Evaluation of reliability on STR typing at leukemic patients used for forensic purposes

Over the past decades, main advances in the field of molecular biology, coupled with benefits in genomic technologies, have led to detailed molecular investigations in the genetic diversity generated by researchers. Short tandem repeat (STR) loci are polymorphic loci found throughout all eukaryotic genome. DNA profiling identification, parental testing and kinship analysis by analysis of STR loci have been widely used in forensic sciences since 1993. Malignant tissues may sometimes be the source of biological material for forensic analysis, including identification of individuals or paternity testing. There are a number of studies on microsatellite instability in different types of tumors by comparing the STR profiles of malignant and healthy tissues on the same individuals. Defects in DNA repair pathways (non-repair or mis-repair) and metabolism lead to an accumulation of microsatellite alterations in genomic DNA of various cancer types that result genomic instabilities on forensic analyses. Common forms of genomic instability are loss of heterozygosity (LOH) and microsatellite instability (MSI). In this study, the applicability of autosomal STR markers, which are routinely used in forensic analysis, were investigated in order to detect genotypes in blood samples collected from leukemic patients to estimate the reliability of the results when malignant tissues are used as a source of forensic individual identification. Specimens were collected from 90 acute and 10 chronic leukemia volunteers with oral swabs as well as their paired peripheral blood samples from the Oncology Centre of the Department of Hematology at Istanbul University, during the years 2010–2011. Specimens were tested and compared with 16 somatic STR loci (CSFIPO, THO1, TPOX, vWA, D2S1338, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D19S433, D21S11 and FGA) widely used in forensic identification and kinship. Only two STR instabilities were encountered among 100 specimens. An MSI in the FGA loci and a LOH in the D2S1338 loci were determined in two individuals separately. Our results demonstrate that the use of the biological samples from leukemia patients in forensic identification and kinship testing is questionable, especially if known microsatellite instability is available. Genetic instabilities may alter the STR polymorphism, leading to potential errors on forensic identification of individuals. Therefore, typing of autosomal STRs from leukemia patients should be performed with both healthy and malignant tissue samples of individual as references.     

Characterization of resistance to clofentezine in populations of European red mite from orchards in Ontario

Resistance to clofentezine was identified in four populations of the European red mite, Panonychus ulmi (Koch), from apple orchards in Ontario after ca. 5 years use. Resistance was expressed at high levels (> 2000-fold at the LC 50) to clofentezine in a population selected in the laboratory. This population was resistant to hexythiazox and to the organotin compounds cyhexatin and fenbutatin-oxide. However, a population selected with fenbutatin-oxide did not show cross resistance to clofentezine. The clofentezine-resistant population was not resistant to pyridaben, propargite or dicofol. The synergists, piperonyl butoxide and DEF, were ineffective in overcoming the resistance. Resistance to clofentezine declined rapidly in a mixed population (most resistance was lost in fewer than three generations). There were several fitness factors (fewer eggs/female, longer egg stage, longer development time for males) associated with the resistant population which may have contributed to the loss of resistant phenotypes. However, in a second test in which selection was removed from a resistant population, resistance persisted for at least 10 generations. Observations on field populations indicated that resistance persisted for at least two seasons. This revised version was published online in July 2006 with corrections to the Cover Date.     

Developing forensic reference database by 18 autosomal STR for DNA identification in Republic of Belarus

For the Republic of Belarus, development of a forensic reference database on the basis of 18 autosomal microsatellites (STR) using a population dataset (N = 1040), “familial” genotypic dataset (N = 2550) obtained from expertise performance of paternity testing, and a dataset of genotypes from a criminal registration database (N = 8756) is described. Population samples studied consist of 80% ethnic Belarusians and 20% individuals of other nationality or of mixed origin (by questionnaire data). Genotypes of 12346 inhabitants of the Republic of Belarus from 118 regional samples studied by 18 autosomal microsatellites are included in the sample: 16 tetranucleotide STR (D2S1338, TPOX, D3S1358, CSF1PO, D5S818, D8S1179, D7S820, THO1, vWA, D13S317, D16S539, D18S51, D19S433, D21S11, F13B, and FGA) and two pentanucleotide STR (Penta D and Penta E). The samples studied are in Hardy–Weinberg equilibrium according to distribution of genotypes by 18 STR. Significant differences were not detected between discrete populations or between samples from various historical ethnographic regions of the Republic of Belarus (Western and Eastern Polesie, Podneprovye, Ponemanye, Poozerye, and Center), which indicates the absence of prominent genetic differentiation. Statistically significant differences between the studied genotypic datasets also were not detected, which made it possible to combine the datasets and consider the total sample as a unified forensic reference database for 18 “criminalistic” STR loci. Differences between reference database of the Republic of Belarus and Russians and Ukrainians by the distribution of the range of autosomal STR also were not detected, corresponding to a close genetic relationship of the three Eastern Slavic nations mediated by common origin and intense mutual migrations. Significant differences by separate STR loci between the reference database of Republic of Belarus and populations of Southern and Western Slavs were observed. The necessity of using original reference database for support of forensic expertise practice in the Republic of Belarus was demonstrated.     

Population Genetic Characteristics of the D1S80 locus in seven human populations

We have analyzed the allele frequency distribution at the highly polymorphic variable number of tandem repeat (VNTR) locus D1S80 (pMCT118) in seven ethnic populations (namely, New Guinea Highlanders of Papua New Guinea, Dogrib Indians of Canada, Pehuenche Indians of Chile, American and Western Samoans, Kacharis of Northeast India, and German Caucasians) using the polymerase chain reaction (PCR) technique. In the pooled sample of 443 unrelated individuals 20 segregating alleles were detected. A trimodal pattern of allelic distribution is present in the majority of populations and is indicative of the evolutionary antiquity of the polymorphism at this locus. In spite of the observed high degree of polymorphism (expected heterozygosity 56%–86%), with a single exception — the marginally significant P value (0.04) of the exact test in American Samoans — the genotype distributions in all populations conform to their respective Hardy-Weinberg expectations. Summary statistics indicate that, in general, the allele frequency distribution at this locus may be approximated by the infinite allele model. The data also demonstrate that alleles that are shared by all populations have the highest average frequency within populations. Furthermore, the kinship bioassay analysis demonstrates that the extensive variation observed at the D1S80 locus is at the interindividual within population level, which dwarfs any interpopulation allele frequency variation, consistent with the population dynamics of hypervariable polymorphisms. These characteristics of the D1S80 locus make it a very useful marker for population genetic research, genetic linkage studies, forensic identification of individuals, and for determination of biological relatedness of individuals.     

The locus for combined factor V-factor VIII deficiency (F5F8D) maps to 18q21, between D18S849 and D18S1103.

Combined factor V-factor VIII deficiency (F5F8D) is a rare, autosomal recessive coagulation disorder in which the levels of both coagulation factor V and coagulation factor VIII are diminished. In order to map and subsequently clone the gene responsible for this phenotype, DNAs from 19 families (16 from Iran, 2 from Pakistan, and 1 from Algeria) with a total of 32 affected individuals were collected for a genomewide linkage search using genotypes of highly informative DNA polymorphisms. All pedigrees except two contained at least one consanguineous marriage. A maximum LOD score (Zmax) of 14.82 for theta = .02 was generated with marker D18S1129 in 18q21; LOD scores > 9 were obtained for several other markers-D18S849, D18S1103, D18S64, and D18S862. Multipoint analysis resulted in Zmax = 18.91 for the interval between D18S1129 and D18S64. Informative recombinants placed the locus for F5F8D between D18S849 and D18S1103, in an interval of approximately 1 cM. These results are similar to the recently reported linkage of this disease to chromosome 18q in Jewish families (Nichols et al. 1997) and provide evidence that the same gene is responsible for all F5F8D among human populations. The difference in clinical severity of the phenotype in unrelated families, as well as the failure to detect a specific haplotype of DNA polymorphisms in the consanguineous Iranian families, suggests the existence of different molecular defects in the F5F8D gene. There exists an apparently gap-free contig with CEPH YACs linking the two markers on either side of the critical region. Positional cloning efforts are now in progress to clone the F5F8D gene.     

Lipid metabolism indices and their correlation with vitamin D levels in indigenous populations of northern European Russia

The level of 25-OH vitamin D in the blood plasma in indigenous residents of northern European Russia has been studied. Vitamin D deficiency has been shown to be widespread. The lowest prevalence of vitamin D deficiencies (15%) has been found in male reindeer herders; the highest (80–100%) one, in adolescents aged 13–15 years. A significant decrease in the 25-OH vitamin D₃ level has been found in the inhabitants of the Far North in March. A relationship has been found between the serum 25-OH vitamin D concentration and lipid metabolism parameters in residents of the Far North, which is confirmed by significant correlations with the total cholesterol, apolipoprotein A, high- and low-density lipoprotein, and vitamin A and E levels.     

Detection of European Peristenus digoneutis loan in mirid populations in southern Ontario

Lygus spp. Hahn (Hemiptera: Miridae) are serious pests of a wide variety of economically important crops in North America. A European parasitoid, Peristenus digoneutis Loan (Hymenoptera: Braconidae), released in the northeastern USA for the biological control of Lygus, has successfully established in nine states and in eastern Canada, including southeastern Ontario, southern Quebec and Nova Scotia. To determine the extent to which P. digoneutis has dispersed and established in mirid populations in Ontario, a single-step multiplex PCR assay, designed to differentiate European species P. digoneutis and P. relictus from native North American Peristenus species, was used to identify parasitoid larvae dissected from field-collected Miridae. A total of 222 parasitoid larvae were analysed with the multiplex assay. Most (172) were identified as members of the P. pallipes Curtis complex; however, three from the Niagara region were identified as P. digoneutis. Specimens that did not amplify using the Peristenus multiplex PCR assay were screened with a PCR primer set designed to detect Leiophron (Hymenoptera: Braconidae) species, and most were confirmed to be Leiophron larvae. This study confirms the presence of P. digoneutis species in southern Ontario, and is another example of the utility of molecular methods for the detection of newly introduced or dispersed parasitoids. The presence of exotic P. digoneutis in southern Ontario may expedite future releases to augment already-established populations. Following such releases, the Peristenus multiplex PCR assay will be a useful component of post-release studies to evaluate the success of the biological control programme for Lygus plant bugs in Canada.     

European population substructure: clustering of northern and southern populations

Using a genome-wide single nucleotide polymorphism (SNP) panel, we observed population structure in a diverse group of Europeans and European Americans. Under a variety of conditions and tests, there is a consistent and reproducible distinction between “northern” and “southern” European population groups: most individual participants with southern European ancestry (Italian, Spanish, Portuguese, and Greek) have >85% membership in the “southern” population; and most northern, western, eastern, and central Europeans have >90% in the “northern” population group. Ashkenazi Jewish as well as Sephardic Jewish origin also showed >85% membership in the “southern” population, consistent with a later Mediterranean origin of these ethnic groups. Based on this work, we have developed a core set of informative SNP markers that can control for this partition in European population structure in a variety of clinical and genetic studies.     

Linkage disequilibrium for DNA haplotypes near the cystic fibrosis locus in two South European populations

Summary Three polymorphic DNA marker loci (INT1L1, D7S23 and D7S399) map to a chromosomal region that is very close to the cystic fibrosis (CF) locus in terms of genetic distance. These marker loci have been used to analyse the linkage disequilibrium in 137CF families from two South European countries (Italy and Spain). The markers can be analysed for differences in linkage disequilibrium more easily in these populations than in North Europeans, in whom the disequilibrium between the allelic systems defined by the probes and CF is much greater and on a plateau through the genetic region. The different levels of disequilibrium found in the studied populations suggest that D7S399 and D7S23 are both closer to CF than INT1L1, and provide additional information on the origins and homogeneity of the CF defect.     

Characterization and fine-mapping of a resistance locus for northern leaf blight in maize bin 8.06

As part of a larger effort to capture diverse alleles at a set of loci associated with disease resistance in maize, DK888, a hybrid known to possess resistance to multiple diseases, was used as a donor in constructing near-isogenic lines (NILs). A NIL pair contrasting for resistance to northern leaf blight (NLB), caused by Setosphaeria turcica, was identified and associated with bin 8.06. This region of the maize genome had been associated in previous studies with both qualitative and quantitative resistance to NLB. In addition, bins 8.05–8.06 had been associated with quantitative resistance to several other diseases, as well as resistance gene analogs and defense response gene homologs. To test the hypothesis that the DK888 allele at bin 8.06 (designated qNLB8.06 _DK888 ) conditions the broad-spectrum quantitative resistance characteristic of the donor, the NILs were evaluated with a range of maize pathogens and different races of S. turcica. The results revealed that qNLB8.06 _DK888 confers race-specific resistance exclusively to NLB. Allelism analysis suggested that qNLB8.06 _DK888 is identical, allelic, or closely linked and functionally related to Ht2. The resistance conditioned by qNLB8.06 was incompletely dominant and varied in effectiveness depending upon allele and/or genetic background. High-resolution breakpoint analysis, using ~2,800 individuals in F₉/F₁₀ heterogeneous inbred families and 98 F₁₀/F₁₁ fixed lines carrying various recombinant events, delimited qNLB8.06 _DK888 to a region of ~0.46 Mb, spanning 143.92–144.38 Mb on the B73 physical map. Three compelling candidate genes were identified in this region. Isolation of the gene(s) will contribute to better understanding of this complex locus.     

Population studies of 17 equine STR for forensic and phylogenetic analysis

As a consequence of the close integration of horses into human society, equine DNA analysis has become relevant for forensic purposes. However, the information content of the equine Short Tandem Repeat (STR) loci commonly used for the identification or paternity testing has so far not been fully characterized. Population studies were performed for 17 polymorphic STR loci (AHT4, AHT5, ASB2, ASB17, ASB23, CA425, HMS1, HMS2, HMS3, HMS6, HMS7, HTG4, HTG6, HTG7, HTG10, LEX3 and VHL20) including 8641 horses representing 35 populations. The power of parental exclusion, polymorphic information content, expected and observed heterozygosity and probability of identity were calculated, showing that the set of 17 STRs has sufficient discriminating power for forensic analysis in almost all breeds. We also explored the reliability of individual assignment tests in identifying the correct breeds of origin for unknown samples. The overall proportion of individuals correctly assigned to a population was 97.2%. Finally, we demonstrate the phylogenetic signal of the 17 STR. We found three clusters of related breeds: (i) the cold-blooded draught breeds Haflinger, Dutch draft and Friesian; (ii) the pony breeds Shetland and Miniature horse with the Falabella, Appaloosa and Icelandic; and (iii) The Warmblood riding breeds, together with the hot-blooded Standard-bred, Thoroughbred and Arabian.     

Haplotype frequencies at the DRD2 locus in populations of the East European Plain

Background

A key to increasing the power of multilocus association tests is to reduce the number of degrees of freedom by suppressing noise from data. One of the difficulties is to decide how much noise to suppress. An often overlooked problem is that commonly used association tests based on genotype data cannot utilize the genetic information contained in spatial ordering of SNPs (see proof in the Appendix), which may prevent them from achieving higher power.

Results

We develop a score test based on wavelet transform with empirical Bayesian thresholding. Extensive simulation studies are carried out under various LD structures as well as using HapMap data from many different chromosomes for both qualitative and quantitative traits. Simulation results show that the proposed test automatically adjusts the level of noise suppression according to LD structures, and it is able to consistently achieve higher or similar powers than many commonly used association tests including the principle component regression method (PCReg).

Conclusion

The wavelet-based score test automatically suppresses the right amount of noise and uses the information contained in spatial ordering of SNPs to achieve higher power.     

Benign recurrent intrahepatic cholestasis (BRIC): evidence of genetic heterogeneity and delimitation of the BRIC locus to a 7-cM interval between D18S69 and D18S64

Benign recurrent intrahepatic cholestasis (BRIC) is an autosomal recessive liver disease characterized by multiple episodes of cholestasis without progression to chronic liver disease. The gene was previously assigned to chromosome 18q21, using a shared segment analysis in three families from the Netherlands. In the present study we report the linkage analysis of an expanded sample of 14 BRIC families, using 15 microsatellite markers from the 18q21 region. Obligate recombinants in two families place the gene in a 7-cM interval, between markers D18S69 and D18S64. All intervening markers had significant LOD scores in two-point linkage analysis. Moreover, we identified one family in which the BRIC gene seems to be unlinked to the 18q21 region, or that represents incomplete penetrance of the BRIC genotype. Received: 6 March 1997 / Accepted: 26 March 1997     

Weak population differentiation in northern European populations of the endangered anadromous clupeid Alosa fallax

Examination of genetic structure among different spawning populations of the endangered twaite shad Alosa fallax showed statistically significant but weak differentiation estimated using nine microsatellite loci. Results point to substantial gene flow among the majority of individual estuary/river spawning locations, and no evidence that habitat disruption has led to increased local genetic drift.     

Genetic variation of three autosomal STR Loci D21S1435, D21S1411, and D21S1412 in Korean population

The autosomal tetranucleotide short tandem repeat loci D21S1435, D21S1411 and D21S1412 were analyzed in samples of unrelated 200 Korean individuals. The loci showed no significant deviations from Hardy–Weinberg equilibrium. Alleles were assigned according to the International Society for Forensic Haemogenetics (ISFH) recommendations. The power of discrimination of the analyzed markers was found to be high for the populations, thereby facilitating the validation and efficiency of these STR markers in forensic human identification and paternity testing. To our knowledge, this is the first report of the nomenclature and the allele frequency data for these three STR loci in Korean population.