Distinct physiological states of Plasmodium falciparum in malaria-infected patients.

Infection with the malaria parasite Plasmodium falciparum leads to widely different clinical conditions in children, ranging from mild flu-like symptoms to coma and death. Despite the immense medical implications, the genetic and molecular basis of this diversity remains largely unknown. Studies of in vitro gene expression have found few transcriptional differences between different parasite strains. Here we present a large study of in vivo expression profiles of parasites derived directly from blood samples from infected patients. The in vivo expression profiles define three distinct transcriptional states. The biological basis of these states can be interpreted by comparison with an extensive compendium of expression data in the yeast Saccharomyces cerevisiae. The three states in vivo closely resemble, first, active growth based on glycolytic metabolism, second, a starvation response accompanied by metabolism of alternative carbon sources, and third, an environmental stress response. The glycolytic state is highly similar to the known profile of the ring stage in vitro, but the other states have not been observed in vitro. The results reveal a previously unknown physiological diversity in the in vivo biology of the malaria parasite, in particular evidence for a functional mitochondrion in the asexual-stage parasite, and indicate in vivo and in vitro studies to determine how this variation may affect disease manifestations and treatment.

Analysing complex genetic traits with chromosome substitution strains.

Many valuable animal models of human disease are known and new models are continually being generated in existing inbred strains,. Some disease models are simple mendelian traits, but most have a polygenic basis. The current approach to identifying quantitative trait loci (QTLs) that underlie such traits is to localize them in crosses, construct congenic strains carrying individual QTLs, and finally map and clone the genes. This process is time-consuming and expensive, requiring the genotyping of large crosses and many generations of breeding. Here we describe a different approach in which a panel of chromosome substitution strains (CSSs) is used for QTL mapping. Each of these strains has a single chromosome from the donor strain substituting for the corresponding chromosome in the host strain. We discuss the construction, applications and advantages of CSSs compared with conventional crosses for detecting and analysing QTLs, including those that have weak phenotypic effects.

Renal disease susceptibility and hypertension are under independent genetic control in the fawn-hooded rat.

Hypertension, diabetes and hyperlipidemia are risk factors for life-threatening complications such as end-stage renal disease, coronary artery disease and stroke. Why some patients develop complications is unclear, but only susceptibility genes may be involved. To test this notion, we studied crosses involving the fawn-hooded rat, an animal model of hypertension that develops chronic renal failure. Here, we report the localization of two genes, Rf-1 and Rf-2, responsible for about half of the genetic variation in key indices of renal impairment. In addition, we localize a gene, Bpfh-1, responsible for about 26% of the genetic variation in blood pressure. Rf-1 strongly affects the risk of renal impairment, but has no significant effect on blood pressure. Our results show that susceptibility to a complication of hypertension is under at least partially independent genetic control from susceptibility to hypertension itself.

Genetic dissection of autoimmune type I diabetes in the BB rat.

The BB rat is among the best models of insulin-dependent diabetes mellitus--with onset and pathogenesis closely resembling the human disease. One unusual feature is a severe T-cell lymphopenia, which appears to be inherited as a recessive trait controlled by a single gene, Lyp. Based on genetic analysis of several crosses, we show that development of diabetes involves at least three genes: Lyp, which is tightly linked to the neuropeptide Y (Npy) gene on chromosome 4, a gene linked to the major histocompatibility complex (MHC) on chromosome 20, and a third unmapped gene for which the Fischer rat strain carries an allele conferring resistance.

High-resolution haplotype structure in the human genome.

Linkage disequilibrium (LD) analysis is traditionally based on individual genetic markers and often yields an erratic, non-monotonic picture, because the power to detect allelic associations depends on specific properties of each marker, such as frequency and population history. Ideally, LD analysis should be based directly on the underlying haplotype structure of the human genome, but this structure has remained poorly understood. Here we report a high-resolution analysis of the haplotype structure across 500 kilobases on chromosome 5q31 using 103 single-nucleotide polymorphisms (SNPs) in a European-derived population. The results show a picture of discrete haplotype blocks (of tens to hundreds of kilobases), each with limited diversity punctuated by apparent sites of recombination. In addition, we develop an analytical model for LD mapping based on such haplotype blocks. If our observed structure is general (and published data suggest that it may be), it offers a coherent framework for creating a haplotype map of the human genome.

CLN5, a novel gene encoding a putative transmembrane protein mutated in Finnish variant late infantile neuronal ceroid lipofuscinosis.

The neuronal ceroid lipofuscinoses (NCLs) represent a group of common recessive inherited neurodegenerative disorders of childhood, with an incidence of 1:12,500 live births. They are characterized by accumulation of autofluorescent lipopigments in various tissues. Several forms of NCLs have been identified, based on age at onset, progression of disease, neurophysiological and histopathological findings and separate genetic loci. All types of NCL cause progressive visual and mental decline, motor disturbance, epilepsy and behavioral changes, and lead to premature death. One of the subtypes, Finnish variant late infantile neuronal ceroid lipofuscinosis (vLINCL; MIM256731) affects children at 4-7 years of age. The first symptom is motor clumsiness, followed by progressive visual failure, mental and motor deterioration and later by myoclonia and seizures. We have previously reported linkage for vLINCL on chromosome 13 (ref. 5) and constructed a long-range physical map over the region. Here, we report the positional cloning of a novel gene, CLN5, underlying this severe neurological disorder. The gene encodes a putative transmembrane protein which shows no homology to previously reported proteins. Sequence analysis of DNA samples from patients with three different haplotypes revealed three mutations; one deletion, one nonsense and one missense mutation, suggesting that mutations in this gene are responsible for vLINCL.

Genetic analysis of non-insulin dependent diabetes mellitus in the GK rat.

Non-insulin dependent diabetes mellitus (NIDDM) is a major public health problem, but its aetiology remains poorly understood. We have performed a comprehensive study of the genetic basis of diabetes in the Goto-Kakizaki (GK) rat, the most widely used animal model of non-obese NIDDM. The genetic dissection of NIDDM using this model has allowed us to map three independent loci involved in the disease. In addition, we identify a major factor affecting body weight, but not glucose tolerance, on chromosome 7 and map a further 10 regions that are suggestive for linkage. We conclude that NIDDM is polygenic and fasting hyperglycaemia and postprandial hyperglycaemia clearly have distinct genetic bases.

Direct isolation of polymorphic markers linked to a trait by genetically directed representational difference analysis.

We describe a technique, genetically directed representational difference analysis (GDRDA), for specifically generating genetic markers linked to a trait of interest. GDRDA is applicable, in principle, to virtually any organism, because it requires neither prior knowledge of the chromosomal location of the gene controlling the trait nor the availability of a pre-existing genetic map. Based on a subtraction technique described recently called representational difference analysis, GDRDA uses the principles of transmission genetics to create appropriate Tester and Driver samples for subtraction. We demonstrate the usefulness of GDRDA by, for example, successfully targeting three polymorphisms to an interval of less than 1 cM of the mouse nude locus of chromosome 11.

A novel member of the F-box/WD40 gene family, encoding dactylin, is disrupted in the mouse dactylaplasia mutant.

Early outgrowth of the vertebrate embryonic limb requires signalling by the apical ectodermal ridge (AER) to the progress zone (PZ), which in response proliferates and lays down the pattern of the presumptive limb in a proximal to distal progression. Signals from the PZ maintain the AER until the anlagen for the distal phalanges have been formed. The semidominant mouse mutant dactylaplasia (Dac) disrupts the maintenance of the AER, leading to truncation of distal structures of the developing footplate, or autopod. Adult Dac homozygotes thus lack hands and feet except for malformed single digits, whereas heterozygotes lack phalanges of the three middle digits. Dac resembles the human autosomal dominant split hand/foot malformation (SHFM) diseases. One of these, SHFM3, maps to chromosome 10q24 (Refs 6,7), which is syntenic to the Dac region on chromosome 19, and may disrupt the orthologue of Dac. We report here the positional cloning of Dac and show that it belongs to the F-box/WD40 gene family, which encodes adapters that target specific proteins for destruction by presenting them to the ubiquitination machinery. In conjuction with recent biochemical studies, this report demonstrates the importance of this gene family in vertebrate embryonic development.

Secretory phospholipase Pla2g2a confers resistance to intestinal tumorigenesis.

Individuals inheriting the same mutation predisposing to cancer may show very different outcomes, ranging from early aggressive cancer to disease-free survival. Experimental mouse models can provide a powerful tool to identify factors in the environment and genetic background that account for such modifications. The Min mouse strain, in which the ApcMin mutation disrupts the mouse homologue of the human familial polyposis gene, develops intestinal neoplasms whose multiplicity is strongly affected by genetic background. We previously mapped a strong modifier locus, Mom1 (modifier of Min-1), to a 4-cM region on mouse chromosome 4 containing a candidate gene Pla2g2a encoding a secretory phospholipase. Here, we report that a cosmid transgene overexpressing Pla2g2a caused a reduction in tumour multiplicity and size, comparable to that conferred by a single copy of the resistance allele of Mom1. These results offer strong evidence that this secretory phospholipase can provide active tumour resistance. The association of Pla2g2a with Mom1 thus withstands a strong functional test and is likely to represent the successful identification of a polymorphic quantitative trait locus in mammals.

Quantitative locus analysis of airway hyperresponsiveness in A/J and C57BL/6J mice.

Airway hyperresponsiveness is a key characteristic of human asthma and a marker for asthma-like conditions in animals. F1 mice derived from A/J and C57BL/6J display a phenotype which resembles the asthma-like phenotype of the A/J mice. Since airway responsiveness failed to segregate as a mendelian trait, we show significant linkage at two loci, Bhr1 (lod = 3.0) and Bhr2 (lod = 3.7) on chromosomes 2 and 15. A third locus, Bhr3 (lod = 2.83), maps to chromosome 17. Each of these loci maps near candidate loci implicated in the pathobiology of asthma. Our study represents the first linkages established through a genome-wide survey of airway hyperresponsiveness in any mammal.

Mutations in the human delta homologue, DLL3, cause axial skeletal defects in spondylocostal dysostosis.

Spondylocostal dysostosis (SD, MIM 277300) is a group of vertebral malsegmentation syndromes with reduced stature resulting from axial skeletal defects. SD is characterized by multiple hemivertebrae, rib fusions and deletions with a non-progressive kyphoscoliosis. Cases may be sporadic or familial, with both autosomal dominant and autosomal recessive modes of inheritance reported. Autosomal recessive SD maps to a 7.8-cM interval on chromosome 19q13.1-q13.3 that is homologous with a mouse region containing a gene encoding the Notch ligand delta-like 3 (Dll3). Dll3 is mutated in the X-ray-induced mouse mutant pudgy (pu), causing a variety of vertebrocostal defects similar to SD phenotypes. Here we have cloned and sequenced human DLL3 to evaluate it as a candidate gene for SD and identified mutations in three autosomal recessive SD families. Two of the mutations predict truncations within conserved extracellular domains. The third is a missense mutation in a highly conserved glycine residue of the fifth epidermal growth factor (EGF) repeat, which has revealed an important functional role for this domain. These represent the first mutations in a human Delta homologue, thus highlighting the critical role of the Notch signalling pathway and its components in patterning the mammalian axial

Radiation hybrid map of the mouse genome.

Radiation hybrid (RH) maps are a useful tool for genome analysis, providing a direct method for localizing genes and anchoring physical maps and genomic sequence along chromosomes. The construction of a comprehensive RH map for the human genome has resulted in gene maps reflecting the location of more than 30,000 human genes. Here we report the first comprehensive RH map of the mouse genome. The map contains 2,486 loci screened against an RH panel of 93 cell lines. Most loci (93%) are simple sequence length polymorphisms (SSLPs) taken from the mouse genetic map, thereby providing direct integration between these two key maps. We performed RH mapping by a new and efficient approach in which we replaced traditional gel- or hybridization-based assays by a homogeneous 5'-nuclease assays involving a single common probe for all genetic markers. The map provides essentially complete connectivity and coverage across the genome, and good resolution for ordering loci, with 1 centiRay (cR) corresponding to an average of approximately 100 kb. The RH map, together with an accompanying World-Wide Web server, makes it possible for any investigator to rapidly localize sequences in the mouse genome. Together with the previously constructed genetic map and a YAC-based physical map reported in a companion paper, the fundamental maps required for mouse genomics are now available.

A susceptibility locus for asthma-related traits on chromosome 7 revealed by genome-wide scan in a founder population.

The genetics of asthma and atopy have been difficult to determine because these diseases are genetically heterogeneous and modified by environment. The pedigrees in our study (n=86) originate in eastern central Finland (Kainuu province). According to census records, this region had only 200 households (2,000 inhabitants) in the mid sixteenth to mid seventeenth centuries. The current population of 100,000 represents the expansion of these founders within the past 400 years. Because this population is relatively homogeneous, we hypothesized that the molecular genetic mechanisms underlying asthma might also have reduced heterogeneity and therefore be easier to dissect than in mixed populations. A recent twin family study supported a strong genetic component for asthma in Finland. We carried out a genome-wide scan for susceptibility loci in asthma in the Kainuu subpopulation. We identified two regions of suggestive linkage and studied them further with higher-density mapping. We obtained evidence for linkage in a 20-cM region of chromosome 7p14-p15 for three phenotypes: asthma, a high level of immunoglobulin E (IgE; atopy) and the combination of the phenotypes. The strongest linkage was seen for high serum IgE (non-parametric linkage (NPL) score 3.9, P=0.0001), exceeding the threshold for genome-wide significance based on simulations. We also observed linkage between this locus and asthma or atopy in two independent data sets.

The mouse pudgy mutation disrupts Delta homologue Dll3 and initiation of early somite boundaries.

Pudgy (pu) homozygous mice exhibit clear patterning defects at the earliest stages of somitogenesis, resulting in adult mice with severe vertebral and rib deformities. By positional cloning and complementation, we have determined that the pu phenotype is caused by a mutation in the delta-like 3 gene (Dll3), which is homologous to the Notch-ligand Delta in Drosophila. Histological and molecular marker analyses show that the pu mutation disrupts the proper formation of morphological borders in early somite formation and of rostral-caudal compartment boundaries within somites. Viability analysis also indicates an important role in early development. The results point to a key role for a Notch-signalling pathway in the initiation of patterning of vertebrate paraxial mesoderm.

ARSACS, a spastic ataxia common in northeastern Québec, is caused by mutations in a new gene encoding an 11.5-kb ORF.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS or SACS) is an early onset neurodegenerative disease with high prevalence (carrier frequency 1/22) in the Charlevoix-Saguenay-Lac-Saint-Jean (CSLSJ) region of Quebec. We previously mapped the gene responsible for ARSACS to chromosome 13q11 and identified two ancestral haplotypes. Here we report the cloning of this gene, SACS, which encodes the protein sacsin. The ORF of SACS is 11,487 bp and is encoded by a single gigantic exon spanning 12,794 bp. This exon is the largest to be identified in any vertebrate organism. The ORF is conserved in human and mouse. The putative protein contains three large segments with sequence similarity to each other and to the predicted protein of an Arabidopsis thaliana ORF. The presence of heat-shock domains suggests a function for sacsin in chaperone-mediated protein folding. SACS is expressed in a variety of tissues, including the central nervous system. We identified two SACSmutations in ARSACS families that lead to protein truncation, consistent with haplotype analysis.

Large-scale discovery and genotyping of single-nucleotide polymorphisms in the mouse.

Single-nucleotide polymorphisms (SNPs) have been the focus of much attention in human genetics because they are extremely abundant and well-suited for automated large-scale genotyping. Human SNPs, however, are less informative than other types of genetic markers (such as simple-sequence length polymorphisms or microsatellites) and thus more loci are required for mapping traits. SNPs offer similar advantages for experimental genetic organisms such as the mouse, but they entail no loss of informativeness because bi-allelic markers are fully informative in analysing crosses between inbred strains. Here we report a large-scale analysis of SNPs in the mouse genome. We characterized the rate of nucleotide polymorphism in eight mouse strains and identified a collection of 2,848 SNPs located in 1,755 sequence-tagged sites (STSs) using high-density oligonucleotide arrays. Three-quarters of these SNPs have been mapped on the mouse genome, providing a first-generation SNP map of the mouse. We have also developed a multiplex genotyping procedure by which a genome scan can be performed with only six genotyping reactions per animal.

Mapping of a gene for type 2 diabetes associated with an insulin secretion defect by a genome scan in Finnish families.

Non-insulin dependent diabetes mellitus (NIDDM) affects more than 100 million people worldwide and is associated with severe metabolic defects, including peripheral insulin resistance, elevated hepatic glucose production, and inappropriate insulin secretion. Family studies point to a major genetic component, but specific susceptibility genes have not yet been identified-except for rare early-onset forms with monogenic or mitochondrial inheritance. We have screened over 4,000 individuals from a population isolate in western Finland, identified 26 families (comprising 217 individuals) enriched for NIDDM and performed a genome-wide scan using non-parametric linkage analysis. We found no significant evidence for linkage when the families were analysed together, but strong evidence for linkage when families were classified according to mean insulin levels in affecteds (in oral glucose tolerance tests). Specifically, families with the lowest insulin levels showed linkage (P = 2 x 10(-6)) to chromosome 12 near D12S1349. Interestingly, this region contains the gene causing the rare, dominant, early-onset form of diabetes MODY3. Unlike MODY3 families, the Finnish families with low insulin have an age-of-onset typical for NIDDM (mean = 58 years). We infer the existence of a gene NIDDM2 causing NIDDM associated with low insulin secretion, and suggest that NIDDM2 and MODY3 may represent different alleles of the same gene.

Determination of ancestral alleles for human single-nucleotide polymorphisms using high-density oligonucleotide arrays.

Here we report the application of high-density oligonucleotide array (DNA chip)-based analysis to determine the distant history of single nucleotide polymorphisms (SNPs) in current human populations. We analysed orthologues for 397 human SNP sites (identified in CEPH pedigrees from Amish, Venezuelan and Utah populations) from 23 common chimpanzee, 19 pygmy chimpanzee and 11 gorilla genomic DNA samples. From this data we determined 214 proposed ancestral alleles (the sequence found in the last common ancestor of humans and chimpanzees). In a diverse human population set, we found that SNP alleles with higher frequencies were more likely to be ancestral than less frequently occurring alleles. There were, however, exceptions. We also found three shared human/pygmy chimpanzee polymorphisms, all involving CpG dinucleotides, and two shared human/gorilla polymorphisms, one involving a CpG dinucleotide. We demonstrate that microarray-based assays allow rapid comparative sequence analysis of intra- and interspecies genetic variation.

Characterization of single-nucleotide polymorphisms in coding regions of human genes.

A major goal in human genetics is to understand the role of common genetic variants in susceptibility to common diseases. This will require characterizing the nature of gene variation in human populations, assembling an extensive catalogue of single-nucleotide polymorphisms (SNPs) in candidate genes and performing association studies for particular diseases. At present, our knowledge of human gene variation remains rudimentary. Here we describe a systematic survey of SNPs in the coding regions of human genes. We identified SNPs in 106 genes relevant to cardiovascular disease, endocrinology and neuropsychiatry by screening an average of 114 independent alleles using 2 independent screening methods. To ensure high accuracy, all reported SNPs were confirmed by DNA sequencing. We identified 560 SNPs, including 392 coding-region SNPs (cSNPs) divided roughly equally between those causing synonymous and non-synonymous changes. We observed different rates of polymorphism among classes of sites within genes (non-coding, degenerate and non-degenerate) as well as between genes. The cSNPs most likely to influence disease, those that alter the amino acid sequence of the encoded protein, are found at a lower rate and with lower allele frequencies than silent substitutions. This likely reflects selection acting against deleterious alleles during human evolution. The lower allele frequency of missense cSNPs has implications for the compilation of a comprehensive catalogue, as well as for the subsequent application to disease association.