Metabolic flexibility in postmenopausal women: Hormone replacement therapy is associated with higher mitochondrial content, respiratory capacity, and lower total fat mass.

AIM: To investigate effects of hormone replacement therapy in postmenopausal women on factors associated with metabolic flexibility related to whole-body parameters including fat oxidation, resting energy expenditure, body composition and plasma concentrations of fatty acids, glucose, insulin, cortisol, and lipids, and for the mitochondrial level, including mitochondrial content, respiratory capacity, efficiency, and hydrogen peroxide emission. METHODS: 22 postmenopausal women were included. 11 were undergoing estradiol and progestin treatment (HT), and 11 were matched non-treated controls (CONT). Peak oxygen consumption, maximal fat oxidation, glycated hemoglobin, body composition, and resting energy expenditure were measured. Blood samples were collected at rest and during 45 min of ergometer exercise (65% VO2peak). Muscle biopsies were obtained at rest and immediately post-exercise. Mitochondrial respiratory capacity, efficiency, and hydrogen peroxide emission in permeabilized fibers and isolated mitochondria were measured, and citrate synthase (CS) and 3-hydroxyacyl-CoA dehydrogenase (HAD) activity were assessed. RESULTS: HT showed higher absolute mitochondrial respiratory capacity and post-exercise hydrogen peroxide emission in permeabilized fibers and higher CS and HAD activities. All respiration normalized to CS activity showed no significant group differences in permeabilized fibers or isolated mitochondria. There were no differences in resting energy expenditure, maximal, and resting fat oxidation or plasma markers. HT had significantly lower visceral and total fat mass compared to CONT. CONCLUSION: Use of hormone therapy is associated with higher mitochondrial content and respiratory capacity and a lower visceral and total fat mass. Resting energy expenditure and fat oxidation did not differ between HT and CONT.

Glaucoma detection with damato multifixation campimetry online.

PurposeTo evaluate Damato Multifixation Campimetry Online (DMCO), a free-of-charge internet-based visual field test. DMCO exists in three versions: DMCO BASIC, DMCO STANDARD, and DMCO ADVANCED. The main focus was (i) to investigate the sensitivity and the specificity of the existing DMCO versions in the detection of glaucomatous visual field loss and (ii) to define and evaluate algorithms for the interpretation of DMCO results.MethodsThe study design was an evaluation of a diagnostic test and included 97 individuals performing DMCO and white-on-white perimetry. Interpretation algorithms were devised to define abnormality, and these were evaluated using the Glaucoma Staging System as gold standard. Receiver operating characteristic (ROC) curves and area under the ROC (AUC) were calculated.ResultsAUCs from 15 algorithms ranged from 0.79 to 0.90. The most promising algorithm combined results from two successive DMCO STANDARD tests. The sensitivity was highly dependent on the severity of glaucoma. Hence, for eyes with mild, moderate, advanced, and severe glaucoma, the DMCO test demonstrated a sensitivity of 11.8, 71.4, 100, and 100%, respectively. The specificity was as high as 98.1%. Median duration per eye to complete the DMCO STANDARD test was 86 s for the control group and 125 s in participants with glaucoma.ConclusionsDMCO shows promise as a free-of-charge online tool to identify glaucomatous visual field defects in a preselected population. Ongoing studies are evaluating the use of DMCO in a nonselected population.

Cognitive impact of genetic variation of the serotonin transporter in primates is associated with differences in brain morphology rather than serotonin neurotransmission.

A powerful convergence of genetics, neuroimaging and epidemiological research has identified the biological pathways mediating individual differences in complex behavioral processes and the related risk for disease. Orthologous genetic variation in non-human primates (NHPs) represents a unique opportunity to characterize the detailed molecular and cellular mechanisms that bias behaviorally and clinically relevant brain function. We report that a rhesus macaque orthologue of a common polymorphism of the serotonin transporter gene (rh5-HTTLPR) has strikingly similar effects on behavior and brain morphology to those in humans. Specifically, the rh5-HTTLPR (S)hort allele broadly affects cognitive choice behavior and brain morphology without observably affecting the 5-hydroxytryptamine (5-HT) transporter or 5-HT(1A) concentrations in vivo. Collectively, our findings indicate that 5-HTTLPR-associated behavioral effects reflect genotype-dependent biases in cortical development rather than static differences in serotonergic signaling mechanisms. Moreover, these data highlight the vast potential of NHP models in advancing our understanding of human genetic variation affecting behavior and neuropsychiatric disease liability.

Human chromosome 19 and related regions in mouse: conservative and lineage-specific evolution.

To illuminate the function and evolutionary history of both genomes, we sequenced mouse DNA related to human chromosome 19. Comparative sequence alignments yielded confirmatory evidence for hypothetical genes and identified exons, regulatory elements, and candidate genes that were missed by other predictive methods. Chromosome-wide comparisons revealed a difference between single-copy HSA19 genes, which are overwhelmingly conserved in mouse, and genes residing in tandem familial clusters, which differ extensively in number, coding capacity, and organization between the two species. Finally, we sequenced breakpoints of all 15 evolutionary rearrangements, providing a view of the forces that drive chromosome evolution in mammals.

Detection of E2A translocations in leukemias via fluorescence in situ hybridization.

Three rearrangements in ALL disrupt E2A and create E2A fusion proteins: the t(1;19)(q23;p13) and E2A-PBX1, t(17;19)(q22;p13) and E2A-HLF and a cryptic inv(19)(p13;q13) and E2A-FB1. While E2A is fused to PBX1 in most ALLs with a t(1;19), 5-10% of cases have translocations that appear identical, but do not affect E2A or PBX1. Because more intensive therapy improves the outcome of patients with E2A-PBX1positive (1;19) translocations, it is critical to identify this subset of patients so that appropriate therapy can be administered. In addition, there are balanced and unbalanced variants of the t(1;19) and controversy exists regarding the clinical significance of this distinction. We have developed a two-color fluorescence in situ hybridization assay that accurately detects E2A translocations in metaphase and interphase cells, distinguishes between balanced and unbalanced variants and identifies patients with a t(1;19) who lack E2A-PBX1 fusion. We found that clonal microheterogeneity is common in patients with E2A translocations and most patients have mixtures of cells with balanced and unbalanced translocations, suggesting that this distinction represents two ends of a continuum rather than distinct biological entities. These reagents should have widespread clinical utility and be useful for translational and basic research studies involving E2A translocations and this region of chromosome 19p13.

Comparative maps of human 19p13.3 and mouse chromosome 10 allow identification of sequences at evolutionary breakpoints.

A cosmid/bacterial artificial chromosome (BAC) contiguous (contig) map of human chromosome (HSA) 19p13.3 has been constructed, and over 50 genes have been localized to the contig. Genes and anonymous ESTs from approximately 4000 kb of human 19p13.3 were placed on the central mouse chromosome 10 map by genetic mapping and pulsed-field gel electrophoresis (PFGE) analysis. A region of approximately 2500 kb of HSA 19p13.3 is collinear to mouse chromosome (MMU) 10. In contrast, the adjacent approximately 1200 kb are inverted. Two genes are located in a 50-kb region after the inversion on MMU 10, followed by a region of homology to mouse chromosome 17. The synteny breakpoint and one of the inversion breakpoints has been localized to sequenced regions in human <5 kb in size. Both breakpoints are rich in simple tandem repeats, including (TCTG)n, (CT)n, and (GTCTCT)n, suggesting that simple repeat sequences may be involved in chromosome breaks during evolution. The overall size of the region in mouse is smaller, although no large regions are missing. Comparing the physical maps to the genetic maps showed that in contrast to the higher-than-average rate of genetic recombination in gene-rich telomeric region on HSA 19p13.3, the average rate of recombination is lower than expected in the homologous mouse region. This might indicate that a hot spot of recombination may have been lost in mouse or gained in human during evolution, or that the position of sequences along the chromosome (telomeric compared to the middle of a chromosome) is important for recombination rates.

A fine physical map of the CACNA1A gene region on 19p13.1-p13.2 chromosome.

The P/Q-type Ca(2+) channel alpha(1A) subunit gene (CACNA1A) was cloned on the short arm of chromosome 19 between the markers D19S221 and D19S179 and found to be responsible for Episodic Ataxia type 2, Familial Hemiplegic Migraine and Spinocerebellar Ataxia type 6. This region was physically mapped by 11 cosmid contigs spanning about 1. 4Mb, corresponding to less than 70% of the whole region. The cosmid contig used to characterize the CACNA1A gene accounted only for the coding region of the gene lacking, therefore, the promoter and possible regulation regions. The present study improves the physical map around and within the CACNA1A by giving a complete cosmid or BAC contig coverage of the D19S221-D19S179 interval. A number of new STSs, whether polymorphic or not, were characterized and physically mapped within this region. Four ESTs were also assigned to cosmids belonging to specific contigs.

Genomic organisation of the human cyclic AMP-specific phosphodiesterase PDE4C gene and its chromosomal localisation to 19p13.1, between RAB3A and JUND.

PDE4C is one of four mammalian genes that encode multiple PDE4 cyclic AMP-specific phosphodiesterase isoforms that are inhibited by rolipram. Fluorescent in situ hybridisation localised PDE4C to the p13.1 region of human chromosome 19. Overlapping cosmid clones spanning the human PDE4C gene were identified and characterised. Analysis of this locus indicated that the PDE4C gene spans at least 38 kb, consists of at least 18 exons, and contains the marker D19S212 within an intron. Comparison of published human PDE4C cDNA sequences with those of the genomic DNA identified four alternatively spliced exons and the possibility that the PDE4C locus contains at least three alternative promoters. PDE4C-containing cosmids also contained the genes for the growth regulatory transcription factor, JUND, and the mini guanine nucleotide regulatory protein, RAB3A. The RAB3A gene was shown to consist of 5 exons spanning 7.9 kb, while the JUND gene was found to contain no introns. Analysis of cosmids containing PDE4C, JUND, and RAB3A showed that 27 kb separate JUND and PDE4C, while only 3.7 kb separate PDE4C and RAB3A. The three genes share the same orientation of transcription and are arranged in the order cen- 5'- JUND-PDE4C-RAB3A-3'-tel.

Identification and characterization of the human homologue of the short PDE4A cAMP-specific phosphodiesterase RD1 (PDE4A1) by analysis of the human HSPDE4A gene locus located at chromosome 19p13.2.

The HSPDE4A gene spans 50 kb, consists of at least 17 exons and is orientated 5'-3', telomere to centromere. It is located at chromosome 19p13.2, being 350 kb proximal to the gene encoding TYK2 and 850 kb distal to the gene encoding the low-density lipoprotein receptor. Its structure is consistent with the production of active 'long' and 'short' isoenzymes as the result of alternative mRNA splicing at two splice junctions. Identified is the single alternatively spliced 5' exon encoding the unique N-terminal region of the long isoenzyme HSPDE4A4B (pde46). The upstream conserved regions, UCR1 and UCR2, which form characteristic domains of PDE4 long forms are each encoded by three exons. The PDE4A-subfamily-specific linker region LR1, which joins UCR1 and UCR2, is encoded by two exons, whereas LR2, which joins UCR2 to the catalytic unit, is encoded by a single exon. Identification of exons encoding an enzymically inactive product of this gene, HSPDE4A8A (2el), indicates that this is an authentic gene product. The 5' exon encoding the unique N-terminal region of the human homologue of the rodent isoform RNPDE4A1A (RD1) was located, and the splice junction used to produce this short PDE4A isoform shown to occur at a different position from that seen in both the rat PDE4B and PDE4D genes. Reverse transcriptase PCR analysis indicates that RD1 homologues are conserved across species, having a conserved membrane-targeting region and a hypervariable LR2 region. Human RD1 was expressed transiently in COS-7 cells and detected as an 83 kDa species primarily associated with the high-speed membrane fraction. Human RD1 exhibited a Km for cAMP of about 3 microM, an IC50 value for inhibition by the PDE4-selective inhibitor rolipram of about 0.3 microM and was considerably more thermostable than rat RD1. Human RD1 was generated as a mature 80 kDa species in an in vitro transcription-translation system and shown to be capable of binding to membranes. Knowledge of the gene structure and the associated sequence information should facilitate analysis of the involvement of PDE4A in hereditary disorders that may result from alterations in enzyme expression, activity, regulation and intracellular targeting and serve as a resource for determining authenticity of cloned PDE4A species.

Localization and genomic structure of human deoxyhypusine synthase gene on chromosome 19p13.2-distal 19p13.1.

The amino acid hypusine is formed post-translationally in a single cellular protein, the eukaryotic translation initiation factor 5A, by two enzymes, namely deoxyhypusine synthase and deoxyhypusine hydroxylase. Hypusine is found in all eukaryotes and in some archaebacteria, but not in eubacteria. The deoxyhypusine synthase cDNA was cloned and mapped by fluorescence in situ hybridization on chromosome 19p13.11-p13.12. Rare cDNAs containing internal deletions were also found. We localized the deoxyhypusine synthase gene on a high resolution cosmid/BAC contig map of chromosome 19 to a region in 19p13.2-distal 19p13.1 between MANB and JUNB. Analysis of the genomic exon/intron structure of the gene coding region showed that it consists of nine exons and spans a length of 6.6kb. From observation of the genomic structure, it seems likely that the internally deleted forms of mature RNA are the result of alternative splicing, rather than of artifacts.

Location of mouse and human genes corresponding to conserved canine olfactory receptor gene subfamilies.

Olfactory receptors are G protein-coupled, seven-transmembrane-domain proteins that are responsible for binding odorants in the nasal epithelium. They are encoded by a large gene family, members of which are organized in several clusters scattered throughout the genomes of mammalian species. Here we describe the mapping of mouse sequences corresponding to four conserved olfactory receptor genes, each representing separate, recently identified canine gene subfamilies. Three of the four canine genes detected related gene clusters in regions of mouse Chromosomes (Chrs) 2, 9, and 10, near previously mapped mouse olfactory genes, while one detected a formerly unidentified gene cluster located on mouse Chr 6. In addition, we have localized two human gene clusters with homology to the canine gene, CfOLF4, within the established physical map of Chr 19p. Combined with recently published studies, these data link the four conserved olfactory gene subfamilies to homologous regions of the human, dog, and mouse genomes.

Regions of sex-specific hypo- and hyper-recombination identified through integration of 180 genetic markers into the metric physical map of human chromosome 19.

The order of and physical distance between 180 polymorphic markers, many from the Généthon and CHLC genetic maps, have been determined through inclusion of probe-positive cosmids in the metric physical map of human chromosome 19. The markers incorporated into the physical map include 38 genetic markers with heterozygosities of > 0.8 and approximately 120 markers with heterozygosities of > 0.60. The average distance between markers in this integrated map is approximately 320 kb. Clustering of markers is noted in several regions of the chromosome; only 11 intervals exist where the distance between markers is greater than 1 Mb, with the largest gap being 1.6 Mb. The ratio of sex-average genetic distance from the Généthon and CHLC genetic linkage maps to physical distance in the metric map is approximately 1.7 cM/Mb for the entire chromosome but ranges from 4 cM/Mb across the telomeric bands to 1 cM/Mb for the centromeric cytogenetic bands. The recombination distance in males is approximately twice that of females in the most telomeric bands but is only 10-25% of the activity observed in females in the more centromeric bands. Seven regions along the chromosome are noted where the recombination distance between markers in one sex is greater than 10 times the recombination distance in the other sex. The integration of genetic markers into the high-resolution physical map of human chromosome 19 provides a framework for isolation of disease genes and resources for studies of genome organization, such as regions of interesting recombinational activity.

A serine/threonine kinase gene defective in Peutz-Jeghers syndrome.

Studies of hereditary cancer syndromes have contributed greatly to our understanding of molecular events involved in tumorigenesis. Here we investigate the molecular background of the Peutz-Jeghers syndrome (PJS), a rare hereditary disease in which there is predisposition to benign and malignant tumours of many organ systems. A locus for this condition was recently assigned to chromosome 19p. We have identified truncating germline mutations in a gene residing on chromosome 19p in multiple individuals affected by PJS. This previously identified but unmapped gene, LKB1, has strong homology to a cytoplasmic Xenopus serine/threonine protein kinase XEEK1, and weaker similarity to many other protein kinases. Peutz-Jeghers syndrome is therefore the first cancer-susceptibility syndrome to be identified that is due to inactivating mutations in a protein kinase.

The human guanidinoacetate methyltransferase (GAMT) gene maps to a syntenic region on 19p13.3, homologous to band C of mouse chromosome 10, but GAMT is not mutated in jittery mice.

Guanindinoacetate methyltransferase (gene symbol, GAMT) catalyses the synthesis of creatine from guanidinoacetate and S-adensylmethionine. Pathological mutations in the coding region of GAMT were recently identified in two children with symptoms of muscular hypotonia, ataxia, seizures, and abnormal extrapyramidal movements. During contig construction in the telomeric region of human chromosome 19 we identified a cosmid clone carrying the entire GAMT gene. This clone was shown to overlap with cosmids from a contig that was previously mapped to chromosome 19p13.3. The human GAMT gene has a size of about 5 kb and consists of six exons which agree with the published cDNA sequence. Since the mouse mutations jittery/hesitant are located on band C of mouse chromosome 10 in a region of conserved synteny with 19p13.3 and jittery mice exhibit ataxia and abnormal movement behaviour, the genomic sequence of GAMT was determined in wild-type and jittery mice. The coding region of the GAMT gene, however, was not mutated in these mutant mice. Our linkage and sequence data will facilitate the identification of new GAMT mutations in patients suffering from an abnormal creatine metabolism.

Chromosomal localization of the human and mouse hyaluronan synthase genes.

We have recently identified a new vertebrate gene family encoding putative hyaluronan (HA) synthases. Three highly conserved related genes have been identified, designated HAS1, HAS2, and HAS3 in humans and Has1, Has2, and Has3 in the mouse. All three genes encode predicted plasma membrane proteins with multiple transmembrane domains and approximately 25% amino acid sequence identity to the Streptococcus pyogenes HA synthase, HasA. Furthermore, expression of any one HAS gene in transfected mammalian cells leads to high levels of HA biosynthesis. We now report the chromosomal localization of the three HAS genes in human and in mouse. The genes localized to three different positions within both the human and the mouse genomes. HAS1 was localized to the human chromosome 19q13.3-q13.4 boundary and Has1 to mouse Chr 17.HAS2 was localized to human chromosome 8q24.12 and Has2 to mouse Chr 15. HAS3 was localized to human chromosome 16q22.1 and Has3 to mouse Chr 8. The map position for HAS1 reinforces the recently reported relationship between a small region of human chromosome 19q and proximal mouse chromosome 17. HAS2 mapped outside the predicted critical region delineated for the Langer-Giedion syndrome and can thus be excluded as a candidate gene for this genetic syndrome.

Physical mapping of receptor type protein tyrosine phosphatase sigma (PTPRS) to human chromosome 19p13.3.

Receptor type protein tyrosine phosphatase sigma (PTPRS), a member of a subfamily of receptor type protein tyrosine phosphatases, has been mapped to human chromosome 19p13.3 using fluorescence in situ hybridization (FISH) analysis. Hybridization analysis of chromosome 19 library cosmids has revealed several positive clones that are part of a contig located in the same region. The location of this contig was verified since one of the cosmid clones is anchored to a high-resolution FISH map of human chromosome 19p. In addition, the location of this gene relative to previously mapped proximal markers reveals a new point in the human-mouse synteny map by extending the mouse chromosome 17 synteny region in the telomeric direction.

Physical and genetic mapping of novel microsatellite polymorphisms on human chromosome 19.

We describe here the identification of 11 novel microsatellite polymorphisms on human chromosome 19. These dinucleotide repeat polymorphisms were detected in chromosome 19-specific cosmids that were physically mapped by fluorescence in situ hybridization. For each repeat, flanking oligonucleotide primers were synthesized and the polymerase chain reaction assay was performed on a panel of 100 unrelated individuals to determine the heterozygosity and allele frequencies. To characterize these markers further, genetic and radiation hybrid maps were constructed. These microsatellite polymorphisms will be valuable in further linkage analysis of inherited diseases on chromosome 19p.

Assembly of a 1-Mb restriction-mapped cosmid contig spanning the candidate region for Finnish congenital nephrosis (NPHS1) in 19q13.1.

We describe the assembly of a 1-Mb cosmid contig and restriction map spanning the candidate region for Finnish congenital nephrosis (NPHS1) in 19q13.1. The map was constructed from 16 smaller contigs assembled by fingerprinting, a BAC and a PAC clone, and 42 previously unmapped cosmids. In most cases, single-step cosmid walks were sufficient to join two previously assembled contigs, and all but one gap was filled from this cosmid contig library. The remaining gap of about 19 kb was spanned with a single BAC and a single PAC clone. EcoRI mapping of a dense set of overlapping clones validated the assembly of the map and indicated a length of 1040 kb for the contig. This high-resolution clone map provides an ideal resource for gene identification through cDNA selection, exon trapping, and DNA sequencing.