Fine Mapping of Best's Macular Dystrophy Localizes the Gene in Close Proximity to but Distinct from the D11S480/ROM1 Loci

The Best's macular dystrophy (BMD) gene has previously been mapped to the 11q13 region. In this study, recombination data localizes the BMD gene to the 6cM genetic interval between the markers FcεRI and D11S480/ROM1 in a large Swedish 12-generation BMD family. Mutation analyses of the candidate gene ROM1 did not reveal any mutations that could explain the disease phenotype. However, one recombination event between intragenic ROM1 polymorphisms and the BMD phenotype was detected. Therefore, it is highly unlikely that mutations in the ROM1 gene cause BMD. Identification of the disease gene will elucidate the pathophysiological mechanism in BMD, which may also be of importance in other retinopathies such as age-related macular degeneration.     

Evaluation of the Best disease gene in patients with age-related macular degeneration and other maculopathies

Vitelliform macular dystrophy (VMD2, Best disease, MIM153700) is an early onset, autosomal, dominant macular degeneration characterized by the deposition of lipofuscin-like material within and below the retinal pigment epithelium (RPE); it is associated with degeneration of the RPE and overlying photoreceptors. Recently, we cloned the gene bestrophin, which is responsible for the disease, and identified a number of causative mutations in families with VMD2. Here, we report that the analysis of bestrophin in a collection of 259 age-related macular degeneration (AMD) patients provides evidence that mutations in the Best disease gene do not play a significant role in the predisposition of individuals to AMD. However, our results suggest that, in addition to Best disease, mutations within the bestrophin gene could be responsible for other forms of maculopathy with phenotypic characteristics similar to Best disease and for other diseases not included in the VMD category. Received: 11 March 1999 / Accepted: 6 April 1999     

North Carolina macular dystrophy is assigned to chromosome 6.

North Carolina macular dystrophy (NCMD) is an autosomal dominant macular dystrophy causing impaired central vision at an early age, is completely penetrant, and is present in a single large family. With the development of the hypervariable microsatellite (CA repeats) markers in the human genome, it was possible to relatively rapidly screen most of the genome for linkage to the NCMD gene. After utilizing 124 genetic markers, which excluded over 95% of the human genome, three Marshfield microsatellites located at 6q13-q21 were linked to the NCMD locus. Marshfield marker (MFD) 131 gave a lod score of Z(theta) = 4.36 at theta = 0.137; MFD 171 gave a Z(theta) = 8.42 at theta = 0.004; and MFD 97 gave a Z(theta) = 13.10 at theta = 0.017. Other retinal diseases have been reported on 6q stressing the importance of this region and possibly suggesting that these diseases may be allelic or located in part of a large macular gene family. Locating and characterizing the NCMD gene may be an important step in understanding this group of maculopathies as well as age-related macular degeneration (AMD), a common cause of blindness in the elderly.     

A recombination event excludes the ROM1 locus from the Best's vitelliform macular dystrophy region

Best's vitelliform macular degeneration has been genetically linked to chromosome 11. Subsequently, the disease locus has been refined to an interval between D11S903 and PYGM and, more recently, between D11S986 and D11S480. The gene encoding ROM1, a photoreceptor-specific membrane protein, has been independently mapped within the Best's disease region and has thus become a strong candidate for the Best's disease gene. In this study, we have mapped ROM1 relative to Best's disease and the loci D11S986, UGB (uteroglobin), and PYGM (human muscle glycogen phosphorylase) in recombinant Best's disease chromosomes. We demonstrate that UGB is localized proximal to ROM1 and that both UGB and ROM1 recombine with the disease phenotype. Thus, this analysis excluded ROM1 as the Best's disease gene.     

A YAC contig encompassing the recessive Stargardt disease gene (STGD) on chromosome 1p.

Stargardt disease (STGD) and fundus flavimaculatus are infrequent autosomal recessive conditions characterized by a juvenile macular dystrophy and variable degrees of peripheral retinal changes. Linkage analysis performed in 47 STGD/fundus flavimaculatus families demonstrated significant linkage to 13 polymorphic DNA markers on chromosome 1p. The maximum combined two-point lod score was 32.7 (maximum recombination fraction [phi max] = .006) with the polymorphic marker D1S188. Our data demonstrate that STGD and fundus flavimaculatus are the same disorder clinically and genetically and provide further evidence for genetic homogeneity of this phenotype. Analysis of recombination events on disease chromosomes placed the STGD gene within a 4-cM interval between markers D1S435 and D1S236. A physical map was constructed of a YAC contig flanking STGD, from markers D1S500 to D1S495, and includes the critical interval delineated by historical recombinants. This contig spans approximately 31 cM, with one gap (3-5 cM) that is outside the 4-cM critical region. Localization of STGD to a single YAC contig will facilitate its positional cloning.     

Mapping of facioscapulohumeral muscular dystrophy gene to chromosome 4q35-qter by multipoint linkage analysis and in situ hybridization

We have recently assigned the facioscapulohumeral muscular dystrophy (FSHD) gene to chromome 4 by linkage to the microsatellite marker Mfd 22 (locus D4S171). We now report that D4S139, a VNTR locus, is much more closely linked to FSHD. Two-point linkage analysis between FSHD and D4S139 in nine informative families showed a maximum combined lod score (Zmax) of 17.28 at a recombination fraction theta of 0.027. Multipoint linkage analysis between FSHD and the loci D4S139 and D4S171 resulted in a peak lod score of 20.21 at 2.7 cM from D4S139. Due to the small number of recombinants found with D4S139, the position of the FSHD gene relative to that of D4S139 could not be established with certainty. D4S139 was mapped to chromosome 4q35-qter by in situ hybridization, thus firmly establishing the location of the FSHD gene in the subtelomeric region of chromosome 4q. One small family yielded a negative lod score for D4S139. In the other families no significant evidence for genetic heterogeneity was obtained. Studies of additional markers and new families will improve the map of the FSHD region, reveal possible genetic heterogeneity, and allow better diagnostic reliability.     

High-resolution meiotic and physical mapping of the best vitelliform macular dystrophy (VMD2) locus to pericentromeric chromosome 11.

Best vitelliform macular dystrophy (VMD2) has previously been linked to several microsatellite markers from chromosome 11. Subsequently, additional genetic studies have refined the Best disease region to a 3.7-cM interval flanked by markers at D11S903 and PYGM. To further narrow the interval containing the Best disease gene and to obtain an estimate of the physical size of the minimal candidate region, we used a combination of high-resolution PCR hybrid mapping and analysis of recombinant Best disease chromosomes. We identified six markers from within the D11S903-PYGM interval that show no recombination with the defective gene in three multigeneration Best disease pedigrees. Our hybrid panel localizes these markers on either side of the centromere on chromosome 11. The closest markers flanking the disease gene are at D11S986 in band p12-11.22 on the short arm and at D11S480 in band q13.2-13.3 on the proximal long arm. This study demonstrates that the physical size of the Best disease region is exceedingly larger than previously estimated from the genetic data, because of the proximity of the defective gene to the centromere of chromosome 11.     

Mapping of facioscapulohumeral muscular dystrophy gene to chromosome 4q35-qter by multipoint linkage analysis and in situ hybridization

We have recently assigned the facioscapulohumeral muscular dystrophy (FSHD) gene to chromosome 4 by linkage to the microsatellite marker Mfd 22 (locus D4S171). We now report that D4S139, a VNTR locus, is much more closely linked to FSHD. Two-point linkage analysis between FSHD and D4S139 in nine informative families showed a maximum combined lod score (Z_max) of 17.28 at a recombination fraction θ of 0.027. Multipoint linkage analysis between FSHD and the loci D4S139 and D4S171 resulted in a peak lod score of 20.21 at 2.7 cM from D4S139. Due to the small number of recombinants found with D4S139, the position of the FSHD gene relative to that of D4S139 could not be established with certainty. D4S139 was mapped to chromosome 4q35-qter by in situ hybridization, thus firmly establishing the location of the FSHD gene in the subtelomeric region of chromosome 4q. One small family yielded a negative lod score for D4S139. In the other families no significant evidence for genetic heterogeneity was obtained. Studies of additional markers and new families will improve the map of the FSHD region, reveal possible genetic heterogeneity, and allow better diagnostic reliability.     

Closing in on a breast cancer gene on chromosome 17q.

Linkage of early-onset familial breast and ovarian cancer to 11 markers on chromosome 17q12-q21 defines an 8-cM region which is very likely to include the disease gene BRCA 1. The most closely linked marker is D17S579, a highly informative CA repeat polymorphism. D17S579 has no recombinants with inherited breast or ovarian cancer in 79 informative meioses in the seven families with early-onset disease (lod score 9.12 at zero recombination). There is no evidence for linkage heterogeneity in the families with early-onset disease. The proportion of older-onset breast cancer attributable to BRCA 1 is not yet determinable, because both inherited and sporadic cases occur in older-onset families.     

Refinement of the locus for autosomal dominant hereditary gingival fibromatosis (GINGF) to a 3.8-cM region on 2p21

Hereditary gingival fibromatosis (HGF, MIM 135300; approved gene symbol GINGF) is an oral disease characterized by enlargement of gingiva. Recently, a locus for autosomal dominant HGF has been mapped to an 11-cM region on chromosome 2p21. In the current investigation, we genotyped four Chinese HGF families using polymorphic microsatellite markers on 2p21. The HOMOG test provided evidence for genetic homogeneity, with evidence for linkage in four families (heterogeneity versus homogeneity test HOMOG, chi(2) = 0. 00). A cumulative maximum two-point lod score of 5.04 was produced with marker D2S390 at a recombination frequency of θ = 0 in the four linked families. Haplotype analysis localized the hereditary gingival fibromatosis locus within the region defined by D2S352 and D2S2163. This region overlaps by 3.8 cM with the previously reported HGF region. Single-strand conformation polymorphism and sequence analysis of the coding region of cytochrome P450 1B1 (CYP1B1) excluded it as a likely candidate gene.     

Genetic analysis of 24 French families with multiple endocrine neoplasia type 2A.

The gene for multiple endocrine neoplasia type 2A (MEN2A) has been mapped to the pericentromeric region of chromosome 10 by linkage analysis. Thirty-four families with multiple cases of medullary carcinoma of the thyroid (MTC), including 24 families with origins in France, have been typed with nine polymorphic markers spanning the centromere of chromosome 10. No recombination was observed between the MEN2A locus and either of the four loci D10Z1 (lod score 12.79), D10S102 (lod score 6.38), D10S94 (lod score 7.76), and D10S34 (lod score 5.94). There was no evidence for genetic linkage heterogeneity in the panel of 34 families. Haplotypes were constructed for a total of 11 polymorphisms in the MEN2A region, for mutation-bearing chromosomes in 24 French families and for 100 spouse controls. One haplotype was present in four MEN2A families but was not observed in any control (P less than .01). Two additional families share a core segment of this haplotype near the MEN2A gene. It is likely that these six families have a common affected ancestor. Because the incidence of pheochromocytoma among carriers varies from 0% to 74% within these six families, it is probable that additional factors modify the expression of the MEN2A gene.     

Linkage studies in a new X-linked myopathy, suggesting exclusion of DMD locus and tentative assignment to distal Xq.

We here report linkage studies in a family suffering from a recently described hereditary muscle disease named X-linked myopathy with excessive autophagy (XMEA). Significant lod scores excluding linkage to the Duchenne-Becker muscular dystrophy locus were found. Several other loci on the short and long arms of the X chromosome produced negative lod scores, whereas probe DX13-7 defining locus DXS15 showed no recombinants and a lod score of z = 0.903 at theta = .0. Further studies should be done to determine whether the gene for XMEA is (1) located at Xq and (2) caused by a mutation of the Emery-Dreifuss muscular dystrophy gene, which has been assigned to the same region.     

Refined genetic localization of the Best disease gene in 11q13 and physical mapping of linked markers on radiation hybrids

Best’s macular dystrophy, also known as vitelliform macular degeneration type 2 (VMD-2), is an autosomal dominant eye disorder that causes reduced visual acuity. It generally manifests itself in the teenage years. The gene mutated in VMD-2 patients may provide valuable insight into the biological mechanisms of the far more common disorder age-related macular degeneration. The VMD-2 gene has been localized to 11q13 between UGB and FcɛRI. In order to clone the gene positionally, a large Swedish VMD-2 family dating back to the 17th century was studied for recombinations. Since the last study, another 40 microsatellite markers have been analyzed in the family; the closest centromeric flanking marker, D11S4076, revealed two recombinations and the closest telomeric flanking marker, UGB, revealed one recombination. The recombinations have occurred in affected individuals, which eliminates the potential problem of reduced penetrance. The order and physical distance between 22 markers located at proximal 11q13 were analyzed on the G3 Stanford radiation-reduced cell hybrids. The data suggest that the VMD-2 region flanked by the microsatellite markers D11S4076 and UGB is approximately 980 kb. Received: 23 April 1997 / Accepted: 15 July 1997     

Refinement of the Locus for Autosomal Dominant Hereditary Gingival Fibromatosis (GINGF) to a 3.8-cM Region on 2p21

Hereditary gingival fibromatosis (HGF, MIM 135300; approved gene symbol GINGF) is an oral disease characterized by enlargement of gingiva. Recently, a locus for autosomal dominant HGF has been mapped to an 11-cM region on chromosome 2p21. In the current investigation, we genotyped four Chinese HGF families using polymorphic microsatellite markers on 2p21. The HOMOG test provided evidence for genetic homogeneity, with evidence for linkage in four families (heterogeneity versus homogeneity test HOMOG, χ² = 0.00). A cumulative maximum two-point lod score of 5.04 was produced with marker D2S390 at a recombination frequency of θ = 0 in the four linked families. Haplotype analysis localized the hereditary gingival fibromatosis locus within the region defined by D2S352 and D2S2163. This region overlaps by 3.8 cM with the previously reported HGF region. Single-strand conformation polymorphism and sequence analysis of the coding region of cytochrome P450 1B1 (CYP1B1) excluded it as a likely candidate gene.     

A gene for late-onset fundus flavimaculatus with macular dystrophy maps to chromosome 1p13.

Fundus flavimaculatus with macular dystrophy is an autosomal recessive disease responsible for a progressive loss of visual acuity in adulthood, with pigmentary changes of the macula, perimacular flecks, and atrophy of the retinal pigmentary epithelium. Since this condition shares several clinical features with Stargardt disease, which has been mapped to chromosome 1p21-p13, we tested the disease for linkage to chromosome 1p. We report here the mapping of the disease locus to chromosome 1p13-p21, in the genetic interval defined by loci D1S435 and D1S415, in four multiplex families (maximum lod score 4.79 at recombination fraction 0 for probe AFM217zb2 at locus D1S435). Thus, despite differences in the age at onset, clinical course, and severity, fundus flavimaculatus with macular dystrophy and Stargardt disease are probably allelic disorders. This result supports the view that allelic mutations produce a continuum of macular dystrophies, with onset in early childhood to late adulthood.     

The ABCR gene in recessive and dominant Stargardt diseases: a genetic pathway in macular degeneration.

Stargardt disease (STGD) is a juvenile-onset macular dystrophy and can be inherited in an autosomal recessive or in an autosomal dominant manner. Genes involved in dominant STDG have been mapped to human chromosomes 13q (STGD2) and 6q (STGD3). Here, we identify a new kindred with dominant STGD and demonstrate genetic linkage to the STGD3 locus. Because of a more severe macular degeneration phenotype of one of the patients in this family, the gene responsible for the recessive STGD1, ABCR, was analyzed for sequence variants in all family members. One allele of the ABCR gene was shown to carry a stop codon-generating mutation (R152X) in three family members, including the one patient who had inherited also the dominant gene. A grandparent of that patient with the same ABCR mutation developed age-related macular degeneration (AMD), consistent with our earlier observation that some variants in the ABCR gene may increase susceptibility to AMD in the heterozygous state. Based on these results, we propose that there is a common genetic pathway in macular degeneration that includes genes for both recessive and dominant STGD.     

Analysis of 7 polymorphic markers at chromosome 11q22-23 in 35 ataxia telangiectasia families; further evidence of linkage

Summary Ataxia telangiectasia (A-T) is an autosomal recessive disorder characterised by progressive neurological degeneration, oculocutaneous telangiectasia, immunodeficiency and a high incidence of lymphoid tumours. A prerequisite to gaining a complete understanding of the basic defect that results in these features is the localization of the gene(s) involved. We report here a linkage analysis using seven polymorphic markers, which map to 11q22–23, on a sample of 35 consecutively obtained families from the British Isles showing this disorder. In a pairwise analysis, the strongest support for linkage was a lod score of 4.01 at zero recombination from Thy-1. This result supports a previous report showing linkage of the A-T gene to 11q22–23. We have also obtained evidence in a multipoint analysis for a more centromeric A-T-linked locus in the region between YNB 3.12/CJ52.208 and 2-7-1D6. This observation is also supported by inspection of the haplotypes of selected recombinants.     

X-linked cone-rod dystrophy (locus COD1): identification of mutations in RPGR exon ORF15

X-linked cone-rod dystrophy (COD1) is a retinal disease that primarily affects the cone photoreceptors; the disease was originally mapped to a limited region of Xp11.4. We evaluated the three families from our original study with new markers and clinically reassessed all key recombinants; we determined that the critical intervals in families 2 and 3 overlapped the RP3 locus and that a status change (from affected to probably unaffected) of a key recombinant individual in family 1 also reassigned the disease locus to include RP3 as well. Mutation analysis of the entire RPGR coding region identified two different 2-nucleotide (nt) deletions in ORF15, in family 2 (delAG) and in families 1 and 3 (delGG), both of which result in a frameshift leading to altered amino acid structure and early termination. In addition, an independent individual with X-linked cone-rod dystrophy demonstrated a 1-nt insertion (insA) in ORF15. The presence of three distinct mutations associated with the same disease phenotype provides strong evidence that mutations in RPGR exon ORF15 are responsible for COD1. Genetic heterogeneity was observed in three other families, including the identification of an in-frame 12-nt deletion polymorphism in ORF15 that did not segregate with the disease in one of these families.     

Linkage analysis of Fanconi anaemia in Italy and mapping of the complementation group A gene

Fanconi anaemia (FA) is an autosomal recessive disease characterised by genetic heterogeneity, with at least five complementation groups (FA-A to FA-E). The FAC gene has been cloned and localised to 9q22.3. The most frequent defective gene, FAA, was recently mapped to chromosome 16q24.3, in a region of 10 cM between D16S498 and the telomere. Eleven FA-A and 16 unclassified Italian families were analysed by microsatellite markers. To define the localisation of the FAA locus further, microsatellites were analysed at 16q24. All the families were consistent with linkage, the highest lod score being observed with D16S1320. Evidence for common haplotypes was obtained in two genetic isolates from the Brenta basin and the Naples region. Autozygosity mapping and haplotype analysis suggest that the FAA locus is distal to D16S305. Received: 29 July 1996     

Pseudoxanthoma elasticum maps to an 820-kb region of the p13.1 region of chromosome 16

We have performed linkage analysis on 21 families with pseudoxanthoma elasticum (PXE) using 10 polymorphic markers located on chromosome 16p13.1. The gene responsible for the PXE phenotype was localized to an 8-cM region of 16p13.1 between markers D16S500 and D16S3041 with a maximum lod score of 8.1 at a recombination fraction of 0.04 for marker D16S3017. The lack of any locus heterogeneity suggests that the major predisposing allele for the PXE phenotype is located in this region. Haplotype studies of a total of 36 PXE families identified several recombinations that further confined the PXE gene to a region (< 1 cM) between markers D16S3060 and D16S79. This PXE locus was identified within a single YAC clone and several overlapping BAC recombinants. From sequence analysis of these BAC recombinants, it is clear that the distance between markers D16S3060 and D16S79 is about 820 kb and contains a total of nine genes including three pseudogenes. We predict that mutations in one of the expressed genes in the locus will be responsible for the PXE phenotype in these families.