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.     

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.     

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.     

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.     

Refining the genetic location of the gene for X linked hydrocephalus within Xq28.

The most common inherited form of hydrocephalus, X linked hydrocephalus (HSAS), is characterised by mental retardation, adducted thumbs, and spastic paraplegia. Genetic analysis has mapped the locus for HSAS to subchromosomal band Xq28 within a region of approximately 2 megabases of DNA. In order to refine the location of the disease gene we have conducted genetic linkage analysis with Xq28 marker loci in four additional HSAS families. A lod score of 4.26 with polymorphic marker DXS52 (St14) confirms the linkage of HSAS to Xq28. Identification of a recombination event between the HSAS gene and Xq28 loci F8C and DXS605 (2-19) reduces the size of the interval likely to contain the disease locus to about 1.5 megabases, the distance between DXS605 and DXS52. The locus for neural cell adhesion molecule, L1CAM, maps within this interval and therefore represents a candidate gene for HSAS.     

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 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.     

Further evidence localising the gene for Hunter''s syndrome to the distal region of the X chromosome long arm.

Cytogenetic re-evaluation of a fibroblast cell line from a female Hunter's syndrome case with a balanced X;autosome translocation, which had previously been reported to have a breakpoint in Xq26 to Xq27, showed the breakpoint to be either between Xq27 and Xq28 or within Xq28. The normal X chromosome was preferentially inactivated, supporting the view that the translocation had disrupted the Hunter gene. The new localisation is now in full agreement with our previous linkage work and other published data. Results of further linkage studies using probes defining the loci DXS86, DXS144, DXS100, DXS102, DXS105, F8C, and DXS134 are also consistent with our original conclusion that the Hunter locus lies within the distal region of the X chromosome long arm.     

Emery-Dreifuss muscular dystrophy: localisation to Xq27.3----qter confirmed by linkage to the factor VIII gene.

Two families with Emery-Dreifuss muscular dystrophy (EMD) have been studied with DNA markers mapping to Xq27.3----qter. No recombination was observed in 11 phase known meioses informative for the factor VIII gene (F8C) and eight phase known meioses informative for DXS15 (DX13), giving maximum lod scores of 3.50 and 2.50 respectively at a recombination fraction of zero. DXS52 (St14) showed one recombinant in 12 phase known meioses giving a maximum lod score of 2.62 at a recombination fraction of 0.07. These results map EMD to the distal end of the long arm of the X chromosome and are an important step in the development of tests for carrier detection and prenatal diagnosis.     

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.     

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.     

Regional localization of two MRX genes to Xq28 (MRX28) and to Xp11.4-Xp22.12 (MRX33)

Two genes responsible for a nonspecific form of X-linked mental retardation (MRX28 and MRX33) were localized by linkage analysis with 40 highly polymorphic DNA markers situated along the entire the X chromosome. In family 1, the gene could be mapped within a 14-cM interval at Xq28, distal to the recombining marker DXS1113 (MRX28). The maximum LOD score was 2.75, with DXS52 at ϕ = .0. In family 2, the gene was localized within a 30-cM interval at Xp11.4-22.12 between the recombining markers DXS365 and MAOB, including the DMD gene (MRX33). Maximum LOD scores of 2.82 were obtained with markers DMD-STR49, DMD-DysII, CYBB, and DXS1068. © 1996 Wiley-Liss, Inc.     

MASA syndrome: new clinical features and linkage analysis using DNA probes.

We describe a two generation family in which two males have the X linked recessive MASA syndrome (mental retardation, aphasia, shuffling gait, and adducted thumbs). A third male in this family died at the age of 15 years from congenital hydrocephalus. In the present family cerebral abnormalities are reported for the first time. Linkage analysis confirms the chromosome localisation at Xq28. A crossover between the coagulation factor VIII locus (F8C) and MASA syndrome, but not with DXS52 and DXS305, locates the gene on the same side of F8C as DXS52 and DXS305. The possible relationship between MASA syndrome and X linked hydrocephalus is discussed.     

New somatic cell hybrids for physical mapping in distal Xq and the fragile X region

Somatic cell hybrids were constructed from 3 patients carrying X chromosome abnormalities with breakpoints in distal Xq: 1) 94-3, from a patient with 46,XX,t(X;15)(q25 or q26;q25), 2) 8121-A1, from a patient with 46,X,del(X)(q26), and 3) 2384-A2, from a patient with 46,X,del(X)(q27). The breakpoint of patient 94 is proximal to HPRT in q26, a significant distance from the fragile X locus. The breakpoint of patient 8121 is distal to F9, but proximal to DXS98, and is thus proximal to the fragile site region. The breakpoint of 2384 is distal to DXS98 but proximal to DXS52, placing it within the region of the fragile site. Use of these physical mapping reference points will aid in the rapid localization of new DNA markers to distal Xq and the fragile X region.     

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%.     

Dyskeratosis congenita: three additional families show linkage to a locus in Xq28.

Dyskeratosis congenita (DC) is a rare inherited disorder with most families being of the X linked recessive type. We describe three families which show linkage to the marker DXS52 on Xq28. The combined maximum lod score was 2.00 at zero recombination. This is further evidence that the X linked DC gene is located at Xq28 and brings the reported maximum lod score for DC and DXS52 to 5.33 at zero recombination fraction, with a supporting recombination fraction interval of 0.00-0.10.     

Loss of heterozygosity on chromosome XP22.2-p22.13 and Xq26.1-q27.1 in human breast carcinomas.

In an attempt to investigate the X chromosome harboring putative tumor suppressor genes (TSGs) in sporadic breast carcinoma, we performed loss of heterozygosity (LOH) studies on 23 breast carcinomas using 15 polymorphic markers covering the whole X chromosomes. Matched DNA extracted from tumor samples and corresponding normal tissues were analyzed by polymerase chain reactions (PCR) using microsatellite markers. In 10 cases (43.5%), LOH was detected for at least 1 of the 15 polymorphic markers of the X chromosome tested. Four cases carried a LOH at Xp, and three cases LOH on Xp and Xq. Three cases carried a LOH Xq. Percentage of LOH was relatively high in DXS987 (26.7%), DXS999(30.0%), HPRT(21.4%), DXS1062(23.1%) loci. Common regions of deletions were found on Xp22.2-p22.13 (30% of LOH) measuring about 4.5Mb and Xq26.1-q27.1 (23.1% of LOH) measuring 10 Mb. The deleted allele was an active copy of the X chromosome. The results indicate the TSGs on the X chromosome are involved in breast cancer.     

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.     

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.