¹Dept of Child Psychiatry, Yale University School of Medicine, New Haven, CT, USA

²FRE 2134 CNRS Genetics, Neurogenetics, Behavior, 3 B Férollerie Street, Orléans cedex 2, France

³Laboratoire d'Hormonologie et de Biologie Moléculaire, CHU de Bicêtre, France

⁴UFR Medecine ifr 53 pôle Biomolecule, Equipe Physiopathologie Dysimmunitaire Humaine, EA 3309, URCA, Reims, France

⁵Fondation Vallée, University Paris-Sud, France

Correspondence to: G M Anderson, PhD, Yale Child Study Center, 230 S Frontage Rd, New Haven, CT 06510, USA. E-mail: george.anderson@yale.edu

The well-replicated platelet hyperserotonemia of autism has stimulated interest in serotonin (5-HT) in autism. We have examined the effects of the serotonin transporter gene (5-HTT, locus SLC6A4) promoter polymorphism (5-HTTLPR) on platelet 5-HT physiology in autism. Platelet 5-HT uptake rates and affinities (V_max and K_m), uptake site densities (B_max) and 5-HT levels were examined in 31 French individuals with autism genotyped with respect to the 5-HTTLPR. Platelet 5-HT uptake and 5-HT levels were measured using HPLC; uptake sites were determined by radioligand binding. A 1.5-fold increased rate (V_max) of platelet 5-HT uptake was observed in ll genotype individuals compared to those with ls and ss genotypes (Mann- Whitney U-test, P = 0.022). However, no significant relationship was observed between genotype and uptake site density (U-test, P = 0.51). Although median levels of platelet 5-HT in platelet-rich plasma were higher in the ll group, only trend level significance was observed (U-test, P= 0.069); platelet 5-HT content measured in whole blood was similar across genotypes. Uptake rates were well correlated with B_max values (r = 0.66, P = 0.002); correlations between uptake and platelet 5-HT levels and between B_max values and 5-HT levels were somewhat lower. While 5-HTTLPR alleles had an appreciable effect on platelet 5-HT uptake rates, effects on 5-HT levels and uptake site density were smaller or absent. Based on these preliminary data and prior studies of allele frequencies, we conclude that the 5-HTTLPR is not a major determinant of the group mean platelet serotonin elevation seen in autism. However, a role for increased uptake in the hyperserotonemia of autism can not be ruled out. In addition, it appears that studies of platelet 5-HT measures in autism and other disorders should take account of the effects of 5-HTTLPR genotype on 5-HT uptake

Molecular Psychiatry (2002) 7, 831-836. doi:10.1038/sj.mp.4001099

serotonin transporter; platelet 5-HT uptake; autism; platelet hyperserotonemia; 5-HTTLPR; SLC6A4 locus

Introduction

The well-replicated platelet hyperserotonemia of autism has focused attention on the possible role of the neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) in the disorder.^1,2,3,4 Observations of other 5-HT-related abnormalities in autism,^5,6 reported effects of selective serotonin reuptake inhibitors (SSRIs) on mood, obsessionality, and social relatedness in some individuals with autism,^7,8,9 and the critical role of 5-HT in neurodevelopment^10,11 have also served to increase interest in the possible involvement of 5-HT in the etiology and pathophysiology of autism.

Although a number of studies have attempted to elucidate the cause or causes of the group mean elevation seen for platelet 5-HT in autism, the mechanism remains unknown. It is likely that the alteration(s) responsible are intrinsic to the platelet, as the platelet does not appear to be exposed to increased levels of 5-HT.¹² The serotonin transporter is a critical component of the serotonergic system and it is the principal site of action of the widely prescribed SSRIs. The 5-HT transporter gene (5-HTT, locus SLC6A4) is of special interest given the nature of the biological findings and the reported effects of SSRIs on autistic symptoms. The reported linkage of an 5-HTT promoter polymorphism with risk to,^13,14 or modified expression of,¹⁵ autism has drawn further attention to the transporter.

The promoter variant in the transporter gene (the 5-HT transporter-linked polymorphic region, 5-HTTLPR) consists of a 44 base-pair deletion/insertion in a repeat region of the promoter. The deletion or short (s) allele occurs with a frequency of approximately 43%, while the long (l) form has an allele frequency of 57% in samples of predominately northern European ancestry.¹⁶ Initial studies of the functional effects of 5-HTTLPR genotype indicated that the deletion form (s) results in lower mRNA levels, reduced transporter protein expression, and decreased uptake rates in transfected and native lymphoblastoid cell lines.¹⁷ Individuals with ls and ss genotypes have been reported to have lower whole blood serotonin levels¹⁸ and reduced 5-HT transporter function in platelets¹⁹ relative to ll genotype individuals. In most, but not all, studies, the s allele has been dominant for reduced levels, expression, or function.

The 5-HTTLPR variant was genotyped in a group of 63 French families with an autistic child. Rates of platelet 5-HT uptake, uptake site density, and levels of platelet 5-HT were examined in a subgroup (n = 31) of the probands in order to assess the influence of transporter promoter genotype on platelet 5-HT physiology in autism. The subject number was determined by scheduling limitations arising from the need to use freshly drawn blood samples for uptake analyses. The major hypotheses were that the l allele would be associated with greater uptake rates and would contribute to elevated 5-HT levels in the autistic group. We have recently reported on allelic effects on the severity of autistic behavior in the larger group.¹⁵

Materials and methods

Patients

Children and adolescents with autism were recruited from child psychiatry day hospitals in Paris and Reims. The study was conducted on 31 individuals with autism (average age in years: 13.9 ± 4.9; 10 prepubertal, five pubertal, 16 postpubertal) and included 19 males (average age in years : 13.9 ± 5.2) and 12 females (average age in years : 13.8 ± 4.7). All subjects were healthy Caucasian individuals with no history of encephalopathy or neuroendocrinological disease. Six autistic subjects had a history of idiopathic epilepsy; all six were receiving anticonvulsant medication at the time of the blood draw. Two subjects were receiving neuroleptics, no other psychoactive medications were being used by any of the subjects. All autistic probands were cognitively impaired with a full-scale IQ range of 40-58. The protocol was approved by the ethics committee of Bicêtre Hospital; written informed consent was obtained before performing assessments or taking specimens.

Controls

Normal controls were recruited from the Reims Preventative Medical Center and were independently evaluated by two pediatricians. Based on clinical examination of the child and parent interview, they were determined to be free of psychopathology or any developmental or neurological disorder; none had a family history of autism and all were attending regular schools. The 77 control subjects had a mean (± SD) age of 12.7 ± 5.9 years (37 males, 40 females).

Diagnostic and clinical assessments

Based on direct clinical observation of the child by two independent child psychiatrists, the diagnosis of autism was made according to the criteria of DSM-IV,²⁰ ICD-10 and CFTMEA,²¹ and was confirmed by Autism Diagnostic Interview Revised (ADI-R²²) ratings. Cognitive functioning of autistic probands was assessed by two psychologists using the age-appropriate intelligence Weschler scales (WPPSI-R, WISC-R, WAIS-R) and the Kaufman K-ABC.²³ Stage of puberty was assessed by a pediatrician using the Tanner scale.²⁴

Biological measures

Genotyping was performed as previously described¹⁵ using PCR primers for the 5-HTT promoter polymorphic region (5-HTTLPR) specified by Gelernter et al¹⁶ Levels of platelet serotonin were determined in whole blood and platelet-rich plasma (PRP) by high performance liquid chromatography (HPLC) with a day-to-day coefficient of variation (CV) of 5.9%.²⁵ Rates of platelet 5-HT uptake were measured using a modification of a previously reported radioisotopic procedure.²⁶ Briefly, PRP was prepared by centrifugation at 100 g for 10 min, adjusted to a platelet count of 2 ´ 10⁸ ml^-1 with autologous plasma, and diluted 10-fold with uptake buffer. Uptake was assessed using the 5-HT analogue 4,6-difluoroserotonin (df-HT, kindly supplied by Kenneth Kirk of the National Institutes of Health) added in concentrations of 0.05-1.5 M.²⁷ Specific uptake was defined using 10 M fluoxetine. After a 2.5 min uptake period (37°C), samples were filtered and analyzed for df-HT and the internal standard, N-methylserotonin, using HPLC with electrochemical detection. Uptake was determined with a within-day CV of less than 10%; Pearson's r values from Eadie-Hofstee plots ranged from -0.85 to -0.99. Density (B_max) of the platelet transporter site in platelet membranes was assessed using [³H]-citalopram at a concentration of 7.5 nM, well above the reported K_d. Platelets were isolated and platelet membranes were prepared as previously described;²⁸ binding (B_max) values for a frozen pooled control sample were determined with a day-to-day CV of 11%. Only whole blood 5-HT content (ng ml^-1) was measured in the control group (n = 77).

Statistical analyses

Uptake rates were defined as the primary dependent measure. In nearly all tests of genotype effects the sl and ss groups were combined due to the small size of the ss group (n = 4) and the Mann-Whitney U-test used for group comparisons. Correlations were calculated using Pearson's r and were not corrected for the number of tests (n = 8). In screening analyses no effect of anticonvulsant (n = 6) or neuroleptic (n = 2) medication, or of pubertal status, was seen; thus all 31 subjects were examined in subsequent analyses. Two-tailed tests were used in all cases despite expectations of increased platelet uptake rates, binding site densities and 5-HT levels in the ll group. A three-way ANOVA was performed in the combined group of autistic and control individuals (n = 108) to examine the effects of group, gender and pubertal status on whole blood 5-HT.

Results

Uptake rates

Platelet 5-HT transport rates determined in 31 autistic patients are presented in Figure 1, with the uptake rates plotted according to 5-HTTLPR genotype. A significant effect of genotype was observed with higher platelet 5-HT uptake rates observed in the ll genotype group (n = 11) compared to the ls group (n = 16) and to the combined ls & ss groups ('S' group, n = 20) (Mann-Whitney U-test, P = 0.032 and 0.022, respectively). Median uptake affinities (K_m values) were similar in the three genotypes (0.64, 0.59, and 0.51 M, respectively; ll vs S, U-test, P = 0.72).

Citalopram binding

Binding site densities (transporter B_max values) did not differ significantly across genotypes, with median values of 306, 395, and 260 fmol 10^-9 platelets observed in the ll, ls, and ss groups (ll vs S, U-test, P = 0.51).

Platelet 5-HT content

Although median (1220, 848, and 984 ng 10^-9 plts) and mean (± SD) (1090 ± 378, 903 ± 336, and 834 ± 471 ng 10^-9 plts) levels of platelet-rich plasma (PRP) platelet 5-HT content were higher in ll individuals compared to ls and ss individuals, the difference was not statistically significant (ll vs S, U-test, P = 0.16). Platelet 5-HT content measured in whole blood (as opposed to PRP) was very similar across genotypes, whether expressed as ng ml^-1 or ng 10^-9 plts: eg, mean 5-HT ng ml^-1 values in the ll, ls and ss groups were 208 ± 109, 179 ± 77.5, and 186 ± 120, respectively (ll vs S, U-test, P = 0.58). A three-way ANOVA was performed in all subjects (n = 108) with group (autistic, control), gender, and pubertal status (prepubertal, pubertal, postpubertal) as factors and whole blood 5-HT as the dependent variable. Significant effects were seen for group (F(1,107) = 4.78, P = 0.031) and for puberty (F(2, 107) = 4.21, P = 0.018); group mean (± SD) 5-HT values of 190 ± 91.5 and 159 ± 57.3 were observed in the autistic and control groups, respectively.

Correlational and multiple linear regression analyses

Inter-relationships between uptake rates, binding site densities, and 5-HT content were examined in the autistic group. Uptake rates (V_max) and binding site densities (B_max) were highly correlated (r = 0.66, P = 0.002). A correlation of 0.55 (P = 0.001) was observed between uptake rate and PRP platelet 5-HT (ng 10^-9 plts) content (the corresponding correlation for uptake rate and whole blood 5-HT content expressed as ng ml^-1 was r = 0.37, P = 0.05). The correlation between binding site density and PRP 5-HT content was 0.47 (P = 0.038) (the corresponding correlation between sites and whole blood 5-HT content was r = 0.44, P = 0.061). The correlation between genotype and uptake was calculated to be r = 0.36, P = 0.044.

Stepwise multiple linear regression was performed in order to clarify the determinants of the dependent variable platelet 5-HT content (ng 10^-9 plts). When uptake rate was entered first, adding genotype produced little increase in r (from 0.546 to 0.552); however entering genotype first, then uptake, produced a substantial increase in r (0.279 vs 0.552). Similar order effects were observed when genotype and binding site density were entered as the indendent variables (binding site entered first, 0.468 vs 0.582; genotype first, 0.279 vs 0.582).

Discussion

The observation of higher rates of 5-HT uptake in platelets of individuals with the ll genotype is consistent with previous data regarding uptake in human lymphoblasts.¹⁷ However, the small size (n = 4) of the ss group does not permit definitive statements regarding the dominant or co-dominant nature of the l and s alleles. It does appear that, on the average, ll individuals have about 50% higher uptake rates than those in the combined ls and ss group. Genotype of the 5-HTTLPR explained about 13% of the group variance in uptake (r² = 0.132). Actual in vivo contribution of genotype to uptake variance may be substantially higher given unavoidable exogenous contributors (including pre-analytical and analytical variances) to the observed group variance.

Significant genotype group differences were not seen in binding site densities or in platelet 5-HT content. The absence of a genotype effect on binding sites is surprising, particularly when viewed alongside the observed effect of genotype on uptake. One might expect that expression of the transporter protein (presumably indexed by binding site density) would be even more highly influenced by the promoter polymorphism than the more multidetermined uptake measure. It can be suggested that difficulties and inaccuracies associated with assaying ligand binding sites may cause some of the apparent dissociation between genotype and binding site values. While unexpected, the data are congruent with the reports of Greenberg et al¹⁹ and Nobile et al²⁹ finding effects of 5-HTTLPR genotype on platelet uptake, but not on binding site density. The results are also consistent with studies demonstrating heritability in twin pairs for platelet 5-HT uptake, but not for uptake sites.^30,31 An imaging study has reported increased 5-HT transporter availability in the dorsal brain stem of ll individuals;³² while studies examining 5-HTTLPR effects on postmortem human brain transporter B_max values are contradictory, with one study finding increased densities in ll individuals³³ and the other reporting no effect of genotype.³⁴

Exploratory examination of inter-relationships between the biochemical measures indicated that about 40% of the variance (r² = 0.43) in uptake rate can be explained on the basis of uptake site density. Substantially less of the variance in platelet 5-HT content was explained on the basis of either uptake rate or site density. The order effects observed in the stepwise multiple linear regression analyses indicate that the effects of genotype were mediated through the highly correlated measures of uptake rate and binding site density.

Unfortunately, few of the studies studying serotonin-related measures in the platelet have examined more than one of the measures at a time. In studies of monkey platelets, Brammer et al³⁵ found a substantial correlation (r = 0.82) between platelet 5-HT content and uptake rate. Similarly, Cook and colleagues⁷ found a significant correlation (r = 0.56) between 5-HT content and uptake rate in relatives of autistic children, and Carrasco and colleagues³⁶ have found both content and uptake to be increased in pre-eclamptic women. In contrast, Stahl et al³⁷ found no correlation between 5-HT content and uptake rate in adult normal control subjects, and Raisman and colleagues³⁸ did not observe a correlation between uptake and binding. It is clear that changes in platelet 5-HT uptake rates (eg, during SSRI administration³⁹) or in the platelet's exposure to 5-HT (eg, as in carcinoid⁴⁰) can greatly affect observed platelet levels of 5-HT. The data presented here support the idea that 5-HTTLPR genotype, through its effects on transporter expression and uptake rate, is an important determinant of platelet 5-HT content. Taking 5-HTTLPR genotype into account should help clarify the factors governing human platelet 5-HT levels.⁴¹

Our finding of higher rates of platelet 5-HT uptake in autistic individuals with the ll genotype needs to be considered in the context of the long-standing observation of platelet hyperserotonemia in autism. If the 5-HTTLPR is actually a quantitative trait locus (QTL) for platelet 5-HT uptake, some of the elevation in platelet 5-HT levels usually seen in autism could be due to the higher l allele frequency that might occur in autism. It should be pointed out that though we¹⁵ and others¹⁴ have found a slight preference for l allele transmission and a slightly increased l allele frequency in autism, this is not a consistent finding.^13,42,43 Even if one: (1) accepts the reports of increased l allele frequency in autism of 63% vs 57% in the general population; (2) accepts a mean 2-fold increase in uptake rate in ll vs ls & ss individuals and; (3) assumes that differences in platelet 5-HT uptake are reflected directly in differences in 5-HT content, the promoter polymorphism would explain only about one quarter of the 25% group mean increase in platelet 5-HT content previously reported in autism.⁴ If one uses values derived from the present investigation (mean uptake increased 1.52-fold in the ll group, uptake rate explaining 30% of the variance in PRP platelet 5-HT content), less than one tenth of the reported platelet 5-HT elevation¾the platelet hyperserotonemia of autism¾could be explained by a possible group increase in l allele frequency from 57 to 63% in autism.¹⁵ Certainly, the promoter polymorphism appears to explain little of the group mean increase in whole blood 5-HT (of 20%) seen in the 31 autistic individuals examined as part of this study.

The l allele effect may be of greater relevance to understanding the group mean increase in platelet 5-HT observed in African-Americans.⁴ The significantly higher l allele frequency of 70% reported for African-Americans¹⁶ suggests that the QTL nature of the 5-HTTLPR may be contributing more substantially to the group mean increase in platelet 5-HT seen in that group. In this vein, if the s and l alleles are in fact associated with susceptibility or modified expression of autism, it is fascinating to consider the possible effects of the widely different l allele frequencies seen in different populations (ranging from 70% in African-Americans to 20% in Japanese¹⁶). However, it should be clearly stated that, to the extent that 5-HTTLPR alleles have been linked with autism (or its modified expression) or with platelet uptake rates, the results pertain only to individuals of European ancestry. Whether such relationships exist in other distinct populations is very much an open question.

In conclusion, understanding the relationship of the 5-HTTLPR alleles to susceptibility and expression of autism, determining the extent of their influence on 5-HT transport and 5-HT physiology, and ascertaining the significance of the substantial ethnic/racial differences in allele frequencies provide clear challenges for further research. Although substantial 5-HTTLPR allelic effects were observed on platelet 5-HT uptake rates, effects on uptake site density and 5-HT levels were considerably less. Taken together with data regarding 5-HTTLPR allele frequencies in autism, the data indicate that the 5-HTTLPR is not a major determinant of the group mean platelet 5-HT elevation seen in autism. Though the 5-HTTLPR probably does not contribute substantially to the elevation in platelet 5-HT, altered uptake may still play an important role. Conclusions derived from the study must be considered preliminary given the sample size, and the need for replication.

Finally, it appears that investigations of platelet 5-HT measures in autism and other neuropsychiatric disorders would benefit from taking into account the effects of 5-HTTLPR genotype on uptake when comparing different subjects and groups.

Acknowledgements

Portions of this paper were presented at the Society for Neuroscience Annual Meeting, Los Angeles, CA, USA, November 7-12, 1998. We thank Laura M Hall and David M Ocame for their expert technical assistance. We gratefully acknowledge the support of the Institut National de la Santé et de la Recherche Médicale (contrat ERCA, Equipe de Recherche Clinique Associée à l'INSERM), CNRS, The French Ministry for Research & Technology, the Fondation pour la Recherche Médicale, the Société d'Etudes et de Soins pour les Enfants Paralysés et Polymalformés; as well as the NIMH (MH30929), the NICHD (HD03008), and the Korczak Foundation for Autism Research.

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Figure 1 Platelet serotonin (5-HT) uptake in individuals with autism grouped according to 5-HT transporter promoter polymorphism genotype (5-HTTLPR long and short alleles, l and s). Median uptake rates in the three genotype groups (ll, ls, ss; n = 11, 16, 4, respectively) were 1.61, 1.14, and 0.81 nmol min^-1 10^-9 platelets, respectively. Calculated mean (± SD) rates were 1.93 ± 1.01, 1.30 ± 0.68, 1.16 ± 0.92 nmol min^-1 10^-9 platelets, respectively. The ll group values were significantly higher than those seen in the combined ll & ss (S) group (Mann-Whitney U-test, U = 2.79, P = 0.022).