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Transcriptome Sequencing Boosts Diagnosis of Patients With Mitochondrial Disorders

NEW YORK (GenomeWeb) – Transcriptome sequencing can provide diagnoses for patients with Mendelian disorders where whole-exome sequencing provided no answer, according to a new study led by researchers in Munich.

In a study published yesterday in Nature Communications, Holger Prokisch, a group leader at the Institute of Human Genetics at the Technical University of Munich (TUM) and the Helmholtz Center Munich, and Julien Gagneur, a professor of computational biology at TUM, together with their colleagues, showed that RNA sequencing allowed them to diagnose 10 percent of patients with mitochondrial disorders for whom exome sequencing had been unsuccessful. In addition, they found candidate genes for most of the cases.

"In conclusion, we predict that RNA-seq will become an essential companion of genome sequencing to address undiagnosed cases of genetic disease," the authors wrote.

The results are similar to those of a study published by US-led researchers in April, in which they used RNA-seq to diagnose 35 percent of patient with previously undiagnosed muscle disorders.

For their present study, the German researchers, who presented their results at the European Society of Human Genetics annual meeting in Copenhagen last month, focused on patients with mitochondrial disorders because they usually have fibroblast cell lines from muscle tissue biopsies available, which can be used for both transcriptome sequencing and for functional assays. 

They performed RNA-seq on 105 fibroblast cell lines from patients with suspected mitochondrial disease, of whom 48 had undergone whole-exome sequencing but did not receive a genetic diagnosis.

To hone in on potentially disease-causing genes, they focused their analysis on genes with aberrant expression levels, genes with aberrant splicing, and genes with allele-specific expression of rare variants.

They found a median of one abnormally expressed gene per patient, among them two — MGST1 in one patient and TIMMDC1 in two patients — that encode mitochondrial proteins and were significantly downregulated in those individuals. Quantitative proteomics confirmed that protein expression was severely affected as well.

In the undiagnosed patients, the researchers also found 175 aberrantly spliced genes, resulting in the differential expression of isoforms, exon skipping, and the creation of new exons. They included two genes encoding mitochondrial proteins, TIMMDC1 and CLPP.

Per sample, they found a median of six abnormally spliced genes, and a median of six mono-allelically expressed rare variants.

Overall, the researchers were able to diagnose five of the previously undiagnosed patients. They also found strong candidate genes, meaning either a known disease-causing gene or a gene encoding a mitochondrial protein, for another 36 patients.

"To our surprise, many newly diagnosed cases were caused by a defective splicing event, which caused loss of function, confirming the increasing recognition of the role of splicing defects in both Mendelian and common disorders," the authors wrote, noting that in one case, the causal variant was intronic and thus not covered by exome sequencing.

Even though mitochondrial disorders usually don't affect skin cells, the genes involved are expressed in most tissues, they explained, allowing them to find the genetic defects by analyzing fibroblasts rather than the affected tissue. This strategy might also work for other diseases, they wrote, because tissue-specificity of a disorder might be related to other factors than expression of the disease gene in that tissue.