BRIEF COMMUNICATION

A 1,100-year-old founder effect mutation in IL12B geneis responsible for Mendelian susceptibility to mycobacterialdisease in Tunisian patients

Imen Ben-Mustapha & Meriem Ben-Ali & Najla Mekki & Etienne Patin &

Christine Harmant & Jihène Bouguila & Houda Elloumi-Zghal &Abdelaziz Harbi & Mohamed Béjaoui & Lamia Boughammoura &

Jalel Chemli & Mohamed-Ridha Barbouche

Received: 12 July 2013 /Accepted: 21 September 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract Mendelian susceptibility to mycobacterial disease(MSMD) is a rare disorder predisposing apparently healthyindividuals to infections caused by weakly virulentmycobacteria such as bacille Calmette–Guerin (BCG), envi-ronmental mycobacteria, and poorly virulent Salmonellastrains. IL-12p40 deficiency is the first reported human dis-ease due to a cytokine gene defect and is one of the deficien-cies that cause MSMD. Nine mutant alleles only have been

identified in the IL12B gene, and three of them are recurrentmutations due to a founder effect in specific populations. IL-12p40 deficiency has been identified especially in countrieswhere consanguinity is high and where BCG vaccination atbirth is universal. We investigated, in such settings, the clin-ical, cellular, and molecular features of six IL-12p40-deficientTunisian patients having the same mutation in IL12B gene(c.298_305del). We found that this mutation is inherited as acommon founder mutation arousing ~1,100 years ago. Thisfinding facilitates the development of a preventive approachby genetic counseling and prenatal diagnosis especially inaffected families.

Keywords Primary immunodeficiency disease . IL12-p40deficiency . Founder effect . Consanguinity . Tunisia

Mendelian susceptibility to mycobacterial disease (MSMD) isa rare primary immunodeficiency that confers an apparentlyselective predisposition to poorly virulent mycobacterial spe-cies such as bacille Calmette–Guérin (BCG) attenuatedsubstrains, environmental mycobacteria (EM), and in almosthalf of the cases, poorly virulent Salmonella strains(Casanova and Abel 2002; MacLennan et al. 2004). Occa-sionally, patients can also develop infections caused by themore virulent Mycobacterium tuberculosis (Alcais et al.2005). Over the last 15 years, the molecular basis of thisgenetic vulnerability has been deciphered with the identifica-tion of nine disease-causing genes, including two X-linked(NEMO and CYBB) and seven autosomal genes (IFNGR1 ,IFNGR2 , STAT1 , IL12B , IL12RB1 , ISG15 , and IRF8 )(Bogunovic et al. 2012; Bustamante et al. 2011; Filipe-Santos et al. 2006; Hambleton et al. 2011). With the exceptionof CYBB , MSMD-causing genes affect the IL-12/IL23-IFNG

Imen Ben-Mustapha and Meriem Ben-Ali contributed equally to thiswork.

I. Ben-Mustapha :M. Ben-Ali :N. Mekki :H. Elloumi-Zghal :M.<R. Barbouche (*)Laboratory of Transmission, Control and Immunobiology ofInfections (LTCII), Institut Pasteur de Tunis, LR11IPT02,Tunis-Belvédère 1002, Tunisiae-mail: ridha.barbouche@pasteur.rns.tn

I. Ben-Mustapha :M. Ben-Ali :N. Mekki :H. Elloumi-Zghal :M.<R. BarboucheUniversité Tunis El Manar, Tunis, Tunisia

E. Patin : C. HarmantUnit of Human Evolutionary Genetics, Institut Pasteur, 75015 Paris,France

E. Patin : C. HarmantCentre National de la Recherche Scientifique, URA 3012,75015 Paris, France

J. Bouguila : L. BoughammouraDepartment of Pediatrics, Farhat Hached Hospital, Sousse, Tunisia

A. Harbi : J. ChemliDepartment of Pediatrics, Sahloul Hospital, Sousse, Tunisia

M. BéjaouiBone Marrow Transplantation Center, Tunis, Tunisia

ImmunogeneticsDOI 10.1007/s00251-013-0739-0

circuit, either in terms of IL-12-dependent induction of IFN-γor cellular responses to IFN-γ.

IL-12p40 deficiency is the first reported human disease dueto a cytokine gene defect (Altare et al. 1998). Along with IL-17F and IL-10 deficiencies (Puel et al. 2011; Engelhardt et al.2013), they consist of the only known cytokine disorders. IL-12 is a cytokine secreted by mononuclear phagocytes anddendritic cells. It comprises two subunits, p35 and p40, re-spectively encoded by IL12A and IL12B , which form thebiologically active p70 heterodimer molecule. IL-12 binds tohigh-affinity beta-1/beta-2 heterodimeric IL-12 receptor com-plex on CD4+ T cells enabling them to differentiate into TH1cells, and promotes the production of high level of IFN-γfrom other cells such as NK cells. These two major effectsemphasize the central role of IL-12 in promoting cell-mediated immunity against intracellular pathogens (Gatelyet al. 1998; Trinchieri 1998). IL-12p40 deficiency is causedby homozygous mutations in the IL12B gene. All knownmutations are recessive, loss of function, leading to a completeIL-12p40 deficiency with an undetectable IL-12p40 secretionby the patient's PBMCs and/or Epstein–Barr virus (EBV) celllines. Interestingly, all previously reported patients originatedfrom the Middle East/North Africa regions or Indian subcon-tinent (Prando et al. 2013). Patients with a given mutationhave the same ethnic origin, providing evidence for underly-ing founder effects in such populations. Indeed, three of thenine mutant IL12B alleles arise from a founder effect: Muta-tion c.320dupA in Saudi Arabia is thought to have occurred~1,100 years ago, mutation g.482+83_856-855del in India/Pakistan was estimated to have occurred ~700 years ago, andfounder event for mutation c.527_528del in Iran has beendated to 600 years ago (Prando et al. 2013).

In the present study, we investigated the clinical, cellular,and molecular features of six IL-12p40-deficient Tunisianpatients having the same mutation in the IL12B gene andprovided evidence that this mutation also arose from a com-mon ancestor. Six IL-12p40-deficient patients (four males:BBH, FLA, YLA and MGA; and two females: FBH andIKO) belonging to four kindreds (BH, LA, GA, and KO)(Fig. 1) and presenting with mycobacterial diseases have beeninvestigated. Three of them (patients BBH, FBH, and IKO)have been reported previously (Elloumi-Zghal et al. 2002),while two others (patients FLA and YLA) were included aspatients 34 and 35 in a recent review (Prando et al. 2013). Twofamilies were consanguineous. Consanguinity was not ex-cluded in the two remaining families since all of them origi-nated from the same small Tunisian town (23,015 inhabitantsaccording to 2008 statistics). The first infection occurred at amean age of 3 months. Indeed, in countries where systematicBCG vaccination is done at birth, MSMD usually present as adisseminated BCG infection early in childhood. Among thefive BCG-vaccinated patients, four developed disseminatedBCG-osis (BBH, IKO, FLA, and MGA) and one developed

localized BCG disease (FBH). None of the BCG-vaccinatedpatients developed EM disease, suggesting that BCG seems toconfer protection against EM. This finding has also beenreported in IL-12Rβ1-deficient patients (de Beaucoudreyet al. 2010). Contrasting with previous studies in which sal-monellosis occurs in more than 25 % of patients (Picard et al.2002; Prando et al. 2013), only patient YLA developed resis-tant salmonella infection with different localizations. The useof broad-spectrum antibiotics for mycobacterial infectiontreatment may lead to underdiagnosed salmonella infection.Interestingly, two patients developed viral infections. Indeed,patient BBH experienced spontaneous fulminant varicellawhereas patient YLA apparently presented with HSE at theage of 8 months followed by varicella at the age of 16 months.The mechanism that accounts for increased susceptibility toviral infections in patients with IL-12p40 deficiency mayconsist of an impairment of either antiviral innate immunityor IL-12-dependent antiviral Th1-adaptive immunity (Torigoet al. 2000). Such susceptibility increases the potential riskassociated with the use of attenuated viral vaccines in suchpatients. Interestingly, both siblings from kindreds BH and LAhad a mycobacterial and/or viral infections but with contrast-ing severity. Indeed, unlike his brother who died from afulminant varicella zoster virus infection, patient FBHcontracted, at the same time, benign varicella. In addition,BCG infection was less severe and had a better outcome forpatients FBH and FLA than for their respective siblings. Thisindicates that even in related individuals with the same cellularand molecular defect, variation in severity and outcome exists,presumably due to other compensatory immunological fac-tors. These factors are not yet fully understood.

The immunological phenotype of IL-12p40-deficient pa-tients is characterized by an impaired secretion of IL-12p40.The IL-12 and IFN-γ responses were assessed in whole bloodcells from 3 IL-12p40-deficient patients belonging to kindreds

Fig. 1 Pedigrees of Tunisian kindreds with IL-12p40 deficiency. Eachkindred is designated by initials of the family's name. Each generation isdesignated by a roman numeral (numerals I–II)

Immunogenetics

LA and GA and 20 Tunisian healthy controls. Patient's cellsstimulated with live BCG alone or BCG plus exogenousIFN-γ produced no IL-12p40, whereas IL-12p40 was pro-duced by the cells of controls (p <0.001) (Fig. 2a). IFN-γproduction was enhanced when cells were added with recom-binant IL-12 to live BCG, reflecting the response of bloodcells to exogenous IL-12 added to the medium. Nevertheless,the level of IFN-γ induced in patients was lower than thatobtained in healthy controls (p <0.05) (Fig. 2b). In contrast toGMA, patients FLA and YLA show detectable levels ofIFN-γ in the supernatant of unstimulated cells or upon BCGstimulation; they probably received recombinant IFN-γtreatment.

Sequencing analysis of IL12B revealed the presence of thesame 8-bp deletion in all the patients (Fig. 3). This mutationdesignated c.298_305del (also known as 297del8) occurs inexon 3 and leads to a frameshift that results in a premature stopcodon at nucleotide position 342. The parents and unaffectedsiblings were heterozygous for the c.298_305del deletion.This mutation was not detected in 50 unrelated healthy Tuni-sian individuals (data not shown). Interestingly, two otherTunisian patients living in France had also the same deletion,supporting evidence that founder effect accounts for the re-currence of this mutation (Prando et al. 2013).

Segregating analysis using eight polymorphic markersoverlapping the IL12B gene and spanning 10 Mb on chromo-some 5 revealed the presence of three different haplotypes. A6.5-Mb homozygous haplotype defined by six microsatellites(D5S410, D5S662, D5S412, D5S2038, D5S403, andD5S529) is shared by patients from kindreds BH and LA.Five patients from kindreds BH, LA, and KO share a 6-Mbcommon homozygous haplotype defined by fivemicrosatellites (D5S410, D5S662, D5S412, D5S2038, andD5S403). These two haplotypes include the complete IL12Bgene. All affected siblings share a 5-Mb common haplotype

defined by four microsatellites (D5S410, D5S662, D5S412,and D5S2038) (Fig. 4). Using the Estiage likelihood method(Genin et al. 2004), which relies on the conserved length ofhaplotypes carrying the mutation, we dated the founder eventresponsible for the c.298_305del mutation. Only the probandsof each kindred were analyzed. Three c.298_305del haplo-types were thus used to estimate the age of the disease muta-tion. Recombination between microsatellites was retrievedfrom deCODE genetic map (Kong et al. 2002) and interpolat-ed from physical distances by a local linear regression, whenrequired. Allele frequencies at microsatellites were retrievedfrom the uniSTS database. A stepwise mutation rate at 10−3

mutations per site per generation (Brinkmann et al. 1998) wasused to model mutations of microsatellites. The estimatednumber of generations since the appearance of the common

Fig. 3 Genetic analysis of IL12B gene. The underlined nucleotidescorrespond to the 8-bp deletion within the IL12B gene. The position ofthe c.298_305del mutation is indicated by an arrow

Fig. 2 Impaired cellular response in IL-12p40-deficient patient. Cyto-kine production in the supernatants of whole blood cells from controls(circles) and three IL-12p40-deficient patients, FLA, YLA, and MGA(squares), unstimulated (NS) or stimulated by BCG alone, BCG plus

IFN-γ, or BCG plus IL-12p70 as detected by ELISA. The horizontalbars indicate the mean. The p value for nonparametric Mann–Whitneytest comparing patients and controls was indicated. **p<0.01, *p <0.05.a IL-12p40 production (pg/ml). b IFN-γ production (pg/ml)

Immunogenetics

ancestor is 44 (95 % confidence interval (CI), 12–86). If it isassumed that a generation is ~25 years, then the c.298_305delmutation in IL12B is ~1,100 years old (95 % CI 300–2,150).

IL-12B genetic defects are rare and have been reportedexclusively in countries where consanguinity is high andBCG vaccination is universal. For severe diseases, inbreedingover several generations may lead to the elimination of dele-terious recessive mutations from the population gene pool(Khoury et al. 1987). Thus, IL12B mutant alleles are probablysubjected to strong negative selection. However, in ethnicalisolates with very high inbreeding level, a limited number ofvariants may be maintained at very low frequency. Indeed,nine mutant alleles only have been identified worldwide in theIL12B gene, and three where shown to be recurrent mutationsdue to founder events in specific populations (Picard et al.2002; Prando et al. 2013). Herein, we have shown that thec.298_305del mutant allele observed in patients originatingfrom the same small town in central Tunisia is also due to afounder effect. Consanguinity is a hallmark of Tunisian aswell as other North African and Middle Eastern populationsreaching more than 50 % in some areas (Tadmouri et al.2009). It has been shown in Tunisian patients with variousgenetic disorders that founder effects are present in 42 % ofthose with identified molecular defect (Romdhane andAbdelhak 2011). In PIDs as well as in other genetic diseasesdescribed in Tunisia, founder mutations can be divided intotwo different categories. The first includes founder mutationsthat occur among Tunisians but are also shared by other NorthAfrican populations. This is, for instance, the case of majorhistocompatibility complex (MHC) class II combined immu-nodeficiency (Ben-Mustapha et al. 2013; Ouederni et al.2011). The second group represents founder haplotypes sofar reported only among Tunisians. This is the case of MSMDdue to IL12B gene mutation which is restricted to a smallvillage in central Tunisia (Akouda). Founder IFNGR1 andCD18 gene mutations have also been found specifically inTunisian patients (Ben-Mustapha et al., unpublished data).However, as these diseases have not yet been investigated in

neighboring countries, specificity of such mutations to Tuni-sian population should be taken with caution.

In summary, herein we present strong evidence to supportthat mutation IL12B c.298_305del is inherited as a commonfounder mutation arising ~1,100 years ago (95 % CI 300–2,150). The identification of a unique mutation helped, in ourresource-limited settings, the establishment of a preventiveapproach by genetic counseling and prenatal diagnosis inaffected families.

Acknowledgments This work has been supported partially by theTunisian Ministry of Higher Education, Research and Technology (grantto the LR11IPT02). The authors would like to thank Beya Larguèche forthe technical assistance and all patients and their families for their kindcooperation.

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