Francesco S. di Giovine

Dating the Origin of the CCR5-Δ32 AIDS-Resistance Allele by the Coalescence of Haplotypes

The CCR5-Delta32 deletion obliterates the CCR5 chemokine and the human immunodeficiency virus (HIV)-1 coreceptor on lymphoid cells, leading to strong resistance against HIV-1 infection and AIDS. A genotype survey of 4,166 individuals revealed a cline of CCR5-Delta32 allele frequencies of 0%-14% across Eurasia, whereas the variant is absent among native African, American Indian, and East Asian ethnic groups. Haplotype analysis of 192 Caucasian chromosomes revealed strong linkage disequilibrium between CCR5 and two microsatellite loci. By use of coalescence theory to interpret modern haplotype genealogy, we estimate the origin of the CCR5-Delta32-containing ancestral haplotype to be approximately 700 years ago, with an estimated range of 275-1,875 years. The geographic cline of CCR5-Delta32 frequencies and its recent emergence are consistent with a historic strong selective event (e.g. , an epidemic of a pathogen that, like HIV-1, utilizes CCR5), driving its frequency upward in ancestral Caucasian populations.

Novel genetic association between ulcerative colitis and the anti-inflammatory cytokine interleukin-1 receptor antagonist

BACKGROUND/AIMS Ulcerative colitis and Crohns disease have well-recognized familial tendencies, but the genetic basis of this clinical observation remains unknown. The cytokine interleukin-1 receptor antagonist is a potent anti-inflammatory protein that can prevent immune-mediated bowel inflammation in animals. We have previously characterized a polymorphism within the gene for this cytokine and others in the genes for the proinflammatory cytokines interleukin 1 alpha, interleukin 1 beta, and tumor necrosis factor alpha. The aim of this study was to determine whether inflammatory bowel disease was associated with particular alleles of these polymorphic cytokine genes. METHODS The allelic frequencies of these polymorphic cytokine genes were determined in patients with ulcerative colitis (n = 113), Crohns disease (n = 78), and healthy controls (n = 261). RESULTS Allele 2 of interleukin-1 receptor antagonist was significantly over-represented in the ulcerative colitis patients: 35% versus 24% in controls (P = 0.007). Carriage of at least one copy of this allele gave an odds ratio of 2.0 for ulcerative colitis compared with controls. This association with allele 2 of interleukin 1 receptor antagonist was greatest in patients with total colitis and was not seen in Crohns disease. There were no associations between UC and any of the other cytokine genes examined. CONCLUSIONS This observation provides evidence that interleukin-1 receptor antagonist may have a role in determining the genetic susceptibility to and pathogenesis of ulcerative colitis.

Functional and ethnic association of allele 2 of the interleukin-1 receptor antagonist gene in ulcerative colitis

BACKGROUND & AIMS The role of the interleukin (IL)-1 receptor antagonist (IL-1ra) in predisposing an individual to inflammatory bowel disease (IBD) is controversial. This study aimed to determine the association between intron 2 IL-1ra polymorphism and IBD by performing a multiethnic case-control study and to assess its functional significance. METHODS A total of 236 patients with ulcerative colitis (UC), 196 patients with Crohns disease (CD), and 338 ethnically matched control patients treated at LAC-USC and Cedars-Sinai Medical Centers and the University of Milan Medical Center were genotyped for a variable length polymorphism in intron 2 of the IL-1ra gene (IL-1RN). Total IL-1ra protein production rates in peripheral blood mononuclear cells (PBMCs) were correlated with carriage of allele 2 of the IL-1RN gene (IL-1RN*2). RESULTS In the LAC-USC group, UC patients (n = 60) had an increased frequency of at least 1 copy of IL-1RN*2 compared with controls (n = 129) (70% vs. 33%; P < 0.01; odds ratio [OR], 4.7). The frequency of IL-1RN*2 carriage in the Cedars-Sinai group was 59% in UC, 45% in CD, and 42% in controls (P < 0.01; OR, 2.0). A significant difference was observed only in the Jewish subgroup (P = 0.003; OR, 5.0). The association was not detected in UC or CD patients treated at the University of Milan. The ORs of 4.7 and 5.0 appear to be the highest reported in any UC population for any genetic markers. Further, carriage of IL-1RN*2 was associated with decreased production of total IL-1ra protein in cultured PBMCs from both UC patients and controls. CONCLUSIONS These results provide further evidence that IL-1ra is important in the predisposition to UC, there may be genetic or pathogenetic heterogeneity between different ethnic groups, and UC and CD are genetically distinct diseases.

A Functional Polymorphism of Toll-like Receptor 4 Is Not Associated with Likelihood or Severity of Meningococcal Disease

Human Toll-like receptor 4 (TLR4) transduces proinflammatory cytokine release by human cells in response to lipopolysaccharide (LPS). This study tested the hypothesis that, if TLR4 is rate limiting for a successful response to bacterial LPS in humans, a human gene polymorphism that results in the amino acid substitution Asp299Gly and causes reduced expression and function of TLR4 should influence susceptibility to or severity of natural gram-negative infection. The allele frequency of the Asp299Gly polymorphism was 5.9% among 879 blood donors, 6.5% among 1047 patients with microbiologically proven meningococcal disease, and 4.1% among 86 patients who died of meningococcal disease. No significant differences were observed, including those analyzed after stratification of the infected population by age and by meningococcal serogroup. Therefore, this functional TLR4 polymorphism does not influence susceptibility to or severity of meningococcal disease.

The Influence of Interleukin Gene Polymorphism on Expression of Interleukin-1β and Tumor Necrosis Factor-α in Periodontal Tissue and Gingival Crevicular Fluid

BACKGROUND A specific composite genotype of the polymorphic interleukin-1 (IL-1) gene cluster has recently been associated with severe periodontitis. One polymorphism of the composite periodontitis-associated genotype (PAG) has been functionally linked with expression of high levels of IL-1. The purpose of this study was to test whether gingival crevicular fluid (GCF) levels of IL-1ß and tumor necrosis factor-alpha (TNFα), and gingival tissue levels of IL- 1α, IL-1β, and TNFα correlate with PAG, and to examine the effec; of conservative periodontal therapy on these levels. METHODS Twenty-two adults with moderate to advanced periodontal disease were enrolled. Polymerase chain reaction amplification and restriction enzymes were used to identify specific polymorphisms from peripheral blood samples. GCF samples were collected at baseline and 3 weeks following conservative treatment and analyzed by ELISA for IL-1ß and TNFα. An interproximal gingival biopsy was collected at baseline and follow-up and analyzed for IL-1α, IL-1β, and TNFα by ELISA. RESULTS The genotyping identified 7 as PAG(+) and 15 as PAG(-). The 2 groups were comparable in terms of existing periodontitis and age. In shallow sites (<4 mm), total IL-1ß in GCF was 2.5 times higher for PAG(+) patients prior to treatment (P = 0.03), and 2.2 times higher after treatment (P = 0.04), while differences were less apparent in deeper sites. Following treatment, a reduction in IL-1β concentration in GCF was seen for PAG(-) but not for PAG(+) patients. While not statistically significant, a trend was observed in mean tissue levels of IL-1β which were 3.6 times higher in PAG(+) versus PAG(-) patients (P = 0.09). CONCLUSIONS These data suggest that PAG(+) patients may demonstrate phenotypic differences as indicated by elevated levels of IL-1ß in GCF. J Periodontol 1999;70:567-573.

Primary biliary cirrhosis shows association with genetic polymorphism of tumour necrosis factor alpha promoter region

BACKGROUND/AIMS Primary biliary cirrhosis is an autoimmune disease in which increased prevalence in first-degree relatives and an association with HLA DR8 suggest a genetic background. TNFalpha is a mediator of inflammation and immunity, and is implicated in the pathogenesis of primary biliary cirrhosis, ex vivo studies having shown reduced production of TNFalpha by lymphocytes from patients. Our group has previously described a biallelic promoter-region polymorphism of the TNFA gene at position -308, and demonstrated that the rare allele, TNF*2, has increased promoter function compared with the common allele, TNF*1. A further biallelic base change has been described in the TNFA gene at -238. We conducted a case-control study to assess association of these gene polymorphisms with primary biliary cirrhosis. METHODS Ninety-one patients and 213 controls were genotyped for both TNFA loci using restriction fragment length polymorphism analysis of PCR products. RESULTS The high production TNFA-308*2 allele was significantly under-represented among subjects with primary biliary cirrhosis (27.5% PBC, 41.6% controls, p=0.02, pc=0.04, OR for carriage of TNF*1/*1 genotype=1.89, CI=1.10-3.32). No association was shown with the TNFA -238 polymorphism. CONCLUSION Primary biliary cirrhosis is associated with reduced carriage of TNF*2. This is in keeping with a protective role of TNFalpha against the disease.

Polymorphic tandem repeat region in interleukin-1α intron 6

Analysis of polymerase chain reaction amplified products from the sixth intron of the human interleukin-1α gene reveals a high polymorphism (polymorphism information content = 0.51) in a Caucasian population. Altogether, seven alleles have been defined ranging from 620 to 1220bp. This polymorphism is probably attributable to a variable number of 46-bp tandem repeats, each containing potential regulatory sequences.

Synovial fluid concentrations of interleukin-1 beta and proteoglycans are inversely related.

Interleukin-1 beta (IL-1 beta) and proteoglycans have been quantified by radioimmunoassay (IL-1 beta) and enzyme linked immunosorbent assay (proteoglycans) in synovial fluids and sera from patients with rheumatoid arthritis (RA) and reactive arthritis. All fluids were also tested for their ability to influence proteoglycan metabolism in a cartilage explant culture system. Synovial fluid IL-1 beta concentrations were inversely related to proteoglycan concentrations in samples from both RA and reactive arthritis patients (P less than 0.002 for all patients). There was no statistically significant relation between immunoreactive IL-1 beta concentration and proteoglycan synthesis or degradation in explants cultured in synovial fluid containing medium. Synovial fluid IL-1 beta concentrations were not related to erythrocyte sedimentation rate or joint total leukocyte count. IL-1 beta was not detectable (limit 250 pg/ml) in any unextracted sera. Although it appears likely that IL-1 beta is involved in the inflammatory and degenerative processes in joint disease, our findings indicate that there is no simple positive relationship between immunoreactive levels of this cytokine in synovial fluid and liberation of proteoglycans from articular cartilage as reflected in synovial fluid proteoglycan concentration.

Biochemical analysis of dermal connective tissue in subjects affected by primary uncomplicated varicose veins

Biochemical analysis of dermal connective tissue was carried out in 14 sub jects affected by primary uncomplicated varicose veins and 14 controls. Skin samples were taken, according to fixed criteria, from operation pieces of total mastectomy for breast cancer. The results suggest that the dermal tissue in these subjects is just thinner than that of controls, confirming previous similar clinical findings. The elective reduction of the collagen content observed, unassociated with changes of other components of the dermal connective tissue, brings evidence for a systemic biochemical defect of the extracellular matrix i.e. a collagen de fect affecting the entire body structure and not only the varicose or pre-varicose veins of the lower limbs.

Long-range DNA interactions at the IL-1/IL-36/IL-37 gene cluster (2q13) are induced by activation of monocytes

The interleukin-1 gene cluster occupies a 360kb region of chromosome 2q13 and contains nine homologous genes. These include agonists and antagonists of the parallel IL-1 and IL-36 systems, and IL1F7, the gene encoding IL-37. As the genes of the cluster are structurally and functionally related and have similar mRNA kinetics, we have sought evidence for gene induction-specific looping of chromatin in the IL-1 cluster by chromatin conformation capture (3C). We show here that IL1A, IL1B and IL1F7 regulatory regions come in close proximity in LPS stimulated cells but not in resting human monocytes. This suggests that IL1A, IL1B and IL1F7 are likely transcribed by the same transcription factory. One cardinal function of transcriptional Locus Control Region (LCR) is bringing map-distant activated genes into close physical proximity within the transcription factory. Our data show distant intergenic DNA segments are also in close proximity to the regulatory regions of the three genes. This may indicate that they are co-regulated and raise the possibility of a LCR within the cluster.