Identification of DNA variants in the SNAP-25 gene and linkage study of these polymorphisms and attention-deficit hyperactivity disorder

The gene for the synaptic vesicle docking fusion protein, synaptosomal-associated protein of 25 kDa (SNAP-25), has been implicated in the etiology of attention-deficit hyperactivity disorder (ADHD) based on the mouse mutant strain coloboma. This neutron-irradiation induced mouse strain is hemizygous for the deletion of the SNAP-25 gene and displays spontaneous hyperactivity that is responsive to dextroamphetamine. Because of these characteristics, this strain has been suggested to be a mouse model for ADHD. We identified using single stranded conformational polymorphism analysis (SSCP) four DNA sequence variants in the 3' untranslated region of the human SNAP-25 gene. We searched for polymorphisms in the 3' untranslated region because the intron/exon structure of this gene has not yet been determined. We tested for linkage of this gene and ADHD using two of the identified polymorphisms that change a restriction enzyme recognition site. We examined the transmission of the alleles of each of these polymorphisms and the haplotypes of both polymorphisms using the transmission disequilibrium test in a sample of 97 small nuclear families consisting of a proband with ADHD, their parents, and affected siblings. We observed biased transmission of the haplotypes of the alleles of these two polymorphisms. Our findings are suggestive of a role of this gene in ADHD.     

Structure and expression of the mouse myelin proteolipid protein gene

The gene for the mouse myelin proteolipid protein has been isolated and the seven exons have been sequenced. Since the sequence of a rat proteolipid protein cDNA and partial sequence of the human proteolipid protein gene have been determined, it was possible to demonstrate a very high degree of conservation for the proteolipid protein gene exons among species. While there are some nucleotide changes, the protein coding region of the mouse gene encodes protein that is totally conserved relative to both rat and human proteolipid proteins. The regulatory and noncoding regions of the proteolipid protein gene are also highly conserved. The upstream regulatory and 5'-noncoding region of the gene is 92% homologous to the comparable region of the human proteolipid protein gene, and the 3'-noncoding region of the mouse gene is approximately 90% homologous to a rat proteolipid protein cDNA through 2,200 nucleotides of 3'-noncoding DNA. S1 nuclease protection experiments indicated that the major 5'-end for proteolipid protein mRNAs from mouse, rat, human, or baboon is approximately 147-160 nucleotides upstream from the initial methionine codon of the protein coding region. Other S1 nuclease protection experiments indicated the possible existence of an alternative splice site within exon 3, which may produce mRNA for DM20. This mRNA is approximately 100 nucleotides shorter than that for the proteolipid protein, and it is missing the latter half of exon 3, that is, amino acids 116-150 of the proteolipid protein sequence.     

Identification and analysis of new sequence variants in the human tryptophan hydroxylase (TpH) gene

The tryptophan hydroxylase (TpH) gene codes for the rate-limiting enzyme in serotonin biosynthesis. It is one of the major candidate genes for psychiatric and behavioral disorders. A polymorphism in TpH intron 7 has been shown to be associated with suicidal attempts, aggressive behavior and psychiatric illnesses. By systematically screening the TpH genomic sequence, we identified and confirmed an earlier report of four variants in the promoter region and localized six new sequence variants, ie two in intron 1b, one in exon 1c, one in intron 8, one in intron 9 and a microsatellite in the 3' region, 5687 bp downstream of the last exon 11. We analyzed these polymorphisms, as well as the one in intron 7, by Single Strand Conformation Analysis, microsatellite or restriction analysis in a collection of 175 West European Caucasian healthy subjects. The four variants in the promoter region are in complete linkage disequilibrium (frequencies of G-T-G-T and T-C-A-G haplotypes are 0. 41 and 0.59, respectively). Deletion of GTT in intron 1b is rare (0. 7%) and so not informative. The rarer allele T of intron 1b polymorphism T3792A has a frequency of 0.34 and is in partial linkage disequilibrium with the more common alleles of intron 7, 8 and 9. The polymorphisms of these three introns are in complete linkage disequilibrium and the frequencies of haplotypes A-T-C and C-C-T are 0.36 and 0.64 respectively. We detected 10 different alleles in the microsatellite localized in the 3' region; allele '194' is in partial linkage disequilibrium with haplotype A-T-C of introns 7, 8, and 9. Analysis of these different polymorphisms will constitute an important tool for future studies between the TpH gene and psychiatric disorders. Molecular Psychiatry (2000) 5, 49-55.     

Characterization of an intronic enhancer that regulates myelin proteolipid protein (Plp) gene expression in oligodendrocytes

The myelin proteolipid protein (Plp) gene is expressed in oligodendrocytes and encodes the most abundant protein (approximately 50%) present in mature myelin from the central nervous system (CNS). Plp gene activity is low to nonexistent early in development but sharply increases, concurrently with the active myelination period of CNS development. Work from our laboratory suggests that the temporal regulation of Plp gene expression in mice is mediated by a positive regulatory element located within Plp intron 1 DNA. We have termed this regulatory element/region ASE (for antisilencer/enhancer). The ASE is situated approximately 1 kb downstream of exon 1 DNA and encompasses nearly 100 bp. To understand the mechanisms by which the ASE augments Plp gene expression in oligodendrocytes, Plp-lacZ constructs were generated and transfected into a mouse oligodendroglial cell line (N20.1). Results presented here demonstrate that upstream regulatory elements in the Plp promoter/5'-flanking DNA are not required for ASE activity; the ASE worked perfectly well when the thymidine kinase (TK) promoter was substituted for the Plp promoter. However, the relative location of the ASE appears to be important. When placed upstream of 2.4 kb of Plp 5'-flanking DNA, or downstream of the lacZ expression cassette, the ASE was no longer effective. Thus, the ASE might have to be in the context of the intron in order to function. To begin to identify the crucial nucleotides within the ASE, orthologous sequences from rat, human, cow, and pig Plp genes were swapped for the mouse sequence. Results presented here demonstrate that the orthologous sequence from rat can substitute for the mouse ASE, unlike those from human, cow, or pig.