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Genomics: The Science and Technology Behind the Human Genome Project.	Charles R. Cantor, Cassandra L. Smith
	Copyright © 1999 John Wiley & Sons, Inc.
	ISBNs: 0-471-59908-5 (Hardback); 0-471-22056-6 (Electronic)

13 Finding Genes and Mutations

DETECTION OF ALTERED DNA SEQUENCES
Genomic DNA maps and sequences are a means to an			end. The end is to use this informa-
tion to understand biological	phenomena. At	the heart of		most	applications of mapping
and sequencing is the search	for altered	DNA	sequences.	These	may be sequences in-

volved in an interesting phenotypic trait, an inherited disease, or a noninherited genetic disease due to a DNA change in somatic (nongermline) cells. The way in which maps and sequences can be used to identify altered DNA sequences very much depends on the con-

text of that alteration. Here we will brieﬂy survey the range of applications of maps and sequences, and then we will cover a few examples in considerable depth. However, the emphasis of much of this chapter will be the development of more efﬁcient methods to ﬁnd any sequence differences between two DNA samples.

Some DNA differences are inherited. There are three levels at which we characterize inherited DNA differences. DNA maps and sequences greatly assist the ﬁnding of genes responsible for inherited diseases or other inherited traits. Once a disease gene has been identiﬁed, we attempt to develop DNA-based tests for the clinical diagnosis of disease risk. The success of these tests will depend on the complexity of the disease and normal alleles. Even before a disease gene has been identiﬁed, DNA-based analyses of linked markers can sometimes offer considerably enhanced presymptomatic or prenatal diagno-

sis, or carrier screening. Finally DNA tests, in principle, provide a way for us to look for new germline mutations, either at the level of sperm (and ova in principle, but not very easily in practice) or anytime after the creation of an embryo. These mutations are referred to, respectively, as gametic mutations and genetic mutations. The distinction is a subtle one. Any mutations that destroy the ability of a gamete to function will not be inheritable because this gamete will produce no progeny.

Some DNA differences are important at the level of organism function, but they do not affect the germ cells, so they are not passed to the offspring. Examples in normal development occur frequently in the immune system. Both the immunoglobulin genes and the

T-cell receptor genes rearrange in lymphocytes, and they also have a high degree of point mutagenesis in certain critical regions. These processes are used to generate the enormous

repertoire of immune	diversity needed to allow the immune system	to detect	and combat
a wide variety of	foreign substances. It has been speculated	that DNA	rearrangements

might also occur in other normal somatic tissues, like the brain, but thus far, evidence for any such functionally signiﬁcant rearrangements is not convincing. DNA changes in abnormal development appear to be commonplace. Most cancer cells contain DNA rearrangements that somehow interfere with the normal control of cell division. As the resulting cells multiply and spread, they frequently accumulate many additional DNA alterations. Other somatic DNA differences occur when chromosomes segregate incorrectly during mitosis.

433

434	FINDING	GENES	AND	MUTATIONS
A ﬁnal example where DNA sequence information plays an important role in clinical
diagnosis is in infectious disease. For example,					strain variations of viruses and bacteria
can be of critical importance in predicting their pathogenicity. Examples include virulent
versus nonvirulent forms of bacteria like					Mycobacterium tuberculosis,		and various	drug-
resistant	strains of	HIV,	the	virus that causes	AIDs. Other examples are quite common
in parasitic protozoa, since these organisms, like HIV, use rapid DNA sequence variation
as a way of escaping the full surveillance of the immune system of the host. Thus DNA
sequence analysis is important in understanding the					biology of		Plasmodium falciparum,
the organism that causes malaria,					Trypanosoma brucii	and	Trypanosoma cruzii,	which
cause sleeping sickness, and many other organisms that pose signiﬁcant public health
hazards.
In this chapter we will describe the sorts of DNA analyses that can be done to detect
genomic changes with present technology, and we will try to extrapolate to see what im-
provements will be likely in the future.
FINDING	GENES
The approach used to ﬁnd genes based on their					location on the genetic map has been
called reverse genetics, but a more accurate term is positional cloning. The basic strategy
is to use the genetic map to approximate the position of the gene (Fig. 13.1). Then a
physical map of the region is constructed if it is not already available. The physical map
should provide a number of potential sequence candidates for the gene of interest. It also
helps to ﬁnd additional useful polymorphic markers that narrow the location of the						de-
sired gene further. Ultimately one is reduced to a search for a particular set of DNA se-
quence differences that correlates with a phenotype					known to be directed by an allele of
the gene. In contrast to positional cloning, genes can sometimes be found by functional
cloning. Here an altered biochemical function is traced to an altered protein. This is se-
quenced, and the resulting string of amino acids is scanned to ﬁnd regions that allow rela-
tively nondegenerate potential DNA coding sequences to be synthesized and used as hy-
bridization probes to screen genomic or cDNA libraries.

Figure 13.1

Contrasting stages in strategies to ﬁnd genes by positional cloning (solid line) and by

functional cloning (dashed line). Adapted from a slide displayed by Hilger Ropers.

FINDING GENES

435

In the past few years there have been many dramatic successes in human positional cloning. Among these are the genes responsible for Duchenne muscular dystrophy, cystic ﬁbrosis, some forms of familial Alzheimers disease, myotonic dystrophy, familial colon cancer, two forms of familial breast cancer, HD, one form of neuroﬁbromatosis, and several genes involved in fragile X-linked mental retardation. Some of the ways in which the genetic map has helped locate and clone these disease genes were discussed in Chapter 6. Here we review, brieﬂy, some of the aspects of this process, with the particular goal of showing where DNA sequencing plays a useful or necessary role. In most gene searches,

thus far there have been	unexpected beneﬁts in	that interesting biological or genetic
mechanisms became apparent	as correlations became	possible between genotype and

phenotype. These serendipitous ﬁndings may have occurred because so few human disease genes were known previously. However, it is still possible that many additional basic biological surprises remain to be uncovered as much larger numbers of human disease

genes are identiﬁed. In Box 13.2 we will illustrate one of the most novel disease mecha-

nisms seen in several of these	diseases which is caused	by unstable	repeating	trinu-
cleotide sequences.
A successful genetic linkage	study within a limited set	of families is	just the	ﬁrst step

in the search for a gene. It reveals that there is a speciﬁc single gene involved in a particu-

lar disease or phenotype, and it provides the approximate location of	that gene in the
genome. However, genetic studies in the human can rarely locate a gene to	better than 1
to 2 cM. In typical regions of the human genome, this corresponds to 1 to 2 Mb; the prob-

lem is that such regions will usually contain 30 to 60 genes. To narrow the search, it is

usually necessary to isolate the DNA of the region (Fig. 13.2). Until the					advent of YACs
and other large insert cloning systems,	this was a very	time-consuming			and	costly
process. It frequently consisted of parallel attempts at chromosome microdissection and
microcloning and attempts at cosmid walking or jumping from the nearest markers ﬂank-
ing the region of interest. Now these steps can usually be carried out much					more efﬁ-
ciently by using the larger size YACs and mega-YACs that		span	most	of	the	human
genome. While these have some limitations, discussed in Chapter 9, the DNA of most re-
gions is available just by a telephone call to the nearest YAC repository.
With the DNA of a particular region in hand, one can search for			additional polymor-
phic markers fairly efﬁciently. For example, simple tandem repeating sequences can be
selected by hybridization screening or	sequence-speciﬁc	puriﬁcation			methods		(see
Chapter 14). These new markers can be used to reﬁne the genetic map in the region.
However, a more effective use of nearby markers, as we discussed in Chapter 6, is to pin-
point the location of any recombinants in	the region. This is	illustrated		in	Figure		13.3.

Figure 13.2 Information and samples usually available at the start of the end game in the search for a disease gene.

436 FINDING GENES AND MUTATIONS

Figure	13.3	The nearest recombination breaking points ﬂank the true	location of the gene.
Hatched	and hollow	bars indicate chromosome segments inherited from different	parental ho-
mologs. D must lie to the right of marker 13 and to the left of marker 15.

Before the gene of interest was successfully linked to markers, any recombinants were damaging, since they subtracted from the statistical power of the linkage tests. Now, however, once the locale of the gene is established beyond doubt, the recombinants are a very valuable resource, and it is often very proﬁtable to search for additional recombinants. As shown in Figure 13.3, the gene location can be narrowed down to a position between the nearest set of available recombinants.

In an ideal case the gene of interest is large, and it occupies a considerable portion of the region. Then frequently a disease allele can be found that contains a large enough size polymorphism to be spotted by PFG analysis of DNA hybridized with available probes in

the region. The polymorphism may arise from an insertion, a deletion, or a translocation. Such an association of a disease phenotype with one or more large-scale rearrangements almost always rapidly pinpoints the location of the gene because ﬁner and ﬁner physical mapping can rapidly be employed to position the actual disrupted gene relative to the precise sites of DNA rearrangements. An example of this approach was the search for the gene for Duchenne muscular dystrophy where roughly half of the disease alleles are large deletions in the DNA of the region.

In typical cases one is not lucky enough to spot the gene of interest by using low-reso- lution mapping approaches. Then it is usually safest to take a number of different approaches simultaneously. This is especially true if, as in many cases of interest, the search for the gene is a competitive one. The genetic approach useful at this point is linkage dis-

equilibrium. This was described in detail in Chapter 6. To reiterate, brieﬂy, if there is a founder effect, that is, if most individuals carrying a disease allele have descended from a common progenitor, they will tend to have similar alleles at other polymorphic sites in the

region. This is true even though the individuals			have	no apparent	familial relationships.
The closer one is to the gene, the	greater the tendency of all individuals with the disease
to share a common haplotype. This	gradient	of genetic		similarity	allows	one	to	narrow
down the location of the gene,	but there	are	many	potential	pitfalls,		as	described in
Chapter 6.
A second useful approach is	to search	for	individuals who		display	multiply genetic

disorders including the particular disease of interest. Such individuals can frequently be found, and they will often be carriers of microscopic DNA deletions. As shown in Figure 13.4, one can use these individuals to narrow the location of the gene. Low-resolution physical maps of each individual can often reveal the size and position of the deletions. Pooling data from several individuals with different deletions will indicate the boundaries

on the possible location of the gene of interest. The process is easiest in cases like X- linked disease, since here, in males, there is only one copy of the region of interest. In somatic disease, there will be two copies, and the altered chromosome will have to be distinguished and analyzed in the presence of the normal one. This general approach can be

very productive because after one gene is found, the genes for the additional inherited disorders must lie nearby, and it will be much easier to ﬁnd them.

FINDING GENES

437

Figure

13.4

The

nearest available chromosome breaking points, frequently seen in patients with

multiple inherited disorders, ﬂank the true location of the gene. Horizontal lines show markers pres-

ent in three individuals with a common genetic disorder. The disease gene must lie between mark-

ers 13.2 and 13.3.

A third parallel approach is to map and characterize the transcripts coded for by the re-

gion. This can be done by using available DNA probes in hybridization against Northern

blots (electrophoretically fractionated mRNAs) or against cDNA libraries. If the disease is

believed to be predominantly localized in particular tissues, this approach can be very ef-

fective, since one can compare mRNAs or cDNAs from tissues believed to be signiﬁcant

sites of expression of the gene of interest with other samples where this gene is not likely

to be expressed. With cystic ﬁbrosis, for example, hypotheses about gene expression in

sweat glands and in the

pancreas were

very

helpful in narrowing the location of the gene

in this way. Alternatively, genes in the target region may already be known as a large num-

ber of expressed sequence tags (ESTs) from known tissues are being added to the EST

database at GenBank daily and are being mapped to chromosomal regions. Note, however,

that considerable pitfalls exist with this approach, since hypotheses about the sites of ex-

pression can easily be wrong, and, even if they are correct, the gene of interest may be ex-

pressed at too low a level to be seen as mRNA or represented in a typical cDNA library.

DNA of the region can be used in a number of different ways to help ﬁnd the location

of the genes in the region, even where no prior hypotheses about sites of likely expression

exist. YACs

have

been

used as hybridization probes to directly isolate corresponding

mRNAs

cDNAs,

technique sometimes referred to as ﬁshing (Lovett, 1994).

Techniques, such as exon trapping, have been developed to allow speciﬁc subcloning of

potentially coding DNA sequences from a region (see Box 13.1). Another frequently ef-

fective strategy is to look for regions of DNA that are conserved in different mammals or

even more distant species. Genes are far more likely to be conserved than noncoding re-

gions. However, this approach is not guaranteed because there is no reason to expect that

every gene will be conserved or even exist among a set of species tested. Even genome

scanning by direct sequencing has revealed the location of genes.

In some types of disease, other strategies become useful. For example, in dominant

lethal disease, most if not all affected

individuals are new mutations. These

will most

likely

occur in regions

of DNA with high intrinsic mutations rates. While we still have

much to learn about how to identify such regions, at least one class of unstable DNA se-

quence has emerged in recent years that appears to play a major role in human disease.

Tandemly repeated DNA sequences have intrinsically high mutation rates because of the

possibilities for polymerase stuttering or unequal sister chromatid exchange, as described

in Chapter

Repeats

like (GAG)

occur

in coding

regions;

(GAA)

n and (GCC)

outside of coding regions. These can shrink or grow rapidly in successive generations and

lead to disease phenotypes. Examples of this were ﬁrst seen in myotonic dystrophy, frag-

ile X-linked mental

retardation,

and

Kennedy’s

disease

(see

Box

13.2). A systematic

now

underway

to map the locations of these and

other

trinucleotide

repeats,

since they may well underlie the cause of

additional human diseases. The repeats appear

to be fairly widespread as shown by the examples already found (see Table 13.1).

n occur

438 FINDING GENES AND MUTATIONS

BOX	13.1
EXON	TRAPPING	METHODS
Exon	trapping	methods	are schemes	for selective cloning	and screening	of coding
DNA sequences. Several different approaches have been described (Duyk et al., 1990;
Buckler et al., 1991; Hamaguchi et al., 1992). Here we will illustrate only the last of
these	because it	seems	to be relatively simple and efﬁcient. The vector used for this
exon trapping scheme is shown in Figure 13.5					a . It contains intron 10 of the p53 gene,
which includes a long pyrimidine tract (which appears to prevent exon skipping), and
consensus sequences for			the 5	- and 3	-splicing sites	(AG/GTGAGT and AG, respec-
tively), and the branch site (TACTCAC) used in an intermediate step in RNA splicing.
The intron contains a			Bgl	II cleavage site used for cloning genomic DNA. Surrounding
the intron are two short p53 exons, ﬂanked by SV40 promoters known to be transcrip-
tionally active in COS-7 cells. Reverse transcriptase is used to make a cDNA copy of
any transcripts, and then PCR with two nested sets of primers is used to detect any
transcripts containing			the two p53 exons. When the vector alone is transfected into
COS-7 cells, only a 72-bp transcript is seen. Cloned inserts containing other complete
exons will produce longer transcripts after transfection. In practice, fragments from 90
to 900 bp are screened for because most exons are shorter than 500 bp. These new
fragments will arise by two splicing events as shown in Figure 13.5						b . For an example
of recent results using exon trapping, see Chen et al. (1996).

(continued)

FINDING GENES

439

BOX 13.1

(Continued)

Figure 13.5	Exon trapping to clone expressed DNA sequences. (	a ) Vector and procedures
used. Adapted from	Hamaguchi et al. (1992). pA, pAB, pRB, and pR are primers	used for
nested PCR. (	b ) Schematic of the PCR product expected from a cloned exon.

TABLE 13.1 Trinucleotide Repeats in Human Genes

Copy

Gene or Encoded Protein	Number	a

Location

znf6 (zinc ﬁnger transcription factor)		8, 3, 3	5	Untranslated region
CENP-B (centromere autoantigen)		5	5	Untranslated region
c- cbl (proto-oncogene)		11	5	Untranslated region
Small subunit of calcium-activated neutral protease	10, 6	Coding region (N-terminal)
CAMIII(calmodulin)		6	5	Untranslated region
BCR (breaking point cluster region)		7	5	Untranslated region
Ferritin H chain		5	5	Untranslated region
Transcription elongation factor SII		7	5	Untranslated region

Early growth response 2 protein		5	Coding region (central)
Androgen receptor		17		Coding region (central)
FMR-1 (fragile X disease)		6–60		Not certain yet
(AGC)	n androgen receptor (Kennedy’s disease)	13–30	Coding region (central)
DM-1 myotonic dystrophy		5–27	3	Untranslated region
IT 15 Huntington’s disease		11–34		Coding region (N-terminal)

Source:	Updated, from Sutherland and Richards (1995).

a In normal individuals

440 FINDING GENES AND MUTATIONS

BOX 13.2
DISEASES CAUSED BY ALTERED TRINUCLEOTIDE REPEATS
Fragile sites on chromosomes have been recognized, cytogenetically, for a long time.
When cells are growth under	metabolically impaired conditions, some	chromosomes,
in metaphase, show defects.	These give the superﬁcial appearance	that the chromo-

some is broken at a speciﬁc locus, as shown in Figure 13.6. Actually it is most unlikely

that a real break has occurred; instead, the chromatin has	failed to condense normally.
A particular fragile spot on the long arm of the human X chromosome, called fraXq27,
shows a genetic association with mental retardation. About	60% of the chromosomes
in individuals with this syndrome show fragile sites; the	incidence in apparently nor-

mal individuals is only 1%. Fragile X-linked mental retardation is actually the second most common cause of inherited mental retardation. It occurs in 1 in 2000 males and 4 in 10,000 females. Earlier genetic studies of fragile X syndrome showed a number of very peculiar features that were inexplicable by any simple classical genetic mechanisms.

Now that the molecular genetics of the fragile X has been revealed, and similar events have been seen in many other diseases, including Kennedy’s disease, myotonic dystrophy, and Huntington’s disease, we can rationalize many of the unusual genetic features of these diseases. A number of fundamental issues, however, remain unresolved. The basic molecular genetic mechanism common to all four diseases and many

others is illustrated in Figure 13.7 (Sutherland and Richards, 1995). In each case, near or in the gene, a repeating trinucleotide sequence occurs. Like other variable number

tandem repeats (VNTRs), this sequence is polymorphic in the population. Normal in-

dividuals are observed to have relatively short repeats: 6 to 60 copies in fragile X syndrome, 13 to 30 in Kennedy’s disease, 5 to 27 copies in myotonic dystrophy, and 11 to 34 in Huntington’s. Individuals affected with the disease have much larger repeats: more than 200 copies in fragile X, more than 39 in Kennedy’s, more than 100 in myotonic dystrophy, and more than 42 in Huntington’s.

The case studied in most detail thus far is the fragile X syndrome, and this will be the focus of our attention here. Individuals who are carriers for fragile X, that is, individuals whose offspring or subsequent descendants display the fragile X phenotype,

have repeats larger than the 60 copies, which represents the maximum in the normal population, but smaller than 200, the lower bound of individuals with discernable disease phenotypes. This progressive growth in the size of the repeat, from normal to car-

Figure 13.6 Appearance of a fragile X chromosome in the light microscope.

(continued)

BOX 13.1	(Continued)
FINDING GENES	441

BOX 13.2

(Continued)

Figure 13.7 Summary of the VNTR expansions seen in four inherited diseases. Shown are repeat sizes to normal alleles and disease-causing alleles. Adapted from Richards and Sutherland (1992).

(continued)

442 FINDING GENES AND MUTATIONS

BOX 13.2	(Continued)

Figure 13.8	Typical fragile X pedigree showing anticipation and a nontransmitting carrier
male (T). Black	symbols denote mentally retarded individuals; gray symbols are carrier fe-

males. Arabic number %’s are the risk of mental retardation based on the general statistics for pedigrees of this type; italic numbers are the copies of CGG repeats present in particular individuals. Adapted from Fu et al. (1991).

rier to affected, explains some of the unusual genetic features of the disease that so puzzled early investigators. Figure 13.8 shows a typical fragile X pedigree. It reveals two nonclassical genetic effects. The individual labeled T in the ﬁgure is called a nontransmitting male. He is unaffected, and yet he is an obligate carrier because two of his


grandchildren developed the disease. One of these is a male						who	must	have received
the	disease-carrying			chromosome from his mother. The second,		more	general feature
of	the	pedigree	in	Figure 13.8	is called anticipation. As the			generations	proceed, a
higher		percentage	of	all the offspring develop the disease phenotype.				This is because
the	number of copies			of the repeated	sequence in the carriers keeps			increasing,	until

the repeat explodes into the full-blown disease allele. Note that in this pedigree, as is usual, the affected males do not have any offspring. This is because the disease is untreatable and severely disabling. It is effectively a genetic lethal, and thus can only ex-

ist	at the high frequency observed because the rate of	acquisition of new mutations
must be high.
	Something about the gradual increase in the size of	the repeat must eventually trig-
ger	a molecular mechanism that leads to a much greater further expansion. Thus above

some critical size the sequence is genetically unstable. Figure 13.9 shows two alternate mechanisms that have been proposed to account for this instability. In the ﬁrst of these,

it is	postulated that somewhere else in the genome, there	is a sequence that normally
has no effect on the trinucleotide repeat. However, in a founder chromosome (one that
will	lead to the carrier state and eventually produce the	disease) a mutation occurs in
this	sequence. This acts, either in cis or in trans, to destabilize the repeat, which then

(continued)

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