Novel carboxypeptidase A6 (CPA6) mutations identified in patients with juvenile myoclonic and generalized epilepsy.

Carboxypeptidase A6 (CPA6) is a peptidase that removes C-terminal hydrophobic amino acids from peptides and proteins. The CPA6 gene is expressed in the brains of humans and animals, with high levels of expression during development. It is translated with a prodomain (as proCPA6), which is removed before secretion. The active form of CPA6 binds tightly to the extracellular matrix (ECM) where it is thought to function in the processing of peptides and proteins. Mutations in the CPA6 gene have been identified in patients with temporal lobe epilepsy and febrile seizures. In the present study, we screened for CPA6 mutations in patients with juvenile myoclonic epilepsy and identified two novel missense mutations: Arg36His and Asn271Ser. Patients harboring these mutations also presented with generalized epilepsy. Neither of the novel mutations was found in a control population. Asn271 is highly conserved in CPA6 and other related metallocarboxypeptidases. Arg36 is present in the prodomain and is not highly conserved. To assess structural consequences of the amino acid substitutions, both mutants were modeled within the predicted structure of the enzyme. To examine the effects of these mutations on enzyme expression and activity, we expressed the mutated enzymes in human embryonic kidney 293T cells. These analyses revealed that Asn271Ser abolished enzymatic activity, while Arg36His led to a ~50% reduction in CPA6 levels in the ECM. Pulse-chase using radio-labeled amino acids was performed to follow secretion. Newly-synthesized CPA6 appeared in the ECM with peak levels between 2-8 hours. There was no major difference in time course between wild-type and mutant forms, although the amount of radiolabeled CPA6 in the ECM was lower for the mutants. Our experiments demonstrate that these mutations in CPA6 are deleterious and provide further evidence for the involvement of CPA6 mutations in the predisposition for several types of epilepsy.

Temperament personality profiles in suicidal behaviour: an investigation of associated demographic, clinical and genetic factors.

BACKGROUND: Personality traits have been suggested as possible risk factors for suicidal behaviours. Cloninger's model of personality (TCI), given its neurobiological background, might provide an ideal tool for the identification of dimensions associated with suicide attempt. METHODS: A number of 1333 suicide attempters and 589 non-suicide attempters suffering from different DSM-IV Axis I disorders were assessed using either the temperament and character inventory (TCI) or the tridimensional personality questionnaire (TPQ), as well as other self-report questionnaires evaluating dimensions associated with suicidal behaviour, such as impulsivity and anger traits. The severity of suicide attempts and the methods used were also assessed. Subjects were genotyped for polymorphisms within the key genes involved in monoaminergic pathways and the HPA axis. RESULTS: Compared with non-suicide attempters, suicide attempters scored higher for harm avoidance (HA) and novelty seeking (NS), and lower for self-directedness (SD). The difference was independent of Axis I disorders. Higher HA and NS scores were associated with a greater severity of suicidal behaviour. A multivariate model showed that HA was the single temperamental dimension independently related to suicide attempt history, beside impulsivity and anger-related traits. The genetic factors investigated did not play a significant role in modulating these temperamental dimensions. LIMITATIONS: The TCI was available for only half of the sample. CONCLUSIONS: Early detection of subjects displaying high HA and low SD, associated with high impulsivity and poor anger control, may help to prevent suicidal behaviours. Physicians should therefore be aware of these risk factors so that they can offer the best primary care intervention.

Naturally occurring carboxypeptidase A6 mutations: effect on enzyme function and association with epilepsy.

Carboxypeptidase A6 (CPA6) is a member of the A/B subfamily of M14 metallocarboxypeptidases that is expressed in brain and many other tissues during development. Recently, two mutations in human CPA6 were associated with febrile seizures and/or temporal lobe epilepsy. In this study we screened for additional CPA6 mutations in patients with febrile seizures and focal epilepsy, which encompasses the temporal lobe epilepsy subtype. Mutations found from this analysis as well as CPA6 mutations reported in databases of single nucleotide polymorphisms were further screened by analysis of the modeled proCPA6 protein structure and the functional role of the mutated amino acid. The point mutations predicted to affect activity and/or protein folding were tested by expression of the mutant in HEK293 cells and analysis of the resulting CPA6 protein. Common polymorphisms in CPA6 were also included in this analysis. Several mutations resulted in reduced enzyme activity or CPA6 protein levels in the extracellular matrix. The mutants with reduced extracellular CPA6 protein levels showed normal levels of 50-kDa proCPA6 in the cell, and this could be converted into 37-kDa CPA6 by trypsin, suggesting that protein folding was not greatly affected by the mutations. Interestingly, three of the mutations that reduced extracellular CPA6 protein levels were found in patients with epilepsy. Taken together, these results provide further evidence for the involvement of CPA6 mutations in human epilepsy and reveal additional rare mutations that inactivate CPA6 and could, therefore, also be associated with epileptic phenotypes.

Rare genotype combination of the serotonin transporter gene associated with treatment response in severe personality disorder.

The insertion deletion (ins/del) polymorphism of the serotonin transporter gene (5-HTTLPR) has been associated with several psychiatric phenotypes and antidepressant's response. We investigated, in a large cohort of 5,608 controls and subjects suffering from various psychiatric disorders, the frequency of haplotypes and corresponding genotypes combining the 5-HTTLPR and the other serotonin transporter promoter functional variant (rs25531). We showed that rs25531 lies 18 bp 5' to the site where the 43 bp (and not 44 bp as previously described) ins/del defines the 14- and 16-repeat alleles. These polymorphisms should therefore be considered as four alleles instead of a triallelic unique locus. The very rare G-14/G-16 genotype was carried on by only three subjects. These are women with a history of suicide attempt with a psychiatric history strongly suggesting a borderline personality disorder. Two of them have shown a non-response to serotoninergic antidepressant. Interestingly, in one of them was observed a spectacular response after the introduction of bupropion. The genotyping droved our therapeutic approach, by preferring a dopaminergic over a serotoninergic agent. This study highlights the usefulness of studying very rare clinical cases as well as rare variants, in order to deal with the biological heterogeneity of spectral disorders. © 2010 Wiley-Liss, Inc.

Simultaneous analysis of serotonin transporter, tryptophan hydroxylase 1 and 2 gene expression in the ventral prefrontal cortex of suicide victims.

Serotonergic signaling abnormalities have been implicated in suicide. Tryptophan hydroxylase (TPH), the rate limiting enzyme of serotonin biosynthesis and the serotonin transporter (SLC6A4), involved in the reuptake of serotonin from the synaptic gap, play major role in serotonergic signaling. In this study, we aimed to compare the levels of expression of these serotonin-related genes between suicide completers and controls and to identify genetic loci involved in their regulation. SLC6A4, TPH1, and TPH2 mRNA levels were measured in the ventral prefrontal cortex (VPFC) of 39 suicide completers and 40 matched controls. To identify the molecular basis of gene expression variation, we performed association studies between cis-acting polymorphisms and SLC6A4, TPH1, and TPH2 transcript levels. Finally, association analyses were carried out between suicide and TPH2 cis-single nucleotide polymorphisms (SNPs) in cohorts of 154 suicide completers and 289 control subjects. Whereas SLC6A4 and TPH1 mRNA expression levels did not differ between suicides and controls, TPH2 levels were found significantly increased (P = 0.003) in suicide completers. We observed that SNP rs10748185 located in the promoter region of TPH2 significantly affect levels of TPH2 mRNA expression. However, we did not find positive association between this eQTL (rs10748185) and suicide. Here, we report the simultaneous analysis of the expression of three serotonin-related genes in the VPFC of suicide victims and controls. This study showed that TPH2 expression levels were increased in the VPFC of suicide victims. Although, we identified a genetic variant that explains variance in TPH2 expression, we did not find evidence associating this cis-regulatory SNP with suicidal behavior.

Genetic and epigenetic analysis of SSAT gene dysregulation in suicidal behavior.

It has recently been proposed that the SSAT gene plays a role in the predisposition to suicidal behavior. SSAT expression was found to be down-regulated in the brain of suicide completers. In addition, a single nucleotide polymorphism (SNP) rs6526342 was associated both with variation in SSAT expression and with suicidal behavior. In this study, we aimed to characterize the relationship between SSAT dysregulation and suicide behavior. To this end, we measured SSAT expression levels in the ventral prefrontal cortex (VPFC) of suicide completers (n = 20) and controls (n = 20) and found them to be significantly down-regulated in suicide victims (P = 0.007). To identify the basis of the regulation of SSAT expression, we performed an association analysis of 309 SNPs with SSAT transcript levels in 53 lymphoblastoid cell lines from the CEPH collection. We then examined the methylation status of the SSAT promoter region in males and females suicide completers and control subjects whose SSAT brain expression had been measured. We found no evidence to support a role for SNPs in controlling the level of SSAT expression. SSAT promoter methylation levels were not different between suicide completers and controls and did not correlate with SSAT expression levels. In addition, we found no indication of a genetic association between suicidal behavior and SNPs located within the SSAT gene. Our study provides new results which show that dysregulation of SSAT expression does play a role in suicide behavior. However, our data do not support any association between rs6526342 and variation in SSAT expression or suicidal behavior.