Origin of the expansion mutation in myotonic dystrophy.

Myotonic dystrophy (DM) is caused by the expansion of a CTG trinucleotide repeat. The mutation is in complete linkage disequilibrium with a nearly two-allele insertion/deletion polymorphism, suggesting a single origin for the mutation or predisposing mutation. To trace this-ancestral event, we have studied the association of CTG repeat alleles in a normal population to alleles of the insertion/deletion polymorphism and of a (CA)n repeat marker 90 kilobases from the DM mutation. The results strongly suggest that the initial predisposing event(s) consisted of a transition from a (CTG)5 allele to an allele with 19 to 30 repeats. The heterogeneous class of (CTG)19-30 alleles which has an overall frequency of about 10%, may constitute a reservoir for recurrent DM mutations.

A gene mutated in X-linked myotubular myopathy defines a new putative tyrosine phosphatase family conserved in yeast.

X-linked recessive myotubular myopathy (MTM1) is characterized by severe hypotonia and generalized muscle weakness, with impaired maturation of muscle fibres. We have restricted the candidate region to 280 kb and characterized two candidate genes using positional cloning strategies. The presence of frameshift or missense mutations (of which two are new mutations) in seven patients proved that one of these genes is indeed implicated in MTM1. The protein encoded by the MTM1 gene is highly conserved in yeast, which is surprising for a muscle specific disease. The protein contains the consensus sequence for the active site of tyrosine phosphatases, a wide class of proteins involved in signal transduction. At least three other genes, one located within 100 kb distal from the MTM1 gene, encode proteins with very high sequence similarities and define, together with the MTM1 gene, a new family of putative tyrosine phosphatases in man.

Reduction of regulatory T cells in skin lesions but not in peripheral blood of patients with systemic scleroderma.

OBJECTIVE: To determine the frequency and suppressive capacity of regulatory T cells (T(reg)) and their association with clinical parameters in patients with systemic scleroderma (SSc). METHODS: Peripheral blood from 25 patients with SSc, 15 patients with localised scleroderma (LS) and 29 healthy controls (HC) was studied. Analysis of CD4(+) forkhead box P3 (Foxp3)(+) and CD4(+)CD25(++)Foxp3(+) T(reg) subpopulations was carried out by flow cytometry and cell proliferation was quantified by (3)H-thymidine incorporation. Quantitative analysis of T(reg) was further performed in skin biopsies from 17 patients with SSc and 21 patients with LS using anti-CD4 and anti-Foxp3 monoclonal antibodies for immunohistochemistry. RESULTS: The frequency of CD4(+)Foxp3(+) and CD4(+)CD25(++)Foxp3(+) T(reg) in peripheral blood from patients with SSc was not significantly different from that of patients with LS or HC. The suppressive capacity of CD4(+)CD25(++) T(reg) in SSc was also found to be similar to that of HC. Phenotypic and functional data revealed no significant difference between the limited or diffuse form of SSc. Moreover, therapy with bosentan showed no significant effect on the frequency of T(reg) during the course of the disease. However, the frequency of T(reg) in skin lesions from patients with SSc or LS, determined as the percentage of CD4(+) cells expressing Foxp3 in the inflammatory infiltrate, was significantly reduced compared with other inflammatory skin diseases. CONCLUSION: These results indicate that although the authors found no defect in the frequency or function of peripheral T(reg) subpopulations, the reduction of CD4(+)Foxp3(+) T(reg) in the skin of patients with SSc may be important in the pathogenesis of the disease.

Characterization of regulatory T cells in patients with dermatomyositis.

The purpose of this study was to characterize regulatory T cells (T(reg)) in skin lesions and peripheral blood from patients with dermatomyositis (DM) and to determine the serum levels of regulatory cytokines in the disease. In skin biopsy specimens from patients with DM, immunohistochemistry was performed for CD4(+), CD25(+), forkhead/winged helix transcription factor (FoxP3)(+), transforming growth factor (TGF)-ß(+) and interleukin (IL)-10(+) cells. Additionally, we defined the number of T(reg) subpopulations in peripheral blood by flow cytometry using monoclonal antibodies against CD4, CD25, FoxP3, CD45RO, CD95, CCR4 and CLA. The levels of TGF-ß and IL-10 were also determined in serum samples from patients with DM by enzyme-linked immunosorbent assays. Controls included patients with cutaneous lupus erythematosus, psoriasis and atopic dermatitis (AD) as well as healthy donors. The frequency of FoxP3(+) cells was significantly reduced in skin lesions from patients with DM (p < 0.001) compared to psoriasis and AD. Moreover, the number of cells positive for TGF-ß was lower in DM than in psoriasis and AD, while IL-10(+) cells were significantly reduced only compared to psoriasis. The number of CD4(+)CD25(++)FoxP3(+) T(reg) in the peripheral blood of patients with DM was significantly reduced compared to healthy controls (p < 0.05), whereas other cell populations showed no significant differences. Finally, TGF-ß and IL-10 serum levels were significantly lower in patients with DM compared to healthy controls (p < 0.05). These data suggest that the depletion of T(reg) and their main effector cytokines in the skin and the serum of patients with DM may be an important factor in the pathogenesis of the disease.

Instability of a 550-base pair DNA segment and abnormal methylation in fragile X syndrome.

The fragile X syndrome, a common cause of inherited mental retardation, is characterized by an unusual mode of inheritance. Phenotypic expression has been linked to abnormal cytosine methylation of a single CpG island, at or very near the fragile site. Probes adjacent to this island detected very localized DNA rearrangements that constituted the fragile X mutations, and whose target was a 550-base pair GC-rich fragment. Normal transmitting males had a 150- to 400-base pair insertion that was inherited by their daughters either unchanged, or with small differences in size. Fragile X-positive individuals in the next generation had much larger fragments that differed among siblings and showed a generally heterogeneous pattern indicating somatic mutation. The mutated allele appeared unmethylated in normal transmitting males, methylated only on the inactive X chromosome in their daughters, and totally methylated in most fragile X males. However, some males had a mosaic pattern. Expression of the fragile X syndrome thus appears to result from a two-step mutation as well as a highly localized methylation. Carriers of the fragile X mutation can easily be detected regardless of sex or phenotypic expression, and rare apparent false negatives may result from genetic heterogeneity or misdiagnosis.

Shifting microbial communities sustain multiyear iron reduction and methanogenesis in ferruginous sediment incubations.

Reactive Fe(III) minerals can influence methane (CH4 ) emissions by inhibiting microbial methanogenesis or by stimulating anaerobic CH4 oxidation. The balance between Fe(III) reduction, methanogenesis, and CH4 oxidation in ferruginous Archean and Paleoproterozoic oceans would have controlled CH4 fluxes to the atmosphere, thereby regulating the capacity for CH4 to warm the early Earth under the Faint Young Sun. We studied CH4 and Fe cycling in anoxic incubations of ferruginous sediment from the ancient ocean analogue Lake Matano, Indonesia, over three successive transfers (500 days in total). Iron reduction, methanogenesis, CH4 oxidation, and microbial taxonomy were monitored in treatments amended with ferrihydrite or goethite. After three dilutions, Fe(III) reduction persisted only in bottles with ferrihydrite. Enhanced CH4 production was observed in the presence of goethite, highlighting the potential for reactive Fe(III) oxides to inhibit methanogenesis. Supplementing the media with hydrogen, nickel and selenium did not stimulate methanogenesis. There was limited evidence for Fe(III)-dependent CH4 oxidation, although some incubations displayed CH4 -stimulated Fe(III) reduction. 16S rRNA profiles continuously changed over the course of enrichment, with ultimate dominance of unclassified members of the order Desulfuromonadales in all treatments. Microbial diversity decreased markedly over the course of incubation, with subtle differences between ferrihydrite and goethite amendments. These results suggest that Fe(III) oxide mineralogy and availability of electron donors could have led to spatial separation of Fe(III)-reducing and methanogenic microbial communities in ferruginous marine sediments, potentially explaining the persistence of CH4 as a greenhouse gas throughout the first half of Earth history.

A new polymorphic marker very closely linked to DXS52 in the q28 region of the human X chromosome.

We have isolated an X chromosome probe, St35.691 (DXS305), which detects two RFLPs with TaqI and PstI, whose combined heterozygosity is about 60%. This probe has been assigned to Xq28 by physical and genetic mapping and is very closely linked to DXS52, DXS15, and the coagulation factor VIII gene (F8C). The best estimate of the recombination fraction for the DXS52-DXS305 interval is 0.014, with a lod score of 50.1. Multipoint analysis places DXS305 on the same side of F8C as DXS52, but complete ordering of the three loci was not possible with our present data. This highly informative marker should be useful in the precise mapping of the many disease genes that have been assigned to the Xq28 band.

Inheritance of the fragile X syndrome: size of the fragile X premutation is a major determinant of the transition to full mutation.

The fragile X mental retardation syndrome is caused by unstable expansion of a CGG repeat. Two main types of mutation have been categorised. Clinical expression is associated with the presence of the full mutation, while subjects who carry only a premutation do not have mental retardation. Premutations have a high risk of transition to full mutation when transmitted by a female. We have used direct detection of the mutations to characterise large families who illustrate the wide variation in penetrance which has been observed in different sibships (a feature often called the Sherman paradox). A family originally found to show tight genetic linkage between the factor 9 gene and the fragile X locus was reanalysed, confirming the original genotype assignments and the observed linkage. The size of premutations was measured by Southern blotting and by using a PCR based test in 102 carrier mothers and this was correlated with the type of mutation found in their offspring. The risk of transition to full mutation was found to be very low for premutations with a size increase (delta) of about 100 bp, increasing up to 100% when the size of premutation was larger than about 200 bp, even after taking into account (at least partially) ascertainment bias. These results confirm and extend those reported by Fu et al (1991) and Yu et al (1992) and explain the Sherman paradox.(ABSTRACT TRUNCATED AT 250 WORDS)

Abnormal pattern detected in fragile-X patients by pulsed-field gel electrophoresis.

The fragile-X syndrome is the most frequent inherited form of mental retardation, with an incidence of 1 in 1,500 males. It is characterized by the presence of a fragile site at Xq27.3 induced in vitro by folate deprivation or by inhibitors of deoxynucleotide synthesis. Its mode of inheritance is unusual for an X-linked trait, with incomplete penetrance in both males and females. Some phenotypically normal males transmit the mutation to all their daughters who rarely express any symptoms, but penetrance is high in sons and daughters of these carrier women. Genetic and physical mapping of the Xq27-q28 region has confirmed that the disease locus is located at or very near the fragile site. Hypotheses proposed to account for the abnormalities in the inheritance of the disease include sequence rearrangements by meiotic recombination or a mutation that affects reactivation of an inactive X chromosome during differentiation of female germ cells. To detect such rearrangements, or methylation changes that may reflect a locally inactive X chromosome, we used pulsed-field gel analysis of DNA from fragile-X patients with probes close to the fragile-X locus. The probe Do33 (DXS465) detected abnormal patterns in fragile-X patients, but not in normal controls or in non-expressing male transmitters.

Cloning and characterization of an alternatively spliced gene in proximal Xq28 deleted in two patients with intersexual genitalia and myotubular myopathy.

We have identified a novel human gene that is entirely deleted in two boys with abnormal genital development and myotubular myopathy (MTM1). The gene, F18, is located in proximal Xq28, approximately 80 kb centromeric to the recently isolated MTM1 gene. Northern analysis of mRNA showed a ubiquitous pattern and suggested high levels of expression in skeletal muscle, brain, and heart. A transcript of 4.6 kb was detected in a range of tissues, and additional alternate forms of 3.8 and 2.6 kb were present in placenta and pancreas, respectively. The gene extends over 100 kb and is composed of at least seven exons, of which two are noncoding. Sequence analysis of a 4.6-kb cDNA contig revealed two overlapping open reading frames (ORFs) that encode putative proteins of 701 and 424 amino acids, respectively. Two alternative spliced transcripts affecting the large open reading frame were identified that, together with the Northern blot results, suggest that distinct proteins are derived from the gene. No significant homology to other known proteins was detected, but segments of the first ORF encode polyglutamine tracts and proline-rich domains, which are frequently observed in DNA-binding proteins. The F18 gene is a strong candidate for being implicated in the intersexual genitalia present in the two MTM1-deleted patients. The gene also serves as a candidate for other disorders that map to proximal Xq28.

Genomic organization of the adrenoleukodystrophy gene.

Adrenoleukodystrophy (ALD), the most frequent peroxisomal disorder, is a severe neurodegenerative disease associated with an impairment of very long chain fatty acids beta-oxidation. We have recently identified by positional cloning the gene responsible for ALD, located in Xq28. It encodes a new member of the "ABC" superfamily of membrane-associated transporters that shows, in particular, significant homology to the 70-kDa peroxisomal membrane protein (PMP70). We report here a detailed characterization of the ALD gene structure. It extends over 21 kb and consists of 10 exons. To facilitate the detection of mutations in ALD patients, we have determined the intronic sequences flanking the exons as well as the sequence of the 3' untranslated region and of the immediate 5' promoter region. Sequences present in distal exons cross-hybridize strongly to additional sequences in the human genome. The ALD gene has been positioned on a pulsed-field map between DXS15 and the L1CAM gene, about 650 kb upstream from the color pigment genes. The frequent occurrence of color vision anomalies observed in patients with adrenomyeloneuropathy (the adult onset form of ALD) thus does not represent a contiguous gene syndrome but a secondary manifestation of ALD.

X-linked myotubular myopathy: refinement of the gene to a 280-kb region with new and highly informative microsatellite markers.

We have recently refined the localization of the myotubular myopathy (MTM1) gene to a 430-kb region between DXS304 and DXS1345 in proximal Xq28. We report two new polymorphic microsatellite markers, DXS8377 and DXS7423, that were physically mapped within the critical interval. A recombination event in a family segregating for MTM1 placed the disease gene telomeric to the trinucleotide polymorphism DXS8377. Together with the recent mapping of two microdeletions associated with MTM1, the recombination refines the critical region to 280 kb. A second recombination event was observed distal to the tetranucleotide repeat DXS7423. This recombination has occurred in the off-spring of a female with a more than 67% probability of being a carrier and very likely restricts the MTM1 gene to a 130-kb region. This physical refinement is significant for positional cloning of the disease gene. The highly polymorphic markers and the precise localization of the MTM1 gene will facilitate genetic diagnosis of the disorder.

Characterization of the myotubularin dual specificity phosphatase gene family from yeast to human.

X-linked myotubular myopathy (XLMTM) is a severe congenital muscle disorder due to mutations in the MTM1 gene. The corresponding protein, myotubularin, contains the consensus active site of tyrosine phosphatases (PTP) but otherwise shows no homology to other phosphatases. Myotubularin is able to hydrolyze a synthetic analogue of tyrosine phosphate, in a reaction inhibited by orthovanadate, and was recently shown to act on both phosphotyrosine and phosphoserine. This gene is conserved down to yeast and strong homologies were found with human ESTs, thus defining a new dual specificity phosphatase (DSP) family. We report the presence of novel members of the MTM gene family in Schizosaccharomyces pombe, Caenorhabditis elegans, zebrafish, Drosophila, mouse and man. This represents the largest family of DSPs described to date. Eight MTM-related genes were found in the human genome and we determined the chromosomal localization and expression pattern for most of them. A subclass of the myotubularin homologues lacks a functional PTP active site. Missense mutations found in XLMTM patients affect residues conserved in a Drosophila homologue. Comparison of the various genes allowed construction of a phylogenetic tree and reveals conserved residues which may be essential for function. These genes may be good candidates for other genetic diseases.

The gene responsible for adrenoleukodystrophy encodes a peroxisomal membrane protein.

Adrenoleukodystrophy is a severe genetic demyelinating disease associated with an impairment of beta-oxidation of very long chain fatty acids (VLCFA) in peroxisomes. Earlier studies had suggested that a deficiency in VLCFA CoA synthetase was the primary defect. A candidate adrenoleukodystrophy gene has recently been cloned and was found unexpectedly to encode a putative ATP-binding cassette transporter. We have raised monoclonal antibodies against this protein, that detect a 75kDa band. This protein was absent in several patients with adrenoleukodystrophy. Immunofluorescence and immunoelectron microscopy showed that the adrenoleukodystrophy protein (ALDP) is associated with the peroxisomal membrane. Distinct immunofluorescence patterns were observed in cell lines from patients with Zellweger syndrome (a peroxisomal biogenesis disorder) belonging to different complementation groups.

Linkage disequilibrium between the fragile X mutation and two closely linked CA repeats suggests that fragile X chromosomes are derived from a small number of founder chromosomes.

In order to investigate the origin of mutations responsible for the fragile X syndrome, two polymorphic CA repeats, one at 10 kb (FRAXAC2) and the other at 150 kb (DXS548) from the mutation target, were analyzed in normal and fragile X chromosomes. Contrary to observations made in myotonic dystrophy, fragile X mutations were not strongly associated with a single allele at the marker loci. However, significant differences in allelic and haplotypic distributions were observed between normal and fragile X chromosomes, indicating that a limited number of primary events may have been at the origin of most present-day fragile X chromosomes in Caucasian populations. We propose a putative scheme with six founder chromosomes from which most of the observed fragile X-linked haplotypes can be derived directly or by a single event at one of the marker loci, either a change of one repeat unit or a recombination between DXS548 and the mutation target. Such founder chromosomes may have carried a number of CGG repeats in an upper-normal range, from which recurrent multistep expansion mutations have arisen.

Direct diagnosis by DNA analysis of the fragile X syndrome of mental retardation.

BACKGROUND: The fragile X syndrome, the most common form of inherited mental retardation, is caused by mutations that increase the size of a specific DNA fragment of the X chromosome (in Xq27.3). Affected persons have both a full mutation and abnormal DNA methylation. Persons with a smaller increase in the size of this DNA fragment (a premutation) have little or no risk of retardation but are at high risk of having affected children or grandchildren. The passage from premutation to full-mutation status occurs only with transmission from the mother. We have devised a method of identifying carriers of these mutations by direct DNA analysis. METHOD: We studied 511 persons from 63 families with the fragile X syndrome. Mutations and abnormal methylation were detected by Southern blotting with a probe adjacent to the mutation target. Analysis of EcoRI and EagI digests of DNA distinguished clearly in a single test between the normal genotype, the premutation, and the full mutation. RESULTS: DNA analysis unambiguously established the genetic status at the fragile X locus for all samples tested. This method was much more powerful and reliable than cytogenetic testing or segregation studies with closely linked polymorphic markers. The frequency of mental retardation in persons with premutations was similar to that in the general population, whereas all 103 males and 31 of 59 females with full mutations had mental retardation. About 15 percent of those with full mutations had some cells carrying only the premutation. All the mothers of affected children were carriers of either a premutation or a full mutation. CONCLUSIONS: Direct diagnosis by DNA analysis is now an efficient and reliable primary test for the diagnosis of the fragile X syndrome after birth, as well as for prenatal diagnosis and genetic counseling.