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Italian Multicentric Pilot Study on MBL2 Genetic Polymorphisms in HIV Positive Pregnant Women and Their Children

Posted on: Saturday, 30 July 2005, 03:01 CDT

Abstract

Objective. We investigated genetic polymorphisms of MBL2 gene, in a cohort of 90 Italian HIV-1 pregnant seropositive women and their children in order to understand whether the MBL2 genotype of HIV-1 positive mothers might be related to their ability to transmit the virus to their children.

Materials and methods. DNA was extracted from Iso Code Stix cards, and MBL2 genoptyping was performed by Melting Temperature Assay.

Results. The frequency of the MBL2 0/0 homozygotes was higher in HIV-1 positive mothers than in healthy controls, the MBL2 0/0 genotype was more frequent in children born from HIV positive mothers than healthy subjects.

Conclusions. We have confirmed the association of polymorphisms involving a gene of the innate immunity with an increased risk of being infected by HIV. These polymorphisms were also evidenced in children born from HIV + mothers, but the risk of infection was strongly reduced by cesarean delivery and by antiretroviral treatment.

Keywords: HIV, pregnancy, genetic polymorphisms, beta defensins, MBL2, innate immunity

Introduction

In the last few years researchers have recognized proteins of the innate immunity as being more and more important in conferring resistance to HIV-I infection, especially in newborns, where the acquired immune response is not fully developed.

Recently, genetic poymorphisms of MBL2 gene, which encode for mannose binding lectin (MBL), have been correlated with susceptibility to HIV-I infection in Brazilian [1] and Italian [2,3] children.

In this pilot study, we investigated genetic polymorphisms of MBL2 gene, in a cohort of 90 HIV-I pregnant seropositive women and their children in order to understand whether the MBL2 genotype of HIV-I positive mothers might be related to their ability to transmit the virus to their children and add new insights about the role of these polymorphisms in mother-to-child HIV-I transmission.

Methods

Patients

We analyzed 90 HIV+ mother/newborns pairs coming from one of the Italian HIV/SIGO Centers (*) since the beginning of 2003. Clinical data of HIV+ mother/newborns (gravidity, parity, previously born HIV- or HIV+ child, important pathologies during or before pregnancy, viremia and number of CD4 + lymphocities), antiretroviral therapy during pregnancy, mode of delivery, presence of Premature Rupture of the Membranes (PROM), before-labor therapies, and complications in the puerperium were evaluated.

HIV+ Mother/Newborns

Samples collection and DNA extraction

A 200 L blood-sample of HIV + mothers and their children were spotted on Iso Code Stix paper.

DNA was extracted from Iso Code Stix cards following standard protocols [4].

MBL2 Genotyping

MBL2 exon I genotyping was performed by using melting temperature assay (MTA) following the protocol of Hladnik and co-workers [5]. As indicated by Garred and co-workers [6], we grouped all three variant alleles of MBL2 (polymorphisms at positions 52, 54 and 57 in the first exon of MBL2 gene) in one category (allele 0} because they have a substantial effect consisting of the reduction of serum MBL concentrations, while the wild-type allele was called A.

Statistics

The significance of differences in allelic and genotype frequencies was calculated by Fisher's exact test using 2 2 and 3 2 contingency tables. Holm's correction for multiple tests was performed and only corrected p values (p^sub corr^) < 0.05 were considered to be significant. All the statistical analyses were carried out with SPSS software (Version 6.1.3).

Results

We evaluated the frequency of the polymorphisms in the first exon of MBL2 gene in a cohort of HIV-I infected mothers and their children, sampled by the Italian group SIGO-HIV in obstetrics and gynecology.

A total of 90 HIV-I infected mothers and their children were included in this study. All children were born to HIV-I positive mothers who had undergone antiretroviral therapy during pregnancy, as well as caesarean section to prevent vertical transmission. Among them there was only one case of maternal-fetal transmission of HIV infection (rate of transmission= 1.1%). A total of 120 uninfected and unexposed children, from the same ethnic origin of the mother- child pairs, were used as controls. The obstetrical features, antiretroviral therapy used during pregnancy as well as obstetrical and neonatal traits of the population are shown respectively in Tables I and II.

Table I. Epidemiological and clinical data of the HIV positive mothers.

Blood count of CD4 + cells and HIV viral load are the same as those observed in the European population of HIV positive pregnancies (see Table III) [7].

The frequency of the MBL-2 0/0 homozygotes (see Table IV) was higher in HIV-I positive mothers (0.10) than in healthy controls (0.05), the MBL2 0/0 genotype was more frequent in children born from HIV positive mothers (0.10) than healthy subjects (0.05) but the difference did not reach statistical significance in both cases. MBL2 A/A homozygotes and A/0 heterozygotes frequencies in HIV-I positive mothers and their exposed children were similar to the healthy controls. The allele O was more frequent in HIV-I positive mothers (0.29) and exposed uninfected children (0.26) when compared with the healthy controls (0.23). Also in this case the difference was not statistically significant.

Discussion

Globally, more than 2 million HIV-infected women give birth annually and in 2002, an estimated 800,000 infants were newly infected with HIV and the transmission rates in breastfeeding women without antiretroviral prophylaxis were between 30 and 40%. Following the USPHS guidelines for prevention of mother-to-child transmission, the US vertical HIV transmission was 1.5% in 2002 (http://www.womenchildrenhiv.org). These results are encouraging, but they could be better, making clear the exact response of the innate immunity system of the HIV-1. For this reason we decided to study the polymorphisms of MBL2 gene which encode for a protein involved in the innate immunity in 90 HIV + mother/child pairs.

Table II. Data about the delivery and the newborns.

Table III. HIV-Infected pregnant women and vertical transmission in Europe since 1986 (European Collaborative Study).

MBL2 0/0 genotype, known for increasing the risk of HIV infection and vertical transmission [1,3], showed higher frequency in HIV + mothers and their children when compared to healthy controls. Even if the difference was not statistically significant, this finding confirms previous results reported in the literature which identify the lack of MBL as a "predisposition" factor to HIV infection in both adults and children [8].

In the cohort of 90 HIV + mother-child pairs we evidenced one case of vertical transmission. The mother to child transmission was consistent (1.1%), but lower than, for example, last updates in the US (1.5%).

A woman with a previous HIV uninfected child, without any important pre- and during-pregnancy pathology except a VZV infection gave birth by caesarean section to a HIV+ child. At the end of the gestation her HIV viral load was 16.900 copy/ml and the count of CD4 lymphocytes was 468 cell/mmc. The woman regularly followed her antiretroviral therapy both during pregnancy (zidovudine, lamivudine, nevirapine) and at delivery (zidovudine according to protocol 076).

The child - a boy weighing 3090 g at birth -presented Apgar scores at 1 and 5 min, of 8 and 9 respectively, and underwent an antiretroviral therapy per os with zidovudine according to protocol 076, though suspended for one week.

The MBL genotypes were A/0 and 0/0, respectively, for the mother and her HIV+ child. The MBL2 newborn genotype was already described to be associated with an increased risk of HIV infection.

Even if it is true that genetic factors could play an important role in HIV infection and vertical transmission, the case described above is too isolated to draw any conclusion, also considering that the other HIV positive mother who gave birth to a HIV negative child was characterized by MBL2 polymorphisms associated with an increased risk of HIV infection.

In this preliminary Italian multicentric pilot study we have confirmed the association of polymorphisms involving a gene of the innate immunity with an increased risk of being infected by HIV. These polymorphisms, known to be associated with vertical HIV transmission, were also evidenced in children born from HIV + mothers, but the risk of infection was strongly reduced by cesarean delivery and by antiretroviral treatment of both mothers and children.

Acknowledgements

(*) Italian Group SIGO HIV in Obstetrics and Gynecology: E. Ferrazzi (Milano), S. Fiore (Milano), M. Ravizza (Milano), V. Savasi (Milano), S. Muggiasca (Milano), C.Tibaldi (Torino), B. Guerra (Bologna), P. Martinelli (Napoli), A. Vimercati (Bari), E. Rubino (Palermo), M. Sansone (Napoli), A. Meloni (Cagliari.), S. Alberico, M. Bernardon (Trieste).

References

1. Boniotto M, Braida L, Pirulli D, et al. MBL2 polymorphisms are involved in HIV-I infection in Brazilian perinatally infected children. AIDS 2003;17:779-780.

2. Amoroso A, Boniotto M, Crovella S, Serra C, Vatta S, Scarlatti G, Palomba E, Berrino M, Tovo PA. Polymorphism at codon 54 of Mannose binding protein gene influences AIDS progression but not HIV infection in exposed children. AIDS \1999;13:863-864.

3. Boniotto M, Crovella S3 Pirulli D, Scarlatti G, Spano A, Vatta L, Zezlina S, Tovo PA, Palomba E, Amoroso A. Polymorphisms in the MBL2 promoter correlated with risk of HIV-I vertical transmission and AIDS progression. Genes and Immunity 2000;l(5):346-348.

4. Walsh PS, Metzger DA, Higuchi R. Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. Biotechniques 1991;10:506-513.

5. Hladnik U, Braida L, Boniotto M, et al. Single-tube genotyping of MBL-2 polymorphisms using melting temperature analysis. Clin Exp Med 2002;2:105-108.

6. Garred P, Madsen HO, Balslev U, et al. Susceptibility to HIV infection and progression of AIDS in relation to variant alleles of mannose-binding lectin. Lancet 1997,349:236-240.

7. HIV-Infected Pregnant Women and Vertical Transmission in Europe since 1986 - European Collaborative Study. AIDS 2001;15:761.

8. Turner MW, Hamvas RM. Mannose-binding lectin: structure, function, genetics and disease associations. Rev Immunogenet 2000;2:305-322.

SERGIO CROVELLA1,2, MARIA BERNARDON3, LAURA BRAIDA1, MICHELE BONIOTTO1,2, SECONDO GUASCHINO4, ENRICO FERRAZZI5, PASQUALE MARTINELLI6, ITALIAN GROUP SIGO HIV IN OBSTETRICS AND GYNECOLOGY7, & SALVATORE ALBERICO3

1 Department of Developmental and Reproductive Sciences, Genetic Unit University of Trieste, Italy, 2 Genetic Service, Children Hospital Burlo Garofolo, Trieste, Italy, 3 Gynecology and Obstetrics Service, Children Hospital Burlo Garofolo, Trieste, Italy, 4 Department of Developmental and Reproductive Sciences, Gynecological Unit University of Trieste, Italy, 5 Department Obstetrics and Gynecology Luigi Sacco Hospital Milan, Italy, 6 Department Obstetrics and Gynecology Federico II University Hospital Naples, Italy, and 7 Italian Group SIGO HIV in Obsterics and Gynecology: Department Obstetrics and Gynecology

Correspondence: Sergio Crovella, Genetic Service, Children Hospital Burlo Garofolo Via dell'Istria 65/1, 34137 Trieste, Italy. Tel: 390403785538. Fax: 39 040 3785540. E-mail: crovella@burlo.trieste.it

Copyright CRC Press Apr 2005

Source: Journal of Maternal - Fetal & Neonatal Medicine

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