A novel locus (RP24) for X-linked retinitis pigmentosa maps to Xq26-27.

Two genetic loci, RP2 and RP3, for X-linked retinitis pigmentosa (XLRP) have been localized to Xp11.3-11.23 and Xp21.1, respectively. RP3 appears to account for 70% of XLRP families; however, mutations in the RPGR gene (isolated from the RP3 region) are identified in only 20% of affected families. Close location of XLRP loci at Xp and a lack of unambiguous clinical criteria do not permit assignment of genetic subtype in a majority of XLRP families; nonetheless, in some pedigrees, both RP2 and RP3 could be excluded as the causative locus. We report the mapping of a novel locus, RP24, by haplotype and linkage analysis of a single XLRP pedigree. The RP24 locus was identified at Xq26-27 by genotyping 52 microsatellite markers spanning the entire X chromosome. A maximum LOD score of 4.21 was obtained with DXS8106. Haplotype analysis assigned RP24 within a 23-cM region between the DXS8094 (proximal) and DXS8043 (distal) markers. Other chromosomal regions and known XLRP loci were excluded by obligate recombination events between markers in those regions and the disease locus. Hemizygotes from the RP24 family have early onset of rod photoreceptor dysfunction; cone receptor function is normal at first, but there is progressive loss. Patients at advanced stages show little or no detectable rod or cone function and have clinical hallmarks of typical RP. Mapping of the RP24 locus expands our understanding of the genetic heterogeneity in XLRP and will assist in development of better tools for diagnosis.     

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 analysis of new French X-linked juvenile retinoschisis kindreds using microsatellite markers closely linked to the RS locus: further narrowing of the RS candidate region

The gene involved in juvenile retinoschisis (RS) has previously been localized, by genetic linkage analyses, to Xp22.1-p22.2, between DXS274 and DXS43/ DXS207; it is closely linked to the latter markers. From our recent data, this interval represents a genetic distance of approximately 10 cM. In the present study, we have studied 14 French families with X-linked juvenile RS by using four CA polymorphisms that are closely linked to the RS locus and that have recently been included in an Xp22.1-p22.2 high-resolution map. Complete cosegregation with the disease locus was observed for three of them, DXS207, DXS418, and DXS999, which further confirms the locus homogeneity for RS and the close linkage to this region. One recombinant was found with the most proximal marker, AFM291wf5, thereby defining this marker as the new proximal boundary of the candidate region for RS. Under the assumption that DXS207 and DXS43 constitute the distal boundary, the present study further reduces the region containing the disease gene to a interval of 3–4 cM. The results reported here should facilitate the eventual cloning of the RS gene.     

注意缺损多动障碍的X染色体基因组扫描分析

摘 要：注意缺损多动障碍（ADHD）是儿童期多见行为障碍。男孩发病多于女孩。家系、双生儿和寄养子研究显示该障碍发生具有遗传基础。但是病因尚不清楚。分子遗传学和药理学研究表明ADHD涉及到多巴胺和去甲肾上腺素等神经递质系统,一系列报告发现ADHD与多巴胺D4受体（DRD4）、多巴胺转运体（DAT1）和儿茶酚-O-甲基转移酶（COMT）等基因相关联。我们以往研究表明ADHD与X染色体上DXS7位点和MAOA基因相关联,而DXS7是紧密连锁于MAO基因。依此假设,我们应用基因组扫描技术探讨ADHD在X染色体上易感位点。采用TDT方法分析X染色体上48个DNA标志的多态性与中国人群中84个ADHD核心家系间的连锁关系,ADHD诊断依据DSM-III-R标准。TDT分析结果观察到如下位点与ADHD相连锁,DXS1214(TDT:χ2=18.1,df=7, P<0.01), DXS8102(TDT: χ2=7.9, df=3, P<0.05),DXS1068(TDT: χ2=21.9, df=9, P<0.01), DXS8015(TDT:χ2=14.6, df=7, P<0.05),DXS1059(TDT: χ2=27.8, df=10, P<0.01) 和DXS8088(TDT:χ2=20.4, df=3, P<0.01).研究资料提示X染色体上Xp11.4-Xp21和Xq23区域可能存在ADHD的易感基因。     

Linkage analysis of two cloned DNA sequences, DXS197 and DXS207, in hypophosphatemic rickets families

The human X-linked hypophosphatemic rickets gene locus (HYP, formerly HPDR) has been previously localized by linkage analysis to Xp22.31-Xp21.3 and the locus order Xpter-DXS43-HYP-DXS41-Xcen established. Recombination between HYP and these flanking markers is frequently observed and additional markers have been sought. The polymorphic loci DXS197 and DXS207 have been localized to Xpter-Xp11 and Xp22-Xp21, respectively. We have further localized DXS197 to Xpter-Xp21.3 by using a panel of rodent-human hybrid cells and have established the map positions of DXS197 and DXS207 in relation to HYP by linkage studies of hypophosphatemic rickets families. Linkage between DXS197 and the loci DXS43, DXS85, and DXS207 was established with peak lod score values of 6.19, 0 = 0.032; 4.14, 0 = 0.000; and 3.01, 0 = 0.000, respectively. Multilocus linkage analysis mapped the DXS197 and DXS207 loci distal to HYP and demonstrated the locus order Xpter-DXS85-(DXS207, DXS43, DXS197)-HYP-DXS41-Xcen. These additional genetic markers DXS197 and DXS207 will be useful as alternative markers in the genetic counseling of some families.     

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).     

Isolation and mapping of discrete DXS101 loci in Xq22 near the X-linked agammaglobulinaemia gene locus

The X-linked agammaglobulinaemia (XLA) gene locus has previously been mapped to Xq22 in genetic linkage studies. The DXS101 locus has shown no recombinations with XLA in the ten informative meioses investigated so far. The DXS101 sequence, recognised by the cX52.5 plasmid, is moderately repeated in Xq22. We have isolated cosmids which contain this sequence; two copies of which have been found to lie near DXS178 and XLA, and a third copy which lies near the PLP gene, distal to these loci. We have used the cosmids to generate probes which should be of use for RFLP analysis, and thus in both prenatal diagnosis and carrier testing for XLA, and in constructing a genetic map of this region. These probes will also be used to complement the genetic map in the construction of a complete physical map of Xq22.     

Refined localization of the gene causing X-linked juvenile retinoschisis

Previous linkage studies in X-linked juvenile retinoschisis (RS) placed the gene between the loci DXS43 and DXS41 in the region Xp22.2-p22.1. Here we have extended our earlier studies by analyzing 31 RS families with the markers DXS16 (pSE3.2-L), DXS274, DXS92, and ZFX. Pairwise linkage analysis revealed significant linkage of the RS gene to all markers used; locus DXS274 (probe CRI-L1391) was tightly-linked to the disorder, with a lod score of 9.02 at a recombination fraction of 0.05. The genetic map around the RS locus was refined by multilocus linkage studies in an expanded database including a large set of normal families (40 CEPH families). The results indicated that the RS gene locus lies between (DXS207, DXS43) and DXS274 with odds of 1.8 x 10(4):1 favoring this most likely location over the second most likely location, i.e., distal to DXS43. Analysis by LINKMAP gave a maximum location score of 136.4 with the order Xpter-DXS16-(DXS207,DXS43)-RS-DXS274-(D XS41,DXS92)-Xcen. To assess the diagnostic value of the markers in Finnish patients, a total of 12 markers were tested for allele frequencies in 126 Finnish unrelated blood donors. With the exception of the markers DXS207, DXS43, and DXS92, allele frequencies did not show any significant deviation from the data published elsewhere. Haplotype analysis was performed with five DNA markers flanking the RS locus. Patients from southwest Finland had a haplotype association that differed from the haplotype association found in the patients from north central Finland, favoring the hypothesis that the mutations in the two groups arose independently.     

Isolation and characterization of a highly polymorphic human locus (DXS455) in proximal Xq28

Human Xq28 is highly gene dense with over 27 loci. Because most of these genes have been mapped by linkage to polymorphic loci, only one of which (DXS52) is informative in most families, a search was conducted for new, highly polymorphic Xq28 markers. From a cosmid library constructed using a somatic cell hybrid containing human Xq27.3----qter as the sole human DNA, a human-insert cosmid (c346) was identified and found to reveal variation on Southern blot analyses with female DNA digested with any of several different restriction endonucleases. Two subclones of c346, p346.8 and p346.T, that respectively identify a multiallelic VNTR locus and a frequent two-allele TaqI polymorphism were isolated. Examination of 21 unrelated females showed heterozygosity of 76 and 57%, respectively. These two markers appeared to be in linkage equilibrium, and a combined analysis revealed heterozygosity in 91% of unrelated females. Families segregating the fragile X syndrome with key Xq28 crossovers position this locus (designated DXS455) between the proximal Xq28 locus DXS296 (VK21) and the more distal locus DXS374 (1A1), which is proximal to DXS52. DXS455 is therefore the most polymorphic locus identified in Xq28 and will be useful in the genetic analysis of this gene dense region, including the diagnosis of nearby genetic disease loci by linkage.     

Structural gene aberrations in mucopolysaccharidosis II (Hunter)

Summary A total of 14 unrelated German patients with X-linked iduronate-2-sulfatase (IDS) deficiency (Hunter syndrome, MPS II) showing variable clinical manifestations was screened for structural gene aberrations by Southern analysis. Using the IDS cDNA clone c2S15 as a probe, no Southern fragments could be detected in blots in the severely affected patient G-65 with respect to DNA digested by HindIII, PstI and TaqI, suggesting a total loss of the IDS structural gene. In this patient, the flanking loci DXS 297, DXS 296 and DXS 466 were tested. The locus DXS 466 is involved in the deletion, whereas both of the other loci are present. A normal 9.0-kb fragment disappeared and an aberrant fragment of 3.5 kb occurred in the HindIII blot of patient G-117. A normal Southern pattern was found in PstI and TaqI blots of this patient. This result can be interpreted as the generation of an additional HindIII restriction site by point mutation in an IDS gene intron.     

Four chromosomal breakpoints and four new probes mark out a 10-cM region encompassing the fragile-X locus (FRAXA)

We report the validation and use of a cell hybrid panel which allowed us a rapid physical localization of new DNA probes in the vicinity of the fragile-X locus (FRAXA). Seven regions are defined by this panel, two of which lie between DXS369 and DXS296, until now the closest genetic markers that flank FRAXA. Of those two interesting regions, one is just distal to DXS369 and defined by probe 2-71 (DXS476), which is not polymorphic. The next one contains probes St677 (DXS463) and 2-34 (DXS477), which are within 130 kb and both detect TaqI RFLPs. The combined informativeness of these two probes is 30%. We cloned from an irradiation-reduced hybrid line another new polymorphic probe, Do33 (DXS465; 42% heterozygosity). This probe maps to the DXS296 region, proximal to a chromosomal breakpoint that corresponds to the Hunter syndrome locus (IDS). The physical order is thus Cen-DXS369-DXS476-(DXS463,DXS477)-(DXS296, DXS465)-IDS-DXS304-tel. We performed a linkage analysis for five of these markers in both the Centre d'Etude du Polymorphisme Humain families and in a large set of fragile-X families. This establishes that DXS296 is distal to FRAXA. The relative position of DXS463 and DXS477 with respect to FRAXA remains uncertain, but our results place them genetically halfway between DXS369 and DXS304. Thus the DXS463-DXS477 cluster defines presently either the closest proximal or the closest distal polymorphic marker with respect to FRAXA. The three new polymorphic probes described here have a combined heterozygosity of 60% and represent a major improvement for genetic analysis of fragile-X families, in particular for diagnostic applications.     

Detection of a molecular deletion at the DXS732 locus in a patient with X-linked hypohidrotic ectodermal dysplasia (EDA), with the identification of a unique junctional fragment.

X-linked hypohidrotic ectodermal dysplasia (EDA) has been localized to the Xq12-q13.1 region. A panel of genomic DNA samples from 80 unrelated males with EDA has been screened for deletions at seven genetic loci within the Xq12-13 region. A single individual was identified with a deletion at the DXS732 locus by hybridization with the mouse genomic probe pcos169E/4. This highly conserved DNA probe is from locus DXCrc169, which is tightly linked to the Ta locus, the putative mouse homologue of EDA. The proband had the classical phenotype of EDA, with no other phenotypic abnormalities, and a normal cytogenetic analysis. A human genomic DNA clone, homologous to pcos169E/4, was isolated from a human X-chromosome cosmid library. On hybridization with the cosmid, the proband was found to be only partially deleted at the DXS732 locus, with a unique junctional fragment identified in the proband and in three of his maternal relatives. This is the first determination of carrier status for EDA in females, by direct mutation analysis. Failure to detect deletion of the other loci tested in the proband suggests that the DXS732 locus is the closest known locus to the EDA gene. Since the DXS732 locus contains a highly conserved sequence, it must be considered to be a candidate locus for the EDA gene itself.     

Definitive localization of Becker muscular dystrophy in Xp by linkage to a cluster of DNA polymorphisms (DXS43 and DXS9)

Summary A study of linkage between Becker muscular dystrophy and four X chromosome-specific DNA polymorphisms in 17 kindreds has indicated that this gene is located in Xp, as already anticipated by single pedigree analysis. In particular the DXS43 and DXS9 loci, identified by probes D2 and RC8, respectively, are closely linked to each other and are both located at approximately 15 cM from the Becker locus. These linkage data, together with the previously established linkage between Becker and the DXS7 locus identified by probe L 1.28, indicate that the Becker gene is located in the same region where Duchenne has been mapped and also yield information about relative genetic distances among different DNA polymorphisms of the X chromosome.     

Identification of a 118-kb DNA fragment containing the locus of blast resistance gene Pi-2(t) in rice

Rice blast disease, caused by the fungal pathogen Pyricularia grisea Sacc., is one of the most devastating crop diseases worldwide. Previous studies have shown that the dominant blast resistance gene Pi-2(t) confers resistance to a broad spectrum of pathogenic strains. Using a population of 292 recombinant inbred lines combined with bioinformatic analysis, we mapped Pi-2(t) between the SSR (simple-sequence repeat) marker SSR140 and the RFLP (restriction fragment length polymorphism) marker JSH12, 0.9 cM from both SSR140 and JSH12. A physical map consisting of six overlapping BAC (bacterial artificial chromosome) clones was anchored to the region containing the Pi-2(t) locus. By analyzing recombination events in this region, the Pi-2(t) locus was localized to a DNA fragment of 118 kb in length. The detailed genetic and physical maps of the Pi-2(t) locus will facilitate both molecular isolation of the gene and marker-assisted transfer of the gene in breeding programs.     

Linkage analysis of X-linked cone-rod dystrophy: localization to Xp11.4 and definition of a locus distinct from RP2 and RP3.

Progressive X-linked cone-rod dystrophy (COD1) is a retinal disease affecting primarily the cone photoreceptors. The COD1 locus originally was localized, by the study of three independent families, to a region between Xp11.3 and Xp21.1, encompassing the retinitis pigmentosa (RP) 3 locus. We have refined the COD1 locus to a limited region of Xp11.4, using two families reported elsewhere and a new extended family. Genotype analysis was performed by use of eight microsatellite markers (tel-M6CA, DXS1068, DXS1058, DXS993, DXS228, DXS1201, DXS1003, and DXS1055-cent), spanning a distance of 20 cM. Nine-point linkage analysis, by use of the VITESSE program for X-linked disorders, established a maximum LOD score (17.5) between markers DXS1058 and DXS993, spanning 4.0 cM. Two additional markers, DXS977 and DXS556, which map between DXS1058 and DXS993, were used to further narrow the critical region. The RP3 gene, RPGR, was excluded on the basis of two obligate recombinants, observed in two independent families. In a third family, linkage analysis did not exclude the RPGR locus. The entire coding region of the RPGR gene from two affected males from family 2 was sequenced and was found to be normal. Haplotype analysis of two family branches, containing three obligate recombinants, two affected and one unaffected, defined the COD1 locus as distal to DXS993 and proximal to DXS556, a distance of approximately 1.0 Mb. This study excludes COD1 as an allelic variant of RP3 and establishes a novel locus that is sufficiently defined for positional cloning.     

Dystonia-parkinsonism syndrome (XDP) locus: flanking markers in Xq12-q21.1.

The study of rare genetic forms of dystonia and parkinsonism permits positional cloning of genes potentially involved in more common, multifactorial forms of these diseases. One movement disorder amenable to molecular genetic analysis is the X-linked dystonia-parkinsonism syndrome (XDP). This disease is endemic to the Philippines where it originated by a genetic founder effect. Linkage analysis was performed with DNA from 14 XDP kindreds by using 12 polymorphic DNA sequences in Xp11-Xq22. Two-point analysis demonstrated maximum lod scores of 5.45, 4.95, 4.28, and 5.99 for DXS106, DXS159, PGK1, and DXS72, respectively, at recombination fractions of zero (DXS106 and DXS159), .01 (PGK1), and .04 (DXS72). Multipoint analysis resulted in a maximum-likelihood score (Zmax) of 8.41 with a (Zmax - 1) support interval of 9 cM between DXS159 and DXS72 (Xq12-q21.1). In 19 XDP kindreds significant linkage disequilibrium was found for loci DXS72 (delta = .47), PGK1 (delta = .36), DXS95 (delta = .30), DXS106 (delta = .28), and DXS159 (delta = .26). These data indicate that the gene mutated in XDP (locus DYT3) is located in Xq12-q21.1.     

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.     

Localization of the gene for the Wiskott-Aldrich syndrome between two flanking markers, TIMP and DXS255, on Xp11.22-Xp11.3.

The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive genetic disease in which the basic molecular defect is unknown. We previously located the WAS gene between two DNA markers, DXS7 (Xp11.3) and DXS14 (Xp11), and mapped it to the proximal short arm of the human X chromosome (Kwan et al., 1988, Genomics 3:39-43). In this study, further mapping was performed on 17 WAS families with two additional RFLP markers, TIMP and DXS255. Our data suggest that DXS255 is closer to the WAS locus than any other markers that have been previously described, with a multipoint maximum lod score of Z = 8.59 at 1.2 cM distal to DXS255 and thus further refine the position of the WAS gene on the short arm of the X chromosome. Possible locations for the WAS gene are entirely confined between TIMP (Xp11.3) and DXS255 (Xp11.22). Use of these markers thus represents a major improvement in genetic prediction in WAS families.     

Identification of CpG islands around the DXS178 locus in the region of the X-linked agammaglobulinaemia gene locus in Xq22

The X-linked agammaglobulinaemia (XLA) gene locus has previously been mapped to Xq22. Genetic linkage analysis has shown tight linkage between the disease and the DXS178 locus and that DXS3 and DXS94 are the closest proximal and distal flanking markers, respectively, separated by a genetic distance of 10–12 cM. We attempted to construct a physical map of Xq22 using pulsed field gel electrophoresis (PFGE) and rare-cutting restriction enzymes in order to obtain a finite physical value for the distance between DXS3 and DXS94. However, these attempts were hampered by the large number of rare-cutting restriction enzyme sites around the DXS178 locus, indicative of the presence of CpG rich regions of DNA. We were able to construct a physical map of the sites close to DXS178 that suggests the presence of at least three, and perhaps as many as five, CpG islands. These are arranged on either side of DXS178, extending over about 550kb of genomic DNA. Each of these regions must be considered as being associated with a potential candidate gene sequence for the XLA gene and we have initiated a chromosome walk from DXS178 to the nearest of these islands.     

A DNA marker closely linked to the factor IX (haemophilia B) gene

Summary We have isolated a DNA segment, pX58dIIIc, from an X-chromosome library which identifies an SstI restriction fragment length polymorphism (RFLP) at locus DXS99. Linkage analysis in six informative families has shown that the DXS99 locus lies close to the factor IX gene (F9). No recombination was detected between these loci in 39 informative meioses (Z=9.79, =0.0). Therefore, DXS99 will be useful as a DNA marker for the assessment of carrier status in families with haemophilia B where intragenic markers are not informative. Heterozygosity at DXS99 is approximately 50% and, in conjunction with the RFLPs at F9, 90% of females at risk for being haemophilia B carriers should be diagnosed.