Allelic heterogeneity at the serotonin transporter locus (SLC6A4) confers susceptibility to autism and rigid-compulsive behaviors

Abstract

Autism is a spectrum of neurodevelopmental disorders with a primarily genetic etiology exhibiting deficits in (1) development of language and (2) social relationships and (3) patterns of repetitive, restricted behaviors or interests and resistance to change. Elevated platelet serotonin (5-HT) in 20%-25% of cases and efficacy of selective 5-HT reuptake inhibitors (SSRIs) in treating anxiety, depression, and repetitive behaviors points to the 5-HT transporter (5-HTT; SERT) as a strong candidate gene. Association studies involving the functional insertion/deletion polymorphism in the promoter (5-HTTLPR) and a polymorphism in intron 2 are inconclusive, possibly because of phenotypic heterogeneity. Nonetheless, mounting evidence for genetic linkage of autism to the chromosome 17q11.2 region that harbors the SERT locus (SLC6A4) supports a genetic effect at or near this gene. We confirm recent reports of sex-biased genetic effects in 17q by showing highly significant linkage driven by families with only affected males. Association with common alleles fails to explain observed linkage; therefore, we hypothesized that preferential transmission of multiple alleles does explain it. From 120 families, most contributing to linkage at 17q11.2, we found four coding substitutions at highly conserved positions and 15 other variants in 5' noncoding and other intronic regions transmitted in families exhibiting increased rigid-compulsive behaviors. In the aggregate, these variants show significant linkage to and association with autism. Our data provide strong support for a collection of multiple, often rare, alleles at SLC6A4 as imposing risk of autism.

Figure 1

Male-biased linkage of autism and novel coding variants at the 17q11.2 SLC6A4 locus. A, Male-biased linkage of autism to 17q11.2. Multipoint linkage analysis on chromosome 17 is shown for the overall 341-family data set (black line), 202 MO families (blue line), or the remaining 138 FC families (red line). HLOD scores were calculated under a recessive model and were plotted as a function of marker position in centimorgans (cM) along chromosome 17. B–D, Sequence detection of novel nonsynonymous SLC6A4 variants in families with autism. Sequence-based detection is shown for each of the three novel coding variants, with corresponding pedigrees. B, Ile425Leu. C, Phe465Leu. D, Leu550Val. Blackened circles or squares reflect individuals with an autism diagnosis, unblackened circles or squares reflect individuals without autism, and allele carriers without autism are indicated by small blackened circles or squares within the larger pedigree symbol. Electropherogram data is shown in either sense (B) or antisense (A and C) orientations, with corresponding coding sequence. Antisense sequences (A and C) indicate the reversed orientation of amino acid codons, represented by lines across each three-base sequence. Variant amino acids are shown in red, and corresponding heterozygous sequence changes are indicated by an arrow. Individual numbers in the respective pedigrees correspond to numbers within each of the sequence frames. E, Schematic representation of the 5-HT transporter. Amino acid substitutions are indicated by location within transmembrane or cytoplasmic domains. F, Dosage-dependent elevated 5-HT transporter activity of Ala56-encoded hSERT in native lymphoblastoid cells. Lymphocytes genotype-matched at 5-HTTLPR (L/L) and the intron 2 VNTR (10/10) and bearing Gly/Gly–, Gly/Ala–, or Ala/Ala–encoding genotypes at residue 56 were assayed for [³H]5-HT transport activity, as described (see the “Subjects and Methods” section). Three independent experiments were performed in triplicate for each line, and the combined basal uptake data were plotted.

Figure 2

ClustalW alignment of hSERT amino acid sequence versus other species of SERT and other biogenic amine transporters. Gly56 is conserved in mammalian species of SERT (A). Ile425 is conserved in all SERT proteins from human to Drosophila (B). Phe465 is conserved in all species shown for all monoamine transporters and is also conserved in glycine, γ-aminobutyric acid, and creatine transporters (C). Leu550 is conserved in all monoamine transporters from human to Drosophila (D).

Figure 3

Altered basal 5-HT transport activity and regulation associated with the Gly56Ala variant. [³H]5-HT (5HT) and [³H]L-glutamate (Glu) transport activities were defined as described in the “Subjects and Methods” section. A, 5-HT transport activity of EBV-transformed lymphocyte cell lines by genotype (G=Gly56; A=Alanine56). B, L-glutamate transport activity of the lines tested in panel A. C, Normalized 5-HT transport activity, with an individual cell lines 5-HT transport activity divided by its L-glutamate transport activity. D, Average by genotype of the values shown in panel C. An asterisk (*) denotes P<.05 (student’s t test) compared with Gly56 cell lines.

Figure 4

SERT is refractory to regulation through PKG and p38 MAP kinase signaling pathways. A, SERT Ala56 lacks sensitivity to 8BrcGMP. Homozygous Ala56 cells (10⁶/tube) were preincubated for various lengths of time at 37°C, with 10 μM 8BrcGMP prior to [³H]5-HT transport assays. B, SERT Ala56 lacks sensitivity to the p38 MAPK activator anisomycin; 8BrcGMP effects on Gly56 SERT are completely blocked by coincubation with the PKG inhibitor H8 (10 μM), whereas the p38 MAPK inhibitor SB203580 (1 μM) prevented anisomycin stimulation (data not shown). Data plotted represent mean data ± SD (n=3) for a single cell line of each genotype assayed in parallel. Findings were replicated with identical results in an additional line for each genotype.