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The Marinesco-Sjogren syndrome (MSS) (MIM 248800) is an autosomal recessive condition characterised by cataracts, ataxia, and growth and mental retardation. Chronic myopathy is a common feature. Peripheral neuropathy and acute [...]

Dystrophin is a very large muscle protein (approximately 400 kd) the deficiency of which is responsible for Duchenne muscular dystrophy. Its function is unknown at present. In order to know whether different domains of the protein are differentially conserved during evolution, we have cloned and sequenced the chicken dystrophin cDNA. The protein coding sequence has almost the same size as in man. The N‐terminal region that resembles the actin binding domain of alpha actinin, as well as the large spectrin like domain show 80% and 75% conservation respectively between chicken and man. In contrast, the C‐terminal region shows 95% identity over 627 aa suggesting that it is an important region of interaction with other proteins. Comparison of the amino acid sequence of this C‐terminal region to other protein sequences shows only marginally significant similarities. Finally we have found a striking conservation of three segments of the 3′ untranslated sequence (85% homology over a total of 920 nt) between chicken and man. These also appear to be conserved in other mammals. This high conservation is not linked to open reading frames.

The adrenoleukodystrophy gene encodes a peroxisomal integral membrane protein (ALDP) consisting of 745 amino acids with a molecular weight of 75 kDa. ALDP expression was studied in fibroblasts from 24 male ALD patients from 17 unrelated ALD kindreds. In four kindreds an identical 2-base-pair deletion was found. We report the absence of ALDP in 12 kindreds carrying nonsense mutations, frame shifts or amino acid substitutions in the carboxy terminus of ALDP, together accounting for 71% of the ALD kindreds. ALDP was present in five kindreds (29%) with amino acid substitutions in the amino terminal half of the protein; in two of these kindreds ALDP was present although at a reduced level. The absence of truncated proteins suggests that the carboxy terminus has a function in the stabilization of ALDP.

The gene for Friedreich ataxia (FA), a severe recessive neurodegenerative disease, has previously been shown to be tightly linked to the polymorphic markers D9S15 and D9S5 on human chromosome 9. In addition, the observation of linkage disequilibrium suggested that D9S15 is within 1 centimorgan (cM) of the disease locus, FRDA. Although D9S5 did not show recombination with FRDA, its localization was less precise (0-5 cM) due to its lower informativeness. We have now identified additional polymorphisms at both marker loci. Two cosmids spanning 50 kilobases around D9S5 were isolated, and a probe derived from one of them detects an informative three-allele polymorphism. We have found a highly polymorphic microsatellite sequence at D9S15 which is rapidly typed by the DNA polymerase chain reaction. The polymorphism information contents at the D9S5 and D9S15 loci have been increased from 0.14 to 0.60 and from 0.33 to 0.74, respectively. With the additional polymorphisms the lod (log10 odds ratio) score for the D9S15-FRDA linkage is now 48.10 at recombination fraction theta = 0.005 and for D9S5-FRDA, the lod score is 27.87 at theta = 0.00. We have identified a recombinant between D9S15 and FRDA. However, due to the family structure, it will be of limited usefulness for more precise localization of FRDA. The linkage disequilibrium previously observed between D9S15 and FRDA is strengthened by analysis of the haplotypes using the microsatellite polymorphism, while weaker but significant disequilibrium is found between D9S5 and FRDA. Extended haplotypes that encompass D9S5 and D9S15 show a strikingly different distribution between chromosomes that carry the FA mutation and normal chromosomes. This suggests that both marker loci are less than 1 cM from the FRDA gene and that a small number of mutations account for the majority of FA cases in the French population studied. D9S5 and D9S15 are thus excellent start points to isolate the disease gene.

The polymorphic DNA marker DXS304 detected by probe U6.2 has recently been shown to be closer to the fragile X locus than previously available markers. Its usefulness has however been limited by its relatively low heterozygosity. We have isolated, by cosmid cloning, a 67 kilobase region around probe U6.2 and have characterized a new probe (U6.2-20E) that detects BanI and BstEII restriction fragment length polymorphisms (RFLPs). The BanI RFLP has a heterozygosity of 0.49 and is in partial linkage disequilibrium with the previously described polymorphism, with a combined heterozygosity of 0.63. Furthermore, we have found that the U6.2 original probe, which probably detects an insertion-deletion polymorphism, is also informative in BanI digests. Thus, the two informative RFLPs at the DXS304 locus can be conveniently tested in a single hybridization with a single digest. An updated linkage analysis confirms that DXS304 is distal to the fragile X locus. This informative locus can now be used effectively for genetic mapping of the Xq27–q28 region, and for diagnostic applications in fragile X or Hunter syndrome families.

A multilocus analysis of the fragile X (fra(X)) syndrome was conducted with 147 families. Two proximal loci, DXS51 and F9, and two distal loci, DXS52 and DXS15, were studied. Overall, the best multipoint distances were found to be DXS51-F9, 6.9%, F9-fra(X), 22.4%; fra(X)-DXS52, 12.7%; DXS52-DXS15, 2.2%. These distances can be used for multipoint mapping of new probes, carrier testing and counseling of fra(X) families. Consistent with several previous studies, the families as a whole showed genetic heterogeneity for linkage between F9 and fra(X).

The q26–q28 region of the human X chromosome contains several important disease loci, including the locus for the fragile X mental retardation syndrome. We have characterized new polymorphic DNA markers useful for the genetic mapping of this region. They include a new BclI restriction fragment length polymorphism (RFLP) detected by the probe St14-1 (DXS52) and which may therefore be of diagnostic use in hemophilia A families. A linkage analysis was performed in fragile X families and in large normal families from the Centre d'Etude du Polymorphisme Humain (CEPH) by using seven polymorphic loci located in Xq26-q28. This multipoint linkage study allowed us to establish the order centromere-DXS100-DXS86-DXS144-DXS51-F9-FRAX-(DXS52-DXS15). Together with other studies, our results define a cluster of nine loci that are located in Xq26-q27 and map within a 10 to 15 centimorgan region. This contrasts with the paucity of markers (other than the fragile X locus) between the F9 gene in q27 and the G6PD cluster in q28, which are separated by about 30% recombination.

Three families with anhidrotic ectodermal dysplasia (AED) have been studied by linkage analysis with seven polymorphic DNA markers from the Xp11-q21 region. Previously reported linkage to DXYS1 (Xq13-q21) has been confirmed (z(?)=4.08 at ?=0.05) and we have also established linkage to another polymorphic locus, DXS159, located in Xq11-q12 (z(?)=4.28 at ?=0.05). Physical mapping places DSX159 proximal to the Xq12 breakpoint of an X autosome translocation found in a female with clinical signs of ectodermal dysplasia. Of all markers that have been used in linkage analysis of AED, DXS159 would appear the closest on the proximal side of the disease locus.

Adrenoleukodystrophy (ALD), a severe demyelinating disease, is caused by mutations in a gene coding for a peroxisomal membrane protein (ALDP), which belongs to the superfamily of ATP binding cassette (ABC) transporters and has the structure of a half transporter. ALDP showed 38% sequence identity with another peroxisomal membrane protein, PMP70, up to now its closest homologue. We describe here the cloning and characterization of a mouse ALD-related gene (ALDR), which codes for a protein with 66% identity with ALDP and shares the same half transporter structure. The ALDR protein was overexpressed in COS cells and was found to be associated with the peroxisomes. The ALD and ALDR genes show overlapping but clearly distinct expression patterns in mouse and may thus play similar but nonequivalent roles. The ALDR gene, which appears highly conserved in man, is a candidate for being a modifier gene that could account for some of the extreme phenotypic variability of ALD. The ALDR gene is also a candidate for being implicated in one of the complementation groups of Zellweger syndrome, a genetically heterogeneous disorder of peroxisome biogenesis, rare cases of which were found to be associated with mutations in the PMP70 (PXMP1) gene.

Friedreich ataxia and ataxia with selective vitamin E deficiency (AVED) share very similar clinical phenotypes. We have mapped the AVED locus to proximal 8q with only three large consanguinous Tunisian families, representing to our knowledge the first use of homozygosity mapping for primary linkage analysis. Subsequently, three additional families showed linkage with the same markers. A maximum lod score of 17.9 was obtained at θ = 0 for the haplotype D8S260–D8S510, consisting of the two closest markers. With only 6 families, the AVED locus is therefore mapped precisely as illustrated by the lod–1 confidence interval of 2.4 cM on either side of D8S260–D8S510. Isolation of a yeast artificial chromosome contig >800 kilobases (kb) showed that D8S260 and D8S510 are less than 400 kb apart.

The ovalbumin gene is split in chicken DNA. Two interruptions in the sequences coding for ovalbumin mRNA have been detected, at least one of them lying in the protein coding sequence. The unexpected gene organisation is present both in oviduct cells highly specialised in ovalbumin synthesis and in erythrocytes.

Hemophilia B is an X-linked disease caused by a functional deficiency in coagulation factor IX. A cDNA clone corresponding to factor IX has been used to detect homologous sequences in the human genome. All DNA fragments hybridizing to the probe, under medium- or high-stringency conditions, are X-linked, and the patterns obtained suggest that a single large (greater than or equal to 20 kilobases) gene is detected. The gene has been mapped to the q26-q27 region of the long arm of the X chromosome by hybridization to DNA from a panel of human-mouse hybrid cell lines. A search for restriction fragment length polymorphisms using seven restriction enzymes has led to the detection of a Taq I polymorphism, with allelic frequencies of about 0.71 and 0.29. This genetic marker should be useful for the detection of carriers of the hemophilia B trait and for prenatal diagnosis in informative families and, more generally, for the establishment of a linkage map of the human X chromosome.

Using a human X chromosome-specific DNA library, we have found arbitrary single-copy DNA sequences that reveal useful restriction fragment length polymorphisms. The inheritance of these and other available polymorphic DNA markers has been studied in a series of unrelated three-generation families with large sibships. These families reveal parental phase and allow determination of recombination frequencies by counting recombinant and nonrecombinant chromosomes. The resulting genetic map indicates that the minimal distance from Xp22 to Xqter is 215 recombination units. The spacing of the marker loci is such that the majority of the loci on the X chromosome, including disease loci, will lie within 20 centimorgans of at least one of these loci.

Impaired expression of the FMR1 gene is responsible for the fragile X mental retardation syndrome. The FMR1 gene encodes a cytoplasmic protein with RNA-binding properties. Its complex alternative splicing leads to several isoforms, whose abundance and specific functions in the cell are not known. We have cloned in expression vectors, cDNAs corresponding to several isoforms. Western blot comparison of the pattern of endogenous FMR1 proteins with these transfected isoforms allowed the tentative identification of the major endogenous isoform as ISO 7 and of a minor band as an isoform lacking exon 14 sequences (ISO 6 or ISO 12), while some other isoforms (ISO 4, ISO 5) were not expressed at detectable levels. Surprisingly, in immunofluorescence studies, the transfected splice variants that exclude exon 14 sequences (and have alternate C-terminal regions) were shown to be nuclear. Such differential localisation was however not seen in subcellular fractionation studies. Analysis of various deletion mutants suggests the presence of a cytoplasmic retention domain encoded in exon 14 and of a nuclear association domain encoded within the first eight exons that appear however to lack a typical nuclear localisation signal.

We have recently described a female patient with myotubular myopathy (MTM1) and an interstitial deletion at Xq28. Characterisation of the deletion allowed us to position the MTM1 gene to a 600 kb region between DXS304 and DXS497. In order to further restrict the region we screened for deletions in a set of 38 patients. We found two overlapping deletions in boys that in addition to MTM1 showed an unexpected abnormal genital development. As the latter phenotype is not found in the other non-deleted MTM1 patients, our observations are best explained by a contiguous gene syndrome. The deletions define a 430 kb region that contains the MTM1 gene and most likely a gene implicated in male sexual development. A high resolution physical map of this region is presented.

Friedreich's ataxia (FA) is an autosomal recessive neurodegenerative disorder, the disease locus (FRDA) of which has been assigned to 9q13-q21.1 by genetic linkage analysis in affected families. We report two large inbred Tunisian families with FA manifestations that did not show the expected linkage. The disease locus could be excluded from a large (12 cMO) region around FRDA. This is the first report providing evidence for nonallelic genetic heterogeneity for the FA clinical phenotype. On subsequent analysis, all patients had very low levels of serum vitamin E whereas the parents and healthy sibs had normal vitamin E levels. This establishes that the selective vitamin E deficiency with normal fat absorption is an autosomal recessive trait, which is associated in the two families reported here with the FA phenotype.

Two large DNA fragments overlapping the chicken ovalbumin gene have been isolated by molecular cloning. Analysis of these fragments provided a map of a 46,000-base pair region of the chicken genome. This region contains the complete ovalbumin gene (including its mRNA leader-coding sequence) and at least two other genes of unknown function. All three genes are orientated in the same direction and their expression in chicken oviduct is under hormonal control. The three genes share some sequence homologies, suggesting that duplications have occurred in the ovalbumin gene region in the course of evolution.

A DNA marker C7, localised Xp21.1-Xp21.3, has been studied in kindreds segregating for Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). In DMD families four crossovers were observed in 38 informative meioses between C7 and the DMD locus (?=0.12, zmax=+2.72). In BMD families no recombinants were observed in the 16 informative meioses studied. These data are consistent with the localisation of the mutations in these disorders being in the same region of Xp21.

We have characterized 19 DNA fragments originating from the human X chromosome. Most of them have been isolated from an X chromosome genomic library (Davies et al. 1981) using a systematic screening procedure. These DNA probes have been used to search for restriction fragment length polymorphisms (RFLP). The frequency of restriction polymorphisms (1 per 350 bp analysed) was lower than expected from data obtained with autosomal fragments. The various probes have been mapped within 12 subchromosomal regions using a panel of human-rodent hybrid cell lines. The validity of the panel was established by hybridization experiments performed with 27 X-specific DNA probes, which yielded information on the relative position of translocation break-points on the X chromosome. The DNAs from the various hybrid lines are blotted onto a reusable support which allows one to quickly map any new X-specific DNA fragment. The probes already isolated should be of use to map unbalanced X chromosome aberrations or to characterize new somatic cell hybrid lines. The probes which detect RFLPs define new genetic markers which will help to construct a detailed linkage map of the human X chromosome, and might also serve for the diagnosis of carriers or prenatal diagnosis.

The coagulation factor IX gene and two other polymorphic loci corresponding to DNA probes 52 A and St 14 have been previously localized in the q27 to qter region of the human X chromosome. In order to study their localization with respect to the fragile site at Xq27-28, we have hybridized the three DNA probes to metaphase chromosomes of a boy with fragile X mental retardation. We show that probe 52A is located in the proximal part of the Xq27 band, while the coagulation factor IX gene is on the distal part of this band, but proximal to the fragile site. The very polymorphic St 14 probe is located in the distal part of the Xq28 band, on the other side of the fragile site.

Recombinant plasmid clone B74 (also named D18S3) containing a human single-copy DNA segment of 6 kilobases (kb) was localized by in situ hybridization on band p113 of chromosome 18. This probe was then used in cytogenetic diagnosis to identify precisely a small supernumerary chromosome as an isochromosome i(18p).

ABSTRACT: Giant axonal neuropathy (GAN) is a rare autosomal recessive disorder described as a symmetrical distal neuropathy, with peripheral axons dilated by accumulation of 10 nm neurofilaments (NF) and a severe course of disease. The observation of kinky or curly hairs is not a constant finding. The GAN1 locus was localized by homozygosity mapping to chromosome 16 q24.1 in a 3 (4) cM interval flanked by the markers D16S3073 and D16S505 (D16S511) in three non-related Tunisian families, showing a genetic homogeneity in these families. Two point lod-score calculation between the linked haplotype and the disease locus was 14.2 at θmax = 0. The patients share a slow course of the disease. The differences in the course of the disease between Tunisian and non-Tunisian patients suggest a possible genetic heterogeneity, which is why the present linkage has been referred to as GAN1. The biochemical defect in GAN1 should help to understand the mechanisms involved in NF accumulations as in other neurological diseases (ALS, SMA).

Adrenoleukodystrophy (ALD), a lethal demyelinating disease of the brain, is caused by mutations of a gene encoding an ATP-binding transporter, called ALDP, localized in the peroxisomal membrane. It is associated with a defective oxidation of very-long-chain fatty acids, leading to their accumulation in many tissues. This study reports that the retroviral-mediated transfer of the ALD cDNA restored very-long-chain fatty acid oxidation in ALD fibroblasts in vitro following abundant expression and appropriate targeting of the vector-encoded ALDP in peroxisomes. The same method may be used in hematopoietic cells as a further step of a gene therapy approach of ALD.

Attempting gene transfer in muscle raises difficult problems: the nuclei of mature muscle fibers do not undergo division, thus excluding strategies involving replicative integration of exogenous DNA. As adenovirus has been reported to be an efficient vector for the transfer of an enzyme encoding gene in mice, we decided to explore its potential for muscle cells. Advantages of adenovirus vectors are their independence of host cell replication, broad host range, and potential capacity for large foreign DNA inserts. We constructed a recombinant adenovirus containing the beta-galactosidase reporter gene under the control of muscle-specific regulatory sequences. This recombinant virus was able to direct expression of the beta-galactosidase in myotubes in vitro. We report its in vivo expression in mouse muscles up to 75 days after infection. The efficiency and stability of expression we obtained compare very favorably with other strategies proposed for gene or myoblast transfer in muscle in vivo.

A DNA fragment (named St14) derived from the human X chromosome reveals a small family of related sequences that have been mapped to the Xq26-Xq28 region by using a panel of rodent-human somatic cell hybrids. The probe detects in human DNA digested by Taq I a polymorphic system defined by a series of at least eight allelic fragments with a calculated heterozygosity in females of 80%. With Msp I, we found three additional restriction fragment length polymorphisms, each of them being defined by two alleles. These polymorphisms are also common in Caucasian populations. The genetic locus defined by probe St14 has been localized more precisely to the distal end of the X chromosome (in band q28) by linkage analysis to other polymorphic DNA markers. The results obtained suggest that the frequency of recombination is distributed very unevenly in the q27-qter region of the X chromosome, with a cluster of seven tightly linked loci in q28 showing about 30% recombination with the gene for coagulation factor IX located in the neighboring q27 band. Probe St14 reveals one of the most polymorphic loci known to date in the human genome, and 17 different genotypes have already been observed. It constitutes the best marker on the X chromosome and should be of great use for the genetic study of three important diseases: hemophilia A, mental retardation with a fragile X chromosome, and adrenoleukodystrophy.

The restriction enzymes HhaI and HpaII, whose activity is inhibited by cytosine methylation within their recognition sites, have been utilised as probes to study methylation in the vicinity of the ovalbumin gene in DNA from various chicken tissues. This was complemented by a preliminary study of methylation in the regions of chicken ovotransferrin (conalbumin), ovomucoid and beta-globin genes. From our data we conclude that HaI or HpaII sites can be divided in 3 classes according to their pattern of methylation in different tissues. In the first class of sites (mV class) the extent of methylation varies in different tissues. The patterns obtained show that methylation at the sites located within and around the 3 genes which code for egg white proteins is in general lowest in oviduct of laying hen, where these genes are expressed. However some sites are not methylated (m- class) and others are 95 to 100% resistant (m+ class) to digestion by HhaI or HpaII in the DNAs of all the tissues which were tested. Our study has also revealed a remarkable number of allelic variants for the presence of HhaI or HpaII sites in the region of the ovalbumin gene.

To analyse the relationship between DNA undermethylation at some sites in the ovalbumin and conalbumin gene regions (1) and the expression of these genes in chick oviduct, digestions with HhaI, which differentiates between methylated and unmethylated HhaI restriction sites, was performed on DNA isolated from chicken erythrocyte or oviduct chromatin treated with DNase I which degrades preferentially "active" chromatin. This was followed by analysis with ovalbumin- and conalbumin-specific hybridization probes. We conclude that the residual DNA methylation found at some sites of the ovalbumin and conalbumin gene regions is derived from the fraction of cells in which the chromatin of these genes is not in an "active" form. On the other hand, the ovalbumin and conalbumin sites which are partially unmethylated in erythrocyte DNA correspond to chromatin regions which are not DNase I-senitive. We have also detected a site about 1 kb downstream from the 3' end of the conalbumin gene that is hypersensitive to DNase I in all tissues tested.

The fragile X-mental retardation syndrome1is defined by a moderate to severe mental retardation associated with a cytogenetic marker, a fragile site localized on the long arm of the X chromosome at band Xq 27 (ref. 2). This syndrome has recently been recognized as one of the major causes of genetically determined mental retardation, and as one of the most important X-linked diseases with respect to its frequency (analogous to that of Duchenne muscular dystrophy or of haemophilia A) and severity3–6. In the absence of treatment, genetic screening for this disease would seem particularly important. Prenatal diagnosis is now feasible although difficult7,8and detection of heterozygous carriers is only possible in ∼50% of cases1,4,9. The recent demonstration of genetic linkage between the glucose 6-phosphate dehydrogenase (G6PD)–colour blindness cluster (at Xq28) and the fragile X locus has suggested that the fragile site is indeed the site of the mutation10. We show here that the fragile X and haemophilia B loci are closely linked, using as genetic marker a polymorphism of the coagulation factor IX gene11. Our study of a large family has demonstrated transmission through a phenotypically normal male, a feature previously described in retrospective analysis of a few other fragile X pedigrees1,6,12,13. Restriction polymorphisms associated with the factor IX gene should be useful for analysing this peculiar aspect of the genetics of the fragile X syndrome, and for genetic screening of the disease.

Friedreich ataxia (FRDA) is an autosomal recessive degenerative disorder that affects the cerebellum, spinal cord, and peripheral nerves. The FRDA gene was localized in 9q13-q21 within 0.7 centimorgan of the D9S5 and D9S15 loci. One recently reported recombination event and haplotype analysis in a population with a founder effect suggested that the FRDA locus is on the D9S5 side. Using a conserved probe from the D9S5 locus, we have now identified an approximately 7-kilobase (kb) transcript and report cloning of its cDNA. The corresponding gene, X11, extends at least 80 kb in a direction opposite D9S15. The gene is expressed in the brain, including the cerebellum, but is not detectable in several nonneuronal tissues and cell lines. In situ hybridization of adult mouse brain sections showed prominant expression in the granular layer of the cerebellum. Expression was also found in the spinal cord. The cDNA contains an open reading frame encoding a 708-amino acid sequence that shows no significant similarity to other known proteins but contains a unique, 24-residue-long, putative transmembrane segment. On the basis of its genomic localization and its neuronal site of expression, particularly in the cerebellum, this "pioneer" gene represents a candidate for FRDA. Direct evidence of its involvement in FRDA will require a search for causative point mutations in patients.

Three DNA-dependent RNA polymerases AI, BI and BII were purified from calf thymus. These enzymes were characterized by their sensitivity to amanitin, their subunit pattern and their template specificities. Enzyme AI is resistant to amanitin, while B enzymes are inhibited by this poison. Each of these enzymes contains two subunits of high molecular weight: A1 (M.W. 197 000) and A2 (M.W. 126 000) for enzyme AI, Bl (M.W. 214 000) and B3 (M.W. 140 000) for enzyme BI, B2 (M.W. 180 000) and B3 for enzyme BII. In addition, each enzyme contains several subunits of lower molecular weight. Purification of B enzymes from rat liver showed the presence of two enzymes similar to calf thymus RNA polymerases BI and BII and suggested the existence of an additional B enzyme in rat liver. The number of RNA polymerase form B molecules was determined in a variety of animal tissues by binding of labelled amanitin. The values ranged from 1 × 104to 6.5 × 104molecules per haploid genome.Studies with derivatives of rifamycin indicated that calf thymus B enzymes were able to recognize specific initiation sites on calf thymus DNA, which were different from those recognized by enzyme AI or E. coliRNA polymerase. These results favour the hypothesis that the multiplicity of animal RNA polymerases could play an active role in the control of transcription.