Familial haemophagocytic lymphohistiocytosis (FHL) is a hyperinflammatory disorder that typically presents in infancy (Filipovich, 2011; Janka, 2012) with an estimated incidence of 1 in 50 000 live births (Henter et al, 1991). Chemo- and immunotherapy can control the disease, but haematopoietic stem cell transplantation (HSCT) is the only cure. FHL is associated with autosomal recessive mutations in PRF1, UNC13D, STX11 and STXBP2 (Pachlopnik Schmid et al, 2010). In this study, we report two novel mutations in UNC13D, one of which is located in intron 1 of the gene, adjacent to a previously reported intronic mutation (c.118-308C > T) causative of FHL3 in a number of patients world-wide (Meeths et al, 2011; Seo et al, 2013).
The index patient (II-3) was a previously healthy 8-year old boy, the third child of non-consanguineous Chinese parents from Singapore. Patients and methods are described in detail in the Supporting Information (Data S1). To determine whether the patient, who rapidly developed haemophagocytic lymphohistiocytosis (HLH), might represent a case of FHL, the phenotype of cytotoxic lymphocytes was examined. This was done on the patient, the parents (father I-1; mother I-2), two brothers (II-1 and II-2), as well as transport- and local controls 2 months after the clinical diagnosis of HLH. In this setting, where samples were transported far afield for functional analyses, conventional natural killer (NK) cell cytotoxicity and degranulation assays using K562 target cells (Bryceson et al, 2012) were difficult to interpret. However, assays quantifying degranulation triggered by engagement of the T cell receptor (CD3) on cytotoxic T cells or the Fc receptor (CD16) on NK cells were more robust (Chiang et al, 2013). Whereas parental and transport controls displayed normal degranulation, the patient (II-3) and remarkably also the middle brother (II-2) displayed defective cytotoxic T cell and NK cell-mediated degranulation in response to anti-CD3 or anti-CD16, respectively (Fig 1A). Upon stimulation with interleukin 2, NK cell and T cell degranulation increased in the two affected brothers (Fig 1B). Thus, the results demonstrate how new read-outs of cytotoxic lymphocyte degranulation excel in the identification of patients with defective cytotoxic lymphocyte function.
Based on the functional results, the FHL genes required for lytic granule exocytosis (UNC13D, STX11, and STXBP2) were bi-directionally sequenced in genomic DNA from the patient. A novel heterozygote UNC13D c.1388A > C; p.Gln463Pro missense mutation in the second last base of exon 15 was identified (Fig 2A, B). Furthermore, sequencing of intron 1 of UNC13D revealed a heterozygous c.118-307G > A mutation (Fig 2A, B), located to an evolutionary conserved region. The oldest brother (II-1) was wild-type for the exon 15 mutation but carried the intron 1 mutation. Importantly, both mutations were also identified in brother II-2, with defective degranulation and cytotoxicity. Neither of the mutations was identified in the Single Nucleotide Polymorphism database, 1000 genomes or in any of the 96 healthy blood donors used as controls.
The p.Gln463Pro amino acid change was not predicted to be damaging with the mutation prediction software Alamut. With the same prediction software, the donor splice site affected by the c.1388A > C mutation was predicted to be functional, albeit with reduced affinity. Analysis of cDNA from the family members carrying the heterozygous mutations revealed normal size and sequence. Notably, in the two affected brothers the mutated c.1388C allele was predominantly amplified, suggesting that transcription of the other allele carrying the intronic c.118-307G > A mutation might be impaired. Thus, allele-specific quantitative real-time polymerase chain reaction (PCR) was performed on the family members carrying this mutation. In exon 11 of UNC13D, a common synonymous polymorphism (c.888G > C) was used to discriminate between the mutated (linked to the c.888G allele) and wild-type allele (linked to the c.888C allele) in carriers of the c.118-307G > A mutation. For comparison, allele-specific transcription was quantified in healthy controls that did not carry the c.118-307G > A mutation but were heterozygous for the c.888G > C polymorphism. In the heterozygous carriers (I-2, II-1, II-2 and II-3) of the c.118-307G > A mutation, transcription of the allele carrying the linked c.888G polymorphism was significantly reduced, representing 10% of total UNC13D transcripts (P = 0·021) (Fig 2C). These data indicate that the c.118-307G > A mutation impairs UNC13D transcription, possibly by disrupting a transcription factor binding-site or enhancer element.
The very low abundance of the transcript with the c.118-307G > A mutation implied that predominantly the transcript with the p.Gln463Pro missense mutation would be translated. To compare expression of mutated and wild-type UNC13D (Munc13-4), Western blot analysis was performed. Whereas UNC13D was clearly present in the parents and brother II-1, its expression was severely reduced in LAK cells from the brother with compound heterozygous mutations in UNC13D (Fig 2D), indicating that the mutated UNC13D was degraded. Expression of STXBP2 (Munc18-2), the protein encoded by STXBP2, was however not affected by the UNC13D mutations. Although the glutamine residue at position 463 is weakly conserved among species (Figure S1), the amino acid change to proline appears to reduce the protein stability. Introduction of proline residues in proteins is often damaging due to imposed structural constraints of the pyrrolidine ring (Pakula & Sauer, 1989). The combination of defective transcription from one UNC13D allele with reduced protein stability encoded by the other allele is a probable cause of the defective cytotoxic lymphocyte function in the two brothers in this study.
At the time of writing, the affected brother (II-2) is 13 years old, Epstein-Barr virus (EBV) IgG-seropositive, IgM-seronegative, EBV-PCR negative, and still asymptomatic. The possible causes of this variable phenotypic expression within the family are not clear. Environmental factors or modifier genes could be involved. Our results imply that a thorough functional evaluation of HLH patients as well as sibling is of great importance since they may be genetically affected and thus have a risk of developing disease late but with rapid-onset and high mortality. There are several arguments in favour of transplanting genetically affected asymptomatic individuals immediately, rather than waiting until they present with disease (Machaczka et al, 2012). However, this is a difficult decision. If affected individuals are not directly scheduled for HSCT, they should be monitored carefully and the identification of a suitable potential donor is of value. This study highlights the necessity of also examining non-coding evolutionary conserved sequences for identification of mutations in inherited haematological disorders, in order to give more patients a correct genetic diagnosis.