Genetic heterogeneity of X-linked mental retardation with fragile X Association of tight linkage to factor IX and incomplete penetrance in males

X-linked mental retardation with fragile X or Martin–Bell Syndrome (MBS) is a frequent cause of mental retardation. So far segregation analysis of MBS in pedigrees ascertained by different, incomplete criteria has produced results, difficult to interpret, which suggest genetic complexity (Sherman et al. 1985). Biochemical and cell biological studies have failed to provide an assay for genetic heterogeneity in MBS and linkage analysis is the only available method. Such analysis, however, is complicated by the incomplete penetrance of the disease in males and the variable penetrance and expression of the defect in heterozygous females. We have used a new approach to test the heterogeneity of recombination between MBS and the coagulation factor IX gene or the anonymous probe 52A in a group of nine families who have sought genetic counselling at Guy's Hospital. We find that both our families alone and our families plus apparently complete samples of pedigrees reported in the literature, separate into two groups: one tightly and one loosely linked to factor IX. In the combined family sample these represent respectively 0·3 and 0·7 of the total and show recombination fractions of 0·0–0·15 and 0·25–0·5. Furthermore, the families with non-penetrant carrier males show tighter linkage to factor IX than the others, thus confirming the suggestion of a systematic difference among MBS families in the recombination between the disease and the factor IX locus. By contrast, no significant differences were found in the recombination between 52A and factor IX in the two groups of MBS families or in these families versus those with Hunter syndrome examined in our laboratory. The causes of the linkage heterogeneity we describe are not known. At least two alternatives can be considered: The existence of two MBS loci or differences in the recombination between a single MBS locus and the factor IX gene. The association between incomplete penetrance and tight, linkage to factor IX as well as the discontinuous variation in recombination fraction we have observed seem to favour the former alternative.     

The genetic distance between the coagulation factor IX gene and the locus for the fragile X syndrome: clinical implications

In 3 families with the fragile-X [fra(X)] syndrome, we have identified a minimum of 4 recombinations in 9 meioses between the syndrome locus and the coagulation Factor IX gene. Two Factor IX intragenic restriction fragment length polymorphisms (RFLPs), produced with TaqI and XmnI, were used as markers. In lod score calculations, incomplete penetrance of the fra(X) mutation in males and females was taken into account by the computer program LIPED. The cumulative maximum lod score calculated from these data and from data previously reported was 2.75 at a recombination frequency of 20% (theta = 0.20). This indicates that the genetic distance between the Factor IX gene and the fra(X) locus is too great for Factor IX probes to be used alone for carrier detection in the fra(X) syndrome. Additional polymorphic loci more tightly linked to the fra(X) syndrome locus are required.     

The Genetic Distance between the Coagulation Factor IX Gene and the Locus for the Fragile X Syndrome: Clinical Implications

In 3 families with the fragile-X [fra(X)] syndrome, we have identified a minimum of 4 recombinations in 9 meioses between the syndrome locus and the coagulation Factor IX gene. Two Factor IX intragenic restriction fragment length polymorphisms (RFLPs), produced with TaqI and XmnI, were used as markers. In lod score calculations, incomplete penetrance of the fra(X) mutation in males and females was taken into account by the computer program LIPED. The cumulative maximum lod score calculated from these data and from data previously reported was 2.75 at a recombination frequency of 20% (θ = 0.20). This indicates that the genetic distance between the Factor IX gene and the fra(X) locus is too great for Factor IX probes to be used alone for carrier detection in the fra(X) syndrome. Additional polymorphic loci more tightly linked to the fra(X) syndrome locus are required.     

Linkage analysis of 7 polymorphic markers at chromosome 11p11. 2-11q13 in 27 multiple endocrine neoplasia type 1 families

The multiple endocrine neoplasia type 1 (MEN1) locus has been previously localized to llq13 by combined tumour deletion mapping and linkage studies. Family linkage analysis has defined the locus order as 11cen-PGA-(PYGM, MENl)-(D11S197, D11S146)-INT2-11qter, and tumour deletion mapping studies have suggested that the MEN1 locus is proximal to D11S146 but distal to PYGM. In order to establish further the location of MEN1, we have utilized the seven polymorphic DNA probes: D118S88, D11S149, PGA, PYGM, D11S97, D11S146 and INT2, in linkage studies of 339 members (116 affected) from 27 MEN 1 families. Linkage between MEN 1 and 6 of the 7 loci was established, and the highest peak lod scores [Z(θ)] were observed with PYGM and D11S97 at Z(θ) = 13–71, θ= 0.047 and Z(θ) = 13.76, θ= 0.076 respectively. Multilocus analysis suggested the most likely locus order as:11pter-(D11S288, D11S149)-11cen-PGA-PYGM-MEN1-D11S97-D11S146-INT2-11qter. In addition, an examination of individual recombinants indicated a centromeric location of D11S149 in relation to D115288. Thus, the results of our study, which favoured a location of MEN1 proximal to D11S97 and distal to PYGM, have established a panel of recombinants that will facilitate further meiotic mapping studies of the MEN 1 locus.     

Fabry disease: comparison of enzymatic, linkage, and mutation analysis for carrier detection in a family with a novel mutation (30delG).

Fabry disease (FD) is an X-linked recessive disorder caused by the deficient activity of the lysosomal enzyme alpha-galactosidase A (alpha-Gal A). Affected males are reliably diagnosed by demonstration of deficient alpha-Gal A activity in plasma or leukocytes. However, identification of female carriers is problematic due to Lyonization, requiring mutation identification and/or linkage studies for accurate carrier detection. Here, we describe a large Brazilian kindred with Fabry disease that permitted comparison of biochemical and molecular diagnostic techniques. Initially, the plasma alpha-Gal A activities were determined in at-risk affected males and potential female carriers; affected males were readily diagnosed, while the females had variable results. To detect carrier females, haplotype analysis using 10 polymorphic markers adjacent to the gene was performed. Subsequently, solid-phase direct sequencing of the alpha-Gal A gene demonstrated a novel single base deletion in exon 1 (30delG). Discrepancies were observed between the enzymatic and molecular diagnoses in two at-risk females. These findings emphasize the need for precise heterozygote diagnosis by mutation and/or haplotype analyses in all families with Fabry disease.     

Physical and genetic mapping of the CDR gene with particular reference to its position with respect to the FRAXA site.

This study narrows down the localization of the gene coding for the cerebellar degeneration-related protein (CDR 34) to the upper boundary of the FRAXA and reports the finding of two common RFLPs respectively identified at an RsaI site flanking the 3' end of the gene and at a Hincll site flanking its 5' end. Segregation analysis carried out in the CEPH-pedigrees for the new CDR/RsaI-RFLP versus other polymorphic loci of the region has established a tight linkage with the markers DXS105/DX98 and absence of measurable linkage with two clusters of markers respectively located proximally to the FRAXA (F9, DXS102, DXS51, and DXS369) or distally to it (DXS52, DXS304). In addition, two recombinants were found among 23 scorable sibs identified in the Sardinian pedigrees segregating for the Martin-Bell Syndrome (MBS) and the CDR/RsaI variants. The overall evaluation of the in situ and genetic data reported suggest that the CDR locus 1) is located at the upper boundary of the FRAXA site; 2) is distal to DXS51 and proximal to DXS 389; and 3) segregates in a close linkage association with the loci DXS98 and DXS105 and, to a lesser extent, with the locus for MBS.     

Fabry disease: Comparison of enzymatic,linkage, and mutation analysis for carrier detection in a family with a novel mutation (30delG)

Fabry disease (FD) is an X-linked recessive disorder caused by the deficient activity of the lysosomal enzyme α-galactosidase A (α-Gal A). Affected males are reliably diagnosed by demonstration of deficient α-Gal A activity in plasma or leukocytes. However, identification of female carriers is problematic due to Lyonization, requiring mutation identification and/or linkage studies for accurate carrier detection. Here, we describe a large Brazilian kindred with Fabry disease that permitted comparison of biochemical and molecular diagnostic techniques. Initially, the plasma α-Gal A activities were determined in at-risk affected males and potential female carriers; affected males were readily diagnosed, while the females had variable results. To detect carrier females, haplotype analysis using 10 polymorphic markers adjacent to the gene was performed. Subsequently, solid-phase direct sequencing of the α-Gal A gene demonstrated a novel single base deletion in exon 1 (30delG). Discrepancies were observed between the enzymatic and molecular diagnoses in two at-risk females. These findings emphasize the need for precise heterozygote diagnosis by mutation and/or haplotype analyses in all families with Fabry disease. Am. J. Med. Genet. 84:420–424, 1999. © 1999 Wiley-Liss, Inc.     

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.     

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.     

Mapping of a cerebellar degeneration related protein and DXS304 around the fragile site.

We have localized the gene encoding a cerebellar degeneration related (CDR) protein to a region proximal to the fragile site close to DXS98 and DXS105. This gene is polymorphic with the enzyme RsaI and therefore also provides a new genetic marker in this region. We have refined the localization of the locus DXS304 distal to the breakpoint in a patient suffering from Hunter disease. This confirms the localization of DXS304 distal to the fragile site previously suggested by linkage studies and localizes the fragile X mutation to a relatively small region between the Hunter breakpoint and the breakpoint in another hybrid B17.     

DNA linkage studies in the fragile X syndrome suggest genetic heterogeneity

Previously, we showed genetic heterogeneity for linkage between the fra(X) locus and a factor IX DNA RFLP (Brown et al, 1985). When fra(X) families were predivided into two classes, one containing those with non-penetrant (NP) males and one with apparent full penetrance (P), evidence of significant heterogeneity was present. We have now extended this analysis by adding DNA linkage information on 2 additional probes, 52A and ST14, studied in 16 fra(X) kindreds. These data were combined with information on 16 published fra(X) families. There were 7 NP families and 25 P families. We confirmed our previous findings of a higher recombination fraction between factor IX and fra(X) in P families (0 = .32 with lod of .67) compared to as NP families (0 = .06 with lod of 6.11) which was significant at p less than .01. In comparing recombination fractions for the additional probes, more recombination between 52A and the other loci was consistently seen in P compared to NP families which suggested that there may be a higher rate of recombination proximal to the fra(X) locus in P kindreds. A strikingly higher recombination fraction between 52A and factor IX was present in comparing all fra(X) families (.18) to normal families (.02) which was significant at p less than .001. These results suggest genetic heterogeneity with respect to recombination is present both among fra(X) pedigrees and between fra(X) and normal pedigrees.     

血友病甲基因分析技术的改进及其在产前诊断中的应用

目的对血友病甲基因分析技术进行改进并应用于携带者检查和产前诊断。方法长距离聚合酶链反应方法直接检测凝血因子Ⅷ第22内含子倒位,对非倒位家系用FⅧ基因内限制酶切位点XbaⅠ、HindⅢ、二核苷酸重复序列多态性位点STR13和STR22,以及基因外可变数目串联重复序列DXS52(St14)位点进行基因连锁分析。结果52个家系共检出71位携带者。21个家系为第22内含子倒位,28个家系经连锁分析得到明确诊断,3个家系无法诊断,可诊断家系占94.2%。为18个家系做胎儿产前诊断,其中10例诊断为血友病甲胎儿;诊断7例正常男胎和1例携带者女胎,随访1年发育正常。结论应用长距离聚合酶链反应和多位点基因连锁分析技术可以快速有效地进行血友病甲携带者检查和产前诊断。     

Cataracts, ataxia, short stature, and mental retardation in a Chinese family mapped to Xpter-q13.1

Six males in a Chinese family affected by congenital cataracts, cerebellar ataxia, short stature, and mental retardation, which were tentatively named CASM syndrome. Eight female carriers in the family had cataracts alone. Linkage analysis demonstrated that the disease is transmitted through X-linked inheritance, either by setting the syndrome in males as an X-linked recessive trait, or by setting cataracts in the family as an X-linked dominant trait. The gene responsible for the syndrome is mapped to Xpter-Xq13.1, with the highest lod score of 3.91 for DXS1226, DXS991, and DXS1213 at θ = 0. Haplotype analysis identified that the allele harboring the disease gene co-segregated with all female carriers as well as affected males in the family. Clinically and genetically, the disease in this family is different from any known disease. Major features of CASM syndrome that distinguish it from other diseases are X-linked inheritance and cataracts in carrier females.Xiangming Guo, Huangxuan Shen, Xueshan Xiao, Qilin Dai, Fielding Hejtmancik, and Qingjiong Zhang contributed equally to this work     

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.     

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.     

Adrenoleucodystrophy: a molecular genetic study in five families.

A genetic study has been performed on five adrenoleucodystrophy families using DNA probes from Xq28. Members of each family had previously been tested for carrier status using the biochemical assay for very long chain fatty acids (VLCFAs), but several persons at risk had equivocal results. DNA analysis with four DNA probes St14-1 (DXS52), DX13 (DXS15), MN12 (DXS33), and hs7 showed no crossovers between them and the disease locus in persons who were clinically affected or had high levels of VLCFA or both. Thus, the genotypes provided by the DNA probes could be used for accurate carrier detection and prenatal diagnosis could be offered. Of the 17 at risk females with VLCFA levels in the normal (1 SD) range, five were defined as carriers and 12 were considered not to be.     

Clasped-thumb mental retardation (MASA) syndrome: confirmation of linkage to Xq28.

We describe a 5-generation Hispanic family with 13 males and 1 female affected with MASA syndrome. The proposita, a 17-year-old female, and her affected male relatives shared many of the cognate manifestations--mental retardation (14/14), aphasia or delayed speech (13/13), shuffling gait (8/13), adduction of thumbs (14/14)--as well as scoliosis (2/13) and increased deep tendon reflexes in the lower extremities (10/13). Southern analysis with the polymorphic DNA probes DXS14 (Xp11), DXS72 (Xq21), and F8C (Xq28) confirmed linkage to the Xq28 region with a maximum lod score of 3.01 for this family.     

X-linked retinoschisis is closely linked to DXS41 and DXS16 but not DXS85

A linkage study was carried out in nine families with 24 males affected by X-linked recessive retinoschisis (RS), using three polymorphic DNA probes from the distal segment of Xp. Close linkage of the disease locus with markers DXS41 (probe p99-6) and DXS16 (pXUT23) was found, confirming the location of the RS gene on the distal short arm of the X chromosome. Lod scores for linkage with DXS85 (probe 782) were negative.     

Choroideremia: close linkage to DXYS1 and DXYS12 demonstrated by segregation analysis and historical-genealogical evidence

Linkage studies using restriction fragment length polymorphisms were conducted in the X-linked disorder, choroideremia, designated TCD for Progressive Tapeto-Choroidal Dystrophy. Previously demonstrated close linkage with locus DXYS1 was confirmed (lod 11.44 at 0 recombination distance). In addition, locus DXYS12 was found to be closely linked with TCD (lod 3.31 at 0 recombination distance). The disease mainly occurs in three large kindreds in remote Northern Finland. While formal genealogical proof is lacking, all presently living (more than 80 affected males and 120 carrier females) probably originate from a common founder couple born in 1644 and 1646, twelve generations ago. All 36 patients and 48 carriers tested from the three kindreds had the same haplotype (TCD/DXYS1, 11kb/DXYS12, 1.6kb). Given that at least 105 female meioses transmitting TCD have occurred since 1650 in these kindreds, extremely close linkage between TCD, DXYS1 and DXYS12 is suggested. The above haplotype is a very useful diagnostic tool in these TCD families. We suggest that our historical-genealogical approach to linkage analysis may be possible elsewhere in similar isolated populations.     

No evidence for linkage between schizophrenia and markers at chromosome 15q13-14

Freedman et al. [1997: Proc Natl Acad Sci USA 94:587-592] reported linkage in nine multiplex schizophrenia families to markers on chromosome 15, using impaired neuronal inhibition to repeated auditory stimuli (P50), a neurophysiological deficit associated with schizophrenia, as the phenotype. The highest LOD score obtained (5.3 at theta = 0) was for marker D15S1360 mapped to chromosome 15q13-14, less than 120 kb from the alpha7-nicotinic receptor (CHRNA7) gene. The study also reported a small positive LOD score for D15S1360 when examined for linkage to the schizophrenia phenotype. Following these findings, we examined three polymorphic markers (D15S1360, L76630, and ACTC) on chromosome 15q13-14 near the CHRNA7 gene for linkage to schizophrenia, using 54 pedigrees from an independent study. Alleles for these three markers were genotyped and analyzed using parametric and nonparametric methods. No LOD score above 1.00 was obtained for any marker, and affected sib-pair analysis likewise showed no evidence for linkage. We conclude that in our families the region around the CHRNA7 locus does not contain a major locus for susceptibility to schizophrenia.