High-density SNP mapping of the HLA region identifies multiple independent susceptibility loci associated with selective IgA deficiency.

Selective IgA deficiency (IgAD; serum IgA<0.07 g/l) is the most common form of human primary immune deficiency, affecting approximately 1∶600 individuals in populations of Northern European ancestry. The polygenic nature of IgAD is underscored by the recent identification of several new risk genes in a genome-wide association study. Among the characterized susceptibility loci, the association with specific HLA haplotypes represents the major genetic risk factor for IgAD. Despite the robust association, the nature and location of the causal variants in the HLA region remains unknown. To better characterize the association signal in this region, we performed a high-density SNP mapping of the HLA locus and imputed the genotypes of common HLA-B, -DRB1, and -DQB1 alleles in a combined sample of 772 IgAD patients and 1,976 matched controls from 3 independent European populations. We confirmed the complex nature of the association with the HLA locus, which is the result of multiple effects spanning the entire HLA region. The primary association signal mapped to the HLA-DQB1*02 allele in the HLA Class II region (combined P = 7.69×10(-57); OR = 2.80) resulting from the combined independent effects of the HLA-B*0801-DRB1*0301-DQB1*02 and -DRB1*0701-DQB1*02 haplotypes, while additional secondary signals were associated with the DRB1*0102 (combined P = 5.86×10(-17); OR = 4.28) and the DRB1*1501 (combined P = 2.24×10(-35); OR = 0.13) alleles. Despite the strong population-specific frequencies of HLA alleles, we found a remarkable conservation of these effects regardless of the ethnic background, which supports the use of large multi-ethnic populations to characterize shared genetic association signals in the HLA region. We also provide evidence for the location of association signals within the specific extended haplotypes, which will guide future sequencing studies aimed at characterizing the precise functional variants contributing to disease pathogenesis.

Molecular characterization of Complement Factor I deficiency in two Spanish families.

Complement Factor I (CFI) is a regulator of the classical and alternative pathways. CFI has enzymatic activity and is able to cleave C3b and C4b. Homozygous Factor I deficiency is associated with infectious and/or autoimmune diseases. Here we describe the biochemical and genetic characterization in two Spanish families with complete Factor I deficiency. In Family 1, the propositus suffered from several episodes of meningitis for more than a year. Biochemical complement studies showed undetectable Factor I levels in the propositus and in her sister, while their parents and a brother had partial Factor I deficiency and were healthy. In Family 2, three out of five children were homozygous for Factor I deficiency, two of whom suffered from meningitis and the third one from several infections. The parents and the other two siblings were healthy and heterozygous for Factor I deficiency. Molecular studies showed that the two families had different mutations at exon 5 of the Factor I gene, which codifies for module LDLr1. One mutation corresponds to a 772G>A change at the donor splice site that was originally found in a family from Northern England. The second is a new missense mutation 739T>G, that generates a Cys to Gly change.

Identification of novel non-pathogenic mutation in SH3 domain of Btk in an XLA patient.

A first report of an XLA patient with a polymorphism in Btk SH3 domain has been identified after sequencing of the entire gene. SH3 domain variants might not be detected due to well characterized mutations outside the domain.

Interleukin-6 gene variation in Spanish patients with immunoglobulin-A deficiency.

Selective immunoglobulin-A deficiency (IgAD) is the most common immunodeficiency in Caucasian populations. Genetic factors are important in its etiology; however human leukocyte antigen (HLA) genes, which explain 40% of the genetic risk for IgAD, are the only susceptibility factors commonly agreed upon at this time. Because interleukin-6 (IL-6) plays an important role in B-lymphocyte differentiation from plasma cells, we aimed to address the IL-6 genetic influence on IgAD susceptibility. We performed a case-control study that included 305 Caucasian Spanish IgAD patients and 529 ethnically matched healthy control subjects, as well as a familial study with 128 IgAD trios. We genotyped the functional promoter polymorphism -174G>C and nine additional single nucleotide polymorphisms. For the case-control analyses the chi(2) test or Fisher's exact test were used, and for the family study the transmission disequilibrium test was used. We observed an increased frequency of the -174C allele in IgAD patients (p = 0.005, odds ratio [OR] = 1.51, 95% confidence interval [CI] = 1.12-2.04) and a protective effect of the rs2069849_C allele (p = 0.007, odds ratio = 0.29, 95% CI = 0.09-0.76). In conclusion, we described for the first time an association between IL6 polymorphisms and IgAD. Although it is not clear which genetic variants are causing susceptibility/protection, this intriguing finding is remarkable because of the role of IL-6 in antibody production.

Interleukin-10 polymorphisms in Spanish IgA deficiency patients: a case-control and family study.

BACKGROUND: IgA deficiency (IgAD) is the most common primary immunodeficiency in Caucasians. Genetic and environmental factors are suspected to be involved in the development of the disease. Interleukin-10 (IL-10) is a cytokine with stimulatory activity on immunoglobulin production and it may be an important regulator in IgAD pathogenesis. The IL-10 gene contains several single nucleotide polymorphisms (SNPs) and two polymorphic microsatellites located in the 5'-flanking region. Our aim was to ascertain if any of these polymorphic markers are associated or linked to IgAD in Spanish patients. METHODS: We genotyped 278 patients with IgAD and 573 ethnically matched controls for the microsatellites IL-10R and IL-10G and for three single nucleotide polymorphisms at positions -1082, -819 and -592 in the proximal promoter of the gene. We also included in this study the parents of 194 patients in order to study the IL-10 haplotypes transmitted and not transmitted to the affected offspring. RESULTS: The only allele where a significant difference was observed in the comparison between IgA deficiency patients and controls was the IL-10G12 allele (OR = 1.58 and p = 0.021). However, this p value could not withstand a Bonferroni correction. None of the IL-10R or promoter SNP alleles was found at a different frequency when patients were compared with controls. CONCLUSION: Our data do not show any significant difference in IL-10 polymorphism frequencies between control and IgAD patient samples. Their haplotype distribution among patients and controls was also equivalent and therefore these microsatellites and SNPs do not seem to influence IgAD susceptibility.

C5 complement deficiency in a Spanish family. Molecular characterization of the double mutation responsible for the defect.

The complement C5 deficiency is a recessive autosomal defect associated with recurrent infectious episodes, generally caused by Gram-negative micro-organisms. To date, only two mutations responsible for C5 deficiency have been characterized, both in heterozygosis. In this paper, we evaluate by immunochemical methods the C5 deficiency in a six-member family, in which one member suffered from meningococcal sepsis and several pneumonia episodes; and a second one with two bacterial meningitis episodes and frequent tonsillitis, pneumonia and herpetic episodes. We also characterize the molecular basis of this deficiency. No C5 protein was found in the serum from three of the children. They were found to be homozygous for a double mutation in the exon 40 of the C5 gene. The parents and the other children have half-normal levels of C5, and they were heterozygotes for the double mutation. This mutation modifies the reading frame, leading to a premature stop codon, and the resulting protein lacks 50 amino acids. As a result, homozygotes and heterozygotes have a total or a partial C5 deficiency respectively. This is the first report of a whole molecular characterization of C5 deficiency.

Hereditary angioedema: the mutation spectrum of SERPING1/C1NH in a large Spanish cohort.

Hereditary angioedema (HAE) is a disease caused by defects in the C1 inhibitor gene (SERPING1/C1NH). We screened the entire C1NH gene for mutations in a large series of 87 Spanish families (77 with type I, and 10 with type II HAE) by SSCP, sequencing, Southern blotting, and quantitative multiplex PCR of short fluorescent fragments (QMPSF), and we characterized several defects at the mRNA level. We found large rearrangements in 13 families, and point mutations or microdeletions/insertions in 74 families. The 13 large rearrangements included nine exon deletions, of which at least eight were distinct, two were distinct exon duplications, and two were rearrangements whose precise nature could not be determined. We confirmed that exon 4 is particularly prone to rearrangements. Thirty-six mutations were unreported, and included 10 microdeletions/insertions, 10 missense, five nonsense, eight splicing, and three splicing or missense mutations. Moreover, we detected six novel uncharacterized sequence variants (USV). RT-PCR studies showed that in addition to several intronic splice site mutations tested, the exonic mutations c.882C>G and c.884T>G, located near the 3' end of exon 5, also produced exon skipping. This is the first evidence of SERPING1/C1NH mutations in coding regions that differ from the canonical splice sites that affect splicing, which suggests the presence of an exonic splicing enhancer (ESE) in exon 5.

Detection of C1 inhibitor (SERPING1/C1NH) mutations in exon 8 in patients with hereditary angioedema: evidence for 10 novel mutations.

Hereditary angioedema (HAE) is caused by mutations in the C1 inhibitor gene (SERPING1, C1NH) and the result is C1 inhibitor deficiency, either in levels or function. We have searched exon 8 for mutations by direct sequencing and analyzed the rest of the exons by SSCP in 87 Spanish families affected by HAE. Out of 87 screened families, we have detected exon 8 mutations in 26. Among these, 17 different mutations were identified: 14 point mutations and 3 frameshift. Seven of the point mutations and the three frameshift were not previously reported. Mutations were: S438P; R444P; V451G; W460X; V468D; G471E; X479R; S417fsX427; I440fsX450; E429fsX450. The rest of the families presented previously reported mutations, 5 missense and two nonsense. In none of the 26 families was an additional change identified in the rest of the exons by SSCP, and, in 20 out of the 22 families with point mutation, we verified that the mutation did not affect a healthy relative. Seven of these families had no history of the disease, and in five of them we were able to verify that the progenitors did not have the mutation. Therefore, they were de novo mutations.

Independent mutational events are rare in the ATM gene: haplotype prescreening enhances mutation detection rate.

Mutations in the ATM gene are responsible for the autosomal recessive disorder ataxia-telangiectasia (A-T). Many different mutations have been identified using various techniques, with detection efficiencies ranging from 57 to 85%. In this study, we employed short tandem repeat (STR) haplotypes to enhance mutation identification in 55 unrelated A-T families of Iberian origin (20 Spanish, 17 Brazilian, and 18 Hispanic-American); we were able to identify 95% of the expected mutations. Allelic sizes were standardized based on a reference sample (CEPH 1347-2). Subsequent mutation screening was performed by PTT, SSCP, and DHPLC, and abnormal regions were sequenced. Many STR haplotypes were found within each population and six haplotypes were observed across several of these populations. Single nucleotide polymorphism (SNP) haplotypes further suggested that most of these common mutations are ancestrally related, and not hot spots. However, two mutations (8977C>T and 8264_8268delATAAG) may indeed be recurring mutational events. Common haplotypes were present in 13 of 20 Spanish A-T families (65%), in 11 of 17 Brazilian A-T families (65%), and, in contrast, in only eight of 18 Hispanic-American families (44%). Three mutations were identified that would be missed by conventional screening strategies. In all, 62 different mutations (28 not previously reported) were identified and their associated haplotypes defined, thereby establishing a new database for Iberian A-T families, and extending the spectrum of worldwide ATM mutations.

[Autoimmune lymphoproliferative syndrome: molecular diagnosis in two families]. / Síndrome linfoproliferativo autoinmune: diagnóstico molecular en dos familias.

BACKGROUND AND OBJECTIVE: The autoimmune lymphoproliferative syndrome (ALPS) is a disorder caused by a defect in lymphocytes' apoptosis and characterized by non malignant lymphoproliferation, autoimmune features and increased TCR alpha + CD4CD8 cells. Most patients have a mutation in the TNFRSF6 gene, which encodes the Fas protein. Our aim was to identify mutations in this gene in two families with possible ALPS cases. PATIENTS AND METHOD: Two patients with suspicion of ALPS, belonging to two unrelated families, were studied. To confirm such a diagnosis, immunoglobulin quantification, cellular phenotypic analysis by flow cytometry, IL-10 quantification, an apoptosis study, and molecular analysis were performed. RESULTS: Both patients showed hypergammaglobulinemia and an increased percentage of TCR alpha + CD4CD8 cells (family A patient: 14%; family B patient: 4.25%). In family A, in vitro Fas-mediated apoptosis was absent in the patient and markedly reduced in his father. In this family, both the patient and his father were heterozygous for the Fas mutation T1045C (Leu 268 Pro). The family B patient and her mother showed the Fas mutation G943T (Arg 234 Leu), both being heterozygous for it too. Both mutations are located in exon 9 of TNFRSF6 gene, affecting the death domain of the Fas protein. CONCLUSIONS: The molecular study of these families confirms a diagnosis of ALPS and suggests that the causing defect of this syndrome is compatible with an autosomal dominant inheritance with incomplete penetrance.

Association of IFIH1 and other autoimmunity risk alleles with selective IgA deficiency.

To understand the genetic predisposition to selective immunoglobulin A deficiency (IgAD), we performed a genome-wide association study in 430 affected individuals (cases) from Sweden and Iceland and 1,090 ethnically matched controls, and we performed replication studies in two independent European cohorts. In addition to the known association of HLA with IgAD, we identified association with a nonsynonymous variant in IFIH1 (rs1990760G>A, P = 7.3 x 10(-10)) which was previously associated with type 1 diabetes and systemic lupus erythematosus. Variants in CLEC16A, another known autoimmunity locus, showed suggestive evidence for association (rs6498142C>G, P = 1.8 x 10(-7)), and 29 additional loci were identified with P < 5 x 10(-5). A survey in IgAD of 118 validated non-HLA autoimmunity loci indicated a significant enrichment for association with autoimmunity loci as compared to non-autoimmunity loci (P = 9.0 x 10(-4)) or random SNPs across the genome (P < 0.0001). These findings support the hypothesis that autoimmune mechanisms may contribute to the pathogenesis of IgAD.

Molecular characterization of three new mutations causing C5 deficiency in two non-related families.

Deficiencies in complement components are rare diseases whose diagnosis is often underestimated. In addition, in only a few cases molecular studies have been carried out for the characterization of the underlying genetic defects. To date, studies involving C5-deficient patients are scarce. The aim of the present report is to characterize the biochemical and molecular complement deficiency in two non-related families with one or more members showing no detectable hemolytic complement activity (CH50<50 U/ml) and reporting a history of several episodes of meningitis. Protein deficiency was assessed by means of hemolytic assays, bi-dimensional double immunodiffusion, ELISA and Western blot of patients' sera. Molecular studies were carried out by PCR and RT-PCR of DNA and RNA, respectively, both extracted from fresh blood samples of each family member. In Family A, only the propositus had complete C5 deficiency. Molecular studies showed that he was heterozygous for two changes in the C5 gene. One of the mutations was also carried by the father (c.1883_1884AGC, Y846H) was a de novo mutation. In Family B, the two C5-deficient members share the homozygous nonsense mutation c.892C>T (Q298X) in exon 9. The characterization of these new mutations is interesting in order to elucidate structure-function relationships in the C5 gene and it also helps to understand the molecular basis of this uncommon deficiency. Moreover, this report highlights the importance of complement screening in cases of repeated meningococcal infections in order to establish its involvement and to consider adequate clinical recommendations such as prophylactic antibiotics or meningococcal vaccines.

Lack of evidence of a role of XBP1 and PRDM1 polymorphisms in Spanish patients with immunoglobulin A deficiency.

The etiology of selective immunoglobulin A deficiency (IgAD) is not yet unraveled, but genetics seem to play an important role. Defects in processes during B-cell differentiation into plasma cells could exist in these patients, turning the genes controlling these processes into interesting candidate genes for IgAD predisposition, as PRDM1 (encoding Blimp-1) and XBP1. We studied the involvement of several polymorphisms located in PRDM1 and XBP1 on IgAD susceptibility. We performed a case-control study with 331 Spanish IgAD patients and 717 healthy controls, by analyzing five single nucleotide polymorphisms (SNPs) in these genes. Genetic frequencies of the studied SNPs did not significantly differ between patients and controls, even after stratifying by the known human leukocyte antigen risk factors or clinical phenotypes. Interaction between PRDM1 and XBP1 to confer disease predisposition was not detected either. In conclusion, the polymorphisms studied in the PRDM1 and XBP1 genes do not seem to be involved in IgAD predisposition in the Spanish population.

A novel Wiskott-Aldrich syndrome protein (WASP) complex mutation identified in a WAS patient results in an aberrant product at the C-terminus from two transcripts with unusual polyA signals.

Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by immunodeficiency, thrombocytopenia and eczema. A broad spectrum of mutations in the WASP gene has been identified as causing the disease. In the present paper, we report on a patient affected by WAS with a novel complex mutation, characterized by a small 9 bp deletion followed by an inversion of 151 bp and a gross deletion of 4.3 kb within the Xp11.23 region. The small deletion and the inverted fragment are found in intron 11. The large deletion initiates downstream of exon 11 of the WASP gene, including exon 12, and a genomic region upstream of the promoter of the contiguous SUV39H1 gene. Expression studies of the mRNA of the patient's sample showed the presence of two aberrant transcripts that code for a protein of 519 amino acids. We demonstrate that these two transcripts differ in the 3' UTR region, and result from the use of two alternative polyadenylation signals. The severe phenotype of the patient correlates with the presence of an aberrant protein.

First case of homozygous C1 inhibitor deficiency.

BACKGROUND: C1 Inhibitor (C1-Inh) deficiency causes angioedema and can be hereditary (HAE), caused by mutations in the C1-Inh gene (C1NH), or acquired (AAE). Patients with HAE show a complement profile different from that of patients with AAE with normal levels of C1 (C1q, C1r, and C1s). OBJECTIVE: We sought to characterize the complement profile of a patient with HAE and a mutation in homozygosis in the C1NH gene (c.1576T>G, Ile462Ser) and study his family. METHODS: Biochemical diagnosis of HAE was confirmed by analyzing the C1NH gene. Further studies on the levels and activation states of the C1q, C1r, C1s, and C1-Inh components of the classical pathway of complement activation were also performed. RESULTS: Another 7 members of the family were given diagnoses of HAE: 1 was homozygous and 6 were heterozygous for the C1NH mutation c.1576T>G. The homozygous patients showed undetectable C1q levels, reduced C1s levels, the circulating active form of C1r, and a C1-Inh mostly in its cleaved inactive form in plasma. CONCLUSION: This is the first report of patients homozygous for a mutation affecting the coding region of C1NH. These patients showed a unique activation and consumption profile of the classical complement activation pathway different from that commonly observed in patients with HAE but similar to that of patients with AAE. CLINICAL IMPLICATIONS: The most common HAE treatment is attenuated androgens, which increase the C1NH gene transcription levels. Because the homozygous patients lack a wild-type allele, long-term prophylactic treatment with attenuated androgens might not be advisable.

Bruton's tyrosine kinase is not essential for LPS-induced activation of human monocytes.

BACKGROUND: X-linked agammaglobulinemia (XLA) is characterized by impaired B-cell differentiation caused by mutations in the Bruton's tyrosine kinase (Btk) gene. The natural disease model, the X-linked immunodeficiency mouse, shows a less severe phenotype, indicating a different requirement of Btk in human and mouse B cells. Btk is also expressed in the myeloid line and participates in LPS signaling. Deficient oxidative burst and myeloid differentiation have been reported in the X-linked immunodeficiency mouse, but the precise mechanism and relevance of Btk activity in human monocytes is poorly understood. OBJECTIVE: The apparent absence in XLA of clinical manifestations of myeloid deficiency prompted us to explore the relevance of complete Btk absence in human myeloid cells. METHODS: Seven patients with XLA with BTK mutations conditioning a null protein expression were included in the study. Monocyte LPS-induced mitogen-activated protein kinase activation, TNF-alpha and IL-6 production in monocytes, and oxidative burst in monocytes and granulocytes were analyzed by means of flow cytometry. RESULTS: We show that in response to LPS, Btk-null monocytes from patients with XLA induce early mitogen-activated protein kinase activation and intracellular TNF-alpha and IL-6 production with the same intensity as cells from age- and sex-matched control subjects. In addition, the oxidative burst in response to LPS and other stimulants was completely normal in Btk-null monocytes and neutrophils. CONCLUSION: Our results indicate that Btk is not essential for early LPS signaling in human monocytes and that different Btk dependency might exist between human and mouse myeloid cells. CLINICAL IMPLICATIONS: These findings provide a better understanding of XLA, and they show the differences between human XLA and murine Xid models.

MHC susceptibility genes to IgA deficiency are located in different regions on different HLA haplotypes.

Familial predisposition to IgA deficiency (IgAD) suggests that genetic factors influence susceptibility. Most studies support a polygenic inheritance with a susceptibility locus (designated IGAD1) in the MHC, but its exact location is still controversial. This study aimed to map the predisposing IGAD1 locus (or loci) within the MHC by investigating the pattern of association of the disease with several markers in the region. DNA-based techniques were used to type individual alleles of four polymorphic HLA genes (HLA-DR, -DQA1, -DQB1, and HLA-B), six microsatellites (all located between HLA-DR and HLA-B), and three single nucleotide polymorphisms on the TNF gene. The frequencies of these alleles were compared among ethnically matched populations comprising 182 patients and 343 controls. Additionally, we investigated parents and siblings of 100 of these patients. All four parental haplotypes were established in each family (n = 400), and transmission disequilibrium tests were performed. Surprisingly, our results did not support the hypothesis of a unique susceptibility gene being shared by all MHC susceptibility haplotypes. On HLA-DR1 and -DR7-positive haplotypes IGAD1 mapped to the class II region, whereas on haplotypes carrying HLA-DR3 the susceptibility locus mapped to the telomeric end of the class III region, as reported previously. Our results show how, in complex diseases, individuals may be affected for different genetic reasons and a single linkage signal to a region of a chromosome may actually be the result of disease-predisposing alleles in different linked genes in different pedigrees.

Polyclonal autoantibodies against C1 inhibitor in a case of acquired angioedema.

BACKGROUND: Angioedema attributable to acquired C1 inhibitor (C1-INH) deficiency is a rare disease related to lymphoproliferative disorders or autoantibodies to Cl inhibitor. We describe a patient with angioedema and autoantibodies to C1 inhibitor. OBJECTIVE: To study the characteristics of autoantibodies to C1-INH in a patient with acquired angioedema. METHODS: Autoantibodies to Cl-INH were measured by enzyme-linked immunoadsorbent assay. Immunoglobulin (Ig)G autoantibody was purified by affinity chromatography on a protein G agarose column. We developed an enzyme-linked immunoadsorbent assay to determine whether the autoantibodies were directed against the C1-INH active center. RESULTS: IgM and mainly C1-INH IgG autoantibodies were detected; both had kappa and lambda chains. No monoclonal component was detected. The autoantibodies were directed against the Cl-INH active center. After various treatment strategies were attempted, an effective clinical response was attained with antifibrinolytic therapy. CONCLUSION: A case of acquired angioedema because of C1-INH deficiency was found to be attributable to the presence of polyclonal autoantibodies to C1-INH.

Síndrome linfoproliferativo autoinmune: diagnóstico molecular en dos familias / Autoimmune lymphoproliferative syndrome: molecular diagnosis in two families

FUNDAMENTO Y OBJETIVO: El síndrome linfoproliferativo autoinmune (SLPA) es una enfermedad debida a un defecto en la apoptosis de los linfocitos, que cursa con linfoproliferación crónica no maligna, manifestaciones autoinmunes e incremento de los linfocitos TCRalfa beta+CD4-CD8-. La mayoría de los casos se deben a mutaciones en el gen TNFRSF6 que codifica para la proteína Fas. Nuestro objetivo fue identificar mutaciones en este gen en dos familias, algunos de miembros presentaban una clínica y una analítica compatibles con SLPA. PACIENTES Y MÉTODO: Estudiamos a dos enfermos con sospecha de SLPA. Para confirmar este diagnóstico realizamos cuantificación de inmunoglobulinas, fenotipado celular por citometría de flujo, cuantificación de interleucina (IL) 10, estudio de apoptosis y análisis molecular. RESULTADOS: Ambos enfermos presentaron hipergammaglobulinemia y un aumento de las células TCRalfa beta+CD4-CD8- (paciente de la familia A: 14 por ciento; enferma de la familia B: 4,25 por ciento). En la familia A se efectuó un estudio de apoptosis, ausente en los linfocitos del paciente y muy disminuida en los linfocitos del padre. Ambos fueron heterocigotos para la mutación T1045C (Leu 268 Pro). La paciente de la familia B y su madre presentaron la mutación G943T (Arg 234 Leu), también en heterocigosis. Las dos mutaciones descritas se localizan en el exón 9 del gen TNFRSF6, que afecta al dominio de muerte de la proteína Fas. CONCLUSIONES: Los resultados del estudio molecular en estas dos familias apoyaron el diagnóstico de SLPA y apuntan a que el defecto causante del síndrome es compatible con un patrón de herencia autosómico dominante con penetrancia incompleta (AU)