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Analysis of the vitamin D receptor Fokl polymorphism.

In the present study we analysed the vitamin D receptor (VDR) Fokl polymorphism distribution in a Caucasian population from the North-West of Spain. This polymorphism has been associated with variation in bone mineral density levels and has been proposed as a factor for the genetic predisposition to osteoporosis. This study was performed in 60 healthy individuals by restriction fragment length polymorphism (RFLP) analysis using the endonuclease Fokl and alternatively using single strand conformation polymorphism (SSCP). The genotype distribution was: 46.7% FF homozygotes, 43.3% Ff heterozygotes, and 10% ff homozygotes. These values do not reveal significant differences in comparison with other available Caucasoid populations, but show clear differences with respect to African-American populations. The analysis of this polymorphism by SSCP reveals the existence of a novel single nucleotide polymorphism (AAT/AAC) located within the DNA binding domain of the VDR gene at position-19 codon. Thus, using a novel 109 bp hVDR gene fragment which excludes the AAT/AAC variant in the vitamin D receptor Fokl allows an easier determination by SSCP analysis of the Fokl polymorphism.

Adult↗

Toll-like receptor 4 gene C119A but not Asp299Gly polymorphism is associated with ischemic stroke among ethnic Chinese in Taiwan.

Stroke is one of the leading causes of death in the world. Most stroke patients are classified as having ischemic stroke. The causes of ischemic stroke are very diverse. Atherosclerosis resulting in cerebral or carotid arterial stenosis/occlusion plays the most important role in the occurrence of ischemic stroke. Inflammatory processes or immune responses are involved in the formation of atherosclerosis. Toll-like receptor 4 (TLR4) is a member of the toll-like receptor (TLR) family. TLRs are pattern-recognition receptors, which initiate innate immune responses after interaction with pattern-specific ligands. A polymorphism of the TLR4 gene, Asp299Gly, is associated with an increased risk for coronary heart diseases in Caucasian populations. In this study, we explored the complete coding regions of TLR4 by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), Single-strand conformation polymorphism (SSCP), and sequencing and found obvious ethnic differences. There was no Asp299Gly polymorphism among the ethnic Chinese examined in this study. We found only one polymorphism on intron 1 (A119C) among our samples. The allele frequencies of 119A were 0.0256 and 0.0022 among the patients and controls, respectively. The odds ratio of 119A of TLR4 in ischemic stroke was 11.71 (95% CI: 1.52-90.01). This polymorphism was significantly associated with ischemic stroke. These data possibly implicate TLR4 as an important genetic factor for stroke in ethnic Chinese populations despite the rarity of the Asp299Gly polymorphism.

Aged↗

GGC and StuI polymorphism on the androgen receptor gene in endometrial cancer patients.

Androgens have an anti-proliferative effect on endometrial cells. Human androgen receptor (AR) gene contains two polymorphic short tandem repeats of GGC and CAG, and a single-nucleotide polymorphism on exon 1 that is recognized by the restriction enzyme, StuI. Prior studies have shown that the lengths of the CAG repeat are inversely and linearly related to AR activity and associated with endometrial cancer. However, little is known about the GGC repeat and the StuI polymorphism of the AR gene. Thus, we investigated whether these AR polymorphisms are risk factors for endometrial cancer. To test this hypothesis, the genetic distributions of these polymorphisms were investigated in blood samples from endometrial cancer patients and healthy controls. The allelic and genotyping profiles were analyzed by polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism (PCR-RFLP), and direct DNA sequencing, and analyzed statistically. The GGC repeat was significantly longer in endometrial cancer patients as compared to normal healthy controls. In general, an increased risk of endometrial cancer was found with increasing GGC repeat. The relative risk for the 17 GGC repeat was greater than 4, as compared to controls. However, the StuI polymorphism was not significantly different between patients and controls. The findings suggest that increased numbers of GGC repeat on the AR gene may be a risk factor for endometrial cancer.

Adult↗

Lack of an intronic Sp1 binding-site polymorphism at the collagen type I alpha1 gene in healthy Korean women.

Osteoporosis is a disease that is strongly genetically influenced. However, the genes responsible for the disease are poorly defined. Recent data show that a G-T transition polymorphism of the Sp1 binding site at the collagen type I alpha1 gene (Sp1 polymorphism) is associated significantly with bone mineral density (BMD) and osteoporotic fracture in British women. To establish the association between the Sp1 genotypes and BMD in Korean women, we examined 200 healthy postmenopausal women of Korean ethnicity, ranging in age from 44 to 66 years (mean+/-SD: 54.7+/-5.3 years). PCR amplification using the same primers as those used previously, with enzyme digestion, revealed no restriction site in our samples. We also performed a single-strand conformational polymorphism (SSCP) analysis in 100 of the 200 samples and could not find any polymorphic sites in the PCR amplification region. Based on our study, the Sp1 polymorphism at the type I collagen alpha1 gene was not found in Korean women. Therefore, we suggest that the Sp1 polymorphism at the type I collagen alpha1 gene is absent or rare in Korean women. Based on the present findings, this polymorphism does not seem to be responsible for the entire genetic contribution to BMD.

Adult↗

Association between the P12A and c1431t polymorphisms in the peroxisome proliferator activated receptor gamma (PPAR gamma) gene and type 2 diabetes.

Variation in the peroxisome proliferator-activated receptor gamma (PPAR gamma) gene may play a role in the development of type 2 diabetes mellitus. Therefore we investigated the association between the P12A and c1431t polymorphisms in the PPAR gamma gene and type 2 diabetes. The incidence of the P12A polymorphism was determined by PCR-RFLP and the c1431t by single-strand conformation polymorphism analysis in 219 patients with, and 429 without type 2 diabetes. The frequency of the A allele of P12A polymorphism was 0.16 and the t allele of c1431t polymorphism, 0.13 in patients with type 2 diabetes, and 0.13 and 0.12 respectively in subjects without diabetes 3.2% of patients with and 1.4% without type 2 diabetes were A12A. Since the polymorphisms are not linked the association of the 9 possible genotypes with type 2 diabetes was determined. All patients with genotype A12A/c1431c had type 2 diabetes (n = 3, p = 0.038). There was no association between A12A/t1431t and diabetes. DNA sequencing revealed no additional mutations in the coding region of the PPAR gamma gene in genotypes A12A/c1431c or A12A/t1431t. The associations found between polymorphisms in the PPAR gamma gene and type 2 diabetes suggest that either the A12 isofrom is functional leading to a predisposition to type 2 diabetes in homozygotes or that there is a third, unknown mutation linked to the A12/c1431 haplotype which is responsible.

Adult↗

Analyses of cross species polymerase chain reaction products to infer the ancestral state of human polymorphisms.

In numerous population genetic and disease association studies decisions about the ancestry of polymorphic alleles are often made based on the relative frequency of the alleles in the extant populations with the most frequent allele being deemed as ancestral. However, the frequency of an allele in a population is generally not a perfect indicator of its ancestral status. A more accurate method to assess ancestral/derived status of polymorphic alleles involves identification of shared alleles between species. We used this strategy to examine genomic regions homologous to several human polymorphisms in four species of non-human primates. Cross species polymerase chain reaction (CS-PCR), with primers designed from human sequence, was used to investigate regions of interest. Nineteen polymorphisms at six loci (DRD2, HOXB@, PAH, D4S10, RBP3, and RET) were examined either by restriction fragment length analysis of PCR products (PCR-RFLP) or by direct sequencing. At seventeen of the eighteen PCR-RFLPs, non-human primates were monomorphic and identical to each other for either lack of restriction enzyme site or presence of the site. Thus, at these seventeen polymorphic sites the shared alleles are most likely to be the ancestral ones in humans. In several cases we have used sequence data to further demonstrate that the nucleotide at the site of the polymorphism is conserved between species confirming the hypothesis of a single ancestral allele. However, not all human alleles can be simply resolved into ancestral and derived; sequence data from one PCR-RFLP (in an intron of the PAH locus) and a single strand conformational polymorphism (SSCP) in the 3' untranslated region (UTR) of the DRD2 gene illustrate this point.

Alleles↗

[Lecithin-cholesterol acyltransferase gene 608C/T polymorphism associated with atherosclerotic cerebral infarction].

OBJECTIVE: To explore the distribution of lecithin-cholesterol acyltransferase gene (LCAT) 608C/T polymorphism in Chinese Han population and the relationship of the polymorphism association with the occurrence of atherosclerotic cerebral infarction. METHODS: The lecithin:cholesterol acyltransferase gene 608C/T polymorphism is identified by polymerase chain reaction (PCR), single-strand conformation polymorphism (SSCP)and restriction fragment length polymorphism (RFLP) in 150 patients with ACI and 122 healthy controls matching age and sex. RESULTS: The distribution of LCAT 608C/T gene polymorphism was in accordance with Hardy-Weinberg equilibrium. The CT genotype frequency (14.0%) and T allele frequency (7.0%) in ACI group were significantly higher than those in control group (P<0.05). The concentration of high density lipoprotein cholesterol (HDL-C) in 608CC subgroups were significantly higher than those in 608CT subgroups both in ACI group and in control group (P<0.05). CONCLUSION: The LCAT 608C/T polymorphism is possibly a predisposing factor in ACI happening of Chinese Han population. T allele frequency is possibly concerned with the metabolism of HDL-C.

Aged↗

Trends in solubility of polymorphs.

Polymorphism of drug substances has been the subject of intense investigation in the pharmaceutical field for over 40 years. Considering the multitude of reports on solubility or dissolution of polymorphs in the literature, an attempt is made in this study to answer the question: How big is the impact of polymorphism on solubility? A large number of literature reports on solubility or dissolution of polymorphs were reviewed and the data were analyzed for trends in solubility ratio of polymorphs. The general trend reveals that the ratio of polymorph solubility is typically less than 2, although occasionally higher ratios can be observed. A similar trend is also observed for anhydrate/hydrate solubility ratios, although anhydrate/hydrate solubility ratios appear to be more spread out and higher than the typical ratio for nonsolvated polymorphs. An attempt is also made in this commentary to estimate the ratio of solubilities of polymorphs from thermal data. The trend in estimated solubility ratio shows good agreement with the one observed with experimentally determined solubility values.

Algorithms↗

Statistical analysis of differences in the Raman spectra of polymorphs.

Raman spectroscopy is a useful tool for identifying polymorphs of pharmaceutical compounds. One limitation of the technique is that the small differences in Raman spectra require confirmation of polymorphs by other methods. Fourteen compounds, both commercial and proprietary pharmaceutical compounds and their polymorphs, were analyzed by Raman microscopy. By using descriptive statistics and analysis of variance (ANOVA), several methods are proposed that provide an approach for comparing the spectra of suspected polymorphs. Because it is difficult to determine the exact amount that a peak may shift before two forms should be considered different; a guideline of a shift greater than 1.6/cm(-1) is proposed. A standard ANOVA analysis is used to compare individual peaks both within and between polymorphs, as well as an alternative method that proposes the use of a total ANOVA table that considers the entire spectrum. Both methods have their advantages and disadvantages, but they provide a starting point for the comparison of a large number of spectra, and effectively differentiate between polymorphs of a given compound. The method accurately identified true polymorphs in all cases, but showed a bias towards misidentifying some samples as polymorphs when they were in fact the same form. This bias was not significant and even in these situations, the magnitude of the calculated F values was a useful indicator of whether the result was a false positive or not.

Analysis of Variance↗

Understanding the polymorphic behavior of sibenadet hydrochloride through detailed studies integrating structural and dynamical assessment.

Three polymorphs of sibenadet hydrochloride (AR-C68397AA, Viozan) have been shown to exist at ambient temperature and have been characterized. Each undergoes a solid-state transition to a common high temperature form, which melts at approximately 220 degrees C. X-ray powder diffraction (XRPD) indicates that Polymorphs I and II share a similar layered structure not exhibited by Polymorph III. All three ambient temperature polymorphs show evidence of varying degrees of dynamic disorder of the terminal phenyl group as shown by solid-state NMR spectroscopy. Furthermore, the carbons alpha to the ether link also undergo different rates of mobility in Polymorphs I and II. This variation in the extent of dynamic disorder results in an alteration in the short-range structure resulting in distinct polymorphs. Polymorph I is the thermodynamically stable form at room temperature as indicated by solution calorimetry and assessment by thermodynamically driven solution mediated phase transition studies. The present study aims to address the types of discriminatory data required to make a clear distinction between physical forms and define, unequivocally, the presence of polymorphism.

Adrenergic beta-2 Receptor Agonists↗

Genetic polymorphism of CYP1A1, CYP2D6, GSTM1 and GSTT1 and susceptibility to acute lymphoblastic leukaemia in Indian children.

BACKGROUND: Biotransformation plays a crucial role in carcinogen activity and many genetic polymorphisms in xenobiotic metabolising enzymes have been associated with an increased risk of cancer. Such polymorphisms can lead to considerable variation in the activities of these enzymes, which are crucial in carcinogen and drug metabolism. These variations could play a role in the risk of developing paediatric acute lymphoblastic leukaemia (ALL) by their varying action on environmental carcinogens. PROCEDURE: The present study looked for two polymorphisms (m1 and m2) in the CYP1A1, CYP2D6*4 genes and deletions of the glutathione S-transferases (GSTM1 and GSTT1) in 118 paediatric ALL patients and 118 age matched control children. The polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) were used to study gene polymorphisms. RESULTS: In children with ALL, CYP1A1 m1 polymorphism was evident in 42.4% of subjects and CYP1A1 m2 in 37.3%. These were significantly different from the results obtained for control children (20.3% for CYP1A1 m1 and 19.5% for m2). Subjects with CYP1A1 m1 homozygous variant had a sixfold risk and CYP1A1 m2 a fourfold risk. In contrast, CYP2D6*4 was more prevalent in the controls than in the cases. Subjects with GSTM1 deletions had increased risk of ALL (OR = 2.1, P = 0.009). The odds ratios for both CYP1A1 m1 and m2 homozygous polymorphisms being associated with childhood ALL was 5.67 (95% CI = 2.11-15.27). The odds ratios for both GSTM1 and GSTT1 deletions being associated with ALL was 2.78 (95% CI = 0.67-11.56). CONCLUSIONS: These results suggest that genetic polymorphisms of xenobiotic metabolising enzymes appear to influence susceptibility to childhood ALL.

Adolescent↗

A comparison of morphology and surface energy characteristics of sulfathiazole polymorphs based upon single particle studies.

The morphological, adhesion and surface energetic properties of three sulfathiazole polymorphs (III, IV and polymorph I prepared from both acetone and methanol, designated I-ace and I-met, respectively) produced using Nektar supercritical fluid (SCF) technology have been characterized using scanning electron microscopy (SEM) and atomic force microscopy (AFM). Surface roughness values for each polymorph were determined at different length scales. At sample sizes less than 1micromx1microm the polymorphs rank in terms of roughness as follows: I-met>I-ace approximately equal to IV>III. At the larger scales the polymorphs rank in terms of roughness as follows: I-met>III>I-ace approximately equal to IV. The surface energies for polymorphs determined against graphite (HOPG) and particles of the same polymorph were, respectively, I-met: 0.99mJm(-2) (S.D. 1.25mJm(-2)), 3.09mJm(-2) (S.D. 2.67mJm(-2)); I-ace: 309mJm(-2) (S.D. 329mJm(-2)), 16mJm(-2) (S.D. 11mJm(-2)); III: 1.17mJm(-2) (S.D. 1.5mJm(-2)), 5.4mJm(-2) (S.D. 3.6mJm(-2)); IV: 20.35mJm(-2) (S.D. 28.5mJm(-2)), 16.8mJm(-2) (S.D. 9.6mJm(-2)). In terms of surface energies the polymorphs hence rank I-ace>IV>III approximately equal to I-met (HOPG adhesion measurements) and IV approximately equal to I-ace>III>I-met (particle cohesion measurements). Consideration of contacting asperities and surface roughness was shown to have limited effect on the surface energies, and instead the differences were ascribed to variations in the surface chemistry as a result of changes in crystallization mechanisms.

Acetone↗

Characterizing the conversion kinetics of carbamazepine polymorphs to the dihydrate in aqueous suspension using Raman spectroscopy.

In an aqueous environment, polymorphic forms I-III of carbamazepine all convert to the dihydrate. This study investigated the conversion of each polymorphic form individually and of a mixture of forms III and I to the dihydrate. Two batches of form I with different crystal morphology were used. Samples were dispersed independently in water at 23+/-1 degrees C and recovered at various timepoints varying from 10 to 210 min. Scanning electron microscopy, X-ray powder diffraction and Raman spectroscopy were used to characterize the initial polymorphic forms and the recovered samples after 210 min. Raman spectroscopy combined with partial least squares analysis was used to generate quantitative models of binary and ternary mixtures of the different polymorphic forms with the dihydrate. On the basis of these models the conversion kinetics of the polymorphic forms I-III were characterized. First-order kinetics with an unconverted portion were used to model the data (R2> or =0.95). The unconverted portions ranged from 16 to 51% after dispersion for 210 min. The conversion kinetics were similar between polymorphic forms with comparable crystal morphology, but differed significantly between batches of the same polymorph (form I) with different crystal morphology. Furthermore, the conversion of forms III and I in the aqueous suspension was not influenced by the presence of the other polymorph when dispersed together.

Anticonvulsants↗

Solubility behavior of polymorphs I and II of mefenamic acid in solvent mixtures.

The dissolution profile and solubility of two polymorphic forms of mefenamic acid were studied in solvent mixtures of ethanol-water and ethyl acetate-ethanol. The solubility parameter (delta) was used to study the effect of polarity on the solubility behavior of the two polymorphs. Differential scanning calorimetry and infrared spectroscopy were performed on the original powders and on the solid phases after contact with the solvent systems for the characterization and identification of the polymorphs. The dissolution rates of both polymorphs is greater in the less polar mixtures (ethyl acetate-ethanol) of lower solubility parameter values. Form II showed larger dissolution rates and saturation concentrations than Form I in all the solvent systems studied. The solid phase of Form II converts totally to Form I after equilibration with the solvents. The rate of conversion was faster in the least polar mixtures. The solubility of both polymorphs reaches a single maximum at 80% ethyl acetate in ethanol, delta = 20.09 MPa1/2. The modified extended Hildebrand method was used to predict the solubility profile of each polymorph. A single equation was obtained for both polymorphs which includes the solubility parameter of the mixtures and the logarithm of the solubility mole fraction of each polymorph in water. The Hildebrand solubility parameter of mefenamic acid is independent of the crystalline form and was determined from two methods giving quite similar values, delta 2 = 20-21 MPa1/2.

Acetates↗

Crystallization of D-mannitol in binary mixtures with NaCl: phase diagram and polymorphism.

PURPOSE: To study the crystallization, polymorphism, and phase behavior of D-mannitol in binary mixtures with NaCl to better understand their interactions in frozen aqueous solutions. METHODS: Differential scanning calorimetry, hot-stage microscopy, Raman microscopy, and variable-temperature X-ray diffractometry were used to characterize D-mannitol-NaCl mixtures. RESULTS: NaCl and D-mannitol exhibited significant melt miscibility (up to 7.5% w/w or 0.20 mole fraction of NaCl) and a eutectic phase diagram (eutectic composition 7.5% w/w NaCl; eutectic temperature 150 degrees C for the alpha and beta polymorphs of D-mannitol and 139 degrees C for the delta). The presence of NaCl did not prevent mannitol from crystallizing but, depending on sample size, affected the polymorph crystallized: below 10 mg, delta was obtained; above 100 mg, alpha was obtained. Pure mannitol crystallized under the same conditions first as the delta polymorph and then as the a polymorph, with the latter nucleating on the former. KCl showed similar eutectic points and melt miscibility with D-mannitol as NaCl. LiCl yielded lower eutectic melting points, inhibited the crystallization of D-mannitol during cooling, and enabled the observation of its glass transition. CONCLUSIONS: Despite their structural dissimilarity, significant melt miscibility exists between D-mannitol and NaCl. Their phase diagram has been determined and features polymorph-dependent eutectic points. NaCl influences the polymorphic behavior of mannitol, and the effect is linked to the crystallization of mannitol in two polymorphic stages.

Algorithms↗

Three polymorphs (alpha, beta, and delta) of D-mannitol at 100 K.

In the monoclinic delta polymorph of D-mannitol, C(6)H(14)O(6), both the molecule and the packing have approximate twofold rotational symmetry. The P2(1) structure thus approximates space group C222(1), and the alpha' polymorph, previously reported in that space group, is almost certainly identical to the delta polymorph. However, torsion angles along the main backbone of the molecule deviate from twofold symmetry by as much as 7.4 (3) degrees and the hydrogen-bonding pattern does not conform to the higher symmetry. The alpha polymorph reported here is identical to the previously reported kappa polymorph, and the low-temperature structure of the beta polymorph agrees well with previously reported room-temperature determinations. The range of C-O bond lengths over the three polymorphs is 1.428 (2)-1.437 (4) A, and the range of C-C distances is 1.515 (4)-1.5406 (19) A. The delta polymorph has the highest density of the three, both at room temperature and at 100 K.

Cold Temperature↗

Concomitant polymorphism and a temperature-dependent phase change in (E)-[1-(4-methoxyphenyl)-3-phenyl-2-propenylideneamino]oxyacetic acid.

The title compound, C(18)H(17)NO(4), crystallizes from ethanol at ambient temperature as two concomitant polymorphs (I) and (II), both monoclinic P2(1)/c with Z' = 1. The less abundant form (I) undergoes a reversible phase-transition at ca 173 K to a third monoclinic polymorph (III), P2(1)/n, with Z' = 2, while the more abundant polymorph (II) is unchanged down to 120 K. In each polymorph of (I)-(III) the molecules are linked by pairs of O-H...O hydrogen bonds into cyclic dimers which are crystallographically centrosymmetric in (I) and (II), and approximately, but not crystallographically, centrosymmetric in (III). There are no direction-specific interactions between the hydrogen-bonded dimers in polymorph (I); in polymorph (II) the dimers are linked into sheets by C-H...N and C-H...pi(arene) hydrogen bonds; in polymorph (III) the dimers are linked into chains by a C-H...pi(arene) hydrogen bond. The interconversion of polymorphs (I) and (III) is a simple displacive phase transition.

Acetates↗

No association of DRD2, DRD3, and tyrosine hydroxylase gene polymorphisms with personality traits in the Japanese population.

BACKGROUND: Dopamine D2 receptor (DRD2) and dopamine D3 receptor (DRD3) genes could be candidates for personality-related genes considering their pharmacological profiles or structures. However, a limited number of studies have investigated the association between these genes and personality traits. In the present study, we investigated the DRD2, DRD3, and tyrosine hydroxylase (TH) genes in relation to personality traits in the Japanese population. Epistasis (gene-gene interaction) among the genes was extensively analyzed, in addition to the analysis based on each gene. METHODS: The -241A/G, -141C Ins/Del, and Ser311Cys polymorphisms in the DRD2 gene, the Ser9Gly polymorphism of the DRD3 gene, and the Val81Met and PstI site polymorphisms in the TH gene were genotyped in 257 healthy Japanese subjects. Personality traits were evaluated by using the Revised NEO Personality Inventory (NEO PI-R) and the State-Trait Anxiety Inventory (STAI). The associations between gene polymorphisms and the scores for NEO PI-R or Trait Anxiety of STAI were statistically analyzed by one-way analysis of covariance (ANCOVA) adjusting sex and age. Epistasis was assessed using two-way ANCOVA between the polymorphisms of independent two genes. RESULTS: In the analysis based on each gene, trends for association were observed between State Anxiety and the DRD2 -141C Ins/Del polymorphism (p = 0.031, uncorrected), and between Trait Anxiety and the DRD2 Ser311Cys or TH PstI site polymorphism (p = 0.048 and 0.041, respectively, uncorrected). In epistatic analysis, a trend for interaction was observed on the scores for Neuroticism and Trait Anxiety between the DRD2 -141C Ins/Del and TH Val81Met polymorphisms (p = 0.015 and 0.010, respectively, uncorrected). However, these differences were insignificant after Bonferroni correction. CONCLUSION: The present study did not provide evidence for the association between these dopamine-related genes and personality traits in the Japanese population.

Journal Article↗