A 475 Years-Old Founder Effect Involving IL12RB1: a Highly Prevalent Mutation Conferring Mendelian Susceptibility to Mycobacterial Diseases in European Descendants
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Infection, Genetics and Evolution 9 (2009) 574–580 Contents lists available at ScienceDirect Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid A 475 years-old founder effect involving IL12RB1: A highly prevalent mutation conferring Mendelian Susceptibility to Mycobacterial Diseases in European descendants J. Yancoski a, C. Rocco a, A. Bernasconi a, M. Oleastro a, L. Bezrodnik b, C. Vra´tnica c, F. Haerynck d, S.D. Rosenzweig a,* a Hospital Nacional de Pediatrı´a J. P. Garrahan, Buenos Aires, Argentina b Hospital de Nin˜os Ricardo Gutierrez, Buenos Aires, Argentina c Hospital de Nin˜os Juan Pablo II, Corrientes, Argentina d Ghent University Hospital, Ghent, Belgium ARTICLE INFO ABSTRACT Article history: Mutations in IFNGR1, IFNGR2, IL12RB1, IL12B, STAT1 and NEMO result in a common clinical phenotype Received 11 December 2008 known as Mendelian Susceptibility to Mycobacterial Diseases (MSMD). Interleukin-12 receptor b1 (IL- Received in revised form 13 February 2009 12Rb1) deficiency is the most common genetic etiology for MSMD. Known mutations affecting IL12RB1 Accepted 16 February 2009 are recessively inherited and are associated with null response to both IL-12 and IL-23. Mutation IL12RB1 Available online 9 March 2009 1623_1624delinsTT was originally described in 5 families from European origin (2 from Germany; 1 from Cyprus, France and Belgium). Interestingly, this same mutation was found in an unexpectedly high Keywords: prevalence among IL-12Rb1 deficient patients in Argentina: 5-out-of-6 individuals born to unrelated Hot spot families carried this particular change. To determine whether mutation 1623_1624delinsTT represents a Salmonella Bacillus Calmette Guerin DNA mutational hotspot or a founder effect, 34 polymorphic markers internal or proximal to IL12RB1 Interferon gamma were studied in the Argentinean and the Belgian patients. A common haplotype spanning 1.45–3.51 Mb Linkage disequilibrium was shared by all chromosomes carrying mutation 1623_1624delinsTT, and was not detected on 100 control chromosomes. Applying a modified likelihood-based method the age of the most recent common ancestor carrying mutation 1623_1624delinsTT was estimated in 475 years (95% CI, 175–1275), which is the time when the Spaniards initiated the colonization of the Americas. Mutation 1623_1624delinsTT represents the first founder effect described on IL-12Rb1, the most frequently affected gene in MSMD, and affecting patients with European ancestors. The reason(s) behind the persistency of this mutation across multiple generations, its relative high prevalence, and any potential selective advantage are yet to be established. Published by Elsevier B.V. 1. Introduction Mutations on the IL12RB1 gene are associated with impaired response to IL-12 and IL-23 (Altare et al., 1998; de Jong et al., 1998; With more than 100 affected patients worldwide, IL-12Rb1 Hoeve et al., 2003). With only one exception, all IL-12Rb1 deficient deficiency is the most common genetic etiology for Mendelian patients described displayed no detectable IL-12Rb1 on their cell susceptibility to Mycobacterial Disease (MSMD). Interleukin-12 surface due to mutations that either interrupt the open reading receptorb1 deficiency causes an illness characterized by a selective frame (nonsense and frameshift mutations) or disrupt folding of susceptibility to poorly pathogenic mycobacteria, and nontyphoid the protein (missense mutations) (Altare et al., 1998; de Jong et al., Salmonella species attesting to the importance of IL-12R in the host 1998; Fieschi et al., 2003; Fieschi et al., 2004; van de Vosse and defense against intracellular pathogens (Altare et al., 1998; de Jong Ottenhoff, 2006; Filipe-Santos et al., 2006). Complex mutation et al., 1998; Fieschi et al., 2003; van de Vosse and Ottenhoff, 2006; 1623_1624delinsTT (GC deletion, TT insertion) results in a silent Filipe-Santos et al., 2006; Rosenzweig et al., 2006). point mutation (V541 V) and the generation of a stop codon (Q542X) in IL12RB. This particular change had been previously described in 5 families from European origin (2 from Germany; and 1 from Cyprus, France and Belgium) (Fieschi et al., 2003; Haerynck * Corresponding author at: Servicio de Inmunologı´a, Hospital Nacional de Pediatrı´a J. P. Garrahan, Combate de los Pozos 1881, Cdad. de Buenos Aires (1245), et al., 2008). Unexpectedly, mutation 1623_1624delinsTT was Argentina. Tel.: +54 9 11 5408 2641; fax: +54 11 4308 4076. found in 5-out-of-6 Argentinean IL-12Rb1 deficient patients (3 E-mail address: [email protected] (S.D. Rosenzweig). homozygous and 2 heterozygous) from 6 unrelated families. 1567-1348/$ – see front matter. Published by Elsevier B.V. doi:10.1016/j.meegid.2009.02.010 J. Yancoski et al. / Infection, Genetics and Evolution 9 (2009) 574–580 575 The finding of identical mutations in apparently unrelated 2.2. Control population patients from different regions in the planet suggested that recurrent mutations could arise (mutational hot spot). Alterna- Genomic DNA was extracted from 50 unrelated healthy tively, it was possible that despite the geographic separation the Argentinean blood donors of European-descent (100 chromo- families shared a common ancestry (mutational founder effect). somes). Samples were screened for SNPs G451C and A705G by Herein, these two alternative hypotheses are explored. restriction fragment length polymorphism (RFLP) analysis and/or direct sequencing. For SNP A705G allele frequency determination, 2. Patients, materials and methods exon 7 IL12RB1 was PCR-amplified with specific primers, the obtained product was PvuII-digested according to manufacturer’s 2.1. Patient cohort recommendations (New England Biolabs, Ipswish, MA), resolved in 2% agarose gels, stained with ethidium bromide and analyzed Six patients with complete IL-12Rb1 deficiency and born to 6 under UV light. Digestion with PvuII in the presence of allele A705 unrelated non-consanguineous Argentinean families were generates 2 fragments of 107 and 91 bp and ruled out the presence referred to the Hospital Nacional de Pediatrı´a J. P. Garrahan in of the mutated haplotype. For G451C allele frequency determina- Buenos Aires, Argentina and evaluated for this study. All the tion, exon 4 IL12Rb1 was PCR-amplified with specific primers, the patients had been assessed for IFNg/IL-12/IL-23 pathway obtained product was digested with restriction enzyme HphI (New integrity after they developed disseminated mycobacterial England Biolabs, Ipswish, MA), PAGE resolved in NOVEX 20% TBE infections early on life. All the patients developed disseminated gels (Invitrogen, Carlsbad, CA), stained with ethidium bromide and M. bovis—BCG infection after routine BCG vaccination. None of the analyzed under UV light. Digestion with HphI generates a set of patients expressed IL-12Rb1 on the surface of their T cells or NK four fragments (320, 172, 112 and 36 bp) in the presence of allele cells as determined by flow cytometry and their lymphocytes did 451C, while a different set of fragments (284, 226, 94 and 36 bp) is not respond to IL-12 stimulation ex vivo. All the patients were observed in the presence of allele G451. Homozygosity for G451 screened for mutations on IL-12Rb1 by PCR amplification of ruled out the presence of the mutated haplotype. Six normal genomic DNA (17 exons and exon/intron boundaries), followed by controls (12 chromosomes) were tested by direct sequencing of the direct sequencing of the PCR products. Among this cohort, entire coding region of IL12Rb1 gene. mutation 1623_1624delinsTT was found in 5 affected individuals, All patients and controls gave written consent for DNA/RNA all of them born to European ancestors. Three patients were extraction and analysis. Primer sequences are shown in the homozygous and 2 heterozygous for this trait. Another patient, Appendix A, Table A1. Belgium-born and heterozygous for the same mutation, was also included in this study. 2.3. Mutation 1623_1624delinsTT age estimation The patients’ ancestry was established by direct questioning for at least 4 generations in each affected family with no evidence of In order to estimate the age to the most recent common consanguinity. However, encrypted relatedness could not be ruled ancestor of haplotypes carrying the 1623_1624delinsTT mutation, out completely. a previously described likelihood method was modified and Thirty-four polymorphic sites on chromosome 19 were applied on a set of six recombination markers located at variant analyzed in these patients: 21 intragenic IL12RB1 (15 exonic, 6 distances on both sides of the disease mutation (Genin et al., 2004). intronic) and 13 extrageneic. All markers were tested on gDNA. In The approximation was introduced by Genin and collaborators as addition, IL-12Rb1 mRNA was extracted, cDNA converted, PCR an interval censored survival analysis in which the mutation locus, amplified, subcloned and sequenced in heterozygous patients for denoted as D, is the starting point, the genetic distance is the time individual-allele IL12RB1-exonic polymorphic markers determina- scale and the occurrence of recombination is the event. The model tion. takes the following basic assumptions: Genomic DNA was obtained by saline extraction from PBMCs. Single nucleotide polymorphisms’ genotyping was performed with monophyletic origin of the disease mutation DYEnamic ET Terminator Cycle Sequencing Kit (Amersham independence of each sampled chromosome and equal time to a Biosciences, Little Chalfont Buckinghamshire, UK) from PCR common ancestor products