Sarah E. Lloyd

Genealogies of mouse inbred strains.

The mouse is a prime organism of choice for modelling human disease. Over 450 inbred strains of mice have been described, providing a wealth of different genotypes and phenotypes for genetic and other studies. As new strains are generated and others become extinct, it is useful to review periodically what strains are available and how they are related to each other, particularly in the light of available DNA polymorphism data from microsatellite and other markers. We describe the origins and relationships of inbred mouse strains, 90 years after the generation of the first inbred strain. Given the large collection of inbred strains available, and that published information on these strains is incomplete, we propose that all genealogical and genetic data on inbred strains be submitted to a common electronic database to ensure this valuable information resource is preserved and used efficiently.

Characterization of Mutations in Patients with Multiple Endocrine Neoplasia Type 1

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by tumors of the parathyroids, pancreatic islets, and anterior pituitary. The MEN1 gene, on chromosome 11q13, has recently been cloned, and mutations have been identified. We have characterized such MEN1 mutations, assessed the reliability of SSCP analysis for the detection of these mutations, and estimated the age-related penetrance for MEN1. Sixty-three unrelated MEN1 kindreds (195 affected and 396 unaffected members) were investigated for mutations in the 2,790-bp coding region and splice sites, by SSCP and DNA sequence analysis. We identified 47 mutations (12 nonsense mutations, 21 deletions, 7 insertions, 1 donor splice-site mutation, and 6 missense mutations), that were scattered throughout the coding region, together with six polymorphisms that had heterozygosity frequencies of 2%-44%. More than 10% of the mutations arose de novo, and four mutation hot spots accounted for >25% of the mutations. SSCP was found to be a sensitive and specific mutational screening method that detected >85% of the mutations. Two hundred and one MEN1 mutant-gene carriers (155 affected and 46 unaffected) were identified, and these helped to define the age-related penetrance of MEN1 as 7%, 52%, 87%, 98%, 99%, and 100% at 10, 20, 30, 40, 50, and 60 years of age, respectively. These results provide the basis for a molecular-genetic screening approach that will supplement the clinical evaluation and genetic counseling of members of MEN1 families.

BSE prions propagate as either variant CJD‐like or sporadic CJD‐like prion strains in transgenic mice expressing human prion protein

Variant Creutzfeldt–Jakob disease (vCJD) has been recognized to date only in individuals homozygous for methionine at PRNP codon 129. Here we show that transgenic mice expressing human PrP methionine 129, inoculated with either bovine spongiform encephalopathy (BSE) or variant CJD prions, may develop the neuropathological and molecular phenotype of vCJD, consistent with these diseases being caused by the same prion strain. Surprisingly, however, BSE transmission to these transgenic mice, in addition to producing a vCJD‐like phenotype, can also result in a distinct molecular phenotype that is indistinguishable from that of sporadic CJD with PrPSc type 2. These data suggest that more than one BSE‐derived prion strain might infect humans; it is therefore possible that some patients with a phenotype consistent with sporadic CJD may have a disease arising from BSE exposure.

Human Prion Protein with Valine 129 Prevents Expression of Variant CJD Phenotype

Variant Creutzfeldt-Jakob disease (vCJD) is a unique and highly distinctive clinicopathological and molecular phenotype of human prion disease associated with infection with bovine spongiform encephalopathy (BSE)–like prions. Here, we found that generation of this phenotype in transgenic mice required expression of human prion protein (PrP) with methionine 129. Expression of human PrP with valine 129 resulted in a distinct phenotype and, remarkably, persistence of a barrier to transmission of BSE-derived prions on subpassage. Polymorphic residue 129 of human PrP dictated propagation of distinct prion strains after BSE prion infection. Thus, primary and secondary human infection with BSE-derived prions may result in sporadic CJD-like or novel phenotypes in addition to vCJD, depending on the genotype of the prion source and the recipient.

Identification of multiple quantitative trait loci linked to prion disease incubation period in mice

Polymorphisms in the prion protein gene are known to affect prion disease incubation times and susceptibility in humans and mice. However, studies with inbred lines of mice show that large differences in incubation times occur even with the same amino acid sequence of the prion protein, suggesting that other genes may contribute to the observed variation. To identify these loci we analyzed 1,009 animals from an F2 intercross between two strains of mice, CAST/Ei and NZW/OlaHSd, with significantly different incubation periods when challenged with RML scrapie prions. Interval mapping identified three highly significantly linked regions on chromosomes 2, 11, and 12; composite interval mapping suggests that each of these regions includes multiple linked quantitative trait loci. Suggestive evidence for linkage was obtained on chromosomes 6 and 7. The sequence conservation between the mouse and human genome suggests that identification of mouse prion susceptibility alleles may have direct relevance to understanding human susceptibility to bovine spongiform encephalopathy (BSE) infection, as well as identifying key factors in the molecular pathways of prion pathogenesis. However, the demonstration of other major genetic effects on incubation period suggests the need for extreme caution in interpreting estimates of variant Creutzfeldt–Jakob disease epidemic size utilizing existing epidemiological models.

Idiopathic low molecular weight proteinuria associated with hypercalciuric nephrocalcinosis in Japanese children is due to mutations of the renal chloride channel (CLCN5).

The annual urinary screening of Japanese children above 3 yr of age has identified a progressive proximal renal tubular disorder characterized by low molecular weight proteinuria, hypercalciuria, and nephrocalcinosis. The disorder, which has a familial predisposition and occurs predominantly in males, has similarities to three X-linked proximal renal tubular disorders that are due to mutations in the renal chloride channel gene, CLCN5. We have investigated four unrelated Japanese kindreds with this tubulopathy and have identified four different CLCN5 mutations (two nonsense, one missense, and one frameshift). These are predicted to lead to a loss of chloride channel function, and heterologous expression of the missense CLCN5 mutation in Xenopus oocytes demonstrated a 70% reduction in channel activity when compared with the wild-type. In addition, single-stranded conformation polymorphism (SSCP) analysis was found to be a sensitive and specific mutational screening method that detected > 75% of CLCN5 mutations. Thus, the results of our study expand the spectrum of clinical phenotypes associated with CLCN5 mutations to include this proximal renal tubular disorder of Japanese children. In addition, the mutational screening of CLCN5 by SSCP will help to supplement the clinical evaluation of the annual urinary screening program for this disorder.

HECTD2 Is Associated with Susceptibility to Mouse and Human Prion Disease

Prion diseases are fatal transmissible neurodegenerative disorders, which include Scrapie, Bovine Spongiform Encephalopathy (BSE), Creutzfeldt-Jakob Disease (CJD), and kuru. They are characterised by a prolonged clinically silent incubation period, variation in which is determined by many factors, including genetic background. We have used a heterogeneous stock of mice to identify Hectd2, an E3 ubiquitin ligase, as a quantitative trait gene for prion disease incubation time in mice. Further, we report an association between HECTD2 haplotypes and susceptibility to the acquired human prion diseases, vCJD and kuru. We report a genotype-associated differential expression of Hectd2 mRNA in mouse brains and human lymphocytes and a significant up-regulation of transcript in mice at the terminal stage of prion disease. Although the substrate of HECTD2 is unknown, these data highlight the importance of proteosome-directed protein degradation in neurodegeneration. This is the first demonstration of a mouse quantitative trait gene that also influences susceptibility to human prion diseases. Characterisation of such genes is key to understanding human risk and the molecular basis of incubation periods.

Identification of genetic loci affecting mouse-adapted bovine spongiform encephalopathy incubation time in mice

Abstract. Prion diseases are fatal neurodegenerative disorders of humans and animals, which include bovine spongiform encephalopathy (BSE) and its human form, variant Creutzfeldt-Jakob disease (vCJD). They are characterized by a prolonged incubation period, which is known to be influenced by polymorphisms in the prion protein gene. Previous studies of inbred mice have demonstrated that additional genetic loci also contribute to the observed variation in incubation period. However, a substantial transmission barrier between cow and mouse complicates studies using BSE. As a result, primary transmissions display large variations in incubation period and not all animals develop clinical signs of disease. To identify quantitative trait loci for BSE without the presence of a transmission barrier, we analysed 124 animals from an F2 intercross between CAST/Ei and NZW/OlaHsd mice and challenged them intracerebrally with a strain of BSE that was passaged twice through C57BL/6OlaHsd mice. Interval mapping identified two highly significant linked regions on chromosomes 2 and 11 with peak lod scores of 6.34 and 4.77, respectively. Composite interval mapping suggests that chromosome 2 includes three linked quantitative trait loci. Loci in the same position on chromosomes 2 and 11 were also identified in a previous study using the same mouse cross but infected with Chandler/RML scrapie prions. If these are the same loci, it suggests that these loci may be influencing incubation time independently of prion strain. This provides hope that it may be possible to identify human quantitative trait loci for prion incubation time using mouse models that may allow identification of at-risk individuals and the discovery of novel therapeutic targets.

Genetics of prion disease.

Prion diseases or transmissible spongiform encephalopathies (TSEs) are neurodegenerative disorders of humans and animals for which there are no effective treatments or cure. They include Creutzfeldt-Jakob disease (CJD) in humans and sheep scrapie, bovine spongiform encephalopathy (BSE) and chronic wasting disease (CWD) in cervids. The prion protein (PrP) is central to the disease process. An abnormal form of PrP is generally considered to be the sole or principal component of the infectious agent and a multimeric isomer (PrP(Sc)) is deposited in affected brains. Inherited prion diseases are caused by over 30 mutations in the prion protein gene (PRNP) and common polymorphisms can have a considerable affect on susceptibility and phenotype. Susceptibility and incubation time are also partly determined by other (non-PRNP) genetic modifiers. Understanding how these other genes modify prion diseases may lead to insights into biological mechanisms. Several approaches including human genome wide association studies (GWAS), mouse mapping and differential expression studies are now revealing some of these genes which include RARB (retinoic acid receptor beta), the E3 ubiquitin ligase HECTD2 and SPRN (Shadoo, shadow of prion protein gene).

Molecular Genetic Characterisation of Frontotemporal Dementia on Chromosome 3

We have previously localized a locus causing familial nonspecific dementia to the centromeric region of chromosome 3 in a pedigree from the Jutland area of Denmark. This pedigree shows anticipation. Here we present further analysis of these anticipation data which are suggestive of trinucleotide repeat expansion involvement. We also outline our strategies to clone the mutant gene via its putative associated trinucleotide repeat sequence.