Microsatellite-based fine mapping of the Van der Woude syndrome locus to an interval of 4.1 cM between D1S245 and D1S414.

Van der Woude syndrome (VWS) is an autosomal dominant craniofacial disorder characterized by lip pits, clefting of the primary or secondary palate, and hypodontia. The gene has been localized, by RFLP-based linkage studies, to region 1q32-41 between D1S65-REN and D1S65-TGFB2. In this study we report the linkage analysis of 15 VWS families, using 18 microsatellite markers. Multipoint linkage analysis places the gene, with significant odds of 2,344:1, in a 4.1-cM interval flanked by D1S245 and D1S414. Two-point linkage analysis demonstrates close linkage of VWS with D1S205 (lod score [Z] = 24.41 at theta = .00) and with D1S491 (Z = 21.23 at theta = .00). The results revise the previous assignment of the VWS locus and show in an integrated map of the region 1q32-42 that the VWS gene resides more distally than previously suggested. When information about heterozygosity of the closely linked marker D1S491 in the affected members of the VWS family with a microdeletion is taken into account, the VWS critical region can be further narrowed, to the 3.6-cM interval between D1S491 and D1S414.     

The gene for spinal cerebellar ataxia 3 (SCA3) is located in a region of approximately 3 cM on chromosome 14q24.3-q32.2.

SCA3, the gene for spinal cerebellar ataxia 3, was recently mapped to a 15-cM interval between D14S67 and D14S81 on chromosome 14q, by linkage analysis in two families of French ancestry. The SCA3 candidate region has now been refined by linkage analysis with four new microsatellite markers (D14S256, D14S291, D14S280, and AFM343vf1) in the same two families, in which 19 additional individuals were genotyped, and in a third French family. Combined two-point linkage analyses show that the new markers, D14S280 and AFM343vf1, are tightly linked to the SCA3 locus, with maximal lod scores, at recombination fraction, (theta) = .00, of 7.05 and 13.70, respectively. Combined multipoint and recombinant haplotype analyses localize the SCA3 locus to a 3-cM interval flanked by D14S291 and D14S81. The same allele for D14S280 segregates with the disease locus in the three kindreds. This allele is frequent in the French population, however, and linkage disequilibrium is not clearly established. The SCA3 locus remains within the 29-cM region on 14q24.3-q32.2 containing the gene for the Machado-Joseph disease, which is clinically related to the phenotype determined by SCA3, but it cannot yet be concluded that both diseases result from alterations of the same gene.     

Linkage of Van der Woude syndrome (VWS) to REN and exclusion of the candidate gene TGFB2 from the disease locus in a large pedigree

Van der Woude syndrome (VWS) is an autosomal dominant disorder associated with one or more of the following features: clefting of the primary or secondary palate, hypodontia or lower lip pits. It has been estimated to account for 2% of all cases of cleft lip and palate. VWS is one of the rare disorders in which clefting of the primary and secondary palate may be seen to segregate as components associated with the same gene. Because of its autosomal dominant inheritance, VWS is readily accessable to linkage analysis as a preliminary step in the identification of the molecular abnormality underlying the clefting effect in the primary and secondary palate. A reported linkage between REN and VWS has promoted us to use pHRnX3.6 (REN) and several markers surrounding REN for a linkage analysis in a large Swiss family. In a second step, linkage analysis was performed to study restriction fragment length polymorphisms for the candidate gene TGFB2 and other loci recently mapped to the candidate region 1q32–1q41. Evidence for linkage ( = 0.00, lod score = 3.01) between REN and VWS could be confirmed in this pedigree. TGFB2 demonstrated recombination with the disease locus and is unlikely to be causative in VWS. The results of a multipoint linkage analysis showed that VWS was flanked by D1S65 and TGFB2 at a maximum location score of 20.3.     

Localization of the Homolog of a Mouse Craniofacial Mutant to Human Chromosome 18q11 and Evaluation of Linkage to Human CLP and CPO

The transgene-induced mutation 9257 and the spontaneous mutation twirler cause craniofacial and inner ear malformations and are located on mouse chromosome 18 near the ataxia locusax.To map the human homolog of 9257, a probe from the transgene insertion site was used to screen a human genomic library. Analysis of a cross-hybridizing human clone identified a 3-kb conserved sequence block that does not appear to contain protein coding sequence. Analysis of somatic cell hybrid panels assigned the human locus to 18q11. The polymorphic microsatellite markers D18S1001 and D18S1002 were isolated from the human locus and mapped by linkage analysis using the CEPH pedigrees. The 9257 locus maps close to the centromeres of human chromosome 18q and mouse chromosome 18 at the proximal end of a conserved linkage group. To evaluate the role of this locus in human craniofacial disorders, linkage to D18S1002 was tested in 11 families with autosomal dominant nonsyndromic cleft lip and palate and 3 families with autosomal dominant cleft palate only. Obligatory recombinants were observed in 8 of the families, and negative lod scores from the other families indicated that these disorders are not linked to the chromosome 18 loci.     

A locus for brachydactyly type A-1 maps to chromosome 2q35-q36

Brachydactyly type A-1 (BDA1) was, in 1903, the first recorded example of a human anomaly with Mendelian autosomal dominant inheritance. Two large families, the affected members of which were radiographed, were recruited in the study we describe here. Two-point linkage analysis for pedigree 1 (maximum LOD score [Zmax] 6.59 at recombination fraction [theta] 0.00) and for pedigree 2 (Zmax=5.53 at straight theta=0.00) mapped the locus for BDA1 in the two families to chromosome 2q. Haplotype analysis of pedigree 1 confined the locus for family 1 within an interval of <8.1 cM flanked by markers D2S2248 and D2S360, which was mapped to chromosome 2q35-q36 on the cytogenetic map. Haplotype analysis of pedigree 2 confined the locus for family 2 within an interval of <28. 8 cM flanked by markers GATA30E06 and D2S427, which was localized to chromosome 2q35-q37. The two families had no identical haplotype within the defined region, which suggests that the two families were not related.     

Genetic and physical mapping of the human cannabinoid receptor gene to chromosome 6q14-q15

A cDNA encoding a G protein-coupled receptor that appears to mediate the behavioral effects of cannabinoids, the psychoactive ingredients of marijuana, has recently been cloned from rat cerebral cortex and expressed. We have now determined the genomic location of the human cannabinoid receptor gene (CNR) by a combination of genetic linkage mapping and chromosomal in situ hybridization. The segregation pattern of a CNR DNA polymorphism was analyzed in 508 individuals from two or three generations of 40 families. Linkage of CNR to chromosome 6 centromeric loci and to DNA markers on the long and short arms was detected. CNR was tightly linked to D6S27, which is known to be located at 6q (log10 odds ratio [lod score, Zmax] of 10.54 at a recombination fraction [theta] of 0.02). Close linkage was suggested between CNR and CGA, the locus for the alpha subunit of human chorionic gonadotropin (Zmax = 2.71 at theta = 0). Moreover, CNR was linked to the two markers 308/BamHI (theta = 0.14) and 308/TaqI (theta = 0.20) defining locus D6Z1, an extended, highly repetitive, and highly conserved sequence localized exclusively to centromeres of all chromosomes and enriched on chromosome 6. In situ hybridization using a biotinylated cosmid probe localizes the gene to 6q14-q15, thereby confirming the linkage analysis and defining a precise alignment of the genetic and cytogenetic maps.     

Identification and regional localization of DNA markers on chromosome 7 for the cloning of the cystic fibrosis gene

To facilitate mapping of the cystic fibrosis locus (CF) and to isolate the corresponding gene, we have screened a flow-sorted chromosome 7-specific library for additional DNA markers in the 7q31-q32 region. Unique ("single-copy") DNA segments were selected from the library and used in hybridization analysis with a panel of somatic cell hybrids containing various portions of human chromosome 7 and patient cell lines with deletion of this chromosome. A total of 258 chromosome 7-specific single-copy DNA segments were identified, and most of them localized to subregions. Fifty three of these corresponded to DNA sequences in the 7q31-q32 region. Family and physical mapping studies showed that two of the DNA markers, D7S122 and D7S340, are in close linkage with CF. The data also showed that D7S122 and D7S340 map between MET and D7S8, the two genetic markers known to be on opposite sides of CF. The study thus reaffirms the general strategy in approaching a disease locus on the basis of chromosome location.     

Genomewide Scan of Multiple Sclerosis in Finnish Multiplex Families

Multiple sclerosis (MS) is a neurological, demyelinating disorder with a putative autoimmune etiology. It is thought to be a multifactorial disease with a complex mode of inheritance. Here we report the results of a two-stage genomewide scan for loci predisposing to MS. The first stage of the screen, with a low-resolution map, was performed in a selection of 16 pedigrees collected from an isolated Finnish population. Multipoint, non-parametric linkage analysis of the 328 markers did not reveal statistically significant results. However, 10 slightly interesting regions (P = .1-.15) emerged, including our previous findings of the HLA complex on 6p21 and a putative locus on 5p14-p12. Eight of these novel regions were further analyzed by use of denser marker maps, in the second stage of the scan. For the chromosomal regions 4cen, 11tel, and 17q, the statistical significance increased, but not conclusively; for 2q32 and 10q21, the statistical significance did not change. Accordingly, genotyping of the high-density markers in these regions was performed, and the data were analyzed by use of two-point, parametric linkage analysis using the complete pedigree information of the 21 Finnish multiplex families. We detected suggestive evidence for a predisposing locus on chromosomal region 17q22-q24. Several markers on 17q22-q24 yielded positive LOD scores, with the maximum LOD score (Zmax) occurring with D17S807 (Zmax = 2.8, theta = .04; dominant model). Interestingly, a suggestive linkage between MS and the markers on 17q22-q24 was also revealed by a recent genomewide scan in MS families from the United Kingdom.     

Regional mapping panel for human chromosome 17: Application to neurofibromatosis type 1

A somatic cell hybrid mapping panel was constructed to localize cloned DNA sequences to any of 15 potentially different regions of human chromosome 17. Relatively high-resolution mapping is possible for 50% of the chromosome length in which 12 breakpoints are distributed over approximately 45 megabases, with an average spacing estimated at 1 breakpoint every 2-7 megabases. This high-resolution capability includes the pericentromeric region of 17 to which von Recklinghausen neurofibromatosis (NF1) has recently been mapped. Using 20 cloned genes and anonymous probes, we have tested the expected order and location of panel breakpoints and confirmed, refined, or corrected the regional assignment of several cloned genes and anonymous probes. Four markers with varying degrees of linkage to NF1 have been physically localized and ordered by the panel: the loosely linked markers myosin heavy chain 2 (25 cM) to p12----13.105 and nerve growth factor receptor (14 cM) to q21.1----q23; the more closely linked pABL10-41 (D17S71, 5 cM) to p11.2; and the tightly linked pHHH202 (D17S33) to q11.2-q12. Thus, physical mapping of linked markers confirms a pericentromeric location of NF1 and, along with other data, suggests the most likely localization is proximal 17q.     

Refining the localization of the PKD2 locus on chromosome 4q by linkage analysis in Spanish families with autosomal dominant polycystic kidney disease type 2.

Autosomal dominant polycystic kidney disease (ADPKD) is a genetically heterogeneous disorder. At least two distinct forms of ADPKD are now well defined. In approximately 86% of affected European families, a gene defect localized to 16p13.3 was responsible for ADPKD, while a second locus has been recently localized to 4q13-q23 as candidate for the disease in the remaining families. We present confirmation of linkage to microsatellite markers on chromosome 4q in eight Spanish families with ADPKD, in which the disease was not linked to 16p13.3. By linkage analysis with marker D4S423, a maximum lod score of 9.03 at a recombination fraction of .00 was obtained. Multipoint linkage analysis, as well as a study of recombinant haplotypes, placed the PKD2 locus between D4S1542 and D4S1563, thereby defining a genetic interval of approximately 1 cM. The refined map will serve as a genetic framework for additional genetic and physical mapping of the region and will improve the accuracy of presymptomatic diagnosis of PKD2.     

Evidence for the localization of a malignant hyperthermia susceptibility locus (MHS2) to human chromosome 17q.

Malignant hyperthermia susceptibility is a lethal autosomal dominant disorder of skeletal muscle metabolism that is triggered by all potent inhalation anesthetic gases. Recent linkage studies suggest a genetic locus for this disorder on 19q13.1. We have previously reported three unrelated families diagnosed with MHS that are unlinked to markers surrounding this locus on 19q13.1. In this report we extend these observations and present linkage studies on 16 MHS families. Four families (25%) were found linked to the region 19q12-q13.2 (Zmax = 2.96 with the ryanodine receptor at theta = 0.0). Five families (31%) were found closely linked to the anonymous marker NME1 (previously designated NM23) on chromosome 17q11.2-q24 (Zmax = 3.26 at theta = 0.0). Two families (13%) were clearly unlinked to either of these chromosomal regions. In five additional families, data were insufficient to determine their linkage status (they were potentially linked to two or more sites). The results of our heterogeneity analyses are consistent with the hypothesis that MHS can be caused in humans by any one of at least three distinct genetic loci. Furthermore, we provide preliminary linkage data suggesting the localization of a gene in human MHS to 17q11.2-q24 (MHS2), with a gene frequency of this putative locus approximately equal to that of the MHS1 locus on 19q.     

Localization of the Krabbe disease gene (GALC) on chromosome 14 by multipoint linkage analysis.

The gene responsible for Krabbe disease, an autosomal recessive disorder caused by deficiency of galactocerebrosidase (GALC), was localized by multipoint linkage analysis on chromosome 14. Eight mapped dinucleotide repeat polymorphisms were tested for linkage to GALC. Two-point linkage analysis demonstrated close linkage of GALC and D14S48, with Z = 13.69 at theta = 0. Multipoint analysis yielded strong support for this finding, with maximum likelihood for GALC located within 1 cM of D14S48. This analysis also identified markers that clearly flank the GALC locus, as the map order of D14S53-GALC-D14S45 is favored by odds greater than 10(6):1. Additional support for close linkage of GALC and D14S48 comes from the apparent linkage disequilibrium between these two loci in a consanguineous Druze community in Israel. These data localize GALC to 14q24.3-q32.1.     

Mapping of a congenital microcoria locus to 13q31-q32.

Congenital microcoria is an autosomal dominant disorder characterized by a pupil with a diameter <2 mm. It is thought to be due to a maldevelopment of the dilator pupillae muscle of the iris, and it is associated with juvenile-onset glaucoma. A total genome search for the location of the congenital microcoria gene was launched in a single large family. We found linkage between the disease and markers located on 13q31-q32 (Zmax = 9.79; theta = 0). Haplotype analysis narrowed the linked region to an interval <8 cM between markers D13S1239 proximally and D13S1280 distally.     

A novel form of "central pouchlike" cataract, with sutural opacities, maps to chromosome 15q21-22

Congenital cataract is a clinically and genetically highly heterogeneous eye disorder, with autosomal dominant inheritance being most common. We investigated a large seven-generation family with 74 individuals affected by autosomal dominant congenital cataract (ADCC). The phenotype in this family can be described as "central pouchlike" cataract with sutural opacities, and it differs from the other mapped cataracts. We performed linkage analysis with microsatellite markers in this family and excluded the known candidate genes. A genomewide search revealed linkage to markers on chromosome 15, with a maximum two-point LOD score of 5.98 at straight theta=0 with marker D15S117. Multipoint analysis also gave a maximum LOD score of 5.98 at D15S117. Multipoint and haplotype analysis narrowed the cataract locus to a 10-cM region between markers D15S209 and D15S1036, closely linked to marker D15S117 in q21-q22 region of chromosome 15. This is the first report of a gene for a clinically new type of ADCC at 15q21-22 locus.     

Genome Scan for Predisposing Loci for Distal Interphalangeal Joint Osteoarthritis: Evidence for a Locus on 2q

The genetic contribution to common forms of osteoarthritis (OA) is well established but poorly understood. We performed a genome scan, using 302 markers for loci predisposing to distal interphalangeal joint (DIP) OA. To minimize genetic heterogeneity in our study sample, we identified siblings with a severe, radiologically defined phenotype from the nationwide registers of Finland. In the initial genome scan, linkage analysis in 27 sibships gave a pairwise LOD score (Z) >1.00 with nine of the screening markers. In the second stage, additional markers and family members were genotyped in these chromosomal regions. On 2q12-q13, IL1R1 resulted in Z=2.34 at recombination fraction (theta) 0, allowing a dominant mode of inheritance. Association analysis of markers D2S2264, IL1R1, D2S373, and D2S1789 jointly provided some evidence for a shared haplotype among the affected individuals (P value of.012). Also, multipoint nonparametric linkage analysis yielded a P value of.0001 near the locus IL1R1 and P=.0007 approximately 20 cM telomeric near marker D2S1399, which, in two-point analysis, gave Z=1.48 (straight theta=. 02). This chromosomal region on 2q harbors the interleukin 1 gene cluster and, thus, represents a good candidate region for inflammatory and autoimmune disorders. Three additional chromosomal regions-4q26-q27, 7p15-p21, and Xcen-also provided some evidence for linkage, and further analyses would be justified to clarify their potential involvement in the genetic predisposition to DIP OA.     

A second locus for familial high myopia maps to chromosome 12q.

Myopia, or nearsightedness, is the most common eye disorder worldwide. "Pathologic" high myopia, or myopia of <=-6.00 diopters, predisposes individuals to retinal detachment, macular degeneration, cataract, or glaucoma. A locus for autosomal dominant pathologic high myopia has been mapped to 18p11.31. We now report significant linkage of high myopia to a second locus at the 12q21-23 region in a large German/Italian family. The family had no clinical evidence of connective-tissue abnormalities or glaucoma. The average age at diagnosis of myopia was 5.9 years. The average spherical-component refractive error for the affected individuals was -9.47 diopters. Markers flanking or intragenic to the genes for the 18p locus, Stickler syndromes type I and II (12q13.1-q13.3 and 6p21.3), Marfan syndrome (15q21.1), and juvenile glaucoma (chromosome 1q21-q31) showed no linkage to the myopia in this family. The maximum LOD score with two-point linkage analysis in this pedigree was 3.85 at a recombination fraction of .0010, for markers D12S1706 and D12S327. Recombination events identified markers D12S1684 and D12S1605 as flanking markers that define a 30.1-cM interval on chromosome 12q21-23, for the second myopia gene. These results confirm genetic heterogeneity of myopia. The identification of this gene may provide insight into the pathophysiology of myopia and eye development.     

Hereditary geniospasm: linkage to chromosome 9q13-q21 and evidence for genetic heterogeneity.

Hereditary geniospasm is an unusual movement disorder causing episodes of involuntary tremor of the chin and the lower lip. Episodes typically start in early childhood and may be precipitated by stress, concentration, and emotion. Hereditary geniospasm is inherited as an autosomal dominant trait, and its cause is not known. We report the results of a genomewide genetic linkage study in a four-generation British family with hereditary geniospasm. Positive two-point LOD scores were obtained for 15 microsatellite markers on the peri-centromeric region of chromosome 9. A maximum two-point LOD score of 5.24 at theta = .00 was obtained for the marker D9S1837. Construction of haplotypes defined an interval of 2.1 cM between the flanking markers D9S1806 and D9S175, thus assigning one locus for hereditary geniospasm to the proximal long arm of chromosome 9q13-q21. Hereditary geniospasm in a second British family is not linked to this region, indicating genetic heterogeneity. These findings may have implications for other inherited focal movement disorders that as yet remain unmapped.     

Linkage of an autosomal dominant clefting syndrome (Van der Woude) to loci on chromosome Iq

Van der Woude syndrome (VWS) is an autosomal dominant disorder in which affected individuals have one or more of the following manifestations: cleft lip, cleft palate, hypodontia, or paramedian lower-lip pits. VWS is a well-characterized example of a single-gene abnormality that disturbs normal craniofacial morphogenesis. As a first step in identifying genes involved in human development, we used a candidate-gene-and-region approach to look for a linkage to VWS. Six families with 3 or more generations of affected individuals were studied. Evidence for linkage (theta = 0.02, lod score = 9.09) was found between the renin (REN) gene on 1q and VWS. Other linked loci included CR1, D1S58, and D1S53. The genes for laminin B2 (LAMB2), a basement-membrane protein, and for decay-accelerating factor (DAF) were studied as possible candidate genes on 1q. Recombinants between VWS and both LAMB2 and DAF excluded these genes from a causal role in the etiology of VWS for the families studied in this report. Multipoint linkage analysis indicated that the VWS locus was flanked by REN and D1S65 at a lod score of 10.83. This tight linkage with renin and other nearby loci provides a first step in identifying the molecular abnormality underlying this disturbance of human development.     

The inhibitory effects of camptothecin, a topoisomerase I inhibitor, on collagen synthesis in fibroblasts from patients with systemic sclerosis

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the production of autoantibodies to a wide range of self-antigens. Recent genome screens have implicated numerous chromosomal regions as potential SLE susceptibility loci. Among these, the 1q41 locus is of particular interest, because evidence for linkage has been found in several independent SLE family collections. Additionally, the 1q41 locus appears to be syntenic with a susceptibility interval identified in the NZM2410 mouse model for SLE. Here, we report the results of genotyping of 11 microsatellite markers within the 1q41 region in 210 SLE sibpair and 122 SLE trio families. These data confirm the modest evidence for linkage at 1q41 in our family collection (LOD = 1.21 at marker D1S2616). Evidence for significant linkage disequilibrium in this interval was also found. Multiple markers in the region exhibit transmission disequilibrium, with the peak single marker multiallelic linkage disequilibrium noted at D1S490 (pedigree disequilibrium test [PDT] global P value = 0.0091). Two- and three-marker haplotypes from the 1q41 region similarly showed strong transmission distortion in the collection of 332 SLE families. The finding of linkage together with significant transmission disequilibrium provides strong evidence for a susceptibility locus at 1q41 in human SLE.     

Genetic linkage and transmission disequilibrium of marker haplotypes at chromosome 1q41 in human systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the production of autoantibodies to a wide range of self-antigens. Recent genome screens have implicated numerous chromosomal regions as potential SLE susceptibility loci. Among these, the 1q41 locus is of particular interest, because evidence for linkage has been found in several independent SLE family collections. Additionally, the 1q41 locus appears to be syntenic with a susceptibility interval identified in the NZM2410 mouse model for SLE. Here, we report the results of genotyping of 11 microsatellite markers within the 1q41 region in 210 SLE sibpair and 122 SLE trio families. These data confirm the modest evidence for linkage at 1q41 in our family collection (LOD = 1.21 at marker D1S2616). Evidence for significant linkage disequilibrium in this interval was also found. Multiple markers in the region exhibit transmission disequilibrium, with the peak single marker multiallelic linkage disequilibrium noted at D1S490 (pedigree disequilibrium test [PDT] global P value = 0.0091). Two- and three-marker haplotypes from the 1q41 region similarly showed strong transmission distortion in the collection of 332 SLE families. The finding of linkage together with significant transmission disequilibrium provides strong evidence for a susceptibility locus at 1q41 in human SLE.