95% CI, 0.24-0.74) and heart failure (OR, 0.27; 95% CI, 0.14-0.50) among individuals harboring truncating TTN variants in exons with high cardiac expression (n = 358). BAG3 C151R was identified as a bidirectional modulator of risk along the DCM-HCM spectrum, as well as an important genetic modifier variant in TTN-mediated DCM. This work expands on the understanding of the etiology and penetrance of DCM, suggesting that BAG3 C151R is an important genetic modifier variant contributing to the variable expressivity of DCM, warranting further exploration of its mechanisms and of genetic modifiers in DCM more broadly.

Genetic and functional analyses of CTBP2 in anorexia nervosa and body weight regulation.

The C-terminal binding protein 2 (CTBP2) gene (translational isoforms: CTBP2-L/S, RIBEYE) had been identified by a cross-trait analysis of genome-wide association studies for anorexia nervosa (AN) and body mass index (BMI). Here, we did a mutation analysis in CTBP2 by performing polymerase chain reactions with subsequent Sanger-sequencing to identify variants relevant for AN and body weight regulation and ensued functional studies. Analysis of the coding regions of CTBP2 in 462 female patients with AN(acute or recovered), 490 children and adolescents with severe obesity, 445 healthy-lean adult individuals and 168 healthy adult individuals with normal body weight detected 24 variants located in the specific exon of RIBEYE.In the initial analysis, three of these were rare non-synonymous variants (NSVs) detected heterozygously in patients with AN (p.Arg72Trp - rs146900874; p.Val289Met -rs375685611 and p.Gly362Arg - rs202010294). Four NSVs and one heterozygousframeshift variant were exclusively detected in children and adolescents with severe obesity (p.Pro53Ser - rs150867595; p.Gln175ArgfsTer45 - rs141864737; p.Leu310Val - rs769811964; p.Pro397Ala - rs76134089 and p.Pro402Ser - rs113477585). Ribeye mRNA was detected in mouse hypothalamus. No effect of fasting or overfeeding on murine hypothalamic Ribeye expression was determined. Yet, increased Ribeye expression was detected in hypothalami of leptin-treated Lepob/ob mice. This increase was not related to reduced food intake and leptin-induced weight loss. We detected rare and frequent variants in the RIBEYE specific exon in both patients with AN and in children and adolescents with severe obesity. Our data suggest RIBEYE as a relevant gene for weight regulation.

The mechanism of Ca2+ independent activation of BKCa channels in mouse inner hair cells and the crucial role of the BK channels in auditory perception

BK channels are expressed in mouse cochlear inner hair cells (IHCs) and exhibit Ca2+-independent activation at negative potentials. However, the mechanism underlying Ca2+-independent activation of the BK channels in mouse IHCs remains unknown. In this study, we found the BK channel expressed in IHCs contains both the STREX-2 (stress axis regulated exon) variant and an alternative splice of exon9 (alt9), which significantly shift the voltage dependence of the BK channels when co-expressed with LRRC52 in 0 [Ca2+]i. Furthermore, we discovered that mechanical force also induces negative shifts in the voltage dependence of IHC-expressed BK channels. Thus, we propose that the additive effects of mechanical force, special isoforms, and LRRC52 co-expression on voltage dependence shifts may account for the Ca2+-independent activation of the BK channel in IHC. Additionally, we found that the IHCs-specific deletion of the BK channels causes hearing damage in mice. Our study suggests a mechanism for Ca2+-independent activation in IHCs and highlights the crucial role of the BK channel in auditory perception.

Updating probability of pathogenicity for RYR1 and CACNA1S exon variants in individuals without malignant hyperthermia after exposure to triggering anesthetics

Objectives We aimed to classify genetic variants in RYR1 and CACNA1S associated with malignant hyperthermia using biobank genotyping data in patients exposed to triggering anesthetics without malignant hyperthermia phenotype. Methods We identified individuals who underwent surgery and were exposed to triggering anesthetics without malignant hyperthermia phenotype and who had RYR1 or CACNA1S genotyping data available in our biobank. We classified all variants in the cohort using a Bayesian framework of the American College of Medical Genetics and Genomics and the Association of Molecular Pathologists guidelines for variant classification and updated the posterior probabilities from this model with the new information from our biobank cohort. Results We identified 253 patients with 95 RYR1 variants and 12 CACNA1S variants. After applying a Bayesian framework, we classified 17 variants as benign (B), 31 as likely benign (LB), 57 as uncertain (VUS), and 2 as likely pathogenic (LP). When we incorporated evidence about unique exposures to malignant hyperthermia triggering anesthetic agents, 48 of 107 (45%) variants were downgraded (9 to B, 37 to LB, and 2 to VUS). Notably, 41 (72%) of 57 VUSs were downgraded to B or LB. When repeat anesthetics in the same individual were counted as one exposure, 42 of 107 (39%) of variants were downgraded (5 to B, 35 to LB, and 2 to VUS). Specifically, 37 (65%) of 57 VUSs were downgraded to LB. Conclusion Deidentified biorepositories linked with anesthetic data offer a new method of integrating clinical evidence into the assessment of variant probability of pathogenicity.

Exon Nomenclature and Classification of Transcripts database (ENACTdb): A resource for analyzing alternative splicing mediated proteome diversity

Abstract Motivation Gene transcripts are distinguished by the composition of their exons and this different exon composition may contribute to advancing proteome complexity. Despite the availability of alternative splicing information documented in various databases, a ready association of exonic variations to the protein sequence remains a mammoth task. Results To associate exonic variation(s) with the protein systematically, we designed the ENACT framework for uniquely annotating exons that tracks their loci in gene architecture context with encapsulating variations in splice site(s) and amino acid coding status. As part of the ENACT annotation, predicted protein features (secondary structure/disorder/Pfam domains) are mapped to exon attributes. Thus, ENACT provides trackable exonic variation(s) association to isoform(s) and protein features enabling the assessment of functional variation due to changes in exon composition. Such analyses can be readily performed through multiple views supported by the server. The exon-centric visualizations of ENACT annotated isoforms could provide insights on functional repertoire of genes due to alternative splicing and its related processes and can serve as important resource for the research community. Availability and implementation The database is publicly available at https://www.iscbglab.in/enactdb/. It contains protein coding genes and isoforms for Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Mus musculus, and Homo sapiens.

Canine Multiple System Degeneration Associated with Sequence Variants in SERAC1

Canine multiple system degeneration (CMSD) is an early onset, progressive movement disorder affecting Kerry Blue Terriers and Chinese Crested dogs. The associated pathologic lesions include degeneration of the cerebellum, caudate nucleus, and substantia nigra. CMSD is inherited as an autosomal recessive trait in both dog breeds. Previous linkage mapping localized the CMSD locus to a 15 MB region on canine chromosome 1 (CFA1). Next-generation sequencing was used to generate whole-genome sequences from the DNA of an affected dog from each breed. The resulting sequence reads were aligned to the NCBI canine reference genome (build 3.1). Among the homozygous sequence variants within the CFA1 target region, a nonsense variant in exon 15 of SERAC1 was identified in the affected Kerry Blue Terrier, while in the Chinese Crested dog, a 4 bp deletion in the SERAC1 exon 4 acceptor splice site was found. RT-PCR showed that this deletion resulted in exon 4 skipping. Genotyping of large cohorts of Kerry Blue Terriers and Chinese Crested dogs for the respective breed-specific SERAC1 variants showed complete concordance between genotype and disease phenotype. Genotype–phenotype concordance was also observed in offspring generated by cross breeding between SERAC1-heterozygous Kerry Blue Terrier and Chinese Crested dogs, with only the compound heterozygotes exhibiting the disease phenotype, further confirming the recessive inheritance of CMSD. Variants in human SERAC1 are associated with disorders with a range of ages of disease onset and patterns of clinical signs, but that are all characterized by movement abnormalities similar to those of the dogs with CMSD. Canine CMSD could serve as a valuable model to elucidate the mechanisms underlying SERAC1-deficiency disorders and to evaluate potential therapeutic interventions.

Genetic Variability in the CPA1 Gene and Its Impact on Acute Pancreatitis Risk: New Insights from a Large-Scale Study.

Acute pancreatitis (AP) is a common and potentially lethal disease. Over the last 10 years, AP has become one of the most important healthcare problems. On a global scale, the incidence has increased by 63% over the last 20 years. AP is usually caused by gallstones and excessive alcohol consumption and genetic factors play an important role in the development of inflammation. Recent studies involving the CPA1 mutations are ambiguous and dependent on the population studied. In this study, the variability of the CPA1 gene in patients with AP was analyzed. Genetic material was isolated from the blood of 301 patients with AP and 184 healthy individuals. Identification of the variants in exons 5, 6, 8, and 9 with introns was performed using molecular biology methods. Mutations were identified by comparison to the reference sequence (NM_001868.4). Statistical analysis included the identification of mutations correlating with the risk of AP, the etiology of inflammation, and family history. Several novel mutations in the CPA1 gene have been identified, along with a high degree of variability within the coding region of the carboxypeptidase gene. A correlation between mutations CPA1:c.1072 + 84del; c.987 + 57G>A and increased risk of developing AP was found. Two protective mutations, CPA1:c.625A>T, c.1072 + 94del, were identified. The CPA1 gene is characterized by high sequence variability and regions in which mutations lead to an increased risk of developing AP. Single or co-occurring mutations of the CPA1 gene can significantly affect the risk of developing AP.

Slowly progressive autosomal dominant Alport Syndrome due to COL4A3 splicing variant.

Alport syndrome is a rare genetic kidney disease caused by variants in the COL4A3/A4/A5 genes. It's characterised by progressive kidney failure, though therapies targeting Renin-Angiotensin System can delay its progression. Additionally, extrarenal manifestations may sometimes coexist. Recent advances in genetic analysis and the necessity to better clarify genotype-phenotype correlations in affected patients raises the importance of detecting even cryptic splicing variants, lying in both canonical and non-canonical splice sites variants such as last exonic nucleotide variants. These variants, often, do not cause an amino acid change but alter the snRNP proteins binding. We studied a big Italian family with Alport syndrome showing a clear dominant pattern of transmission with younger family members having only haematuria and older individuals presenting with End-Stage Kidney Failure (ESKF). Kidney biopsy showed the typical disease hallmarks. We deeply mined the data for SNV and CNV through exome sequencing on DNA from both peripheral blood samples and patients' podocytes-lineage cells. We identified an already reported synonymous variant, c.765G>A (p.(Thr255Thr)), in the last exonic nucleotide of exon 13 of the COL4A3 gene. Employing the patient's podocytes we demonstrated that this variant results in exon skipping leading to an in-frame deletion of 28 amino acids without leaky effect. According to the pattern of transmission, to the kidney biopsy and to the exome data analysis we provided further evidence that autosomal dominant Alport syndrome is a well-defined clinical entity. We also confirmed the pathogenicity of the synonymous COL4A3 variant for the first time demonstrating its role in a dominant pattern of transmission.

OpenASO: RNA Rescue - designing splice-modulating antisense oligonucleotides through community science.

Splice-modulating antisense oligonucleotides (ASOs) are precision RNA-based drugs that are becoming an established modality to treat human disease. Previously, we reported the discovery of ASOs that target a novel, putative intronic RNA structure to rescue splicing of multiple pathogenic variants of F8 exon 16 that cause hemophilia A. However, the conventional approach to discovering splice-modulating ASOs is both laborious and expensive. Here, we describe an alternative paradigm that integrates data-driven RNA structure prediction and community science to discover splice-modulating ASOs. Using a splicing-deficient pathogenic variant of F8 exon 16 as a model, we show that 25% of the top-scoring molecules designed in the Eterna OpenASO challenge have a statistically significant impact on enhancing exon 16 splicing. Additionally, we show that a distinct combination of ASOs designed by Eterna players can additively enhance the inclusion of the splicing-deficient exon 16 variant. Together, our data suggests that crowdsourcing designs from a community of citizen scientists may accelerate the discovery of splice-modulating ASOs with potential to treat human disease.

Clinical and genetic analysis of a Chinese pedigree affected with Familial focal epilepsy with variable foci due to variant of NPRL3 gene

To explore the clinical manifestations and genetic etiology of a Chinese pedigree affected with Familial focal epilepsy with variable foci (FFEVF). A FFEVF pedigree presented at the Department of Medical Genetics of Linyi Maternal and Child Health Care Hospital on March 14, 2023 was selected as the study subject. The proband was subjected to whole exome sequencing (WES). Candidate variants were verified by Sanger sequencing of the proband and other affected members and bioinformatic analysis. This study was approved by the Linyi Maternal and Child Health Care Hospital (Ethics No. QTL-YXLL-2023048). WES revealed that the proband has harbored a heterozygous c.1642C>T (p.Arg548Cys) missense variant in exon 15 of the NPRL3 gene. Sanger sequencing confirmed that the variant was inherited from the proband's father, and multiple members of the pedigree had also harbored the same variant. Based on guidelines from the American College of Medical Genetics and Genomics (ACMG), the variant was classified as variant of unknown significance (PM2_supporting + PP3). The clinical phenotype of FFEVF patients caused by variants of NPRL3 gene is extensive, and the patients may present with neurological abnormality of autism spectrum disorder in addition to seizures.

A Novel Loss of Function Variant in HCN1 Gene Underlies Early Infantile Epileptic Encephalopathy 24 [EIEE24]

Background: Early infantile epileptic encephalopathy (EIEE) is a rare neurological condition characterized by frequent seizures in the early stages of life, resulting in severely impaired cognitive and motor development. Although the specific causes of EIEE remain unknown, one of the primary causes is gene pathogenicity (even in the absence of consanguinity). Hyperpolarization-activated cyclic nucleotide-gated channels (HCNs) are essential for proper brain function. They are regulated by multiple genes, and mutations in these genes induce channel malfunction, which can result in various severe conditions, including EIEE. Herein, we have reported a patient presenting hallmarks of EIEE. Methods: The patient underwent clinical, radiographic, and genetic analysis. A thorough clinical examination and molecular study were conducted utilizing whole exome sequencing and Sanger sequencing. Results: Whole exome sequencing of the proband revealed a novel de novo nonsynonymous/nonsense variant (c.1468A>T; (p.Lys490Ter) in exon 6 of the HCN1 gene. This variant may cause channel dysfunction via nonsynonymous/nonsense-mediated decay or aberrant protein, which may be associated with EIEE phenotypes. Conclusions: This evidence backs the idea that HCN1 has a vital role in brain development and lose of function can cause a range of debilitating conditions. Still, the functional characterization study of the HCN1 variants will be the fundamental tool for a better understanding of EIEE in the near future.

Non-Hotspot PIK3CA Variants Have Higher Variant Allele Frequency and are More Common in Syndromic Vascular Malformations.

PIK3CA variants are known to cause vascular malformations. We were interested in studying the phenotypic spectrum, the location within the PIK3CA gene, and the variant allele frequency (VAF) of somatic PI3KCA variants in vascular malformations. Clinical data of consecutive patients with extracranial/extraspinal vascular malformations were collected in the context of the VASCOM cohort (2008-2022, n = 558). Starting October 2020, biopsy samples were tested with the TSO500 gene panel (Illumina). All consenting patients with PIK3CA variants were included in this study. Eighty-nine patients had available genetic results by June 2022. PIK3CA variants (n = 25) were found in 16 simple/combined (nonsyndromic) vascular malformations and in nine vascular malformations associated with other anomalies (syndromic). Four hotspot variants in exons 9 and 20 (c.1624G>A, c.1633G>A, c.3140A>G, c.3140A>T) were identified in 16/25 patients (VAF 0.9%-9.7%). Six non-hotspot variants (c.328_330del, c.323_337del, c.353G>A, c.1258T>C, c.3132T>A, c.3195_3203delinsT) were detected in nine patients (VAF 3.6%-31.7%). Non-hotspot variants were more frequent in syndromic than nonsyndromic vascular malformations (p = 0.0034) and exhibited a higher VAF than hotspot variants (p = 0.0253). Our study contributes to the growing body of knowledge of the genetic background in vascular malformations. Further studies will enrich the ever-growing list of pathogenic PIK3CA variants associated with vascular malformations.

8428 Rare Variants in the MECP2 Gene in Two Boys with Central Precocious Puberty

Abstract Disclosure: A.P. Canton: None. J.B. Mebarak: None. M. Magnuson: None. S. Roberts: None. N. Mauras: None. M. Benson: None. S. Witchel: None. R.S. Carroll: None. A. Latronico: None. U.B. Kaiser: None. A. Abreu: None. Introduction: Central precocious puberty (CPP) occurs more frequently in girls and is usually labelled as idiopathic; however, in boys organic causes are more frequent. Identification of imprinted genes causing CPP have revealed epigenetic mechanisms underlying puberty. MECP2, an X-linked gene, encodes a methylated DNA reader protein with a role in gene transcription. MECP2 loss-of-function mutations usually cause Rett syndrome, a severe neurodevelopmental disorder that may be associated with early pubertal development. Recently, rare variants in MECP2 have been recognized in girls with sporadic CPP with or without mild neurodevelopmental disorders. In our cohort of 78 patients with idiopathic CPP, no MECP2 mutations were identified in 73 girls. In this study, five boys with CPP were evaluated for potential MECP2 sequence variants. Methods and Results: Five boys with CPP were screened for MECP2 sequence variants using Sanger sequencing. At the time of CPP diagnosis, they had median (interquartile range) chronological age 9.2 yr (4.3), bone age advancement 2.1 yr (3.5), height SDS 2.3 (1.5), basal LH levels 1.6 IU/L (0.9), and testosterone levels 370 ng/dL (570). Organic causes of CPP were excluded. No MKRN3 or DLK1 mutations were identified. Familial segregation analysis was performed when appropriate. We identified a hemizygous MECP2 variant in a boy (Patient 1) who presented at age 2.7 yr with sporadic CPP. In addition, he had speech delay and behavioral changes, defined as autism spectrum disorder by neuropsychological assessment. He harbored an extremely rare (gnomAD AF=0.000004956) missense variant (p.Val312Ile) in exon 3 of MECP2, encoding the transcriptional repression domain, critical for protein function. The p.Val312Ile mutation was classified as likely pathogenic (ACMG criteria) with a potential association with the phenotype. Another hemizygous MECP2 variant was identified in a boy (Patient 2) who presented at age 8.0 yr with sporadic CPP; he had no neurodevelopmental conditions. He harbored a rare (gnomAD AF=0.00008) missense variant (p.Arg366Cys) in exon 3, encoding the C-terminal domain, classified as a variant of uncertain significance (ACMG criteria). Familial segregation analysis revealed that both boys inherited the MECP2 variants from their unaffected mothers, consistent with a pattern of clinical variability described in women with defects in X-linked genes.Conclusions: We identified rare MECP2 variants in two boys with sporadic CPP without classic features of Rett syndrome, expanding the phenotype of patients with MECP2 mutations, as described in girls. The MECP2 p.Val312Ile variant was likely pathogenic, whereas the functional significance of the p.Arg366Cys variant in still indeterminate. These findings provide additional evidence for a role of MECP2, an epigenetic factor, in the hypothalamic control of pubertal timing. Presentation: 6/1/2024

Personal Genetic-Hypertension Odyssey From Phenotypes to Genotypes and Targets.

Hypertension requires increased systemic vascular resistance. Thus far, Mendelian hypertension-related genes are related to salt retention, an indirect regulatory effect. With the identification of mutated, overactive, PDE3A (phosphodiesterase 3A), we have uncovered a more direct vasoconstrictive mechanism. The autosomal-dominant syndrome features another specific phenotype, brachydactyly type E. Hypertension and the bony phenotype invariably occur together. We distinguished between these phenotypes by examining individual pedigrees. We implicated the gene encoding the parathyroid hormone-related peptide in the brachydactyly. We identified the hypertensive mechanisms as involving regulatory-region, gain-of-function, exon 4 rare pathogenic variants, in the cAMP-cGMP-catabolizing enzyme, PDE3A. We generated rodent models that recapitulate all human phenotypes. Comparisons not only allowed pathogenic insights into the human condition but also provided intervention models. Moreover, we identified rare pathogenic variants in exon 13 encoding the enzymatic pocket. These patients had identical phenotypes, also corroborated in a rodent model, which produced the same human phenotypes. These data could allow the differentiation between a target organ and blood pressure phenotype. The research allows visualization of enzymatic processes at the intracellular nanodomain level. The scope of this project has elucidated genetic mechanisms important to cartilage development, possibly cancer metastases, and findings relevant to cardiovascular regulation via systemic vascular resistance. For our team, the project was an educational/scientific adventure over a professional lifetime.

The first Japanese case of autosomal dominant cutis laxa with a frameshift mutation in exon 30 of the elastin gene complicated by small airway disease with 8 years of follow-up

BackgroundCutis laxa constitutes a diverse group of connective tissue diseases, both inherited and acquired, characterized by loose skin and varying systemic involvement, including pulmonary lesions. While cutis laxa has been linked to conditions like emphysema, asthma, and bronchiectasis, the specific pathological and radiological characteristics underlying pulmonary complications related to cutis laxa remain unclear.Case presentationA 36-year-old woman, diagnosed with cutis laxa at birth, presented to our outpatient clinic with severe obstructive ventilatory impairment, evident in pulmonary function tests (expiratory volume in one second (FEV1)/forced vital capacity (FVC): 34.85%; %residual volume [RV]: 186.5%; %total lung capacity [TLC]: 129.2%). Pulmonary function tests also indicated small airway disease (%FEF50%, 7.9%; %FEF75%, 5.7%; and %FEF25–75%, 6.8%). Computed tomography (CT) revealed the lack of normal increase in lung attenuation on expiratory CT scan, with no discernible emphysematous changes. Exome sequencing was performed to confirm the association between the pulmonary lesions and cutis laxa, revealing a frameshift variant in exon 30 of the elastin gene (ELN). Further analysis employing a parametric response map revealed a longitudinal increase in the percentage of functional small airway disease (fSAD) from 37.84% to 46.61% over the 8-year follow-up, despite the absence of overt changes in CT findings, specifically the lack of normal increase in lung attenuation on expiratory CT scan. Over the same follow-up interval, there was a modest reduction of 25.6 mL/year in FEV1 coupled with a significant increase in %RV. Pulmonary function test metrics, reflective of small airway disease, exhibited a continual decline; specifically, %FEF50%, %FEF75%, and %FEF25–75% diminished from 7.9% to 7.0%, 5.7% to 4.6%, and 6.8% to 5.4%, respectively.ConclusionsThis case highlighted an instance of autosomal dominant cutis laxa arising from a frameshift variant in exon 30 of ELN, accompanied by small airway disease. Comprehensive investigation, utilizing quantitative CT analysis, revealed a longitudinal increase in fSAD percentage with a mild reduction in FEV1. These findings indicate that elastin deficiency may not only diminish elastic fibers in the skin but also be implicated in small airway disease by impacting components of the extracellular matrix in the lungs.

Novel unreported mutations in two cases of X-linked retinoschisis in Indian eye

We report two cases of X-linked retinoschisis (XLRS) with novel gene mutation. The first case revealed a hemizygous nonsense variant (c.440G>A) in exon 5 of the RS1 gene and the second case revealed a hemizygous missense variant (c.286T>G) in exon 4 of the RS1 gene. These gene mutations in XLRS are yet to be reported in published literature. Genetic mutations can have ethnic variations, and understanding novel mutations can help in genotype–phenotype correlation in a better way.

Identification of the Novel HLA-A*33:03:68 Allele in a Chinese Platelet Donor.

The novel HLA-A*33:03:68 allele differs from HLA-A*33:03:01:01 by 1 variation in exon 3.

Patient-derived induced pluripotent stem cells to study non-canonical splicing variants associated with Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is the most prevalent inherited cardiomyopathy and a leading cause of sudden death. Genetic testing and familial cascade screening play a pivotal role in the clinical management of HCM patients. However, conventional genetic tests primarily focus on the detection of exonic and canonical splice site variation. Oversighting intronic non-canonical splicing variants potentially contributes to a proportion of HCM patients remaining genetically undiagnosed. Here, using a non-integrative reprogramming strategy, we generated induced pluripotent stem cell (iPSC) lines from four individuals carrying one of two variants within intronic regions of MYBPC3: c.1224-52G > A and c.1898-23A > G. Upon differentiation to iPSC-derived cardiomyocytes (iPSC-CMs), mis-spliced mRNAs were identified in cells harbouring these variants. Both abnormal mRNAs contained a premature termination codon (PTC), fitting the criteria for activation of nonsense mediated decay (NMD). However, the c.1898-23A > G transcripts escaped this mRNA quality control mechanism, while the c.1224-52G > A transcripts were degraded. The newly generated iPSC lines represent valuable tools for studying the functional consequences of intronic variation and for translational research aimed at reversing splicing abnormalities to prevent disease progression.

Vitamin B12 responsive developmental and epileptic encephalopathy due to a novel mutation in the FUT2 gene: a case report

BackgroundVitamin B12 deficiency is a recognised cause of neurological manifestations, including peripheral neuropathy, behavioural changes, and seizures. However, developmental and epileptic encephalopathy due to vitamin B12 deficiency is very rare. Here, we report an infant with vitamin B12-responsive developmental and epileptic encephalopathy due to a novel mutation in the fucosyltransferase 2 (FUT2) gene responsible for vitamin B12 absorption.Case presentationAn 11-month-old girl of non-consanguineous parents presented with recurrent episodes of seizures since four months. Her seizures started as flexor epileptic spasms occurring in clusters resembling infantile epileptic spasms syndrome with hypsarrhythmia in the electroencephalogram. She was treated with multiple drugs, including high-dose prednisolone, vigabatrin, sodium valproate, levetiracetam and clobazam, without any response, and she continued to have seizures at 11 months. She had an early developmental delay with maximally achieving partial head control and responsive smile at four months. Her development regressed with the onset of seizure; at 11 months, her developmental age was below six weeks. On examination, she was pale and had generalised hypotonia with normal muscle power and reflexes.Her full blood count and blood picture revealed macrocytic anaemia with oval and round macrocytes. Bone marrow aspiration showed hypercellular marrow erythropoiesis with normoblastic and megaloblastic maturation. Due to the unusual association of refractory epilepsy and megaloblastic anaemia, a rare genetic disease of the vitamin B12 or folate pathways was suspected. The whole exome sequencing revealed a homozygous missense variant in exon 2 of the FUT2 gene associated with reduced vitamin B12 absorption and low plasma vitamin B12 levels, confirming the diagnosis of vitamin B12 deficiency related developmental and epileptic encephalopathy. She was started on intramuscular hydroxocobalamin, for which she showed a marked response with reduced seizure frequency.ConclusionWe report a novel variant in the FUT2 gene associated with vitamin B12-responsive developmental and epileptic encephalopathy and megaloblastic anaemia. This case report highlights the importance of timely genetic testing in children with refractory developmental and epileptic encephalopathy to identify treatable causes.

A pseudo-homozygous missense variant and Alu-mediated exon 5 deletion in FARS2 causing spastic paraplegia 77.

FARS2-associated hereditary spastic paraplegia, later onset spastic paraplegia type 77, is a rarely neurodegenerative disease. Here, we reported two affected siblings in an autosomal recessive spastic paraplegia family with a pseudo-homozygous missense variant and Alu-mediated exon 5 deletion in FARS2. Both patients gradually developed altered gaits and weakness in both lower limbs. In our literature review, spastic paraplegia type 77 shows high heterogeneity in clinical manifestations. Our study broadens the scope of pathogenic mechanisms of SPG77 resulting from compound heterozygous mutations in FARS2 and provides strong evidence that deletion in FARS2 due to recombination event mediated by Alu element.