👤 C M Lewis

Inconsistent effects of fish oil supplementation on plasma lipids may be influenced by genetic variation. We investigated 12 single nucleotide polymorphisms (SNPs) associated with dyslipidaemia in genome-wide association studies, in 310 participants randomised to treatment with placebo or 0.45, 0.9 and 1.8 g/day eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA) (1.51:1) in a 12-month parallel controlled trial. Effects of risk alleles were assessed as trait-specific genetic predisposition scores (GPS) and singly. GPS were positively associated with baseline concentrations of plasma total cholesterol, low-density-lipoprotein cholesterol and triglyceride (TG) and negatively with high-density-lipoprotein cholesterol. The TG-GPS was associated with 0.210 mmol/L higher TG per risk allele (P < 0.0001), but no effects of single TG SNPs were significant at baseline. After treatment with EPA and DHA, TG-GPS was associated with 0.023 mmol/L lower TG per risk allele (P = 0.72). No interactions between GPS and treatment were significant; however, FADS1 SNP rs174546 C/T interaction with treatment was a significant determinant of plasma TG concentration (P = 0.047, n = 267). Concentration differed between genotype groups after the 1.8 g/day dose (P = 0.026), decreasing by 3.5 (95 % CI -15.1 to 8.2) % in non-carriers of the risk T-allele (n = 30) and by 21.6 (95 % CI -32.1 to -11.2) % in carriers (n = 37), who showed a highly significant difference between treatments (P = 0.007). Carriers of the FADS1 rs174546 risk allele could benefit from a high intake of EPA and DHA in normalising plasma TG. Show less

Intracranial germ cell tumours (IGCTs) are a group of rare heterogeneous brain tumours that are clinically and histologically similar to the more common gonadal GCTs. IGCTs show great variation in their geographical and gender distribution, histological composition and treatment outcomes. The incidence of IGCTs is historically five- to eightfold greater in Japan and other East Asian countries than in Western countries, with peak incidence near the time of puberty. About half of the tumours are located in the pineal region. The male-to-female incidence ratio is approximately 3-4:1 overall, but is even higher for tumours located in the pineal region. Owing to the scarcity of tumour specimens available for research, little is currently known about this rare disease. Here we report the analysis of 62 cases by next-generation sequencing, single nucleotide polymorphism array and expression array. We find the KIT/RAS signalling pathway frequently mutated in more than 50% of IGCTs, including novel recurrent somatic mutations in KIT, its downstream mediators KRAS and NRAS, and its negative regulator CBL. Novel somatic alterations in the AKT/mTOR pathway included copy number gains of the AKT1 locus at 14q32.33 in 19% of patients, with corresponding upregulation of AKT1 expression. We identified loss-of-function mutations in BCORL1, a transcriptional co-repressor and tumour suppressor. We report significant enrichment of novel and rare germline variants in JMJD1C, which codes for a histone demethylase and is a coactivator of the androgen receptor, among Japanese IGCT patients. This study establishes a molecular foundation for understanding the biology of IGCTs and suggests potentially promising therapeutic strategies focusing on the inhibition of KIT/RAS activation and the AKT1/mTOR pathway. Show less

Although primary cilia are well established as important sensory and signaling structures, their function in most tissues remains unknown. Obesity is a feature associated with some syndromes of cilia dysfunction, such as Bardet-Biedl syndrome (BBS) and Alström syndrome, as well as in several cilia mutant mouse models. Recent data indicate that obesity in BBS mutant mice is due to defects in leptin receptor trafficking and leptin resistance. Furthermore, induction of cilia loss in leptin-responsive proopiomelanocortin neurons results in obesity, implicating cilia on hypothalamic neurons in regulating feeding behavior. Here, we directly test the importance of the cilium as a mediator of the leptin response. In contrast to the current dogma, a longitudinal study of conditional Ift88 cilia mutant mice under different states of adiposity indicates that leptin resistance is present only when mutants are obese. Our studies show that caloric restriction leads to an altered anticipatory feeding behavior that temporarily abrogates the anorectic actions of leptin despite normalized circulating leptin levels. Interestingly, preobese Bbs4 mutant mice responded to the anorectic effects of leptin and did not display other phenotypes associated with defective leptin signaling. Furthermore, thermoregulation and activity measurements in cilia mutant mice are inconsistent with phenotypes previously observed in leptin deficient ob/ob mice. Collectively, these data indicate that cilia are not directly involved in leptin responses and that a defect in the leptin signaling axis is not the initiating event leading to hyperphagia and obesity associated with cilia dysfunction. Show less

The study of the genetic regulation of metabolism in human serum samples can contribute to a better understanding of the intermediate biological steps that lead from polymorphism to disease. Here, we conducted a genome-wide association study (GWAS) to discover metabolic quantitative trait loci (mQTLs) utilizing samples from a study of prostate cancer in Swedish men, consisting of 402 individuals (214 cases and 188 controls) in a discovery set and 489 case-only samples in a replication set. A global nontargeted metabolite profiling approach was utilized resulting in the detection of 6,138 molecular features followed by targeted identification of associated metabolites. Seven replicating loci were identified (PYROXD2, FADS1, PON1, CYP4F2, UGT1A8, ACADL, and LIPC) with associated sequence variants contributing significantly to trait variance for one or more metabolites (P = 10(-13) -10(-91)). Regional mQTL enrichment analyses implicated two loci that included FADS1 and a novel locus near PDGFC. Biological pathway analysis implicated ACADM, ACADS, ACAD8, ACAD10, ACAD11, and ACOXL, reflecting significant enrichment of genes with acyl-CoA dehydrogenase activity. mQTL SNPs and mQTL-harboring genes were over-represented across GWASs conducted to date, suggesting that these data may have utility in tracing the molecular basis of some complex disease associations. Show less

Delta-5 and delta-6 desaturases (D5D and D6D) are key enzymes in endogenous synthesis of long-chain PUFAs. In this sample of healthy subjects (n = 310), genotypes of single nucleotide polymorphisms (SNPs) rs174537, rs174561, and rs3834458 in the FADS1-FADS2 gene cluster were strongly associated with proportions of LC-PUFAs and desaturase activities estimated in plasma and erythrocytes. Minor allele carriage associated with decreased activities of D5D (FADS1) (5.84 × 10(-19) ≤ P ≤ 4.5 × 10(-18)) and D6D (FADS2) (6.05 × 10(-8) ≤ P ≤ 4.20 × 10(-7)) was accompanied by increased substrate and decreased product proportions (0.05 ≤ P ≤ 2.49 × 10(-16)). The significance of haplotype association with D5D activity (P = 2.19 × 10(-17)) was comparable to that of single SNPs, but haplotype association with D6D activity (P = 3.39 × 10(-28)) was much stronger. In a randomized controlled dietary intervention, increasing eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) intake significantly increased D5D (P = 4.0 × 10(-9)) and decreased D6D activity (P = 9.16 × 10(-6)) after doses of 0.45, 0.9, and 1.8 g/day for six months. Interaction of rs174537 genotype with treatment was a determinant of D5D activity estimated in plasma (P = 0.05). In conclusion, different sites at the FADS1-FADS2 locus appear to influence D5D and D6D activity, and rs174537 genotype interacts with dietary EPA+DHA to modulate D5D. Show less

Genome-wide association studies (GWASs) primarily performed in European-ancestry (EA) populations have identified numerous loci associated with body mass index (BMI). However, it is still unclear whether these GWAS loci can be generalized to other ethnic groups, such as African Americans (AAs). Furthermore, the putative functional variant or variants in these loci mostly remain under investigation. The overall lower linkage disequilibrium in AA compared to EA populations provides the opportunity to narrow in or fine-map these BMI-related loci. Therefore, we used the Metabochip to densely genotype and evaluate 21 BMI GWAS loci identified in EA studies in 29,151 AAs from the Population Architecture using Genomics and Epidemiology (PAGE) study. Eight of the 21 loci (SEC16B, TMEM18, ETV5, GNPDA2, TFAP2B, BDNF, FTO, and MC4R) were found to be associated with BMI in AAs at 5.8 × 10(-5). Within seven out of these eight loci, we found that, on average, a substantially smaller number of variants was correlated (r(2) > 0.5) with the most significant SNP in AA than in EA populations (16 versus 55). Conditional analyses revealed GNPDA2 harboring a potential additional independent signal. Moreover, Metabochip-wide discovery analyses revealed two BMI-related loci, BRE (rs116612809, p = 3.6 × 10(-8)) and DHX34 (rs4802349, p = 1.2 × 10(-7)), which were significant when adjustment was made for the total number of SNPs tested across the chip. These results demonstrate that fine mapping in AAs is a powerful approach for both narrowing in on the underlying causal variants in known loci and discovering BMI-related loci. Show less

Major depressive disorder (MDD) is a common complex disorder with a partly genetic etiology. We conducted a genome-wide association study of the MDD2000+ sample (2431 cases, 3673 screened controls and >1 M imputed single-nucleotide polymorphisms (SNPs)). No SNPs achieved genome-wide significance either in the MDD2000+ study, or in meta-analysis with two other studies totaling 5763 cases and 6901 controls. These results imply that common variants of intermediate or large effect do not have main effects in the genetic architecture of MDD. Suggestive but notable results were (a) gene-based tests suggesting roles for adenylate cyclase 3 (ADCY3, 2p23.3) and galanin (GAL, 11q13.3); published functional evidence relates both of these to MDD and serotonergic signaling; (b) support for the bipolar disorder risk variant SNP rs1006737 in CACNA1C (P=0.020, odds ratio=1.10); and (c) lack of support for rs2251219, a SNP identified in a meta-analysis of affective disorder studies (P=0.51). We estimate that sample sizes 1.8- to 2.4-fold greater are needed for association studies of MDD compared with those for schizophrenia to detect variants that explain the same proportion of total variance in liability. Larger study cohorts characterized for genetic and environmental risk factors accumulated prospectively are likely to be needed to dissect more fully the etiology of MDD. Show less

Leber congenital amaurosis (LCA) is one of the most severe eye dystrophies characterized by severe vision loss at an early stage and accounts for approximately 5% of all retinal dystrophies. The purpose of this study was to identify a novel LCA disease allele or gene and to develop an approach combining genetic mapping with whole exome sequencing. Three patients from King Khaled Eye Specialist Hospital (KKESH205) underwent whole genome single nucleotide polymorphism genotyping, and a single candidate region was identified. Taking advantage of next-generation high-throughput DNA sequencing technologies, whole exome capture sequencing was performed on patient KKESH205#7. Sanger direct sequencing was used during the validation step. The zebrafish model was used to examine the function of the mutant allele. A novel missense mutation in Bardet-Biedl syndrome 4 protein (BBS4) was identified in a consanguineous family from Saudi Arabia. This missense mutation in the fifth exon (c.253G>C;p.E85Q) of BBS4 is likely a disease-causing mutation as it segregates with the disease. The mutation is not found in the single nucleotide polymorphism (SNP) database, the 1000 Genomes Project, or matching normal controls. Functional analysis of this mutation in zebrafish indicates that the G253C allele is pathogenic. Coinjection of the G253C allele cannot rescue the mislocalization of rhodopsin in the retina when BBS4 is knocked down by morpholino injection. Immunofluorescence analysis in cell culture shows that this missense mutation in BBS4 does not cause obvious defects in protein expression or pericentriolar localization. This mutation likely mainly reduces or abolishes BBS4 function in the retina. Further studies of this allele will provide important insights concerning the pleiotropic nature of BBS4 function. Show less

Primary cilia are ubiquitous cellular appendages that provide important yet not well understood sensory and signaling functions. Ciliary dysfunction underlies numerous human genetic disorders. However, the precise defects in cilia function and the basis of disease pathophysiology remain unclear. Here, we report that the proteins disrupted in the human ciliary disorder Bardet-Biedl syndrome (BBS) are required for the localization of G protein-coupled receptors to primary cilia on central neurons. We demonstrate a lack of ciliary localization of somatostatin receptor type 3 (Sstr3) and melanin-concentrating hormone receptor 1 (Mchr1) in neurons from mice lacking the Bbs2 or Bbs4 gene. Because Mchr1 is involved in the regulation of feeding behavior and BBS is associated with hyperphagia-induced obesity, our results suggest that altered signaling caused by mislocalization of ciliary signaling proteins underlies the BBS phenotypes. Our results also provide a potential molecular mechanism to link cilia defects with obesity. Show less

Bardet-Biedl syndrome (BBS) is a heterogeneous genetic disorder that comprises numerous features, including renal cystic disease. Twelve BBS genes have been identified (BBS1-12). Although the exact functions of the BBS proteins are unknown, evidence suggests that they are involved in cilia assembly, maintenance and/or function. Renal primary cilia dysfunction can lead to cystic kidney disease. To test whether lacking Bbs4 affects cilia assembly and structure, we analyzed primary cilia in Bbs4-null (Bbs4(-/-)) mice. Renal tubule cultures from wild-type (Bbs4(+/+)) and Bbs4(-/-) mice were examined by immunocytochemistry and scanning and transmission electron microscopy. Our culture conditions generated ciliated epithelial cells that were mostly of collecting duct origin. The microtubule ultrastructure of cilia and basal bodies did not appear disrupted in Bbs4(-/-) cells. In control cells, cilia length was maximal at 7 days in culture. In cells cultured from Bbs4(-/-) mice, cilia were shorter initially, but surpassed the length of control cilia by 10 days. Renal primary cilia were also longer in Bbs4(-/-) kidneys. Lacking Bbs4 does not lead to aberrant cilia or basal body structure. However, the dynamics of cilia assembly is altered in Bbs4(-/-) cells, suggesting a role for Bbs4 in the regulation of ciliary assembly. Show less

Recognition of membrane proteins by the Nedd4/Rsp5 ubiquitin ligase family is a critical step in their targeting to the multivesicular body pathway. Some substrates contain "PY" motifs (PPxY), which bind to WW domains in the ligase. Others lack PY motifs and instead rely on adaptors that recruit the ligase to them. To investigate the mechanism of adaptor-mediated ubiquitination, we have characterized the interactions between the adaptor Bsd2, the ubiquitin ligase Rsp5, and the membrane proteins Cps1, Tre1, and Smf1 from Saccharomyces cerevisiae. We have reconstituted adaptor-mediated modification of Cps1 and Tre1 in vitro, and we show that two PY motifs in Bsd2 and two WW domains (WW2 and WW3) in Rsp5 are crucial for this. The binding of a weak noncanonical DMAPSY motif in Bsd2 to WW3 is an absolute requirement for Bsd2 adaptor function. We show that sorting of the manganese transporter Smf1, which requires both Bsd2 and Tre1, depends upon two PY motifs in Bsd2 and one motif in Tre1 but only two WW domains in Rsp5. We suggest that sequential assembly of first a Bsd2/Rsp5 complex, then a Tre1/Bsd2/Rsp5 complex followed by a rearrangement of PY-WW interactions is required for the ubiquitination of Smf1. Show less

Genetic variation across inbred and outbred mouse strains have been observed for intake of sweet solutions, salts, bitter tastants and a high-fat diet. Our laboratory recently reported marked strain differences in the amounts and/or percentages of kilocalories of sucrose consumed among 11 inbred and one outbred mouse strains exposed to a wide range of nine sucrose concentrations (0.0001-5%) in two-bottle 24-h preference tests. To assess whether differences in fat intake were similarly associated with genetic variation, the present study examined intake of chow, water and an emulsified fat source (Intralipid) across nine different concentrations (0.00001-5%) in the same 11 inbred and 1 outbred mouse strains using two-bottle 24-h preference tests, which controlled for Intralipid concentration presentation effects, Intralipid and water bottle positions, and measurement of kilocalorie intake consumed as Intralipid or chow. Strains displayed differential increases in Intralipid intake relative to corresponding water with significant effects observed at the seven (BALB/cJ: 0.001% threshold sensitivity), four (AKR/J, C57BL/6J, DBA/2J, SWR/J: 0.5% threshold sensitivity), three (CD-1, C57BL/10J, SJL/J: 1% threshold sensitivity) and two (A/J, CBA/J, C3H/HeJ, 129P3/J: 2% threshold sensitivity) highest concentrations. In assessing the percentage of kilocalories consumed as Intralipid, SWR/J mice consumed significantly more at the three highest concentrations to a greater degree than BALB/cJ, C57BL/6J, CD-1, C3H/HeJ, DBA/J and 129P3/J strains which in turn consumed more than A/J, AKR/J, CBA/J, C57BL/10J and SJL/J mice. Relatively strong (h2 = 0.73-0.79) heritability estimates were obtained for weight-adjusted Intralipid intake at those concentrations (0.001-1%) that displayed the largest strain-specific effects in sensitivity to Intralipid. The identification of strains with diverging abilities to regulate kilocalorie intake when presented with high Intralipid concentrations may lead to the successful mapping of genes related to hedonics and obesity. Show less

Bardet-Biedl syndrome (BBS) is a rare oligogenic disorder exhibiting both clinical and genetic heterogeneity. Although the BBS phenotype is variable both between and within families, the syndrome is characterized by the hallmarks of developmental and learning difficulties, post-axial polydactylia, obesity, hypogenitalism, renal abnormalities, retinal dystrophy, and several less frequently observed features. Eleven genes mutated in BBS patients have been identified, and more are expected to exist, since about 20-30% of all families cannot be explained by the known loci. To investigate the etiopathogenesis of BBS, we created a mouse null for one of the murine homologues, Bbs4, to assess the contribution of one gene to the pleiotropic murine Bbs phenotype. Bbs4 null mice, although initially runted compared to their littermates, ultimately become obese in a gender-dependent manner, females earlier and with more severity than males. Blood chemistry tests indicated abnormal lipid profiles, signs of liver dysfunction, and elevated insulin and leptin levels reminiscent of metabolic syndrome. As in patients with BBS, we found age-dependent retinal dystrophy. Behavioral assessment revealed that mutant mice displayed more anxiety-related responses and reduced social dominance. We noted the rare occurrence of birth defects, including neural tube defects and hydrometrocolpos, in the null mice. Evaluations of these null mice have uncovered phenotypic features with age-dependent penetrance and variable expressivity, partially recapitulating the human BBS phenotype. Show less

Brain-derived neurotrophic factor (BDNF) has been reported to be critical for the development of cortical inhibitory neurons. However, the effect of BDNF on the expression of transcripts whose protein products are involved in gamma amino butric acid (GABA) neurotransmission has not been assessed. In this study, gene expression profiling using oligonucleotide microarrays was performed in prefrontal cortical tissue from mice with inducible deletions of BDNF. Both embryonic and adulthood ablation of BDNF gave rise to many shared transcriptome changes. BDNF appeared to be required to maintain gene expression in the SST-NPY-TAC1 subclass of GABA neurons, although the absence of BDNF did not alter their general phenotype as inhibitory neurons. Furthermore, we observed expression alterations in genes encoding early-immediate genes (ARC, EGR1, EGR2, FOS, DUSP1, DUSP6) and critical cellular signaling systems (CDKN1c, CCND2, CAMK1g, RGS4). These BDNF-dependent gene expression changes may illuminate the biological basis for transcriptome changes observed in certain human brain disorders. Show less

The feeding response following administration of the free fatty acid oxidation inhibitor, mercaptoacetate (MA) is conceptualized as an experimental model of lipoprivation, which may contribute to the understanding of inter-individual differences in the modulation of this homeostatic response. Although variation in the intake of food, water and glucoprivation as well as intake of several nutrients is known to be associated with genetic variation, it is not known whether MA-induced feeding is similarly dependent upon genotype. The present study therefore examined MA-induced feeding in mice of 11 inbred (A/J, AKR/J, BALB/cJ, CBA/J, C3H/HeJ, C57BL6/J, C57BL10/J, DBA/2J, SJL/J, SWR/J, 129P3/J) and one outbred (CD-1) strains across a wide range of previously determined effective MA doses (5, 35, 70, 100 mg/kg) and test times (1-4 h). MA produced significant dose-dependent and strain-dependent increases in food intake with strong responses noted in DBA/2J, outbred CD-1 and AKR/J mice. More limited dose-specific increases in food intake following MA occurred in C3H/HeJ, BALB/cJ, CBA/J, SJL/J, SWR/J and C57BL/6J mice. In contrast, MA failed to significantly increase food intake in A/J, C57BL/10J and 129P/3J mice. MA-induced food intake correlated significantly across strains only following the two highest doses, and intake following only the highest MA dose correlated significantly across strains with intake following only a moderate glucoprivic dose of 2-deoxy-d-glucose. Thus, these inter-strain differences suggest that lipoprivic (e.g., MA intake) and glucoprivic (e.g., 2-deoxy-d-glucose intake) responsivity operate via only partially overlapping genetic mechanisms of action. The demonstration of genotype-dependent variability in this lipoprivic response may provide the basis for the subsequent identification of trait-relevant genes. Show less

Mouse strain differences for intake of sucrose and saccharin have been reported across studies, and some of these differences have been related to variants of the Tas1r3 taste receptor gene. However, several methodological concerns remain, including use of relatively few strains and/or a limited number of palatable concentrations in previous analyses. The present study examined strain differences in sucrose intake among 11 inbred (A/J, AKR/J, BALB/cJ, CBA/J, C3H/HeJ, C57BL6/J, C57BL10/J, DBA/2J, SJL/J, SWR/J, 129P3/J) and one outbred (CD-1) mouse strains across nine different sucrose concentrations (0.0001-20%) using two-bottle 24-h preference tests which controlled for sucrose concentration presentation effects, sucrose and water bottle positions, and measurement of kilocalorie intake as sucrose or chow. A/J, C57BL/6J, CD-1 and SWR/J strains consumed the greatest (11.6-22 ml) amount of sucrose, whereas the A/J, C57BL/10J, SJL/J and SWR/J strains consumed the greatest (44-56%) percentages of kilocalories as sucrose. The AKR/J, CBA/J, C3H/HeJ and DBA/2J strains consumed the least (6.9-7.9 ml) amount of sucrose, and displayed lower (20-30%) percentages of kilocalories consumed as sucrose. Whereas A/J, C57BL/6J, C57BL/10J, CD-1, SWR/J and SJL/J strains all displayed the most pronounced compensatory decreases in chow intake as the percentage of kilocalories consumed as sucrose increased, the AKR/J, C3H/HeJ and DBA/2J strains failed to significantly alter chow intake even at high sucrose concentrations. There was a paucity of significant correlations in the percentage of sucrose intake between sucrose concentrations, but percentage of sucrose intake at lower concentrations did correlate with previous descriptions of saccharin intake and variants of the Tas1r3 taste receptor gene. These data demonstrate clear mouse strain differences across a range of measures in sucrose intake across a wide range of concentrations, but caution against extrapolating between extremely high and low concentrations. The identification of strains with diverging abilities to regulate kilocalorie intake when presented with high sucrose concentrations may lead to the successful QTL mapping of this trait. Show less

The increasing identification of disease genes is revealing a growing number of traits that fail to conform to traditional Mendelian paradigms, thereby creating new challenges to both genetic investigators and clinicians. Bardet-Biedl syndrome (BBS) is one such disorder that has helped to define 'oligogenic' inheritance, a term that implies that some diseases are not inherited as simple single-gene Mendelian disorders and yet are not classic complex traits, but rather fit a model in which mutations in a small number of genes may interact genetically to manifest the phenotype. BBS is a pleiotropic disorder characterized by postnatal obesity, post-axial polydactyly, and progressive retinal dystrophy. Eight BBS loci have been identified to date and six of these genes have been cloned. Mutation analysis of these BBS genes in a cohort of patients has led to the description of the novel phenomenon of 'triallelic inheritance', wherein families were identified in which three mutations from genes at two different BBS loci segregate with expression of the disease. Modeling the cooperative ability of alleles of different genes at distinct loci to give rise to a particular phenotype will facilitate the understanding of complex multifactorial and polygenic traits. Show less

Bardet-Biedl syndrome is a genetically and clinically heterogeneous disorder caused by mutations in at least seven loci (BBS1-7), five of which are cloned (BBS1, BBS2, BBS4, BBS6, and BBS7). Genetic and mutational analyses have indicated that, in some families, a combination of three mutant alleles at two loci (triallelic inheritance) is necessary for pathogenesis. To date, four of the five known BBS loci have been implicated in this mode of oligogenic disease transmission. We present a comprehensive analysis of the spectrum, distribution, and involvement in non-Mendelian trait transmission of mutant alleles in BBS1, the most common BBS locus. Analyses of 259 independent families segregating a BBS phenotype indicate that BBS1 participates in complex inheritance and that, in different families, mutations in BBS1 can interact genetically with mutations at each of the other known BBS genes, as well as at unknown loci, to cause the phenotype. Consistent with this model, we identified homozygous M390R alleles, the most frequent BBS1 mutation, in asymptomatic individuals in two families. Moreover, our statistical analyses indicate that the prevalence of the M390R allele in the general population is consistent with an oligogenic rather than a recessive model of disease transmission. The distribution of BBS oligogenic alleles also indicates that all BBS loci might interact genetically with each other, but some genes, especially BBS2 and BBS6, are more likely to participate in triallelic inheritance, suggesting a variable ability of the BBS proteins to interact genetically with each other. Show less

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous disorder, the primary features of which include obesity, retinal dystrophy, polydactyly, hypogenitalism, learning difficulties, and renal malformations. Conventional linkage and positional cloning have led to the mapping of six BBS loci in the human genome, four of which (BBS1, BBS2, BBS4, and BBS6) have been cloned. Despite these advances, the protein sequences of the known BBS genes have provided little or no insight into their function. To delineate functionally important regions in BBS2, we performed phylogenetic and genomic studies in which we used the human and zebrafish BBS2 peptide sequences to search dbEST and the translation of the draft human genome. We identified two novel genes that we initially named "BBS2L1" and "BBS2L2" and that exhibit modest similarity with two discrete, overlapping regions of BBS2. In the present study, we demonstrate that BBS2L1 mutations cause BBS, thereby defining a novel locus for this syndrome, BBS7, whereas BBS2L2 has been shown independently to be BBS1. The motif-based identification of a novel BBS locus has enabled us to define a potential functional domain that is present in three of the five known BBS proteins and, therefore, is likely to be important in the pathogenesis of this complex syndrome. Show less

Bardet-Biedl syndrome (BBS) is an uncommon multisystemic disorder characterized primarily by retinal dystrophy, obesity, polydactyly, and renal dysfunction. BBS has been modeled historically as an autosomal recessive trait, under which premise six independent BBS loci (BBS1-BBS6) have been mapped in the human genome. However, extended mutational analyses of BBS2 and BBS6, the first two BBS genes cloned, suggest that BBS exhibits a more complex pattern of inheritance, in which three mutations at two loci simultaneously are necessary and sufficient in some families to manifest the phenotype. We evaluated the spectrum of mutations in the recently identified BBS4 gene with a combination of haplotype analysis and mutation screening on a multiethnic cohort of 177 families. Consistent with predictions from previous genetic analyses, our data suggest that mutations in BBS4 contribute to BBS in <3% of affected families. Furthermore, integrated mutational data from all three currently cloned BBS genes raise the possibility that BBS4 may participate in triallelic inheritance with BBS2 and BBS1, but not the other known loci. Establishment of the loci pairing in triallelism is likely to be important for the elucidation of the functional relationships among the different BBS proteins. Show less

A novel GIP receptor antagonist was developed to evaluate the acute role of glucose-dependent insulinotropic polypeptide (GIP) in the insulin response to oral glucose in rats. Antisera to an extracellular epitope of the GIP receptor (GIPR) detected immunoreactive GIPR on rat pancreatic beta-cells. Purified GIPR antibody (GIPR Ab) specifically displaced GIP binding to the receptor and blocked GIP-mediated increases in intracellular cAMP. When delivered to rats by ip injection, GIPR Ab had a half-life of approximately 4 days. Treatment with GIPR Ab (1 microg/g BW) blocked the potentiation of glucose-stimulated insulin secretion by GIP (60 pmol) but not glucagon-like peptide-1 (GLP-1, 60 pmol) in anesthetized rats. The insulin response to oral glucose was delayed in conscious unrestrained rats that were pretreated with GIPR Ab. Plasma insulin levels were approximately 35% lower at 10 min in GIPR Ab treated animals compared with controls. As a result, the glucose excursion was greater in the GIPR Ab treated group. Fasting plasma glucose levels were not altered by GIPR Ab. We conclude that release of GIP following oral glucose may act as an anticipatory signal to pancreatic beta-cells to promote rapid release of insulin for glucose disposal. Show less

Deletion analysis offers a powerful alternative to linkage and karyotypic approaches for human chromosome mapping. A panel of deletion hybrids has been derived by mutagenizing J1, a hamster cell line that stably retains chromosome 11 as its only human DNA, and selecting for loss of MIC1, a surface antigen encoded by a gene in band 11p13. A unique, self-consistent map was constructed by analyzing the pattern of marker segregation in 22 derivative cells lines; these carry overlapping deletions of 11p13, but selectively retain a segment near the 11p telomere. The map orders 35 breakpoints and 36 genetic markers, including 3 antigens, 2 isozymes, 12 cloned genes, and 19 anonymous DNA probes. The deletions span the entire short arm, dividing it into more than 20 segments and define a set of reagents that can be used to rapidly locate any newly identified marker on 11p, with greatest resolution in the region surrounding MIC1. The approach we demonstrate can be applied to map any mammalian chromosome. To test the gene order, we examined somatic cell hybrids from five patients, whose reciprocal translocations bisect band 11p13; these include two translocations associated with familial aniridia and two with acute T-cell leukemia. In each patient, the markers segregate in telomeric and centromeric groups as predicted by the deletion map. These data locate the aniridia gene (AN2) and a recurrent T-cell leukemia breakpoint (TCL2) in the marker sequence, on opposite sides of MIC1. To provide additional support, we have characterized the dosage of DNA markers in a patient with Beckwith-Wiedemann syndrome and an 11p15-11pter duplication. Our findings suggest the following gene order: TEL - (HRAS1, MER2, CTSD, TH/INS/IGF2, H19, D11S32) - (RRM1, D11S1, D11S25, D11S26) - D11S12 - (HBBC, D11S30) - D11S20 - (PTH, CALC) - (LDHA, SAA, TRPH, D11S18, D11S21) - D11S31 - D11S17 - HBVS1 - (FSHB, D11S16) - AN2 - MIC1 - TCL2 - delta J - CAT - MIC4 - D11S9 - D11S14 - ACP2 - (D11S33, 14L) - CEN. We have used the deletion map to show the distribution on 11p of two centromeric repetitive elements and the low-order interspersed repeat A36Fc. Finally, we provide evidence for an allelic segregation event in the hamster genome that underlies the stability of chromosome 11 in J1. The deletion map provides a basis to position hereditary disease loci on 11p, to distinguish the pattern of recessive mutations in different forms of cancer and, since many of these genes have been mapped in other mammalian species, to study the evolution of a conserved syntenic group. Show less

The concentration of transferrin in fluids collected by micropuncture techniques from the rete testis and zones 1A, 2, 5A, and 6B of the rat ductus epididymidis was 44, 527, 113, and 49 ng/microliter, respectively. When changes in fluid volume were taken into account, it was found that transferrin was concentrated by the efferent ducts, whereas the amount of endogenous luminal transferrin declined from the caput to the cauda epididymidis. Using transferrin-gold as an electron dense marker, we showed that the decline in the concentration of transferrin along the epididymis could be attributed to its cumulative receptor-mediated endocytosis by the lining principal cells. No significant difference in the net receptor-mediated endocytosis of transferrin-gold was found between the proximal caput and corpus epididymidis in vivo. Show less

Micropuncture techniques were used to study receptor-mediated endocytosis of alpha 2-macroglobulin bound to colloidal gold (alpha 2M-gold) by principal cells in the proximal caput epididymidis of control and efferent duct-ligated rats. The pathway of receptor-mediated endocytosis of alpha 2-macroglobulin-gold in vivo was similar to that which occurs in vitro. Alpha 2-macroglobulin-gold was taken up and internalized in coated pits and coated vesicles and was localized sequentially in uncoated vesicles (endosomes), tubular-vesicular structures, multivesicular bodies, and lysosomes. However, a 100-fold excess of alpha 2-macroglobulin did not displace the uptake of alpha 2-macroglobulin-gold in principal cells from control rats. In contrast, uptake of alpha 2-macroglobulin-gold by principal cells from efferent duct-ligated rats was six-fold greater than in control rats, and could be displaced to control levels by a 100-fold excess of alpha 2-macroglobulin. It is suggested that the inability of a 100-fold excess of alpha 2-macroglobulin to displace uptake of alpha 2-macroglobulin-gold in control rats was due to the normal saturation of apparent alpha 2-macroglobulin receptors on principal cells. The effect of efferent duct ligation was to remove the high levels of endogenous alpha 2-macroglobulin, which depleted the receptors of alpha 2-macroglobulin, thereby allowing a higher uptake of alpha 2-macroglobulin-gold in the efferent duct-ligated rats. Show less