The CODATwins Project: The Current Status and Recent Findings of COllaborative Project of Development of Anthropometrical Measures in Twins.

The COllaborative project of Development of Anthropometrical measures in Twins (CODATwins) project is a large international collaborative effort to analyze individual-level phenotype data from twins in multiple cohorts from different environments. The main objective is to study factors that modify genetic and environmental variation of height, body mass index (BMI, kg/m2) and size at birth, and additionally to address other research questions such as long-term consequences of birth size. The project started in 2013 and is open to all twin projects in the world having height and weight measures on twins with information on zygosity. Thus far, 54 twin projects from 24 countries have provided individual-level data. The CODATwins database includes 489,981 twin individuals (228,635 complete twin pairs). Since many twin cohorts have collected longitudinal data, there is a total of 1,049,785 height and weight observations. For many cohorts, we also have information on birth weight and length, own smoking behavior and own or parental education. We found that the heritability estimates of height and BMI systematically changed from infancy to old age. Remarkably, only minor differences in the heritability estimates were found across cultural-geographic regions, measurement time and birth cohort for height and BMI. In addition to genetic epidemiological studies, we looked at associations of height and BMI with education, birth weight and smoking status. Within-family analyses examined differences within same-sex and opposite-sex dizygotic twins in birth size and later development. The CODATwins project demonstrates the feasibility and value of international collaboration to address gene-by-exposure interactions that require large sample sizes and address the effects of different exposures across time, geographical regions and socioeconomic status.

Heritability of arterial stiffness and carotid intima-media thickness: an Italian twin study.

BACKGROUND AND AIMS: Carotid intima-media thickness (IMT) and arterial stiffness parameters, including aortic augmentation index (AIx) and pulse wave velocity (PWV), are independent predictors of stroke and cardiovascular disease. Genetic effects on these traits were never explored in a Mediterranean country. The present study aims to quantify the contribution of genes, environment and age to carotid IMT and aortic Aix and PWV. METHODS AND RESULTS: The twin design was used. A total of 348 adult twins from the Italian Twin Register underwent measurements of carotid IMT and aortic PWV and AIx in three university hospitals located in Rome, Padua and Perugia. Carotid IMT was measured by B-mode ultrasound, aortic PWV and AIx by Arteriograph. Genetic modelling was performed to decompose total variance of traits into genetic, shared and unshared environmental and age components. For each phenotype, the best-fitting model included additive genetic, unshared environmental and age effects. For IMT, heritability was 0.32 (95% confidence interval (CI): 0.25-0.38), unshared environmental component was 0.25 (0.18-0.32) and age contribution was 0.44 (0.39-0.49). For AIx and PWV, heritabilities were 0.42 (0.29-0.55) and 0.49 (0.35-0.62), unshared environmental components were 0.31 (0.22-0.44) and 0.37 (0.26-0.51) and age contributions were 0.27 (0.16-0.39) and 0.14 (0.06-0.24), respectively. CONCLUSION: This study shows substantial genetic and unshared environmental influences on carotid intima-media thickness and arterial stiffness and confirms the relevant role of age in the aetiology of these traits. Further support is provided for prevention and health promotion strategies based on modifiable factors.

Linkage disequilibrium mapping of a type 1 diabetes susceptibility gene (IDDM7) to chromosome 2q31-q33.

The role of human chromosome 2 in type 1 diabetes was evaluated by analysing linkage and linkage disequilibrium at 21 microsatellite marker loci, using 348 affected sibpair families and 107 simplex families. The microsatellite D2S152 was linked to, and associated with, disease in families from three different populations. Our evidence localizes a new diabetes susceptibility gene, IDDM7, to within two centiMorgans of D2S152. This places it in a region of chromosome 2q that shows conserved synteny with the region of mouse chromosome 1 containing the murine type 1 diabetes gene, Idd5. These results demonstrate the utility of polymorphic microsatellites for linkage disequilibrium mapping of genes for complex diseases.

Insulin VNTR allele-specific effect in type 1 diabetes depends on identity of untransmitted paternal allele. The IMDIAB Group.

The IDDM2 type 1 diabetes susceptibility locus was mapped to and identified as allelic variation at the insulin gene (INS) VNTR regulatory polymorphism. In Caucasians, INS VNTR alleles divide into two discrete size classes. Class I alleles (26 to 63 repeats) predispose in a recessive way to type 1 diabetes, while class III alleles (140 to more than 200 repeats) are dominantly protective. The protective effect may be explained by higher levels of class III VNTR-associated INS mRNA in thymus such that elevated levels of preproinsulin protein enhance immune tolerance to preproinsulin, a key autoantigen in type 1 diabetes pathogenesis. The mode of action of IDDM2 is complicated, however, by parent-of-origin effects and possible allelic heterogeneity within the two defined allele classes. We have now analysed transmission of specific VNTR alleles in 1,316 families and demonstrate that a particular class I allele does not predispose to disease when paternally inherited, suggestive of polymorphic imprinting. But this paternal effect is observed only when the father's untransmitted allele is a class III. This allelic interaction is reminiscent of epigenetic phenomena observed in plants (for example, paramutation; ref. 17) and in yeast (for example, trans-inactivation; ref. 18). If untransmitted chromosomes can have functional effects on the biological properties of transmitted chromosomes, the implications for human genetics and disease are potentially considerable.

Adenoid reservoir for pathogenic biofilm bacteria.

Biofilms of pathogenic bacteria are present on the middle ear mucosa of children with chronic otitis media (COM) and may contribute to the persistence of pathogens and the recalcitrance of COM to antibiotic treatment. Controlled studies indicate that adenoidectomy is effective in the treatment of COM, suggesting that the adenoids may act as a reservoir for COM pathogens. To investigate the bacterial community in the adenoid, samples were obtained from 35 children undergoing adenoidectomy for chronic OM or obstructive sleep apnea. We used a novel, culture-independent molecular diagnostic methodology, followed by confocal microscopy, to investigate the in situ distribution and organization of pathogens in the adenoids to determine whether pathogenic bacteria exhibited criteria characteristic of biofilms. The Ibis T5000 Universal Biosensor System was used to interrogate the extent of the microbial diversity within adenoid biopsy specimens. Using a suite of 16 broad-range bacterial primers, we demonstrated that adenoids from both diagnostic groups were colonized with polymicrobial biofilms. Haemophilus influenzae was present in more adenoids from the COM group (P = 0.005), but there was no significant difference between the two patient groups for Streptococcus pneumoniae or Staphylococcus aureus. Fluorescence in situ hybridization, lectin binding, and the use of antibodies specific for host epithelial cells demonstrated that pathogens were aggregated, surrounded by a carbohydrate matrix, and localized on and within the epithelial cell surface, which is consistent with criteria for bacterial biofilms.

HLA-DQA1 and DQB1 gene polymorphisms in type I diabetic patients from central Italy and their use for risk prediction.

Susceptibility to type I diabetes has been shown to be highly correlated with the presence of an amino acid other than Asp at position 57 of the DQ beta-chain (non-Asp57) and also with the presence of an Arg at position 52 of the DQ alpha-chain (Arg52). In this study we analyzed the DQA1 and DQB1 gene polymorphisms in 65 patients from central Italy and 93 randomly selected control subjects. Polymerase chain reaction amplification of DNA encoding the first polymorphic domain of the DQB1 and DQA1 chains was performed, and DQB1 gene polymorphism was evaluated by dot blot analysis using 11 sequence-specific oligonucleotide probes. For DQA1 typing, a new simple procedure based on allele-specific amplification and analysis of heteroduplex DNA molecules formed by the annealing of mismatched allelic strands was used. This technique allows the discrimination of Arg52 and non-Arg52 DQA1 alleles. We then calculated by logistic regression the contribution of these genetic markers to the development of diabetes. Frequencies and odds ratios relative to the amino acid in position 57 of the DQ beta-chain and the amino acid in position 52 of the DQ alpha-chain showed that the highest odds ratio (odds ratio = 161; 95% confidence interval 19-1386) was that of the homozygous combination of the two susceptibility markers (non-Asp57 and Arg52).(ABSTRACT TRUNCATED AT 250 WORDS)

An explanation for the neutral effect of DR2 on IDDM susceptibility in central Italy.

Susceptibility to IDDM is strongly associated with HLA. Some HLA allelic combinations (haplotypes) can be found in most patients, whereas other haplotypes are encountered only rarely. It has been proposed that this difference in susceptibility depends on the absence (in the DR3 and DR4 haplotypes) or the presence (in the DR2 haplotype) of Asp57 in the DQ beta-chain. Data on southern European populations challenge this hypothesis because the DR2 haplotype has not been associated negatively with IDDM, as reported in northern European populations. This study on a selected panel of DR2-positive Italian IDDM patients shows that 19 of 21 (90.5%) DR2 haplotypes possess a non-Asp57 DQB allele. Moreover, the same non-Asp57 subtype has a comparatively high frequency (9/28, or 32.1%, DR2 haplotypes) also in the DR2-positive healthy Italian population. The difference between patients and control subjects is significant (P less than 0.0001). This is the largest series of DR2-positive patients analyzed so far. Comparison with cumulated data in various white populations shows a distinct northern European-to-southern European gradient. Toward southern Europe, the relative frequency of the non-Asp57 DR2 subtype increases. Concomitantly, the apparent protective effect of the DR2 haplotype disappears. Therefore, the observed differences in DR2-IDDM association in white populations can be explained adequately by the Asp57 hypothesis, which this study's data strongly support.

Subacromial Space Width: Does Overuse or Genetics Play a Greater Role in Determining It? An MRI Study on Elderly Twins.

BACKGROUND: Age and peripheral microcirculation disorders are the main causes of rotator cuff degeneration. Acromion variants may affect subacromial space width, causing a pathological narrowing of the space that may compromise the cuff integrity. However, it is not clear if the subacromial space width is genetically determined or if it changes according to loading conditions. To clarify this unresolved question, we performed an MRI (magnetic resonance imaging) study with the aim of evaluating the acromiohumeral distance in a group of elderly monozygotic and dizygotic twins, and we analyzed the obtained data using the twin design to separate the contributions of shared and unique environments. METHODS: We identified twenty-nine pairs of elderly twins. On MRI scans, we evaluated the acromiohumeral distance and health status of the rotator cuff tendons. Heritability, defined as the proportion of total variance of a specific characteristic in a particular population due to a genetic cause, was estimated as twice the difference between the intraclass correlation coefficients for monozygotic and dizygotic pairs. The influence of shared environment, due to environmental factors that contribute to twin and sibling similarity, was calculated as the difference between the monozygotic correlation coefficient and the heritability index. One-way ANOVA (analysis of variance) was used to estimate the differences among job categories, both in the total cohort and within zygosity groups. RESULTS: The intraclass correlation coefficient was substantially higher for monozygotic than for dizygotic twins, indicating a high degree of concordance of the acromiohumeral distance in pairs of individuals who shared 100% of their genes. The heritability index was 0.82, and shared and unique environmental contributions were both 0.09. There were no significant differences among subjects in different job categories, either in the total cohort (p = 0.685) or within the monozygotic (p = 0.719) and dizygotic groups (p = 0.957). CONCLUSIONS: The acromiohumeral distance is mainly genetically determined and only marginally influenced by external factors.

Linkage analysis of multiple sclerosis with candidate region markers in Sardinian and Continental Italian families.

Previous genome screens in multiple sclerosis have shown some evidence of linkage in scattered chromosomal regions. Although in no case the evidence of each single study was compelling and although in general the linkage 'peaks' of the different studies did not coincide, some regions can be considered likely candidates for the presence of MS risk genes because of the clustering of MLS scores and homology with eae loci. We performed a linkage analysis of markers in these regions and of intragenic markers of some individual candidate genes (HLA-DRB1, CTLA-4, IL9, APOE, BCL2, TNFR2). For the first time, Southern European populations were targeted, namely Continental Italians and Sardinians. A total of 69 multiplex families were typed for 67 markers by a semi-automatic fluorescence-based assay. Results were analysed for linkage by two non-parametric tests: GENEHUNTER and SimIBD. In general, the linkage scores obtained were low, confirming the conclusion that no gene is playing a major role in the disease. However, some markers, in 2p11, 3q21.1, 7p15.2 and 22q13.1 stood out as promising since they showed higher scores with one or the other test. This stimulates further association analysis of a large number of simplex families from the same populations.

Vitamin A modifies the glycopeptide composition of cultured Sertoli cells.

Sertoli cells obtained from prepubertal rat testes were cultured in the presence or absence of retinol. Incorporation of monosaccharides and glycopeptide composition of the cells were studied under two experimental conditions. The results indicate that retinol increases the amount of mannose and glucosamine incorporated into cellular glycoconjugates. The labeled glycopeptides obtained from control and retinol-treated cells were separated by size and lectin affinity. Gel filtration analysis showed no size differences between the glycopeptides obtained from control and vitamin A-treated cells. Affinity chromatography on Concanavalin A and Wheat Germ Agglutinin of 3H-mannose-labeled glycopeptides showed that Sertoli cells cultured in the presence of retinol contain a higher percentage of high mannose-type glycopeptides compared with control cells. The effect of retinol on Sertoli cell glycopeptide composition is partially reversed by the administration of FSH.

Association of DRB1*04-DQB1*0301 haplotype and lack of association of two polymorphic sites at CTLA-4 gene with Hashimoto's thyroiditis in an Italian population.

Hashimoto's thyroiditis (HT) is an autoimmune disease resulting from a complex interaction between genetic and environmental factors. The genetic loci conferring susceptibility need to be still defined. The aim of the present study was to determine whether Cytotoxic T-Lymphocyte-Associated Antigen-4 (CTLA-4), HLA DRB1, and DQB1 genes were associated to HT in an Italian population. We evaluated the allele distribution of the following loci: CTLA-4 exon 1 A49G dimorphism, which resulted in an amino acidic exchange (Thr/Ala) in the leader peptide, CTLA-4 3' microsatellite, HLA DRB1 and DQB1 in 126 patients with HT and in 301 control subjects from an Italian population (Lazio region). CTLA-4 exon 1 A49G dimorphism was typed by Polymerase Chain Reaction and Restriction Fragment Length Polymorphism (PCR-RFLP); CTLA-4 3' microsatellite alleles were defined using a fluorescence-based method. HLA DRB1 and DQB1 alleles were typed using a SSO reverse line blot method and a probeless procedure based on allele group-specific amplification followed by DNA heteroduplex analysis, respectively. Data were initially analyzed by chi2 test or Fisher's exact test. Multiple logistic regression analysis was then applied on factors with significant crude odds ratios and on CTLA-4 exon 1 A49G dimorphism to investigate their independent effects. The two polymorphic sites at CTLA-4 gene did not increase the risk for HT. The distribution of HLA DRB1 and DQB1 alleles did not show any significant difference between patients and controls, however, the DRB1*04-DQB1*0301 haplotype was significantly increased in patients. Other factors that increase the risk of disease were gender and age. Females showed approximately 18 times more risk than males; subjects older than 50 years had an odds ratio of 6.6. These data suggest that these two polymorphic sites at CTLA-4 do not play a major role in the susceptibility of the disease in an Italian population while female gender, age over 50 years, HLA DRB1*04-DQB1*0301 haplotype increase the risk of developing HT.

The HLA system and insulin dependent diabetes: recent findings and prospects for disease prediction.

Our knowledge of the genetics of insulin dependent diabetes (IDDM), and in particular the HLA system, has gained considerable expansion thanks to the application of molecular biology. The genetic susceptibility to the disease is linked to the HLA region, particularly at DQ-alpha and DQ-beta chain genes. Particular amino acid other than aspartic acid in position 57 of the DQ beta chain and the presence of an arginine in position 52 of the DQ alpha chain and to how these markers can be used to identify subjects at risk for developing IDDM. The identification of such subjects may be useful for the development of strategies aimed to prevent the disease and in addition may offer a new insight into population screening.

Clinical implications of power toothbrushing on fluoride delivery: effects on biofilm plaque metabolism and physiology.

Dental biofilms are implicated in the formation of caries and periodontal disease. A major constituent of the supragingival biofilm is Streptococcus mutans, which produces lactic acid from sucrose fermentation, enhancing enamel demineralization and eventual caries development. Caries prevention through F inhibits enamel demineralization and promotes remineralization. Fluoride also exerts effects on metabolic activities in the supragingival biofilm such as aerobic respiration, acid fermentation and dentrification. In experimental S. mutans biofilms, adding 1000 ppm F to an acidogenic biofilm resulting from 10% sucrose addition increased pH to pre-sucrose levels, suggesting inhibition of acid fermentation. F effects on metabolic activity and sucrose utilization in interproximal plaque biofilms were also recorded. Addition of 10% sucrose reduced pH from neutral to 4.2, but subsequent addition of 1000 ppm F increased pH by 1 unit, inhibiting acid fermentation. 10% Sucrose addition also stimulated denitrification, increasing production of nitrous oxide (N(2)O). Addition of 1000 ppm F suppressed denitrification, indicating an additional mechanism by which F exerts effects in the active interproximal biofilm. Finally, fluid dynamic activity by power tooth brushing enhanced F delivery and retention in an experimental S. mutans biofilm, suggesting a potential novel benefit for this intervention beyond mechanical plaque removal.

Vascular endothelial growth factor gene polymorphisms increase the risk to develop psoriasis.

We investigated the relationship between eight polymorphisms in the gene encoding for vascular endothelial growth factor (VEGF) (-1540C > A, -1512Ins18, -1451C > T, -460T > C, -160C > T, -152G > A, -116G > A and +405G > C) and plaque-type psoriasis stratified for age at onset, gender and family history of dermatosis. For this purpose, 117 patients with chronic plaque-type psoriasis and 215 healthy subjects were enrolled. We found that being homozygous -1540AA, -1512InsIns, -1451TT, -460CC and -152AA conferred a significant risk in developing psoriasis compared with heterozygous (-1540CA, -1512 + Ins, -1451CT, -460CT and -152AG) and homozygous genotypes (-1540CC, -1512 + +-1451CC, -460TT and -152GG) grouped together [odds ratio (ORs) = 1.73, 1.73, 1.73, 1.77 and 1.87, respectively]. Conversely, having the -116AA or +405GG genotype did not significantly increase the risk of disease expression compared with other genotypes of the same loci. Interestingly, we found that -1540AA, -1512InsIns, -1451TT, -460CC and -152AA homozygous genotypes have a significant two-fold increased risk in developing psoriasis after the age of 40 years (late-onset psoriasis) (ORs = 2.19, 2.19, 2.19, 2.05 and 2.26; P = 0.02, 0.02, 0.02, 0.04 and 0.02, respectively) as compared with controls. On the contrary, we found no phenotype-genotype association of the same magnitude among the patients in whom psoriasis developed at or before the age of 40 years (early-onset psoriasis) compared with controls. Genotype distributions were not significantly different when cases and controls were stratified either by gender or family history of psoriasis. Finally, VEGF plasma concentration was not significantly different between patients and controls and was not correlated with the severity of the disease.

Concordance, disease progression, and heritability of coeliac disease in Italian twins.

BACKGROUND AND AIMS: We adopted the twin method to disentangle the genetic and environmental components of susceptibility to coeliac disease (CD). We estimated disease concordance rate by zygosity and HLA genotypes, discordance times, progression rates to disease, and heritability. METHODS: We crosslinked the Italian Twin Registry with the membership lists of the Italian Coeliac Disease Association and recruited 23 monozygotic (MZ) and 50 dizygotic (DZ) twin pairs with at least one affected member. Zygosity was assigned by DNA fingerprinting, and HLA-DQ and DR alleles were genotyped. Disease status was ascertained by antiendomysial, anti-human tissue transglutaminase antibodies, and bowel biopsy. RESULTS: Concordance was significantly higher in MZ (83.3% probandwise, 71.4% pairwise) than in DZ (16.7% probandwise, 9.1% pairwise) pairs. Concordance was not affected by sex or HLA genotype of the co-twin and being MZ was significantly associated with the occurrence of CD (Cox adjusted hazard ratio 14.3 (95% confidence interval 4.0-50.3)). In 90% of concordant pairs the discordance time was

Retinoids regulate gonadotropin action in cultured rat Sertoli cells.

The effect of retinoids on cultured rat Sertoli cells was studied by evaluation of cAMP and estradiol production after gonadotropin stimulation in the presence or absence of the retinoid. Sertoli cells cultured in the presence of FSH produce a high amount of cAMP and increase their aromatase activity. The addition of retinol alone has no effect on cAMP and estradiol production; however, the presence of retinol in the culture medium exerts an inhibitory effect on Sertoli cell response to FSH stimulation. In particular, FSH-induced cAMP production of rat Sertoli cells was significantly reduced (50-60% decrease) both by retinol and by retinoic acid. This effect was observable during the first ten days of culture and was also evident when Sertoli cells were cultured in the presence of retinol and methylisobutylxanthine, an inhibitor of phosphodiesterase activity. Cholera toxin-stimulated cAMP levels were reduced by retinol, whereas forskolin-induced elevation of cAMP levels was not affected by vitamin treatment. The inhibitory effect of retinoids on FSH-stimulated aromatase activity of Sertoli cells, which is cAMP mediated, was also evident. In conclusion, the present study demonstrates that retinoids modulate FSH action on cultured rat Sertoli cells and decrease cAMP production.

Cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase activity in the somatic cells of the seminiferous tubules. I. Isoform analysis.

In this work firstly are reported the chromatographic analysis of cAMP dependent protein kinases present in the cytosols obtained from rat Sertoli cells and peritubular cells. In both cell types two different isoenzymes have been detected, one eluting at 40-80 mM KC1 (type I) and a second one eluting at 150-200 mM KC1 (type II). Only the type I was strongly stimulated by cAMP whereas the type II was slightly cAMP dependent both in the Sertoli cells and in the peritubular cells.

Cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase activity in the somatic cells of the seminiferous tubules. II. Effect of retinol.

The effect of retinol on cyclic AMP dependent protein kinase activity of Sertoli cells and peritubular cells isolated from prepubertal rats has been investigated. Treatments longer than six hours induced a significant inhibition of type I protein kinase activity of Sertoli cells without appreciable variation of type II protein kinase. Short time treatments with the vitamin did not affect the Sertoli cell protein kinase activity. The vitamin A addition did not induce any appreciable variation of peritubular cell protein kinase activity.