Homozygosity mapping of Alström syndrome to chromosome 2p
Homozygosity mapping of Alström syndrome to chromosome 2p Gayle B. Collin, Jan D. Marshall, Lon R. Cardon1 and Patsy M. Nishina*
The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609-1500, USA and 1Sequana Therapeutics, 11099 North Torrey Pines Road, La Jolla, CA 92037, USA
Received July 26, 1996;Revised and Accepted November 27, 1996
Alström syndrome is a rare autosomal recessive disorder characterized by pigmentary retinal degeneration, sensorineural hearing loss, childhood obesity, non-insulin-dependent diabetes mellitus, hyperlipidemia and chronic nephropathy. Features occasionally observed include acanthosis nigricans, hypogonadism, hypothyroidism, alopecia, short stature and cardiomyopathy. We report here the results of a linkage study in a large French Acadian kindred, as a first step in identifying the molecular basis of Alström syndrome. Evidence of a founder effect made it feasible to use a homozygosity mapping strategy to identify the chromosomal location of the Alström gene. In a genome-wide screen, haplotype sharing for a region on chromosome 2 was observed in all affected individuals. Two point linkage analysis resulted in a maximum lod score of 3.84 ([theta] = 0.00) for marker D2S292. By testing additional markers, the disease gene was localized to a 14.9 cM region on chromosome 2p.
Alström syndrome is an autosomal recessive disorder in which the earliest manifestations are pigmentary retinal degeneration, sensorineural hearing loss and childhood obesity (1 ,2 ). Chronic nephritis, non-insulin-dependent diabetes mellitus (NIDDM) and hepatic dysfunction occur in the final stages of the disease, between the second and fourth decade of life. Other features observed in some, but not all, affected individuals include acanthosis nigricans, male hypogonadism, hypothyroidism, alopecia, short stature and infantile cardiomyopathy (3 -8 ). With the exception of studies showing normal chromosomal karyotypes in Alström patients (8 ,9 ), no attempt has been made to genetically define this syndrome.
A number of diseases with founder effects have been identified in the French Acadian population (10 ). This is due in part to the small number of individuals that were sent to colonize French Canada and the rapid expansion of the population thereafter (11 ). As a first step in determining the molecular basis for Alström syndrome, we began a linkage study in a large kindred of French Acadian ancestry. The identification of a common founder pair and the high degree of consanguinity in this kindred (average kinship coefficient = 0.01) allowed us to use the homozygosity mapping strategy to identify the chromosomal location of Alström syndrome (12 ). Assuming that the chromosomal region flanking the Alström gene was likely to be homozygous-by-descent, we performed a candidate gene and genome-wide search and identified a 14.9 cM interval on chromosome 2p segregating with Alström syndrome.
Individuals with Alström syndrome and the mouse mutant tubby share marked phenotypic similarities including obesity, insulin resistance and retinal and cochlear degeneration. We hypothesized that tubby was a homolog of Alström and initially tested for linkage of Alström to human chromosome 11p15, the homologous human region to mouse chromosome 7 where tub maps (13 ). Additionally, linkage to homologous chromosomal regions of other mouse obesity genes (i.e. fat, ob, Ayand db) and to growth-associated candidate genes [i.e. growth hormone (GH), GH receptor and GH-releasing factor] were tested. No linkage was observed at any of the loci examined.
Consequently, we performed a genome-wide scan using the homozygosity mapping strategy with 226 polymorphic markers distributed at 15-25 cM intervals throughout the genome (14 ,15 ). To screen the genome rapidly, five affected individuals and one obligate heterozygote (carrier) were genotyped. A bias for homozygous alleles among the affected individuals was observed for seven markers on six chromosomes (Fig. 1 ). Subsequently, DNAs from parents and unaffected siblings were tested for each of these and nearby flanking marker loci. The only haplotype that co-segregated with the disease phenotype was a region on chromosome 2. To investigate further this apparent linkage to Alström syndrome, additional flanking markers were evaluated on all available DNAs from this kindred and haplotypes of family members were constructed (Fig. 2 ). Homozygosity for common alleles at markers D2S136, D2S292, D2S2113, D2S327 and D2S145 was observed in all affected individuals. The conserved founder haplotype for markers D2S393, D2S136, D2S292, D2S2113, D2S327, D2S145 and D2S286 appears to be 6-5-5-1-4-1-5, respectively. Ancestral recombination events in subjects 353 and 373 place the disease gene distal to D2S286, while an ancestral recombination event in subject 500 places the disease gene proximal to D2S393. Additionally, a maternal meiotic event between D2S145 and D2S286 (subject 372) further defines the proximal boundary of the Alström region. Altogether, these informative recombinational events delineate the Alström region to a 14.9 cM interval flanked by markers D2S393 and D2S286 (Fig. 3 ).
Pairwise linkage analysis between the Alström locus and the seven chromosome 2p markers was performed using the MLINK program of the LINKAGE 5.1 computer package (16 ). Although a founder effect is indicated by examination of >1600 individuals in this pedigree, inclusion of all relationships would have exceeded the capacity of the LINKAGE program. Additionally, identification of all consanguineous loops, especially in the earlier generations, may be incomplete. Therefore, linkage analysis was only done on those generations where most, if not all, genealogical relationships were established (Fig. 2 ).
The use of incorrect marker allele frequencies in linkage analysis may result in erroneous lod score calculations (17 ). Ideally, marker allele frequencies should be obtained from the same genetic population as the diseased individuals. In this study, pairwise analysis was completed using marker allele frequencies from unrelated individuals in the general population (GDB) and in the French Acadian population (Table 1 ). Although some variation in the frequencies of alleles was observed among the two populations, there was no significant effect on the calculated lod scores. All data included in this report, therefore, are based on the estimated French Acadian marker allele frequencies. Two point analysis indicated linkage of Alström syndrome to the chromosome 2 loci tested, with a maximum lod score of 3.84 ([theta] = 0.00) with marker D2S292 (Table 2 ). We expect that the calculated lod scores are underestimated since two point and multipoint linkage analyses could not be performed with all genealogical relationships linking affected individuals to a common ancestor.
Frequency of transmitted (T) and untransmitted (U) chromosomes in affected individuals. Associated alleles and their sizes (bp) are presented, as well as estimates of the number of ancestral recombinants using unaffected frequencies from the observed untransmitted chromosomes ([theta]obs) and the control sample ([theta]control). Probability values for the [chi]2 statistics are derived from Fisher's exact test to accommodate the small sample sizes.
The region homozygous-by-descent on chromosome 2 was evaluated further for linkage disequilibrium. Simple contingency tables were used to compare the frequencies of associated alleles on parental transmitted (T) chromosomes and untransmitted (U) chromosomes. Fisher's exact test demonstrated a significant association between the alleles shared by Alström subjects and the transmitted parental chromosome at several markers tested (Table 3 ).
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