Marcello Morgutti

Association between HLA-G 3'UTR 14-bp polymorphism and HIV vertical transmission in Brazilian children.

Objectives:The aim of our study was to verify the possible association between an HLA-G 14-bp deletion/insertion polymorphism and perinatal HIV transmission in Brazilian children. Design:We analyzed the 14-bp deletion/insertion polymorphisms in seronegative (i.e., exposed uninfected, N = 71) and seropositive (exposed infected, N = 175) Brazilian children born from HIV-positive mothers and in healthy controls (n = 175). Methods:HLA-G 14-bp deletion/insertion polymorphism (rs16375) was detected by PCR amplification of the target sequence followed by agarose gel electrophoresis. All the samples were also analyzed by direct sequencing in order to validate the genotyping results. Results:HIV-exposed uninfected children showed significant differences in their allele and genotype frequencies of the HLA-G 14-bp polymorphism when compared to both seropositive children and healthy controls. The 14-bp-deleted (D) allele was more frequent in exposed uninfected children (79%) than in healthy controls (60%) and HIV-positive children (58%); the higher percentage of the D allele found in the exposed uninfected children with respect to HIV-positive individuals was significantly associated with a reduced risk of vertical transmission. This effect was ascribable to the presence of the D/D homozygous genotype. Conclusion:Our findings support the possible role for the HLA-G 14-bp deletion/insertion polymorphism in the HIV vertical transmission in Brazilian children. The presence of the D allele and D/D genotype is associated with a protective effect toward HIV perinatal infection.

Factor V Leiden and prothrombin gene G20210A mutation and in vitro fertilization: prospective cohort study

BACKGROUND The influence of thrombophilia on fertility and on IVF outcome is very controversial. The objectives of this study were: (i) to compare the prevalence of Factor V Leiden (FVL) and prothrombin gene G20210A mutation (PGM) in women undergoing IVF to women with spontaneous pregnancy; (ii) to compare the IVF outcomes and the risk of complications in FVL and PGM carrier to non-carrier women. METHODS From March 2005 to December 2009, a total of 510 women requiring IVF were recruited in a prospective cohort study. A separate population of 490 nulliparous women who conceived naturally was also evaluated as fertile controls. All women were tested for the presence of FVL and PGM. RESULTS The prevalence of thrombophilic mutations was the same among women requiring IVF (6.9%) and women with spontaneous pregnancy (6.9%). A total of 480 patients underwent 1105 IVF cycles. There were 30 women carriers (86 IVF cycles) and 450 non-carriers for thrombophilic mutations (1019 IVF cycles). No significant differences in the mean number of oocytes retrieved and the number of good quality embryos transferred were found between the mutation carrier and non-mutation carrier women; likewise the reproductive outcome and the IVF complications were not statistically different between the two groups. The cumulative live birth rate after six IVF cycles was similar in the mutation carrier and non-mutation carrier women. For the mutation carrier women, the optimistic estimate of cumulative live birth rate after six IVF cycles was 60.8% and the conservative estimate was 50.0%. Corresponding rates for the non-mutation carrier women were 56.8 and 36.2%, respectively. CONCLUSIONS The results of this study suggest that FVL and PGM presence in asymptomatic women and in the absence of other risk factors do not influence IVF outcome, or represent risk factors for ovarian hyperstimulation syndrome (OHSS), or favour thrombosis after IVF. Screening for FVL and PGM does not appear to be justified to identify the patients at the risk for IVF failure, and/or for OHSS, and/or for thrombotic complications.

Does epidermal thickening explain GJB2 high carrier frequency and heterozygote advantage

In the post-human-genome area, the challenge is to derive details of heritable variation in relation to how human variation reflects adaptation to the different environments. Heterozygote advantage represents a superior genetic adaptation presumably explaining the presence of the allele at frequencies above those expected from a simple replacement of a homozygous lethal allele by mutation alone. A successful adaptation requires natural selection acting on that part of the body that makes a difference in survival. There is ongoing discussion as to what extent inherited diseases may be phenotypically balanced by a selective heterozygous advantage. We know for sure that sickle cell anemia and some other red blood cell disorders are selected for by malaria,1 whereas a selective carrier advantage for cystic fibrosis has been recently shown.2 For other common inherited diseases counterbalancing mechanisms have been only hypothesized. Congenital hearing loss accounts for about 1 in 1000 infants and approximately 80% of the cases are inherited as an autosomal recessive trait. Connexin 26 (GJB2) gene, a component of a gap junction, is a major gene for these forms, and a large proportion of recessive cases are due to mutations in this gene.3 Different common population-specific mutations have been so far described.4, 5 Among them, mutation 35delG explains up to 70% of the alleles detected in Caucasians, with carrier frequencies ranging from 1/30 in the Mediterranean area to 1/70 in northern Europe.5 The high carrier frequency of the GJB2 mutations in many ethnic groups strongly suggests that there may be a heterozygous advantage. Why GJB2 carriers are so frequent? Quite recently, it has been reported that carriers of R134W allele, present in some African countries such as Ghana,6, 7 show a thicker epidermis than wild-type ones.8 Moreover, in vitro studies have further supported this finding showing that cells expressing the R134W allele: (a) form a significantly thicker epidermis in an organotypic co-culture skin model, (b) show an increased migration, (c) are significantly less susceptible to cellular invasion by the enteric pathogen Shigella flexneri than wild-type cells.9 Another study has shown that the transfection of some deafness-associated mutant constructs resulted in a statistically significant reduction in cell death when compared with wild-type ones.10 Thus, researchers hypothesized that an increased cell survival may explain a thicker epidermis due to an extended terminal differentiation program leading to an improved barrier against infection. Moreover, some specific GJB2 mutated alleles lead to skin diseases in which abnormal thickening of the skin is present.11 If epidermal thickening is the phenomenon underlying heterozygote advantage, should we be able to find an increased thickness of the epidermis in GJB2 carriers? To answer this question and possibly to explain the increased 35delG carrier frequency, we have developed an accurate sonographic protocol to measure epidermal thickness able to discriminate between normal controls and GJB2 carriers.12 Table 1 report the data on epidermal thickness in a series of 240 Italian normal controls (134 females and 106 males) aged from 20 to 86 years. They have been then compared with those obtained in a series of fifty-four 35delG obligate carriers (29 females and 25 males). Results of the analysis are shown in Figure 1, in which two regression lines are calculated for each of the two groups (normal controls and carriers). A clear tendency to thickening along with age is present in both sexes (P<2.0e−16), being more relevant in males. Two clusters are easily distinguishable, one defined by controls and one by carriers. An ANCOVA analysis showed that three variables (GJB2 status, age and gender) explain up to 75% of epidermal thickness variability (P<2.1e−16). Removing the GJB2 status, the impact of the remaining two variables (ie, gender and age) is of only 26%. The difference between controls and GJB2 carriers cannot be explained with any social or occupational reason (ie, lifestyle, job and so on).

Detection of Epidermal Thickening in GJB2 Carriers with Epidermal US

PURPOSE To measure epidermal thickness by using skin ultrasonography (US) in a series of healthy control subjects and obligate carriers for the worldwide most frequent form of congenital hearing loss owing to the mutated alleles of the connexin 26 gene (GJB2). MATERIALS AND METHODS The patent for the protocol, coupled with a new sonographic probe specifically designed to analyze epidermal thickness and a dedicated algorithm to classify individuals in groups, is pending. Institutional ethics committee approval and patient consent were obtained. After a preliminary study in 23 subjects aimed to define the best body site and instrument and protocol for US, a total of 303 individuals (237 healthy subjects, 51 carriers, and 15 homozygotes) were tested at midline forehead by using a linear large-band probe with a frequency ranging from 6 to 15 MHz to determine epidermal thickness. Variance and linear regression analyses were performed. Regression coefficients were then used to obtain measurements of thickness corrected for age and sex. RESULTS GJB2 obligate carriers had a significant increase in epidermal thickness compared with control subjects. GJB2 status explains about 50.0% of this variability, whereas an additional 25.0% is explained by sex and age. Results led to the development of a possible screening protocol with a 98.0% sensitivity and 92.8% specificity in subjects aged 2080 years, with a likelihood ratio of a positive test of 14:1. Even better results (100% sensitivity and 98.9% specificity) were obtained in an analysis of people of only reproductive age. CONCLUSION Epidermal thickening in the white population owing to GJB2 carrier status can be detected by using US. This measurement could provide a simple, noninvasive, rapid, and sensitive test for carrier screening.

HLA-G 3' UTR haplotypes and HIV vertical transmission

We evaluated the possible association of human leukocyte antigen-G (HLA-G) 3777G>C and 14-bp deletion/insertion (D/I) polymorphisms haplotypes and combined genotypes with perinatal HIV transmission in Brazilian children. The 3777G>C polymorphism alone has no effect on HIV vertical transmission but, when linked with the D allele, exerts a positive role in the protection. Indeed, we identified the DC HLA-G haplotype as significantly associated with a protective effect towards HIV vertical transmission.

HLA-G 14 bp deletion/insertion polymorphism in celiac disease.

OBJECTIVES:Nonclassical major histocompatibility class I HLA-G antigen is a tolerogenic molecule that inhibits lytic activity of natural killer (NK) cells and cytotoxic T lymphocytes. Because of its immunomodulatory and tolerogenic properties, HLA-G molecules may have a role in celiac disease (CD). We analyzed the HLA-G 14 bp deletion/insertion polymorphism, known to have a functional effect on mRNA stability, in a group of 522 CD patients, stratified for the presence of HLA-DQ2 genotype, and 400 healthy individuals to evaluate the possible effect of the polymorphism on the risk to develop the disease.METHODS:HLA-G 14 bp deletion/insertion polymorphism (rs1704) was detected by polymerase chain reaction and double-checked by direct sequencing.RESULTS:The 14bp inserted (I) allele and the homozygous I/I genotype were significantly more frequent in CD patients than in healthy controls. The presence of I allele was associated with an increased risk of CD (OR 1.35) and the effect of I allele was consistent with a recessive genetic model (P<0.001).CONCLUSIONS:Our results also indicate that the effect of the HLA-G D/I polymorphism is restricted for HLA-DQ2, and not simply due to the presence of linkage disequilibrium with the major known risk factor; moreover we found that the presence of the I allele confers an increased risk of CD in addition to the risk conferred by HLA-DQ2 alone and that subjects that carry both DQ2 and HLA-G I alleles have an increased risk of CD than subjects that carry DQ2 but not the I allele.

Five new OTOF gene mutations and auditory neuropathy

OBJECTIVE Purpose of this paper is to analyse OTOF gene in a series of subjects affected by auditory neuropathy. METHODS Four children showing mild to profound prelingual deafness, confirmed by the absence of a clear and detectable responses at auditory brainstem responses (ABR), associated with the presence of bilateral OAE, were enrolled in the study. RESULTS AND CONCLUSIONS Genetic analysis identified five new mutations (a nonsense, a small and a large deletion and two splicing site mutations), and one missense mutation (F1795C) previously described. These results further confirm the role of OTOF gene in auditory neuropathy. In the absence of a context of neurological syndrome, the combination of absent ABR and positive OAE responses should lead to an auditory neuropathy diagnosis and to a mutational screening in OTOF.

HLA-G*0105N allele is associated with augmented risk for HIV infection in white female patients.

We analyzed HLA-G 3777G > C, HLA-G 14 bp deletion/insertion and HLA-G*0105N polymorphisms in HIV-positive white adult participants, infected through horizontal heterosexual transmission, and unexposed uninfected individuals, all from north eastern Italy. We report a new association between the HLA-G*0105N allele and HIV infection in adult white female participants, being HLA-G*0105N null allele correlated with an augmented risk (odds ratio = 4.35, 95% confidence interval = 1.38–18.07, P = 0.005) for HIV infection.

ORIGINAL ARTICLE: MBL2 Genetic Screening in Patients with Recurrent Vaginal Infections

Problem  Mannose‐binding lectin (MBL) is an important component of the innate immunity, present at the mucosal level in vagina: a common pathogen’s entry point.

A polymorphism in the 5' UTR of the DEFB1 gene is associated with the lung phenotype in F508del homozygous Italian cystic fibrosis patients

Abstract Background: The identification of cystic fibrosis (CF) patients who are at greater risk of lung damage could be clinically valuable. Thus, we attempted to replicate previous findings and verify the possible association between three single nucleotide polymorphisms (SNPs c.–52G>A, c.–44C>G and c.–20G>A) in the 5’ untranslated region (5′ UTR) of the β defensin 1 (DEFB1) gene and the CF pulmonary phenotype. Methods: Genomic DNA from 92 Italian CF patients enrolled in different regional CF centres was extracted from peripheral blood and genotyped for DEFB1 SNPs using TaqMan® allele specific probes. In order to avoid genetic confounding causes that can account for CF phenotype variability, all patients were homozygous for the F508del CFTR mutation, and were then classified on the basis of clinical and functional data as mild lung phenotype (Mp, n=50) or severe lung phenotype patients (Sp, n=42). Results: For the c.–20G>A SNP, the frequency of the A allele, as well as the AA genotype, were significantly more frequent in Mp than in Sp patients, and thus this was associated with a protective effect against severe pulmonary disease (OR=0.48 and 0.28, respectively). The effect of the c.–20G>A A allele is consistent with a recessive model, and the protective effect against Sp is exerted only when it is present in homozygosis. For the other two SNPs, no differences were observed as allelic and genotypic frequency in the two subgroups of CF patients. Conclusions: Our results, although necessary to be confirmed in larger and multiethnic populations, reinforce DEFB1 as a candidate modifier gene of the CF pulmonary phenotype.