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.     

X linked neonatal centronuclear/myotubular myopathy: evidence for linkage to Xq28 DNA marker loci.

We have studied the inheritance of several polymorphic Xq27/28 DNA marker loci in two three generation families with the X linked neonatal lethal form of centronuclear/myotubular myopathy (XL MTM). We found complete linkage of XLMTM to all four informative Xq28 markers analysed, with GCP/RCP (Z = 3.876, theta = 0.00), with DXS15 (Z = 3.737, theta = 0.00), with DXS52 (Z = 2.709, theta = 0.00), and with F8C (Z = 1.020, theta = 0.00). In the absence of any observable recombination, we are unable to sublocalise the XLMTM locus further within the Xq28 region. This evidence for an Xq28 localisation may allow us to carry out useful genetic counselling within such families.     

Tightly linked polymorphic markers for fragile X syndrome and prenatal cytogenetic diagnostic experience.

Linkage analysis using the polymorphic loci DXS369, DXS296, DXS297 and DXS306 was carried out on a cohort of 17 families segregating for fragile X syndrome. The observed recombination fractions at: DXS369 (Zmax = 3.02; theta = 0.06), DXS297 (Zmax = 2.92; theta = 0.0), DXS296 (Zmax = 3.82; theta = 0.0), DXA306 (Zmax = 4.55; theta = 0.05) confirm that these loci are tightly linked to FRAXA. Our experience in the cytogenetic analysis of 58 at risk pregnancies by chorionic villus or fetal blood sample examination documents a false negative rate in obligate carrier male pregnancies for CVS of 11% and for FBS of 3%.     

Tightly linked polymorphic markers for fragile X syndrome and prenatal cytogenetic diagnostic experience

Linkage analysis using the polymorphic loci DXS369, DXS296, DXS297 and DXS306 was carried out on a cohort of 17 families segregating for fragile X syndrome. The observed recombination fractions at: DXS369 (Zmax = 3. 02; theta=0. 06), DXS297 (Zmax= 2. 92; theta = 0.0), DXS296 (Zmax = 3. 82; theta = 0.0), DXA306 (Zmax = 4. 55; theta = 0.05) confirm that these loci are tightly linked to FRAXA. Our experience in the cytogenetic analysis of 58 at risk pregnancies by chorionic villus or fetal blood sample examination documents a false negative rate in obligate carrier male pregnancies for CVS of 11% and for FBS of 3%.     

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.     

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 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 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%.     

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 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.     

Localisation of the MRX3 gene for non-specific X linked mental retardation.

A family is described with five affected males segregating a new gene for non-specific X linked mental retardation (MRX). Linkage analysis localised the gene at Xq28-qter. The maximum lod score was 2.89 with DXS52 (St14) at theta = 0.0. A recombinant was observed with DXS304 (U6.2) defining the proximal limit to the localisation. No evidence for linkage was determined using markers at several points along the remainder of the X chromosome, including the regions known to contain MRX1 and MRX2. This delineates the third gene for non-specific X linked mental retardation, MRX3.     

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 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.     

Mapping to distal Xq28 of nonspecific X-linked mental retardation MRX72: linkage analysis and clinical findings in a three-generation Sardinian family

Families with mentally retarded males found to be negative for FRAXA and FRAXE mutations are useful in understanding the genetic basis of X-linked mental retardation. According to the most recent data (updated to 1999), 69 MRX loci have been mapped and 6 genes cloned. Here we report on a linkage study performed on 20 subjects from a 4-generation Sardinian family segregating a non-specific X-linked recessive mental retardation (XLMR)(MRX72) associated with global delay of all psychomotor development. Five of 8 affected males have been tested for mental age, verbal and performance skills and behavioral anomalies; mental impairment ranged from mild to severe. Only minor anomalies were present in the affected subjects. Two-point linkage analysis based on 28 informative microsatellites spanning the whole X chromosome demonstrated linkage between the disorder and markers DXS1073 and F8c in Xq28 (maximum Lod score of 2. 71 at straight theta = 0.00). Multipoint linkage analysis confirmed the linkage with a Z(max) of 3.0 at straight theta = 0.00 at DXS1073 and F8c. Recombination in an affected male at DXS1073 and F8c allowed us to delimit centromerically and telomerically the region containing the putative candidate gene. The region, where MRX72 maps, overlaps that of another MRX families previously mapped to Xq28, two of which harbored mutations in GDI. Involvement of this gene was excluded in our family, suggesting another MRX might reside in Xq28.     

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.     

Fragile X syndrome: genetic localisation by linkage mapping of two microsatellite repeats FRAXAC1 and FRAXAC2 which immediately flank the fragile site.

We report the genetic localisation of the fragile site at Xq27.3 associated with fragile X syndrome. The position of the fragile site within the multipoint linkage map was determined using two polymorphic microsatellite AC repeat markers FRAXAC1 and FRAXAC2. These markers were physically located within 10 kilobases and on either side of the p(CCG)n repeat responsible for the fragile site. FRAXAC1 has five alleles with heterozygosity of 44% and is in strong linkage disequilibrium with FRAXAC2 which has eight alleles and a heterozygosity of 71%. No recombination was observed either between these markers in 40 normal CEPH pedigrees or with the fragile X in affected pedigrees. These markers provide the means for accurate diagnosis of the fragile X genotype in families by rapid polymerase chain reaction analysis and were used to position the fragile X within the multipoint map of the X chromosome to a position 3.7 cM distal to DXS297 and 1.2 cM proximal to DXS296.     

Close linkage of a gene for X linked deafness to three microsatellite repeats at Xq21 in radiologically normal and abnormal families.

We have used three highly polymorphic microsatellite repeats from Xq21 to type families in whom a gene for X linked deafness with perilymphatic gusher (DFN 3) was segregating. All three markers were tightly linked to the disease in its radiologically normal and abnormal forms, with a maximum lod score of 10.37 with DXS995 and 8.44 with DXS986 at zero recombination, and 14.03 with DXS1002 at theta = 0.01. In an isolated case of deafness of this type, DXS995 indicated either the first recombination observed between the marker and the disease gene or a new mutation in the proband. Southern blotting using a cosmid fragment from the candidate region has confirmed a de novo mutation by showing a deletion in the proband which is not present in his mother as judged by dosage analysis. We also describe a family with a paracentric inversion associated with a microdeletion and discuss how deletion mapping using these and other markers in the region has helped to define a candidate region for the gene.