Recombination sites in the HLA class II region are haplotype dependent

We have analyzed DNA sequence polymorphisms of DQ alpha and DQ beta chains from three haplotypes from the DRw52 family: DR5 DQw1 (FPA, GM3106), DRw6 DQw1 (CB6B, 10w9060), and DRw6 DQw3 (AMALA, 10w9064). The results indicate that the DR5 DQw1 and DRw6 DQw1 haplotypes have arisen by recombination between the DR beta 1 and DQ alpha loci. This contrasts with our previous analysis of DR4 DQ"Wa", DR3 DQ"Wa", and DR7 DQw3 haplotypes, all of which appear to have arisen by virtue of recombination between DQ alpha and DQ beta. Thus, there appear to be at least two different sites where recombination has occurred within the DR and DQ subregions. These differing patterns of recombination were interpreted in the context of the three major family groups of class II haplotypes, the DRw53, DRw52, and DR1/2 haplotype families. The data indicate that haplotypes from these family groups tend to undergo recombination at different locations. We propose that these differences in site of recombination are a reflection of differences in the molecular organization of the haplotypes belonging to each family group.     

Molecular characterization of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency: identification of a lys329 to glu mutation in the MCAD gene,and expression of inactive mutant enzyme protein in E. coli

Summary A series of experiments has established the molecular defect in the medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCAD) gene in a family with MCAD deficiency. Demonstration of intra-mitochondrial mature MCAD indistinguishable in size (42.5-kDa) from control MCAD, and of mRNA with the correct size of 2.4 kb, indicated a point-mutation in the coding region of the MCAD gene to be disease-causing. Consequently, cloning and DNA sequencing of polymerase chain reaction (PCR) amplified complementary DNA (cDNA) from messenger RNA of fibroblasts from the patient and family members were performed. All clones sequenced from the patient exhibited a single base substitution from adenine (A) to guanine (G) at position 985 in the MCAD cDNA as the only consistent base-variation compared with control cDNA. In contrast, the parents contained cDNA with the normal and the mutated sequence, revealing their obligate carrier status. Allelic homozygosity in the patient and heterozygosity for the mutation in the parents were established by a modified PCR reaction, introducing a cleavage site for the restriction endonuclease NcoI into amplified genomic DNA containing G⁹⁸⁵. The same assay consistently revealed A⁹⁸⁵ in genomic DNA from 26 control individuals. The A to G mutation was introduced into an E. coli expression vector producing mutant MCAD, which was demonstrated to be inactive, probably because of the inability to form active tetrameric MCAD. All the experiments are consistent with the contention that the G⁹⁸⁵ mutation, resulting in a lysine to glutamate shift at position 329 in the MCAD polypeptide chain, is the genetic cause of MCAD deficiency in this family. We found the same mutation in homozygous form in 11 out of 12 other patients with verified MCAD deficiency.     

Acyl-CoA: glycine N-acyltransferase: in vitro studies on the glycine conjugation of straight- and branched-chained acyl-CoA esters in human liver

Apparent kinetic constants (Km and Vmax values) were determined for human liver acyl-CoA: glycine acyltransferase (glycine-N-acylase) towards isobutyryl-CoA, 2-methyl butyryl-CoA, isovaleryl-CoA, butyryl-CoA, hexanoyl-CoA, octanoyl-CoA, and decanoyl-CoA. These acyl-CoA esters were selected because of their relevance to the human diseases with cellular accumulation of these esters, i.e., especially to metabolic defects in the acyl-CoA dehydrogenation steps of the branched-chain amino acids, lysine, 5-hydroxy lysine, tryptophan, and fatty acid oxidation pathways. With the acyl-CoA ester as the fixed substrate, the Km value for glycine ranged from 0.5 to 2.9 mole/liter, and with glycine as fixed substrate, the Km values for the acyl-CoA esters varied from 0.3 to 5.6 mmole/liter. It is concluded that the substrate concentration is decisive for the glycine conjugate formation and that the occurrence in urine of acylglycines reflects an intramitochondrial accumulation of the corresponding acyl-CoA ester.     

A novel association of DQ alpha and DQ beta genes in the DRw10 haplotype. Determination of a DQw1 specificity by the DQ beta-chain

The association of the class II genes of the DRw10 haplotype from a cell line, NASC, initiated from a member of a well characterized family, was analyzed by sequencing cDNA clones corresponding to DR beta I, DQ alpha, and DQ beta genes. An identical haplotype was also identified in the Raji cell line. In addition to typing as DRw10 and DQw1 with HLA typing sera both, the NASC and Raji cell lines were shown to react strongly with the monoclonal antibodies 109d6 (specific for DRw10 beta 1 and DRw53 beta 2 gene products) and Genox 3.5.3 (specific for DQw1) and exhibited the restriction fragment length polymorphism indicative of a DRw10, DQw1 haplotype. The DR beta 1 gene corresponding to the DRw10 specificity was found to have a first domain sequence different from all other DR beta I genes. Sequence analysis of the 3'-untranslated region of this DR beta-chain gene showed a significant divergence from the 3' untranslated region of the DRw53 family of haplotypes and a lesser divergence from that of the DRw52 and DR1/DR2 families. The sequence of the DQ beta genes corresponding to the DQw1 specificity in the DRw10 haplotype was found to be identical to the DQ beta gene from a DR1, DQw1 haplotype. Surprisingly, however, the DQ alpha gene did not resemble other DQw1-like DQ alpha genes, but was identical in sequence to the DQ alpha gene found in DR4 haplotypes. The novel association of DQ alpha and DQ beta genes in the DRw10 haplotype revealed in these studies may result from a double recombinational event. More consequentially, these studies strongly suggest that the DQw1 specificity recognized by Genox 3.5.3 is determined by the DQ beta chain and is not affected by the DQ alpha-chain.     

Characteristics of spontaneous and evoked motility in the isolated perfused porcine duodenum.

The aims of the study were to evaluate characteristics of spontaneous motility and of the ascending excitatory peristaltic reflex (AEPR) and intraluminal cross-sectional area in the isolated perfused porcine duodenum. The parameters were measured by an intraluminal catheter by use of the perfused side-hole technique and impedance planimetry. Respiratory parameters such as pH and oxygen consumption and the arterial perfusion pressure were monitored and did not vary significantly throughout the study time. Spontaneous motility was intense at the beginning but declined and disappeared within 45-90 min. It was abolished by atropine, epinephrine, and UK-14,304 (an alpha 2-adrenoceptor agonist). Secondary motility was evoked by intraluminal balloon distensions by raising the balloon pressure to 1.5 kPa for 1-min periods. Reproducible results regarding the AEPR, external balloon diameters to elicit the AEPR, and intraluminal cross-sectional area were obtained. The order of potency (pD2 values) for inhibition of the AEPR was the selective M3-receptor antagonist 4-DAMP greater than atropine greater than the selective M2-receptor antagonist AFDX-116 greater than the selective M1-receptor antagonist pirenzepine greater than hexamethonium. 4-DAMP was 16 and 29 times more potent than AFDX-116 (P less than 0.02) and pirenzepine (P less than 0.02). None of the drugs altered the intraluminal cross-sectional area during the balloon distensions. The model provides the opportunity for physiological and pharmacological studies of duodenal motility and duodenal cross-sectional area devoid of extrinsic neural and endocrine effects. The abolishment of the AEPR by atropine is caused by blockade of the M3-receptor in the porcine duodenum.     

Quantifying Missing Heritability at Known GWAS Loci

Recent work has shown that much of the missing heritability of complex traits can be resolved by estimates of heritability explained by all genotyped SNPs. However, it is currently unknown how much heritability is missing due to poor tagging or additional causal variants at known GWAS loci. Here, we use variance components to quantify the heritability explained by all SNPs at known GWAS loci in nine diseases from WTCCC1 and WTCCC2. After accounting for expectation, we observed all SNPs at known GWAS loci to explain more heritability than GWAS-associated SNPs on average (). For some diseases, this increase was individually significant: for Multiple Sclerosis (MS) () and for Crohn''s Disease (CD) (); all analyses of autoimmune diseases excluded the well-studied MHC region. Additionally, we found that GWAS loci from other related traits also explained significant heritability. The union of all autoimmune disease loci explained more MS heritability than known MS SNPs () and more CD heritability than known CD SNPs (), with an analogous increase for all autoimmune diseases analyzed. We also observed significant increases in an analysis of Rheumatoid Arthritis (RA) samples typed on ImmunoChip, with more heritability from all SNPs at GWAS loci () and more heritability from all autoimmune disease loci () compared to known RA SNPs (including those identified in this cohort). Our methods adjust for LD between SNPs, which can bias standard estimates of heritability from SNPs even if all causal variants are typed. By comparing adjusted estimates, we hypothesize that the genome-wide distribution of causal variants is enriched for low-frequency alleles, but that causal variants at known GWAS loci are skewed towards common alleles. These findings have important ramifications for fine-mapping study design and our understanding of complex disease architecture.     

Remodelling of the zero-stress state and residual strains in apoE-deficient mouse aorta

Atherosclerosis is the most frequent cause of death and severe chronic disability in North America and Europe. The atherosclerosis-prone apolipoprotein E (apoE)-deficient mice contain the entire spectrum of lesions observed during atherogenesis. Significant remodelling of the artery occurs in atherosclerosis. The aim was to study the remodelling of the zero-stress state of the aorta in apoE-deficient mice up to 56 weeks of age. Normal wild-type mice served as control groups. The mice were euthanised at ages 10, 28 and 56 weeks and tissue rings where excised from several locations along the aorta. The rings where photographed in the no-load state (without any external forces applied), then cut radially to obtain the zero-stress state and photographed again. The cross-sectional wall area and wall thickness increased over time in apoE-deficient mice compared to controls (P<0.001). The residual strains at the inner and outer surface varied as function of aortic location both in controls and apoE-deficient mice (P<0.001). From age 28 to age 56 weeks a gradual increase in positive strain at the outer surface and negative strain at the inner surface was found in the apoE-deficient mice when compared to age-matched control mice (P<0.001). Furthermore, the inner residual strain in the plaque location was significantly smaller than in the non-plaque location in the rings with atherosclerotic plaques (P<0.001). The change over time of the opening angle was especially pronounced in the aortic arch. The opening angle increased to app. 200 degrees in the aortic arch in apoE-deficient mice at 56 weeks of age whereas it in age-matched controls was app. 125 degrees. Correspondingly, atherosclerotic plaques were prominent in the apoE-deficient mice, especially at week 56 in the ascending aorta and the aortic arch. In conclusion, a pronounced remodelling of the biomechanical properties in aorta was found in apoE-deficient mice. The stress gradient across the vessel wall in the plaque region is likely larger in vivo due to the smaller residual strain in the plaque area.