T I G - - ] u n e 1985

A new class of genetic markers for the human genome, based on DNA sequence polymor- phisms, has stimulated a renewed interest in linkage approaches to several funda- mental problems in human genetics. The human genetic system has been unwieldy because, for practical as well as social reasons, specified geno- types cannot be constructed to test hypotheses. Progress in human genetics has therefore been constrained by our inability to determine the geno- types of individuals. Although the relevant genotype could be inferred from a family study if the gene of interest were known to be linked to a genetic marker, this approach was not generally applicable because of a scarcity of good genetic markers.

Now, however, advances in DNA technology have made it possible to detect many genetic markers. As a result, extensive linkage mapping of some human chromosomes is underway and the locations of a number of genetic disease loci have already been determined, albeit with varying degrees of precision. Furthermore, mapping promises to provide useful information about a variety of human traits. For example, familial predispositions to such common dis- orders as coronary heart disease, cancer, Alzheimer's disease and psychiatric disorders have been sug- gested. If a predisposing gene can be mapped for any of these disorders, then the significance of a genetic component will be established.

DNA sequence polymorphisms Although the DNA sequences at homologous loci

are very nearly identical from one copy to the next, sequence variations are often found. New techniques allow us to detect these polymorphic variants in DNA sequence and use them as genetic markers. We can directly determine the inheritance of each variant and, therefore, track the inheritance of specific regions of chromosomes. At present, DNA sequence variants are detected by the method of Southern ], in which recombinant DNA probes define the locus of interest and restriction enzymes define the sequence variant.

Most variants observed thus far are polymorphisms which create or destroy a recognition site for a restric- tion enzyme; the presence or absence of the enzyme recognition site affects the length of the resulting restriction fragment. The restriction enzymes MspI and TaqI have proven especially valuable in detecting polymorphisms. The CG rimers in the recognition sequences for these enzymes appear to be hotspots for mutation and, therefore, for polymorphism; apparently this is because their abundant methylation in mammalian DNA prevents the repair of spon- taneously deaminated cytosines 2.

Regions containing tandem repeats of short DNA sequences are another common source of detectable sequence variation 3,4. The number of tandem repeats in the region can vary from one chromosome to the next. If a restriction fragment encompasses such a

© 1~5, Elsevier ~ Publishers B.V. Amsterdam 0168- 95~B.5/~2.00

review DNA sequence

polymorphisms revitalize linkage approaches in

human genetics Ray White

Linkage approaches using genetic markers based on DNA sequence polymorphisms represent an emerging trend in human genetic analysis. A well-developed map of arln'lrary loci derived from normal DNA polymorphisms, now well underway, is a

promising potential tool for linkage studies on the etiology of many human diseases.

region, it will have many length variants within a population, and will therefore define many alleles. Such regions of tandem repeats are the best genetic markers so far characterized for the human genome because the quality of a human genetic marker depends largely on the number and frequency of its alleles.

Several hundred DNA-based human genetic markers have been characterized. Many were found during the characterization of cloned genes, but the majority stem from systematic searches of arbitrary cloned DNA segments. Recently, it has become pos- sible to choose arbitrary segments from chromosome~ specific libraries, or even from libraries of subregions of specific chromosomes.

The genetic problems For many genetic systems, rapid development and

analysis have depended upon good mapping data. Furthermore, genetic maps can, especially for the human, have considerable practical value. Expression of the mutant phenotype is not required, because the possible inheritance of a mutant allele at a locus that has been mapped can be examined using linked genetic markers. Although specific protocols and further technical development are still required, map location could (in principle) lead to the isolation of genes of considerable interest. The development of these technologies could be very important in isolating genes such as those for Huntington's disease or Duchenne muscular dystrophy. At present, no other approaches are apparent.

Probably most intriguing, however, are specula- tions as to whether the inheritance of a mutant allele is involved at all in a given disease or condition, i.e. is there a genetic component to the disease? That question is still unresolved for many disorders because we cannot determine the genotypes of indi- viduals. If we had the answer, it would then be reason- ably easy to ascertain whether a specific genotype affects phenotype; in statistical terms, do individuals with the same genotype share the same phenotype?

Linkage tests with candidate genes Several factors tend to confound linkage tests: lack

of complete penetrance of the mutant allele, false posi- tives (individuals who show the phenotype for reasons other than inheritance of the gene) and recombination.

r iews a@ A:I 2

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(a) (b)

2

A:1,1 B:5,5

A II B:5 5

b.

T I G - - J u n e 1985

A:2,2 B:7,8

:1: l: ( )

Fig. 1. (a) A n exaraple of ambiguity in the determination of allele distn'bution (phase) at two loa; A and B, when only parental genotypes are known. The two possibilities for phase in the mother are shown as (a) and (b ). (b) When the genoO~es of both of a set of grandparents are known, then phase in a parent may often be determined with certainty. Reprinted by permission from Nature 313, 102.

The first two can be dealt with only in a statistical sense. The disassociation of marker allele and disease allele due to recombination can be overcome by using markers so close to the disease gene that recombina- tion will be very infrequent. Close markers can be developed when we have a preliminary indication of linkage (see below).

However, this process could be shortened if we had some idea which mutant gene might cause a disorder. The cloned gene could then be used to develop poly- morphism patterns and to define the genetic marker locus. For example, polymorphisms of the cloned beta-hemoglobin gene could easily be used as genetic markers in family studies to show that most beta- thalassemias result from mutations in or near the beta- hemoglobin locus. If the polymorphisms detected were so close to the mutation site that recombination occurred only at a frequency < 10-*, the statistical cor- relation would be very strong.

Not all gene-disease pairs are equally interesting for such a test. In the case of the thalassemias it would not be especially enlightening to demonstrate that muta- tions in the beta-hemaglobin gene cause the disorder; alterations in hemoglobin protein concentrations have already strongly inmcated this. Furthermore, nucleic acid sequencing has indicated sequence alterations sufficient to reduce protein concentrations s. However, the fact that most alleles carrying the thalassemia mutations produce beta-hemoglobin with normal amino acid sequence reminds us that human mutations do not always alter the amino acid sequence of the product. Sometimes the mutational change in DNA sequence affects only the amount of the gene product by causing defects in transcriptional regula- tion or RNA processing.

The defect in thalassemia products is obvious because beta-hemoglobin is a highly abundant protein in a known and accessible tissue. For many gene and disorder pairs, however, we think it will be far less obvious whether the gene causes the disease. For example, some mutations might affect gene expres- sion in a temporal or a tissue-specific fashion. To

observe their effects we might have to look in just the right tissue at just the fight time. Such mutants have been characterized in the mouse, but in the human they would go undetected by a gross examination of the amino acid sequence or by quantification of the protein product. Moreover, in other instances indi- viduals affected by a disorder may show markedly reduced concentrations of a gene product; but is this a cause or an effect of the disease? However, because their phenotypes are revealed in the intact organism, regulatory mutants can be detected by linkage approaches in family studies if an appropriate cloned gene is available as a probe to define the locus.

In a linkage analysis with a candidate gene, the alleles of the gene will be defined by DNA sequence polymorphisms revealed when we use the cloned gene as probe. If we fail to see tight linkage and if obligatory recombinants must be invoked, we will be sure that the disorder in question is not caused by mutations in or near the candidate gene. On the other hand, if no recombinants are seen among 20 or more possibilities, then we will have a strong argument that a mutant allele of the candidate gene cause the disease. Furthermore, if a marker with several common alleles can be made from the gene, the tests can be done with quite limited family studies; a single family with perhaps half a dozen affected individuals and no more than 20 members should be sufficient. It is important to note that, because the study can be done within a single family, concerns over heterogeneity are virtu- ally eliminated.

The evidence is mounting that familial predisposi- tion is one of the most important risk factors in coronary heart disease and hypertension, and deep suspicion exists that several genes may be involved. Defects in the low-density lipoprotein (LDL) receptor gene are already known in some families 6, and mutations in apolipoprotein genes have been impli- cated in others 7. The general linkage approach described below is, however, likely to be difficult for these diseases for two main reasons: misclassifi- cation of the genotype at the hypothesized gene locus;

TIG - - June 1985

because of variable penetrance, likely to be influenced by lifestyle; and the frequency of the diseases means that cases within a family could have different causes. Nevertheless, since several relevant genes have been cloned recently, this family of dis- orders is very suitable for the candidate gene approach.

Linkage with arbitrary markers The most global approach to genetic etiology and

mapping genetic disease loci is the general linkage study. As mentioned earlier, if a trait can be mapped to a specific chromosomal location by linkage to a genetic marker, it provides persuasive evidence for a strong genetic component to the trait. It is a truism that if a gene is involved, the gene must have a location somewhere in the genome. Therefore, given a clear phenotype, adequate family samples and enough genetic markers, it should often be possible to map the hypothesized gene by linkage methods.

Although this approach holds great promise for many well characterized genetic diseases, and has already proven successful for several 8'9, we may encounter serious difficulties in applying it to the common diseases. The confounding effects of incom- plete penetrance, high backgrounds due to sporadic cases, and the less than optimal family structures characteristic of many disorders reduce the statistical correlation between inheritance at the marker locus and the disease phenotype. Better knowledge of pre- cise and definitive phenotypic characteristics of the disease, as well as new computer approaches, may be needed. For genetic diseases such as myotonic dys- trophy, retinitis pigrnentosa and cystic fibrosis, how- ever, linkage studies should work; at present, they may often be the only possible and useful approaches.

review We have argued for some time that the most - " "

effective approach to mapping a disease locus by linkage methods is to use a well developed linkage map for the human. Such a map would provide the genetic markers needed for mapping the diseases and provide valuable information about the relationships of the marker loci in terms of order and distance.

The human linkage map Because genetic mapping has a long and honorable

history, one would imagine that most of the problems had already been solved. Indeed, the problems have been well defined, but some special characteristics of the human system have raised new and interesting dif- ficulties. These stem from the same uniquely human cause, our inability to arrange matings to suit the needs of the investigator.

The first problem in a human genetic system is that the phase of the alleles is often not known. Even though our markers are codominant, they define only which alleles are present at each locus (see Fig. la). The distribution of the alleles over the two chromo- somes for the several loci with respect to one another (the marker phase) is not directly revealed. With mice, or Drosophila, the distribution of alleles on the two parental chromosomes is known because the test animal usually comes from two homozygous parents.

The problem of unknown phase is addressed in two ways. Sampling the grandparents together with good (i.e. multiallelic) genetic markers (Fig. lb), can explicitly define the phase of the alleles in the parents. For this reason, the structure of the families (number of children and living grandparents) in the study is of the utmost importance. If the phase is still not explicitly defined, both possibilities must be taken into account. Even with fairly large pedigrees, sophis-

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I I I t

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Fig. 2. Bookkeeping with partial infor, nativow~ in a multzf ~ l analysis. Data from partially informalive loci can be integrated by considen'ng several loci simultaneously in the analysis. (r,s,t = rearmbination fmaions in the indicated intervals; k = coefficient of coincidence.) 1 7 ~

reviews Table 1 Relative order o f loa" 6 and

(0.012) 3 . . . . . . . . . . (0.253) . . . . . 5--7-6- . . . . . . (0.323) . . . . . . . . . 8

(0.066) Relative likelihood

7-6-8 2/1 6-7-8

5-7-6 25/1 5-6-7

5-7-6-8 50/1 5-6-7-8

3-5-7-6-8 160/1 3-5-6-7-8

*Reprinted by permission from The Harvey Lectures (1985, in press), The Harvey Society, New York.

ticated, computerized bookkeeping can keep track of all the possibilities for phase and weight them appro- priately.

The second problem is that some meioses in a data set will be only partially informative (Fig. 2). Again, this is because the investigator cannot define the parental genotypes; therefore, not all the marker loci will be heterozygous.

To extract as much information as possible from the data set, we must combine data from overlapping intervals. This can only be done if we consider all the progeny chromosomes jointly and make some pre- sumptions about the independence (or lack thereof) of recombination events with respect to one another in adjacent intervals (interference). Although this topic interests eukaryotic geneticists, Lalouel 1° argues persuasively that in almost any feasible human genetic data set, the numbers will be too small to detect interference even if it exists, and we should not complicate our analyses by worrying about it. We can, therefore, consider that for a set of three marker loci A, B and C, with intervals r, s and t, the probability of chromosomes informative for only the outside markers can be written as t (N) = r + s - 2rs .

Chromosomes informative for all three loci can be written as r x s if they are doubly recombinant, or r x (1 - s) and (1 - r) x s if they are singly recombin- ant.

The bookkeeping is adjudicated by the method of maximum likelihoodU: it follows the 'test of reason- ableness' thai the best values of r and s will be those which give the highest probability to the data set. Obviously some very fancy computerized bookkeep- ing is required to keep both phase and multilocus analyses straight. Fortunately, programs exist which can organize not just two- and three-locus analyses of data sets but, because they are written recursively, can consider simultaneously as many loci as the computer's memory will allow ~2. We have achieved joint analysis of as many as six loci with a DEC VAX 750, and analysis of three with an IBM PC. The ability to examine many loci jointly can make a considerable difference to the precision of estimate of recombina- tion, and to the estimates of support for gene order. Increasing the number of alleles included in an analysis can raise the support for a specific gene order from 2/1 to 160/1, as shown in Table 1 (Ref. 13).

T I G - - J u n e 1 9 8 5

The two major and interrelated parameters of a linkage map are gene order and distance, with order being the most critical. As pointed out by Bridges and Morgan in 1923 (Ref. 14):

'The serial order of the loci is the most important information represented by the map. . . The dis- tances between the loci are also represented, but this type of information is by no means so unques- tionable as is the serial order. The serial order can be established absolutely...

Genetic orders can be determined by examining linkage between loci taken two at a time, provided the variances on the estimates of the recombination fraction are small enough. For a set of three loci, A, B and C, the two showing the highest recombination fraction become the outside markers. However, for studies with humans sample sizes are often inade- quate for statistically significant discrimination among the recombination fractions.

Much more powerful linkage evidence for gene order comes from multiply informative chromosomes because the drastically reduced likelihood of close multiple exchanges can provide good evidence for the elimination of most of the possible gene orders. The number of multiply informative chromosomes increases primarily as a function of the quality of the genetic markers and the size of the sample set. Note that the proportion of multiply informative chromo- somes goes down exponentially with the quality of the markers.

However, such tests can give good support for a specific gene order. For example, a comparison for a specific set of data indicated that if the data had been gathered by independent two factor tests, the support for gene order would be only 3/1 in favor of the most favored gene order. When examined as multi-factor crosses the support became 300 000/1 (Ref. 15).

Implications Powerful new technologies are being brought to

bear on the problems of human genetics and we can anticipate a rapid growth in our knowledge of the implications of individual genotypes. We can hope that this knowledge will unravel genetic components of disease and resolve heterogeneity, to make possible more specific therapies. Certainly individuals will have access to a great deal more information as to their predispositions than they do now.

In the current legal climate, however, such knowledge has significant risks: insurance companies and prospective employers are often permitted to require full disclosure of risk factors known to the individual. In genetic disorders where there may be little gain to the individual to know his risks, it might be appropriate to withhold such information, for fear of compromising his future prospects. The challenge is much greater, however, for situations such as pre- disposition to coronary heart disease. We might well imagine, indeed we would hope, that alterations in lifestyle as a result of the knowledge of the predisposi- tion could be very useful to the individual; it would be very important that he be informed. At present, however, such information could also be very damaging for the reasons discussed above. The time may be rapidly approaching for the initiation of legis-

TIG - - June 1985

lation that will protect an individual's right to privacy with respect to his genetic heritage.

R e f e r e n c e s 1 Southern, E. (1978) Detection of specific sequences

among DNA fragments separated by gel electra phoresis. J. Mol. Biol. 98, 503-517

2 Barker, D., Schafer, M. and White, R. (1984) Restric- tion sites containing CpG show a higher frequency of polymorphism in human DNA. Cell 36, 131-138

3 Bell, G., Solby, J. and Rutter, W. (1982) The highly polymorphic region near the insulin gene is composed of single tandemly repeated sequences. Nature 295, 31-35

4 Jeffreys, A., Wilson, V. and Thein, S. (1985) Hyper- variable 'minisatellite' regions in human DNA. Nature 314, 67-73

5 Antonarakis, S., Kazazian, H. and Orkin, S. (1985) DNA polymorphism and molecular pathology of the human globin gene clusters. Hum. Genet. 69, 1-14

6 Goldstein, J. and Brown, M. (1979) The LDL receptor locus and the genetics of familial hypercholes- terolemia. Annu. Rev. Genet. 13,259-289

7 Utermann, G. (1983) Coronary heart disease. In Principles and Practice of Medical Genetics (Emery, A. and Rimoin, D., eds), pp. 956-978, Churchill Living- stone, New York

8 Davies, K., Pearson, P., Harper, P., Murray, J., O'Brien, T., Sarfazazi, M. and Williamson, R. (1983) Linkage analysis of two cloned DNA sequences flanking the Duchenne muscular dystrophy locus on the short arm of the human X chromosome. Nucl.

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13

14

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review Ac~s Res. 11, 2303-2312 GuseUa, J., Wexler, N., Conneally, P., Naylor, S., Anderson, M., Tanzi, R., Watkins, P., Ottina, K., Wallace, M., Sakaguchi, A., Young, A., Shoulson, I., BoniUa, E. and Martin, J. (1983) A polymorphic DNA marker genetically linked to Huntington's disease. Nature 306, 234-238 Lathrop, G., Lalouel, J., Julier, C. and Ott, J. Multilocus linkage analysis in humans: detection of linkage and estimation of recombination. Am. J. Hum. Genet. (in press) Edwards, A. (1972) Likelihood: An Account of the Statistical Concept of Likelihood and its Application to Scientific Inference, Cambridge University Press Lathrop, G., Lalouel, J., Juliet, C. and Ott, J. (1984) Strategies for multilocus linkage analysis in humans. Proc. Natl Acad. Sci. USA 81, 3443-3446 White, R. Mapping human chromosomes. In The Haney Lectures, The Harvey Society, New York (in press) Bridges, C. and Morgan,T. (1923) The third chromo- some group of mutant characters of Drosophi/a me/ano- gaster, Carnegie Institute, Washington DC White, R., Leppert, D., Bishop, T., Barker, D., Berko- wit.z, J., Brown, C., Callahan, P., Holm, T. and Jerominski, L. (1985) Construction of linkage maps with DNA markers for human chromosomes. Nature 313, 101-105

R. White is at the Howard Hughes Medical Institute and Department of Human Genetics, University of Utah, Salt Lake City, UT, USA.

Segregation distortion in Drosophila is the observation that a fraction of chromosomes 2 (referred to as SD chro- mosomes) extracted from vir- tually any natural population of Drosophila melanogaster in the world, when made hetero- zygous in males with a standard laboratory-stock second chromosome (most typically one marked by the recessive eye color mutations cn and bw), will be transmitted to the offspring of those males with a frequency vastly exceeding the expected 50% 1 . A typical frequency distribution of segregation values (k values) for an SD chromosome collected in Madison, Wisconsin is shown in Fig. 1. The reciprocal .':ross which, owing to inversions that eliminate recombination, is comparable, yielded a mean k value of 0.52. This provides both a control for the relative viabilities of the two progeny classes and illustrates that segregation distortion operates only in males.

The elementary genetics and physiology of distor- tion are now fairly well understood. (The experi- mental basis of this understanding, along with original references, is reviewed in Refs 2-7.) Briefly, segrega- tion distortion occurs as follows. Sometime in sperma-

Segregation distortion in Drosophila

L. Sandier and Kent Golic

Segregation distortion in Drosophila occurs in SD/SD + ma/es because SD induces a developmental anomaly in SD'-bearing spennatocytes that results in the normal maturation of SD-bearing sperm only. This was interpreted to mean that distortion resulted from a lesion in the normal genetic control of spermiogene, ds. Recent umte suggests, instead, that the genetic elements that cause d i s ~ a n may be adventitious, and are detected as segregation distorters because, owing to the resultant meiotic drive, they

are frequently recovered frora natural lx~1mlations.

tngenesis, perhaps during prophase I of meiosis (but see Ref. 8), elements on the SD and cn bw second chromosomes interact in such a way that the cn bw chromosome 2 becomes a dominant gametic lethal. This gametic lethality manifests itself, after comple- tion of a cytologically normal meiosis, in failure of the

bw-bearing spermatid nuclei to effect proper chromatin condensation and individualization, along with (or possibly as a consequence of) failure to achieve the requisite transition from lysine-rich chromosomally associated histones to arginine-rich histone proteins 9J°. Whatever the ultimate cause, the result of these failures of proper sperm maturation in

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