Intronic Variant Research Articles

Genetic investigation of the ubiquitin-protein ligase E3A gene as putative target in Angelman syndrome.

Angelman syndrome (AS) is caused by maternal chromosomal deletions, imprinting defects, paternal uniparental disomy involving chromosome 15 and the ubiquitin-protein ligase UBE3A gene mutations. However the genetic basis remains unclear for several patients. To investigate the involvement of UBE3A gene in AS and identifying new potential genes using exome sequencing. We established a cohort study in 50 patients referred to Farhat Hached University Hospital between 2006 and 2021, with a strong suspicion of AS and absence of chromosomal aberrations. The UBE3A gene was screened for mutation detection. Two unrelated patients issued from consanguineous families were subjected to exome analysis. We describe seven UBE3A variants among them 3 none previously described including intronic variants c.2220+14T>C (intron14), c.2507+43T>A (Exon15) and insertion in Exon7: c.30-47_30-46. The exome sequencing revealed 22 potential genes that could be involved in AS-like syndromes that should be investigated further. Screening for UBE3A mutations in AS patients has been proven to be useful to confirm the diagnosis. Our exome findings could rise to new potential alternative target genes for genetic counseling.

Whole genome sequencing enables new genetic diagnosis for inherited retinal diseases by identifying pathogenic variants

Inherited retinal diseases (IRDs) are a group of common primary retinal degenerative disorders. Conventional genetic testing strategies, such as panel-based sequencing and whole exome sequencing (WES), can only elucidate the genetic etiology in approximately 60% of IRD patients. Studies have suggested that unsolved IRD cases could be attributed to previously undetected structural variants (SVs) and intronic variants in IRD-related genes. The aim of our study was to obtain a definitive genetic diagnosis by employing whole genome sequencing (WGS) in IRD cases where the causative genes were inconclusive following an initial screening by panel sequencing. A total of 271 unresolved IRD patients and their available family members (n = 646) were screened using WGS to identify pathogenic SVs and intronic variants in 792 known ocular disease genes. Overall, 13% (34/271) of IRD patients received a confirmed genetic diagnosis, among which 7% were exclusively attributed to SVs, 4% to a combination of single nucleotide variants (SNVs) and SVs while another 2% were linked to intronic variants. 22 SVs, 3 deep-intronic variants, and 2 non-canonical splice-site variants across 14 IRD genes were identified in the entire cohort. Notably, all of these detected SVs and intronic variants were novel pathogenic variants. Among those, 74% (20/27) of variants were found in genes causally linked to Retinitis Pigmentosa (RP), with the gene EYS being the most frequently affected by SVs. The identification of SVs and intronic variants through WGS enhances the genetic diagnostic yield of IRDs and broadens the mutational spectrum of known IRD-associated genes.

Putative SNPs in Ovar-DRB1 and GALNTL6 Genes Conferring Susceptibility to Natural Infection of Haemonchus Contortus in Southern Indian Sheep.

To explore associations between phenotypic traits and polymorphisms in the DRB1 and GALNT6 gene in Nellore, Deccani and Kenguri sheep naturally infected with Haemonchus contortus. Blood and faecal samples were collected to evaluate fecal worm egg counts (FEC), packed cell volume (PCV), hemoglobin (Hb), eosinophilia and for DNA isolation. Animals were grouped into susceptible and resistant groups based on EPG counts. FEC and circulating eosinophilia were higher in a susceptible group. Log FEC was negatively correlated (P < 0.01) with PCV, and Hb estimates. The second exon of DRB1 and intron variant of GALNTL6 genes were amplified from DNA samples of resistant and susceptible sheep. Characterization of Ovar-DRB1 amplicon by RFLP revealed two genotypes ('bb' and 'ab'). The genotype frequencies differed significantly between both groups (P < 0.05). The 'bb' genotypes had higher (P < 0.05) log FEC value than 'ab' genotypes and 'b' allele was linked with susceptibility to haemonchosis in sheep. The mean FEC of Nellore sheep was high indicating susceptibility of the breed and also in which the frequency of 'b' allele was more compared to the other two breeds. OVAR-DRB1 genotypes associated with FEC did not affect PCV and Hb. PCR-RFLP assay developed to determine the genotypes with respect to SNP rs424521894 of GALNTL6 revealed monomorphic nature at the locus in the breeds studied. MHC polymorphism could be used as a genetic marker for the selection of sheep resistant to H. contortus. However, a more intensive study, involving controlled infections and other GALNTL6 SNPs may be enforced to make any decisive assertion.

Identification of a novel intronic mutation of MAGED2 gene in a Chinese family with antenatal Bartter syndrome

BackgroundAntenatal Bartter syndrome is a life-threatening disease caused by a mutation in the MAGED2 gene located on chromosome Xp11. It is characterized by severe polyhydramnios and extreme prematurity. While most reported mutations are located in the exon region, variations in the intron region are rarely reported.MethodsIn our study, we employed whole exome sequencing and Sanger sequencing to genotype members of this family. Additionally, a minigene assay was conducted to evaluate the impact of genetic variants on splicing.ResultsOur findings reveal a novel intronic variant (NM_177433.3:c.1271 + 4_1271 + 7delAGTA) in intron 10 of the MAGED2 gene. Further analysis using the minigene assay demonstrated that this variant activated an intronic cryptic splice site, resulting in a 96 bp insertion in mature mRNA.ConclusionsOur results indicate that the novel intronic variant (c.1271 + 4_1271 + 7delAGTA) in intron 10 of the MAGED2 gene is pathogenic. This expands the mutation spectrum of MAGED2 and highlights the significance of intronic sequence analysis.

Proof-of-concept for multiple AON delivery by a single U7snRNA vector to restore splicing defects in ABCA4

The high allelic heterogeneity in Stargardt disease (STGD1) complicates the design of intervention strategies. A significant proportion of pathogenic intronic ABCA4 variants alters thepre-mRNA splicing process. Antisense oligonucleotides (AONs) are an attractive yet mutation-specific therapeutic strategy to restore these splicing defects. In this study, we experimentally assessed the potential of a splicing modulation therapy to target multiple intronic ABCA4 variants. AONs were inserted into U7snRNA gene cassettes and tested in midigene-based splice assays. Five potent antisense sequences were selected to generate a multiple U7snRNA cassette construct, and this combination vector showed substantial rescue of all of the splicing defects. Therefore, the combination cassette was used for viral synthesis and assessment in patient-derived photoreceptor precursor cells (PPCs). Simultaneous delivery of several modified U7snRNAs through a single AAV, however, did not show substantial splicing correction, probably due to suboptimal transduction efficiency in PPCs and/or a heterogeneous viral population containing incomplete AAV genomes. Overall, these data demonstrate the potential of the U7snRNA system to rescue multiple splicing defects, but also suggest that AAV-associated challenges are still a limiting step, underscoring the need for further optimization before implementing this strategy as a potential treatment for STGD1.

Novel compound heterozygous ABCA2 variants cause IDPOGSA, a variable phenotypic syndrome with intellectual disability.

The gene for ATP binding cassette subfamily A member 2 (ABCA2) is located at chromosome 9q34.3. Biallelic ABCA2 variants lead to intellectual developmental disorder with poor growth and with or without seizures or ataxia (IDPOGSA). In this study, we identified novel compound heterozygous ABCA2 variants (NM_001606.5:c.[5300-17C>A];[6379C>T]) by whole exome sequencing in a 28-year-old Korean female patient with intellectual disability. These variants included intronic and nonsense variants of paternal and maternal origin, respectively, and are absent from gnomAD. SpliceAI predicted that the intron variant creates a cryptic acceptor site. Reverse transcription-PCR using RNA extracted from a lymphoblastoid cell line of the patient confirmed two aberrant transcripts. Her clinical features are compatible with those of IDPOGSA.

Detection of hidden intronic DDC variant in aromatic L-amino acid decarboxylase deficiency by adaptive sampling.

Aromatic l-amino acid decarboxylase (AADC) deficiency is an autosomal recessive neurotransmitter disorder caused by pathogenic DOPA decarboxylase (DDC) variants. We previously reported Japanese siblings with AADC deficiency, which was confirmed by the lack of enzyme activity; however, only a heterozygous missense variant was detected. We therefore performed targeted long-read sequencing by adaptive sampling to identify any missing variants. Haplotype phasing and variant calling identified a novel deep intronic variant (c.714+255 C > A), which was predicted to potentially activate the noncanonical splicing acceptor site. Minigene assay revealed that wild-type and c.714+255 C > A alleles had different impacts on splicing. Three transcripts, including the canonical transcript, were detected from the wild-type allele, but only the noncanonical cryptic exon was produced from the variant allele, indicating that c.714+255 C > A was pathogenic. Target long-read sequencing may be used to detect hidden pathogenic variants in unresolved autosomal recessive cases with only one disclosed hit variant.

The glucocorticoid receptor gene (NR3C1) is linked to and associated with polycystic ovarian syndrome in Italian families

ObjectivesComponents of the hypothalamic-pituitary axis (HPA) pathway are potential mediators of the genetic risk of polycystic ovarian syndrome (PCOS). Impaired glucocorticoid receptor (NR3C1) expression and function may underlie impaired HPA-axis cortisol activity, thereby also contributing to the increased adrenal cortisol and androgen production present in women with PCOS. In this study, we aimed to identify whether NR3C1 is linked or in linkage disequilibrium (LD), that is, linkage joint to association, with PCOS in Italian peninsular families.MethodIn 212 Italian families with type 2 diabetes (T2D) from the Italian peninsula, previously recruited for a T2D study and phenotyped for PCOS, we used microarray to genotype 25 variants in the NR3C1 gene. We analyzed the 25 NR3C1 variants by Pseudomarker parametric linkage and LD analysis.ResultsWe found the novel implication in PCOS risk of two intronic variants located within the NR3C1 gene (rs10482672 and rs11749561), thereby extending the phenotypic implication related to impaired glucocorticoid receptor.ConclusionsTo the best of our knowledge, this is the first study to report NR3C1 as a risk gene in PCOS.

Genetic variants in the mTOR pathway with renal cancer risk and subtypes in East Indian population.

Renal Cell Carcinoma (RCC), which accounts for 2%-3% of all adult malignant neoplasms with a male-to-female predominance of 1.9 to 1 with typical presentation between 55 and 75 years. The phosphoinositide-3-kinase-protein kinase B/Akt (PI3KPKB/Akt) pathway is a main pathway in control of cell growth. mTOR pathway plays a key role in the pathogenesis of RCC. Its a prospective observational study. Tissue samples were collected and processed and DNA isolation and sequencing was done to see for any association and expression. Polymorphism analysis of the sequence of three genes MTOR, AKT1, and PIK3CA done and found an intronic variant of the MTOR gene (rs3737611) and AKT1 gene (rs2498797) to be significantly associated with clear cell Renal Cell Carcinoma tumor samples. This study will help to understand the pathogenesis better and the information can be used to develop new drugs and personalized treatment strategies that are tailored to an individual's genetic makeup. The study identify individuals who are at heightened risk for developing renal cancer and could benefit from targeted screening or preventative measures. Some sample size and definite geographical sample pool remains the main limitation of the study which may not be externally validate the study results.

Genetic analysis of a Chinese pedigree with an allele dropout at the HLA-B locus

To delineate a deletional mutation of the HLA-B gene in a Chinese pedigree. A female patient with acute myeloid leukemia who had visited Liuzhou People's Hospital in April 2022 was selected as the study subject. Routine human leukocyte antigen (HLA) was determined by using PCR-sequence specific oligonucleotide polymorphism (PCR-SSOP) and PCR-sequence-based typing (PCR-SBT) methods. Next generation sequencing (NGS) was used to validate the candidate variant in the HLA-B gene. The PCR-SBT and SSOP results for the HLA-B locus were inconsistent for the patient and her daughter. The SSOP results of the two individuals were HLA-B*35:01, 40:02 and HLA-B*35:01, 40:01, respectively. However, the PCR-SBT results has indicated a mismatch with the nearest HLA-B*35:01 at exon 4. NGS results showed that the HLA-B*35:01 had a 9 bp deletion in the intron 5. The patient's husband was HLA-B*40:01, 58:01, which was normal. The variant in intron 5 of the HLA-B gene in this pedigree has mapped to a primer-binding region for the SBT reagent, which has affected the accuracy of PCR-SBT results.

X-Linked Lymphoproliferative Syndrome: A Spectrum of Clinical and Immunological Profile and Novel Pathogenic Variants from Chandigarh, India

Introduction: X-linked lymphoproliferative syndrome (XLP) is a rare primary immune deficiency. Two types of XLP have been described: XLP-1 and XLP-2. Methods: We found 7 patients with XLP (3 had XLP-1 and 4 had XLP-2) after reviewing the data from Pediatric Immunodeficiency Clinic from 1997 to 2021. Results: Mean age at diagnosis was 3.8 years, and mean delay in diagnosis was 2.6 years. Five patients had recurrent episodes of infections. Four patients developed at least one episode of hemophagocytic lymphohistiocytosis (HLH) (2 with XLP-1 and 2 with XLP-2). Of these, 2 had recurrent HLH (both with XLP-2). Epstein-Barr virus (EBV) infection was detected in 2 (1 with XLP-1 and 1 with XLP-2). Both these patients had HLH. One child with XLP-2 had inflammatory bowel disease. Hypogammaglobulinemia was seen in 3 (2 with XLP-1 and 1 with XLP-2). Genetic analysis showed previously reported variants in 5, while 2 had novel variants (one in exon 7 of XIAP gene [c.1370dup p.Asn457Lysfs Ter16] and other had splice site variant in intron 1 of SH2D1A gene [c.138-2_138-1insG]). Episodes of HLH were managed with intravenous immunoglobulin (IVIg), methylprednisolone, oral prednisolone, cyclosporine, and rituximab. Inflammatory bowel disease was managed using oral prednisolone and azathioprine. One patient underwent haploidentical hematopoietic stem cell transplantation. One child with XLP-2 and WAS died because of fulminant pneumonia. Discussion/Conclusions: XLP should be considered as a strong possibility in any patient with features of HLH, repeated infections with hypogammaglobulinemia, persistent EBV infection, and early-onset IBD.

Integration of multi-omics technologies for molecular diagnosis in ataxia patients.

Background: Episodic ataxias are rare neurological disorders characterized by recurring episodes of imbalance and coordination difficulties. Obtaining definitive molecular diagnoses poses challenges, as clinical presentation is highly heterogeneous, and literature on the underlying genetics is limited. While the advent of high-throughput sequencing technologies has significantly contributed to Mendelian disorders genetics, interpretation of variants of uncertain significance and other limitations inherent to individual methods still leaves many patients undiagnosed. This study aimed to investigate the utility of multi-omics for the identification and validation of molecular candidates in a cohort of complex cases of ataxia with episodic presentation. Methods: Eight patients lacking molecular diagnosis despite extensive clinical examination were recruited following standard genetic testing. Whole genome and RNA sequencing were performed on samples isolated from peripheral blood mononuclear cells. Integration of expression and splicing data facilitated genomic variants prioritization. Subsequently, long-read sequencing played a crucial role in the validation of those candidate variants. Results: Whole genome sequencing uncovered pathogenic variants in four genes (SPG7, ATXN2, ELOVL4, PMPCB). A missense and a nonsense variant, both previously reported as likely pathogenic, configured in trans in individual #1 (SPG7: c.2228T>C/p.I743T, c.1861C>T/p.Q621*). An ATXN2 microsatellite expansion (CAG32) in another late-onset case. In two separate individuals, intronic variants near splice sites (ELOVL4: c.541 + 5G>A; PMPCB: c.1154 + 5G>C) were predicted to induce loss-of-function splicing, but had never been reported as disease-causing. Long-read sequencing confirmed the compound heterozygous variants configuration, repeat expansion length, as well as splicing landscape for those pathogenic variants. A potential genetic modifier of the ATXN2 expansion was discovered in ZFYVE26 (c.3022C>T/p.R1008*). Conclusion: Despite failure to identify pathogenic variants through clinical genetic testing, the multi-omics approach enabled the molecular diagnosis in 50% of patients, also giving valuable insights for variant prioritization in remaining cases. The findings demonstrate the value of long-read sequencing for the validation of candidate variants in various scenarios. Our study demonstrates the effectiveness of leveraging complementary omics technologies to unravel the underlying genetics in patients with unresolved rare diseases such as ataxia. Molecular diagnoses not only hold significant promise in improving patient care management, but also alleviates the burden of diagnostic odysseys, more broadly enhancing quality of life.

Overcoming challenges associated with identifying FBN1 deep intronic variants through whole-genome sequencing.

Marfan syndrome (MFS), caused by pathogenic variants of FBN1 (fibrillin-1), is a systemic connective tissue disorder with variable phenotypes and treatment responsiveness depending on the variant. However, a significant number of individuals with MFS remain genetically unexplained. In this study, we report novel pathogenic intronic variants in FBN1 in two unrelated families with MFS. We evaluated subjects with suspected MFS from two unrelated families using Sanger sequencing or multiplex ligation-dependent probe amplification of FBN1 and/or panel-based next-generation sequencing. As no pathogenic variants were identified, whole-genome sequencing was performed. Identified variants were analyzed by reverse transcription-PCR and targeted sequencing of FBN1 mRNA harvested from peripheral blood or skin fibroblasts obtained from affected probands. We found causative deep intronic variants, c.6163+1484A>T and c.5788+36C>A, in FBN1. The splicing analysis revealed an insertion of in-frame or out-of-frame intronic sequences of the FBN1 transcript predicted to alter function of calcium-binding epidermal growth factor protein domain. Family members carrying c.6163+1484A>T had high systemic scores including prominent skeletal features and aortic dissection with lesser aortic dilatation. Family members carrying c.5788+36C>A had more severe aortic root dilatation without aortic dissection. Both families had ectopia lentis. Variable penetrance of the phenotype and negative genetic testing in MFS families should raise the possibility of deep intronic FBN1 variants and the need for additional molecular studies. This study expands the mutation spectrum of FBN1 and points out the importance of intronic sequence analysis and the need for integrative functional studies in MFS diagnosis.

Combined bioinformatic and splicing analysis of likely benign intronic and synonymous variants reveals evidence for pathogenicity

PurposeClinical variant analysis pipelines likely have poor sensitivity to the effects on splicing from variants beyond 10 to 20 bases of exon-intron boundaries. Here, we demonstrate the value of SpliceAI to inform curation of rare variants previously classified as benign/likely benign (B/LB) under current guidelines. MethodsExome sequencing data from 576 pediatric cancer patients enrolled in the Texas KidsCanSeq study were filtered for intronic or synonymous variants absent from population databases, predicted to alter splicing via SpliceAI (>0.20), and scored >10 by combined annotation-dependent depletion. Rare synonymous or intronic B/LB variants in 61 genes submitted to ClinVar were also evaluated and RNA further assessed in monocyte-derived messenger RNA and/or an in vitro splice reporter assay in HEK-293T cells. ResultsSpliceAI-supplemented analysis of the KidsCanSeq cohort revealed a DICER1 intronic variant that resulted in missplicing in RNA from a proband with a personal and family history of pleuropulmonary blastoma but negative clinical exome and panel reports. Analysis of 34,188 B/LB ClinVar variants yielded 18 variants predicted to cause disrupted reading frames. Assessment of 8 variants (DICER1 n = 4, CDH1 n = 2, PALB2 n = 2) by in vitro splicing assay demonstrated abnormal splice products (mean 66%; range 6% to 100%). When available, phenotypic information from submitting laboratories demonstrated DICER1-associated tumors in 2 families (1 variant) and breast cancer in 3 families (2 PALB2 variants). ConclusionIncorporation of SpliceAI in variant curation pipelines may improve classification of B/LB intronic and synonymous variants and highlight putative pathogenic variants for functional assays and RNA analysis, thereby increasing diagnostic yield for rare diseases.

Marginal Contribution of Pathogenic RAD51D Germline Variants to Pakistani Early-Onset and Familial Breast/Ovarian Cancer Patients.

RAD51D has been reported as a breast cancer (BC) and ovarian cancer (OC) predisposition gene, particularly among Caucasian populations. We studied the prevalence of RAD51D variants in Pakistani BC/OC patients. In total, 371 young or familial BC/OC patients were thoroughly analyzed for RAD51D sequence variants using denaturing high-performance liquid chromatography pursued by DNA sequencing of differentially eluted amplicons. We also assessed the pathogenic effects of novel variants using in-silico algorithms. All detected RAD51D variants were investigated in 400 unaffected controls. No pathogenic RAD51D variant was detected. However, we identified nine unique heterozygous variants. Of these, two missense variants (p.Pro10Leu and p.Ile311Asn) and one intronic variant (c.481-26_23delGTTC) were classified as in silico-predicted variants of uncertain significance, with a frequency of 0.8% (3/371). The p.Pro10Leu variant was detected in a 28-year-old female BC patient of Punjabi ethnic background, whose mother and maternal cousin had BCs at ages 53 and 40, respectively. This variant was also detected in 1/400 (0.25%) healthy controls, where the control subject's daughter had acute lymphoblastic leukemia. The p.Ile311Asn variant was identified in a female BC patient at age 29 of Punjabi ethnicity and in 1/400 (0.25%) healthy controls, where the control subject's daughter had Hodgkin's disease at age 14. A novel intronic variant, c.481-26_-23delGTTC, was found in a 30-year-old Punjabi female BC patient but not in 400 healthy controls. No pathogenic RAD51D variant was identified in the current study. Our study data suggested a negligible association of RAD51D variants with BC/OC risk in Pakistani women.

A Study on the Retrospective Reinterpretation of BRCA1 and BRCA2 Variants.

Hereditary breast/ovarian cancer is associated with BRCA gene mutations. As large volumes of clinical data on BRCA variants are continuously updated, their clinical interpretation may change, leading to their reclassification. This study analyzed the class and proportion of the changed clinical interpretations of BRCA variants to validate the need for periodic reviews of these variants. This retrospective study reinterpreted previously reported BRCA1 and BRCA2 exon variants according to the 2015 American College of Medical Genetics and Genomics guidelines and the clinical significance of the recent public genomic database. Reanalyzed results were obtained for patients tested for BRCA genetic mutation for 10 years and 4 months. We included data from 4,058 patients, with 595 having at least one pathogenic variant (P), likely pathogenic variant (LP), or variant of uncertain significance (VUS) at a detection rate of 14.66%. The numbers of exon and intron variants were 562 (87.81%) and 78 (12.19%), respectively. BRCA1 exhibited a significantly higher P/LP detection rate of 6.96% compared to that of BRCA2 at 6.89% (p < 0.001). Conversely, BRCA2 demonstrated a significantly higher VUS rate of 10.38% compared to that of BRCA1 at 5.08% (p < 0.001). Among BRCA1 mutations, substitutions were the most prevalent in P/LP and VUS. Among BRCA2 mutations, deletions were most prevalent in P/LP, and substitutions were most prevalent in VUS. Among the 131 patients with P/LP in BRCA1 exons, the clinical interpretation was reclassified in two cases (1.53%), one VUS and one benign/likely benign (B/LB), and 48 cases (48.00%) with VUS were reclassified; one to P/LP and 47 to B/LB. Among the 138 patients with P/LP in BRCA2 exons, the clinical interpretation was reclassified in six (4.35%), five to VUS, and one to B/LB, and all 74 with VUS were reclassified to B/LB. We determined the class and proportion of reclassified BRCA variants. In conclusion, reviews are required to provide clinical guidance, such as determining treatment direction and preventive measures in the future.

Genomic analysis of venous thrombosis in children with acute lymphoblastic leukemia from diverse ancestries.

Venous thrombosis is a common adverse effect of modern therapy for acute lymphoblastic leukemia (ALL). Prior studies to identify risks of thrombosis in pediatric ALL have been limited by genetic screens of pre-identified genetic variants or genome- wide association studies (GWAS) in ancestrally uniform populations. To address this, we performed a retrospective cohort evaluation of thrombosis risk in 1,005 children treated for newly diagnosed ALL. Genetic risk factors were comprehensively evaluated from genome-wide single nucleotide polymorphism (SNP) arrays and were evaluated using Cox regression adjusting for identified clinical risk factors and genetic ancestry. The cumulative incidence of thrombosis was 7.8%. In multivariate analysis, older age, T-lineage ALL, and non-O blood group were associated with increased thrombosis while non-low-risk treatment and higher presenting white blood cell count trended toward increased thrombosis. No SNP reached genome-wide significance. The SNP most strongly associated with thrombosis was rs2874964 near RFXAP (G risk allele; P=4x10-7; hazard ratio [HR] =2.8). In patients of non-European ancestry, rs55689276 near the α globin cluster (P=1.28x10-6; HR=27) was most strongly associated with thrombosis. Among GWAS catalogue SNP reported to be associated with thrombosis, rs2519093 (T risk allele, P=4.8x10-4; HR=2.1), an intronic variant in ABO, was most strongly associated with risk in this cohort. Classic thrombophilia risks were not associated with thrombosis. Our study confirms known clinical risk features associated with thrombosis risk in children with ALL. In this ancestrally diverse cohort, genetic risks linked to thrombosis risk aggregated in erythrocyte-related SNP, suggesting the critical role of this tissue in thrombosis risk.

Genome Sequencing Unveils the Role of Copy Number Variants in Hearing Loss and Identifies Novel Deletions With Founder Effect in the DFNB1 Locus

Sensorineural hearing loss is a prevalent disorder with significant genetic involvement, which is often challenging to diagnose due to genetic heterogeneity. Exome sequencing (ES) has been a standard diagnostic tool for sensorineural hearing loss, but its limitations in detecting copy number variants (CNVs) and intronic variants have prompted the exploration of genome sequencing (GS) for improved diagnostic yield. We conducted GS on 46 hearing loss families with previously negative ES results and an additional cohort of 36 patients with a monoallelic pathogenic variant in GJB2 (the most common deafness gene). Additionally, the impact of a previously unrecognized novel 125‐kb deletion in the DFNB1 locus on GJB2 expression was assessed using quantitative polymerase chain reaction (qPCR), and haplotype analysis was performed to characterize the deletion. GS diagnosed eight cases (17%, 8/46) in the ES‐negative cohort, primarily attributed to CNVs (6/8). Notably, a previously unrecognized 125 kb deletion in the DFNB1 region was identified, affecting GJB2 expression and characterizing it as a founder effect in East Asian. In 47 patients with a monoallelic GJB2 variant, 15% (95% CI, 7.4%–28%) were diagnosed with DFNB1 deletions. Analysis of the gnomAD database revealed the prevalence and ethnic diversity of DFNB1 deletions, with the novel 125 kb deletion emerging as a prominent pathogenic variant in East Asian, non‐Finnish European, and admixed American populations. Our study highlights the utility of GS in diagnosing sensorineural hearing loss. The identification of DFNB1 deletions underscores their significant contribution to hearing loss etiology, advocating for their inclusion in routine diagnostic testing. We propose GS as a primary genetic testing approach for patients with hearing loss, offering comprehensive genomic analysis and the potential for improved diagnostic accuracy.

A homozygous structural variant of RPGRIP1 is frequently associated with achromatopsia in Japanese patients with IRD

PurposeAchromatopsia (ACHM) is an early-onset cone dysfunction caused by 5 genes with cone-specific functions (CNGA3, CNGB3, GNAT2, PDE6C, and PDE6H) and by ATF6, a transcription factor with ubiquitous expression. To improve the relatively low variant detection ratio in these genes in a cohort of exome-sequenced Japanese patients with inherited retinal diseases (IRD), we performed genome sequencing to detect structural variants and intronic variants in patients with ACHM. MethodsGenome sequencing of 10 ACHM pedigrees was performed after exome sequencing. Structural, non-coding, and coding variants were filtered based on segregation between the affected and unaffected in each pedigree. Variant frequency and predicted damage scores were considered in identifying pathogenic variants. ResultsA homozygous deletion involving exon 18 of RPGRIP1 was detected in 5 of 10 ACHM probands, and variant inheritance from each parent was confirmed. This deletion was relatively frequent (minor allele frequency = 0.0023) in the Japanese population but was only homozygous in patients with ACHM among the 199 Japanese IRD probands analyzed by the same genome sequencing pipeline. ConclusionThe deletion involving exon 18 of RPGRIP1 is a prevalent cause of ACHM in Japanese patients and contributes to the wide spectrum of RPGRIP1-associated IRD phenotypes, from Leber congenital amaurosis to ACHM.

Investigating differences in the GWAS-based protein-protein interaction network of blood pressure regulation due to ancestry or transcript consequence severity

Genome-wide association studies (GWAS) have been valuable for the identification of genetic factors associated with complex diseases or traits. GWAS findings can be enhanced if analysed in the context of protein-protein interaction (PPI) networks, as the related pathophysiology results from dysfunction of interacting polyprotein pathways. In a recent study, we reconstructed the PPI network of blood pressure regulation (BP) based on a systematically-curated GWAS meta-database and network extension principles. Our meta-database enables the selection of GWAS-data of different ancestries and/or different Ensembl-defined transcript consequence severities. Thus, this study aimed at investigating any differences in the BP PPI network, due to (a) ancestry, by comparing the most-abundant European and Asian ancestry GWAS datasets, and (b) variant consequence severity, by excluding single nucleotide polymorphisms (SNPs) involved only in “modifier” categories, of difficult to predict impact. We identified that 82% of the collected BP-SNPs are from European-specific studies, with only 11% from Asian-specific, validating the need to augment the GWAS-data from other than European ancestries. Thus, only 7% vs 83% of the 1170 BP GWAS-proteins originate from Asian- and European-specific studies, respectively, with 2% (24) identified as Asian-specific vs ∼45% (524) as European-specific GWAS-proteins. In the second part of the study, we found that the vast majority (85%) of the BP-SNPs with protein-coding transcript consequences are only intron variants (‘modifier’ category). Hence, the PPI network based on the SNPs in other than “modifier” categories included 142 proteins, with 12 in the largest connected component. However, while the relevant interactome is much smaller than the full, when extended based on our shortest-path approach, it revealed the same BP-significant pathways. This result supports the need to upgrade the information content of GWAS-data through network analysis.