Pier Franco Pignatti

Sequence variants affecting eosinophil numbers associate with asthma and myocardial infarction

Eosinophils are pleiotropic multifunctional leukocytes involved in initiation and propagation of inflammatory responses and thus have important roles in the pathogenesis of inflammatory diseases. Here we describe a genome-wide association scan for sequence variants affecting eosinophil counts in blood of 9,392 Icelanders. The most significant SNPs were studied further in 12,118 Europeans and 5,212 East Asians. SNPs at 2q12 (rs1420101), 2q13 (rs12619285), 3q21 (rs4857855), 5q31 (rs4143832) and 12q24 (rs3184504) reached genome-wide significance (P = 5.3 × 10−14, 5.4 × 10−10, 8.6 × 10−17, 1.2 × 10−10 and 6.5 × 10−19, respectively). A SNP at IL1RL1 associated with asthma (P = 5.5 × 10−12) in a collection of ten different populations (7,996 cases and 44,890 controls). SNPs at WDR36, IL33 and MYB that showed suggestive association with eosinophil counts were also associated with atopic asthma (P = 4.2 × 10−6, 2.2 × 10−5 and 2.4 × 10−4, respectively). We also found that a nonsynonymous SNP at 12q24, in SH2B3, associated significantly (P = 8.6 × 10−8) with myocardial infarction in six different populations (6,650 cases and 40,621 controls).

Consensus on the use and interpretation of cystic fibrosis mutation analysis in clinical practice

It is often challenging for the clinician interested in cystic fibrosis (CF) to interpret molecular genetic results, and to integrate them in the diagnostic process. The limitations of genotyping technology, the choice of mutations to be tested, and the clinical context in which the test is administered can all influence how genetic information is interpreted. This paper describes the conclusions of a consensus conference to address the use and interpretation of CF mutation analysis in clinical settings. Although the diagnosis of CF is usually straightforward, care needs to be exercised in the use and interpretation of genetic tests: genotype information is not the final arbiter of a clinical diagnosis of CF or CF transmembrane conductance regulator (CFTR) protein related disorders. The diagnosis of these conditions is primarily based on the clinical presentation, and is supported by evaluation of CFTR function (sweat testing, nasal potential difference) and genetic analysis. None of these features are sufficient on their own to make a diagnosis of CF or CFTR-related disorders. Broad genotype/phenotype associations are useful in epidemiological studies, but CFTR genotype does not accurately predict individual outcome. The use of CFTR genotype for prediction of prognosis in people with CF at the time of their diagnosis is not recommended. The importance of communication between clinicians and medical genetic laboratories is emphasized. The results of testing and their implications should be reported in a manner understandable to the clinicians caring for CF patients.

Contribution of Gene Sequence Variations of the Hepatic Cytochrome P450 3A4 Enzyme to Variability in Individual Responsiveness to Clopidogrel

Objectives—Metabolic activity of cytochrome P450 (CYP) 3A4 has been associated with clopidogrel response variability. Because metabolic activity of CYP3A4 is genetically regulated, we hypothesized that genetic variations of this enzyme may contribute to clopidogrel response variability. Methods and Results—The CYP3A4*1B, CYP3A4*3, IVS7+258A>G, IVS7+894C>T, and IVS10+12G>A polymorphisms of the CYP3A4 gene were assessed in 82 patients in a steady phase of clopidogrel therapy. Glycoprotein (platelet glycoprotein (GP) IIb/IIIa receptor activation and platelet aggregation were assessed. A cohort of 45 clopidogrel-naïve patients was studied to determine the modulating effects of these polymorphisms after loading dose (300 mg) administration. Only the IVS7+258A>G, IVS7+894C>T, and IVS10+12G>A polymorphisms were sufficiently polymorphic. During the steady phase of clopidogrel treatment, IVS10+12A allele carriers had reduced GP IIb/IIIa activation (P=0.025) and better responsiveness (P=0.02); similarly, clopidogrel-naïve patients carriers of the IVS10+12A allele had reduced GP IIb/IIIa activation during the first 24 hours after a loading dose (P=0.025), increased platelet inhibition (P=0.006), and a more optimal drug response (P=0.003). This polymorphism did not influence platelet aggregation profiles. No association was observed between the other polymorphisms and clopidogrel responsiveness. Conclusions—The IVS10+12G>A polymorphism of the CYP3A4 gene modulates platelet activation in patients treated with clopidogrel and may therefore contribute to clopidogrel response variability.

Variation in a Repeat Sequence Determines Whether a Common Variant of the Cystic Fibrosis Transmembrane Conductance Regulator Gene Is Pathogenic or Benign

An abbreviated tract of five thymidines (5T) in intron 8 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene is found in approximately 10% of individuals in the general population. When found in trans with a severe CFTR mutation, 5T can result in male infertility, nonclassic cystic fibrosis, or a normal phenotype. To test whether the number of TG repeats adjacent to 5T influences disease penetrance, we determined TG repeat number in 98 patients with male infertility due to congenital absence of the vas deferens, 9 patients with nonclassic CF, and 27 unaffected individuals (fertile men). Each of the individuals in this study had a severe CFTR mutation on one CFTR gene and 5T on the other. Of the unaffected individuals, 78% (21 of 27) had 5T adjacent to 11 TG repeats, compared with 9% (10 of 107) of affected individuals. Conversely, 91% (97 of 107) of affected individuals had 12 or 13 TG repeats, versus only 22% (6 of 27) of unaffected individuals (P<.00001). Those individuals with 5T adjacent to either 12 or 13 TG repeats were substantially more likely to exhibit an abnormal phenotype than those with 5T adjacent to 11 TG repeats (odds ratio 34.0, 95% CI 11.1-103.7, P<.00001). Thus, determination of TG repeat number will allow for more accurate prediction of benign versus pathogenic 5T alleles.

A degradation-sensitive anionic trypsinogen (PRSS2) variant protects against chronic pancreatitis

Chronic pancreatitis is a common inflammatory disease of the pancreas. Mutations in the genes encoding cationic trypsinogen (PRSS1) and the pancreatic secretory trypsin inhibitor (SPINK1) are associated with chronic pancreatitis. Because increased proteolytic activity owing to mutated PRSS1 enhances the risk for chronic pancreatitis, mutations in the gene encoding anionic trypsinogen (PRSS2) may also predispose to disease. Here we analyzed PRSS2 in individuals with chronic pancreatitis and controls and found, to our surprise, that a variant of codon 191 (G191R) is overrepresented in control subjects: G191R was present in 220/6,459 (3.4%) controls but in only 32/2,466 (1.3%) affected individuals (odds ratio 0.37; P = 1.1 × 10−8). Upon activation by enterokinase or trypsin, purified recombinant G191R protein showed a complete loss of trypsin activity owing to the introduction of a new tryptic cleavage site that renders the enzyme hypersensitive to autocatalytic proteolysis. In conclusion, the G191R variant of PRSS2 mitigates intrapancreatic trypsin activity and thereby protects against chronic pancreatitis.

IRAK-M Is Involved in the Pathogenesis of Early-Onset Persistent Asthma

Asthma is a multifactorial disease influenced by genetic and environmental factors. In the past decade, several loci and >100 genes have been found to be associated with the disease in at least one population. Among these loci, region 12q13-24 has been implicated in asthma etiology in multiple populations, suggesting that it harbors one or more asthma susceptibility genes. We performed linkage and association analyses by transmission/disequilibrium test and case-control analysis in the candidate region 12q13-24, using the Sardinian founder population, in which limited heterogeneity of pathogenetic alleles for monogenic and complex disorders as well as of environmental conditions should facilitate the study of multifactorial traits. We analyzed our cohort, using a cutoff age of 13 years at asthma onset, and detected significant linkage to a portion of 12q13-24. We identified IRAK-M as the gene contributing to the linkage and showed that it is associated with early-onset persistent asthma. We defined protective and predisposing SNP haplotypes and replicated associations in an outbred Italian population. Sequence analysis in patients found mutations, including inactivating lesions, in the IRAK-M coding region. Immunohistochemistry of lung biopsies showed that IRAK-M is highly expressed in epithelial cells. We report that IRAK-M is involved in the pathogenesis of early-onset persistent asthma. IRAK-M, a negative regulator of the Toll-like receptor/IL-1R pathways, is a master regulator of NF- kappa B and inflammation. Our data suggest a mechanistic link between hyperactivation of the innate immune system and chronic airway inflammation and indicate IRAK-M as a potential target for therapeutic intervention against asthma.

Complete mutational screening of the CFTR gene in 120 patients with pulmonary disease

In order to determine the possible role of the cystic fibrosis transmembrane regulator (CFTR) gene in pulmonary diseases not due to cystic fibrosis, a complete screening of the CFTR gene was performed in 120 Italian patients with disseminated bronchiectasis of unknown cause (DBE), chronic bronchitis (CB), pulmonary emphysema (E), lung cancer (LC), sarcoidosis (S) and other forms of pulmonary disease. The 27 exons of the CFTR gene and their intronic flanking regions were analyzed by denaturing gradient gel electrophoresis and automatic sequencing. Mutations were detected in 11/23 DBE (P=0.009), 7/25 E, 5/27 CB, 5/26 LC, 5/8 S (P=0.013), 1/4 tuberculosis, and 1/5 pneumonia patients, and in 5/33 controls. Moreover, the IVS8–5T allele was detected in 6/25 E patients (P=0.038). Four new mutations were identified: D651N, 2377C/T, E826K, and P1072L. These results confirm the involvement of the CFTR gene in disseminated bronchiectasis of unknown origin, and suggest a possible role for CFTR gene mutations in sarcoidosis, and for the 5T allele in pulmonary emphysema.

PlA polymorphism and platelet reactivity following clopidogrel loading dose in patients undergoing coronary stent implantation.

The PlA polymorphism (Leu33Pro) of the platelet glycoprotein (GP) IIIa gene has been suggested to play an important role in coronary thrombosis. In vitro studies have shown differences for this polymorphism in platelet sensitivity towards antiplatelet drugs (aspirin and abciximab), suggesting a pharmacogenetic modulation. The aim of the study was to assess the modulatory effect of the PlA polymorphism on clopidogrel-induced antiplatelet effects in 38 patients undergoing coronary stent implantation receiving a 300 mg clopidogrel loading-dose. Platelet reactivity was assessed as GPIIb/IIIa activation and P-selectin expression in platelets stimulated with 2 μmol/l adenosine diphosphate using whole blood flow cytometry. The distribution of the homozygous PlA1/A1 and heterozygous PlA1/A2 genotypes were 74 and 26%, respectively. PlA2 carriers had a higher degree of GPIIb/IIIa activation (P = 0.05) and P-selectin expression (P = 0.02) during the overall study time course and a lower antiplatelet effect to a 300 mg clopidogrel loading-dose up to 24 h following intervention (P < 0.05). In conclusion, the PlA polymorphism of the GPIIIa gene modulates platelet reactivity towards clopidogrel front loading in patients undergoing coronary stenting. This suggests the need for individualized antithrombotic regimens to optimally inhibit platelet reactivity.

Gross genomic rearrangements involving deletions in the CFTR gene: characterization of six new events from a large cohort of hitherto unidentified cystic fibrosis chromosomes and meta-analysis of the underlying mechanisms

Gross genomic rearrangements involving deletions in the CFTR gene have recently been found to account for ∼20% of unidentified cystic fibrosis (CF) chromosomes in both French and Italian patients. Using QMPSF and walking quantitative DHPLC, six novel mutations (three simple deletions, two complex deletions with short insertions of 3–6 bp, and a complex deletion with a 182 bp inverted downstream sequence) were characterized by screening 274 unidentified CF chromosomes from 10 different countries. These lesions increase the total number of fully characterized large CFTR genomic rearrangements involving deletions to 21. Systematic analysis of the 42 associated breakpoints indicated that all 21 events were caused by nonhomologous recombination. Whole gene complexity analysis revealed a significant correlation between regions of low sequence complexity and the locations of the deletion breakpoints. Known recombination-promoting motifs were noted in the vicinity of the breakpoints. A total of 11 simple deletions were potentially explicable in terms of the classical model of replication slippage. However, the complex deletions appear to have arisen via multiple mechanisms; three of the five complex deletions with short insertions and both examples of large inverted insertions (299 and 182 bp, respectively) can be explained by either a model of serial replication slippage in cis (SRScis) or SRS in trans (SRStrans). Finally, the nature and distribution of large genomic rearrangements in the CFTR gene were compared and contrasted with those of two other genes, DMD and MSH2, with a view to gaining a broader understanding of DNA sequence context in mediating the diverse underlying mutational mechanisms.

The MTHFR 1298A>C Polymorphism and Genomic DNA Methylation in Human Lymphocytes

Methylenetetrahydrofolate reductase (MTHFR) balances the pool of folate coenzymes in one-carbon metabolism for DNA synthesis and methylation, both implicated in carcinogenesis. Epidemiologic studies have shown that two functional polymorphisms in MTHFR gene, 677C>T and 1298A>C, are related to increased cancer risk. We aimed to analyze lymphocyte DNA from 198 subjects to evaluate the MTHFR 1298A>C polymorphism and folate status affecting genomic DNA methylation as a possible mechanism underlying the relationship between MTHFR polymorphisms and cancer susceptibility. Carriers of the 1298AA wild-type genotype showed lower genomic DNA methylation compared with 1298AC or 1298CC genotypes [3.72 versus 8.59 or 6.79 ng 5-methyl-2′-deoxycytidine (5-mCyt)/μg DNA, P < 0.0001 and P = 0.007, respectively]. When DNA methylation was evaluated according to plasma folate status, only 1298AA with low folate levels revealed diminished DNA methylation (P < 0.0001). Moreover, when the two MTHFR polymorphisms were concomitantly evaluated at the low folate status, DNA methylation was reduced only in 1298AA/677TT compared with 1298AA/677CC (3.11 versus 7.29 ng 5-mCyt/μg DNA, P = 0.001) and 1298CC/677CC genotypes (3.11 versus 7.14 ng 5-mCyt/μg DNA, P = 0.004). However, the high prevalence of 677TT mutants within the 1298AA group (79%) and the similar biochemical features of 1298AA/677CC and 1298CC/677CC combined genotypes suggest that the gene-nutrient interaction affecting DNA methylation in 1298AA is mainly due to the coexistence of the 677TT genotype and that the 1298A>C polymorphism may convey its protective effect not through this interaction but through another pathway in one-carbon metabolism. Further mechanistic studies are warranted to investigate how single polymorphisms as well as MTHFR combined genotypes exert their effect on cancer susceptibility.