Dataset Information

High density SNP array and reanalysis of genome sequencing uncovers CNVs associated with neurodevelopmental disorders in KOLF2.1J iPSCs.

ABSTRACT: The KOLF2.1J iPSC line was recently proposed as a reference iPSC to promote the standardization of research studies in the stem cell field. Due to overall good performance differentiating to neural cell lineages, high gene editing efficiency, and absence of genetic variants associated to neurological disorders KOLF2.1J iPSC line was particularly recommended for neurodegenerative disease modeling. However, our work uncovers that KOLF2.1J hPSCs carry heterozygous small copy number variants (CNVs) that cause DTNBP1, JARID2 and ASTN2 haploinsufficiencies, all of which are associated with neurological disorders. We further determine that these CNVs arose in vitro over the course of KOLF2.1J iPSC generation from a healthy donor-derived KOLF2 iPSC line and affect the expression of DNTBP1, JARID2 and ASTN2 proteins in KOLF2.1J iPSCs and neural progenitors. Therefore, our study suggests that KOLF2.1J iPSCs carry genetic variants that may be deleterious for neural cell lineages. This data is essential for a careful interpretation of neural cell studies derived from KOLF2.1J iPSCs and highlights the need for a catalogue of iPSC lines that includes a comprehensive genome characterization analysis.

SUBMITTER: Gracia-Diaz C

PROVIDER: S-EPMC10327134 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

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High density SNP array and reanalysis of genome sequencing uncovers CNVs associated with neurodevelopmental disorders in KOLF2.1J iPSCs.

Gracia-Diaz Carolina C Perdomo Jonathan E JE Khan Munir E ME Disanza Brianna B Cajka Gregory G GG Lei Sunyimeng S Gagne Alyssa A Maguire Jean Ann JA Roule Thomas T Shalem Ophir O Bhoj Elizabeth J EJ Ahrens-Nicklas Rebecca C RC French Deborah D Goldberg Ethan M EM Wang Kai K Glessner Joseph J Akizu Naiara N

bioRxiv : the preprint server for biology 20230626

The KOLF2.1J iPSC line was recently proposed as a reference iPSC to promote the standardization of research studies in the stem cell field. Due to overall good performance differentiating to neural cell lineages, high gene editing efficiency, and absence of genetic variants associated to neurological disorders KOLF2.1J iPSC line was particularly recommended for neurodegenerative disease modeling. However, our work uncovers that KOLF2.1J hPSCs carry heterozygous small copy number variants (CNVs) ...[more]

PMID: 37425875

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Project description:This study aims to inform future genetic reanalysis management by evaluating the yield of whole-exome sequencing (WES) reanalysis in standard patient care in the Netherlands. Single-center data of 159 patients with a neurodevelopmental disorder (NDD), in which WES analysis and reanalysis were performed between January 1, 2014, and December 31, 2021, was retrospectively collected. Patients were included if they were under the age of 18 years at initial analysis and if this initial analysis did not result in a diagnosis. Demographic, phenotypic, and genotypic characteristics of patients were collected and analyzed. The primary outcomes of our study were (i) diagnostic yield at reanalysis, (ii) reasons for detecting a new possibly causal variant at reanalysis, (iii) unsolicited findings, and (iv) factors associated with positive result of reanalysis. In addition, we conducted a questionnaire study amongst the 7 genetic department in the Netherlands creating an overview of used techniques, yield, and organization of WES reanalysis. The single-center data show that in most cases, WES reanalysis was initiated by the clinical geneticist (65%) or treating physician (30%). The mean time between initial WES analysis and reanalysis was 3.7 years. A new (likely) pathogenic variant or VUS with a clear link to the phenotype was found in 20 initially negative cases, resulting in a diagnostic yield of 12.6%. In 75% of these patients, the diagnosis had clinical consequences, as for example, a screening plan for associated signs and symptoms could be devised. Most (32%) of the (likely) causal variants identified at WES reanalysis were discovered due to a newly described gene-disease association. In addition to the 12.6% diagnostic yield based on new diagnoses, reclassification of a variant of uncertain significance found at initial analysis led to a definite diagnosis in three patients. Diagnostic yield was higher in patients with dysmorphic features compared to patients without clear dysmorphic features (yield 27% vs. 6%; p = 0.001).ConclusionsOur results show that WES reanalysis in patients with NDD in standard patient care leads to a substantial increase in genetic diagnoses. In the majority of newly diagnosed patients, the diagnosis had clinical consequences. Knowledge about the clinical impact of WES reanalysis, clinical characteristics associated with higher yield, and the yield per year after a negative WES in larger clinical cohorts is warranted to inform guidelines for genetic reanalysis. These guidelines will be of great value for pediatricians, pediatric rehabilitation specialists, and pediatric neurologists in daily care of patients with NDD.What is known• Whole exome sequencing can cost-effectively identify a genetic cause of intellectual disability in about 30-40% of patients. • WES reanalysis in a research setting can lead to a definitive diagnosis in 10-20% of previously exome negative cases.What is new• WES reanalysis in standard patient care resulted in a diagnostic yield of 13% in previously exome negative children with NDD. • The presence of dysmorphic features is associated with an increased diagnostic yield of WES reanalysis.

Project description:BackgroundThe X chromosome is enriched with genes related to brain development, and the hemizygous state of these genes in men causes some difficulties in the clinical interpretation of copy number variations (CNVs). In this study, we present data on the frequency and spectrum of CNVs on the X chromosome in a cohort of patients with neurodevelopmental disorders (NDDs).MethodsChromosomal microarray analysis was performed for 1175 patients with NDDs. CNVs were confirmed by real-time quantitative PCR. X chromosome inactivation was analysed by methyl-sensitive PCR. To determine the pathogenic significance of the CNVs, several criteria, including the origin (inherited or de novo), variant type (microdeletion or microduplication), and X chromosome inactivation pattern in asymptomatic and symptomatic carriers, were considered. Additionally, the spectrum, size and molecular bases of copy number changes in genes or gene regions involved in the development of the pathological phenotype in each patient were considered.ResultsCNVs on the X chromosome were identified in 33 patients (2.8%). Duplications and triplications (27 cases) were four times more common than deletions (6 cases). In 74% of patients, CNVs were of maternal origin; in 10% they were of paternal origin; and in 16% they arose de novo. The frequency of skewed X inactivation among family members who were healthy carriers of pathogenic and likely pathogenic CNVs and variants of uncertain significance (VUSs) on the X chromosome was 23%. For the first time, we reported several CNVs, including a pathogenic microdeletion at Xq26.1q26.2 involving the ARHGAP36 gene and a microduplication at Xp22.2 involving the OFD1 gene, CONCLUSIONS: This study expands on the frequency and spectrum of CNVs in patients with NDDs. Pathogenic variants on the X chromosome were present in 15% of cases, LP in 12%, VUS in 57%, and LB in 16% of cases. Previously unreported CNVs aid in the identification of new structural variants and genes associated with X-linked intellectual disability. We propose to consider the X-chromosome inactivation status when assessing the pathogenetic significance of CNVs using the ACMG algorithm (American College of Medical Genetics).

Project description:BackgroundHigh density (HD) SNP genotyping arrays are an important tool for genetic analyses of animals and plants. Although the chicken is one of the most important farm animals, no HD array is yet available for high resolution genetic analysis of this species.ResultsWe report here the development of a 600 K Affymetrix® Axiom® HD genotyping array designed using SNPs segregating in a wide variety of chicken populations. In order to generate a large catalogue of segregating SNPs, we re-sequenced 243 chickens from 24 chicken lines derived from diverse sources (experimental, commercial broiler and layer lines) by pooling 10-15 samples within each line. About 139 million (M) putative SNPs were detected by mapping sequence reads to the new reference genome (Gallus_gallus_4.0) of which ~78 M appeared to be segregating in different lines. Using criteria such as high SNP-quality score, acceptable design scores predicting high conversion performance in the final array and uniformity of distribution across the genome, we selected ~1.8 M SNPs for validation through genotyping on an independent set of samples (n = 282). About 64% of the SNPs were polymorphic with high call rates (>98%), good cluster separation and stable Mendelian inheritance. Polymorphic SNPs were further analysed for their population characteristics and genomic effects. SNPs with extreme breach of Hardy-Weinberg equilibrium (P < 0.00001) were excluded from the panel. The final array, designed on the basis of these analyses, consists of 580,954 SNPs and includes 21,534 coding variants. SNPs were selected to achieve an essentially uniform distribution based on genetic map distance for both broiler and layer lines. Due to a lower extent of LD in broilers compared to layers, as reported in previous studies, the ratio of broiler and layer SNPs in the array was kept as 3:2. The final panel was shown to genotype a wide range of samples including broilers and layers with over 100 K to 450 K informative SNPs per line. A principal component analysis was used to demonstrate the ability of the array to detect the expected population structure which is an important pre-investigation step for many genome-wide analyses.ConclusionsThis Affymetrix® Axiom® array is the first SNP genotyping array for chicken that has been made commercially available to the public as a product. This array is expected to find widespread usage both in research and commercial application such as in genomic selection, genome-wide association studies, selection signature analyses, fine mapping of QTLs and detection of copy number variants.

Dataset Information

High density SNP array and reanalysis of genome sequencing uncovers CNVs associated with neurodevelopmental disorders in KOLF2.1J iPSCs.

Publications

High density SNP array and reanalysis of genome sequencing uncovers CNVs associated with neurodevelopmental disorders in KOLF2.1J iPSCs.

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