Elizabeth M. McCormick

Primary Respiratory Chain Disease Causes Tissue-Specific Dysregulation of the Global Transcriptome and Nutrient-Sensing Signaling Network

Primary mitochondrial respiratory chain (RC) diseases are heterogeneous in etiology and manifestations but collectively impair cellular energy metabolism. Mechanism(s) by which RC dysfunction causes global cellular sequelae are poorly understood. To identify a common cellular response to RC disease, integrated gene, pathway, and systems biology analyses were performed in human primary RC disease skeletal muscle and fibroblast transcriptomes. Significant changes were evident in muscle across diverse RC complex and genetic etiologies that were consistent with prior reports in other primary RC disease models and involved dysregulation of genes involved in RNA processing, protein translation, transport, and degradation, and muscle structure. Global transcriptional and post-transcriptional dysregulation was also found to occur in a highly tissue-specific fashion. In particular, RC disease muscle had decreased transcription of cytosolic ribosomal proteins suggestive of reduced anabolic processes, increased transcription of mitochondrial ribosomal proteins, shorter 5′-UTRs that likely improve translational efficiency, and stabilization of 3′-UTRs containing AU-rich elements. RC disease fibroblasts showed a strikingly similar pattern of global transcriptome dysregulation in a reverse direction. In parallel with these transcriptional effects, RC disease dysregulated the integrated nutrient-sensing signaling network involving FOXO, PPAR, sirtuins, AMPK, and mTORC1, which collectively sense nutrient availability and regulate cellular growth. Altered activities of central nodes in the nutrient-sensing signaling network were validated by phosphokinase immunoblot analysis in RC inhibited cells. Remarkably, treating RC mutant fibroblasts with nicotinic acid to enhance sirtuin and PPAR activity also normalized mTORC1 and AMPK signaling, restored NADH/NAD+ redox balance, and improved cellular respiratory capacity. These data specifically highlight a common pathogenesis extending across different molecular and biochemical etiologies of individual RC disorders that involves global transcriptome modifications. We further identify the integrated nutrient-sensing signaling network as a common cellular response that mediates, and may be amenable to targeted therapies for, tissue-specific sequelae of primary mitochondrial RC disease.

AGC1 Deficiency Causes Infantile Epilepsy, Abnormal Myelination, and Reduced N -Acetylaspartate

BACKGROUND Whole exome sequencing (WES) offers a powerful diagnostic tool to rapidly and efficiently sequence all coding genes in individuals presenting for consideration of phenotypically and genetically heterogeneous disorders such as suspected mitochondrial disease. Here, we report results of WES and functional validation in a consanguineous Indian kindred where two siblings presented with profound developmental delay, congenital hypotonia, refractory epilepsy, abnormal myelination, fluctuating basal ganglia changes, cerebral atrophy, and reduced N-acetylaspartate (NAA). METHODS Whole blood DNA from one affected and one unaffected sibling was captured by Agilent SureSelect Human All Exon kit and sequenced on the Illumina HiSeq2000. Mutations were validated by Sanger sequencing in all family members. Protein from wild-type and mutant fibroblasts was isolated to assess mutation effects on protein expression and enzyme activity. RESULTS A novel SLC25A12 homozygous missense mutation, c.1058G>A; p.Arg353Gln, segregated with disease in this kindred. SLC25A12 encodes the neuronal aspartate-glutamate carrier 1 (AGC1) protein, an essential component of the neuronal malate/aspartate shuttle that transfers NADH and H(+) reducing equivalents from the cytosol to mitochondria. AGC1 activity enables neuronal export of aspartate, the glial substrate necessary for proper neuronal myelination. Recombinant mutant p.Arg353Gln AGC1 activity was reduced to 15% of wild type. One prior reported SLC25A12 mutation caused complete loss of AGC1 activity in a child with epilepsy, hypotonia, hypomyelination, and reduced brain NAA. CONCLUSIONS These data strongly suggest that SLC25A12 disease impairs neuronal AGC1 activity. SLC25A12 sequencing should be considered in children with infantile epilepsy, congenital hypotonia, global delay, abnormal myelination, and reduced brain NAA.

Assessing genotype-phenotype correlation in Costello syndrome using a severity score

Purpose:Costello syndrome, a rare genetic disorder with multisystemic involvement, is caused by germline HRAS mutations. Because several different missense mutations have been reported, a severity scoring system was developed to assess a possible genotype–phenotype correlation.Methods:Records of 78 individuals with Costello syndrome were scored in early childhood, childhood, and young adulthood by a reviewer blinded to the individuals’ specific mutations. These scores were based on certain medically relevant feeding, neurologic, orthopedic, endocrine, cardiac, malignancy, and mortality manifestations. Individuals’ severity scores were then grouped by the particular HRAS mutation. The mixed-model approach for repeated-measures analysis of variance with unstructured within-subject correlation, pairwise comparisons, and contrast were used to determine whether the severity scores differed by mutation.Results:Although the sample size was small, individuals with the p.G12A or p.G12C HRAS change were more severely affected than those with other HRAS mutations. Regardless of the mutation, severity did not increase significantly over time.Conclusion:Despite its limitations, including the small number of individuals with rare mutations and possibly incomplete medical records, this work providing the first quantitative assessment of phenotypic severity in a Costello syndrome cohort supports a medically relevant genotype–phenotype correlation.Genet Med 2013:15(7):554–557

Loss-of-Function Mutations in FRRS1L Lead to an Epileptic-Dyskinetic Encephalopathy

Glutamatergic neurotransmission governs excitatory signaling in the mammalian brain, and abnormalities of glutamate signaling have been shown to contribute to both epilepsy and hyperkinetic movement disorders. The etiology of many severe childhood movement disorders and epilepsies remains uncharacterized. We describe a neurological disorder with epilepsy and prominent choreoathetosis caused by biallelic pathogenic variants in FRRS1L, which encodes an AMPA receptor outer-core protein. Loss of FRRS1L function attenuates AMPA-mediated currents, implicating chronic abnormalities of glutamatergic neurotransmission in this monogenic neurological disease of childhood.

Mutations in SPECC1L, encoding sperm antigen with calponin homology and coiled-coil domains 1-like, are found in some cases of autosomal dominant Opitz G/BBB syndrome

Background Opitz G/BBB syndrome is a heterogeneous disorder characterised by variable expression of midline defects including cleft lip and palate, hypertelorism, laryngealtracheoesophageal anomalies, congenital heart defects, and hypospadias. The X-linked form of the condition has been associated with mutations in the MID1 gene on Xp22. The autosomal dominant form has been linked to chromosome 22q11.2, although the causative gene has yet to be elucidated. Methods and results In this study, we performed whole exome sequencing on DNA samples from a three-generation family with characteristics of Opitz G/BBB syndrome with negative MID1 sequencing. We identified a heterozygous missense mutation c.1189A>C (p.Thr397Pro) in SPECC1L, located at chromosome 22q11.23. Mutation screening of an additional 19 patients with features of autosomal dominant Opitz G/BBB syndrome identified a c.3247G>A (p.Gly1083Ser) mutation segregating with the phenotype in another three-generation family. Conclusions Previously, SPECC1L was shown to be required for proper facial morphogenesis with disruptions identified in two patients with oblique facial clefts. Collectively, these data demonstrate that SPECC1L mutations can cause syndromic forms of facial clefting including some cases of autosomal dominant Opitz G/BBB syndrome and support the original linkage to chromosome 22q11.2.

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

The synthesis of all 13 mitochondrial DNA (mtDNA)-encoded protein subunits of the human oxidative phosphorylation (OXPHOS) system is carried out by mitochondrial ribosomes (mitoribosomes). Defects in the stability of mitoribosomal proteins or mitoribosome assembly impair mitochondrial protein translation, causing combined OXPHOS enzyme deficiency and clinical disease. Here we report four autosomal-recessive pathogenic mutations in the gene encoding the small mitoribosomal subunit protein, MRPS34, in six subjects from four unrelated families with Leigh syndrome and combined OXPHOS defects. Whole-exome sequencing was used to independently identify all variants. Two splice-site mutations were identified, including homozygous c.321+1G>T in a subject of Italian ancestry and homozygous c.322-10G>A in affected sibling pairs from two unrelated families of Puerto Rican descent. In addition, compound heterozygous MRPS34 mutations were identified in a proband of French ancestry; a missense (c.37G>A [p.Glu13Lys]) and a nonsense (c.94C>T [p.Gln32∗]) variant. We demonstrated that these mutations reduce MRPS34 protein levels and the synthesis of OXPHOS subunits encoded by mtDNA. Examination of the mitoribosome profile and quantitative proteomics showed that the mitochondrial translation defect was caused by destabilization of the small mitoribosomal subunit and impaired monosome assembly. Lentiviral-mediated expression of wild-type MRPS34 rescued the defect in mitochondrial translation observed in skin fibroblasts from affected subjects, confirming the pathogenicity of MRPS34 mutations. Our data establish that MRPS34 is required for normal function of the mitoribosome in humans and furthermore demonstrate the power of quantitative proteomic analysis to identify signatures of defects in specific cellular pathways in fibroblasts from subjects with inherited disease.

A novel HSD17B10 mutation impairing the activities of the mitochondrial RNase P complex causes X-linked intractable epilepsy and neurodevelopmental regression

ABSTRACT We report a Caucasian boy with intractable epilepsy and global developmental delay. Whole-exome sequencing identified the likely genetic etiology as a novel p.K212E mutation in the X-linked gene HSD17B10 for mitochondrial short-chain dehydrogenase/reductase SDR5C1. Mutations in HSD17B10 cause the HSD10 disease, traditionally classified as a metabolic disorder due to the role of SDR5C1 in fatty and amino acid metabolism. However, SDR5C1 is also an essential subunit of human mitochondrial RNase P, the enzyme responsible for 5′-processing and methylation of purine-9 of mitochondrial tRNAs. Here we show that the p.K212E mutation impairs the SDR5C1-dependent mitochondrial RNase P activities, and suggest that the pathogenicity of p.K212E is due to a general mitochondrial dysfunction caused by reduction in SDR5C1-dependent maturation of mitochondrial tRNAs.

Muscle oxidative phosphorylation quantitation using creatine chemical exchange saturation transfer (CrCEST) MRI in mitochondrial disorders

Systemic mitochondrial energy deficiency is implicated in the pathophysiology of many age-related human diseases. Currently available tools to estimate mitochondrial oxidative phosphorylation (OXPHOS) capacity in skeletal muscle in vivo lack high anatomic resolution. Muscle groups vary with respect to their contractile and metabolic properties. Therefore, muscle group-specific estimates of OXPHOS would be advantageous. To address this need, a noninvasive creatine chemical exchange saturation transfer (CrCEST) MRI technique has recently been developed, which provides a measure of free creatine. After exercise, skeletal muscle can be imaged with CrCEST in order to make muscle group-specific measurements of OXPHOS capacity, reflected in the recovery rate (τCr) of free Cr. In this study, we found that individuals with genetic mitochondrial diseases had significantly (P = 0.026) prolonged postexercise τCr in the medial gastrocnemius muscle, suggestive of less OXPHOS capacity. Additionally, we observed that lower resting CrCEST was associated with prolonged τPCr, with a Pearsons correlation coefficient of -0.42 (P = 0.046), consistent with previous hypotheses predicting that resting creatine levels may correlate with 31P magnetic resonance spectroscopy-based estimates of OXPHOS capacity. We conclude that CrCEST can noninvasively detect changes in muscle creatine content and OXPHOS capacity, with high anatomic resolution, in individuals with mitochondrial disorders.

Desmin common mutation is associated with multi-systemic disease manifestations and depletion of mitochondria and mitochondrial DNA

Desmin (DES) is a major muscle scaffolding protein that also functions to anchor mitochondria. Pathogenic DES mutations, however, have not previously been recognized as a cause of multi-systemic mitochondrial disease. Here, we describe a 45-year-old man who presented to The Childrens Hospital of Philadelphia Mitochondrial-Genetics Diagnostic Clinic for evaluation of progressive cardiac, neuromuscular, gastrointestinal, and mood disorders. Muscle biopsy at age 45 was remarkable for cytoplasmic bodies, as well as ragged red fibers and SDH positive/COX negative fibers that were suggestive of a mitochondrial myopathy. Muscle also showed significant reductions in mitochondrial content (16% of control mean for citrate synthase activity) and mitochondrial DNA (35% of control mean). His family history was significant for cardiac conduction defects and myopathy in multiple maternal relatives. Multiple single gene and panel-based sequencing studies were unrevealing. Whole exome sequencing identified a known pathogenic p.S13F mutation in DES that had previously been associated with desmin-related myopathy. Desmin-related myopathy is an autosomal dominant disorder characterized by right ventricular hypertrophic cardiomyopathy, myopathy, and arrhythmias. However, neuropathy, gastrointestinal dysfunction, and depletion of both mitochondria and mitochondrial DNA have not previously been widely recognized in this disorder. Recognition that mitochondrial dysfunction occurs in desmin-related myopathy clarifies the basis for the multi-systemic manifestations, as are typical of primary mitochondrial disorders. Understanding the mitochondrial pathophysiology of desmin-related myopathy highlights the possibility of new therapies for this otherwise untreatable and often fatal class of disease. We postulate that drug treatments aimed at improving mitochondrial biogenesis or reducing oxidative stress may be effective therapies to ameliorate the effects of desmin-related disease.

Mitochondrial disease patient motivations and barriers to participate in clinical trials

Background Clinical treatment trials are increasingly being designed in primary mitochondrial disease (PMD), a phenotypically and genetically heterogeneous collection of inherited multi- system energy deficiency disorders that lack effective therapy. We sought to identify motivating factors and barriers to clinical trial participation in PMD. Methods A survey study was conducted in two independent mitochondrial disease subject cohorts. A discovery cohort invited subjects with well-defined biochemical or molecularly- confirmed PMD followed at a single medical center (CHOP, n = 30/67 (45%) respondents). A replication cohort included self-identified PMD subjects in the Rare Disease Clinical Research Network (RDCRN) national contact registry (n = 290/1119 (26%) respondents). Five-point Likert scale responses were analyzed using descriptive and quantitative statistics. Experienced and prioritized symptoms for trial participation, and patient attitudes toward detailed aspects of clinical trial drug features and study design. Results PMD subjects experienced an average of 16 symptoms. Muscle weakness, chronic fatigue, and exercise intolerance were the lead symptoms encouraging trial participation. Motivating trial design factors included a self-administered study drug; vitamin, antioxidant, natural or plant-derivative; pills; daily treatment; guaranteed treatment access during and after study; short travel distances; and late-stage (phase 3) participation. Relative trial participation barriers included a new study drug; discontinuation of current medications; disease progression; daily phlebotomy; and requiring participant payment. Treatment trial type or design preferences were not influenced by population age (pediatric versus adult), prior research trial experience, or disease severity. Conclusions These data are the first to convey clear PMD subject preferences and priorities to enable improved clinical treatment trial design that cuts across the complex diversity of disease. Partnering with rare disease patient communities is essential to effectively design robust clinical trials that engage patients and enable meaningful evaluation of emerging treatment interventions.