The 1993-94 Généthon human genetic linkage map.

In 1992, we described a second-generation genetic linkage map of the human genome. Using 1,267 new microsatellite markers, we now present a new genetic linkage map containing a total of 2,066 (AC)n short tandem repeats, 60% of which show a heterozygosity of over 0.7. Statistical linkage analysis based on the genotyping of eight large CEPH families placed these markers in the 23 linkage groups. The map includes 1,266 intervals and spans a total distance of 3690 centiMorgans (cM). A total of 1,041 markers could be ordered with odds ratios greater than 1000:1. About 56% of this map is at a distance of 1 cM or less from one of its markers.

A radiation hybrid transcript map of the mouse genome.

Expressed-sequence tag (EST) maps are an adjunct to sequence-based analytical methods of gene detection and localization for those species for which such data are available, and provide anchors for high-density homology and orthology mapping in species for which large-scale sequencing has yet to be done. Species for which radiation hybrid-based transcript maps have been established include human, rat, mouse, dog, cat and zebrafish. We have established a comprehensive first-generation-placement radiation hybrid map of the mouse consisting of 5,904 mapped markers (3,993 ESTs and 1,911 sequence-tagged sites (STSs)). The mapped ESTs, which often originate from small-EST clusters, are enriched for genes expressed during early mouse embryogenesis and are probably different from those localized in humans. We have confirmed by in situ hybridization that even singleton ESTs, which are usually not retained for mapping studies, may represent bona fide transcribed sequences. Our studies on mouse chromosomes 12 and 14 orthologous to human chromosome 14 show the power of our radiation hybrid map as a predictive tool for orthology mapping in humans.

Effects of postfire salvage logging on deadwood-associated beetles.

In Canada and the United States pressure to recoup financial costs of wildfire by harvesting burned timber is increasing, despite insufficient understanding of the ecological consequences of postfire salvage logging. We compared the species richness and composition of deadwood-associated beetle assemblages among undisturbed, recently burned, logged, and salvage-logged, boreal, mixed-wood stands. Species richness was lowest in salvage-logged stands, largely due to a negative effect of harvesting on the occurrence of wood- and bark-boring species. In comparison with undisturbed stands, the combination of wildfire and logging in salvage-logged stands had a greater effect on species composition than either disturbance alone. Strong differences in species composition among stand treatments were linked to differences in quantity and quality (e.g., decay stage) of coarse woody debris. We found that the effects of wildfire and logging on deadwood-associated beetles were synergistic, such that the effects of postfire salvage logging could not be predicted reliably on the basis of data on either disturbance alone. Thus, increases in salvage logging of burned forests may have serious negative consequences for deadwood-associated beetles and their ecological functions in early postfire successional forests.

An STS-based map of the human genome.

A physical map has been constructed of the human genome containing 15,086 sequence-tagged sites (STSs), with an average spacing of 199 kilobases. The project involved assembly of a radiation hybrid map of the human genome containing 6193 loci and incorporated a genetic linkage map of the human genome containing 5264 loci. This information was combined with the results of STS-content screening of 10,850 loci against a yeast artificial chromosome library to produce an integrated map, anchored by the radiation hybrid and genetic maps. The map provides radiation hybrid coverage of 99 percent and physical coverage of 94 percent of the human genome. The map also represents an early step in an international project to generate a transcript map of the human genome, with more than 3235 expressed sequences localized. The STSs in the map provide a scaffold for initiating large-scale sequencing of the human genome.

A physical map of 30,000 human genes.

A map of 30,181 human gene-based markers was assembled and integrated with the current genetic map by radiation hybrid mapping. The new gene map contains nearly twice as many genes as the previous release, includes most genes that encode proteins of known function, and is twofold to threefold more accurate than the previous version. A redesigned, more informative and functional World Wide Web site (www.ncbi.nlm.nih.gov/genemap) provides the mapping information and associated data and annotations. This resource constitutes an important infrastructure and tool for the study of complex genetic traits, the positional cloning of disease genes, the cross-referencing of mammalian genomes, and validated human transcribed sequences for large-scale studies of gene expression.

A gene map of the human genome.

The human genome is thought to harbor 50,000 to 100,000 genes, of which about half have been sampled to date in the form of expressed sequence tags. An international consortium was organized to develop and map gene-based sequence tagged site markers on a set of two radiation hybrid panels and a yeast artificial chromosome library. More than 16,000 human genes have been mapped relative to a framework map that contains about 1000 polymorphic genetic markers. The gene map unifies the existing genetic and physical maps with the nucleotide and protein sequence databases in a fashion that should speed the discovery of genes underlying inherited human disease. The integrated resource is available through a site on the World Wide Web at http://www.ncbi.nlm.nih.gov/SCIENCE96/.

Pangenomic changes induced by DHEA in the skin of postmenopausal women.

The objective of this study was to explore, for the first time, the changes in the pangenomic profile induced in human skin in women treated with dehydroepiandrosterone (DHEA) applied locally. Sixty postmenopausal women participated in this phase II prospective, randomized, double-blind and placebo-controlled study. Women were randomized to the twice daily local application of 0% (placebo), 0.3%, 1% or 2% DHEA cream. Changes in the pangenomic expression profile were studied using Affymetrix Genechips. Significant changes (p<0.05) in sixty-six DHEA-responsive probe sets corresponding to 52 well-characterized genes and 9 unknown gene sequences were identified. A dose-dependent increase in the expression of several members of the collagen family was observed, namely COL1, COL3 and COL5 as well as the concomitant modulation of SPARC, a gene required for the normal deposition and maturation of collagen fibrils in the dermis. Several genes involved in the proliferation and differentiation of keratinocytes were also modulated. In addition, topical DHEA reduced the expression of genes associated with the terminal differentiation and cornification of keratinocytes. Our results strongly suggest the possibility that DHEA could exert an anti-aging effect in the skin through stimulation of collagen biosynthesis, improved structural organization of the dermis while modulating keratinocyte metabolism.

Higher sensitivity of primary thyrotropin in screening for congenital hypothyroidism: a myth?

We have simultaneously measured T4 and TSH concentrations from 93,000 consecutive filter paper blood samples received by the Quebec network for Genetic Medicine. T4 was measured using the Micromedic Automated System and TSH was measured by RIA kits from Pharmacia and Becton Dickinson, each for a 6-month period. Statistical analyses to assess quality control parameters for the various methods revealed that the T4 assay had greater precision and reproducibility than either of the 2 commercially available TSH kits. The number of false negative results was similar for both methods. Eight infants with ectopic thyroid glands were correctly detected by the T4 method. One infant with secondary hypothyroidism was detected by the T4 approach and missed by the TSH methodology. Three infants overall would have been missed using either the T4 or TSH approach. These data indicate that primary T4 screening has similar sensitivity compared to TSH kits for mass screening programs for congenital hypothyroidism.

Genetic linkage mapping of the CYP11A1 gene encoding the cholesterol side-chain cleavage P450scc close to the CYP1A1 gene and D15S204 in the chromosome 15q22.33-q23 region.

The first rate-limiting step in the biosynthesis of all mammalian steroid hormones is the conversion of cholesterol to pregnenolone by the cholesterol side-chain cleavage enzyme, P450scc. The human CYP11A1 gene, encoding the mitochondrial P450scc, has been previously assigned by in situ hybridization to 15q23-q24 region. To map the CYP11A1 gene with linkage analysis, a novel TAAAA repeat polymorphism, found in its promoter region, was genotyped in the eight largest CEPH reference families, which included 91 children, 16 parents and 26 grandparents. Two-point linkage analysis was performed between this tetra-allelic polymorphism and the chromosome 15 microsatellite markers of Généthon as well as the tetranucleotide polymorphism of the CYP19 gene and a MspI RFLP of the CYP1A1 gene. A close linkage was observed with D15S204 (Z max = 27.33; theta max = 0.009) and CYP1A1 gene (Z max = 6.62; theta max = 0.001), but not with the CYP19 gene (Z max = 4.06; theta max = 0.26). The CYP19 gene that encodes the P450arom was rather closely linked with D15S123 (Z max = 31.04; theta max = 0.001). A framework map, including Généthon markers flanking the polymorphic CYP11A1 and CYP19 genes, was built by multipoint linkage analysis. Thereafter, a high-resolution genetic map of the 15q15-q25.3 region was constructed, yielding to the following order: cen-D15S214-[D15S123; CYP19]-D15S117-D15S159-D15S153-D15S983-[++ +D15S204; CYP11A1; CYP1A1]-D15S211-D15S152-D15S199-tel. Thus, the CYP11A1 gene is closely linked with the CYP1A1 gene, whereas it is located approximately 27.4 cM telomeric to the CYP19 gene.

A 10-cM YAC contig spanning GLC1A, the primary open-angle glaucoma locus at 1q23-q25.

Primary open-angle glaucoma is a complex of ocular disorders characterized by irreversible lesions of the optic nerve, open angle of the anterior chamber of the eye and elevated intraocular pressures. GLC1A, a locus involved in one form of this disease, has been mapped to an approximately 9-cM interval within 1q23-q25, between markers D1S445 and D1S416/D1S480. A 10-cM yeast artificial chromosome (YAC) contig spanning the whole region is described. This contig is based on 67 YACs, and 41 sequence tagged sites comprising 23 genetic markers, 16 YAC ends and 2 expressed sequence tags. The reagents reported in this study should be useful tools for the identification of the GLC1A gene by positional cloning.

Developmental plasticity in cerebellar tactile maps: fractured maps retain a fractured organization.

Plasticity following deafferentation has been repeatedly demonstrated in topographic sensory maps in the mammalian brain. In this paper we investigated the developmental plasticity of the fractured somatotopic map found in the tactile regions of the rat cerebellum. At various stages of postnatal development between postnatal days 1 and 30, we cauterized the infraorbital branch of the trigeminal nerve, which innervates the upper lip, furry buccal pad, and vibrissae that are represented within cerebellar folium crus IIa. The organization of the crus IIa map was then examined 2 to 3 months after denervation. We found that tactile receptive fields had reorganized throughout the denervated area but maintained a fractured somatotopy. Comparison of the reorganization in different animals showed that the denervated upper lip region was consistently and predominantly replaced by representation of the upper incisors. Analysis of evoked field potentials revealed an alteration, in denervated animals, of the response of the granule cell layer to brief tactile stimulation. This response in normal animals consists of two components at different latencies. Animals lesioned later in development were less likely to have the short latency component. This result suggests a difference in the developmental sensitivity of different cerebellum-related pathways to nerve lesions.

Plasticity in cerebellar tactile maps in the adult rat.

We previously demonstrated that the fractured tactile cerebellar map within the crus IIa folia of the cerebellar hemispheres reorganizes after deafferentation of the upper lip in neonatal rats (postnatal day [PND] 1-30). The present study examined the capacity of this map to reorganize after deafferentation in adults and animals late in development (PND 30-89). Several months after cauterization of the infraorbital branch of the trigeminal nerve, the tactile map in the granule cell layer of crus IIa reorganized, with representations of intact structures expanding into the denervated area. The pattern of reorganization was similar to reorganization after neonatal lesions in that (1) all representations were from perioral structures, (2) the reorganized map maintained a fractured somatotopy, and (3) the denervated area was predominantly and consistently invaded by the upper incisor representation. We conclude that the spatial pattern of reorganization is essentially the same regardless of the age of deafferentation. However, we also observed developmental differences in reorganization. First, more areas of crus IIa were nonresponsive in animals lesioned later in development (PND 30-89). Second, we found a surprising degree of variability in the pattern of tactilely evoked cerebellar field potentials of PND 30-40 animals compared with neonates and adults, suggesting that this time period differs from other stages. The pattern of evoked potentials reflects the two primary inputs to the map. Our data show that, although both afferent pathways are capable of reorganization throughout development, their relative contribution to the map appears to differ, depending on the age at which lesion occurs.

Osteogenesis imperfecta type VII maps to the short arm of chromosome 3.

We have identified a novel form of autosomal recessive osteogenesis imperfecta (OI) in a small First Nations community from northern Quebec. Mutation screening of the COL1A1/COL1A2 genes revealed no detectable mutations, and type I collagen protein analyses were also normal. By linkage analysis, we mapped this unique autosomal recessive variant of osteogenesis imperfecta to chromosome 3p22-24.1. Based on the assumption of a founder effect, genome-wide screening was performed on a DNA sample pooled from seven affected individuals. Familial as well as historical recombinations identified within an extended haplotype of 19 markers localized the disease between markers D3S2324 and D3S1561, separated by <5 cM. Based on chromosomal localization to 3p22-24.1, the transforming growth factor-beta receptor 2 gene and the parathyroid hormone/parathyroid hormone-related peptide receptor were tested, but were excluded as being associated with the phenotype. This study excludes type I collagen mutations in the pathogenesis of the disease and assigns this form of OI to a locus other than the ones containing the type I collagen genes.

Fine mapping of low-density lipoprotein receptor gene by genetic linkage on chromosome 19p13.1-p13.3 and study of the founder effect of four French Canadian low-density lipoprotein receptor gene mutations.

Familial hypercholesterolemia (FH) is one of the most common autosomal codominant diseases. FH is caused by mutations in the low-density lipoprotein receptor (LDLR) gene and is characterized by raised plasma LDL-cholesterol, tendon xanthomas, and premature coronary heart disease. The frequency of FH among French Canadians in northeastern Quebec is higher than in most other populations, 1:154 vs. 1:500 due to high prevalence of few recurrent mutations in the LDLR gene. In the French Canadian population, 11 mutations in the LDLR gene have been found to occur in geographically diverse areas and account for > 90% of cases. We have first constructed a high-resolution genetic map to locate several highly polymorphic markers close to LDLR locus, thus providing the necessary tools to study the origin of the four most common mutations which account for approximately 80% of our FH patients. We have then genotyped five markers (D19S413, D19S865, D19S221, D19S914, D19S586) in 102 heterozygotes (38 del > 15kb; 36 W66G; 16 C646Y; 12 E207K), two compound heterozygotes (del > 15kb/W66G; del > 15kb/C646Y) and seven homozygotes (three del > 15 kb; three W66G: one E207K) with FH unrelated to the first and second degree. We have found that patients bearing the same LDLR gene mutation carry a common haplotype at the LDLR locus although there is evidence for the early occurrence of a recombinational event between the LDLR and the D19S221 locus in the French Canadian patients bearing the W66G mutation. The fine mapping of LDLR gene close to several highly informative microsatellite markers provide fine mapping details of the LDLR region and additional tools for studies of association between plasma lipoprotein levels and LDLR gene.

Effects of dihydrotestosterone on adipose tissue measured by serial analysis of gene expression.

Intra-abdominal fat accumulation is related to several diseases, especially diabetes and heart disease. Molecular mechanisms associated with this independent risk factor are not well established. Through the serial analysis of gene expression (SAGE) strategy, we have studied the transcriptomic effects of castration and dihydrotestosterone (DHT) in retroperitoneal adipose tissue of C57BL6 male mice. Approximately 50,000 SAGE tags were isolated in intact and gonadectomized mice, as well as 3 and 24 h after DHT administration. Transcripts involved in energy metabolism, such as glyceraldehyde-3-phosphate dehydrogenase, malic enzyme supernatant, fatty acid synthase, lipoprotein lipase, hormone-sensitive lipase and monoglyceride lipase, were upregulated by DHT. Transcripts involved in adipogenesis, and cell cycle and cell shape organization, such as DDX5, C/EBPalpha, cyclin I, procollagen types I, III, IV, V and VI, SPARC and matrix metalloproteinase 2, were upregulated by DHT. Cell defense, division and signaling, protein expression and many novel transcripts were regulated by castration and DHT. The present results provide global genomic evidence for a stimulation of glycolysis, fatty acids and triacylglycerol production, lipolysis and cell shape reorganization, as well as cell proliferation and differentiation, by DHT. The novel transcripts regulated by DHT may contribute to identify new mechanisms involved in the action of sex hormones and their potential role in obesity.

Type I protein C deficiency in French Canadians: evidence of a founder effect and association of specific protein C gene mutations with plasma protein C levels.

Protein C (PROC) deficiency is one of the most common autosomal codominant diseases. Although more than 150 germline mutations in the PROC gene have been described around the world, the spectrum of mutations among French Canadians is unknown. We have identified one frameshift (3363 ins C) and two missense mutations (R178Q and T298M) in 7 French Canadian families with type I PROC deficiency. In order to demonstrate a possible founder effect for the 3363 ins C mutation, we have constructed a high-resolution genetic map to locate several highly polymorphic markers close to PROC locus. We have then genotyped five markers in 36 heterozygotes for the 3363 ins C mutation. Our data suggest that these patients carry a common haplotype at the PROC locus. Immunologic plasma PROC levels of heterozygotes and genetically normal relatives were also correlated with the nature of the mutation in the coding sequence and with the genotype of three polymorphisms in the PROC promoter. We found that the mean immunologic plasma PROC levels were lower in heterozygotes for the frameshift mutation 3363 ins C compared to heterozygotes for one of the two missense mutations R178Q and T298M (0.46 vs 0.61; P = 0.0004). Moreover, this difference cannot be explained by the genetic variation of the three polymorphisms in the PROC promoter which accounts for only 10.4% of the variation of immunologic PROC levels in non-deficient subjects. These results suggest that the nature of the mutation in the coding sequence of PROC gene may modulate immunologic plasma PROC levels.

Chromosome 13 workshop report.

Evidence was presented that provided support for linkage in a relatively broad telomeric region of chromosome 13. A significant overlap for positive markers linked to both bipolar disorder and schizophrenia occurred in this area.

Genome-wide search for linkage of bipolar affective disorders in a very large pedigree derived from a homogeneous population in quebec points to a locus of major effect on chromosome 12q23-q24.

We completed a genome-wide scan for susceptibility loci for bipolar affective disorders in families derived from a rather homogeneous population in the Province of Québec. The genetic homogeneity of this population stems from the migration of founding families into this relatively isolated area of Québec in the 1830s. A possible founder effect, combined with a prevalence of very large families, makes this population ideal for linkage studies. Genealogies for probands can be readily constructed from a population database of acts of baptism and marriage from the early 1830s up to the present time (the BALSAC register). We chose probands with a DSM III diagnosis of bipolar affective disorder and who may be grouped within large families having genealogical origins with the founding population of the Saguenay-Lac-St-Jean area. Living members (n approximately 120) of a very large pedigree were interviewed using the Structured Clinical Interview for DSM III (SCID I), SCID II, and with a family history questionnaire. A diagnostic panel evaluated multisource information (interview, medical records, family history) and pronounced best-estimate consensus diagnoses on all family members. Linkage, SimAPM, SimIBD, and sib-pair analyses have been performed with 332 microsatellite probes covering the entire genome at an average spacing of 11 cM. GENEHUNTER and haplotype analyses were performed on regions of interest. Analysis of a second large pedigree in the same regions of interest permitted confirmation of presumed linkages found in the region of chromosome 12q23-q24.

Intimal hyperplasia and thrombosis of the visceral arteries in a young woman: possible relation with oral contraceptives and smoking.

This is a report of a 26-year-old woman who died of a massive intestinal infarction caused by occlusion of the celiac axis and the superior and inferior mesenteric arteries. Autopsy showed intimal hyperplasia and an overlying thrombus that obstructed the lumen of the vessel. As shown by Irey et al., exogenous or endogenous female reproductive steroids can act on the vascular system as a target organ and induce intimal hyperplasia and thrombus formation. The effects of contraceptive estrogens and progestogens are discussed, but the catalytic effect of heavy cigarette smoking appears to be the factor that induces, in predisposed women with hyperplasia, thrombosis of visceral arteries. In this case report we want to emphasize that the association between smoking and oral contraceptives can cause cardiovascular disease in young women. Failure to recognize this fact could result in delayed diagnosis and worsen the prognosis.

PIP: To further understand the pathophysiology of arterial diseases induced by oral contraceptives (OCs), a case report is presented of a young woman who died of extensive visceral artery thrombosis. The possible role of estrogens and progestogens and of cigarette smoking as the predisposing factors in this patient are discussed. A 26-year-old woman, who complained of progressive abdominal pain and whose past medical and surgical history was negative, was admitted to the general surgery service. She was the mother of 1 child and had had 2 previous spontaneous abortions. She had received ethinyl estradiol 35 mcg with norethindrone 500 mcg and 1000 mcg for 3 months, but because of a problem with breakthrough bleeding the medication was changed to mestranol 50 mcg with norethindrone 1000 mcg. She had been taking Ortho-Novum 1/50 for 2 1/2 years. She had smoked 25-35 cigarettes daily for about 10 years but denied use of alcohol or other drugs. She was not known to be diabetic, hypertensive, or dyslipidemic, and had no history of atherosclerosis in her family. For 7 months prior to her admission, the patient complained of abdominal pain, which progressively increased in intensity and duration, interrupted by periods of well-being. The patient reported 2 recent, isolated episodes of mild proctalgia but no tenesmus or melena. There had been no fever, but the patient had been anorexic for the past 2 weeks and reported losing 10 kg in the past month. She had no complaints apart from those related to the gastrointestinal system. At an emergency laparotomy, gangrenous acalculous cholecystitis and infarction of the terminal ileum were discovered. A cholecystectomy with resection of the terminal ileum and the right colon was performed. An end-to-end primary anastomosis was performed. On exploration of the superior mesenteric artery, a thrombus was discovered at its origin. As a transverse arteriotomy showed a good retrograde flow, a thrombectomy was performed. There appeared to be an unsatisfactory antegrade flow. The superior mesenteric artery then was transposed in an end-to-end fashion on the abdominal aorta. An immediate postoperative arteriogram showed thrombosis of the celiac axis at its origin. Revascularization failed to improve the condition of the intestine. The patient died. The intent of this case report is to emphasize that the association between smoking and oral contraceptives can cause cardiovascular disease in young women, and a failure to recognize this association can result in delayed diagnosis and worsen the prognosis.