L-histidine decarboxylase and Tourette's syndrome

Abstract

Tourette's syndrome is a common developmental neuropsychiatric disorder characterized by chronic motor and vocal tics. Despite a strong genetic contribution, inheritance is complex, and risk alleles have proven difficult to identify. Here, we describe an analysis of linkage in a two-generation pedigree leading to the identification of a rare functional mutation in the HDC gene encoding L-histidine decarboxylase, the rate-limiting enzyme in histamine biosynthesis. Our findings, together with previously published data from model systems, point to a role for histaminergic neurotransmission in the mechanism and modulation of Tourette's syndrome and tics.

Figure 1. The Two-Generation Pedigree and Results of Haplotype Analysis

Squares indicate male family members; circles, female family members; solid symbols, persons with Tourette's syndrome; the open circle, the unaffected mother; and gray triangles, miscarriages. The haplotypes illustrated below the pedigree symbols correspond to the 8.13-Mb segment that cosegregates with Tourette's syndrome at 15q21.1–15q21.3. The short-tandem-repeat markers used to confirm linkage are listed to the left, with the corresponding genotypes at these markers represented as numerals 1 through 4 for each chromosome of each proband. The haplotype that segregates with Tourette's syndrome is indicated by the dark blue bar and is bounded by markers D15S126 and GATA153F11. Detailed clinical information is presented in the Supplementary Appendix.

Figure 2. Mutation in HDC within the Lod – 2 Linkage Interval

Panel A shows the results of the genomewide parametric linkage analysis, expressed as lod scores. The maximum theoretical lod score for this family is 2.1, as indicated by the horizontal dashed line. The vertical dashed lines show the boundaries of each chromosome. Genotyping was performed on the GeneChip Human Mapping 100K Array (Affymetrix). The analysis modeled dominant inheritance of a rare allele (allele frequency, 0.001); a rate of phenocopy of 1% (i.e., 1% of affected persons may not carry the genetic risk factor), which is equal to the population prevalence of Tourette's syndrome; and 99% penetrance. Panel B shows the lod scores for chromosome 15q; the horizontal dashed line indicates the maximum theoretical lod score for this family (2.1). Panel C shows the sequence chromatograms at the nucleotide position 951 of the HDC transcript. The affected father and an affected child have a G-to-A substitution (indicated by red arrows), which is predicted to result in a change from the wild-type tryptophan to a stop codon at amino acid 317 (W317X). The sequence of the mother, who is unaffected, is the wild-type sequence. Resequencing of both DNA strands, as well as a reverse-transcriptase–polymerase-chain-reaction assay of messenger RNA, revealed this variation in all the affected family members.

Figure 3. Results of Functional Analysis of Mutant HDC (l-Histidine Decarboxylase) Activity

The wild-type HDC protein (Panel A, top), 74 kD (662 amino acids) in size, contains a pyridoxal 5′-phosphate (PLP) domain at the N-terminal end (NH₂), two PEST domains (PEST1 and PEST2), and a C-terminal (COOH) intracellular targeting domain (ER2). The mutant HDC (Panel A, bottom) is predicted to be truncated at the site of the W317X mutation (red diamond), resulting in a 35-kD product (316 amino acids) lacking a portion of the PLP domain, one of two PEST domains, and the entire ER2 domain. The wild-type 54-kD isoform (representing the active, processed form of HDC) and the truncated mutant HDC (35-kD) were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. The resulting proteins were largely insoluble, primarily found in inclusion bodies, and therefore were isolated under denaturing conditions and refolded. These isolated proteins were analyzed with the use of sodium dodecyl sulfate–polyacrylamide-gel electrophoresis (SDS-PAGE) and Coomassie staining. Both proteins were the expected size (after accounting for the size of the glutathione S-transferase tag) (Panel B). A fluorescence-quenching–based assay was then performed to assess HDC activity. The turnover of histidine to histamine was measured by means of displacement of the fluorescent dye (2-aminoethyl)sulfonamide (Dapoxyl), which serves as a histamine substitute. A steady decline in fluorescence was seen with the wild-type HDC, reflecting enzymatic activity, whereas no activity was observed in the W317X mutant HDC (Panel C). Expression of HDC proteins generated by in vitro translation (Panel D) showed that the wild-type HDC and W317X mutant HDC were translated separately as well as cotranslated. Biotinylated transfer RNA was added to the reaction to allow for visualization of the resulting proteins by means of SDS-PAGE and streptavidin–horseradish peroxidase. The wild type and the W317X mutant were the expected 54-kD and 35-kD sizes, respectively. Evaluation of dominant negative activity by means of liquid chromatography–mass spectrometry was performed on the cotranslated protein products (Panel E). The amount of wild-type DNA template used in the in vitro translation is listed in the first column and the amount of W317X mutant DNA template added to the cotranslation is listed in the second. The resulting ratio of the amounts of W317X mutant DNA to wild-type DNA is noted in the third column. After the reactions were incubated with l-histidine, the resulting mean (±SD) amount of histamine produced was measured by means of liquid chromatography–mass spectrometry. The dose–response curve for HDC activity (Panel F) shows the percent activity, as calculated by setting to 100% the amount of histamine produced with wild-type HDC DNA template only (23,251 fmol per microliter). As more W317X mutant DNA template was added, a decrease in overall activity was noted, according to the amount of W317X mutant added to a 0.5-g sample of wild-type HDC. The red square indicates a sample of W317X mutant only. The data are the means of three technical replicates of a single cotranslation experiment (see the Supplementary Appendix). The I bars indicate standard deviations.