A set of eleven pig breeds originating from six European countries, and including a small sample of wild pigs, was chosen for this study of genetic diversity. Diversity was evaluated on the basis of ...18 microsatellite markers typed over a total of 483 DNA samples collected. Average breed heterozygosity varied from 0.35 to 0.60. Genotypic frequencies generally agreed with Hardy-Weinberg expectations, apart from the German Landrace and Schwäbisch-Hällisches breeds, which showed significantly reduced heterozygosity. Breed differentiation was significant as shown by the high among-breed fixation index (overall F(ST)= 0.27), and confirmed by the clustering based on the genetic distances between individuals, which grouped essentially all individuals in 11 clusters corresponding to the 11 breeds. The genetic distances between breeds were first used to construct phylogenetic trees. The trees indicated that a genetic drift model might explain the divergence of the two German breeds, but no reliable phylogeny could be inferred among the remaining breeds. The same distances were also used to measure the global diversity of the set of breeds considered, and to evaluate the marginal loss of diversity attached to each breed. In that respect, the French Basque breed appeared to be the most "unique" in the set considered. This study, which remains to be extended to a larger set of European breeds, indicates that using genetic distances between breeds of farm animals in a classical taxonomic approach may not give clear resolution, but points to their usefulness in a prospective evaluation of diversity.
Plasma cobalamin is requested in order to diagnose cobalamin deficiency and low levels confirm a deficient state. Here, we present three family members with unexpected high levels of cobalamin.
We ...included a patient referred for cobalamin measurement due to neurological symptoms, her son and her daughter. Mother and son both suffered from myotonic dystrophy type II, while the daughter tested negative for this disease. Blood samples were analyzed for cobalamin, haptocorrin, transcobalamin, holoTC, and sCD320. We employed gel filtration and antibody precipitation for further characterization. The protein coding region of the TCN2 gene, encoding transcobalamin, was sequenced.
The patient, her {son} and daughter all had cobalamin levels above the measurement range of the routine method employed (>1476 pmol/L). Total transcobalamin and (holoTC) were 5980 (1500), {5260 (2410)} and 5630 (1340) pmol/L, which is well above the upper reference limits of 1500 (160) pmol/L. The sCD320 concentration was also well above the upper reference limit of 97 arb.u.: 1340, {1510} and 1090 arb.u. Haptocorrin levels were within the reference range and no signs of cobalamin deficiency were found. DNA sequencing of the TCN2 gene revealed several known polymorphisms not associated with highly elevated transcobalamin levels. Upon gel filtration, sCD320 eluted as a larger molecule than previously reported. By incubation with anti-transcobalamin antibodies, we precipitated both transcobalamin and part of sCD320.
The high cobalamin levels were mainly explained by high levels of holoTC, possibly caused by complex formation with its soluble receptor, sCD320. The family occurrence points to a genetic explanation.
The serum-angiotensin I converting enzyme (s-ACE) activity is influenced by a genetic insertion/deletion (I/D) polymorphism in the ACE gene, and the resulting large interindividual variation in s-ACE ...limits the use of normal reference intervals in the evaluation of sarcoidosis. In this study, we developed a new method for genotyping the I/D polymorphism in ACE and established genotype-specific reference intervals in order to improve the diagnostic accuracy and the value for treatment of sarcoidosis.
The new genotyping assay is based on high-resolution melting (HRM) using LCGreen + and was used to genotype 400 healthy Danish individuals. The assay was compared to a real-time polymerase chain reaction (RT-PCR) assay in a validation set of 86 samples. Enzyme activity in serum was measured using the Infinity™ ACE Liquid Stable Reagent from Thermo adapted for the ABX Pentra analyzer.
There was full concordance between genotyping assays. The three genotypes II, ID and DD were present with a frequency of 0.23, 0.51 and 0.26. The distribution of s-ACE values in the total population was non-Gaussian (non-parametric 95% reference interval 12.0-60.0 U/L). The median activities of the genotypes differed significantly (P<0.0001). Ninety-five per cent non-parametric reference intervals for the subpopulations were determined to 6.3-38.5, 14.0-56.0 and 23.3-71.2 U/L for II, ID and DD, respectively.
We have developed a simple and robust method for ACE genotyping and determined genotype-specific reference intervals for s-ACE concentrations in the Danish population. The new reference intervals may increase the value of s-ACE measurements.
This investigation examined the interaction between carotid baroreflex (CBR) responsiveness during head-up tilt (HUT)-induced
central hypovolaemia and aerobic fitness. Seven average fit (AF) ...individuals, with a mean maximal oxygen uptake ( ) of 49 ± 1 (ml O 2 ) kg â1 min â1 , and seven high fit (HF) individuals, with a of 61 ± 1 (ml O 2 ) kg â1 min â1 , voluntarily participated in the investigation. After 10â15 min supine, each subject was exposed to nine levels of progressively
increasing HUT by 10 deg increments from â20 deg to +60 deg. During the final 3 min of each stage of HUT, the CBR responsiveness
was measured using a rapid pulse (500 ms) train of neck pressure (NP) and neck suction (NS) ranging from +40 to â80 Torr.
The maximal gain of the carotid-HR ( G max -HR) and carotid-MAP ( G max -MAP) baroreflex function curves was identified as measures of CBR responsiveness. During HUT-induced decreases in thoracic
admittance, an index of central blood volume (CBV), the G max -HR and G max -MAP of the AF subjects increased more than the G max -HR and G max -MAP of the HF subjects ( P < 0.05). The data demonstrate that the increase in the CBR responsiveness during a tilt-induced progressive unloading of
the cardiopulmonary baroreceptors was attenuated in endurance-trained subjects. These findings provide an explanation for
the predisposition to orthostatic hypotension and intolerance in well-trained athletes.
Transcobalamin (TC) deficiency (OMIM# 275350) is a rare, autosomal recessive disorder that presents in early infancy with a broad spectrum of symptoms, including failure to thrive, megaloblastic ...anemia, immunological deficiency, and neurological symptoms. Here we report a study of a family (parents and three children) with two children suffering from TC deficiency caused by two different mutations in the
TCN2
gene. Initially, molecular genetic analysis of genomic DNA revealed a heterozygous mutation in the +1 position of exon 7 (c.1106+1 G > A) in the father and all three children. Bioinformatic analysis indicates that this mutation causes exon skipping, and further experiments supported this hypothesis and suggested that the mutant allele undergoes nonsense-mediated messenger RNA (mRNA) decay. We did not identify further mutations in genomic DNA that could explain TC deficiency in the two children. However, further efforts using complementary DNA (cDNA) derived from RNA from blood leukocytes identified a large deletion removing the entire exon 8, resulting in a frameshift and a premature stop codon (p.E371fsX372) in the mother and the two affected children. Our data indicate that if exon-by-exon DNA sequencing of genomic DNA does not uncover mutations corresponding to the phenotype, a systematic search for other mutations should be initiated by sequencing cDNA or using semiquantitative methods to detect large deletions in
TCN2
.
Bovine spinal dysmyelination (BSD) is a recessive congenital neurodegenerative disease in cattle (
Bos taurus
) characterized by pathological changes of the myelin sheaths in the spinal cord. The ...occurrence of BSD is a longstanding problem in the American Brown Swiss (ABS) breed and in several European cattle breeds upgraded with ABS. Here, we show that the disease locus on bovine chromosome 11 harbors the
SPAST
gene that, when mutated, is responsible for the human disorder hereditary spastic paraplegia (HSP). Initially,
SPAST
encoding Spastin was considered a less likely candidate gene for BSD since the modes of inheritance as well as the time of onset and severity of symptoms differ widely between HSP and BSD. However, sequence analysis of the bovine
SPAST
gene in affected animals identified a R560Q substitution at a position in the ATPase domain of the Spastin protein that is invariant from insects to mammals. Interestingly, three different mutations in human
SPAST
gene at the equivalent position are known to cause HSP. To explore this observation further, we genotyped more than 3,100 animals of various cattle breeds and found that the glutamine allele exclusively occurred in breeds upgraded with ABS. Furthermore, all confirmed BSD carriers were heterozygous, while all affected calves were homozygous for the glutamine allele consistent with recessive transmission of the underlying mutation and complete penetrance in the homozygous state. Subsequent analysis of recombinant Spastin in vitro showed that the R560Q substitution severely impaired the ATPase activity, demonstrating a causal relationship between the
SPAST
mutation and BSD.
Summary
Background Mutation screening of the CASR by DNA sequencing is commonly used in the diagnosis of disorders of calcium metabolism, such as familial hypocalciuric hypercalcaemia (FHH). Exon ...copy number variation is not detected by currently used molecular genetic screening methods, and might be a genetic cause of inherited forms of hyper‐ or hypocalcaemia caused by the CASR.
Objective We wanted to further evaluate possible genetic causes for disorders of calcium metabolism, by investigating the prevalence of exon copy number variations, such as large deletions or duplications of the CASR.
Patients and methods The study included 257 patient samples referred to our laboratory for molecular genetic analysis of the CASR gene. A total of 245 were patients suspected to have FHH, while the remaining 12 samples represent patients with a phenotype of idiopathic hypocalcaemia/hypoparathyroidism. All samples were previously found negative for CASR mutations. Multiplex ligation‐dependent probe amplification was used to screen the patients for exon copy number variations.
Results All exons were amplified with mean normalised ratios between 0·98 and 1·06. We did not identify any exon copy number variation in the CASR. Bioinformatic analyses revealed that the CASR gene contains 52% repeated elements, of which approximately 6% consist of Alu elements.
Conclusions The present study indicates that including CASR MLPA analysis as a routine part of the diagnostic setup is not necessary, but could still be of interest in cases with a clear family history and no evidence of missense mutations in the CASR gene.
Background & Aims Acute liver failure (ALF) often results in cardiovascular instability, renal failure, brain oedema and death either due to irreversible shock, cerebral herniation or development of ...multiple organ failure. High-volume plasma exchange (HVP), defined as exchange of 8–12 or 15% of ideal body weight with fresh frozen plasma in case series improves systemic, cerebral and splanchnic parameters. Methods In this prospective, randomised, controlled, multicentre trial we randomly assigned 182 patients with ALF to receive either standard medical therapy (SMT; 90 patients) or SMT plus HVP for three days (92 patients). The baseline characteristics of the groups were similar. The primary endpoint was liver transplantation-free survival during hospital stay. Secondary-endpoints included survival after liver transplantation with or without HVP with intention-to-treat analysis. A proof-of-principle study evaluating the effect of HVP on the immune cell function was also undertaken. Results For the entire patient population, overall hospital survival was 58.7% for patients treated with HVP vs. 47.8% for the control group (hazard ratio (HR), with stratification for liver transplantation: 0.56; 95% confidence interval (CI), 0.36–0.86; p = 0.0083). HVP prior to transplantation did not improve survival compared with patients who received SMT alone (CI 0.37 to 3.98; p = 0.75). The incidence of severe adverse events was similar in the two groups. Systemic inflammatory response syndrome (SIRS) and sequential organ failure assessment (SOFA) scores fell in the treated group compared to control group, over the study period ( p <0.001). Conclusions Treatment with HVP improves outcome in patients with ALF by increasing liver transplant-free survival. This is attributable to attenuation of innate immune activation and amelioration of multi-organ dysfunction.
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