Alison G. Compton

Molecular Diagnosis of Infantile Mitochondrial Disease with Targeted Next-Generation Sequencing

Applying next-generation sequencing to 42 infants with mitochondrial disease highlights both the potential and the challenge of using this technology in clinical diagnosis. Getting to the Genetic Root of Mitochondrial Disease Next-generation DNA sequencing is being applied with great success in research settings to uncover new disease genes. Despite these successes, it is unclear how useful the technology will be for routine clinical diagnosis given the challenge of interpreting DNA variations in individual patients. In a new study, Calvo and colleagues apply next-generation sequencing to infants with mitochondrial disorders, a large collection of inherited diseases that are notoriously difficult to diagnose because of the multitude of candidate genes and the highly variable nature of the clinical presentation. First, the authors selected 42 unrelated infants with mitochondrial diseases that were refractory to standard clinical genetic testing. Then, for each child, they sequenced the DNA of the mitochondrial genome, the 100 genes previously linked to mitochondrial disease, and the ~1000 additional genes that are known to participate in mitochondrial biology. Of all the DNA differences present in the patients, the researchers prioritized those that were rare in the general population, predicted to disrupt protein function, and inherited in a recessive fashion. Such variants showed fivefold enrichment in the patients compared to that in healthy control individuals. In 10 patients (24%), firm molecular diagnoses were made in genes previously linked to mitochondrial diseases; 13 patients (31%) had prioritized recessive mutations in genes not previously linked to disease. For two of these genes, the authors were able to show that the mutations caused the mitochondrial disorder. These results suggest that next-generation sequencing may be able to provide a molecular diagnosis for ~25% of currently unsolved cases of infantile mitochondrial disease. An additional 25% of cases could be solved in the coming few years as more genes are formally proven to be linked to mitochondrial disease. The remaining 50% of patients in whom diagnosis was not possible underscores the challenge of interpreting DNA sequence data for clinical diagnosis. Nevertheless, the study by Calvo and colleagues will help to calibrate clinicians’ expectations regarding the diagnostic use of next-generation sequencing. Advances in next-generation sequencing (NGS) promise to facilitate diagnosis of inherited disorders. Although in research settings NGS has pinpointed causal alleles using segregation in large families, the key challenge for clinical diagnosis is application to single individuals. To explore its diagnostic use, we performed targeted NGS in 42 unrelated infants with clinical and biochemical evidence of mitochondrial oxidative phosphorylation disease. These devastating mitochondrial disorders are characterized by phenotypic and genetic heterogeneity, with more than 100 causal genes identified to date. We performed “MitoExome” sequencing of the mitochondrial DNA (mtDNA) and exons of ~1000 nuclear genes encoding mitochondrial proteins and prioritized rare mutations predicted to disrupt function. Because patients and healthy control individuals harbored a comparable number of such heterozygous alleles, we could not prioritize dominant-acting genes. However, patients showed a fivefold enrichment of genes with two such mutations that could underlie recessive disease. In total, 23 of 42 (55%) patients harbored such recessive genes or pathogenic mtDNA variants. Firm diagnoses were enabled in 10 patients (24%) who had mutations in genes previously linked to disease. Thirteen patients (31%) had mutations in nuclear genes not previously linked to disease. The pathogenicity of two such genes, NDUFB3 and AGK, was supported by complementation studies and evidence from multiple patients, respectively. The results underscore the potential and challenges of deploying NGS in clinical settings.

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

Discovering the molecular basis of mitochondrial respiratory chain disease is challenging given the large number of both mitochondrial and nuclear genes that are 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 disorders. We created seven pools of DNA from a cohort of 103 cases and 42 healthy controls and then performed deep sequencing of 103 candidate genes to identify 151 rare variants that were predicted to affect protein function. We established genetic diagnoses in 13 of 60 previously unsolved cases using confirmatory experiments, including cDNA complementation to show that mutations in NUBPL and FOXRED1 can cause complex I deficiency. Our study illustrates how large-scale sequencing, coupled with functional prediction and experimental validation, can be used to identify causal mutations in individual cases.

Mutation of the Mitochondrial Tyrosyl-tRNA Synthetase Gene, YARS2, Causes Myopathy, Lactic Acidosis, and Sideroblastic Anemia—MLASA Syndrome

Mitochondrial respiratory chain disorders are a heterogeneous group of disorders in which the underlying genetic defect is often unknown. We have identified a pathogenic mutation (c.156C>G [p.F52L]) in YARS2, located at chromosome 12p11.21, by using genome-wide SNP-based homozygosity analysis of a family with affected members displaying myopathy, lactic acidosis, and sideroblastic anemia (MLASA). We subsequently identified the same mutation in another unrelated MLASA patient. The YARS2 gene product, mitochondrial tyrosyl-tRNA synthetase (YARS2), was present at lower levels in skeletal muscle whereas fibroblasts were relatively normal. Complex I, III, and IV were dysfunctional as indicated by enzyme analysis, immunoblotting, and immunohistochemistry. A mitochondrial protein-synthesis assay showed reduced levels of respiratory chain subunits in myotubes generated from patient cell lines. A tRNA aminoacylation assay revealed that mutant YARS2 was still active; however, enzyme kinetics were abnormal compared to the wild-type protein. We propose that the reduced aminoacylation activity of mutant YARS2 enzyme leads to decreased mitochondrial protein synthesis, resulting in mitochondrial respiratory chain dysfunction. MLASA has previously been associated with PUS1 mutations; hence, the YARS2 mutation reported here is an alternative cause of MLASA.

Mutations in MTFMT Underlie a Human Disorder of Formylation Causing Impaired Mitochondrial Translation

The metazoan mitochondrial translation machinery is unusual in having a single tRNA(Met) that fulfills the dual role of the initiator and elongator tRNA(Met). A portion of the Met-tRNA(Met) pool is formylated by mitochondrial methionyl-tRNA formyltransferase (MTFMT) to generate N-formylmethionine-tRNA(Met) (fMet-tRNA(met)), which is used for translation initiation; however, the requirement of formylation for initiation in human mitochondria is still under debate. Using targeted sequencing of the mtDNA and nuclear exons encoding the mitochondrial proteome (MitoExome), we identified compound heterozygous mutations in MTFMT in two unrelated children presenting with Leigh syndrome and combined OXPHOS deficiency. Patient fibroblasts exhibit severe defects in mitochondrial translation that can be rescued by exogenous expression of MTFMT. Furthermore, patient fibroblasts have dramatically reduced fMet-tRNA(Met) levels and an abnormal formylation profile of mitochondrially translated COX1. Our findings demonstrate that MTFMT is critical for efficient human mitochondrial translation and reveal a human disorder of Met-tRNA(Met) formylation.

Leigh syndrome: One disorder, more than 75 monogenic causes

Leigh syndrome is the most common pediatric presentation of mitochondrial disease. This neurodegenerative disorder is genetically heterogeneous, and to date pathogenic mutations in >75 genes have been identified, encoded by 2 genomes (mitochondrial and nuclear). More than one‐third of these disease genes have been characterized in the past 5 years alone, reflecting the significant advances made in understanding its etiological basis. We review the diverse biochemical and genetic etiology of Leigh syndrome and associated clinical, neuroradiological, and metabolic features that can provide clues for diagnosis. We discuss the emergence of genotype–phenotype correlations, insights gleaned into the molecular basis of disease, and available therapeutic options. Ann Neurol 2016;79:190–203

Respiratory chain complex I deficiency caused by mitochondrial DNA mutations.

Defects of the mitochondrial respiratory chain are associated with a diverse spectrum of clinical phenotypes, and may be caused by mutations in either the nuclear or the mitochondrial genome (mitochondrial DNA (mtDNA)). Isolated complex I deficiency is the most common enzyme defect in mitochondrial disorders, particularly in children in whom family history is often consistent with sporadic or autosomal recessive inheritance, implicating a nuclear genetic cause. In contrast, although a number of recurrent, pathogenic mtDNA mutations have been described, historically, these have been perceived as rare causes of paediatric complex I deficiency. We reviewed the clinical and genetic findings in a large cohort of 109 paediatric patients with isolated complex I deficiency from 101 families. Pathogenic mtDNA mutations were found in 29 of 101 probands (29%), 21 in MTND subunit genes and 8 in mtDNA tRNA genes. Nuclear gene defects were inferred in 38 of 101 (38%) probands based on cell hybrid studies, mtDNA sequencing or mutation analysis (nuclear gene mutations were identified in 22 probands). Leigh or Leigh-like disease was the most common clinical presentation in both mtDNA and nuclear genetic defects. The median age at onset was higher in mtDNA patients (12 months) than in patients with a nuclear gene defect (3 months). However, considerable overlap existed, with onset varying from 0 to >60 months in both groups. Our findings confirm that pathogenic mtDNA mutations are a significant cause of complex I deficiency in children. In the absence of parental consanguinity, we recommend whole mitochondrial genome sequencing as a key approach to elucidate the underlying molecular genetic abnormality.

Mutations in LYRM4, encoding iron-sulfur cluster biogenesis factor ISD11, cause deficiency of multiple respiratory chain complexes

Iron-sulfur clusters (ISCs) are important prosthetic groups that define the functions of many proteins. Proteins with ISCs (called iron-sulfur or Fe-S proteins) are present in mitochondria, the cytosol, the endoplasmic reticulum and the nucleus. They participate in various biological pathways including oxidative phosphorylation (OXPHOS), the citric acid cycle, iron homeostasis, heme biosynthesis and DNA repair. Here, we report a homozygous mutation in LYRM4 in two patients with combined OXPHOS deficiency. LYRM4 encodes the ISD11 protein, which forms a complex with, and stabilizes, the sulfur donor NFS1. The homozygous mutation (c.203G>T, p.R68L) was identified via massively parallel sequencing of >1000 mitochondrial genes (MitoExome sequencing) in a patient with deficiency of complexes I, II and III in muscle and liver. These three complexes contain ISCs. Sanger sequencing identified the same mutation in his similarly affected cousin, who had a more severe phenotype and died while a neonate. Complex IV was also deficient in her skeletal muscle. Several other Fe-S proteins were also affected in both patients, including the aconitases and ferrochelatase. Mutant ISD11 only partially complemented for an ISD11 deletion in yeast. Our in vitro studies showed that the l-cysteine desulfurase activity of NFS1 was barely present when co-expressed with mutant ISD11. Our findings are consistent with a defect in the early step of ISC assembly affecting a broad variety of Fe-S proteins. The differences in biochemical and clinical features between the two patients may relate to limited availability of cysteine in the newborn period and suggest a potential approach to therapy.

Recent Advances in the Genetics of Mitochondrial Encephalopathies

Mitochondrial encephalopathy, the most common neurometabolic disorder, may be caused by mutations in approximately 100 different genes and may present with various symptoms, such as seizures, ataxia, myopathy, cognitive impairment, blindness, and stroke. Fewer than 50% of patients with mitochondrial encephalopathy receive a molecular diagnosis, primarily because of the large degree of clinical and genetic heterogeneity among patients and the limited knowledge of the genes involved in mitochondrial function. Here we review the most recent discoveries of genes associated with mitochondrial disease with variable neuropathology. All these genes have been identified via homozygosity mapping or linkage analysis; however, advances in sequencing technology indicate that the future of genetic diagnosis and disease gene discovery likely lies in high-throughput sequencing.

Mutations in the UQCC1-Interacting Protein, UQCC2, Cause Human Complex III Deficiency Associated with Perturbed Cytochrome b Protein Expression

Mitochondrial oxidative phosphorylation (OXPHOS) is responsible for generating the majority of cellular ATP. Complex III (ubiquinol-cytochrome c oxidoreductase) is the third of five OXPHOS complexes. Complex III assembly relies on the coordinated expression of the mitochondrial and nuclear genomes, with 10 subunits encoded by nuclear DNA and one by mitochondrial DNA (mtDNA). Complex III deficiency is a debilitating and often fatal disorder that can arise from mutations in complex III subunit genes or one of three known complex III assembly factors. The molecular cause for complex III deficiency in about half of cases, however, is unknown and there are likely many complex III assembly factors yet to be identified. Here, we used Massively Parallel Sequencing to identify a homozygous splicing mutation in the gene encoding Ubiquinol-Cytochrome c Reductase Complex Assembly Factor 2 (UQCC2) in a consanguineous Lebanese patient displaying complex III deficiency, severe intrauterine growth retardation, neonatal lactic acidosis and renal tubular dysfunction. We prove causality of the mutation via lentiviral correction studies in patient fibroblasts. Sequence-profile based orthology prediction shows UQCC2 is an ortholog of the Saccharomyces cerevisiae complex III assembly factor, Cbp6p, although its sequence has diverged substantially. Co-purification studies show that UQCC2 interacts with UQCC1, the predicted ortholog of the Cbp6p binding partner, Cbp3p. Fibroblasts from the patient with UQCC2 mutations have deficiency of UQCC1, while UQCC1-depleted cells have reduced levels of UQCC2 and complex III. We show that UQCC1 binds the newly synthesized mtDNA-encoded cytochrome b subunit of complex III and that UQCC2 patient fibroblasts have specific defects in the synthesis or stability of cytochrome b. This work reveals a new cause for complex III deficiency that can assist future patient diagnosis, and provides insight into human complex III assembly by establishing that UQCC1 and UQCC2 are complex III assembly factors participating in cytochrome b biogenesis.

Next-generation sequencing in molecular diagnosis: NUBPL mutations highlight the challenges of variant detection and interpretation.

Next‐generation sequencing (NGS) is transitioning from being a research tool to being used in routine genetic diagnostics, where a major challenge is distinguishing which of many sequence variants in an individual are truly pathogenic. We describe some limitations of in silico analyses of NGS data that emphasize the need for experimental confirmation. Using NGS, we recently identified an apparently homozygous missense mutation in NUBPL in a patient with mitochondrial complex I deficiency. Causality was established via lentiviral correction studies with wild‐type NUBPL cDNA. NGS data, however, provided an incomplete understanding of the genetic abnormality. We show that the maternal allele carries an unbalanced inversion, while the paternal allele carries a branch‐site mutation in addition to the missense mutation. We demonstrate that the branch‐site mutation, which is present in approximately one of 120 control chromosomes, likely contributes to pathogenicity and may be one of the most common autosomal mutations causing mitochondrial dysfunction. Had these analyses not been performed following NGS, the original missense mutation may be incorrectly annotated as pathogenic and a potentially common pathogenic variant not detected. It is important that locus‐specific databases contain accurate information on pathogenic variation. NGS data, therefore, require rigorous experimental follow‐up to confirm mutation pathogenicity. Hum Mutat 33:411–418, 2012.