Copyright 0 1992 by the Genetics Society of America

Human Population Genetic StudiesUsing Hypervariable Loci. I. Analysis of Assamese, Australian, Cambodian, Caucasian, Chinese and Melanesian Populations Ivan Balazs,* John Neuweiler,* Peter Gunn; Judith Kidd,f Kenneth K. Kidd? Joy Kuh15 and Liu Mingjun’ *L$ecodes Corporation, Valhalla, New York, TGenetic Technologies, Crows Nest, New South Wales, Australia; XDepartment of Human Genetics, Yale University, New Haven, Connecticut 06510, “orensic Science Section, Winnellie, Northern Territory, Australia, and ’Department of Forensic Biology, Xian Medical School, Xian, China Manuscript received August 13, 1991 Accepted for publication January 6, 1992

ABSTRACT Population genetic studies, in Australian, Assamese, Cambodian, Chinese, Caucasian and Melanesian populations, were performed with several highlypolymorphic DNA loci. Results showedthat the Caucasian and Chinese had the highest level of heterozygosity. The size range of the majority of the polymorphic DNA fragments of a locus was the same in the different populations. The distinguishing feature of each ethnic group was the relative frequency of a particular set or group of alleles. For example, alleles >9.0 kb in size, in D14S13, or from 4.5 to 4.7 kb, in D18S27, were less than half as frequent in Caucasians than in the other populations. Overall, there were groups of alleles, at one or more loci, whose frequencies were different among some of the ethnic groups and therefore could be used to differentiate one groupfrom the other.

D

NA polymorphic loci containing variable number of tandem repeats (VNTR) have been used in populationgeneticstudies of theNorth American Caucasian and black populations (BAIRDet al. 1986; BALAZS et al. 1989; CHIMERA, HARRIS and LITT 1989), indigenous populations from Polynesia (FLINTet al. 1989), amerindians(KIDDet al. 1991), Assam, Canada and Papua New Guinea (DEKA, CHAKRABORTY and FERRELL 199 1). Results of these studies showed that the range of DNA fragment sizeswas the same in different ethnic groups, with some differencesin allele frequency distributions. Therefore, it is possible that some of these genetic markers may be useful in the study of genetic variation in different populations. Toward thatgoal, studies were carried out on a variety of populations throughoutthe world. Thisreport compares the results obtained for Assamese, Australian aborigines, Cambodian, Caucasian, Chinese and Melanesian populations. MATERIALS AND METHODS

Samples from human populations: The Assamese samples represent 18 unrelated individuals collected by R. DEKA with R. FERRELL (University of Pittsburgh) and R. CHAKRABORTY (University of Texas). The Australian aborigines samples were collected by J. KUHL from unrelated individualsin Northern Territory. They consistof 82 samples derived from allegedly full-heritage aborigines and 8 samples from half-heritage individuals. The 25 Cambodian samples were from unrelated individuals collected by K. DuMARS (University of California, Irvine) from the individuals living in the Los Angeles area of California. The Chinese Genetics 131: 191-198 (May, 1992)

samples were collected by L. MINCJUN in Xian from about 200 unrelated individuals belonging to the Han population. The Melanesian (Nasioi) samples are from 12 unrelated and 9 related individuals and were collected from the eastcentral highlands of Bougainvilleby J. FRIEDLANDER (Temple University, Philadelphia). Caucasian populations from two countries, the United States and Australia, were from unrelated individuals received for paternity testing at Lifecodes Corporation andIntegrated Technologies, respectively. DNA polymorphic loci: The properties of the loci and the probes used for their detection have already been described for D2S44, D14S1, D14S13, D17S79 (BALAZS et al. 1989). Locus D18S27 was analyzed with a synthetic oligonucleotide probe (Sli251) consisting of two tandem copies of the consensus sequence found in the clone pAC404 (IP et al. 1989). Locus D4S163wasanalyzed with the probeSli479 which is a synthetic oligonucleotide containing two tandem copies of the consensus sequence present in a 0.92-kb HaeIII fragment of pAC407 (NEUWEILERet al. 1990). Analysis of samplesforrestrictionfragmentlength polymorphism (RFLP):DNA was isolated from peripheral blood cells (Australian aborigine, Caucasian and Chinese samples) or from transformed lymphoblastcelllines (Assamese, Cambodian and Melanesian samples) and digested with 10-fold excess of PstI restriction endonuclease (International Biotechnologies Inc., IBI) according to the manufacturer’s specifications. The conditions for fractionation of DNA by agarose gel electrophoresis, transfer ofDNA to nylon membrane and hybridization to radioactively labeled probes has been described previously (BALAS et al. 1989). The size of the polymorphic DNA fragments detected for each locus was determined relative to that of DNAsize fragments ofknownsize. The measurements were performed using a digitizing tablet connected to a computer (BAIRDet al. 1986).

I. Balazs et al.

192

TABLE 1 Heterozygosity of VNTR loci in various populations Heterozygosity at locus: ~~~

D2S44 Ethnic group

~

~~

Dl 7379

D14S13D14S1D4S 163

D18S27 ~~

Caucasian Chinese Cambodian Assamese Melanesian Australian aborigine

0.86 (2349) 0.75 (153) 0.65 (23) 0.67 ( 1 8 ) 0.50 (20) 0.68 (90)

0.94 (530) 0.94 (179) 0.87 (23) 0.78 (18) 0.80 (20) 0.94 (70)

0.91 (602) 0.89 (149) 0.78 (23) 0.89 (18) 0.85 (20) 0.72 (24)

0.93 (1555) 0.92 (166) 0.96 (23) 0.89 (18) 0.90 (20) 0.80 (59)

Average

~~~

0.85 (2285) (1085) 0.87 0.89 0.76 (172) (166) 0.76 0.74 (23) (23)0.83 0.67 ( 1 8 ) 0.61 (18) 0.70 (20) 0.80 0.76 (20) 0.72 (87) 0.77(76)0.76

0.84 0.80 0.75

The number of individuals examined is shown in parentheses.

RESULTS AND DISCUSSION

Heterozygosity of the polymorphic loci:The definition of heterozygosity for RFLP is strictly functional. It indicates the fraction of individuals with two distinct size alleles. A relative measure of the heterogeneity within different populations can be obtained by comparing their averageheterozygosities at several polymorphic loci. T h e results obtainedfromthe analysis of six polymorphic lociwith PstI-digested DNA from six populations are summarized in Table 1. Caucasians had the highest heterozygosity (89%), followed by Chinese (84%) and Cambodians (80%). Australian aborigines, Assamese and Melanesians had an average heterozygosity of about 76%. T h e general interpretation of heterozygosity is that it reflects the levelof genetic variation in the populations. The results show that the variations were not very large and may mostly represent sampling error. To determine whether the differences in heterozygosity were significant, at the 95%confidence level, pairwise comparisons of the loci from each population were performed. The results indicate that Caucasians, when compared with the otherfive populations, show statistically significant differences atonetothree loci. Comparisons of the heterozygosity values for Cambodian, Assamese and Chinese did not reveal statistically significant differences at any loci. This type of VNTR locus contains a large number of alleles. Therefore, theobserved heterozygosity will be affected by the resolution of the gel and thesize of the polymorphic DNA fragments. In turn, thesize of the DNA fragments will vary depending on the choice of restriction endonuclease used to digest the DNA samples. An extreme example is the case of D2S44. T h e alleles for PstI-digested DNA are about 9kb larger than the alleles of HueIII-digested DNA. As a result,the heterozygosity of D2S44 with Hue111is 94% vs. 86% with PstI. However, for the other loci, these two restriction enzymes produced only small differences in size, hence similar values of heterozygosity. Analysis of the VNTR locus DlS58 by the polymerase chain reaction (KASAI, NAKAMURA and WHITE

1989) shows that this type of polymorphism can vary by intervals of a single repeat unit. The size of the repeat units, for the loci used in this study, ranges from 14 bp (e.g., D14S13) to 32 bp (e.g., D17S79). Clearly, given the resolution of the gels used in this study, the vast majority of closely spaced alleles will coalesce and will be scored as homozygotes (DEVLIN, RISCHand ROEDER1990). Therefore, the observed heterozygosity value for each locus is an underestimate of the real value. Calculations to determine whether VNTRloci (ie., D2S44, D14S13, D17S79, D18S27) met Hardy-Weinberg expectations were performed using the method of DEVLIN,RISCHand ROEDER(1 990), forCaucasians and Chinese. No excess or deficiency of heterozygotes was detected at these loci for these two populations. T h e number of individuals in the other populations was not enough to perform the calculations. Allele frequency distribution: The distribution of polymorphic DNA fragments was determined in the various populations for six VNTR loci. Since the method used to fractionate DNA does not allow for the separation of every DNA fragment, it is not possible to obtain directly the truefrequency of individual alleles. For some of these loci it may be possible to use nonparametricmethodstoobtain maximum likelihood estimates for allele frequency (DEVLIN,RISCH and ROEDER199 1).Unfortunately,thenumber of individuals available, for most of the populations examined, was too small for this type of analysis. Each of these polymorphic locishows the same broad range of DNA fragment sizes in all the populations examined. The frequencydistribution,for each locus (Figures 1through 6),is shown for Chinese, Australian aborigines and Caucasians. Frequency values, shown in these figures, were calculated at increments of 0.005% of fragment size. Since the number of individuals tested from the other three populations ( i e . , Assamese, Cambodian, Melanesian) were 25 or fewer, the data points were too sparse to make this type of graphic illustration meaningful. Instead, comparisons of the various populations were performed by measuring the combined frequencyof polymorphic

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FIGURE2.-Frequency distribution of PstI-DNA fragments in random individuals, at the D4S163 locus; (A) Australian aborigines, (B) Caucasians, (C) Chinese. The number of individuals tested were 70, 530 and 179, respectively.

DNA fragments at different size ranges (see below). Comparison of allele frequency distributions in Caucasians from the United States and Australia: In addition to studies of allele frequency distribution for these six populations, two geographicallydistinct Caucasian populations (ie., Australia and North America) were analyzed with several of the polymorphic loci. The purpose of this study was to determine whether any major differences could be found between them. The origins within Europe of these twopopulations is different. More than 80% of the Australian population is ofEnglish/Irish/Scottish descent while the American population is mixed European with an estimated frequency of English/Irish/Scottish of only 25-30%.

So far four locihavebeen compared D2S44, D14S13, D17S79 and D18S27. Examples of the distributions obtained for the two populations for locus D17S79 and D2S44 are shown superimposed in Figure 7, A and B. Although the number of individuals tested, from the two populations, was very different and the analysis was performed in different laboratories (ie., the United States and Australia), the superimposed profiles look very similar. The number and size of the most common DNA fragments appear to be the same and theminor variations in frequency are likely to reflect measurement and sampling errors. A more rigorous method to compare the frequency distributions of these Caucasian populations would be

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to incorporate the measurement error into the allele frequency estimates. Therefore, the two distributions were comparedafter calculating the allele frequencies as sliding windows encompassing f 3 SD of the measurement error (measurement error: 0.6%of fragment size). The sum of eventsobserved within this size range was used as an estimate forthe frequency of an allele (ie., +1.8% of the DNA fragment size). T h e results obtained forD l 7879 and D2S44 are shown in Figure 8, A and B. T h e frequency distribution patterns of each of these loci are very similar for thetwo Caucasian populations and they do not show statisti-

FIGURE4.-Frequency distribution of PstI-DNA fragments in random individuals, at the Dl 4S13 locus; (A) Australian aborigines, (B) Caucasians, (C) Chinese. The number of individuals tested were 59, 1555 and 166, respectively.

cally significant differences by the Kolmogorov-Smirnov test. One of the uses ofallele frequency estimates, for single locus DNA polymorphisms, is to calculate the frequency of particular genotypes in identity testing. The conclusion, based in this comparison, is that calculations using either database would be comparable. Estimation of admixture in Australian aborigines: There is the possibility that the distribution patterns obtained for Australian aborigines were affected by Caucasian admixture. An estimate of the amount of admixture could be calculated using a locus with alleles that are very common in Caucasians and rare in Australian aborigines.D14S13 contains a group of

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alleles at about 5 kb that is very common in Caucasians. The sum of the frequencies for DNA fragments from4.82 to 5.18kb is 34.8% in Caucasians and, when excluding the 8 half-caste individuals, 5.9% in aborigines. Using these values, and assuming that the ancestor population did not have alleles in that size range, the admixture of genes would be 17%. However, of the more than 15 populations from Africa, America, Asia and Europe, the lowest frequency observed, of these DNA fragments, was 5%. Using that value, the admixturewould be only 3%. Interestingly, an analysis of the eight mixed aborigines shows that the frequency of DNA fragments within the same size range is 19%. This intermediate value is consistent with what is expectedfrom half-caste heritage. Although this result suggests that the level of Caucasian admixture in the aboriginepopulation is low, this

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conclusion needs to be confirmed by supporting evidence from additional loci before a firm conclusion can be reached. Comparison of the frequency distributions,of allele groups, among Assamese, Australian, Cambodian, Caucasian, Chinese and Melanesian populations: In general, foreach locus, the overall size range of polymorphic DNA fragments detected was similar for ali the populations examined in this study. These results were analogous to those obtained in earlier comparisons between North American black, Caucasian and Hispanic populations (BALAZSet al., 1989). The major difference between populations was not the size range of the alleles at a locus but their relative frequencies. One approach used to study these variations was to subdivide the frequency distribution into

I. Balazs et al.

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size ranges or allele groups representing the major frequency peaks. Although the selection of these size ranges was seemingly arbitrary ( i e . , they do not equate to alleles), it serves as a simple methodto perform comparisons given the small sample size of many of these populations. For the locus D2S44, the data were subdivided into four groups or sizeclasses. The most common size group was from 10 to 11.5 kb (Figure 9A). Since the frequency estimates are affected by sampling error, a range of values representing a 95% confidence interval was calculated for each size range. T h e second most common size group of alleles was from 11.5 to 13 kb. In most populationsthese two size ranges accounted for more than 80% of all DNA fragment sizes detected. Even though the frequency values in these bar graphs correspond to a broad range of DNA fragment sizes,in many instances, the small differences observed were statistically significant. Analysis of the allele distribution of D4S163, in the various populations, shows thatthe most common DNA fragments are between 7.5 and 8.5 kb. The frequencies of these common alleles vary between populations and in some cases the differences are statistically significant. Forexample, the totalfre-

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quency of the group of DNA fragments, 7.5 to 8.5 kb, is about 15% higher in Australian aborigines than in Caucasians (Figure 9B). The main feature of D14S1 was that the majority of the DNA fragments were found between 3.7 and 4.5 kb (Figure 9C). The polymorphic DNA fragmentsdetectedfor D14S13, from 4.5 to 5.5 kb, were two to six times more frequent in Caucasians than in the other populations used in this study. The other region showing major differences in allele frequency has fragments larger than 9.0 kb. These alleles are relatively rare in Caucasians while they are 10 to 20 times more frequent in several of the otherpopulations (Figure OA). 1 The predominant feature of the polymorphic DNA fragments for D17S79 and D18S27 is that they are found mostly in clusters at various size ranges. Several of these size ranges show statistically significant differences in frequency between populations. For example, the D17S79 alleles, 3.3-3.4kb, areabout half as common in Caucasians than in Assamese, Cambodians or Chinese (Figure 1OB). Similarly for Dl 8S27 alleles, 4.5-4.7 kb, the frequency in Caucasians is about one

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FIGURElO.-Comparison of the frequencies of the major group of alleles in different ethnic groups: (A) D14S1.7, (H)D17S79, (C) D18S27. The sampling error associated with each frequency was calculated for a 95% confidence limit and it is represented by the vertical line over each bar.

half to a third lower than in the otherfive populations (Figure 1OC). Although it is not possible to show that two DNA fragments of the same size are identical by descent, a consistent similarity forthefrequency of several groups of alleles, across multiple polymorphic loci, may reflect a common ancestral origin for those populations. For example, a comparison of the frequency of these allele groups between the three Mongoloid populations, Assamese, Cambodian and Chinese, show few groups with statistically significant differences. Another method used to study these populations was to compare the frequency of the most common alleles, for the six loci, in the various populations (Table 2). These values were generated, as previously described, by summing the frequency of the DNA fragments within a +1.8% size range. The results show that the frequenciesof the most common alleles may vary up to two to three fold among the different populations. The locus with the highest frequencies was D17S79 which has the narrowest distribution of allele sizes of the six loci tested. It is interesting to note thateven in this locus there is large heterogeneity of alleles in all populations, as indicated by the fact that the frequency of the most common alleles (Table

2) vary only between 29 and 56%. In addition, many of these differences may be the result of the small number of individuals tested from most of the populations. Pairwise comparison of the highest frequency values for each locus, in the various populations, show that in the vast majority of cases the differences were not statistically significant (ie., 95% confidence limit). For example, the difference in values for D 18S27 in the six populations were not statistically significant. However, for locus Dl 7S79, the maximum allele frequency was lower in Caucasians than in theother populations and the difference was statistically significant. Most of the analysis presented here is semiquantitative in nature. However, more rigorous methods are being developed for the analysis of populations using this type of genetic marker (our manuscript in preparation). Thus,as more information is gathered from different populations it may be possible to establish a correlation between the frequency distributions for different loci and theorigin and/or migration patterns of human populations. This work was pxtially supported by National Science Foundation grant BNS 8813234 to K.K.K.

198

I. Balazs et al. TABLE 2 Frequency of the most commonalleles per locus in various populations Locus

Assamese

Australian

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Chinese

Melanesian

D2S44 No. chromosomes D4S163

0.31 f 0.1 51 36 0.31 f 0.1 51 36 0.28 f 0.1 47 36 0.22 -c 0.1 35 36 0.56 f 0.162 36 0.31 f 0.151 36

0.35 +. 0.070 180 0.30 f 0.076 140 0.22 f 0.1 17 48 0.10 f 0.054

0.48 f 0.144 46 0.22 f 0.120 46 0.37 f 0.140 46 0.17 k 0.109 46 0.46 f 0.144 46 0 . 2 8 f 0.130 46

0.23 f 0.012 4698 0.18 f 0.023 1060 0.27 f 0.025 1204 0.26 f 0.015 3110 0.29 f 0.013 4570 0 . 2 5 k 0.018 2170

0.36 f 0.054 306 0.17 k 0.039 358 0.20 f 0.045 298 0.08 f 0.029 332 0 . 4 4 k 0.052 344 0.28 k 0.048 332

0.45 f 0.154 40 0.28 f 0.139 40 0.21 f 0.126 40 0.20 f 0.124 40 0.45 k 0.154 40 0.25 k 0.134 40

No. chromosomes D14S1 No. chromosomes D14S13 No. chromosomes Dl 7S79 No. chromosomes D 18S27 No. chromosomes

118

0.46 f 0.074 174 0.21 f 0.065 152

T h e values after f represent at 95% confidence interval.

LITERATURE CITED BAIRD,M., I. BALAZS,A. GIUSTI,G. L. MIYASAKI, L. NICHOLAS,K. F. ALLEN, P.RUBINSTEIN WEXLER,E. KANTER,J. GLASSBERG, and L. SUSSMAN,1986 Allelefrequencydistributionoftwo highly polymorphic DNA sequences in three ethnic groups and its application to the determinationof paternity. Am.J. Hum. Genet. 3 9 489-501. BALAZS,I., M. BAIRD,M. CLYNEand E. MEADE,1989 Human population genetic studies offive hypervariable DNA loci. Am. J. Hum. Genet. 44: 182-190. CHIMERA, J. A., C. R. HARRISand M. LITT, 1989 Population genetics of the highly polymorphic locus D16S7 and its use in paternity evaluation. Am. J. Hum. Genet. 45: 926-93 1. DEKA,R., R. CHAKRABORTY and R. E. FERRELL,1991 A population genetic study ofsix V N T R loci in three ethnically defined populations. Genomics 11: 83-92. DEVLIN,B., N. RISCHand K. ROEDER,1990 No excess of homo249: zygosity at loci used for DNAfingerprinting.Science 1416-1420. DEVLIN,B., N. RISCHand K. ROEDER,1991 Estimation of allele

frequency for VNTR loci. Am. J. Hum. Genet. 48: 662-676. FLINT, J., A. J. BOYCE,J. J. MARTINSONand J. B. CLEGG, 1989 Populationbottlenecks in Polynesia revealed by minisatellites. Hum. Genet. 83: 257-263. lp, N., I. VANDE STADT,Z. LOEWY,S. LEARY,K. H. GRZESCHIK and I. BALAZS,1989 Identification and characterization of a hypervariableregion [D18S27] on chromosome 18. Nucleic Acids Res. 17: 8404 KASAI,K., Y . NAKAMURA and WHITE, R. 1990 Amplification of a variable number of tandem repeats (VNTR) locus (pMCT118) by the polymerase chain reaction (PCR) and its application to forensic science. J. Forensic Sci. 35: 1 196-1 200. KIDD,J. R., F. L. BLACK,K. M. WEISS,1. BALAZSand K. K. KIDD, 1991 Studies of three amerindian populations using nuclear DNA polymorphisms. Hum. Biol. 63: 775-794. NEUWEILER, J., V.RUVOLO, H. BAUM,K. H. GRZESCHIK and 1. BALAZS,1990 Isolation and characterization of a hypervariable region [D4S163] on chromosome4. Nucleic Acids Res. 18: 691. Communicating editor: B. S. Weir