Genetic Approaches for Definitive Diagnosis of Agammaglobulinemia in Consanguineous Families.

Autosomal recessive agammaglobulinemia (ARA) is a primary immunodeficiency characterized by absent peripheral B cells, severe hypogammaglobulinemia, and absent BTK gene mutations. In ARA, mutations occur in genes encoding the pre-B cell receptor (pre-BCR) or downstream signaling proteins. In this work, we used candidate gene and whole-exome sequencing to investigate the molecular basis of ARA in 6 patients from 4 consanguineous North-African families. Sanger sequencing of candidate genes encoding the pre-BCR components (ΙGΗΜ, CD79A, CD79B, IGLL1, and VPREB1) was initially performed and determined the genetic defect in five patients. Two novel mutations in IGHM (p.Val378Alafs*1 and p.Ile184Serfs*21) were identified in three patients from two unrelated kindred and a novel nonsense mutation was identified in CD79A (p.Trp66*) in two siblings from a third kindred. Whole-exome sequencing (WES) was performed on the sixth patient who harbored a homozygous stop mutation at position 407 in the RAG2 gene (p.Glu407*). We concluded that conventional gene sequencing, especially when multiple genes are involved in the defect as is the case in ARA, is costly and time-consuming, resulting in delayed diagnosis that contributes to increased morbidity and mortality. In addition, it fails to identify the involvement of novel and unsuspected gene defects when the phenotype of the patients is atypical. WES has the potential to provide a rapid and more accurate genetic diagnosis in ARA, which is crucial for the treatment of the patients.

Activation induced cytidine deaminase mutant (AID-His130Pro) from Hyper IgM 2 patient retained mutagenic activity on SHM artificial substrate.

Activation induced cytidine deaminase (AID) is an essential enzyme for class switch recombination (CSR) and somatic hypermutation (SHM) during secondary immune response. Mutations in the AICDA gene are responsible for Hyper IgM 2 syndrome where both CSR and SHM or only CSR are affected. Indeed, triggering either of the two mechanisms requires the DNA deamination activity of AID. Besides, different domains of AID may be differentially involved in CSR and SHM through their interaction with specific cofactors. Herein, we studied the AID-induced SHM activity of the AID-His130Pro mutant identified in a patient with Hyper IgM 2 syndrome. AID mutagenic activity was monitored by the reversion of nonsense mutations of the EGFP gene assessed by flow cytometry. We found that the His130Pro mutation, which affects CSR, preserves AID mutagenic activity. Indeed, the His130 residue is located in a putative specific CSR region in the APOBEC-like domain, known to involve CSR specific cofactors that probably play a major role in AID physiological activities.

A Founder Effect of c.257 + 2T > C Mutation in NCF2 Gene Underlies Severe Chronic Granulomatous Disease in Eleven Patients.

Chronic granulomatous disease (CGD) is the prototypic functional neutrophil disorder caused by genetic defects in one of the five genes encoding the superoxide-generating nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase subunits of phagocytes. Mutations causing the most prevalent form of CGD in western populations are located in the X-linked-CYBB gene. The four remaining autosomal recessive (AR) forms collectively account for one-third of CGD cases. We investigated the clinical and molecular features of eleven patients with CGD from 6 consanguineous families, originating from contiguous regions in the west of Tunisia. The patients' clinical phenotype is characterized by a high incidence of mycobacterial infections. Five out of the eleven patients died despite treatment arguing in favor of a severe clinical form of CGD. These findings correlated with the absence of functional p67phox protein as well as the absence of residual reactive oxygen intermediates (ROI) production. Genetic analysis showed the presence, in all patients, of a unique mutation (c.257 + 2T > C) in NCF2 gene predicted to affect RNA splicing. Segregating analysis using nine polymorphic markers overlapping the NCF2 gene revealed a common haplotype spanning 4.1 Mb. The founder event responsible for this mutation was estimated to have arisen approximately 175 years ago. These findings will facilitate the implementation of preventive approaches through genetic counseling in affected consanguineous families.

Novel and recurrent AID mutations underlie prevalent autosomal recessive form of HIGM in consanguineous patients.

Immunoglobulin class switch recombination deficiencies (Ig-CSR-D) are characterized by normal or elevated serum IgM level and absence of IgG, IgA, and IgE. Most reported cases are due to X-linked CD40L deficiency. Activation-induced cytidine deaminase deficiency is the most frequent autosomal recessive form, whereas CD40 deficiency is more rare. Herein, we present the first North African study on hyper IgM (HIGM) syndrome including 16 Tunisian patients. Phenotypic and genetic studies allowed us to determine their molecular basis. Three CD40LG mutations have been identified including two novels (c.348_351dup and c.782_*2del) and one already reported mutation (g.6182G>A). No mutation has been found in another patient despite the lack of CD40L expression. Interestingly, three AICDA mutations have been identified in 11 patients. Two mutations were novel (c.91T>C and c.389A>C found in one and five patients respectively), and one previously reported splicing mutation (c.156+1T>G) was found in five patients. Only one CD40-deficient patient, bearing a novel mutation (c.109T>G), has been identified. Thus, unlike previous reports, AID deficiency is the most frequent underlying molecular basis (68%) of Ig-CSR-D in Tunisian patients. This finding and the presence of specific recurrent mutations are probably due to the critical role played by inbreeding in North African populations.

Report of the Tunisian Registry of Primary Immunodeficiencies: 25-Years of Experience (1988-2012).

PURPOSE: Primary immunodeficiencies (PIDs) are a large group of diseases characterized by susceptibility to not only recurrent infections but also autoimmune diseases and malignancies. The aim of this study was to describe and analyze the distribution, clinical features and eventual outcome of PID among Tunisian patients. METHODS: We reviewed the record of 710 patients diagnosed with Primary Immunodeficiency Diseases (PIDs) from the registry of the Tunisian Referral Centre for PIDs over a 25-year period. RESULTS: The male-to-female ratio was 1.4. The median age at the onset of symptoms was 6 months and at the time of diagnosis 2 years. The estimated prevalence was 4.3 per 100,000 populations. The consanguinity rate was found in 58.2 % of families. According to the International Union of Immunological Societies classification, spectrums of PIDs were as follows: combined T-cell and B-cell immunodeficiency disorders account for the most common category (28.6 %), followed by congenital defects of phagocyte (25.4 %), other well-defined immunodeficiency syndromes (22.7 %), predominant antibody deficiency diseases (17.7 %), diseases of immune dysregulation (4.8 %), defect of innate immunity (0.4 %) and complement deficiencies (0.4 %). Recurrent infections, particularly lower airway infections (62.3 %), presented the most common manifestation of PID patients. The overall mortality rate was 34.5 %, mainly observed with combined immunodeficiencies. CONCLUSION: The distribution of PIDs was different from that reported in Western countries, with a particularly high proportion of Combined Immunodeficiencies and phagocyte defects in number and/or function. More is needed to improve PID diagnosis and treatment in our country.

Clinical, immunological and genetic findings of a large tunisian series of major histocompatibility complex class II deficiency patients.

INTRODUCTION: Major histocompatibility complex class II (MHC-II) expression deficiency is a combined primary immunodeficiency leading to the impairment of the cellular and humoral immune responses. A majority of affected patients belong to consanguineous families particularly from the Maghreb, where a founder effect for a highly frequent mutation (named c.338-25_338del26) in the RFXANK gene was reported. Herein, we report the largest single Maghrebian country series of MHC-II deficient patients. PATIENTS AND METHODS: In Tunisia, among 551 PIDs diagnosed from 1993 to 2011, 54 had an MHC-II deficiency. The clinical features and immunological investigations were retrospectively analyzed in 34 children of them belonging to 28 kindred. The genetic study included the c.338-25_338del26 screening by the amplification of the affected region using polymerase chain reaction (PCR) followed by direct sequencing. RESULTS: Consanguinity was present in 22 out of 28 families. Mean age at the first infection was 6.1 months. Chronic diarrhea with failure to thrive and pulmonary infections were the most common manifestations occurring in 26 and 28 patients respectively. The most specific laboratory findings were the defect of MHC-II (HLA-DR) expression in all patients. The c.338-25_338del26 mutation was identified in 25 of them. CONCLUSION: In Maghrebian settings, pediatricians should definitely consider this diagnosis in the presence of an early onset of severe and recurrent infections of the respiratory and intestinal tracts, particularly protracted diarrhea with a failure to thrive. The founder effect for the c.338-25_338del26 mutation in the RFXANK gene is also confirmed, facilitating prenatal diagnosis as a preventive approach in the Tunisian affected families with severe forms, particularly in the context of limited access to bone marrow transplantation.