Haplotype association analysis of discrete and continuous traits using mixture of regression models.

We present a regression-based method of haplotype association analysis for quantitative and dichotomous traits in samples consisting of unrelated individuals. The method takes account of uncertain phase by initially estimating haplotype frequencies and obtaining the posterior probabilities of all possible haplotype combinations in each individual, then using these as weights in a finite mixture of regression models. Using this method, different combinations of marker loci can be modeled, to find a parsimonious set of marker loci that are most predictive and therefore most likely to be closely associated with the a quantitative trait locus. The method has the additional advantage of being able to use individuals with some missing genotype data, by considering all possible genotypes at the missing markers. We have implemented this method using the SNPHAP and Mx programs and illustrated its use on published data on idiopathic generalized epilepsy.

Evidence that the N-methyl-D-aspartate subunit 1 receptor gene (GRIN1) confers susceptibility to bipolar disorder.

There is evidence for the involvement of glutamatergic transmission in the pathogenesis of major psychoses. The two most commonly used mood stabilizers (ie lithium and valproate) have been found to act via the N-methyl-D-aspartate receptor (NMDAR), suggesting a specific role of NMDAR in the pathogenesis of bipolar disorder (BP). The key subunit of the NMDAR, named NMDA-1 receptor, is coded by a gene located on chromosome 9q34.3 (GRIN1). We tested for the presence of linkage disequilibrium between the GRIN1 (1001-G/C, 1970-A/G, and 6608-G/C polymorphisms) and BP. A total of 288 DSM-IV Bipolar I, Bipolar II, or schizoaffective disorder, manic type, probands with their living parents were studied. In all, 73 triads had heterozygous parents for the 1001-G/C polymorphism, 174 for the 1970-A/G, and 48 for the 6608-G/C. These triads were suitable for the final analyses, that is, the transmission disequilibrium test (TDT) and the haplotype-TDT. For the 1001-G/C and the 6608-G/C polymorphisms, we found a preferential transmission of the G allele to the affected individuals (chi(2)=4.765, df=1, P=0.030 and chi(2)= 8.395, df=1, P=0.004, respectively). The 1001G-1970A-6608A and the 1001G-1970A-6608G haplotypes showed the strongest association with BP (global chi(2)=14.12, df=4, P=0.007). If these results are replicated there could be important implications for the involvement of the GRIN1 in the pathogenesis of BP. The role of the gene variants in predicting the response to mood stabilizers in BP should also be investigated.

Suggestive evidence for association of two potassium channel genes with different idiopathic generalised epilepsy syndromes.

Several potassium channel genes have been implicated in epilepsy. We have investigated three such genes, KCNJ3, KCNJ6 and KCNQ2, by association studies using a broad sample of idiopathic generalised epilepsy (IGE) unselected by syndrome. One of the two single nucleotide polymorphisms (SNPs) examined in one of the inward rectifying potassium channel genes, KCNJ3, was associated with IGE by genotype (P=0.0097), while its association by allele was of borderline significance (P=0.051). Analysis of the different clinical subgroups within the IGE sample showed more significant association with the presence of absence seizures (P=0.0041) and which is still significant after correction for multiple testing. Neither SNP in the other rectifying potassium channel gene, KCNJ6, was associated with IGE or any subgroup. None of the three SNPs in the voltage-gated potassium channel gene, KCNQ2, was associated with IGE. However, one SNP was associated with epilepsy with generalised tonic clonic seizures only (P=0.016), as was an SNP approximately 56 kb distant in the closely linked nicotinic acetylcholine gene CHRNA4 (P=0.014). These two SNPs were not in linkage disequilibrium with each other, suggesting that if they are not true associations they have independently occurred by chance. Neither association remains significant after correcting for multiple testing.

Investigation of promoter variants of the histamine 1 and 2 receptors in schizophrenia and clozapine response.

We report the identification of four novel histamine 1 (H1-17-C/T, -974-C/A, -1023-A/G and -1536-G/C) and four novel histamine 2 promoter polymorphisms (H2-294-A/G, -592-A/G, -1018-G/A and -1077-G/A) which we have investigated for involvement in susceptibility to schizophrenia, and in clinical response to clozapine treatment. We identified a weak independent association between variants at the H1-1536-G/C locus and schizophrenia, where an excess of the H1-1536-C allele was observed amongst such patients (P = 0.036). However upon correction for multiple testing this relationship was no longer statistically significant. Similar investigation of the H2 receptor polymorphisms revealed association between genotype at the H2-1018-G/A locus and clinical response to clozapine treatment (P = 0.027), though upon correction for multiple testing this difference was no longer significant. We have concluded that the participation of these variants in the disorder is unlikely, particularly in view of their apparent lack of function and unlikely influence on receptor expression. However their nature alludes to the potential presence of other more important alterations further along these regions, where sequences encoding alternate promoters have recently been identified for each receptor that may yet be found to harbour such polymorphisms.

Association of mu-opioid receptor subunit gene and idiopathic generalized epilepsy.

OBJECTIVE: To replicate and extend the previously reported association between the opioid receptor mu subunit gene (OPRM1) and idiopathic absence epilepsy (IAE), using a sample of 230 probands with idiopathic generalized epilepsy (IGE). BACKGROUND: In humans and in animal models, several lines of evidence implicate opioid receptors with seizures. The G118 allele of OPRM1 was associated with IAE (p = 0.019). METHODS: Three single nucleotide polymorphisms (SNP) of OPRM1 were investigated by association studies with IGE using a case/control design, one of which also used a within-family design. RESULTS: Association was found for G118 with IGE (p = 0.00027, odds ratio [OR] = 1.86), replicating the previous association. Within-family tests of linkage and association (haplotype-based haplotype relative risk and transmission disequilibrium test) confirmed this result. Further evidence for involvement of OPRM1 in IGE was provided by an association with G-172T, located in the 5' untranslated region (p = 0.0015, OR = 2.36). Haplotypes of the two SNPs were associated with IGE with a greater level of significance (p = 0.000087) suggesting that both SNPs might be in linkage disequilibrium with a single functional variant. Analysis of the results by subgroups of IGE showed association with all subgroups tested. CONCLUSIONS: These results confirm the previous association and support the hypothesis of a role for OPRM1 in IGE, including absence syndromes. However, the authors found no evidence for a specific association between OPRM1 and idiopathic absence epilepsy. The data suggest that the functional variant predisposing to IGE is located within 60kb of exon 1.

Antidepressants enhance glucocorticoid receptor function in vitro by modulating the membrane steroid transporters.

1. Previous data demonstrate that the tricyclic antidepressant, desipramine, induces glucocorticoid receptor (GR) translocation from the cytoplasm to the nucleus in L929 cells and increases dexamethasone-induced GR-mediated gene transcription in L929 cells stably transfected with the mouse mammary tumour virus-chloramphenicol acetyltransferase (MMTV-CAT) reporter gene (LMCAT cells) (Pariante et al., 1997). 2. To extend these findings, the present study has investigated the effects of 24 h coincubation of LMCAT cells with dexamethasone and amitriptyline, clomipramine, paroxetine, citalopram or fluoxetine. 3. All antidepressants, except fluoxetine, enhanced GR-mediated gene transcription, with clomipramine having the greatest effect (10 fold increase). Twenty-four hours coincubation of cells with desipramine, clomipramine or paroxetine, also enhanced GR function in the presence of cortisol, but not of corticosterone. 4. It is proposed that these effects are due to the antidepressants inhibiting the L929 membrane steroid transporter, which actively extrudes dexamethasone and cortisol from the cell, but not corticosterone. This is further confirmed by the fact that clomipramine failed to enhance GR-mediated gene transcription in the presence of dexamethasone when the membrane steroid transporter was blocked by verapamil. 5. The membrane steroid transporters that regulate access of glucocorticoids to the brain in vivo, like the multiple drug resistance p-glycoprotein, could be a fundamental target for antidepressant action.

RNA editing of the 5-HT(2C) receptor is reduced in schizophrenia.

5-HT(2C) receptor (5HT(2C)R, serotonin-2C) RNA undergoes editing to produce several receptor variants, some with pharmacological differences. This investigation comprised two parts: the characterisation of 5-HT(2C)R RNA editing in a larger human control sample than previously examined, and a comparative study in subjects with schizophrenia. Secondary structure analysis of the putative edited region of the human 5-HT(2C)R gene predicted the existence of a double stranded (ds) RNA loop, essential for RNA editing in this receptor. RNA was then extracted from frontal cortex of five controls and five subjects with schizophrenia. RT-PCR products of the edited region were cloned and sequenced (n = 100). Reduced RNA editing, increased expression of the unedited 5-HT(2C-INI) isoform in schizophrenia (P = 0.001) and decreased expression of the 5-HT(2C-VSV) and 5-HT(2C-VNV) isoforms were detected in the schizophrenia group. In addition, two novel mRNA edited variants were identified: 5-HT(2C-MNI) and 5-HT(2C-VDI). Screening of the 5-HT(2C)R gene did not reveal any mutations likely to disrupt the dsRNA loop, suggesting that the reduced RNA editing in schizophrenia may instead be caused by altered activity of the editing enzyme(s). Since the unedited 5-HT(2C-INI) is more efficiently coupled to G proteins than the other isoforms, its increased expression in schizophrenia may lead to enhanced 5-HT(2C)R-mediated effects. The results also illustrate that potentially important receptor alterations may occur in schizophrenia which are not detectable merely in terms of receptor abundance.

Dishevelled regulates the metabolism of amyloid precursor protein via protein kinase C/mitogen-activated protein kinase and c-Jun terminal kinase.

Alzheimer's disease (AD) is a disorder of two pathologies: amyloid plaques, the core of which is a peptide derived from the amyloid precursor protein (APP), and neurofibrillary tangles composed of highly phosphorylated tau. Protein kinase C (PKC) is known to increase non-amyloidogenic alpha-secretase cleavage of APP, producing secreted APP (sAPPalpha), and glycogen synthase kinase (GSK)-3beta is known to increase tau phosphorylation. Both PKC and GSK-3beta are components of the wnt signaling cascade. Here we demonstrate that overexpression of another member of this pathway, dishevelled (dvl-1), increases sAPPalpha production. The dishevelled action on APP is mediated via both c-jun terminal kinase (JNK) and protein kinase C (PKC)/mitogen-activated protein (MAP) kinase but not via p38 MAP kinase. These data position dvl-1 upstream of both PKC and JNK, thereby explaining the previously observed dual signaling action of dvl-1. Furthermore, we show that human dvl-1 and wnt-1 also reduce the phosphorylation of tau by GSK-3beta. Therefore, both APP metabolism and tau phosphorylation are potentially linked through wnt signaling.

Polymorphism analysis of JRK/JH8, the human homologue of mouse jerky, and description of a rare mutation in a case of CAE evolving to JME.

Disruption of the function of the mouse jerky gene by transgene insertion causes generalized recurrent seizures reminiscent of human idiopathic generalized epilepsy (IGE). A human homologue, JRK/JH8, has been cloned, which maps to 8q24, a chromosomal region associated with several forms of IGE. JRK/JH8 is, therefore, a candidate locus for at least some forms of IGE. We report corrected cDNA sequences and extended open reading frames for the mouse jerky and human JRK/JH8 genes, which add 48 amino acids to the N-terminus of the Jerky protein and which extends the region of homology with the N-terminal DNA-binding domain of the centromere-binding protein, CENP-B. Systematic sequencing of the coding region of the extended JRK/JH8 gene identified single nucleotide polymorphisms that define three haplotypes, which were used for association studies in patients with idiopathic generalized epilepsy. We report one subject with childhood absence epilepsy (CAE) that evolved to juvenile myoclonic epilepsy (JME) that has a unique de novo mutation that results in a non-conservative amino acid change at a potential protein glycosylation site. Familial analysis supports a causal role for this mutation in the disease.

Polymorphisms in the genes for mGluR types 7 and 8: association studies with schizophrenia.

A dysfunctional glutamatergic system has been implicated in the pathophysiology of schizophrenia. The group III metabotropic glutamate receptor (mGluR) types 7 and 8 presynaptically inhibit glutamate release, thereby modulating glutamatergic transmission in the brain. We conducted association studies to investigate the novel Tyr433Phe (mGluR7) variant and the 2846-C/T (mGluR8) polymorphism in schizophrenia. Both variants, present at high frequencies, failed to demonstrate any significant association with schizophrenia (mGluR7 [Tyr433Phe] allele: P=0.33; genotype: P=0.63; mGluR8 [2846-C/T] allele: P=0.72; genotype: P=0.63).

Failure to replicate association between the gene for the neuronal nicotinic acetylcholine receptor alpha 4 subunit (CHRNA4) and IGE.

The gene for the neuronal nicotinic acetylcholine receptor alpha4 subunit (CHRNA4) was identified as a gene underlying a rare idiopathic partial epilepsy syndrome in humans, autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). In a recent study, one of four silent polymorphisms (594 C/T) in CHRNA4 showed association with the common subtypes of idiopathic generalised epilepsy (IGE). In the present study, three of these polymorphisms were investigated for association in 182 Caucasian patients with IGE, but not categorised by subtype. They were compared with 178 controls in a case/control study. Further analyses were performed using a family-based design. None of the three polymorphisms exhibited any association with IGE. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:814-816, 2000.

Haplotype transmission disequilibrium and evidence for linkage of the CHRNA7 gene region to schizophrenia in Southern African Bantu families.

Recent reports have strongly linked markers near the alpha-7 nicotinic cholinergic receptor subunit gene on human chromosome 15q13-q14 to a sensory gating deficit common in schizophrenics, and have shown positive though non-significant results linking this region to the primary phenotype of schizophrenia in a sample of North American families. We therefore tested for linkage between markers in this region of chromosome 15q and schizophrenia in a sample of 15 multiply affected and 5 single case families with schizophrenia drawn from the Bantu-speaking black population of South Africa. An initial replication using markers from the original study gave an affected-only LOD score maximum of 1.08 under a recessive model at Theta=0.00 for D15S1360, a dinucleotide polymorphism found on the same YAC as the alpha-7 receptor gene. Nonparametric affected-only multipoint analysis gave a Z-score of 1. 29, P=0.098, for D15S1360, and Z=1.45, p=0.075 for D15S118. We then increased the resolution of the map with an extended set of 20 markers. Again, two peaks were observed, with NPL scores of 1.81, p=0.037, at D15S1043 and 1.79 at D15S1360 and 1.80 at D15S1010, both p=0.037. Transmission disequilibrium testing of data from D15S1360 gave an allele-wise and genotype-wise chi(2) of 6.59, 2 df, p=0.037. Haplotype transmission disequilibrium testing using a restricted allele and haplotype set from D15S1043 and D15S1360 gave a global chi(2) of 10.647, 4 df, P=0.007, and a maximum chi(2) of 6.567, 1 df, P=0.004 for excess transmission of the 1.2 haplotype into affected offspring. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:196-201, 2000.

Association study of dopamine receptor gene polymorphisms with drug-induced hallucinations in patients with idiopathic Parkinson's disease.

Some patients with idiopathic Parkinson's disease experience hallucinations as a result of treatment with levodopa and dopamine agonists. There is evidence for some heterogeneity in these hallucinating patients based on duration of Parkinson's disease at onset of hallucinations. We compared the frequency of polymorphisms in the dopamine D2 and D3 receptor genes between patients with drug-induced hallucinations and non-hallucinating patients. Two polymorphisms close to DRD2 and one in DRD3 were studied. No association was found with the whole group of hallucinating patients and their controls. However, an association was found with late-onset hallucinations and the C allele of the TaqIA polymorphism, 10.5 kb 3' to DRD2. This polymorphism may be in linkage disequilibrium with a mutation in DRD2 or a nearby gene that predisposes to drug-induced hallucinations which occur later in the course of idiopathic Parkinson's disease.

No association found between polymorphisms in genes encoding mGluR7 and mGluR8 and idiopathic generalised epilepsy in a case control study.

The genes of two group III metabotropic glutamate receptors, mGluR7 and 8, are candidate susceptibility genes for epilepsy. The Tyr433Phe polymorphism of mGluR7 and a novel polymorphism in the mGluR8 gene located 29 bp after the termination codon (2756C/T) were investigated in case control association studies performed on DNA from more than 100 patients with idiopathic generalised epilepsy (IGE). No significant association was found with IGE for either polymorphism.

Failure to respond to treatment with typical antipsychotics is not associated with CYP2D6 ultrarapid hydroxylation.

AIMS: To investigate whether or not there is a correlation between failure to respond to typical antipsychotics and CYP2D6 ultrarapid metaboliser status. METHODS: CYP2D6 phenotype (metaboliser status) was assigned following genotyping for gene duplication, as well as for the CYP2D6*3, CYP2D6*4, and CYP2D6*5 null alleles in 235 treatment-refractory patients and 73 nonrefractory patients. RESULTS: Four (1.7%) of the 235 treatment-refractory subjects were positive on the duplication assay, but, of these, two were found to represent duplications of a null allele (CYP2D6*4 ), therefore leaving only two (0.85%) positive for duplication of a wild type allele (ultrarapid metabolisers). Three (4.1%) of the nonrefractory subjects had a genotype consistent with ultrarapid metaboliser status. Fisher's exact test gave a two-tailed P value of 0.091, i.e. a trend towards an excess of ultrarapid metabolisers in the nonrefractory group, which was in the opposite direction to that predicted by our hypothesis. CONCLUSIONS: Although the results show a trend towards an excess of ultrarapid metabolisers in the nonrefractory group, the percentages in the two groups of patients are both within the range for ultrarapid metabolisers in Caucasian populations. Our data are not consistent with ultrarapid metaboliser status being a major cause of failure to respond to typical antipsychotics.

Molecular cloning of human GABABR1 and its tissue distribution.

GABABR1 clones were isolated from a human cerebellum library. The human sequence is very similar to rat GABABR1 with the cDNAs sharing 91.3% sequence identity and the receptors sharing 98.6% amino acid sequence identity. Northern blotting has shown that the receptor is brain-specific with a widespread distribution throughout the brain but none detected in the spinal cord.

Immunohistochemical localisation of mGluR7 protein in the rodent and human cerebellar cortex using subtype specific antibodies.

Metabotropic glutamate receptors (mGluRs) are a heterogeneous family of G protein coupled receptors that are linked to multiple second messenger systems to regulate neuronal excitability and synaptic transmission. To characterise the protein expression of the two mGluR7 receptor splice variants in human and rat cerebellar cortex, antibodies specific to mGluR7 were generated. Antibodies were raised against a glutathione-S-transferase fusion protein containing amino acid residues located in the extracellular domain common to both the human and rat mGluR7 splice variants. These antibodies specifically detected human mGluR7a in mammalian cells transfected with this receptor. In agreement with mGluR7 in situ hybridisation studies, immunohistochemistry performed at the light microscope level revealed that mGluR7 protein expression occurred most prominently in a particular population of nerve cells common to both the human and rat, located within the cerebellar cortex of gray matter contained within transverse folia. Moreover, strong mGluR7-like immunoreactivity was seen in Purkinje cell body cytoplasm of the Purkinje cell layer. In the most superficial cerebellar cortical layer, the molecular layer, immunostaining was observed in Purkinje cell associated proximal and distal dendritic trees. No detectable labelling was evident in intrinsic deep cerebellar nuclei known to contain GABAergic terminals of projecting Purkinje cell axons. These data are suggestive of a post-synaptic location of mGluR7 in this central nervous system structure. In the rodent, additional non-Purkinje cells thought to represent inhibitory interneurones were labelled at all levels in the molecular layer. mGluR7-like immunoreactivity was not associated with glial cells.