Twist exome capture allows for lower average sequence coverage in clinical exome sequencing

Background Exome and genome sequencing are the predominant techniques in the diagnosis and research of genetic disorders. Sufficient, uniform and reproducible/consistent sequence coverage is a main determinant for the sensitivity to detect single-nucleotide (SNVs) and copy number variants (CNVs). Here we compared the ability to obtain comprehensive exome coverage for recent exome capture kits and genome sequencing techniques. Results We compared three different widely used enrichment kits (Agilent SureSelect Human All Exon V5, Agilent SureSelect Human All Exon V7 and Twist Bioscience) as well as short-read and long-read WGS. We show that the Twist exome capture significantly improves complete coverage and coverage uniformity across coding regions compared to other exome capture kits. Twist performance is comparable to that of both short- and long-read whole genome sequencing. Additionally, we show that even at a reduced average coverage of 70× there is only minimal loss in sensitivity for SNV and CNV detection. Conclusion We conclude that exome sequencing with Twist represents a significant improvement and could be performed at lower sequence coverage compared to other exome capture techniques

A Solve-RD ClinVar-based reanalysis of 1522 index cases from ERN-ITHACA reveals common pitfalls and misinterpretations in exome sequencing

Purpose Within the Solve-RD project (https://solve-rd.eu/), the European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies aimed to investigate whether a reanalysis of exomes from unsolved cases based on ClinVar annotations could establish additional diagnoses. We present the results of the “ClinVar low-hanging fruit” reanalysis, reasons for the failure of previous analyses, and lessons learned. Methods Data from the first 3576 exomes (1522 probands and 2054 relatives) collected from European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies was reanalyzed by the Solve-RD consortium by evaluating for the presence of single-nucleotide variant, and small insertions and deletions already reported as (likely) pathogenic in ClinVar. Variants were filtered according to frequency, genotype, and mode of inheritance and reinterpreted. Results We identified causal variants in 59 cases (3.9%), 50 of them also raised by other approaches and 9 leading to new diagnoses, highlighting interpretation challenges: variants in genes not known to be involved in human disease at the time of the first analysis, misleading genotypes, or variants undetected by local pipelines (variants in off-target regions, low quality filters, low allelic balance, or high frequency). Conclusion The “ClinVar low-hanging fruit” analysis represents an effective, fast, and easy approach to recover causal variants from exome sequencing data, herewith contributing to the reduction of the diagnostic deadlock

Molecular Biology of Huntington’s Disease

Huntington’s Disease (HD) is the most common among nine known polyglutamine disorders. Its prevalence is estimated to be 3-7/100 000 in populations of Western European descent. HD is an autosomal dominantly inherited neurodegenerative disorder of the central nervous system, characterized by involuntary movements, impaired motor coordination, cognitive loss and various psychiatric abnormalities. The most prominent pathological finding is the selective neuron death in basal ganglia. The disease gene (IT-15), localized to chromosome 4 in 1993 and 180kb long, is composed of 67 exons. The gene product is a 348 kDa protein, called huntingtin, whose function is not known yet. The mutation causing HD is the expansion of the CAG triplet repeat in the first exon of the IT-15 gene. Huntington’s Disease Working Group has identified four repeat intervals: People who carry 26 or less CAG repeats in the IT-15 gene are healthy, alleles with 27-35 repeats may show intergenerational instability, people carrying 36-39 CAG repeats may or may not develop the disease , however 40 or more CAG repeats definitely cause HD, if people live long enough. The molecular diagnosis of HD with direct mutation analysis has been available since 1993. In this method, the CAG repeat region on the IT15 gene is PCR-amplified, and the repeat number is determined using radioactive or non-radioactive methods. Although the genetic mutation leading to HD has been identified ten years ago, the underlying molecular mechanism leading to selective neurodegeneration is not clear yet. Proteolytic cleavage and aggregate formation of the mutant protein, aberrant protein interactions and transcriptional dysregulation are possible pathogenic mechanisms. The understanding of HD pathogenesis will enlighten the way to a cure for several other neurodegenerative disorders, which are thought to share a common mechanism

Molecular Biology of Alzheimer’s Disease

Alzheimer’s disease (AD) is the most common cause of dementia in the elderly. The disease, clinically characterized by a progressive decline in intellectual functions, becomes increasingly distressing for the patient and for his/her caregivers, since it is also associated with psychosis, depresion, agitation, anxiety and loss of personality. The two neuropathological hallmarks of AD are senile plaques and neurofibrillary tangles. Following diagnosis, the course of AD varies considerably from a few years to over 20 years, with a mean survival of 2-8 years according to age of onset. A positive family history, previous head injury depression, atherosclerosis low education level are found to be risk factors for AD besides age, which is the most effective risk factor. AD usually onsets after age of 65 years and its prevalence increases with age. Late-onset AD occurs after the age of 65 and 70% of those cases are sporadic; 60 % of early-onset AD, occuring before 65 years are familial. In a small number of pedigrees, AD segregates with a fully penetrant autosomal dominant trait, which indicates that AD has a genetic ethiology. To date, three genes are identified that when mutated cause early-onset AD: β-amyloid precursor protein gene (APP), Presenilin 1 gene (PS1) and Presenilin 2 gene (PS2). Together these mutations, which are by themselves sufficient to cause early-onset AD, account for 2-10% of the whole AD population. The mutations located in the coding region of these genes, increase the production of Aβ42, thereby leading to an increased Aβ deposition, which is the major component of the senile plaques. Apolipoprotein E (APOE) gene is an other indication for the genetic ethiology of AD. ApoE4 allele has been shown to be a risk factor for sporadic and familial AD in multiple populations. To date more than 100 genes, including the promoter regions of PS1, PS2 and APOE, have been considered to contribute to sporadic AD pathology; however the majority of these studies showed contradictory results. The linkage and association analysis data provide evidence for the high complexity of the disease, rather than shedding light on the disease mechanism. Full genome screens revealed many different loci on different chromosomes, but only some of them yielded positive results in independent studies, which may be promising for the discovery of new AD genes. Although there is enough of evidence that Aβ is central to AD pathogenesis, there are many complex secondary events that all contribute to the final outcome of neurodegeneration. Ultimately, the disease process in man has to be prevented through the appropriate use of therapeutic drugs. In this regard Aβ is leading the way as a target for therapeutic agents in an attempt to reduce its production, inhibit aggregation and neurotoxicity. When in future advanced treatment options become available for the early prevention of AD, individuals at risk can make use of predictive DNA testing; the DNA test has to be performed according to strictly approved international guidelines, which asure the confidentiality of the test results

TDP-43 Proteinopathies: A New Player in Neurodegenerative Diseases with Defective Protein Folding

The proteome is the sum of all proteins inside a cell, and proteostasis (protein homeostasis) is the stable condition of the proteome. Proteostasis is essential for the cellular and organismal health. Stress, aging and the chronic expression of misfolded proteins challenge the proteostasis machinery and the vitality of the cell. There is increasing evidence that the accumulation of damaged proteins not only has direct consequences on the efficiency and fidelity of cellular processes but, when not corrected, that they initiate a cascade of dysfunction, which in humans is associated with a plethora of diseases of protein conformation, referred to as proteinopathies. Alzheimer’s Disease (AD), Parkinson’s Disease (PD), Huntington’s Disease (HD), Amyotrophic Lateral Sclerosis (ALS), cancer and diabetes, whose frequencies have drastically increased in countries with aging populations, are all consequences of misfolded proteins. This paper focuses on TDP-43, which excelled as a key protein in neurodegenerative processes because of its association with different diseases, especially with ALS and Frontotemporal Lobar Dementia (FTLD), the two best studied examples of TDP-43 proteinopathies

Brait-fahn-schwarz disease: Parkinson's disease and amyotrophic lateral sclerosis complex

WOS: 000381980000033PubMed: 26319125Neurodegenerative diseases such as Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS) are sometimes present together, as in the Parkinsonism-dementia complex of Guam. Outside the specific geographical regions, the combination of ALS and PD is rare [1]. This neurodegenerative complex was first described by Brait et al. [2] and clinically presents with levodopa responsive parkinsonism followed by AL

Revisiting the complex architecture of ALS in Turkey: Expanding genotypes, shared phenotypes, molecular networks, and a public variant database

The last decade has proven that amyotrophic lateral sclerosis (ALS) is clinically and genetically heterogeneous, and that the genetic component in sporadic cases might be stronger than expected. This study investigates 1,200 patients to revisit ALS in the ethnically heterogeneous yet inbred Turkish population. Familial ALS (fALS) accounts for 20% of our cases. The rates of consanguinity are 30% in fALS and 23% in sporadic ALS (sALS). Major ALS genes explained the disease cause in only 35% of fALS, as compared with similar to 70% in Europe and North America. Whole exome sequencing resulted in a discovery rate of 42% (53/127). Whole genome analyses in 623 sALS cases and 142 population controls, sequenced within Project MinE, revealed well-established fALS gene variants, solidifying the concept of incomplete penetrance in ALS. Genome-wide association studies (GWAS) with whole genome sequencing data did not indicate a new risk locus. Coupling GWAS with a coexpression network of disease-associated candidates, points to a significant enrichment for cell cycle- and division-related genes. Within this network, literature text-mining highlightsDECR1, ATL1, HDAC2, GEMIN4, andHNRNPA3as important genes. Finally, information on ALS-related gene variants in the Turkish cohort sequenced within Project MinE was compiled in the GeNDAL variant browser (www.gendal.org).TUBITAKTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [109S075]; Bogazici University Research FundsBogazici University [15B01P1]; Suna and Inan Kirac Foundation [2005-2020]TUBITAK, Grant/Award Number: 109S075; Bogazici University Research Funds, Grant/Award Number: 15B01P1; Suna and Inan Kirac Foundation, Grant/Award Number: 2005-202

FBXW8 is shifted into insolubility in <i>Atxn2</i>-CAG42-KIN mice due to interaction with expanded ATXN2.

<p>(A) Pulling either with anti-ATXN2 or anti-FBXW8 antibody, ATNX2 and FBXW8 show an interaction in the cerebellum of 18-month-old <i>Atxn2</i>-CAG42-KIN mice independent of the polyQ length (experiment repeated three times for anti-ATXN2 and once with anti-FBXW8, representative images). (B) In cerebellar tissue of 18-month-old <i>Atxn2</i>-CAG42-KIN mice FBXW8 protein level is downregulated in the RIPA-soluble fraction while it is upregulated in the SDS-soluble fraction (two independent experiments each with 4 <i>Atxn2</i>^CAG1/CAG1 vs. 4 <i>Atxn2</i>^CAG42/CAG42 mice).</p

PARK2 interacts with FBXW8 and is recruited into insolubility in <i>Atxn2</i>-CAG42-KIN mice.

<p>(A) In HeLa cells overexpressing Cherry-GFP-PARK2 and FBXW8-HA, pulling with anti-FBXW8 antibody resulted in the detection of FBXW8 as well as of PARK2 in Co-IP lysates, demonstrating their interaction (experiment repeated twice, representative image). (B) PARK2 interacts with FBXW8 in Co-IP samples of <i>Atxn2</i>-CAG42-KIN mice independent of the polyQ length. Lower bands represent PARK2 protein (experiment repeated once). (C) PARK2 protein level is decreased in the RIPA-soluble fraction while it is increased in the SDS-soluble fraction (8 <i>Atxn2</i>^CAG1/CAG1 mice vs. ≥ 6 <i>Atxn2</i>^CAG42/CAG42 mice).</p

FBXW8 protein levels are dysregulated in SCA2 patient material.

<p>FBXW8 expression is upregulated at the transcript level in SCA2 patient skin fibroblasts (A; 4 CTL individuals vs. 4 SCA2 patients) as well as in SCA2 patient blood samples (B; 5 CTL individuals vs. 3 SCA2 patients). (C) At the protein level FBXW8 is decreased in the RIPA-soluble fraction while it is increased in the SDS-soluble fraction in SCA2 patient fibroblasts (4 CTL individuals vs. 4 SCA2 patients).</p