Advancing allele group-specific amplification of the complete HLA-C gene--isolation of novel alleles from three allele groups (C*04, C*07 and C*08).

A variety of strategies have been designed for sequence-based HLA typing (SBT) and for the isolation of new human leucocyte antigen (HLA) alleles, but unambiguous characterization of complete genomic sequences remains a challenge. We recently reported a simple method for the group-specific amplification (GSA) and sequencing of a full-length C*04 genomic sequence in isolation from the accompanying allele. Here we build on this strategy and present homologous methods that enable the isolation of HLA-C alleles belonging to another two allele groups. Using this approach, which can be applied to sequence-based typing in some clinical settings, we have successfully characterized three novel HLA-C alleles (C*04:128, C*07:01:01:02, and C*08:62).

X-linked thrombocytopenia in a female with a complex familial pattern of X-chromosome inactivation.

The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by thrombocytopenia, eczema and various degrees of immune deficiency caused by mutations in the WAS gene, which encodes the WASP protein, the expression of which is restricted to haematopoietic cells. Mild allelic variants are associated with X-linked thrombocytopenia (XLT). Female carriers tend in general to be asymptomatic as a consequence of a positive selection of cells with an active normal X chromosome, which results in a non-random inactivation of the mutated gene in affected cell lineages. We report on six female members of the same family carrying the mutated WAS allele p.V332A, which is known to be associated with XLT. One of them had presented severe thrombocytopenia from birth. Western blotting showed the WASP protein in peripheral blood cells to be normal in size and expression, and scanning electron microscopy revealed a normal distribution of microvilli on T cells. X-chromosome inactivation-pattern analysis showed total inactivation of the non-mutated paternal X chromosome in the patient's peripheral blood cells. All the other female family members were healthy and presented varying X-chromosome inactivation patterns, ranging from random X chromosome inactivation to total X-chromosome inactivation of the mutated chromosome. Our results in these female carriers of p.V332A show that manifestation of the disease requires a total inactivation of the non-mutated X chromosome and allow us to confirm that clinical manifestations in female carriers are highly dependent not only on the mutation characteristics but also on the X-chromosome inactivation pattern of affected line.

Elevated serum interleukin (IL)-12p40 levels in common variable immunodeficiency disease and decreased peripheral blood dendritic cells: analysis of IL-12p40 and interferon-gamma gene.

Common variable immunodeficiency disease (CVID) is a heterogeneous syndrome characterized by low immunoglobulin serum levels and recurrent bacterial infections. Several studies suggest that CVID patients have a polarized immune response towards a T helper type 1 phenotype (TH1). However, the factors causing the TH1 polarization remain to be determined in this disease. In the present study, serum interleukin (IL)-12, interferon (IFN)-gamma levels and the IL-12p40 and IFN-gamma gene were studied in CVID patients. Furthermore, we evaluate dendritic cells (DCs) compartment, myeloid dendritic cells (mDCs) and plasmocytoid dendritic cells (pDCs), which help to differentiate naive T cells preferentially into TH1 and TH2, respectively. The serum IL-12p40 subunit levels were increased significantly in CVID patients compared to healthy controls. We examined whether these elevated serum IL-12p40 levels are associated with IFN-gamma or IL-12p40 gene polymorphisms, or with new mutations in the IL-12p40 promoter gene. In our hands, no new mutations were found and gene polymorphisms frequencies in CVID patients were similar to the control population. In conclusion, the elevated serum levels of IL-12p40 found in our CVID patients were not related to these genetic variations. The DC compartment analysis did not show an imbalance between pDCs and mDCs, but revealed the presence of low numbers and percentage of both DC populations in CVID.

Costimulatory molecules and cytokine production by T lymphocytes in common variable immunodeficiency disease.

Common variable immunodeficiency (CVID) is a heterogeneous syndrome characterized by hypogammaglobulinaemia and recurrent infections. Although early works pointed to a primary B-lymphocyte defect as a cause of the disease, a failure in T-lymphocyte cooperation has also been suggested. T cells exert their costimulatory function through either membrane costimulatory molecules or secreted cytokines, both having an influence in the development of the humoral response. The aim of our study was to evaluate whether an abnormal expression and induction of costimulatory molecules or alterations in the production of cytokines by T cells cause deficient T/B cooperation in CVID patients. We studied the expression and upregulation of costimulatory molecules (CD28, CD40L/CD154 and CTLA-4/CD152) and production of cytokines (IL-2, IL-4, IL-6, IL-10, IFN-gamma and TNF-alpha) in purified T lymphocytes from CVID patients stimulated with optimal doses of anti-CD3 or suboptimal doses of anti-CD3 and anti-CD28. Stimulated T cells from CVID patients expressed normal levels of CD28, CD40L/CD154 and CTLA-4/CD152 when compared with controls. Except for higher production of IL-4 after stimulation with anti-CD3, T cells of CVID patients produced similar amounts of cytokines compared with controls. An imbalance between costimulatory molecules expression (CD28, CD40L/CD154 and CTLA-4/CD152) and cytokine production by T cells does not explain a deficient cooperation between T and B cells in this group of CVID patients.

Estudio de incontinentia pigmenti en España. Nueva mutación del gen NEMO asociada a inmunodeficiencia transitoria / No dispnible

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Estudio de incontinentia pigmentien España. Nueva mutación del gen NEMO asociada a inmunodeficiencia transitoria / No disponible

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HLA polymorphism in a Majorcan population of Jewish descent: comparison with Majorca, Minorca, Ibiza (Balearic Islands) and other Jewish communities.

'Chueta' was the name given to the Catholic descendants of Jewish victims of the last Spanish Inquisition process in Majorca Island in the western Mediterranean. We have studied the allele distribution of HLA-A, -B, -Cw, -DRB1 and -DQB1 loci of 103 random, healthy, unrelated individuals belonging to the ancient Majorcan Jewish community, known locally as Chuetas, and 589 individuals from the Balearic population selected because of their typical Balearic - Majorca, Minorca or Ibiza - lineages and according to their ancestor's place of birth. Our aim was to establish the genetic relationship between Majorcan Chuetas, and Balearic and other Jewish and Mediterranean populations. Our results have shown that, to a remarkable extent, they have retained their biological identity, with a unique pattern, in terms of gene and haplotype frequencies, separate from the other populations of Majorca. The Chuetas were found to be more related to Moroccan and Libyan Jews than other Majorcans. Characteristic Jewish haplotypes, A26-B38-DRB1*13, A24-B38-DRB1*11, A1-B52-DRB1*15/16, were found in our study. Some peculiarities were observed in the distribution of common haplotypes among the three main Balearic Islands. The Ibizan population was genetically different from the other Balearic populations, with a high frequency of some haplotypes, for example, A29-Cw*16-B44-DRB1*07-DQB1*03; A1-Cw*07-B8-DRB1*03-DQB1*02. We also found a new haplotype, A25-Cw*12-B39-DRB1*11-DQB1*03(3.5%), in Ibizans and a more limited variability in the HLA alleles that were expressed, perhaps because of genetic isolation. The genetic diversity of the populations from Majorca and Minorca were similar and more related to the mainland Spanish population.