Nikolaos Vamvakopoulos

TLR4 gene polymorphisms: evidence for protection against type 2 diabetes but not for diabetes-associated ischaemic heart disease

OBJECTIVE Several factors either predisposing or protecting from the onset of diabetes mellitus type 2 (DM2) have been proposed. Two specific polymorphisms of toll-like receptor 4 (TLR4; Asp299Gly and Thr399Ile) have recently been identified either as candidate protector genes against DM2 and associated neuropathy or risk alleles for the manifestation of diabetic retinopathy. The impact of these alleles on the risk for ischaemic heart disease (IHD) is controversial while their role in diabetes-associated IHD has never been studied. DESIGN AND METHODS In order to clarify the potential impact of TLR4 polymorphisms on the predisposition for DM2 as well as on diabetes-related IHD vulnerability, the distribution of the mutant TLR4 Asp299Gly and Thr399Ile alleles in 286 DM2 patients and 413 non-DM2 controls with or without IHD, was examined. RESULTS Mutant alleles were predominantly detected in 79/413 non-diabetic individuals versus 15/286 DM2 patients (P<0.0001). The rates of positivity for mutant alleles were similar among diabetic patients with or without IHD (7/142 vs 8/144, P>0.1), whereas they proved different among non-diabetic individuals with or without IHD (39/145 vs 40/268, P=0.004). Following multivariate analysis, the difference between diabetic and non-diabetic subjects, with regard to TLR4 mutations alone, remained significant (P=0.04). CONCLUSIONS Mutant TLR4 alleles confer protection against DM2. However, their presence does not seem to play any role, protective or aggravating, in the manifestation of IHD either in diabetic or in non-diabetic individuals.

The 5′ Regulatory Region of the Human Fetal Globin Genes is a Gene Conversion Hotspot

The human fetal globin genes consist of the first mammalian genomic loci for which gene conversion was reported. To date, 14 gene conversions have been described in the human Gγ- and Aγ-globin genes, the vast majority of which are restricted to the coding sequences. Here, we provide evidence for three new gene conversion events in the 5′ regulatory region of the human fetal globin genes, identified during a large genetic screening effort in adult individuals with high fetal hemoglobin (Hb) levels. The sequence variations, resulting from these conversion events, are transcriptionally silent polymorphisms that do not contribute to increased fetal Hb levels. Our results suggest that the 5′ regulatory region of the human fetal globin genes is a gene conversion hotspot that prevent globin gene promoter sequence diversification, further underlining the need for two functional fetal globin genes in fetal erythropoiesis.

Impact of TLR‐4 Polymorphisms on Circulating Levels of Antibodies Against Helicobacter pylori

Dear Editor, Over the years, Helicobacter pylori infection has been associated with a variety of intestinal, as well as extraintestinal disorders [1], thus underlining the need for the development and application of successful diagnostic and therapeutic strategies [2]. Among the procedures used for the diagnosis of infection from this Gram-negative bacterium, serology antibodies against H. pylori is one of the most commonly applied in research and clinical practice [2]. On the other hand, various factors capable of modifying immune responses against pathogens are currently known, including polymorphisms of toll-like receptors (TLR) [3,4]. These molecules and their polymorphic alleles have been implicated in the recognition of several bacterial components i.e. lipopolysaccharides of Gram-negative bacteria from TLR-4 [3–5]. To investigate whether the presence of polymorphic alleles could influence the production of antibodies against H. pylori, antibody titers were compared between H. pylori-infected individuals, with either wild-type or mutant TLR-4 genotypes. The TLR-4 Asp299Gly and Thr399Ile polymorphic alleles were genotyped using a polymerase chain reaction (PCR) and direct sequencing, in 246 individuals with documented, via both campylobacter-like organism (CLO) test and histology, H. pylori infection. For the purposes of the PCR amplification, a forward, 5¢-TCTAGAGGGCCTGTGCAATT-3¢, and a reverse primer, 5¢-TGAAACTCACTCATTTGTTTCAA-3¢, were used. Using an enzyme-linked immunosorbent assay (ELISA, Enzygnost; Dade Behring Marburg GmbH, Marburg, Germany), IgG and IgA antibodies against H. pylori were determined. All study participants lacked any disorder that could alter the immune response and were not under immuno-modulatory or -suppressive medication. A total of 40 individuals were tested positive for both mutant alleles, heterozygotes, while the rest 206 were homozygotes for the wild-type allele. The simultaneous presence –cosegregationof TLR-4 Asp299Gly and Thr399Ile alleles, in our study was 100%. The mean ± SEM of anti-H. pylori IgG titers were 51.90 ± 4.927 U ⁄ mL, in individuals with the wild-type genotype, and 48.94 ± 9.878 U ⁄ mL, in the group carrying the mutant alleles (p > .05). On the contrary, a statistically significant difference was recorded when antiH. pylori IgA titers were compared between carriers of wild-type (11.61 ± 1.330 U ⁄ mL) and mutant polymorphic alleles (5.887 ± 0.6506 U ⁄ mL) (p < .001). Evidently, lower IgA levels were found in the group of TLR-4 Asp299Gly and Thr399Ile gene carriers. This observation does not come entirely as a surprise, because interactions between TLR-4 and Gram-negative bacteria have already been described [3–5]. The impact of the TLR-4 Asp299Gly and Thr399Ile polymorphic alleles, however, on immune responses and especially those triggered by pathogens, has been debated [3]. One of the major reasons for the lack of unanimity regarding this matter was that the simultaneous presence of the two alleles did not seem to increase susceptibility or alter the cytokine response to microorganisms [3]. The results from this study seem to contradict the notion that the cosegregated mutant alleles ‘‘behave’’ pretty much like the wild-type allele. As for the rather selective downregulation of IgA response against H. pylori, it could be hypothesized that it reflects a defect or a dysregulation in the mucosal defense against H. pylori. Besides, the production of IgAs, circulating and mucosal, which represent the most common immunoglobulin subclass in the mucosa, can be triggered by TLR-facilitated recognition of microorganisms, in the intestinal epithelium [4,5]. In addition, a similar blunt antibody production has been reported for anti-chitobioside (ACCA) and anti-outer membrane porin (Omp) IgA, in patients with inflammatory bowel disease carrying the TLR-4 Asp299Gly allele [6]. This, however, is the first attempt to record a potential interaction between widely studied polymorphisms, tightly linked to the host’s immune responses toward Gram-negative bacteria, and H. pylori. In view of the notion that the presence of these specific TLR-4 polymorphic alleles could reduce the diagnostic utility of H. pylori serology, our findings are not consistent with this remark, because the IgG response against H. pylori remained intact. All in all, it seems that the idea that the host’s genetic background can ‘‘carve’’ the reaction toward environmental challenges is once more verified this time via the suppression of anti-H. pylori Helicobacter ISSN 1523-5378

The Hellenic type of nondeletional hereditary persistence of fetal hemoglobin results from a novel mutation (g.-109G>T) in the HBG2 gene promoter

Nondeletional hereditary persistence of fetal hemoglobin (nd-HPFH), a rare hereditary condition resulting in elevated levels of fetal hemoglobin (Hb F) in adults, is associated with promoter mutations in the human fetal globin (HBG1 and HBG2) genes. In this paper, we report a novel type of nd-HPFH due to a HBG2 gene promoter mutation (HBG2:g.-109G>T). This mutation, located at the 3′ end of the HBG2 distal CCAAT box, was initially identified in an adult female subject of Central Greek origin and results in elevated Hb F levels (4.1%) and significantly increased Gγ-globin chain production (79.2%). Family studies and DNA analysis revealed that the HBG2:g.-109G>T mutation is also found in the family members in compound heterozygosity with the HBG2:g.-158C>T single nucleotide polymorphism or the silent HBB:g.-101C>T β-thalassemia mutation, resulting in the latter case in significantly elevated Hb F levels (14.3%). Electrophoretic mobility shift analysis revealed that the HBG2:g.-109G>T mutation abolishes a transcription factor binding site, consistent with previous observations using DNA footprinting analysis, suggesting that guanine at position HBG2/1:g.-109 is critical for NF-E3 binding. These data suggest that the HBG2:g-109G>T mutation has a functional role in increasing HBG2 transcription and is responsible for the HPFH phenotype observed in our index cases.

Low-Grade Appendiceal Mucinous Neoplasm Presenting as a Surgical Emergency: A Case Report

We present the case of a female patient admitted to our University Hospital with acute abdominal pain mimicking an intraperitoneal septic condition caused possibly by acute appendicitis. CT and ultrasound scan showed a mass situated in the right iliac fossa. The patient was submitted to laparotomy and right hemicolectomy. The operative findings were suggestive of an appendiceal mucocele. The histology report revealed a low-grade appendiceal mucinous neoplasm. The patient had no clinical, biochemical or visual signs of disease recurrence 6 months postoperatively.