Flexible, Scalable, and Efficient Targeted Resequencing on a Benchtop Sequencer for Variant Detection in Clinical Practice

Leeneer, Kim De; Hellemans, Jan; Steyaert, Wouter; Lefever, Steve; Vereecke, Inge; Debals, Eveline; Crombez, Brecht; Baetens, Machteld; Heetvelde, Mattias Van; Coppieters, Frauke; Vandesompele, Jo; Jaegher, Annelies De; Baere, Elfride De; Coucke, Paul; Claes, Kathleen

doi:10.1002/humu.22739

Cited by 45 publications

(38 citation statements)

References 32 publications

(34 reference statements)

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“…Starting from 2012, a targeted NGS panel was introduced using a flexible protocol, consisting of singleplex PCR followed by NexteraXT library preparation and sequencing on a MiSeq instrument [8]. The CLC Genomics Workbench v.6 (Qiagen) was employed for read mapping against the hg19 human reference genome and variant calling.…”

Section: Methodsmentioning

confidence: 99%

Mutations in Splicing Factor Genes Are a Major Cause of Autosomal Dominant Retinitis Pigmentosa in Belgian Families

et al. 2017

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PurposeAutosomal dominant retinitis pigmentosa (adRP) is characterized by an extensive genetic heterogeneity, implicating 27 genes, which account for 50 to 70% of cases. Here 86 Belgian probands with possible adRP underwent genetic testing to unravel the molecular basis and to assess the contribution of the genes underlying their condition.MethodsMutation detection methods evolved over the past ten years, including mutation specific methods (APEX chip analysis), linkage analysis, gene panel analysis (Sanger sequencing, targeted next-generation sequencing or whole exome sequencing), high-resolution copy number screening (customized microarray-based comparative genomic hybridization). Identified variants were classified following American College of Medical Genetics and Genomics (ACMG) recommendations.ResultsMolecular genetic screening revealed mutations in 48/86 cases (56%). In total, 17 novel pathogenic mutations were identified: four missense mutations in RHO, five frameshift mutations in RP1, six mutations in genes encoding spliceosome components (SNRNP200, PRPF8, and PRPF31), one frameshift mutation in PRPH2, and one frameshift mutation in TOPORS. The proportion of RHO mutations in our cohort (14%) is higher than reported in a French adRP population (10.3%), but lower than reported elsewhere (16.5–30%). The prevalence of RP1 mutations (10.5%) is comparable to other populations (3.5%-10%). The mutation frequency in genes encoding splicing factors is unexpectedly high (altogether 19.8%), with PRPF31 the second most prevalent mutated gene (10.5%). PRPH2 mutations were found in 4.7% of the Belgian cohort. Two families (2.3%) have the recurrent NR2E3 mutation p.(Gly56Arg). The prevalence of the recurrent PROM1 mutation p.(Arg373Cys) was higher than anticipated (3.5%).ConclusionsOverall, we identified mutations in 48 of 86 Belgian adRP cases (56%), with the highest prevalence in RHO (14%), RP1 (10.5%) and PRPF31 (10.5%). Finally, we expanded the molecular spectrum of PRPH2, PRPF8, RHO, RP1, SNRNP200, and TOPORS-associated adRP by the identification of 17 novel mutations.

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Section: Methodsmentioning

confidence: 99%

Mutations in Splicing Factor Genes Are a Major Cause of Autosomal Dominant Retinitis Pigmentosa in Belgian Families

et al. 2017

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“…Still, normal protein expression does not exclude a functional defect. If only one or a limited number of CVID genes are suspected based on the patient's phenotype (table 1), we recommend protein expression analysis (if applicable) in combination with targeted testing of a single CVID gene or of a gene panel112 (in case of more than one possible gene). However, taking into account the large genetic heterogeneity of monogenic CVID as well as the large phenotypical overlap with other PID syndromes, clinical WES113 will be likely recommended in the majority of suspected monogenic CVID cases.…”

Section: Genes Associated With Monogenic Forms Of Cvidmentioning

confidence: 99%

Genes associated with common variable immunodeficiency: one diagnosis to rule them all?

et al. 2016

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Common variable immunodeficiency (CVID) is a primary antibody deficiency characterised by hypogammaglobulinaemia, impaired production of specific antibodies after immunisation and increased susceptibility to infections. CVID shows a considerable phenotypical and genetic heterogeneity. In contrast to many other primary immunodeficiencies, monogenic forms count for only 2-10% of patients with CVID. Genes that have been implicated in monogenic CVID include ICOS, TNFRSF13B (TACI), TNFRSF13C (BAFF-R), TNFSF12 (TWEAK), CD19, CD81, CR2 (CD21), MS4A1 (CD20), TNFRSF7 (CD27), IL21, IL21R, LRBA, CTLA4, PRKCD, PLCG2, NFKB1, NFKB2, PIK3CD, PIK3R1, VAV1, RAC2, BLK, IKZF1 (IKAROS) and IRF2BP2. With the increasing number of disease genes identified in CVID, it has become clear that CVID is an umbrella diagnosis and that many of these genetic defects cause distinct disease entities. Moreover, there is accumulating evidence that at least a subgroup of patients with CVID has a complex rather than a monogenic inheritance. This review aims to discuss current knowledge regarding the molecular genetic basis of CVID with an emphasis on the relationship with the clinical and immunological phenotype

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“…Variant classification was performed following the American College of Medical Genetics and Genomics (ACMG) guidelines of 2015. 16 Whole-exome sequencing was performed to exclude the presence of other likely pathogenic variants. Exome enrichment was performed with the Nextera Rapid Capture Exome kit (Illumina), followed by paired-end sequencing on HiSeq 2000 (2 × 100 cycles) (Illumina).…”

Section: Sanger Sequencing and Targeted Next-generation Sequencingmentioning

confidence: 99%

Biallelic and monoallelic ESR2 variants associated with 46,XY disorders of sex development

Baetens

Güran

Mendonça

et al. 2018

Genetics in Medicine

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Purpose: Disorders or differences of sex development (DSDs) are rare congenital conditions characterized by atypical sex development. Despite advances in genomic technologies, the molecular cause remains unknown in 50% of cases.Methods: Homozygosity mapping and whole-exome sequencing revealed an ESR2 variant in an individual with syndromic 46,XY DSD. Additional cases with 46,XY DSD underwent whole-exome sequencing and targeted next-generation sequencing of ESR2. Functional characterization of the identified variants included luciferase assays and protein structure analysis. Gonadal ESR2 expression was assessed in human embryonic data sets and immunostaining of estrogen receptor-β (ER-β) was performed in an 8-week-old human male embryo.

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Flexible, Scalable, and Efficient Targeted Resequencing on a Benchtop Sequencer for Variant Detection in Clinical Practice

Cited by 45 publications

References 32 publications

Mutations in Splicing Factor Genes Are a Major Cause of Autosomal Dominant Retinitis Pigmentosa in Belgian Families

Mutations in Splicing Factor Genes Are a Major Cause of Autosomal Dominant Retinitis Pigmentosa in Belgian Families

Genes associated with common variable immunodeficiency: one diagnosis to rule them all?

Biallelic and monoallelic ESR2 variants associated with 46,XY disorders of sex development

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