A wave of deep intronic mutations in X-linked Alport syndrome.

X-linked Alport syndrome (XLAS) is an inherited kidney disease caused exclusively by pathogenic variants in the COL4A5 gene. In 10-20% of cases, DNA sequencing of COL4A5 exons or flanking regions cannot identify molecular causes. Here, our objective was to use a transcriptomic approach to identify causative events in a group of 19 patients with XLAS without identified mutation by Alport gene panel sequencing. Bulk RNAseq and/or targeted RNAseq using a capture panel of kidney genes was performed. Alternative splicing events were compared to those of 15 controls by a developed bioinformatic score. When using targeted RNAseq, COL4A5 coverage was found to be 23-fold higher than with bulk RNASeq and revealed 30 significant alternative splicing events in 17 of the 19 patients. After computational scoring, a pathogenic transcript was found in all patients. A causative variant affecting COL4A5 splicing and absent in the general population was identified in all cases. Altogether, we developed a simple and robust method for identification of aberrant transcripts due to pathogenic deep-intronic COL4A5 variants. Thus, these variants, potentially targetable by specific antisense oligonucleotide therapies, were found in a high percentage of patients with XLAS in whom pathogenic variants were missed by conventional DNA sequencing.

Rapid Methodologies for Assessing Pseudomonas syringae pv. actinidiae Colonization and Effector-Mediated Hypersensitive Response in Kiwifruit.

The infection of Pseudomonas syringae pv. actinidiae in kiwifruit is currently assessed by numerous methodologies, each with their own limitations. Most studies are based on either a laborious method of growth quantification of the pathogen or qualitative assessments by visual scoring following stem or cutting inoculation. Additionally, when assessing for resistance against specific pathogen effectors, confounding interactions between multiple genes in the pathogen can make mapping resistance phenotypes nearly impossible. Here, we present robust alternative methods to quantify pathogen load based on rapid bacterial DNA quantification by PCR, the use of Pseudomonas fluorescens, and a transient reporter eclipse assay for assessing resistance conferred by isolated bacterial avirulence genes. These assays compare well with bacterial plate counts to assess bacterial colonization as a result of plant resistance activation. The DNA-based quantification, when coupled with the P. fluorescens and reporter eclipse assays to independently identify bacterial avirulence genes, is rapid, highly reproducible, and scalable for high-throughput screens of multiple cultivars or genotypes. Application of these methodologies will allow rapid and high-throughput identification of resistant cultivars and the bacterial avirulence genes they recognize, facilitating resistance gene discovery for plant breeding programs.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Maple syrup urine disease mutation spectrum in a cohort of 40 consanguineous patients and insilico analysis of novel mutations.

Maple syrup urine disease is the primary aminoacidopathy affecting branched-chain amino acid (BCAA) metabolism. The disease is mainly caused by the deficiency of an enzyme named branched-chained α-keto acid dehydrogenase (BCKD), which consist of four subunits (E1α, E1ß, E2, and E3), and encoded by BCKDHA, BCKDHB, DBT, and DLD gene respectively. BCKD is the main enzyme in the catabolism pathway of BCAAs. Hight rate of autosomal recessive disorders is expected from consanguineous populations like Iran. In this study, we selected two sets of STR markers linked to the four genes, that mutation in which can result in MSUD disease. The patients who had a homozygous haplotype for selected markers of the genes were sequenced. In current survey, we summarized our recent molecular genetic findings to illustrate the mutation spectrum of MSUD in our country. Ten novel mutations including c.484 A > G, c.834_836dup CAC, c.357del T, and c. (343 + 1_344-1) _ (742 + 1_743-1)del in BCKDHB, c.355-356 ins 7 nt ACAAGGA, and c.703del T in BCKDHA, and c.363delCT/c.1238 T > C, c. (433 + 1_434-1) _ (939 + 1_940-1)del, c.1174 A > C, and c.85_86ins AACG have been found in DBT gene. Additionally, structural models of MSUD mutations have been performed to predict the pathogenicity of the newly identified variants.

Identification of a novel mutation in the HACD1 gene in an Iranian family with autosomal recessive congenital myopathy, with fibre-type disproportion.

Congenital fibre-type disproportion (CFTD) with myopathy, is a genetically heterogeneous disease in which there is relative hypotrophy of type-1-muscle-fibres compared to type-2-fibres on skeletal muscle biopsy. The classical characteristics of CFTD are infantile hypotonia and nonprogressive muscle weakness with a broad range of clinical manifestations. Pathogenic mutations in the HACD1 gene encoding 3-hydroxyacyl-CoA-dehydratase-1 have recently been reported to be associated with this disease. Whole-exome sequencing (WES) was conducted in a 12-year-old girl born to consanguineous parents from the Iranian-Azeri-Turkish population. She was diagnosed with congenital myopathy at the age of 4-month-old due to hypotonia and abnormal electromyography. DNAs were extracted from the blood samples of the proband and her parents, and subjected to PCR-Sanger-sequencing to confirm the WES result. WES data analysis identified a homozygous single nucleotide change (A>T) at position c.785-2 located in intron 6 of the HACD1 gene (NC_000010.11(NM_014241.4):c.785-2A>T). This novel mutation located at the splice-acceptor site is disturbing the splicing procedure. The absence of this mutation among our control group (100 normal healthy adults from the same ethnic group) and not being reported in any other population database confirms the pathogenicity of this mutation. Bioinformatics analysis also classified this variant as a pathogenic mutation. PCR-Sanger-sequencing data analysis confirmed the WES result in the proband and showed that the parents were carriers for the mutation. A substitution (NC_000010.11(NM_014241.4):c.785-2A>T) mutation resulted in the removal of the splicing acceptor site at the HACD1 gene. This pathogenic mutation is associated with CFTD disease.

VNtyper enables accurate alignment-free genotyping of MUC1 coding VNTR using short-read sequencing data in autosomal dominant tubulointerstitial kidney disease.

The human genome comprises approximately 3% of tandem repeats with variable length (VNTR), a few of which have been linked to human rare diseases. Autosomal dominant tubulointerstitial kidney disease-MUC1 (ADTKD-MUC1) is caused by specific frameshift variants in the coding VNTR of the MUC1 gene. Calling variants from VNTR using short-read sequencing (SRS) is challenging due to poor read mappability. We developed a computational pipeline, VNtyper, for reliable detection of MUC1 VNTR pathogenic variants and demonstrated its clinical utility in two distinct cohorts: (1) a historical cohort including 108 families with ADTKD and (2) a replication naive cohort comprising 2,910 patients previously tested on a panel of genes involved in monogenic renal diseases. In the historical cohort all cases known to carry pathogenic MUC1 variants were re-identified, and a new 25bp-frameshift insertion in an additional mislaid family was detected. In the replication cohort, we discovered and validated 30 new patients.

Identification of novel cis-mutations in the GJA8 gene in a 3-generation Iranian family with autosomal dominant congenital nuclear cataract.

BACKGROUND: Cataract is mainly due to the presence of high molecular weight protein, which disrupts the normal function of the lens. Pathogenic variants in Gap Junction protein alpha-8 (GJA8) have been associated with autosomal dominant congenital nuclear cataract. In general, mutations in those genes that have important functions in lens development lead to congenital cataract. METHODS: We conducted whole-exome sequencing (WES) in a four-year-old male patient referred to the genetic center for genetic analysis. He had developed cataract at an early age. DNAs were extracted from the blood samples of all family members and subjected to PCR-Sanger sequencing to confirm the WES results. RESULTS: WES analysis on the proband revealed two mutations in the GJA8 gene (c.G12C, c.G58A). His mother, alongside several other members of the third-generation family, had developed cataract. Sanger sequencing of the interested regions showed that these two mutations were co-segregated in all affected members. However, none of the healthy individuals carried these mutations confirming that these two mutations are located in the same allele (complex allele). Bioinformatics analysis of the mutated GJA8 RNA and protein structure confirmed the pathogenicity of the cis-mutations. CONCLUSIONS: Genetic segregation analysis in a three-generation family and also bioinformatics analysis showed that the complex-allele containing c.G12C+c.G58A mutations in the GJA8 gene is a pathogenic variant that causes autosomal-dominant congenital nuclear cataract.

Comprehensive transcriptome mining identified the gene expression signature and differentially regulated pathways of the late-onset preeclampsia.

Preeclampsia (PE) is categorized as a pregnancy-related hypertensive disorder and is a serious concern in pregnancies. Several factors, including genetic factors (placenta gene expression, and imprinting), oxidative stress, the inaccurate immune response of the mother, and the environmental factors are responsible for PE development, but still, the exact mechanism of the pathogenesis has remained unknown. The main aim of the present study is to identify the gene expression signature in placenta tissue, to unveil disease etiology mechanisms. The GEO, PubMed, and ArrayExpress databases have selected to identify gene expression datasets on placenta samples of both preeclampsia and the normotensive controls. A comprehensive gene expression meta-analysis of fourteen publicly available microarray data of preeclampsia disease has performed to identify gene expression signature and responsible biological pathways and processes. Using two different meta-analysis pipeline (in-house and INMEX) we have identified a total of 1234 differentially expressed genes (DEGs) with in-house method, including 713 overexpressed and 356 under-expressed genes whereas 272 DEGs (131 over and 141 under-expressed) have identified with INMEX, across PEs and healthy controls. Comprehensive functional enrichment and pathway analysis was performed by EnrichR library, whic revealed "Asparagine N-linked glycosylation Homo sapiens", "Nef and signal transduction", "Hemostasis", and "immune system" among the most enriched terms. The present study sets out to explain a novel database of candidate genetic markers and biological pathways that play a critical role in PE development, which might aid in the identification of diagnostic, prognostic, and therapeutic informative molecules.

Whole-Exome Sequencing Uncovers Novel Causative Variants and Additional Findings in Three Patients Affected by Glycogen Storage Disease Type VI and Fanconi-Bickel Syndrome.

Glycogen storage diseases (GSDs) are the heterogeneous group of disorders caused by mutations in at least 30 different genes. Different types of GSDs, especially liver GSDs, take overlapping symptoms and can be clinically indistinguishable. This survey evaluated the use of whole-exome sequencing (WES) for the genetic analysis of the liver GSD-suspected patients in three unrelated families. An in-house filtering pipeline was used to assess rare pathogenic variants in GSD-associated genes, autosomal recessive/mendelian disorder genes (carrier status for genetic counseling subjects), and the ACMG's list of 59 actionable genes. For the interpretation of the causative variants and the incidental/secondary findings, ACMG guidelines were applied. Additionally, we have explored PharmGKB class IA/IB pharmacogenetic variants. The segregation analysis was performed using Sanger sequencing for the novel causative variants. Bioinformatics analysis of the exome data in three individuals revealed three novel homozygous causative variants in the GSD-associated genes. The first variant, c.298_307delATGATCAACC in PYGL gene has related to HERS disease (GSD VI). Both variants of c.1043dupT and c.613-1G > C in SLC2A2 gene have been associated with Fanconi-Bickel syndrome (GSDXI). Eight pathogenic/likely pathogenic medical actionable findings in Mendelian disease genes and 10 pharmacogenetic variants with underlying drug response phenotypes have been identified. No known/expected pathogenic variants were detected in the ACMG's list of 59 actionable genes. The logical filtering steps can help in finding other medical actionable secondary/incidental findings as well as effectively identifying the causative variants in heterogeneous conditions such as GSDs. Three novel variants related to GSD genes recognized in liver GSD-suspected patients with early infantile and childhood-age onset.