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Map and Link Human Genetic Disorders with SSLP Analysis


Quickly identify closely related alleles with the CastAway system

Michelle L. Mack Douglas J. Wilkin
Medical Genetics Branch, National Human Genome Research Institute National Institutes of Health, Bethesda, MD

Stratagenes CastAway system is ideal for analyzing SSLPs (simple sequence-length polymorphisms), or microsatellites. Different alleles that contain these polymorphisms can vary by just a few bases. With the CastAway denaturing polyacrylamide gels,* it is now easy to separate the PCR products containing these polymorphisms. The system consistently produces high-quality band resolution.

The field of human genetics has greatly accelerated due in part to recent advances in the development of tools to map human genetic disorders. New methods generated to discover the chromosomal location of disease genes, and identify the gene after the map location is known, have aided the rapid identification of new disease genes. Recognition of the heterogeneity of genetic diseases, the allelic nature of certain disorders, and mutational heterogeneity in the cause of genetic conditions may ultimately lead to understanding the relationship between phenotype and gene.

Simple Sequence-Length Polymorphism Analysis

To identify disease loci, small fragments of genomic DNA must be analyzed. These markers, SSLPs or microsatellites, are derived from unique stretches of DNA that contain very short, simple-sequence repeats.1 Each microsatellite marker is made up of a variable number of di-, tri-, or tetranucleotide repeats at a particular location.1 Often these markers are (CA)n repeat polymorphisms.1 These polymorphisms can easily be genotyped by PCR with primers that anneal to single-copy DNA, which flanks the repetitive element.

Genotyping, using microsatellite markers, at specific locations throughout the genome allows inheritance patterns of specific pieces of chromosomes within a family to be determined. Haplotypes are generated by analyzing individual members of a pedigree with a number of closely linked markers. These haplotypes allow the transmission of a particular locus through a family to be analyzed; furthermore, they determine whether that locus is linked to a particular phenotype, as well as aid in identifying recombinational events, which define a critical region for a disease locus. Statistical programs, such as FASTLINK,2,3 are then employed to determine if a particular haplotype occurs only with the disease state; if a statistically significant correlation can be made between a particular marker at a given location and a particular disease, then the disease is linked to this marker at a known location within the genome.

Use the CastAway System for Mapping Studies

Employing polymorphic markers in a mapping study relies upon the method used to separate the particular bands generated by PCR. In a novel approach, we used CastAway precast polyacrylamide gels instead of pouring our own gels. The latter traditional method is not only tedious and labor intensive but is also open to problems such as bubble formation and leaking. Because CastAway gels are precast, they eliminate the time needed to prepare traditional polyacrylamide gels. These prepoured gels are available with or without preformed wells. In addition, the thinner gel format (0.25 mm), as well as the various options of polyacrylamide compositions, allowed us to effectively resolve PCR products, which often ranged in size from 120 bp to 400 bp and sometimes differed by only a few base pairs. The CastAway system permitted us to generate reproducible data; hence, variability between the gels, which may affect band resolution, was minimized. As a result, our studies, which easily c ould have taken many months, were completed in a just a few days or weeks.

Linkage Studies Analyzed

We used Stratagenes CastAway system to analyze linkage studies of many families with inherited disorders. Stickler syndrome is one of the milder phenotypes that results from mutations in the gene encoding type II collagen, COL2A1.4,5 Genetic heterogeneity has been demonstrated for Stickler syndrome, with mutations in two genes that encode type XI collagen, COL11A1 and COL11A2, and in at least one additional unidentified gene, resulting in Stickler syndrome.6,7 To determine if the phenotype in a family with Stickler syndrome is linked to the COL11A1 or COL11A2 locus (COL2A1 was previously excluded as the Stickler syndrome gene in this family7), polymorphic markers were analyzed by PCR amplification of the genomic DNA. A (CA)n repeat microsatellite marker, D1S206, was analyzed as one of a number of polymorphic markers in chromosome 1p21. The markers were analyzed to generate haplotypes across the region of chromosome 1 containing COL11A1. Analysis of polymorphic markers within the COL11A1 gene and a-amylase (AMY2B) gene completed the haplotypes. Analysis of these haplotypes excluded COL11A1 as a candidate for Stickler syndrome in this pedigree. Haplotype analysis, using markers in chromosome 6p21.3, also excluded COL11A2 as a candidate for Stickler syndrome in this pedigree.

In a genotyping experiment using the marker D1S206, PCR products were analyzed on a denaturing polyacrylamide gel to resolve allelic differences that were greater than 2 bp (Figure 1, Panel A). Five alleles, labeled A through E (Figure 1, Panel A) were identified for this marker, with each allele differing by at least 2 bp, representing multiples of (CA) dinucleotides. Each allele is represented by multiple bands (stutter bands), which may be produced from Taq DNA polymerase slippage during the amplification process. These bands resemble a 2-bp ladder, with the most common band often being 2 bp shorter than a major band. Artifact bands can be distinguished from real bands by identifying a heterozygous individual, with two sets of multiple bands. We observed heterozygous patterns (Figure 1, Panel A, Lanes 1, 3-7, 9, and 13-17), 2-bp differences between the upper alleles (Figure 1, Panel A, Lane 6 compared to Lane 7 and Lane 13 compared to Lane 14), and homozygous individuals with only one set of bands (Figure 1, Panel B, Lanes 3, 6, 8, 11, 12, 15, and 16). For each marker, the dominant band is designated the true allele. Because the pattern of stutter bands is usually consistent for each marker, the genotype of each individual is deduced by scoring the dominant bands only.

We also used the CastAway system to analyze linkage studies of families with cartilage hair hypoplasia (CHH), an autosomal recessive skeletal dysplasia characterized by dwarfism, decreased immunity, and fine, sparse hair.8 CHH was previously linked to chromosome 9p13.9 D9S165 was analyzed as one of a number of markers in chromosome 9p13 to further define the segment of chromosome 9p13 to which the phenotype is linked. In a genotyping experiment using the marker D9S165, three alleles, labeled A through C (Figure 1, Panel B), were identified. Additional examples of heterozygous patterns were observed in Figure 1, Panel B, Lanes 1, 4, 5, 7, 10, 17, and 20.

By analyzing this marker and others using the CastAway system, haplotypes were generated, w hich further defined a critical region of chromosome 9 linked to CHH in this pedigree (not shown). Experiments to identify the CHH gene continue.

Conclusions

Use the CastAway system as a convenient, time-saving alternative to traditional poured polyacrylamide gels for mapping studies. Because linkage mapping relies upon a reproducible resolution of bands, which are only minutely different in size, the CastAway system greatly increases the likelihood of correctly identifying alleles and provides more time to test additional markers.

Methods

Polymorphic markers were analyzed by PCR amplification of genomic DNA in the presence of a-[35S]dATP, followed by analysis on CastAway 4.5% or 6% denaturing polyacrylamide gels. PCR amplifications were performed in 25-l reactions containing 50-ng of genomic DNA; 2 units of Taq DNA polymerase; 2.5 pMol of each primer; 200 M of dCTP, dTTP, and dGTP; 2.5 M dATP; 2 mM MgCl2; 10 mM Tris-HCl pH 8.3; 50 mM KCl; and 2 Ci of a-[35S]dATP. Amplification conditions consisted of an initial incubation of 2 minutes at 94C followed by 35 cycles of 94C for 1 minute, 60C for 1 minute, and 72C for 1 minute followed by a 9-minute incubation at 72C. Oligonucleotide primers were purchased from Research Genetics.

REFERENCES
  1. Weber, J.L. and May, P.E. (1989) Am. J. Hum. Genet. 44: 388-396.

  2. Cottingham Jr., R.W., Idury, R.M., and Schaffer, A.A. (1993) Am. J. Hum. Genet. 53: 252-263.

  3. Schaffer, A.A., et al. (1994) Hum. Hered. 44: 225-237.

  4. Stickler, G.B., et al. (1965) Mayo Clin. Proc. 40: 433-455.

  5. Ahmad, N.N., et al. (1991) Proc. Natl. Acad. Sci. USA 88: 6624-6627.

  6. Richards , A.J., et al. (1996) Hum. Mol. Genet. 5: 1339-1343.

  7. Wilkin, D.J., et al. (1998) Am. J. Med. Genet. 80: 121-127.

  8. McKusick, V.A., et al. (1965) Bull. Johns Hopkins Hospital 116: 285-326.

  9. Sulisalo, T., et al. (1994) Genomics 20: 347-353.

* U.S. Patent No. 5,837,288


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