Proline Dehydrogenase Gene Research Articles

Oncogenic human herpesvirus hijacks proline metabolism for tumorigenesis

Three-dimensional (3D) cell culture is well documented to regain intrinsic metabolic properties and to better mimic the in vivo situation than two-dimensional (2D) cell culture. Particularly, proline metabolism is critical for tumorigenesis since pyrroline-5-carboxylate (P5C) reductase (PYCR/P5CR) is highly expressed in various tumors and its enzymatic activity is essential for in vitro 3D tumor cell growth and in vivo tumorigenesis. PYCR converts the P5C intermediate to proline as a biosynthesis pathway, whereas proline dehydrogenase (PRODH) breaks down proline to P5C as a degradation pathway. Intriguingly, expressions of proline biosynthesis PYCR gene and proline degradation PRODH gene are up-regulated directly by c-Myc oncoprotein and p53 tumor suppressor, respectively, suggesting that the proline-P5C metabolic axis is a key checkpoint for tumor cell growth. Here, we report a metabolic reprogramming of 3D tumor cell growth by oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV), an etiological agent of Kaposi's sarcoma and primary effusion lymphoma. Metabolomic analyses revealed that KSHV infection increased nonessential amino acid metabolites, specifically proline, in 3D culture, not in 2D culture. Strikingly, the KSHV K1 oncoprotein interacted with and activated PYCR enzyme, increasing intracellular proline concentration. Consequently, the K1-PYCR interaction promoted tumor cell growth in 3D spheroid culture and tumorigenesis in nude mice. In contrast, depletion of PYCR expression markedly abrogated K1-induced tumor cell growth in 3D culture, not in 2D culture. This study demonstrates that an increase of proline biosynthesis induced by K1-PYCR interaction is critical for KSHV-mediated transformation in in vitro 3D culture condition and in vivo tumorigenesis.

Proline dehydrogenase (PRODH) gene polymorphisms and the risk of schizophrenia in Iranian populations

Proline dehydrogenase (PRODH) gene polymorphisms and the risk of schizophrenia in Iranian populations

Leaf status and environmental signals jointly regulate proline metabolism in winter oilseed rape.

Proline metabolism is an essential component of plant adaptation to multiple environmental stress conditions that is also known to participate in specific developmental phases, particularly in reproductive organs. Recent evidence suggested a possible role for proline catabolism in Brassica napus for nitrogen remobilization processes from source leaves at the vegetative stage. Here, we investigate transcript levels of Δ1-PYRROLINE-5-CARBOXYLATE SYNTHASE (P5CS) and PROLINE DEHYDROGENASE (ProDH) genes at the vegetative stage with respect to net proline biosynthesis and degradation fluxes in leaves having a different sink/source balance. We showed that the underexpression of three P5CS1 genes in source leaves was accompanied by a reduced commitment of de novo assimilated 15N towards proline biosynthesis and an overall depletion of free proline content. We found that the expression of ProDH genes was strongly induced by carbon starvation conditions (dark-induced senescence) compared with early senescing leaves. Our results suggested a role for proline catabolism in B. napus, but acting only at a late stage of senescence. In addition, we also identified some P5CS and ProDH genes that were differentially expressed during multiple processes (leaf status, dark to light transition, and stress response).

IRAP Analysis of Transgenic Wheat Plants with a Double-Stranded RNA Suppressor of the Proline Dehydrogenase Gene

The polymorphism level of DNA regions flanked by inverted LTR retrotransposon repeats has been analyzed by the IRAP method in genetically modified wheat plants that contained a double-stranded RNA suppressor of the proline dehydrogenase gene and that had been obtained by Agrobacterium-mediated transformation in an in vitro culture. No DNA polymorphism was detected in the transgenic plants when highly efficient primers for Sukkula, Sabrina, Wham, Nikita, and Wilma1 retrotransposons were used. We did not register the disappearance of amplicons in the DNA profiles of PCR in the experiment, and this may be indicative of the absence of rearrangements in the primer annealing sites and in the loci studied. The emergence of new amplicons was not observed in the spectra of DNA amplification products, which is indicative of the absence of activation of the transposon activity of mobile genetic elements in transgenic plants with a double-stranded RNA suppressor of the proline dehydrogenase gene. To expand the spectrum of amplicons in PCR products of the samples studied, we tested a method that involved the combined use of IRAP primers for different retrotransposons in a single reaction. IRAP primer pairs were selected experimentally, but the use of this method did not reveal the disappearance or emergence of polymorphic fragments. The absence of DNA polymorphism in transgenic plants with a double-stranded RNA suppressor of the proline dehydrogenase gene may be due to the phenomenon of RNA interference that suppresses retrotransposon activity.

Physiological and biochemical analysis of transgenic wheat plants of seed generation T2 with double-stranded RNA suppressor of the proline dehydrogenase gene

Aim. To carry out physiological and biochemical analysis of genetically modified plants of bread wheat of seed generation T2 with the double-stranded RNA suppressor of the proline dehydrogenase gene. Methods. Biochemical determination of free L-proline content and activity of the enzyme proline dehydrogenase; physiological examination of plant growth in vitro and in vivo. Results. It has been shown that transgenic plants, in contrast to control groups, grow more intensively in a selective medium with mannitol, maintaining a green color. It has been established that under normal conditions and under conditions of water deficit, plants of the T2 have an elevated level of free proline in the leaves, compared with the control genotypes. It was found that transformants are characterized by reduced activity of the enzyme of proline dehydrogenase, which manifests itself when the norm-stress-norm conditions change. Transgenic T2 plants had a higher resistance to aqueous deficiency compared to baseline, which was reflected in the nature of their growth. In conditions of soil moisture shortage, the yield of the most of the transformed lines was significantly higher than non-transformed plants. Conclusions. The obtained results allow us to conclude that the use of the pBi2E vector construct with the double-stranded RNA suppressor of the pdh gene is effective for the production of transgenic bread wheat plants with a high level of resistance to water deficiency.
 Keywords: Triticum aestivum L., Agrobacterium-mediated transformation, RNA suppressor of the proline dehydrogenase gene, T2 plants, physiological-biochemical analysis.

Changes in the ionome of genetically modified corn plants with double-stranded RNA-suppressor of proline dehydrogenase gene

Changes in the ionome of genetically modified corn plants with double-stranded RNA-suppressor of proline dehydrogenase gene

Employing exogenous melatonin applying confers chilling tolerance in tomato fruits by upregulating ZAT2/6/12 giving rise to promoting endogenous polyamines, proline, and nitric oxide accumulation by triggering arginine pathway activity

In the present study, the mechanisms employed by exogenous melatonin applying for conferring chilling tolerance in tomato fruits during storage at 4 °C for 28 days were investigated. Conferring chilling tolerance in tomato fruits in response to exogenous melatonin applying at 100 µM may arise from upregulating SlZAT2/6/12 giving rise to triggering CBF1 gene expression. Employing higher arginine pathway activity in tomato fruits by exogenous melatonin applying demonstrating by higher endogenous polyamines accumulation arising from higher ornithine decarboxylase (ODC) and arginine decarboxylase (ADC) genes expression and enzymes activity, higher endogenous proline accumulation arising from higher pyroline 5-carboxylate synthetase (P5CS) and ornithine aminotransferase (OAT) genes expression and enzymes activity accompanying by lower proline dehydrogenase (PDH) gene expression and enzyme activity and higher endogenous nitric oxide (NO) accumulation arising from higher nitric oxide synthase (NOS) gene expression and enzyme activity may be responsible for keeping safe membrane integrity.

Enhanced synthesis of poly gamma glutamic acid by increasing the intracellular reactive oxygen species in the Bacillus licheniformis Δ1-pyrroline-5-carboxylate dehydrogenase gene ycgN-deficient strain.

Poly gamma glutamic acid (γ-PGA) is an anionic polyamide with numerous applications. Previous studies revealed that L-proline metabolism is implicated in a wide range of cellular processes by increasing intercellular reactive oxygen species (ROS) generation. However, the relationship between L-proline metabolism and γ-PGA synthesis has not yet been analyzed. In this study, our results confirmed that deletion of Δ1-pyrroline-5-carboxylate dehydrogenase gene ycgN in Bacillus licheniformis WX-02 increased γ-PGA yield to 13.91gL-1, 85.22% higher than that of the wild type (7.51gL-1). However, deletion of proline dehydrogenase gene ycgM had no effect on γ-PGA synthesis. Furthermore, a 2.92-fold higher P5C content (19.24μmolgDCW-1) was detected in the ycgN deficient strain WXΔycgN, while the P5C levels of WXΔycgM and the double mutant strain WXΔycgMN showed no difference, compared to WX-02. Moreover, the ROS level of WXΔycgN was increased by 1.18-fold, and addition of n-acetylcysteine (antioxidant) decreased its ROS level, which further reduced γ-PGA synthesis capability of WXΔycgN. Collectively, our results demonstrated that proline catabolism played an important role in maintaining ROS homeostasis, and deletion of ycgN-enhanced P5C accumulation, which induced a transient ROS signal to promote γ-PGA synthesis in B. licheniformis.

Obtaining of transgenic potato plants (Solanum tuberosum L.) that contain antisense sequence of prolindehydrogenase gene

Aim. The aim of our work was to obtain transgenic potato plants of Ukrainian varieties with the expression of a double-stranded RNA suppressor of proline dehydrogenase gene. We propose the decrease of proline degradation level and increase of overall proline concentration in obtained transgenic plants. Methods. The Agrobasterium tumefaciens-mediated method of genetic transformation to obtain transgenic plants of potato was used. Internodes of aseptic potato plants were transformed with a binary vector pBi2E containing an inverted repeats of two copies of proline dehydrogenase gene’s first exon and the gene of neomycin phosphotransferase II (nptII). Results. As a result of experiments kanamycin resistant transgenic potato lines of Deseiree, Belarusian 12 and Slavianka varieties were obtained. The transgenic nature of the obtained plants was confirmed by PCR with primers specific to the first exon of proline dehydrogenase and to nptII genes. Conclusions. The optimized conditions of genetic transformation and used agrobacterial strain allow to obtain the transgenic plants of a model potato variety Désirée, as well as varieties Belorussian 12 and Slovyanka which are of practical interest for cultivation in Ukraine. Keywords: transgenic plants, potatoes (Solanum tuberosum L.), stress resistance, proline.

Activity of antioxidant enzymes in chloroplasts of transgenic plants of wheat with double-stranded RNA-suppressor of the prolindehydrogenase gene

Aim. To investigate the activity of antioxidant enzymes in chloroplasts of transgenic wheat plants variety Zymoyarka containing a double-stranded RNA-suppressor of the proline dehydrogenase gene. Methods. Spectrophotometrical determination of the activity of enzymes. Results. Under physiological conditions, the activity of superoxide dismutase (SOD) and ascorbate peroxidase (APX) in transgenic plants significantly exceeded these indices in non-transgenic forms. Under soil drought, the activity of SOD in transgenic plants decreased significantly and amounted to only 70 % of the activity of this enzyme in control plants, the activity of APX was 12 % lower than that of control. The water deficit increased sharply (4–5 times) the level of free L-proline in transgenic wheat plants with reduced activity pdh. Conclusions. Biotechnological plants of wheat with increased activity of SOD and APX under optimum conditions can be characterized by increased adaptive plasticity compared to plants of original variety with lower activity of the enzymes. The increased activity of antioxidant enzymes may be a prerequisite for increasing the tolerance of these plants to stressors of different origins.
 Keywords: Triticum aestivum L., transgenic plants, double-stranded RNA-suppressor of the proline dehydrogenase gene, L-proline, antioxidant enzymes.

Abscisic acid cross-talking with hydrogen peroxide and osmolyte compounds may regulate the leaf rolling mechanism under drought

Leaf rolling observed in some crops such as maize, rice, wheat and sorghum is an indicator of decreased water status. Moderate leaf rolling not tightly or early increases the photosynthesis and grain yield of crop cultivars under environmental stresses. Moreover, the effects of exogenous abscisic acid (ABA) on stomatal conductance, water status and synthesis of osmotic compounds are a well-known issue in plants subjected to water deficit. However, it is not clear how the cross-talk of ABA with H2O2 and osmolyte compounds affects the leaf rolling mechanism. Regulation mechanism of leaf rolling by ABA has been first studied in maize seedlings under drought stress induced by polyethylene glycol 6000 (PEG 6000) in this study. ABA treatment under drought stress reduced hydrogen peroxide (H2O2) content and the degree of leaf rolling (%) while the treatment-induced ABA synthesis, osmolyte levels (proline, polyamine and total soluble sugars) and some antioxidant enzyme activities in comparison to the plants that were not treated with ABA. Furthermore, exogenous ABA up-regulated the expression levels of arginine decarboxylase (ADC) and pyrroline-5-carboxylate synthase (P5CS) genes and down-regulated polyamine oxidase (PAO), diamine oxidase (DAO) and proline dehydrogenase (ProDH) gene expressions. When endogenous ABA content was decreased by the treatment of fluoridone (FLU) that is an ABA inhibitor, leaf rolling degree (%), H2O2 content and antioxidant enzyme activities increased, but osmolyte levels, ADC and P5CS gene expressions decreased. Finally, the treatment of ABA to maize seedlings exposed to drought stress resulted in the stimulation of the antioxidant system, osmotic adjustment and reduction of leaf rolling. We concluded that ABA can be a signal compound cross-talking H2O2, proline and polyamines and thus involved in the leaf rolling mechanism by providing osmotic adjustment. The results of this study can be used to provide data for the molecular breeding of maize hybrids with high grain yield by means of moderately rolled leaves.

Proline dehydrogenase gene (PRODH) polymorphisms and schizophrenia susceptibility: a meta-analysis.

Previous studies have been conducted to explore the association between proline dehydrogenase gene (PRODH) polymorphisms and schizophrenia (SZ) susceptibility, but providing the controversial results. Here we performed this meta-analysis to determine whether PRODH variants were associated with SZ risk. Relevant studies were screened by retrieving online database PubMed, Web of Science, Embase, China National Knowledge Infrastructure (CNKI) and SZGene from inception to December 2016. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated based on genotype data or allele frequency to evaluate the strength of this association. For rs372055, eleven studies with 3398 SZ patients and 3171 controls were included and the results indicated that people carrying the T allele was not associated with SZ risk in allele frequency model (C vs T, OR=1.12, 95%CI=0.96-1.32). However, results from subgroup analysis showed a significant relationship between rs372055 and SZ risk in dominant genetic model (CC+CT vs TT, OR=1.26, 95% CI=1.05-1.50) and heterogeneous model (CT vs TT, OR=1.26, 95% CI=1.05-1.52) in Asian, but not in Caucasian. For polymorphisms rs383964, rs450046, rs385440 and rs2870983, no associations were found between these polymorphisms and SZ susceptibility in allele frequency. This meta-analysis suggests that rs372055 (C/T) polymorphism in PRODH gene is associated with increased SZ risk only in Asian.

Transgenic plants as genetic models for studying functions of plant genes

Transgenic plants are widely used for the investigation of functions of particular genes and for reconstruction of complex gene networks controlling plant morphology, biochemistry, and physiology during different development stages and in response to various external stimuli. Gene engineering instruments for the design of transgenic plants with either an elevated or suppressed expression of the target genes are discussed. Genetic constructs for protein synthesis or antisense RNA/self-complementary double-stranded RNA transcription are described. Transgenic plants with elevated or decreased levels of expression of S-like ribonucleases and a decreased expression of the proline dehydrogenase gene are considered as examples. It was believed that S-like RNase functions concern mainly phosphate remobilization from senescent plant organs. However, the expression patterns of some genes coding for S-like RNases were similar to some pathogen- responsive genes (both local and systemic induction after damage or pathogen inoculation). In addition, some pathogenesis-related proteins (PR-4 family) possess RNase activity and can inhibit the growth of pathogenic fungi. Investigation of transgenic tobacco plants revealed that high ribonuclease activity in the apoplast is correlated with increased resistance against the tobacco mosaic virus. Thus, S-like RNases may have a new function as a part of a plant’s basal antiviral defense mechanism. Another set of transgenic plants carries an antisense suppressor of the proline dehydrogenase gene (PDH) constructed with an Arabidopsis target gene segment. Tobacco, maize, and sunflower plants with this heterologous suppressor were characterized by a moderate decrease in PDH activity and a moderate (1.5–3-fold) increase in the proline content under normal conditions. It was also found that these plants were more tolerant to various abiotic stresses (drought, NaCl, cold, and toxic heavy metals), which may result from the protective proline effect early in exposure to stress, preventing the cellular gene expression machinery from damage by stress-generated free radicals.

Proline dehydrogenase promotes senescence through the generation of reactive oxygen species

Cellular senescence is a complex stress response characterized by permanent loss of proliferative capacity and is implicated in age-related disorders. Although the transcriptional activity of p53 (encoded by TP53) is known to be vital for senescence induction, the downstream effector genes critical for senescence remain unsolved. Recently, we have identified the proline dehydrogenase gene (PRODH) to be upregulated specifically in senescent cells in a p53-dependent manner, and the functional relevance of this to senescence is yet to be defined. Here, we conducted functional analyses to explore the relationship between PRODH and the senescence program. We found that genetic and pharmacological inhibition of PRODH suppressed senescent phenotypes induced by DNA damage. Furthermore, ectopic expression of wild-type PRODH, but not enzymatically inactive forms, induced senescence associated with the increase in reactive oxygen species (ROS) and the accumulation of DNA damage. Treatment with N-acetyl-L-cysteine, a ROS scavenger, prevented senescence induced by PRODH overexpression. These results indicate that PRODH plays a causative role in DNA damage-induced senescence through the enzymatic generation of ROS.

Ethanolamine induced modification in glycine betaine and proline metabolism in Nicotiana rustica under salt stress

The present study aimed to investigate the effects of ethanolamine on glycine betaine and proline metabolism in Nicotiana rustica under salt stress. The in vitro grown tobacco (Nicotiana rustica) plants were pretreated with ethanolamine (at concentrations 70, 130, 270, and 530 μM for biochemical analysis and only at the concentration of 530 μM for molecular analysis) and then transferred to Murashige and Skoog medium containing 200 mM NaCl for 3 weeks. Our results showed that ethanolamine promoted glycine betaine biosynthesis by an increase in betaine aldehyde dehydrogenase (BADH) gene expression and BADH enzymatic activity. Moreover, ethanolamine pretreatment possibly reduced proline content in salt stressed plants via its negative effect on Δ-pyrroline-5-carboxylate synthase (P5CS) gene expression and P5CS enzymatic activity and its positive effect on proline dehydrogenase (PDH) gene expression and PDH activity.

SlgA, encoded by the homolog of the human schizophrenia-associated gene PRODH, acts in clock neurons to regulate Drosophila aggression.

ABSTRACTMutations in the proline dehydrogenase gene PRODH are linked to behavioral alterations in schizophrenia and as part of DiGeorge and velo-cardio-facial syndromes, but the role of PRODH in their etiology remains unclear. Here, we establish a Drosophila model to study the role of PRODH in behavioral disorders. We determine the distribution of the Drosophila PRODH homolog slgA in the brain and show that knockdown and overexpression of human PRODH and slgA in the lateral neurons ventral (LNv) lead to altered aggressive behavior. SlgA acts in an isoform-specific manner and is regulated by casein kinase II (CkII). Our data suggest that these effects are, at least partially, due to effects on mitochondrial function. We thus show that precise regulation of proline metabolism is essential to drive normal behavior and we identify Drosophila aggression as a model behavior relevant for the study of the mechanisms that are impaired in neuropsychiatric disorders.

Differential control and function of Arabidopsis ProDH1 and ProDH2 genes on infection with biotrophic and necrotrophic pathogens.

Arabidopsis contains two proline dehydrogenase (ProDH) genes, ProDH1 and ProDH2, encoding for homologous and functional isoenzymes. Although ProDH1 has been studied extensively, especially under abiotic stress, ProDH2 has only started to be analysed in recent years. These genes display distinctive expression patterns and show weak transcriptional co-regulation, but are both activated in pathogen-infected tissues. We have demonstrated previously that Arabidopsis plants with silenced ProDH1/2 expression fail to trigger defences against the hemibiotrophic bacterial pathogen Pseudomonas syringae pv. tomato AvrRpm1 (Pst-AvrRpm1), and that ProDH1 and ProDH2 are differentially regulated by salicylic acid (SA). In the current work, we used prodh1 and prodh2 single-mutant plants to assess the particular contribution of each gene to resistance against Pst-AvrRpm1 and the necrotrophic fungal pathogen Botrytis cinerea. In addition, we studied the sensitivity of ProDH1 and ProDH2 to the jasmonic acid (JA) defence pathway. We found that ProDH1 and ProDH2 are both necessary to achieve maximum resistance against Pst-AvrRpm1 and B. cinerea. However, ProDH2 has a major effect on early restriction of B. cinerea growth. Interestingly, ProDH1 is up-regulated by SA and JA, whereas ProDH2 is only activated by JA, and both genes display transcriptional inter-regulation at basal and infection conditions. These studies provide the first evidence of the contribution of ProDH2 to disease resistance, and describe the differential regulation and non-redundant but complementary function of both enzyme isoforms in infected tissues, providing support for a fundamental role of ProDH in the control of biotrophic and necrotrophic pathogens.

Evidence that COMT genotype and proline interact on negative-symptom outcomes in schizophrenia and bipolar disorder.

Elevated peripheral proline is associated with psychiatric disorders, and there is evidence that proline is a neuromodulator. The proline dehydrogenase (PRODH) gene, which encodes the enzyme that catalyzes proline catabolism, maps to human chromosome 22q11.2, a region conferring risk of schizophrenia. In the Prodh-null mouse, an interaction between elevated peripheral proline and another 22q11.2 gene, catechol-O-methyltransferase (COMT), on neurotransmission and behavior has been reported. We explored the relationship between fasting plasma proline levels and COMT Val158Met genotype on symptoms (positive, negative and total) in schizophrenia patients. In an exploratory study we also examined symptom change in patients with bipolar disorder. There was a significant interaction between peripheral proline and COMT on negative symptoms in schizophrenia (P<0.0001, n=95). In COMT Val/Val patients, high proline was associated with low Scale for the Assessment of Negative Symptom (SANS) scores. In contrast, high proline was associated with high SANS scores in patients carrying a Met allele. The relationship between proline and COMT also appears to modify negative symptoms across psychiatric illness. In bipolar disorder, a significant interaction was also observed on negative-symptom change (P=0.007, n=43). Negative symptoms are intractable and largely unaddressed by current medications. These data indicate a significant interaction between peripheral proline and COMT genotype, influencing negative symptoms in schizophrenia and bipolar disorder. That high proline has converse effects on symptoms by COMT genotype, may have implications for therapeutic decisions.

Proteomic and functional analysis of proline dehydrogenase 1 link proline catabolism to mitochondrial electron transport in Arabidopsis thaliana.

Proline accumulates in many plant species in response to environmental stresses. Upon relief from stress, proline is rapidly oxidized in mitochondria by proline dehydrogenase (ProDH) and then by pyrroline-5-carboxylate dehydrogenase (P5CDH). Two ProDH genes have been identified in the genome of the model plant Arabidopsis thaliana To gain a better understanding of ProDH1 functions in mitochondria, proteomic analysis was performed. ProDH1 polypeptides were identified in Arabidopsis mitochondria by immunoblotting gels after 2D blue native (BN)-SDS/PAGE, probing them with an anti-ProDH antibody and analysing protein spots by MS. The 2D gels showed that ProDH1 forms part of a low-molecular-mass (70-140kDa) complex in the mitochondrial membrane. To evaluate the contribution of each isoform to proline oxidation, mitochondria were isolated from wild-type (WT) and prodh1, prodh2, prodh1prodh2 and p5cdh mutants. ProDH activity was high for genotypes in which ProDH, most likely ProDH1, was strongly induced by proline. Respiratory measurements indicate that ProDH1 has a role in oxidizing excess proline and transferring electrons to the respiratory chain.

The 22q11 PRODH/DGCR6 deletion is frequent in hyperprolinemic subjects but is not a strong risk factor for ASD.

The proline dehydrogenase (PRODH) gene maps to 22q11.2 in the region deleted in the velo-cardio-facial syndrome (VCFS). A moderate to severe reduction (>50%) in PRODH activity resulting from recessive deletions and/or missense mutations has been shown to cause type 1 hyperprolinemia (HPI). Autistic features have been reported as a common clinical manifestation of HPI. Here we studied the frequency of a recurrent small 22q11.2 deletion encompassing PRODH and the neighboring DGCR6 gene in three case-control studies, one comprising HPI patients (n = 83), and the other two comprising autism spectrum disorder (ASD) patients (total of n = 2800), analyzed with high-resolution microarrays. We found that the PRODH deletion is a strong risk factor for HPI (OR = 50.7; 95%CI = 7.5-2147) but not for ASD (P = 0.4, OR = 0.6-3.3). This result indicates either that the suggested association between ASD and HPI is spurious and results from a bias leading to the preferential inclusion of patients with autistic features in HPI series, or that HPI is present in only a very small subset of ASD patients. In this latter case, a very large sample size would be required to detect an association between the PRODH deletion and ASD in a case-control study.