Leukoencephalopathy with Brain stem and Spinal cord involvement and Lactate elevation (LBSL): Report of a new family and a novel DARS2 mutation.

Background: LBSL is a mitochondrial disorder caused by mutations in the mitochondrial aspartyl-tRNA synthetase gene DARS2, resulting in a distinctive pattern on brain magnetic resonance imaging (MRI) and spectroscopy. Clinical presentation varies from severe infantile to chronic, slowly progressive neuronal deterioration in adolescents or adults. Most individuals with LBSL are compound heterozygous for one splicing defect in an intron 2 mutational hotspot and a second defect that could be a missense, non-sense, or splice site mutation or deletion resulting in decreased expression of the full-length protein. Aim: To present a new family with two affected members with LBSL and report a novel DARS2 mutation. Results: An 8-year-old boy (Patient 1) was referred due to headaches and abnormal MRI, suggestive of LBSL. Genetic testing revealed a previously reported c.492 + 2 T > C mutation in the DARS2 gene. Sanger sequencing uncovered a novel variant c.228-17C > G in the intron 2 hotspot. Family studies found the same genetic changes in an asymptomatic 4-year-old younger brother (Patient 2), who was found on follow-up to have an abnormal MRI. mRNA extracted from patients' fibroblasts showed that the c.228-17C > G mutation caused skipping of exon 3 resulting in lower DARS2 mRNA level. Complete absence of DARS2 protein was also found in both patients. Summary: We present a new family with two children affected with LBSL and describe a novel mutation in the DARS2 intron 2 hotspot. Despite findings of extensive white matter disease in the brain and spine, the proband in this family presented only with headaches, while the younger sibling, who also had extensive white matter changes, was asymptomatic. Our in-vitro results confirmed skipping of exon 3 in patients and family members carrying the intron 2 variant, which is consistent with previous reported mutations in intron 2 hotspots. DARS2 mRNA and protein levels were also reduced in both patients, further supporting the pathogenicity of the novel variant.

Population-level deficit of homozygosity unveils CPSF3 as an intellectual disability syndrome gene.

Predicting the pathogenicity of biallelic missense variants can be challenging. Here, we use a deficit of observed homozygous carriers of missense variants, versus an expected number in a set of 153,054 chip-genotyped Icelanders, to identify potentially pathogenic genotypes. We follow three missense variants with a complete deficit of homozygosity and find that their pathogenic effect in homozygous state ranges from severe childhood disease to early embryonic lethality. One of these variants is in CPSF3, a gene not previously linked to disease. From a set of clinically sequenced Icelanders, and by sequencing archival samples targeted through the Icelandic genealogy, we find four homozygous carriers. Additionally, we find two homozygous carriers of Mexican descent of another missense variant in CPSF3. All six homozygous carriers of missense variants in CPSF3 show severe intellectual disability, seizures, microcephaly, and abnormal muscle tone. Here, we show how the absence of certain homozygous genotypes from a large population set can elucidate causes of previously unexplained recessive diseases and early miscarriage.

ECHS1 disease in two unrelated families of Samoan descent: Common variant - rare disorder.

Mutations in the short-chain enoyl-CoA hydratase (SCEH) gene, ECHS1, cause a rare autosomal recessive disorder of valine catabolism. Patients usually present with developmental delay, regression, dystonia, feeding difficulties, and abnormal MRI with bilateral basal ganglia involvement. We present clinical, biochemical, molecular, and functional data for four affected patients from two unrelated families of Samoan descent with identical novel compound heterozygous mutations. Family 1 has three affected boys while Family 2 has an affected daughter, all with clinical and MRI findings of Leigh syndrome and intermittent episodes of acidosis and ketosis. WES identified a single heterozygous variant in ECHS1 at position c.832G > A (p.Ala278Thr). However, western blot revealed significantly reduced ECHS1 protein for all affected family members. Decreased SCEH activity in fibroblasts and a mild increase in marker metabolites in urine further supported ECHS1 as the underlying gene defect. Additional investigations at the DNA (aCGH, WGS) and RNA (qPCR, RT-PCR, RNA-Seq, RNA-Array) level identified a silent, common variant at position c.489G > A (p.Pro163=) as the second mutation. This substitution, present at high frequency in the Samoan population, is associated with decreased levels of normally spliced mRNA. To our understanding, this is the first report of a novel, hypomorphic allele c.489G > A (p.Pro163=), associated with SCEH deficiency.

Novel mutations in the mitochondrial complex I assembly gene NDUFAF5 reveal heterogeneous phenotypes.

Primary mitochondrial complex I deficiency is the most common defect of the mitochondrial respiratory chain. It is caused by defects in structural components and assembly factors of this large protein complex. Mutations in the assembly factor NDUFAF5 are rare, with only five families reported to date. This study provides clinical, biochemical, molecular and functional data for four unrelated additional families, and three novel pathogenic variants. Three cases presented in infancy with lactic acidosis and classic Leigh syndrome. One patient, however, has a milder phenotype, with symptoms starting at 27 months and a protracted clinical course with improvement and relapsing episodes. She is homozygous for a previously reported mutation, p.Met279Arg and alive at 19 years with mild neurological involvement, normal lactate but abnormal urine organic acids. We found the same mutation in one of our severely affected patients in compound heterozygosity with a novel p.Lys52Thr mutation. Both patients with p.Met279Arg are of Taiwanese descent and had severe hyponatremia. Our third and fourth patients, both Caucasian, shared a common, newly described, missense mutation p.Lys109Asn which we show induces skipping of exon 3. Both Caucasian patients were compound heterozygotes, one with a previously reported Ashkenazi founder mutation while the other was negative for additional exonic variants. Whole genome sequencing followed by RNA studies revealed a novel deep intronic variant at position c.223-907A>C inducing an exonic splice enhancer. Our report adds significant new information to the mutational spectrum of NDUFAF5, further delineating the phenotypic heterogeneity of this mitochondrial defect.

Activation of a cryptic splice site in the mitochondrial elongation factor GFM1 causes combined OXPHOS deficiency.

We report the clinical, biochemical, and molecular findings in two brothers with encephalopathy and multi-systemic disease. Abnormal transferrin glycoforms were suggestive of a type I congenital disorder of glycosylation (CDG). While exome sequencing was negative for CDG related candidate genes, the testing revealed compound heterozygous mutations in the mitochondrial elongation factor G gene (GFM1). One of the mutations had been reported previously while the second, novel variant was found deep in intron 6, activating a cryptic splice site. Functional studies demonstrated decreased GFM1 protein levels, suggested disrupted assembly of mitochondrial complexes III and V and decreased activities of mitochondrial complexes I and IV, all indicating combined OXPHOS deficiency.

Mutations in TFAM, encoding mitochondrial transcription factor A, cause neonatal liver failure associated with mtDNA depletion.

In humans, mitochondrial DNA (mtDNA) depletion syndromes are a group of genetically and clinically heterogeneous autosomal recessive disorders that arise as a consequence of defects in mtDNA replication or nucleotide synthesis. Clinical manifestations are variable and include myopathic, encephalomyopathic, neurogastrointestinal or hepatocerebral phenotypes. Through clinical exome sequencing, we identified a homozygous missense variant (c.533C>T; p.Pro178Leu) in mitochondrial transcription factor A (TFAM) segregating in a consanguineous kindred of Colombian-Basque descent in which two siblings presented with IUGR, elevated transaminases, conjugated hyperbilirubinemia and hypoglycemia with progression to liver failure and death in early infancy. Results of the liver biopsy in the proband revealed cirrhosis, micro- and macrovesicular steatosis, cholestasis and mitochondrial pleomorphism. Electron microscopy of muscle revealed abnormal mitochondrial morphology and distribution while enzyme histochemistry was underwhelming. Electron transport chain testing in muscle showed increased citrate synthase activity suggesting mitochondrial proliferation, while respiratory chain activities were at the lower end of normal. mtDNA content was reduced in liver and muscle (11% and 21% of normal controls respectively). While Tfam mRNA expression was upregulated in primary fibroblasts, Tfam protein level was significantly reduced. Furthermore, functional investigations of the mitochondria revealed reduced basal respiration and spare respiratory capacity, decreased mtDNA copy number and markedly reduced nucleoids. TFAM is essential for transcription, replication and packaging of mtDNA into nucleoids. Tfam knockout mice display embryonic lethality secondary to severe mtDNA depletion. In this report, for the first time, we associate a homozygous variant in TFAM with a novel mtDNA depletion syndrome.

Mitochondrial and ion channel gene alterations in autism.

To evaluate the potential importance in autistic subjects of copy number variants (CNVs) that alter genes of relevance to bioenergetics, ionic metabolism, and synaptic function, we conducted a detailed microarray analysis of 69 autism probands and 35 parents, compared to 89 CEU HapMap controls. This revealed that the frequency CNVs of≥100kb and CNVs of≥10 Kb were markedly increased in probands over parents and in probands and parents over controls. Evaluation of CNVs≥1Mb by chromosomal FISH confirmed the molecular identity of a subset of the CNVs, some of which were associated with chromosomal rearrangements. In a number of the cases, CNVs were found to alter the copy number of genes that are important in mitochondrial oxidative phosphorylation (OXPHOS), ion and especially calcium transport, and synaptic structure. Hence, autism might result from alterations in multiple bioenergetic and metabolic genes required for mental function. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).