Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease

Crohn's disease and ulcerative colitis, the two main types of chronic inflammatory bowel disease, are multifactorial conditions of unknown aetiology. A susceptibility locus for Crohn's disease has been mapped to chromosome 16. Here we have used a positional-cloning strategy, based on linkage analysis followed by linkage disequilibrium mapping, to identify three independent associations for Crohn's disease: a frameshift variant and two missense variants of NOD2, encoding a member of the Apaf-1/Ced-4 superfamily of apoptosis regulators that is expressed in monocytes. These NOD2 variants alter the structure of either the leucine-rich repeat domain of the protein or the adjacent region. NOD2 activates nuclear factor NF-kB; this activating function is regulated by the carboxy-terminal leucine-rich repeat domain, which has an inhibitory role and also acts as an intracellular receptor for components of microbial pathogens. These observations suggest that the NOD2 gene product confers susceptibility to Crohn's disease by altering the recognition of these components and/or by over-activating NF-kB in monocytes, thus documenting a molecular model for the pathogenic mechanism of Crohn's disease that can now be further investigated.     

Type 1 diabetes in mice is linked to the interleukin-1 receptor and Lsh/Ity/Bcg genes on chromosome 1.

Human type 1 (insulin-dependent) diabetes is a common auto-immune disease of the insulin-producing beta cells of the pancreas which is caused by both genetic and environmental factors. Several features of the genetics and immunopathology of diabetes in nonobese diabetic (NOD) mice are shared with the human disease. Of the three diabetes-susceptibility genes, Idd-1 -3 and -4 that have been mapped in mice to date, only in the case of Idd-1 is there any evidence for the identity of the gene product: allelic variation within the murine immune response I-A beta gene and its human homologue HLA-DQB1 correlates with susceptibility, implying that I-A beta is a component of Idd-1. We report here the mapping of Idd-5 to the proximal region of mouse chromosome 1. This region contains at least two candidate susceptibility genes, the interleukin-1 receptor gene and Lsh/Ity/Bcg, which encodes resistance to bacterial and parasitic infections and affects the function of macrophages.     

Insulin-IGF2 region on chromosome 11p encodes a gene implicated in HLA-DR4-dependent diabetes susceptibility

A class of alleles at the VNTR (variable number of tandem repeat) locus in the 5' region of the insulin gene (INS) on chromosome 11p is associated with increased risk of insulin-dependent diabetes mellitus (IDDM), but family studies have failed to demonstrate linkage. INS is thought to contribute to IDDM susceptibility but this view has been difficult to reconcile with the lack of linkage evidence. We thus investigated polymorphisms of INS and neighbouring loci in random diabetics, IDDM multiplex families and controls. HLA-DR4-positive diabetics showed an increased risk associated with common variants at polymorphic sites in a 19-kilobase segment spanned by the 5' INS VNTR and the third intron of the gene for insulin-like growth factor II (IGF2). As INS is the major candidate gene from this region, diabetic and control sequence were compared to identify all INS polymorphisms that could contribute to disease susceptibility. In multiplex families the IDDM-associated alleles were transmitted preferentially to HLA-DR4-positive diabetic offspring from heterozygous parents. The effect was strongest in paternal meioses, suggesting a possible role for maternal imprinting. Our results strongly support the existence of a gene or genes affecting HLA-DR4 IDDM susceptibility which is located in a 19-kilobase region of INS-IGF2. Our results also suggest new ways to map susceptibility loci in other common diseases.     

Identification and mapping to chromosome 1 of a susceptibility locus for periinsulitis in non-obese diabetic mice

Insulin-dependent diabetes mellitus (IDDM) is a polygenic disease caused by autoimmune destruction of insulin-producing beta cells in the islets of Langerhans. Its onset is preceded by a long and variable period in which lymphoid cells infiltrate the pancreas but first remain outside the islets (peri-insulitis) before invading them (insulitis). Among susceptibility loci, only the major histocompatibility complex (MHC) has been clearly assigned. Genetic study of the nonobese diabetic (NOD) mouse model for insulin-dependent diabetes mellitus has revealed genetic linkage of insulitis and of early onset diabetes with two non-MHC loci mapping to chromosome 3 and 11 respectively. Here we report a close association of periinsulitis with a third non-MHC locus mapping to chromosome 1. Successive stages in the progression of diabetic disease thus appear to be controlled by distinct genes or sets of genes.     

Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer's disease.

A locus segregating with familial Alzheimer's disease (AD) has been mapped to chromosome 21, close to the amyloid precursor protein (APP) gene. Recombinants between the APP gene and the AD locus have been reported which seemed to exclude it as the site of the mutation causing familial AD. But recent genetic analysis of a large number of AD families has demonstrated that the disease is heterogeneous. Families with late-onset AD do not show linkage to chromosome 21 markers. Some families with early-onset AD show linkage to chromosome 21 markers, but some do not. This has led to the suggestion that there is non-allelic genetic heterogeneity even within early onset familial AD. To avoid the problems that heterogeneity poses for genetic analysis, we have examined the cosegregation of AD and markers along the long arm of chromosome 21 in a single family with AD confirmed by autopsy. Here we demonstrate that in this kindred, which shows linkage to chromosome 21 markers, there is a point mutation in the APP gene. This mutation causes an amino-acid substitution (Val----Ile) close to the carboxy terminus of the beta-amyloid peptide. Screening other cases of familial AD revealed a second unrelated family in which this variant occurs. This suggests that some cases of AD could be caused by mutations in the APP gene.     

The genetic defect in familial Alzheimer's disease is not tightly linked to the amyloid beta-protein gene

Amyloid beta-protein (AP) is a peptide of relative molecular mass (Mr) 42,000 found in the senile plaques, cerebrovascular amyloid deposits, and neurofibrillary tangles of patients with Alzheimer's disease and Down's syndrome (trisomy 21). Recent molecular genetic evidence has indicated that AP is encoded as part of a larger protein by a gene on chromosome 21 (refs 5-7). The defect in the inherited autosomal dominant form of Alzheimer's disease, familial Alzheimer's disease (FAD), has been mapped to the same approximate region of chromosome 21 by genetic linkage to anonymous DNA markers, raising the possibility that this gene product, which could be important in the pathogenesis of Alzheimer's disease, is also the site of the inherited defect in FAD (ref. 5). We have determined the pattern of segregation of the AP gene in FAD pedigrees using restriction fragment length polymorphisms. The detection of several recombination events with FAD suggests that the AP gene is not the site of the inherited defect underlying this disorder.     

Ryanodine receptor gene is a candidate for predisposition to malignant hyperthermia

Malignant hyperthermia (MH) is a potentially lethal condition in which sustained muscle contracture, with attendant hypercatabolic reactions and elevation in body temperature, are triggered by commonly used inhalational anaesthetics and skeletal muscle relaxants. In humans, the trait is usually inherited in an autosomal dominant fashion, but in halothane-sensitive pigs with a similar phenotype, inheritance of the disease is autosomal recessive or co-dominant. A simple and accurate non-invasive test for the gene is not available and predisposition to the disease is currently determined through a halothane- and/or caffeine-induced contracture test on a skeletal muscle biopsy. Because Ca2+ is the chief regulator of muscle contraction and metabolism, the primary defect in MH is believed to lie in Ca2+ regulation. Indeed, several studies indicate a defect in the Ca2+ release channel of the sarcoplasmic reticulum, making it a prime candidate for the altered gene product in predisposed individuals. We have recently cloned complementary DNA and genomic DNA encoding the human ryanodine receptor (the Ca2(+)-release channel of the sarcoplasmic reticulum) and mapped the ryanodine receptor gene (RYR) to region q13.1 of human chromosome 19 (ref. 14), in close proximity to genetic markers that have been shown to map near the MH susceptibility locus in humans and the halothane-sensitive gene in pigs. As a more definitive test of whether the RYR gene is a candidate gene for the human MH phenotype, we have carried out a linkage study with MH families to determine whether the MH phenotype segregates with chromosome 19q markers, including markers in the RYR gene. Co-segregation of MH with RYR markers, resulting in a lod score of 4.20 at a linkage distance of zero centimorgans, indicates that MH is likely to be caused by mutations in the RYR gene.     

Susceptibility to leprosy is associated with PARK2 and PACRG

Leprosy is caused by Mycobacterium leprae and affects about 700,000 individuals each year. It has long been thought that leprosy has a strong genetic component, and recently we mapped a leprosy susceptibility locus to chromosome 6 region q25-q26 (ref. 3). Here we investigate this region further by using a systematic association scan of the chromosomal interval most likely to harbour this leprosy susceptibility locus. In 197 Vietnamese families we found a significant association between leprosy and 17 markers located in a block of approx. 80 kilobases overlapping the 5' regulatory region shared by the Parkinson's disease gene PARK2 and the co-regulated gene PACRG. Possession of as few as two of the 17 risk alleles was highly predictive of leprosy. This was confirmed in a sample of 975 unrelated leprosy cases and controls from Brazil in whom the same alleles were strongly associated with leprosy. Variants in the regulatory region shared by PARK2 and PACRG therefore act as common risk factors for leprosy.     

Prevention of insulin-dependent diabetes mellitus in non-obese diabetic mice by transgenes encoding modified I-A beta-chain or normal I-E alpha-chain

Insulin-dependent diabetes mellitus (IDDM) is a disease with an autoimmune aetiology. The inbred non-obese diabetic (NOD) mouse strain provides a good animal model of the human disease and genetic analysis suggests that, as in man, at least one of the several genes controlling the development of IDDM is linked to the major histocompatibility complex. The NOD mouse does not express I-E owing to a deletion in the promoter region of the I-E alpha-chain gene, and the sequence of NOD I-A beta-chain in the first external domain is unique with His 56 and Ser 57 replacing Pro and Asp, respectively, at these positions. There has been considerable interest in the role amino acid 57 might have in conferring susceptibility to autoimmune diseases, including IDDM. The presence of a charged residue (such as Asp) at this position might affect the conformation of the peptide binding groove. But it could be assumed that Pro 56 gives rise to a different conformation of I-A beta-chain than does His 56. We therefore constructed transgenic NOD mice in which the transgene encoded a modified A beta nod with Pro 56, and studied its effect on the development of IDDM in this mouse strain. Previous studies have suggested that NOD mice expressing I-E as a result of the introduction of an I-E alpha-chain (E alpha) transgene are protected from the development of insulitis and hence IDDM. To explore further the protective effect of this molecule we constructed a second class of transgenic NOD mouse carrying an E alpha d transgene. Both transgenes protected the mice from IDDM, but this was not associated with a complete deletion of any T cells expressing commonly used T-cell receptor V beta genes.     

A three-base-pair deletion in the peripherin-RDS gene in one form of retinitis pigmentosa.

The group of retinopathies termed retinitis pigmentosa (RP) greatly contribute to visual dysfunction in man with a frequency of roughly 1 in 4,000. We mapped the first autosomal dominant RP (adRP) gene to chromosome 3q, close to the gene encoding rhodopsin, a rod photoreceptor pigment protein. Subsequently, mutations in this gene have been implicated as responsible for some forms of adRP. Another adRP gene has been mapped to chromosome 8p. A third adRP gene in a large Irish pedigree has been mapped to chromosome 6p, showing tight linkage with the gene for peripherin, a photoreceptor cell-specific glycoprotein, which is thus a strong candidate for the defective gene. We have now identified a three-base-pair deletion which results in the loss of one of a pair of highly conserved cysteine residues in the predicted third transmembrane domain of peripherin. This deletion segregates with the disease phenotype but is not present in unaffected controls, and suggests that mutant peripherin gives rise to retinitis pigmentosa.     

Human dopamine D1 receptor encoded by an intronless gene on chromosome 5

Receptors for dopamine have been classified into two functional types, D1 and D2. They belong to the family of receptors acting through G (or guanine nucleotide-binding) proteins. D2 receptors inhibit adenylyl cyclase, but D1 receptors stimulate adenylyl cyclase and activate cyclic AMP-dependent protein kinases. Dopamine D1 and D2 receptors are targets of drug therapy in many psychomotor disorders, including Parkinson's disease and schizophrenia, and may also have a role in drug addiction and alcoholism. D1 receptors regulate neuron growth and differentiation, influence behaviour and modify dopamine D2 receptor-mediated events. We report here the cloning of the D1 receptor gene, which resides on an intronless region on the long arm of chromosome 5, near two other members of the G-linked receptor family. The expressed protein, encoded by 446 amino acids, binds drugs with affinities identical to the native human D1 receptor. The presence of a D1 receptor gene restriction fragment length polymorphism will be helpful for future disease linkage studies.     

No linkage of chromosome 5q11-q13 markers to schizophrenia in Scottish families

Recent work suggests that an autosomal dominant gene for schizophrenia may be located on the 5q11-q13 region of chromosome 5 (refs 1 and 2): a report of schizophrenia associated with trisomy 5q11-q13 in two members of a family of Chinese origin prompted the discovery of linkage with markers p105-599Ha and p105-153Ra in five Icelandic and two English schizophrenic families. The strongest linkage was observed when the phenotype was broadly defined to include minor psychiatric diagnoses not traditionally considered part of the schizophrenia spectrum. By contrast, no evidence was found of linkage in a single multiplex Swedish schizophrenic pedigree. To determine whether these conflicting results arise from genetic and/or uncertainties in defining the schizophrenic phenotype, we examined fifteen Scottish schizophrenic families with restriction fragment length polymorphisms that span this region. We found no evidence for linkage, regardless of how broadly or narrowly the schizophrenic phenotype is defined, and conclude that a susceptibility locus, whose presence awaits confirmation, on the proximal portion of the long arm of chromosome 5 can be responsible for only a minority of cases of familial schizophrenia.     

Assignment of multiple endocrine neoplasia type 2A to chromosome 10 by linkage

Multiple endocrine neoplasis type 2A (MEN2A) is one of several kinds of cancers that appear to be inherited in an autosomally dominant fashion. We have assigned the MEN2A locus to chromosome 10 by linkage with a new DNA marker (D10S5). The linkage led us to investigate other chromosome 10 markers and demonstrate linkage between the disease locus and the interstitial retinol-binding protein (IRBP) gene. The D10S5 locus was sublocalized to 10q21.1 by hybridization in situ and the IRBP gene to p11.2----q11.2 with a secondary site at q24----q25. The linkages were established using 292 members of five families, three different restriction fragment length polymorphisms (RFLPs) at D10S5 and two RFLPs recognized by the IRBP probe. The recombination frequencies from pairwise linkage analysis between the disease and two marker loci D10S5 and IRBP were 0.19 and 0.11, with maximum lod scores of 3.6 and 8.0 respectively. Ordering of the three loci by multipoint analysis placed the IRBP gene approximately midway between the disease and D10S5 loci.     

Construction and characterization of a bacterial artificial chromosome library of rice 5460F

Thermo-sensitive genie male sterile (TGMS) rice has a number of desirable characteristics for hybrid rice production. Many studies have demonstrated that the sterility of TGMS rice is controlled by a single recessive gene. It has been mapped for the first time on chromosome 8 and namedtms 1. Several AFLP markers which tightly linked to thetms 1 gene have been identified recently. In order to develop a detailed physical map of thetms1 gene-encompassing region and finally clone thetms1 gene, a bacterial artificial chromosome (BAC) library of rice 5460F (the fertile mutant line of TGMS rice 5460S) using a modified vector pECBAC1 has been constructed. The constructed 5460F BAC library consists of 16 896 clones with an average insert size of 119 kb, which represents about 4.7 times rice haploid genome equivalents. Neither chloroplast nor mitochondrial DNA was detected from the library. The library was screened with three single copy sequence amplified fragment length polymorphism (AFLP) markers which tightly linked totms1 gene as probes and eight positive clones were identified.     

Deletion of Huntington's disease-linked G8 (D4S10) locus in Wolf-Hirschhorn syndrome

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by progressive involuntary movements and dementia. The symptoms of the disease, although devastating in severity, do not usually appear until the third to fourth decade of life. The gene defect is highly penetrant, and results in the loss of neurones in the basal ganglia, globus pallidus, and more diffusely in the cortex. A DNA marker, G8 (or D4S10), is tightly linked to Huntington's disease and this gene has been localized to chromosome 4 (ref. 3). The discovery of this linkage marker raises the possibility of developing a presymptomatic test for the disorder, and of eventually isolating the disease gene based on its map position. We have now regionally localized the DNA marker G8 to the terminal band of the short arm of the chromosome, a region representing approximately 0.5% of the total human genome. The assignment was made by examining DNA from patients with Wolf-Hirschhorn syndrome, a birth defect resulting from partial heterozygous deletion of the short arm of chromosome 4.     

Requirement of the RNA helicase-like protein PRP22 for release of messenger RNA from spliceosomes

The product of the yeast PRP22 gene acts late in the splicing of yeast pre-messenger RNA, mediating the release of the spliced mRNA from the spliceosome. The predicted PRP22 protein sequence shares extensive homology with that of PRP2 and PRP16 proteins, which are also involved in nuclear pre-mRNA splicing. The homologous region contains sequence elements characteristic of several demonstrated or putative ATP-dependent RNA helicases. A putative RNA-binding motif originally identified in bacterial ribosomal protein S1 and Escherichia coli polynucleotide phosphorylase has also been found in PRP22.     

Fine mapping of the awn gene on chromosome 4 in rice by association and linkage analyses

Awnness is a key trait in rice domestication, yet no studies have been conducted on fine mapping or association mapping of the rice awn gene. In this study, we investigated the awnness and genotype of a core collection of 303 cultivated rice varieties and a BC5F2 segregating population of 200 individuals. Combining association and linkage analyses, we mapped the awnness related genes to chromosome 4. Primary association analysis using 24 SSR markers revealed five loci significantly associated with awnness on chromosome 4. The associated markers cover previously identified regions. Fine association mapping was conducted using another 29 markers within a 4-Mb region, covering the associated marker in34, which is close to the awn gene Awn4.1. Seven associated markers were revealed, distributed over an 870-kb region. Combining the fine association mapping and linkage analysis of awnness in the 200 BC5F2 segregating population, we finally identified a 330-kb region as the candidate region for Awn4.1. The results indicate that combining association mapping and linkage mapping provides an efficient and precise approach to both genome-wide mapping and fine mapping of rice genes.     

Close linkage of glucokinase locus on chromosome 7p to early-onset non-insulin-dependent diabetes mellitus.

Non-insulin-dependent diabetes mellitus (NIDDM) is a major health problem, affecting 5% of the world population. Genetic factors are important in NIDDM, but the mechanisms leading to glucose intolerance are unknown. Genetic linkage has been investigated in multigeneration families to localize, and ultimately identify, the gene(s) predisposing to NIDDM. Here we report linkage between the glucokinase locus on chromosome 7p and diabetes in 16 French families with maturity-onset diabetes of the young, a form of NIDDM characterized by monogenic autosomal dominant transmission and early age of onset. Statistical evidence of genetic heterogeneity was significant, with an estimated 45-95% of the 16 families showing linkage to glucokinase. Because glucokinase is a key enzyme of blood glucose homeostasis, these results are evidence that a gene involved in glucose metabolism could be implicated in the pathogenesis of NIDDM.     

Mutations in progranulin cause tau-negative frontotemporal dementia linked to chromosome 17

Frontotemporal dementia (FTD) is the second most common cause of dementia in people under the age of 65 years. A large proportion of FTD patients (35-50%) have a family history of dementia, consistent with a strong genetic component to the disease. In 1998, mutations in the gene encoding the microtubule-associated protein tau (MAPT) were shown to cause familial FTD with parkinsonism linked to chromosome 17q21 (FTDP-17). The neuropathology of patients with defined MAPT mutations is characterized by cytoplasmic neurofibrillary inclusions composed of hyperphosphorylated tau. However, in multiple FTD families with significant evidence for linkage to the same region on chromosome 17q21 (D17S1787-D17S806), mutations in MAPT have not been found and the patients consistently lack tau-immunoreactive inclusion pathology. In contrast, these patients have ubiquitin (ub)-immunoreactive neuronal cytoplasmic inclusions and characteristic lentiform ub-immunoreactive neuronal intranuclear inclusions. Here we demonstrate that in these families, FTD is caused by mutations in progranulin (PGRN) that are likely to create null alleles. PGRN is located 1.7 Mb centromeric of MAPT on chromosome 17q21.31 and encodes a 68.5-kDa secreted growth factor involved in the regulation of multiple processes including development, wound repair and inflammation. PGRN has also been strongly linked to tumorigenesis. Moreover, PGRN expression is increased in activated microglia in many neurodegenerative diseases including Creutzfeldt-Jakob disease, motor neuron disease and Alzheimer's disease. Our results identify mutations in PGRN as a cause of neurodegenerative disease and indicate the importance of PGRN function for neuronal survival.