Association between a common CYP17A1 haplotype and anxiety in female anorexia nervosa.

Dehydroepiandrosterone (DHEA), the main brain neurosteroid, has been implicated in various psychiatric disorders especially those including gender differences. We studied genetic variability in the DHEA-producing enzyme CYP17A1 in relation to anorexia nervosa (AN) susceptibility and AN-related co-morbidities. We performed analysis of 100 Israeli AN family trios accounting for CYP17A1 haplotypes characteristic of populations of European origin and studied genotype-phenotype relationships using correlation analyses and transmission disequilibrium test. Although our analysis revealed no evidence of association between CYP17A1 and AN per se, it revealed an association between specific CYP17A1 haplotypes and AN co-morbidity, specifically anxiety. We found that a common CYP17A1 haplotype (H1) was associated with higher anxiety in AN patients (Clinical Global Impression; CGI-anxiety ≥4). Moreover, H1 homozygotes were at higher risk for expressing high CGI-anxiety levels (OR = 3.7), and H1 was preferentially transmitted to AN patients with high CGI-anxiety levels (P = 0. 037). We suggest that CYP17A1 H1 haplotype may contribute to genetic predisposition to higher CGI-anxiety levels in AN patients and that this predisposition may be mediated by reduced CYP17A1 enzymatic activity and corresponding lower DHEA production.

Epilepsy in Rett syndrome---the experience of a National Rett Center.

PURPOSE: Rett syndrome (RTT), an X-linked, dominant neurodevelopmental disorder caused by mutations in the methyl-CpG-binding protein 2 (MECP2) gene, presents with acquired microcephaly, autistic regression, hand usage loss, and stereotypies. Epilepsy is frequent and has been reported to correlate with mutation type, general disease severity, and BDNF polymorphism. Our purpose was a comprehensive description of epilepsy features and course in RTT. METHODS: Retrospective review of charts and electroencephalography (EEG) studies in 97 patients with RTT. RESULTS: Seventy-two percent of patients had epilepsy, appearing at a median age of 3 years. According to age of onset, we divided patients into three groups: 6 with early epileptic variant (0-1 year), 42 with early epilepsy (1-5 years), and 20 with late epilepsy (after 5 years). Early epileptic variant had severe seizure types in the first year of life, followed by a typical RTT picture; all were MECP2 negative. Early epilepsy and late epilepsy groups were similar with respect to Rett-related symptoms, but seizures were better controlled in the second group (p < 0.05). Epileptiform activity appeared earlier and was more confluent in the early epilepsy group, including nine patients with electrical status epilepticus during sleep (ESES) versus one in the late epilepsy group (p < 0.05). No correlation was found between epilepsy onset or severity and genotype. BDNF val/met polymorphism correlated with earlier onset of seizures (p < 0.05). DISCUSSION: Epilepsy appears earlier than described previously, frequently during the regression stage. Early age of onset predicts a more severe course of seizures. ESES is common among those with early onset epilepsy. BDNF polymorphism was the only genetic correlate with seizure onset, whereas MECP2 mutation type and location did not influence epilepsy.

Dual contribution of NR2B subunit of NMDA receptor and SK3 Ca(2+)-activated K+ channel to genetic predisposition to anorexia nervosa.

Since identification of the genetic component in anorexia nervosa (AN), genes that partake in serotonergic and dopaminergic systems and in hormonal and weight regulation have been suggested as potential candidates for AN susceptibility. We propose another set of candidate genes. Those are genes that are involved in the signaling pathway using NMDA-R and SK channels and have been suggested as possible effectors of NMDA-R driven signaling. The role of NMDA-R in the etiology of schizophrenia has already been substantiated on various levels. Several studies based on population and family groups have implicated SK3 in schizophrenia and more recently in AN as well. Our study group consisted of 90 AN family trios. We examined the transmission of two potentially functional polymorphisms, 5073T>G polymorphism in the gene encoding the NR2B subunit of NMDA-R and CAG repeats in the coding region of SK3 channel gene. Using HHRR and TDT approaches, we found that both polymorphisms were preferentially transmitted to AN offspring (TDT yielded chi(2)=5.01, p=0.025 for NR2B 5073G alleles and chi(2)=11.75, p<0.001 for SK3 L alleles including >19 repeats). Distribution analysis of the combined NR2B/SK3 genotypes suggests that the contribution of both polymorphisms to AN risk is independent and cumulative (OR=2.44 for NR2B GG genotype and OR=3.01 for SK3 SL and LL genotypes, and OR=6.8 for the combined NR2B/SK3 genotypes including high-risk alleles). These findings point to the contribution of genes associated with the NMDA-R signaling pathway to predisposition and development of AN.

Combined genetic profiles of components and regulators of the vitamin K-dependent gamma-carboxylation system affect individual sensitivity to warfarin.

We examined the influence of combined genotypes on interindividual variability in warfarin dose-response. In 100 anticoagulated patients we quantified the effects of polymorphisms in: CYP2C9, VKORC1, calumenin (CALU), gamma-glutamyl carboxylase (GGCX) and microsomal epoxide hydrolase (EPHX1) on warfarin dose requirements. The G(1542)C VKORC1 polymorphism was associated with decreased warfarin doses in the hetero- and homozygous mutant patients (21% and 50% lower, respectively; p < 0.0001). Warfarin daily dose was predominantly determined by VKORC1 and CYP2C9 genotypes (partial r(2) = 0.21; 0.20, respectively). Together with age and body weight, these two genotypes explained 63% of the dose variance. A single patient, homozygous for G(11)A CALU mutant allele, required an exceptionally high warfarin dose (20 mg/day) and the prevalence of heterozygous (11)A allele carriers in the upper 10(th) dose percentile was significantly higher (0.27 vs. 0.18, p < 0.02). Combined genotype analysis revealed that CYP2C9 andVKORC1 wild type and CALU mutant patients required the highest warfarin doses (7.8 +/- 1.5mg/day; n = 9) as compared to the CYP2C9 and VKORC1 mutant and CALU wild type genotypes (2.8 +/- 0.3 mg/day; n = 18; p < 0.01). The odds ratio for doses <3mg/day was 5.9 (1.9-18.4) for this genotype. Compound genetic profiles comprising VKORC1, CALU and CYP2C9 improve categorization of individual warfarin dose requirements in more than 25% of patients at steady-state anticoagulation.

Common genetic variants of microsomal epoxide hydrolase affect warfarin dose requirements beyond the effect of cytochrome P450 2C9.

BACKGROUND: Warfarin dose response is partially explained by the polymorphisms in the cytochrome P450 (CYP) 2C9 gene, affecting S -warfarin clearance, as well as by age and body weight. We examined the influence on warfarin dose requirements of candidate genes encoding microsomal epoxide hydrolase (mEH), as well as glutathione S -transferase A1 (GSTA1) components of vitamin K epoxide reductase and the gamma-glutamylcarboxylase (GGCX) gene. METHODS: We studied the effects of CYP2C9, mEH, GSTA1, and GGCX genotypes on warfarin maintenance doses, accounting for age, weight, vitamin K plasma concentrations and concurrent medications, in 100 patients undergoing therapeutic anticoagulation. RESULTS: Allele frequencies were 76.5%, 12.5%, and 11% for CYP2C9*1 , *2 , and *3 , respectively; 75% and 25% for mEH T 612 C; 75.8% and 24.2% for mEH A 691 G; 73.5% and 26.5% for GSTA1 T 631 G; and 70.5% and 29.5% for GGCX G 8762 A. Warfarin doses differed among the CYP2C9 ( 2C9*1 , 2C9*2 , and 2C9*3 ) genotype groups: 6.3 +/- 1.9 mg/d, 5.3 +/- 1.8 mg/d, and 3.8 +/- 1.7 mg/d, respectively (F = 4.83, P < .01). There were no differences in any of the other genotype groups. Among the 62 wild-type CYP2C9 patients, variant mEH T 612 C homozygotes required higher doses than heterozygotes and wild-type patients (7.5 +/- 2.9 mg/d, 6.5 +/- 4.2 mg/d, and 6.0 +/- 2.6 mg/d, respectively [F = 3.57, P = .03]). The odds ratio for requiring greater than 7 mg/d in variant mEH T 612 C patients versus wild-type patients was 3.14 (95% confidence interval, 1.47-6.67), accounting for CYP2C9. CONCLUSIONS: Variant mEH T 612 C genotypes are associated with warfarin doses of greater than 50 mg/wk beyond the effect of CYP2C9.

Association study of CAG repeats in the KCNN3 gene in Israeli patients with major psychosis.

OBJECTIVES: Several studies reported contradictory findings regarding the association of major psychosis with CAG repeats in the KCNN3 gene. We investigated the contribution of the CAG repeat at the KCNN3 gene, localized to chromosome 1q21.3, to the genetic susceptibility for schizophrenia, schizoaffective and bipolar disorders. METHODS: Analysis of the number of CAG repeats and the differences in allele length were performed for Israeli Ashkenazi Jews, non-Ashkenazi Jews, and Arabs diagnosed with major psychosis (n=181) versus matched ethnic controls (n=207). RESULTS: We found no significant difference in the number of CAG repeats between the entire sample of patients and controls. However, an analysis of the differences of allele length revealed a significantly greater number of patients with identical allele length (43.1%) when compared with normal controls (30.4%). Furthermore, an earlier age of non-paranoid schizophrenia onset was found associated with differences in allele sizes. There were no significant differences in the number of CAG repeats and the differences in allele length when subjects were grouped according to gender, ethnic origins of their parents, family history, and diagnostic groups. CONCLUSIONS: Our results support the hypothesis that a contribution of the KCNN3 gene to genetic susceptibility to major psychosis and their phenotypic polymorphism may be related to the difference of allele length rather than to the number of CAG repeats.

The VKORC1 Asp36Tyr variant and VKORC1 haplotype diversity in Ashkenazi and Ethiopian populations.

The vitamin K epoxide reductase (VKORC1) is a key enzyme in the vitamin K cycle impacting various biological processes. VKORC1 genetic variability has been extensively studied in the context of warfarin pharmacogenetics revealing different distributions of VKORC1 haplotypes in various populations. We previously identified the VKORC1 Asp36Tyr mutation that was associated with warfarin resistance and with distinctive ethnic distribution. In this study, we performed haplotype analysis using Asp36Tyr and seven other VKORC1 markers in Ashkenazi and Ethiopian-Jewish and non-Jewish individuals. The VKORC1 variability was represented by nine haplotypes (V1-V9) that could be grouped into two distinct clusters (V1-V3 and V4-V9) with intra-cluster difference limited to two nucleotide changes. Phylogeny analysis suggested that these haplotypes could have developed from an ancestral variant, the common V8 haplotype (40 % in all population samples), after ten single mutation events. Asp36Tyr was exclusive to the V5 haplotype of the second cluster. Two haplotypes V5 and V4, distinguished only by Asp36Tyr, were prevalent in both Ethiopian population samples. The V2 haplotype, belonging to the first cluster, was the second most prevalent haplotype in the Ashkenazi population sample (15.8 %) but relatively uncommon in the Ethiopian origin (4.5-4.7 %). We discuss the genetic diversity among studied populations and its potential impact on warfarin-dose management in certain populations of African and European origin.

Effect of the VKORC1 D36Y variant on warfarin dose requirement and pharmacogenetic dose prediction.

Pharmacogenetic dosing algorithms help predict warfarin maintenance doses, but their predictive performance differs in different populations, possibly due to unsuspected population-specific genetic variants. The objectives of this study were to quantify the effect of the VKORC1 D36Y variant (a marker of warfarin resistance previously described in 4% of Ashkenazi Jews) on warfarin maintenance doses and to examine how this variant affects the performance of the International Warfarin Pharmacogenetic Consortium (IWPC) dose prediction model. In 210 Israeli patients on chronic warfarin therapy recruited at a tertiary care centre, we applied the IWPC model and then added D36Y genotype as covariate to the model (IWPC+D36Y) and compared predicted with actual doses. Median weekly warfarin dose was 35 mg (interquartile range [IQR], 24.5 to 52.5 mg). Among 16 heterozygous D36Y carriers (minor allele frequency = 3.8%), warfarin weekly dose was increased by a median of 43.7 mg (IQR, 40.5 to 47.2 mg) compared to non-carriers after adjustment for all IWPC parameters, a greater than two-fold dose increase. The IWPC model performed suboptimally (coefficient of determination R²=27.0%; mean absolute error (MAE), 14.4 ± 16.2 mg/week). Accounting for D36Y genotype using the IWPC+D36Y model resulted in a significantly better model performance (R²=47.2%, MAE=12.6 ± 12.4 mg/week). In conclusion, even at low frequencies, variants with a strong impact on warfarin dose may greatly decrease the performance of a commonly used dose prediction model. Unexpected discrepancies of the performance of universal prediction models in subpopulations should prompt searching for unsuspected confounders, including rare genetic variants.

Benign hereditary chorea: an update.

Benign hereditary chorea (BHC, MIM 118700) is a rare autosomal dominant disorder manifesting with chorea in conjunction with hypothyroidism and respiratory problems, a triad also named "brain-lung-thyroid syndrome". BHC is characterized by childhood onset with minimal or no progression into adult life and normal cognitive function. The genetic basis of BHC has been partially resolved, when mutations in the TTF1 gene on chromosome 14q13 encoding the thyroid transcription factor-1 have been identified in a number of BHC patients, suggesting that aberration of TTF1 transcriptional function or haploinsufficiency is associated with this disorder. TTF1 (also known as TITF1, TEBP or NKX2-1), belonging to the NKX2 homeodomain transcription factor family, has been implicated in several important molecular pathways essential for brain, thyroid and lung morphogenesis. Clinical evaluation of TTF1 gene mutations carrier patients exposed the involvement of each of the triad's components characterized by heterogeneity between index cases and even within families. This review highlights the current updates on expanded clinical aspects of BHC, imaging and treatment experience, its genetic markers, proposed molecular mechanisms, animal models and link to cancer.

Ex vivo treatment with a novel synthetic aminoglycoside NB54 in primary fibroblasts from Rett syndrome patients suppresses MECP2 nonsense mutations.

BACKGROUND: Nonsense mutations in the X-linked methyl CpG-binding protein 2 (MECP2) comprise a significant proportion of causative MECP2 mutations in Rett syndrome (RTT). Naturally occurring aminoglycosides, such as gentamicin, have been shown to enable partial suppression of nonsense mutations related to several human genetic disorders, however, their clinical applicability has been compromised by parallel findings of severe toxic effects. Recently developed synthetic NB aminoglycosides have demonstrated significantly improved effects compared to gentamicin evident in substantially higher suppression and reduced acute toxicity in vitro. RESULTS: We performed comparative study of suppression effects of the novel NB54 and gentamicin on three MECP2 nonsense mutations (R294X, R270X and R168X) common in RTT, using ex vivo treatment of primary fibroblasts from RTT patients harboring these mutations and testing for the C-terminal containing full-length MeCP2. We observed that NB54 induces dose-dependent suppression of MECP2 nonsense mutations more efficiently than gentamicin, which was evident at concentrations as low as 50 µg/ml. NB54 read-through activity was mutation specific, with maximal full-length MeCP2 recovery in R168X (38%), R270X (27%) and R294X (18%). In addition, the recovered MeCP2 was translocated to the cell nucleus and moreover led to parallel increase in one of the most important MeCP2 downstream effectors, the brain derived neurotrophic factor (BDNF). CONCLUSION: Our findings suggest that NB54 may induce restoration of the potentially functional MeCP2 in primary RTT fibroblasts and encourage further studies of NB54 and other rationally designed aminoglycoside derivatives as potential therapeutic agents for nonsense MECP2 mutations in RTT.

MeCP2 deficiency downregulates specific nuclear proteins that could be partially recovered by valproic acid in vitro.

MeCP2, the major causative factor of Rett syndrome and related phenotypes including autism, is a two-face nuclear modulator acting via transcriptional and chromatin remodeling mechanisms. This study investigated the expression of several nuclear proteins and their dependence on MeCP2 dose and presence of the Rett causative R306C mutation. To this end, we developed in vitro models representing MeCP2 deficiency induced by siRNAs, and cells expressing the R306C mutation. Using an extended antibody microarray validated by specific assays, revealed that MeCP2 dose was correlated with specific nuclear proteins profiles including the BRM/SNF2 component of SWI/SNF complex, PRMT1 methyl transferase and HDAC2. Furthermore, while exposing the MeCP2 knock-down system to therapeutic concentrations of valproic acid (VPA), a known HDACs inhibitor, we observed a partial restoration of MeCP2 expression levels. Exposure to VPA also increased the levels of BRM, as well as of BDNF, an important co-factor in MeCP2-mediated pathway. Our findings provide additional evidence of diverse mechanisms of MeCP2 function as transcriptional repressor and activator of specific genes. As it has been recently demonstrated that post-natal restoration of MeCP2 deficiency may reverse neurological defects in a mouse model of Rett syndrome, we suggest to study the restorative effect of HDAC inhibitors in MeCP2-deficient mouse model.

10 years of oral anticoagulant pharmacogenomics: what difference will it make? A critical appraisal.

Since the first report on warfarin pharmacogenetics in 1999, genetic variants have emerged as an important predictor of warfarin maintenance doses before therapy is initiated, raising expectations of greatly improved clinical outcomes. However, much of the information on warfarin sensitivity conveyed by genetic variants is captured by early international normalized ratio values traditionally used to guide dose titration. Thus, inclusion of early international normalized ratios in prediction models reduces the contribution of genetics. Moreover, in large population cohorts, genetics explained only 20-30% of variance in warfarin doses. Finally, even pharmacogenetic prediction models did not predict doses reliably in the majority of at-risk patients with warfarin requirements at the low or high end of the dose range. Currently, the clinical utility and cost-effectiveness of pharmacogenetic-based dosing are being assessed in large prospective trials in various settings. In the interim, enthusiasm for warfarin pharmacogenetics should not supersede strict adherence to traditional measures used to optimize coumarin anticoagulation.

A novel missense mutation in the NDP gene in a child with Norrie disease and severe neurological involvement including infantile spasms.

Norrie disease (ND) is a rare X-linked recessive disorder characterized by congenital blindness and in some cases, mental retardation and deafness. Other neurological complications, particularly epilepsy, are rare. We report on a novel mutation identified in a patient with ND and profound mental retardation. The patient was diagnosed at the age of 6 months due to congenital blindness. At the age of 8 months he developed infantile spasms, which were diagnosed at 11 months as his EEG demonstrated hypsarrhythmia. Mutation analysis of the ND gene (NDP) of the affected child and his mother revealed a novel missense mutation at position c.134T > A resulting in amino acid change at codon V45E. To the best of our knowledge, such severe neurological involvement has not been previously reported in ND patients. The severity of the phenotype may suggest the functional importance of this site of the NDP gene.

A coding VKORC1 Asp36Tyr polymorphism predisposes to warfarin resistance.

CYP2C9 and VKORC1 genetic variants are associated with low and intermediate warfarin dose requirements, but markers of high doses are less well characterized. We analyzed the VKORC1 coding sequence and known CYP2C9 and VKORC1 polymorphisms in 15 selected warfarin-resistant (dose, 80 to 185 mg/wk) and 8 warfarin-sensitive patients (7 to 13 mg/wk) and 99 unselected controls (8 to 105 mg/wk). We identified a coding VKORC1 Asp36Tyr polymorphism in 7 of 15 resistant compared with 0 of 8 sensitive patients (P = .026) Carriers of Asp36Tyr in the control group (8 of 99) required significantly higher warfarin doses of 80.9 +/- 10.1 mg/wk compared with 42.7 +/- 7.5 mg/wk in noncarriers (F = 9.79, P = .002). Asp36Tyr was significantly associated with doses of more than 70 mg/wk (odds ratio, 13.0; 95% confidence limit, 1.3 to 124.2), while doses of 20 to 70 mg/wk were associated with Asp36Tyr (partial r(2) = .11; P = .004), CYP2C9*2 and *3 (r(2) = .08; P = .01), and VKORC1*2 and *3 markers (r(2) = .05; P = .05). All Asp36Tyr carriers also had VKORC1*1 tag-single nucleotide polymorphisms (tag-SNPs) indicating a new haplotype. Asp36Tyr was common in Jewish ethnic groups of Ethiopian (15%) and Ashkenazi (4%) origin. We suggest that Asp36Tyr is a new marker of the high end of the warfarin dosing range.

Splicing mutation associated with Rett syndrome and an experimental approach for genetic diagnosis.

Around 80% of Rett syndrome (RS) cases have a mutation or deletion within the coding sequence of the MeCP2 gene. The other RS patients remain genetically undiagnosed. A significant fraction (10-15%) of disease-causing mutations in humans, affect pre-mRNA splicing. Two potential splice mutations were found in the MeCP2 gene in RS patients, however it was not clear whether these mutations in fact interfere with splicing and consequently cause RS. One such mutation is a deletion of the GT dinucleotide at the 5' donor splice site of exon 1 and the other a deletion of the T nucleotide in the polypyrimidine tract (PPT) of intron 3. Here we experimentally assess the effects exerted by these mutations on the expression of MeCP2 in patients' blood samples and on splicing of the MeCP2 transcript using a hybrid minigene in transient transfection experiments. The results revealed that the Delta T mutation in the PPT is a benign polymorphism and that the GT deletion in intron 1 is a bona fide splicing mutation that causes a complete skipping of exon 1 in the minigene transfection experiment. This explains the observed complete elimination of the MeCP2 message and protein in the lymphoblast clones of the RS patient that carry the mutation on the active X. An analysis of the MeCP2 transcript and protein production in lymphoblast clones, as described here, can be used to confirm clinically diagnosed RS patients with no mutation in the MeCP2 coding sequence. This will enable RS diagnosis without specifically identifying a mutation.

CAG repeat polymorphism within the KCNN3 gene is a significant contributor to susceptibility to anorexia nervosa: a case-control study of female patients and several ethnic groups in the Israeli Jewish population.

The human small-conductance Ca(2+)-activated potassium channel gene KCNN3 has been involved in mechanisms underlying neuronal function and plasticity. A multiallelic CAG repeat polymorphism within the KCNN3 has been associated with schizophrenia and bipolar disorder. We have previously reported in a family-based study that longer CAG repeats are preferentially transmitted to patients with anorexia nervosa (AN). The present study extends the analysis of KCNN3 allele distribution to a larger series of AN female patients and control groups, incorporating information on ethnicity and co-morbidities associated with AN. The data analysis is presented while considering separately the two alleles of each individual, namely a minor (shorter) and a major (longer) allele. This study has found that the KCNN3 allele distribution in the general Israeli population does not differ significantly in at least four Jewish ethnic groups of Ashkenazi, North African, Iraqi, and Yemenite origin. These have been used as control groups in a matched case-control analysis that has demonstrated a significant over-representation of KCNN3 alleles with longer CAG repeats among AN patients (P < 0.001 for the major allele and P = 0.035 for allele sum). Under dichotomization, a significantly higher prevalence of the L allele (>19 repeats) has been observed among AN patients (P < 0.001). While considering AN and co-morbid phenotypes, a tendency towards longer (L) alleles has been observed in the subset of patients with obsessive-compulsive disorder (OCD) co-morbidity. These findings further implicate KCNN3 as a significant contributor to predisposition to AN.