The effect of exercise and metformin treatment on circulating free DNA in pregnancy.

INTRODUCTION: Some pregnancy complications are characterized by increased levels of cell-free fetal (cffDNA) and maternal DNA (cfmDNA), the latter may also be elevated during physical strain. This study aims at assessing the impact of exercise and metformin intervention in pregnancy, and to compare the levels of cell free DNA in pregnant women with or without PCOS diagnosis. METHODS: Consecutive women from two previous randomized controlled trials in pregnancy were included. Women came from a trial with organized exercise vs. standard antenatal care in pregnancy and a trial of metformin vs. placebo in PCOS women. Levels of cffDNA, cfmDNA and cell-free total DNA (cftDNA) were measured by qPCR. RESULTS: Training in pregnancy did not affect the levels of cffDNA, cfmDNA or cftDNA. PCOS-women treated with metformin had lower levels of cfmDNA and cftDNA at week 32 (mean ± SD: 301 ± 162 versus 570 ± 337, p = 0.012, 345 ± 173 versus 635 ± 370, p = 0.019); otherwise the levels were comparable to PCOS-controls. Metformin-treated PCOS-women had higher cffDNA at inclusion, in the 1st trimester; later on in pregnancy the levels in the metformin and placebo groups were equal. A comparison of pregnant women in the exercise study (TRIP) to placebo-treated pregnant PCOS-women, showed the levels of cffDNA, cfmDNA or cftDNA during mid-pregnancy (weeks 18-36) to be equal. DISCUSSION: Training during pregnancy was not associated with altered levels of cffDNA cfmDNA or cftDNA, but metformin treatment may reduce cfmDNA and cftDNA in pregnant PCOS women.

Multiple alpha-synuclein gene polymorphisms are associated with Parkinson's disease in a Norwegian population.

OBJECTIVES: Previous studies have found associations between Parkinson's disease (PD) and polymorphisms located within both the alpha-synuclein gene (SNCA) promoter and other gene regions. Our aim was to study SNCA gene markers in a closely matched Norwegian PD population to examine the genetic relationship between different polymorphisms associated with the disease. METHODS: We genotyped seven single nucleotide polymorphisms (SNPs) located in the SNCA promoter and two SNPs in the 3' gene region and seven microsatellite markers located across the gene in a closely matched series of 236 PD patients and 236 controls. Linkage disequilibrium (LD) structure was examined, and association of single markers and gene haplotypes analyzed. RESULTS: Several markers located across the SNCA gene were associated with PD, including marker alleles associated with disease in previous studies (Rep1 263-bp allele, rs356165 and rs356219). CONCLUSION: LD between associated marker alleles located across the SNCA gene suggests that a single genetic effect might explain the previous reported association in the promoter and 3' regions.

Pigmentary switches in domestic animal species.

Although homogeneous pigmentation usually is observed in wild animals, most domestic animal species display a wide variety of coat colors. In fur animals, the coat color is an important production trait, and in other species such as cattle and sheep, the coat color is a major breed characteristic. Variability in coat color is seen both within and between breeds, and makes domesticated species unique for studying gene function and gene regulation of loci affecting pigmentation. In several species, mutations in the MC1-R gene have been shown to cause the dominant expression of black pigment. In fox, alleles of both the agouti and the MC1-R gene could cause eumelanin synthesis. In addition, a nonepistatic interaction between MC1-R and agouti has been observed, resulting in several different coat color phenotypes expressing a mixture of red and black pigmentation. Also in cattle and sheep, amino acid substitutions within the MC1-R explain the dominant inheritance of black pigmentation. Unlike the constitutively activated MC1-R found in the Alaska silver fox, dominant variants of the MC1-R found in cattle and sheep seem to be completely dominant with no antagonizing effect of agouti. MC1-R variants with premature stop codons are widespread in several cattle populations, indicating that this well-conserved gene has no other fundamental function beside pigmentation. Other well-established breed characteristics include distinct coat color patterns in which the distribution of melanocytes, partly regulated by the c-kit gene, seems to be involved.

Consensus and comprehensive linkage maps of bovine chromosome 24.

This study describes development of a consensus genetic linkage map of bovine chromosome 24 (BTA24). Eight participating laboratories contributed data for 58 unique markers including a total of 25 409 meioses. Eighteen markers, which were typed in more than one reference population, were used as potential anchors to generate a consensus framework map. The framework map contained 16 loci ordered with odds greater than 1000:1 and spanned 79.3 cM. Remaining markers were included in a comprehensive map relative to these anchors. The resulting BTA24 comprehensive map was 98.3 cM in length. Average marker intervals were 6.1 and 2.5 cM for framework and comprehensive maps, respectively. Marker order was generally consistent with previously reported BTA24 linkage maps. Only one discrepancy was found when comparing the comprehensive map with the published USDA-MARC linkage map. Integration of genetic information from different maps provides a high-resolution BTA24 linkage map.

A genome scan for quantitative trait loci affecting milk production in Norwegian dairy cattle.

An autosomal genome scan for quantitative trait loci (QTL) affecting milk production traits was carried out on the Norwegian Dairy Cattle population. Six half-sibling families with a total of 285 sons organized according to a granddaughter design were analyzed by a multiple marker regression method. Suggestive QTL for one or several of the five milk traits (milk yield, protein percentage, protein yield, fat percentage and fat yield) were detected on chromosomes 3, 5, 6, 11, 13, 18 and 20. Among these results, the findings on chromosomes 3, 6, and 20 are highly supported by literature. The most convincing result was found close to marker FBN9 on chromosome 6, where a QTL was detected with alleles that cause a marked reduction in both protein and fat percentages and an increase in milk yield. The results for fat and protein percentage were highly significant even after accounting for multiple testing across the genome. Using bootstrapping, a 95% confidence interval for the position of the QTL for the percentage traits on chromosome 6 was estimated to 16 cM.

Quantitative trait loci affecting clinical mastitis and somatic cell count in dairy cattle.

Norway has a field recording system for dairy cattle that includes recording of all veterinary treatments on an individual animal basis from 1978 onwards. Application of these data in a genome search for quantitative trait loci (QTL) verified genome-wise significant QTL affecting clinical mastitis on Chromosome (Chr) 6. Additional putative QTL for clinical mastitis were localized to Chrs. 3, 4, 14, and 27. The comprehensive field recording system includes information on somatic cell count as well. This trait is often used in selection against mastitis when direct information on clinical mastitis is not available. The absence of common QTL positions for the two traits in our study indicates that the use of somatic cell count data in QTL studies aimed for reducing the incidence of mastitis should be carefully evaluated.

A primary screen of the bovine genome for quantitative trait loci affecting twinning rate.

An autosomal genome scan for quantitative trait loci (QTL) affecting twinning rate was carried out in the Norwegian Cattle population. Suggestive QTL were detected on Chromosomes (Chr) 5, 7, 12, and 23. Among these, the QTL positions on both Chr 5 and Chr 23 are strongly supported by literature in the field. Our results also confirm previous mapping of a QTL for twinning to Chr 7, but definitely suggest a different location of the QTL on this chromosome. The most convincing QTL peak was observed for a region in the middle part of Chr 5 close to the insulin-like growth factor 1 (IGF1) gene. Since IGF1 plays an important role in the regulation of folliculogenesis, a mutation search was performed by sequencing more than 3.5 kb of the gene in actual families. The sequencing revealed three polymorphisms in noncoding regions of the gene that will be important in fine structure mapping and characterization of the QTL.

Resolution of conflicting assignments for the bovine casein kinase II alpha (CSNK2A2) gene.

The casein kinase II alpha' gene (CSNK2A2), which physically maps to human chromosome 16 (HSA16), has previously been mapped to bovine chromosome 5 (BTA5). Based on these results, a new segment of homology between the human and bovine genomes was suggested. In this paper we demonstrate linkage between CSNK2A2 and several markers on BTA18. Our result is supported by the extensive conservation of synteny between HSA16q and BTA18. Bovine chromosome 18 markers used in this study included several microsatellites, as well as the MC1R gene previously mapped to HSA16q24.3. Sequencing of the PCR-fragment mapped to BTA5 reveals that a CSNK-like retroposon was responsible for the conflicting assignments. The present results further extend the observed conservation of synteny between HSA16q and BTA18.

Presence of the dominant extension allele E(D) in red and mosaic cattle.

The melanocyte-stimulating hormone receptor (MC1-R) is a central regulator of mammalian coat colour, encoded by the extension locus. In cattle, the dominant extension allele E(D) is associated with the production of black pigment in coloured areas. Genotyping of the MC1-R gene in a bull with mosaic expression of red vs. black pigment verified the existence of the E(D) allele, in spite of the fact that the majority of the animal is red coloured. No further mutations were found within the E(D) variant of the MC1-R gene, which was inherited from a completely red mother (genotype E(D)/e).

Bovine chromosome 4 workshop: consensus and comprehensive linkage maps.

The report of the bovine chromosome 4 (BTA4) workshop is presented. Six laboratories contributed a total of 30,168 informative meioses from 62 loci. Twenty-two loci were typed by at least two independent laboratories and were used to construct a consensus linkage map of BTA4. The remaining 40 loci were subsequently incorporated into a comprehensive map. The sex-averaged consensus map covered 131.4 cM. The female map was 124.3 cM in length, while the male map was 134.3 cM. The comprehensive sex-averaged map spanned 141.6 cM. The length of the female and male comprehensive maps were 123.1 cM and 156.4 cM, respectively. Average genetic distance between loci was 6 and 2.3 cM for the consensus and comprehensive linkage maps, respectively.

High-resolution gametic map of the sheep callipyge region: linkage heterogeneity among rams detected by sperm typing.

The callipyge locus (CLPG) causing muscular hypertrophy in domestic sheep has previously been mapped to the distal part of ovine chromosome 18. In this study, an accurate multipoint linkage map consisting of six microsatellite markers in this chromosomal region was constructed based on the analysis of 1145 single sperm cells. The best supported order of markers was OARHH47-ILSTS54-MCM38-CSSM18-IDVGA30-BM S1561. The log odds against the second most likely order, which has a reversal of the closely linked markers CSSM18 and IDVGA30, was 5.026. Sperm typing can be used to examine a large number of meioses in single individuals, and therefore, was exploited to study individual variability of recombination rate in rams of different callipyge genotypes. The results revealed statistically significant linkage heterogeneity among rams (P < 0.05) for marker interval OARHH47-CSSM18, with individual recombination fractions varying from 0.209 to 0.357.

Molecular and pharmacological characterization of dominant black coat color in sheep.

Dominant black coat color in sheep is predicted to be caused by an allele ED at the extension locus. Recent studies have shown that this gene encodes the melanocyte stimulating hormone receptor (MC1-R). In mouse and fox, naturally occurring mutations in the coding region of MC1-R produce a constitutively activated receptor that switches the synthesis from phaeomelanin to eumelanin within the melanocyte, explaining the black coat color observed phenotypically. In the sheep, we have identified a Met-->Lys mutation in position 73 (M73K) together with a Asp --> Asn change at position 121 (D121N) showing complete cosegregation with dominant black coat color in a family lineage. Only the M73K mutation showed constitutive activation when introduced into the corresponding mouse receptor (mMC1-R) for pharmacological analysis; however, the position corresponding to D121 in the mouse receptor is required for high affinity ligand binding. The pharmacological profile of the M73K change is unique compared to the constitutively active E92K mutation in the sombre mouse and C123R mutation in the Alaska silver fox, indicating that the M73K change activates the receptor via a mechanism distinct from these previously characterized mutations.

The melanocyte-stimulating hormone receptor (MC1-R) gene as a tool in evolutionary studies of artiodactyles.

The complete coding region of the melanocyte-stimulating hormone receptor (MC1-R) gene was characterized in species belonging to the two families Bovidae and Cervidae; cattle (Bos taurus), sheep (Ovis aries), goat (Capra hircus), muskox (Ovibos moschatus), roe deer (Capreolus capreolus), reindeer (Rangifer tarandus), moose (Alces alces), red deer (Cervus elaphus) and fallow deer (Dama dama). This well conserved gene is a central regulator of mammalian coat colour. Examination of the interspecies variability revealed a 5.3-6.8% divergence between the Cervidae and Bovidae families, whereas the divergence within the families were 1.0-3.1% and 1.2-4.6%, respectively. Complete identity was found when two subspecies of reindeer, Eurasian tundra reindeer (R.t. tarandus) and Svalbard reindeer (R.t. platvrhynehus), were analyzed. An rooted phylogenetic tree based on Bovidae and Cervidae MC1-R DNA sequences was in complete agreement with current taxonomy, and was supported by bootstrapping analysis. Due to different frequencies of silent vs. replacement mutations, the amino acid based phylogenetic tree contains several dissimilarities when compared to the DNA based phylogenetic tree.

Characterisation of porcine peroxisome proliferator-activated receptors gamma 1 and gamma 2: detection of breed and age differences in gene expression.

Two isoforms of peroxisome proliferator-activated receptor gamma (PPAR gamma) cDNAs, gamma 1 and gamma 2, have been isolated and characterised in swine. The relative expression of the two transcripts was studied by northern blot analysis using total RNA isolated from several porcine tissues taken at three different ages (day 1, after 5 weeks and at 100 kg weight). Hybridisation were carried out with two different probes, one binding to both PPAR gamma transcripts and the other being PPAR gamma 2 specific. Strongest hybridisation signals with the PPAR gamma probe binding both variants were detected in adipose tissues and spleen at all three ages, whereas only faint or no signals were detected in other tissues. The tissue distribution pattern of PPAR gamma 1 and gamma 2 suggests a modulation of tissue distribution for the two transcripts and obvious age and breed differences in gene expression in swine.

Mapping of bovine FcgammaR (FCGR) genes by sperm typing allows extended use of human map information.

Polymorphic sites within the bovine FcgammaRI (FCGR1), FcgammaRII (FCGR2), and FcgammaRIII (FCGR3) genes were used for proximal mapping of these genes to bovine Chromosome (Chr) 3 (BTA3) with paternal half-sib families from Norwegian Cattle. A fine-structure genetic map of the region was obtained by the analysis of 288 sperm cells from three bulls that were heterozygous for the loci included in the study. No recombinants were observed between FCGR2 and FCGR3 (242 sperm cells). Considering FCGR2 and FCGR3 as a single locus, a three-point linkage analysis for [FCGR2/FCGR3], FCGR1, and INRA003 was carried out. The best-supported order of the loci was found to be INRA003-FCGR1-[FCGR2/FCGR3]. Map distances in a two-point linkage analysis were 10.3 cM between [FCGR2/FCGR3] and FCGR1, and 25.5 cM between FCGR1 and INRA003, respectively. This linkage mapping of the bovine FCGR gene family resembles the human situation where all FCGR genes are located at Chr 1 (HSA1), at position q21-q24. Moreover, the results locate the evolutionary breakpoint between HSA1q and BTA3 within the human 1q24 region.

A non-epistatic interaction of agouti and extension in the fox, Vulpes vulpes.

Agouti and extension are two genes that control the production of yellow-red (phaeomelanin) and brown-black (eumelanin) pigments in the mammalian coat. Extension encodes the melanocyte-stimulating hormone receptor (MC1R) while agouti encodes a peptide antagonist of the receptor. In the mouse, extension is epistatic to agouti, hence dominant mutants of the MC1R encoding constitutively active receptors are not inhibited by the agouti antagonist, and animals with dominant alleles of both loci remain darkly pigmented. In the fox the proposed extension locus is not epistatic to the agouti locus. We have cloned and characterized the MC1R and the agouti gene in coat colour variants of the fox (Vulpes vulpes). A constitutively activating C125R mutation in the MC1R was found specifically in darkly pigmented animals carrying the Alaska Silver allele (EA). A deletion in the first coding exon of the agouti gene was found associated with the proposed recessive allele of agouti in the darkly pigmented Standard Silver fox (aa). Thus, as in the mouse, dark pigmentation can be caused by a constitutively active MC1R, or homozygous recessive status at the agouti locus. Our results, demonstrating the presence of dominant extension alleles in foxes with significant red coat colouration, suggest the ability of the fox agouti protein to counteract the signalling activity of a constitutively active fox MC1R.