Independence of VNTR alleles defined as floating bins.

Data bases of VNTR fragments determined for Caucasians and blacks by Cellmark Diagnostics and Lifecodes Corporation are analyzed for independence of variants within and between loci. Floating bins are constructed around specific fragment lengths and are used to define discrete genotypes. Simple chi 2 test statistics for independence of bins within and between loci are described and applied to large sets of randomly generated four-locus profiles. The proportions of significant test statistics were about as expected under the hypotheses of independence, suggesting the absence of both Hardy-Weinberg and linkage disequilibrium. In any particular forensic application, however, these tests need to be performed on the fragments in question.     

Correlation of DNA fragment sizes within loci in the presence of non-detectable alleles

At present most forensic databases of DNA profiling of individuals consist of DNA fragment sizes measured from Southern blot restriction fragment length polymorphism (RFLP) analysis. Statistical studies of these databases have revealed that, when fragment sizes are measured from RFLP analysis, some of the single-band patterns of individuals may actually be due to heterozygosity of alleles in which fragment size resulting from one allele remains undetected. In this work, we evaluate the effect of such allelic non-detectability on correlation of fragment sizes within individuals at a locus, and its impact on the inference of independence of fragment sizes within loci. We show that when non-detectable alleles are present in a population at a locus, positive correlations of fragment sizes are expected, which increase with the proportion of non-detectable alleles at the locus. Therefore, a non-zero positive correlation is not a proof of allelic dependence within individuals. Applications of this theory to the current forensic RFLP databases within the US show that there is virtually no evidence of significant allelic dependence within any of the loci. Therefore, the assumption that DNA fragment sizes within loci are independent is valid, and hence, the population genetic principles of computing DNA profile frequencies by multiplying binned frequencies of fragment sizes are most likely to be appropriate for forensic applications of DNA typing data.Editor's commentsThe presence of non-detectable alleles for VNTR loci has plagued the use of these highly-discriminating systems in human identification. The authors take explicit account of these alleles, and are able to show independence of the frequencies of detectable alleles. They raise the troubling issue of how to account for occasional significant results when multiple tests are performed. By invoking Bonferroni corrections, they regard all tests, even those performed on different loci, as addressing the same hypothesis—the absence of dependence at any VNTR locus.     

VNTR loci reveal differentiation between and structure within populations of the eastern barred bandicoot Perameles gunnii

The Eastern Barred Bandicoot Perameles gunnii has declined in abundance within mainland south-eastern Australia, to a relict wild population of less than 100 individuals in Hamilton, Victoria. It is more common, but is also declining in Tasmania. Genomic DN A variability was compared within and between surviving populations of P. gunnii using variable number of tandem repeat (VNTR) markers in one of two ways. First, average percentage differences (APDs) were determined between profiles for two VNTR probe—endonuclease combinations. Secondly, because one of these combinations revealed two multiallelic VNTR loci, genotypes were assigned and analysed for homogeneity of allele frequencies among subpopulations, for deviation of heterozygosity from Hardy-Weinberg equilibrium within populations and for genetic structuring among individuals from different subpopulations. The results of both the APD and defined locus approaches showed consistent trends within and between populations. Genetic variability was higher among mainland P. gunnii than in Tasmanian populations (higher APDs, number of alleles, and heterozygosity at one locus), despite the known decline and subdivision of the Hamilton population. Eleven per cent of the variability detected in Hamilton was attributed to genetic differentiation between east and west subdivisions of the population. Departure from random mating indicating local inbreeding within collecting localities was evident for one locus in both north and south Tasmania, particularly at one locality. AH alleles at both loci were unique to either Hamilton or Tasmanian P. gunnii. The initial captive colony contains high heterozygosity for these loci. It is concluded that VNTR markers can be of benefit for use in studies of population differentiation and for conservation management.     

Nondetectability of restriction fragments and independence of DNA fragment sizes within and between loci in RFLP typing of DNA.

We provide experimental evidence showing that, during the restriction-enzyme digestion of DNA samples, some of the HaeIII-digested DNA fragments are small enough to prevent their reliable sizing on a Southern gel. As a result of such nondetectability of DNA fragments, individuals who show a single-band DNA profile at a VNTR locus may not necessarily be true homozygotes. In a population database, when the presence of such nondetectable alleles is ignored, we show that a pseudodependence of alleles within as well as across loci may occur. Using a known statistical method, under the hypothesis of independence of alleles within loci, we derive an efficient estimate of null allele frequency, which may be subsequently used for testing allelic independence within and across loci. The estimates of null allele frequencies, thus derived, are shown to agree with direct experimental data on the frequencies of HaeIII-null alleles. Incorporation of null alleles into the analysis of the forensic VNTR database suggests that the assumptions of allelic independence within and between loci are appropriate. In contrast, a failure to incorporate the occurrence of null alleles would provide a wrong inference regarding the independence of alleles within and between loci.     

Violations of the ceiling principle: exact conditions and statistical evidence.

The National Research Council recommended the use of the ceiling principle in forensic applications of DNA testing on the grounds that the ceiling principle was believed to be "conservative," giving estimates greater than or equal to the actual genotype frequencies in the appropriate reference population. We show here that the ceiling principle can fail to be conservative in a population with two subpopulations and two loci, each with two alleles at Hardy-Weinberg equilibrium, if there is some linkage disequilibrium between loci. We also show that the ceiling principle can fail in a population with two subpopulations and a single locus with two alleles if Hardy-Weinberg equilibrium does not hold. We give explicit analytical formulas to describe when the ceiling principle fails. By showing that the ceiling principle is not always mathematically reliable, this analysis gives users of the ceiling principle the responsibility of demonstrating that it is conservative for the particular data with which it is used. Our reanalysis of VNTR data bases of the FBI provides compelling evidence of two-locus associations within three major ethnic groups (Caucasian, black, and Hispanic) in the United States, even though the loci tested are located on different chromosomes. Before the ceiling principle is implemented, more research should be done to determine whether it may be violated in practice.     

Independence tests for VNTR alleles defined as quantile bins.

VNTR fragment lengths in three databases maintained by the FBI for forensic purposes were partitioned into quantile bins, and tests for independence of the two bins at each of six loci were conducted. Whether independence was declared depended on the number of quantiles used. For a large number of quantile bins, equal to the number of fixed bins used by the FBI, 10 of 18 likelihood-ratio tests showed significant departures from independence when all genotypes were considered, and this changed to 7 of 18 when only heterozygotes were tested. This is in contrast to likelihood-ratio tests on fixed bins, when there were five significant departures over all genotypes and two departures for heterozygotes.     

Worldwide genetic relationships among Francisella tularensis isolates determined by multiple-locus variable-number tandem repeat analysis

The intracellular bacterium Francisella tularensis is the causative agent of tularemia and poses a serious threat as an agent of bioterrorism. We have developed a highly effective molecular subtyping system from 25 variable-number tandem repeat (VNTR) loci. In our study, multiple-locus VNTR analysis (MLVA) was used to analyze genetic relationships and potential population structure within a global collection of 192 F. tularensis isolates, including representatives from each of the four subspecies. The VNTR loci displayed between 2 and 31 alleles with Nei's diversity values between 0.05 and 0.95. Neighbor-joining cluster analysis of VNTR data revealed 120 genotypes among the 192 F. tularensis isolates, including accurate subspecies identification. F. tularensis subsp. tularensis (type A) isolates showed great diversity at VNTR loci, while F. tularensis subsp. holarctica (type B) isolates showed much lower levels despite a much broader geographical prevalence. The resolution of two distinct clades within F. tularensis subsp. tularensis (designated A.I and A.II) revealed a previously unrecognized genetic division within this highly virulent subspecies. F. tularensis subsp. holarctica appears to have recently spread globally across continents from a single origin, while F. tularensis subsp. tularensis has a long and complex evolutionary history almost exclusively in North America. The sole non-North American type A isolates (Slovakian) were closely related to the SCHU S4 strain. Significant linkage disequilibrium was detected among VNTR loci of F. tularensis consistent with a clonal population structure. Overall, this work greatly augments the study of tularemia ecology and epidemiology, while providing a framework for future forensic analysis of F. tularensis isolates.     

PERMANENT GENETIC RESOURCES: Isolation and characterization of 12 microsatellite markers in the middle-spotted woodpecker (Dendrocopos medius)

Twelve polymorphic microsatellite loci were developed for Dendrocopos medius. Polymorphism was assessed for 27 individuals from the southwesternmost population of this woodpecker species. The number of alleles per locus ranged from three to seven, with observed heterozygosity values from 0.444 to 0.852. Genotypic frequencies conformed to Hardy-Weinberg equilibrium, and no evidence for linkage disequilibrium was observed. Multilocus genotypes resulting from this set of markers will be useful to determine genetic diversity and differentiation within and among habitat patches inhabited by D. medius. Three of the loci were polymorphic for Picoides articus.     

Inference of population subdivision from the VNTR distributions of New Zealanders

A population sample from people of diverse ethnic origins living in New Zealand serves as a database to test methods for inference of population subdivision. The initial null hypothesis, that the population sample is homogeneous across ethnic groups, is easily rejected by likelihood ratio tests. Beyond this, methods for quantifying subdivision can be based on the probability of drawing alleles identical by descent (F _ST ), probabilities of matching multiple locus genotypes, and occurrence of unique alleles. Population genetic theory makes quantitative predictions about the relation betweenF _ST , population sizes, and rates of migration and mutation. Some VNTR loci have mutation rates of 10^–2 per generation, but, contrary to theory, we find no consistent association between the degree of population subdivision and mutation rate. Quantification of population substructure also allows us to relate the magnitudes of genetic distances between ethnic groups in New Zealand to the colonization history of the country. The data suggests that the closest relatives to the Maori are Polynesians, and that no severe genetic bottleneck occurred when the Maori colonized New Zealand. One of the central points of contention regarding the application of VNTR loci in forensics is the appropriate means for estimating match probabilities. Simulations were performed to test the merits of the product rule in the face of subpopulation heterogeneity. Population heterogeneity results in large differences in estimates of multilocus genotype frequencies depending on which subpopulation is used for reference allele frequencies, but, of greater importance for forensic purposes, no five locus genotype had an expected frequency greater than 10^–6. Although this implies that a match with an innocent individual is unlikely, in a large urban area such chance matches are going to occur.Editor's commentsA side-benefit of the collection of DNA data from human populations is the light it may shed on human evolution. The authors discuss the colonization history of New Zealand in the light of such data. From a forensic viewpoint, too much should not be made of the differences between the major ethnic groups within New Zealand, as the forensic community in that country maintains separate databases for Caucasian, Maori and Pacific Islander (Buckletonet al., 1987). It will be of interest in the future to examine subdivision within these groups, as opposed to within the country as a whole. The authors' comments on testing for independence will need to read along with the findings of Zaykinet al. and Maiste and Weir in this volume. The authors had not seen the Budowleet al. (1994) rebuttal to the paper of Kraneet al. (1992).     

Effects of subpopulation structure on probability calculations of DNA profiles from forensic PCR analysis

DNA typing for forensic identification is a two-step process. The first step involves determining the profiles of samples collected at the crime scene and comparing them with the profiles obtained from suspects and the victims. In the case of a match that includes the suspect as the potential source of the material collected at the crime scene, the last step in the process is to answer the question, what is the likelihood that someone in addition to the suspect could match the profile of the sample studied? This likelihood is calculated by determining the frequency of the suspect's profile in the relevant population databases. The design of forensic databases and the criteria for comparison has been addressed by the NRC report of 1996 (National Research Council, 1996). However, the fact that geographical proximity, migrational patterns, and even cultural and social practices have effects on subpopulation structure establishes the grounds for further study into its effects on the calculation of probability of occurrence values. The issue becomes more relevant in the case of discrete polymorphic markers that show higher probability of occurrence in the reference populations, where several orders of magnitude difference between the databases may have an impact on the jury. In this study, we calculated G values for all possible pairwise comparisons of allelic frequencies in the different databases from the races or subpopulations examined. In addition, we analyzed a set of 24 unrelated Caucasian, 37 unrelated African-American, and 96 unrelated Sioux/Chippewa individuals for seven polymorphic loci (DQA1, LDLR, GYPA, HBGG, D7S8, GC, and D1S80). All three sets of individuals where sampled from Minnesota. The probability of occurrence for all seven loci were calculated with respect to nine different databases: Caucasian, Arabic, Korean, Sioux/Chippewa, Navajo, Pueblo, African American, Southeastern Hispanic, and Southwestern Hispanic. Analysis of the results demonstrated marked differences in the probabilities of occurrence when individuals were compared to the different populations and subpopulation databases. The possible genetic and forensic consequences of subpopulation structure on probability calculations are discussed.     

Testing independence of fragment lengths within VNTR loci.

Methods that were devised to test independence of the bivariate fragment lengths obtained from VNTR loci are applied to several population databases. It is shown that for many of the probes independence (Hardy-Weinberg equilibrium) cannot be sustained.     

Diversity of ethnic and racial VNTR RFLP fixed-bin frequency distributions.

To examine the impact that intra- and interracial genetic diversities have on VNTR RFLP-fragment-size distributions, a multiracial (East Asian, African American, U.S. Southwest Hispanic, and European Caucasian) and multiethnic (Chinese, Japanese, Korean, and Vietnamese) database has been constructed for the following loci: D1S7, D2S44, D4S139, and D10S28. Homogeneity between samples was examined using the Komologorov-Smirnov two-sample test for RFLP fragment sizes and a log-likelihood test for fixed-bin frequencies with theoretical and Monte Carlo empirical significance levels. Small but significant differences between theoretical and empirical significance-level distributions were observed with both procedures, particularly with the latter. The significance levels of the two types of tests were poorly correlated. Statistically significant differences in fragment-size and fixed-bin distributions were found within and between races, with greater differences occurring between races. Cluster analysis and principal components analysis, using different similarity measures, did not support the hypothesis of greater intra- than interracial diversity, which suggests that ethnic variation can be conservatively estimated by racial variation.     

Frequency of the hypervariable DNA loci D18S849, D3S1744, D12S1090 and D1S80 in a mixed ancestry population of Chilean blood donors

Blood donors (N = 150) at San José Hospital (Santiago, Chile) were typed for one VNTR locus (D1S80) and three STR loci (D18S849, D3S1744, D12S1090). A questionnaire was used to determine the socioeconomic level of the donors, because it is known that some genetic markers (e.g., the ABO and Rh groups) are differentially distributed between different socioeconomic strata. This methodology revealed that two of the three socioeconomic strata distinguishable in Santiago were present in our sample of blood donors, with stratum II representing the middle strata and stratum III the low strata. Allele frequency was determined for each locus and socioeconomic stratum, and it was found that the allele distributions of each locus in socioeconomic strata II and III were statistically similar. All loci conformed to the Hardy-Weinberg law and there was no evidence for association between the alleles of the four loci, allelic frequencies being similar to those found in North American Hispanic populations. The results support the view that the analysis of these loci may have useful applications in population genetics as well as in identity tests.     

Linkage Disequilibrium between Allozymes in Natural Populations of Lodgepole Pine

Pairwise linkage disequilibrium values (D) were estimated for 14 allozyme loci in two natural populations of lodgepole pine (Pinus contorta ssp. latifolia). Maternal multilocus genotypes were inferred from samples of (haploid) megagametophytic seed-endosperms. Coupling/repulsion double heterozygotes were distinguished for closely linked pairs of loci. Assays of seven of the loci in seed embryos allowed estimates of D for these loci in the outcross pollen pool (estimates of outcrossing rates indicate no significant departures from random mating in either population). No disequilibrium was observed between unlinked loci in either maternal genotypes or outcross pollen. However, significant disequilibrium was observed within and between gametes for some allelic combinations of four tightly linked loci; the assumption of random association of gamete types within individuals is thus invalid for some loci in lodgepole pine. Possible causes of the observed D were examined using the noncentrality parameter of the general noncentral chi square distribution. We concluded, from estimates of population size, linkage and measurements of population substructure, that neither drift nor population subdivision was responsible for the significant values of D which were observed and that epistatic selection was the most likely cause of the disequilibrium observed.     

Enzyme variability in natural populations of Daphnia magna. IV. Ecological differentiation and frequency changes of genotypes at Audley End.

Genotypic frequencies were analysed for two years in a permanent population of the cladoceran crustacean, Daphnia magna, which was polymorphic for an esterase and for malate dehydrogenase. Large temporal changes in genotypic frequencies occurred at both loci. There was no evidence of a seasonal pattern in the frequency changes. In most samples, genotypes at the two enzyme loci were non-randomly associated; these associations showed temporal changes. On some occasions marked spatial heterogeneity in genotypic frequencies existed within the population. Genotypic differences in parthenogenetic and sexual egg production were observed. In a primarily parthenogenetically reproducing population, non-random associations between genotypes of structural and regulatory loci will be the rule. The allozyme variants themselves may or may not be under selection. The relevance of these observations to ecological studies on Daphnia is considered.     

Population genetics of the HRAS1 minisatellite locus.

Several years ago it was reported that rare HRAS1 VNTR alleles occurred more frequently in U.S. Caucasian cancer patients than in unaffected controls. Such an association, in theory, could be caused by undetected population heterogeneity. Also, in a study clearly relevant to this issue, it was recently reported that significant deviations from Hardy-Weinberg equilibrium exist at this locus in a sample of U.S. Caucasians. These considerations motivate our population genetic analysis of the HRAS1 locus. From published studies of the HRAS1 VNTR locus, which classified alleles into types, we found only small differences in the allele frequency distributions of samples from various European nations, although there were larger differences among ethnic groups (African American, Caucasian, and Oriental). In an analysis of variation of rare-allele frequencies among samples from four European nations, most of the variance was attributable to molecular methodology, and very samples from four European nations, most of the variance was attributable to molecular methodology, and very little of the variance was accounted for by nationality. In addition, we showed that mixture of European subpopulations should result in only minor deviations from expected genotype proportions in a Caucasian database and demonstrated that there was no significant deviation from Hardy-Weinberg equilibrium in our HRAS1 data.     

A comparison of tests for independence in the FBI RFLP data bases

Several tests of independence of alletic frequencies within and between loci have been compared, and it has been found that Fisher's exact test is the best test to use. When this test is applied to RFLP databases established by the FBI, paying no attention to the single-band problem, there is generally evidence for independence at one locus but not at two loci. When the test is restricted to double-banded entries in the databases; there is overall evidence for independence.     

Study of human SP-A,SP-B and SP-D loci: allele frequencies,linkage disequilibrium and heterozygosity in different races and ethnic groups

Background  

SP-A, SP-B, and SP-D are pulmonary surfactant proteins. Several linkage and association studies have been done using these genes as markers to locate pulmonary disease susceptibility genes, but few have studied the markers systematically in different ethnic groups. Here we studied eight markers in SP-A, SP-B, and SP-D genes in seven ethnic groups from three races (Caucasian, Black and Hispanic). We measured the similarity of the marker distribution among the ethnic groups in order to see whether people in different ethnic groups or races could be mixed together for linkage and association studies. To evaluate the usefulness of these markers, we estimated the informativeness of each marker loci in the seven ethnic groups by assessing their heterozygosity and PIC values. We also conducted linkage disequilibrium (LD) analysis to identify associated marker loci and to estimate the haplotype frequencies in each of the seven ethnic groups in an attempt to find valuable haplotypes so that the level of polymorphism of the "markers" could be increased.     

Five polymorphisms of the apolipoprotein B gene in healthy Bulgarians

Five APOB polymorphisms (I/D in the promoter region, XbaI [codon 24881, MspI [codon 3611], EcoRI [codon 41541, and 3' VNTRs) were studied in a population sample of 147 healthy normolipemic Bulgarians. For all biallelic loci, the observed genotype distributions do not deviate from Hardy-Weinberg equilibrium. In Bulgaria the insertion allele and the MspI+ allele of APOB presented the highest allelic frequencies (0.793 +/- 0.024 and 0.959 +/- 0.012, respectively) among the European population groups studied so far. The allele frequencies of the other two biallelic polymorphisms (XbaI and EcoRI) found in the Bulgarian population are similar to those previously described in other Caucasian populations. Analysis of the 3' VNTR polymorphism revealed 11 different alleles. Like studies in other Caucasian populations, this study found bimodal allele-size distribution and a high level of heterozygosity. The frequency of allele *31 (0.162 +/- 0.022) among Bulgarians is higher than that of any other European population group studied so far. Genetic distances between Bulgarians and each of six populations from southeastern Europe for which 3' VNTR allele frequencies are available showed an increase in the order: Albanians相似文献