Multipoint linkage analysis in X-linked Alport syndrome

Summary In order to localize the gene for the X-linked form of Alport syndrome (ATS) more precisely, we performed restriction fragment length polymorphism analysis with nine different X-chromosomal DNA markers in 107 members of twelve Danish families segregating for classic ATS or progressive hereditary nephritis without deafness. Two-point linkage analysis confirmed close linkage to the markers DXS17(S21) (Z _max = 4.44 at = 0.04), DXS94(pXG-12) (Z _max=8.07 at =0.04), and DXS101(cX52.5) (Z _max=6.04 at =0.00), and revealed close linkage to two other markers: DXS88(pG3-1) (Z _max =6.36 at =0.00) and DXS11(p22–33) (z _max=3.45 at =0.00). Multipoint linkage analysis has mapped the gene to the region between the markers DXS17 and DXS94, closely linked to DXS101. By taking into account the consensus map and results from other studies, the most probable order of the loci is: DXYS1(pDP34)-DXS3(p19-2)-DXS17-(ATS, DXS101)-DXS94-DXS11-DXS42(p43-15)-DXS51(52A). DXS88 was found to be located between DXS17 and DXS42, but the order in relation to the ATS locus and the other markers used in this study could not be determined.     

Linkage relationship between retinoschisis and four marker loci

Summary The linkage relationship between the locus for juvenile retinoschisis (RS) and four X-chromosomal marker loci DXS9 (RC8), DXS16 (XUT23), DXS41 (99-6), and DXS43 (D2) has been studied in six families showing a history of this disease. Recombination with RS was found for all marker loci except DXS9. The maximum lod score is =2.66 for RS vs. SXS9 at a recombination fraction of =0.0. Multipoint linkage analysis was performed and the locus order best supported by our data is: RS-DXS9-DXS43-DXS16-DXS41.     

New markers for linkage analysis of X-linked hypophosphataemic rickets

Three polymorphic markers have been used to improve the genetic map of the region Xp22.1-p22.2, which contains the HYP (hypophosphataemic rickets) locus. DXS365 gave no recombinants with HYP, with a peak Lod score of 5.4 at = 0.0. A microsatellite marker mPA274 was derived for the DXS274 locus; it detects five alleles with a polymorphism information content of 0.55. Combining information from this microsatellite and the original DXS274 marker, probe CRI-L1391, the peak Lod score for DXS274 against HYP was 9.6 at = 0.02. A microsatellite associated with the DXS207 locus (mPA207) gave a peak lod score against HYP of 4.7 at = 0.14. A consideration of key recombinants and multilocus analysis suggests the gene order: Xpter-DXS207-DXS43-DXS197-(DXS365, HYP)-DXS274-DXS41-Xcen.     

Multilocus analysis of the fragile X syndrome

Summary A multilocus analysis of the fragile X (fra(X)) syndrome was conducted with 147 families. Two proximal loci, DXS51 and F9, and two distal loci, DXS52 and DXS15, were studied. Overall, the best multipoint distances were found to be DXS51-F9, 6.9%, F9-fra(X), 22.4%; fra(X)-DXS52, 12.7%; DXS52-DXS15, 2.2%. These distances can be used for multipoint mapping of new probes, carrier testing and counseling of fra(X) families. Consistent with several previous studies, the families as a whole showed genetic heterogeneity for linkage between F9 and fra(X).     

Linkage of PGK1 to X-linked severe combined immunodeficiency (IMD4) allows predictive testing in families with no surviving male

Summary We present a linkage map of DNA probes around the X-linked severe combined immunodeficiency (IMD4) locus at Xq11-13. DXS159 and PGK1 show no cross-overs with the disease locus (Lod 3.01 at = 0.00). The order of loci is DXS1-DXS106-(DXS159-PGK1-IMD4)-DXS72-DXYS1. Members of families whose carrier status has been established by X-inactivation patterns were included in the analysis. As the probe (pSPT/PGK), which is used for investigation of X-inactivation patterns, has been shown to be linked to the disease itself, it is possible to assign phase in mothers of sporadic cases who have been shown to be carriers, even when they have no surviving male offspring.     

Heterogeneity in X-linked recessive Charcot-Marie-Tooth neuropathy.

Three families presenting with X-linked recessive Charcot-Marie-Tooth neuropathies (CMT) were studied both clinically and genetically. The disease phenotype in family 1 was typical of CMT type 1, except for an infantile onset; two of five affected individuals were mentally retarded, and obligate-carrier females were unaffected. Families 2 and 3 showed distal atrophy with weakness, juvenile onset, and normal intelligence. Motor-nerve conduction velocities were significantly slowed, and electromyography data were consistent with denervation in affected CMT males in all three families. Thirty X-linked RFLPs were tested for linkage studies against the CMT disease loci. Family 1 showed tight linkage (recombination fraction [theta] = 0) to Xp22.2 markers DXS16, DXS143, and DXS43, with peak lod scores of 1.75, 1.78, and 2.04, respectively. A maximum lod score of 3.48 at DXS16 (theta = 0) was obtained by multipoint linkage analysis of the map DXS143-DXS16-DXS43. In families 2 and 3 there was suggestion of tight linkage (theta = 0) to Xq26 markers DXS86, DXS144, and DXS105, with peak lod scores of 2.29, 1.33, and 2.32, respectively. The combined maximum multipoint lod score of 1.81 at DXS144 (theta = 0) for these two families occurred in the map DXS10-DXS144-DXS51-DXS105-DXS15-DXS52++ +. A joint homogeneity analysis including both regions (Xp22.2 and Xq26-28) provided evidence against homogeneity (chi 2 = 9.12, P less than .005). No linkage to Xp11.12-q22 markers was observed, as was reported for X-linked dominant CMT and the Cowchock CMT variant. Also, the chromosomes 1 and 17 CMT loci were excluded by pairwise linkage analysis in all three families.     

Refinement of linkage of human severe combined immunodeficiency (SCIDX1) to polymorphic markers in Xq13.

The most common form of human severe combined immunodeficiency (SCID) is inherited as an X-linked recessive genetic defect, MIM 300400. The disease locus, SCIDX1, has previously been placed in Xq13.1-q21.1 by demonstration of linkage to polymorphic markers between DXS159 and DXS3 and by exclusion from interstitial deletions of Xq21.1-q21.3. We report an extension of previous linkage studies, with new markers and a total of 25 SCIDX1 families including female carriers identified by nonrandom X chromosome inactivation in their T lymphocytes. SCIDX1 was nonrecombinant with DXS441, with a lod score of 17.96. Linkage relationships of new markers in the SCIDX1 families were consistent with the linkage map generated in the families of the Centre d'Etude du Polymorphisme Humain (CEPH) and with available physical map data. The most likely locus order was DXS1-(DXS159,DXS153)-DXS106-DXS132-DXS4 53-(SCIDX1,PGK1, DXS325,DXS347,DXS441)-DXS447-DXS72-DXYS 1X-DXS3. The SCIDX1 region now spans approximately 10 Mb of DNA in Xq13; this narrowed genetic localization will assist efforts to identify gene candidates and will improve genetic management for families with SCID.     

Genetic mapping of the Xq27-q28 region: new RFLP markers useful for diagnostic applications in fragile-X and hemophilia-B families.

We have characterized and genetically mapped new polymorphic DNA markers in the q27-q28 region of the X chromosome. New informative RFLPs have been found for DXS105, DXS115, and DXS152. In particular, heterozygosity at the DXS105 locus has been increased from 25% to 52%. We have shown that DXS105 and DXS152 are contained within a 40-kb region. A multipoint linkage analysis was performed in fragile-X families and in large normal families from the Centre d'Etudes du Polymorphisme Humain (CEPH). This has allowed us to establish the order centromere-DXS144-DXS51-DXS102-F9-DXS105-FRAX A-(F8, DXS15, DXS52, DXS115). DXS102 is close to the hemophilia-B locus (z[theta] = 13.6 at theta = .02) and might thus be used as an alternative probe for diagnosis in Hemophila-B families not informative for intragenic RFLPs. DXS105 is 8% recombination closer to the fragile-X locus than F9 (z[theta] = 14.6 at theta = .08 for the F9-DXS105 linkage) and should thus be a better marker for analysis of fragile-X families. However, the DXS105 locus appears to be still loosely linked to the fragile-X locus in some families. The multipoint estimation for recombination between DXS105 and FRAXA is .16 in our set of data. Our data indicate that the region responsible for the heterogeneity in recombination between F9 and the fragile-X locus is within the DXS105-FRAXA interval.     

Linkage of Wiskott-Aldrich syndrome with three marker loci,DXS426, SYP and TFE3, which map to the Xp11.3–p11.22 region

Linkage analysis was performed in 19 families segregating for the Wiskott-Aldrich syndrome (WAS) and in 1 family with X-linked thrombocytopenia using nine polymorphic DNA markers spanning the interval DXS7-DXS14. The results confirm close linkage of WAS to the DXS7, TIMP, OATL1, DXS255, DXS146, and DXS14 loci and reveal three additional marker loci, DXS426, SYP, and TFE3, to be closely linked to WAS. The linkage data are also consistent with the localization of X-linked thrombocytopenia to the same chromosomal region as WAS and support localization of the WAS gene between the TIMP and DXS 146 loci. However, the data were insufficient for positioning these disease genes with respect to the four marker loci that map within this latter interval. Analysis of recombination events between the marker loci place the TFE3 gene distal to DXS255 and favor the marker loci order Xpter-DXS7-(DXS426, TIMP)-(OATL1, SYP, TFE3)-DXS255-DXS146-DXS14.     

Linkage relationships and gene order around the locus for X-linked retinoschisis.

X-linked recessive retinoschisis (RS) is a hereditary disorder with variable clinical features. The main symptoms are poor sight; radial, cystic macula degeneration; and peripheral superficial retinal detachment. The disease is quite common in Finland, where at least 300 hemizygous males have been diagnosed. We used nine polymorphic DNA markers to study the localization of RS on the short arm of the X chromosome in 31 families comprising 88 affected persons. Two-point linkage results confirmed close linkage of the RS gene to the marker loci DXS43, DXS16, DXS207, and DXS41 and also revealed close linkage to the marker loci DXS197 and DXS9. Only one recombination was observed between DXS43 and RS in 59 informative meioses, giving a maximum lod score of 13.87 at the recombination fraction .02. No recombinations were observed between the RS locus and DXS9 and DXS197 (lods between 3 and 4), but at neither locus was the number of informative meioses sufficient to provide reliable estimates of recombination fractions. The most likely gene order on the basis of multilocus analysis was Xpter-DXS85-(DXS207,DXS43)-RS-DXS41-DXS 164-Xcen. Because multilocus linkage analysis indicated that the most probable location of RS is proximal to DXS207 and DXS43 and distal to DXS41, these three flanking markers are the closest and most informative markers currently available for carrier detection.     

Allele and Haplotype Diversity of 26 X-STR Loci in Four Nationality Populations from China

Background

Haplotype analysis of closely associated markers has proven to be a powerful tool in kinship analysis, especially when short tandem repeats (STR) fail to resolve uncertainty in relationship analysis. STR located on the X chromosome show stronger linkage disequilibrium compared with autosomal STR. So, it is necessary to estimate the haplotype frequencies directly from population studies as linkage disequilibrium is population-specific.

Methodology and Findings

Twenty-six X-STR loci including six clusters of linked markers DXS6807-DXS8378-DXS9902(Xp22), DXS7132-DXS10079-DXS10074-DXS10075-DXS981 (Xq12), DXS6801-DXS6809-DXS6789-DXS6799(Xq21), DXS7424-DXS101-DXS7133(Xq22), DXS6804-GATA172D05(Xq23), DXS8377-DXS7423 (Xq28) and the loci DXS6800, DXS6803, DXS9898, GATA165B12, DXS6854, HPRTB and GATA31E08 were typed in four nationality (Han, Uigur, Kazakh and Mongol) samples from China (n = 1522, 876 males and 646 females). Allele and haplotype frequency as well as linkage disequilibrium data for kinship calculation were observed. The allele frequency distribution among different populations was compared. A total of 5–20 alleles for each locus were observed and altogether 289 alleles for all the selected loci were found. Allele frequency distribution for most X-STR loci is different in different populations. A total of 876 male samples were investigated by haplotype analysis and for linkage disequilibrium. A total of 89, 703, 335, 147, 39 and 63 haplotypes were observed. Haplotype diversity was 0.9584, 0.9994, 0.9935, 0.9736, 0.9427 and 0.9571 for cluster I, II, III, IV, V and VI, respectively. Eighty-two percent of the haplotype of cluster IIwas found only once. And 94% of the haplotype of cluster III show a frequency of <1%.

Conclusions

These results indicate that allele frequency distribution for most X-STR loci is population-specific and haplotypes of six clusters provide a powerful tool for kinship testing and relationship investigation. So it is necessary to obtain allele frequency and haplotypes data of the linked loci for forensic application.     

Genetic mapping of new RFLPs at Xq27-q28.

The development of the human gene map in the region of the fragile X mutation (FRAXA) at Xq27 has been hampered by a lack of closely linked polymorphic loci. The polymorphic loci DXS369 (detected by probe RN1), DXS296 (VK21A, VK21C), and DXS304 (U6.2) have recently been mapped to within 5 cM of FRAXA. The order of loci near FRAXA has been defined on the basis of physical mapping studies as cen-F9-DXS105-DXS98-DXS369-DXS297-FRAXA-++ +DXS296-IDS-DXS304-DXS52-qter. The probe VK23B detected HindIII and XmnI restriction fragment length polymorphisms (RFLPs) at DXS297 with heterozygote frequencies of 0.34 and 0.49, respectively. An IDS cDNA probe, pc2S15, detected StuI and TaqI RFLPs at IDS with heterozygote frequencies of 0.50 and 0.08, respectively. Multipoint linkage analysis of these polymorphic loci in normal pedigrees indicated that the locus order was F9-(DXS105, DXS98)-(DXS369, DXS297)-(DXS293,IDS)-DXS304-DXS52. The recombination fractions between adjacent loci were F9-(0.058)-DXS105-(0.039)-DXS98-(0.123)-DXS369-(0.00)- DXS297-(0.057)-DXS296- (0.00)-IDS-(0.012)-DXS304-(0.120)-DXS52. This genetic map will provide the basis for further linkage studies of both the fragile X syndrome and other disorders mapped to Xq27-q28.     

Multipoint genetic mapping of the Xq26-q28 region in families with fragile X mental retardation and in normal families reveals tight linkage of markers in q26–q27

Summary The q26–q28 region of the human X chromosome contains several important disease loci, including the locus for the fragile X mental retardation syndrome. We have characterized new polymorphic DNA markers useful for the genetic mapping of this region. They include a new BclI restriction fragment length polymorphism (RFLP) detected by the probe St14-1 (DXS52) and which may therefore be of diagnostic use in hemophilia A families. A linkage analysis was performed in fragile X families and in large normal families from the Centre d'Etude du Polymorphisme Humain (CEPH) by using seven polymorphic loci located in Xq26-q28. This multipoint linkage study allowed us to establish the order centromere-DXS100-DXS86-DXS144-DXS51-F9-FRAX-(DXS52-DXS15). Together with other studies, our results define a cluster of nine loci that are located in Xq26-q27 and map within a 10 to 15 centimorgan region. This contrasts with the paucity of markers (other than the fragile X locus) between the F9 gene in q27 and the G6PD cluster in q28, which are separated by about 30% recombination.     

DNA linkage analysis of X-linked retinoschisis

Summary Four families with juvenile retionoschisis (RS) have been studied by linkage analysis utilizing eleven polymorphic X-chromosomal markers. The results suggest a close linkage between DXS43, DXS41, and DXS208 and the RS locus at Xp22. The RS locus is distal to the OTC locus, DXS84, and the DMD locus but proximal to DXS85. No recombination events were observed between the RS locus and DXS43 and DXS41. The maximum likelihood estimate of the recombination fraction () was thus zero and the peak lod scores () were 4.98 (DXS43) and 4.09 (DXS41). The linkage data suggest that the gene order on Xp is DXS85-(DXS43, RS, DXS41)-DMD-DXS84-OTC.     

Identification of a closely linked DNA marker, DXS178, to further refine the X-linked agammaglobulinemia locus

X-linked agammaglobulinemia (XLA) is an inherited recessive disorder in which the primary defect is not known and the gene product has yet to be identified. Utilizing genetic linkage analysis, we previously localized the XLA gene to the map region of Xq21.3-Xq22 with DNA markers DXS3 and DXS17. In this study, further mapping was performed with two additional DNA probes, DXS94 and DXS178, by means of multipoint analysis of 20 families in which XLA is segregating. Thirteen of these families had been previously analyzed with DXS3 and DXS17. Three crossovers were detected with DXS94 and no recombinations were found between DXS178 and the XLA locus in 9 informative families. Our results show that XLA is closely linked to DXS178 with a two-point lod score of 4.82 and a multipoint lod score of 10.24. Thus, the most likely gene order is DXS3-(XLA,DXS178)-DXS94-DXS17, with the confidence interval for location of XLA lying entirely between DXS3 and DXS94. In 2 of these families, we identified recombinants with DXS17, a locus with which recombination had not previously been detected by others in as many as 40 meiotic events. Furthermore, DXS178 is informative in both of these families and does not show recombination with the disease locus. Therefore, our results indicate that DXS178 is linked tightly to the XLA gene.     

Genetic mapping of nine DNA markers in the q11----q22 region of the human X chromosome

We have ordered nine polymorphic DNA markers within detailed map of the proximal part of the human X chromosome long arm, extending from band q11 to q22, by use of both physical mapping with a panel of rodent-human somatic hybrids and multipoint linkage analysis. Analysis of 44 families (including 17 families from the Centre d'Etude du Polymorphisme Humain) provided highly significant linkage data for both order and estimation of map distances between loci. We have obtained the following order: DXS1-DXS159-DXYS1-DXYS12-DXS3-(DXS94 , DXS178)-DXYS17. The most probable location of DXYS2 is between DXS159 and DXS3, close to DXYS1 and DXYS12. The high density of markers (nine loci within 30 recombination units) and the improvement in the estimation of recombination frequencies should be very useful for multipoint mapping of disease loci in this region and for diagnostic applications.     

Genetic mapping of two new DNA markers in Xq26-q28 relative to the fragile-X syndrome locus.

We have characterized and genetically mapped two new DNA markers (DXS311 and DXS312) with respect to 10 existing loci in Xq26----Xq28 in a set of 15 families in which the fragile-X [fra(X)] syndrome was segregating. Two-point and multipoint linkage analyses were performed taking into account the incomplete penetrance of the fra(X) mutation. The most likely order on the basis of these data is centromere-DXS79-DXS10-DXS311-DXS86-(F9-DXS99 )-(DXS98-DXS312)-fra(X)-DXS52- DXS15-F8C-telomere. DXS98 and one of the new loci, DXS312, were found to be the proximal markers closest to the fra(X) locus. The order F9-(DXS98-DXS312)-fra(X) was found to be 5.9 x 10(4) times more likely than the order (DXS98-DXS312)-F9-fra(X).     

Localization of the highly polymorphic microsatellite DXS456 on the genetic linkage map of the human X chromosome

The CA repeat microsatellite DXS456, with a heterozygosity of 77%, has been localized by multipoint linkage analysis in relation to 20 other genetic markers. DXS456 mapped to a 4.2-cM interval defined by the flanking markers DXS178 and DXS287. The maximum likelihood order of markers, cen-(DXYS1X/DXYS13X/DXYS2X/DXYS12X)-DXS366 -DXS178-DXS456-DXS287-DXS358-DXS267- qter, is favored by odds greater than 1000:1 over the subset of most likely alternative orders. Linkage of DXS456 can be inferred for at least six disease genes that are known to be linked to markers in the region Xq21.31-Xq25 and the marker will serve as an important index point for orienting these and other disease and marker loci in the region.     

Mapping of the gene for X-linked amelogenesis imperfecta by linkage analysis.

X-linked Amelogenesis imperfecta (AI) is a genetic disorder affecting the formation of enamel. In the present study two families, one with X-linked dominant and one with X-linked recessive AI, were studied by linkage analysis. Eleven cloned RFLP markers of known regional location were used. Evidence was obtained for linkage between the AI locus and the marker p782, defining the locus DXS85 at Xp22, by using two-point analysis. No recombination was scored between these two loci in 15 informative meioses, and a peak lod score (Zmax) of 4.45 was calculated at zero recombination fraction. Recombination was observed between the more distal locus DXS89 and AI, giving a peak lod score of 3.41 at a recombination fraction of .09. Recombination was also observed between the AI locus and the more proximal loci DXS43 and DXS41 (Zmax = 0.09 at theta max = 0.31 and Zmax = 0.61 at theta max = 0.28, respectively). Absence of linkage was observed between the AI locus and seven other loci, located proximal to DXS41 or on the long arm of the X chromosome. On the basis of two-point linkage analysis and analysis of crossover events, we propose the following order of loci at Xp22: DXS89-(AI, DXS85)-DXS43-DXS41-Xcen.     

Assignment of the gene for complete X-linked congenital stationary night blindness (CSNB1) to Xp11.3

X-linked congenital stationary night blindness (CSNB) is a nonprogressive retinal disorder characterized by a presumptive defect of neurotransmission between the photoreceptor and bipolar cells. Carriers are not clinically detectable. A new classification for CSNB includes a complete type, which lacks rod function by electroretinography and dark adaptometry, and an incomplete type, which shows some rod function on scotopic testing. The refraction in the complete CSNB patients ranges from mild to severe myopia; the incomplete ranges from moderate hyperopia to moderate myopia. To map the gene responsible for this disease, we studied eight multigeneration families, seven with complete CSNB (CSNB1) and one with incomplete CSNB, by linkage analysis using 17 polymorphic X-chromosome markers. We found tight genetic linkage between CSNB1 and an Xp11.3 DNA polymorphic site, DXS7, in seven families with CSNB1 (LOD 7.35 at theta = 0). No recombinations to CSNB1 were found with marker loci DXS7 and DXS14. The result with DXS14 may be due to the small number of scored meioses (10). No linkage could be shown with Xq loci PGK, DXYS1, DXS52, and DXS15. Pairwise linkage analysis maps the gene for CSNB1 at Xp11.3 and suggests that the CSNB1 locus is distal to another Xp11 marker, TIMP, and proximal to the OTC locus. Five-point analysis on the eight families supported the order DXS7-CSNB1-TIMP-DXS225-DXS14. The odds in favor of this order were 9863:1. Removal of the family with incomplete CSNB (F21) revealed two most favored orders, DXS7-CSNB1-TIMP-DXS255-DXS14 and CSNB1-DXS7-TIMP-DXS255-DXS14. Heterogeneity testing using the CSNB1-M27 beta and CSNB1-TIMP linkage data (DXS7 was not informative in F21) was not significant to support evidence of genetic heterogeneity (P = 0.155 and 0.160, respectively).