Carrier Noncarrier

▲ FIGURE 9-44 Three common inheritance patterns for human genetic diseases. Wild-type autosomal (A) and sex chromosomes (X and Y) are indicated by superscript plus signs. (a) In an autosomal dominant disorder such as Huntington's disease, only one mutant allele is needed to confer the disease. If either parent is heterozygous for the mutant HD allele, his or her children have a 50 percent chance of inheriting the mutant allele and getting the disease. (b) In an autosomal recessive disorder such as cystic fibrosis, two mutant alleles must be present to confer the disease. Both parents must be heterozygous carriers of the mutant CFTR gene for their children to be at risk of being affected or being carriers. (c) An X-linked recessive disease such as Duchenne muscular dystrophy is caused by a recessive mutation on the X chromosome and exhibits the typical sex-linked segregation pattern. Males born to mothers heterozygous for a mutant DMD allele have a 50 percent chance of inheriting the mutant allele and being affected. Females born to heterozygous mothers have a 50 percent chance of being carriers.

ure 9-44b). Related individuals (e.g., first or second cousins) have a relatively high probability of being carriers for the same recessive alleles. Thus children born to related parents are much more likely than those born to unrelated parents to be homozygous for, and therefore affected by, an autosomal recessive disorder.

The third common pattern of inheritance is that of an X-linked recessive allele. A recessive allele on the X-chromo-some will most often be expressed in males, who receive only one X chromosome from their mother, but not in females who receive an X chromosome from both their mother and father. This leads to a distinctive sex-linked segregation pattern where the disease is exhibited much more frequently in males than in females. For example, Duchenne muscular dystrophy (DMD), a muscle degenerative disease that specifically affects males, is caused by a recessive allele on the X chromosome. DMD exhibits the typical sex-linked segregation pattern in which mothers who are heterozygous and therefore phenotypically normal can act as carriers, transmitting the DMD allele, and therefore the disease, to 50 percent of their male progeny (Figure 9-44c).

Recombinational Analysis Can Position Genes on a Chromosome

The independent segregation of chromosomes during meio-sis provides the basis for determining whether genes are on the same or different chromosomes. Genetic traits that segregate together during meiosis more frequently than expected from random segregation are controlled by genes located on the same chromosome. (The tendency of genes on the same chromosome to be inherited together is referred to as genetic linkage.) However, the occurrence of recombination during meiosis can separate linked genes; this phenomenon provides a means for locating (mapping) a particular gene relative to other genes on the same chromosome.

Recombination takes place before the first meiotic cell division in germ cells when the replicated chromosomes of each homologous pair align with each other, an act called synapsis (see Figure 9-3). At this time, homologous DNA sequences on maternally and paternally derived chromatids can exchange with each other, a process known as crossing over. The sites of recombination occur more or less at random along the length of chromosomes; thus the closer together two genes are, the less likely that recombination will occur between them during meiosis (Figure 9-45). In other words, the less frequently recombination occurs between two genes on the same chromosome, the more tightly they are linked and the closer together they are. The frequency of recombination between two genes can be determined from the proportion of recombinant progeny, whose phenotypes differ from the parental phenotypes, produced in crosses of parents carrying different alleles of the genes.

The presence of many different already mapped genetic traits, or markers, distributed along the length of a chromosome facilitates the mapping of a new mutation by assessing its possible linkage to these marker genes in appropriate crosses. The more markers that are available, the more precisely a mutation can be mapped. As more and more mutations are mapped, the linear order of genes along the length of a chromosome can be constructed. This ordering of genes along a chromosome is called a genetic map, or linkage map. By convention, one genetic map unit is defined as the distance between two positions along a chromosome that results in one recombinant individual in 100 progeny. The distance corresponding to this 1 percent recombination frequency is called a centimorgan (cM). Comparison of the actual physical distances between known genes, determined by molecular analysis, with their recombination frequency indicates that in humans 1 centimorgan on average represents a distance of about 7.5 X 105 base pairs.

(a) Homologous chromosomes undergoing crossing over

To separate a and b, crossover must occur in this narrow stretch



To separate a and e, crossover can occur anywhere in this stretch

▲ FIGURE 9-45 Recombination during meiosis. (a) Crossing over can occur between chromatids of homologous chromosomes before the first meiotic division (see Figure 9-3). (b) The longer the distance between two genes on a chromatid, the more likely they are to be separated by recombination.

DNA Polymorphisms Are Used in Linkage-Mapping Human Mutations

Many different genetic markers are needed to construct a high-resolution genetic map. In the experimental organisms commonly used in genetic studies, numerous markers with easily detectable phenotypes are readily available for genetic mapping of mutations. This is not the case for mapping genes whose mutant alleles are associated with inherited diseases in humans. However, recombinant DNA technology has made available a wealth of useful DNA-based molecular markers. Because most of the human genome does not code for protein, a large amount of sequence variation exists between individuals. Indeed, it has been estimated that nucleotide differences between unrelated individuals can be detected on an average of every 103 nucleotides. If these variations in DNA sequence, referred to as DNA polymorphisms, can be followed from one generation to the next, they can serve as genetic markers for linkage studies. Currently, a panel of as many as 104 different known polymorphisms whose locations have been mapped in the human genome is used for genetic linkage studies in humans.

Restriction fragment length polymorphisms (RFLPs) were the first type of molecular markers used in linkage studies. RFLPs arise because mutations can create or destroy the sites recognized by specific restriction enzymes, leading to variations between individuals in the length of restriction fragments produced from identical regions of the genome. Differences in the sizes of restriction fragments between individuals can be detected by Southern blotting with a probe specific for a region of DNA known to contain an RFLP (Figure 9-46a). The segregation and meiotic recombination of such DNA polymorphisms can be followed like typical genetic markers. Figure 9-46b illustrates how RFLP analysis of a family can detect the segregation of an RFLP that can be used to test for statistically significant linkage to the allele for an inherited disease or some other human trait of interest.

The amassing of vast amounts of genomic sequence information from different humans in recent years has led to identification of other useful DNA polymorphisms. Single nucleotide polymorphisms (SNPs) constitute the most abundant type and are therefore useful for constructing highresolution genetic maps. Another useful type of DNA polymorphism consists of a variable number of repetitions of a one- two-, or three-base sequence. Such polymorphisms, known as simple sequence repeats (SSRs), or microsatellites, presumably are formed by recombination or a slippage mechanism of either the template or newly synthesized strands during DNA replication. A useful property of SSRs is that different individuals will often have different numbers of repeats. The existence of multiple versions of an SSR makes it more likely to produce an informative segregation pattern in a given pedigree and therefore be of more general use in mapping the positions of disease genes. If an SNP or SSR alters a restriction site, it can be detected by RFLP analysis. More commonly, however, these polymorphisms do not alter restriction fragments and must be detected by PCR amplification and DNA sequencing.

Linkage Studies Can Map Disease Genes with a Resolution of About 1 Centimorgan

Without going into all the technical considerations, let's see how the allele conferring a particular dominant trait (e.g., familial hypercholesterolemia) might be mapped. The first step is to obtain DNA samples from all the members of a family containing individuals that exhibit the disease. The DNA from each affected and unaffected individual then is analyzed to determine the identity of a large number of known DNA polymorphisms (either SSR or SNP markers can be used). The segregation pattern of each DNA polymorphism within the family is then compared with the segregation of the

Chromosomal arrangement

lle Al

Mutation at site a2 prevents cleavage lle Al

Restriction endonuclease A ^ Restriction endonuclease B I I Probed single-copy region

Hybridization banding pattern from individual with both allele 1 and allele 2

Enzyme Enzyme A B







Fragment lengths

10 kb

▲ EXPERIMENTAL FIGURE 9-46 Restriction fragment length polymorphisms (RFLPs) can be followed like genetic markers. (a) In the example shown, DNA from an individual is treated with two different restriction enzymes (A and B), which cut DNA at different sequences (a and b). The resulting fragments are subjected to Southern blot analysis (see Figure 9-26) with a radioactive probe that binds to the indicated DNA region (green) to detect the fragments. Since no differences between the two homologous chromosomes occur in the sequences recognized by the B enzyme, only one fragment is recognized by the probe, as indicated by a single hybridization band. However, treatment with enzyme A produces fragments of two different lengths (two bands are seen), indicating that a mutation has caused the loss of one of the a sites in one of the two chromosomes. (b) Pedigree based on RFLP analysis of the DNA from a region known to be present on chromosome 5. The DNA samples were cut with the restriction enzyme TaqI and analyzed by Southern blotting. In this family, this region of the genome exists in three allelic forms characterized by TaqI sites spaced 10, 7.7, or 6.5 kb apart. Each individual has two alleles; some contain allele 2 (7.7 kb) on both chromosomes, and others are heterozygous at this site. Circles indicate females; squares indicate males. The gel lanes are aligned below the corresponding subjects. [After H. Donis-Keller et al., 1987, Cell 51:319.]

disease under study to find those polymorphisms that tend to segregate along with the disease. Finally, computer analysis of the segregation data is used to calculate the likelihood of linkage between each DNA polymorphism and the disease-causing allele.

In practice, segregation data are collected from different families exhibiting the same disease and pooled. The more families exhibiting a particular disease that can be examined, the greater the statistical significance of evidence for linkage that can be obtained and the greater the precision with which the distance can be measured between a linked DNA polymorphism and a disease allele. Most family studies have a maximum of about 100 individuals in which linkage between a disease gene and a panel of DNA polymorphisms can be tested. This number of individuals sets the practical upper limit on the resolution of such a mapping study to about 1 centimorgan, or a physical distance of about 7.5 X 105 base pairs.

A phenomenon called linkage disequilibrium is the basis for an alternative strategy, which in some cases can afford a higher degree of resolution in mapping studies. This approach depends on the particular circumstance in which a genetic disease commonly found in a particular population results from a single mutation that occurred many generations in the past. This ancestral chromosome will carry closely linked DNA polymorphisms that will have been conserved through many generations. Polymorphisms that are farthest away on the chromosome will tend to become separated from the disease gene by recombination, whereas those closest to the disease gene will remain associated with it. By assessing the distribution of specific markers in all the affected individuals in a population, geneticists can identify DNA markers tightly associated with the disease, thus localizing the disease-associated gene to a relatively small region. The resolving power of this method comes from the ability to determine whether a polymorphism and the disease allele were ever separated by a meiotic recombination event at any time since the disease allele first appeared on the ancestral chromosome. Under ideal circumstances linkage disequilibrium studies can improve the resolution of mapping studies to less than 0.1 centimorgan.

Further Analysis Is Needed to Locate a Disease Gene in Cloned DNA

Although linkage mapping can usually locate a human disease gene to a region containing about 7.5 X 105 base pairs, as many as 50 different genes may be located in a region of this size. The ultimate objective of a mapping study is to locate the gene within a cloned segment of DNA and then to determine the nucleotide sequence of this fragment.

One strategy for further localizing a disease gene within the genome is to identify mRNA encoded by DNA in the region of the gene under study. Comparison of gene expression in tissues from normal and affected individuals may suggest tissues in which a particular disease gene normally is expressed. For instance, a mutation that phenotypically affects muscle, but no other tissue, might be in a gene that is expressed only in muscle tissue. The expression of mRNA in both normal and affected individuals generally is determined by Northern blotting or in situ hybridization of labeled DNA or RNA to tissue sections. Northern blots permit comparison of both the level of expression and the size of mRNAs in mutant and wild-type tissues (see Figure 9-27). Although the sensitivity of in situ hybridization is lower than that of Northern blot analysis, it can be very helpful in identifying an mRNA that is expressed at low levels in a given tissue but at very high levels in a subclass of cells within that tissue. An mRNA that is altered or missing in various individuals affected with a disease compared with wild-type individuals would be an excellent candidate for encoding the protein whose disrupted function causes that disease.

In many cases, point mutations that give rise to disease-causing alleles may result in no detectable change in the level of expression or electrophoretic mobility of mRNAs. Thus if comparison of the mRNAs expressed in normal and affected individuals reveals no detectable differences in the candidate mRNAs, a search for point mutations in the DNA regions encoding the mRNAs is undertaken. Now that highly efficient methods for sequencing DNA are available, researchers frequently determine the sequence of candidate regions of DNA isolated from affected individuals to identify point mutations. The overall strategy is to search for a coding sequence that consistently shows possibly deleterious alterations in DNA from individuals that exhibit the disease. A limitation of this approach is that the region near the affected gene may carry naturally occurring polymorphisms unrelated to the gene of interest. Such polymorphisms, not functionally related to the disease, can lead to misidentification of the DNA fragment carrying the gene of interest. For this reason, the more mutant alleles available for analysis, the more likely that a gene will be correctly identified.

Many Inherited Diseases Result from Multiple Genetic Defects

Most of the inherited human diseases that are now understood at the molecular level are monogenetic traits. That is, a clearly discernible disease state is produced by the presence of a defect in a single gene. Monogenic diseases caused by mutation in one specific gene exhibit one of the characteristic inheritance patterns shown in Figure 9-44. The genes associated with most of the common monogenic diseases have already been mapped using DNA-based markers as described previously.

However, many other inherited diseases show more complicated patterns of inheritance, making the identification of the underlying genetic cause much more difficult. One type of added complexity that is frequently encountered is genetic heterogeneity. In such cases, mutations in any one of multiple different genes can cause the same disease. For example, retinitis pigmentosa, which is characterized by degeneration of the retina usually leading to blindness, can be caused by mutations in any one of more than 60 different genes. In human linkage studies, data from multiple families usually must be combined to determine whether a statistically significant linkage exists between a disease gene and known molecular markers. Genetic heterogeneity such as that exhibited by retinitis pigmentosa can confound such an approach because any statistical trend in the mapping data from one family tends to be canceled out by the data obtained from another family with an unrelated causative gene.

Human geneticists used two different approaches to identify the many genes associated with retinitis pigmentosa. The first approach relied on mapping studies in exceptionally large single families that contained a sufficient number of affected individuals to provide statistically significant evidence for linkage between known DNA polymorphisms and a single causative gene. The genes identified in such studies showed that several of the mutations that cause retinitis pigmentosa lie within genes that encode abundant proteins of the retina. Following up on this clue, geneticists concentrated their attention on those genes that are highly expressed in the retina when screening other individuals with retinitis pigmentosa. This approach of using additional information to direct screening efforts to a subset of candidate genes led to identification of additional rare causative mutations in many different genes encoding retinal proteins.

A further complication in the genetic dissection of human diseases is posed by diabetes, heart disease, obesity, predisposition to cancer, and a variety of mental disorders that have at least some heritable properties. These and many other diseases can be considered to be polygenic traits in the sense that alleles of multiple genes, acting together within an individual, contribute to both the occurrence and the severity of disease. A systematic solution to the problem of mapping complex polygenic traits in humans does not yet exist. Future progress may come from development of refined diagnostic methods that can distinguish the different forms of diseases resulting from multiple causes.

Models of human disease in experimental organisms may also contribute to unraveling the genetics of complex traits such as obesity or diabetes. For instance, large-scale controlled breeding experiments in mice can identify mouse genes associated with diseases analogous to those in humans. The human orthologs of the mouse genes identified in such studies would be likely candidates for involvement in the corresponding human disease. DNA from human populations then could be examined to determine if particular alleles of the candidate genes show a tendency to be present in individuals affected with the disease but absent from unaffected individuals. This "candidate gene" approach is currently being used intensively to search for genes that may contribute to the major polygenic diseases in humans.

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