Nimbus: a design-driven analyses suite for amplicon-based NGS data

Brouwer, Rutger W. W.; Hout, Mirjam C G N van den; Kockx, Christel; Brosens, Erwin; Eussen, Bert; Klein, Annelies de; Sleutels, Frank; IJcken, Wilfred F. J. van

doi:10.1093/bioinformatics/bty145

Cited by 6 publications

(5 citation statements)

References 19 publications

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“…Furthermore, if a specific monogenetic syndrome ( S3 Table ) was suspected, based on the phenotypic spectrum observed, the suspected gene(s) were evaluated using a targeted NGS panel or whole exome sequencing. In four HSCR patients with associated anomalies (Group 1) and nine HSCR patients without associated anomalies (Group 3), the involvement of other known disease genes was excluded [ 8 , 46 , 95 , 96 ] using whole exome sequencing (WES) with previously described pipelines [ 97 , 98 ] and variant prioritization methods [ 99 ].…”

Section: Methodsmentioning

confidence: 99%

Size matters: Large copy number losses in Hirschsprung disease patients reveal genes involved in enteric nervous system development

et al. 2021

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Hirschsprung disease (HSCR) is a complex genetic disease characterized by absence of ganglia in the intestine. HSCR etiology can be explained by a unique combination of genetic alterations: rare coding variants, predisposing haplotypes and Copy Number Variation (CNV). Approximately 18% of patients have additional anatomical malformations or neurological symptoms (HSCR-AAM). Pinpointing the responsible culprits within a CNV is challenging as often many genes are affected. Therefore, we selected candidate genes based on gene enrichment strategies using mouse enteric nervous system transcriptomes and constraint metrics. Next, we used a zebrafish model to investigate whether loss of these genes affects enteric neuron development in vivo. This study included three groups of patients, two groups without coding variants in disease associated genes: HSCR-AAM and HSCR patients without associated anomalies (HSCR-isolated). The third group consisted of all HSCR patients in which a confirmed pathogenic rare coding variant was identified. We compared these patient groups to unaffected controls. Predisposing haplotypes were determined, confirming that every HSCR subgroup had increased contributions of predisposing haplotypes, but their contribution was highest in isolated HSCR patients without RET coding variants. CNV profiling proved that specifically HSCR-AAM patients had larger Copy Number (CN) losses. Gene enrichment strategies using mouse enteric nervous system transcriptomes and constraint metrics were used to determine plausible candidate genes located within CN losses. Validation in zebrafish using CRISPR/Cas9 targeting confirmed the contribution of UFD1L, TBX2, SLC8A1, and MAPK8 to ENS development. In addition, we revealed epistasis between reduced Ret and Gnl1 expression and between reduced Ret and Tubb5 expression in vivo. Rare large CN losses—often de novo—contribute to HSCR in HSCR-AAM patients. We proved the involvement of six genes in enteric nervous system development and Hirschsprung disease.

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Section: Methodsmentioning

confidence: 99%

Size matters: Large copy number losses in Hirschsprung disease patients reveal genes involved in enteric nervous system development

et al. 2021

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“…Furthermore, if a specific monogenetic syndrome (S6) was suspected based on the phenotypic spectrum observed, the suspected gene(s) were evaluated using a targeted NGS panel or whole exome sequencing. In four HSCR patients with associated anomalies (Group 1) and nine HSCR patients without associated anomalies (Group 3), the involvement of other known disease genes was excluded[2, 36, 37, 58] using whole exome sequencing (WES) with previously described pipelines[59, 60] and variant prioritization methods[61].…”

Section: Methodsmentioning

confidence: 99%

Size matters: large copy number losses reveal novel Hirschsprung disease genes

Kuil

MacKenzie

et al. 2020

Preprint

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Background Hirschsprung disease (HSCR) is characterized by absence of ganglia in the intestine. Approximately 18% of patients have additional anatomical malformations or neurological symptoms (HSCR-AAM). HSCR is a complex genetic disease in which the loss of enteric ganglia stems from a combination of genetic alterations: rare coding variants, predisposing haplotypes and Copy Number Variation (CNV). Pinpointing the responsible culprits within a large CNV is challenging as often many genes are affected. We investigated if we could find deleterious CNVs and if we could identify the genes responsible for the aganglionosis. Results Deleterious CNVs were detected in three groups of patients: HSCR-AAM, HSCR patients with a confirmed causal genetic variant and HSCR-isolated patients without a known causal variant and controls. Predisposing haplotypes were determined, confirming that every HSCR subgroup had increased contributions of predisposing haplotypes, but their contribution was highest in isolated HSCR patients without RET coding variants. CNV profiling proved that HSCR-AAM patients had larger copy number losses. Gene enrichment strategies using mouse enteric nervous system transcriptomes and constraint metrics were used to determine plausible candidate genes in Copy Number Losses. Validation in zebrafish using CRISPR/Cas9 targeting confirmed the contribution of UFD1L, TBX2, SLC8A1 and MAPK8 to ENS development. In addition, we revealed epistasis between reduced Ret and Gnl1 expression in vivo. Conclusion Rare large Copy Number losses - often de novo - contribute to the disease in HSCR-AAM patients specifically. We proved the involvement of five genes in enteric nervous system development and Hirschsprung disease.

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“…Bioinformatics Analysis. Bioinformatic analysis was performed as described previously [10,11]. See Supplementary Information, Methods for details.…”

Section: 4mentioning

confidence: 99%

Targeted Genomic Sequencing of TSC1 and TSC2 Reveals Causal Variants in Individuals for Whom Previous Genetic Testing for Tuberous Sclerosis Complex Was Normal

et al. 2023

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Tuberous sclerosis complex (TSC) is caused by inactivating variants in TSC1 and TSC2. Somatic mosaicism, as well as the size and complexity of the TSC1 and TSC2 loci, makes variant identification challenging. Indeed, in some individuals with a clinical diagnosis of TSC, diagnostic testing fails to identify an inactivating variant. To improve TSC1 and TSC2 variant detection, we screened the TSC1 and TSC2 genomic regions using targeted HaloPlex custom capture and next-generation sequencing (NGS) in genomic DNA isolated from peripheral blood of individuals with definite, possible or suspected TSC in whom no disease-associated variant had been identified by previous diagnostic genetic testing. We obtained >95% target region coverage at a read depth of 20 and >50% coverage at a read depth of 300 and identified inactivating TSC1 or TSC2 variants in 83/155 individuals (54%); 65/113 (58%) with clinically definite TSC and 18/42 (43%) with possible or suspected TSC. These included 19 individuals with deep intronic variants and 54 likely cases of mosaicism (variant allele frequency 1-28%; median 7%). In 13 cases (8%), we identified a variant of uncertain significance (VUS). Targeted genomic NGS of TSC1 and TSC2 increases the yield of inactivating variants found in individuals with suspected TSC.

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Nimbus: a design-driven analyses suite for amplicon-based NGS data

Cited by 6 publications

References 19 publications

Size matters: Large copy number losses in Hirschsprung disease patients reveal genes involved in enteric nervous system development

Size matters: Large copy number losses in Hirschsprung disease patients reveal genes involved in enteric nervous system development

Size matters: large copy number losses reveal novel Hirschsprung disease genes

Targeted Genomic Sequencing of TSC1 and TSC2 Reveals Causal Variants in Individuals for Whom Previous Genetic Testing for Tuberous Sclerosis Complex Was Normal

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