L. De Meirleir

Respiratory chain complex V deficiency due to a mutation in the assembly gene ATP12

In patients with mitochondrial encephalomyopathies an increasing number of causative gene defects have been detected. The number of identified pathogenic mitochondrial DNA mutations has largely increased over the past 15 years. Recently, much attention has turned to the investigation of nuclear oxidative phosphorylation (OXPHOS) gene defects. Within the OXPHOS defects, complex V deficiency is rarely found and, so far, these defects have only been attributed to mutations in the mitochondrial MTATP6 gene. Mutation analysis of the complete coding regions at the cDNA level of the nuclear ATP11, ATP12, ATPα, ATPβ and ATPγ genes and the mitochondrial MTATP6 and MTAT8 genes was undertaken in two unrelated patients. Blue Native polyacrylamide gel electrophoresis followed by catalytic staining had already documented their complex V decreased activity. Extensive molecular analysis of five nuclear and two mitochondrial genes revealed a mutation in the ATP12 assembly gene in one patient. This mutation is believed to be the cause of the impaired complex V activity. To our knowledge, this is the first report of a pathogenic mutation in a human nuclear encoded ATPase assembly gene.

Mutations in SACS cause atypical and late-onset forms of ARSACS

Background: Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a complex neurodegenerative disorder caused by mutations in SACS. The phenotype consists of a childhood-onset triad of cerebellar ataxia, peripheral neuropathy, and pyramidal tract signs. Objective: To provide more insight into the prevalence of SACS mutations and the variability of the associated phenotype. Methods: Mutation screening of SACS by direct sequencing and multiplex amplicon quantification for detection of intragenic copy number variations in a cohort of 85 index patients with phenotypes suggestive for ARSACS. Additional short tandem repeat (STR) marker analysis was performed for haplotype sharing. Results: In 11 families,18 new SACS mutations were found (12.9% of total cohort). Five patients displayed onset ages in adulthood, a feature not known to be associated with ARSACS. The remaining index patients displayed a classic early onset phenotype. Initial phenotypic presentation was atypical in several patients, obscuring the clinical diagnosis. A founder mutation in SACS was identified in 3 Belgian families. In one isolated patient, an intragenic SACS deletion of exons 3–5 was detected. Partial SACS deletions were not previously described. Conclusions: In this study, we enlarge the ARSACS phenotype and the underlying genetic spectrum of SACS mutations. Patients with ARSACS are more common than previously known and risk underdiagnosis due to late onset age and unusual presentation.

Fulminant Leigh syndrome and sudden unexpected death in a family with the T9176C mutation of the mitochondrial ATPase 6 gene.

We report an Italian family in which the T-to-C point mutation at nucleotide 9176 of the mitochondrial adenosine triphosphate synthetase (mtATPase) 6 gene is associated with an early-onset fulminant form of Leigh syndrome and with sudden unexpected death in two siblings, respectively. Polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis and direct sequencing revealed that the mutation was homoplasmic in mitochondrial DNA of the proband. The T9176C mutation changes a highly conserved leucine to a proline in subunit 6 of the mtATPase gene and is maternally inherited, but the maternal relatives are asymptomatic. This point mutation was initially described in two brothers with bilateral striatal necrosis, a milder variant of Leigh syndrome.

TUBA1A mutations From isolated lissencephaly to familial polymicrogyria

Background: Mutations in the TUBA1A gene have been reported in patients with lissencephaly and perisylvian pachygyria. Methods: Twenty-five patients with malformations of cortical development ranging from lissencephaly to polymicrogyria were screened for mutations in TUBA1A. Results: Two novel heterozygous missense mutations in TUBA1A were identified: c.629A>G (p.Tyr210Cys) occurring de novo in a boy with lissencephaly, and c.13A>C (p.Ile5Leu) affecting 2 sisters with polymicrogyria whose mother presented somatic mosaicism for the mutation. Conclusions: Mutations in TUBA1A have been described in patients with lissencephaly and pachygyria. We report a mutation in TUBA1A as a cause of polymicrogyria. So far, all mutations in TUBA1A have occurred de novo, resulting in isolated cases. This article describes familial recurrence of TUBA1A mutations due to somatic mosaicism in a parent. These findings broaden the phenotypic spectrum associated with TUBA1A mutations and have implications for genetic counseling.

Biochemical and genetic studies of four patients with pyruvate dehydrogenase E1α deficiency

Abstract We report studies of four patients with pyruvate dehydrogenase complex (PDH) deficiency caused by mutations in the E1α subunit. Two unrelated male patients presented with Leigh syndrome and a R263G missense mutation in exon 8. This mutation has previously been described in males with the same phenotype. The two other patients had different novel mutations: (1) an 8-bp deletion at the C-terminus (exon 11) was found in one allele of a young girl suffering from microcephaly and (2) a C88S missense mutation (exon 3) in a boy who only presented with motor neuropathy. These mutations were not found in the mothers of any of the four cases. Immunoblot analysis revealed decreased immunoreactivity for the E1α and E1β subunits in three out of the four patients. These findings confirm that: (1) PDH deficiencies are genetically heterogeneous, (2) the R263G mutation is more frequent in male cases than are other mutations and this amino acid is a hot spot for gene mutations, (3) the last eight amino acids may be important for the conformation of the tetrameric E1-PDH enzyme, and (4) the amino acids at positions 88, 263 and 382–387 are essential for the linking of the α subunit with the β subunit and for the activity of the holoenzyme.

Mutation analysis of the pyruvate dehydrogenase E1 alpha gene in eight patients with a pyruvate dehydrogenase complex deficiency.

Most of the mutations causing deficiency of the pyruvate dehydrogenase (PDH) complex are in the X‐linked E1α gene. We have developed a rapid screening method for the detection of mutations in this gene using reverse transcription of total RNA, polymerase chain reaction amplification of the whole coding region of the gene and single‐strand conformation polymorphism (SSCP) analysis. With this method, we studied eight patients with a PDH complex deficiency, using cultured fibroblasts. In all patients, aberrant SSCP patterns were found and, after sequencing of the corresponding fragments, we were able to identify six new mutations and two mutations already described previously. The mutations are point mutations leading to amino acid substitutions (5) and direct repeat insertions (3). The presence of the mutations was confirmed in genomic fibroblast DNA. The 4 female patients were shown to carry both a normal and a mutated E1α gene.

Mutational study in the PDHA1 gene of 40 patients suspected of pyruvate dehydrogenase complex deficiency

Quintana E, Gort L, Busquets C, Navarro‐Sastre A, Lissens W, Moliner S, Lluch M, Vilaseca MA, De Meirleir L, Ribes A, Briones P, PDH Working Group. Mutational study in the PDHA1 gene of 40 patients suspected of pyruvate dehydrogenase complex deficiency.

Clinical and biochemical spectrum of mitochondrial complex III deficiency caused by mutations in the BCS1L gene

To the Editor: We report on a baby who presented neonatal severe hypotonia, food intake intolerance and vomiting. She soon developed signs of proximal renal tubulopathy (glucosuria, phosphaturia, and aminoaciduria), metabolic lactic acidosis and hepatic involvement. Bilateral cataracts were also noted. Cranial and abdominal radiological studies were normal. At 4 months, she showed nystagmus, hypertonia, microcephaly, developmental delay and failure to thrive. Her neurological condition and metabolic acidosis worsened rapidly and she died at 6 months of age. Biochemical muscle studies demonstrated mitochondrial complex III (CIII) impaired activity. Direct sequencing of the BCS1L gene showed that the baby harboured a paternal 246C.T (p.R45C) and a maternal 279C.T (p.R56X) transitions in exon 1. Most previously reported patients with CIII deficiency due to BCS1L mutations (Table 1) share a phenotype consisting of low birth weight, proximal tubulopathy, hepatopathy, and a progressive neurological picture characterized by hypotonia, developmental delay and postnatal microcephaly (1–5). However, some have a more restricted phenotype affecting either the renal/ hepatic organs or the neurological system. Two patients (5) showed encephalopathy without visceral manifestations, while other had only hepatic manifestations (1). This picture resembles that of Growth Retardation, Aminoaciduria, Cholestasis, Iron Overload, Lactacidosis, Early Death (GRACILE) syndrome, another BCS1L-associated disorder described in the Finnish population (2, 6, 7). Children with GRACILE syndrome present growth retardation, severe renal and liver disease and early death without neurological symptoms. The biochemical picture is also similar to that of impaired CIII caused by BCS1L mutations. In addition, they have liver hemosiderosis and increased ferritin and iron concentrations, a defect of the iron metabolism also described in patients with decreased CIII activity (3). Besides CIII deficiency and GRACILE syndrome, BCS1L mutations can cause Björnstad syndrome, a disorder characterized by a restricted phenotype consisting of hearing loss and twisted hair (5). Ear and hair defects have been described in 3 of 14 cases with BCS1L-associated CIII deficiency. Interestingly, these patients presented a severe neurological picture without renal or hepatic signs, characteristic of the impaired CIII phenotypic picture. This clinical variability observed in children with CIII deficiency caused by BCS1L mutations indicates that its associated phenotype is more heterogeneous than previously thought, ranging from a multisystemic early lethal disorderwith cerebral andvisceral involvement and iron metabolism defects, resembling the GRACILE syndrome, to a predominant neurological picture with ear and hair involvement, overlapping the phenotype of Björnstad syndrome. The underlining mechanism of BCS1L gene defects has not been fully elucidated. Clinically severe mutations are mainly located in the sorting sequence of the gene, while mutations in residues on the external chaperone surface produce a less severe phenotype (5). However, clinical differences have also been observed among patients with CIII deficiency carrying the same BCS1L changes. Our case is the third reported patient carrying the p.R56X and p.R45C mutations. Although all three cases present a severe metabolic acidosis with liver and cerebral dysfunction, involvement of other organs is heterogeneous. One patient did not show signs of tubulopathy and two had altered iron metabolism (3). In addition, our baby showed bilateral congenital cataracts, a defect not previously described in this disorder. CIII activity can range from mild or no defect in fibroblasts to severe deficiency in liver and muscle. Additionally, BCS1L mutations increase the production of reactive oxygen species, which

A mitochondrial DNA microdeletion in a newborn girl with transient lactic acidosis.

The human mitochondrial DNA (mtDNA) genome (16.6 kb) is exclusively involved in the mitochondrial energy generation pathway of the cell. Therefore, knowledge of the mitochondrial genome and its DNA alterations is essential in identifying functional defects of the respiratory chain. During the last decade a growing list of mitochondrial point mutations, deletions and duplications have been associated with various degenerative diseases. We have identified a microdeletion in the mtDNA of lymphocytes and cultured skin fibroblasts of a baby girl. The newly discovered deletion is located at nucleotides 9204-9205 of the mt-ATPase subunit 6 gene and removes the cleavage site between the ATPase 6 and COIII mRNAs. This is of interest because these mRNAs are normally cleaved apart from a polycistronic transcript in the absence of an intervening tRNA, in what might be a novel mechanism for control of gene expression.

Progressive myoclonic epilepsy as an adult-onset manifestation of Leigh syndrome due to m.14487T > C

Background: m.14487T>C, a missense mutation (p.M63V) affecting the ND6 subunit of complex I of the mitochondrial respiratory chain, has been reported in isolated childhood cases with Leigh syndrome (LS) and progressive dystonia. Adult-onset phenotypes have not been reported. Objectives: To determine the clinical–neurological spectrum and associated mutation loads in an extended m.14487T>C family. Methods: A genotype–phenotype correlation study of a Belgian five-generation family with 12 affected family members segregating m.14487T>C was carried out. Clinical and mutation load data were available for nine family members. Biochemical analysis of the respiratory chain was performed in three muscle biopsies. Results: Heteroplasmic m.14487T>C levels (36–52% in leucocytes, 97–99% in muscle) were found in patients with progressive myoclonic epilepsy (PME) and dystonia or progressive hypokinetic-rigid syndrome. Patients with infantile LS were homoplasmic (99–100% in leucocytes, 100% in muscle). We found lower mutation loads (between 8 and 35% in blood) in adult patients with clinical features including migraine with aura, Leber hereditary optic neuropathy, sensorineural hearing loss and diabetes mellitus type 2. Despite homoplasmic mutation loads, complex I catalytic activity was only moderately decreased in muscle tissue. Interpretation: m.14487T>C resulted in a broad spectrum of phenotypes in our family. Depending on the mutation load, it caused severe encephalopathies ranging from infantile LS to adult-onset PME with dystonia. This is the first report of PME as an important neurological manifestation of an isolated mitochondrial complex I defect.