High-throughput, pooled sequencing identifies mutations in NUBPL and FOXRED1 in human complex I deficiency

Abstract

Discovering the molecular basis of mitochondrial respiratory chain disease is challenging given the large number of both mitochondrial and nuclear genes involved. We report a strategy of focused candidate gene prediction, high-throughput sequencing, and experimental validation to uncover the molecular basis of mitochondrial complex I (CI) disorders. We created five pools of DNA from a cohort of 103 patients and then performed deep sequencing of 103 candidate genes to spotlight 151 rare variants predicted to impact protein function. We used confirmatory experiments to establish genetic diagnoses in 22% of previously unsolved cases, and discovered that defects in NUBPL and FOXRED1 can cause CI deficiency. Our study illustrates how large-scale sequencing, coupled with functional prediction and experimental validation, can reveal novel disease-causing mutations in individual patients.

Combined defects in oxidative phosphorylation and fatty acid β-oxidation in mitochondrial disease

“…The COPP gene list also contains genes that have been confirmed experimentally as bona fide CI assembly factors, including FOXRED1 [53], NDUFAF6 [52] and NDUFAF5 [51,144], substantiating the validity of the COPP list in predicting the involvement of HADH and ECI1 in CI biogenesis.…”

“…The first pathogenic human mutation in a CI assembly factor was found in NDUFAF2 [47]. Since this initial discovery, several other pathogenic mutations have been identified in CI assembly factor genes, including NDUFAF1 [48], NDUFAF3 [49], NDUFAF4 [50], NDUFAF5 [51], NDUFAF6 [52], FOXRED1 [53], ACAD9 [54] and NUBPL [53] (Table 1). The identification of these mutations, and the examination of how they disrupt CI assembly, have greatly aided our understanding of CI biogenesis.…”

Biallelic Mutations in MRPS34 Lead to Instability of the Small Mitoribosomal Subunit and Leigh Syndrome

“…22 For cDNA studies of subject 1, cultured fibroblasts were grown with and without cycloheximide treatment, as described previously. 32 For cDNA studies of subjects from families 2 and 3, cultured fibroblasts or Epstein-Barr virus-transformed lymphoblasts were grown without cycloheximide treatment. Total RNA was extracted using the miRNeasy Mini kit (QIAGEN) as per manufacturer's protocol and as described previously.…”

“…32 Synthesis of cDNA was performed using the SuperScript IV First-Strand Synthesis System (ThermoFisher Scientific) as per manufacturer's protocol and as described previously. 32 To examine the effect of the c.321þ1G>T and the c.322À10G>A mutations on mRNA splicing, PCR primers were designed to amplify exons 1-3 of MRPS34 from cDNA (using either pair 1 [forward primer 5 0 -CGGGAGCAACT GAACAGG-3 0 , reverse primer 5 0 -TGCGTATCCTCTGCACATTC-3 0 ] or pair 2 [forward primer 5 0 -AGCTCTACGCGGTGGACTAC-3 0 , reverse primer 5 0 -GATCCAGGCAGAGAGAGCAC-3 0 ], respectively). PCR products amplified from the MRPS34 transcript containing the c.322À10G>A mutation were cloned into the pCRTM4-TOPO TA vector using the TOPO TA cloning kit (ThermoFisher Scientific).…”

Clinical Severity of PGK1 Deficiency Due To a Novel p.E120K Substitution Is Exacerbated by Co-inheritance of a Subclinical Translocation t(3;14)(q26.33;q12), Disrupting NUBPL Gene

“…2), a member of the Mrp/NBP35 ATPbinding protein family, and critical for the assembly of human respiratory mitochondrial CI (Sheftel et al 2009). Recently, recessive mutations in NUBPL have been implicated in the genetic etiology of CI deficiency [OMIM #252010], a mitochondrial respiratory chain defect that is caused by mutations in multiple different nuclear, mitochondrial, or X-linked genes (Calvo et al 2010;Fassone and Rahman 2012). Additionally, in subjects with pediatric neurological disorders, three deletions encompassing this gene were reported (p ¼ 0.135) (Cooper et al 2011).…”