Next generation sequence analysis for mitochondrial disorders

Valeria Vasta , Sarah B Ng , Emily H Turner , Jay Shendure and Si Houn Hahn
Genome Medicine 2009, 1:100doi:10.1186/gm100
Published: 23 October 2009
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Background
Mitochondrial disorders can originate from mutations in one of many nuclear genes controlling the organelle function or in the mitochondrial genome (mitochondrial DNA (mtDNA)). The large numbers of potential culprit genes, together with the little guidance offered by most clinical phenotypes as to which gene may be causative, are a great challenge for the molecular diagnosis of these disorders.

Methods
We developed a novel targeted resequencing assay for mitochondrial disorders relying on microarray-based hybrid capture coupled to next-generation sequencing. Specifically, we subjected the entire mtDNA genome and the exons and intron-exon boundary regions of 362 known or candidate causative nuclear genes to targeted capture and resequencing. We here provide proof-of-concept data by testing one HapMap DNA sample and two positive control samples.

Results
Over 94% of the targeted regions were captured and sequenced with appropriate coverage and quality, allowing reliable variant calling. Pathogenic mutations blindly tested in patients' samples were 100% concordant with previous Sanger sequencing results: a known mutation in Pyruvate dehydrogenase alpha 1 subunit (PDHA1), a novel splicing and a known coding mutation in Hydroxyacyl-CoA dehydrogenase alpha subunit (HADHA) were correctly identified. Of the additional variants recognized, 90 to 94% were present in dbSNP while 6 to 10% represented new alterations. The novel nonsynonymous variants were all in heterozygote state and mostly predicted to be benign. The depth of sequencing coverage of mtDNA was extremely high, suggesting that it may be feasible to detect pathogenic mtDNA mutations confounded by low level heteroplasmy. Only one sequencing lane of an eight lane flow cell was utilized for each sample, indicating that a cost-effective clinical test can be achieved.

Conclusions
Our study indicates that the use of next generation sequencing technology holds great promise as a tool for screening mitochondrial disorders. The availability of a comprehensive molecular diagnostic tool will increase the capacity for early and rapid identification of mitochondrial disorders. In addition, the proposed approach has the potential to identify new mutations in candidate genes, expanding and redefining the spectrum of causative genes responsible for mitochondrial disorders.


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Focus: Genes targeted for capture and sequencing....Enzymes...FXN,
New variants and mutations identified in the samples..... FXN [Genbank:NM_000144.3]:c.626A>G (p.Asp209Gly) het

The conserved Trp-155 in human frataxin as a hotspot for oxidative stress related chemical modifications

Biochemical and Biophysical Research Communications

Ana R. Correiaa, Saw Y. Owb, Phillip C. Wrightb and Cláudio M. Gomesa, Corresponding Author Contact Information, E-mail The Corresponding Author

aInstituto Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2780-756 Oeiras, Portugal

bDepartment of Chemical and Process Engineering, ChELSI, University of Sheffield, Sheffield S10 2TN, UK

Received 15 October 2009.
Available online 22 October 2009.

Keywords: Friedreich’s Ataxia, Frataxin, Protein Folding, Protein Flexibility, Metallochaperone, Oxidative Stress, Fenton reactions, carbonylation, nitration.