Clinical and laboratory interpretation of mitochondrial mRNA variants.

Interpretation of mitochondrial protein-encoding (mt-mRNA) variants has been challenging due to mitochondrial characteristics that have not been addressed by American College of Medical Genetics and Genomics guidelines. We developed criteria for the interpretation of mt-mRNA variants via literature review of reported variants, tested and refined these criteria by using our new cases, followed by interpreting 421 novel variants in our clinical database using these verified criteria. A total of 32 of 56 previously reported pathogenic (P) variants had convincing evidence for pathogenicity. These variants are either null variants, well-known disease-causing variants, or have robust functional data or strong phenotypic correlation with heteroplasmy levels. Based on our criteria, 65.7% (730/1,111) of variants of unknown significance (VUS) were reclassified as benign (B) or likely benign (LB), and one variant was scored as likely pathogenic (LP). Furthermore, using our criteria we classified 2, 12, and 23 as P, LP, and LB, respectively, among 421 novel variants. The remaining stayed as VUS (91.2%). Appropriate interpretation of mt-mRNA variants is the basis for clinical diagnosis and genetic counseling. Mutation type, heteroplasmy levels in different tissues of the probands and matrilineal relatives, in silico predictions, population data, as well as functional studies are key points for pathogenicity assessments.

Correction: Interpretation of mitochondrial tRNA variants.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Correction: Interpretation of mitochondrial tRNA variants.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Interpretation of mitochondrial tRNA variants.

PURPOSE: To develop criteria to interpret mitochondrial transfer RNA (mt-tRNA) variants based on unique characteristics of mitochondrial genetics and conserved structural/functional properties of tRNA. METHODS: We developed rules on a set of established pathogenic/benign variants by examining heteroplasmy correlations with phenotype, tissue distribution, family members, and among unrelated families from published literature. We validated these deduced rules using our new cases and applied them to classify novel variants. RESULTS: Evaluation of previously reported pathogenic variants found that 80.6% had sufficient evidence to support phenotypic correlation with heteroplasmy levels among and within families. The remaining variants were downgraded due to the lack of similar evidence. Application of the verified criteria resulted in rescoring 80.8% of reported variants of uncertain significance (VUS) to benign and likely benign. Among 97 novel variants, none met pathogenic criteria. A large proportion of novel variants (84.5%) remained as VUS, while only 10.3% were likely pathogenic. Detection of these novel variants in additional individuals would facilitate their classification. CONCLUSION: Proper interpretation of mt-tRNA variants is crucial for accurate clinical diagnosis and genetic counseling. Correlations with tissue distribution, heteroplasmy levels, predicted perturbations to tRNA structure, and phenotypes provide important evidence for determining the clinical significance of mt-tRNA variants.

MPV17-related mitochondrial DNA maintenance defect: New cases and review of clinical, biochemical, and molecular aspects.

Mitochondrial DNA (mtDNA) maintenance defects are a group of diseases caused by deficiency of proteins involved in mtDNA synthesis, mitochondrial nucleotide supply, or mitochondrial dynamics. One of the mtDNA maintenance proteins is MPV17, which is a mitochondrial inner membrane protein involved in importing deoxynucleotides into the mitochondria. In 2006, pathogenic variants in MPV17 were first reported to cause infantile-onset hepatocerebral mtDNA depletion syndrome and Navajo neurohepatopathy. To date, 75 individuals with MPV17-related mtDNA maintenance defect have been reported with 39 different MPV17 pathogenic variants. In this report, we present an additional 25 affected individuals with nine novel MPV17 pathogenic variants. We summarize the clinical features of all 100 affected individuals and review the total 48 MPV17 pathogenic variants. The vast majority of affected individuals presented with an early-onset encephalohepatopathic disease characterized by hepatic and neurological manifestations, failure to thrive, lactic acidemia, and mtDNA depletion detected mainly in liver tissue. Rarely, MPV17 deficiency can cause a late-onset neuromyopathic disease characterized by myopathy and peripheral neuropathy with no or minimal liver involvement. Approximately half of the MPV17 pathogenic variants are missense. A genotype with biallelic missense variants, in particular homozygous p.R50Q, p.P98L, and p.R41Q, can carry a relatively better prognosis.

Novel insights into the functional metabolic impact of an apparent de novo m.8993T>G variant in the MT-ATP6 gene associated with maternally inherited form of Leigh Syndrome.

In this study, we report a novel perpective of metabolic consequences for the m.8993T>G variant using fibroblasts from a proband with clinical symptoms compatible with Maternally Inherited Leigh Syndrome (MILS). Definitive diagnosis was corroborated by mitochondrial DNA testing for the pathogenic variant m.8993T>G in MT-ATP6 subunit by Sanger sequencing. The long-range PCR followed by massively parallel sequencing method detected the near homoplasmic m.8993T>G variant at 83% in the proband's fibroblasts and at 0.4% in the mother's fibroblasts. Our results are compatible with very low levels of germline heteroplasmy or an apparent de novo mutation. Our mitochondrial morphometric analysis reveals severe defects in mitochondrial cristae structure in the proband's fibroblasts. Our live-cell mitochondrial respiratory analyses show impaired oxidative phosphorylation with decreased spare respiratory capacity in response to energy stress in the proband's fibroblasts. We detected a diminished glycolysis with a lessened glycolytic capacity and reserve, revealing a stunted ability to switch to glycolysis upon full inhibition of OXPHOS activities. This dysregulated energy reprogramming results in a defective interplay between OXPHOS and glycolysis during an energy crisis. Our study sheds light on the potential pathophysiologic mechanism leading to chronic energy crisis in this MILS patient harboring the m.8993T>G variant.

FARS2 deficiency; new cases, review of clinical, biochemical, and molecular spectra, and variants interpretation based on structural, functional, and evolutionary significance.

An increasing number of mitochondrial diseases are found to be caused by pathogenic variants in nuclear encoded mitochondrial aminoacyl-tRNA synthetases. FARS2 encodes mitochondrial phenylalanyl-tRNA synthetase (mtPheRS) which transfers phenylalanine to its cognate tRNA in mitochondria. Since the first case was reported in 2012, a total of 21 subjects with FARS2 deficiency have been reported to date with a spectrum of disease severity that falls between two phenotypes; early onset epileptic encephalopathy and a less severe phenotype characterized by spastic paraplegia. In this report, we present an additional 15 individuals from 12 families who are mostly Arabs homozygous for the pathogenic variant Y144C, which is associated with the more severe early onset phenotype. The total number of unique pathogenic FARS2 variants known to date is 21 including three different partial gene deletions reported in four individuals. Except for the large deletions, all variants but two (one in-frame deletion of one amino acid and one splice-site variant) are missense. All large deletions and the single splice-site variant are in trans with a missense variant. This suggests that complete loss of function may be incompatible with life. In this report, we also review structural, functional, and evolutionary significance of select FARS2 pathogenic variants reported here.

Molecular and clinical spectra of FBXL4 deficiency.

F-box and leucine-rich repeat protein 4 (FBXL4) is a mitochondrial protein whose exact function is not yet known. However, cellular studies have suggested that it plays significant roles in mitochondrial bioenergetics, mitochondrial DNA (mtDNA) maintenance, and mitochondrial dynamics. Biallelic pathogenic variants in FBXL4 are associated with an encephalopathic mtDNA maintenance defect syndrome that is a multisystem disease characterized by lactic acidemia, developmental delay, and hypotonia. Other features are feeding difficulties, growth failure, microcephaly, hyperammonemia, seizures, hypertrophic cardiomyopathy, elevated liver transaminases, recurrent infections, variable distinctive facial features, white matter abnormalities and cerebral atrophy found in neuroimaging, combined deficiencies of multiple electron transport complexes, and mtDNA depletion. Since its initial description in 2013, 36 different pathogenic variants in FBXL4 were reported in 50 affected individuals. In this report, we present 37 additional affected individuals and 11 previously unreported pathogenic variants. We summarize the clinical features of all 87 individuals with FBXL4-related mtDNA maintenance defect, review FBXL4 structure and function, map the 47 pathogenic variants onto the gene structure to assess the variants distribution, and investigate the genotype-phenotype correlation. Finally, we provide future directions to understand the disease mechanism and identify treatment strategies.

Next generation deep sequencing corrects diagnostic pitfalls of traditional molecular approach in a patient with prenatal onset of Pompe disease.

Pompe disease is a rare inherited metabolic disorder of glycogen metabolism caused by mutations in the GAA gene, encoding the acid α-1,4 glucosidase. Successful diagnosis of Pompe disease is achieved by clinical and biochemical evaluation followed by confirmation with DNA testing. Here, we report a male infant with a prenatal onset of cardiac symptoms and enzyme testing consistent with Pompe disease, but DNA testing by Sanger sequencing revealed no pathogenic variants. Due to the strong indication from clinical, enzymatic, and histological studies (despite the absence of molecular confirmation by traditional Sanger sequencing), enzyme replacement therapy (ERT) for Pompe disease was initiated. Reanalysis of the patient's DNA sample using next generation sequencing (NGS) of a panel of target genes causing glycogen storage disorders demonstrated compound heterozygosity for a point mutation and an exonic deletion in the GAA gene. This case illustrates the value of astute clinical judgement in patient management as well as the power of target capture deep NGS in the simultaneous detection of both a point mutation and a heterozygous exonic deletion by correcting pitfalls of the traditional PCR based sequencing, namely; allele dropout and the inability to detect exonic deletions.

ADIPOR1 Is Mutated in Syndromic Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is a genetically heterogeneous retinal disorder. Despite the numerous genes associated with RP already identified, the genetic basis remains unknown in a substantial number of patients and families. In this study, we performed whole-exome sequencing to investigate the molecular basis of a syndromic RP case that cannot be solved by mutations in known disease-causing genes. After applying a series of variant filtering strategies, we identified an apparently homozygous frameshift mutation, c.31delC (p.Q11Rfs*24) in the ADIPOR1 gene. The reported phenotypes of Adipor1-null mice contain retinal dystrophy, obesity, and behavioral abnormalities, which highly mimic those in the syndromic RP patient. We further confirmed ADIPOR1 retina expression by immunohistochemistry. Our results established ADIPOR1 as a novel disease-causing gene for syndromic RP and highlight the importance of fatty acid transport in the retina.

Identification of KLHL40 mutations by targeted next-generation sequencing facilitated a prenatal diagnosis in a family with three consecutive affected fetuses with fetal akinesia deformation sequence.

BACKGROUND: Fetal akinesia deformation sequence (FADS) refers to a broad spectrum of disorder with the absent fetal movement as the unifying feature. The etiology of FADS is heterogeneous, and the majority remains unknown. Prenatal diagnosis of FADS because of neuromuscular origin has relied on clinical features and fetal muscle pathology, which can be unrevealing. The recent advance of next-generation sequencing (NGS) can provide definitive molecular diagnosis effectively. METHODS AND RESULTS: An 18-week-old fetus presented with akinesia and multiple contractures of joints. The mother had two previously aborted similarly affected fetuses. Clinical diagnosis of FADS was made. Molecular diagnosis using cord blood by NGS of genes related to neuromuscular diseases revealed two compound heterozygous mutations; c.602G > A(p.W201*) and c.1516A > C(p.T506P), in the Kelch-like 40 (KLHL40) gene. Based on this information, prenatal diagnosis was performed on the CVS of the subsequent pregnancy that resulted in an unaffected female baby, heterozygous for the c.1516A > C(p.T506P) mutation. CONCLUSION: Identification of KLHL40 mutations in one of the aborted fetuses provided a confirmative diagnosis of FADS, facilitating the prenatal diagnosis of the subsequent pregnancy. This report underscores the importance of target NGS in providing FADS families with an affordable, precise molecular diagnosis for genetic counseling and options of prenatal diagnosis. © 2016 John Wiley & Sons, Ltd.

Improved molecular diagnosis by the detection of exonic deletions with target gene capture and deep sequencing.

PURPOSE: We aimed to demonstrate the detection of exonic deletions using target capture and deep sequencing data. METHODS: Sequence data from target gene capture followed by massively parallel sequencing were analyzed for the detection of exonic deletions using the normalized mean coverage of individual exons. We compared the results with those obtained from high-density exon-targeted array comparative genomic hybridization and applied similar analysis to examine samples from patients with pathogenic exonic deletions. RESULTS: Thirty-eight samples, each containing 2,134, 2,833, or 4,688 coding exons from different panels, with a total of 103,863 exons, were analyzed by capture-massively parallel sequencing and array comparative genomic hybridization. Ten deletions detected by array comparative genomic hybridization were all detected by massively parallel sequencing, whereas only two of three duplications were detected. We were able to detect all pathogenic exonic deletions in 11 positive cases. Thirty-one exonic copy number changes from nine perspective clinical samples were also identified. CONCLUSION: Our results demonstrated the feasibility of using the same set of sequence data to detect both point mutations and exonic deletions, thus improving the diagnostic power of massively parallel sequencing-based assays.

Arginine:glycine amidinotransferase (AGAT) deficiency: Clinical features and long term outcomes in 16 patients diagnosed worldwide.

BACKGROUND: Arginine:glycine aminotransferase (AGAT) (GATM) deficiency is an autosomal recessive inborn error of creative synthesis. OBJECTIVE: We performed an international survey among physicians known to treat patients with AGAT deficiency, to assess clinical characteristics and long-term outcomes of this ultra-rare condition. RESULTS: 16 patients from 8 families of 8 different ethnic backgrounds were included. 1 patient was asymptomatic when diagnosed at age 3 weeks. 15 patients diagnosed between 16 months and 25 years of life had intellectual disability/developmental delay (IDD). 8 patients also had myopathy/proximal muscle weakness. Common biochemical denominators were low/undetectable guanidinoacetate (GAA) concentrations in urine and plasma, and low/undetectable cerebral creatine levels. 3 families had protein truncation/null mutations. The rest had missense and splice mutations. Treatment with creatine monohydrate (100-800 mg/kg/day) resulted in almost complete restoration of brain creatine levels and significant improvement of myopathy. The 2 patients treated since age 4 and 16 months had normal cognitive and behavioral development at age 10 and 11 years. Late treated patients had limited improvement of cognitive functions. CONCLUSION: AGAT deficiency is a treatable intellectual disability. Early diagnosis may prevent IDD and myopathy. Patients with unexplained IDD with and without myopathy should be assessed for AGAT deficiency by determination of urine/plasma GAA and cerebral creatine levels (via brain MRS), and by GATM gene sequencing.

Recurrent ACADVL molecular findings in individuals with a positive newborn screen for very long chain acyl-coA dehydrogenase (VLCAD) deficiency in the United States.

Very long chain acyl-coA dehydrogenase deficiency (VLCADD) is an autosomal recessive inborn error of fatty acid oxidation detected by newborn screening (NBS). Follow-up molecular analyses are often required to clarify VLCADD-suggestive NBS results, but to date the outcome of these studies are not well described for the general screen-positive population. In the following study, we report the molecular findings for 693 unrelated patients that sequentially received Sanger sequence analysis of ACADVL as a result of a positive NBS for VLCADD. Highlighting the variable molecular underpinnings of this disorder, we identified 94 different pathogenic ACADVL variants (40 novel), as well as 134 variants of unknown clinical significance (VUSs). Evidence for the pathogenicity of a subset of recurrent VUSs was provided using multiple in silico analyses. Surprisingly, the most frequent finding in our cohort was carrier status, 57% all individuals had a single pathogenic variant or VUS. This result was further supported by follow-up array and/or acylcarnitine analysis that failed to provide evidence of a second pathogenic allele. Notably, exon-targeted array analysis of 131 individuals screen positive for VLCADD failed to identify copy number changes in ACADVL thus suggesting this test has a low yield in the setting of NBS follow-up. While no genotype was common, the c.848T>C (p.V283A) pathogenic variant was clearly the most frequent; at least one copy was found in ~10% of all individuals with a positive NBS. Clinical and biochemical data for seven unrelated patients homozygous for the p.V283A allele suggests that it results in a mild phenotype that responds well to standard treatment, but hypoglycemia can occur. Collectively, our data illustrate the molecular heterogeneity of VLCADD and provide novel insight into the outcomes of NBS for this disorder.

Application of next generation sequencing to molecular diagnosis of inherited diseases.

Recent development of high throughput, massively parallel sequencing (MPS or next generation sequencing, NGS) technology has revolutionized the molecular diagnosis of human genetic disease. The ability to generate enormous amount of sequence data in a short time at an affordable cost makes this approach ideal for a wide range of applications from sequencing a group of candidate genes, all coding regions (known as exome sequencing) to the entire human genome. The technology brings about an unprecedented application to the identification of the molecular basis of hard-to-diagnose genetic disorders. This chapter reviews the up-to-date published application of next generation sequencing in clinical molecular diagnostic laboratories. We also emphasize the various target gene enrichment methods and their advantages and shortcomings. Obstacles to compliance with regulatory authorities like CLIA/CAP in clinical settings are also briefly discussed.

Transition to next generation analysis of the whole mitochondrial genome: a summary of molecular defects.

The diagnosis of mitochondrial disorders is challenging because of the clinical variability and genetic heterogeneity. Conventional analysis of the mitochondrial genome often starts with a screening panel for common mitochondrial DNA (mtDNA) point mutations and large deletions (mtScreen). If negative, it has been traditionally followed by Sanger sequencing of the entire mitochondrial genome (mtWGS). The recently developed "Next-Generation Sequencing" (NGS) technology offers a robust high-throughput platform for comprehensive mtDNA analysis. Here, we summarize the results of the past 6 years of clinical practice using the mtScreen and mtWGS tests on 9,261 and 2,851 unrelated patients, respectively. A total of 344 patients (3.7%) had mutations identified by mtScreen and 99 (3.5%) had mtDNA mutations identified by mtWGS. The combinatorial analyses of mtDNA and POLG revealed a diagnostic yield of 6.7% in patients with suspected mitochondrial disorders but no recognizable syndromes. From the initial mtWGS-NGS cohort of 391 patients, 21 mutation-positive cases (5.4%) have been identified. The mtWGS-NGS provides a one-step approach to detect common and uncommon point mutations, as well as deletions. Additionally, NGS provides accurate, sensitive heteroplasmy measurement, and the ability to map deletion breakpoints. A new era of more efficient molecular diagnosis of mtDNA mutations has arrived.

Comprehensive next-generation sequence analyses of the entire mitochondrial genome reveal new insights into the molecular diagnosis of mitochondrial DNA disorders.

PURPOSE: The application of massively parallel sequencing technology to the analysis of the mitochondrial genome has demonstrated great improvement in the molecular diagnosis of mitochondrial DNA-related disorders. The objective of this study was to investigate the performance characteristics and to gain new insights into the analysis of the mitochondrial genome. METHODS: The entire mitochondrial genome was analyzed as a single amplicon using a long-range PCR-based enrichment approach coupled with massively parallel sequencing. The interference of the nuclear mitochondrial DNA homologs was distinguished from the actual mitochondrial DNA sequences by comparison with the results obtained from conventional PCR-based Sanger sequencing using multiple pairs of primers. RESULTS: Our results demonstrated the uniform coverage of the entire mitochondrial genome. Massively parallel sequencing of the single amplicon revealed the presence of single-nucleotide polymorphisms and nuclear homologs of mtDNA sequences that cause the erroneous and inaccurate variant calls when PCR/Sanger sequencing approach was used. This single amplicon massively parallel sequencing strategy provides an accurate quantification of mutation heteroplasmy as well as the detection and mapping of mitochondrial DNA deletions. CONCLUSION: The ability to quantitatively and qualitatively evaluate every single base of the entire mitochondrial genome is indispensible to the accurate molecular diagnosis and genetic counseling of mitochondrial DNA-related disorders. This new approach may be considered as first-line testing for comprehensive analysis of the mitochondrial genome.Genet Med 2013:15(5):388-394.

Molecular and clinical characterization of the myopathic form of mitochondrial DNA depletion syndrome caused by mutations in the thymidine kinase (TK2) gene.

Mitochondrial DNA (mtDNA) depletion syndromes (MDSs) are a clinically and molecularly heterogeneous group of mitochondrial cytopathies characterized by severe mtDNA copy number reduction in affected tissues. Clinically, MDSs are mainly categorized as myopathic, encephalomyopathic, hepatocerebral, or multi-systemic forms. To date, the myopathic form of MDS is mainly caused by mutations in the TK2 gene, which encodes thymidine kinase 2, the first and rate limiting step enzyme in the phosphorylation of pyrimidine nucleosides. We analyzed 9 unrelated families with 11 affected subjects exhibiting the myopathic form of MDS, by sequencing the TK2 gene. Twelve mutations including 4 novel mutations were detected in 9 families. Skeletal muscle specimens were available from 7 out of 11 subjects. Respiratory chain enzymatic activities in skeletal muscle were measured in 6 subjects, and enzymatic activities were reduced in 3 subjects. Quantitative analysis of mtDNA content in skeletal muscle was performed in 5 subjects, and marked mtDNA content reduction was observed in each. In addition, we outline the molecular and clinical characteristics of this syndrome in a total of 52 patients including those previously reported, and a total of 36 TK2 mutations are summarized. Clinically, hypotonia and proximal muscle weakness are the major phenotypes present in all subjects. In summary, our study expands the molecular and clinical spectrum associated with TK2 deficiency.

SURF1-associated Leigh syndrome: a case series and novel mutations.

Leigh syndrome (LS) is a mitochondrial disease that typically presents in infancy with subacute neurodegenerative encephalopathy. It is genetically heterogeneous, but mutations in the complex IV assembly genes, particularly SURF1, are an important cause. In this study, SURF1 gene was sequenced in 590 patients with clinical suspicion of LS, complex IV deficiency, or clinical features of mitochondrial disorders. We identified 21 patients with clinical features of LS who are either homozygous or compound heterozygous for SURF1 mutations. Twenty-two different mutations were identified, including 13 novel mutations. Of the 42 mutant alleles, 36 (86%) are null mutations (frameshift, splicing, or nonsense) and 6 (14%) are missense. We have also reviewed the previously reported SURF1 mutations and observed a clustering of mutation in exon 8 of SURF1, suggesting a vital function for this region. Although mutations in SURF1 have been mainly associated with typical LS, five of the patients in this report had an atypical course of LS. There is no definite genotype-phenotype correlation; however, frameshift mutations resulting in protein truncation closer to the C-terminus may carry a better prognosis.