I. Liebaers

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.

Molecular cytogenetic analysis of XX males using Y-specific DNA sequences, including SRY

SummaryXX maleness is the most common condition in which testes develop in the absence of a cytogenetically detectable Y chromosome. Using molecular techniques, it is possible to detect Yp sequences in the majority of XX males. In this study, we could detect Y-specific sequences, including the sex-determining region of the Y chromosome (SRY), using fluorescence in situ hybridization. In 5 out of 6 previously unpublished XX males, SRY was translocated onto the terminal part of an X chromosome. This is the first report in which translocation of an SRY-bearing fragment to an X chromosome in XX males could be directly demonstrated.

Neonatal myotubular myopathy with a probable X-linked inheritance: observations on a new family with a review of the literature

SummaryDuring the 3 weeks of his life, an infant born at term presented pronounced hypotonia, areflexia and generalized paresis with severe respiratory and feeding problems. He was the fourth male in two generations to die in the perinatal period, therefore suggesting an X-linked inheritance. Postmortem examination revealed a centronuclear or myotubular myopathy. The difficulty in distinguishing the signs due to muscle disorder from those due to hypoxaemic encephalopathy is stressed. Infants with centronuclear myopathy have in any case a high risk for hypoxaemic encephalopathy.The literature concerning neonatal centronuclear myopathy with X-linked inheritance is reviewed.

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 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.

The role of fluorinated pyrimidine analogues in the induction of the in vitro expression of the fragile X chromosome

SummaryThe modes of action of 5-fluoro-2′-deoxyuridine (FdUrd) and 5-fluoro-2′-deoxycytidine (FdCyd) were studied in PHA-stimulated lymphocytes from normal volunteer donors and a fragile X patient. In both cell types, FdUrd and FdCyd inhibited cell proliferation at concentrations of 3x10-8M. Thymidylate synthetase was identified as the decisive target for the action of both FdUrd and FdCyd, as judged from the following observations: First, addition of thymidine to the culture medium was able to counteract both FdUrd and FdCyd toxicities, whereas addition of dCyd had no observable effect. Second, inhibition of the in situ thymidylate synthetase activity measured as an increase in the level of [3H]-dThd incorporation coincided with the inhibition of cell proliferation. Third, the inhibition of the thymidylate synthetase-dependent incorporation of [3H]-dUrd into newly synthesized DNA coincided with the inhibition of cell proliferation. The effects of FdUrd and FdCyd on the in vitro expression of fragile site Xq27 of fragile X chromosomes was shown to be based on the depletion of the intracellular pool of thymidine-5′-monophosphate (dTMP), as fudged from the following observations: First, both the FdUrd- and FdCyd-dependent induction of site Xq27 coincided with the antiproliferative effects of the respective fluoropyrimidines. Second, addition of thymidine (dThd) to the culture medium both prevented the expression of site Xq27 and neutralized the cytotoxicity of FdUrd and of FdCyd. On the basis of these findings, we provide further evidence for the concept that the fragile X site is located in an AT-rich region.

Importance of sequence analysis in NARP syndrome

We read with interest the report presented by Klement et al (1994) on the restrictionsite analysis of mitochondrial DNA (mtDNA) in a family with 3 patients suffering from NARP syndrome. NARP (neurogenic muscle weakness, ataxia, retinitis pigmentosa) was originally described by Holt et al (1990): the authors identified a T-to-G transition at nucleotide 8993 resulting in the substitution of an arginine residue for leucine in the mtATPase 6 subunit. Since then, several reports have presented strong evidence that the same mutation can also be the cause of Leigh disease (Tatuch et al 1992). The mtDNA 8993 mutation can be identified by enzymatic digestion because the mutation creates a new site for the AvaI restriction enzyme. Southern blot analysis performed with mtDNA of a patient suffering from NARP shows two bands of approximately 4 kilobases (kb) and 10kb. In normal control mtDNA only one band of 14kb is detected. We wish to comment on this restriction-digest analysis relying on unpublished data from our own laboratory. For some time we have been analysing mtDNA of patients suspected of mitochondrial myopathy. Recently, Southern blot analysis of mtDNA of one of our patients revealed two bands of approximately 4 kb and t0kb after cleavage with AvaI restriction enzyme. Unlike the family Klement and colleagues described, our patient was homoplasmic for the 4kb and 10kb restriction fragments, as was his asymptomatic mother. The index case himself presented a clinical pattern with multiple cerebral infarcts resembling MELAS syndrome. Sequence analysis now confirms that there is a G-to-A transition at bp 8784 that does not cause an amino acid substitution. However, this alteration also creates an AvaI restriction site: the sizes of the mtDNA restriction pattern are almost identical with those of the 8993 NARP/Leigh mutation (plus 209bp on a 4kb DNA band and minus 209bp on a 10kb DNA band). Therefore, AvaI restriction analysis by Southern blotting is unable to make a clear distinction between the 8993 mutation with pathogenic importance and the 8784 polymorphism. In summary, our findings strongly suggest caution since single basepair alterations leading to new AvaI restriction sites in the region of the 8993 mutation can interfere with a diagnosis of the NARP/Leigh mutation at position 8993 based only on AvaI restriction analysis by Southern blotting.