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  • Although ALPS type is very rare


    Although ALPS type 0 is very rare with few published cases, we herein report the clinical and immunogenetic characteristics of two additional unrelated patients from highly inbred North African population. Both patients present two rare splicing defects mechanisms in FAS gene inducing the skipping of exon 6, which encodes the transmembrane domain of CD95, thus precluding normal Fas expression.
    Materials and methods
    Discussion The two patients we report in the present study have developed the full clinical features of ALPS early in life beginning at 3days and 7months respectively. This suggested a severe phenotype compared to the majority of reported ALPS patients presenting a median age of symptom onset at 3years [16]. The clinical presentation and the biological characteristics including autoimmune cytopenias and important hypergammaglobulinemia prompted the determination of DNT NPS-2143 synthesis percentage which was found to be significantly high at 6,4% and 9,6% in patients 1 and 2 respectively. Although DNT cells have recently been demonstrated to be increased in a subset of pediatric patients with autoimmune diseases [17], they remain a reliable immunological marker of ALPS. Moreover, the two patients showed the concomitant increase in the plasma levels of IL-10 and sFasL as in virtually all ALPS patients [18]. The severe early clinical onset and the highly elevated ALPS markers were suggestive of a severe form of ALPS with the possibility of a defective Fas expression. Immunophenotyping on PBMCs showed an undetectable Fas surface expression in both patients in contrast to healthy controls and to patient 2 parents who did not show any clinical features. Stimulation of PBMCs did not induce Fas protein expression on generated PHA-blasts from T cells in patient 2 suggesting that Fas protein cannot be expressed even with strong stimuli in vitro. The Fas detected intracellularly in both patients corresponds presumably to the truncated form. This is supported by the presence in patient 1 immunoblot of the Fas truncated protein only, and by the absence of the full length Fas transcript in the two patients by RQ-PCR analysis experiments. In line with these findings, high levels of sFas were also detected in culture supernatants (EBV cell line) and in plasma from these patients. The clinical and immunological phenotype of both patients is consistent with the few described patients with homozygous FAS mutations [6], [12], [13], [14]. The first patient showed a homozygous mutation 16 nucleotides upstream of the 3′ acceptor splice site of intron 5 (c.506-16A>G). The mutation was predicted to break a potential branch point sequence (BPS). Indeed, it falls at position −16 in the presumed branch point consensus region extending from 5 to 50 nucleotides upstream of the 3′ end of an intron. The mutation affects a highly conserved adenosine “A” in a stretch of five nucleotides (CCAAT) with strong resemblance to the consensus BPS “yUnAy” [19]. Most splicing mutations affect the splice donor or acceptor sites, however few ones disrupt BPS inducing aberrant splicing [19]. The branch site A residue acts as the nucleophile in the first catalytic step of splicing [20] and is bound by the splicing factor 1 (SF1), also called branch point binding protein (BBP) [21]. A recent study confirms the major role for SF1 in early stages of the splicing reaction and its implication for efficient spliceosome assembly [22]. The mutation we report here could disrupt the BPS recognition by SF1 thus precluding correct splicing of pre-mRNA. Consistently, nucleotide substitution of the BP adenosine in the conserved BPS of yeast “UACUAAC”, which is also the optimal BPS for mammals [23], reduced the SF1 binding 25- fold [24]. All mutations that affect BPS typically lead to exon skipping [25], [26], intron retention [27] or the use of a cryptic 3′ splice site [28], [29], [30] and a majority affect the branch site A residue and induces mainly A-to-G mutations [19]. This is consistent with the mutation described here and supports the notion reported in a previous study that A-to-G transitions are more deleterious than A-to-T or A-to-C transversions. Indeed, the overrepresentation of disease-causing adenosine to guanosine BP substitutions observed in Mendelian disorders is due to a more profound defect of gene expression at the level of pre-mRNA splicing [31]. Therefore, the mutation c.506-16A>G could account for the severe splicing defect observed in the first patient and the complete absence of the Fas transcript encoding for the membrane isoform.