Whole-genome DNA/RNA sequencing identifies truncating mutations in RBCK1 in a novel Mendelian disease with neuromuscular and cardiac involvement - PubMed (original) (raw)

doi: 10.1186/gm471. eCollection 2013.

Cecilia Kim 2, Jonathan Bradfield 2, Yunfei Guo 3, Elina Toskala 2, Frederick G Otieno 2, Cuiping Hou 2, Kelly Thomas 2, Christopher Cardinale 2, Gholson J Lyon 4, Ryan Golhar 2, Hakon Hakonarson 5

Affiliations

Whole-genome DNA/RNA sequencing identifies truncating mutations in RBCK1 in a novel Mendelian disease with neuromuscular and cardiac involvement

Kai Wang et al. Genome Med. 2013.

Abstract

Background: Whole-exome sequencing has identified the causes of several Mendelian diseases by analyzing multiple unrelated cases, but it is more challenging to resolve the cause of extremely rare and suspected Mendelian diseases from individual families. We identified a family quartet with two children, both affected with a previously unreported disease, characterized by progressive muscular weakness and cardiomyopathy, with normal intelligence. During the course of the study, we identified one additional unrelated patient with a comparable phenotype.

Methods: We performed whole-genome sequencing (Complete Genomics platform), whole-exome sequencing (Agilent SureSelect exon capture and Illumina Genome Analyzer II platform), SNP genotyping (Illumina HumanHap550 SNP array) and Sanger sequencing on blood samples, as well as RNA-Seq (Illumina HiSeq platform) on transformed lymphoblastoid cell lines.

Results: From whole-genome sequence data, we identified RBCK1, a gene encoding an E3 ubiquitin-protein ligase, as the most likely candidate gene, with two protein-truncating mutations in probands in the first family. However, exome data failed to nominate RBCK1 as a candidate gene, due to poor regional coverage. Sanger sequencing identified a private homozygous splice variant in RBCK1 in the proband in the second family, yet SNP genotyping revealed a 1.2Mb copy-neutral region of homozygosity covering RBCK1. RNA-Seq confirmed aberrant splicing of RBCK1 transcripts, resulting in truncated protein products.

Conclusions: While the exact mechanism by which these mutations cause disease is unknown, our study represents an example of how the combined use of whole-genome DNA and RNA sequencing can identify a disease-predisposing gene for a novel and extremely rare Mendelian disease.

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Figures

Figure 1

Figure 1

The loss-of-function mutations within RBCK1 in two families. (a,b) Pedigree structure for the two families, respectively. (c) Genome browser shots illustrating the location of the mutations within RBCK1. Multiple zooming levels are shown from the chromosome to the gene structure, and then to three exons harboring the mutations.

Figure 2

Figure 2

Validation of mutations in RBCK1 in two families. (a,b) Validation on the nonsense mutation and frameshift indel in family 1. The identified mutations are labeled with arrows. The nucleotide sequences of the insertion were resolved from the sequence trace. (c) SNP array showed a 1.2 Mb copy-neutral region-of-homozygosity (ROH) on chromosome 20p in the proband from family 2. The upper panel (log R ratio) represents normalized total signal intensity, demonstrating the lack of copy number changes in the 1.2 Mb region. The lower panel (B allele frequency) represents normalized allelic intensity ratio, demonstrating the lack of heterozygous SNPs in the 1.2 Mb region. The location of RBCK1 is marked by the grey vertical line. (d,e) Validation of a homozygous intronic variant and a homozygous splice variant in the proband from family 2.

Figure 3

Figure 3

Integrative Genomics Viewer screen shots of the RNA-Seq data on three subjects from two families. The RNA-Seq experiments were performed using RNAs extracted from lymphoblastoid cell lines. The results validated the presence of a splice variant at the exon-intron boundary in the proband from family 2 (subject II-3), and that the intronic regions are transcribed between exon 5 and exon 6. However, the variant is not present in family 1, and exons 5 and 6 are correctly spliced in two subjects (subjects II-1 and I-2) in family 1.

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