Haplogroup diversity in the Indian population using 23 Y-STRs.

BACKGROUND: A Y-STR polymorphism study is a convenient tool in molecular anthropology and forensic DNA analysis. AIM: Through standard ethical procedures, the proposed study explored the genetic scenario in male lineage in Madhya Pradesh, a central Indian state, by Y-STR genotyping and haplogroup studies. SUBJECTS AND METHODS: Five hundred and eleven unrelated male blood samples were directly amplified, and fragment separation was done using capillary electrophoresis to generate a Y-STR profile for 23 forensic relevant markers through PowerPlex® Y 23 multiplex system. The different statistical methods were applied for studying the forensic and genetics parameters. Subsequently, population comparison was performed by AMOVA, PCoA, and MDS plot, and Haplogroups were predicted with Whit Athey's haplogroup predictor tool. CONCLUSION: These data represented the potential value of the PowerPlex® Y-23 multiplex system for the forensic and human genetics application in the population of Madhya Pradesh, India. Simultaneously the Haplogroup analysis revealed information about the multi-geographic origin as well as multi-ethnic genetic affinities of the Madhya Pradesh population.

Forensic characterization of 124 SNPs in the central Indian population using precision ID Identity Panel through next-generation sequencing.

With the advent of next-generation sequencing technology, SNP markers are being explored as a useful alternative to conventional capillary electrophoresis-based STR typing. Low mutation rate and short-sized amplicons are added advantages of SNP markers over the STRs. However, to achieve a sufficient level of discrimination among individuals, a higher number of SNPs need to be characterized simultaneously. Hence, the NGS technique is highly useful to analyze a sufficiently higher number of SNPs simultaneously. Though the technique is in its nascent stage, an attempt has been made to assess its usability in the central Indian population by analyzing 124 SNPs (90 autosomal and 34 Y-chromosome) in 95 individuals. Various quality parameters such as locus balance, locus strand balance, heterozygosity balance, and noise level showed a good quality sequence obtained from the Ion GeneStudio S5 instrument. Obtained frequency of SNP alleles ranged from 0.001 to 0.377 in autosomal SNPs. rs9951171 was found to be the most informative SNP in the studied population with the highest PD and lowest MP value. The cumulative MP of 90 SNPs was found to be 4.76698 × 10-37. Analysis of 34 Y-chromosome SNPs reveals 11 unique haplogroups in 54 male samples with R1a1 as the most frequent haplogroup found in 22.22% of samples. Interpopulation comparison by FST analysis, PCA plot, and STRUCTURE analysis showed genetic stratification of the studied population suggesting the utility of SNP markers present in the Precision ID Identity Panel for forensic demands of the Indian population.

Sequence variations, flanking region mutations, and allele frequency at 31 autosomal STRs in the central Indian population by next generation sequencing (NGS).

Capillary electrophoresis-based analysis does not reflect the exact allele number variation at the STR loci due to the non-availability of the data on sequence variation in the repeat region and the SNPs in flanking regions. Herein, this study reports the length-based and sequence-based allelic data of 138 central Indian individuals at 31 autosomal STR loci by NGS. The sequence data at each allele was compared to the reference hg19 sequence. The length-based allelic results were found in concordance with the CE-based results. 20 out of 31 autosomal STR loci showed an increase in the number of alleles by the presence of sequence variation and/or SNPs in the flanking regions. The highest gain in the heterozygosity and allele numbers was observed in D5S2800, D1S1656, D16S539, D5S818, and vWA. rs25768 (A/G) at D5S818 was found to be the most frequent SNP in the studied population. Allele no. 15 of D3S1358, allele no. 19 of D2S1338, and allele no. 22 of D12S391 showed 5 isoalleles each with the same size and with different intervening sequences. Length-based determination of the alleles showed Penta E to be the most useful marker in the central Indian population among 31 STRs studied; however, sequence-based analysis advocated D2S1338 to be the most useful marker in terms of various forensic parameters. Population genetics analysis showed a shared genetic ancestry of the studied population with other Indian populations. This first-ever study to the best of our knowledge on sequence-based STR analysis in the central Indian population is expected to prove the use of NGS in forensic case-work and in forensic DNA laboratories.

Genetic diversity and forensic characterization of 15 autosomal microsatellite markers in the North-east Indian Tripuri population.

This study was conducted to explore the genomic diversity and forensic characterization of 15 autosomal microsatellite markers in the East Indian Tripuri population. In the studied population, we observed 158 different alleles with the average 10.53 alleles per locus. The locus D2S1338 (PIC= 0.862) was found to be the most polymorphic wheres locus TPOX (PIC= 0.647) as the least polymorphic, among all the studied loci. The locus FGA was found with the highest number of effective alleles (Nall=19) whereas locus TH01 showed least number of effective alleles (Nall=6). The cumulative values for matching probability (CPm), power of discrimination (CPD), power of exclusion CPE), and paternity index (CPI) were found as 1.94×10-18, 1, 0.999998, and 4.8×105 respectively. The studied population showed genetic closeness with the Gorkha population. In neighbor-joining tree, Tripura population pooled with the population of Nepal and Tibet. The genetic data obtained from the present study will not only enrich the existing autosomal STR database but will also be useful for forensic DNA application and genealogical studies.

A study of genomic diversity in populations of Maharashtra, India, inferred from 20 autosomal STR markers.

OBJECTIVE: This study was planned to evaluate the genetic diversity in the admixed and Teli (a Hindu caste) populations of Maharashtra, India using 20 autosomal Short Tandem Repeat (STR) genetic markers. We further investigated the genetic relatedness of the studied populations with other Indian populations. RESULTS: The studied populations showed a wide range of observed heterozygosity viz. 0.690 to 0.918 for the admixed population and 0.696 to 0.942 for the Teli population. This might be due to the multi-directional gene flow. The admixed and Teli populations also showed a high degree polymorphism which ranged from 0.652 to 0.903 and 0.644 to 0.902, respectively. Their combined value of matching probability for all the studied loci was 4.29 × 10-25 and 5.01 × 10-24, respectively. The results of Neighbor-Joining tree and Principal Component Analysis showed that the studied populations clustered with the general populations of Jharkhand, UttarPradesh, Rajasthan and Central Indian States, as well as with the specific populations of Maharashtra (Konkanastha Brahmins) and Tamil Nadu (Kurmans). Overall, the obtained data showed a high degree of forensic efficacy and would be useful for forensic applications as well as genealogical studies.

A case of "false tri-allelic pattern" on D7S820, caused by invasion of a short SE33 allele into the bins of D7S820.

Autosomal short tandem repeats (asSTR) serve as genetic markers for discriminating individuals and have been extensively used for criminal investigations as well as the establishment of genetic relationships. Tri-allelic pattern usually occurs due to chromosomal duplication, trisomy, and chimerism during mitotic division, but a false tri-allelic pattern at the D7S820 locus was encountered in our laboratory during the analysis of a case exhibit. DNA isolation from exhibit for profiling was done as per manufacturer's protocol. This is the first report which observed false tri-allelic pattern (10, 11, 14.1 allele) on D7S820 locus by analysis with GlobalFiler™ PCR Amplification Kit in Indian population. Findings were re-confirmed using other available asSTR kits in the laboratory, viz., AmpFLSTR™ Identifiler™ Plus PCR Amplification Kit and PowerPlex® Fusion 6C System. Two alleles (10, 11) found at D7S820, apart from SE33 marker, showed homozygous condition, but one Off Marker (OMR) peak was observed before start of SE33 marker region with the analysis using PowerPlex® Fusion 6C System. As it has been confirmed that the OMR allele belongs to the SE33 locus, this could be possible because of the adjacent locations of the D7S820 and the SE33 in the GlobalFiler® PCR amplification kit. 14.1 allele appeared within the allelic window of D7S820. The false tri-allelic pattern was due to the overlapping of SE33 marker allele (1.2 repeat) with bin window of D7S820 Marker. This finding might create confusion for the establishment of genetic relationships. We, therefore, conclude that such uncommon observations with rare events should be carefully investigated and interpreted.

Forensic characterization and genetic evaluation in the Central Indian population using 27 Y-STRs.

INTRODUCTION: Forensic characterization and genetic evaluation study in the 539 randomly selected unrelated adult healthy individuals belonging to the Central Indian population was undertaken. METHODS: The study was performed using a multiplex of 27 Y-STRs incoporated in Yfiler™ Plus multiplex kit. RESULTS: Out of 539 samples, 6 samples were observed for large deletion and tri-allelic patterns, which were removed from the analysis, and out of 533 samples, a total of 507 haplotypes were found, and out of these haplotypes, 482 unique haplotypes were found in this piece of work. The forensically important parameters, i.e., gene diversity (GD) and discrimination capacity (DC), were found to be 0.669 and 0.951, respectively, for the tested Y STR loci. The genetic data of this study will enrich the Y STR data bank and being used as a potential tool for forensic DNA and various genetic studies.

Novel molecular diagnostic technique for detecting the different species of Plasmodium.

BACKGROUND: Sensitive diagnostic techniques are needed for timely detection of malaria parasite and disease control. Molecular diagnostic techniques involving Polymerase chain reaction (PCR) with 18 s rRNA as a known diagnostic target with an overall sensitivity of 10 parasites per microliter is used as a gold standard. Till date, no attempt has been undertaken to develop a technique for the identification of four Plasmodium species in a single step PCR combined with restriction digestion with enzymes. METHOD: Plasmodium species-specific polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assays have been developed, based on RFLP of amplified PCR product of mitochondrial gene as a target. This approach identifies Plasmodium species in two steps involving amplification of mitochondrial (Mt) gene by PCR followed by digestion with restriction enzymes. RESULT: A total of 36 clinical samples were subjected to PCR-RFLP for the diagnosis and detection of malaria parasites targeting mitochondrial gene (Mt). The findings of the method were compared with gold standard methods (Microscopy, RDTs and Nested PCR) and was able to detect mixed infection with a sensitivity of 100% and specificity of 93.8% with respect to nested PCR. The results obtained by PCR-RFLP were validated with Sanger sequencing (n = 32) and were found to be consistent with the method. CONCLUSION: This method identifies and distinguishes four species of human malaria parasite namely P. falciparum (Pf), P. vivax (Pv), P. malariae (Pm) and P. ovale (Po) in approximately 4 h. To overcome and address PCR difficulties, continuous efforts are needed for the development of newer diagnostic techniques.

Forensic effectiveness and genetic distribution of 23 autosomal STRs included in Verifiler PlusTM multiplex in a population sample from Madhya Pradesh, India.

We report here the first ever global study on genetic polymorphism using a Verifiler PlusTM autosomal STR multiplex system. The study evaluated genetic characteristics of 23 autosomal STRs in 200 unrelated residents of Guna district of Madhya Pradesh, India. Allele frequencies and forensic parameters are reported. Population comparison analysis was also performed using NJ tree and PCA plot. Penta E marker showed highest power of discrimination (0.938) among all 23 studied markers. The study also presents the first ever global forensic assessment in Indian population on D6S1043 marker (PD 0.937). The results demonstrated that all the 23 markers were highly polymorphic and the Verifiler PlusTM kit is suitable for forensic purposes in Indian population.

Haplotype data for 17 Y-STR loci in the population of Himachal Pradesh, India.

We report here the data of Y chromosome haplotypes of 259 unrelated males from the population of Himachal Pradesh, India, using the Yfiler® multiplex kit. A total of 188 haplotypes were detected, out of which 148 were unique. Three samples showed bi-allelic pattern on locus DYS448. The observed genetic diversity and discrimination capacity were 0.996 and 0.73, respectively. In order to compare the genetic distance of the studied population with the published populations, multidimensional scaling (MDS) plot was constructed. The reported data is expected to be valuable for both forensic and population genetics.

Forensic genetic analysis of population of Madhya Pradesh with PowerPlex Fusion 6C™ Multiplex System.

Performance of PowerPlex Fusion 6C kit (PP F6C) was assessed in 374 unrelated individuals belonging to Madhya Pradesh, an Indian state. The study evaluated the forensic parameters for the loci included in PP F6C Multiplex System. The combined discrimination power (CPD) and combined exclusion power (CPE) were 1 and 0.999999995, respectively, for all 23 autosomal STR loci. SE33 showed the greatest power of discrimination (0.990) in the studied population, whereas TPOX showed the lowest (0.843). The availability of three Y-STR loci in the Multiplex System is suitable for assessing male contribution and amelogenin deletion in a single Multiplex PCR simultaneously. The study also presents the first global report on polymorphism in the Indian population on SE 33 autosomal STR loci and PP Fusion 6C Multiplex System. The results revealed that the studied STR Multiplex System is highly polymorphic and suitable for forensic purposes.

Genetic data for PowerPlex 21™ autosomal and PowerPlex 23 Y-STR™ loci from population of the state of Uttar Pradesh, India.

In the present study, the statistical forensic parameters were evaluated for the loci present in PowerPlex 21 autosomal and PowerPlex 23 Y-STR multiplex systems in 168 unrelated individuals living in the state of Uttar Pradesh, India. The combined discrimination power (CPD) and combined exclusion power (CPE) was 1 and 0.999999 respectively for all 20 autosomal STR loci. Penta E showed the greatest (0.980) and CSF1PO showed the lowest (0.855) power of discrimination in the studied population. The haplotype diversity for 23 Y-STR loci was observed to be 0.999. The study also presents the first global report on polymorphism on D1S1656, D6S1043 and D12S391 autosomal STR loci in the Indian population. The resulting data revealed that these STR multiplex systems are highly polymorphic and can be used for forensic purposes.

In vitro sensitivity pattern of chloroquine and artemisinin in Plasmodium falciparum.

Artemisinin (ART) and its derivatives form the mainstay of antimalarial therapy. Emergence of resistance to them poses a potential threat to future malaria control and elimination on a global level. It is important to know the mechanism of action of drug and development of drug resistance. We put forwards probable correlation between the mode of action of chloroquine (CQ) and ART. Modified trophozoite maturation inhibition assay, WHO Mark III assay and molecular marker study for CQ resistance at K76T codon in Plasmodium falciparum CQ-resistant transporter gene were carried out on cultured P. falciparum. On comparing trophozoite and schizont growth for both CQ-sensitive (MRC-2) and CQ-resistant (RKL-9) culture isolates, it was observed that the clearance of trophozoites and schizonts was similar with both drugs. The experiment supports that CQ interferes with heme detoxification pathway in food vacuoles of parasite, and this may be correlated as one of the plausible mechanisms of ART.

Monitoring the efficacy of antimalarial medicines in India via sentinel sites: Outcomes and risk factors for treatment failure.

BACKGROUND & OBJECTIVES: To combat the problem of antimalarial drug resistance, monitoring the changes in drug efficacy over time through periodic surveillance is essential. Since 2009, systematic and continuous monitoring is being done through nationwide sentinel site system. Potential early warning signs like partner drug resistance markers were also monitored in the clinical samples from the study areas. METHODS: A total of 1864 patients with acute uncomplicated malaria were enrolled in therapeutic efficacy studies of artesunate plus sulphadoxine-pyrimethamine (AS+SP) for Plasmodium falciparum; those infected with P. vivax were given chloroquine (CQ). Polymerase chain reaction (PCR) was used to distinguish post-treatment reinfection from treatment failures. Isolates of P. falciparum were also analysed for dihydropteroate synthase (dhps) and dihydrofolate reductase (dhfr) gene mutations. RESULTS: Overall, 1687 (91.7%) patients completed the follow-up. In most of the falciparum patients the parasitaemia was cleared within 24 h of treatment, except 12 patients who remained parasite positive after 72 h. Presence of dhfr and dhps quintuple mutation was observed predominantly in treatment failure samples. A daily dose of artesunate of < 3 mg/kg of body weight, age of <5 yr, and fever at enrolment were associated with an increased risk of treatment failure. The AS+SP in P. falciparum was effective in > 95% cases in all the sentinel sites except in Northeastern region (NE). Chloroquine remained 100% efficacious in case of P. vivax infections. INTERPRETATION & CONCLUSION: Till 2012, India's national antimalarial drug resistance monitoring system proved highly efficacious and safe towards first-line antimalarials used in the country, except in Northeastern region where a decline in efficacy of AS+SP has been observed. This led to change in first-line treatment for P. falciparum to artemether-lumefantrine in Northeastern region.

Surveillance of artemisinin resistance in Plasmodium falciparum in India using the kelch13 molecular marker.

Malaria treatment in Southeast Asia is threatened with the emergence of artemisinin-resistant Plasmodium falciparum. Genome association studies have strongly linked a locus on P. falciparum chromosome 13 to artemisinin resistance, and recently, mutations in the kelch13 propeller region (Pfk-13) were strongly linked to resistance. To date, this information has not been shown in Indian samples. Pfk-13 mutations were assessed in samples from efficacy studies of artemisinin combination treatments in India. Samples were PCR amplified and sequenced from codon 427 to 727. Out of 384 samples, nonsynonymous mutations in the propeller region were found in four patients from the northeastern states, but their presence did not correlate with ACT treatment failures. This is the first report of Pfk-13 point mutations from India. Further phenotyping and genotyping studies are required to assess the status of artemisinin resistance in this region.

Declining efficacy of artesunate plus sulphadoxine-pyrimethamine in northeastern India.

BACKGROUND: Anti-malarial drug resistance in Plasmodium falciparum in India has historically travelled from northeast India along the Myanmar border. The treatment policy for P. falciparum in the region was, therefore, changed from chloroquine to artesunate (AS) plus sulphadoxine-pyrimethamine (SP) in selected areas in 2005 and in 2008 it became the first-line treatment. Recognizing that resistance to the partner drug can limit the useful life of this combination therapy, routine in vivo and molecular monitoring of anti-malarial drug efficacy through sentinel sites was initiated in 2009. METHODS: Between May and October 2012, 190 subjects with acute uncomplicated falciparum malaria were enrolled in therapeutic efficacy studies in the states of Arunachal Pradesh, Tripura, and Mizoram. Clinical and parasitological assessments were conducted over 42 days of follow-up. Multivariate analysis was used to determine risk factors associated with treatment failure. Genotyping was done to distinguish re-infection from recrudescence as well as to determine the prevalence of molecular markers of antifolate resistance among isolates. RESULTS: A total of 169 patients completed 42 days of follow-up at three sites. The crude and PCR-corrected Kaplan-Meier survival estimates of AS + SP were 60.8% (95% CI: 48.0-71.4) and 76.6% (95% CI: 64.1-85.2) in Gomati, Tripura; 74.6% (95% CI: 62.0-83.6) and 81.7% (95% CI: 69.4-89.5) in Lunglei, Mizoram; and, 59.5% (95% CI: 42.0-73.2) and 82.3% (95% CI: 64.6-91.6) in Changlang, Arunachal Pradesh. Most patients with P. falciparum cleared parasitaemia within 24 hours of treatment, but eight, including three patients who failed treatment, remained parasitaemic on day 3. Risk factors associated with treatment failure included age < five years, fever at the time of enrolment and AS under dosing. No adverse events were reported. Presence of dhfr plus dhps quintuple mutation was observed predominantly in treatment failure samples. CONCLUSION: AS + SP treatment failure was widespread in northeast India and exceeded the threshold for changing drug policy. Based on these results, in January 2013 the expert committee of the National Vector Borne Disease Control Programme formulated the first subnational drug policy for India and selected artemether plus lumefantrine as the new first-line treatment in the northeast. Continued monitoring of anti-malarial drug efficacy is essential for effective malaria control.

Monitoring antimalarial drug resistance in India via sentinel sites: outcomes and risk factors for treatment failure, 2009-2010.

OBJECTIVE: To describe India's National Antimalarial Drug Resistance Monitoring System, measure the efficacy of first-line malaria treatments, and determine risk factors for treatment failure. METHODS: In 2009-2010, prospective studies with 28 days of follow-up were conducted at 25 sentinel sites. Patients infected with Plasmodium falciparum were given artesunate plus sulfadoxine-pyrimethamine (AS+SP); those infected with P. vivax were given chloroquine. Polymerase chain reaction was used to distinguish post-treatment reinfection from treatment failure. Isolates of P. falciparum were checked for dhfr and dhps mutations. FINDINGS: Overall, 1664 patients were enrolled. Kaplan-Meier survival analysis showed an efficacy of 98.8% for AS+SP. Most patients with P. falciparum parasitaemia cleared their parasitaemias within 24 hours of treatment initiation, but six, including four with treatment failure, remained parasitaemic after 72 hours. Double mutants in dhfr were found in 68.4% of the genotyped isolates. Triple or quadruple mutants in dhfr and mutations in dhps were rare. A daily dose of artesunate of < 3 mg per kg of body weight, age of less than 5 years, and fever at enrolment were associated with an increased risk of treatment failure. Chloroquine remained 100% efficacious and generally cleared P. vivax parasitaemias within 48 hours. Vomiting (seen in 47 patients) was the most common adverse event. CONCLUSION: India's National Antimalarial Drug Resistance Monitoring System provides wide coverage. The first-line antimalarials used in the country remain safe and efficacious. The treatment of malaria in young children and the relative benefits of age- and weight-based dosing need further exploration.