Genetic mapping of Xp22.12-p22.31, with a refined localization for spondyloepiphyseal dysplasia (SEDL).

Spondyloepiphyseal dysplasia tarda (SEDL) is an X-linked recessive disorder characterized in affected males by short stature resulting from a growth defect of the vertebral bodies. We have extended our earlier studies by analyzing 15 families with newly identified microsatellite DNA markers; analysis of recombination events with these markers indicates that the gene responsible for SEDL is located in Xp22 between DXS 16 and DXS 987 on an interval spanning approximately 2 Mb.

The gene for Bazex-Dupré-Christol syndrome maps to chromosome Xq.

Bazex-Dupré-Christol syndrome is an inherited condition with skin cancer predisposition characterized by follicular atrophoderma, hypotrichosis, and early onset of multiple basal cell carcinomas. Previous reports suggested an X-linked mode of inheritance. We therefore performed linkage analysis with microsatellite markers of the X chromosome in three families. We obtained evidence for X-linkage and regional assignment to Xq24-q27 of this syndrome (maximal lod score = 5.26 with a recombination fraction of 0% at the DXS1192 locus). This represents a first step towards the identification of a gene involved in hair follicle development and skin tumor formation.

Fine deletion mapping of the p22 region of the human X chromosome using a radiation-induced hybrid panel.

Radiation-induced somatic cell hybrids containing fragments of the human X chromosome were constructed. A panel of 17 hybrids was selected with the help of known markers in the Xp22 region. These hybrids identified 11 different breakpoints between Xp22.2 and Xp21.3. Eight markers were located in eight of the nine corresponding intervals, resulting in the following physical map: tel...DXS89-DXS278-DXS85-(DXS1224, DXS16)-(GLRA2, DXS987)-DXS207-(DXS-197, DXS1053)-(DXS43, DXS1195)-(DXS1229, DXS-999)-(DXS1052, DXS92, DXS274)-(DXS41, DXS1226)-DXS1198-DXS28...cen.

Localization of a new type of X-linked liver glycogenosis to the chromosomal region Xp22 containing the liver alpha-subunit of phosphorylase kinase (PHKA2).

We describe here a new type of X-linked liver glycogen storage disease. The main symptoms include liver enlargement and growth retardation. The clinical and biochemical abnormalities of this glycogenosis are similar to those of classical X-linked liver glycogenosis due to phosphorylase kinase deficiency (XLG). However, in contrast to patients with XLG, the patients described here have no reduced phosphorylase kinase activity in erythrocytes and leukocytes, and no enzyme deficiency could be found. Linkage analysis of four families with this X-linked type of liver glycogenosis assigned the disease gene to Xp22. Lod scores obtained with the markers DXS987, DXS207, and DXS999 were 3.97, 2.71, and 2.40, respectively, all at 0% recombination. Multipoint linkage analysis localized the disease gene between DXS143 and DXS989 with a maximum lod score of 4.70 at theta = 0, relative to DXS987. As both the classical XLG gene and the liver alpha-subunit of PHK (PHKA2) are also located in Xp22, this variant type of XLG may be allelic to classical XLG, and both diseases may be caused by mutations in PHKA2. Therefore, we propose to classify XLG as XLG type I (the classical type of XLG) and XLG type II (the variant type of XLG).

Deletions in the COL4A5 collagen gene in X-linked Alport syndrome. Characterization of the pathological transcripts in nonrenal cells and correlation with disease expression.

The type IV collagen alpha 5 chain (COL4A5) gene of 88 unrelated male patients with X-linked Alport syndrome was tested for major gene rearrangements by Southern blot analysis, using COL4A5 cDNA probes. 14 different deletions were detected, providing a 16% deletion rate in the COL4A5 gene in the patient population. The deletions are dispersed all over the gene with different sizes, ranging from 1 kb to the complete absence of the gene (> 250 kb) in one patient. In four patients with intragenic deletions, absence of the alpha 3 (IV) chain in the glomerular basement membrane was demonstrated by immunohistochemical studies. This finding supports the hypothesis that abnormalities in the alpha 5 (IV) chain may prevent normal incorporation of the alpha 3 (IV) chain into the glomerular basement membrane. Direct sequencing of cDNA amplified from lymphoblast mRNA of four patients with internal gene deletions, using appropriate combinations of primers amplifying across the predicted boundaries of the deletions, allowed us to determine the effect of the genomic rearrangements on the transcripts and, by inference, on the alpha 5 (IV) chain. Regardless of the extent of deletion and of the putative protein product, the 14 deletions occur in patients with juvenile-type Alport syndrome.

Rearrangements between irradiated chromosomes in three-species radiation hybrid cell lines revealed by two-color in situ hybridization.

A human-hamster hybrid cell line containing only the human X chromosome (GM06318B) was exposed to 6,000-7,000 rad of X-rays and fused with a mouse cell line (CL1D,TK-). Three radiation hybrids, LXKC40, LXKC50, and LXKC56, were selected among 39 independent clones containing human material. Two-color in situ hybridization with total genomic DNA probes (cot1 human DNA and hamster total genomic DNA) was used to analyse the irradiated chromosome rearrangements. With this three-species model system (human-hamster-mouse) and the chromosome painting process it was possible to determine the origin of each chromosomal fragment in metaphase and interphase. The results obtained indicate preferential rearrangement between irradiated human and hamster chromosomes. Whole, apparently intact hamster chromosomes were observed in all the mitoses. We suggest that these chromosomes could be neoformated from random fragments after irradiation. Hamster and human "minichromosomes" were also detected. While the integration of human material into the mouse genome was exceptional, the integration of hamster material into mouse chromosomes was more frequent. During interphase the irradiated chromosome domains were often at the periphery of the nucleus. Irradiated material protruded at the periphery of the nuclei. Micronuclei containing hamster material were detected in the vicinity of these protrusions.

Isolation and characterisation of five new anonymous X-specific probes. One of them detecting a TaqI RFLP.

The authors isolated five single-copy X-specific probes from an X-enriched library. These probes were regionally localized on the X chromosome by using somatic hybrid cell lines obtained from patients carrying different X-autosome translocations. Three clones were located between Xq23 and Xq26, the two others were mapped between Xp21 and Xp11.2. Analyses with different restriction enzymes indicated that one of them detects a TaqI polymorphism with a polymorphism information content (PIC) of 0.28.

An irradiation-reduced hybrid panel for fine-structure mapping of the Xq28 region in the human genome.

Irradiation-reduced somatic cell hybrids containing fragments of the human X chromosome were constructed. Analysis of 16 hybrids that retained the Xq28 region with 12 Xq28-specific markers identified at least six different breakpoints, supporting the order cen-DXS304-DXS374-(DXS33, DXS134, DXS52, DXS15)-RCP-(DXS254, G6PD, F8C)-(DXS115, DXYS64)-qter. The generated panel of hybrids provides a useful tool for fine mapping of probes in the Xq28 region.

Alport syndrome and diffuse leiomyomatosis: deletions in the 5' end of the COL4A5 collagen gene.

Alport syndrome (AS) is an hereditary glomerulonephritis that is mainly inherited as a dominant X-linked trait. Structural abnormalities in the type IV collagen alpha 5 chain gene (COL4A5), which maps to Xq22, have recently been detected in several patients with AS. The association of AS with diffuse esophageal leiomyomatosis (DL) has been reported in 24 patients, most of them also suffering from congenital cataract. The mode of transmission and the location of the gene(s) involved in this association have not been elucidated. Southern blotting using cDNA probes spanning the whole COL4A5 and a 5' end COL4A5 genomic probe showed that three out of three patients with the DL-AS association had a deletion in the 5' part of the COL4A5 gene extending beyond its 5' end. This indicates that the same gene, COL4A5, is involved in classical AS and in DL-AS and that the transmission of DL-AS is X-linked dominant. These results also suggest that leiomyomatosis might be due to the alteration of a second gene involved in smooth muscle cell proliferation, which is located upstream of the COL4A5 gene, and that there might be a contiguous gene deletion syndrome, involving at least the genes coding for congenital cataract, DL and AS.

Isolation of new probes from Xq12-->q13: an example of the screening of reference libraries with Alu-PCR products from radiation hybrids.

In order to isolate new probes from the juxtacentromeric region of the long arm of the human X chromosome, we used Alu-mediated polymerase chain reaction (Alu-PCR) products as probes to directly screen a chromosome X-specific gridded cosmid library. These Alu-PCR products were synthesized from radiation hybrids containing the loci DXS159, PGK1, and PGK1P1. This approach allowed us to select 18 cosmids capable of hybridizing with at least two Alu-PCR products. Four cosmids hybridized to more than three Alu-PCR products. Three of these four cosmids were contiguous, and the fourth was independent. Two cosmids that hybridized with two Alu-PCR products were further characterized. Physical mapping indicated that all of these clones are located in the expected region on Xq, confirming the validity of our approach.

Mapping of human chromosome 22 with a panel of somatic cell hybrids.

The adenylosuccinate lyase (ADSL) which is essential for generating adenylate, maps to the long arm of chromosome 22. By using a Chinese hamster ovary cell line deficient in ADSL activity, we have constructed a set of 17 somatic cell hybrids containing defined regions of human chromosome 22. This panel was extended with six additional hybrids, obtained in other laboratories using various methods of selection. Southern analysis of the hybrids with 38 chromosome 22 probes defined 14 different subregions which could be linearly organized on the long arm of chromosome 22. The order of the probes thus deduced is fully compatible with their previous localization and with the genetic map. The ADSL gene was further sublocalized between the MB and D22S22. This panel, which enables the rapid assignment of chromosome 22 single copy probes to small subregions, will be an important tool in the construction of a detailed physical map of this part of the genome.

New polymorphism and a new chromosome breakpoint establish the physical and genetic mapping of DXS369 in the DXS98-FRAXA interval.

Recently some of us cloned a new probe RN1 (DXS369), which appears a close marker for the fragile X locus (FRAXA) [Oostra et al.: Genomics 1990]. We present here new evidence for its physical and genetic mapping in the DXS98--FRAXA interval. We used 2 different somatic cell hybrid lines with breakpoints in the Xq27-q28 region: L10B Rea and PeCHN, and we established the order: (DXS105, DXS98)-L10B Rea-DXS369-PeCHN- (DXS304, DXS52). We detected an additional TaqI RFLP at the DXS369 locus which increases its informativeness up to 57%. Two point linkage analysis in a large set of families gave high lod scores for the FRAXA-DXS369 linkage (z(theta) = 10.1 at theta = 0.044) and for DXS369-DXS304, a marker distal to FRAXA (z = 19.2 at theta = 0.070). By multipoint analyses we established the localization of DXS369 in the DXS98-FRAXA interval. DXS369 is a much closer proximal marker for FRAXA than DXS105 or DXS98 and any new probe mapping between the breakpoints in L10B Rea and PeCHN will be of potential interest as a marker for FRAXA.

A new DNA marker tightly linked to the fragile X locus (FRAXA).

The fragile X syndrome is the most common cause of familial mental retardation. Genetic counseling and gene isolation are hampered by a lack of DNA markers close to the disease locus. Two somatic cell hybrids that each contain a human X chromosome with a breakpoint close to the fragile X locus have been characterized. A new DNA marker (DXS296) lies between the chromosome breakpoints and is the closest marker to the fragile X locus yet reported. The Hunter syndrome gene, which causes iduronate sulfatase deficiency, is located at the X chromosome breakpoint that is distal to this new marker, thus localizing the Hunter gene distal to the fragile X locus.

The gene for incontinentia pigmenti is assigned to Xq28.

A linkage study of eight families with incontinentia pigmenti (IP) has been performed, and linkage to site DXS52 has been established. We suggest that the IP locus lies in the Xq terminal region on the long arm of the X chromosome.

The anonymous PAS45 probe detects RFLPs in 13q31.

An anonymous DNA probe PAS45 was isolated. This probe detects an RFLP with two alleles 1 and 2 at the same locus, with the different restriction enzymes (Bg1II, EcoRI, HindIII, PstI, MspI, XbaI). The observed polymorphism is explained by a chromosome rearrangement involving these enzyme cleavage sites. The frequency of alleles 1 and 2 was 0.875 and 0.125, respectively, in a sample of 48 unrelated individuals in France. Codominant inheritance of alleles 1 and 2 was demonstrated in 13 families with 30 offspring. The PAS45 probe was localized on chromosome 13 by somatic cell hybrid analysis and on 13q31 by in situ hybridization. The rearrangement on 13q31 is present in one out of four healthy individuals in France.

Incontinentia pigmenti: Xp breakpoint is not the same in a case of r(X) and in X/autosome translocations.

X-specific DNA probes were used to characterize the r(X) of a 45,X/46,X,r(X) female patient with Incontinentia pigmenti. It was found to be of maternal origin. Breakpoints were shown to be in or distal to p11.22 and between q12.2 and q13.1. When considering all known cases of Incontinentia pigmenti and X rearrangements at least four different break sites on the X have been shown.

Spondyloepiphyseal dysplasia tarda: linkage with genetic markers from the distal short arm of the X chromosome.

A linkage study of six families with spondyloepiphyseal dysplasia tarda (SEDL) has been performed. A linkage to site DXS41 (theta = 0.08; z = 3.07) and DXS92 (theta = 0.05; z = 2.95) has been established. We propose that the SEDL locus lies on the distal part of the short arm of the X chromosome.

Linkage studies in X-linked Alport's syndrome.

Four kindreds segregating for Alport's syndrome (ASLN) compatible with a X-linked inheritance were studied for linkage with polymorphic markers of the human X chromosome. No recombinant was observed between the ASLN locus and the DXS101 and DXS94 loci, the maximum lod scores were z = 3.93 and 3.50 respectively. Linkage data between the ASLN locus and the other genetic markers used in the present study are in keeping with the assignment of the mutation to the proximal Xq arm.

Time dependence of X gene reactivation induced by 5-azacytidine: possible progressive restructuring of chromatin.

According to the theoretical mechanism of DNA demethylation by 5-azacytidine, the complete demethylation of one site will require two cell divisions. If reexpression is directly related to demethylation, a maximal reexpression is expected after two cell divisions. In a hamster X human hybrid cell line containing an inactive human X chromosome treated by 5-azacytidine, we show that HPRT reactivation frequency is increased more than 10-fold when cells are allowed to divide 14 times before the selection for the HPRT reactivants. We suggest that the delay corresponds to changes in chromatin conformation.