Human DNA polymorphisms and methods of analysis James L. Weber Marshfield Medical Research Foundation, Marshfield, Wisconsin 54449-5790, USA Current Opinion in Biotechnology 1990, 1:166-171

Introduction DNA polymorphisms can be defined as DNA sequences that vary between homologous chromosomes. The primary application of human DNA polymorphisms is in the construction of linkage maps and the mapping of genetic disease genes. For thousands of genetic diseases and other heritable traits, the gene products responsible are unknown, and the only practical means of isolating the relevant genes is firstly to map them to specific chromosomal sites by demonstrating co-inheritance (linkage) of the genes to one or more polymorphic DNA markers. Once a disease gene is mapped, presymptomatic diagnostic tests are usually possible using flanking DNA polymorphisms. Other important applications of human DNA polymorphisms are in the identification of individuals in forensic testing and the determination of ancestry in paternity and immigration cases. Polymorphisms are also used to detect the loss of heterozygosity that occurs in various tumors, to distinguish donors from recipients in transplants and chromosome transfer experiments, and to determine the parental origin of inactivated X chromosomes and of chromosome non-disjunction. In this review, the major types of human DNA polymorphisms and the various methods for their analysis will be described and compared. Although a few known human DNA polymorphisms are based upon insertions, deletions or other rearrangements of non-repeated sequences, the vast majority are based either upon single base substitutions or upon variations in the number of tandem repeats [1,2]. Base substitutions for which the most frequent variant (allele) is present on no more than 99% of the chromosomes (the standard definition of DNA polymorphism [3]) are very abundant in the human genome, occurring on average once every 200-500 bp. Length variations in blocks of tandem repeats are also common in the genome, with at least tens of thousands of interspersed polymorphic sites (loci). Repeat lengths for tandem repeat polymorphisms range from I bp in (dA)n'(dT) n sequences to at least 170bp in m-satellite DNA. Tandem repeat polymorphisms can be divided into two:,major groups: minisatellites/variable number of tandem repeats (VNTRs), with typical repeat lengths of tens of base pairs and with tens to thousands

of total repeat units [4,5]; and microsatellites, with repeat lengths of up to 6 bp and with maximum total lengths of about 70 bp [6oo,7o,8.°]. Most of the microsatellite polymorphisms identified to date have been based on (dC-dA)n'(dG-dT) n dinucleotide repeat sequences.

Methods of analysis A wide variety of techniques have been developed for the analysis of DNA polymorphisms. The most widely used method, the restriction fragment length polymorphism (RFLP) approach, combines restriction enzyme digestion, agarose gel electrophoresis, blotting to a membrane and hybridization to a (usually) cloned DNA probe (Fig. 1). Polymorphisms are detected as variations in the lengths of the labeled fragments on the blots. The RFIP approach can be used to analyze base substitutions when the sequence change falls within a restriction enzyme site or to analyze minisatellites/VNTRs by choosing restriction enzymes that cut outside the repeat units. The agarose gels do not usually afford the resolution necessary to distinguish minisatellite/VNTR aUeles differing by a single repeat unit, but many of the minisatellites/VNTRs are so variable that highly informative markers can still be obtained. Analysis of microsatellite polymorphisms involves amplification by the polymerase chain reaction (PCR) of a small fragment of DNA containing a block of repeats followed by electrophoresis of the amplified DNA on denaturing polyacrylamide gels (Fig. 1). Typically, the amplified DNA is labeled by incorporating radioactive nucleotides into the newly synthesized strands [6 "°] or by end-labeling one of the PCR primers [8°°,9o]. The PCR primers are complementary to unique sequences that flank the blocks of repeats. Polyacrylamide gels, rather than agarose gels, are generally required for microsatellites because the alleles often only differ in size by a single repeat. Some minisatellite/VNTR polymorphisms have been analysed using PCR, much as with the microsatellites [10,11,12 °]. However, many of the minisatellite/VNTR alleles are too large for efficient PCR amplification using current technology. A technique for the simultaneous

Abbreviations CEPH--Centre d'l~tude du Polymorphisme Humain; PCR--polymerase chain reaction; PIC--polymorphism information content; RFLP--restriction fragment length polymorphism; VNTR--variable number of tandem repeats. 166

~ Current Biology Ltd ISSN 0958-1669

Human DNA polymorphisms and methods of analysis Weber

Restriction enzyme digestion ]

Agarose gel electrophoresis ]

I

4 0,ott,ng 4

I

] Probe acquisition and preparation ]

Primer synthesis ]

I o' me--e chain--ctioo [

Fig. 1. The steps involved in the two most common approaches for analyzing human DNA polymorphisms. Base substitution and minisatellite/variable number of tandem repeats polymorphisms are usually analyzed using the restriction fragment length polymorphism (RFLP)approach and microsatellite polymorphisms are analyzed using the polymerase chain reaction (PCR) approach.

Acrylamide gel electrophoresis~

utoradiography [ ]

1 " '0'zat,on 1 [ Autoradiography ]

analysis of multiple minisatellite/VNTR polymorphisms using two-dimensional gel electrophoresis was recently described [13°]. This technique involves the separation of restriction enzyme fragments on agarose gels in the first dimension and on polyacrylamide denaturating gradient gels in the second. A number of methods in addition to the RFLP approach are available for analyzing base substitution polymorphisms. Orita et al. [14.,15°°] have devised a promising new way of analyzing these polymorphisms on the basis of conformational differences in denatured DNA. Restriction enzyme digestion or PCR is used to produce relatively small DNA fragments which are then denatured and resolved by electrophoresis on non-denaturing polyacrylamide gels. Conformational differences in the single-stranded DNA fragments resulting from base substitutions are detected by electrophoretic mobility shifts. In principle, the method could also be used to analyze polymorphisms based on short insertions or deletions. Another set of methods use allele-specific oligodeoxynucleotides to distinguish base substitution variants. Oligomers can be used as hybridization probes under Conditions that allow perfectly matched duplexes to be distinguished from those with single base mis-

matches; altematively, oligomers that anneal to adjacent sites along a cDNA strand can be linked in a sequencedependent reaction catalysed by DNA ligase [16]. Both these approaches are usually carried out after prior target DNA amplification with PCR. Under appropriate conditions, successful PCR amplification itself can be dependent upon perfect sequence complementarity between the primer and template [17o,18oo,19 °]. Amplified DNA can also be tested for sequence variation using restriction enzyme digestion, denaturating gradient gel electrophoresis, RNAse cleavage of RNA-DNA hybrids, or direct sequencing [20]. Criteria for selecting optimal types of polymorphisms and methods of analysis differ somewhat depending on the application. For example, linkage mapping of genetic disease genes requires markers that are distributed uniformly throughout the genome, whereas for forensic testing, the chromosomal location of the markers is a minor consideration. Nevertheless, there are some basic properties of the polymorphisms and the methods of analysis that that are of general importance. These include informativeness, genomic distribution, availability and genotyping rate.

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Mammalian gene studies Informativeness In general, the usefulness of a polymorphism increases with increasing marker informativeness. Informativeness is measured using either heterozygosity, which is simply the fraction of unrelated individuals who are heterozygous, or polymorphism information content (PIC), which is similar except that it takes into account the loss of information that occurs in a family when both parents are heterozygous with identical genotypes [21]. Two-allele polymorphisms, including most base substitution polymorphisms, are limited in informativeness to maximum heterozygosities of 0.50 and PIC values of 0.375. Roughly 75% of known human DNA polymorphisms fall into this category [22]. Hybridization probes for RFLP markers can sometimes detect two or more different base substitution polymorphisms at a single locus, thereby increasing the informativeness of a locus. Multiallelic polymorphisms, in contrast to two-allele markers, have few limits on informativeness. Some hypervariable minisatellites have heterozygosities exceeding 0.99 [23]. Average heterozygosity for VNTR markers is 0.68 [5,24]. Although microsatellite polymorphisms cannot match the high informativeness values of hypervariable minisatellltes/VNTRs, the average heterozygosity for 60 recently developed (dC~lA)n'(dG-dT) n polymorphisms was 0.67 (Weber, unpublished results). The fact that the average informativeness of multiallelic polymorphisms is double that for two-allele polymorphisms means that about twice as much usable data can be obtained for a fixed sample size with multiallelic markers. For linkage studies of rare genetic disorders, this difference in informativeness can spell the difference between overall project success and failure.

Genomic distribution Ideally, a given class of polymorphisms should be densely and uniformly distributed throughout the genome. Base substitution polymorphisms fit these criteria extremely well; RFLP linkage maps based on substitution polymorphisms show no bias to particular chromosomes or chromosomal regions. The entire human genome contains of the order of 107 base substitution polymorphisms. In contrast, minisatellite/VNTR loci are preferentially situated near chromosome telomeres [25]. The total number of potential minisatellites/VNTRs is uncertain, but probably is at least in the thousands. The genomic distribution of microsatellite polymorphisms remains uncertain, but preliminary evidence favors a wide and approximately uniform distribution. I n situ hybridization of an (AC)25 oligodeoxynucleotide to human metaphase chromosomes strongly labeled all chromosomal regions with the exception of the centromeric regions (J Meyne, personal communication), and subchromosomal mapping of a limited number of microsatellite polymorphisms has shown them to be located throughout the lengths of specific chromosomes [26,27 oo]. The number of relatively informative (dC--dA)n'(dG-dT) n sequences

in the human genome (PIC >_ 0.50) was estimated recently at 12 000 [28"°]. Potentially informative microsatellites with other repeat sequences (including (dA)n'(dT) n sequences) probably number at least in the tens of thousands.

Availability Existing human linkage maps are based almost exclusively on RFLP polymorphisms. Although the process of collecting probes for RFLP markers can be time-consuming and expensive, probes are generally available from individual laboratories, from the American Type Tissue Collection (Rockville, Maryland, USA) and from Collaborative Research, Inc. (Bedford, Massachussets, USA). For microsatellite polymorphisms and for other polymorphisms whose analysis is based on PCR, pruner sequences and other marker information can be transmitted electronically. Aliquots of primers for about 50 PCR markers will also soon be available form the American Type Tissue Collection. At the time of writing (October 1990) at least 200 human microsatellite DNA polymorphisms had been identified worldwide, and primer sequences for roughly half of these had been published. Identification of additional microsatellite markers is proceeding rapidly, but there will undoubtedly be a lag before the new markers are incorporated into existing linkage maps.

Genotyping rate Faster genotyping means earlier completion of projects and, at least in most cases, lower project costs. To date, sutticient numbers of markers and sutt]cient genotyping results to estimate genotyping rates are only available for RFLP and microsatellite polymorphisms. For situations in which a set of DNA samples need to be typed with only one or a few markers, the microsatellite PCR-based approach is much faster than the RFLP approach because it only involves about half as many steps (Fig. 1). For cases such as linkage mapping projects, in which a set of samples must be typed with many different markers, the RFLP approach becomes relatively faster as blots can generally be reused. In order to compare the two approaches in this sort of case, I obtained estimates of genotyping rates from a number of different laboratories, including some of the largest linkage mapping ones. The results are presented in Table 1. Despite considerable variation between laboratories, the average rate for the microsatellite polymorphisms, 650 genotypes/week/person, was nearly twice the average rate of 360 genotypes/week/person for RFLPs. Perhaps even more convincing is the unanimous opinion, from groups who have compared the two techniques directly, that the PCR-based approach is considerably faster [29-31]. The prospects for signiticant improvements in genotyping rates for microsatellite DNA polymorphisms are also good. One new ap-

Human DNA polymorphisms and methods of analysis Weber proach would be to label PCR primers with fluorescent dyes and then analyze the amplified DNA on automated DNA sequencers. Another approach would be to apply the electroblotting and successive hybridization technology of multiplex DNA sequencing [32].

Table 1. Genotyping rates for restriction fragment length polymorphisms and microsatellite polymorphisms. RFLP approach

PCR approach

Laboratory

Rate

Laboratory

Rate

A

320

H

600

B

125

I

850

C

200

J

50O

D

800

K

-

E

200

-

-

F

500

-

-

G

400

-

-

Average

360

Average

650

Genotyping rates are shown as genotypes/week/person for each of the laboratories A-J. PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism.

Genotyping rates for other methods of polymorphism analysis are difficult to estimate. The method developed by Orita et al. [14-,15..] involves PCR and polyacrylamide gel electrophoresis and so should produce a similar rate to microsatellite analysis. Several of the other techniques also involve PCR and gel electrophoresis and would therefore probably have genotyping rates similar to those of microsatellite analysis (although the fact that base substitution polymorphisms are being detected may allow more markers to be packed onto each gel lane) [33,34 °]. Eventually, it may be possible to automate completely a polymorphism analysis technique that does not involve any electrophoresis, such as allele-specific oligomer hybridization or allele-specific PCR using fluorescently labeled primers. It remains to be determined whether such an approach would be faster than existing methods.

Other factors One of the virtues of PCR is that it allows minute amounts of template DNA to be detected, even down to a single template molecule [34 °]. All methods for analyzing polymorphisms that use PCR have this sensitivity. As hundreds of PCR polymorphisms can be typed with a few I.tg of genomic DNA and as about 400 I~g of DNA can be isolated from a 40 ml blood sample, it may soon become possible to omit the expensive process of establishing lymphoblastoid cell lines for each individual in genetic disease families.

The number of oligodeoxynucleotides required per marker varies between the different analysis techniques. Microsatellite and single-strand conformation polymorphisms only require two oligomers, but allele-specific PCR polymorphisms require three. Markers based on allele-specific oligomer hybridization require four oligomers, two for PCR and two to act as hybridization probes. The ligase-mediated technique requires five oligomers, two for PCR and three for the detection of the base substitution (one common oligomer and a pair differing in sequence at the 3' end). The more oligomers that are needed for analysis of a marker, the higher will be the costs of development and application. Genotyping error rates, where known, do not seem to be appreciably different for the different types of polymorphisms and methods of analysis, although individual markers vary considerably in typing difficulty. Estimates of error rates for the typing of Centre d't2tude du Polymorphisme Humain (CEPH) reference families, as determined through the process of map construction, range from one to a few percent. Despite the relative complexity of the banding patterns on autoradiographs for microsatellite markers, initial results for typing within the CEPH families do not indicate any appreciable difference in error rates compared with RFLP markers [26]. LOD scores of more than 50 in CEPH families between pairs of microsatellite markers have been reported [35]. Most of the complexity of the microsatellite banding pattems appears to be due to PCR artefacts, particularly skipping or addition of repeat units as a result of strand slippage during the primer elongation steps. The artefacts seem to cause fewer problems as the repeat length of the sequence increases. Sequences of the form (A2_4 N)n , where N is A, C or G, appear to be relatively abundant and may be easier to type than the dinucleotide repeat sequences [36",37"]. On the other hand, (dA)n'(dT) n sequences, which are found at the 3' ends of most Alu elements and other retroposons, are currently quite difficult to type and, like minisatellites/VNTRs, may only be useful when alleles differ by several repeats [36"]. With the exception of a few hypervariable minisatellite polymorphisms [23], mutation rates for human DNA polymorphisms, including microsatellite polymorphisms, do not seem to be high enough to limit their use in genetic studies. New mutations within disease or reference famih'es are only detected very rarely. Although this review has been confined to human DNA polymorphisms, most of the conclusions are applicable to other species. In particular, mouse microsatellite polymorphisms are being detected in large numbers. Love et al. [38"'] recently described the development of 50 microsatellite markers from the mouse genome.

Summary The current predominant method of analyzing base substitution polymorphisms, RFLP analysis, is likely to be gradually supplanted by methods based on PCR because

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Mammaliangene studies of the improved sensitivity and genotyping rate. The most promising PCR methods for analysis appear to be allelespecific PCR and single-stranded conformational analysis. The single-stranded conformation approach has already been applied to the scanning of cystic fibrosis exons for new mutations [39]. Linkage mapping projects that cover large segments of the human genome will probably rely, in the coming years, primarily on tandem repeat polymorphisms, particularly microsatellite polymorphisms. Microsatellite polymorphisms have at least a fourfold advantage over base substitution RFLPs because they are twice as informative and can be typed at at least twice the rate. The facioscapulohumeral muscular dystrophy gene was recently mapped in just 6 weeks using microsatellite polymorphisms [29]. Because of the informativeness handicap, it will be difficult for base substitution polymorphisms to overtake tandem repeat markers for large-scale linkage mapping. Methods that allow base substitution polymorphisms to be typed at two or three times the rate of microsatellite markers would have to be developed. Most of the other applications of DNA polymorphisms described in the introduction are also increasingly likely to rely on highly informative tandem repeat markers in the future. Methods for analysis will probably be based on PCR. It is easy to envisage, for example, an automated method for large-scale DNA fingerprinting of individuals based upon a standard set of highly informative, dependable microsatellite polymorphisms. Methods for analyzing base substitution polymorphisms will continue to be important for the diagnostic detection of disease-gene alleles. Also, as the hunts for particular disease-genes progress beyond the large-scale linkage mapping phase, the more abundant base substitution polymorphisms may be more useful than tandem repeat markers for the purpose of narrowing the region where the gene may be located. A particularly difficult problem facing human geneticists in the coming decades will be the task of identifying genes that influence complex disorders such as schizophrenia or hypertension. The pathway to these genes is unclear, but family, sibling pair or related studies using DNA polymorphisms are one obvious possibility. Because these mapping projects are likely to involve many more individuals and be much more costly than simple disease-gene-mapping projects, it will be essential to use highly informative markers that can be typed very efficiently. An important challenge to investigators working on human DNA polymorphisms will be to provide the markers and techniques needed for successful completion of these large-scale projects.

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6. ••

WEBERJL, MAy PE: Abundant class of human DNA polymorp h i s m s w h i c h can be typed using t h e polymerase chain reaction. A m J H u m Genet 1989, 44:388-396. One of the original reports showing that (dC~lA)n'(dCr~T) n sequences exhibit length polymorphisms. The properties of 10 polymorphisms are reported. Multiple markers can be amplified and analyzed simultaneously. 7. •

SMEETSHJM, BRUNNER FIG, ROPERS HH, WIERINGA B: Use of variable simple sequence motifs as genetic markers: application to study of myotonic dystrophy. H u m Genet 1989, 83:245-251. Another original report on (dC-dA)n'(dG-dT) n sequences. A microsatellite polymorphism at the APOC2 locus on chromosome 19 is linked to the myotonic dystrophy gene. 8. •,

TAUTZD: Hypervariability of simple sequences as a general source for polymorphic DNA markers. Nucleic Acids Res 1989, 17:6463~471. Length polymorphisms in simple sequence tandem repeats are extended to (dA~lG)n'(dT~lC) n and trinucleotide repeat sequences, and to species other than humans. 9. •

LtTF M, LUTY JA: A hypervariable microsatellite revealed by in vitro amplification of a dinucleotide repeat within the cardiac muscle actin gene. A m J H u m Genet 1989, 44:397-401. A particularly informative (dC~lA)n'(dG-dT)n polymorphism within the cardiac actin gene is described. The total number of (dC~IA) n. (dG-dT) n sequences in the h u m a n genome is estimated to be 35 000, on the basis of hybridization of poly(dC-dA).poly(dG-dT) to a cosmid library. 10.

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1,

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ECONOMOUEP, BERGEN A, WARRENAC, ANTONARAKISSE: The polydeoxyadenylate tract of Alu repetitive e l e m e n t s is polym o r p h l c in t h e h u m a n g e n o m e . Proc Natl Ac ad Sci USA 1990, 87:2951-2954. The A-rich sequences at the 3' ends of Alu elements exhibit length potymorphisms. A fraction of the A-rich sequences have more complex repeats such as (TAD)n. One PCR primer within the Alu element is permissible. 37. •

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DEANM, WHITE MB, AMOSJ, GERRARDB, STEWARTC, KHAWKT, LEPPERT M: Multiple

Human DNA polymorphisms and methods of analysis.

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