Somatic Cell and Molecular Genetics, Vol. 13, No. 4, 1987, pp. 361-363

Polymorphic DNA Markers on the Human Genomic Map: Signposts for Localization of Unknown Genes

Ray White

Howard Hughes Medical Institute, University o f Utah Medical Center, Salt Lake City, Utah 84132

A major direction of research among geneticists is toward development of a detailed map of the human genome. The rationale for this effort is to cover each chromosome with markers at close enough intervals that any gene can be located by its coinheritance with a mapped marker within a family. Genetic link- age mapping is sometimes the only way to locate the gene causing an inherited disease when the biochemical defect is unknown; sim- ilarly, linkage mapping can establish or elimi- nate genetic etiology for a disease or a trait, and confirm or deny the role of a "candidate" gene whose product is suspected of playing a role in a given condition.

A panel of several dozen normal families has been established, to provide genomic DNA for genotypic analysis as new markers for the map become available. These refer- ence families are ideally constituted for link- age studies: each has four living grandparents and a sibship of eight or more. Cell lines from each individual in the family panel are main- tained by the Centre d'Etude du Polymor- phisme Humain (CEPH) in Paris, to provide DNA to researchers involved in the worldwide mapping effort.

The markers that serve to identify chro- mosome regions must be polymorphic in order to be useful for linkage studies in families, because only when the parents are heterozy- gous at the marker locus can linkage to a gene be followed in the children. Fortunately, nor- mal variations in DNA sequence seem to be common. Most of the polymorphie markers that have been mapped so far, over 300 of

them, are arbitrary segments of DNA that have no known coding function. Polymor- phism among these segments is detected by treating them with bacterial endonucleases (restriction enzymes); when a recognition site for a given enzyme has been altered, cleavage of that portion of DNA will not occur and the fragments that result will vary in length from those in which the cleavage site remains. These differences in fragment lengths are easily detected by autoradiography after sepa- ration in electrophoretic gels, by a technique known as Southern blotting. DNA markers detected in this way, however, have only two alleles (the cleavage site is either present or absent), so heterozygosity will never exceed 50% in the population; in consequence, many individuals will be uninformative for linkage at these loci. Nevertheless, primary linkage maps of several chromosomes have been developed using DNA markers; from our own laboratory we have published genetic linkage maps of chromosomes 6p (1), 1 lp (2), 12 (2), 13q (3), and X (4).

An intriguing result of our linkage map- ping studies has been the realization that the frequency of recombination events between loci usually varies according to sex and appar- ently not in a constant ratio from one chromo- some to the next. For example, on chromo- some 13q we found recombination in female meioses to be about 3.9 times more frequent than in males over the length of the chromo- some arm; on chromosome l lp, however, recombination events were twice as frequent in males as in females in the interval between

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362 White

the loci for beta-globin and the Harvey-rasl oncogene; other intervals showed different ratios. On chromosome 12, we see a higher rate of female recombination on the long arm, and a male excess on the short arm.

Is the phenomenon of sex effect on recombination in fact localized? If so, a possi- ble interpretation might be that different sites for initiation of recombination are recognized in each sex. If we assume that recombination frequency between two loci is proportional to the number of recognition sequences in the interval separating them, we could hypothe- size that separate male and female initiation sequences, differently concentrated in a given chromosome segment, account for the obser- vations described. Significant variations in the ratios of male to female recombination fre- quencies within chromosome regions would imply that the recombination frequency per DNA distance for at least one recombination system, the male or the female, or both, must also vary. In any event, as a practical matter we conclude that two genetic linkage maps must be prepared for each chromosome, one for each sex, showing a constant gene order but different distances between loci. The maps we are publishing reflect this constraint.

For the enormous task of mapping the entire genome, markers that have a higher degree of polymorphism within the population than the two-allele systems described earlier would yield much more rapid progress. Recently a series of new markers has been developed, representing loci that consist of tandemly repeated short sequences of DNA. By their nature, these loci can exist in a number of sizes (alleles), depending on how many repeats of the sequence are present. Several loci containing a variable number of tandem repeats (VNTR) have been recently identified, such as the "minisatellites" asso- ciated with the insulin gene (5), the myoglo- bin gene (6), the zeta-globin pseudogene (7), and others. VNTR loci are, predictably, highly polymorphic; so much so that they, and

sequences scattered throughout the genome that show some homology to them, combine to provide a unique genetic "fingerprint" of any individual.

Recently in our laboratory we have detected a large number of VNTR loci by screening cosmid libraries to identify clones having homology to known short nucleotide sequences. Polymorphism is revealed at these loci by variations in the lengths of restriction fragments produced in a similar pattern by a number of different enzymes, in contrast to the enzyme-specific nature of the polymor- phism that affects only one restriction site. Some of the VNTR loci we have identified show more than a dozen alleles in a population sample of unrelated individuals. The tech- nique can now be used to develop similar highly polymorphic markers for the genetic map. For example, we have characterized a hypervariable locus on chromosome 1 that contains repeats of a 40-base-pair sequence organized into a region 250-500 kilobases long, which we have designated a "midisatel- lite" because of its large size (8). The origin and the possible function of hypervariable loci are intriguing subjects, with implications for basic research into such matters as evolution and the mechanism of genetic recombination.

Among the genetic traits that are not understood biochemically and can only be recognized by their effect on phenotype when they are inherited in mutant form are medi- cally significant conditions such as certain inherited cancers and neurological disorders. Localization of such important genes cannot wait for the completion of the genomic map; already the linkage approach has identified the chromosomal regions harboring the genes for Huntington's chorea, for example, and cystic fibrosis. The known markers that are closely linked to these diseases are already proving valuable for prenatal diagnosis, but the long-term significance of the localization of disease genes lies in the opportunity it provides to close in on the gene itself and

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ultimately to isolate and clone it. Genetic linkage can provide resolution only down to about a million base pairs (1000 kilobases), which is a long distance; but new technologies that permit "walking" over large segments of D N A are becoming available, as are methods of obtaining overlapping D N A fragments one of which will eventually contain the gene. Once a gene is cloned, its product can be determined and its defect(s) identified; spe- cific therapies may then be defined and applied.

On a more general level, completion of the human genomic map, in which linkage mapping will play a major part, will lead to fundamental insights into the human organ- ism, by making specific the characteristics that genes prescribe and proscribe for us.

L I T E R A T U R E C I T E D

1. Leach, R., DeMars, R., Hasstedt, S., and White, R. (1986). Proc. Natl. Acad. Sci. U.S.A. 83:3909 3913.

2. White, R., and Lalouel, J.-M. (1987). Adv. Hum. Genet. 16"121-228.

3. Leppert, M., Cavenee, W., Callahan, P., Holm, T., O'Connell, P., Thompson, K., Lathrop, G.M., Lalouel, J.-M., and White, R. (1986). Am. J. Hum. Genet. 39:425 437.

4. Drayna, D., and White, R. (1985). Science 230:753- 758.

5. Bell, G.I., Selby, M.J., and Rutter, W.J. (1982). Nature 295:31 35.

6. Jeffreys, A.J., Wilson, V., and Thein, S.L. (1985). Nature 314:67 73.

7. Proudfoot, N.J., Gil, A., and Maniatis, T. (1982). Cell 31:553 563.

8. Nakamura, Y., Julier, C., Wolff, R., Holm, T., O'Connell, P., Leppert, M., and White, R. (1987). Nucleic Acids Res. 15:2537-2547.