An apoptotic defect in lens differentiation caused by human p53 is rescued by a mutant allele.

If deprived of wild-type p53 function, the body loses a guardian that protects against cancer. Restoration of p53 function has, therefore, been proposed as a means of counteracting oncogenesis. This concept of therapy requires prior knowledge with regard to proper balance of p53 function in a given target tissue. We have addressed this problem by targeting expression of the wild-type human p53 gene to the lens, a tissue entirely composed of epithelial cells that differentiate into elongated fiber cells. Transgenic mice expressing wild-type human p53 develop microphthalmia as a result of a defect in fiber formation that sets in shortly after birth. We see apoptotic cells that fail to undergo proper differentiation. In an effort to directly link the observed lens phenotype to the activity of the wild-type human p53 transgene, we also generated mice expressing a mutant human p53 allele that lacks wild-type function. A normal lens phenotype is restored in double transgenic animals that carry both wild-type and mutant human p53 alleles. Our study highlights the difficulties that can arise if p53 levels are improperly balanced in a differentiating tissue.

An n-allele model for progressive amplification in the FMR1 locus.

An n-allele model is developed for the FMR1 locus, which causes the fragile X syndrome, where n is the number of triplet repeats in the first exon. Frequencies in the general population and in index families are used to generate an n to n + delta transition matrix that predicts specific risks in satisfactory agreement with observation. However, until sequencing distinguishes between stable and unstable alleles with the same value of n, it is premature to infer whether allelic frequencies at the FMR1 locus are at equilibrium or, as some have suggested, are evolving toward higher frequencies of the pathogenic allele.

Interactions between quantitative trait loci in soybean in which trait variation at one locus is conditional upon a specific allele at another.

A large recombinant inbred population of soybean has been characterized for 220 restriction fragment-length polymorphism (RFLP) markers. Values for agronomic traits also have been measured. Quantitative trait loci (QTL) for height, yield, and maturity were located by their linkage to RFLP markers. QTL controlling large amounts of trait variation were analyzed for the dependence of trait variation on particular alleles at a second locus by comparing cumulative distributions of the trait for each genotype (four genotypes per pair of loci). Interesting pairs of loci were analyzed statistically with maximum likelihood and Monte Carlo comparison of additive and epistatic models. For each locus affecting height, variation was conditional upon the presence of a particular allele at a second unlinked locus that itself explained little or no trait variation. The results show that interactions between QTL are frequent and control large effects. Interactions distinguished between different QTL in a single linkage group and between QTL that affect different traits closely linked to one RFLP marker--i.e., distinguished between pleiotropy and closely linked genes. The implications for the evolution of inbreeding plants and for the construction of agronomic breeding strategies are discussed.

Uber die W-Flachen mit der Relation 2(R1 - R2) = sin 2 (R1 + R2) zwischen den Hauptkrummungsradien R1 und R2.

Thesis (doctoral)--K. Technische Hochschule Munchen.

Patched 1 conditional null allele in mice

The patched gene (Ptc) is a member of the hedgehog signaling pathway which plays a central role in the development of many invertebrate and vertebrate tissues. In addition, Ptc and a number of other pathway members are mutated in some common human cancers. Patched is the receptor for the hedgehog ligand and in the mouse ablation of the Ptc gene leads to developmental defects and an embryonic lethal phenotype. Here we describe a conditional Ptc allele in mice which will have utility for the temporospatial ablation of Ptc function. genesis 36:158-161, 2003. (C) 2003 Wiley-Liss, Inc.

Low frequency of CHEK2 1100delC allele in Australian multiple-case breast cancer families: functional analysis in heterozygous individuals

A protein-truncating variant of CHEK2, 1100delC, is associated with a moderate increase in breast cancer risk. We have determined the prevalence of this allele in index cases from 300 Australian multiple-case breast cancer families, 95% of which had been found to be negative for mutations in BRCA1 and BRCA2. Only two (0.6%) index cases heterozygous for the CHEK2 mutation were identified. All available relatives in these two families were genotyped, but there was no evidence of co-segregation between the CHEK2 variant and breast cancer. Lymphoblastoid cell lines established from a heterozygous carrier contained approximately 20% of the CHEK2 1100delC mRNA relative to wild-type CHEK2 transcript. However, no truncated CHK2 protein was detectable. Analyses of expression and phosphorylation of wild-type CHK2 suggest that the variant is likely to act by haploinsufficiency. Analysis of CDC25A degradation, a downstream target of CHK2, suggests that some compensation occurs to allow normal degradation of CDC25A. Such compensation of the 1100delC defect in CHEK2 might explain the rather low breast cancer risk associated with the CHEK2 variant, compared to that associated with truncating mutations in BRCA1 or BRCA2.

HLA and Genomewide Allele Sharing in Dizygotic Twins

Gametic selection during fertilization or the effects of specific genotypes on the viability of embryos may cause a skewed transmission of chromosomes to surviving offspring. A recent analysis of transmission distortion in humans reported significant excess sharing among full siblings. Dizygotic (DZ) twin pairs are a special case of the simultaneous survival of two genotypes, and there have been reports of DZ pairs with excess allele sharing around the HLA locus, a candidate locus for embryo survival. We performed an allele-sharing study of 1,592 DZ twin pairs from two independent Australian cohorts, of which 1,561 pairs were informative for linkage on chromosome 6. We also analyzed allele sharing in 336 DZ twin pairs from The Netherlands. We found no evidence of excess allele sharing, either at the HLA locus or in the rest of the genome. In contrast, we found evidence of a small but significant (P = .003 for the Australian sample) genomewide deficit in the proportion of two alleles shared identical by descent among DZ twin pairs. We reconciled conflicting evidence in the literature for excess genomewide allele sharing by performing a simulation study that shows how undetected genotyping errors can lead to an apparent deficit or excess of allele sharing among sibling pairs, dependent on whether parental genotypes are known. Our results imply that gene-mapping studies based on affected sibling pairs that include DZ pairs will not suffer from false-positive results due to loci involved in embryo survival.

The variant rpoS allele of S. enteritidis strain 27655R does not affect virulence in a chick model nor constitutive curliation but does generate a cold-sensitive phenotype

Adherence of pathogenic Escherichia coli and Salmonella spp. to host cells is in part mediated by curli fimbriae which, along with other virulence determinants, are positively regulated by RpoS. Interested in the role and regulation of curli (SEF17) fimbriae of Salmonella enteritidis in poultry infection, we tested the virulence of naturally occurring S. enteritidis PT4 strains 27655R and 27655S which displayed constitutive and null expression of curli (SEF17) fimbriae, respectively, in a chick invasion assay and analysed their rpoS alleles. Both strains were shown to be equally invasive and as invasive as a wild-type phage type 4 strain and an isogenic derivative defective for the elaboration of curli. We showed that the rpoS allele of 27655S was intact even though this strain was non-curliated and we confirmed that a S. enteritidis rpoS::strr null mutant was unable to express curli, as anticipated. Strain 27655R, constitutively curliated, possessed a frameshift mutation at position 697 of the rpoS coding sequence which resulted in a truncated product and remained curliated even when transduced to rpoS::strr. Additionally, rpoS mutants are known to be cold-sensitive, a phenotype confirmed for strain 27655R. Collectively, these data indicated that curliation was not a significant factor for pathogenesis of S. enteritidis in this model and that curliation of strains 27655R and 27655S was independent of RpoS. Significantly, strain 27655R possessed a defective rpoS allele and remained virulent. Here was evidence that supported the concept that different naturally occurring rpoS alleles may generate varying virulence phenotypic traits.

Peptide binding prediction for the human class II MHC allele HLA-DP2:a molecular docking approach

MHC class II proteins bind oligopeptide fragments derived from proteolysis of pathogen antigens, presenting them at the cell surface for recognition by CD4+ T cells. Human MHC class II alleles are grouped into three loci: HLA-DP, HLA-DQ and HLA-DR. In contrast to HLA-DR and HLA-DQ, HLA-DP proteins have not been studied extensively, as they have been viewed as less important in immune responses than DRs and DQs. However, it is now known that HLA-DP alleles are associated with many autoimmune diseases. Quite recently, the X-ray structure of the HLA-DP2 molecule (DPA*0103, DPB1*0201) in complex with a self-peptide derived from the HLA-DR a-chain has been determined. In the present study, we applied a validated molecular docking protocol to a library of 247 modelled peptide-DP2 complexes, seeking to assess the contribution made by each of the 20 naturally occurred amino acids at each of the nine binding core peptide positions and the four flanking residues (two on both sides).

Toward prediction of class II mouse major histocompatibility complex peptide binding affinity:in silico bioinformatic evaluation using partial least squares, a robust multivariate statistical technique

The accurate identification of T-cell epitopes remains a principal goal of bioinformatics within immunology. As the immunogenicity of peptide epitopes is dependent on their binding to major histocompatibility complex (MHC) molecules, the prediction of binding affinity is a prerequisite to the reliable prediction of epitopes. The iterative self-consistent (ISC) partial-least-squares (PLS)-based additive method is a recently developed bioinformatic approach for predicting class II peptide−MHC binding affinity. The ISC−PLS method overcomes many of the conceptual difficulties inherent in the prediction of class II peptide−MHC affinity, such as the binding of a mixed population of peptide lengths due to the open-ended class II binding site. The method has applications in both the accurate prediction of class II epitopes and the manipulation of affinity for heteroclitic and competitor peptides. The method is applied here to six class II mouse alleles (I-Ab, I-Ad, I-Ak, I-As, I-Ed, and I-Ek) and included peptides up to 25 amino acids in length. A series of regression equations highlighting the quantitative contributions of individual amino acids at each peptide position was established. The initial model for each allele exhibited only moderate predictivity. Once the set of selected peptide subsequences had converged, the final models exhibited a satisfactory predictive power. Convergence was reached between the 4th and 17th iterations, and the leave-one-out cross-validation statistical terms - q2, SEP, and NC - ranged between 0.732 and 0.925, 0.418 and 0.816, and 1 and 6, respectively. The non-cross-validated statistical terms r2 and SEE ranged between 0.98 and 0.995 and 0.089 and 0.180, respectively. The peptides used in this study are available from the AntiJen database (http://www.jenner.ac.uk/AntiJen). The PLS method is available commercially in the SYBYL molecular modeling software package. The resulting models, which can be used for accurate T-cell epitope prediction, will be made freely available online (http://www.jenner.ac.uk/MHCPred).

A loss-of-function allele of OsHMA3 associated with high cadmium accumulation in shoots and grain of Japonica rice cultivars

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Novel Genetic Determinants of Diabetic Kidney Disease

Diabetic kidney disease (DKD) is a devastating diabetes complication, with known heritability not fully revealed by previous genetics studies. We performed the largest genome-wide association study of type 1 DKD to date, in a 13-cohort consortium of 15,590 individuals of European ancestry genotyped on the Illumina HumanCoreExome Beadchip, which allows exploration of coding variation in addition to genomic markers.

As prior work has shown that different characterizations of the DKD phenotype highlight distinct genetic associations, we investigated a spectrum of DKD definitions based on proteinuria and renal function criteria. Controls were DKD-free after a minimum of 15 years diabetes duration; cases had diabetes for at least 10 years prior to DKD diagnosis. We also performed a quantitative trait analysis of estimated glomerular filtration rate in all participants.

Our top finding was a missense mutation in COL4A3, rs55703767 (Asp326Tyr); the minor allele is common in Europeans (20%) and East Asians (13%) but not Africans (2%). This SNP had a genome-wide significant association with traditionally defined DKD (macroalbuminuria or end-stage renal disease [ESRD], (OR= 0.79, P=1.9×10-9), and a suggestive association with macroalbuminuria (OR= 0.79, P=1.6×10-6) and ESRD (OR= 0.79, P=4.5×10-5) individually. Though its PolyPhen score is 0.3 (benign), this SNP has been implicated as a splice site disruptor.

The COL4A3 gene encodes the alpha 3 subunit of Type IV collagen, the major structural component of basement membranes. Pathogenic mutations in COL4A3 have been identified in thin basement membrane nephropathy, familial focal segmental glomerulosclerosis, and Alport syndrome. A proxy (r2=0.6) for rs55703767 had no significant associations in the CKDGen consortium, suggesting its pathogenicity occurs solely in the setting of hyperglycemia.

By significantly increasing sample size we have discovered a novel locus underlying DKD risk, paving the way for better understanding of pathology, prevention, and treatment.