Familial hemophagocytic Lymphohistiocytosis (FLH) is a rare disorder characterized by fever, hepatosplenomegaly, cytopenia, hypertriglyceridemia, hypofibrinogenemia and reduced natural killer (NK) cell activity. Several genetic defects have been recognized as associated with FHL. After identification of perforin gene (PRF1) mutation as the cause of about one third of cases of FHL (Stepp,1999), in 2003 the same group reported that another gene involved in cellular cytotoxicity, Munc13-4, encoding for a protein involved in granule release in cytotoxic lymphocyte, is associated with a subset of patients with FHL. We have analyzed for Munc13-4 mutations 16 families of patients diagnosed with FHL according to current criteria, in whom PRF1 mutations had been excluded; 14 were Italian, 1 from USA, one from UK. The 32 exons and their proximal flanking regions of Munc13-4 gene were amplified from genomic DNA and amplification products were directly sequenced by cycle sequencing approach (BigDye Terminator Applied Biosystems). Sequences of primers utilized for the amplification reactions were designed with dedicated software (Primer Express) according to the gene sequence retrived from Genebank. A total of 13 mutations were identified in 12 families; 3 had homozygous mutations, 5 had combined heterozygous mutations, and 4 had only one detectable mutated allele. Mutation 753+IG in exon 9 (splice donor site) had been already reported (Feldman 2003, zurStadt 2005); The following novel mutations were found: G175A in exon 3 (A59T), delC532 in exon 6 (178 fs), A610G in exon 7 (M204V), G1241T in exon 14 (R414L), A1847G in exon 20 (E616G), del GGAG 2346 (782 fs) in exon 24, A2599G (K867E) in exon 27, C2650T in exon 28 (Q884stop), C2782T in exon 29 (R928C), C2896T in exon 30 (R966W), 3082delC in exon 31 (1028fs), insG3226 in exon 32 (1076fs). Mutation A2599 was observed in 6 families; mutation A1847G in 3 families; C2782T and G1241T in 2 families. We have also identified the polymorphisms C279T, and A3198G. Mutations were scattered over different exons and almost entirely different from those reported so far. A2599G mutation was frequently observed and its role deserves further investigation. Screening of Munc13-4 mutations is a powerful tool to confirm the diagnosis, to refine the therapeutic choice including indication to HSCT, selection of familiar donor, and identification of carriers and prenatal diagnosis in most of the families with FHL not associated with PRF1 mutations. Due to lack of immunologic or flow-cytometric tools for rapid screening of Munc13-4 defective patients, the high number of coding exons and lack of mutation clustering, screening of Munc13-4 mutations, although expensive and time consuming, remains necessary and allowed us to identify the genetic defect in the majority of our patients with FHL not associated with PRF1 mutations.

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