scholarly journals Functional Analyses of HNF1A-MODY Variants Refine the Interpretation of Identified Sequence Variants

2020 ◽  
Vol 105 (4) ◽  
pp. e1377-e1386
Author(s):  
Jana Malikova ◽  
Alba Kaci ◽  
Petra Dusatkova ◽  
Ingvild Aukrust ◽  
Janniche Torsvik ◽  
...  
Keyword(s):  
Dna Binding ◽  
Rare Variants ◽  
Functional Evaluation ◽  
Functional Protein ◽  
Functional Studies ◽  
Uncertain Significance ◽  
Hnf1a Gene ◽  
Nuclear Factor 1 ◽  
Functional Analyses

Abstract Context While rare variants of the hepatocyte nuclear factor-1 alpha (HNF1A) gene can cause maturity-onset diabetes of the young (HNF1A-MODY), other variants can be risk factors for the development of type 2 diabetes. As has been suggested by the American College of Medical Genetics (ACMG) guidelines for variant interpretation, functional studies provide strong evidence to classify a variant as pathogenic. Objective We hypothesized that a functional evaluation can improve the interpretation of the HNF1A variants in our Czech MODY Registry. Design, Settings, and Participants We studied 17 HNF1A variants that were identified in 48 individuals (33 female/15 male) from 20 Czech families with diabetes, using bioinformatics in silico tools and functional protein analyses (transactivation, protein expression, DNA binding, and nuclear localization). Results Of the 17 variants, 12 variants (p.Lys120Glu, p.Gln130Glu, p.Arg131Pro, p.Leu139Pro, p.Met154Ile, p.Gln170Ter, p.Glu187SerfsTer40, p.Phe215SerfsTer18, p.Gly253Arg, p.Leu383ArgfsTer3, p.Gly437Val, and p.Thr563HisfsTer85) exhibited significantly reduced transcriptional activity or DNA binding (< 40%) and were classified as (likely) pathogenic, 2/17 variants were (likely) benign and 3/17 remained of uncertain significance. Functional analyses allowed for the reclassification of 10/17 variants (59%). Diabetes treatment was improved in 20/29 (69%) carriers of (likely) pathogenic HNF1A variants. Conclusion Functional evaluation of the HNF1A variants is necessary to better predict the pathogenic effects and to improve the diagnostic interpretation and treatment, particularly in cases where the cosegregation or family history data are not available or where the phenotype is more diverse and overlaps with other types of diabetes.

scholarly journals Arrhythmia variant associations and reclassifications in the eMERGE-III sequencing study

2021 ◽  
Author(s):  
Andrew M Glazer ◽  
Giovanni Davogustto ◽  
Christian M Shaffer ◽  
Carlos G Vanoye ◽  
Reshma R Desai ◽  
...  
Keyword(s):  
Rare Variants ◽  
Disease Risk ◽  
Disease Genes ◽  
Functional Evaluation ◽  
Functional Studies ◽  
Large Numbers ◽  
Inherited Arrhythmia ◽  
Genomic Screening ◽  
Uncertain Significance

In 21,846 eMERGE-III participants, sequencing 10 arrhythmia syndrome disease genes identified 123 individuals with pathogenic or likely pathogenic (P/LP) variants. Compared to non-carriers, P/LP carriers had a significantly higher burden of arrhythmia phenotypes in their electronic health records (EHRs). Fifty one participants had variant results returned. Eighteen of these 51 participants had inherited arrhythmia syndrome diagnoses (primarily long QT syndrome), and 11/18 of these diagnoses were made only after variant results were returned. After in vitro functional evaluation of 50 variants of uncertain significance (VUS), we reclassified 11 variants: 3 to likely benign and 8 to P/LP. As large numbers of people are sequenced, the disease risk from rare variants in arrhythmia genes can be assessed by integrating genomic screening, EHR phenotypes, and in vitro functional studies.

scholarly journals Novel insights into genetics and clinics of the HNF1A-MODY

2019 ◽  
Vol 53 (2) ◽  
pp. 110-134 ◽  
Author(s):  
Terezia Valkovicova ◽  
Martina Skopkova ◽  
Juraj Stanik ◽  
Daniela Gasperikova
Keyword(s):  
Dna Analysis ◽  
Gene Mutations ◽  
Hereditary Disease ◽  
Actual Problem ◽  
Functional Studies ◽  
Uncertain Significance ◽  
New Gene ◽  
Phenotype Heterogeneity

AbstractMODY (Maturity Onset Diabetes of the Young) is a type of diabetes resulting from a pathogenic effect of gene mutations. Up to date, 13 MODY genes are known. Gene HNF1A is one of the most common causes of MODY diabetes (HNF1A-MODY; MODY3). This gene is polymorphic and more than 1200 pathogenic and non-pathogenic HNF1A variants were described in its UTRs, exons and introns. For HNF1A-MODY, not just gene but also phenotype heterogeneity is typical. Although there are some clinical instructions, HNF1A-MODY patients often do not meet every diagnostic criteria or they are still misdiagnosed as type 1 and type 2 diabetics. There is a constant effort to find suitable biomarkers to help with in distinguishing of MODY3 from Type 1 Diabetes (T1D) and Type 2 Diabetes (T2D). DNA sequencing is still necessary for unambiguous confirmation of clinical suspicion of MODY. NGS (Next Generation Sequencing) methods brought discoveries of multiple new gene variants and new instructions for their pathogenicity classification were required. The most actual problem is classification of variants with uncertain significance (VUS) which is a stumbling-block for clinical interpretation. Since MODY is a hereditary disease, DNA analysis of family members is helpful or even crucial. This review is updated summary about HNF1A-MODY genetics, pathophysiology, clinics functional studies and variant classification.

scholarly journals Impact of functional studies on exome sequence variant interpretation in early-onset cardiac conduction system diseases

2020 ◽  
Vol 116 (13) ◽  
pp. 2116-2130 ◽  
Author(s):  
Kenshi Hayashi ◽  
Ryota Teramoto ◽  
Akihiro Nomura ◽  
Yoshihiro Asano ◽  
Manu Beerens ◽  
...  
Keyword(s):  
Rare Variants ◽  
Electrophysiological Study ◽  
Conduction System ◽  
Cardiac Conduction ◽  
Cardiac Conduction System ◽  
Functional Studies ◽  
Pathogenic Variants ◽  
Uncertain Significance ◽  
Standards And Guidelines

Abstract Aims The genetic cause of cardiac conduction system disease (CCSD) has not been fully elucidated. Whole-exome sequencing (WES) can detect various genetic variants; however, the identification of pathogenic variants remains a challenge. We aimed to identify pathogenic or likely pathogenic variants in CCSD patients by using WES and 2015 American College of Medical Genetics and Genomics (ACMG) standards and guidelines as well as evaluating the usefulness of functional studies for determining them. Methods and results We performed WES of 23 probands diagnosed with early-onset (<65 years) CCSD and analysed 117 genes linked to arrhythmogenic diseases or cardiomyopathies. We focused on rare variants (minor allele frequency < 0.1%) that were absent from population databases. Five probands had protein truncating variants in EMD and LMNA which were classified as ‘pathogenic’ by 2015 ACMG standards and guidelines. To evaluate the functional changes brought about by these variants, we generated a knock-out zebrafish with CRISPR-mediated insertions or deletions of the EMD or LMNA homologs in zebrafish. The mean heart rate and conduction velocities in the CRISPR/Cas9-injected embryos and F2 generation embryos with homozygous deletions were significantly decreased. Twenty-one variants of uncertain significance were identified in 11 probands. Cellular electrophysiological study and in vivo zebrafish cardiac assay showed that two variants in KCNH2 and SCN5A, four variants in SCN10A, and one variant in MYH6 damaged each gene, which resulted in the change of the clinical significance of them from ‘Uncertain significance’ to ‘Likely pathogenic’ in six probands. Conclusion Of 23 CCSD probands, we successfully identified pathogenic or likely pathogenic variants in 11 probands (48%). Functional analyses of a cellular electrophysiological study and in vivo zebrafish cardiac assay might be useful for determining the pathogenicity of rare variants in patients with CCSD. SCN10A may be one of the major genes responsible for CCSD.

scholarly journals Identification and Computational Analysis of Rare Variants of Known Hearing Loss Genes Present in Five Deaf Members of a Pakistani Kindred

Genes ◽  
2021 ◽  
Vol 12 (12) ◽  
pp. 1940
Author(s):  
Irum Badshah Saleem ◽  
Muhammad Shareef Masoud ◽  
Muhammad Qasim ◽  
Muhammad Ali ◽  
Zubair M. Ahmed
Keyword(s):  
Hearing Loss ◽  
Rare Variants ◽  
Family Members ◽  
Genetic Diagnosis ◽  
Low Frequencies ◽  
Genetic Traits ◽  
3 Dimensional ◽  
Functional Studies ◽  
Whole Exome ◽  
Uncertain Significance

Hearing loss (HL) is the most common neurosensory defect in humans that affects the normal communication. Disease is clinically and genetically heterogeneous, rendering challenges for the molecular diagnosis of affected subjects. This study highlights the phenotypic and genetic complexity of inherited HL in a large consanguineous Pakistan kindred. Audiological evaluation of all affected individuals revealed varying degree of mild to profound sensorineural HL. Whole exome (WES) of four family members followed by Sanger sequencing revealed candidate disease-associated variants in five known deafness genes: GJB2 (c.231G>A; p.(Trp77 *)), SLC26A4 (c.1337A>G; p.(Gln446Arg)), CDH23 (c.2789C>T; p.(Pro930Leu)), KCNQ4 (c.1672G>A; p.(Val558Met)) and MPDZ (c.4124T>C; p.(Val1375Ala)). All identified variants replaced evolutionary conserved residues, were either absent or had low frequencies in the control databases. Our in silico and 3-Dimensional (3D) protein topology analyses support the damaging impact of identified variants on the encoded proteins. However, except for the previously established “pathogenic” and “likely pathogenic” categories for the c.231G>A (p.(Trp77 *)) allele of GJB2 and c.1377A>G (p.(Gln446Arg)) of SLC26A4, respectively, all the remaining identified variants were classified as “uncertain significance” based on the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) variant pathogenicity guidelines. Our study highlights the complexity of genetic traits in consanguineous families, and the need of combining the functional studies even with the comprehensive profiling of multiple family members to improve the genetic diagnosis in complex inbred families.

scholarly journals Comparison of in silico strategies to prioritize rare genomic variants impacting RNA splicing for the diagnosis of genomic disorders

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Charlie Rowlands ◽  
Huw B. Thomas ◽  
Jenny Lord ◽  
Htoo A. Wai ◽  
Gavin Arno ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Messenger Rna ◽  
Rare Variants ◽  
Diagnostic Testing ◽  
Substantial Impact ◽  
Splicing Variants ◽  
Uncertain Significance ◽  
Single Strategy ◽  
Functional Analyses ◽  
Genomic Disorders

AbstractThe development of computational methods to assess pathogenicity of pre-messenger RNA splicing variants is critical for diagnosis of human disease. We assessed the capability of eight algorithms, and a consensus approach, to prioritize 249 variants of uncertain significance (VUSs) that underwent splicing functional analyses. The capability of algorithms to differentiate VUSs away from the immediate splice site as being ‘pathogenic’ or ‘benign’ is likely to have substantial impact on diagnostic testing. We show that SpliceAI is the best single strategy in this regard, but that combined usage of tools using a weighted approach can increase accuracy further. We incorporated prioritization strategies alongside diagnostic testing for rare disorders. We show that 15% of 2783 referred individuals carry rare variants expected to impact splicing that were not initially identified as ‘pathogenic’ or ‘likely pathogenic’; one in five of these cases could lead to new or refined diagnoses.

scholarly journals Comparison of in Silico Strategies to Prioritize Rare Genomic Variants Impacting RNA Splicing for the Diagnosis of Genomic Disorders

2021 ◽  
Author(s):  
Charlie Rowlands ◽  
Huw B Thomas ◽  
Jenny Lord ◽  
Htoo A Wai ◽  
Gavin Arno ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Messenger Rna ◽  
Rare Variants ◽  
Diagnostic Testing ◽  
Substantial Impact ◽  
Splicing Variants ◽  
Uncertain Significance ◽  
Single Strategy ◽  
Functional Analyses ◽  
Genomic Disorders

Abstract The development of computational methods to assess pathogenicity of pre-messenger RNA splicing variants is critical for diagnosis of human disease. We assessed the capability of eight algorithms, and a consensus approach, to prioritize 250 variants of uncertain significance (VUSs) that underwent splicing functional analyses. It is the capability of algorithms to differentiate VUSs away from the immediate splice site as being ‘pathogenic’ or ‘benign’ that is likely to have the most substantial impact on diagnostic testing. We show that SpliceAI is the best single strategy in this regard, but that combined usage of tools using a weighted approach can increase accuracy further. We incorporated prioritization strategies alongside diagnostic testing for rare disorders. We show that 15% of 2783 referred individuals carry rare variants expected to impact splicing that were not initially identified as ‘pathogenic’ or ‘likely pathogenic’; 1 in 5 of these cases could lead to new or refined diagnoses.

scholarly journals Novel MSX1 variants identified in families with nonsyndromic oligodontia

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Jinglei Zheng ◽  
Miao Yu ◽  
Haochen Liu ◽  
Tao Cai ◽  
Hailan Feng ◽  
...  
Keyword(s):  
Current Data ◽  
Phenotypic Characterization ◽  
Functional Evaluation ◽  
Gene Variants ◽  
Chinese Families ◽  
Whole Exome ◽  
Bioinformatics Databases ◽  
Uncertain Significance ◽  
Vitro Analysis

AbstractThe goal of this study was to identify MSX1 gene variants in multiple Chinese families with nonsyndromic oligodontia and analyse the functional influence of these variants. Whole-exome sequencing (WES) and Sanger sequencing were performed to identify the causal gene variants in five families with nonsyndromic oligodontia, and a series of bioinformatics databases were used for variant confirmation and functional prediction. Phenotypic characterization of the members of these families was described, and an in vitro analysis was performed for functional evaluation. Five novel MSX1 heterozygous variants were identified: three missense variants [c.662A>C (p.Q221P), c.670C>T (p.R224C), and c.809C>T (p.S270L)], one nonsense variant [c.364G>T (p.G122*)], and one frameshift variant [c.277delG (p.A93Rfs*67)]. Preliminary in vitro studies demonstrated that the subcellular localization of MSX1 was abnormal with the p.Q221P, p.R224C, p.G122*, and p.A93Rfs*67 variants compared to the wild type. Three variants (p.Q221P, p.G122*, and p.A93Rfs*67) were classified as pathogenic or likely pathogenic, while p.S270L and p.R224C were of uncertain significance in the current data. Moreover, we summarized and analysed the MSX1-related tooth agenesis positions and found that the type and variant locus were not related to the severity of tooth loss. Our results expand the variant spectrum of nonsyndromic oligodontia and provide valuable information for genetic counselling.

scholarly journals Janus-faced EPHB4-associated disorders: novel pathogenic variants and unreported intrafamilial overlapping phenotypes

2021 ◽  
Author(s):  
Silvia Martin-Almedina ◽  
Kazim Ogmen ◽  
Ege Sackey ◽  
Dionysios Grigoriadis ◽  
Christina Karapouliou ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Fetal Hydrops ◽  
Clinical Phenotypes ◽  
Functional Studies ◽  
Pathogenic Variants ◽  
Disease Mechanisms ◽  
Novel Variants ◽  
Uncertain Significance ◽  
Clinical Phenotyping

Abstract Purpose Several clinical phenotypes including fetal hydrops, central conducting lymphatic anomaly or capillary malformations with arteriovenous malformations 2 (CM-AVM2) have been associated with EPHB4 (Ephrin type B receptor 4) variants, demanding new approaches for deciphering pathogenesis of novel variants of uncertain significance (VUS) identified in EPHB4, and for the identification of differentiated disease mechanisms at the molecular level. Methods Ten index cases with various phenotypes, either fetal hydrops, CM-AVM2, or peripheral lower limb lymphedema, whose distinct clinical phenotypes are described in detail in this study, presented with a variant in EPHB4. In vitro functional studies were performed to confirm pathogenicity. Results Pathogenicity was demonstrated for six of the seven novel EPHB4 VUS investigated. A heterogeneity of molecular disease mechanisms was identified, from loss of protein production or aberrant subcellular localization to total reduction of the phosphorylation capability of the receptor. There was some phenotype–genotype correlation; however, previously unreported intrafamilial overlapping phenotypes such as lymphatic-related fetal hydrops (LRFH) and CM-AVM2 in the same family were observed. Conclusion This study highlights the usefulness of protein expression and subcellular localization studies to predict EPHB4 variant pathogenesis. Our accurate clinical phenotyping expands our interpretation of the Janus-faced spectrum of EPHB4-related disorders, introducing the discovery of cases with overlapping phenotypes.

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