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 analysis of the fragile X syndrome using a new DNA marker U6.2 defining locus DXS304.

A new RFLP marker U6.2 defining the locus DXS304 was recently mapped to the distal long arm of the X chromosome. In the present study we report the results of genetic linkage analysis of 13 fragile X [fra(X)] families that were informative for the new marker. Analysis of the recombinants for F9-FRAXA, DXS105-FRAXA, DXS98-FRAXA, DXS52-FRAXA, DXS15-FRAXA, and F8C-FRAXA, places DXS304 distal and near to the FRAXA locus. Combined with results from previous studies, our results support the order Xcen.-F9-DXS105-DXS98-FRAXA-DXS304-DXS5 2-DXS15-F8C-Xqter. Close linkage was observed between DXS304 and the disease locus with a peak lod score of 5.12 at theta = 0.04 from the present study and, with a peak lod score of 17.45 at theta = 0.035 when our data are combined with published data from 2 other studies. The present study confirms that U6.2 is useful for prenatal diagnosis and carrier testing in families affected by fra(X) syndrome.     

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%.     

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.     

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.     

Emery-Dreifuss muscular dystrophy: linkage to markers in distal Xq28.

Emery-Dreifuss muscular dystrophy (EMD) is characterised by (1) early contractures of the Achilles tendons, elbows, and postcervical muscles, (2) slowly progressive muscle wasting and weakness with a predominantly humeroperoneal distribution in the early stages, and (3) cardiomyopathy with conduction defects and risk of sudden death. Inheritance is usually X linked recessive but can be autosomal dominant. Family linkage studies have mapped X linked EMD to the distal long arm of the X chromosome but precise genetic localisation has been hampered by the rarity of this condition. We report three new families with X linked Emery-Dreifuss muscular dystrophy studied with DNA markers from Xq27-qter and three previously published families typed for additional markers. No recombination was observed with the red/green cone pigment locus, RGCP (lod score, Z = 2.46), the factor VIII coagulant gene locus, F8C (Z = 6.39), or with DXS115 (Z = 4.94). Two recombinants were observed which mapped EMD distal to DXS15 (DX13) and DXS52 (St14) respectively. Multipoint linkage analysis gave odds exceeding 200:1 for EMD being distal to these markers. A multipoint analysis incorporating published data gave the map cen-DXS304-9cM-DXS15-3cM-DXS52-2 cM-(RGCP,EMD)-3cM-F8C-2cM-DXS115 with odds of 120:1 in favour of a location for EMD between DXS52 and F8C as compared to the next best position distal to F8C.     

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.     

Multipoint linkage of 9 anonymous probes to HPRT, factor 9, and fragile X

We have analyzed the segregation of restriction fragment length polymorphisms (RFLPs) associated with 9 anonymous probes detecting loci DXS10, DXS15, DXS19, DXS37, DXS51, DXS52, DXS98, DXS99, and DXS100 and probes for HPRT and F9 in a set of 40 families segregating fragile X (fra(X]. Using two-point and multipoint analysis, we have established their relative genetic locations. The results indicate that DXS99 and DXS10, unlike previous reports, are not tightly linked to F9. A new locus was found to map within the F9 - fra(X) region. DXS98 showed 6% recombination with fra(X) and appeared to be the closest locus to fra(X). These results will be useful for mapping the relative position of newly defined X probes in this region and for future genetic studies of families with fra(X), hemophilia B, or Lesch-Nyhan mutations.     

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.     

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.     

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.     

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.     

Assignment of the locus order DXS28- DXS67-DMD as a spin-off from diagnostic DNA marker analysis in a family with Duchenne muscular dystrophy

During diagnostic segregation analysis for seven DNA markers, linked to and flanking the locus for Duchenne muscular dystrophy (DMD), a family was identified in which a boy with a recombinant X chromosome had inherited his maternal grandmother's alleles at the loci DXS43 (D2/Pvu II) and DXS28 (C7/Eco RV), and his maternal grandfather's alleles at DXS67 (B24/Msp I) and DXS84 (754/Pst I). Combined with earlier data this finding strongly suggests the locus order DXS28-DXS67-DMD. Another recombination event, identified in the same family, supports the previously established order DMD-DXS84-OTC. The diagnostic importance of flanking markers, and the likelihood of false diagnostic conclusions due to possible double crossovers, with and without demonstrable neighbouring single crossover events, are discussed.     

Linkage analysis using multiple Xq DNA polymorphisms in normal families, families with the fragile X syndrome, and other families with X linked conditions.

Multipoint linkage analysis was undertaken with eight Xq cloned DNA sequences which identify one or more restriction fragment length polymorphisms in 26 families. These families comprise seven phase known normal families with three or more males in the third generation, seven families segregating for haemophilia B, one large family with dyskeratosis congenita, and 11 families with the fragile X syndrome. Phase known meioses informative for three or more loci supported the order centromere--DXYS1--DXS107--DXS102, DXS51--F9--FRAXA--DXS15, DXS52, F8--Xqter in each group of families studied. One of the normal families was segregating for protan colour blindness and showed a phase known recombination which would support the order centromere--F9--DXS52--CBP--Xqter. With the exception of DXYS1, all of these sequences have been localised to Xq27----qter by in situ hybridisation or hybridisation to Xq fragment panels, and on this basis should lie within 20 cM of one another. No recombination was observed between the sequences localised to Xq28, namely DXS52, F8, and DXS15 (between DXS15 and DXS52 Z = 12.25 at theta = 0 with confidence limits of 0 to 5 cM). However, an excess of recombination was apparent in the region of FRAXA with maximal lod scores as follows: F9 versus FRAXA (Z = 2.05, theta = 0.19), DXS52 versus FRAXA (Z = 1.85, theta = 0.26), and DXS15 versus FRAXA (Z = 1.33, theta = 0.27). No consistent differences were observed in the frequency of recombination when families with the fragile X syndrome were compared with normal families or families segregating for other X linked conditions. These results are compared with other published work and support the conclusion that although measurable linkage exists between these flanking markers and FRAXA, the intervals as measured by the frequency of meiotic recombination will seriously limit their clinical usefulness.     

An assessment of the use of flanking DNA markers for fra(X) syndrome carrier detection and prenatal diagnosis

One hundred and three individuals in 11 unrelated families with the fragile-X [fra(X)] syndrome were tested for polymorphisms identified by probes flanking the fra(X) site at Xq27.3. Two probes distal and 2 proximal to the fra(X) site were used. Thirteen known female carriers were analyzed retrospectively. DNA markers gave probabilities of carrying the mutation of 99% in 1 female, 89% in 8 females, and 10-55% in the other 4 females. We also estimated the probability of having inherited the mutation for 16 individuals of unknown fra(X) status using DNA markers and corrections for incomplete penetrance. The DNA marker test gave risks for females of 1-6% (7 females), 15% (1 female), and 97% (1 female). In males the risks were 1-3% (6 males) and 91% (1 male). In 3 families, DNA marker data were used to calculate probabilities of greater than or equal to 98.5% that transmission of the fra(X) mutation had occurred through normal males. In the retrospective studies, only 1 of 7 retarded males could have been diagnosed prenatally as having the fra(X) mutation with a probability of 99%. DNA marker analysis was uninformative in 5 of these males. When fra(X) carrier status cannot be established by chromosome analysis, DNA marker studies provide an alternative test that can be used to calculate individual risks more precisely. However, linkage analysis of the probe loci in these 11 families suggests that the recombination frequency between the fra(X) locus and the factor IX gene (F9) and DXS52 may be greater than previously suggested. Until the true recombination frequencies are established and the question of heterogeneity among families is fully analyzed, caution in using DNA markers as a predictive test is advised.     

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.     

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 spastic paraplegia (SPG2): clinical heterogeneity at a single gene locus.

X linked hereditary spastic paraplegia is a rare condition that has been divided into two forms (the pure spastic form and the complicated form) as a function of clinical course and severity. A gene for pure hereditary spastic paraplegia (SPG2) has been mapped to the proximal long arm of the X chromosome (Xq21) by linkage to the DXS17 locus, while a gene for a complicated form of the disease has been mapped to the distal long arm by linkage to the DXS52 locus (Xq28). Here we report on the mapping of a gene for complicated hereditary spastic paraplegia to the Xq21 region by linkage to the probe S9 at the DXS17 locus (Z = 5 at theta = 0.04) in a three generation pedigree. Multipoint linkage analysis supports the distal location of the disease gene with respect to the DXYS1-DXS17 block (cen-DXYS1-DXS3-DXS17-SPG2-tel). The observation of a complicated form of spastic paraplegia mapping to Xq21 raises the difficult issue of variable phenotypic expression, allelic heterogeneity, or even close proximity of two genes for hereditary spastic paraplegia in this region. However, since our study provides clinical evidence for intrafamilial heterogeneity in complicated X linked spastic paraplegia, the present data support the hypothesis of variable clinical expression of a single gene at the SPG2 locus, as previously suggested for SPG1. Finally, we report here what we believe to be the first evidence of clinical expression in heterozygous carriers, a feature that is relevant to genetic counselling in at risk females.     

DNA-based genetic testing in fifty fragile X families.

During the past 4 years (1985-1989), we have analyzed 171 cases in 50 fragile X [fra(X)] families by DNA linkage methods. Most (140 cases; 81%) were for carrier detection, both female (98 cases; 57%) and male (41 cases; 24%). Women who were obligate carriers of the fra(X) mutation accounted for an additional 6 "prior-to-pregnancy" cases. Four pregnancies have subsequently occurred with 3 having been successfully monitored (one male, 2 females). One pregnancy miscarried early prior to testing. Prenatal diagnoses (26 cases; 15%) accounted for the remainder of cases (15 males, 11 females). These will be discussed in the companion paper by Shapiro et al. (Am J Med Genet, 1991). A diagnosis in the cytogenetically uninformative carrier cases was reached in greater than 75% of analyses with a panel of 5 probes: 3 proximal (F9, DXS105, DXS98) and 2 distal (F8, DSX52). Five additional probes, 3 proximal (DXS10, DSX51, DSX102) and 2 distal (DSX15, DXS33), were used in cases that were resistant to analysis with the standard panel. In 60% of cases, flanking markers were identified (proximal and distal). Given this panel, only 5% of cases did not have any informative markers identified. Thus, molecular methods can provide a useful adjunct to cytogenetic analysis in most situations. An unusual association between the rare allele (A1) of DXS10 with the X chromosome carrying the fra(X) mutation was observed. This occurred in both male and female carriers in the uppermost generation tested. The basis for this association is uncertain at the present time.     

Genetic mapping of loci for X-linked retinitis pigmentosa

Linkage analysis was performed in three Swedish families segregating for X-linked retinitis pigmentosa (XLRP), using five polymorphic DNA markers assigned to Xp. Individual recombination events were analyzed and two- and five-point linkage analysis was undertaken. In one family, a XLRP locus was mapped to the same position as OTC corresponding to RP3. In two families, a disease locus linked to OTC was excluded. In one family, recombination events indicate a locus for XLRP outside the interval (DXS84-OTC-DXS255-DXS14), most likely on the centromeric side of DXS14.