Sequence analysis of the human genome: implications for the understanding of nervous system function and disease.

The recent publication of the sequence of the human genome will accelerate the discovery of new genetic susceptibility factors for human disease, leading to the development of novel diagnostics and therapeutics. The exhaustive analysis of the human genome sequence will be the focus of the biomedical research community for many years to come. In particular, comparative analysis of the available eukaryotic genome sequences is an important approach to further our understanding of gene structure, function, and evolution. Our initial analysis of the human genome sequence has revealed many interesting features that are relevant to nervous system function, evolution, and disease. We analyzed the prominent features of predicted human proteins involved in neuronal function and prepared a comparative analysis of 146 human genes that have alleles (or mutations) conferring susceptibility for 168 neurologic diseases.

Genetic diversity of the human serotonin receptor 1B (HTR1B) gene.

We systematically and comprehensively investigated polymorphisms of the HTR1B gene as well as their linkage disequilibrium and ancestral relationships. We have detected the following polymorphisms in our sample via denaturing gradient gel electrophoresis, database comparisons, and/or previously published assays: G-511T, T-261G, -182INS/DEL-181, A-161T, C129T, T371G, T655C, C705T, G861C, A1099G, G1120A, and A1180G. The results of the intermarker analyses showed strong linkage disequilibrium between the C129T and the G861C polymorphisms and revealed four common haplotypes: ancestral (via chimpanzee comparisons), 129T/861C, -161T, and -182DEL-181. The results of association tests with schizophrenia were negative, although A-161T had a nominal P = 0.04 via ASPEX/sib_tdt. The expressed missense substitutions, Phe124Cys, Phe219Leu, Ile367Val, and Glu374Lys, could potentially affect ligand binding or interaction with G proteins and thus modify drug response in carriers of these variants. On average, the human cSNPs and differences among other primates clustered in the more thermodynamically unstable regions of the mRNA, which suggests that the evolutionary survival of nucleotide sequence variation may be influenced by the mRNA structure of this gene.

A chemosensory gene family encoding candidate gustatory and olfactory receptors in Drosophila.

A novel family of candidate gustatory receptors (GRs) was recently identified in searches of the Drosophila genome. We have performed in situ hybridization and transgene experiments that reveal expression of these genes in both gustatory and olfactory neurons in adult flies and larvae. This gene family is likely to encode both odorant and taste receptors. We have visualized the projections of chemosensory neurons in the larval brain and observe that neurons expressing different GRs project to discrete loci in the antennal lobe and subesophageal ganglion. These data provide insight into the diversity of chemosensory recognition and an initial view of the representation of gustatory information in the fly brain.

Neurochemical individuality: genetic diversity among human dopamine and serotonin receptors and transporters.

Behavioral variation in human beings encompasses wide differences in personality and susceptibility to psychiatric illness arising from both genotype and experience. Long-lasting behavioral differences generally have heritabilities of 30% or more, and such inheritance is ultimately attributable to functional variants of genes programming brain development and function. The sequencing of the human genome is revealing a pattern of gene sequence variation. The ability of sequence variants to affect neural function either alone or in concert may reveal effects of behavioral selection on the human genome over evolutionary time frames. Dopamine and serotonin are phylogenetically ancient neurotransmitters intrinsic to brain function and behavior. Dopamine and serotonin receptor and transporter genes have been an early focus for efforts to identify and functionally characterize sequence variation. The purpose of this article is to present a preview of a developing new perspective in human behavior: the genetic variation of the brain or neurochemical individuality. Arch Gen Psychiatry. 2000;57:1105-1114.

Comparative genomics of the eukaryotes.

A comparative analysis of the genomes of Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae-and the proteins they are predicted to encode-was undertaken in the context of cellular, developmental, and evolutionary processes. The nonredundant protein sets of flies and worms are similar in size and are only twice that of yeast, but different gene families are expanded in each genome, and the multidomain proteins and signaling pathways of the fly and worm are far more complex than those of yeast. The fly has orthologs to 177 of the 289 human disease genes examined and provides the foundation for rapid analysis of some of the basic processes involved in human disease.

Functional analysis of the human D5 dopamine receptor missense and nonsense variants: differences in dopamine binding affinities.

The functional analysis of expressed human gene variants is important in the study of genetic susceptibility to diseases, pharmacogenetic traits and for the investigation of the human genetic diversity at the molecular level. We have performed the analysis of sequence polymorphisms in the human D5 dopamine receptor gene (DRD5) predicting missense and nonsense amino acid changes in the receptor protein. The amino acid substitutions in the human D5 dopamine receptor are: Leu88 to Phe in the putative second transmembrane domain, Ala269 to Val in the third intracellular and Pro330 to Gln in the third extracellular loops, Asn351 to Asp in the seventh transmembrane and Ser453 to Cys in the C-terminal domains and Cys335 to Stop in the third extracellular loop. The two amino acid substitutions in the transmembrane domains had an effect on agonist binding to the human D5 dopamine receptor. Asn351 to Asp resulted in an approximately 10-fold decrease in dopamine and threefold decrease in R(+)-SKF-38393 binding affinities. Leu88 to Phe resulted in a small increase in dopamine binding affinity. Antagonist binding affinities were mostly unaffected by the amino acid substitutions with the exception of Leu88 to Phe, which showed small reductions in binding affinities of SCH-23390 and risperidone. The existence of functionally different variants of the human dopamine receptors might have phenotypic consequences given their importance in central nervous system physiology and pharmacology.

Analysis of neuroleptic binding affinities and potencies for the different human D2 dopamine receptor missense variants.

Neuroleptics, or antipsychotics, are widely used for the treatment of psychotic symptoms such as hallucinations and delusions in schizophrenia and other psychiatric disorders. Pharmacotherapy of these diseases is frequently complicated by a great variability in the clinical response to neuroleptics and by the development of serious and potentially life-threatening side-effects. Brain D2 dopamine receptors are one of the major targets of neuroleptic treatment. The human D2 dopamine receptor (DRD2) gene has three variants predicting the amino acid substitutions Ser311Cys, Pro310Ser and Val96Ala in the receptor protein. We show that several typical and atypical neuroleptics commonly used in the treatment of psychotic disorders have differences in binding affinities and potencies for the D2 dopamine receptor variants. Functional differences between dopamine receptor variants might be related to genetically determined differences in response to neuroleptic treatment.

Closing in on genes for manic-depressive illness and schizophrenia.

Advances in the human genetic map, and in genetic analysis of linkage and association in complex inheritance traits, have led to genetic progress in the major psychoses. For chromosome 6 in schizophrenia, and chromosomes 18 and 21 in manic-depressive illness, there are reports of linkage in several independent data sets. These are small effect genes, best detected with affected-relative-pair linkage methods. Association with candidate genes is an alternative strategy to uncovering susceptibility genes for these illnesses, but convincing associations remain to be demonstrated. New clinical and laboratory investigation methods are being developed. Testing every gene in the human genome for association with illness has recently been proposed (Risch and Merikangas 1996). This would require further progress in characterizing the genome and in automated large-scale genotyping. The best type of pedigree sampling for common disease studies, whether for linkage or association, is not yet established. An endophenotype hybrid strategy can combine genetic linkage, association, and pathophysiologic studies. As clinical molecular investigation methods advance, identification of disease susceptibility mutations and delineation of their pathophysiological roles may be expected.

Suggestive evidence for a schizophrenia susceptibility locus on chromosome 6q and a confirmation in an independent series of pedigrees.

We have investigated whether there is a locus on chromosome 6 that confers an increased susceptibility to schizophrenia using a two-stage approach and nonparametric linkage analysis. Allele sharing identical by descent (IBD) and multipoint maximum likelihood score (MLS) statistics were employed. Results from two tested data sets, a first data set, or genome scanning data set, and a second replication data set, show excess allele sharing for multiple markers in 6q, a chromosomal region not previously reported as linked to schizophrenia. In our genome scanning data set, excess allele sharing was found for markers on 6q13-q26. The greatest allele sharing was at interval 6q21-q22.3 at marker D6S416 (IBD percentage 69; P = 0.00024). The multipoint MLS values were greater than 2.4 in the 11.4-cM interval delimited by D6S301 and D6S303, with a maximum value of 3.06 close to D6S278 and of 3.05 at D6S454/D6S423. We did not confirm, however, the previously described linkage in 6p, when tested in the systematic genome scanning data set. The replication data set also showed excess allele sharing in chromosomal area 6q13-q26, which overlapped with the aforementioned positive linkage area of the genome scanning data set. The highest sharing of the second data set was at D6S424 (IBD percentage 64; P = 0.0004), D6S283 (IBD percentage 62; P = 0.0009), and D6S423 (IBD percentage 63; P = 0.0009). Multipoint MLS analysis yielded MLS values greater than 1 in an area of about 35 cM, which overlaps with the MLS multipoint area of linkage from the genome scanning data set. The multipoint MLS at the D6S454/D6S423 locus was 2.05. In the second data set, the maximum multipoint MLS was located about 10 cM centromeric from the maximum of the genome scanning data set, at the interval D6S424-D6S275 (2.35). Our results provide very suggestive evidence for a susceptibility locus for schizophrenia in chromosome 6q from two independent data sets.

Antagonist binding characteristics of the Ser311-->Cys variant of human dopamine D2 receptor in vivo and in vitro.

We report in vivo and in vitro antagonist binding characteristics of the naturally occurring Ser311-->Cys variant of the human D2 dopamine receptor. Striatal receptor binding characteristics in vivo were measured with positron emission tomography and the D2 antagonist [11C]raclopride. The in vitro affinity of raclopride for the Ser311-->Cys variant and the wild type receptor was studied in membrane binding assays from stably transfected cell lines. One healthy male carrying the heterozygous Ser311-->Cys (TCC-->TGC) substitution was identified with denaturing gradient gel electrophoresis and DNA sequencing. The striatal D2 receptor binding characteristics in vivo in this subject were normal. This was supported by the in vitro data as the Ki values of raclopride for the Ser311-->Cys variant and the wild type receptor were identical. Our data suggest that the Ser311-->Cys variant of the human D2 receptor does not influence antagonist-receptor recognition in vivo or in vitro.

No abnormality in the gene for the G protein stimulatory alpha subunit in patients with bipolar disorder.

BACKGROUND: The available evidence for an involvement of the heterotrimeric guanine-nucleotide-binding proteins (G proteins) in bipolar disorder relies primarily on the effects of lithium salts on G protein function and on alterations in the concentration or function of G proteins (most notably Gs-alpha) in peripheral leukocytes and in postmortem tissues of patients with bipolar disorder. METHODS: The hypothesis that a mutation in Gs-alpha gene confers an increased susceptibility to bipolar disorder was tested by the following strategies: (1) mutational screening of the Gs-alpha subunit gene coding sequences and promoter sequences by denaturing gradient gel electrophoresis in unrelated individuals with bipolar disorder and (2) association and linkage analyses with a common silent exonic polymorphism, using genetic allelic information from American families with at least 1 affected child. For association analysis, the transmission test for linkage disequilibrium was used; for linkage analysis, nonparametric methods were used. RESULTS: No structural or regulatory mutations in this gene were found in bipolar disorder; the results of association and genetic linkage were negative. CONCLUSION: Our results do not support the speculation that the Gs-alpha protein gene has a role in the genetic predisposition to bipolar disorder.

Functional analysis of the human D2 dopamine receptor missense variants.

The human dopamine D2 receptor gene (DRD2) has three polymorphic variants that predict the amino acid substitutions Val96 --> Ala, Pro310 --> Ser, and Ser311 --> Cys in the receptor protein. We have investigated the ligand binding and signal transduction properties of these human D2 receptor variants by stably expressing them in cultured mammalian cells. The Cys311 and Ser310 variants of the human D2 receptor, which involve substitutions located in the third cytoplasmic loop, were markedly less effective in inhibiting cAMP synthesis than the most prevalent form (Pro310, Ser311). Despite this difference, the Cys311 and Ser310 variants couple to G proteins in CHO-K1 (Chinese hamster ovary) cells. The impairment of the Cys311 and Ser310 variants to inhibit cAMP levels thus appears to result from a reduced ability of those variant receptors to activate the appropriate Gi-like protein. The demonstration of substantial functional differences between DRD2 gene variants found in the human population might have important pharmacological implications given the widespread use of D2 receptor blocking drugs in the treatment of schizophrenia and other psychiatric disorders.

No association detected between a D3 receptor gene-expressed variant and schizophrenia.

A missense polymorphism (glycine to serine) in the first exon of the dopamine D3 (DRD3) gene was examined in the sib-pairs schizophrenia collection by the transmission test for linkage disequilibrium (TDT). No association due to linkage disequilibrium was detected using TDT. Additionally, no evidence for excess homozygosity was found.

Identification of a novel microtubule-binding domain in microtubule-associated protein 1A (MAP1A).

Several microtubule-associated proteins (MAPs) have been shown to bind to microtubules via short sequences with repeated amino acids motifs. A microtubule-binding domain has hitherto not been defined for the adult brain microtubule-associated protein 1A (MAP1A). We have searched for a microtubule-binding domain by expressing different protein regions of MAP1A in cultured cell lines using cDNA constructs. One construct included an area with homology to the microtubule-binding domain of MAP1B (Noble et al. (1989) J. Cell Biol. 109, 437-448), but this did not bind to microtubules in transfected cells. Further investigation of other areas of MAP1A revealed a protein domain, capable of autonomously binding to microtubules, which bears no homology to any previously described microtubule-binding sequence. This MAP1A domain is rich in charged amino acids, as are other mammalian microtubule-binding domains, but unlike them has no identifiable sequence repeats. Whereas all previously described mammalian microtubule-binding domains are basic, this novel microtubule-binding domain of MAP1A is acidic. The expression of this polypeptide in cultured cell lines led to a rearrangement of the microtubules in a pattern distinct from that produced by MAP2 or tau, and increased their resistance to treatment with the microtubule depolymerising agent nocodazole. When the MAP1A microtubule-binding domain was co-expressed in cultured cell lines together with MAP2c, the MAP1A microtubule-binding domain was able to bind to the MAP2c-induced microtubule bundles. These results suggest that different microtubule-binding sequences have a common ability to stabilise microtubules but differ in their influence on microtubule arrangement in the cell. This may be significant in neurons, where microtubule-associated proteins with different microtubule-binding sequences are expressed in different cell compartments and at different times during development.

A novel strategy for the immunological tagging of cDNA constructs.

We describe the construction of pBact-myc, an expression vector that incorporates an immunological 'tag' into the produced polypeptide. When transfected into recipient cell lines, tagged protein fragments derived from any source can be visualised using a single monoclonal antibody (mAb). The neuronal-associated protein 2c (MAP2c) was tagged with a sequence encoding a peptide from the human c-myc gene. The preservation of normal function of the tagged protein was shown by transfecting it into cultured cell lines. No difference in binding ability to cellular microtubules could be observed between the myc-tagged MAP2c and the wild-type forms, and both produced the same characteristic changes in microtubule organisation. This approach is being used to study the biological function of selected fragments of MAP2c and other MAP-encoding genes. The pBact-myc expression vector represents a fast and convenient way to produce tagged polypeptides of selected sequences encoding whole proteins or fragments, for the analysis of their function in living cells.

Responses elicited by species-specific models in the cichlid Crenicichla lepidota (Heckel).

Species recognition by the cichlid fish C. lepidota was studied by measuring species-specific aggressive behavior toward either live conspecifics (control) or toward different two-dimensional models. A plain fish-shaped model elicited aggressive behavior, but the responses were mostly absent when the model was a rectangle. Adding heavy stripes to the fish-shaped form increased the responses, which were further enhanced by adding a spot. Comparable aggressive responses were also induced by adding spots and eye to dummies with thin stripes. We conclude that in our experimental conditions, fish-like shape and contrast seem to be of great significance for recognition of conspecifics in the cichlid C. lepidota (Heckel).