Genetic variants of the human obesity (OB) gene in subjects with and without Prader—Willi syndrome: comparison with body mass index and weight

We investigated whether an association exists between genetic variants of the human obesity ( OB or leptin) gene and body mass index (BMI) or weight in subjects with Prader-Willi syndrome (PWS) and in age-and gender-matched lean and obese subjects without PWS. The study included 51 subjects with PWS (mean age = 17.7 ± 9.5 years, BMI = 29.7 ± 8.3 kg/m²); 50 non-PWS obese subjects (mean age = 18.2 ± 10.8 years, BMI = 33.3 ± 9.5 kg/m²); and 53 non-PWS lean subjects (mean age = 17.8 ± 9.5 years, BMI = 19.5 ± 2.9 kg/m²). Allele sizes were determined via standard polymerase chain reaction of the D7S1875 locus, a dinucleotide repeat polymorphism close to the OB gene and classified as trichotomous (homozygous < 208 bp, heterozygous < 208/≥ 208 bp, homozygous < 208 bp) or dichotomous (homozygous < 208 bp or not). Non-PWS males showed a marked decrease in weight with larger alleles while females did not (interaction effect, p < 0.05). Comparable effects were not observed among the PWS subjects. Associations between BMI and genotype were statistically significant (r = 0.22, one-tailed p < 0.05) and comparable to previous research among the non-PWS subjects < 18 years, but not the adults (r = 0.05, one-tailed p = 0.38). Correlations were not statistically significant among either the adult or non-adult PWS subjects.     

Association of the G-2548A polymorphism in the 5' region of the LEP gene with overweight

Mutations in the translated part of the leptin gene ( LEP ) have been found in only two families. Nevertheless DNA polymorphisms in the LEP region are linked to extreme obesity. We previously found in the 5' region of LEP a polymorphism, G-2548A, associated with a difference in BMI reduction following a low calorie diet in overweight women. Recently, this polymorphism was associated with extreme obesity in women. In this work, we genotyped a new sample from the general population including 314 normal weight (BMI < 27 kg/m²) and 109 overweight subjects (BMI ≥ 27 kg/m²). The genotype and allele frequencies were significantly different between groups, with the G-2548 allele being more frequent in the overweight subjects ( p < 0.01). In men, carriers of this allele had lower leptin concentrations adjusted for fat mass ( p = 0.05). Our results indicate that variations at the leptin locus are associated with common obesity phenotypes, and not only with extreme obesity or the rare mendelian obesity syndromes.     

Allelic association discriminates draft orders

A year ago there was hope that a finished sequence of the human genome would soon be publicly available and would give a more reliable locus order than an unconstrained radiation hybrid or genetic map. Alas, there are now different draft orders for each region, none of which may be correct because of gaps, uncertain polarity of contigs, and errors in assembly. Shortly before these drafts became available, we analysed allelic association (also called linkage disequilibrium, LD) in the FRAX region in a large sample of haplotypes (Ennis et al . 2000). We demonstrate here that this material discriminates among alternative draft orders. To express support for discrimination between two values of χ²=−2 ln L we use the Akaike criterion AIC = df[χ²/min χ²−1]. Excluding premutations and full mutations at FMR1, all maps have 715 degrees of freedom (df) among 717 pairs of alleles after accepting L = 0 and estimating M, ∈ in the Malecot equation E(ρ) = Me ^−∈d, where ρ is the association between a pair of alleles at distance d. An AIC in excess of 2 provides evidence against a map with the larger χ².     

Obesity is Associated with Genetic Variants That Alter Dopamine Availability

Human and animal studies have implicated dopamine in appetite regulation, and family studies have shown that BMI has a strong genetic component. Dopamine availability is controlled largely by three enzymes: COMT, MAOA and MAOB, and by the dopamine transporter SLC6A3, and each gene has a well-characterized functional variant. Here we look at these four functional polymorphisms together, to investigate how heritable variation in dopamine levels influences the risk of obesity in a cohort of 1150, including 240 defined as obese (BMI ≥ 30). The COMT and SLC6A3 polymorphisms showed no association with either weight, BMI or obesity risk. We found, however, that both MAOA and MAOB show an excess of the low-activity genotypes in obese individuals ( MAOA: χ²= 15.45, p = 0.004; MAOB: χ²= 8.05, p = 0.018). Additionally, the MAOA genotype was significantly associated with both weight (p = 0.0005) and BMI (p = 0.001). When considered together, the 'at risk genotype' - low activity genotypes at both the MAOA and MAOB loci - shows a relative risk for obesity of 5.01. These results have not been replicated and, given the experience of complex trait genetics, warrant caution in interpretation. In implicating both the MAOA and MOAB variants, however, this study provides the first indication that dopamine availability (as opposed to other effects of MAOA) is involved in human obesity. It is therefore a priority to assess the associations in replication datasets.     

Association of HincII RFLP of low density lipoprotein receptor gene with obesity in essential hypertensives

Obese (BMI 26 kg/m²; n=51) and lean (BMI <26 kg/m²; n=61) Caucasian patients with severe, familial essential hypertension, were compared with respect to genotype and allele frequencies of a Hin cII RFLP of the low density lipoprotein receptor gene ( LDLR ). A similar analysis was performed in obese (n=28) and lean (n=68) nonmotensives. A significant association of the C allele of the T → C variant responsible for this RFLP was seen with obesity ( x ²=4.6, P =0.029) in the hypertensive, but not in the normotensive, group (odds ratio=3.0 for the CC genotype and 2.7 for CT ). Furthermore, BMI tracked with genotypes of this allele in the hypertensives ( P =0.046). No significant genotypic relationship was apparent for plasma lipids. Significant linkage disequilibrium was, moreover, noted between the Hin cII RFLP and an Apa LI RFLP ( x ²=33, P <0.0005) that has previously shown even stronger association with obesity (odds ratio 19.6 for cases homozygous for the susceptibility allele and 15.2 for heterozygotes). The present study therefore adds to our previous evidence implicating LDLR as a locus for obesity in patients with essential hypertension.     

Donor-recipient sharing of HLA class II alleles predicts earlier recurrence and accelerated progression of hepatitis C following liver transplantation

Abstract: Both direct viral cytopathic effects and host immune responses appear to be important in the pathogenesis of hepatitis C virus (HCV) infection. Liver transplantation provides a means to explore the role of the immune system in the development of HCV-related liver damage through comparing the natural history of HCV in patients with different degrees of donor-recipient human leukocyte antigen (HLA) matching. We evaluated 36 patients with recurrent hepatitis C viremia following liver transplantation to determine whether hepatocellular injury or progression to bridging fibrosis occur more rapidly when donor and recipients share HLA alleles. HLA typing for the HLA-A and HLA-B loci was performed by serological techniques and PCR-based oligotyping was used to type alleles of the DRB1, DRB3, DQA1, and DQB1 loci. A median of eight liver biopsies, obtained during a median follow-up of 36 months, were reviewed per patient. Donor-recipient sharing of alleles of HLA-DQB1 or DRB1 was associated with more rapid development of recurrent hepatitis by univariate analysis (χ²=5.7, P =0.02 and χ²=5.54, P =0.02 respectively). However, only sharing of HLA-DRB1 alleles was identified as an independent predictor of reduced time to recurrent histologic injury by multivariate analysis (χ² =5.74, P = 0.02). Furthermore, sharing of HLA-DRB3 and histologic evidence of rejection were associated with more rapid progression to bridging fibrosis both by univariate methods (χ²=4.12, P =0.04 and χ²=4.66, P =0.03 respectively), and by multivariate analysis (χ²= 13.01, P =0.001). These findings suggest that HLA class II-restricted immune responses may contribute to the pathogenesis of HCV-related liver injury in liver transplant recipients.     

Slow removal of Na+ channel inactivation underlies the temporal filtering property in the teleost thalamic neurons

It has been previously shown that the 'large cell' in the corpus glomerulosum (CG) of a teleost brain has a low-pass temporal filtering property. It fires a single spike only in response to temporally sparse synaptic inputs and thus extracts temporal aspects of afferent activities. To explore the ionic mechanisms underlying this property, we quantitatively studied voltage-gated Na⁺ channels of the large cell in the CG slice preparation of the marine filefish by means of whole-cell patch clamp recordings in the voltage-clamp mode. Recorded Na⁺ current was well described using the Hodgkin-Huxley ' m³h ' model. It was revealed that the Na⁺ channels have a novel feature: remarkably slow recovery from inactivation. In other words, the time constant for the ' h ' gate was extremely large (∼100 ms at −80 to −50 mV). In order to test whether the analysed Na⁺ current serves as a mechanism for filtering, the behaviour of the membrane model incorporating the Na⁺ channel was simulated using a computer program called NEURON. In response to current injections, the membrane model displayed low-pass filtering and firing properties similar to those reported in real cells. The present results suggest that slow removal of Na⁺ channel inactivation serves as a crucial mechanism for the low-pass temporal filtering property of the large cell. The simulation study also suggested that velocity and/or amplitude of a spike propagating though an axon expressing Na⁺ channels of this type could potentially be modulated depending on the preceding activities of the cells.     

KCNQ1 is the luminal K+ recycling channel during stimulation of gastric acid secretion

Parietal cell (PC) proton secretion via H⁺/K⁺-ATPase requires apical K⁺ recycling. A variety of K⁺ channels and transporters are expressed in the PC and the molecular nature of the apical K⁺ recycling channel is under debate. This study was undertaken to delineate the exact function of KCNQ1 channels in gastric acid secretion. Acid secretory rates and electrophysiological parameters were determined in gastric mucosae of 7- to 8-day-old KCNQ1^+/+, ^+/– and ^−/− mice. Parietal cell ultrastructure, abundance and gene expression levels were quantified. Glandular structure and PC abundance, and housekeeping gene expression did not differ between the KCNQ1^−/− and ^+/+ mucosae. Microvillar secretory membranes were intact, but basal acid secretion was absent and forskolin-stimulated acid output reduced by ∼90% in KCNQ1^−/− gastric mucosa. Application of a high K⁺ concentration to the luminal membrane restored normal acid secretory rates in the KCNQ1^−/− mucosa. The study demonstrates that the KCNQ1 channel provides K⁺ to the extracellular K⁺ binding site of the H⁺/K⁺-ATPase during acid secretion, and no other gastric K⁺ channel can substitute for this function.     

Chi-squared tests with small numbers

The usual advice given in standard texts is to combine classes when expected numbers are too small. It is suggested that an alternative would be to use the exact values of teh mean and variance of χ², for which algebraic expressions are provided. The reliability of the test can be improved by using √χ² instead of χ² as the test criterion.     

Molecular scanning for mutations in the melanocortin-4 receptor gene in obese/diabetic Japanese

Decreased function of the melanocortin-4 receptor (MC4R) was reported to cause late-onset obesity and insulin resistance in rodents. Thus mutations in the MC4R gene drew strong attention as a possible cause of obesity and diabetes. We screened for mutations in the MC4R gene in extremely obese [body mass index (BMI) ≥ 35 kg/m²] Japanese with diabetes by direct sequencing. A heterozygous mutation (V103I) was detected in one case (2.0 %), however the frequency was not significantly different from that in non-obese (BMI ≤ 24 kg/m²) and non-diabetic subjects (2.7 %). No other mutations were detected. These results suggest that mutations including V103I in the MC4R gene are not a major cause of obesity or diabetes in Japanese.     

Insulin hypersensitivity in mice lacking the V1b vasopressin receptor

We have reported that [Arg⁸]-vasopressin-stimulated insulin release is blunted in islet cells isolated from V1b receptor-deficient ( V1bR ^−/−) mice. In this study, we used V1bR ^−/− mice to examine the physiological role of the V1b receptor in regulating blood glucose levels in vivo , and we found that the fasting plasma glucose, insulin and glucagon levels were lower in V1bR ^−/− mice than in wild-type ( V1bR ^+/+) mice. Next, we evaluated glucose tolerance by performing an intraperitoneal glucose tolerance test (GTT). The plasma glucose and insulin levels during the GTT were lower in V1bR ^−/− mice than in V1bR ^+/+ mice. An insulin tolerance test (ITT) revealed that, after insulin administration, plasma glucose levels were lower in V1bR ^−/− mice than in V1bR ^+/+ mice. In addition, a hyperinsulinaemic–euglycaemic clamp study showed that the glucose infusion rate was increased in V1bR ^−/− mice, indicating that insulin sensitivity was enhanced at the in vivo level in V1bR ^−/− mice. Furthermore, we found that the V1b receptor was expressed in white adipose tissue and that insulin-stimulated phosphorylation of Akt as an important signaling molecule was increased in adipocytes isolated from V1bR ^−/− mice. Thus, the blockade of the V1b receptor could result, at least in part, in enhanced insulin sensitivity by altering insulin signalling in adipocytes.     

Tumor Necrosis Factor-α-Associated Mechanisms Affecting the Embryonic Response to Cyclophosphamide

Problem  We have previously shown that TNF-α^−/− embryos are more sensitive to the exposure to cyclophosphamide (CP) compared with TNF-α^+/+ embryos; however, the underlying mechanisms are not fully understood. Thus, in our present study, we tried to identify those molecules that might be responsible for the protective effect of the cytokine.
Method of study  CP-treated TNF-α^−/− and TNF-α^+/+ embryos were analyzed for changes in apoptosis by TUNEL and flow cytometry, while cell proliferation was analyzed by BrdU incorporation. The expression of Bax, bcl-2, p53, the p65 subunit of NF-κB and IκBα was assessed by Western blotting and immunohistochemistry.
Results  CP-treated TNF-α^−/− embryos exhibited a more profound decrease in their weight, which was accompanied by an earlier appearance of cellular damage and apoptotic cells and an earlier decrease in cell proliferation in the embryonic brain compared with TNF-α^+/+ embryos. Also, an increased percentage of Bax-positive cells and a decreased percentage of bcl-2-positive cells were detected in TNF-α^−/− embryos 48 hr after exposure, which were accompanied by a decreased percentage of p53-positive cells.
Conclusion  Our data implicate TNF-α to be involved in the protection of the embryo against CP teratogenicity, possibly via alteration in Bax, bcl-2 or p53 expression.     

Regulatory T cells in spontaneous autoimmune encephalomyelitis

Summary: Spontaneous experimental autoimmune encephalomyelitis (EAE) develops in 100% of mice harboring a monoclonal myelin basic protein (MBP)-specific CD4⁺αβ T-cell repertoire. Monoclonality of the αβ T-cell repertoire can be achieved by crossing MBP-specific T-cell receptor (TCR) transgenic mice with either RAG^−/− mice or TCR α^−/−/TCR β^−/− double knockout mice. Spontaneous EAE can be prevented by a single administration of purified CD4⁺ splenocytes or thymocytes obtained from wild-type syngeneic mice. The regulatory T cells (T-reg) that protect from spontaneous EAE need not express the CD25 marker, as effective protection can be attained with populations depleted of CD25⁺ T cells. Although the specificity of the regulatory T cells is important for their generation or regulatory function, T cells that protect from spontaneous EAE can have a diverse TCR α and β chain composition. T-reg cells expand poorly in vivo , and appear to be long lived. Finally, precursors for T-reg are present in fetal liver as well as in the bone marrow of aging mice. We propose that protection of healthy individuals from autoimmune diseases involves several layers of regulation, which consist of CD4⁺CD25⁺ regulatory T cells, CD4⁺CD25⁻ T-reg cells, and anti-TCR T cells, with each layer potentially operating at different stages of T-helper cell-mediated immune responses.     

Association of angiotensin II type 1 receptor gene polymorphism with essential hypertension

The renin-angiotensin system is involved in control of blood pressure and salt and fluid homeostasis. Genes for components of this system have been of major focus in research on the causation of the common, complex, polygenic trait, essential hypertension (HT). Association of an A→C variant at nucleotide 1166 of the angiotensin II type 1 receptor (AT₁R) gene with HT, but an absence of linkage of this locus with this disease, has been reported recently. Since confirmation in a different setting is imperative, we performed a cross-sectional case-control study of the A1166C variant in a well-characterized group of 108 Caucasian HT subjects with a strong family history (two affected parents) and early onset disease. Genotyping was by mismatch polymerase chain reaction/ Bfr I restriction fragment length polymorphism analysis. Frequency of the C¹¹⁶⁶allele was 0.40 in HTs and 0.29 in normotensives. The difference in genotype (χ²= 13, P = 0.0015) and allele (χ²= 5.3, P = 0.02) frequencies between the two groups was significant (odds ratio for CC vs AA+AC = 7.3 [95% CI, 1.9–31.9). The present results implicate the AT₁R gene, or a locus in linkage disequilibrium with the variant tested, in the causation of essential HT.     

CTLA-4 +49A/G polymorphism is associated with Behçet's disease in a Tunisian population

The involvement of excessive T-helper cell functions in the pathogenesis of Behçet's disease (BD) has been reported. Cytotoxic T-lymphocyte antigen-4 (CTLA-4) plays a role in T-cell downregulation. In this report, we investigated the possible association between BD patients and the CTLA-4 +49A/G polymorphism in Tunisian population. A total of 135 Tunisian BD patients and 151 healthy blood donors from the same geographic area were genotyped by polymerase chain reaction for the CTLA-4 +49 A/G polymorphism. A highly significant difference between Tunisian BD patients and healthy controls was found regarding the distribution of CTLA-4 +49 A allele [ P  < 10⁻⁷; χ² = 75.63; odds ratio (OR) = 4.63; 95% confidence interval (CI) = 3.20–6.72] and genotype frequencies ( P  < 10⁻⁷; χ² = 71.02). Furthermore, in the BD group, the A allele was predominant in males (76.3%) when compared with females (62%), ( P  = 0.014; χ² = 5.97; OR = 1.99; 95% CI = 1.10–3.59). No relationship was found between the studied genotype and clinical manifestations. Our results show a gene dose effect of the A allele on the BD. The A allele exerts a stronger effect on disease susceptibility in males compared with females.     

Association of the renin gene polymorphism with essential hypertension in a Chinese population

To study the association of renin gene polymorphism with essential hypertension in the Chinese population, 86 hypertensive and 107 normotensive subjects were enrolled from an epidemiologic survey. Leukocyte DNA was extracted and digested with Hind HI and Bgl I restriction enzymes. Southern hybridization was done with digoxigenin-incorporated renin gene probes generated by polymerase chain reaction. The restriction fragments were detected by anti-digoxigenin antibody and enzyme methods. Two Hind III polymorphysms of the renin gene (8.7kb and 6.2kb) were identified. The allele frequences were 129(75%) and 43(25%), respectively, in hypertensives; they were 139(65%) and 75(35%), respectively, in normotensives (χ²= 4.074, p = 0.044). The genotypes of 8.7/8.7,8.7/6.2 and 6.2/6.2 were significantly different between hypertensives and normotensives, being 45(52%), 39(45%), 2(3%) and 48(45%), 43(40%), and 16(15%), respectively (χ²= 9.002, p = 0.011). The Bgl I polymorphism did not show a difference between hypertensives and normotensives. Thus, we conclude that the renin gene Hind III polymorphysm is associated with hypertension in this Chinese population.     

New variants of Ps salivary polymorphic proteins

Electrophoretic analysis of the Ps protein demonstrated the existence of phenotypes additional to those described by Azen & Denniston (1980). A hypothesis that the polymorphism of the Ps protein is determined by five expressed and one unexpressed alleles was supported by family studies. The gene frequencies in a Japanese population were Ps ^1F= 0.0016, Ps ¹= 0.2983, Ps ^2F= 0.0288, Ps ^2S= 0.0079, Ps ³= 0.0111, Ps ⁰= 0.6523.     

The structural effect of systemic NGF treatment on permanently axotomised dorsal root ganglion cells in adult rats

The effect of systemic NGF treatment on loss and shrinkage of dorsal root ganglion cells was studied in adult male rats after permanent axotomy. Nineteen 16 to 18-wk-old rats had their right 5th lumbar spinal nerve ligated and cut approximately 7 mm peripheral to the ganglion. Two days before the operation, treatment with subcutaneous injections of human recombinant NGF (1.0–0.5 mg/kg/day) was started in 9 test rats; 10 controls were given saline injections. After 1 mo the levels of substance P (SP) and calcitonin gene related peptide (CGRP) were significantly increased in intact sciatic nerve. The number and mean volume of perikarya were estimated using assumption-free stereological techniques including vertical sections, the Cavalieri principle, optical disectors, the planar rotator and systematic sampling techniques. Systemic NGF administration had no influence on survival of primary sensory neurons after axotomy. The number of perikarya was 14300 ( S.D. =1800) in axotomised ganglia in control rats versus 14700 ( S.D. =2100) in axotomised ganglia of NGF treated rats. The reduction of perikarya volume after axotomy was significantly less after NGF treatment (11600 μm³ in the control group versus 8000 μm³ in the NGF treated group). However, the apparent protection of NGF-treatment on perikaryal volume is explained by a hitherto unrecognised size effect on nonaxotomised dorsal root ganglion cells. The untreated rats had a mean volume of 24700 μm³ ( S.D. =2700 μm ³) whereas rats treated with NGF had a volume of 20400 μm³ ( S.D. =1700 μm³) on the nonaxotomised side. In conclusion, systemic NGF treatment in adult rats has no effect on dorsal root ganglion cell loss in permanent axotomy whereas perikaryal size of intact nonaxotomised cells is reduced.     

Polymorphisms of tumor necrosis factor receptor 2 are not associated with insulin-dependent diabetes mellitus or Graves' disease

Insulin-dependent diabetes mellitus (IDDM) and Graves' disease (GD) are autoimmune endocrinopathies and associated with distinct HLA-DR and -DQ alleles as well as several tumor necrosis factor a (TNF-α) and β (TNF-β) alleles. TNF-α and TNF-β interact with TNF receptor (TNF-R), of which two subtypes have been described: TNF-R1 and TNF-R2. We investigated TNF-R2 alleles in 90 patients with IDDM, 101 with GD and 70 healthy controls. Genomic DNA was amplified with specific flanking primers for the untranslated 3 region of TNF-R2. SSCP analysis revealed two alleles by different fragment patterns: TNF-R2*1 and TNF-R2*2. Patients with IDDM or Graves' disease and controls did not differ significantly: TNF-R2*1/*1:IDDM(8%)/GD(2%)/KO(4%); TNF-R2*2/*2:IDDM(34%)/GD(48%)/KO(42%), heterozygosity TNF-R2*1/*2:IDDM(58%)/GD(50%)/KO(54%) (IDDM vs KO: P =0.46, χ²=1.57; GD vs KO: P =0.59, χ²=1.05). In conclusion, the studied polymorphism of TNF-R2 was associated with neither IDDM nor GD in a German population.