Genomics- The Science and Technology Behind the Human Genome Project. Charles R. Cantor, Cassandra L / genomics11-15 / 13

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					DETECTION		OF	NEW MUTATIONS	463
body ﬂuids may be very small. PCR				is very helpful in amplifying whatever DNA is
present, but the real problem is distinguishing a true positive signal from the frequent, un-
wanted background caused by PCR side reactions. Several strategies have been used to
enhance the DNA analysis of infection. If				the type of target cell is known, one can often
use immobilized monoclonal antibodies against this particular cell type to purify it away
from the rest of the sample. This will substantially reduce subsequent PCR background.
Another approach is to screen for the ribosomal RNA of the infectious agent. This is
applicable to all agents except viruses, since they do not have ribosomes. Some regions of
rRNA vary	sufﬁciently to allow a wide variety of organisms to be distinguished easily.
However, the major advantage of looking				at rRNA directly is that there are typically 10					4

to 10 5 copies per cell versus only a few copies of most DNA sequences. An alternative,
undoubtedly worth exploring for protozoa, will be to use repeating DNA sequences spe-
ciﬁc to a given species. This is unlikely to work for most simpler organisms because they
have very few repeats.
The potential utility of DNA analysis in infectious disease is staggering. We will con-
sider in detail the case of HIV, the virus that causes AIDS. One difﬁculty in the clinical
management of this disease is that the virus has a very high mutation rate. Thus most peo-
ple have different viral mixtures, and some components of these mixtures are resistant to
particular drugs, because of mutations within the HIV reverse transcriptase or protease
genes. A brute force approach recently described, which appeared to have some success
(although the generality of this success is now disputed), is to treat with a mixture of sev-
eral different		drugs simultaneously. However, each of the drugs has potentially				serious
side reactions. Furthermore, by treatment				with all of the effective agents	at once,	there is
a real possibility of selecting for a viral variant resistant to them all. The complete DNA
sequence of the virus is known, and the sequence of many drug-resistant variants has also
been determined. Thus we know which sites in the viral reverse transcriptase and protease
are likely to mutate and confer resistance to particular drugs.
By direct PCR cycle sequencing of blood samples from AIDs patients undergoing drug
therapy, Mathias Uhlen and his coworkers have been able to monitor the course of the
disease with a precision not before obtainable (Wahlberg et al., 1992). The single-color,
four-lane ﬂuorescent sequencing used by Uhlen is sufﬁciently quantitative that it not only
can distinguish pure viruses, it also can analyze the composition of mixtures of viruses as
seen as apparent fractional populations				of particular bases at given sequence locations
(Fig. 13.20). When the analysis shows that the population of a particular drug-resistant
variant is	beginning		to climb, the physician is alerted to alter the therapy		by switching to
a different drug. After a while, it is typical to see a relapse in the viral					population		back	to
the original major strain, and since this is sensitive to the original					drug used,		one	can
switch back to that drug to help control the infection. As this kind of precise diagnostics
becomes more affordable and more readily available, it could have a major impact on the
practice of medicine.
DETECTION	OF	NEW	MUTATIONS
By deﬁnition, a new mutation can occur at any site within a target DNA sequence. To de-
tect new mutations by current methods is very difﬁcult, and it is much more demanding
than most of the problems we have discussed earlier in this chapter. New mutations must
be detected in		the	analysis of autosomal	dominant lethal diseases, as we have discussed

464 FINDING GENES AND MUTATIONS

Figure 13.20	DNA sequence analysis of changes in the HIV-1 population in azidothymidine-
treated AIDs patients.	Shown is the raw sequence data A’s (dotted dashed line) and G’s (solid line)

for a portion of the reverse transcriptase gene before treatment and at various times after treatment. Corresponding amino acid changes are shown below. Adapted from Wahlberg et al. (1992).

before. It is also necessary to detect new mutations if we are to be able to estimate the in-

trinsic, basal human mutation rate, and how	this may be inﬂuenced by exposure to vari-
ous agents in our environment including	radioactivity, sunlight, exposure to various

chemicals, diet, and various types of radiation such as emissions from electrical power lines, microwave ovens, and color televisions. These types of environmental damage raise serious issues of liability and responsibility which can only be properly assessed if we can monitor, directly and quantitatively, their effect on our genes. Hence the motivation to be able to monitor human new mutations is very high. The sensitivity of different animal species to many of these environmental agents is known to be quite variable. Thus, unfortunately, here we have a case where humans must be studied directly.

There are actually three different types of mutation rates that have to be considered in judging the relative effects of various environmental agents. The three are illustrated in Figure 13.21. Genetic mutations are the type of events we have been discussing throughout most of this text. A new genetic mutation means a change in the genomic DNA of a

child resulting in the presence of a sequence that could not have been inherited from either parent. One obvious way that this can come about is mis-paternity. Clearly this trivial explanation must be ruled out for any putative new mutation. Fortunately the power of current DNA personal identity testing makes such screening quite easy and accurate. The second type of mutation one must consider is a gametic mutation. Here one can look in a

sperm cell for a DNA sequence not	present in the father’s genomic DNA. Alternatively,
one can compare single sperm, by	methods described in Chapter 7, and look for the oc-

DETECTION OF NEW MUTATIONS

465

Figure 13.21	Three different types of new	mutations that one would like to	be able to detect. (		a )
Genetic mutations are inherited by the offspring. (		b ) Gametic mutations are present		in the	gametes
but are lethal, so no offspring are produced. (		c ) Somatic	mutations are present in a	subset	of cells
and are not passed to offspring.

currence of sequences that could not have arisen by simple meiotic recombination or gene conversion. The third form of mutation we must consider is at the somatic level. Here we need to look for DNA sequences that are present in some cells but not others. A key ele-

ment of the problem is that effects of			some environmental	agents may	be different			for
each type of	mutagenesis. For	example, an agent that was lethal to			gametes		would	not
show up as a genetic mutagen, and yet it would be a source of considerable damage if a
signiﬁcant decrease in fertility resulted. Speciﬁc types of cells may be particularly sus-
ceptible to certain agents. A few generalities can be made—it seems that rapidly dividing
cells are more sensitive to agents that damage DNA; highly transcribed genes seem to be
more easily mutagenized. However, in general, our current knowledge of these effects is
very slight.
The major difﬁculty in studying new mutations is that, unless they are accompanied by
an easily scored phenotype, the		target is a small needle in a very			large	haystack. The
basal, spontaneous mutation rate in the human is estimated to be 10								8 per base per meio-
sis. This means only 30 new mutations per haploid genome. What fraction of these are
point mutations or more complex DNA rearrangements is unknown at the present time,
although a guess that about half are in each category is probably reasonable. Since the lo-
cation of the new mutants is unknown a priori, the magnitude of the					search	required to
ﬁnd them is staggering. Environmental agents will raise the rate of mutations above the
basal level. What little evidence we have for typical agents of concern indicates that the
increases in	mutation caused by	typical	exposure levels	are small.	Thus,	to	quantitate

these, we will either need a very good strategy or extraordinary sensitivity.

466 FINDING GENES AND MUTATIONS

Two different basic scenarios must be considered that can arise and demand a search for new mutations. In the ﬁrst of these one may have a small number of individuals exposed to a local, and perhaps very high, dose of toxic agent. An example would be a chemical waste spill. This is a very difﬁcult situation to analyze. One choice is to examine a large percentage of the genomes of the exposed individuals in order to have sufﬁcient sensitivity to see an effect. This is not practical with existing methods. The alternative approach would be to look at potential hot spots for action of the particular agent if enough is known for us to be able to identify such hot spots. Cases where this may eventually be possible are agents that cause very speciﬁc kinds of tumors such as acetyl-aminoﬂuorene, an aromatic hydrocarbon that is a selective hepatic carcinogen.

The second scenario is a more favorable case, at least from the limited point of view of DNA analysis. Here one wants to look for mutations in a large number of people at risk (or a large number of sperm at risk) by exposure to a particular agent. An example may be

to screen for the genetic effects of depletion of the ozone in the earth’s atmosphere. Here a sensible approach is to design assays around regions that are easily tested in large numbers of different samples. Then one can apply these assays to a large population of individuals (or sperm). A risk in this approach is that the region selected may not be representative of the genome as a whole. Some portions of the genome are known to be mutation hot spots, such as VNTRs, and the mitochondrial D loop (origin of replication) because there are no genes there. Many other regions are likely to be identiﬁed as we learn more about both the sequence of the human genome and the molecular mechanisms of mutage-

nesis.

Two types of easy assays can be imagined. In the ﬁrst strategy, one needs to ﬁnd a region of DNA that is homozygous in a male, or in both parents. Then a mixture of DNA

from parent and child (or sperm) is melted and reannealed. Any heterozygotes are puriﬁed away from perfect DNA duplexes. These heterozygotes must represent new mutations. This can now be tested more conclusively by sequence comparisons of the DNAs

of interest. The important feature is that a physical puriﬁcation step is used to examine a very complex mixture of DNA species simultaneously and select just a small fraction of it

for subsequent analysis. In this way one begins to approach	the	ability	to handle	the
amounts of DNA needed to see effects in the 10		8 range.
An alternative approach which is potentially very powerful	but	is only	applicable	to

very particular regions of the genome is shown in Figure 13.22. Here PCR is used to assay for mutations in a restriction enzyme recognition site. Ampliﬁcation will occur only when the site is not intact. By starting with a set of DNA sequences that contain restriction sites in between PCR primers, only DNA that contains mutations will be ampliﬁed.

Figure 13.22 A potentially very sensitive PCR method for detecting a mutation in the site recognized by a restriction endonuclease.

	SOURCES	AND ADDITIONAL READINGS	467
This	is a most effective way to purify the DNA of interest. However, the difﬁculty with
this	approach is that in each sample one only looks at the small numbers of	bases that

make up a single restriction site. For the assay to be effective at very low mutation frequencies, it will probably be necessary to perform the initial PCR from a large mixture of different sequences, and this makes things potentially complex and noisy. Nevertheless,

this sort of approach is methodologically quite attractive, and perhaps variants can be conceived that will be even more sensitive.

SOURCES AND	ADDITIONAL READINGS
Birkenkamp, K., and Kemper, B. 1995. In vitro processing of heteroduplex loops and mismatches
by endonuclease VII.		DNA Research	2: 9–14.
Broude, N. E., Chandra, A., and Smith, C. L. 1997. Differential display of genome subsets contain-
ing speciﬁc interspersed repeats.			Proceedings of	the National Academy of	Sciences USA	94:
4548–4553.
Buckler, A. J., Chang, D. D., Graw, S. L., Brook, J. D., Haber, D. A., Sharp, P. A., and Houseman,
D. E. 1991. Exon ampliﬁcation: A strategy to isolate mammalian genes based on RNA splicing.
Proceedings of the National Academy of Sciences USA				88: 4005–4009.
Chen, X., and Kwok, P.-Y. 1997. Template-directed dye-terminator incorporation (TDI) assay: A
homogeneous DNA diagnostic method based on ﬂuorescence resonance energy transfer.						Nucleic
Acids Research		25: 347–353.
Chi, N.-W., and Kolodner, R. D. 1994. Puriﬁcation and characterization of MSH1, a yeast mito-
chondrial protein that		binds to DNA mismatches.		Journal of	Biological Chemistry	269:
29984–29992.
Cotton, R. G. H. 1997. Slowly but surely towards better scanning for mutations.					Trends in Genetics
13:43–46.
Duyao, M., et al. 1993. Trinucleotide repeat length instability and age of onset in Huntington’s dis-
ease.	Nature Genetics	4: 387–392.
Duyk, G. M., Kim, S., Myers, R. M., and Cox, D. R. 1990. Exon trapping: A genetic screen to iden-
tify candidate transcribed sequences in cloned mammalian genomic DNA.					Proceedings of	the
National Academy of Sciences USA			87: 8995–8999.
Fu, Y.-H., Kuhl, D. P. A., Pizzuti, A., Pieretti, M., Sutcliffe, J. S., Richards, S., Verkerk, A. J. M. H.,
Holden, J. J. A., Fenwick Jr., R. G., Warren, S. T., Oostra, B. A. Nelson, D. L., and Caskey, C. T.
1991. Variation of CGG repeat at the fragile X site results				in genetic instability: Resolution of
the Sherman paradox.		Cell 67: 1047–1058.
Ganguly, A., and Prockop, D. J. 1990. Detection of single-base mutations by reaction of DNA het-
erduplexes with a water-soluble carbodimide followed by primer extension: Application to prod-
ucts from the polymerase chain reaction.			Nucleic Acids Research		18: 3933–3939.
Hacia, J. G., Brody, L. C., Chee, M. S., Fodor, S. P. A., and Collins, F. S. 1996. Detection of het-
erozygous mutations in BRCA1 using high density oligonucleotide arrays and two-colour ﬂuo-
rescence analysis.		Nature Genetics	14: 447.
Hamaguchi, M., Sakamoto, H., Tsuruta, H., Sasaki, H., Muto, T., Sugimura, T., and Terada, M.
1992. Establishment of a highly sensitive and speciﬁc exon-trapping system.					Proceedings of the
National Academy of Sciences USA			89: 9779–9783.
Hattori, M., Shibata, A., Yoshioka, K., and Sakaki, Y. 1993. Orphan peak analysis: A novel method
for detection of point mutations using an automated ﬂuorescence DNA sequencer.						Genomics
15:415–417.

468	FINDING GENES AND MUTATIONS
Ketterling, R. P., Vielhaber, E., and Sommer, S. S. 1994. The rates of G : C								: T: A		: C : G trans-
versions at CpG dinucleotides in the human				factor IX gene.				American Journal of		Human
Genetics		54: 831–835.
Khrapko, K., Hanekamp, J. S., Thilly, W. G., Belenkii, A., Foret, F., and Karger, B. L. 1994.
Constant denaturant capillary electrophoresis (CDCE): A high resolution approach to mutational
analysis.		Nucleic Acids Research		22: 364–369.
Knight, S. J. L., Flannery, A. V., Hirst, M. C., Campbell, L., Christodoulou, Z., Phelps, S. R.,
Pointon, J., Middleton-Price, H. R., Barnicoat, A., Pembrey, M. E., Holland, J., Oostra, B. A.,
Bobrow, M., and Davies, K. E. 1993. Trinucleotide repeat ampliﬁcation						and hypermethylation
of a CpG island in			FRAXE mental retardation.		Cell	74: 127–134.
Lishanski, A., Ostrander, E. A., and Rine, J. 1994. Mutation detection by mismatch binding protein,
MutS, in ampliﬁed DNA: Application to the cystic ﬁbrosis gene.								Proceedings of	the National
Academy of Sciences USA			91: 2674–2678.
Liu, Q., and Sommer, S.			S. 1995.	Restriction	endonuclease ﬁngerprinting (REF): A sensitive
method for screening mutations in long, contigous segments of DNA.									BioTechniques		18:
470–477.
Lovett, M. 1994. Fishing for complements: Finding genes by direct selection.									Trends	in Genetics
10: 352–357.
Miyoshi, Y., Nagase, H., Ando, H., Horii, A., Ichii, S., Nakatsuru, S., Aoki,							T., Miki, Y., Mori, T.,
and Nakamura, Y. 1992. Somatic mutations of the APC gene in colorectal tumours: Mutation
cluster in the APC gene.			Human Molecular Genetics				1: 229–233.
Nawroz, H., Koch, W., Anker, P., Stroun, M., and Sidransky, D. 1996. Microsatellite alterations in
serum DNA of head and neck cancer patients.					Nature Medicine			2: 1035–1037.
Oliveira, R. P., Broude, N. E., Macedo, A. M., Cantor, C. R., Smith, C. L., and Pena, S. D. 1998.
Probing the genetic population structure of					Trypanosoma		cruzi	with polymorphic	microsatel-
lites. Proceedings of the National Academy of Sciences USA							95: 3776–3780.
Pena, S. D. J., Barreto, G., Vago, A. R., De Marco, L., Reinach, F. C., Dias Neto, E., and Simpson,
A. J. G. 1994. Sequence-speciﬁc “gene signatures” can be obtained by PCR with single speciﬁc
primers	at low stringency.		Proceedings of the National Academy of Sciences USA; Biochemistry
91: 1946–1949.
Petruska, J., Arnheim, N., and Goodman, M. F. 1996. Stability of intrastrand hairpin structures
formed by the CAG/CTG class of DNA triplet repeats associated with neurological diseases.
Nucleic Acids Research			24: 1992–1998.
Richards, R. I., and Sutherland, G. R. 1992. Dynamic mutations: a new class of mutations causing
human disease.		Cell 70: 709–712.
Sarkar, G., Yoon, H.-S., and Sommer, S. S. 1992. Dideoxy ﬁngerprinting (ddF): A rapid and efﬁ-
cient screen for the presence of mutations.					Genomics		13: 441–443.
Schalling, M., Hudson, T. J., Buetow, K. H., and Housman, D. E. 1993. Direct detection of novel
expanded trinucleotide repeats in the human genome.							Nature Genetics	4: 135–138.
Smith, J., and Modrich, P. 1996. Mutation detection with MutH, MutL, and MutS mismatch repair
proteins.		Proceedings of the National Academy of Sciences USA						93: 4374–4379.
Snell, R. G., MacMillan, J. C., Cheadle, J. P., Fenton, I., Lazarou, L. P., Davies, P., MacDonald, M.
E., Gusella, J. F., Harper, P. S., and Shaw, D. J. 1993. Relationship between trinucleotide repeat
expansion and phenotypic variation in Huntington’s disease.							Nature Genetics		4: 393–397.
Sommer, S. S. 1992. Assessing the underlying pattern of human germline mutations: Lessons from
the factor IX gene.			FASEB Journal	6: 2767–2774.
Soto, D., and Sukumar, S. 1992. Improved detection of mutations in the p53 gene in human tumors
as single-stranded conformation polymorphs and double-stranded heteroduplex							DNA.				PCR
Methods and Applications			2: 96–98.

		SOURCES	AND ADDITIONAL	READINGS				469
Sutherland, G., and Richards, R. I. 1995. Simple tandem DNA repeats and human genetic disease.
Proceedings of the National Academy of Sciences USA			92: 3636–3641.
Wahlberg, J., Albert, J., Lundeberg, J., Cox, S., Wahren, B., and Uhlen, M. 1992. Dynamic changes
in HIV-1 quasispecies from azidothymidine (AZT)-treated patients.					FASEB		Journal	6:
2843–2847.
Wells, R. D. 1996. Molecular basis of genetic instability of triplet repeats.					Journal	of	Biological
Chemistry	271: 2875–2878.
Youil, R., Kemper, B. W., and Cotton, R. G. H. 1995. Screening for mutations by enzyme mismatch
cleavage with T4 endonuclease VII.		Proceedings of the National Academy of Sciences USA						92:
87–91.
Yu, A., Dill, J., Wirth, S. S., Huang, G., Lee, V. H., Haworth, I. S., and Mitas, M. 1995. The trinu-
cleotide repeat	sequence d(GTC)	15 adopts a hairpin conformation.		Nucleic	Acids	Research		23:
2706–2714.

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