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PubMed1999

[Polymorphisms of five short tandem repeat systems in Chinese Han population in Chengdu]

OBJECTIVE: To get preliminary genotype and allele frequency distributions of D6S366, D6S477, D12S375, D12S391 and PLA2A loci in Chinese Han population in Chengdu area and to validate more short Tandem repeat (STR) systems for forensic application. METHODS: The polymerase chain reaction (PCR) was used. Five STR systems (D6S366, D6S477, D12S375, D12S391 and PLA2A) were amplified on DNA samples, which were extracted from 108 unrelated individuals EDTA-blood by Phenol/Chloroform method. The PCR products were analyzed by PAG vertical electrophoresis. RESULTS: Seven alleles were found at D6S366 locus, nine alleles at D6S477 locus, five alleles at D12S375 locus, nine alleles at D12S391 locus and seven alleles at PLA2A locus. No deviations from Hardy-Weinberg equilibrium were observed. The heterozygosities observed were 0.69, 0.78, 0.81, 0.68 and 0.79 for D6S366, D6S477, D12S375, D12S391 and PLA2A respectively. The chances of exclusion were 0.3621, 0.6004, 0.4581, 0.7014 and 0.5589 and the discriminating powers were 0.79, 0.93, 0.86, 0.96 and 0.91. CONCLUSION: All of the five loci in this study were useful markers for individual identification and paternity test and for genetics purposes.

Journal Article↗
PubMed2001

[Analysis of short tandem repeats with fluorescence labeling polymerase chain reaction to document engraftment following allogeneic bone marrow transplantation].

OBJECTIVE: In order to determine the status of engraftment following allogeneic bone marrow transplantation (allo-BMT) and predict the outcome, short tandem repeats (STR) were analysed by fluorescence labeling polymerase chain reaction (PCR). METHOD: Fluorescence labeling PCR detecting STR 9 loci was established. DNA was extracted by monoclonal magnetic beads from recipients peripheral blood nucleated cells and labeled with four color fluorescences before and from 7 days to six months after allogeneic bone marrow transplantation. RESULTS: 1. STR and clinical feature of 12 patients after allogeneic bone marrow transplantation were analysed. In the ten patients with full donor chimeras, 8 received marrow grafts from HLA identical sibling donors, 1 from HLA identical unrelated donor and 1 from HLA haploidentical mother. All of the 10 patients had durable engraftment and survived disease-freely for 3-25 months of follow-up. 2. One patient received a HLA-AB loci mismatched graft and his STR showed mixed chimeras at day 30, and converted into full donor chimeras at day 60 after transplantation. 3. One patient received HLA haploidentical sibling donor stem cells failed to be engrafted and died 4 months later. CONCLUSION: Analysis of multi-loci of STR after allo-BMT provided a real indication of engraftment. Mixed chimeras after transplantation suggested that donor lymphocytes or bone marrow cells be re-infused to enhance the conversion from mixed chimeras to full donor chimeras. For the documentation of engraftment, analysis of STR was better than any other available methods.

Adolescent↗
PubMed2003

[Genotype reliability of short tandem repeats typing from minute DNA].

OBJECTIVE: To understand the forensic practice of DNA profiling from minimal amount of DNA. METHODS: Serial dilutions of DNA were amplified with the PowerPlex 16 System Kit, then the genotyping of short tandem repeat(STR) was performed by ABI 377 DNA automated Sequencer. RESULTS: When the mount of DNA template was less than 250 pg, allelic drop-out apparently occurred at several loci. Other disturbed peaks, such as artefact bands and imbalanced heterozygote, also presented. CONCLUSION: The anomalous results may result in incorrect genotyping. Careful and comprehensive considerations are needed to interpret the STR profile of minute DNA.

Alleles↗
PubMed1995

Short tandem repeat typing of bodies from a mass disaster: high success rate and characteristic amplification patterns in highly degraded samples.

We have used a PCR-based DNA-typing method, involving the coamplification of four tetrameric short tandem repeat loci, in the analysis of a large number of severely degraded tissue samples taken from the scene of a mass disaster in which bodies were exposed to extreme thermal, physical and chemical insult. Analysis of the amplified DNA in a number of the samples revealed uniquely sized artifact PCR products resulting from the amplification of degraded genomic DNA as well as characteristic patterns in the amounts of PCR products generated from differently sized loci. This system has proved to be very reliable and robust, and we were successful in typing all of the four loci in 66% of the samples tested and at least one locus in 83% of the cases. A PCR-based sex test also proved to be very effective when applied to the degraded samples.

Alleles↗
PubMed1999

Analysis and interpretation of short tandem repeat microvariants and three-banded allele patterns using multiple allele detection systems.

The Palm Beach County Sheriffs Office (PBSO) Crime Laboratory and the Alabama Department of Forensic Sciences (ADFS) have validated and implemented analysis of short tandem repeat (STR) sequences on casework using silver staining kit and SYBR Green I detection systems and are presently validating fluorescently tagged STR alleles using the Hitachi FMBIO 100 instrument. Concurrently, the Broward County Sheriff's Office (BSO) Crime Laboratory is validating the ABI Prism310 Genetic Analyzer capillary electrophoresis STR detection system (ABI CE310) from Perkin Elmer Applied BioSystems. During the course of analyzing over 10,000 individuals for the STR loci CSF1PO, TPOX and THO1 (CTT) using silver staining for allele detection, 42 samples demonstrated alleles that were "off ladder," contained three-banded patterns at a single locus, or exhibited an apparent THO1 "9.3,10" allele pattern. PBSO, ADFS and BSO Crime Laboratories have collaborated on the verification of the allele patterns observed in these 42 samples using the following allele detection systems: (1) manual silver staining, (2) SYBR Green I staining, and/or (3) fluorescently tagged amplified products separated by polyacrylamide gel electrophoresis or capillary electrophoresis followed by laser detection. Regardless of the CTT allele detection system utilized, concordant results were obtained for 41 of the 42 samples. The only exception was a sample in which a wide band within the THO1 locus was identified as a THO1 "9.3, 10" genotype by silver staining kit and SYBR Green I staining but was verified to be a THO1 "9.3" homozygote by all other allele detection systems. Manual allele detection could readily identify microvariants, as a visual assessment of stained gels clearly shows that alleles do not migrate coincident with well-characterized allele size standards. As would be predicted, however, the manual detection systems did not provide adequate resolution to approximate the basepair size for off-ladder variants. All fluorescent software program systems were consistent in designating alleles "not in range" or "off ladder," thereby indicating true microvariants. All single-locus three-banded patterns were detected using all of the STR multiplex systems. In addition, individual locus-specific primers verified multiplexed amplified products were specific for the locus in question.

Alleles↗
PubMed1995

Genetic typing with HUMTH01, HUMVWA31A and HUMFES/FPS short tandem repeat loci, D1S80 variable number tandem repeat locus and HLA-DQ alpha of recent and from XII-XIII centuries spongy bone.

The genetic analysis of ancient populations through DNA from bone remains, requires use of short sized loci that can be amplified by polymerase chain reaction (PCR) for which the short tandem repeat (STR) loci are most suitable. These techniques can also be applied to genetic identification in forensic casework. In this study three STR loci, HUMFES/FPS, HUMTH01, and HUMVWA31A, were selected to estimate their usefulness when applied to recent and ancient spongy bone DNA typing. In addition, loci D1S80 and HLA DQ alpha were also tested in the analysis of recent spongy bone DNA. The recent remains studied were constituted by ten spongy bone samples of postmortem material from one individual buried for 1 year. The ancient remains are composed by 8 spongy bone samples from the heads of left femurs from a XII-XIII Centuries Basque Country population. Adequate amplification and typing results could only be obtained with cetyltrimethyl ammonium bromide (CTAB)-extracted DNA, without any further purification after precipitation. Genotypes of the one year post-mortem material and those of his son and his wife were obtained at the D1S80, HLA-DQ alpha, and STR loci. In all these systems, no exclusion was observed, with a combined probability of paternity of 0.9997. This demonstrates the reliability of the obtained results. The genetic typing of HUMTH01 in spongy bone from the XII-XIII Centuries Basque Country individuals was also performed. This will allow the genetic analysis of ancient bone remains and therefore, to carry out evolutionary population studies.

Base Sequence↗
PubMed1995

Intra- and inter-population diversity at short tandem repeat loci in diverse populations of the world.

To study the level of intra- and inter-population variation at hypervariable DNA loci, we have characterized 15 human populations of diverse ethnic and geographic origins at six short tandem repeat loci by using the polymerase chain reaction. Even though the spectrum of allelic variation is quite broad and there are substantial differences in allele frequency distributions among populations, in general, population within a major racial group show a greater degree of similarity. This observation is reflected in the analysis of gene diversity. When the total diversity is apportioned, the maximum variation becomes attributable to inter-individual differences within a population; of the variation that is attributed to differences between populations within a racial group and differences between racial groups, namely, African, Caucasian, and Mongoloid, than the American Indians and the Pacific Islanders. As expected, a reciprocal relationship between gene diversity and FST levels is observed. Higher values of FST in the American Indian and the Pacific Islanders may reflect smaller population size and a higher level of isolation. An analysis of genetic distance encompassing the populations belonging to the three major racial groups recognizes three distinct clusters - all the populations of African affiliation cluster together, as do the Caucasian affiliated and the Mongoloid groups, in two distinct clusters. Interestingly, three broadly classified cosmopolitan US populations, namely, US White, US Black and US Asian, cluster with their ancestrally related populations. This study dispels some of the concerns regarding the applicability of DNA typing data for forensic use.

Alleles↗
PubMed1995

PCR analysis of the short tandem repeat (STR) system HUMVWA31. Allele and genotype frequencies in an Italian population sample.

A population study in a sample of 211 unrelated individuals from 2 cities in North and Central Italy was carried out to investigate the short tandem repeat (STR) system HUMVWA31. Separation of PCR- amplified DNA fragments was performed by high-resolution horizontal denaturing polyacrylamide gel electrophoresis (PAGE) followed by silver staining. The 7 common alleles were found, together with a new smaller allele. Distribution of the observed genotypes did not deviate from Hardy-Weinberg (H-W) equilibrium. The power of discrimination for this locus was 0.93 and the chance of exclusion was 0.61. Good agreement was found between the allele frequencies in 2 Italian population samples and previous studies on Caucasians. The results of this study suggest that this STR system may be a useful tool in forensic investigations.

Alleles↗
PubMed1999

Carrier detection and presymptomatic identification of Wilson disease in Chinese by non-isotopic linkage analysis with four short tandem repeat polymorphisms.

Wilson disease (WD) is an autosomal recessive disorder of copper metabolism. To establish an efficient, accurate and fast diagnostic method for carrier detection and presymptomatic identification of WD in Chinese population, we studied haplotypes of short tandem repeat (STR) polymorphisms flanking the WD gene in 40 Chinese WD families. The results suggested that this genetic diagnosis system based on the four STR polymorphisms is of high value for the detection of potential carriers and WD homozygotes in families with at least one previously affected child. It is an efficient, accurate and fast diagnostic method that can be well suited for routine use in clinical laboratories.

Asian People↗
PubMed2004

Feasibility of a cost-effective approach to evaluate short tandem repeat markers suitable for chimerism follow-up.

BACKGROUND: Precise chimerism monitoring is important for the prediction of the success of allogeneic bone marrow transplantation (BMT). Most of the current procedures employed for chimerism follow-up with short tandem repeat (STR) markers are either time-consuming, labor-intensive, or use expensive assays, making it burdensome to perform large-scale studies of transplanted patients. AIM: To set-up a simple nonradioactive method to investigate a set of STR markers that could be used in the evaluation of chimerism status after allogeneic BMT. METHOD: Six dinucleotide STRs (D2S123, D5S107, CRTL1, D7S500, D11S1356, and TP53) were analyzed by touchdown (TD)-PCR followed by medium size non-denaturing polyacrylamide gel electrophoresis and silver staining. The sensitivity of the approach was evaluated by dilution competition assays. Peripheral blood samples were taken from a group of 50 healthy Argentinean donors, two transplanted patients, and their respective bone marrow donors. Buccal mucosa samples were also obtained from the BMT recipients. RESULTS: Four markers, D2S123, D7S500, D11S1356, and TP53, presented the highest heterozygosities (0.67-0.88) under our experimental system. A sensitivity of 0.8-1.6% for chimerism detection was consistently found for the different STR. The usefulness of these STR in chimerism analysis was illustrated with the screening of related siblings analyzing two transplanted patients with persistent mixed chimerism, which were previously studied by fluorescence in situ hybridization (FISH). Similar proportions of mixed chimerism were obtained with STR analysis compared with those estimated by FISH. DISCUSSION: To our knowledge, this was the first study of mixed chimerism using TD-PCR to achieve a highly specific STR amplification. This approach allows simple and accurate chimerism quantification because it avoids slippage of Taq polymerase on repeat stretches and prevents the differential amplification of the shorter allele. STR heterozygosities and the high level of sensitivity of this method demonstrated that this approach is not only very informative in this population, but is also rapid (taking less than 14 hours) and cost-efficient. CONCLUSION: The data confirms that this method is a useful tool applicable to routine large-scale STR genotyping and mixed chimerism analysis in low-complexity laboratories worldwide.

Bone Marrow Transplantation↗
PubMed2000

Population frequency for the short tandem repeat loci D18S849, D3S1744, and D12S1090 in Caucasian-Mestizo and African descent populations of Colombia.

Blood samples from 489 unrelated Caucasian Mestizo and 252 individuals of African descent in Colombia were amplified and typed for three short tandem repeat (STR) markers (D12S1090, D3S1744, and D18S849). All markers conformed to Hardy-Weinberg equilibrium expectations in both populations studied. In addition, heterozygosity, mean exclusion chance, polymorphism information content, discrimination power, and the assumption of independence within and between loci were determined. The mean exclusion chance for all three STR markers is 0.9750 in the Caucasian Mestizo population and 0.9731 in the African Colombian Population. The discrimination power is 0.999925 and 0.999911 in the Caucasian Mestizo and African Colombian respectively.

Black People↗
PubMed2004

[Genetic polymorphism of short tandem repeat loci D1S549, D3S1754 and D12S375 in Qingdao Han population].

OBJECTIVE: To know the genotype and allele frequency distribution of D1S549, D3S1754 and D12S375 in Chinese Han population in the Qingdao area and to study the three short tandem repeat(STR) loci for genetic application. METHODS: ACD-blood specimens were collected from the unrelated individuals in Qingdao. The DNA samples were extracted with the use of Chelex method and were amplified by polymerase chain reaction (PCR) technique. The PCR products were analyzed by polyacrylamide gel electrophoresis and were visualized by silver staining. RESULTS: Eight alleles were found at D1S549 locus, eight alleles at D3S1754 locus and five alleles at D12S375 locus, and 22, 19 and 14 genotypes were identified respectively. No deviation from Hardy-Weinberg equilibrium was observed in the three loci. The heterozygosities expected of them were 0.7988, 0.7087 and 0.75 respectively. The exclusion probability was calculated as 0.6592 for D1S549, and 0.5605 for D3S1754, and 0.5864 for D12S375. The discriminating power of the three loci were 0.9143, 0.8382 and 0.8861. Comparison of the allelic frequencies in Qingdao area with those in Hans of Chengdu area by chi-square test showed a difference statistically significant at D1S549 locus but no difference at D3S1754 and D12S375 loci. CONCLUSION: This study reveals the structure of the three loci and the obtained data are beneficial to understanding the population genetics in Chinese Han population. All of the three loci have higher chance of exclusion and higher discriminating power, and they will be useful markers for individual identification, paternity test and genetics purposes.

China↗
PubMed2002

Rapid carrier screening using short tandem repeats in the phenylalanine hydroxylase gene.

Phenylketonuria (PKU) is an autosomal recessive genetic disorder caused by defects in the phenylalanine hydroxylase (PAH) system. Our work aimed to screen the PAH locus for the presence of potentially useful short tandem repeats (STR) as markers for carrier detection in PKU families in Egypt, and to determine the level of PAH heterozygosity within the Egyptian population. The system contains at least eight independent alleles in the Egyptian population, transmitted in a Mendelian fashion. Variations in the number of STR in the 16 families studied gave rise to polymorphisms that proved to be suitable markers for PKU carrier detection and prenatal diagnosis. The most frequent allelic fragment size in PKU patients was 246 bp (35.7%), which together with a fragment of 254 bp accounted for 60.7% of the mutant chromosomes.

Asian People↗
PubMed1998

Genetic analysis of the short tandem repeat system D12S391 in the German and three Asian populations.

Genomic DNA samples from 222 individuals from Southern China, 154 individuals from Thailand, 100 individuals from Japan as well as from 124 German individuals were analysed for the short tandem repeat (STR) locus D12S391. Typing was carried out by polymerase chain reaction (PCR) amplification and subsequent polyacryramide gel electrophoresis and silver staining. In total, 12 alleles could be distinguished in two of the populations. Among Chinese, allele 19 is the most common with a frequency of 0.225, and among Germans, allele 18 with a frequency of 0.186. In the Thai population only 11 alleles could be distinguished and allele 19 is the most common with a frequency of 0.198. In Japanese, two previously unknown alleles 27 and 28 were detected, 14 alleles could be distinguished, and allele 18 is the most common with a frequency of 0.295. The expected exclusion chance for Chinese, Thai, Japanese and Germans in paternity cases is 0.67, 0.71, 0.67 and 0.75, respectively, and the discrimination power in identification cases is 0.95, 0.96, 0.95 and 0.97, respectively. Testing of the observed genotype distributions for Hardy-Weinberg equilibrium did not reveal any significant deviations. Segregation studies of 124 meioses among German families did not reveal any mutations at the D12S391 locus. In casework studies two variant alleles were detected with a trimeric repeat each (17.3 and 18.3), which have been confirmed by sequencing.

Alleles↗
PubMed2001

Standardisation of multiplex fluorescent short tandem repeat analysis for chimerism testing.

To evaluate the origin of cells after allogeneic haematopoietic stem cell transplantation we optimised and evaluated two commercially available systems (AmpFlSTR Profiler Plus and GenePrint Powerplex-16) which are based on multiplex fluorescent short tandem repeat (STR) analysis. A standard procedure for interpretation of electropherographs was found essential to obtain reproducible results. On the basis of the relative length of donor and recipient alleles, TYPE-I (no shared alleles are used to calculate chimerism), TYPE-II (one shared and one unshared allele is used to calculate chimerism) or TYPE-III (not informative) allelic distribution types were distinguished. Also, stutter peaks were recognised as an important criterion to exclude a marker for analysis. Intralaboratory and multicentre evaluation of the AmpFlSTR Profiler Plus system showed that mixed blood samples could be determined with an absolute deviation of less than 2%. A sensitivity threshold was set at 5% for TYPE-I and 10% for TYPE-II markers since relative imprecision increases at low chimerism values. No significant difference of calculated chimerism values was observed between STR markers shared between both systems. By monitoring 26 allogeneic peripheral blood stem cell transplants, the applicability of the proposed method was demonstrated.

Alleles↗
PubMed2000

Short tandem repeat methodology for genotypic identification of single-person versus multi-person use of syringes.

OBJECTIVE: To develop laboratory methods to differentiate between single- versus multi-person use of syringes by injection drug users. METHODS: Forensic short tandem repeat (STR) genetic analysis was undertaken to cross-validate a test panel of trace blood contents from syringes representing single- versus multi-person syringe use. Laboratory-simulated scenarios of needle sharing generated 34 syringe washes that were blinded for evaluation. Polymerase chain reaction was used to amplify the polymorphic STR locus D6S502 from blood trace contents in used syringes. Alleles were sized and quantified using a commercial gene sequencer. A statistical algorithm was developed to determine the number of alleles present in the amplified DNA fragments. Syringes with more than two expected alleles were considered to represent multi-person syringe use. Sensitivity, specificity and the kappa coefficient were calculated. RESULTS: Allelic matrix-based analysis of alleles from the single STR successfully characterized single-use (n = 12) and multiple-use (n = 22) syringes with 68% sensitivity and 100% specificity upon re-analysis. The extent of agreement over and above chance (kappa = 0.6; P < 0.0001) indicated good agreement for differentiating single- versus multi-person syringe use. CONCLUSIONS: These findings suggest that improved genotypic STR analysis of syringe material could be an adjunct to methods for validating self-reported needle sharing, conducting behavioral surveillance of needle-sharing behaviors, and evaluating interventions such as needle-exchange programs. Assays based on multiple STR loci will undoubtedly improve upon the promising results obtained from laboratory simulations of needle sharing.

Alleles↗
PubMed2006

Quantification of chimerism within peripheral blood, bone marrow and purified leukocyte subsets: comparison of singleplex and multiplex PCR amplification of short tandem repeat (STR) loci.

OBJECTIVE AND METHODS: Chimerism analysis has become a routine diagnostic procedure after haematopoietic allogeneic stem cell transplantation for early detection of relapse of disease or graft failure. Whereas some centres developed individual in-house short tandem repeat (STR) systems, others prefer commercial multiplex PCR systems. However, little is known about inter-assay variation, which could have a significant impact on treatment decision. We therefore compared two commercial multiplex PCR kits with our in-house STR system using different sample sources, such as peripheral blood (PB), bone marrow (BM) and specific leukocyte subsets. RESULTS: Fifty samples of eighteen paediatric patients were analysed. For neither material, PB, BM and leukocyte subtypes, a significant difference between the STR systems tested was observed. Chimerism analyses of each single STR primer, which is component of both the in-house and the commercial STR system, did not reveal significant differences. CONCLUSION: Our analysis demonstrates that similar results can be obtained with both assays, even when using various sample sources. Further evaluation of different test systems will help to increase interlaboratory standardisation of chimerism analyses for early clinical intervention.

Adolescent↗
PubMed2004

Short tandem repeat analysis to monitor chimerism in macaca fascicularis.

Chimerism assessment following bone marrow transplantation (BMT) in cynomolgus monkeys (cynos) has been hampered by the lack of good engraftment markers. In human BMT, such markers have been provided by short tandem repeat (STR) loci. We tested the idea that techniques effective for detecting human STR could be readily adapted to cynos. Genomic DNA was extracted from cyno unseparated blood or peripheral cell subsets. With only slight modifications, reagents for detecting human STR alleles were used to amplify and detect cyno STRs and to quantitate allelic mixtures on an automated sequencer. Of the 15 STR loci tested, only CSF1PO, D18S51, and FGA successfully amplified, with seven, seven and two alleles, respectively. CSF1PO and D18S51 heterozygosity (80% and 55%, respectively) allowed use of these two loci for chimerism quantitation after BMT. The successful adaptation of human STR reagents to monitor chimerism in transplanted cynos will facilitate the use of this species in preclinical tolerance studies.

Animals↗