Heterozygous Type Research Articles

Founder mutation Arg485Pro led to recurrent compound heterozygous GGCX genotypes in two German patients with VKCFD type 1

Congenital combined deficiency of the vitamin-K-dependent coagulation factors (VKCFD) represents a rare autosomal recessive inherited bleeding disorder caused by mutations in either the gamma-glutamyl carboxylase gene (VKCFD type 1) or the vitamin K epoxide reductase gene (VKCFD type 2). Four different mutations of the gamma-glutamyl carboxylase gene (GGCX) have so far been reported in three unrelated patients with VKCFD type 1. Here we report on a fourth patient who presented with two compound heterozygous missense mutations of the GGCX gene, His404Pro and Arg485Pro. The His404Pro mutation has not been described previously, while the Arg485Pro mutation has been reported in another compound heterozygous VKCFD type 1 patient from Germany. Most interestingly, haplotype analysis revealed that Arg485Pro is due to a founder mutation, suggesting that this mutation is present in the German population at some low frequency. The founder mutation explains that the only two compound heterozygous VKCFD type 1 patients known today originated from Germany.

Effects of Polymorphisms of Chemokine Receptors on Neurodevelopment and the Onset of Encephalopathy in Children with Perinatal HIV-1 Infection

This study examined the effects of chemokine receptor polymorphisms on neurodevelopment and the onset of encephalopathy in children with perinatal HIV-1 infection. Infected children (N = 121) between the ages of 1 and 72 months were categorized into dichotomous groups (heterozygous or homozygous mutant vs. homozygous wild type) for each chemokine receptor 2 (CCR2) and chemokine receptor 5 (CCR5) allele. Neurodevelopmental measures included the Bayley Scales of Infant Development (BSID) for children age equal to or less than 30 months and the McCarthy Scales of Children's Abilities (MSCA) for children aged > 30 months. A basic linear spline was used to model the mean value at each visit for the relevant test index, with determination of the slope between 4-12 months, 12-30 months, and 31-72 months of age. A mixed model analysis of variance was used to compare differences between slopes (Δβ) and intercepts (Δα) according to the presence or absence of the specified CCR2 or CCR5 polymorphism. Survival analyses were used to compare the onset of encephalopathy by chemokine receptor allelic grouping. After adjusting for potential confounds, statistically significant differences emerged in CCR5-39353, 39356, and 39402. Although the protective effects appeared to be discrete and transient, children with mutant CCR5 genotypes exhibited better neurodevelopmental outcomes than children with the wild type alleles. Chemokine polymorphisms did not appear to impact the onset of encephalopathy. Although possibly a temporary effect, HIV-1 infected children with selected mutant chemokine receptor polymorphims CCR5-39353, 39356, and 39402 may exhibit better neurodevelopmental outcome than children with the wild type allele.

Food motivated behavior of melanocortin-4 receptor knockout mice under a progressive ratio schedule

Melanocortin-4 receptor knockout (MC4RKO) mice are hyperphagic and develop obesity under free feeding conditions. We reported previously that MC4RKO mice did not maintain hyperphagia and as a result lost weight when required to press a lever to obtain food on a fixed ratio procurement schedule. To assess the generality of this result, we tested MC4RKO mice and their heterozygous and wild type littermates using progressive ratio (PR) schedules that are believed to be sensitive indicators of motivation. Mice lived in operant chambers and obtained all of their food (20 mg pellets) via lever press responding. Food was available according to a PR schedule so that within a meal, food became progressively more costly, and we expected this would provide a stringent test of mechanisms controlling meal size. The schedule reset after either 3 or 20 min of no responding, so defining meals, and the highest ratio completed before the reset was defined as the breakpoint. The average daily number of meals was lower and mean size of meals was higher at the 20 compared with the 3 min reset condition. Mean daily food intake did not differ between the two reset criteria but did differ as a function of genotype, with MC4RKO mice eating about 25% more than heterozygous or wild type mice. Hyperphagia in the MC4RKO mice was characterized primarily by larger meals (higher breakpoints) and they emitted about twice as many responses as wild type mice. Thus, using a PR schedule, MC4RKO mice exhibit hyperphagia, and show a high level of motivation to support large meal sizes.

The putative SWI/SNF complex subunit BRAHMA activates flower homeotic genes in Arabidopsis thaliana

Arabidopsis thaliana BRAHMA (BRM, also called AtBRM) is a SNF2 family protein homolog of Brahma, the ATPase of the Drosophila SWI/SNF complex involved in chromatin remodeling during transcription. Here we show that, in contrast to its Drosophila counterpart, BRM is not an essential gene. Thus, homozygous BRM loss of function mutants are viable but exhibit numerous defects including dwarfism, altered leaf and root development and several reproduction defects. The analysis of the progeny of self-fertilized heterozygous brm plants and reciprocal crosses between heterozygous and wild type plants indicated that disruption of BRM reduced both male and female gametophyte transmission. This was consistent with the presence of aborted ovules in the self-fertilized heterozygous flowers that contained arrested embryos predominantly at the two terminal cells stage. Furthermore, brm homozygous mutants were completely sterile. Flowers of brm loss-of-function mutants have several developmental abnormalities, including homeotic transformations in the second and third floral whorls. In accordance with these results, brm mutants present reduced levels of APETALA2, APETALA3, PISTILLATA and NAC-LIKE, ACTIVATED BY AP3/PI. We have previously shown that BRM strongly interacts with AtSWI3C. Now we extend our interaction studies demonstrating that BRM interacts weakly with AtSWI3B but not with AtSWI3A or AtSWI3D. In agreement with these results, the phenotype described in this study for brm plants is very similar to that previously described for the AtSWI3C mutant plants, suggesting that both proteins participate in the same genetic pathway or form a molecular complex.

Prevalence of Prothrombin Gene Mutation (G-A 20210 A) in General Population: A Pilot Study

The objective of this study was to determine the prevalence of prothrombin gene mutation in a sample population from Pakistan. Two hundred apparently healthy unrelated adults (older than 18 years) were included in the study. The sample population comprised 100 Punjabis (male 50, female 50) and 100 Pathans (male 50, female 50). Patients with a history of previous thromboembolism were excluded from the study. Five milliliters (5 mL) of whole blood was drawn in an EDTA bottle. The DNA was extracted by the standard phenol-chloroform method. The DNA was amplified between exon number 14 and the 3'-untranslated region of the prothrombin gene by a polymerase chain reaction in a thermal cycler. Amplified products were digested overnight with HindIII at 37 degrees C. The digested products were electrophoresed on 6% polyacrylamide gel. The fragments were visualized by silver nitrate staining. A heterozygous wild type and an uncut amplified product were included in the electrophoresis strip for quality control. The wild type of DNA ran as a 350-bp fragment and internal control was cut as 550- and 150-bp fragments. The abnormal prothrombin gene was cut into 350-, 322-, and 28-bp fragments. Only two cases of heterozygous prothrombin gene mutation G-A 20210A were found in the sample studied, giving an overall carrier rate of 01% (95% CI 0.4-2.4%) in the target population. Prothrombin gene mutation is present in our population but at a lower frequency than in the white population.

Severe hearing loss in Dlx1 mutant mice

The Dlx homeobox gene family participates in regulating middle and inner ear development. A significant role for Dlx1, in particular, has been demonstrated in the development of the middle ear ossicles, but the functional consequences of Dlx1 gene mutation on hearing thresholds has not been assessed. The present study characterizes auditory brainstem responses to click and tonal stimuli in a non-lethal variant of a Dlx1 gene knockout. We found that peripheral hearing thresholds for click and tonal stimuli were significantly elevated in homozygous Dlx1 knockout ( Dlx1 −/−) compared to both heterozygous (Dlx1 +/−) and wild type ( Dlx1 +/+) mice. Thus, abnormal morphogenesis of the incus and stapes that has been documented previously with histological measures is now known to result in a severe peripheral hearing deficit.

Determination of thiopurine S-methyltransferase (TPMT) activity by comparing various normalization factors: Reference values for Estonian population using HPLC-UV assay

Thiopurine S-methyltransferase (TPMT; EC 2.1.1.67) is the key enzyme in the metabolism of thiopurine drugs. Determination of TPMT activity has been used for the individualization of thiopurine dose. We developed HPLC-UV assay for the determination of TPMT activity in human erythrocytes using 6-mercaptopurine as a substrate. Various extraction and chromatographic conditions were compared. In-house developed extraction with acetonitrile provided the lowest limit of quantification. TPMT activity was determined in 99 previously genotyped healthy Estonians. TPMT activity was expressed as the formation of 6-methylmercaptopurine ng/ml/h and normalized either to haemoglobin, haematocrit, erythrocyte count or protein content. The receiver-operating characteristic curve analysis revealed similar accuracy values for TPMT activity in predicting heterozygous and wild type individuals for each method of calculation. In healthy Estonians, TPMT activity varied from 21.5 to 129.6 ng/ml/h. For heterozygous individuals (n = 18), TPMT activity was 48.1 +/- 11.7 ng/ml/h. Wild type individuals (n = 81) revealed significantly higher TPMT activity 79.3 +/- 20.7 ng/ml/h (P < 0.001). This sensitive HPLC assay for quantitative determination of TPMT activity could easily be used in clinical settings. Under constant experimental conditions for haemolysate preparation no normalization is required.

Lack of association between mannose-binding lectin gene polymorphisms and juvenile idiopathic arthritis in a Han population from the Hubei province of China

Many studies have reported that polymorphisms of the mannose-binding lectin (MBL) gene are associated with autoimmune disease. Here, we investigate the relationship between MBL gene polymorphisms and susceptibility to juvenile idiopathic arthritis (JIA) in a Han-nationality population from the Hubei province of China. PCR-restriction fragment length polymorphism was used to investigate polymorphisms of codons 54 and 57 in exon 1 of the MBL gene in 93 patients with JIA and 48 control children. Neither group showed codon 57 polymorphisms. There was no significant difference in the genotypic frequencies of codon 54 between patients with JIA and healthy controls (wild type, 71.0% versus 75.0%, respectively; heterozygous type, 25.8% versus 25.0%, respectively; and homozygous type, 3.2% versus 0.0%, respectively). In addition, no association was found between the subgroups of patients with JIA and control individuals. Our results provide no evidence for a relationship between MBL gene mutation and susceptibility to JIA.

The mop1 (mediator of paramutation1) Mutant Progressively Reactivates One of the Two Genes Encoded by the MuDR Transposon in Maize

Transposons make up a sizable portion of most genomes, and most organisms have evolved mechanisms to silence them. In maize, silencing of the Mutator family of transposons is associated with methylation of the terminal inverted repeats (TIRs) surrounding the autonomous element and loss of mudrA expression (the transposase) as well as mudrB (a gene involved in insertional activity). We have previously reported that a mutation that suppresses paramutation in maize, mop1, also hypomethylates Mu1 elements and restores somatic activity to silenced MuDR elements. Here, we describe the progressive reactivation of silenced mudrA after several generations in a mop1 background. In mop1 mutants, the TIRA becomes hypomethylated immediately, but mudrA expression and significant somatic reactivation is not observed until silenced MuDR has been exposed to mop1 for several generations. In subsequent generations, individuals that are heterozygous or wild type for the Mop1 allele continue to exhibit hypomethylation at Mu1 and mudrA TIRs as well as somatic activity and high levels of mudrA expression. Thus, mudrA silencing can be progressively and heritably reversed. Conversely, mudrB expression is never restored, its TIR remains methylated, and new insertions of Mu elements are not observed. These data suggest that mudrA and mudrB silencing may be maintained via distinct mechanisms.

Differential effects of the loss of intrachain- versus interchain-disulfide bonds in the cystine-knot domain of von Willebrand factor on the clinical phenotype of von Willebrand disease

Von Willebrand factor (VWF) contains a large number of cysteine residues, which all form disulfide bonds. Mutations of cysteines located in the cystine-knot (CK) domain of VWF have been identified in both qualitative type 2A (IID) and quantitative type 3 von Willebrand disease (VWD). Our objective was to test the hypothesis that the difference in phenotype is related to whether the mutated cysteine residue is involved in either interchain- or intrachain-disulfide-bond formation. The effects of three cysteine mutations which are all located in the CK-domain of VWF, C2773S (type 2A(IID)), C2739Y (type 3), and C2754W (type 3), were studied by transient expression in 293T cells. Cotransfection of wild-type (wt) and C2773S VWF constructs reproduced the plasma phenotype of heterozygous type 2A(IID) patients, with normal to high levels of VWF antigen (VWF:Ag), absence of high-molecular-weight multimers, and the presence of intervening bands between the normal multimers. In contrast, single transfections of C2739Y or C2754W resulted in a quantitative VWF defect with low VWF:Ag levels, and co-transfections of wt and mutant constructs resulted in a 50% reduction of VWF:Ag and only a minor effect on VWF multimerization. We demonstrated N-terminal dimerization of VWF-C2773S and both N- and C-terminal dimerization of VWF-C2754W. Our data suggest that loss of a single disulfide bond in the CK-domain of VWF leads to a recessive, quantitative VWF deficiency if an intrachain-disulfide bond is involved, and to a dominant-negative, qualitative defect of VWF if an interchain-disulfide bond is involved.

Trypanosoma cruzi 5S rRNA arrays define five groups and indicate the geographic origins of an ancestor of the heterozygous hybrids

Isolates of the etiological agent of Chagas disease, Trypanosoma cruzi, have been subdivided into six subgroups referred to as discrete typing units. The subgroups are related through two distinct hybridisation events: representatives of homozygous discrete typing units I and IIb fused to form discrete typing units IIa and IIc, whose homozygous genotypes have features of both ancestral types; a second fusion between strains of homozygous discrete typing units IIb and IIc created the heterozygous hybrid strains discrete typing units IId and IIe. The intergenic region of the tandemly repeated 5S rRNA array displays four variant sequence classes, allowing the discrimination of five discrete typing units. The genome project reference strain, CL Brener, is a hybrid discrete typing unit IIe strain that contains both discrete typing unit IIb and IIc classes of 5S rRNA repeats in distinct arrays present on different chromosomes. The CL Brener discrete typing unit IIb-type array contains approximately 193 repeated units, of which about one-third contain a 129 bp sequence that replaces a majority of the 5S rRNA sequence. The 129 bp ‘invader’ sequence was detected within the arrays of all hybrid discrete typing unit IId and IIe strains and in a subset of discrete typing unit IIb strains. This array invader replaces the internal promoter elements conserved in 5S rRNA. The discrete typing unit IIb Esmeraldo strain contains approximately 135 repeats and shows a region of homology to the array invader in the 5′ flank of the array, but no evidence of the invading sequence element within the array. A survey of additional discrete typing unit IIb strains revealed a split within the subgroup, in which some strains contained invaded arrays and others were homogeneous for the 5S rRNA. The putative discrete typing unit IIb ancestor of the hybrid discrete typing units IId and IIe more closely resembles the extant Bolivian/Chilean IIb isolates than the Brazilian IIb isolates based on the correlation with the array invader.

MARKED BILE DUCT PROLIFERATION IN MICE HETEROZYGOUS FOR LUNATIC FRINGE AND JAG1 MUTATIONS

Background: The Notch signaling pathway is involved in cell fate determination in many organs. JAG1, encoding a ligand in the Notch pathway, has been identified as the disease gene for Alagille Syndrome (AGS), characterized by bile duct paucity and other manifestations. Mice heterozygous for mutations in both the Notch2 receptor and Jag1 ligand have a phenotype similar to AGS. Notch signaling is regulated by three mammalian Fringe genes. These genes encode glycosyltransferases that modify the activation of the Notch receptor by the ligand. To examine the role of the Lunatic Fringe (Lfng) gene in modifying Jag1 ligand function, we carried out genetic analysis of double heterozygous mice (Jag1tm1Grid/+; Lfngtm1Rjo/+, abbreviated as J1LF). Methods: Liver sections from both J1LF and control mice were examined by H&E staining. Bile ducts were identified by cytokeratin staining, and blood vessels were stained with Factor VIII antibody. Mice were evaluated as newborns and 5-week old adults. Quantitative real time RT-PCR for Hes1, a downstream effector in the Notch pathway, was performed to measure Notch signaling. Results: Adult J1LF mice had a significant increase in the number of bile ducts in the portal tracts, when compared to their single heterozygous and wild type littermates. Numbers of blood vessels in portal tracts were not significantly different between J1LF and control mice. Gross examination revealed that the extrahepatic biliary tree was intact. In adult J1LF livers with bile duct proliferation, real time PCR demonstrated a 4 to 8 fold increase in Hes1 expression levels over wild type and single heterozygous controls. However, in the newborn period, J1LF mice did not display a significant difference in the numbers of bile ducts. Conclusions: Jag1/Lfng double heterozygous mice have a striking proliferation of bile ducts in the adult liver. These differences arise postnatally, pointing to a role for Lfng in regulating Jag1-Notch signaling in bile duct growth and maturation and LFNG as a potential modifier gene for AGS.

Ethanol self-administration and ethanol conditioned place preference are reduced in mice lacking cannabinoid CB1 receptors

Cannabinoids are postulated to play a role in modulating the reinforcing effects of abused drugs, including alcohol. Experiment 1 examined alcohol self-administration in cannabinoid CB1 receptor knockout (KO), heterozygous (HT) and wild type (WT) mice in a two-bottle choice paradigm. Mice were trained in a limited 8 h access/day to 10% (v/v) EtOH (EtOH) versus water. After baseline drinking levels (% EtOH preference and total EtOH intake (g/kg)), results indicated that the CB1 knockout mice displayed significantly lower baseline EtOH consumption compared to wild type mice. Subsequently, treatment with SR141716A (5 mg/kg) significantly attenuated EtOH intake in the WT and HT mice but had little effect on the knockout mice. Experiment 2 examined the CB1 WT and CB1 KO strains in a conditioned place preference (CPP) procedure between saline and 2 g/kg EtOH. The CB1 WT mice spent significantly more time in the EtOH-paired versus saline-paired chambers, whereas no significant preference was observed in the CB1 KO mice. Finally, we observed that CB1 KO mice were significantly lighter than WT and HT and that SR141716A did not significantly alter body weight. These results demonstrate that the cannabinoid CB1 receptor is an essential component of the molecular pathways underlying the reinforcing effects of alcohol. Thus, medications targeting the CB1 receptors may be beneficial for the treatment of alcoholism.

362. Using Real-Time (TaqMan®) PCR to Genotype Offspring of Transgenic CF-null Mice — A Core Facility of the UK Cystic Fibrosis Gene Therapy Consortium

Cystic fibrosis (CF) is a common and fatal recessive disease, which is caused by dysfunction of a chloride ion channel, termed the CF transmembrane conductance regulator (CFTR). Several transgenic mouse models have been created to assess the effectiveness of a variety of gene transfer vectors for CF in the respiratory epithelium of the nose and lung (Snouwaert et al, 1992). Use of these animal models requires a sensitive, accurate and fast method for genotyping neonatal mice in order that individual animals may be selected for experiments. The speed of the assay is important as many CF transgenic mouse models have poor survival rates under standard housing conditions. We have established a Core Testing Service using the ABI7700 Sequence Detector (TaqMan|[reg]|, Applied Biosystems Inc, Foster City, USA) to differentiate wild-type, homozygous Cftrtm1UNC and heterozygous littermates by simultaneously detecting mCFTR exon 10, and the vector that replaces exon 10 in the transgenic mice. These sequences were confirmed by cloning of the target DNA to ensure primer specificity. Genotyping can be performed on a range of samples, including ear clips, tail clips and blood, from immediately after birth. Samples are collected in the core facility and the DNA extracted using the DNEasy mini kit (Qiagen Ltd, Crawley, UK). DNA is amplified using the TaqMan|[reg]| instrument. Fluorescent detection avoids the use of gel electrophoresis and potential observer error. The sensitivity of the assay is such that only 20ng DNA is necessary for reproducible genotyping. Up to 28 mice can be assayed at once, and results are typically available in less than 24h. The assay is capable of differentiating between allele copy numbers in homozygotes and heterozygotes, and Fig. 1 shows the differing Cts in detection of the wild type allele in CF-null, heterozygous and wild type mice. Detection of the Cftrtm1UNC allele followed a similar pattern. The assay has been seen to be in complete agreement with other assessments of genotype including weight abnormality. PCR in real time is a rapid, reliable and sensitive method to assess genotype and well suited for genotyping a range of transgenic animal models.

An Analysis of North American Specialized Coagulation Laboratory Association Proficiency Testing

To assess current laboratory practice and the performance of different reagent-instrument combinations for protein S testing, protein S results from the North American Specialized Coagulation Laboratory Association (NASCOLA) proficiency testing surveys for 2002 and the first half of 2003 were analyzed. A written survey of NASCOLA laboratories also was performed to further assess current laboratory practices for protein S testing. The free protein S antigen assays and the Diagnostica Stago Staclot protein S assay were extremely accurate in detecting a heterozygous type I protein S deficiency. Another functional protein S assay and most total protein S assays were less reliable, depending to some extent on the instrument. All assays used by NASCOLA laboratories appropriately identified normal protein S specimens. NASCOLA laboratories performed at least as well as European Concerted Action on Thrombosis laboratories in the proficiency tests. The results suggest that the diagnosis of heterozygous protein S deficiency may be problematic with some currently available assays. Many total protein S antigen assays do not add to the diagnosis and can be unreliable for protein S deficiency subtyping. Better standardization of functional and antigenic assays is needed.

Protein S Assays

To assess current laboratory practice and the performance of different reagent-instrument combinations for protein S testing, protein S results from the North American Specialized Coagulation Laboratory Association (NASCOLA) proficiency testing surveys for 2002 and the first half of 2003 were analyzed. A written survey of NASCOLA laboratories also was performed to further assess current laboratory practices for protein S testing. The free protein S antigen assays and the Diagnostica Stago Staclot protein S assay were extremely accurate in detecting a heterozygous type I protein S deficiency. Another functional protein S assay and most total protein S assays were less reliable, depending to some extent on the instrument. All assays used by NASCOLA laboratories appropriately identified normal protein S specimens. NASCOLA laboratories performed at least as well as European Concerted Action on Thrombosis laboratories in the proficiency tests. The results suggest that the diagnosis of heterozygous protein S deficiency may be problematic with some currently available assays. Many total protein S antigen assays do not add to the diagnosis and can be unreliable for protein S deficiency subtyping. Better standardization of functional and antigenic assays is needed.

Genetic deletion of the norepinephrine transporter decreases vulnerability to seizures

Norepinephrine (NE) has been reported to modulate neuronal excitability and act as endogenous anticonvulsant. In the present study we used NE transporter knock-out mice (NET-KO), which are characterized by high levels of extracellular NE, to investigate the role of endogenous NE in seizure susceptibility. Seizure thresholds for cocaine (i.p.), pentylenetetrazol (i.v.) and kainic acid (i.v.) were compared in NET-KO, heterozygous (NET-HT) and wild type (NET-WT) female mice. The dose–response curve for cocaine-induced convulsions was significantly shifted to the right in NET-KO mice, indicating higher seizure thresholds. The threshold doses of pentylenetetrazol that induced clonic and tonic seizures were also significantly higher in NET-KO when compared to NET-WT mice. Similarly, NET-KO mice displayed higher resistance to convulsions engendered by kainic acid. For all drugs tested, the response of NET-HT mice was always intermediate. These data provide further support for a role of endogenous NE in the control of seizure susceptibility.

Serological Screening for MHC (B)-Polymorphism in Indigenous Chickens

As part of a series of studies to characterize innate and specific immune responses of indigenous chicken lines, birds from Bolivia and India were screened serologically for MHC class IV (BG) polymorphism by direct haemagglutination using haplotype-specific antisera (B2, B4, B12, B13, B14, B15, B19, B21). The sample consisted of 95 Bolivian indigenous chickens and 119 hens from the four most common North Indian 'back-yard' chicken lines: Yellow Aseel (AP), Kadaknath (KN), frizzled typed (Ff-) and naked neck (NN). Of all chickens tested, the majority were haplotyped as B2, B15, B19 and B21. Of the Bolivian chickens, 89.5% could be haplotyped: 54.9% were homozygous (including 43.3% B15), and 34.6% were heterozygous (including 15.7% B15). B2-like haplotypes were not found among the Bolivian hens, and only 3.2% of these birds showed homozygous B21-like proteins. Of the Indian hens, MHC (BG)-like proteins could be detected in 60.0% of the AP birds, 6.7% of the KN birds; 40.0% of the Ff- birds; and 10.3% of the NN birds. In these lines, a total of 40.1% (AP), 6.7% (KN), 30.1% (Ff-) and 10.3% (NN) were homozygous for the B-haplotype. Only in the AP line (19.9%), and the Ff- line (9.9%) were heterozygous B-haplotypes types found. The B2 haplotype was found in all Indian chicken lines. Most Indian birds have completely unknown haplotypes, indicating a potentially interesting genetic pool. Subgrouping the Bolivian and Indian indigenous hens into monomorphic BG populations revealed individual differences based on the B-types.

Effect of CYP2C8 genotype on rosiglitazone pharmacokinetics

Background/Aims The purpose of this study was to investigate the effect of the CYP2C8 genetic polymorphisms CYP2C8*2, CYP2C8*3, and CYP2C8*4 on the pharmacokinetics of rosiglitazone, a PPARγ agonist and CYP2C8 substrate. Methods This single dose pharmacokinetics study involved thirty-eight healthy subjects with the following CYP2C8 genotypes: CYP2C8*1/*1 (n= 14), *1/*2 (n= 5), *1/*3 (n= 11), *1/*4 (n= 6), *2/*2 (n= 1), and *3/*3 (n= 1). Subjects were administered a single dose of rosiglitazone 8 mg. Rosiglitazone concentrations were determined by HPLC and noncompartmental pharmacokinetic parameters were compared between wild type and heterozygous genotype groups. Results The pharmacokinetics of rosiglitazone did not differ between CYP2C8 homozygous wild type and heterozygous individuals. The mean AUC was 3161±625, 3290±866, 2928±519, and 3230±658, μg×h/L in the *1/*1, *1/*2, *1/*3, and *1/*4 genotype groups, respectively. The subject with the CYP2C8*3/*3 genotype had the second lowest rosiglitazone AUC. Conclusions The pharmacokinetics of rosiglitazone were not different in individuals carrying a single variant CYP2C8 allele, as the pharmacokinetic parameters of rosiglitazone did not differ between heterozygous and homozygous wild type individuals. Further study is needed to determine whether homozygous carriers of the CYP2C8*2 and CYP2C8*3 alleles have altered rosiglitazone pharmacokinetics. Clinical Pharmacology & Therapeutics (2005) 77, P36–P36; doi: 10.1016/j.clpt.2004.12.029

A null mutation for Fmr1 in female mice: Effects on regional cerebral metabolic rate for glucose and relationship to behavior

As a measure of functional activity we determined regional cerebral metabolic rate for glucose (rCMR glc) in adult, female wild type and fragile X ( Fmr1 null) mice homozygous and heterozygous for the null mutation. To ascertain if the sexes differ with respect to the severity of the effects of the mutation we compared our results with results of our previous study on male Fmr1 null mice [Qin M, Kang J, Smith CB (2002) Increased rates of cerebral glucose metabolism in a mouse model of fragile X mental retardation. Proc Natl Acad Sci U S A 99:15758–15763.]. In contrast to the male Fmr1 null mouse, rCMR glc was unchanged in the homozygous female except in the dorsal raphe where rCMR glc was increased by 36%. There were no differences in rCMR glc between heterozygous and wild type female mice. We compared male and female mice for effects of the null mutation on behavior. We found that the female Fmr1 null mouse is similar to the male with deficits in performance on a passive avoidance task, general hyperactivity, and increased susceptibility to audiogenic seizures. Both homozygous and heterozygous female mice exhibited hyperactivity and increased susceptibility to seizures, whereas only the homozygous mice had a deficit on the passive avoidance test. Male Fmr1 null mice had a tendency for lower anxiety-like behavior in an open field, whereas this was not evident in females. Compared with male wild type, male Fmr1 null mice also had a diminished acoustic startle response at higher stimulus intensities, whereas all three female genotypes had responses similar to those of male Fmr1 null mice. Whether estrogen affords female Fmr1 null mice some protection from the effects of the mutation remains to be determined.