S Seneca

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.

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.

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.

Immunohistochemical analysis of the oxidative phosphorylation complexes in skeletal muscle from patients with mitochondrial DNA encoded tRNA gene defects.

Background: Mitochondrial diseases display a heterogeneous spectrum of clinical phenotypes and therefore the identification of the underlying gene defect is often a difficult task. Aims: To develop an immunohistochemical approach to stain skeletal muscle for the five multi-protein complexes that organise the oxidative phosphorylation (OXPHOS) in order to improve the diagnostic workup of mitochondrial defects. Methods: OXPHOS complexes were visualised in skeletal muscle tissue using antibodies directed against different subunits. The staining patterns of patients with heteroplasmic defects in mtDNA tRNA genes were compared with those of normal and disease controls. Results: Normal skeletal muscle displayed a checkerboard staining pattern for complexes I to V due to the higher mitochondrial content of slow muscle fibres versus fast fibres. In patients with tRNA defects, a much more heterogeneous staining pattern was observed for complex I (all six patients) and complex IV (4 of 6 patients): a mosaic staining pattern in which individual fibres displayed staining intensities that ranged from strong to negative. Ragged red fibres (RRFs) in patients with MERRF (myoclonic epilepsy and ragged red fibres) were all complex I and IV negative, while in patient with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) the majority of RRFs were complex I negative and complex IV positive. Conclusion: Immunohistochemical detection of OXPHOS complexes could represent a valuable additional diagnostic tool for the evaluation of mitochondrial cytopathy. The technique helps to detect heteroplasmic mtDNA defects. Staining for complex I in particular was able to identify two tRNA patients that stayed undetected with routine histochemical evaluation.

Intractable ulcerative colitis of infancy in a child with mitochondrial respiratory chain disorder.

Inflammatory bowel diseases are rare in young infants. Most cases of colitis are associated with necrotizing enterocolitis, bacterial infection, or intolerance to dietary proteins. Some cases are associated with immune disorders, Behçet disease, or vascular ischemic causes (systemic vasculitis, hemolytic uremic syndrome) (1). There is one report of five infants with intractable ulcerative enterocolitis of infancy (2). We report a 2-month-old child with a presentation suggesting this last diagnosis. However, elevated lactic acidosis and subsequent onset of a cardiomyopathy led to the discovery of a defect of mitochondrial respiratory chain complex I.

Pitfalls in the diagnosis of mtDNA mutations.

1 Combarros 0, Calleja J, Polo JM, Berciano J. Prevalence of hereditary motor and sensory neuropathy in Cantabria. Acta Neurol Scand 1987;75:9-12. 2 Lupski JR, Montes de Oca-Luna R, Slaugenhaupt S, et al. DNA duplication associated with Charcot-Marie-Tooth disease type ia. Cell 1991;66:219-32. 3 Raeymaekers P, Timmerman V, Nelis E, et al and the HMSN Collaborative Research Group. Duplication in chromosome 17pIl .2 in Charcot-Marie-Tooth neuropathy type 1 a (CMT la). Neuromusc Disord 199 1;1:93-7. 4 Brice A, Ravise N, Stevanin G, et al. Duplication within chromosome 17pl 1.2 in 12 families of French ancestry with Charcot-Marie-Tooth disease type lA. Med Genet 1992;29:807-12. 5 Pentao L, Wise CA, Chinault AC, Patel PI, Lupski JR. Charcot-Marie-Tooth type IA duplication appears to arise from recombination at repeat sequences flanking the 1.5 Mb monomere unit. Nat Genet 1992;2:292-300. 6 Chance PF, Abbas N, Lensch NW, et al. Two autosomal dominant neuropathies result from reciprocal DNA duplication/deletion of a region on chromosome 17. Hum Mol Genet 1994;3:223-8. 7 Reiter LT, Murakami T, Koeuth T, et al. A recombination hotspot responsible for two inherited peripheral neuropathies is located near a mariner transposon-like element. Nat Genet 1996;12:288-97. 8 Timmerman V, Rautenstrauss B, Reiter LT, et al. Detection of the CMTlA/HNPP recombination hotspot in unrelated patients of European descent. JMed Genet 1997;34:43-9. 9 Haupt A, Schols L, Przuntek H, Epplen JT. Polymorphisms in the PMP-22 gene region (1 7pll .2-12) are crucial for simplified diagnosis of duplications/deletions. Hum Genet 1997; 99:688-91. 10 Wise CA, Garcia CA, Davis SN, et al. Molecular analyses of unrelated Charcot-Marie-Tooth (CMT) disease patients suggest a high frequency of the CMT1A duplication. Am 7Hum Genet 1993;53:853-63. 11 Navon R, Timmerman V, L6fgren A, et al. Prenatal diagnosis of Charcot-Marie-Tooth disease type 1A (CMT1A) using molecular genetic techniques. Prenat Diagn 1995;15:63340. 12 Kiyosawa H, Lensch W, Chance PF. Analysis of the CMTlA-REP repeat: mapping crossover breakpoints in CMT1A and HNPP. Hum Mol Genet 1995;4:2327-34.

Pyruvate dehydrogenase E1α deficiency in a family: Different clinical presentation in two siblings

pyruvate dehydrogenase (PDH) complex (PDHc) is responsible for the irreversThe ible conversion of pyruvate to acetyl-CoA. PDHc is a multienzyme complex consisting of three catalytic subunits, pyruvate decarboxylase (E1), dihydrolipoamide acetyltransferase (E2), dihydrolipoamide dehydrogenase (E3), and two regulatory subunits, E1 kinase and phospho-E1 phosphatase. An abnormal E1a subunit, whose gene is located on the X chromosome, is the most frequent cause of PDH deÐciency. The clinical presentation of a PDH-E1a deÐciency (McKusick 312170) is variable. We have analysed a family with a mutation (36 bp insertion in exon 10) in the PDH-E1a gene in which the male member had a di†erent and less severe clinical picture than his a†ected sister.

Pyruvate dehydrogenase complex deficiency and absence of subunit X

The pyruvate dehydrogenase complex (PDHc) is a multienzyme complex consisting of three catalytic and two regulatory enzymes, as well as a less well defined subunit called protein X. PDHc deficiency is a common cause of congenital lactic acidosis. Most patients with PDH deficiency have a mutation in the α chain of the PDH E1 enzyme. Very few patients have been described in whom the basic defect of a PDH deficiency is situated in the X protein. We studied a boy with severe lactic acidosis and developmental delay in whom a deficiency of PDH activity led to further investigations. Immunochemical analysis with anti-PDHc antibodies demonstrated an absence of the X component. This report is the fourth family in which an abnormal protein X has been found. In cases with PDH deficiency where no mutation of the PDHE1α gene is found, further investigations by means of immunoblotting with specific antibodies against the different subunits should be performed.