The Glass syndrome is a rare genetic disorder that is associated with a multisystem disorder due to a SATB2 gene mutation. The intellectual development delay and the delay in speech are the ...predominant characteristics of the disorder. The reports on Glass syndrome are very few and have not been reported in India. In the present study, we report the Glass syndrome disorder in an Indian mother and her child, both affected by the same likely pathogenic mutation c.1741-2A>C intronic variant at intron 11 of the SATB2 gene. The mutation was observed as a heterozygous and autosomal dominant missense mutation through focused exome sequencing analysis. The variant identified has not been observed in the general population, and the clinical features like cleft palate, speech delay, and intellectual delay match the causative mutation. The present study is novel in exhibiting the intronic variant and identifying Glass syndrome in the Indian population.
PurposeTo identify the missing heritability of patients with Wolfram syndrome 1 (WFS1) in a Chinese cohort and to report their clinical and genetic features. MethodsWe recruited 24 unrelated patients ...with suspected WFS1 who carried at least one variant in WFS1. All patients underwent ophthalmic examinations and comprehensive molecular genetic analyses, including Sanger-DNA sequencing of WFS1 and next-generation sequencing of the whole WFS1 sequence. ResultsWe identified 38 distinct pathogenic variants of WFS1 in the 24 probands, comprising 23 patients with biallelic variants and one patient with a monoallelic variant. Sanger-DNA sequencing of WFS1 initially detected 35 variants, and subsequent whole genome sequencing revealed three missing variants: one novel deep intronic variant (DIV), one copy number variant (CNV), and one variant in the promoter region. Minigene assays showed that the DIV activated cryptic splice sites, leading to the insertion of pseudoexons. Optic atrophy was observed in all patients, and diabetes mellitus (DM) was revealed in 21 patients (91.3%), hearing loss in nine patients (39.1%), renal tract abnormalities in nine patients (39.1%), and diabetes insipidus in five patients (21.7%). The mean onset age for DM was significantly younger in the patients with biallelic null variants than in the patients with biallelic missense variants. ConclusionsOur results extend the pathogenic variant spectrum of WFS1. DIVs and CNVs explained rare unresolved Chinese cases with WFS1. The patients showed a wide and variable clinical spectrum, supporting the importance of genetic analysis for patients with atypical WFS1.
PurposeTo describe the genetic landscape of BEST1 for a large Chinese cohort with autosomal recessive bestrophinopathy (ARB), identify the missing heritability, and report a common Chinese founder ...variant. MethodsWe recruited 65 patients from 63 families with a clinical diagnosis of ARB. All patients underwent ophthalmic examinations and comprehensive genetic analyses, including Sanger DNA sequencing of BEST1 and whole genome sequencing (WGS). The effects of deep intronic variants (DIVs) on splicing were assessed using in vitro splicing assays in HEK293T cells and patient-derived peripheral blood mononuclear cells. Haplotype mapping was performed for 17 unrelated patients harboring variant c.867+97G>A. ResultsWe identified 54 distinct disease-causing variants of BEST1 in 63 pedigrees, 62 probands with biallelic variants, and one family with monoallelic variants. Sanger DNA sequencing of BEST1 initially detected 51 variants in 61 pedigrees, including 19 probands with one heterozygous variant. Subsequent WGS, combined with supplementary Sanger sequencing, revealed three missing DIVs (c.1101-491A>G, c.867+97G>A, and c.867+97G>T) in 20 families. The novel DIV c.1101-491A>G caused an abnormal splicing resulting in a 204-nt pseudoexon (PE) insertion, whereas c.867+97G>A/T relatively strengthened an alternative donor site, resulting in a 203-nt intron retention (IR). The PE and IR generated a premature termination codon downstream. Haplotype analysis identified c.867+97G>A as a common founder variant with an allele frequency of 16%. ConclusionsOur results expand the pathogenic variant spectrum of BEST1, and DIVs can explain almost all of the missing heritability. The c.867+97G>A DIV is a common founder variant for Chinese patients with ARB.
To identify the missing heritability of ABCA4-related retinopathy in a Chinese cohort.
We recruited 33 unrelated patients with ABCA4-related retinopathy carrying a monoallelic variant in ABCA4. All ...patients underwent ophthalmic examinations. Next-generation sequencing of the whole ABCA4 sequence, including coding and noncoding regions, was performed to detect deep intronic variants (DIVs) and copy number variations (CNVs).
We identified eight missing pathogenic ABCA4 variants in 60.6% of the patients (20/33), which comprised five DIVs and three CNVs. The five DIVs, including four novel (c.1555-816T>G, c.2919-169T>G, c.2919-884G>T, and c.5461-1321A>G) and one reported (c.4539+1100A>G), accounted for the missing alleles in 51.5% of the patients. Minigene assays showed that four novel DIVs activated cryptic splice sites leading to the insertions of pseudoexons. The three novel CNVs consisted of one gross deletion of 1273 bp (exon 2) and two gross duplications covering 25.2 kb (exons 28-43) and 9.4 kb (exons 38-44). The microhomology domains were identified at the breakpoints and revealed the potential mechanisms of CNV formation.
DIVs and CNVs explained approximately two-thirds of the unresolved Chinese cases with ABCA4-related retinopathy. Combining results from phenotypic-directed screening, targeting the whole ABCA4 sequencing and in silico tools can help to identify the missing heritability.
Using exome sequencing, the underlying variants in many persons with autosomal recessive diseases remain undetected. We explored autosomal recessive Stargardt disease (STGD1) as a model to identify ...the missing heritability.
Sequencing of ABCA4 was performed in 8 STGD1 cases with one variant and p.Asn1868Ile in trans, 25 cases with one variant, and 3 cases with no ABCA4 variant. The effect of intronic variants was analyzed using in vitro splice assays in HEK293T cells and patient-derived fibroblasts. Antisense oligonucleotides were used to correct splice defects.
In 24 of the probands (67%), one known and five novel deep-intronic variants were found. The five novel variants resulted in messenger RNA pseudoexon inclusions, due to strengthening of cryptic splice sites or by disrupting a splicing silencer motif. Variant c.769-784C>T showed partial insertion of a pseudoexon and was found in cis with c.5603A>T (p.Asn1868Ile), so its causal role could not be fully established. Variant c.4253+43G>A resulted in partial skipping of exon 28. Remarkably, antisense oligonucleotides targeting the aberrant splice processes resulted in (partial) correction of all splicing defects.
Our data demonstrate the importance of assessing noncoding variants in genetic diseases, and show the great potential of splice modulation therapy for deep-intronic variants.
Type IV collagen is an integral component of basement membranes. Mutations in COL4A1, one of the key genes encoding Type IV collagen, can result in a variety of diseases. It is clear that a ...significant proportion of mutations that affect splicing can cause disease directly or contribute to the susceptibility or severity of disease. Here, we analyzed exonic mutations and intronic mutations described in the COL4A1 gene using bioinformatics programs and identified candidate mutations that may alter the normal splicing pattern through a minigene system. We identified seven variants that induce splicing alterations by disrupting normal splice sites, creating new ones, or altering splice regulatory elements. These mutations are predicted to impact protein function. Our results help in the correct molecular characterization of variants in COL4A1 and may help develop more personalized treatment options.
Whole genome sequencing (WGS) is a comprehensive genetic testing approach that reports most types of nucleotide variants.
This study sought to assess WGS for hypertrophic cardiomyopathy (HCM) in ...which prior genetic testing did not establish a molecular diagnosis, and as a first-line genetic test.
WGS was performed on 58 unrelated patients with HCM, 14 affected family members, and 2 unaffected parents of a severely affected proband. The authors searched for nucleotide variants in coding regions of 184 candidate cardiac hypertrophy genes. They also searched for nucleotide variants in deep intronic regions that alter RNA splicing, large genomic rearrangements, and mitochondrial genome variants. RNA analysis was performed to validate splice-altering variants.
The authors found a pathogenic or likely pathogenic variant in 9 of 46 families (20%) for which prior genetic testing was inconclusive. Three families had variants in genes not included in prior genetic testing. One family had a pathogenic variant that was filtered out with prior exome sequencing. Five families had pathogenic variants in noncoding regions, including 4 with deep intronic variants that activate novel splicing, and 1 mitochondrial genome variant. As a first-line genetic test, WGS identified a pathogenic variant in 5 of 12 families (42%) that had never received prior genetic testing.
WGS identified additional genetic causes of HCM over targeted gene sequencing approaches. Extending genetic screening to deep intronic regions identified pathogenic variants in 9% of gene-elusive HCM. These findings translate to more accurate diagnosis and management in HCM families.
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Background
An accurate genetic diagnosis of Becker muscular dystrophy (BMD) can be sometimes challenging due to deep intronic DMD variants. Here, we report on the genetic diagnosis of a BMD patient ...with a novel deep‐intronic splice‐altering variant in DMD.
Methods
The index case was a 3.8‐year‐old boy who was suspected of having a diagnosis of BMD based on his clinical, muscle imaging, and pathological features. Routine genomic detection approaches did not detect any disease‐causing variants in him. Muscle‐derived DMD mRNA studies, followed by genomic Sanger sequencing and in silico bioinformatic analyses, were performed in the patient.
Results
DMD mRNA studies detected a cryptic exon‐containing transcript and normally spliced DMD transcript in the patient. The cryptic exon‐containing transcript encoded a frameshift and premature termination codon (NP_003997.1:p.=,Asp2740Valfs*52). Further genomic Sanger sequencing and bioinformatic analysis identified a novel deep‐intronic splice‐altering variant in DMD (c.8217 + 23338A > G). The novel variant strengthened a cryptic donor splice site and activated a cryptic acceptor splice site in the deep‐intronic region of DMD intron 55, resulting in the activation of a new dystrophin cryptic exon found in the patient.
Conclusion
Our case report expands the genetic spectrum of BMD and highlights the essential role of deep‐intronic cryptic exon‐activating variants in genetically unsolved BMD patients.
Our study successfully identified a novel deep‐intronic splice‐altering variant in DMD via the combined application of dystrophin mRNA analysis, genomic Sanger sequencing, and in silico bioinformatic analyses. The novel deep‐intronic variant (c.8217 + 23338A > G) resulted in the activation of a new dystrophin cryptic exon, which confirmed the genetic diagnosis of dystrophinopathy in our patient.
Pre-messenger RNA splicing is initiated with the recognition of a single-nucleotide intronic branchpoint (BP) within a BP motif by spliceosome elements. Forty-eight rare variants in 43 human genes ...have been reported to alter splicing and cause disease by disrupting BP. However, until now, no computational approach was available to efficiently detect such variants in massively parallel sequencing data. We established a comprehensive human genome-wide BP database by integrating existing BP data and generating new BP data from RNA sequencing of lariat debranching enzyme DBR1-mutated patients and from machine-learning predictions. We characterized multiple features of BP in major and minor introns and found that BP and BP-2 (two nucleotides upstream of BP) positions exhibit a lower rate of variation in human populations and higher evolutionary conservation than the intronic background, while being comparable to the exonic background. We developed BPHunter as a genome-wide computational approach to systematically and efficiently detect intronic variants that may disrupt BP recognition. BPHunter retrospectively identified 40 of the 48 known pathogenic BP variants, in which we summarized a strategy for prioritizing BP variant candidates. The remaining eight variants all create AG-dinucleotides between the BP and acceptor site, which is the likely reason for missplicing. We demonstrated the practical utility of BPHunter prospectively by using it to identify a novel germline heterozygous BP variant of
in a patient with critical COVID-19 pneumonia and a novel somatic intronic 59-nucleotide deletion of
in a lymphoma patient, both of which were validated experimentally. BPHunter is publicly available from https://hgidsoft.rockefeller.edu/BPHunter and https://github.com/casanova-lab/BPHunter.
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