Examining the Distribution and Impact of Single-Nucleotide Polymorphisms in the Capsular Locus of Streptococcus pneumoniae Serotype 19A.

Streptococcus pneumoniae serotype 19A prevalence has increased after the implementation of the PCV7 and PCV10 vaccines. In this study, we have provided, with high accuracy, the genetic diversity of the 19A serotype in a cohort of Dutch invasive pneumococcal disease patients and asymptomatic carriers obtained in the period from 2004 to 2016. The whole genomes of the 338 pneumococcal isolates in this cohort were sequenced and their capsule (cps) loci compared to examine their diversity and determine the impact on the production of capsular polysaccharide (CPS) sugar precursors and CPS shedding. We discovered 79 types with a unique cps locus sequence. Most variation was observed in the rmlB and rmlD genes of the TDP-Rha synthesis pathway and in the wzg gene, which is of unknown function. Interestingly, gene variation in the cps locus was conserved in multiple alleles. Using RmlB and RmlD protein models, we predict that enzymatic function is not affected by the single-nucleotide polymorphisms as identified. To determine if RmlB and RmlD function was affected, we analyzed nucleotide sugar levels using ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC-MS). CPS precursors differed between 19A cps locus subtypes, including TDP-Rha, but no clear correlation was observed. Also, significant differences in multiple nucleotide sugar levels were observed between phylogenetically branched groups. Because of indications of a role for Wzg in capsule shedding, we analyzed if this was affected. No clear indication of a direct role in shedding was found. We thus describe genotypic variety in rmlB, rmlD, and wzg in serotype 19A in the Netherlands, for which we have not discovered an associated phenotype.

Molecular basis for activation and biased signaling at the thrombin-activated GPCR proteinase activated receptor-4 (PAR4).

Proteinase-activated receptor (PAR)-4 is a member of the proteolytically-activated PAR family of G-protein-coupled receptors (GPCR) that represents an important target in the development of anti-platelet therapeutics. PARs are activated by proteolytic cleavage of their receptor N terminus by enzymes such as thrombin, trypsin, and cathepsin-G. This reveals the receptor-activating motif, termed the tethered ligand that binds intramolecularly to the receptor and triggers signaling. However, PARs are also activated by exogenous application of synthetic peptides derived from the tethered-ligand sequence. To better understand the molecular basis for PAR4-dependent signaling, we examined PAR4-signaling responses to a peptide library derived from the canonical PAR4-agonist peptide, AYPGKF-NH2, and we monitored activation of the Gαq/11-coupled calcium-signaling pathway, ß-arrestin recruitment, and mitogen-activated protein kinase (MAPK) pathway activation. We identified peptides that are poor activators of PAR4-dependent calcium signaling but were fully competent in recruiting ß-arrestin-1 and -2. Peptides that were unable to stimulate PAR4-dependent calcium signaling could not trigger MAPK activation. Using in silico docking and site-directed mutagenesis, we identified Asp230 in the extracellular loop-2 as being critical for PAR4 activation by both agonist peptide and the tethered ligand. Probing the consequence of biased signaling on platelet activation, we found that a peptide that cannot activate calcium signaling fails to cause platelet aggregation, whereas a peptide that is able to stimulate calcium signaling and is more potent for ß-arrestin recruitment triggered greater levels of platelet aggregation compared with the canonical PAR4 agonist peptide. These findings uncover molecular determinants critical for agonist binding and biased signaling through PAR4.

Genome-wide association study of body morphological traits in Sudanese goats.

Long-term selection of goats for a certain production system and/or different environmental conditions will be reflected in the body morphology of the animals under selection. To investigate the variation contributing to different morphological traits and to identify genomic regions that are associated with body morphological traits in Sudanese goats, we genotyped 96 females belonging to four Sudanese goat breeds with the SNP52 BeadChip. After quality control of the data, the genome-wide association study was performed using 95 goats and 24 027 informative single nucleotide polymorphisms (SNPs). Bicoastal diameter was significantly associated (LOD = 6.32) with snp10185-scaffold1365-620922 on chromosome 2. The minor allele has an additive effect, increasing the bicoastal diameter by 2.6 cm. A second significant association was found between body length and snp56482-scaffold89-467312 on chromosome 3 (LOD = 5.65). The minor allele is associated with increased body length. Additionally, five regions were suggestive for cannon bone, head width, rump length and withers height (LOD > 5). Only one gene (CNTNAP5) is located within the 1-Mb region surrounding the significant SNP for bicoastal diameter on chromosome 2. The body length QTL on chromosome 3 harbors 49 genes. Further research is required to validate the observed associations and to prioritize candidate genes.

Reducing the interval of a growth QTL on chromosome 4 in laying hens.

In our previous research, we identified a QTL with an interval of 3.4 Mb for growth on chicken chromosome (GGA) 4 in an advanced intercross population of an initial cross between the New Hampshire inbred line (NHI) and the White Leghorn inbred line (WL77). In the current study, an association analysis was performed in a population of purebred white layers (WLA) with White Leghorn origin. Genotypic data of 130 SNPs within the previously identified 3.4-Mb region were obtained using a 60K SNP chip. In total, 24 significant SNPs (LOD ≥ 4.44) on GGA4 were detected for daily weigh gain from 8 to 14 weeks and two SNPs (LOD ≥ 4.80) for body weight at 14 weeks. The QTL interval was reduced by 1.9 Mb to an interval of 1.5 Mb (74.6-76.1 Mb) that harbors 15 genes. Furthermore, to identify additional loci for chicken growth, a genome-wide association study (GWAS) was carried out in a WLA population. The GWAS identified an additional QTL on GGA6 for body weight at six weeks (19.8-21.2 Mb). Our findings showed that by using a WLA population we were able to further reduce the QTL confidence interval previously detected using a NHI × WL77 advanced intercross population.

Genetic diversity of Syrian Arabian horses.

Although Arabian horses have been bred in strains for centuries and pedigrees have been recorded in studbooks, to date, little is known about the genetic diversity within and between these strains. In this study, we tested if the three main strains of Syrian Arabian horses descend from three founders as suggested by the studbook. We examined 48 horses representing Saglawi (n = 18), Kahlawi (n = 16) and Hamdani (n = 14) strains using the Equine SNP70K BeadChip. For comparison, an additional 24 Arabian horses from the USA and three Przewalski's horses as an out group were added. Observed heterozygosis (Ho ) ranged between 0.30 and 0.32, expected heterozygosity (He ) between 0.30 and 0.31 and inbreeding coefficients (Fis ) between -0.02 and -0.05, indicating high genetic diversity within Syrian strains. Likewise, the genetic differentiation between the three Syrian strains was very low (Fst  < 0.05). Hierarchical clustering showed a clear distinction between Arabian and Przewalski's horses. Among Arabian horses, we found three clusters containing either horses from the USA or horses from Syria or horses from Syria and the USA together. Individuals from the same Syrian Arabian horse strain were spread across different sub-clusters. When analyzing Syrian Arabian horses alone, the best population differentiation was found with three distinct clusters. In contrast to expectations from the studbook, these clusters did not coincide with strain affiliation. Although this finding supports the hypothesis of three founders, the genetic information is not consistent with the currently used strain designation system. The information can be used to reconsider the current breeding practice. Beyond that, Syrian Arabian horses are an important reservoir for genetic diversity.

Fine mapping of a distal chromosome 4 QTL affecting growth and muscle mass in a chicken advanced intercross line.

In our previous research, QTL analysis in an F2 cross between the inbred New Hampshire (NHI) and White Leghorn (WL77) lines revealed a growth QTL in the distal part of chromosome 4. To physically reduce the chromosomal interval and the number of potential candidate genes, we performed fine mapping using individuals of generations F10 , F11 and F12 in an advanced intercross line that had been established from the initial F2 mapping population. Using nine single nucleotide polymorphism (SNP) markers within the QTL region for an association analysis with several growth traits from hatch to 20 weeks and body composition traits at 20 weeks, we could reduce the confidence interval from 26.9 to 3.4 Mb. Within the fine mapped region, markers rs14490774, rs314961352 and rs318175270 were in full linkage disequilibrium (D' = 1.0) and showed the strongest effect on growth and muscle mass (LOD ≥ 4.00). This reduced region contains 30 genes, compared to 292 genes in the original region. Chicken 60 K and 600 K SNP chips combined with DNA sequencing of the parental lines were used to call mutations in the reduced region. In the narrowed-down region, 489 sequence variants were detected between NHI and WL77. The most deleterious variants are a missense variant in ADGRA3 (SIFT = 0.02) and a frameshift deletion in the functional unknown gene ENSGALG00000014401 in NHI chicken. In addition, five synonymous variants were discovered in genes PPARGC1A, ADGRA3, PACRGL, SLIT2 and FAM184B. In our study, the confidence interval and the number of potential genes could be reduced 8- and 10- fold respectively. Further research will focus on functional effects of mutant genes.

Milk protein polymorphisms and casein haplotypes in Butana cattle.

Butana is a Bos indicus dairy cattle breed that is well adapted to the local environment of Sudan. The breed has been gradually declining in number due to breed substitution. Therefore, conservation and improvement strategies are required to maintain this breed. The aim of the present study was to assess genetic variation that is characteristic for Butana cattle in the milk protein genes CSN1S1, CSN2, CSN1S2, CSN3, LALBA, and LGB. In a first step, genomic DNA of five unrelated individuals was comparatively sequenced across all exon and flanking sequences. Ninety-three single nucleotide polymorphisms (SNPs) were identified in Butana cattle compared with the Bos taurus reference sequence at Ensembl. We confirmed the recently identified protein variants CSN2*J, CSN2*L, and LALBA*E. Fifty-two SNPs in non-coding regions are novel. Among the novel SNPs, five are located in promoter regions, three of them are in putative transcription factor binding sites (TFBSs) of the CSN1S2 promoter. Fifteen SNPs potentially affect miRNA target sites. In a second step, 50 unrelated Butana cattle were genotyped. This allowed deriving haplotypes for the casein gene cluster on BTA6. The most frequent haplotype was CSN1S1*C-CSN2*A 2 -CSN1S2*A-CSN3*A (C-A 2 -A-A, frequency 0.1546). Considering the newly identified CSN1S2 promoter variants, the most frequent haplotype was C-A 2 -TTC-A-A (0.1046), with TTC as the promoter variant. The information on protein and promoter variants can be used for the development of conservation and breeding strategies for this local breed.

Fine mapping a major obesity locus (jObes1) using a Berlin Fat Mouse × B6N advanced intercross population.

BACKGROUND/OBJECTIVES: The Berlin Fat Mouse Inbred line 860 is a model for juvenile obesity. Previously, a recessive major effect locus (jObes1) on chromosome 3 between 34 and 44 Mb has been found to be responsible for 39% of the variance of total fat mass at 10 weeks in a (BFMI860 x C57BL/6NCrl) F2 population. The aim of this study was fine mapping of the jObes1 locus. SUBJECTS/METHODS: An advanced intercross line (AIL) was generated from the initial F2 mapping population. Three hundred and forty-four male mice of generation 28 were excessively phenotyped and genotyped using the MegaMuga mouse chip containing 22 164 informative single-nucleotide polymorphisms. Expression of candidate genes was investigated in gonadal adipose tissue, liver and whole brain from mice of different genotype classes. Classical genetic complementation tests were performed to test candidate genes. RESULTS: The high mapping resolution of the AIL reduced the confidence interval for jObes1 from 10 to 0.37 Mb between 36.48 and 36.85 Mb. This region was highly significantly (logarithm (base 10) of odds (LOD) score after Benjamini and Hochberg correction (LOD(BH))>50) associated with total fat mass starting at puberty (6 weeks). Male homozygous carriers of the jObese1 BFMI allele had 3 g more fat than the other genotypes. Surprisingly, this genotype class showed lower body mass until weaning at 3 weeks (LOD(BH)=3.2). The mapped interval contains four genes. Bbs7, the most likely candidate gene that also caused obesity in the complementation test was differentially expressed in all tissues examined, whereas the neighboring cyclin A2 (Ccna2) gene showed differential expression in gonadal adipose tissue. CONCLUSIONS: Using an AIL, the confidence interval for jObes1 could be 27-fold reduced by finding chromosomal recombinations. Although Bbs7 is the most likely obesity gene in the jObes1 region, neighboring genes cannot be entirely excluded. Further examinations are needed to enlighten the mechanism leading to physiological consequences on body mass and fat mass in juvenile animals.

High Variability of Insulin Sensitivity in Closely Related Obese Mouse Inbred Strains.

Obesity is one of several risk factors for insulin resistance and type 2 diabetes. Here we examined males of 6 obese mouse inbred lines derived from the Berlin Fat Mouse (BFM) outbred population with respect to insulin sensitivity and factors of the metabolic syndrome with focus on the skeletal muscle as a major target of insulin dependent glucose uptake.Males were kept on a rodent standard diet and several approaches were carried out to address insulin sensitivity, adiposity and lipids in the serum. Transcript and protein levels of several genes in the insulin signalling pathway were measured. 2 of the lines, BFMI860-12 and in particular BFMI861-S1, showed a markedly reduced insulin sensitivity already at the age of 20 weeks. BFMI861-S1 mice also displayed elevated liver triglyceride levels as a sign of lipid overload and ectopic fat storage. The analysis of the insulin signalling pathway in skeletal muscle provided evidence for low insulin receptor (INSR) and normal glucose 4 transporter (GLUT4) protein amounts in BFMI861-S1 mice, while BFMI860-12 mice showed increased INSR and very low GLUT4 protein amounts. Interestingly, the sublines BFMI860-S2 and BFMI861-S2, which are highly related to the former 2 lines, respectively, were inconspicuously insulin sensitive. The expected few genetic differences among the BFMI lines facilitate the identification of causal genetic variation. This study identified 2 mouse lines with different impairments of insulin signalling. These lines resemble useful models for studying mechanisms leading to the pathophysiology of the metabolic syndrome, in particular insulin resistance.

Importance of the left ventricular filling pressure on diastolic filling in idiopathic dilated cardiomyopathy.

Both mitral regurgitation and elevated left ventricular (LV) filling pressures may normalize or enhance rapid filling in patients with idiopathic dilated cardiomyopathy. To assess the individual effects of the LV filling pressure and mitral regurgitation, 33 normal subjects, 14 patients with cardiomyopathy and normal LV filling pressures (measured as mean pulmonary capillary pressure) and 26 patients with elevated LV filling pressures (greater than 15 mm Hg) were studied with transmitral spectral tracings derived from pulsed Doppler echocardiography. Both cardiomyopathy groups demonstrated similarly dilated left ventricles with reduced systolic dysfunction. Patients with cardiomyopathy and normal LV filling pressures had prolonged isovolumic relaxation periods and a reduced ratio of the rapid filling to atrial filling integrals. Patients with cardiomyopathy and elevated LV pressures demonstrated an increased peak rapid filling velocity (97 +/- 21 cm/s) and rapid filling fraction (74.8 +/- 16.2%) compared with normal subjects (80 +/- 16 cm/s, p less than 0.01; 62.4 +/- 12.5%, p less than 0.05) and patients with cardiomyopathy and normal LV filling pressures (81 +/- 27 cm/s, p less than 0.05; 59.3 +/- 8.8%, p less than 0.05). Conversely, the atrial filling fraction was decreased in the cardiomyopathy group with elevated LV filling pressures compared with normal subjects and patients with cardiomyopathy and normal LV filling pressures. Mitral regurgitation increased the peak rapid filling velocity in both cardiomyopathy groups without altering the distribution of diastolic filling. In conclusion, elevated LV filling pressures appear to affect the distribution of diastolic filling, whereas mitral regurgitation affects the peak rate of rapid filling.