Genetic mapping of two new DNA markers in Xq26-q28 relative to the fragile-X syndrome locus.

We have characterized and genetically mapped two new DNA markers (DXS311 and DXS312) with respect to 10 existing loci in Xq26----Xq28 in a set of 15 families in which the fragile-X [fra(X)] syndrome was segregating. Two-point and multipoint linkage analyses were performed taking into account the incomplete penetrance of the fra(X) mutation. The most likely order on the basis of these data is centromere-DXS79-DXS10-DXS311-DXS86-(F9-DXS99 )-(DXS98-DXS312)-fra(X)-DXS52- DXS15-F8C-telomere. DXS98 and one of the new loci, DXS312, were found to be the proximal markers closest to the fra(X) locus. The order F9-(DXS98-DXS312)-fra(X) was found to be 5.9 x 10(4) times more likely than the order (DXS98-DXS312)-F9-fra(X).     

Genetic mapping of new DNA probes at Xq27 defines a strategy for DNA studies in the fragile X syndrome

The fragile X syndrome is the most common cause of familial mental retardation and is characterized by a fragile site at the end of the long arm of the X chromosome. The unusual genetics and cytogenetics of this X-linked condition make genetic counseling difficult. DNA studies were of limited value in genetic counseling, because the nearest polymorphic DNA loci had recombination fractions of 12% or more with the fragile X mutation, FRAXA. Five polymorphic loci have recently been described in this region of the X chromosome. The positions of these loci in relation to FRAXA were defined in a genetic linkage study of 112 affected families. The five loci--DXS369, DXS297, DXS296, IDS, and DXS304--had recombination fractions of 4% or less with FRAXA. The closest locus, DXS296, was distal to FRAXA and had a recombination fraction of 2%. The polymorphisms at these loci can be detected in DNA enzymatically digested with a limited number of restriction endonucleases. A strategy for DNA studies which is based on three restriction endonucleases and on five probes will detect one or more of these polymorphisms in 94% of women. This strategy greatly increases the utility of DNA studies in providing genetic advice to families with the fragile X syndrome.     

Genetic heterogeneity in X-linked hydrocephalus: linkage to markers within Xq27.3.

X-linked hydrocephalus is a well-defined disorder which accounts for > or = 7% of hydrocephalus in males. Pathologically, the condition is characterized by stenosis or obliteration of the aqueduct of Sylvius. Previous genetic linkage studies have suggested the likelihood of genetic homogeneity for this condition, with close linkage to the DXS52 and F8C markers in Xq28. We have investigated a family with typical X-linked aqueductal stenosis, in which no linkage to these markers was present. In this family, close linkage was established to the DXS548 and FRAXA loci in Xq27.3. Our findings demonstrate that X-linked aqueductal stenosis may result from mutations at two different loci on the X chromosome. Caution is indicated in using linkage for the prenatal diagnosis of X-linked hydrocephalus.     

Mapping of DNA markers close to the fragile site on the human X chromosome at Xq27.3.

We report the identification of a new RFLP detected by the DNA probe MN12, which is linked to both the fragile site on the X chromosome at Xq27.3 and the highly polymorphic locus detected by St14 (DXS52). In situ mapping confirms the localisation of MN12 distal to the fragile site. A detailed physical analysis of this region of the X chromosome using pulsed-field gel electrophoresis has shown that MN12, St14 and DX13 (DXS15) are physically linked within a region of 470kb. A long range restriction map around the MN12 locus reveals at least two candidate HTF islands, suggesting the existence of expressed sequences in this region.     

Linear order of new and established DNA markers around the fragile site at Xq27.3

We have used recombinant clones derived from microdissection of the fragile X region to characterize breakpoints around the fragile site at Xq27.3. So far, no microdissection markers derived from Xq28 material have been found, thus allowing a rapid screening for clones surrounding the fragile site by their presence in a somatic cell hybrid containing Xq27.2-Xqter. A total of 43 new DNA markers from Xq27 have been sublocalized within this chromosome band. Of these new DNA markers, 5 lie in an interval defined as containing the fragile X region. The saturation of Xq27 with DNA markers by microdissection demonstrates the power of this technique and provides the resources for generating a complete physical map of the region.     

Genetic mapping of new RFLPs at Xq27-q28.

The development of the human gene map in the region of the fragile X mutation (FRAXA) at Xq27 has been hampered by a lack of closely linked polymorphic loci. The polymorphic loci DXS369 (detected by probe RN1), DXS296 (VK21A, VK21C), and DXS304 (U6.2) have recently been mapped to within 5 cM of FRAXA. The order of loci near FRAXA has been defined on the basis of physical mapping studies as cen-F9-DXS105-DXS98-DXS369-DXS297-FRAXA-++ +DXS296-IDS-DXS304-DXS52-qter. The probe VK23B detected HindIII and XmnI restriction fragment length polymorphisms (RFLPs) at DXS297 with heterozygote frequencies of 0.34 and 0.49, respectively. An IDS cDNA probe, pc2S15, detected StuI and TaqI RFLPs at IDS with heterozygote frequencies of 0.50 and 0.08, respectively. Multipoint linkage analysis of these polymorphic loci in normal pedigrees indicated that the locus order was F9-(DXS105, DXS98)-(DXS369, DXS297)-(DXS293,IDS)-DXS304-DXS52. The recombination fractions between adjacent loci were F9-(0.058)-DXS105-(0.039)-DXS98-(0.123)-DXS369-(0.00)- DXS297-(0.057)-DXS296- (0.00)-IDS-(0.012)-DXS304-(0.120)-DXS52. This genetic map will provide the basis for further linkage studies of both the fragile X syndrome and other disorders mapped to Xq27-q28.     

Concurrence of the triple-X syndrome and expression of the fragile site Xq27.3

Summary The Xq27.3 fragile site was found to be expressed in an XXX woman, who was mentally and physically normal, and in her son who was mentally retarded and showed behavioural and physical features characteristic of the fragile X syndrome.     

Genetic mapping of the Xq27-q28 region: new RFLP markers useful for diagnostic applications in fragile-X and hemophilia-B families.

We have characterized and genetically mapped new polymorphic DNA markers in the q27-q28 region of the X chromosome. New informative RFLPs have been found for DXS105, DXS115, and DXS152. In particular, heterozygosity at the DXS105 locus has been increased from 25% to 52%. We have shown that DXS105 and DXS152 are contained within a 40-kb region. A multipoint linkage analysis was performed in fragile-X families and in large normal families from the Centre d'Etudes du Polymorphisme Humain (CEPH). This has allowed us to establish the order centromere-DXS144-DXS51-DXS102-F9-DXS105-FRAX A-(F8, DXS15, DXS52, DXS115). DXS102 is close to the hemophilia-B locus (z[theta] = 13.6 at theta = .02) and might thus be used as an alternative probe for diagnosis in Hemophila-B families not informative for intragenic RFLPs. DXS105 is 8% recombination closer to the fragile-X locus than F9 (z[theta] = 14.6 at theta = .08 for the F9-DXS105 linkage) and should thus be a better marker for analysis of fragile-X families. However, the DXS105 locus appears to be still loosely linked to the fragile-X locus in some families. The multipoint estimation for recombination between DXS105 and FRAXA is .16 in our set of data. Our data indicate that the region responsible for the heterogeneity in recombination between F9 and the fragile-X locus is within the DXS105-FRAXA interval.     

Genetic mapping of X-linked albinism-deafness syndrome (ADFN) to Xq26.3-q27.1

X-linked albinism-deafness syndrome (ADFN) was described in one Israeli Jewish family and is characterized by congenital nerve deafness and piebaldness. The ADFN mutation probably affects the migration of neural crest-derived precursors of the melanocytes. As a first step toward identifying the ADFN gene, a linkage study was performed to localize the disease locus on the X chromosome. The family was found to be informative for 11 of 107 RFLPs along the X, and two-point analysis showed four of them--factor 9 (F9), DXS91, DXS37, and DNF1--to have definite or suggestive linkage with ADFN. Multipoint linkage analysis indicated two possible orders within this cluster of loci, neither of which was preferable. In both orders F9 was the most distal, and the best estimate for the location of ADFN was between F9 and the next proximal marker (8.6 cM from F9 [Z = 8.1] or 8.3 cM from F9 [Z = 7.9]). These results suggest that the ADFN is at Xq26.3-q27.1. Disagreement between our data and previous localization of DXS91 at Xq11-q13 was resolved by hybridization of the probe pXG-17, which detects the DXS91 locus, to a panel of somatic cell hybrids containing different portions of the X chromosome. This experiment showed that this locus is definitely at Xq24-q26. Together with the linkage data, our results place DXS91 at Xq26 and underscore the importance of using more than one mapping method for the localization of molecular probes.     

Fine mapping of the nail-patella syndrome locus at 9q34.

Nail-patella syndrome (NPS), or onychoosteodysplasia, is an autosomal dominant, pleiotropic disorder characterized by nail dysplasia, absent or hypoplastic patellae, iliac horns, and nephropathy. Previous studies have demonstrated linkage of the nail-patella locus to the ABO and adenylate kinase loci on human chromosome 9q34. As a first step toward isolating the NPS gene, we present linkage analysis with 13 polymorphic markers in five families with a total of 69 affected persons. Two-point linkage analysis with the program MLINK showed tight linkage of NPS and the anonymous markers D9S112 (LOD = 27.0; theta = .00) and D9S315 (LOD = 22.0; theta = .00). Informative recombination events place the NPS locus within a 1-2-cM interval between D9S60 and the adenylate kinase gene (AK1).     

Evidence for an iduronate-sulfatase pseudogene near the functional Hunter syndrome gene in Xq27.3-q28

We are currently characterizing mutations of the iduronate-2-sulfatase (IDS) gene in patients with Hunter syndrome (mucopolysaccharidosis type II). Surprisingly, all 17 patients with a mutation in exon III of the IDS gene identified by us were found to carry both the mutant and wild-type sequences in polymerase chain reaction (PCR) products amplified from genomic DNA. Similarly, two unaffected male controls showed a heterozygous pattern for two different point mutations in exon III. Collectively, the data suggest that at least intron 2, exon III, and the 3-half of exon II of the functional IDS gene are present in the human genome as (part of) a non-expressed IDS gene. Deletion mapping further suggests that the pseudogene is in distal Xq in physical proximity to the functional IDS gene. The high degree of sequence homology observed between the functional IDS gene and pseudogene results in permanent co-amplification in PCR-based screening methods and makes mutation analysis at the genomic DNA level difficult.     

Genetic and physical mapping of Xq24-q26 markers flanking the Lowe oculocerebrorenal syndrome

The Lowe oculocerebrorenal syndrome (OCRL) is characterized by congenital cataract, mental retardation, and renal tubular dysfunction. We are using the approaches of linkage analysis, mapping with somatic cell hybrids, and long-range restriction mapping to determine the order of Xq24-q26 markers with respect to each other and to the OCRL locus. DXS42 and DXS100 are proximal to the translocation breakpoint in a female patient with OCRL and a de novo translocation t(X;3)(q25;q27). DXS10, DXS86, HPRT, and DXS177 are distal to the breakpoint. These flanking markers show tight linkage to the disease locus in 11 families segregating for OCRL. Results from field inversion gel analysis show that DXS86 and DXS10 share a 460-kb BssHII fragment. Multipoint analysis to determine the position of HPRT with respect to (DXS10,DXS86) suggests that HPRT is proximal to (DXS10,DXS86). We propose the following order for markers in Xq24-q26: Xcen-(DXS42,DXS37,DXS100)-OCRL-DXS53 -HPRT-[(DXS10,DXS86),DXS177]-Xqter. The identification of additional tightly linked flanking markers extends the number of markers available for use in genetic counseling and begins to define the physical map of the region containing the gene for OCRL.     

Genetic mapping of the Salmonella typhimurium pncB locus.

The nicotinic acid phosphoribosyltransferase locus pncB was located on the Salmonella typhimurium linkage map counterclockwise relative to pyrC. P22 and P1 transductional analyses revealed linkage of pncB with aroA and pyrD, indicating a pncB map position of approximately 20 map units. The results of these cotransduction experiments also indicated that the genetic map distance between gal and pyrD is greater than the published 2.2 map units.     

Genetic mapping of the Salmonella typhimurium pepB locus.

Transposon technology has been used to map the pepB locus of Salmonella typhimurium. This locus is cotransducible by phage P22 with glyA and strB at min 56 on the Salmonella genetic map. The gene order is strB pepB glyA.     

Oto-palato-digital syndrome type I: further evidence for assignment of the locus to Xq28

Summary The oto-palado-digital syndrome (OPD) is a rare X-linked disease with diagnostic skeletal features, conduction deafness, cleft palate and mild mental retardation. Differences in clinical presentation between families have led investigators to classify OPD into two subtypes: type I and type II. A linkage study performed in one family segregating for OPD I has recently suggested linkage to three marker loci: DXS15, DXS52 at Xq28, and DXS86 at Xq26. We have investigated an additional OPD I family for linkage by using distal chromosome Xq DNA probes. The linkage data and the analysis of recombination events that have occurred in this family excluded, definitively, the Xq26 region for OPD I, and provide further support for mapping the mutant gene close to the cluster of tightly linked markers DXS15, DXS52 and DXS305 at Xq28.     

The human genes for hemophilia A and hemophilia B flank the X chromosome fragile site at Xq27.3.

Two DNA recombinant clones, shown by separate studies to contain DNA sequences homologous to the genes coding for the human blood coagulation Factors VIII and IX, were hybridized in situ to metaphases or prometaphases derived from patients with the fragile-X syndrome and from a normal control. The results of these experiments indicate that (i) both genes are located in the subtelomeric region of the long arm of the human X chromosome flanking the fragile site at Xq27.3, (ii) the resolution of this localization is approximately 0.5% the length of the human haploid genome, i.e., 1.8 X 10(7) bp, (iii) the linear order of loci within the above region is Factor IX-fragile site-Factor VIII-Xqter. Both the localization and the linear order of these loci have been confirmed by Southern blotting studies using the same molecular probes and a panel of rodent-human somatic cell hybrids known to have retained different segments of the human X chromosome. The findings described herein and the knowledge that Factor IX deficiency recombines freely with at least two loci of the G6PD cluster support our hypothesis that the chromosomal region which includes the fragile-X site is normally a region of high meiotic recombination.     

Allan-Herndon syndrome. II. Linkage to DNA markers in Xq21.

The original family with the Allan-Herndon type of X-linked mental retardation has been investigated for linkage by using DNA probes spanning the length of the X chromosome. Available for study, over 3 generations, were 13 affected males, three obligate carriers, and three normal sons of the obligate carriers. Initial disease-to-marker analysis suggested linkage to three markers (DXYS2 [7b], DXS250 [GMGX22], and DXS3 [p19-2]) located in Xq21. All three exhibited the same maximum lod score of 2.3 at a maximum theta of .05. Multipoint analysis using LINKMAP and a set of four DNA markers (DXYS1-DXYS2-DXS3-DXS94) gave a multipoint lod score of 3.58 for a location of the Allan-Herndon syndrome near locus DXYS1 (pDP34). Therefore, our data indicate that the gene for the Allan-Herndon syndrome is likely located in Xq21.     

Waisman syndrome, a human X-linked recessive basal ganglia disorder with mental retardation: localization to Xq27.3-qter.

Linkage of the gene responsible for an X-linked early onset parkinsonism disorder with mental retardation (McKusick 311510) to DNA probes that detect restriction fragment length polymorphisms is described. The disease gene is linked to the F8C gene, and to DNA probes detecting polymorphic loci DXS52, DXS15, DXS134, and DXS374 with maximum lod scores at theta = 0 of 5.08, 5.19, 5.00, 5.03, and 4.46, respectively. Multipoint linkage analysis gives a maximum multipoint lod score of 6.75 at the F8C gene. This places the disease gene in chromosomal region Xq27.3-qter.     

Genetic mapping of the mouse X chromosome in the region homologous to human Xq27–Xq28

The four loci Gabra3, DXPas8, CamL1, and Bpa, located near the murine X-linked visual pigment gene (Rsup), have been ordered using 248 backcross progeny from an interspecific mating of (B6CBA-A^w-J/A-Bpa) and Mus spretus. One hundred twenty backcross progeny have been analyzed at seven anchor loci spanning the X chromosome and form a regional mapping panel. An additional 128 progeny have been screened for recombination events between Cf-9 and Dmd. Eighteen recombinants between these loci have been detected in the 248 animals; all of the recombinants were screened at the other anchor loci to identify any double crossovers. Pedigree analysis using these recombinants strongly favors a gene order of (Cf-9)-Gabra3-(DXPas8, Bpa)-CamL1-(Rsvp, P3, Cf-8)-Dmd for the loci studied. Synteny with human Xq27–Xq28 is retained, although the relative order of some loci may differ between the two species.     

Genetic mapping of the dentinogenesis imperfecta type II locus.

Dentinogenesis imperfecta type II (DGI-II) is an autosomal dominant disorder of dentin formation, which has previously been mapped to chromosome 4q12-21. In the current study, six novel short tandem-repeat polymorphisms (STRPs) have been isolated, five of which show significant evidence of linkage to DGI-II. To determine the order of the STRPs and define the genetic distance between them, nine loci (including polymorphisms for two known genes) were mapped through the CEPH reference pedigrees. The resulting genetic map encompasses 16.3 cM on the sex-averaged map. To combine this map with a physical map of the region, all of the STRPs were mapped through a somatic cell hybrid panel. The most likely location for the DGI-II locus within the fixed marker map is in the D4S2691-D4S2692 interval of 6.6 cM. The presence of a marker that shows no recombination with the DGI-II phenotype between the flanking markers provides an important anchor point for the creation of physical continuity across the DGI-II candidate region.