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.     

Mapping of a new RFLP marker RN1 (DXS369) close to the fragile site FRAXA on Xq27-q28.

A new polymorphic DNA marker RN1, defining locus DXS369, was recently isolated. Using different somatic cell hybrids, RN1 was mapped between markers 4D-8 and U6.2. We have narrowed the localization of RN1 to the region between 4D-8 and FRAXA by genetic mapping in fragile X [fra(X)] families. Combined with information from other reports, the following order of loci on Xq27-q28 is suggested: cen-F9-(DXS105-DXS152)-DXS98-DXS369-FRAXA- DXS304-(DXS52-DXS15-F8)-tel. The locus DXS369 is closely linked to FRAXA, with a peak lodscore of 18.5 at a recombination fraction of 0.05. Therefore, RN1 is a useful probe for carrier detection and prenatal diagnosis in fra(X) families.     

Linkage relationships between DXS105, DXS98, and other polymorphic DNA markers flanking the fragile X locus.

We report on linkage data between DXS105, DXS98, the locus for the fragile X syndrome (FRAXA), and 3 other polymorphic loci that flank the FRAXA locus. An analysis was undertaken to determine the relative positions of DXS105 and DXS98 and to test the assignment of DXS105 to a location proximal and closely linked to FRAXA. In this study of fragile X fra(X) syndrome families, the DXS105 locus was calculated to be proximal to FRAXA with a maximum lod score of 10.36 at theta = 0.08. DXS105 was also shown to be closely linked to the gene for factor IX (F9)(Z = 11.84 at theta = 0.08) and to DXS98 (Z = 4.91 at theta = 0.04). The order of the loci proximal to FRAXA is most likely centromere-factor IX-DXS105-DXS98-FRAXA-telomere. The use of DXS105 and DXS98 in clinical investigations should significantly increase the accuracy of risk assessment in informative fragile X families.     

Multipoint linkage analysis of DXS369 and DXS304 in fragile X families

Diagnosis of carriers of the fragile-X mental retardation gene is hampered by the paucity of tightly linked DNA markers. Recently, 2 new DNA markers RN1 (DXS369) and U6.2 (DXS304) have become available. Both markers are tightly linked to the fragile-X locus, but their location relative to the fragile site was not known with certainty. We have tested these new markers in a multipoint linkage analysis of 26 fragile-X families typed for DXS105 as a proximal marker and DXS52 as a distal marker. Our results establish the order DXS105-DXS369-fra(X)-DXS304-DXS52, which is in agreement with physical mapping results.     

Linkage in fragile X families of three distal flanking markers: ST14, DX13, and F8.

The use of linked DNA markers and linkage analysis in the fragile X [fra(X)] syndrome allows for improved genetic counseling and prenatal diagnosis. In order to provide the most accurate information, it is important to determine the order and location and position of flanking markers. Conflicting results have been reported for the order of 3 DNA markers distal to the fra(X) locus. We analyzed the linkage relationships of the distal markers ST14 (DXS52), DX13 (DXS15), and F8 (F8C) in 102 fra(X) families. The results indicated that the 3 DNA markers were closely linked to one another and mapped approximately 11 to 15% recombination units away from the fra(X) locus. The most likely order was fra(X)-DXS52-DXS15-F8. The order fra(X)-DXS52-F8 and 728 times more likely than the order fra(X)-F8-DXS52. One family showed a probable double recombinant: in one individual there was recombination between fra(X)-DXS52 and between DXS52-F8. The low probability of this occurring, 0.3%, raises the possibility of an alternate chromosome arrangement or an unusual recombinant mechanism in some individuals.     

Recombination between the factor VIII gene and the DXS52 locus gives the most probable genetic order as centromere-fra(X)-DXS15-DXS52-F8C-telomere

The linkage relationship between the factor VIII gene (F8C) and the DXS52 locus was examined in 8 families. Two recombinations were identified in 35 informative meioses (Zmax = 5.67; theta = 0.05), one in a family with hemophilia A, the other in a family with the fra(X) syndrome. Based on the latter recombination, the most probable order of loci was determined to be centromere-fra(X)-DXS15-DXS52-F8C-telomere. When these data are added to those reported previously the most probable genetic distance between F8C and DXS52 is 3 cM (Z = 14.62). Identification of these and other recombinations suggests that the use of DXS52 as a genetic marker for carrier detection and prenatal diagnosis of hemophilia A has an error rate between 3-5%.     

Linkage analysis in the fragile X syndrome using multiple distal Xq polymorphic DNA markers.

Linkage data using the polymorphic loci F9, DXS105, DXS98, DXS52, DXS15, and F8 and the DNA probe 1A1 are presented from 14 families segregating for fragile X [fra(X)] syndrome. Recombination fractions corresponding to the maximum LOD scores obtained by two-point linkage analysis suggest that DXS98 (Zmax = 3.23, theta = 0.0) and DXS105 (Zmax = 2.09, theta = 0.0) are the closest markers proximal to FRAXA and that DXS52 is the closest distal marker (Zmax = 3.55, theta = 0.16). FRAXA is located within a 25 cM interval between F9 and DXS52, coincident with DXS98, on multipoint linkage analysis. Phase-known three way crossover information places F8 outside the cluster (DXS52, DXS15, 1A1). Confidence limits for the markers DXS98 and DXS52 are relatively wide (0.0-0.15 and 0.06-0.31, respectively), but when used in combination with cytogenetic examination offer improved carrier detection in comparison with cytogenetic analysis alone.     

Bridging markers defining the map position of X linked hypophosphataemic rickets.

Hypophosphataemic rickets is commonly an X linked dominant hereditary disorder associated with a renal tubular defect in phosphate transport and bone deformities. The gene causing this disorder has been mapped to Xp22.31----p21.3 by using cloned human X chromosome sequences identifying restriction fragment length polymorphisms (RFLPs) in linkage studies of affected families. The hypophosphataemic rickets gene locus (HPDR) was previously mapped distal to the X linked polymorphic locus DXS41 (99.6) but its position in relation to the distal loci DXS43 (D2) and DXS85 (782) was not established. In order to obtain a precise mapping of the disease locus in relation to these genetic loci, additional affected families informative for these X linked markers have been investigated. The combined results from the two studies have established linkage with the loci DXS41 (99.6) and DXS43 (D2); peak lod score for DXS41 (99.6) = 7.35, theta = 0.09, and peak lod score for DXS43 (D2) = 4.77, theta = 0.16. Multilocus linkage analysis mapped the hypophosphataemic rickets gene distal to the DXS41 (99.6) locus and proximal to the DXS43 (D2) locus, thereby revealing two bridging genetic markers for the disease.     

Carrier detection of the fragile X syndrome using flanking loci DXS98, DXS105, and DXS304.

Diagnosis of the carrier status of the fragile X [fra(X)] syndrome was made in 2 unrelated women who did not express the fragile site. Both were related to several individuals with a typical fra(X) phenotype and the marker X chromosome. A restriction fragment length polymorphism (RFLP) approach was used with probes that flank the fra(X) locus (FRAXA). The loci used for risk calculations of the fra(X) genotype were DXS98 and DXS105 on the centromeric side and a recently characterized locus, DXS304, on the telomeric side. Coincidence correction for the distances between marker loci and FRAXA was made according to the Kosambi function. The DNA marker test gave the risk for one female to be a carrier of 99.7-99.9%. In another family a female was excluded from being a carrier with a probability of greater than 99.7%. The DNA marker U6.2, defining the locus DXS304, has increased the reliability of DNA based diagnosis of carrier status for females-at-risk. It is concluded that DNA analysis can serve as a valuable complement to chromosome analysis in families informative for the more closely linked flanking markers.     

Improved genetic mapping of X linked retinoschisis.

X linked retinoschisis (RS) causes poor vision in affected males owing to radial cystic changes at the macula. Genetic linkage analysis was carried out in 16 British families with X linked retinoschisis using markers from the Xp22 region. Linkage was confirmed between the RS locus and the markers DXS207 (lod score, Zmax = 17.9 at recombination fraction theta = 0.03; confidence interval for theta = 0.007-0.09), DXS1053 (Zmax = 18.0 at theta = 0.01, CI = 0.001-0.06), DXS43 (Zmax = 12.9 at theta = 0.03, CI = 0.004-0.09), DXS1195 (Zmax = 6.4 at theta = 0.00), DXS418 (Zmax = 8.2 at theta = 0.00), DXS999 (Zmax = 21.2 at theta = 0.01, CI = 0.001-0.05), DXS443 (Zmax = 14.2 at theta = 0.03, CI = 0.004-0.09), DXS365 (Zmax = 24.5 at theta = 0.008, CI = 0.001-0.04). Key recombinants placed RS between DXS43 distally and DXS999 proximally. Multipoint linkage analysis gave odds of 344:1 in favour of this location for RS and supported the map Xpter-(DXS207, DXS1053)-DXS43-1 cM-RS-1 cM-DXS999-DXS443-DXS365-DXS1052-Xcen.     

Genetic mapping of the Kallmann syndrome and X linked ocular albinism gene loci.

The X linked form of Kallmann syndrome (KAL) and X linked ocular albinism (OA1) have both been mapped to Xp22.3. We have used a dinucleotide repeat polymorphism at the Kallmann locus to type 17 X linked ocular albinism families which had previously been typed for the Xg blood group (XG) and the DNA markers DXS237 (GMGX9), DXS143 (dic56), and DXS85 (782). Close linkage was found between KAL and OA1 with a maximum lod score (Zmax) of 30.14 at a recombination fraction (theta max) of 0.06 (confidence interval for theta: 0.03-0.10). KAL was also closely linked to DXS237 (Zmax = 15.32; theta max = 0.05; CI 0.02-0.12) and DXS143 (Zmax = 14.57; theta max = 0.05; CI 0.02-0.13). There was looser linkage to the Xg blood group (XG) and to DXS85 (782). Multipoint linkage analysis gave the map: Xpter-XG-0.13-DXS237-0.025-KAL-0.025-DXS143-0.01 5-OA1-0.09-DXS85-Xcen. Placement of OA1 proximal to DXS143 was supported by odds of 2300:1 compared to other orders. This confirms our previous localisation of OA1 and improves the genetic mapping of both disease loci.     

Linkage and risk assessment in fragile X families using new DNA probes at Xq27.

Until recently few polymorphic loci had been genetically mapped close to the fragile X syndrome locus [FRAXA]. Six polymorphic loci, DXS369, DXS297, DXS296, DXS304, IDS and DXS374, have now been mapped closer to the fragile X FRAXA than in the present study. We report the results of genetic linkage analysis of 32 fragile X [fra(X)] families using 12 polymorphic loci including these new markers. Cytogenetic and molecular data were combined in two-point linkage analysis for the estimation of lod scores and carrier probabilities in potential carriers. Combined with results from previous studies, recombination fractions (0) corresponding to the maximum lod scores (Z max) were obtained for each of the 12 loci versus FRAXA. Recombination fractions between marker loci in the families were also calculated. The data were evaluated to determine the efficacy of using the strategy suggested by Suthers et al. (1991a) for molecular studies in fra(X) families. The large proportion of females heterozygous for at least one locus (83%) and of females heterozygous for flanking loci (60%) indicate that this is a very useful diagnostic strategy. Use of these new marker loci substantially changed the carrier risk estimates for members of 7 of the 32 families from the risk estimates previously calculated on the basis of less closely linked probes available prior to 1989.     

Linkage between the fragile X and F9, DXS52 (St14), DXS98 (4D-8) and DXS105 (cX55.7)

Linkage data using the markers F9, DXS105 (cX55.7), DXS98 (4D-8) and DXS52 (St14) are presented from 22 kindreds segregating with the fragile X. Two-point linkage analysis was carried out taking into account cytogenetic results and penetrance classes defined by mental impairment status of mothers. Recombination frequencies (theta) corresponding to the maximum z scores (z) were obtained between F9 (z = 3.48, theta = 0.18), DXS105 (z = 5.06, theta = 0.07), DXS98 (z = 4.79, theta = 0.01) and DXS52 (z = 6.44, theta = 0.09) and the fragile X. Recombination frequencies between marker loci in fragile X families are also presented. These recombination frequencies need to be combined with those from other studies in order to determine the best estimates of map distances for use in genetic counselling, until markers closer to the fragile X, or at the fragile X, can be used. Most potential fra(X) heterozygotes were informative for flanking markers using the above 4 probes. Carrier risks were determined by 3-point analysis using informative flanking markers, taking into account cytogenetic results. Low level fra(X) expression occurred in 2 probable non-carriers; emphasising the need for extreme caution in the interpretation of low rates of expression.     

X linked neonatal myotubular myopathy: one recombination detected with four polymorphic DNA markers from Xq28.

A three generation family with X linked myotubular myopathy (MTM1) was studied with several polymorphic markers from the distal long arm of the X chromosome. A recombination between the disease gene and four markers (loci DXS52, DXS134, DXS15, F8C) from the Xq28 cluster was detected. A new polymorphic marker (U6.2) defining the locus DXS304 in the Xq27-28 region proximal to the Xq28 cluster did not show any recombination with MTM1. These results suggest the following order of loci in distal Xq: cen-DXS42-DXS105-(DXS304, MTM1)-(DXS52, DXS134, DXS15, F8C)-tel.     

Hemophilia A: genetic prediction and linkage studies in all available families in Finland

RFLP studies were done in 82 (75%) of all known hemophilia A families in the Finnish population (approximately 5 million). Two intragenic RFLPs (Bc1I/F8A, XbaI/p482.6) and two extragenic markers (TaqI/St14, Bg1II/DX13) were used. Among 263 females at risk, carriership could be evaluated with an intragenic marker in 47% and with an extragenic marker in 26%. In 27% of the females, carriership could be neither excluded nor confirmed; 68% of these females were relatives of an isolated patient. Eight recombinations between the factor VIII gene (F8C) and DXS52 (lod 25.02 at theta max 0.06), eight recombinations between F8C and DXS15 (lod 21.91 at theta max 0.05), and two recombinations between DXS52 and DXS15 (lod 33.56 at theta max 0.01) were found. Using multipoint linkage analysis, the most likely order of loci supported by the data was: F8C-DXS15-DXS52-DXS134. RFLP segregation analysis provides a highly useful method of carrier detection and prenatal diagnosis of hemophilia A, but its limitations must be carefully taken into account.     

Probable localisation of the Coffin-Lowry locus in Xp22.2-p22.1 by multipoint linkage analysis

The Coffin-Lowry syndrome (McKusick No. 30360) is a rare genetically transmitted disorder characterized by severe mental retardation, "coarse" facial appearance, thick soft skin, tapering fingers, and progressive skeletal abnormalities. X-linked inheritance is implied since the males are severely affected with variably mild manifestations in carrier women. We have performed a linkage analysis with many X-linked RFLP markers in 4 families. Positive two-point lod scores were obtained with DXS28 (z(theta) = 2.00 at theta = 0.05) and DXS41 (z(theta) = 1.26 at theta = 0.10). We performed a 5-point linkage analysis using the LINKMAP program assuming that DXS16 and DXS43 are a single locus and using the following fixed map (distances in centimorgans): DXS85 - 18cM - (DXS16, DXS43) - 13cM - DXS41 - 5cM -DXS28. This gave a multipoint lod score of 3.41 for a localisation in Xp22.2-p22.1, between DXS43 and DXS41.     

Characterisation of a highly polymorphic microsatellite at the DXS207 locus: confirmation of very close linkage to the retinoschisis disease gene.

Juvenile retinoschisis (RS) is an X linked recessive vitreoretinal disorder for which the basic molecular defect is unknown. The gene for RS has been previously localised by linkage analysis to Xp22.1-p22.2 and the locus order Xpter-DXS16-(DXS43, DXS207)-RS-DXS274-DXS41-Xcen established. To improve the resolution of the genetic map in the RS region, we have isolated a highly polymorphic microsatellite at DXS207, which displays at least nine alleles with a heterozygosity of 0.83. Using this microsatellite and four other Xp22.1-p22.2 marker loci, DXS16, DXS43, DXS274, and DXS41, we performed pairwise and multilocus linkage analysis in 14 kindreds with RS. The microsatellite was also typed in the CEPH (Centre d'Etude du Polymorphisme Humain) reference families. Tight linkage was found between RS and DXS207 (Z(theta) = 14.32 at theta = 0.0), RS and DXS43 (Z(theta) = 8.10 at theta = 0.0), and DXS207 and DXS43 (Z(theta) = 40.31 at theta = 0.0). Our linkage results combined with data previously reported suggest that the DXS207-DXS43 cluster is located less than 2 cM telomeric to the RS locus. The microsatellite reported here will be a very useful marker for further linkage studies with retinoschisis as well as with other diseases in this region of the X chromosome.     

Linkage analysis of two Canadian families segregating for X linked spondyloepiphyseal dysplasia.

X linked spondyloepiphyseal dysplasia (SED) is caused by a growth defect of the vertebral bodies leading to characteristic changes in the vertebral bodies and a short trunk. The gene responsible for this disorder has previously been mapped to Xp22, with a maximum likelihood location between markers DXS16 and DXS92. We present linkage data using microsatellite markers on two Canadian X linked SED families, one of Norwegian descent and the other from Great Britain. In the Xp22 region, three recombination events have occurred in these families, two between markers DXS996 and DXS1043 and one between DXS999 and DXS989. One family shows a maximal lod score of 3.0 at theta = 0 with marker DXS1043 and the other family has a maximal lod score of 1.2 at theta = 0 with markers DXS1224 and DXS418. Both families therefore support the previously reported gene localisation.     

Carrier detection of the fragile X syndrome with flanking RFLP markers and linkage analysis.

Linkage analysis was performed in 34 fragile X (fra(X)) families in order to study the efficiency of carrier detection using the restriction fragment length polymorphisms (RFLPs) closely linked to fra(X) locus (FRAXA). The marker loci used were F9, DXS105, DXS98, DXS369, DXS297 and DXS477 proximally and DXS465, DXS296, DXS304, DXS52 and F8C distally to FRAXA. Flanking heterozygosity was achieved in 60% of the females with a combination of 3 restriction enzymes and 6 closest RFLP markers. When adding more distant markers and other restriction enzymes to the analysis, the proportion of females heterozygous for flanking polymorphisms increased to 96%. With RFLP-analysis most (85/91) females at high risk of being a carrier could be separated clearly into 2 groups: those with a very low and those with a very high risk. The 6 cases with a recombination between flanking markers did not benefit from RFLP-analysis.     

X linked myotubular myopathy (MTM1) maps between DXS304 and DXS305, closely linked to the DXS455 VNTR and a new, highly informative microsatellite marker (DXS1684).

The locus for X linked recessive myotubular myopathy (MTM1) has previously been mapped to Xq28 by linkage analysis. We report two new families that show recombination between MTM1 and either DXS304 or DXS52. These families and a third previously described recombinant family were analysed with two highly polymorphic markers in the DXS304-DXS52 interval, the DXS455 VNTR and a newly characterised microsatellite, DXS1684 (82% heterozygosity). These markers did not recombine with MTM1 in the three families. Together with the recent mapping of an interstitial X chromosome deletion in a female patient with moderate signs of myotubular myopathy, our data suggest the following order of loci in Xq28: cen-DXS304-(DXS455, MTM1)-DXS1684-DXS305-DXS52-tel. This considerably refined localisation of the MTM1 locus should facilitate positional cloning of the gene. The availability of highly polymorphic and very closely linked markers will markedly improve carrier and prenatal diagnosis of MTM1.