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 and genetic counselling for the fragile X using DNA probes 52A, F9, DX13, and ST14

Linkage data using the markers DXS51, F9, DXS15, and DXS52 are presented from 14 pedigrees segregating with the fragile X. Cytogenetic and DNA data were combined by two- or three-point linkage analysis for estimation of lod scores and carrier probabilities in potential carriers. Recombination frequencies (theta) corresponding to maximum z scores (zeta) were obtained for DXS51 (zeta = 3.45, theta = 0.0), DXS15 (zeta = 0.40, theta = 0.06), F9 (zeta = 3.15, theta = 0.09), and DXS52 (zeta = 3.60, theta = 0.11) with the fragile X. Considerable alterations to carrier probabilities occurred in some cases, especially when flanking markers were informative. The chance of mentally impaired offspring was reduced to 1% for five of eight women with prior carrier probabilities of 32%. Three pedigrees were identified in which mutation had possibly occurred. An alternative explanation for two of these was inheritance of the fragile X from normal males and for the other inheritance from a clinically normal woman. Probabilities were computed for each of these alternatives.     

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.     

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.     

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.     

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.     

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.     

The fragile X syndrome in a large family. III. Investigations on linkage of flanking DNA markers with the fragile site Xq27.

In a large family with the fragile X syndrome, we performed linkage investigations with six probes, detecting RFLPs at both sides of the fragile site Xq27. The nearest flanking markers were cX55.7 (DXS105) on the centromeric side (theta = 0.04, lod 5.0) and St14 (DXS52) on the telomeric side (theta = 0.08, lod 4.0). Non-penetrance could be shown by the presence of the grandpaternal X chromosome in three mentally retarded fra(X) positive males. A second non-penetrant male in this family had inherited an abnormal grandmaternal X chromosome. His carrier mother had two retarded fra(X) positive brothers. Intermediate between the non-penetrant and fully penetrant males was a non-retarded male, who expressed the fragile site in 6% of his cells. His X chromosome showed the same polymorphisms as were found in his seven severely retarded brothers. In five fra(X) negative females the presence of an abnormal X chromosome could be demonstrated. Despite the existence of non-penetrance in this pedigree, there was no close linkage between a factor IX polymorphism and the fragile site (theta = 0.16, lod 1.9). However, in six descendants of a non-penetrant male, the change to penetrance appeared to be accompanied by a low recombination frequency for flanking markers.     

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.     

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.     

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 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.     

Improved DNA markers for efficient analysis of fragile X families

We report the characteristics of two new probes that detect BclI RFLPs useful for analysis of fragile X families. With these two probes and a single blot, 34% of women are heterozygous both for the proximal marker DXS105 (closer to the fragile X locus than the factor IX gene) and for the distal markers DXS52 or the factor VIII gene. Combined with the analysis of previously described polymorphic markers, it is possible to have a majority of families fully informative for flanking markers using a limited number of probes and restriction digests.     

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.     

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.     

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.     

New X-linked syndrome of mental retardation, gynecomastia, and obesity is linked to DXS255.

We describe 14 males from 3 successive generations in a family who have X-linked mental retardation (XLMR), obesity, gynecomastia, speech difficulties, emotional lability, tapering fingers, and small feet. Linkage analysis using markers spread along the X chromosome demonstrated a gene localisation close to the centromere. Maximum lod scores for markers near the centromere, all at theta = 0.00, were 1.36 for DXS72, and 1.46 for DXYS1. The closest flanking markers which showed recombination were DXS84 and DXS94, defining the physical localisation within Xp21.1-q22. DXS255 was fully informative with lod-1 confidence interval for theta of 0.00-0.12. Clinical findings and linkage data in this family distinguish it from the B?rjeson-Forssman-Lehmann syndrome and other previously described XLMR syndromes.     

Infantile autism, fragile (X) (q27.3) and RFLP analysis in an extended Swedish family

In an extended family with eight individuals with infantile autism, in association with other developmental disorders and fragile (X) (q27.3), DNA techniques were used to investigate linkage between X chromosomal probes and the disorder. F9 was not informative and recombination was found between fragile X and DXS15, DXS51 and DXS52.     

A non-syndromal form of X-linked mental retardation (XLMR) is linked to DXS14

An analysis of the linkage of a non-syndromal form of X-linked mental retardation (MRX1) with a number of markers on the X chromosome was performed in a large pedigree. The affected males had moderate mental retardation; in all other clinical respects and cytogenetically they were normal. No recombinants were observed between the MRX1 gene and the marker DXS14 (p58.1) located at Xp11-cen (Z (max.) = 2.12 at theta = 0.00). Recombination was observed between the MRX1 gene and the markers DXS7 and DXYS1 which flank DXS14. This form of XLMR maps to the centromeric portion of the X-chromosome.     

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.