The role of nuclear-encoded subunit genes in mitochondrial complex 1 deficiency

BACKGROUND: Mitochondrial complex I deficiency often leads to a devastating neurodegenerative disorder of childhood. In most cases, the underlying genetic defect is unknown. Recessive nuclear gene mutations, rather than mitochondrial DNA mutations, account for the majority of cases. AIM: Our aim was to identify the genetic basis of complex I deficiency in 34 patients with isolated complex I deficiency, by studying six of the 39 nuclear encoded complex I subunit genes (NDUFV1, NDUFS1, NDUFS2, NDUFS4, NDUFS7 and NDUFS8). These genes have been conserved throughout evolution and carry out essential aspects of complex I function. METHODS: RNA was extracted from patient fibroblasts and cDNA made by reverse transcription. Overlapping amplicons that together spanned the entire coding area of each gene were amplified by PCR. The genes were screened for mutations using denaturing High Performance Liquid Chromatography (dHPLC). Patient samples with abnormal dHPLC profiles underwent direct DNA sequencing. RESULTS: Novel mutations were identified in six of 34 (18%) patients with isolated complex I deficiency. Five patients had two mutations identified and one patient had a single mutation in NDUFS4 identified. All patients with mutations had a progressive encephalopathy and five out of six had Leigh syndrome or Leigh like syndrome. Mutations were found in three nuclear encoded subunit genes, NDUFV1, NDUFS2 and NDUFS4. Three novel NDUFV1 mutations were identified (R386H, K111E and P252R). The R386H mutation was found in two apparently unrelated patients. Four novel NDUFS2 mutations were identified (R221X, M292T, R333Q and IVS9+4A&ltG). The novel NDUFS4 mutation c.221delC was found in two patients - one in homozygous form and the other heterozygous. Specific genotype and phenotype correlations were not identified. CONCLUSIONS: Nuclear encoded complex I subunit gene mutations are an important contributor to the aetiology of isolated complex I deficiency in childhood. Screening of these genes is an essential part of the investigation of complex I deficiency

Biochemical and molecular diagnosis of mitochondrial respiratory chain disorders

AbstractBiochemical diagnosis of mitochondrial respiratory chain disorders requires caution to avoid misdiagnosis of secondary enzyme defects, and can be improved by the use of conservative diagnostic criteria. Pathogenic mutations causing mitochondrial disorders have now been identified in more than 30 mitochondrial DNA (mtDNA) genes encoding respiratory chain subunits, ribosomal- and t-RNAs. mtDNA mutations appear to be responsible for most adult patients with mitochondrial disease and approximately a quarter of paediatric patients. A family history suggesting maternal inheritance is the exception rather than the norm for children with mtDNA mutations, many of whom have de novo mutations. Prenatal diagnosis and pre-implantation genetic diagnosis can be offered to some women at risk of transmitting a mtDNA mutation, particularly those at lower recurrence risk. Mutations in more than 30 nuclear genes, including those encoding for respiratory chain subunits and assembly factors, have now been shown to cause mitochondrial disorders, creating difficulties in prioritising which genes should be studied by mutation analysis in individual patients. A number of approaches offer promise to guide the choice of candidate genes, including Blue Native-PAGE immunoblotting and microarray expression analysis

The p.M292T NDUFS2 mutation causes complex I-deficient Leigh syndrome in multiple families

Isolated complex I deficiency is the most frequently observed oxidative phosphorylation defect in children with mitochondrial disease, leading to a diverse range of clinical presentations, including Leigh syndrome. For most patients the genetic cause of the biochemical defect remains unknown due to incomplete understanding of the complex I assembly process. Nonetheless, a plethora of pathogenic mutations have been described to date in the seven mitochondrial-encoded subunits of complex I as well as in 12 of the nuclear-encoded subunits and in six assembly factors. Whilst several mitochondrial DNA mutations are recurrent, the majority of these mutations are reported in single families. We have sequenced core structural and functional nuclear-encoded subunits of complex I in a cohort of 34 paediatric patients with isolated complex I deficiency, identifying pathogenic mutations in 6 patients. These included a novel homozygous NDUFS1 mutation in an Asian child with Leigh syndrome, a previously identified NDUFS8 mutation (c.236C>T, p.P79L) in a second Asian child with Leigh-like syndrome and six novel, compound heterozygous NDUFS2 mutations in four white Caucasian patients with Leigh or Leigh-like syndrome. Three of these children harboured an identical NDUFS2 mutation (c.875T>C, p.M292T), which was also identified in conjunction with a novel NDUFS2 splice site mutation (c.866+4A>G) in a fourth Caucasian child who presented to a different diagnostic centre, with microsatellite and single nucleotide polymorphism analyses indicating that this was due to an ancient common founder event. Our results confirm that NDUFS2 is a mutational hotspot in Caucasian children with isolated complex I deficiency and recommend the routine diagnostic investigation of this gene in patients with Leigh or Leigh-like phenotypes

Leadership of primary school foundation subjects: a mixed-methods case study of perceptions

This research examines the role foundation subject leaders in a primary school context, understanding varying factors that support or limit fulfilment of the responsibility. A case study approach investigated knowledge and experiences of participant subject leaders, operating within the context of a primary school Multi-Academy Trust. A mixed methodology was applied, gathering data from questionnaires and 1:1 semi-structured interviews. Findings suggest that whilst accountability measures, and distribution of leadership responsibilities both impacted subject leaders, volume of time and professional development were perceived to be the most influencing factors to those in middle leadership positions

A novel mutation in GMPPA in siblings with apparent intellectual disability, epilepsy, dysmorphism, and autonomic dysfunction.

GMPPA encodes the GDP-mannose pyrophosphorylase A protein (GMPPA). The function of GMPPA is not well defined, however it is a homolog of GMPPB which catalyzes the reaction that converts mannose-1-phosphate and guanosine-5'-triphosphate to GDP-mannose. Previously, biallelic mutations in GMPPA were reported to cause a disorder characterized by achalasia, alacrima, neurological deficits, and intellectual disability. In this study, we report a female proband with achalasia, alacrima, hypohydrosis, apparent intellectual disability, seizures, microcephaly, esotropia, and craniofacial dysmorphism. Exome sequencing identified a previously unreported homozygous c.853+1G>A variant in GMPPA in the proband and her affected sister. Their unaffected parents were heterozygous, and unaffected brother homozygous wild type for this variant. Lymphoblast cells from the affected sisters showed complete loss of the GMPPA protein by Western blotting, and increased levels of GDP-mannose in lymphoblasts on high performance liquid chromatography. Based on our findings and the previous report describing patients with an overlapping phenotype, we conclude that this novel variant in GMPPA, identified by exome sequencing in the proband and her affected sister, is the genetic cause of their phenotype and may expand the known phenotype of this recently described glycosylation disorder

Recessive Inactivating Mutations in TBCK, Encoding a Rab GTPase-Activating Protein, Cause Severe Infantile Syndromic Encephalopathy

Infantile encephalopathies are a group of clinically and biologically heterogeneous disorders for which the genetic basis remains largely unknown. Here, we report a syndromic neonatal encephalopathy characterized by profound developmental disability, severe hypotonia, seizures, diminished respiratory drive requiring mechanical ventilation, brain atrophy, dysgenesis of the corpus callosum, cerebellar vermis hypoplasia, and facial dysmorphism. Biallelic inactivating mutations in TBCK (TBC1-domain-containing kinase) were independently identified by whole-exome sequencing as the cause of this condition in four unrelated families. Matching these families was facilitated by the sharing of phenotypic profiles and WES data in a recently released web-based tool (Geno2MP) that links phenotypic information to rare variants in families with Mendelian traits. TBCK is a putative GTPase-activating protein (GAP) for small GTPases of the Rab family and has been shown to control cell growth and proliferation, actin-cytoskeleton dynamics, and mTOR signaling. Two of the three mutations (c.376C>T [p.Arg126(∗)] and c.1363A>T [p.Lys455(∗)]) are predicted to truncate the protein, and loss of the major TBCK isoform was confirmed in primary fibroblasts from one affected individual. The third mutation, c.1532G>A (p.Arg511His), alters a conserved residue within the TBC1 domain. Structural analysis implicated Arg511 as a required residue for Rab-GAP function, and in silico homology modeling predicted impaired GAP function in the corresponding mutant. These results suggest that loss of Rab-GAP activity is the underlying mechanism of disease. In contrast to other disorders caused by dysregulated mTOR signaling associated with focal or global brain overgrowth, impaired TBCK function results in progressive loss of brain volume