Specific Short Tandem Repeats Research Articles

STRsensor: a computationally efficient method for STR allele-typing from massively parallel sequencing data.

Short tandem repeats (STRs) represent one of the most polymorphic variations in the human genome, finding extensive applications in forensics, population genetics and medical genetics. In contrast to the traditional capillary electrophoresis (CE) method, genotyping STRs using massive parallel sequencing technology offers enhanced sensitivity and accuracy. However, current methods are mainly designed for target sequencing with higher coverage for a specific STR locus, thereby constraining the utility of STRs in low- and medium-coverage whole genome sequencing (WGS) data. Here, we introduce STRsensor, a method designed to type STR alleles in low-coverage WGS data and target sequencing data, achieving a significant high detection ratio and accuracy. STRsensor employs two methods for STR allele-typing: the Kmers-based method and the CIGAR-based method. Furthermore, by incorporating a model for PCR stutters, STRsensor greatly enhances the accuracy of STR allele typing. With simulation data, we demonstrate that STRsensor achieves a detection ratio of 100$\%$ and an accuracy of 99.37$\%$ for a 30$\times $ WGS data, outperforming the existing methods, such as STRait Razor, STRinNGS, and HipSTR. When applied to real target sequencing data from 687 individuals, STRsensor achieves a detection ratio of 99.64$\%$ and an accuracy of 99.99$\%$. Moreover, STRsensor is a computationally efficient method that runs 79 times faster than HipSTR and 10 000 times faster than STRinNGS. STRsensor is freely available on GitHub: https://github.com/ChenHuaLab/STRsensor.

Optimization of Autosomal STR Markers for Equine Genotyping Using Multiplex PCR

The investigation of horse lineage was of paramount importance in the registration of different breeds, trade, and formulation of studbooks. The pioneering technique of DNA fingerprinting emerged as the first highly responsive method reliant on DNA for individual identification and the examination of genetic affiliations. Microsatellites were a valuable tool for analyzing the genetic variations present among different horse breeds. The International Society for Animal Genetics (ISAG) has endorsed a set of 17 specific Short Tandem Repeats (STRs) for the equine identification, although these can be quite expensive to obtain through commercially available multiplex kits. Objective: To determine five autosomal STR markers (HMS6, HMS7, ASB23, VHL20, and LEX14) were optimized using multiplex PCR for equine genotyping. Methods: DNA was extracted from a Thoroughbred horse blood sample via an organic extraction method. Sensitivity analysis determined the optimal PCR concentration. Genotyping was performed on the ABI PRISM® 3100XL, and data were processed with Gene Mapper ID 3.2v software. Results: The optimal conditions for multiplex PCR of HMS6, HMS7, ASB23, VHL20, and LEX14 primers were 60°C annealing temperature, 3ng DNA concentration and 6μM primer concentration. A 12.5μL PCR reaction volume was recommended for cost efficiency. Conclusions: The results of this research have the potential to create a cost-effective, regionally produced multiplex PCR kit. This kit would be designed for analyzing parentage lineage within the Equine family in Pakistan, incorporating ISAG-recommended markers: VHL20, HMS6, HMS7, ASB23, and additionally LEX14. It could significantly streamline the import and export of horses in Pakistan.

Pinpointing the Short-tandem Repeats Alleles for Ethnic Inferencing in Forensic Identification by K-medoids Approach

Abstract Background: The role of DNA analysis for ethnicity inferencing is a topic that attracts much interest from researchers in forensic identification, especially for identifying unknown bodies and trace evidence. So far, the approaches considered effective for ethnic inferencing are autosomal single-nucleotide polymorphisms, Y-chromosome short-tandem repeats (STRs), and mitochondrial DNA haplotyping, which successfully demonstrates the association of specific nucleotides or patterns with population groups. Ethnic inferencing based on autosomal STRs is complex due to the nature of recombination in gamete formation. Aim: This study attempts to use clustering analysis to associate alleles and loci of autosomal STRs with population groups. Materials and Methods: We examined the allele frequency data from 19 STRs loci from the Malay Indonesian population (n = 470) to compare with other populations, namely, Chinese Indonesian (n = 133) and four reference populations (Malay Malaysian, Filipino, Chinese, and Caucasian). K-Medoids clustering analysis was carried out to pinpoint alleles and loci affecting the population clustering process. Results: The first stage of clustering results placed Malay Indonesians and four other Asian populations, namely, Chinese Indonesian, Malay Malaysian, Filipino, and Chinese, in Cluster 1, whereas the Caucasian group was in Cluster 2. It indicates that the CSF1PO, D5S818, and D8S1179 loci significantly distinguished the five Asian population groups from the Caucasian group, whereas D2S441, D8S1179, and D22S1045 were the three loci that significantly influenced the separation between Malay Indonesians and other groups. Conclusions: We conclude that K-medoids clustering analysis has the potential to play a role in ethnicity estimation by pinpointing specific STRs alleles.

STR genotyping by real-time PCR using QueSTR probes

QueSTR probes provide a feasible alternative for implementing forensic Short Tandem Repeat (STR) genotyping on lab-on-a-chip (LoC) devices. Unlike capillary electrophoresis (CE), which poses challenges for LoC implementation, QueSTR probes have been demonstrated to accurately genotype STRs in a hybridization curve assay after PCR amplification. In this study, we modified the QueSTR probe assay for use as a hydrolysis probe assay during qPCR. An asymmetric real-time PCR was performed with QueSTR probes and RNase H2 in the master mix, during which the fluorescence was recorded. Designed to hybridize with specific STR alleles, QueSTR probes contain a fluorescent dye and quencher molecule, enabling detection of probe hybridization through RNase H2-mediated cleavage of the RNA:DNA duplex, which releases the quencher. Matching probes yielded lower threshold cycle values and steeper incline of fluorescence curves compared to non-matching probes, indicating accurate genotype. The QueSTR qPCR assay was used to successfully genotype four CODIS core loci (D16S539, D7S820, TPOX, and TH01) in 12 samples, with one exception. The integration of amplification and detection in a single reaction supports the use of QueSTR probes for miniaturizing STR genotyping, thereby complementing CE-based analyses in centralized labs.

Specific Short Tandem Repeat Loci Detection in Prenatal Diagnosis of Fetal Chromosomal Diseases.

It was to investigate the value of quantitative fluorescence PCR (QF-PCR) for the selection of specific short tandem repeat (STR) in prenatal diagnosis of fetal chromosomal diseases. Amniotic fluid (AF) and villus samples were obtained from 80 pregnant women at 16-20 weeks of gestation, and venous blood samples were obtained from 60 normal individuals to extract and prepare peripheral blood chromosome, AF cell chromosome, and villus cell chromosome samples for specific STR locus detection. It showed that the area ratio of AMX peak to AMY peak in the Genescan typing map of peripheral blood DNA of normal males was close to 1:1, while the Genescan typing map of peripheral blood DNA of normal females had only AMX peak and no AMY peak. Normal heterozygous individuals had an area ratio between 1 and 1.45 for venous blood, 1.002 and 1.27 for villous samples, and 1 and 1.35 for AF samples. The karyotype of a male fetus was 46, XY, inv [9] (p11: q13), and the structure of fetal chromosome 9 was inverted (interarm), and the site of structural inversion was band 1 in the short breech 1 region and band 3 in the long arm 1 region of chromosome 9. It suggested that QF-PCR can effectively identify the normal human body and cases by selecting specific STR locus detection, which has a good application value for prenatal diagnosis of fetal chromosomal diseases.

Development and Application of a Short Tandem Repeat Multiplex Typing Assay for Candida tropicalis.

Candida tropicalis is a clinically important yeast that causes candidemia in humans with a high mortality rate. The yeast primarily infects immunocompromised patients, and causes outbreaks in health care facilities. Antifungal resistant isolates have been reported. We developed a short tandem repeat (STR) typing scheme for C. tropicalis to enable fast, cost-effective, and high-resolution genotyping. For the development of the typing scheme, 6 novel STR markers were selected, combined into 2 multiplex PCRs. In total, 117 C. tropicalis isolates were typed, resulting in the identification of 104 different genotypes. Subsequently, the outcome of STR typing of 10 isolates was compared to single nucleotide polymorphism (SNP) calling from whole-genome sequencing (WGS). Isolates with more than 111 SNPs were differentiated by the typing assay. Two isolates, which were identical according to SNP analysis, were separated by STR typing in 1 marker. To test specificity, the STR typing was applied to 15 related yeast species, and we found no amplification of these targets. For reproducibility testing, 2 isolates were independently typed five times, which showed identical results in each experiment. In summary, we developed a reliable and multiplex STR genotyping for C. tropicalis, which was found to correlate well to SNP calling by WGS. WGS analysis from and extensive collection of isolates is required to establish the precise resolution of this STR assay. IMPORTANCE Candida tropicalis frequently causes candidemia in immunocompromised patients. C. tropicalis infections have a high mortality rate, and the yeast is able to cause outbreaks in health care facilities. Further, antifungal resistant isolates are on the rise. Genotyping is necessary to investigate potential outbreaks. Here, we developed and applied a STR genotyping scheme in order to rapidly genotype isolates with a high-resolution. WGS SNP outcomes were highly comparable with STR typing results. Altogether, we developed a rapid, high-resolution, and specific STR genotyping scheme for C. tropicalis.

Stool is a sensitive and noninvasive source of DNA for monitoring expansion in repeat expansion disease mouse models.

ABSTRACTRepeat expansion diseases are a large group of human genetic disorders caused by expansion of a specific short tandem repeat tract. Expansion in somatic cells affects age of onset and disease severity in some of these disorders. However, alleles in DNA derived from blood, a commonly used source of DNA, usually show much less expansion than disease-relevant cells in the central nervous system in both humans and mouse models. Here we examined the extent of expansion in different DNA sources from mouse models of the fragile X-related disorders, Huntington's disease, spinocerebellar ataxia type 1 and spinocerebellar ataxia type 2. We found that DNA isolated from stool is a much better indicator of somatic expansion than DNA from blood. As stool is a sensitive and noninvasive source of DNA, it can be useful for studies of factors affecting the risk of expansion, or the monitoring of treatments aimed at reducing expansion in preclinical trials, as it would allow expansions to be examined longitudinally in the same animal and allow significant changes in expansion to be observed much earlier than is possible with other DNA sources.

Genetic diversity and forensic parameters of 12 X-STR included in Argus X-12® marker panel in the population of the Russian Federation

Background X-chromosome specific short tandem repeats (X-STR) are indispensable for parentage analysis, kinship determination and forensic casework. Aim To determine allelic frequencies and forensic parameters of the 12 X-STR markers from the Investigator® Argus X-12® kit panel for the population of the Russian Federation. Subjects and methods We analysed 803 unrelated individuals from 58 localities across the Russian Federation. Intra-population genetic diversity was evaluated and comparison of the Russian population with 19 geographically distant populations performed. Results Heterozygosity values across all the loci ranged from 0.685 for DXS7423 to 0.939 for DXS10135. Deviations from the Hardy-Weinberg equilibrium were observed for DXS10074 (p < 0.05) and DXS10146 (p < 0.01). The value for power of discrimination across all the loci in males was 0.9999999996, while in females it equalled 0.9999999999. Combined mean exclusion chances MECKruger, MECKishida, MECD-trio, and MECD-duo were 0.9999988168, 0.9999999983, 0.9999999983, and 0.9999995797, respectively. Phylogenetic analysis indicated small genetic differences between the Russian population and the populations of neighbouring Lithuania, Belarus and the Slavic-speaking countries (Czech Republic, Serbia and Croatia). Conclusions This paper is the first report of forensically important parameters and allele frequencies for the 12 X-STR loci included in Argus X-12® marker system in the population of the Russian Federation.

Short tandem repeat typing technologies used in paternity testing: a case study

Background: In forensic field, for establishing the paternity of disputed offspring, STR typing (autosomal, and Y typing) are commonly used to dissolve the cases. Short tandem repeat (STR) technology is used to evaluate specific regions (loci) within nuclear DNA. Variability in STR regions can be used to distinguish one DNA profile from another. The Federal Bureau of Investigation (FBI) uses a standard set of 13 specific STR regions for CODIS. Aim of the study: The present work was done to find out the biological father of child in a case where mother was killed by her husband for the doubt of her pregnancy. Methods: After taking blood samples from the murdered mother, the baby and the killer husband, Autosomal STRs Using AmpFlSTR Identifiler Plus PCR Amplification Kit and Y-STR analysis using AmpFlSTR Yfiler PCR Amplification Kit were done to solve the disputed paternity case. Results: After the analysis of DNA STRs profiles of autosomal and Y-chromosome markers, the dead fetus was proved to be the son of the killer father. Conclusions: Autosomal STR multiplex analysis combined with Y-STR analysis help to reach a conclusion in disputed paternity cases with greater confidence. Keywords: Disputed; Paternity; Multiplex; Short tandem reprats; Y-STR; Analysis

Predicting Outcomes of Rat Vascularized Composite Allotransplants through Quantitative Measurement of Chimerism with PCR-Amplified Short Tandem Repeat.

Chimerism has been associated with the induction and maintenance of tolerance to vascularized composite allotransplants (VCA). Although most VCA studies have examined chimerism using flow cytometry, we proposed that precision in the measurement of chimerism may be better approximated when complimentary polymerase chain reaction (PCR) is applied to a specific short tandem repeat (STR). We identified a STR, D10Rat25, which exhibited a ~20 bp difference in length between two rat strains (BN and LEW) often utilized as the donor and recipient in many allotransplantation studies. D10Rat25 was PCR-amplified and quantified with capillary electrophoresis. With pure LEW and BN DNA, a standard curve was constructed to measure chimerism with good linearity. When applied to rat VCA, the relationship between systematic (in peripheral blood) or local (at specific organ/tissues) chimerism to allograft outcomes was noted. We found that peripheral chimerism was elevated by up to ~9% postoperative month 1 (POM 1) but then reduced regardless of the final VCA outcome. However, differences in VCA skin chimerism between early rejection and POM 1 (shown as ΔChimerismPOM1-ER) were notable with respect to VCA outcomes. ROC analysis identified the optimum cutoff value as 17.7%. In summary, we have developed a reliable method to quantify the percentage of BN cells/DNA in BN-LEW chimeras. The detection limit was characterized, and the acquired data were comparable with flow cytometry. This method can be applied to solid organ and composite tissue allotransplantation studies.

Mutation in Y STRs: Repeat motif gains vs. losses

Y chromosome specific short tandem repeats (Y-STRs) are widely used in population genetics and forensics. Since these markers do not recombine, mutation is the only source of diversity. The primary mutational mechanism leading to length changes in STRs is thought to be polymerase template slippage, and the most common change is the gain or the loss of one repeat motif. In this work, we aim to study 19 Y-STR alleles’ contraction and expansion. Alleles were grouped into tertiles: short (1st tertile), intermediate (2nd tertile) and long alleles (3rd tertile). Significant differences between repeat gains and losses were found at four markers - DYS19, DYS439 for intermediate alleles, and DYS570 and DYS626 for long alleles. When the average number is computed for the pooled loci, for short alleles, the number of repeat motif gains is higher than of repeat losses, and the opposite happens for long alleles. For intermediate alleles, the proportion between the number of repeat gains and losses is close to one. Generally, the rate of expansion decreases from the first tertile to the third, and conversely, the rate of contraction increases from the first tertile to the third. The pooled loci tertiles’ mutation rate increases from short to long alleles. Our results demonstrate that the mutation direction and rate depend on alleles’ length. The longer the allele the greater the mutation and contraction rates.

Regulatory Architecture of the Neuronal Cacng2/Tarp\u03b32 Gene Promoter: Multiple Repressive Domains, a Polymorphic Regulatory Short Tandem Repeat, and Bidirectional Organization with Co-regulated lncRNAs

CACNG2 (TARPγ2, Stargazin) is a multi-functional regulator of excitatory neurotransmission and has been implicated in the pathological processes of several brain diseases. Cacng2 function is dependent upon expression level, but currently, little is known about the molecular mechanisms that control expression of this gene. To address this deficit and investigate disease-related gene variants, we have cloned and characterized the rat Cacng2 promoter and have defined three major features: (i) multiple repressive domains that include an array of RE-1 silencing transcription factor (REST) elements, and a calcium regulatory element-binding factor (CaRF) element, (ii) a (poly-GA) short tandem repeat (STR), and (iii) bidirectional organization with expressed lncRNAs. Functional activity of the promoter was demonstrated in transfected neuronal cell lines (HT22 and PC12), but although selective removal of REST and CaRF domains was shown to enhance promoter-driven transcription, the enhanced Cacng2 promoter constructs were still about fivefold weaker than a comparable rat Synapsin-1 promoter sequence. Direct evidence of REST activity at the Cacng2 promoter was obtained through co-transfection with an established dominant-negative REST (DNR) construct. Investigation of the GA-repeat STR revealed polymorphism across both animal strains and species, and size variation was also observed in absence epilepsy disease model cohorts (Genetic Absence Epilepsy Rats, Strasbourg [GAERS] and non-epileptic control [NEC] rats). These data provide evidence of a genotype (STR)-phenotype correlation that may be unique with respect to proximal gene regulatory sequence in the demonstrated absence of other promoter, or 3′ UTR variants in GAERS rats. However, although transcriptional regulatory activity of the STR was demonstrated in further transfection studies, we did not find a GAERS vs. NEC difference, indicating that this specific STR length variation may only be relevant in the context of other (Cacna1h and Kcnk9) gene variants in this disease model. Additional studies revealed further (bidirectional) complexity at the Cacng2 promoter, and we identified novel, co-regulated, antisense rat lncRNAs that are paired with Cacng2 mRNA. These studies have provided novel insights into the organization of a synaptic protein gene promoter, describing multiple repressive and modulatory domains that can mediate diverse regulatory inputs.

Correction to: Dual-Purpose Bioreactors to Monitor Noninvasive Physical and Biochemical Markers of Kidney and Liver Scaffold Recellularization, by Uzarski JS, Bijonowski BM, Wang B, Ward HH, Wandinger-Ness A, Miller WM, and Wertheim JA. Tissue Eng Part C Methods 2015;21(10):1032–1043. DOI: 10.1089/ten.tec.2014.0665

[This corrects the article DOI: 10.1089/ten.tec.2014.0665.].

Evaluation of Chimerism Dynamics after Allogeneic Hematopoietic Stem Cell Transplantation in Children with Nonmalignant Diseases

It is recognized that chimerism following hematopoietic stem cell transplantation (HSCT) is a dynamic process. The aims of this study were to describe the evolution of chimerism in children with nonmalignant diseases who underwent allogeneic HSCT, and to analyze the risk factors influencing chimerism status. A total of 101 HSCTs were performed in 85 patients with nonmalignant diseases. The donor was unrelated in 62.4% of HSCTs. Reduced-intensity conditioning (RIC) regimen was administered in 48.5% of patients. Acute graft-versus-host disease (aGVHD) occurred in 51.7% and chronic GVHD (cGVHD) in 39.7% of patients. Analysis of chimerism was performed through amplification of 9 specific short tandem repeats by polymerase chain reaction at engraftment and 1, 6, and 12 months after HSCT. Upon first evaluation, complete chimerism (CC) was detected in 34.7% and mixed chimerism (MC) in 55.4%, whereas graft failure occurred in 9.9% of patients. Severe aGVHD was associated with CC (P = .031). The last chimerism evaluation showed CC in 72.1%, stable MC in 12.8%, and progressive MC in 3.5%. CC was associated with a higher incidence of aGVHD (P = .016) and cGVHD (P = .022), whereas the RIC regimen was associated with graft failure (P = .026). One- and 3-year overall survival (OS) was 87.4% and 80.5%, respectively, with a lower OS at 3 years in patients with CC compared with those with MC (P = .008). aGVHD and cGVHD represent factors favoring CC, thus close, careful follow-up of chimerism is recommended in patients affected by nonmalignant disease.

Assessment of the most reliable sites in mandibular bone for the best deoxyribonucleic acid yield for expeditive human identification in forensics.

Background:In recent years, the techniques used to identify human remains post accidents, trauma or in case of criminal investigation have been expanded, improved and rendered more complex by the emergence of technologies based on deoxyribonucleic acid (DNA) analysis. In the head and neck area, tooth has been proven to be the best quantitative source for DNA but in certain cases where the mandible specimen is edentulous or the tooth is extensively destroyed with caries, large dental restorations, mobile, or if they show any perimortem or postmortem fractures, sampling of such tooth specimen is usually avoided. In such situations, bone is considered the next best site for DNA analysis. Mandible being the largest, strongest and dense cortical bone is the most prominent facial bone that can be easily disarticulated. It can be analyzed for the best short tandem repeat (STR) segment qualitative amplification using polymerase chain reaction (PCR) technique for forensic analysis which can be used for gender and age determination.Aim:The aim of this study is to determine the best site for optimum quantitative and qualitative yield of DNA for amplification using specific and standard STR segment by conventional PCR technique.Methodology:Fifteen mandibular samples exposed to different environmental conditions were collected. Bone pieces of 1 cm × 1 cm were cut from each mandible from three sites, i.e., the ramus, angle and body, wherein the genomic DNA was isolated and was subjected to PCR using restricted number of 25 cycles.Results:The STR segment D3S1358 from clone RP11-438F9 used for the study showed very good amplification in restricted number of PCR cycles in the ramus region with number of repeats in every 15th genomic region.Conclusion:This study highlights the use of mandibular bone for the expeditive human identification. As per the study, the ramus of the mandible gave high quantitative and qualitative yield of DNA with thick amplification band of the STR segment as compared to the body and angle of the mandible. Thus ramus of the mandible can be preferred over other sites for molecular forensic investigations.

Association of Parental Origin with Phenotypic Profile in Turner Syndrome

Abstract Objective: The objective of the study is to report the association between the phenotype of the Turner syndrome with that of its karyotype and the parental origin of the X. 181 female referred to division of human genetics, St. John’s Medical College with different referral reasons are included in 1st phase of the study to observe the karyotype, upon obtaining the consent. Families of probands with confirmed pure or mosaic (more than 10% of mosaic status) karyotype of Turner stigmata are requested to participate in 2nd phase of study to determine the parent of origin. 20 families agreed and gave the consent for the study. Material and Method: The Karyotype analsyis was carried by GTG banding method automated karyotyping system. The parental origin was determined by X chromosome specific short tandem repeat (STR) markers. 23 Turner stigmata features were listed in the proforma. The secondary measure is to observe the associated Turner stigmata with the determined karyotype and the parental origin. Results: In 11 female probands, one had X structural abnormality (45,X/46,X,i(Xq)); 2 had X-mosaicism (45,X/ 46,XX) and 8 had 45,X. The determined parental origin of X was maternal in 8 (72.7%) and paternal in 3 (27.2%). 23 phenotypic features of Turner stigmata were tabulated. The features were further categorized as general observations, classical features of TS, features based on ultrasound findings and behavior. Percentage analysis indicated that a higher concordance of phenotype and behavior in proband with maternal X. Conclusion: In the present study, the Turner syndrome features were seemed to be associated with the maternal origin of X in Turner syndrome.

Association of parental origin with clinical profile in klinefelter syndrome.

Several genomic imprinting mechanisms have been postulated to report the parent-of-origin in Klinefelter syndrome. It was stated in the literature, parental origin has an effect on behavioral phenotype of Klinefelter individuals, but the association of the same on clinical profile was less reported. The detailed clinical phenotype when studied with the known origin of extra X may possibly explain the imprinting effect that may be helpful to derive diagnostic criteria in the syndrome. In the present study, we investigated the parental-of-origin of extra X chromosome in Klinefelter syndrome probands with an aim to report the association between the phenotype with that of its karyotype and the parental origin of supernumerary X. Seventy two probands that were referred to division of Human Genetics, St.John's Medical College, Bangalore with variable complaints and phenotypic features were diagnosed with informed consent as Klinefelter syndrome with a confirmed karyotype. The Karyotype was prepared by peripheral lymphocyte culture and GTG banding method. The parental origin was studied in 9 families of Klinefelter probands with standard protocol for GENE SCAN using X-chromosome specific Short Tandem Repeat markers. The outcome was analyzed to determine the parental origin by GENE MAPPER. STATISTICAL ANALYSIS was conducted to ascertain the significance of parental origin of supernumerary X with the phenotypic profile with confirmed karyotype. Seven of nine probands had 47, XXY karyotype and 2 were mosaic with 47,XXY/46,XY karyotype. Five probands had their supernumerary X from maternal side and four were paternally derived. Sixteen features as framed proforma were tabulated against the originated X in Klinefelter probands. 55.56% of Klinefelter stigmata were seen in prob and who had maternally derived X and the rest were with paternal X. The findings of the present study points on parent-of-origin effect on clinical profile and indicate that the imprinted X chromosome genes show differential effect general and systemic traits.

Application of Short Tandem Repeat markers in diagnosis of chromosomal aneuploidies and forensic DNA investigation in Pakistan

Short Tandem Repeat (STR) genetic markers hold great potential in forensic investigations, molecular diagnostics and molecular genetics research. AmpFlSTR® Identifiler™ PCR amplification kit is a multiplex system for co-amplification of 15 STR markers used worldwide in forensic investigations. This study attempts to assess forensic validity of these STRs in Pakistani population and to investigate its applicability in quick and simultaneous diagnosis and tracing parental source of common chromosomal aneuploidies. Samples from 554 healthy Pakistani individuals from 5 different ethnicities were analyzed for forensic parameters using Identifiler STRs and 74 patients' samples with different aneuploidies were evaluated for diagnostic strengths of these markers. All STRs hold sufficient forensic applicability in Pakistani population with paternity index between 1.5 and 3.5, polymorphic information content from 0.63 to 0.87 and discrimination power ≥0.9 (except TPOX locus). Variation from Hardy-Weinberg equilibrium was observed at some loci reflecting selective breeding and intermarriages trend in Pakistan. Among aneuploidic samples, all trisomies were precisely detectable while aneuploidies involving sex chromosomes or missing chromosomes were not clearly detectable using Identifiler STRs. Parental origin of aneuploidy was traceable in 92.54% patients. The studied STR markers are valuable tools for forensic application in Pakistan and utilizable for quick and simultaneous identification of some common trisomic conditions. Adding more sex chromosome specific STR markers can immensely increase the diagnostic and forensic potential of this system.

Association between allelic variation due to short tandem repeats in tRNA gene of Entamoeba histolytica and clinical phenotypes of amoebiasis

Genotypes of Entamoeba histolytica (E. histolytica) may contribute clinical phenotypes of amoebiasis such as amoebic liver abscess (ALA), dysentery and asymptomatic cyst passers state. Hence, we evaluated allelic variation due to short tandem repeats (STRs) in tRNA gene of E. histolytica and clinical phenotypes of amoebiasis. Asymptomatic cyst passers (n=24), patients with dysentery (n=56) and ALA (n=107) were included. Extracted DNA from stool (dysentery, asymptomatic cyst passers) and liver aspirate was amplified using 6 E. histolytica specific tRNA-linked STRs (D-A, A-L, N-K2, R-R, S-Q, and STGA-D) primers. PCR products were subjected to sequencing. Association between allelic variation and clinical phenotypes was analyzed. A total of 9 allelic variations were found in D-A, 8 in A-L, 4 in N-K2, 5 in R-R, 10 in STAG-D and 7 in S-Q loci. A significant association was found between allelic variants and clinical phenotypes of amoebiasis. This study reveals that allelic variation due to short tandem repeats (STRs) in tRNA gene of E. histolytica is associated different clinical outcome of amoebiasis.

A reliable assay for rapidly defining transplacental metastasis using quantitative PCR.

To choose the most appropriate treatment for children affected by a transplacental metastasis, it is crucial to ascertain the maternal origin of the tumor. Up-to-date conclusive diagnosis is generally achieved through fluorescence in situ hybridization or karyotyping analysis. Herein, we report an alternative, reliable assay for rapidly defining vertical cancer transmission to the fetus by using quantitative polymerase chain reaction. Our assay indicates that quantification of the copy number of the sex chromosomes by specific short tandem repeats markers, in genomic DNA purified from the tumor biopsy cells, could be used to correctly evaluate transplacental metastasis events.