In vitro transcription analysis of the Drosophila tropomyosin and other muscle genes.

Nuclear transcription extracts were prepared from embryos of Drosophila melanogaster to study the in vitro transcription of the tropomyosin genes. Several non-muscle gene promoters, including the non-muscle promoter of the Tropomyosin II gene, were shown to be efficiently transcribed in vitro. The Tropomyosin I gene and the muscle promoter of the Tropomyosin II gene, as well as two other contractile protein muscle genes, were not transcribed in vitro. The embryonic extract did, however, contain developmental-specific proteins that bound to the muscle enhancer regulatory region of the Tropomyosin I gene.

The impact of pharmacogenetics on the future of healthcare.

Pharmacogenetics has been promoted as potentially providing benefits to patients, managed care organizations and pharmaceutical companies. This has not translated into products that benefit healthcare developers, providers or consumers. The reasons for this are many, but this will change as the financial incentives become clear for the pharmaceutical industry to develop products that use genetic susceptibility as part of the rationale for products, healthcare providers have increasing incentive to reduce costs, and patients demand up-to-date technologies to optimize healthcare. Recent studies have established genetic contributions that alter the response to therapy for some disease entities, and more will follow as pharmacogenetics becomes increasingly accepted as an important consideration in the therapeutic decision-making process.

Vitamin D receptor alleles and rates of bone loss: influences of years since menopause and calcium intake.

A genetic marker for the 1,25-dihydroxyvitamin D receptor (VDR) is reported to account for much of the heritable component of bone density. It is not known whether VDR genotype influences bone accretion or loss, or how it is related to calcium metabolism. The VDR genotype was determined in 229 healthy postmenopausal women who previously participated in a calcium trial. VDR alleles were designated according to presence (b) or absence (B) of the BsmI restriction enzyme cutting site. There were 83 bb, 102 Bb, and 44 BB individuals. Two-thirds of the women took 500 mg of calcium supplement (mean calcium intake = 892 mg/day) and one-third a placebo (mean = 376 mg/day). Bone mineral density (BMD) at the femoral neck, spine, and radius were measured by dual- and single-photon absorptiometry at baseline and after 1 and 2 years. Among women more than 10 years postmenopausal, those with the BB genotype had the lowest femoral neck BMD. Rates of bone loss over 2 years were greater in the BB group at all sites (e.g., at the femoral neck, bb, 0.45 +/- 0.43; Bb, -0.01 +/- 0.40; BB, -0.99 +/- 0.50%/year; BB vs. bb, p = 0.01), and this trend was found both in women < 10 years since menopause (e.g., at the radius, bb, 0.43 +/- 0.47; Bb, -0.37 +/- 0.42; BB, -1.20 +/- 0.59% per year; BB vs. bb, p = 0.02) and those > or = 10 years (radius, bb, -0.71 +/- 0.41; Bb, 0.08 +/- 0.39; BB, -1.41 +/- 0.49% per year; BB vs. Bb, p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Presymptomatic testing using DNA markers for individuals at risk for familial multiple endocrine neoplasia 2A.

The carrier status of 39 at-risk individuals in 6 multiple endocrine neoplasia 2A families was determined using a DNA based test. We were able to calculate a virtual diagnosis (probability greater than 95%) for 77% of the individuals and a probable diagnosis (probability greater than 90%) for 90% of the individuals. This study points out some of the problems of specific pedigree structures that can affect the risk calculation. This study further shows that no single test based on either biochemistry, pathology, or genetics can consistently and unambiguously produce a presymptomatic diagnosis. We also describe two specific examples where DNA testing has helped to resolve clinical uncertainties in at-risk individuals.

Alleles at the dopamine D4 receptor locus do not contribute to the genetic susceptibility to schizophrenia in a large Swedish kindred.

The discovery of a functional polymorphism within the dopamine D4 receptor gene (DRD4) has not only strengthened the hypotheses implicating DRD4 in the etiology of neuropyschiatric disorders, but also provided a genetic marker for testing these hypotheses. The possibility of the dopamine D4 receptor as a candidate gene for schizophrenia was investigated in a large Swedish kindred segregating for schizophrenia. Linkage to schizophrenia was tested by linkage analyses of 6 polymorphic markers (at 4 loci) in chromosome 11p15.5 including the dopamine D4 receptor (DRD4) and the tyrosine hydroxylase (TH) loci. Schizophrenia was excluded from close linkage to the DRD4 locus using two of the polymorphisms located within the dopamine D4 receptor gene. The first DRD4 polymorphism consists of variation in the number of a 48 bp imperfect direct repeat in the third exon; the second consists of a variable number of repeated G nucleotides in the first intron. In addition, some of the individuals homozygous for four or seven copies of 48 bp repeat alleles were tested for previously reported sequence variation among repeats. No single haplotype of the DRD4 alleles or haplotype of other markers in chromosome 11p15.5 was found to be common to the schizophrenic individuals in this family. Therefore, we find no evidence for linkage of the D4 receptor, or this region of 11p15.5, with genetic susceptibility to schizophrenia in this kindred.

Variability of dopamine D4 receptor (DRD4) gene sequence within and among nonhuman primate species.

The dopamine D4 receptor is one of five receptors known to function in mammalian dopaminergic pathways. The DNA sequence of the human dopamine D4 receptor gene (DRD4) has previously been investigated in several populations and found to be highly polymorphic at both the DNA and amino acid levels, exhibiting at least 25 alleles. This variation results from differences in the number and DNA sequence of a 48-bp (16-amino acid) repeat unit in the coding region of DRD4. In the present study, DRD4 DNA sequence was examined in at least two individuals from each of five nonhuman primate species. All five species exhibit intraspecies variability, including both single nucleotide substitutions and variation in the number of 48-bp repeat units. No differences were found between the two alleles of one individual from a sixth nonhuman species. Within each species, all of the DRD4 alleles share species-specific features, indicating that while repeat-unit variation is nearly ubiquitous, ancestral variation has been lost and subsequently regenerated in each of the evolutionary lineages studied. Chimpanzees and gorillas share a unique 12-bp deletion in the coding region of DRD4, outside the repeat-unit segment of the gene. This suggest that the extant chimpanzee DRD4 is more closely related to the gorilla DRD4 than either is to the human DRD4.

A high-resolution meiotic mapping panel for the pericentromeric region of chromosome 10.

Familial multiple endocrine neoplasia type 2A (MEN 2A) is a dominantly inherited cancer syndrome characterized by tumors in tissues derived from the neural crest. The disease manifests as medullary carcinoma of the thyroid, pheochromocytoma, and hyperparathyroidism. The MEN2A locus has been mapped near the centromere of chromosome 10 by linkage analysis. Statistical analyses have not resolved the location of MEN2A among several close markers. We have used our family material to refine the positions of 36 identified and confirmed crossovers among the markers most closely linked to MEN2A. This high-resolution meiotic mapping panel will help order loci in this pericentromeric region and narrow the region in which MEN2A maps.

The study of candidate genes in drug trials: sample size considerations.

With discovery of an increasing number of candidate genes that may affect inter-individual variability in response to drugs, the design of drug trials that incorporate their study has become relevant. We discuss the determination of sample size for such studies when the number of tests to perform is given, or, alternatively, the number of tests to perform when the sample size is given. In many cases, a uniformly most powerful test does not exist and normal approximations are not sufficiently accurate to determine sample size. We discuss briefly various tests of interest and we give simple examples to illustrate some of the problems that arise.

Physical and genetic maps for chromosome 10.

A fluorescence in situ hybridization (FISH) physical map of 14 polymorphic loci on chromosome 10 covers over 62% of the fractional length of chromosome 10. The positions of three previously mapped loci are confirmed, nine more are refined, and two new loci are cytogenetically mapped. The order of loci determined by FISH agrees with that obtained by genetic linkage studies. When the distance estimates for the physical map are compared to the distance estimates of our existing linkage map, the sex average ratio of the fractional length (FL) per centimorgan (cM) for this portion of chromosome 10 is 0.004 (or 0.4% FL/cM). However, the average ratios for male- and female-specific genetic distances are quite different, in agreement with an overall higher rate of recombination in females (0.008 FL/cM and 0.003 FL/cM, respectively). Moreover, the ratio across the centromere is larger for both the male (0.031 FL/cM) and the female (0.009 FL/cM) than the ratio encompassing the q arm (0.006 FL/cM for males and 0.002 FL/cM for females), suggesting that there is reduced recombination at the centromere in both the male and the female maps when compared to the physical distance generated from FISH on metaphase chromosomes.

Computer-assisted restriction mapping: an integrated approach to handling experimental uncertainty.

Building a map of restriction sites from double-digest gel data can be a complex and frustrating task, especially when many DNA fragments are detected or when the gel results are ambiguous. 'Double Digester' is an interactive, graphical computer program which helps researchers understand and resolve such data. It explicitly represents the experimental data, the associated uncertainties, the researcher's hypotheses and possible map interpretations. Alternative solutions are frequently possible, and the differences between them may help determine which additional experiments might resolve ambiguities. Initial use has confirmed the benefits of this approach, and has suggested ways in which it can be refined and extended. Double Digester meets the need for a practical tool to help build restriction maps, and also illustrates how a computer-based tool can confront experimental uncertainty in an integrated fashion.

Genomic and yeast artificial chromosome long-range physical maps linking six loci in 10q11.2 and spanning the multiple endocrine neoplasia type 2A (MEN2A) region.

Multiple endocrine neoplasia types 2A and 2B (MEN 2A and MEN 2B) and familial medullary thyroid carcinoma (FMTC) are dominantly inherited cancers that have in common the clinical feature of medullary thyroid carcinoma (MTC). We have performed both genomic long-range restriction mapping and yeast artificial chromosome (YAC) contig assembly and restriction mapping to establish physical linkage, order, and distances between six loci in 10q11.2 near the genes responsible for these hereditary cancers. RET, D10S94, D10S182, and D10S102 have been mapped in genomic DNA. RET, D10S94, D10S182, D10F38S3, and the 10q11.2 sequences detected by DNA marker DM124 are encompassed by a 1-Mb YAC contig. Six physically linked loci are within 1.4 Mb and have an order and orientation of 10cen, D10F38S3, DM124, RET, D10S94, D10S182, D10S102, 10qter. Mutations in the RET proto-oncogene have recently been demonstrated to be associated with MEN 2A and FMTC. RET is located within a genetically defined MEN2A candidate interval between D10S141 and D10S94; MEN2B has been mapped to a larger, overlapping region between D10S141 and a more distal locus, RBP3. Both our genomic physical map and our YAC contig span the entire MEN2A candidate region and overlap with that of MEN2B. These maps will facilitate the identification of genes that can be considered candidates for MEN2B and the identification of tumor-specific alterations important in sporadic MTC.

Isolation and high-resolution mapping of new DNA markers from the pericentromeric region of chromosome 10.

The gene responsible for multiple endocrine neoplasia type 2A (MEN 2A) has been localized to the pericentromeric region of chromosome 10. Several markers that fail to recombine with MEN2A have been identified, including D10Z1, D10S94, D10S97, and D10S102. Meiotic mapping in the MEN2A region is limited by the paucity of critical crossovers identified and by the dramatically reduced rates of recombination in males. Additional approaches to mapping loci in the pericentromeric region of chromosome 10 are required. We have undertaken the generation of a detailed physical map by radiation hybrid mapping. Here we report the development of a radiation hybrid panel and its use in the mapping of new DNA markers in pericentromeric chromosome 10. The radiation-reduced hybrids used for mapping studies all retain small subchromosomal fragments that include both D10S94 and D10Z1. One hybrid was selected as the source of DNA for cloning. One hundred five human recombinant clones were isolated from a lambda library made with pp11A DNA. We have completed regional mapping of 22 of those clones using our radiation hybrid mapping panel. Seven markers have been identified and, when taken together with previously meiotically mapped markers, define eight radiation hybrid map intervals between D10S34 and RBP3. The identical order is found for a number of these using either the radiation hybrid mapping panel or the meiotic mapping panel. We believe that this combination cloning and mapping approach will facilitate the precise positioning of new markers in pericentromeric chromosome 10 and will help in refining further the localization of MEN2A.

Localization of the gene for MEN 2A.

The search for the gene that causes the multiple endocrine neoplasia type 2A (MEN 2A) syndrome is entering a new phase. Genetic linkage studies have localized the gene to the pericentromeric region of chromosome 10. The statistical portion of mapping the gene for MEN 2A is nearly complete and now classical molecular biological/gene mapping techniques will be employed. We have used fluorescence in situ hybridization to estimate the size of the MEN2A region to be about 2 to 5 mb, using some liberal assumptions; at worst the region should contain no more than about 10 mb of non-alphoid DNA. Our mapping panels (meiotic recombinant and radiation reduced hybrid) give consistent orders of markers in this small region. We describe our initial attempts to clone the region using yeast artificial chromosomes.

A hypervariable segment in the human dopamine receptor D4 (DRD4) gene.

The human dopamine D4 receptor contains a novel polymorphism within the putative third cytoplasmic loop of the protein. The polymorphism is characterized by a varying number of direct imperfect 48-bp repeats in the gene. Pharmacological characterization has suggested that this receptor is the site through which the atypical neuroleptic clozapine exerts its antipsychotic action and that some polymorphic variants display different pharmacological properties. Further analysis of the repeat region using innovative technologies indicates that the alleles vary not only in the number of repeats (2-8 or 10 repeat units) but also in the sequence of the repeats and the order in which they appear. In 178 unrelated chromosomes we have identified 19 different repeats in 25 different haplotypes coding for 18 different predicted amino acid sequences, making this one of the most variable functional proteins currently described.

A new polymorphic marker (D10S97) tightly linked to the multiple endocrine neoplasia type 2A (MEN2A) locus.

Familial multiple endocrine neoplasia type 2A (MEN 2A) is a cancer syndrome that is inherited as an autosomal dominant with high penetrance. Its clinical features are medullary carcinoma of the thyroid, pheochromocytomas, and hyperparathyroidism. A new polymorphic locus D10S97 (probe: KW6 delta SacI) detects a codominant EcoRI polymorphism that is tightly linked to the MEN2A locus. The peak lod score for linkage between D10S97 with MEN2A is 13.03 at theta = 0.00. The polymorphic locus D10S97 maps, by linkage analysis, into the previously defined interval between FNRB and RBP3 to which MEN2A has been assigned. We present physical mapping data showing that the probe pKW6 originates from 10p13 and that the polymorphic locus D10S97 in 10q11.2 is detected by cross-hybridization.

A refined linkage map for DNA markers around the pericentromeric region of chromosome 10.

A refined genetic linkage map for the pericentromeric region of human chromosome 10 has been constructed from data on 12 distinct polymorphic DNA loci as well as the locus for multiple endocrine neoplasia type 2A (MEN 2A), a dominantly inherited cancer syndrome. The map extends from D10S24 (at 10p13-p12.2) to D10S3 (at 10q21-q23) and is about 70 cM long. Overall, higher female than male recombination frequencies were observed for this region, with the most remarkable female excess in the immediate vicinity of the centromere, as previously reported. Most of the DNA markers in this map are highly informative for linkage and the majority of the interlocus intervals are no more than 6 cM apart. Thus this map should provide a fine framework for future efforts in more detailed mapping studies around the centromeric area. A set of ordered cross-overs identified in this work is a valuable resource for rapidly and accurately localizing new DNA clones isolated from the pericentromeric region.

A preliminary analysis of consortium data for markers tightly linked to multiple endocrine neoplasia type 2A.

We have analyzed DNA marker typing data contributed by six independent groups to estimate the pairwise genetic distances between these markers and the locus for multiple endocrine neoplasia type 2A (MEN 2A). We used LIPED to calculate these distances for female, male, and sex-average linkage maps and to determine the corresponding LOD scores. The preliminary analyses of this large data set (89 MEN 2A families and five non-MEN 2A references families, with 1,934 total individuals) are reported here. These refined estimates of the genetic map in this region will aid in the assignment of presymptomatic diagnoses. This study clearly points out the limitation of pairwise linkage analysis in further refining the position of MEN2A in this small region of chromosome 10. Further refinement of the genetic map position of MEN2A will be best accomplished by finding, verifying, and accurately mapping crossovers in specific families.