The Localization of a Gene Causing X-Linked Cleft Palate and Ankyloglossia (CPX) in an Icelandic Kindred Is between DXS326 and DXYS1X

The locus responsible for X-linked, nonsyndromic cleft palate and/or ankyloglossia (CPX) has previously been mapped to the proximal long arm of the human X chromosome between Xq21.31 and q21.33 in an Icelandic kindred. We have extended these studies by analyzing an additional 14 informative markers in the family as well as including several newly investigated family members. Recombination analysis indicates that the CPX locus is more proximal than previously thought, within the interval Xq21.1-q21.31. Two recombinants place DXYS1X as the distal flanking marker, while one recombinant defines DXS326 as the proximal flanking marker, an interval of less than 5 cM. Each of the flanking markers recombines with the CPX locus, giving 2-point lod scores of Z_max = 4.16 at θ = 0.08 (DXS326) and Z_max = 5.80 at θ = 0.06 (DXYS1X).     

Refinement of the X-linked cleft palate and ankyloglossia (CPX) localisation by genetic mapping in an Icelandic kindred

The gene responsible for X-linked cleft palate and ankyloglossia (CPX) has previously been localized to the proximal region of the q arm of the X chromosome in both Icelandic and North American Indian kindreds. In this study, further linkage analysis has been performed on the Icelandic family and has resulted in a significant reduction in the size of the interval containing the mutated gene. A new polymorphism at DXS95, together with DXS1002 and DXS349, defines the proximal boundary of the CPX interval, whereas DXYS1X defines the distal boundary. Multipoint analysis supports this localisation with a peak lod score of 12.7, more than 2 lod score units higher than the next most likely position. In order to assess the physical size of the CPX interval prior to initiating yeast artificial chromosome cloning, metaphase fluorescence in situ hybridisation analysis was performed with the closest flanking markers. The size of the interval between DXS95 and DXYS1X was estimated to be approximately 2–3 Mb.     

Three DNA markers for hypophosphataemic rickets

Summary This paper presents three markers, 16D/E, pHMAI (DXS208), and CRI-L1391 (DXS274), that show close linkage for X-linked hypophosphataemic rickets (HYP). DXS274 is closely linked to HYP ( _max= 0.00, Z_max = 4.20), and DXS41 (99.6), ( _max= 0.00, Z_max = 5.20). Marker 16D/E maps distal to the disease locus ( _max= 0.05, Z_max = 3.11). The pHMAI probe recognises the same restriction fragment length polymorphism (RFLP) as 99.6. Multipoint analysis suggests that the most probable order of loci is Xpter-(DXS43, 16D/E)-HYP-DXS274-(DXS208, DXS41)-Xcen. The location of DXS274 distal to HYP cannot be excluded, as no recombinants were observed between DXS274 and HYP, or between DXS274 and DXS41/DXS208. One of the families contains a large number of recombinants, four of which are double recombinants. This most probably means that the disease in this family maps elsewhere on the X chromosome or on an autosome, indicating locus heterogeneity.     

Localization of a mutant gene for cleft palate and ankyloglossia in an X-linked Icelandic family.

Common congenital malformations such as cleft lip and cleft palate are in most cases multifactorial in origin, involving both environmental and genetic components. Molecular biology techniques have enabled the successful chromosomal localization of many mutant genes from disorders that exhibit simple Mendelian segregation, whether autosomally dominant (e.g., Huntington's disease), autosomal recessive (e.g., cystic fibrosis), or X-linked (e.g., Duchenne muscular dystrophy). Studying the genetic aspect of multifactorial disorders is more complex. It requires a model family or families within which the common multifactorial phenotype is displayed as a single gene defect. Such a model has been recently exploited in the form of a large Icelandic family (over 280 members) exhibiting X-linked secondary cleft palate (CP) and ankyloglossia (A) (tongue-tied) as a single gene mutation. Using this family and the large bank of well-characterized DNA probes available for the human X chromosome, the gene for CP + A was localized by linkage analysis to Xq13-q21.1 (LOD score = 3.07, linked to anonymous probe DXYS1). Further fine mapping, using other X probes from this region (confirmed by analysis of DNA from a deletion cell-line) has placed the gene between markers DXYS12 and DXS17 (LOD score = 4.1) at Xq21.3-q22. The approximate distance between these two probes is 5 centimorgans (cM), equivalent to approximately 5 million base pairs. Now that the limits of genetic linkage have been fully tested and there are two markers flanking the defect locus, strategies are being pursued to clone the gene responsible.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physical and transcriptional mapping of the X-linked cleft palate and ankyloglossia (CPX) critical region

Cleft palate most commonly occurs as a sporadic multifactorial disorder with a clear but difficult to define genetic component. As a semi-dominant disorder, X-linked cleft palate (CPX) provides a useful model to investigate a congenital defect that is little influenced by non-genetic factors. By using an Icelandic kindred, CPX has been localised between DXS1196 and DXS1217 and mapped, in a 3-Mb yeast artificial chromosome contig, at Xq21.3. Markers generated from this physical map have now been used to construct a contig of P1 and bacterial artificial chromosome clones for genomic DNA sequencing. Genomic DNA sequence analysis has revealed two novel expressed genes and two pseudogenes in the order Cen-KLHL4-LAMRL5-CAPZA1P-CPXCR1-Tel. KLHL4 and CPXCR1 are widely expressed in fetal tissues, including the tongue, mandible and palate. DNA mutation screening of CPXCR1 has revealed several sequence variants present on all affected CPX chromosomes. However, these variants have also been detected at a lower frequency on unaffected chromosomes, indicating that they are polymorphisms that are unlikely to cause the CPX phenotype.     

Amplification of human polymorphic sites in the X-chromosomal region q21.33 to q24: DXS17, DXS87, DXS287, and alpha-galactosidase A.

Methods for the PCR amplification of five polymorphic sites in the region Xq21.33 to Xq24 were developed and used to predict heterozygosity for Fabry disease in informative families. Clones containing polymorphic sites associated with DNA segments DXS17, DXS87, and DXS287, and the alpha-galactosidase A gene were isolated from genomic libraries. Surrounding nucleotide sequences and optimal conditions for amplification of each polymorphic site were determined. These amplifiable polymorphisms provided predictions of heterozygosity for Fabry disease and should be useful for diagnostic linkage analyses in Alport syndrome, X-linked cleft palate and ankyloglossia, Pelizaeus-Merzbacher disease, and X-linked agammaglobulinemia as well as sequence-tagged sites for gene mapping.     

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.     

Cloning and expression analysis of the chick ortholog of TBX22, the gene mutated in X-linked cleft palate and ankyloglossia

T-box genes constitute a conserved gene family with important roles in many developmental processes. Several family members have been implicated in human congenital diseases. Recently, mutations in TBX22 were found to cause X-linked cleft palate (CPX and ankyloglossia), a semidominant X-linked disorder affecting formation of the secondary palate. Here, we have cloned the chick ortholog of human TBX22 and have analyzed its expression during embryogenesis. Expression is very prominent in the somites and in the myotome, and in the mandible and maxilla of the developing jaw. Other sites of expression include the limbs, the cranial mesenchyme and the eye. Hence, Tbx22 expression domains encompass the regions important for the development of the disease phenotype.     

Assignment of the gene for dyskeratosis congenita to Xq28

Summary Dyskeratosis congenita is an X-linked recessive disorder with diagnostic dermatological features, bone marrow hypofunction, and a predisposition to neoplasia in early adult life. Linkage analysis was undertaken in an extensive family with the condition using the Xg blood group and 17 cloned X chromosomal DNA sequences which recognise restriction fragment length polymorphisms (RFLPs). No recombination was observed between the locus for dyskeratosis congenita (DKC) and the RFLPs identified by DXS52 (St 14-1) (Z_max=3.33 at _max=0 with 95% confidence limits of 0 to 14 cM). Similarly no recombination was observed for the disease locus and F8 (Z_max=1.23 at _max=0) nor for DXS15 (Z_max=1.62 at _max=0), but both of these markers were only informative in part of the family whereas DXS52 was fully informative. DXS52, DXS15, and F8 are known to be tightly linked and have previously been assigned to Xq28. Thus the gene for dyskeratosis congenita can be assigned to Xq28. These DNA sequence polymorphisms will be of clinical value for carrier detection and prenatal diagnosis.     

X-linked megalocornea: close linkage to DXS87 and DXS94

Summary In a family in which X-linked megalocornea is segregating, the disease locus was found to be closely linked to DXS87 (z_max=3.91, _max=0.00) and DXS94 (z_max=3.34, _max=0.00) in Xq21.3-q22.     

Linkage analysis using multiple DNA polymorphic markers in normal families and in families with fragile X syndrome

Summary Linkage data, using the polymorphic markers 52A (DXS51), F9, 4D-8(DXS98), and St14(DXS52), are presented from 14 fragile X pedigrees and from 7 normal pedigrees derived from the collection of the Centre d'Étude du Polymorphisme Humaine. A multipoint linkage analysis indicates that the most probable order of these four loci in normal families is DXS51-F9-DXS98-DXS52. Recombination frequencies ( ) corresponding to maximum LOD scores ( ) were obtained by two-point linkage analysis for a nuber of linkage groups, including: DXS51-F9 ( =5.94, =0.03), F9-DXS98 ( =0.51, =0.26), F9-DXS52 ( =0.84, =0.27), and DXS98-DXS52 ( =0.32, =0.20). A multipoint linkage analysis of these loci, including the fragile X locus, was also performed for the fragile X population and the data support the relative order (DSX51, F9, DXS98)-FRAXA-DXS52. Recombination frequencies and maximum LOD scores, which again were derived from two-point linkage analyses, were obtained for the linkage groups DXS51-F9 ( =9.96, =0) and F9-DXS52 ( =0.07, =0.45), as well as for the groups DXS51-FRAXA ( =2.42, =0.15), F9-FRAXA ( =1.30, =0.18), DXS98-FRAXA ( =0.05 =0.36), and DXS52-FRAXA ( =2.42 =0.15). The linkage data was further tested for the presence of genetic heterogeneity both within and between the fragile X and normal families for the intervals DXS51-F9, F9-DXS52, F9-FRAXA, and DXS52-FRAXA using a modification of the A test. Except for the interval F9-FRAXA (P<0.10) there was no evidence of genetic heterogeneity for each of the various linkage groups examined. The heterogeneity detected for the interval F9-FRAXA, however, was most likely due to one family (Fx-28) that displayed very tight linkage between these two loci.     

Phenotypic variability in X-linked ocular albinism: relationship to linkage genotypes.

One hundred nineteen individuals from 11 families with X-linked ocular albinism (OA1) were studied with respect to both their clinical phenotypes and their linkage genotypes. In a four-generation Australian family, two affected males and an obligatory carrier lacked cutaneous melanin macroglobules (MMGs); ocular features were identical to those of Nettleship-Falls OA1. Four other families had more unusual phenotypic features in addition to OA1. All OA1 families were genotyped at DXS16, DXS85, DXS143, STS, and DXS452 and for a CA-repeat polymorphism at the Kallmann syndrome locus (KAL). Separate two-point linkage analyses were performed for the following: group A, six families with biopsy-proved MMGs in at least one affected male; group B, four families whose biopsy status was not known; and group C, OA-9 only (16 samples), the family without MMGs. At the set of loci closest to OA1, there is no clear evidence in our data set for locus heterogeneity between groups A and C or among the four other families with complex phenotypes. Combined multipoint analysis (LINKMAP) in the 11 families and analysis of individual recombination events confirms that the major locus for OA1 resides within the DXS85-DXS143 interval. We suggest that more detailed clinical evaluations of OA1 individuals and families should be performed for future correlation with specific mutations in candidate OA1 genes.     

Choroideremia: linkage analysis with physically mapped close DNA-markers

Summary We report linkage studies in 18 choroideremia (TCD) families using four closely linked polymorphic markers. Probe pZ11, which is known to be deleted in several unrelated patients with TCD, showed no recombinations (z _max 15.63 at = 0.00). In contrast, one recombination was observed with DXS367, which is also physically very close to TCD. Loci DXS95 and DXYS69 each showed more than one recombination with TCD. Moreover, these analyses revealed a double crossover between TCD and DXYS1, changing the previously reported very close linkage to a recombination fraction of 0.04 with a lod score of 9.93. Multipoint linkage analysis placed TCD proximal to DXS95-DXYS69 and very close to DXS367-pZ11 with almost identical multipoint lod score maxima either proximal to DXS367 (z _max= 23.43) or proximal to pZ11 (z _max=23.36). These results provide a refined linkage map around TCD and will also be useful in DNA diagnostics of the disease.     

Oto-palato-digital syndrome type I: further evidence for assignment of the locus to Xq28

Summary The oto-palado-digital syndrome (OPD) is a rare X-linked disease with diagnostic skeletal features, conduction deafness, cleft palate and mild mental retardation. Differences in clinical presentation between families have led investigators to classify OPD into two subtypes: type I and type II. A linkage study performed in one family segregating for OPD I has recently suggested linkage to three marker loci: DXS15, DXS52 at Xq28, and DXS86 at Xq26. We have investigated an additional OPD I family for linkage by using distal chromosome Xq DNA probes. The linkage data and the analysis of recombination events that have occurred in this family excluded, definitively, the Xq26 region for OPD I, and provide further support for mapping the mutant gene close to the cluster of tightly linked markers DXS15, DXS52 and DXS305 at Xq28.     

Anderson Fabry disease,a close linkage with highly polymorphic DNA markers DXS17, DXS87 and DXS88

Summary Anderson Fabry disease is an X-linked lysosomal storage disorder caused by α-galactosidase A deficiency. Hemizygous males and some heterozygous females develop renal failure and cardiovacular complications in early adult life. We have investigated six large UK families to assess the possible linkage of five polymorphic DNA probes to the Anderson Fabry locus, previously localised to Xq21-24. No recombination was found between Anderson Fabry disease and DXS87, DXS88 and DXS17, which gave lod_max=6.4,6.4 and 5.8 respectively at θ=0.00, (upper confidence limit 0.10). DXS3 gave lod_max 2.9 at θ=0.10 (upper confidence limit 0.25). DXYS1 was excluded from linkage. The best fit map (DXYS1/DXS3) θ=0.192 (DXS17/DXS87/DXS88/Anderson Fabry locus) provided no information about the order of loci in parentheses due to the absence of recombinants. The close linkage of DXS17, DXS87 and DXS88, together with α-galactosidade A estimation, can be used for antenatal diagnosis and carrier detection until the application of a gene specific probe has been evaluated.     

Mapping of the gene for X-chromosomal split-hand/split-foot anomaly to Xq26–q26.1

A large inbred kindred from Pakistan in which an isolated type of split-hand/split-foot anomaly is transmitted as an X-chromosomal trait has previously been described. An X/autosomal translocation and an X-chromosomal rearrangement have been excluded by cytogenetic studies. In order to map the gene responsible for this disorder, linkage analysis has been performed by using 14 highly polymorphic DNA markers distributed over the whole X chromosome. Two-point linkage analysis between the disease locus and X-chromosomal marker loci gives maximal lod scores at = 0.00 with the loci DXS294 (Z _max= 5.13) and HPRT (Z _max= 4.43), respectively, suggesting that the gene for the X-chromosomal split-hand/split-foot anomaly is localized at Xq26–q26.1.     

Multipoint linkage analysis and heterogeneity testing in 20 X-linked retinitis pigmentosa families

Using multipoint linkage analysis in 20 families segregating for X-linked retinitis pigmentosa (XLRP), the lod scores on a map of eight RFLP loci were obtained. Our results indicate that under the hypothesis of homogeneity the maximal multipoint lod score supports one disease locus located slightly distal to OTC at Xp21.1. Heterogeneity testing for two XLRP loci suggested that a second XLRP locus may be located 8.5 cM proximal to DXS28 at Xp21.3. Further heterogeneity testing for three disease loci failed to detect a third XLRP locus proximal to DXS7 in any of our 20 XLRP families.     

Spondyloepiphyseal dysplasia tarda: linkage with genetic markers from the distal short arm of the X chromosome

Summary A linkage study of six families with spondyloepiphyseal dysplasia tarda (SEDL) has been performed. A linkage to site DXS41 ( =0.08; =3.07) and DXS92 ( =0.05; =2.95) has been established. We propose, that the SEDL locus lies on the distal part of the short arm of the X chromosome.     

Genetic heterogeneity in X-linked amelogenesis imperfecta.

The AMELX gene located at Xp22.1-p22.3 encodes for the enamel protein amelogenin and has been implicated as the gene responsible for the inherited dental abnormality X-linked amelogenesis imperfecta (XAI). Three families with XAI have been investigated using polymorphic DNA markers flanking the position of AMELX. Using two-point linkage analysis, linkage was established between XAI and several of these markers in two families, with a combined lod score of 6.05 for DXS16 at theta = 0.04. This supports the involvement of AMELX, located close to DXS16, in the XAI disease process (AIH1) in those families. Using multipoint linkage analysis, the combined maximum lod score for these two families was 7.30 for a location of AIH1 at 2 cM distal to DXS16. The support interval around this location extended about 8 cM proximal to DXS92, and the AIH1 location could not be precisely defined by multipoint mapping. Study of recombination events indicated that AIH1 lies in the interval between DXS143 and DXS85. There was significant evidence against linkage to this region in the third family, indicating locus heterogeneity in XAI. Further analysis with markers on the long arm of the X chromosome showed evidence of linkage to DXS144E and F9 with no recombination with either of these markers. Two-point analysis gave a peak lod score at DXS144E with a maximum lod score of 2.83 at theta = 0, with a peak lod score in multipoint linkage analysis of 2.84 at theta = 0. The support interval extended 9 cM proximal to DXS144E and 14 cM distal to F9.(ABSTRACT TRUNCATED AT 250 WORDS)