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Several angiosperm plant genomes, including Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), poplar (Populus trichocarpa), and grapevine (Vitis vinifera), have been sequenced, but the lack of reference genomes in gymnosperm phyla reduces our understanding of plant evolution and restricts the potential impacts of genomics research. A gene catalog was developed for the conifer tree Picea glauca (white spruce) through large-scale expressed sequence tag sequencing and full-length cDNA sequencing to facilitate genome characterizations, comparative genomics, and gene mapping. The resource incorporates new and publicly available sequences into 27,720 cDNA clusters, 23,589 of which are represented by full-length insert cDNAs. Expressed sequence tags, mate-pair cDNA clone analysis, and custom sequencing were integrated through an iterative process to improve the accuracy of clustering outcomes. The entire catalog spans 30 Mb of unique transcribed sequence. We estimated that the P. glauca nuclear genome contains up to 32,520 transcribed genes owing to incomplete, partially sequenced, and unsampled transcripts and that its transcriptome could span up to 47 Mb. These estimates are in the same range as the Arabidopsis and rice transcriptomes. Next-generation methods confirmed and enhanced the catalog by providing deeper coverage for rare transcripts, by extending many incomplete clusters, and by augmenting the overall transcriptome coverage to 38 Mb of unique sequence. Genomic sample sequencing at 8.5% of the 19.8-Gb P. glauca genome identified 1,495 clusters representing highly repeated sequences among the cDNA clusters. With a conifer transcriptome in full view, functional and protein domain annotations clearly highlighted the divergences between conifers and angiosperms, likely reflecting their respective evolutionary paths.

Pediatric glioblastomas (GBM) including diffuse intrinsic pontine gliomas (DIPG) are devastating brain tumors with no effective therapy. Here, we investigated clinical and biological impacts of histone H3.3 mutations. Forty-two DIPGs were tested for H3.3 mutations. Wild-type versus mutated (K27M-H3.3) subgroups were compared for HIST1H3B, IDH, ATRX and TP53 mutations, copy number alterations and clinical outcome. K27M-H3.3 occurred in 71 %, TP53 mutations in 77 % and ATRX mutations in 9 % of DIPGs. ATRX mutations were more frequent in older children (p

Glioblastoma multiforme (GBM) is a lethal brain tumour in adults and children. However, DNA copy number and gene expression signatures indicate differences between adult and paediatric cases. To explore the genetic events underlying this distinction, we sequenced the exomes of 48 paediatric GBM samples. Somatic mutations in the H3.3-ATRX-DAXX chromatin remodelling pathway were identified in 44% of tumours (21/48). Recurrent mutations in H3F3A, which encodes the replication-independent histone 3 variant H3.3, were observed in 31% of tumours, and led to amino acid substitutions at two critical positions within the histone tail (K27M, G34R/G34V) involved in key regulatory post-translational modifications. Mutations in ATRX (α-thalassaemia/mental retardation syndrome X-linked) and DAXX (death-domain associated protein), encoding two subunits of a chromatin remodelling complex required for H3.3 incorporation at pericentric heterochromatin and telomeres, were identified in 31% of samples overall, and in 100% of tumours harbouring a G34R or G34V H3.3 mutation. Somatic TP53 mutations were identified in 54% of all cases, and in 86% of samples with H3F3A and/or ATRX mutations. Screening of a large cohort of gliomas of various grades and histologies (n = 784) showed H3F3A mutations to be specific to GBM and highly prevalent in children and young adults. Furthermore, the presence of H3F3A/ATRX-DAXX/TP53 mutations was strongly associated with alternative lengthening of telomeres and specific gene expression profiles. This is, to our knowledge, the first report to highlight recurrent mutations in a regulatory histone in humans, and our data suggest that defects of the chromatin architecture underlie paediatric and young adult GBM pathogenesis.

Introduction: When a pilot is referred for vestibular schwannoma (VS), his or her fitness to fly may be questioned. The objective of this retrospective study was to describe a series of VS cases in a pilot population and to discuss their fitness to fly options. Methods: Between September 2002 and March 2010, the ENT/Head and Neck Surgery Department of the National Pilot Expertise Center conducted nearly 120,000 expert consultations for 40,000 pilots. We examined the files of 10 pilots who were referred to our 2 national experts for VS. Results: At the time of the expert consultation, hypoacusis was present in nine cases (four with total deafness), tinnitus in one case, and vertigo in nine cases. In our series, only 2 of the 10 pilots experienced a negative impact on their fitness to fly. Discussion: Decisions on fitness to fly were based on several factors: minimally disturbed audition, i.e., less than a 35-dB hearing loss with a good speech discrimination score; good balance, i.e., no reported difficulties; no spontaneous nystagmus recorded on videonystagmography (VNG); no postural deviation; and a normal head-shaking test. The delay and the VS's evolution between diagnosis and expert consultation are important because the selection of a treatment to control VS is critical in minimizing the possible associated complications. When a pilot is referred for VS, his or her fitness to fly is determined by the size of the tumor, balance, auditory status, and the follow-up results of these findings. The complications that may arise from VS treatments must also be considered.

Spartacus is our robot entry in the 2005 AAAI Mobile Robot Challenge, making a robot attend the National Conference on Artificial Intelligence. Designing robots that are capable of interacting with humans in real-life settings can be considered the ultimate challenge when it comes to intelligent autonomous systems. One key issue is the integration of multiple modalities (e.g., mobility, physical structure, navigation, vision, audition, dialogue, reasoning). Such integration increases the diversity and also the complexity of interactions the robot can generate. It also makes it difficult to monitor how such increased capabilities are used in unconstrained conditions, whether it is done while the robot is in operation of afterwards. This paper reports solutions and findings resulting from our hardware, software and decisional integration work on Spartacus. It also outlines perspectives in making intelligent and interaction capabilities evolve for autonomous robots.

Methylmalonic aciduria, cblB type (OMIM 251110) is an inborn error of vitamin B(12) metabolism that occurs due to mutations in the MMAB gene. MMAB encodes the enzyme ATP:cobalamin adenosyltransferase, which catalyzes the synthesis of the coenzyme adenosylcobalamin required for the activity of the mitochondrial enzyme methylmalonyl CoA mutase (MCM). MCM catalyzes the isomerization of methylmalonyl CoA to succinyl CoA. Deficient MCM activity results in methylmalonic aciduria and a susceptibility to life-threatening acidotic crises. The MMAB gene was sequenced from genomic DNA from a panel of 35 cblB patients, including five patients previously investigated. Nineteen MMAB mutations were identified, including 13 previously unknown mutations. These included 11 missense mutations, two duplications, one deletion, four splice-site mutations, and one nonsense mutation. None of these mutations was identified in 100 control alleles. Most of the missense mutations (9/11) were clustered in exon 7; many of these affected amino acid residues that are part of the probable active site of the enzyme. One previously described mutation, c.556C >T (p.R186W), was particularly common, accounting for 33% of pathogenic alleles. It was seen almost exclusively in patients of European background and was typically associated with presentation in the first year of life.

Methylmalonic aciduria and homocystinuria, cblC type (OMIM 277400), is the most common inborn error of vitamin B(12) (cobalamin) metabolism, with about 250 known cases. Affected individuals have developmental, hematological, neurological, metabolic, ophthalmologic and dermatologic clinical findings. Although considered a disease of infancy or childhood, some individuals develop symptoms in adulthood. The cblC locus was mapped to chromosome region 1p by linkage analysis. We refined the chromosomal interval using homozygosity mapping and haplotype analyses and identified the MMACHC gene. In 204 individuals, 42 different mutations were identified, many consistent with a loss of function of the protein product. One mutation, 271dupA, accounted for 40% of all disease alleles. Transduction of wild-type MMACHC into immortalized cblC fibroblast cell lines corrected the cellular phenotype. Molecular modeling predicts that the C-terminal region of the gene product folds similarly to TonB, a bacterial protein involved in energy transduction for cobalamin uptake.

Other than from its sensing and processing capabilities, a mobile robotic platform can be limited in its use by its ability to move in the environment. Legs, tracks and wheels are all efficient means of ground locomotion that are most suitable in different situations. Legs allow to climb over obstacles and change the height of the robot, modifying its viewpoint of the world. Tracks are efficient on uneven terrains or on soft surfaces (snow, mud, etc.), while wheels are optimal on flat surfaces. Our objective is to work on a new concept capable of combining different locomotion mechanisms to increase the locomotion capabilities of the robotic platform. The design we came up with, called AZIMUT, is symmetrical and is made of four independent leg-track-wheel articulations. It can move with its articulations up, down or straight, allowing the robot to deal with three-dimensional environments. AZIMUT is also capable of moving sideways without changing its orientation, making it omnidirectional. By putting sensors on these articulations, the robot can also actively perceive its environment by changing the orientation of its articulations. Designing a robot with such capabilities requires addressing difficult design compromises, with measurable impacts seen only after integrating all of the components together. Modularity at the structural, hardware and embedded software levels, all considered concurrently in an iterative design process, reveals to be key in the design of sophisticated mobile robotic platforms.

Mutations in the MMAA gene on human chromosome 4q31.21 result in vitamin B12-responsive methylmalonic aciduria (cblA complementation group) due to deficiency in the synthesis of adenosylcobalamin. Genomic DNA from 37 cblA patients, diagnosed on the basis of cellular adenosylcobalamin synthesis, methylmalonyl–coenzyme A (CoA) mutase function, and complementation analysis, was analyzed for deleterious mutations in the MMAA gene by DNA sequencing of exons and flanking sequences. A total of 18 novel mutations were identified, bringing the total number of mutations identified in 37 cblA patients to 22. A total of 13 mutations result in premature stop codons; three are splice site defects; and six are missense mutations that occur at highly conserved residues. Eight of these mutations were common to two or more individuals. One mutation, c.433C>T (R145X), represents 43% of pathogenic alleles and a common haplotype was identified. Restriction endonuclease or heteroduplex diagnostic tests were designed to confirm mutations. None of the sequence changes identified in cblA patients were found in 100 alleles from unrelated control individuals. Hum Mutat 24:509–516, 2004. © 2004 Wiley-Liss, Inc.

Myelin basic protein (MBP) is required for normal myelin compaction and is implicated in both experimental and human demyelinating diseases. In this study, as an initial step in defining the regulatory network controllingMBPtranscription, we located and characterized the function of evolutionarily conserved regulatory sequences. Long-range human-mouse sequence comparison revealed over 1 kb of conserved noncodingMBP5′ flanking sequence distributed into four widely spaced modules ranging from 0.1 to 0.4 kb. We demonstrate first that a controlled strategy of transgenesis provides an effective means to assign and compare qualitative and quantitativein vivoregulatory programs. Using this strategy, single-copy reporter constructs, designed to evaluate the regulatory significance of modular and intermodular sequences, were introduced by homologous recombination into the mousehprt(hypoxanthine-guanine phosphoribosyltransferase) locus. The proximal modules M1 and M2 confer comparatively low-level oligodendrocyte expression primarily limited to early postnatal development, whereas the upstream M3 confers high-level oligodendrocyte expression extending throughout maturity. Furthermore, constructs devoid of M3 fail to target expression to newly myelinating oligodendrocytes in the mature CNS. Mutation of putative Nkx6.2/Gtx sites within M3, although not eliminating oligodendrocyte targeting, significantly decreases transgene expression levels. High-level and continuous expression is conferred to myelinating or remyelinating Schwann cells by M4. In addition, when isolated from surroundingMBPsequences, M3 confers transient expression to Schwann cells elaborating myelin. These observations define thein vivoregulatory roles played by conserved noncodingMBPsequences and lead to a combinatorial model in which different regulatory modules are engaged during primary myelination, myelin maintenance, and remyelination.

Identifying the genes responsible for human diseases requires combining information about gene position with clues about biological function. The recent availability of whole-genome data sets of RNA and protein expression provides powerful new sources of functional insight. Here we illustrate how such data sets can expedite disease-gene discovery, by using them to identify the gene causing Leigh syndrome, French-Canadian type (LSFC, Online Mendelian Inheritance in Man no. 220111), a human cytochrome c oxidase deficiency that maps to chromosome 2p16-21. Using four public RNA expression data sets, we assigned to all human genes a “score” reflecting their similarity in RNA-expression profiles to known mitochondrial genes. Using a large survey of organellar proteomics, we similarly classified human genes according to the likelihood of their protein product being associated with the mitochondrion. By intersecting this information with the relevant genomic region, we identified a single clear candidate gene, LRPPRC . Resequencing identified two mutations on two independent haplotypes, providing definitive genetic proof that LRPPRC indeed causes LSFC. LRPPRC encodes an mRNA-binding protein likely involved with mtDNA transcript processing, suggesting an additional mechanism of mitochondrial pathophysiology. Similar strategies to integrate diverse genomic information can be applied likewise to other disease pathways and will become increasingly powerful with the growing wealth of diverse, functional genomics data.

The incidence of renal cell carcinoma (RCC) is increasing worldwide, and its prevalence is particularly high in some parts of Central Europe. Here we undertake whole-genome and transcriptome sequencing of clear cell RCC (ccRCC), the most common form of the disease, in patients from four different European countries with contrasting disease incidence to explore the underlying genomic architecture of RCC. Our findings support previous reports on frequent aberrations in the epigenetic machinery and PI3K/mTOR signalling, and uncover novel pathways and genes affected by recurrent mutations and abnormal transcriptome patterns including focal adhesion, components of extracellular matrix (ECM) and genes encoding FAT cadherins. Furthermore, a large majority of patients from Romania have an unexpected high frequency of A:T>T:A transversions, consistent with exposure to aristolochic acid (AA). These results show that the processes underlying ccRCC tumorigenesis may vary in different populations and suggest that AA may be an important ccRCC carcinogen in Romania, a finding with major public health implications.

En 1914, il est possible de définir le plan de transport que doit mettre en œuvre l’état-major général français comme un espace vectoriel de dimension 3 : moyens, espace, temps. Les moyens sont constitués surtout par le matériel humain appuyé pour l’essentiel par le canon de 75. L’espace assigné aux opérations militaires relève de considérations diplomatiques : une action offensive doit-elle être dirigée vers la frontière de l’Est, en Alsace-Lorraine, ou vers le Nord ? La décision influence les délais d’acheminement des troupes et la capacité des réseaux ferrés à les respecter. Le troisième facteur, le temps, est régulé par l’aptitude du chemin de fer à concentrer sur les frontières plus de 800 000 hommes dans un délai au plus égal à 14 jours sitôt la mobilisation décrétée. Sachant que la vitesse moyenne du déplacement des trains d’un corps d’armée est de 30 kilomètres à l’heure, le réseau ferroviaire français est-il en mesure de répondre à cette problématique ? Il faut pour cela innover dans les domaines du matériel, des dépôts, des installations fixes, de l’exploitation. Il faut surtout améliorer les infrastructures, créer les quais d’embarquement, élargir le gabarit des tunnels, concevoir des itinéraires de détournement. Grâce à ces mesures judicieuses, la concentration s’avère un succès. Dès le 15 août 1914, l’objectif est atteint. En 1939, le chemin de fer s’inspire des mêmes principes qu’en 1914 et bénéficie de l’expérience acquise et des effets de la modernisation ; il assume son rôle plus complexe d’une façon très satisfaisante comme le reconnaissent les autorités civiles et militaires. Malheureusement, en dépit d’un succès logistique indéniable, la jeune SNCF est la cible d’une nouvelle dimension de la guerre, la suprématie aérienne. In 1914, one can define the transportation plan that the General Staff is to implement as a vectorial space in three dimensions: means, space, time. Means are mostly human material, essentially supported by the 75 mm cannon. Space devoted to military operations depends on diplomatic considerations: where should an offensive action be directed, toward the eastern border, in Alsace-Lorraine, or toward the North? The decision has an impact on the time limit necessary for the troop’s transportation and the capacity of railway networks to comply with it. The third factor, time, is regulated by the ability of railway to concentrate at the border more than 800.000 men in a deadline at most equal to 14 days as soon as the mobilisation proclaimed. Given that the average speed of the trains of an Army Corp is 30 km/h, is the French railway network up to the problem? For that one has to innovate in the domain of rolling stock, depots, fixe facilities, exploitation. Overall, infrastructures had to be improved, departure platforms created, tunnel template enlarged, alternative routes set up. Thanks to these well-advised decisions, the concentration is a success. From August the 15th, the goal is achieved. In 1939, the railway takes inspiration of the same principles as in 1914, takes advantage of the acquired experience and of modernisation effects. Its more complex part is quite well carried out as acknowledged by civilian and military authorities. Unfortunately, in spite of an undisputable success, the young SNCF falls prey to a new dimension of the war, air supremacy.

In peripheral nerves, large caliber axons are ensheathed by myelin-elaborating Schwann cells. Multiple lines of evidence demonstrate that expression of the genes encoding myelin structural proteins occurs in Schwann cells in response to axonal instructions. To gain further insight into the mechanisms controlling myelin gene expression, we used reporter constructs in transgenic mice to search for the DNA elements that regulate themyelin basic protein(MBP) gene. Through thisin vivoinvestigation, we provide evidence for the participation of multiple, widely distributed, positive and negative elements in the overall control ofMBPexpression. Notably, all constructs bearing a 0.6 kb far-upstream sequence, designated Schwann cell enhancer 1 (SCE1), expressed at high levels in myelin-forming Schwann cells. In addition, robust targeting activity conferred by SCE1 was shown to be independent of otherMBP5′ flanking sequence. These observations suggest that SCE1 will make available a powerful tool to drive transgene expression in myelinating Schwann cells and that a focused analysis of the SCE1 sequence will lead to the identification of transcription factor binding sites that positively regulateMBPexpression.

We compared the frequency of PPM1D mutation in the white blood cells from 1295 ovarian cancer case patients and 834 control subjects. We found a truncating mutation in 20 case patients vs 1 control subject (odds ratio [OR] = 13.07; 95% confidence interval [CI] = 1.75 to 97.55; P < .001). The 12-year mortality of the PPM1D-positive case patients was higher than that of the PPM1D-negative case patients (hazard ratio = 2.02; 95% CI = 1.21 to 3.39; P = .007). Three of the 20 PPM1D carrier case patients had a past history of breast cancer compared with 29 of 1129 noncarriers (OR = 6.69; 95% CI = 1.86 to 24.11; P = .007). The lifetime risks for breast or ovarian cancer among female first-degree relatives of PPM1D mutation carriers were not increased compared with that of case patients without mutations. These observations suggest PPM1D mutations in the mosaic state predispose women to breast and ovarian cancer in the absence of a family history of cancer.

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.

We have previously reported that keratinocytes defective in glycosylphosphatidylinositol (GPI)-anchor biosynthesis display enhanced TGF-beta responses. These studies implicated the involvement of a 150 kDa GPI-anchored TGF-beta1 binding protein, r150, in modulating TGF-beta signaling. Here, we sought to determine the molecular identity of r150 by affinity purification and microsequencing. Our results identify r150 as CD109, a novel member of the alpha2-macroglobulin (alpha2M)/complement superfamily, whose function has remained obscure. In addition, we have identified a novel CD109 isoform that occurs in the human placenta but not keratinocytes. Biochemical studies show that r150 contains an internal thioester bond, a defining feature of the alpha2M/complement family. Loss and gain of function studies demonstrate that CD109 is a component of the TGF-beta receptor system, and a negative modulator of TGF-beta responses in keratinocytes, as implicated for r150. Our data suggest that CD109 can inhibit TGF-beta signaling independently of ligand sequestration and may exert its effect on TGF-beta signaling by direct modulation of receptor activity. Together, our results linking CD109 function to regulation of TGF-beta signaling suggest that CD109 plays a unique role in the regulation of isoform-specific TGF-beta signaling in keratinocytes.

An “in-house” radioimmunoassay (RIA) procedure utilizing commercial sources of antiserum and radiolabel was developed for the simultaneous measurement of cortisol in serum and saliva from swine. A commercial RIA kit (Incstar) for cortisol was used to validate the in-house procedure for serum measurements. The correlation coefficient (r) between the Incstar kit and the in-house method for serum cortisol was 0.94. The Incstar kit was modified to accommodate measurement of salivary cortisol concentrations and compared to the in-house procedure. The correlation between these procedures was r = 0.81 and best described by a logarithmic (Ln) curve. Salivary cortisol measures by the in-house RIA were significantly higher than those obtained from the modified Incstar kit (p < 0.01), a probable consequence of the different relative cross-reactions of the respective antisera to cortisone and a dilution effect on the kit standards. Keywords: Radioimmunoassay; salivary cortisol; pigs; stress

The mouse chromosome 6 locus Cmv1 controls replication of mouse Cytomegalovirus (MCMV) in the spleen of the infected host. In our effort to clone Cmv1, we have constructed a high-resolution genetic linkage map in the proximity of the gene. For this, a total of 45 DNA markers corresponding to either cloned genes or microsatellites were mapped within a 7.9-cM interval overlapping the Cmv1 region. We have followed the cosegregation of these markers with respect to Cmv1 in a total of 2248 backcross mice from a preexisting interspecific backcross panel of 281 (Mus spretus x C57BL/6J)F1 x C57BL/6J and 2 novel panels of 989 (A/ J x C57BL6)F1 x A/J and 978 (BALB/c x C57BL/6J)F1 x BALB/c segregating Cmv1. Combined pedigree analysis allowed us to determine the following gene order and intergene distances (in cM) on the distal region of mouse chromosome 6: D6Mit216-(1.9)-D6Mit336-(2.2)- D6Mit218-(1.0)-D6Mit52-(0.5)-D6Mit194-(0.2)-Nkrp 1/ D6Mit61/135/257/289/338-(0.4)-Cmv1/Ly49A/D6Mit370++ +- (0.3)-Prp/Kap/D6Mit13/111/219-(0.3)-Tel/D6Mit374/290/ 220/196/195/110-(1.1)-D6Mit25. Therefore, the minimal genetic interval for Cmv1 of 0.7 cM is defined by 13 tightly linked markers including 2 markers, Ly49A and D6Mit370, that did not show recombination with Cmv1 in 1967 meioses analyzed; the proximal limit of the Cmv1 domain was defined by 8 crossovers between Nkrp1/ D6Mit61/135/257/289/338 and Cmv1/Ly49A/D6Mit370, and the distal limit was defined by 5 crossovers between Cmv1/Ly49A/D6Mit370 and Prp/Kap/D6Mit13/111/219. This work demonstrates tight linkage between Cmv1 and genes from the natural killer complex (NKC), such as Nkrp1 and Ly49A, suggesting that Cmv1 may represent an NK cell recognition structure encoded in the NKC region.

Whole exome sequencing (WES) has greatly facilitated the identification of causal mutations for diverse human genetic disorders. We applied WES as a molecular diagnostic tool to identify disease-causing genes in consanguineous families in Qatar. Seventeen consanguineous families with diverse disorders were recruited. Initial mutation screening of known genes related to the clinical diagnoses did not reveal the causative mutations. Using WES approach, we identified the definitive disease-causing mutations in four families: (i) a novel nonsense homozygous (c.1034C>G) in PHKG2 causing glycogen storage disease type 9C (GSD9C) in a male with initial diagnosis of GSD3; (ii) a novel homozygous 1-bp deletion (c.915del) in NSUN2 in a male proband with Noonan-like syndrome; (iii) a homozygous SNV (c.1598C>G) in exon 11 of IDUA causing Hurler syndrome in a female proband with unknown clinical diagnosis; (iv) a de novo known splicing mutation (c.1645+1G>A) in PHEX in a female proband with initial diagnosis of autosomal recessive hypophosphatemic rickets. Applying WES as a diagnostic tool led to the unambiguous identification of disease-causing mutations in phenotypically complex disorders or correction of the initial clinical diagnosis in ˜25% of our cases.

The efficacy of salivary cortisol analyses for assessment of the hypothalamic-pituitary-adrenal (HPA) response to stimulation is compared to serum cortisol measurement. Matched samples of serum and saliva were collected from pigs (n = 6) subjected to exogenous adrenocorticotrophic hormone (ACTH) stimulation (i.v. 200 IU) and snaring. Salivary cortisol responses to handling and transport (1 h) were measured in a further 10 pigs. Saliva samples were collected before and after handling and transport. There were significant correlations between serum and salivary cortisol values, following ACTH stimulation (r = 0.8813, P

Objective: To identify rare variants contributing to multiple sclerosis (MS) susceptibility in a family we have previously reported with up to 15 individuals affected across 4 generations. Methods: We performed exome sequencing in a subset of affected individuals to identify novel variants contributing to MS risk within this unique family. The candidate variant was genotyped in a validation cohort of 2,104 MS trio families. Results: Four family members with MS were sequenced and 21,583 variants were found to be shared among these individuals. Refining the variants to those with 1) a predicted loss of function and 2) present within regions of modest haplotype sharing identified 1 novel mutation (rs55762744) in the tyrosine kinase 2 ( TYK2 ) gene. A different polymorphism within this gene has been shown to be protective in genome-wide association studies. In contrast, the TYK2 variant identified here is a novel, missense mutation and was found to be present in 10/14 (72%) cases and 28/60 (47%) of the unaffected family members. Genotyping additional 2,104 trio families showed the variant to be transmitted preferentially from heterozygous parents (transmitted 16: not transmitted 5; χ 2 = 5.76, p = 0.016). Conclusions: Rs55762744 is a rare variant of modest effect on MS risk affecting a subset of patients (0.8%). Within this pedigree, rs55762744 is common and appears to be a modifier of modest risk effect. Exome sequencing is a quick and cost-effective method and we show here the utility of sequencing a few cases from a single, unique family to identify a novel variant. The sequencing of additional family members or other families may help identify other variants important in MS.

Objective: Multiple sclerosis (MS) is a complex neurological disease. Genetic linkage analysis and genotyping of candidate genes in families with 4 or more affected individuals more heavily loaded for susceptibility genes has not fully explained familial disease clustering. Methods: We performed whole exome sequencing to further understand the heightened prevalence of MS in these families. Results: Forty-three individuals with MS (1 from each family) were sequenced to find rare variants in candidate MS susceptibility genes. On average, >58,000 variants were identified in each individual. A rare variant in the CYP27B1 gene causing complete loss of gene function was identified in 1 individual. Homozygosity for this mutation results in vitamin D-dependent rickets I (VDDR1), whereas heterozygosity results in lower calcitriol levels. This variant showed significant heterozygous association in 3,046 parent-affected child trios (p = 1 × 10−5). Further genotyping in >12,500 individuals showed that other rare loss of function CYP27B1 variants also conferred significant risk of MS, Peto odds ratio = 4.7 (95% confidence interval, 2.3–9.4; p = 5 × 10−7). Four known VDDR1 mutations were identified, all overtransmitted. Heterozygous parents transmitted these alleles to MS offspring 35 of 35× (p = 3 × 10−9). Interpretation: A causative role for CYP27B1 in MS is supported; the mutations identified are known to alter function having been shown in vivo to result in rickets when 2 copies are present. CYP27B1 encodes the vitamin D-activating 1-alpha hydroxylase enzyme, and thus a role for vitamin D in MS pathogenesis is strongly implicated. ANN NEUROL 2011;70:881–886

The incidence of esophageal squamous cell carcinoma (ESCC) is very high in northeastern Iran. Previously, we reported a strong familial component of ESCC among Turkmens, who constitute approximately one-half of the population of this region. We hypothesized that the genes which cause Fanconi anemia might be candidate genes for ESCC. We sequenced the entire coding regions of 12 Fanconi anemia genes in the germline DNA of 190 Turkmen cases of ESCC. We identified three heterozygous insertion/deletion mutations: one in FANCD2 (p.Val1233del), one in FANCE (p.Val311SerfsX2), and one in FANCL (p.Thr367AsnfsX13). All three patients had a strong family history of ESCC. In addition, four patients (out of 746 tested) were homozygous for the FANCA p.Ser858Arg mutation, compared to none of 1,373 matched controls (OR = 16.7, 95% CI = 6.2–44.2, P = 0.01). The p. Lys3326X mutation in BRCA2 (also known as Fanconi anemia gene FANCD1) was present in 27 of 746 ESCC cases and in 16 of 1,373 controls (OR = 3.38, 95% CI = 1.97–6.91, P = 0.0002). In summary, both heterozygous and homozygous mutations in several Fanconi anemia-predisposing genes are associated with an increased risk of ESCC in Iran.

The most common ground locomotion method to make a mobile robot move is to use two-wheel drive with differential steering and a rear balancing caster. Controlling the two motors independently makes the robot non-holonomic in its motion. Such robots can work well indoor on flat surfaces and in environments adapted for wheelchairs. But the benefit of providing mobility to a robot directly relies on its locomotion capability, for handling different types of terrains (indoors or outdoors) and situations such as moving slowly or rapidly, with or without the presence of moving objects (living or not), climbing over objects and potentially having to deal with hazardous conditions. It is with this objective in mind that we designed AZIMUT. AZIMUT is a legged tracked wheeled robot capable of changing the orientation of its four articulations. Each articulation has three degrees of freedom (DOF): it can rotate 360deg around its point of attachment to the chassis, can change its orientation over 180deg, and rotate to propulse the robot.

Mucolipidosis (ML) II (I-cell disease) is a lysosomal storage disorder caused by a deficiency of UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase. MLII is an autosomal recessive disease with a carrier rate estimated at 1/39 in Saguenay-Lac-Saint-Jean (SLSJ) (Quebec, Canada), which is the highest frequency documented worldwide. To identify the causing mutation, we sequenced GNPTAB exons in 27 parents of 16 MLII-deceased children from the SLSJ region as obligatory and potential carriers. We also performed a genealogical reconstruction for each parent to evaluate consanguinity levels and genetic contribution of ancestors. Our goal was to identify which parameters could explain the high MLII frequency observed in the SLSJ population. A single mutation (c.3503_3504delTC) was found in all obligatory carriers. In addition, 11 apparent polymorphisms were identified. The mutation was not detected in genomic DNA of 50 unrelated controls. Genealogical data show six founders (three couples) with a higher probability of having introduced the mutation in the population. The frequency of the mutation was increased as a consequence of this founder effect and of the resulting population structure. We suggest that c.3503_3504delTC is the allele causing MLII in the SLSJ population, and its high carrier rate is most likely explained by a founder effect.

No more than ≈30% of hereditary breast cancer has been accounted for by mutations in known genes. Most of these genes, such as BRCA1 , BRCA2 , TP53 , CHEK2 , ATM , and FANCJ/BRIP1 , function in DNA repair, raising the possibility that germ line mutations in other genes that contribute to this process also predispose to breast cancer. Given its close relationship with BRCA2 , PALB2 was sequenced in affected probands from 68 BRCA1/BRCA2 -negative breast cancer families of Ashkenazi Jewish, French Canadian, or mixed ethnic descent. The average BRCAPRO score was 0.58. A truncating mutation (229delT) was identified in one family with a strong history of breast cancer (seven breast cancers in three female mutation carriers). This mutation and its associated breast cancers were characterized with another recently reported but unstudied mutation (2521delA) that is also associated with a strong family history of breast cancer. There was no loss of heterozygosity in tumors with either mutation. Moreover, comparative genomic hybridization analysis showed major similarities to that of BRCA2 tumors but with some notable differences, especially loss of 18q, a change that was previously unknown in BRCA2 tumors and less common in sporadic breast cancer. This study supports recent observations that PALB2 mutations are present, albeit not frequently, in breast cancer families. The apparently high penetrance noted in this study suggests that at least some PALB2 mutations are associated with a substantially increased risk for the disease.

Telehealth is defined as the use of information and communication technologies (ICT) to extend health care service delivery across distance. Remote vital sign monitoring is a common example for home telehealth and one promising market, but lacks the breadth, comforting and subtleties of face-to-face assessments usually done by having either the patient go to the clinic or the clinician going to see the patient at home. To conduct more lively and interactive sessions, virtual visits involve the use of video and audio for live and remote consultations between clinicians and patients [1] . These consultations are done through fixed workstations or mobile devices using web videoconferencing systems, which may not always be sufficient to the practice of clinicians [2] . Objectives To provide flexibility to the interactions that would normally take place in face-to-face consultations, we are developing a multi-point, multi-view and multi-data (video, audio, medical devices) videoconferencing system, called VIGIL, to conduct live sessions between health caregivers (e.g., physiotherapists, occupational therapists, doctors, nurses) and patients at home. Methods Like Skype and OmniJoin/Nefsis, VIGIL is a cloud-based videoconferencing system working on mobile devices, laptops and workstations. VIGIL has customized and simple interfaces to improve usability for specific telehealth needs. For instance, it can transmit images from one or multiple cameras, control pan-tilt-zoom (PTZ) cameras from a distance, and interface a mobile communication badge and a mobile PTZ camera for interaction away from the workstation. It can also receive data from wireless medical devices. Results VIGIL is currently hosted at Sherbrooke Techno-Centre and exploited in trials are underway at the Centre de readaptation de l’Estrie. Discussion Technologically, it is possible to provide a diversity of interaction modalities in support of telehealth practice. It is now more a matter of finding out what is necessary to address those needs.

This paper presents the kinematical analysis of AZIMUT-2, a four steered wheeled mobile robot. The utilization of a new wheel concept called the AZIMUT wheel allowed us to create an innovative omnidirectional non-holonomic robot. Novelty of this wheel concept resides in the non-conventional positioning of the steering axis and the wheel axis. We propose a kinematical model based on the geometrical constraints of these wheels. The degree of mobility, steerability and maneuverability are studied. Additionally, we describe a special design implementation of the wheel mechanism to overcome a hyper-motorization issue inherent to the wheel's geometrical properties. Finally, we describe AZIMUT-2's two operational and seven locomotion modes, along with a control algorithm based on the kinematical model of the robot

The adipokine visfatin (PBEF1) exhibits insulin-mimetic effects and correlates strongly with visceral adiposity. We sequenced visfatin gene exons and 1,480 bp of the promoter in 23 individuals, including 18 individuals from the Quebec Family Study (QFS) with varying degrees of abdominal visceral fat, assessed by computed tomography, and 5 individuals from the Saguenay-Lac-Saint-Jean region of Québec. We identified a synonymous polymorphism in exon 7 (SER301SER) but no nonsynonymous mutations. We observed an additional 10 polymorphisms, including 5 intronic, 4 within the promoter, and 1 within the 3′ untranslated region. Further promoter sequencing (816 bp) identified five additional single nucleotide polymorphisms (SNPs) in the QFS population. To investigate the role of visfatin gene variants in obesity-related phenotypes, we genotyped a total of 13 SNPs in the promoter region of the gene. From these, we analyzed the seven common SNPs in the QFS sample (918 participants from 208 families). A significant association was found between two SNPs (rs9770242 and rs1319501), in perfect linkage disequilibrium, and fasting insulin levels (P = 0.002). These SNPs were also associated with fasting glucose (P ≤ 0.02). In addition, a more distal SNP (rs7789066) was significantly associated with the apolipoprotein B component of VLDL (P = 0.012).

This paper presents the kinematical analysis of AZIMUT-2. The utilization of a new wheel concept called the AZIMUT wheel allowed us to create an innovative omnidirectional non-holonomic robot. Novelty of this wheel concept resides in the non-conventional positioning of the steering axis and the wheel axis. We propose a kinematical model based on the geometrical constraints of these wheels. The degree of mobility, steerability and maneuverability are studied. Additionally, we describe a special design implementation of the wheel mechanism to overcome an hyper-motorization issue inherent to this wheel geometrical properties. Finally, we describe AZIMUT-2's two operational modes and its seven locomotion modes, along with a control algorithm based on the kinematical model of the robot.

Contains fulltext : 50018.pdf (Publisher’s version ) (Closed access) Mucopolysaccharidosis IIIC (MPS IIIC, or Sanfilippo C syndrome) is a lysosomal storage disorder caused by the inherited deficiency of the lysosomal membrane enzyme acetyl-coenzyme A: alpha -glucosaminide N-acetyltransferase (N-acetyltransferase), which leads to impaired degradation of heparan sulfate. We report the narrowing of the candidate region to a 2.6-cM interval between D8S1051 and D8S1831 and the identification of the transmembrane protein 76 gene (TMEM76), which encodes a 73-kDa protein with predicted multiple transmembrane domains and glycosylation sites, as the gene that causes MPS IIIC when it is mutated. Four nonsense mutations, 3 frameshift mutations due to deletions or a duplication, 6 splice-site mutations, and 14 missense mutations were identified among 30 probands with MPS IIIC. Functional expression of human TMEM76 and the mouse ortholog demonstrates that it is the gene that encodes the lysosomal N-acetyltransferase and suggests that this enzyme belongs to a new structural class of proteins that transport the activated acetyl residues across the cell membrane.

Myelin basic protein (MBP) gene expression is conferred in oligodendrocytes and Schwann cells by different upstream enhancers. In Schwann cells, expression is controlled by a 422 bp enhancer lying -9 kb from the gene. We show here that it contains 22 mammalian conserved motifs ≥6 bp. To investigate their functional significance, different combinations of wild-type or mutated motifs were introduced into reporter constructs that were inserted in single copy at a common hypoxanthine phosphoribosyltransferase docking site in embryonic stem cells. Lines of transgenic mice were derived, and the subsequent qualitative and quantitative expression phenotypes were compared at different stages of maturation. In the enhancer core, seven contiguous motifs cooperate to confer Schwann cell specificity while different combinations of flanking motifs engage, at different stages of Schwann cell maturation, to modulate expression level. Mutation of a Krox-20 binding site reduces the level of reporter expression, whereas mutation of a potential Sox element silences reporter expression. This potential Sox motif was also found conserved in other Schwann cell enhancers, suggesting that it contributes widely to regulatory function. These results demonstrate a close relationship between phylogenetic footprints and regulatory function and suggest a general model of enhancer organization. Finally, this investigation demonstrates thatin vivofunctional analysis, supported by controlled transgenesis, can be a robust complement to molecular and bioinformatics approaches to regulatory mechanisms.

Inter-individual variation in gene expression has proven to be in part governed by genetic determinants, which may be trans- or cis-acting. The underlying cause of cis-acting regulatory variation has been identified in only a handful of the hundreds of genes shown to display differential allelic expression. In this report, we describe a systematic effort to map common cis-acting variants in 64 genes, using association methods in HapMap samples. We identified 16 loci (25%), each of which harbors common haplotypes that affect total expression of a gene, and a further 17 loci (27%) with evidence of haplotypes affecting relative allelic expression in heterozygote samples. Our survey suggests that detailed mapping of allele-specific in vivo expression will provide a rich source of regulatory SNPs or haplotypes that should be given high priority in association studies of human phenotypes.

Cobalamin nonresponsive methylmalonic acidemia (MMA, mut complementation class) results from mutations in the nuclear gene MUT, which codes for the mitochondrial enzyme methylmalonyl CoA mutase (MCM). To better elucidate the spectrum of mutations that cause MMA, the MUT gene was sequenced in 160 patients with mut MMA. Sequence analysis identified mutations in 96% of disease alleles. Mutations were found in all coding exons, but predominantly in exons 2, 3, 6, and 11. A total of 116 different mutations, 68 of which were novel, were identified. Of the 116 different mutations, 53% were missense mutations, 22% were deletions, duplications or insertions, 16% were nonsense mutations, and 9% were splice-site mutations. Sixty-one of the mutations have only been identified in one family. A novel mutation in exon 2, c.322C>T (p.R108C), was identified in 16 of 27 Hispanic patients. SNP genotyping data demonstrated that Hispanic patients with this mutation share a common haplotype. Three other mutations were seen exclusively in Hispanic patients: c.280G>A (p.G94R), c.1022dupA, and c.970G>A (p.A324T). Seven mutations were seen almost exclusively in black patients, including the previously reported c.2150G>T (p.G717V) mutation, which was identified in 12 of 29 black patients. Two mutations were seen only in Asian patients. Some frequently identified mutations were not population-specific and were identified in patients of various ethnic backgrounds. Some of these mutations were found in mutation clusters in exons 2, 3, 6, and 11, suggesting a recurrent mutation.

Cis-acting allelic variation in gene regulation is a source of phenotypic variation. Consequently, recent studies have experimentally screened human genes in an attempt to initiate a catalog of genes possessing cis-acting variants. In this study, we use human EST data in dbEST as the source of allelic expression data, and the HapMap database to provide expected allele frequencies in human populations. We demonstrate a greater concordance of allele frequencies estimated from human ESTs in dbEST with those derived from the CEPH HapMap sample representing Caucasians from northern and western Europe, than population samples obtained in Asia and Africa. Deviations between allele frequencies observed in EST databases and the ones obtained from the CEPH HapMap samples may result from common heritable cis-acting variants altering the relative allele distribution in RNA. We provide in silico as well as experimental evidence that this strategy does allow significant enrichment of genes harboring common heritable cis-acting polymorphisms in linkage disequilibrium with expressed alleles.

Mutations in the MMAA gene on human chromosome 4q31.21 result in vitamin B12-responsive methylmalonic aciduria (cblA complementation group) due to deficiency in the synthesis of adenosylcobalamin. Genomic DNA from 37 cblA patients, diagnosed on the basis of cellular adenosylcobalamin synthesis, methylmalonyl-coenzyme A (CoA) mutase function, and complementation analysis, was analyzed for deleterious mutations in the MMAA gene by DNA sequencing of exons and flanking sequences. A total of 18 novel mutations were identified, bringing the total number of mutations identified in 37 cblA patients to 22. A total of 13 mutations result in premature stop codons; three are splice site defects; and six are missense mutations that occur at highly conserved residues. Eight of these mutations were common to two or more individuals. One mutation, c.433CT (R145X), represents 43% of pathogenic alleles and a common haplotype was identified. Restriction endonuclease or heteroduplex diagnostic tests were designed to confirm mutations. None of the sequence changes identified in cblA patients were found in 100 alleles from unrelated control individuals.

The identification of human sequence polymorphisms that regulate gene expression is key to understanding human genetic diseases. We report a survey of human genes that demonstrate allelic differences in gene expression, reflecting the presence of putative allele-specific cis-acting factors of either genetic or epigenetic nature. The expression of allelic transcripts in heterozygous samples is assessed directly by relative quantitation of intragenic marker alleles in messenger or heteronuclear RNA derived from cells or tissues. This survey used 193 single-nucleotide polymorphisms (SNPs) from 129 genes expressed in lymphoblastoid cell lines, to identify 23 genes (18%) with common allele-specific transcripts whose expression deviated from the expected equimolar ratio. A subset of these deviations, or “allelic imbalances,” can be observed in multiple samples derived from reference CEPH (“Centre d’Etude du Polymorphisme Humain”) pedigrees and demonstrate a spectrum of patterns of transmission, including cosegregation of allelic skewing across generations compatible with Mendelian inheritance as well as random monoallelic expression for three genes ( IL1A, HTR2A, and FGB). Additional studies for BTN3A2 provide evidence of SNPs and haplotypes in complete linkage disequilibrium with high- and low-expressing transcripts. The pipeline described herein offers tools for efficient identification and characterization of allelic expression allowing identification of regulatory sequence variants as well as epigenetic variation affecting human gene expression.

Other than from its sensing and processing capabilities, a mobile robotic platform can be limited in its use by its ability to move in the environment. A wheeled robot works well on flat surfaces. Tracks are useful over rough terrains, while legs allow a robot to move over obstacles. In this paper we present a new concept of mobile robot with the objective of combining different locomotion mechanisms on the same platform to increase its locomotion capabilities. After presenting a review of multi-modal robotic platforms, we describe the design of our robot called AZIMUT. AZIMUT combines wheels, legs and tracks to move in three-dimensional environments. The robot is symmetrical and is made of four independent leg-track-wheel articulations. It can move with its articulations up, down or straight, or move sideways without changing the robot's orientation. The robot could be used in surveillance and rescue missions, exploration or operation in hazardous environments.

Leprosy is caused by Mycobacterium leprae and affects about 700,000 individuals each year1. It has long been thought that leprosy has a strong genetic component2, 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.

The glycan moiety of human recombinant gonadotrophins (r-hFSH, r-hLH, and r-hCG) produced in CHO cell lines has been characterized by a combination of chromatographic and mass spectrometric techniques, including both matrix-assisted laser desorption ionization and electrospray. Two glycan mapping methods have been developed for the three gonadotrophins that allow separation of the glycans according to either their charge or sialylation level or their antennarity. A method was also developed for r-hCG that permits the complete resolution of the N-glycan from the O-glycan species. Whereas the structure found for the N-glycans of the gonadotrophins was in agreement with the complex type model, the structure for an O-glycan of r-hCG, not yet described, has been unambiguously determined using nanoelectrospray ion trap mass spectrometry. Using these two glycan mapping methods, the high level of batch-to-batch consistency achieved for the glycosylation of the three recombinant gonadotrophins in commercial production has been shown. These data demonstrate the tight control that can be achieved in the manufacturing of complex recombinant therapeutic glycoproteins, which is a prerequisite to the delivering of a guaranteed dose of drug from vial to vial, and in turn to ensuring the clinical efficacy of the product.

AZIMUT is a mobile robotic platform that combines wheels, legs and tracks to move in three-dimensional environments. Its design is the result of an interdisciplinary effort combining expertise in mechanical engineering, electrical engineering, computer engineering and industrial design. After presenting AZIMUT, this paper describes the challenges of designing such a robot, outlining the interdependencies between the disciplines and the difficult compromises that have to be made during the iterative design process of a mobile robotic platform. Modularity at the structural, hardware and embedded software levels, all considered concurrently in an iterative design process, reveals to be key in the design of sophisticated mobile robotic platforms.Copyright © 2003 by ASME

Diabetes and obesity have long been known to be related. The recently characterized adipocyte hormone resistin (also called FIZZ3/ADSF) has been implicated as a molecular link between impaired glucose tolerance (IGT) and obesity in mice. A search for sequence variants at the human resistin locus identified nine single-nucleotide polymorphisms (SNPs) but no coding variants. An investigation into the association of these SNPs with diabetes and obesity revealed two 5′ flanking variants (g.-537 and g.-420), in strong linkage disequilibrium, that are associated with BMI. In nondiabetic individuals from the Quebec City area and the Saguenay-Lac-St-Jean region of Quebec, the g.-537 mutation (allelic frequency = 0.04) was significantly associated with an increase in BMI (P = 0.03 and P = 0.01, respectively). When the data from these two populations were combined and adjusted for age and sex, both the g.-537 (odds ratio [OR] 2.72, 95% CI 1.28–5.81) and the g.-420 variants (1.58, 1.06–2.35) were associated with an increased risk for a BMI ≥30 kg/m2. In contrast, in case/control and family-based study populations from Scandinavia, we saw no effect on BMI with either of these promoter variants. No association was seen with diabetes in any of the population samples.

Several lines of independent evidence suggest that human Natural Resistance Associated Macrophage Protein 1 gene (NRAMP1) is an important regulator of susceptibility to infectious diseases caused by certain intracellular pathogens. Here, we report the nucleotide sequence of 32198 bp of genomic DNA overlapping NRAMP1 on chromosomal region 2q35. The NRAMP1 gene spans 13604 bp. The gene and its 5′ genomic region are highly enriched for DNA repeat sequences. A second gene was identified in the immediate vicinity of NRAMP1 and was tentatively named Nuclear LIM Interactor-Interacting Factor (NLI-IF) by analogy to its closest ortholog. The human NLI-IF gene begins 4721 bp downstream of the NRAMP1 stop codon and is composed of seven exons varying in size from 57 bp to 1644 bp. The gene gives rise to a 2655-bp mRNA transcript that contains a 783-bp open reading frame. The predicted molecular weight of the 261-amino acid NLI-IF protein is 29.2 kDa. Several putative gene regulatory elements were identified in the 5′ upstream region of NLI-IF, including consensus binding sequences for Sp1, AP-2, NF-kappa B, and PU 1. The NLI-IF amino acid sequence has homology to proteins that have a high degree of homology with the NLI-interacting factor from Gallus gallus and are found in divergent species ranging from yeast to plants. NLI-IF is part of a human gene family encoding four related proteins of unknown function. Northern blot analysis of 15 different human tissues revealed a 2.6-kb NLI-IF mRNA that was ubiquitously expressed, but at varying levels. A second transcript with estimated size of 7 kb was expressed only in the placenta. Our data provide new sequence information about the NRAMP1 gene region that will be useful in the search for genetic variants causally involved in altered susceptibility to infectious diseases.

The role of lysine residues in the catalytic mechanism of myo-inositol monophosphatase (EC 3.1.3.25) was investigated. The enzyme was completely inactivated by amidination with ethyl acetimidate or reductive methylation with formaldehyde and cyanoborohydride. Activity was retained when the active site was protected with Mg2+, Li+, and D,L-myo-inositol 1-phosphate. Using radiolabeling, peptide mapping, and sequence analysis, Lys-36 was shown to be the protected residue, which is responsible for inactivation. Replacing Lys-36 with glutamine produced a mutant protein, K36Q, with similar affinities for the substrate and the activator Mg2+, but a 50-fold lower turnover number as compared to the wild-type enzyme. Crystallographic studies did not indicate any gross structural changes in the mutant as compared to the native form. Initial velocity data were best described by a rapid equilibrium ordered mechanism with two Mg2+ binding before and a third one binding after the substrate. Inhibition by calcium was unaffected by the mutation, but inhibition by lithium was greatly reduced and became noncompetitive. The pH dependence of catalysis and the solvent isotope effect on kcat are altered in the mutant enzyme. D,L-myo-Inositol 1-phosphate, 4-nitrophenyl phosphate, and D-glucose 6-phosphate are cleaved at different rates by the wild-type enzyme, but with similar efficiency by K36Q. All data taken together are consistent with the hypothesis that modifying or replacing the lysine residue in position 36 decreases its polarizing effect on one of the catalytic metal ions and prevents the efficient deprotonation of the metal-bound water nucleophile.

Multiple sclerosis (MS) is a complex neurological disease. Genetic linkage analysis and genotyping of candidate genes in families with four or more affected individuals more heavily loaded for susceptibility genes has not fully explained familial disease clustering. We performed whole exome sequencing to further understand the heightened prevalence of MS in these families. Forty-three individuals with MS (one from each family) were sequenced to find rare variants in candidate MS susceptibility genes. On average, >58 000 variants were identified in each individual. A rare variant in the CYP27B1 gene causing complete loss of gene function was identified in one individual. Homozygosity for this mutation results in vitamin D-dependent rickets I (VDDR1), whereas heterozygosity results in lower calcitriol levels. This variant showed significant heterozygous association in 3046 parent-affected child trios (p=1 ×10 5 ). Further genotyping in >12 500 individuals showed that other rare loss of function CYP27B1 variants also conferred significant risk of MS, Peto OR=4.7 (95% CI 2.3–9.4; p=5 ×10 −7 ). Four known VDDR1 mutations were identified, all overtransmitted. Heterozygous parents transmitted these alleles to MS offspring 35 of 35×(p=3×10 −9 ). A causative role for CYP27B1 in MS is supported; the mutations identified are known to alter function having been shown in vivo to result in rickets when two copies are present. CYP27B1 encodes the vitamin D-activating 1-α hydroxylase enzyme, and thus a role for vitamin D in MS pathogenesis is strongly implicated.

A candidate gene for the mouse chromosome 1 host resistance locus Bcg/Ity/Lsh was recently cloned and designated Nramp (natural resistance-associated macrophage protein). Nramp is part of a small family of at least two genes, Nramp1 and Nramp2. Primer extension and cDNA cloning were used to isolate the complete 5' end of the Nramp1 mRNA. Analysis of genomic cosmid and bacteriophage clones overlapping the complete Nramp1 gene revealed that the gene was composed of 15 exons and spanned 11.5 kb of genomic DNA. Positioning of introns on the coding portion of the mRNA revealed a modular relationship between coding exons and predicted structural domains of the protein, with 8 of the 12 transmembrane (TM) domains encoded by individual exons. Northern blotting analysis indicated that Nramp1 expression was restricted to J774A.1 and RAW 264.7 macrophage lines and was dramatically increased by treatment with interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS). Primer extension and S1 nuclease mapping experiments were used to locate the transcription initiation site of Nramp1 and revealed the presence of one major and several minor initiation sites. Nucleotide sequencing of the corresponding region failed to detect classical TATA and CAAT elements, but identified two putative initiator sequences located near the major initiation site. Consensus sequences for binding of the macrophage and B-cell-specific transcription factor PU.1, as well as several LPS (NF-IL6) and IFN-gamma response elements, were also identified.

Nature 482, 226–231 (2012) Sequence reads for paediatric glioblastoma multiforme (GBM) samples have been deposited in the European Genome Archive under the accession number EGAS00001000226. This has been corrected in the HTML and PDF versions online.