Eva Rudd

Spectrum and clinical implications of syntaxin 11 gene mutations in familial haemophagocytic lymphohistiocytosis: association with disease-free remissions and haematopoietic malignancies

Objective: To determine the frequency and spectrum of mutations in the gene encoding syntaxin 11 (STX11) in familial haemophagocytic lymphohistiocytosis (FHL), a rare autosomal recessive disorder of immune dysregulation characterised by a defect in natural killer cell function. Methods: Mutational analysis of STX11 by direct sequencing was done in 28 FHL families that did not harbour perforin mutations, previously identified in some FHL patients. A detailed investigation of clinical features of these patients was also undertaken. Results: Two different STX11 mutations were identified, one nonsense mutation and one deletion, affecting six of 34 children in four of 28 unrelated PRF1 negative families. Both mutations have been reported before. Three patients experienced long periods (⩾1 year) in remission without specific treatment, which is very uncommon in this disease. Despite the milder phenotype, some children with STX11 mutations developed severe psychomotor retardation. Two of the six patients harbouring STX11 gene defects developed myelodysplastic syndrome (MDS) or acute myelogenous leukaemia (AML). Conclusions:STX11 gene mutations were found in 14% of the PRF1 negative FHL families included in the present cohort. These results suggest that STX11 gene mutations may be associated with secondary malignancies (MDS/AML), and that there is segregation of specific clinical features in FHL patients with an underlying genotype.

Clinical presentation of Griscelli syndrome type 2 and spectrum of RAB27A mutations.

Griscelli syndrome type 2 (GS2) is an autosomal‐recessive immunodeficiency caused by mutations in RAB27A, clinically characterized by partial albinism and haemophagocytic lymphohistocytosis (HLH). We evaluated the frequency of RAB27A mutations in 21 unrelated patients with haemophagocytic syndromes without mutations in familial HLH (FHL) causing genes or an established diagnosis of GS2. In addition, we report three patients with known GS2. Moreover, neurological involvement and RAB27A mutations in previously published patients with genetically verified GS2 are reviewed.

Characterization of PRF1, STX11 and UNC13D genotype-phenotype correlations in familial hemophagocytic lymphohistiocytosis

Familial hemophagocytic lymphohistiocytosis (FHL) is a rare autosomal recessive lethal condition characterized by fever, cytopenia, hepatosplenomegaly and hemophagocytosis. The hallmark of FHL is defect apoptosis triggering and lymphocyte cellular cytotoxicity. Thus far three disease‐causing genes (PRF1, UNC13D, STX11) have been identified. We performed a genotype‐phenotype study in a large, multi‐ethnic cohort of 76 FHL patients originating from 65 unrelated families. Biallelic mutations in PRF1, UNC13D and STX11 were demonstrated in 13/74 (18%), 6/61 (10%) and 14/70 (20%) patients, respectively. In 27/60 (45%) patients analyzed for all three genes, no molecular diagnosis was established. STX11 mutations were most common in Turkish families (7/28, 25%), whereas in Middle East families, PRF1 mutations were most frequent (6/13, 46%). No biallelic mutation was identified in most families of Nordic origin (13/14, 93%). Patients carrying PRF1 mutations had higher risk of early onset (age <6 months) compared to patients carrying STX11 mutations [adjusted odds ratio 8·23 (95% confidence interval [CI] = 1·20–56·40), P = 0·032]. Moreover, patients without identified mutations had increased risk of pathological cerebrospinal fluid (CSF) at diagnosis compared to patients with STX11 mutations [adjusted odds ratio 26·37 (CI = 1·90–366·82), P = 0·015]. These results indicate that the disease‐causing mutations in FHL have different phenotypes with regard to ethnic origin, age at onset, and pathological CSF at diagnosis.

Syntaxin 11 marks a distinct intracellular compartment recruited to the immunological synapse of NK cells to colocalize with cytotoxic granules

The syntaxin 11 (STX11) gene is mutated in a proportion of patients with familial haemophagocytic lymphohistiocytosis (FHL) and exocytosis of cytotoxic granules is impaired in STX11‐deficient NK cells. However, the subcellular localization, regulation of expression and molecular function of STX11 in NK cells and other cytotoxic lymphocytes remain unknown. Here we demonstrate that STX11 expression is strictly controlled by several mechanisms in a cell‐type‐specific manner and that the enzymatic activity of the proteasome is required for STX11 expression in NK cells. In resting NKL cells, STX11 was localized in the cation‐dependent mannose‐6‐phosphate receptor (CD‐M6PR)‐containing compartment, which was clearly distinct from cytotoxic granules or Rab27a‐expressing vesicles. These subcellular structures appeared to fuse at the contact area with NK‐sensitive target cells as demonstrated by partial colocalization of STX11 with perforin and Rab27a. Although STX11‐deficent allo‐specific cytotoxic T‐lymphocytes efficiently lysed target cells and released cytotoxic granules, they exhibited a significantly lower extent of spontaneous association of perforin with Rab27a as compared with STX11‐expressing T cells. Thus, our results suggest that STX11 promotes the fusion of Rab27a‐expressing vesicles with cytotoxic granules and reveal an additional level of complexity in the spatial/temporal segregation of subcellular structures participating in the process of granule‐mediated cytotoxicity.

Fatal hemophagocytic lymphohistiocytosis in X-linked chronic granulomatous disease associated with a perforin gene variant

A patient with previously unrecognized X‐linked chronic granulomatous disease (X‐CGD) died of multi‐organ failure, secondary to ongoing infection and hemophagocytic lymphohistiocytosis (HLH). Post mortem histological investigations were compatible with X‐CGD, and a CYBB gene mutation was confirmed. No homozygous mutations in the genes encoding perforin (PRF1), MUNC 13‐4 or syntaxin‐11 (STX11) were found; however, there was a heterozygous alteration c.1471G>A in the PRF1 gene causing a p.Asp491Asn substitution. Although this substitution has not been reported to cause primary or secondary HLH, we speculate that it may have made the patient more susceptible for HLH under the circumstances of ongoing infection associated with X‐CGD. Pediatr Blood Cancer 2009;52:527–529.

Severe bacteria‐associated hemophagocytic lymphohistiocytosis in an extremely premature infant

Hemophagocytic lymphohistiocytosis (HLH) is a rare condition with high mortality. We report an extremely premature girl, born in the 24th gestational week (BW 732 g), that during her second month developed a severe HLH subsequent to a Serratia marcescens septicemia, with hepatosplenomegaly, cytopenias, hyperbilirubinemia (mostly conjugated, total bilirubin 916 μmol/L), hypertriglyceridemia, hypofibrinogenemia, hyperferritinemia (21266 μg/L), and elevated sIL‐2 receptor levels. Genetic analysis revealed no PRF1, STX11 or UNC13D gene mutations. Treatment was provided according to the HLH‐2004 protocol with etoposide, dexamethasone, and immunoglobulin, but no cyclosporin because of immature kidneys. She recovered fully from the HLH but developed a severe retinopathy as well as green teeth secondary to the hyperbilirubinemia. We conclude that secondary, bacteria‐associated HLH can develop in premature infants, and that HLH can be treated with cytotoxic therapy also in premature infants. It is important to be aware of HLH in premature infants, since it is treatable.

Multiple inherited sequence variations in two disease-causing genes in familial haemophagocytic lymphohistiocytosis.

Familial haemophagocytic lymphohistiocytosis (FHL) is a rare childhood disease of autosomal recessive inheritance characterized by a pronounced deregulation of immune homeostasis (Henter et al, 1998). FHL is genetically heterogeneous, and four different disease loci have been identified to date: 9q21.222 (FHL1), 10q21 (FHL2), 17q25 (FHL3), and 6q24 (FHL4). While the gene defect responsible for FHL1 remains unknown, mutations in the perforin gene (PRF1) are found in FHL2 patients (Stepp et al, 1999) and UNC13D (Munc13-4) mutations result in FHL3 (Feldmann et al, 2003). In addition, mutations in the gene encoding syntaxin-11 (STX11) were identified in FHL4 patients (zur Stadt et al, 2005). Perforin is a major constituent of natural killer (NK) cell and cytotoxic T lymphocyte (CTL) granules, whereas Munc134, a SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-regulatory protein, is required for priming of perforin-containing granules for membrane fusion and exocytosis. Syntaxin-11 is also expressed in cytotoxic cells, and a degranulation defect has recently been demonstrated in FHL4 patients (Bryceson et al, 2007). Here we report two cases of FHL with central nervous system (CNS) involvement and concurrent inheritance of homozygous mutations in two different disease-causing genes (UNC13D and STX11). The index case is a girl, born to consanguineous parents of Turkish origin. She is the second of four children; there are two healthy sisters, aged 6 and 10 years, respectively, at the time of writing of this report, and a younger brother who has also been diagnosed with FHL. At 5 months of age, she presented with typical clinical features and laboratory signs of FHL. At the time of diagnosis, she also suffered from psychomotor retardation and an inability to control her head movements. Treatment with the haemophagocytic lymphohistiocytosis (HLH)-94 protocol (Henter et al, 2002) was initiated and the clinical HLH findings resolved, but the psychomotor and growth retardation persisted. At 14 months of age, the patient developed a fever and died in her home. Her brother was admitted at 5 months of age, at which time he was unable to control his head movements. He had low NK cell activity and HLH-94 treatment was initiated. He improved and underwent stem cell transplantation (SCT) from his younger sister, and at the time of writing he is 5 years old. He currently has no clinical signs of HLH; however, his growth, mental, and motor retardation continues. We have previously reported that our patients both carry a homozygous deletion of 2 + 3 base-pairs (c.369_370delAG;c.374_376delCGC) in the STX11 gene (Rudd et al, 2006). The mutation was inherited from the parents (Fig 1) and causes a frame-shift at position V124 and a premature stop codon (zur Stadt et al, 2005). The oldest sister harboured a wild-type sequence in this position, and the other healthy sibling carried the STX11 mutation in heterozygous form. Our previous studies have suggested that patients with STX11 mutations have milder clinical features when compared to other FHL patients, with long periods of disease-free remission without specific treatment (Rudd et al, 2006). Moreover, our recent genotype–phenotype studies have shown that neurological symptoms are less common at onset of disease in FHL patients with STX11 mutations (Horne et al, 2008). On the other hand, other investigators have reported that CNS disease is a major feature of FHL3 patients with UNC13D mutations (Santoro et al, 2006). Our index patient suffered from severe psychomotor retardation, and the more severe phenotype in this patient and in her brother compared to other patients with the same STX11 mutation therefore prompted us to sequence other genes. We found two homozygous sequence variations in the UNC13D gene in our patients (Fig 1). The first variation was a point mutation in exon 3 (175G fi A) causing a change from Alanine to Threonine at position 59 (A59T). The mutation was inherited from the parents, and was also present in heterozygous form in the oldest sister, and in homozygous form in the other healthy sister (Fig 1). In addition, one out of 50 healthy controls carried the same sequence variation in heterozygous form (data not shown). This sequence variation has been described previously in FHL3 patients (Santoro et al, 2006), and does not fall within any of the known functional domains of the Munc13-4 protein (Rudd et al, 2008). The second variation was a point mutation in exon 13 (1061G fi T) causing a change from Tryptophan to Leucine at position 354 (W354L) and has not previously been reported. The mutation was inherited from the parents, and was also present in heterozygous form in the oldest sister, and in homozygous form in the other healthy sister (Fig 1). This mutation occurs in a region between the Munc-homology domain 1 and the Caand phospholipid-binding domain that is required for the interaction of Munc13-4 with Rab27a, and was not found in 50 healthy controls (data not shown). UNC13D mutations were not identified in other patients with STX11 mutations and milder clinical features (patient histories described in Rudd et al, 2006) (data not shown). correspondence

Sequence analysis of the SRGN, AP3B1, ARF6, and SH2D1A genes in familial hemophagocytic lymphohistiocytosis

In the present study, DNA sequencing of the genes SRGN, ARF6, AP3B1, and SH2D1A was performed in a well defined cohort of 18 families with familial hemophagocytic lymphohistiocytosis (FHL). A heterozygous nucleotide change (C > T) in the 3′untranslated region of the SRGN gene and a monoallelic 3‐base pair deletion (c.2409_2411delGAA) in exon 21 of the AP3B1 gene were observed in two different families. Additionally, two novel polymorphisms, one in intron 17 of AP3B1 and another in intron 2 of SH2D1A were identified. We conclude that mutations in SRGN, ARF6, and AP3B1 are not likely to be common in patients fulfilling the FHL criteria. Pediatr Blood Cancer 2008;50:1067–1069.