The reliability of haplotyping inference in nuclear families: misassignment rates for SNPs and microsatellites

Single nucleotide polymorphisms (SNPs) are widely used when investigators try to map complex disease genes. Although biallelic SNP markers are less informative than microsatellite markers, one can increase their information content by using haplotypes. However, assigning haplotypes (i.e., assigning phase) correctly can be problematic in the presence of SNP heterozygosity. For example, a doubly heterozygous individual, with genotype 12, 12, could have haplotypes 1-1/2-2 or 1-2/2-1 with equal probability; in the absence of additional information, there is no way to determine which haplotype is correct. Thus an algorithm that assigns haplotypes to such an individual will assign the wrong one 50% of the time. We have studied the frequency of haplotype misassignments, i.e., haplotypes that are misassigned solely because of inherent marker ambiguity (not because of errors in genotyping or calculation). We examined both SNPs and microsatellite markers. We used the computer programs GENEHUNTER and SIMWALK to assign the haplotypes. We simulated (a) families with 1-5 children, (b) haplotypes involving different numbers of marker loci (3, 5, 7 and 10 loci, all in linkage equilibrium), and (c) different allele frequencies. Misassignment rates are highest (a) in small families, (b) with many SNP loci, and (c) for loci with the greatest heterozygosity (i.e., where both alleles have frequency 0.5). For example, for triads (i.e., one-child families with both parents genotyped), misassignment rates for SNPs can reach almost 50%. Family sizes of 4-5 children are required in order to ensure a misassignment frequency of < or = 5% for ten-SNP haplotypes with allele frequencies of 0.25-0.5. For microsatellites, a family size of at least 2-3 children is necessary to keep haplotyping misassignments < or = 5%. Finally, we point out that it is misleading for a computer program to yield haplotype assignments without indicating that they may have been misassigned, and we discuss the implications of these misassignments for association and linkage analysis.     

Cell differentiation: reciprocal regulation of Apaf-1 and the inhibitor of apoptosis proteins

The molecular mechanisms by which differentiated cells combat cell death and injury have remained unclear. In the current issue, it has been shown in neurons that cell differentiation is accompanied by a decrease in Apaf-1 and the activity of the apoptosome with an increased ability of the inhibitor of apoptosis proteins (IAPs) to sustain survival (Wright et al., 2004). These results, together with earlier ones, deepen our understanding of how cell death and the apoptosome are regulated during differentiation and in tumor cells.     

Project DyAdd: Fatty acids in adult dyslexia, ADHD, and their comorbid combination

In project DyAdd, we compared the fatty acid (FA) profiles of serum phospholipids in adults with attention deficit hyperactivity disorder (ADHD) (n=26), dyslexia (n=36), their comorbid combination (n=9), and healthy controls (n=36). FA proportions were analyzed in a 2×2 design with Bonferroni corrected post hoc comparisons. A questionnaire was used to assess dietary fat quality and use of supplements. Results showed that ADHD and dyslexia were not associated with total saturated FAs, monounsaturated FAs, or n-3 polyunsaturated FAs (PUFAs). However, those with ADHD had elevated proportions of total n-6 PUFAs (including γ-linolenic and adrenic acids) as compared to those without ADHD. Dyslexia was related to a higher proportion of monounsaturated nervonic acid and a higher ratio of n-6/n-3 PUFAs. Among females none of the associations were significant. However in males, all the original associations observed in all subjects remained and ADHD was associated with elevated nervonic acid and n-6/n-3 PUFA ratio like dyslexia. Controlling for poorly diagnosed reading difficulties, education, dietary fat quality, or use of FA supplements did not generally remove the originally observed associations.     

Polymorphisms within the HLA-DR3 haplotypes

HLA-DR molecules were isolated from eight different HLA-DR3 homozygous B-cell lines by immunoprecipitation with monoclonal antibodies, and they were subsequently analyzed by two-dimensional gel electrophoresis. We found that HLA-DR3 homozygous B-cell lines of consanguineous origin express two types of HLA-DR molecules. One type of HLA-DR molecule was present in all the cell lines tested, whereas the second DR molecule appears to be polymorphic. DNA isolated from the different HLA-DR3 homozygous cell lines was studied by Southern blot analysis to determine whether any DR restriction fragment length polymorphism could be observed. Polymorphisms detected at both the product and genomic level have been compared to each other, and their relations to the serological (HLA-DR) and cellular (HLA-D and LB-Q1) typing data will be discussed.No reprints available     

Life-stage-specific differences in exploitation of food mixtures: diet mixing enhances copepod egg production but not juvenile development

Development, egg production and hatching success of the calanoidcopepods Temora longicornis and Pseudocalanus elongatus weremeasured in food mixtures to test their ability to obtain acomplete nutrition by combining different nutritionally poorfood species. In all the food mixtures used, the copepods failedto moult past the first copepodite stage, and the mortalitywas high. In sharp contrast, mixing two nutritionally poor foodspecies often resulted in egg production which was not significantlydifferent from nutritionally high quality food, although hatchingsuccess in many mixtures was low. Whereas egg production wassignificantly correlated with particulate organic nitrogen inthe diet, and independent of the highly unsaturated fatty acids(HUFAs), eicosapentaenoic acid (EPA) and docosahexaenoic acid(DHA), hatching increased with increasing DHA and EPA concentration.Growth and juvenile mortality were, however, independent ofeither nitrogen or HUFAs in the diet. Our results show thatadult copepods are effective in combining their nutrition fromseveral food sources, whereas juveniles are not. We suggestthat there are species- and life-stage-specific differencesin nutritional requirements and/or in the ability to digestand/or assimilate essential nutrients from food mixtures, whichmay significantly contribute to the success of copepod populationsin nature.     

Increased production of xylanase by expression of a truncated version of the xyn11A gene from Nonomuraea flexuosa in Trichoderma reesei

We have previously shown that the Nonomuraea flexuosa Xyn11A polypeptides devoid of the carbohydrate binding module (CBM) have better thermostability than the full-length xylanase and are effective in bleaching of pulp. To produce an enzyme preparation useful for industrial applications requiring high temperature, the region encoding the CBM was deleted from the N. flexuosa xyn11A gene and the truncated gene was expressed in Trichoderma reesei. The xylanase sequence was fused to the T. reesei mannanase I (Man5A) signal sequence or 3' to a T. reesei carrier polypeptide, either the Man5A core/hinge or the cellulose binding domain (CBD) of cellobiohydrolase II (Cel6A, CBHII). The gene and fusion genes were expressed using the cellobiohydrolase 1 (cel7A, cbh1) promoter. Single-copy isogenic transformants in which the expression cassette replaced the cel7A gene were cultivated and analyzed. The transformants expressing the truncated N. flexuosa xyn11A produced clearly increased amounts of both the xylanase/fusion mRNA and xylanase activity compared to the corresponding strains expressing the full-length N. flexuosa xyn11A. The transformant expressing the cel6A CBD-truncated N. flexuosa xyn11A produced about 1.9 g liter-1 of the xylanase in laboratory-scale fermentations. The xylanase constituted about 25% of the secreted proteins. The production of the truncated xylanase did not induce the unfolded protein response (UPR) pathway. However, the UPR was induced when the full-length N. flexuosa xyn11A with an exact fusion to the cel7A terminator was expressed. We suggest that the T. reesei folding/secretion machinery is not able to cope properly with the bacterial CBM when the mRNA of the full-length N. flexuosa xyn11A is efficiently translated.     

Effects of weaning age and formic acid-based feed additives on pigs from weaning to slaughter

Two hundred and forty piglets were used in a 2 x 6 factorial experiment to study the effects of weaning age (26 or 36 d) and four formic acid-based feed additives on the performance of pigs from weaning to slaughter. Either formic acid (F) or a mixture of formic acid, propionic acid, and potassium sorbate (FPS) or a mixture of formic acid, propionic acid, and sodium benzoate (FPB) or formic acid in a diatomaceous earth carrier (FD) were added to the diets of weaned piglets (from weaning to 60 d of age) and growing (18-46 kg) and finishing pigs (46-107 kg) to provide 8, 6, and 6 g acid per kg feed, respectively. The negative control treatment's (C) diets contained no growth promoters, whereas the positive control treatment's weaner and grower diets were supplemented with 40 mg/kg of avilamycin (A). The piglets weaned at the age of 26 and 36 d weighed 7.6 and 10.7 kg at weaning (p < 0.001), and 18.5 and 17.9 kg at the age of 60 d (p > 0.05), respectively. There was a weaning age x feed additive interaction in the weight gain of piglets after weaning (p < 0.05). The weight gain of piglets weaned on day 26 was enhanced by A, FPS, and FD (p < 0.05), and that of piglets weaned on day 36 by A and FPB (p < 0.05). The feed conversion ratio was not affected by weaning ages but was decreased in groups A, F, FBS, and FPB (p < 0.05). The severity of post-weaning diarrhoea was less in groups A, F, FPS, and FD than in C (p < 0.05). In piglets weaned on day 26, faecal water content and the total Escherichia coli count were highest 9 d after weaning. The total E. coli count was reduced only by FD (p < 0.05). Increased faecal water content was characterized by increased faecal Na+ and decreased K+ concentrations. Weaning age did not influence performance or carcass quality in the growing-finishing pigs. Feed additives did not affect weight gain in the growing pigs, but FPS and FPB enhanced weight gain during finishing period and total fattening (p < 0.05). In summary, the pigs' growth performance from weaning to slaughter was not affected by weaning age but it was enhanced by mixtures of formic and propionic acids with small amounts of sorbate or benzoate.     

Binding specificity of monoclonal antibodies to ganglioside, Fuc-GM1

The binding specificity of thirteen mouse monoclonal antibodies reacting with Fuc-GM1, Fuc alpha 1-2Gal beta 1-3GalNAc beta 1-4(NeuAc alpha 2-3)-Gal beta 1-4Glc beta 1-1Cer, a ganglioside found to be associated with small cell lung carcinoma (O. Nilsson et al. (1984) Glycoconjugate J. 1, 43-49) was studied. The results are based upon radioimmunodetection of their binding to structurally related glycolipids adsorbed to microtiter plates or chromatographed on thin-layer plates. Four of thirteen antibodies reacted only with Fuc-GM1 and both the fucose and the sialic residues were necessary for binding. Optimal binding was obtained when the sialic acid was N-acetylneuraminic acid. When this sialic acid residue was substituted with N-glycoloylneuraminic acid the binding activity was reduced and up to 10-times more Fuc-GM1 was needed for detection. The ceramide composition did not influence the binding. The other nine monoclonal antibodies cross-reacted with glycolipids containing structures closely related to Fuc-GM1 and differed from the specific ones by recognizing a smaller portion of the carbohydrate moiety in Fuc-GM1. These results indicate that anticarbohydrate monoclonal antibodies, recognizing structures involving a large proportion of the sugar in the glycolipid, possess a high specificity and might be useful for detection of tumor-associated ganglioside antigen.     

Detecting early kidney damage in horses with colic by measuring matrix metalloproteinase -9 and -2, other enzymes,urinary glucose and total proteins

Background  

The aim of the study was to investigate urine matrix metalloproteinase (MMP-2 and -9) activity, alkaline phosphatase/creatinine (U-AP/Cr) and gamma-glutamyl-transpeptidase/creatinine (U-GGT/Cr) ratios, glucose concentration, and urine protein/creatinine (U-Prot/Cr) ratio and to compare data with plasma MMP-2 and -9 activity, cystatin-C and creatinine concentrations in colic horses and healthy controls. Horses with surgical colic (n = 5) were compared to healthy stallions (n = 7) that came for castration. Blood and urine samples were collected. MMP gelatinolytic activity was measured by zymography.