Frequency of CCR5 Gene 32-bp deletion in Pakistani ethnic groups

CCR5 is a G-protein-coupled chemokine receptor that is used as a co-factor by macrophage-tropic (M-tropic) isolates of human immunodeficiency virus-1 (HIV-1) to gain entry into host cells. A 32-bp deletion in the CCR5 gene (CCR5-Delta32) leads to the production of an altered gene product that prevents HIV-1 from entering the host cell. This study was carried out to determine prevalence of CCR5-Delta32 allele frequency in a large Pakistani population sample (n = 821) representing 10 ethnic groups. No individual was homozygous for the mutant allele and the frequency of the CCR5-Delta32 allele ranged from 0.62% to 3.57%. The CCR5-Delta32 allele frequency was generally lower in populations from southern Pakistan. The overall frequency of the CCR5-Delta32 allele in Pakistan was 2.31%, which is much lower than that found in European populations and similar to that in the Middle East. This is consistent with the historical records and genetic data that indicate a close genetic affinity among these populations. This study demonstrates that the Pakistani population is highly susceptible to M-tropic isolates of HIV-1 and public health measures need to be enforced with urgency if Pakistan is to avoid an HIV epidemic.     

Comparison of Univariate and Multivariate Models of 13C SSNMR and XRPD Techniques for Quantification of Nimodipine Polymorphs

The focus of the present investigation was to explore the use of solid-state nuclear magnetic resonance (¹³C ssNMR) and X-ray powder diffraction (XRPD) for quantification of nimodipine polymorphs (form I and form II) crystallized in a cosolvent formulation. The cosolvent formulation composed of polyethylene glycol 400, glycerin, water, and 2.5% drug, and was stored at 5°C for the drug crystallization. The ¹³C ssNMR and XRPD data of the sample matrices containing varying percentages of nimodipine form I and form II were collected. Univariate and multivariate models were developed using the data. Least square method was used for the univariate model generation. Partial least square and principle component regressions were used for the multivariate models development. The univariate models of the ¹³C ssNMR were better than the XRPD as indicated by statistical parameters such as correlation coefficient, R², root mean square error, and standard error. On the other hand, the XRPD multivariate models were better than the ¹³C ssNMR as indicated by precision and accuracy parameters. Similar values were predicted by the univariate and multivariate models for independent samples. In conclusion, the univariate and multivariate models of ¹³C ssNMR and XRPD can be used to quantitate nimodipine polymorphs.KEY WORDS: nimodipine polymorphs, X-ray powder diffraction, solid-state nuclear magnetic resonance, univariate, multivariate     

N-acetyltransferase polymorphism among northern Sudanese

Interindividual and interethnic differences in allele frequencies of N-acetyltransferase (NAT2) single nucleotide polymorphisms (SNPs) are responsible for phenotypic variability of adverse drug reactions and susceptibility to cancer. We genotyped the seven NAT2 common SNPs in 127 randomly selected unrelated northern Sudanese subjects using allele-specific and RFLP polymerase chain reaction (PCR) based methods. Molecular genotyping was enough to designate alleles for 41 individuals unambiguously, whereas 63 individuals' alleles were inferred from haplotypes previously described. In the remaining 23 individuals, however, the phase of the SNPs could not be decided because of multiple SNP heterozygotes. Using computational methods in the HAP and Phase programs, we confirmed the inferred alleles of the 62 individuals and predicted the remaining 23 ambiguous alleles. Twelve NAT2 alleles were identified. Four alleles coded for rapid acetylators (18%), and eight alleles coded for slow acetylators (82%). Two genotypes coded for rapid acetylation (3.9%), 10 for intermediate acetylation (27.6%), and 13 for slow acetylation (68.5%). The G191A African SNP and the G857A predominantly Asian SNP were each detected at a low frequency of 3.1%. The combination of molecular and computational analysis was useful in resolving ambiguous genotypes of NAT2 in multiple SNP heterozygotes. Among the northern Sudanese the SNPs associated with slow acetylation are more prevalent than in Caucasians and Asians. This and other African studies are suggestive of an African origin for NAT2-associated polymorphism.     

Comparative genomic hybridization arrays in clinical pathology: progress and challenges

Array-based comparative genomic hybridization (array CGH) genome scanning is a powerful method for the global detection of gains and losses of genetic material in both congenital and neoplastic disorders. When used as a clinical diagnostic test, array CGH combines the whole genome perspective of traditional G-banded cytogenetics with the targeted identification of cryptic chromosomal abnormalities characteristic of fluorescence in situ hybridization (FISH). However, the presence of structural variants in the human genome can complicate analysis of patient samples, and array CGH does not provide morphologic information about chromosome structure, balanced translocations, or the actual chromosomal location of segmental duplications. Identification of such anomalies has significant diagnostic and prognostic implications for the patient. We therefore propose that array CGH should be used as a guide to the presence of genomic structural rearrangements in germline and tumor genomes that can then be further characterized by FISH or G-banding, depending on the clinical scenario. In this article, we share some of our experiences with diagnostic array CGH and discuss recent progress and challenges involved with the integration of array CGH into clinical laboratory medicine.     

RNAi-mediated male sterility of tobacco by silencing TA29

The superior performance of F₁ hybrids has a significant impact on agricultural productivity. For commercial application, the availability of an efficient system for obtaining male-sterile lines of crops is an essential prerequisite. Here we have investigated the use of RNA interference (RNAi) technology to silence a male-specific gene in the model host tobacco. TA29 is expressed exclusively in anthers at the time of microspore development. About 10 out of 13 tobacco lines transformed with a hairpin RNAi construct containing TA29 sequences were male sterile. Transgenic plants were phenotypically indistinguishable from non-transgenic plants. At the anthesis stage, pollen grains from transgenic, male-sterile plants were aborted and lysed in comparison to the round and fully developed pollen in non-transgenic plants. Microscopic analysis of anthers showed selective degradation of tapetum in transgenic plants with no microspore development. One week after self-pollination, the ovules of non-transgenic plants were double the size of those in transgenic plants, due to successful self-fertilization. Male sterile transgenic plants set seed normally, when cross-pollinated with pollen from non-transgenic plants, confirming no adverse effect on the female parts of the flower. These results show that silencing of male-specific genes by RNAi is potentially a useful tool for generating male-sterile lines for producing hybrid seed.     

A QbD Case Study: Bayesian Prediction of Lyophilization Cycle Parameters

As stipulated by ICH Q8 R2 (1), prediction of critical process parameters based on process modeling is a part of enhanced, quality by design approach to product development. In this work, we discuss a Bayesian model for the prediction of primary drying phase duration. The model is based on the premise that resistance to dry layer mass transfer is product specific, and is a function of nucleation temperature. The predicted duration of primary drying was experimentally verified on the lab scale lyophilizer. It is suggested that the model be used during scale-up activities in order to minimize trial and error and reduce costs associated with expensive large scale experiments. The proposed approach extends the work of Searles et al. (2) by adding a Bayesian treatment to primary drying modeling.