W. Lissens

Preimplantation diagnosis for Huntington's disease (HD): clinical application and analysis of the HD expansion in affected embryos

Huntingtons disease (HD) is an autosomal dominant disease characterized by motor disturbance, cognitive loss and psychiatric manifestations, starting between the fourth and the fifth decade, followed by death within 10–20 years of onset of the disease. The disease‐causing mutation is an expansion of a CAG triplet repeat at the 5′ coding end of the Huntington gene. We have developed a single‐cell PCR assay for the HD gene in order to propose preimplantation genetic diagnosis (PGD) for the couples at risk. We present here our first results with our first nine PGD cycles and also discuss the behaviour of the disease‐causing expansion in pre‐implantation embryos. Copyright

Preimplantation genetic diagnosis for sickle‐cell anemia and for β‐thalassemia

We developed single‐cell polymerase chain reaction (PCR) assays for preimplantation genetic diagnosis (PGD) in couples carrying mutations in the β‐globin gene. With PGD the genetic status of an embryo obtained after intracytoplasmic sperm injection (ICSI) is determined by PCR analysis in single blastomeres, allowing only healthy embryos to be transferred to the uterus. We carried out nine PGD cycles using fluorescent PCR for two couples in whom the partners carried sickle‐cell trait. Both couples achieved pregnancies, one of which was spontaneously aborted. We have developed two β‐thalassemia PGD protocols: one for the analysis of the 25‐26delAA and the IVS2+1G>A mutation, and the other for the simultaneous detection of the IVS1+6T>C and the IVS1+110G>A mutations. For the second protocol, both non‐labelled PCR and later fluorescent PCR were used. Both protocols were applied in clinical cycles (two non‐labelled PCR cycles and one fluorescent PCR cycle) for two couples. The patient with the fluorescent PCR‐PGD cycle became pregnant. Overall, the three fluorescent PCR assays were accurate and reliable with amplification efficiencies of minimum 93% and allele dropout (ADO) rates between 0 and 12%. Copyright

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.

Clinical application of preimplantation genetic diagnosis for cystic fibrosis.

Cystic fibrosis (CF) is an autosomal recessive disease characterized by obstruction and chronic infections of the respiratory tract and pancreatic insufficiency. The gene was cloned in 1989 and the most frequent mutation was shown to be the ΔF508 mutation. During PGD, embryos obtained in vitro are checked for the presence or absence of the mutation, after which only embryos shown to be free of the mutation are returned to the mother. Up to 1999, 48 intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) cycles had been carried out for PGD for CF in 24 couples, and different diagnostic tests had been used to select non‐affected embryos. Thirteen patients became pregnant and 12 healthy babies have been born. Copyright

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.

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.

Pitfalls in the diagnosis of mtDNA mutations.

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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.

A D255H substitution in the arylsulphatase A gene of two unrelated Belgian patients with late-infantile metachromatic leukodystrophy

SummaryMetachromatic leukodystrophy (MLD) is an autosomal recessive disease of myelin metabolism caused by a deficiency in the lysosomal enzyme arylsulphatase A (ARSA). We have identified a new mutation in exon 4 of the ARSA gene of two unrelated Belgian patients with late-infantile MLD. The mutation predicts an aspartic acid-to-histidine substitution at position 255 in arylsulphatase A (D255H), in a highly conserved region among sulphatases. Transient expression of the mutation in COS cells did not show an increase in ARSA activity. Both patients were compound heterozygotes carrying the frequent splice site mutation in intron 2 (459+1G→A) on the other allele.