Yixi Zhong

Development of a Highly Polymorphic STR Marker for Identity Testing Purposes at the Human Androgen Receptor Gene (HUMARA)

We developed a non-isotopic method which improves the technical quality of the X-linked HUMARA locus typing process. The use of formamide and a low concentration of acrylamide increased resolution and sharpness of HUMARA alleles in silver-stained polyacrylamide gels. In addition, the construction of an allelic ladder containing amplified sequence of 9 alleles (even-numbered alleles) of the HUMARA locus, allows confident, rapid and precise assignment of discretely defined alleles. Allele and genotype frequencies for the HUMARA locus were determined in a French Canadian population sample. Observed genotype frequencies in females conformed to Hardy-Weinberg expectations. Furthermore, the HUMARA locus is highly polymorphic with 18 observed alleles and an heterozygosity value of 89.3%. Also, this locus has average powers of discrimination of 97.8% and 88.7% for testing samples of female and male origin, respectively. In the French Canadian population, the average probability of excluding a random man as the father in paternity analysis when both mother and daughter are tested for this locus is 88.0%. Together, the results indicate that the HUMARA locus provides a highly discriminatory system that is appropriate for the purposes of forensic identification and paternity testing involving a female child.

The utility of short tandem repeat loci beyond human identification: Implications for development of new DNA typing systems

Since the first characterization of the population genetic properties of repeat polymorphisms, the number of short tandem repeat (STR) loci validated for forensic use has now grown to at least 13. Worldwide variations of allele frequencies at these loci have been studied, showing that variations of interpopulation diversity at these loci do not compromise the power of identification of individuals. However, data collected for validation of these loci for forensic use has utility beyond human identification; the origin and past migration history of modern humans can be reconstructed from worldwide variations at these loci. Furthermore, complex forensic cases previously unresolvable can now be investigated with the help of the validated STR loci. Here, we provide the absolute power of the validated set of 13 STR loci for addressing these issues using multilocus genotype data on 1,401 individuals belonging to seven populations (US European‐American, US African‐American, Jamaican, Italian, Swiss, Chinese and Apache Native‐American). Genomic research is discovering new classes of polymorphic loci (such as the single nucleotide polymorphisms, SNPs) and lineage markers (such as the mitochondrial DNA and Y‐chromosome markers); our aim, therefore, was to determine how many SNP loci are needed to match the power of this set of 13 STR loci. We conclude that the current set of STR loci is adequate for addressing most problems of human identification (including interpretations of DNA mixtures). However, if suitable number of SNPs are used that would match the power of the STR loci, they alone cannot resolve more complex cases unless they are supplemented by the validated STR loci.

Global genetic variation at nine short tandem repeat loci and implications on forensic genetics

We have studied genetic variation at nine autosomal short tandem repeat loci in 20 globally distributed human populations defined by geographic and ethnic origins, viz., African, Caucasian, Asian, Native American and Oceanic. The purpose of this study is to evaluate the utility and applicability of these nine loci in forensic analysis in worldwide populations. The levels of genetic variation measured by number of alleles, allele size variance and heterozygosity are high in all populations irrespective of their effective sizes. Single- as well as multi-locus genotype frequencies are in conformity with the assumptions of Hardy-Weinberg equilibrium. Further, alleles across the entire set of nine loci are mutually independent in all populations. Gene diversity analysis shows that pooling of population data by major geographic groupings does not introduce substructure effects beyond the levels recommended by the National Research Council, validating the establishment of population databases based on major geographic and ethnic groupings. A network tree based on genetic distances further supports this assertion, in which populations of common ancestry cluster together. With respect to the power of discrimination and exclusion probabilities, even the relatively reduced levels of genetic variation at these nine STR loci in smaller and isolated populations provide an exclusionary power over 99%. However, in paternity testing with unknown genotype of the mother, the power of exclusion could fall below 80% in some isolated populations, and in such cases use of additional loci supplementing the battery of the nine loci is recommended.

Genetic variation at twentythree microsatellite loci in sixteen human populations

We have analysed genetic variation at 23 microsatellite loci in a global sample of 16 ethnically and geographically diverse human populations. On the basis of their ancestral heritage and geographic locations, the studied populations can be divided into five major groups, viz. African, Caucasian, Asian Mongoloid, American Indian and Pacific Islander. With respect to the distribution of alleles at the 23 loci, large variability exists among the examined populations. However, with the exception of the American Indians and the Pacific Islanders, populations within a continental group show a greater degree of similarity. Phylogenetic analyses based on allele frequencies at the examined loci show that the first split of the present-day human populations had occurred between the Africans and all of the non-African populations, lending support to an African origin of modern human populations. Gene diversity analyses show that the coefficient of gene diversity estimated from the 23 loci is, in general, larger for populations that have remained isolated and probably of smaller effective sizes, such as the American Indians and the Pacific Islanders. These analyses also demonstrate that the component of total gene diversity, which is attributed to variation between groups of populations, is significantly larger than that among populations within each group. The empirical data presented in this work and their analyses reaffirm that evolutionary histories and the extent of genetic variation among human populations can be studied using microsatellite loci.

Estimation of mutation rates from parentage exclusion data: applications to STR and VNTR loci

Nonpaternity is a common source of bias in estimating mutation rates when they are obtained from family data showing discordance of parental and childrens genotypes. With the availability of hypervariable DNA markers, this source of bias can be largely eliminated. However, the proportion of cases where parentage exclusion is caused by presumed mutation(s) of parental alleles must be adjusted to obtain a valid mutation rate estimate. The present work derives the basis of this adjustment factor, called the proportional bias. This proportional bias depends upon the allele frequency distribution at the locus. The maximum and minimum bounds of the proportional bias depend on the number of alleles at the locus. Using data from Caucasian populations at tandem repeat loci commonly used for parentage testing and forensic identification purposes, we show that when mutation rates are estimated at these loci, the proportional bias is generally very close to the maximum possible value for the observed number of alleles (or binned fragment sizes) at each locus. The expected proportional bias decreases with increasing mutation rate at a locus. For the short tandem repeat loci, without bias correction, the direct count method can result in an underestimation of up to 60% of their true value. In contrast, for the minisatellite VNTR loci, even with crude measurements on allele sizes, we show that the absolute proportional bias is generally below the coefficient of variation of the direct estimates.

Patterns of instability of expanded CAG repeats at the ERDA1 locus in general populations.

A highly polymorphic CAG repeat locus, ERDA1, was recently described on human chromosome 17q21.3, with alleles as large as 50-90 repeats and without any disease association in the general population. We have studied allelic distribution at this locus in five human populations and have characterized the mutational patterns by direct observation of 731 meioses. The data show that large alleles (>/=40 CAG repeats) are generally most common in Asian populations, less common in populations of European ancestry, and least common among Africans. We have observed a high intergenerational instability (46. 3%+/-5.1%) of the large alleles. Although the mutation rate is not dependent on parental sex, paternal transmissions have predominantly resulted in contractions, whereas maternal transmissions have yielded expansions. Within this class of large alleles, the mutation rate increases concomitantly with increasing allele size, but the magnitude of repeat size change does not depend on the size of the progenitor allele. Sequencing of specific alleles reveals that the intermediate-sized alleles (30-40 repeats) have CAT/CAC interruptions within the CAG-repeat array. These results indicate that expansion and instability of trinucleotide repeats are not exclusively disease-associated phenomena. The implications of the existence of massively expanded alleles in the general populations are not yet understood.

Paternity evaluation in cases lacking a mother and nondetectable alleles

In parentage testing the formulae for computing paternity index and exclusion probability generally ignores the presence of nondetectable alleles at the loci tested. In contrast, it is now known that even when paternity testing is done with hypervariable DNA markers, nondetectable alleles should not be ignored. This work presents simple formulae needed with this consideration, to analyze paternity evaluation from DNA markers in cases where the mother of the disputed child is unavailable for testing. It is shown that even a modest frequency of nondetectable alleles (e.g., 2–5% per locus) may have a substantial impact on the paternity index when the child and/or the alleged father exhibits a single-banded DNA profile at a locus. Use of such formulae can generate a high probability of exclusion and a high paternity index when multiple independently segregating hypervariable DNA markers are used.ZusammenfassungBei der Vaterschaftsbestimmung ignorieren im allgemeinen die Formeln für die Berechnung des Paternitätsindex und der Ausschlußchance die Anwesenheit nicht-nachweisbarer Allele an den untersuchten Loci. Im Gegenteil, es ist jetzt bekannt, daß selbst, wenn Vaterschaftsuntersuchungen mit hypervariablen DNA-Markern durchgeführt werden, nicht nachweisbare Allele nicht ignoriert werden sollten. Diese Untersuchung präsentiert einfach Formeln, welche unter dieser Bedingung benötigt werden, um die Vaterschaftsbestimmung mit DNA-Markern in solchen Fällen durchzuführen, in denen die Mutter des zu untersuchenden Kindes nicht verfügbar ist. Es wird gezeigt, daß sogar eine mäßige Frequenz nicht detektierbarer Allele (z.B. 2–5% per Locus) einen substantiellen Einfluß auf den Paternitätsindex haben kann, wenn das Kind und/oder der Putativvater ein Einzelbandenmuster an einem Locus haben. Die Benutzung solcher Formeln kann eine hohe Wahrscheinlichkeit des Ausschlusses und einen hohen Paternitätsindex generieren, wenn zahlreiche, unabhängig voneinander segregierende hypervariable DNA-Marker benutzt werden.

ANALYSIS OF ALLELE DISTRIBUTION FOR SIX SHORT TANDEM REPEAT LOCI IN THE FRENCH CANADIAN POPULATION OF QUEBEC

Short tandem repeat (STR) loci represent a rich source of highly polymorphic markers in the human genome which are useful for the purposes of forensic identification and determination of biological relatedness of individuals. Here, as a part of an ongoing extensive study, we report the analysis of a multilocus genotype survey of 642 to 870 chromosomes in the French Canadian Caucasian population of Québec at six STR loci. The loci HUMCSF1PO, HUMTPOX, HUMTH01, HUMF13A01, HUMFESFPS, and HUMvWA were typed using two multiplex polymerase chain reactions (PCR). Amplified DNA samples were subsequently analyzed by polyacrylamide gel electrophoresis followed by silver staining. The heterozygote frequencies of the loci range from 0.614 to 0.820 (0.661 to 0.818 expected) and the number of alleles from 7 to 12 per locus. Although statistically significant deviation from Hardy-Weinberg expectations of genotype frequencies was noted at some loci by one or more tests, in general, the genotype frequencies are well estimated from the product of allele frequencies at all loci. The most frequent six-locus genotype is expected to occur in the French Canadian population with a frequency of 3.50 by 10(-5) and together, these six loci have an average probability of discrimination of 0.9999985. The study presented here indicates that these six STR loci are informative genetic markers for identity testing purposes in the French Canadian Caucasian population of Québec.

Worldwide genetic diversity at the HLA-DQA1 locus

Genotype, gene, or phenotype frequency data, obtained by PCR analysis with sequence specific oligonucleotide probes at the HLA‐DQA1 locus, in 176 population samples, each consisting of 30 or more individuals, from all around the world were analyzed. The sampled populations were assigned to the following nine groups: African, Afro‐American, American Native, American Mestizo, Asian, American Caucasian, European Caucasian, Caucasian from other regions, and Pacific Islanders. Observed genotype proportions agreed with Hardy‐Weinberg expectations (HWE) in 90 of the 102 populations for which genotype data are available. The 12 discordant population samples were of substantial mixed origin. For these discordant populations, 41 of the 252 (population by genotype) data points showed significant departures of the observed frequencies in comparison to their HWE predictions. The deviations, by and large (38 of the 41), were in the direction of HWE over‐estimating the actual genotype frequencies. Total heterozygosity (HT) at a worldwide level was 79%, and varied from 56% in American Natives to >80% in Caucasians. Of the total diversity, 94.4% was due to intra‐population and 5.6% to inter‐population variation (FST), partitioned into 3.0% inter‐population within groups and 2.6% inter‐group variation. FST for the locus was high among Africans and American Natives (>9%) and low in Afro‐Americans and American Caucasians (<0.5%). The range for allele specific FST was 0.2% to 5.9%, and the lowest value did not correspond to the same allele in all nine groups. A variance component analysis of allele frequencies showed no relationship of the ratio of between/within group variation with the world average frequency of the respective alleles. The first three principal components explained 36.2%, 27.5%, and 22.9% of the total allelic diversity, respectively. For Caucasian and Afro‐American samples, the first two PCs formed clusters by groups. In contrast, the American Native, Asian, and Pacific Islander groups showed a greater inter‐population diversity, while the corresponding principal component scores of the American Mestizo samples were between American Natives and Caucasians. In aggregate, the analyses indicate that genetic drift in contrast to natural selection, more readily explains the pattern of worldwide diversity at the HLA‐DQA1 locus. Am. J. Hum. Biol. 9:735–749, 1997.

A Comparative Study of Genetic Variation at Five VNTR Loci in Three Ethnic Groups of Houston, Texas

Following the technique of Southern blot restriction fragment length polymorphisms (RFLP) analysis, we generated a database of DNA profiles at five Variable Number of Tandem Repeats loci (D1S7, D2S44, D4S139, D10S28, and D17S79) for 669 individuals of three major ethnic populations (Caucasians, Blacks, and Hispanics) of Houston, Texas. Analysis of fragment sizes at these loci within each sample, as well as their fixed-bin analyses, reveal that the assumptions of independence of allelic occurrences within and between loci are valid for this database. Fixed-bin allele frequency tables, therefore, are the best descriptors of this database for conservative forensic calculations. Finally, we demonstrate that this regional database from Houston, Texas, does not yield any meaningfully different forensic inference than the one obtained from the National database of the respective ethnic groups.