The core clock transcription factor BMAL1 drives circadian ß-cell proliferation during compensatory regeneration of the endocrine pancreas.

Circadian clocks in pancreatic islets participate in the regulation of glucose homeostasis. Here we examined the role of these timekeepers in ß-cell regeneration after the massive ablation of ß cells by doxycycline-induced expression of diphtheria toxin A (DTA) in Insulin-rtTA/TET-DTA mice. Since we crossed reporter genes expressing α- and ß-cell-specific fluorescent proteins into these mice, we could follow the fate of α- and ß cells separately. As expected, DTA induction resulted in an acute hyperglycemia, which was accompanied by dramatic changes in gene expression in residual ß cells. In contrast, only temporal alterations of gene expression were observed in α cells. Interestingly, ß cells entered S phase preferentially during the nocturnal activity phase, indicating that the diurnal rhythm also plays a role in the orchestration of ß-cell regeneration. Indeed, in arrhythmic Bmal1-deficient mice, which lack circadian clocks, no compensatory ß-cell proliferation was observed, and the ß-cell ablation led to aggravated hyperglycemia, hyperglucagonemia, and fatal diabetes.

Pancreatic α- and ß-cellular clocks have distinct molecular properties and impact on islet hormone secretion and gene expression.

A critical role of circadian oscillators in orchestrating insulin secretion and islet gene transcription has been demonstrated recently. However, these studies focused on whole islets and did not explore the interplay between α-cell and ß-cell clocks. We performed a parallel analysis of the molecular properties of α-cell and ß-cell oscillators using a mouse model expressing three reporter genes: one labeling α cells, one specific for ß cells, and a third monitoring circadian gene expression. Thus, phase entrainment properties, gene expression, and functional outputs of the α-cell and ß-cell clockworks could be assessed in vivo and in vitro at the population and single-cell level. These experiments showed that α-cellular and ß-cellular clocks are oscillating with distinct phases in vivo and in vitro. Diurnal transcriptome analysis in separated α and ß cells revealed that a high number of genes with key roles in islet physiology, including regulators of glucose sensing and hormone secretion, are differentially expressed in these cell types. Moreover, temporal insulin and glucagon secretion exhibited distinct oscillatory profiles both in vivo and in vitro. Altogether, our data indicate that differential entrainment characteristics of circadian α-cell and ß-cell clocks are an important feature in the temporal coordination of endocrine function and gene expression.

Resveratrol potentiates glucose-stimulated insulin secretion in INS-1E beta-cells and human islets through a SIRT1-dependent mechanism.

Resveratrol, a polyphenol compound, is known for its effects on energy homeostasis. With properties of energy sensors mediating effects of calorie restriction, sirtuins are targets of resveratrol. The mammalian sirtuin homolog SIRT1 is a protein deacetylase playing a role in glucose metabolism and islet function. Here, we investigated the effects of resveratrol and possible link with SIRT1 in ß-cells. Insulinoma INS-1E cells and human islets were cultured with resveratrol before analyzing their physiological responses. Treatment of INS-1E cells for 24 h with 25 µM resveratrol resulted in marked potentiation of glucose-stimulated insulin secretion. This effect was associated with elevated glycolytic flux, resulting in increased glucose oxidation, ATP generation, and mitochondrial oxygen consumption. Such changes correlated with up-regulation of key genes for ß-cell function, i.e. Glut2, glucokinase, Pdx-1, Hnf-1α, and Tfam. In human islets, chronic resveratrol treatment similarly increased both the glucose secretory response and expression of the same set of genes, eventually restoring the glucose response in islets obtained from one type 2 diabetic donor. Overexpression of Sirt1 in INS-1E cells potentiated resveratrol effects on insulin secretion. Conversely, inhibition of SIRT1 achieved either by expression of an inactive mutant or by using the EX-527 inhibitor, both abolished resveratrol effects on glucose responses. Treatment of INS-1E cells with EX-527 also prevented resveratrol-induced up-regulation of Glut2, glucokinase, Pdx-1, and Tfam. Resveratrol markedly enhanced the glucose response of INS-1E cells and human islets, even after removal of the compound from the medium. These effects were mediated by and fully dependent on active SIRT1, defining a new role for SIRT1 in the regulation of insulin secretion.

Frequencies of promoter pentanucleotide (TTTTA)n of CYP11A gene in European and North African populations.

The present work attempts to determine the distribution of CYP11A (TTTTA)n genotype and allele frequencies in 10 European and North African populations. This polymorphism has been associated with hyperandrogenism by several association studies. To our knowledge, this is the first study investigating the ethnic variation of this polymorphism. DNA was extracted from 868 whole-blood samples with the standard phenol-chloroform technique, and PCR reactions were carried out using fluorescent primers as described previously. PCR products were analyzed by an ABI 3,730 DNA Analyzer. A total of six alleles were identified, ranging from 220 bp (4 repeats [4R]) to 250 bp (10R). The most frequent allelic fragment size in all populations was 4R, with frequencies ranging from 47.9% (Sicily) to 62.8% (Tuscany and Germany). Allelic frequencies showed high heterogeneity between analyzed populations. We detected a significant gradient for alleles 4R and 8R. In this study, we report the allele frequency distribution of CYP11A (TTTTA)n showing a north-south geographic gradient. This result could be useful for epidemiological studies about hyperandrogenism.

Population genetic data on four STR loci, PAI (CA)n, GpIIIa (CT)n, PLAT (TG)14 (CA)12, and NOS2A (CCTTT)n, in Mediterranean populations.

In the present study, we have analyzed four highly polymorphic regions (STRs) chosen from four candidate genes involved in: (1) Platelet aggregation: alpha subunit of the platelet GpIIb/GpIIIa integrin complex (GpIIIa (CT)n; 17q21.31), (2) Coagulation fibrinolysis: Plasminogen Activator Tissue (PLAT5 (TG)14(TA)12; 8p12-q11.2) and Plasminogen Activator Inhibitor-1 (PAI-1 (CA)n; 7q21.3-q22), (3) Oxidative metabolism: the inducible nitric oxide (NO) synthase (iNOS) gene (NOS2A (CCTTT)n; 17cen-q11.2). Allele frequencies for these four STR loci were investigated in several Mediterranean populations. The population data deviate from the Hardy-Weinberg equilibrium in all populations for GpIIIa (CT)n polymorphism.

Enhancement of islet engraftment and achievement of long-term islet allograft survival by Toll-like receptor 4 blockade.

BACKGROUND: Toll-like receptors are key players in sterile inflammation phenomena and can link the innate and adaptive immune systems by enhancing graft immunogenicity. They are also considered mediators of types 1 and 2 diabetes development. The aim of the present study was to assess the role of Toll-like receptor-4 (TLR4) in mediating the inflammatory and immune responses to pancreatic islets, thereby promoting inflammatory destruction and immune rejection of islet grafts. METHODS: Experiments were conducted in murine and human in vitro systems and in vivo murine islet transplant models, using species-specific anti-TLR4 monoclonal antibodies. In vitro, mixed lymphocyte-islet reaction experiments were performed to assess T-cell activation and proliferation. In vivo, both a syngeneic (B6-to-B6) marginal mass islet transplant model to assess the impact of TLR4 blockade on islet engraftment and an allogeneic (DBA1-to-B6) model were used. RESULTS: In vitro TLR4 blockade decreased lipopolysaccharide-mediated ß-cell apoptosis and T-cell activation and proliferation against allogeneic islets. In vivo, TLR4 blockade resulted in significantly better syngeneic marginal mass islet engraftment and in indefinite allogeneic islet graft survival. Tolerance was not observed because donor-specific skin graft rechallenge in nonrejecting animals resulted in rejection of both skin and islets, but without accelerated rejection as compared to naive animals. CONCLUSION: Taken together, our data indicate that TLR4 blockade leads to a significant improvement of syngeneic islet engraftment and of allogeneic islet graft survival. A mechanism of graft accommodation with concurrent inhibition of donor-specific immune memory is likely to be involved.

Survival of free and encapsulated human and rat islet xenografts transplanted into the mouse bone marrow.

Bone marrow was recently proposed as an alternative and potentially immune-privileged site for pancreatic islet transplantation. The aim of the present study was to assess the survival and rejection mechanisms of free and encapsulated xenogeneic islets transplanted into the medullary cavity of the femur, or under the kidney capsule of streptozotocin-induced diabetic C57BL/6 mice. The median survival of free rat islets transplanted into the bone marrow or under the kidney capsule was 9 and 14 days, respectively, whereas that of free human islets was shorter, 7 days (bone marrow) and 10 days (kidney capsule). Infiltrating CD8+ T cells and redistributed CD4+ T cells, and macrophages were detected around the transplanted islets in bone sections. Recipient mouse splenocytes proliferated in response to donor rat stimulator cells. One month after transplantation under both kidney capsule or into bone marrow, encapsulated rat islets had induced a similar degree of fibrotic reaction and still contained insulin positive cells. In conclusion, we successfully established a small animal model for xenogeneic islet transplantation into the bone marrow. The rejection of xenogeneic islets was associated with local and systemic T cell responses and macrophage recruitment. Although there was no evidence for immune-privilege, the bone marrow may represent a feasible site for encapsulated xenogeneic islet transplantation.

Endocrine secretory reserve and proinsulin processing in recipients of islet of langerhans versus whole pancreas transplants.

OBJECTIVE: ß-Cells have demonstrated altered proinsulin processing after islet transplantation. We compare ß-cell metabolic responses and proinsulin processing in pancreas and islet transplant recipients with respect to healthy control subjects. RESEARCH DESIGN AND METHODS: We studied 15 islet and 32 pancreas transplant recipients. Islet subjects were subdivided into insulin-requiring (IR-ISL, n = 6) and insulin-independent (II-ISL, n = 9) groups. Ten healthy subjects served as control subjects. Subjects were administered an intravenous arginine stimulation test, and insulin, C-peptide, total proinsulin, intact proinsulin, and proinsulin fragment levels were determined from serum samples. Acute insulin response (AIR) and proinsulin processing rates were calculated. RESULTS: We found that basal insulin and C-peptide levels were higher in the pancreas group than in all other groups. II-ISL patients had basal insulin and C-peptide levels similar to healthy control subjects. The IR-ISL group had significantly lower AIRs than all other groups. Basal processing rates were higher in the pancreas and II-ISL groups than in healthy control subjects and the IR-ISL group. After arginine stimulation, all groups had elevated processing rates, with the exception of the IR-ISL group. CONCLUSIONS: Our data suggest that II-ISL transplant recipients can maintain basal metabolic parameters similar to healthy control subjects at the cost of a higher rate of proinsulin processing. IR-ISL transplant recipients, on the other hand, demonstrate both lower insulin response and lower basal rates of proinsulin processing even after arginine stimulation.

Quantification of islet loss and graft functionality during immune rejection by 3-tesla MRI in a rat model.

BACKGROUND: Because metabolic markers are not suitable for early diagnosis of islet graft dysfunction, magnetic resonance imaging (MRI) has been used to study islets that were labeled pretransplantation with superparamagnetic iron oxide nanoparticles. However, the relation between graft functionality assessed by glycemia, and MRI signal remains unclear. METHODS: We transplanted hyperglycemic rats intraportally with 2500 ferucarbotran-labeled syngeneic (n=10) or allogeneic (n=12) islet equivalents or normoglycemic rats with 5000 xenogeneic human islet equivalents. Images were acquired on a clinical 3-Tesla MRI scanner. RESULTS: When rejection occurred on days 4 and 8 in xenogeneic and allogeneic recipients, 60% (57-68) and 55% (46-73) of the initial signal remained compared to 93% (71-104) and 82% (59-90) in syngeneic controls (P=0.006 and 0.03). With a cutoff value of 84% on day 4 for the diagnosis of allogeneic rejection, sensitivity of 91% and specificity of 70% were obtained. Based on MRI signal on day 4, treatment with antilymphocytic serum from day 4 allowed graft rescue in 75% of recipients. CONCLUSIONS: In this model, MRI of pretransplantation superparamagnetic iron oxide nanoparticle-labeled islet grafts allows timely diagnosis of immune rejection.

Rapamycin impairs proliferation of transplanted islet ß cells.

BACKGROUND: The cause for a progressive attrition of islet graft function observed over the years after islet transplantation is not well defined but may be in part the result of adverse effects of immunosuppressive agents. In this study, we examined the effect of rapamycin, a key component of the immunosuppressive regimen, on ß-cell replication of transplanted islets. METHODS: Mice transplanted with rat islets under kidney capsule received bromodeoxyuridine for 7 days. Mice were treated with rapamycin or appropriate vehicle. ß-cell replication was determined by double immunofluorescence staining for insulin and bromodeoxyuridine. For in vitro studies, apoptosis, glucose-stimulated insulin secretion, and proliferation were determined in islet cells incubated with EdU in the presence or absence of rapamycin. RESULTS: In our islet transplant model, rapamycin impaired glucose tolerance and ß-cell proliferation of transplanted and host islets. In vitro, rapamycin reduced glucose-stimulated insulin secretion and reversibly decreased ß-cell replication. The inhibitory effect of rapamycin on ß-cell proliferation was not due to the decrease in insulin release. Additionally, in islet cells, expression of cell cycle proteins was significantly modified by rapamycin, suggesting a blockade of cell cycle progression. Inhibition of p38MAPK partially reverted rapamycin effect on ß-cell proliferation. CONCLUSION: Rapamycin, at concentration usually used to prevent islet graft rejection, is able to reduce the rate of ß-cell proliferation in transplanted rat islets but also in host murine islets. These data suggest that the progressive islet graft dysfunction observed under immunosuppressive therapy may result in part from an impairment of ß-cell regeneration.

Cadherin engagement protects human ß-cells from apoptosis.

The aim of this study was to assess the expression of different types of cadherins in human islets and their role in human ß-cell apoptosis. Expression of E-, N-, and P-cadherins was studied by immunofluorescence on pancreas sections and islet cells, and by Western blotting on protein extracts of isolated islets and islet cells. The effects of specific cadherins on cell adhesion and apoptosis were studied using chimeric proteins containing functional E-, N-, or P-cadherin ectodomains fused to Fc fragment of Ig (E-cad/Fc, N-cad/Fc, and P-cad/Fc) and immobilized on glass substrate. ß-Cells were identified by immunofluorescence for insulin and apoptotic cells by terminal deoxynucleotide transferase-mediated 2'-deoxyuridine, 5'-triphosphate nick-end labeling. By immunofluorescence, we showed that E- and N-, and not P-, cadherins were expressed at the surface of islet cells. By triple staining, we showed that E-cadherin was expressed at similar extent in ß- and α-cells, whereas N-cadherin was preferentially expressed in ß-cells. These results were confirmed by Western blot analysis using protein extracts from fluorescence-activated cell sorting-sorted ß- and non-ß-cells. Adhesion tests showed that the affinity of islet cells for E-cad/Fc and N-cad/Fc and not for P-cad/Fc was increased compared with control. By terminal deoxynucleotide transferase-mediated 2'-deoxyuridine, 5'-triphosphate nick-end labeling, we showed that the percentage of apoptotic cells was lower in aggregated ß-cells compared with single ß-cells and that attachment to E-cad/Fc and N-cad/Fc and not to P-cad/Fc decreased apoptosis of single ß-cells compared with control. Our results show that at least E- and N-cadherins are expressed at the surface of human ß-cells and that these adhesion molecules are involved in the maintenance of ß-cell viability.

Influence of donor age on islet isolation and transplantation outcome.

BACKGROUND: It has been suggested that the age of human organ donors might influence islet isolation and transplantation outcome in a negative way due to a decrease of in vivo function in islets isolated from older donors. METHODS: We retrospectively analyzed 332 islet isolations according to donor age. We determined isolation outcome by islet yields, transplantation rates, and [beta]-cell function in vitro. Transplanted patients were divided into two groups depending on donor age (n=25 and n=31 patients for <=45- and >45-year-old donors, respectively). We assessed islet graft function by C-peptide/glucose ratio, [beta] score, secretory units of islets in transplantation index, and insulin independence rate at 1, 6, and 12 months after transplantation. RESULTS: There was no difference in islet yields between the two groups (251,900+/-14,100 and 244,600+/-8400 islet equivalent for <=45- and >45-year-old donors, respectively). Transplantation rates and stimulation indices were similar in both groups as well. All islet graft function parameters were significantly higher at 1-month follow-up in patients who had received islets from younger donors. At 6-month follow-up after second or third injection and at 12-month follow-up, secretory units of islets in transplantation indices and C-peptide/glucose ratios were significantly higher in patients with donors aged 45 years or younger. CONCLUSIONS: These data suggest that, despite similar outcomes of the isolation procedure, islet graft function is significantly influenced by donor age. These results may have important consequences in the definition of pancreas allocation criteria.

Immunosuppressive effects of streptozotocin-induced diabetes result in absolute lymphopenia and a relative increase of T regulatory cells.

OBJECTIVE: Streptozotocin (STZ) is the most widely used diabetogenic agent in animal models of islet transplantation. However, the immunomodifying effects of STZ and the ensuing hyperglycemia on lymphocyte subsets, particularly on T regulatory cells (Tregs), remain poorly understood. RESEARCH DESIGN AND METHODS: This study evaluated how STZ-induced diabetes affects adaptive immunity and the consequences thereof on allograft rejection in murine models of islet and skin transplantation. The respective toxicity of STZ and hyperglycemia on lymphocyte subsets was tested in vitro. The effect of hyperglycemia was assessed independently of STZ in vivo by the removal of transplanted syngeneic islets, using an insulin pump, and with rat insulin promoter diphtheria toxin receptor transgenic mice. RESULTS: Early lymphopenia in both blood and spleen was demonstrated after STZ administration. Direct toxicity of STZ on lymphocytes, particularly on CD8(+) cells and B cells, was shown in vitro. Hyperglycemia also correlated with blood and spleen lymphopenia in vivo but was not lymphotoxic in vitro. Independently of hyperglycemia, STZ led to a relative increase of Tregs in vivo, with the latter retaining their suppressive capacity in vitro. The higher frequency of Tregs was associated with Treg proliferation in the blood, but not in the spleen, and higher blood levels of transforming growth factor-ß. Finally, STZ administration delayed islet and skin allograft rejection compared with naive mice. CONCLUSIONS: These data highlight the direct and indirect immunosuppressive effects of STZ and acute hyperglycemia, respectively. Thus, these results have important implications for the future development of tolerance-based protocols and their translation from the laboratory to the clinic.

Impact of the number of infusions on 2-year results of islet-after-kidney transplantation in the GRAGIL network.

BACKGROUND: Insulin independence after islet transplantation is generally achieved after multiple infusions. However, single infusion would increase the number of recipients. Our aim was to evaluate the results of islet-after-kidney transplantation according to the number of infusions. METHODS: Islets were isolated at the Geneva University, shipped, and transplanted into French patients from the Swiss-French GRAGIL network, on the "Edmonton" immunosuppression protocol between 2004 and 2010. RESULTS: Nineteen patients were transplanted with 33 preparations. Fifteen patients reached 24 months follow-up; eight subjects were single-graft recipients and seven were double-graft recipients. Finally, single-graft recipients received a median of 5312 islet equivalents/kg (5186-6388) vs. 10,564 (10,054-11,375) for double-graft recipients (P=0.0003) with similar islet mass at first infusion. Insulin independence was achieved in five of eight single-graft subjects (62.5%) versus five of seven in double-graft subjects (71.4%), not significant. Median insulin independence duration was 4.7 (3.1-15.2) months after one infusion vs. 19 (9.6-20.8) months after two infusions (not significant). At 24 months posttransplant, comparing single- with double-graft patients, insulin doses were 0.23 (0.11-0.34) U/kg vs. 0.02 (0.0-0.23) U/kg, P=0.11; HbA1c was 6.5% (5.9%-6.8%) vs. 6.2% (5.9%-6.3%), P=0.16; and basal C-peptide was 302 (143-480) pmol/L vs. 599 (393-806) pmol/L, P=0.05. Only 37.5% of single-graft patients had a ß-score ≥4 compared with 100% of double-graft patients (P=0.03). Two recipients experienced postinfusion bleeding, and two patients (13%) showed renal dysfunction in the absence of biopsy-proven rejection. CONCLUSIONS: One infusion achieves good glycemic control and sometimes insulin independence. However, double-graft patients remain insulin-free longer, tend to have lower HbA1c, and show better graft function 24 months after transplant.

Unique arrangement of alpha- and beta-cells in human islets of Langerhans.

OBJECTIVE: It is generally admitted that the endocrine cell organization in human islets is different from that of rodent islets. However, a clear description of human islet architecture has not yet been reported. The aim of this work was to describe our observations on the arrangement of human islet cells. RESEARCH DESIGN AND METHODS: Human pancreas specimens and isolated islets were processed for histology. Sections were analyzed by fluorescence microscopy after immunostaining for islet hormones and endothelial cells. RESULTS: In small human islets (40-60 mum in diameter), beta-cells had a core position, alpha-cells had a mantle position, and vessels laid at their periphery. In bigger islets, alpha-cells had a similar mantle position but were found also along vessels that penetrate and branch inside the islets. As a consequence of this organization, the ratio of beta-cells to alpha-cells was constantly higher in the core than in the mantle part of the islets, and decreased with increasing islet diameter. This core-mantle segregation of islet cells was also observed in type 2 diabetic donors but not in cultured isolated islets. Three-dimensional analysis revealed that islet cells were in fact organized into trilaminar epithelial plates, folded with different degrees of complexity and bordered by vessels on both sides. In epithelial plates, most beta-cells were located in a central position but frequently showed cytoplasmic extensions between outlying non-beta-cells. CONCLUSIONS: Human islets have a unique architecture allowing all endocrine cells to be adjacent to blood vessels and favoring heterologous contacts between beta- and alpha-cells, while permitting homologous contacts between beta-cells.

Assessment of human islet labeling with clinical grade iron nanoparticles prior to transplantation for graft monitoring by MRI.

Ex vivo labeling of islets with superparamagnetic iron oxide (SPIO) nanoparticles allows posttransplant MRI imaging of the graft. In the present study, we compare two clinical grade SPIOs (ferucarbotran and ferumoxide) in terms of toxicity, islet cellular uptake, and MRI imaging. Human islets (80-90% purity) were incubated for 24 h with various concentrations of SPIOs (14-280 µg/ml of iron). Static incubations were performed, comparing insulin response to basal (2.8 mM) or high glucose stimulation (16.7 mM), with or without cAMP stimulation. Insulin and Perl's (assessment of iron content) staining were performed. Electronic microscopy analysis was performed. Labeled islets were used for in vitro or in vivo imaging in MRI 1.5T. Liver section after organ removal was performed in the same plane as MRI imaging to get a correlation between histology and radiology. Postlabeling islet viability (80 ± 10%) and function (in vitro static incubation and in vivo engraftment of human islets in nude mice) were similar in both groups. Iron uptake assessed by electron microscopy showed iron inclusions within the islets with ferucarbotran, but not with ferumoxide. MRI imaging (1.5T) of phantoms and of human islets transplanted in rats, demonstrated a strong signal with ferucarbotran, but only a weak signal with ferumoxide. Signal persisted for >8 weeks in the absence of rejection. An excellent correlation was observed between radiologic images and histology. The hepatic clearance of intraportally injected ferucarbotran was faster than that of ferumoxide, generating less background. A rapid signal decrease was observed in rejecting xenogeneic islets. According to the present data, ferucarbotran is the most appropriate of available clinical grade SPIOs for human islet imaging.

TNFA locus is associated with beta degrees 39 thalassemia in Corsica and Sardinia.

Malaria causes more than one million deaths annually, worldwide. Understanding the genetic defenses against this disease is an important challenge for science. We know that the long-term presence of endemic malaria has led to a prevalence of the beta degrees 39 heterozygous thalassemia mutation in the two islands of Corsica and Sardinia. The populations of both islands are isolated, which could make it easier to find other genetic traits selected by disease pressure. We chose to investigate genes implicated in the primary defenses against Plasmodium falciparum: oxidative metabolism and the immune response. We indeed selected genes coding for nitric oxide synthase 2 (NOS2 promoter, polymorphisms NOS2(AAAT) I/D and NOS2(CCTTT)n) and genes coding for tumor necrosis factor-alpha (TNFA 3'UTR, polymorphisms TNFd(GA)n and TNFe(GA)n). Some associations of TNFA alleles or haplotypes were found either with or without the beta degrees 39 mutation, suggesting a complex link originally between TNF-alpha and resistance or susceptibility to infection.

Genetic history of some western Mediterranean human isolates through mtDNA HVR1 polymorphisms.

The existence of a genetic gradient across continents has often been highlighted. Comparisons among several genetic markers have suggested that most genes of current Europeans are descended from the Near East. During the Paleolithic period, populations were confined in refuges by the last glaciation. At the end of the Paleolithic period, European migrations began from these refuges. Our objective was to highlight these various flows, starting from well-defined isolated populations, originating mainly from western Mediterranean islands. We investigated polymorphisms in the hypervariable 1 (HVR1) zone of mitochondrial DNA (mtDNA) in many Mediterranean isolates: Andalusia, Balearic Islands, southern Corsica, Morocco, Sant' Antioco Island, San Pietro Island, Gallura, Nuoro and Trexenta (Sardinia) and Tuscany. We have compared our findings with those from other Mediterranean populations. Occupation of the Mediterranean area from the Middle East began in the Upper Paleolithic period around 40,000 years ago, with a population diversity determined by geographical and historical factors. Of the isolates studied, the population of the Balearic Islands show genetic characteristics correlated with various European flows initiated about 5,000 years ago. The island of San Pietro, in southwest Sardinia, still preserves the genetic traces of settlement by Ligurian migrants in 1736.

beta-globin gene cluster haplotypes associated with beta-thalassemia on Corsica island.

In the Corsican population, the incidence of beta-thalassemia traits is reported to be 3.1%. We have investigated the 2 more important beta-thalassemia mutations present in the Corsican population: beta0-39 and beta+IVS1-110. Seven polymorphic sites in the beta-globin gene cluster were analyzed from a sample of 43 non-related beta-thalassemia heterozygotes and of 47 nonrelated healthy individuals, from Central Corsica (Corte). Among the 43 Corsican patients analyzed, the nonsense codon is predominant (88.40%), whereas the beta+IVS1-110 mutation, the most common of beta-thalassemia in the eastern part of the Mediterranean basin, is underrepresented (2.33%). The other individuals did not show positive for the two tested mutations (9.27%). The beta0-39 mutation in the studied population shows a strong association with haplotype II (18.7%) and a weaker association with haplotypes I (2.3%) and VII (2.1%). The strong association of the beta0-39 mutation with haplotype II was also found in Sardinia, suggesting that the mutation on the two islands have the same origin. In the present study all the data concerning frequencies of the mutations and of sequence haplotypes, support the hypothesis of a western Mediterranean origin of the beta0-39 mutation. For the first time, this paper analyzes the association of beta-globin gene cluster haplotypes with the 2 more frequent beta-thalassemia mutations in an isolated population in the centre of Corsica (Corte), which presents certain genetic peculiarities. However, the analysis of beta-haplotypes will be very useful for the genetic epidemiological study in this region.