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Human Molecular Genetics Advance Access originally published online on November 29, 2006
Human Molecular Genetics 2007 16(1):83-91; doi:10.1093/hmg/ddl443
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Involvement of hyperprolinemia in cognitive and psychiatric features of the 22q11 deletion syndrome

Grégory Raux1, Emilie Bumsel1, Bernadette Hecketsweiler2, Therese van Amelsvoort3, Janneke Zinkstok3, Sylvie Manouvrier-Hanu4, Carole Fantini4, Georges-Marie M. Brévière5, Gabriella Di Rosa6, Giuseppina Pustorino6, Annick Vogels7, Ann Swillen7, Solenn Legallic1, Jacqueline Bou1, Gaelle Opolczynski8, Valérie Drouin-Garraud1, Marie Lemarchand1, Nicole Philip9, Aude Gérard-Desplanches10, Michèle Carlier11, Anne Philippe12, Marie Christine Nolen12, Delphine Heron13, Pierre Sarda14, Didier Lacombe15, Cyril Coizet15, Yves Alembik16, Valérie Layet17, Alexandra Afenjar18, Didier Hannequin1, Caroline Demily1,8, Michel Petit1,8, Florence Thibaut1,8, Thierry Frebourg1 and Dominique Campion1,8,*

1 Department of Genetics and Inserm U614, IFRMP, Faculty of Medicine, Rouen, France, 2 Laboratory of Biochemistry, CHRU, Rouen, France, 3 Department of Psychiatry, AMC, Amsterdam, The Netherlands, 4 Department of Genetics and 5 Department of Cardio-Pediatrics, CHRU, Lille, France, 6 Department of Medical and Surgical Pediatrics, Unit of Infantile Neuropsychiatry, University Hospital, Messina, Italy, 7 Center for Human Genetics, University Hospital, Leuven, Belgium, 8 Units of Psychiatry, CH du Rouvray and CHRU, Rouen, France, 9 Department of Genetics, CHRU Marseille, France, 10 Research Center PsyCLE (EA3273) and 11 Cognitive Psychology UMR 6146 CNRS, Aix Marseille 1 University, France, 12 INSERM U781 and Department of Genetics, Hôpital Necker-enfants malades, Paris, France, 13 Department of Genetics, Hôpital Pitié Salpétrière, Paris, France, 14 Department of Genetics, CHRU Montpellier, France, 15 Department of Genetics, CHRU Bordeaux, France, 16 Department of Genetics, CHRU Strasbourg, France, 17 Department of Genetics, CHR Le Havre, France and 18 Department of Neuropediatry, Hopital Trousseau, Paris, France

* To whom correspondence should be addressed at: Inserm U614, Faculté de Médecine, 22 bd Gambetta, 76183 Rouen, France. Tel: +33 235148280; Fax: +33 235148237; Email: dominique.campion{at}univ-rouen.fr

Received September 28, 2006; Accepted November 16, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 METHOD
 REFERENCES
 
Microdeletions of the 22q11 region, responsible for the velo-cardio-facial syndrome (VCFS), are associated with an increased risk for psychosis and mental retardation. Recently, it has been shown in a hyperprolinemic mouse model that an interaction between two genes localized in the hemideleted region, proline dehydrogenase (PRODH) and catechol-o-methyl-transferase (COMT), could be involved in this phenotype. Here, we further characterize in eight children the molecular basis of type I hyperprolinemia (HPI), a recessive disorder resulting from reduced activity of proline dehydrogenase (POX). We show that these patients present with mental retardation, epilepsy and, in some cases, psychiatric features. We next report that, among 92 adult or adolescent VCFS subjects, a subset of patients with severe hyperprolinemia has a phenotype distinguishable from that of other VCFS patients and reminiscent of HPI. Forward stepwise multiple regression analysis selected hyperprolinemia, psychosis and COMT genotype as independent variables influencing IQ in the whole VCFS sample. An inverse correlation between plasma proline level and IQ was found. In addition, as predicted from the mouse model, hyperprolinemic VCFS subjects bearing the Met-COMT low activity allele are at risk for psychosis (OR = 2.8, 95% CI = 1.04–7.4). Finally, from the extensive analysis of the PRODH gene coding sequence variations, it is predicted that POX residual activity in the 0–30% range results into HPI, whereas residual activity in the 30–50% range is associated either with normal plasma proline levels or with mild-to-moderate hyperprolinemia.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 METHOD
 REFERENCES
 
Type I hyperprolinemia (HPI MIM 239500 [OMIM] ), an autosomal recessive inborn error, results from inherited deficiency of proline oxidase (POX), a mitochondrial enzyme expressed in kidney, liver and brain, which is encoded by the proline dehydrogenase (PRODH) gene (1). Clinically, HPI is recognized by elevation of the plasma proline value above 550 µmol/l without excretion of the degradation product of proline, {Delta}1-Pyrroline-5-carboxylate (P5C). This condition was initially considered as benign (1). Nevertheless, we had previously reported evidence that HPI, resulting either from a homozygous 350 kb deletion removing the entire PRODH gene and the neighboring DGCR6 gene, or from a PRODH homozygous L441P missense mutation, was associated with seizures, mental retardation and psychiatric symptoms in children (2–3). In addition, we had shown that mild-to-moderate hyperprolinemia (i.e. plasma proline levels between 350–500 µmol/l) was a weak risk factor for schizoaffective disorder (4). The PRODH gene, which is located in the 22q11 chromosomal region, is hemideleted in 22q11 deletion syndrome (22q11DS MIM 192430 [OMIM] ) also known as velo-cardio-facial syndrome (VCFS). It has been reported that{approx}50% of the 22q11DS patients are hyperprolinemic (5), whereas 30–45% of them present with mental retardation (6–7) and 12–30% have psychosis (8–10). The Pro/Re mouse hyperprolinemic strain (11) harbors a mutation in the ortholog of the human PRODH gene that introduces a premature termination (E453X) and results in a drastic reduction in enzymatic activity. These knockdown mice have regional neurochemical alterations in the brain accompanied by a deficit in sensorimotor gating, similar to that seen in individuals with schizophrenia and other neuropsychiatric disorders (12). The catechol-O-methyl transferase (COMT) gene, which encodes for the enzyme responsible for the degradation of dopamine (DA) in the prefrontal cortex (PFC), is also located in the 22q11 region. Recently, transcriptional and behavioral interaction between PRODH and COMT have been documented (13). Expression profiling with microarrays revealed that PRODH-deficient mice had selective upregulation of COMT in the PFC, which is likely to represent a homeostatic response to enhanced dopaminergic signaling induced by PRODH deficiency. According to this model, hyperprolinemic 22q11 individuals with low COMT activity are those who are the less efficient to compensate the dopaminergic hyperactivity and, therefore, are those who might have the higher risk to develop PFC-mediated cognitive and/or psychiatric phenotype. Interestingly, the COMT gene contains in its coding sequence a functional polymorphism (p.Val158Met) that determines high or low activity of this enzyme (14).

The present study was undertaken to characterize the relationship between PRODH gene variations, hyperprolinemia, cognitive and psychiatric symptoms in HPI and VCFS subjects. More specifically, our goal was to test the hypothesis that the high neuropsychiatric disease risk associated with the 22q11 locus results from an interaction between the PRODH and COMT genes.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 METHOD
 REFERENCES
 
HPI is associated with epilepsy, mental retardation and psychosis
Table 1 presents the clinical features of eight children with HPI, as determined by plasma proline values above 550 µmol/l without P5C excretion. Mental retardation was a frequent feature (5/8), generalized tonic-clonic seizures were also present in 6/8 patients and autistic features including stereotypies, lack of responsiveness to people and deficits in language development were found in 4/8 patients. Three patients had psychomotor delay since birth, in other patients mental retardation appeared during early childhood.


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  		Table 1. Phenotype and genotype of HPI patients

 
The VCFS sample
In the VCFS sample, 2/92 subjects were unwilling to participate to the IQ assessment. Sixty-two subjects (69%) had mental retardation (IQ <70) including 23 subjects (25%) with an IQ < 55. Thirty-three subjects (36%) were rated as psychotic (VCFS-P) and 59 as non-psychotic (VCFS-NP). The mean full scale IQ was 64 ± 14 (VIQ = 67 ± 17, PIQ = 64 ± 13). For each individual, significant difference between VIQ and PIQ was determined, taking age into account, at the 0.05 level. Twenty-two subjects performed significantly better to VIQ than PIQ and 14 subjects had significantly better performance to PIQ. In this sample of 92 VCFS patients, plasma proline levels ranged from 138 to 1275 µmol/l. Using the thresholds previously defined for the adult population (4) (i.e. 316 µmol/l in females and 377 µmol/l in males), we found that 34 subjects (37%) were hyperprolinemic.

A subset of VCFS patients has a phenotype reminiscent of HPI subjects
Plasma proline value was in the range of HPI (i.e. >550 µmol/l) in seven subjects (Table 2). These subjects differed from the rest of the sample for Full Scale IQ (51 ± 7 versus 65 ± 14, P = 0.007, Mann–Whitney U), prevalence of epileptic seizures—not related to hypocalcemia—(4/7 versus 4/85 P = 0.0008, Fisher's exact test) and frequency of psychotic symptoms (6/7 versus 27/85, P = 0.007 Fisher's exact test).


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  		Table 2. Phenotype and genotype of 92 VCFS patients according to plasma proline levels

 
Factors influencing IQ in VCFS patients
We next conducted a series of analysis in order to assess the contribution of five variables, namely age, sex, plasma proline level, COMT genotype and psychosis to IQ value. We first examined the interrelationships between these variables. These analyses revealed an interaction between plasma proline level and gender. The mean plasma proline values were significantly higher in males (416 ± 227 µmol/l) than in females (302 ± 185 µmol/l) (P = 0.000009, Mann–Whitney U), as classically reported (4). In subsequent analyses, this factor was controlled because the plasma proline level was used as a categorical variable (i.e. subjects were considered as hyperprolinemic or not) and different thresholds were used to define hyperprolinemia in males and females. The first factor that influenced IQ was psychosis: the mean full scale IQ in non-psychotic patients was 67 ± 14 when compared with 59 ± 11 in psychotic patients (P = 0.01, Mann–Whitney U). Detailed analysis of subscales was realized in 80 subjects and showed that VCFS-P patients performed significantly worse on Digit Symbol Coding, Arithmetics, Similarities, Digit Span and Picture Arrangement, when compared with VCFS-NP subjects (Table 3). The second factor that influenced IQ was hyperprolinemia: the mean full scale IQ in hyperprolinemic patients was 59 ± 12 when compared with 67 ± 14 in patients with normal plasma proline levels (P = 0.01, Mann–Whitney U). IQ was inversely correlated to the proline level (r = –0.24, P = 0.02, Spearman's test). There was also a tendency toward higher full scale IQ in Met-COMT hemizygous patients, which remained below statistical significance (P = 0.07, Mann–Whitney).


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  		Table 3. Scores on cognitive measures of 90 VCFS patients

 
Forward stepwise multiple regression analysis selected hyperprolinemia, psychosis and COMT genotype as independent variables influencing IQ. Sex and age did not appear as independent predictors of IQ. The regression coefficients were: hyperprolinemia;–7, 35 (±2, 8), P < 0.011; psychosis;–7, 51 (±2, 88), P < 0.01; COMT genotype; 6, 33 (±2, 7), P < 0.023. When hyperprolinemia and COMT genotype were considered jointly, no significant effect on IQ was detected (Table 3). Likewise, there was no significant effect of hyperprolinemia and Met-COMT allele, either independently or conjointly, on the Digit Span and Arithmetics tasks (two subscales of WAIS relying on PFC mediated working memory performances) (Table 3).

Hyperprolinemia associated with Met allele of COMT gene is a risk factor for psychosis in VCFS patients
Among the 33 VCFS-P subjects, 18 satisfied DSM IIIR criteria for schizophrenia (paranoid n = 15, disorganized n = 3), three had schizophreniform (SZF) disorder, six had atypical psychosis (A-PS), four had schizotypal (SZT) personality and two had autism. Mean age of onset for schizophrenia was 16.7 ± 3.5 years (range 12–27). Among schizophrenic patients, the most prominent symptoms were ideas of reference, paranoid ideation and auditory hallucinations. Atypical psychosis was diagnosed in six subjects who reported auditory hallucinations that did not significantly interfere with social functioning. Mean age of onset for atypical psychosis was 14.6 ± 0.8 years (range 14–16). Two subjects who received a diagnosis of autism were severely impaired and failed to acquire any language. Neither hyperprolinemia nor the Met-COMT allele was associated with psychosis, although a trend was observed in both cases. However, when these two factors were considered jointly, hyperprolinemic subjects bearing the low activity Met-COMT allele were at risk for psychosis (P = 0.03, OR = 2.8, 95% CI 1.04–7.4) (Table 4).


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  		Table 4. Distribution of hyperprolinemia+Met-COMT allele in VCFS-P and VCFS-NP patients

 
Hyperprolinemia and PRODH genotype
A detailed account of phenotypic and genotypic characteristics for each HPI and VCFS subject is given in Tables 1 and 3. In all HPI and VCFS subjects, we searched for PRODH deletions, using QMPSF, and we sequenced the coding sequence of PRODH. The 350 kb deletion removing the PRODH gene was detected in two HPI patients and was present at the homozygous state in one patient. According to the in vitro functional data of Bender et al. (15), PRODH missense mutations were divided in two groups with moderate (30–70%) or severe (>70%) reduction in POX activity, respectively. For the sake of simplicity, mutations with no or mild effect, as well as silent mutations, were not included in Tables 1 and 3. Several unreported DNA variations with unknown functional consequences (P8L, P30S, A58T, T275N and G444D) were also found during this screening. In 6/8 HPI patients (Table 1), missense mutations with severe effect were found in association with a deletion, a stop codon or a mutation with moderate effect on the second allele. The second allele bore a mutation with unknown functional consequence (T275N) in one HPI patient. The HPI subject with the lowest plasma proline level was homozygous for a mutation (Q19P) with moderate effect, but also harbored in one allele a mutation (A58T) with unknown functional consequence. In the VCFS sample, in which all subjects were hemizygous for the PRODH gene, only one mutation with severe effect was detected in one hyperprolinemic patient (Table 3). Mutations with a moderate effect were detected in 66/92 VCFS subjects (72%), either with or without hyperprolinemia. These mutations were equally distributed between subjects with and without hyperprolinemia, with the exception of the R431H variation that was more frequent in hyperprolinemic subjects (P = 0.0001, Fisher's exact test). Four-to-seven VCFS subjects with the highest plasma proline levels and two HPI subjects bore this variation, indicating that besides its moderate effect on catalytic activity, it could have a more drastic effect on protein structure.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
METHOD
 REFERENCES
 
Despite the well-known association of type II hyperprolinemia with mental retardation (1), the impact of less severe increase in plasma proline levels on cognitive functions and psychiatric illness has largely escaped attention. Here, we provide further evidence that HP1 is indeed a cause of mental retardation associated with neurological and, in some cases, psychiatric features in children. To better understand the consequences of hyperprolinemia, we have constituted a sample of 92 VCFS subjects, including 33 patients with psychosis. This high proportion is not representative from that found in a randomly ascertained population, since we actively searched for psychotic patients, in order to achieve maximum statistical efficiency. Likewise, a higher proportion of subjects with mental retardation were included in this study, when compared with the proportion found in randomly ascertained populations (6–7). We show that a small subset of VCFS subjects had severe hyperprolinemia in the range of HPI, and that the phenotype of these subjects was distinguishable from that of other VCFS subjects. Notably, the mean IQ of these patients was significantly lower than that of other VCFS subjects, and the prevalence of psychosis was high. Epilepsy—not related to hypocalcemia—was frequent in patients with severe hyperprolinemia, although it is an uncommon feature in VCFS patients (16). Interestingly, comorbidity between seizures and psychosis has recently been reported in two VCFS subjects, one of them with an IQ of 46 (17). Overall, these findings have important potential therapeutic consequences for HPI as well as VCFS subjects with hyperprolinemia, since it is conceivable to lower plasma proline levels by reduction of proline intake (18) or by pharmacological treatment early during early childhood in these patients.

Increased plasma proline level, including both severe and mild/moderate hyperprolinemia, was not significantly associated with psychosis in the whole VCFS sample. Likewise, in agreement with a previous study of adult psychotic VCFS patients (8) and in contrast with a recent report (19), no association between COMT genotype and psychosis was found in the present study. However, as predicted from the PRODH deficient mouse model, individuals with both hyperprolinemia and the Met-COMT allele were at risk for a broad spectrum of psychotic diagnoses, including schizophrenia. Consistent with a previous report (20), schizophrenic VCFS subjects experienced more often positive than negative symptoms during lifetime. Auditory hallucinations were also a key symptom in patients with SZF disorder and atypical psychosis suggesting that, despite different categorical diagnoses, most VCFS-P subjects shared prominent positive symptoms. Another notable feature in schizophrenic patients was the early age of onset, which is in part inherent to the age structure of our sample, but could also reflect the strong association between childhood-onset schizophrenia and VCFS (21). Recently, two studies have reported that a high proportion of VCFS adolescents experienced transient psychotic episodes that could be predictive of schizophrenia in adulthood (22–23). The symptoms of these patients are similar to those found in subjects diagnosed as atypical psychosis or SZT personality in the present study.

Stepwise multiple regression analysis indicated that three factors, psychosis, hyperprolinemia and COMT genotype had an independent effect on IQ in the VCFS sample. A similar trend toward a relationship between psychosis and lower IQ in VCFS subjects has previously been reported in three studies (8,22,24). Although relying on a less complete battery of tests, our data are also consistent with the results obtained by Chow et al. (24) and van Amelsvoort et al. (25), showing that adult VCFS-P subjects performed significantly worse on a large number of cognitive tasks, when compared with other VCFS subjects. In the present study, increased plasma proline level, including both severe and mild/moderate hyperprolinemia, was the second factor associated with lower mean full scale IQ in VCFS subjects. Moreover, an inverse correlation between plasma proline level and IQ was found. Although it is recognized that a high level of proline has a detrimental effect on neuronal integrity (26) and is associated with a pattern of global cognitive impairment in HPI and HPII, there is, to our knowledge, no previous study addressing the involvement of mild-to-moderate hyperprolinemia in cognitive impairment in humans. The third factor influencing IQ was the COMT genotype. In contrast to the lack of proline studies, the impact of COMT genotype on IQ has previously been investigated in several studies, that yielded conflicting results. In VCFS children, the Met-COMT low activity allele has been associated either with higher verbal and full scale IQ (27), as in the present study, or inversely with a tendency toward a decline in verbal (19) and full scale IQ (28).

The PRODH deficient mouse model predicts a specific impairment of PFC-mediated cognitive functions, related to an excess of dopaminergic transmission, in VCFS patients with both hyperprolinemia and Met-COMT genotype. The neuropsychological evaluation performed in this study does not allow to address properly this question. Indeed, IQ accounts for a very modest proportion of the variance in executive functions (29) and the subtests of WAIS are lacking specificity to adequately capture working memory deficits. Similarly, literature data do not deal efficiently with this issue. It should be stressed that, although the impact of COMT genotype on PFC cognition has been investigated in numerous studies, hyperprolinemia and COMT genotype have never been studied together, thus limiting the value of these reports. In the general population or in schizophrenic patients, most studies (30–34) have concluded that the Met-COMT low activity allele was associated with better prefrontal functioning, although a large study failed to detect any effect (35) and another concluded that the main effect was on processing speed and attention (36). In VCFS subjects, two studies (28–29) have reported better PFC cognition in Met-COMT hemizygotes, whereas one study only found a trend (37) and another one reported no effect (38). In contrast, in a recent study, it was reported that the Met-COMT allele was a risk factor for decline in PFC volume and cognition (19). Thus, although most studies have concluded that low COMT activity (i.e. increased level of DA transmission) was associated with fewer perseverative errors on frontal tests, some discrepancies persist and the problem is probably more complex than initially thought. There is now evidence that either a reduction (39) or an excess (such as in PRODH-deficient mice) of DA signalling have detrimental effects on PFC cognition and that different neuronal process (i.e. phasic and tonic DA release) affect the capacity to built efficiently working memory representations (40–42). Therefore, future studies aimed to delineate the pattern of cognitive impairment in VCFS subjects with both hyperprolinemia and the Met-COMT allele, should rely on more sensitive tasks (43–44) allowing to discriminate between these different aspects of dopaminergic transmission in the PFC.

In the present study, we have progressed in our attempt to establish a link between PRODH genotypes and plasma proline levels, but the picture remains incomplete, since (i) functional data are still lacking for some individual variations or some haplotypes with multiple mutations in cis and (ii) regulatory regions have not been examined. Nevertheless, on the basis of available data, our working hypothesis is that predicted POX residual activity in the 0–30% range results into HPI, whereas residual activity in the 30–50% range is associated either with normal plasma proline level or with mild-to-moderate hyperprolinemia.

Finally, two limitations of our study have to be underlined. First, few studies have addressed the question of the reproducibility of plasma proline determination. We had previously assessed this issue in 25 controls or mildly hyperprolinemic subjects. The maximum variation between two independent measures was 12% and the mean variation was 8% (unpublished data). In contrast, for an unknown reason, there are often considerable variations in plasma proline levels in HPI patients over time, but the mean value remains generally above 550 µmol/l. Secondly, it should be stressed that 40 VCFS-NP subjects had less than 30 years of age. Therefore, it is conceivable that some of these subjects may yet develop psychotic symptoms. To avoid the risk of such misclassification, future studies should rely on older VCFS-NP populations that have escaped to a larger part of the risk period for schizophrenia. Owing to the scarcity of such subjects, this will require larger collaborative studies.


   METHOD
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 METHOD
REFERENCES
 
Subjects
Eight children with HP1, ascertained in France and Italy from medical genetics centers, and 92 VCFS participants, aged 15 and older (46% males, mean age 25 ± 10 years, range 15–58) were included in this study. Eighty-one VCFS subjects were recruited from nine medical centers from France, Belgium and in The Netherlands, five were ascertained from psychiatric sources and six via the French association ‘Generation 22’. In all patients, the diagnosis of VCFS was confirmed by fluorescence in situ hybridization and Quantitative Multiplex PCR of Short Fragments (2). Information about behavioral, somatic, neurological, pharmacological and psychiatric history were collected. Consensus diagnoses for psychiatric cases were established by two raters according to DSM IIIR criteria from all available information following examination of case notes and in most cases, direct interview of patients with the appropriate sections of the Schedule for Affective Disorder and Schizophrenia (45). In addition, the Schedule for Schizotypal Personality (SSP) (46) was administered when necessary. Clinical data were compiled into case vignettes and diagnoses were made blind of proline data. Intellectual functioning was assessed by trained psychologists using the Wechsler Intelligence Scale for Children 3rd edition (WISC III) for subjects younger than age 17 and either Wechsler Adult intelligence Scale-Revised (WAIS-R) or Wechsler Adult intelligence Scale 3rd edition (WAIS III) for older subjects. The proportion of subjects assessed with WAIS-R was not significantly different between VCFS-P and VCFS-NP subjects. All VCFS-P subjects were assessed during a stable or remitted phase of their psychotic illness. At the time of assessment, 12 VCFS-P subjects received atypical neuroleptic drugs, 11 received conventional neuroleptics and six had anticholinergic medications. Seven epileptic subjects were treated by valproate. Informed consent was obtained from all participants and the Local Research Ethics Committee approved the study protocol.

Determination of plasma proline levels
In VCFS patients, plasma proline levels were determined in the morning after overnight fasting. All samples were analyzed by the same laboratory using ion exchange chromatography on a BIOTRONIK LC 3000 system. As previously reported (4), subjects whose plasma proline levels were two standard deviations above the controls' mean values (i.e. 316 µmol/l in females and 377 µmol/l in males) were considered to be hyperprolinemic.

Quantitative multiplex PCR of short fluorescent fragments
Ten exons (1, 2, 4, 5, 9, 10, 11, 12, 14, 15) of the PRODH gene were analyzed by quantitative multiplex PCR of short fluorescent fragments (QMPSF) in all subjects. When a rearrangement involving the PRODH gene was detected, four different QMPSF assays covering the entire 22q11.2 region were performed, as previously described (2).

Sequence analysis
Exons 2–15 of PRODH were PCR-amplified from peripheral blood lymphocytes. PCR conditions and primers were identical to those previously described (4), except the sense primer of exon 12 (5'-TTGGGGACAGAGGTTGGAGGC-3') and exon 11 sense (5'-GCTCGGGCAGAGGGTACC-3') and antisense (5'-CCGCTGGGTGAGCTGTGCTG-3') primers. After purification by low-melt agarose gel electrophoresis, PCR products were directly sequenced on both strands using the ABI PRISM BigDye Terminator v3.1 Cycle Sequencing kit (PE Applied Biosystems, Foster city, CA, USA) and an Applied Biosystems model 3100 automated sequencer (PE Applied Biosystems).

Screening for specific DNA variation
The Val158Met polymorphism in the COMT gene was detected using the ABI Prism SNaPshot multiplex kit.

Statistical analyses
Quantitative data were expressed as mean ± SD. Comparisons for continuously distributed variables were performed using the Mann–Whitney non-parametric test. Statistical comparisons for categorical variables were performed using {chi}2 test or Fisher's exact test, when the cells size was small. Correlations between quantitative variables were assessed by the Spearman's test. All tests reported were two-tailed and significance level was set to 5%. OR are given with their 95% CI. One-way analysis of variance (ANOVA) was used to detect group differences for neuropsychological measures.

To determine the relative contribution of five variables (age, sex, hyperprolinemia, psychosis and COMT genotype) to the full scale IQ, we submitted the data to a forward stepwise multiple regression analysis, using a selection criterion of P < 0.05.


   ACKNOWLEDGEMENTS
 
Supported by a grant from Programme Hospitalier de Recherche Clinique. A.G.-D. held a Jerôme Lejeune Foundation fellowship. We are indebted to our colleagues S. Lebreton, O. Boespflug-Tanguy, C. Francannet, M. Lacambre, M.O. Krebs, N. Bazin, who referred patients for this study and to J. Benichou and J.F. Ménard for statistical advice. We would like to thank the patients who took part in this study and the support of the French association of parents Génération 22.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 METHOD
 REFERENCES
 

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