Frequency of mitochondrial transfer RNA mutations and deletions in 225 patients presenting with respiratory chain deficiencies.

OBJECTIVE: To evaluate the frequency of pathogenic mtDNA transfer RNA mutations and deletions in biochemically demonstrable respiratory chain (RC) deficiencies in paediatric and adult patients. METHODS: We screened for deletions and sequenced mitochondrial transfer RNA genes in skeletal muscle DNA from 225 index patients with clinical symptoms suggestive of a mitochondrial disorder and with biochemically demonstrable RC deficiency in skeletal muscle. RESULTS: We found pathogenic mitochondrial DNA mutations in 29% of the patients. The detection rate was significantly higher in adults (48%) than in the paediatric group (18%). Only one pathogenic mutation was detected in the neonatal group. In addition, we describe seven novel transfer RNA sequence variations with unknown pathogenic relevance (six homoplasmic and one heteroplasmic) and 13 homoplasmic polymorphisms. One heteroplasmic transfer RNA(Leu(UUR)) A>G mutation at position 3274 is associated with a distinct neurological syndrome. CONCLUSIONS: We provide an estimation of the frequency of mitochondrial transfer RNA mutations and deletions in paediatric and adult patients with respiratory chain deficiencies.

Limb girdle and facial weakness in female carriers of X-linked myotubular myopathy mutations.

Although X-linked myotubular myopathy (XLMTM) is a recessive disorder, heterozygous female carriers of MTM1 mutations may present with limb girdle and facial weakness. It is proposed that manifesting heterozygote females with XLMTM have a skewed pattern of X-chromosome inactivation. However, skewed X-chromosome inactivation was not detected in either the lymphocyte or muscle DNA of a woman who presented with limb girdle/facial weakness and was found to be heterozygous for the R224X mutation.

MTM1 mutations in X-linked myotubular myopathy.

X-linked myotubular myopathy (XLMTM; MIM# 310400) is a severe congenital muscle disorder caused by mutations in the MTM1 gene. This gene encodes a dual-specificity phosphatase named myotubularin, defining a large gene family highly conserved through evolution (which includes the putative anti-phosphatase Sbf1/hMTMR5). We report 29 mutations in novel cases, including 16 mutations not described before. To date, 198 mutations have been identified in unrelated families, accounting for 133 different disease-associated mutations which are widespread throughout the gene. Most point mutations are truncating, but 26% (35/133) are missense mutations affecting residues conserved in the Drosophila ortholog and in the homologous MTMR1 gene. Three recurrent mutations affect 17% of the patients, and a total of 21 different mutations were found in several independent families. The frequency of female carriers appears higher than expected (only 17% are de novo mutations). While most truncating mutations cause the severe and early lethal phenotype, some missense mutations are associated with milder forms and prolonged survival (up to 54 years).

Buccal cell DNA analysis in premature and term neonates: screening for mutations of the complete coding region for the cystic fibrosis transmembrane conductance regulator.

UNLABELLED: Traditionally, cystic fibrosis (CF) is diagnosed either by measuring sweat electrolyte levels or by screening for mutations using genomic DNA isolated from leucocytes. The aim of this work was to develop a modified fast and non-invasive tool for the collection of cell samples and the genetic analysis of the entire coding region for the cystic fibrosis transmembrane conductance regulator (CFTR) in newborns, especially premature infants. Cell samples were taken by scraping the buccal mucus with tiny dental brushes, followed by DNA isolation and mutation analysis using SSCP-heteroduplex (single-strand conformation polymorphism) screening and sequencing. We have demonstrated that buccal cell DNA collected from premature and term newborns yields sufficient DNA (at least 60 ng) to perform a mutation screening of the complete CFTR coding region, independently of the patients' weight (mean 2200 g) or gestational age (mean 35 weeks). The high stability of the samples at room temperature admits the possibility of dry shipment of samples collected elsewhere to the diagnostic laboratory. CONCLUSION: This fast, non-invasive sampling and DNA isolation method allows for early diagnosis of CF, initiation of therapy and minimisation of parental uncertainty and offers a technique for mutation analysis in any other monogenic disorder.

[New advances in muscular dystrophy: an up-to-date diagnostic plan]. / Neue Erkenntnisse bei den Muskeldystrophien: aktualisierter Abklärungsplan.

During the last few years new findings from molecular studies have much advanced the understanding of muscular dystrophies. A unifying concept has been put forward to explain many forms of such disorders. A complex of several proteins associated to dystrophin has been described which links the cytoskeleton to the extracellular matrix. The components of this complex can be examined at molecular and protein levels. This allows a search for an exact diagnosis in many such patients. Relevant new facts are reviewed in the frame of a traditional approach to the diagnosis of muscular dystrophies.

Two buffer PAGE system-based SSCP/HD analysis: a general protocol for rapid and sensitive mutation screening in cystic fibrosis and any other human genetic disease.

The large size of many disease genes and the multiplicity of mutations complicate the design of an adequate assay for the identification of disease-causing variants. One of the most successful methods for mutation detection is the single strand conformation polymorphism (SSCP) technique. By varying temperature, gel composition, ionic strength and additives, we optimised the sensitivity of SSCP for all 27 exons of the CFTR gene. Using simultaneously SSCP and heteroduplex (HD) analysis, a total of 80 known CF mutations (28 missense, 22 frameshift, 17 nonsense, 13 splicesite) and 20 polymorphisms was analysed resulting in a detection rate of 97.5% including the 24 most common mutations worldwide. The ability of this technique to detect mutations independent of their nature, frequency, and population specificity was confirmed by the identification of five novel mutations (420del9, 1199delG, R560S, A613T, T1299I) in Swiss CF patients, as well as by the detection of 41 different mutations in 198 patients experimentally analysed. We present a three-stage screening strategy allowing analysis of seven exons within 5 hours and analysis of the entire coding region within 1 week, including sequence analysis of the variants. Additionally, our protocol represents a general model for point mutation analysis in other genetic disorders and has already been successfully established for OTC deficiency, collagene deficiency, X-linked myotubular myopathy (XLMTM), Duchenne and Becker muscular dystrophy (DMD, BMD), Wilson disease (WD), Neurofibromatosis I and II, Charcot-Marie-Tooth disease, hereditary neuropathy with liability to pressure palsies, and defects in mitochondrial DNA. No other protocol published so far presents standard SSCP/HD conditions for mutation screening in different disease genes.

Skewed X-inactivation in a manifesting carrier of X-linked myotubular myopathy and in her non-manifesting carrier mother.

X-linked recessive myotubular myopathy (XLMTM) is a muscle disorder usually affecting newborn males. In the majority of cases, muscle weakness and hypotonia lead to a rapid demise at neonatal age. The responsible MTM1 gene is located in proximal Xq28. Heterozygous carriers are described as being asymptomatic but, in a few cases, mild facial weakness has been reported. We report a family in which a 39-year old female showed severe progressive muscle weakness. XLMTM was initially diagnosed in the male offspring of one of the patient's sisters. The patient, one of her sisters, and their mother were heterozygous carriers for a common MTM1 gene mutation. We found an extremely skewed X-inactivation pattern in the patient and, in the opposite direction, in her non-manifesting carrier mother, thus explaining her normal phenotype and indicating a possible inheritance of skewed X-inactivation. Linkage analysis excluded a possible involvement of the XIST locus at Xq13.

Characterization of 34 novel and six known MTM1 gene mutations in 47 unrelated X-linked myotubular myopathy patients.

X-linked myotubular myopathy (XLMTM) is a congenital muscle disorder mainly affecting newborn males. Neonatal muscle weakness and hypotonia usually leads to a rapid demise. The responsible gene, MTM1, was isolated in 1996, and mutational data derived from 90 patients have been published. We report on our findings in a further 53 patients, using genomic DNA and mRNA screening protocols. Thirty-four novel mutations were identified in 37 cases, and six known mutations found in 10 other patients. The 34 new mutations include five large deletions, eight nonsense, six frameshift, five missense, and eight splice-site mutations, whereas two intronic variants causing partial exon skipping represent the first report on such a mechanism in MTM1. Two deletions, one involving exon 1, and the second exon 15, are the first defects to be identified in these exons. The heterogeneity of the mutations, their mutational origins, and the varied ethnic backgrounds of the patients, indicate that the majority of XLMTM families are affected by unique MTM1 mutations.

Association of two silent polymorphisms of platelet glycoprotein Ia/IIa receptor with risk of myocardial infarction: a case-control study.

BACKGROUND: The platelet membrane glycoprotein Ia/IIa plays a major part in platelet function as a primary receptor for collagen. A previous report showed a variation of glycoprotein Ia/IIa receptor density and function associated with two silent and linked polymorphisms (807C/T and 873G/A) within the glycoprotein Ia gene. Because platelet thrombus formation is implicated in the pathogenesis of acute myocardial infarction, we investigated these polymorphisms among patients who had had a myocardial infarction. METHODS: We did a 2/1 case-control study including 177 patients (median age 57.0 [range 32-72] years) and 89 controls with same age and sex. Distributions of the 807C/T and 873G/A polymorphisms were investigated by genotyping DNA by PCR, single-strand conformation polymorphism analysis, and sequencing. FINDINGS: The prevalence of the homozygous 807T/873A genotype was 2.9 times higher among patients with myocardial infarction than among controls (16.4% vs 5.6%, p=0.022). There was an association between patients homozygous for the 807T/873A allele and myocardial infarction (odds ratio 3.3 [95% CI 1.2-8.8]), which was strongest in a subgroup of smokers. The homozygous 807T/873A genotype remained an independent risk factor for myocardial infarction (p=0.005) when age, sex, smoking, hypertension, diabetes, body-mass index, LDL-cholesterol and HDL-cholesterol, and fibrinogen were adjusted for by logistic regression. INTERPRETATION: The 807T/873A homozygosity of the platelet glycoprotein Ia/IIa gene polymorphism, associated with differences in surface collagen receptor density and activity, appears to be an independent risk factor for acute myocardial infarction. Our findings need to be confirmed in a larger, prospective study that includes patients from different populations and cardiovascular risk groups.

A systematic mutation screen of 10 nuclear and 25 mitochondrial candidate genes in 21 patients with cytochrome c oxidase (COX) deficiency shows tRNA(Ser)(UCN) mutations in a subgroup with syndromal encephalopathy.

COX deficiency is believed to be the most common defect in neonates and infants with mitochondrial diseases. To explore the causes of this group of disorders, we examined 25 mitochondrial genes (three COX subunit genes and 22 tRNA genes) and 10 nuclear COX subunit genes for disease associated mutations using PCR-SSCP and direct sequencing of polymorphic SSCP fragments. DNA from one patient with severe COX deficiency and with consanguineous parents was entirely sequenced. The patient population consisted of 21 unrelated index patients with mitochondrial disorders and predominant (n=7) or isolated (n=14) COX deficiency. We detected two distinct tRNA(Ser)(UCN) mutations, which have been recently described in single kindreds, in a subgroup of four patients with COX deficiency, deafness, myoclonic epilepsy, ataxia, and mental retardation. Besides a number of nucleotide variants, a single novel missense mutation, which may contribute to the disease phenotype, was found in the mitochondrial encoded COX 1 gene (G6480A). Mutations in nuclear encoded COX subunit genes were not detected in this study.

Genotype-phenotype association in infants with cystic fibrosis at the time of diagnosis.

The relationship between the most common disease-causing mutations, the clinical manifestation, and lung function was prospectively assessed in 60 infants (33 females, 27 males) with cystic fibrosis at time of diagnosis (age: 7.2 months; range: 0.8-23.8 months). Lung function was assessed by infants whole-body plethysmography. Age at time of diagnosis was independent from the genotype. Weight gain from birth until the time of diagnosis expressed in percent predicted of a normal population was lower in the 3905insT group (57.9 +/- 19.0%) compared with deltaF508 homozygotes (62.5 +/- 20.6%; n.s.) and the R553X group (85.9 +/- 10.9%; p < 0.005). Differences regarding lung function within the genetic groups are mainly related to pulmonary hyperinflation, measured by thoracic gas volume (TGV), present in 8 of 9 infants with 3905insT, differentiating this frameshift mutation (TGV of 7.0 +/- 3.6 SD-S) from the R553X mutation (TGV 2.1 +/- 4.6 SD-S; p < 0.02). It is concluded that the variable disease findings in infants with cystic fibrosis is clinically and functionally reflected by features already present at time of diagnosis. The degree of pulmonary hyperinflation is, at least partly, influenced by the genotype.

Genomic organization of the MTM1 gene implicated in X-linked myotubular myopathy.

X-linked recessive myotubular myopathy (XLMTM) is a very severe congenital muscular disease characterised by an impaired maturation of muscle fibres, and caused by defects in the MTM1 gene. This gene defines a new family of putative tyrosine phosphatases conserved through evolution. We have determined intronic flanking sequences for all the 15 exons to facilitate the detection of mutations in patients and genetic counselling. We characterised a new polymorphic marker in the immediate vicinity of the gene, which might prove useful for linkage analysis. Sequencing of the TATA-less predicted promoter provides the basis for transcriptional regulatory studies.

A novel mitochondrial tRNA(Phe) mutation inhibiting anticodon stem formation associated with a muscle disease.

We have identified a novel mitochondrial (mt) DNA mutation in the tRNA(Phe)-gene in a patient with an isolated mitochondrial myopathy. This T to C transition at position 618 disrupts a strictly conserved base pair within the anticodon stem of tRNA(Phe). Computer analysis showed that the affected base pair is essential for anticodon stem formation of tRNA(Phe). The mutant mtDNA was heteroplasmic in skeletal muscle (95% mutant) and peripheral blood cells (20% mutant) from the patient but was undetectable in blood cells from his healthy sister. The patient presented with ragged red fibers and reduced activities of complex I and complex III in skeletal muscle. The T618C mutation described here is the second found in this region. Both mutations affect the same base pair of the tRNA(Phe) anticodon stem substantiating the pathogenic nature of both mutations.

A new polymorphic restriction site in the human 11 beta-hydroxysteroid dehydrogenase type 2 gene.

The 11 beta-hydroxysteroid dehydrogenase type II enzyme (11 beta HSD2) inactivates glucocorticoids in the kidney and thus prevents glucocorticoids from occupying the non-selective mineralocorticoid receptor in epithelial tissues. Mutations in the HSD11B2 gene have been found to cause the syndrome of apparent mineralocorticoid excess, a rare autosomal recessive disease characterized by severe hypertension. Thus, this locus could also be an ideal candidate involved in the etiology of primary hypertension. We identified a polymorphism in exon 3 characterized by a GAG to GAA transition at codon 178, with the loss of an Alu I restriction site and analysed it in an association study using end-stage renal disease patients, diabetic or essential hypertensive patients and control subjects. Two-hundred and eighty nine subjects and patients were analysed; the genotype was determined by amplification of genomic DNA and subsequent digestion with Alu I restriction enzyme. The prevalence of the Alu I allele was 8.6% in healthy control subjects (n = 116). This prevalence was lower (chi 2 P = 0.035 vs. controls) than the 18.0% in a group of renal transplant patients (n = 61). The corresponding values for patients with diabetes mellitus (n = 25), hypertension (n = 41) and patients on dialysis (n = 46) were 4.0%, 4.8% and 4.3%, respectively. There was no correlation between blood pressure and the marker in non-ESRD subjects. These data indicate the presence of a polymorphic marker in exon 3 of the HSD11B2 gene; this marker is associated with end-stage renal disease but not with essential hypertension in humans.

Confirmation of prenatal diagnosis results of X-linked recessive myotubular myopathy by mutational screening, and description of three new mutations in the MTM1 gene.

X-linked recessive myotubular myopathy (XLMTM; MTM1) is a severe neonatal disorder often causing perinatal death of the affected males. The responsible gene, designated MTM1, was localized to proximal Xq28 and recently isolated. The characterization of MTM1 allowed us to screen for causing mutations in three families, previously investigated by linkage analysis. Using exon amplification, single strand conformation polymorphism, and subsequent sequencing analysis, three new mutations and their mutational origin were characterized by analyzing 10 exons. An acceptor splice site and a frameshift mutation were correlated with the concurrent appearance of XLMTM in two families. A third intronic mutation was also analyzed by reverse transcription PCR and revealed a cryptic splice site mutation cosegregating with the presumed XLMTM haplotype in the third family. These results further support the implication of the MTM1 gene in XLMTM and allow efficient and reliable carrier and prenatal diagnosis in these families. Direct mutational diagnosis of families at risk in combination with haplotype analysis avoid the drawbacks using only linkage analysis, make genetic counselling far more reliable, and early clinical management of this disease more appropriate. Moreover, pedigree analyses provide first information on de novo mutation frequency in this newly identified human disease gene.

Screening of the Fc epsilon RI-beta-gene in a Swiss population of asthmatic children: no association with E237G and identification of new sequence variations.

BACKGROUND: The gene of the beta subunit of the high affinity receptor for IgE (Fc epsilon RI-beta) encoded on chromosome 11q13 has recently been identified as a candidate gene for asthma and atopy. Two coding variations, E237G and I181L have been described as being associated with asthma and atopy. Our aim was to investigate a Swiss population of atopic and asthmatic children for variations in this gene. METHODS: We screened all 7 exons of the Fc epsilon RI-beta-gene in 224 atopic/asthmatic, 68 relatives and 159 control subjects using exon amplification by PCR and single strand conformation polymorphism (SSCP) analysis followed by fluorescence based DNA sequencing. RESULTS: The sequence variant E237G was found in 3.7% in atopics and in 2.6% in the control population. None of the samples carried the I181L mutation. In addition, we characterised nine novel mutations (1 nonsense mutation, 2 missense mutations, mutation, 2 silent mutations, 4 intronic mutations). CONCLUSIONS: Our results suggest that the E237G does not have a primary effect on the development of atopy and asthma, and thus excludes the Fc epsilon RI-beta locus from being a candidate gene directly involved in these diseases.

Detection and characterization of mitochondrial DNA rearrangements in Pearson and Kearns-Sayre syndromes by long PCR.

We used a strategy based on long PCR (polymerase chain reaction) for detection and characterization of mitochondrial DNA (mtDNA) rearrangements in two patients with clinical signs suggesting Pearson syndrome and Kearns-Sayre syndrome (KSS), respectively, and one patient with myopathic symptoms of unidentified origin. Mitochondrial DNA rearrangements were detected by amplification of the complete mitochondrial genome (16.6 kb) using long PCR with primers located in essential regions of the mitochondrial genome and quantified by three-primer PCR. Long PCR with deletion-specific primers was used for identification and quantitative estimation of the different forms of rearranged molecules, such as deletions and duplications. We detected significant amounts of a common 7.4-kb deletion flanked by a 12-bp direct repeat in all tissues tested from the patient with Pearson syndrome. In skeletal muscle from the patient with clinical signs of KSS we found significant amounts of a novel 3.7-kb rearrangement flanked by a 4-bp inverted repeat that was present in the form of deletions as well as duplications. In the patient suffering from myopathic symptoms of unidentified origin we did not detect rearranged mtDNA in blood but found low levels of two rearranged mtDNA populations in skeletal muscle, a previously described 7-kb deletion flanked by a 7-bp direct repeat and a novel 6.6-kb deletion with no repeat. These two populations, however, were unlikely to be the cause of the myopathic symptoms as they were present at low levels (10-40 ppm). Using a strategy based on screening with long PCR we were able to detect and characterize high as well as low levels of mtDNA rearrangements in three patients.

Mutations in the MTM1 gene implicated in X-linked myotubular myopathy. ENMC International Consortium on Myotubular Myopathy. European Neuro-Muscular Center.

X-linked recessive myotubular myopathy (XLMTM) is characterized by severe hypotonia and generalized muscle weakness, with impaired maturation of muscle fibres. The gene responsible, MTM1, was identified recently by positional cloning, and encodes a protein (myotubularin) with a tyrosine phosphatase domain (PTP). Myotubularin is highly conserved through evolution and defines a new family of putative tyrosine phosphatases in man. We report the identification of MTM1 mutations in 55 of 85 independent patients screened by single-strand conformation polymorphism for all the coding sequence. Large deletions were observed in only three patients. Five point mutations were found in multiple unrelated patients, accounting for 27% of the observed mutations. The possibility of detecting mutations and determining carrier status in a disease with a high proportion of sporadic cases is of importance for genetic counselling. More than half of XLMTM mutations are expected to inactivate the putative enzymatic activity of myotubularin, either by truncation or by missense mutations affecting the predicted PTP domain. Additional mutations are missenses clustered in two regions of the protein. Most of these affect amino acids conserved in the homologous yeast and Caenorhabditis elegans proteins, thus indicating the presence of other functional domains.

A new mutation, 3905insT, accounts for 4.8% of 1173 CF chromosomes in Switzerland and causes a severe phenotype.

We have analysed 1173 cystic fibrosis (CF) chromosomes from Switzerland for eight mutations in the CF transmembrane conductance regulator (CFTR) gene. This permitted the identification of 88.5% of all mutations present. A novel insertion mutation in exon 20 of the CFTR gene, 3905insT, was discovered. This mutation accounted for 4.8% of CFTR gene mutations in Switzerland and has since been identified in other populations of probable Swiss descent. It is associated with a highly variable clinical phenotype but always with pancreatic insufficiency. Haplotype analysis with three intragenic microsatellites in the CFTR gene showed that the mutation is associated with a haplotype rarely identified on other CFTR alleles and, therefore, that the frequency of the mutation in Switzerland is explained by a founder effect of a relatively recent mutation event.