Molecular Biotechnology: Principles and Applications - Glick B., Pasternak J. 2002

Molecular Biotechnology of Microbiological Systems
Human Molecular Genetics
Mapping a genetic disease locus to a specific chromosomal region

Pedigree analysis does not allow for determining the chromosomal localization of a disease-causing Gene unless that gene resides on the X chromosome. However, it is possible to investigate linkage between the disease gene and polymorphic RFLP or STRP loci by identifying the latter using appropriate probes. This approach yields the best results when a disease has clear-cut symptoms, its inheritance pattern is straightforward, and its penetrance level is known.

To perform linkage analysis, Blood samples are first collected from members of several families spanning two to three generations, or from members of a single large multi-generational family affected by the genetic disorder (all participants must be informed of the study's purpose and give their informed consent). The Blood Cells are cultured to provide a continuous source of DNA for subsequent Procedures without requiring repeated blood draws. DNA from each individual is then genotyped for several polymorphic markers. Some studies utilize over 250 markers distributed across different regions of all autosomes. For each informative family, a two-point (two-locus) lod score is calculated for every polymorphic locus and the disease locus. A lod score of Z ≥ +3.00 indicates linkage, whereas Z ≤ -2.00 excludes it.

To determine the chromosomal Location OF THE gene responsible for benign familial neonatal convulsions (BFNC), a large multi-generational family affected by this disorder was studied (Fig. 20.16). This condition manifests as seizures involving involuntary twitching of the face, trunk, and limbs During the first six months of life. In approximately 90% of cases, symptoms disappear after 1 year of age. The seizures apparently have no impact on neurological and intellectual status. BFNC is a rare condition with distinct Clinical Features, inherited in an autosomal dominant manner with high penetrance.

In the multi-generational pedigree examined, two of all tested polymorphic markers, D20S19 and D20S20, were found to be linked to the BFNC locus (Table 20.4; Fig. 20.16). The alleles of the D20S19 and D20S20 loci for each genotyped pedigree member shown in Fig. 20.16 are designated by numbers stacked vertically. The upper numbers correspond to the alleles of the D20S19 locus, and the lower numbers to D20S20. A vertical line separates the alleles of loci on the same chromosome.

Using the D20S19 probe, 10 alleles of the RFLP locus were identified, and 2 alleles were identified using the D20S20 probe. In some RFLP loci, multiple recognition sites for a single restriction enzyme are clustered within a small DNA segment (approximately 20 kb) and are collectively considered a single locus. Four closely spaced sites for a single restriction enzyme can generate 20 fragments of varying lengths, which are detected by a single probe. STRP loci may also possess more than two alleles.

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Fig. 20.16. Haplotype analysis of two Chromosomes across 20 polymorphic loci in a pedigree affected by BFNC. Numbers below the symbols represent alleles of two polymorphic loci, D20S19 and D20S20 (upper and lower numbers, respectively). Haplotypes of an individual's two chromosomes are separated by a vertical line. Crossed-out symbols denote deceased individuals, and shaded symbols indicate affected individuals. The symbol with one-quarter of the square shaded represents a case where an individual's "clinical" phenotype differed from those of other affected family members. II-8 and II-9 are fraternal (dizygotic) twins. Asterisks mark cases of possible incomplete penetrance (III-18, IV-4, IV-14). Affected family members show cosegregation of the haplotype (8,2) with the disease; all of them carry this chromosome inherited from a common ancestor. (Based on Leppert et al., Nature [London] 337: 647–648, 1989.)

Among the members of the pedigree presented in Fig. 20.16, the haplotype (8,2) largely cosegregates with the disease, suggesting that in this family the BFNC locus resides precisely on chromosome 8,2. One would expect that individuals III-18, IV-4, and IV-14, who inherited chromosome (8,2) from an affected parent, would also be affected, but this was not the case. These exceptions are likely due to incomplete penetrance of the disorder. In other words, individuals III-18, IV-4, and IV-14 carry the BFNC gene, but it is unexpressed. Similar cases in linkage analyses of other diseases can be attributed to diagnostic errors or to the fact that some gene carriers have not yet developed symptoms.

Table 20.4. Two-locus lod scores for the BFNC locus and two polymorphic loci on chromosome 20¹⁾

¹⁾ Based on Leppert et al., Nature (London) 337: 647–648, 1989.

In a few instances, children of a parent with BFNC carry chromosome (8,2) (e.g., IV-7 and IV-11) yet exhibit no symptoms of the disease. In both aforementioned cases, THE ORIGIN OF this chromosome can be traced. For example, individual IV-7 inherited chromosome (14,1) from the affected father, and chromosome (8,2) bearing the normal BFNC gene from the unaffected mother. The genotypes of individuals IV-9 and V-1 are more difficult to explain. On the one hand, they could have received chromosome (8,2) with the normal BFNC gene from unaffected ancestors.

For instance, individual IV-9 could have inherited chromosome (8,2) through his mother (III-15) from his grandmother (II-6). On the other hand, the absence of disease signs in IV-9 and V-1 could be explained by incomplete penetrance if they inherited chromosome (8,2) carrying the BFNC*D gene from an affected parent. It should be emphasized that in other families with BFNC, alleles D20S19*8 and D20S20*2 may not show linkage to the disease allele. It just so happens that in the case we examined, these specific polymorphic alleles reside on the same chromosome that carries the BFNC*D allele inherited from a single common ancestor. In general, loci are linked, not alleles.

The data in Table 20.4 suggest that the distance from loci D20S19 and D20S20 to the BFNC locus does not exceed 5 cM (<5 ∙ 10⁶ bp). Generally speaking, linkage analysis cannot resolve two loci if the distance between them is less than 1–2 cM. Because loci D20S19 and D20S20 are located within the 13.2–13.3 region of the long arm (q) of chromosome 20 (20q13.2-13.3), the BFNC locus must also lie near or within this chromosomal region. To date, more than one hundred disease-associated genes have been mapped to specific chromosomal regions using lod score-based Methods and polymorphic markers.