Familial hemophagocytic lymphohistiocytosis (FHL) is a rare autosomal recessive disorder characterized by hyperactive phagocytes and defects in natural killer cell function. It has been shown previously that mutations in the perforin 1 gene (PRF1) and in UNC13D are associated with FHL2 and FHL3, respectively, indicating genetic heterogeneity. We performed genome-wide homozygosity mapping in a large consanguineous Kurdish kindred with five children affected with FHL. Linkage to a 10 cM region on chromosome 6q24 between D6S1569 and D6S960 defined a novel FHL locus. By screening positional candidate genes, we identified a homozygous deletion of 5 bp in the syntaxin 11 gene (STX11) in this family. We could demonstrate that syntaxin 11 protein was absent in the mononuclear cell fraction of patients with the homozygous 5 bp deletion. In addition to this family, we found homozygous mutations in STX11 in five consanguineous Turkish/Kurdish FHL kindreds including two families with the 5 bp deletion, one family with a large 19.2 kb genomic deletion spanning the entire coding region of STX11 (exon 2) and two families with a nonsense mutation that leads to a premature stop codon in the C-terminal end of the protein. As both STX11 and UNC13D are involved in vesicle trafficking and membrane fusion, we conclude that, besides mutations in perforin 1, defects in the endocytotic or the exocytotic pathway may be a common mechanism in FHL.

INTRODUCTION

Familial hemophagocytic lymphohistiocytosis (FHL) is a genetically heterogeneous disorder typically first manifesting in early childhood with a rapidly fatal outcome if untreated (1). At diagnosis, patients present with a hyperinflammatory syndrome characterized by persistent fever, hepatosplenomegaly, cytopenia and, less frequently, central nervous system involvement. Common laboratory findings include low levels of fibrinogen and high concentrations of triglycerides, ferritin and the α-chain of the soluble interleukin 2 receptor (sCD25) secreted by activated T-cells. Defective natural killer (NK) cell activity is a hallmark of the disease (2). Hemophagocytosis in bone marrow, cerebrospinal fluid or lymph nodes by activated histiocytes may be absent initially but can be observed later in most of the cases. Chemoimmunotherapy based on corticosteroids, epipodophyllotoxins, cyclosporin A and antithymocyte globulins results in control of the disease in the majority of patients; however, remission is rarely sustained (3,4). Most patients suffer an early death unless treated by hematopoietic stem cell transplantation (HSCT), which is the only curative approach so far (5).

Linkage analysis in FHL patients has identified a genomic region with a defect in a still unknown gene on chromosome 9q21 (FHL1; MIM 603552) (6), and a defect in the perforin 1 gene (PRF1) on chromosome 10q21 (FHL2; MIM 603553) (7,8). In FHL2, perforin mutations lead to significant reduction or complete absence of protein synthesis resulting in a defective cytolytic activity of T-cells and NK cells (9) [reviewed by Arico et al. (10)]. The incidence and type of PRF1 mutations vary in different ethnic groups (1115). Very recently, FHL3 was identified on chromosome 17q25 with mutations in the gene UNC13D (16). The gene product of UNC13D, hMunc13-4, is a member of a protein family involved in vesicle priming, which prepares cytolytic granules at the plasma membrane for vesicle membrane fusion. Lymphocytes of FHL patients with UNC13D mutations were shown to lack the ability to deliver cytotoxic substances like perforin or granzyme B to the target cells. Both proteins, perforin and granzyme B, are directly involved in the cytotoxic response of the immune system to exo- or endogenous pathogens (17). It can be assumed that defects in other genes involved in the release of cytotoxic proteins could also lead to the clinical picture of FHL. In fact, two other related disorders characterized by hemophagocytosis and immune defects are the Chédiak–Higashi Syndrome (CHS) and the Griscelli-Syndrome (GS2) with defects in the genes CHS1 and RAB27A, respectively (18,19). Patients with these disorders have defective NK and/or T-cell cytotoxicity and, besides the overall secretion deficiency in immune cells, defects in the transport of pigment proteins that result in a partial albinism, not seen in the classical FHL forms (20). In all types of genetically based hemophagocytosis syndromes, the immune defect leads to the same severe clinical picture as in FHL. As all primary forms of FHL should rapidly undergo HSCT, early identification of the underlying genetic defect is crucial for patients without family history.

In the present study, we excluded mutations in PRF1 by genomic sequencing of the entire coding region, and performed a genome-wide homozygosity analysis in a large consanguineous FHL kindred of Kurdish descent. Linkage analysis led to identification of a novel FHL locus on chromosome 6q24, designated FHL4. By analyzing several genes residing in this region, we identified a homozygous 5 bp deletion in STX11 in all affected patients of this family. We could show that the 5 bp deletion, resulting in a premature stop codon, leads to abrogation of syntaxin 11 protein synthesis. We identified three different mutations in STX11 in a total of 10 patients from six different families with FHL, all of them with a common ethnic background.

RESULTS

Identification of FHL4 by linkage and homozygosity analysis

A genome-wide homozygosity analysis was performed with 19 members of the large consanguineous FHL family of Kurdish origin described earlier (family 1). By analyzing 380 markers, all known loci for FHL, namely FHL1, FHL2 (PRF1) and FHL3 (UNC13D) were clearly excluded. We obtained a maximum two-point LOD score of 4.89 at Θ=0.00 to D6S311 on chromosome 6q24 representing the only region with an LOD score significant for linkage (Table 1). We confirmed the localization by construction of likely haplotypes and demonstrated homozygosity in a 10 cM interval between D6S1569 and D6S960 in all affected individuals, and a heterozygous mutant haplotype in all obligate carriers (Fig. 1). A maximum multi-point LOD score of 4.94 was obtained between D6S1649 and D6S311. We have designated this novel locus for FHL FHL4.

Detection of homozygous mutations in STX11

The candidate genomic region is 11 Mb in length and harbors more than 50 different known or predicted genes (Fig. 2A). Among other candidates, we analyzed the gene encoding syntaxin 11 (STX11) as syntaxins are known for their function in vesicle transport. The STX11 gene consists of two exons and covers a genomic interval of 37 kb (Fig. 2B), of which exon 2 contains all of the coding sequence. We identified a homozygous deletion of two plus three nucleotides, c.369_370delAG and c.374_376delCGC, in exon 2 of STX11 (Fig. 2C). Both deletions completely cosegregated in the pedigree. The total deletion of five basepairs leads to a frameshift and a premature termination codon after 59 altered residues (Fig 2D). The homozygous mutation, Val124fsX60, was found in all affected children of the family, the heterozygous mutation was present in the obligate carriers. Neither the complete mutation nor the 2 bp or the 3 bp deletion alone was identified in samples from more than 200 control subjects of Kurdish or Turkish descent. Subsequently, additional FHL patients of Turkish or Kurdish descent without PRF1 mutations were analyzed for mutations in STX11. In two patients from different families of Kurdish origin (families 2 and 3, respectively), the homozygous mutation Val124fsX60 was also present (Table 2).

The analysis of marker loci around STX11, namely GATA129G07, D6S1569, D6S308, D6S971, D6S1649, GATA184A08 and D6S311, revealed the same mutant haplotypes in the patients from families 1, 2 and 3 in a 5.9 cM interval between D6S1569 and D6S311. As these families had the same ethnic background and originated from the north eastern part of Turkey, mutation Val124fsX60 probably had a single, common origin.

In family 4, mutations in PRF1 or UNC13D were excluded by direct sequencing of the entire coding regions. Amplification of genomic DNA from the patient yielded no product for exon 2 of STX11. Normal amplification was observed with DNA from other family members (father, mother and one sister; data not shown). To characterize a possible homozygous deletion within STX11, we established a semi-quantitative DHPLC assay to detect heterozygosity in obligate carriers. In a first approach, duplex PCR was performed with an interchromosomal control (exon 3 of VWF on chromosome 12) and fragment 1 of exon 2 (Ex2-1) of STX11. To detect differences in the amount of PCR product between individuals, reactions were stopped in the logarithmic phase after 27 cycles. DHPLC analysis revealed no STX11 PCR product in the sample from the patient and ∼50% DNA amount in parents when compared with controls (Fig. 3A). Amplification of the internal control was at comparable levels in all four samples.

Several fragments, each 300–400 bp in length, were analyzed in order to identify the deletion breakpoints. They covered a total of 37.6 kb genomic DNA from exon 2 and adjacent regions (Fig. 3B). Whereas all reactions from a control DNA resulted in expected products, reactions c–f from DNA of the patient failed, indicating a homozygous genomic deletion in this region (Fig. 3B, upper panel). PCR with primers flanking this interval and subsequent sequence analysis identified a breakpoint spanning fragment, which revealed a deletion of 19 189 bp including part of intron 1 and the entire coding region of STX11. The breakpoint was located between nucleotide 25560 and nucleotide 44750 of the corresponding genomic clone (Fig. 3B, lower panel). Both parents and the brother of the patient were heterozygous carriers of this deletion. In families 5 and 6, mutation screening revealed a C to T exchange at nucleotide 802 that leads to a premature termination codon at amino-acid 268 (Table 2). No STX11 mutation was detected in more than 30 FHL patients of non-Kurdish/Turkish origin.

Synthesis and distribution of syntaxin 11

The gene product of STX11, syntaxin 11, consists of 287 amino acid residues, completely encoded by exon 2. It has previously been demonstrated that STX11 is predominantly expressed in cells of the immune system, including thymus, spleen and lymph nodes (21), and is strongly associated with intracellular membranes (21). In control samples, we detected syntaxin 11 in the Triton X-100 soluble membrane fraction from monocytes but not from lymphocytes (Fig. 4A, left and middle panels). Syntaxin 11 was not detectable in any cytosolic fraction. In membrane fractions from B- or T-cell lines only weak or absent signals were found (Fig. 4A, right panel, lanes 2 and 3). Syntaxin 11 was clearly present in the myelo-monocytic cell line HL-60 (Fig. 4A, right panel, lane 1). In mononuclear cells (MNCs) from two FHL families with mutations in STX11 (Fig. 4B, panels 1 and 2), we could detect syntaxin 11 in membrane lysates from the heterozygous parents (samples M and F), as well as in lysates from healthy donors (HD). However, lysates prepared from patients (Pt) carrying the homozygous mutation in STX11 were entirely negative. As a control, we also investigated STX11 expression in a patient with GS2 (19) without mutations in either syntaxin 11 or perforin 1 (Fig. 4B, panel 3). This patient presented with a clinical picture of FHL and carried a 27.8 kb intragenic deletion in combination with an A87P missense mutation in RAB27A (zur Stadt, unpublished data). Moreover, we investigated SNAP23 (22), the syntaxin 11-binding synaptosomal-associated protein 23 that is essential for membrane fusion and protein transport (23). We found SNAP23 at comparable levels in all samples tested, regardless of whether the syntaxin 11 (or Rab-27A) mutation was present or not (Fig. 4B). Thus, our studies revealed that STX11 was expressed in peripheral MNCs from healthy controls, heterozygous parents and a patient with a related disease (i.e. Griscelli syndrome), but was negative in samples from individuals carrying the homozygous mutation. At least by western blot analysis, a consequence of Val124fsX60 on the expression of STX11 in heterozygous carriers could not be detected. Western blot analysis in families 3–6 was not feasible because of insufficient material. However, the genomic deletion of exon 2 in family 4 should obviously result in complete absence of the protein and the premature termination codon in families 5 and 6 should lead to a loss of regular protein function. These findings demonstrate that the mutations have a truly recessive effect and can be supposed to directly cause the lack of syntaxin 11 in type-4 FHL patients.

DISCUSSION

FHL is a severe disease of uncontrolled inflammation characterized by fever, hepatosplenomegaly, cytopenia, hemophagocytosis and characteristic laboratory values. Diagnostic guidelines were published by the Histiocyte Society in 1991 (24), and revised criteria were summarized in 2004 by Janka and Schneider (2). The uncontrolled activation of immune cells is thought to occur because of an incomplete eradication of infectious organisms or an inadequate response to endogenous factors like tissue damage, autoantigens or other stimuli. Nearly all FHL patients exhibit a complete deficiency of NK cell activity as measured in a standard 4 h chromium release assay against K562 cells (25).

Two different molecular defects have been identified earlier, indicating genetic heterogeneity. Mutations in PRF1 (FHL2) and UNC13D (FHL3) disrupt the ability of cytotoxic cells to kill their targets. Whereas mutations in PRF1 lead to reduced amounts or absent perforin 1 in cytotoxic granules, defects in UNC13D inhibit the priming step of these granules prior to vesicle membrane fusion.

In addition to mutations in PRF1 (FHL2) and UNC13D (FHL3), we here present a novel genetic defect in FHL, in STX11 on chromosome 6q24, which we identified after homozygosity mapping in a large Kurdish FHL family with altogether six affected individuals. The homozygous mutation, Val124fsX60, found in all five investigated patients from this family was predicted to result in a premature termination codon leading to complete loss of syntaxin 11. We further detected the 5 bp deletion, a 19 kb deletion including exon 2 or a nonsense mutation in five additional families with FHL, all of them with the same ethnic background.

Syntaxin 11 is a member of soluble N-ethylmaleimide sensitive factor attachment protein receptors present on target membranes (t-SNAREs) (26,27). In contrast to other syntaxins, it harbors a short cystein-rich C-terminal tail instead of a hydrophobic membrane anchor that serves as a putative palmitoylation site (21,28). Despite the lack of this membrane anchor, syntaxin 11 is retained in membrane protein fractions solubilized by Triton X-100 treatment (23). As expected from the SNARE hypothesis of regulated intracellular vesicle transport, which specified the complex organization within different intracellular membrane fusion processes (29), the non-neuronal SNAP25 homolog SNAP23 was clearly identified as one of the binding partners in vivo (23). A SNARE complex harbors a four-helix bundle including a vesicle-associated v-SNARE (synaptobrevin/VAMP), a t-SNARE (syntaxin) and two molecules of either SNAP25 involved in neuronal complexes or SNAP23 in complexes associated with fusions in non-neuronal cell types (26). All syntaxin family members harbor an N-terminal HABC domain that regulates SNARE complex formation (30).

In FHL3, the priming step at the immunological synapse during exocytosis of cytotoxic vesicles is abrogated because of mutations in UNC13D as recently described by Feldmann et al. (16). It is unlikely that syntaxin 11 is involved in these steps during exocytosis because of the different intracellular localization. Confocal immunofluorescence microscopy analysis showed a strong association of syntaxin 11 with the intermediate compartment, a region between late endosomal areas and the trans-Golgi network suggesting a regulatory role in cycling between these two compartments. Syntaxin 11 may actively be involved in the transport of yet unknown vesicles from intracellular regions to the cell surface rather than being directly involved in exocytotic processes as other homologues such as syntaxins 1–4.

As we do not know the exact role of syntaxin 11 in the intracellular vesicle transport of the phagocytic system, a clear functional link to the characteristic features of the disease is still elusive. The genes known previously to underlie FHL, PRF1 and UNC13D, are directly associated with the effector cells, cytotoxic T, or NK-cells. The PRF1/granzyme dependent cytotoxic pathway is essential for the elimination of target cells transformed or infected with viral proteins (31). On the other hand, NK cells are stimulated by cell-to-cell interactions with DCs. This direct DC to NK cell contact seems to have an impact on the activation of the cytolytic activity of NK cells and their IFN-γ production (32). Thus, a reduced or absent NK cell activity may be caused not only by mutations in PRF1 or UNC13D but also indirectly by an incomplete activation of NK cells by DCs (33). Future studies have be initiated to investigate these complex interactions.

Clinically, the age at onset of the disease and the time between diagnosis and time point of transplantation in the seven patients from three different families with homozygous mutation Val124fsX60 varied markedly (Table 2). The patient from family 4 with a complete deletion of the STX11 coding region had the first signs of FHL at the age of 33 months (Table 2). This is strikingly different from our findings regarding the most common PRF1 mutation detected in patients who originated from Turkey. All patients with a homozygous mutation 1122G>A (Trp374X) had a very early onset of the disease between 1 and 3 months of age (34). These Trp374X patients should also undergo HSCT as soon as possible to avoid an early death. Both types of mutations result in a premature stop codon in either PRF1 or STX11, and both groups of patients were otherwise clinically very similar. Insufficient defense against a pathogen may result more efficiently and more rapidly in the typical signs of FHL in perforin 1-deficient patients than in those with a defect in STX11, because syntaxin 11 is not directly involved in the ‘killing’ machinery. Nevertheless, the described patient groups are too small to make a statistically significant conclusion. The identification of STX11 as a novel gene involved in the development of FHL will provide new insights into the mechanisms of the disease and will also further facilitate genetic counseling and prenatal diagnosis (35) in affected families.

MATERIALS AND METHODS

Families

All patients specified in this study are children of Kurdish origin with consanguineous parents. They all fulfilled the diagnostic criteria for FHL including decreased NK cell activity in the standard 4 h 51Cr release assay (24). Patients' characteristics are given in Table 2 and the index family analyzed for linkage (family 1) is described in more detail subsequently.

In family 1, FHL was first diagnosed in three members of the family, two of whom were included in the study described here (IV : 8, IV : 9 in Fig. 1). The age at onset of the disease in the three brothers was 3, 12 and 39 months, respectively. The oldest brother died shortly after diagnosis, and samples were not available for further studies. The other two underwent HSCT after several relapses, and they are both alive. Clinical data from these patients have been described previously (36). The first signs of FHL in one of the monozygotic female twins (V : 2) occurred at 5 months of age. Haploidentical HSCT with the father as a donor was performed at the age of 15 months. After primary graft failure and no engraftment of the autologous back-up, the child died. At the age of 25 months, the brother (V : 1) was referred to the hospital with a history of fever and diarrhea. In the other twin sister (V : 3), onset of disease did not occur until the age of 17 months. Both children received HSCT from unrelated donor and are presently doing well.

Samples

For linkage analysis, DNA was extracted from peripheral whole blood. In case of patients who had undergone HSCT, we prepared DNA from mouth wash material and from fibroblast cultures. Once the gene had been identified, additional patients with FHL from Turkey were investigated. We obtained written informed consent from all patients and family members before the study was performed.

Homozygosity mapping and linkage analysis

A whole-genome scan was performed with 380 microsatellite markers (37), with an average distance of 11 cM. Markers were amplified in singleplex reactions in a final reaction volume of 10 µl containing 6 ng of genomic DNA. DNA amplification was carried out in PTC-225 thermal cyclers (MJ Research). Products were then pooled, and semi-automated genotyping was performed with MegaBACE-1000 analysis systems (Amersham Biosciences). Data were analyzed with Genetic Profiler software, version 1.5. Two-point LOD score calculations were performed with the program package LINKAGE version 5.2 (38) using an autosomal recessive model with full penetrance. Haplotypes were constructed either manually or with the program SIMWALK (39). SIMWALK was also used for the calculation of multi-point LOD scores, assuming equal allele frequencies.

Mutation analysis

The genomic structure of STX11 was determined by alignment of the cDNA sequence (NM_003764) with the appropriate sequence of a genomic chromosome 6 contig (AL135917). Primers used for amplification of the coding exon were as follows: Ex2F (5′-ACTTATTGCCCACACCGAGGAATAC) and Ex2R (5′-TGGGCTTCTGTCAAGACGGTAAGAG) with two internal sequencing primers, Ex2-2F and Ex2-3F. For subsequent analysis of additional samples and for DHPLC analysis of control samples, exon 2 of STX11 was divided into three PCR fragments using the following primer pairs:

  • Ex2-1F (5′-ACTTATTGCCCACACCGAGGAATAC) and Ex2-1R (5′-TTGGCGATGGAGTTGGTGTCGC);

  • Ex2-2F (5′-AGAACGCCCGCTTCCTCACGTCC) and Ex2-2R (5′-TGGCGGCTCTCGATCTCGTTGAG);

  • Ex2-3F (5′-ACCAGATCGAGGACATGTTCGAG) and Ex2-3R (5′-AGAGCTCCCGGCTTTGGTGCGTC).

DHPLC analysis

As control, for the presence or absence of the 5 bp deletion in STX11 230 samples of healthy blood donors from Turkey were analyzed. General DHPLC conditions were described elsewhere (40). DHPLC-based mutation analysis in fragments 1–3 of exon 2 (Ex2-2) was performed at 66 and 68°C column oven temperature. DHPLC-based semi-quantitative PCR analysis was performed at a column oven temperature of 50°C. DHPLC was performed on a WAVE system (Transgenomic, Crewe, UK).

Antibodies and inhibitors

Polyclonal rabbit antiserum raised against human SNAP23 was purchased from Novus Biochemicals (Littleton, CO, USA), and monoclonal antibody directed against amino acids 12–41 of human syntaxin 11 (clone 32) was from BD Transduction Laboratories (Heidelberg, Germany). Alexa680-conjugated goat anti-mouse IgG was obtained from MoBiTec (Göttingen, Germany) and IRDye800-conjugated goat anti-rabbit IgG was from Biotrend (Köln, Germany). Complete and Pefabloc SC were obtained from Roche (Mannheim, Germany) and okadaic acid from Alexis (Grünberg, Germany).

Preparation of mononuclear cells

MNC were routinely isolated from ethylenediaminetetraacetic acid (EDTA) blood of healthy controls and patients, respectively, by dextran sedimentation (Plasmasteril; Fresenius, Oberursel, Germany) followed by Ficoll-Hypaque (Pharmacia; Freiburg, Germany) density centrifugation as described previously (41). More than 98% of the cells were viable as assessed by trypan blue exclusion.

Isolation of detergent-soluble membranes

MNC were collected by rapid centrifugation. Cells were lysed in hypotonic lysis buffer (42 mM KCl, 10 mM HEPES, pH 7.4, 5 mM MgCl2) supplemented with inhibitors (2 mM sodium ortho-vanadate, 2 mM NaF, 500 nM okadaic acid, 4 mM Pefabloc, and 1× Complete). Lysates were incubated for 30 min on ice, followed by sonication. Thereafter, lysates were cleared by centrifugation at 250g for 10 min at 4°C to remove nuclei and intact cells. The supernatant was additionally centrifuged at 150 000g for 30 min at 4°C to separate the cytoplasm from the membrane fraction. The membrane fraction was then lysed for 30 min on ice in the same volume of 1% Triton X-100 in TNE buffer (10 mM Tris–HCl pH 7.5, 150 mM NaCl, 5 mM EDTA) supplemented with inhibitors. Lysates were centrifuged at 150 000g for 30 min at 4°C to remove Triton-insoluble material, and protein concentrations were determined in the supernatant by the method of Bradford (42), using BSA as a standard (Biorad, München, Germany). For western blotting, 50 µg protein were diluted in 3-fold concentrated sample buffer, and the samples were boiled for 5 min prior to electrophoresis.

Western blot analysis

Proteins derived from cell lysates were separated by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) (43) using a 10% polyacrylamide gel and blotted onto polyvinylidene fluoride (PVDF) membranes (Biorad, München, Germany) with a tank-blot transfer unit (Transphor II; Amersham Pharmacia Biotech, Freiburg, Germany). Immunodetection was performed as described in detail elsewhere (44) with some modifications. Briefly, membranes were blocked in Roti-ImmunoBlock (Roth, Karlsruhe, Germany), incubated with the respective primary antibodies and Alexa680-conjugated goat anti-mouse IgG or IRDye800-conjugated goat anti-rabbit IgG secondary antibodies according to the manufacturer's recommendations. Bands were visualized by Odyssey infrared imaging system (LICOR, Bad Homburg, Germany).

ELECTRONIC DATABASE INFORMATION

GenBank, http://www.ncbi.nlm.nih.gov/Genbank/ Center for Medical Genetics, Marshfield Clinic Research Foundation, http://research.marshfieldclinic.org/genetics/ Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim/

ACKNOWLEDGEMENTS

We are grateful to the families involved for their participation in this research project. We thank the following physicians for their generous cooperation in this study: P. Gutjahr (Mainz, Germany); A. Kulozik (Heidelberg, Germany); Th. Kühne (Basel, Switzerland); T. Celkan (Istanbul, Turkey); A. Gulen (Izmir, Turkey). We thank F. Oyen, B. Weber and N. Wittstruck for excellent technical assistance. This work was supported by grants in aid from the ‘Fördergemeinschaft Kinderkrebszentrum Hamburg e.V.’ and the National Genome Research Network.

The authors wish it to be known that, in their opinion, the first three authors should be regarded as joint First Authors.

Figure 1. Pedigree of the large Kurdish FHL family used for the mapping study and construction of haplotypes in the FHL4 region on chromosome 6. The filled box denotes the mutant haplotype completely cosegregating with the FHL4 mutation. Obligatory recombination events and lack of homozygosity, respectively, define the critical interval between D6S1569 and D6S960.

Figure 1. Pedigree of the large Kurdish FHL family used for the mapping study and construction of haplotypes in the FHL4 region on chromosome 6. The filled box denotes the mutant haplotype completely cosegregating with the FHL4 mutation. Obligatory recombination events and lack of homozygosity, respectively, define the critical interval between D6S1569 and D6S960.

Figure 2. Sequence analysis and detailed map of STX11. (A) The 11 Mb candidate region on chromosome 6q24 containing several positional candidate genes including STX11. (B) Genomic organization of STX11. The coding region (shaded) comprises only exon 2. (C) DNA sequence of STX11 in patient V : 1 and his parents (IV : 5 and IV : 6) from the large Kurdish family with FHL. The homozygous mutation c.369_370delAG and c.374_376delCGC in exon 2 was detected in samples from the patients, the respective parents showed the heterozygous mutation. (D) Corresponding section of the peptide sequence. The mutation leads to a frameshift in codon 124 (altered residues shown in italics) and a premature termination codon. WT, wild-type.

Figure 2. Sequence analysis and detailed map of STX11. (A) The 11 Mb candidate region on chromosome 6q24 containing several positional candidate genes including STX11. (B) Genomic organization of STX11. The coding region (shaded) comprises only exon 2. (C) DNA sequence of STX11 in patient V : 1 and his parents (IV : 5 and IV : 6) from the large Kurdish family with FHL. The homozygous mutation c.369_370delAG and c.374_376delCGC in exon 2 was detected in samples from the patients, the respective parents showed the heterozygous mutation. (D) Corresponding section of the peptide sequence. The mutation leads to a frameshift in codon 124 (altered residues shown in italics) and a premature termination codon. WT, wild-type.

Figure 3. Identification of a large homozygous deletion including exon 2 of syntaxin 11. (A) Inheritance from father and mother was shown by a semi-quantitative DHPLC assay. Duplex amplification with an interchromosomal control (exon 3 of VWF on chromosome 12) and part one of STX11 was stopped in the logarithmic phase after 27 cycles. (B) Deletion boundaries were mapped by amplification of various fragments of 300–400 bp around exon 2, from nucleotide 22500 (sample a) to nucleotide 60140 (sample k). Amplification and subsequent sequencing of a fragment spanning the deletion identified the breakpoint.

Figure 3. Identification of a large homozygous deletion including exon 2 of syntaxin 11. (A) Inheritance from father and mother was shown by a semi-quantitative DHPLC assay. Duplex amplification with an interchromosomal control (exon 3 of VWF on chromosome 12) and part one of STX11 was stopped in the logarithmic phase after 27 cycles. (B) Deletion boundaries were mapped by amplification of various fragments of 300–400 bp around exon 2, from nucleotide 22500 (sample a) to nucleotide 60140 (sample k). Amplification and subsequent sequencing of a fragment spanning the deletion identified the breakpoint.

Figure 4. Synthesis and distribution of syntaxin 11 and SNAP23 in different cell types. (A) Analysis of cytosolic (cyt) and Triton-soluble membrane proteins (mem) from monocytes and lymphocytes of normal controls, and of membrane proteins from three different cell lines (1, HL-60; 2, Jurkat; 3, B cell line). Proteins (50 µg) were separated by SDS–PAGE and detection was performed by western blot analysis with anti-syntaxin 11 or anti-SNAP23 antibodies. (B) Analysis of membrane proteins from peripheral MNCs of various probands. The left two panels (1 and 2) represent two families with STX11 mutations, the right panel (3) shows a family with Griscelli syndrome type 2 and mutations in RAB27A. M, mother; Pt, patient; F, father; HD, healthy donor.

Figure 4. Synthesis and distribution of syntaxin 11 and SNAP23 in different cell types. (A) Analysis of cytosolic (cyt) and Triton-soluble membrane proteins (mem) from monocytes and lymphocytes of normal controls, and of membrane proteins from three different cell lines (1, HL-60; 2, Jurkat; 3, B cell line). Proteins (50 µg) were separated by SDS–PAGE and detection was performed by western blot analysis with anti-syntaxin 11 or anti-SNAP23 antibodies. (B) Analysis of membrane proteins from peripheral MNCs of various probands. The left two panels (1 and 2) represent two families with STX11 mutations, the right panel (3) shows a family with Griscelli syndrome type 2 and mutations in RAB27A. M, mother; Pt, patient; F, father; HD, healthy donor.

Table 1.

Two-point LOD scores for each analyzed marker from the FHL4 interval and FHL in the large, consanguineous Kurdish family used for the genome-wide scan

Marker  Recombination fraction 
0.000  0.001  0.010  0.050  0.100  0.200  0.300  0.400 
D6S1009  −1.873  −0.321  0.623  1.121  1.164  0.930  0.570  0.221 
D6S1569  −∞  1.257  2.197  2.626  2.576  2.127  1.487  0.747 
D6S308  1.516  1.512  1.481  1.339  1.157  0.779  0.403  0.105 
D6S971  0.902  0.901  0.888  0.828  0.751  0.587  0.409  0.216 
D6S1649  3.038  3.033  2.990  2.794  2.541  2.001  1.409  0.751 
GATA184A08  3.663  3.656  3.596  3.322  2.966  2.213  1.420  0.644 
D6S311  4.891  4.883  4.813  4.495  4.079  3.179  2.184  1.105 
D6S960  −∞  −4.084  −2.133  −0.948  −0.598  −0.430  −0.380  −0.259 
D6S2436  −∞  −1.325  −0.357  0.200  0.326  0.297  0.187  0.084 
Marker  Recombination fraction 
0.000  0.001  0.010  0.050  0.100  0.200  0.300  0.400 
D6S1009  −1.873  −0.321  0.623  1.121  1.164  0.930  0.570  0.221 
D6S1569  −∞  1.257  2.197  2.626  2.576  2.127  1.487  0.747 
D6S308  1.516  1.512  1.481  1.339  1.157  0.779  0.403  0.105 
D6S971  0.902  0.901  0.888  0.828  0.751  0.587  0.409  0.216 
D6S1649  3.038  3.033  2.990  2.794  2.541  2.001  1.409  0.751 
GATA184A08  3.663  3.656  3.596  3.322  2.966  2.213  1.420  0.644 
D6S311  4.891  4.883  4.813  4.495  4.079  3.179  2.184  1.105 
D6S960  −∞  −4.084  −2.133  −0.948  −0.598  −0.430  −0.380  −0.259 
D6S2436  −∞  −1.325  −0.357  0.200  0.326  0.297  0.187  0.084 
Table 2.

Characteristics of patients reported with mutations in syntaxin 11

Family (patient)a  Mutation in STX11  Deduced peptide change  Sex  Age at onset (months)  Age at HSCTd (months)  Outcome 
1 (IV : 8)b  c.369_370delAG/c.374_376delCGC  Val124fsX60  12  125  Alive (>10 years) 
1 (IV : 9)b  c.369_370delAG/c.374_376delCGC  Val124fsX60  39  113  Alive (>10 years) 
1 (V : 1)  c.369_370delAG/c.374_376delCGC  Val124fsX60  24  43  Alive (>1 year) 
1 (V : 2)c  c.369_370delAG/c.374_376delCGC  Val124fsX60  16  Dead (graft rejection) 
1 (V : 3)c  c.369_370delAG/c.374_376delCGC  Val124fsX60  17  27  Alive (>1.5 years) 
c.369_370delAG/c.374_376delCGC  Val124fsX60  13  30  Dead (Liver failure) 
c.369_370delAG/c.374_376delCGC  Val124fsX60  16  Alive (∼2 months) 
AL135917:g.25561_44749del  deletion  33  41  Dead (TRM)e 
c.802C→T  Gln268X  16  Not transplanted  Alive 
c.802C→T  Gln268X  1.5  Not transplanted  Alive 
Family (patient)a  Mutation in STX11  Deduced peptide change  Sex  Age at onset (months)  Age at HSCTd (months)  Outcome 
1 (IV : 8)b  c.369_370delAG/c.374_376delCGC  Val124fsX60  12  125  Alive (>10 years) 
1 (IV : 9)b  c.369_370delAG/c.374_376delCGC  Val124fsX60  39  113  Alive (>10 years) 
1 (V : 1)  c.369_370delAG/c.374_376delCGC  Val124fsX60  24  43  Alive (>1 year) 
1 (V : 2)c  c.369_370delAG/c.374_376delCGC  Val124fsX60  16  Dead (graft rejection) 
1 (V : 3)c  c.369_370delAG/c.374_376delCGC  Val124fsX60  17  27  Alive (>1.5 years) 
c.369_370delAG/c.374_376delCGC  Val124fsX60  13  30  Dead (Liver failure) 
c.369_370delAG/c.374_376delCGC  Val124fsX60  16  Alive (∼2 months) 
AL135917:g.25561_44749del  deletion  33  41  Dead (TRM)e 
c.802C→T  Gln268X  16  Not transplanted  Alive 
c.802C→T  Gln268X  1.5  Not transplanted  Alive 

aNumbering according to Figure 1.

bFirst described by Henter et al. (36).

cV : 2 and V : 3 were monozygotic twins.

dHSCT, hematopoietic stem cell transplantation.

eTRM, transplantation related mortality.

1
Janka, G.E. (
1983
) Familial hemophagocytic lymphohistiocytosis.
Eur. J. Pediatr.
 ,
140
,
221
–230.
2
Janka, G.E. and Schneider, E.M. (
2004
) Modern management of children with haemophagocytic lymphohistiocytosis.
Br. J. Haematol.
 ,
124
,
4
–14.
3
Henter, J.I., Samuelsson-Horne, A., Arico, M., Egeler, R.M., Elinder, G., Filipovich, A.H., Gadner, H., Imashuku, S., Komp, D., Ladisch, S. et al. (
2002
) Treatment of hemophagocytic lymphohistiocytosis with HLH-94 immunochemotherapy and bone marrow transplantation.
Blood
 ,
100
,
2367
–2373.
4
Stephan, J.L., Donadieu, J., Ledeist, F., Blanche, S., Griscelli, C. and Fischer, A. (
1993
) Treatment of familial hemophagocytic lymphohistiocytosis with antithymocyte globulins, steroids, and cyclosporin A.
Blood
 ,
82
,
2319
–2323.
5
Durken, M., Horstmann, M., Bieling, P., Erttmann, R., Kabisch, H., Loliger, C., Schneider, E.M., Hellwege, H.H., Kruger, W., Kroger, N. et al. (
1999
) Improved outcome in haemophagocytic lymphohistiocytosis after bone marrow transplantation from related and unrelated donors: a single-centre experience of 12 patients.
Br. J. Haematol.
 ,
106
,
1052
–1058.
6
Ohadi, M., Lalloz, M.R., Sham, P., Zhao, J., Dearlove, A.M., Shiach, C., Kinsey, S., Rhodes, M. and Layton, D.M. (
1999
) Localization of a gene for familial hemophagocytic lymphohistiocytosis at chromosome 9q21.3–22 by homozygosity mapping.
Am. J. Hum. Genet.
 ,
64
,
165
–171.
7
Dufourcq-Lagelouse, R., Jabado, N., Le Deist, F., Stephan, J.L., Souillet, G., Bruin, M., Vilmer, E., Schneider, M., Janka, G., Fischer, A. et al. (
1999
) Linkage of familial hemophagocytic lymphohistiocytosis to 10q21-22 and evidence for heterogeneity.
Am. J. Hum. Genet.
 ,
64
,
172
–179.
8
Stepp, S.E., Dufourcq-Lagelouse, R., Le Deist, F., Bhawan, S., Certain, S., Mathew, P.A., Henter, J.I., Bennett, M., Fischer, A., de Saint Basile, G. et al. (
1999
) Perforin gene defects in familial hemophagocytic lymphohistiocytosis.
Science
 ,
286
,
1957
–1959.
9
Kogawa, K., Lee, S.M., Villanueva, J., Marmer, D., Sumegi, J. and Filipovich, A.H. (
2002
) Perforin expression in cytotoxic lymphocytes from patients with hemophagocytic lymphohistiocytosis and their family members.
Blood
 ,
99
,
61
–66.
10
Arico, M., Danesino, C., Pende, D. and Moretta, L. (
2001
) Pathogenesis of haemophagocytic lymphohistiocytosis.
Br. J. Haematol.
 ,
114
,
761
–769.
11
Suga, N., Takada, H., Nomura, A., Ohga, S., Ishii, E., Ihara, K., Ohshima, K. and Hara, T. (
2002
) Perforin defects of primary haemophagocytic lymphohistiocytosis in Japan.
Br. J. Haematol.
 ,
116
,
346
–349.
12
Feldmann, J., Le Deist, F., Ouachee-Chardin, M., Certain, S., Alexander, S., Quartier, P., Haddad, E., Wulffraat, N., Casanova, J.L., Blanche, S. et al. (
2002
) Functional consequences of perforin gene mutations in 22 patients with familial haemophagocytic lymphohistiocytosis.
Br. J. Haematol.
 ,
117
,
965
–972.
13
Ericson, K.G., Fadeel, B., Nilsson-Ardnor, S., Soderhall, C., Samuelsson, A., Janka, G., Schneider, M., Gurgey, A., Yalman, N., Revesz, T. et al. (
2001
) Spectrum of perforin gene mutations in familial hemophagocytic lymphohistiocytosis.
Am. J. Hum. Genet.
 ,
68
,
590
–597.
14
Clementi, R., zur Stadt, U., Savoldi, G., Varoitto, S., Conter, V., De Fusco, C., Notarangelo, L.D., Schneider, M., Klersy, C., Janka, G. et al. (
2001
) Six novel mutations in the PRF1 gene in children with haemophagocytic lymphohistiocytosis.
J. Med. Genet.
 ,
38
,
643
–646.
15
Molleran Lee, S., Villanueva, J., Sumegi, J., Zhang, K., Kogawa, K., Davis, J. and Filipovich, A.H. (
2004
) Characterisation of diverse PRF1 mutations leading to decreased natural killer cell activity in North American families with haemophagocytic lymphohistiocytosis.
J. Med. Genet.
 ,
41
,
137
–144.
16
Feldmann, J., Callebaut, I., Raposo, G., Certain, S., Bacq, D., Dumont, C., Lambert, N., Ouachee-Chardin, M., Chedeville, G., Tamary, H. et al. (
2003
) Munc13-4 is essential for cytolytic granules fusion and is mutated in a form of familial hemophagocytic lymphohistiocytosis (FHL3).
Cell
 ,
115
,
461
–473.
17
Lord, S.J., Rajotte, R.V., Korbutt, G.S. and Bleackley, R.C. (
2003
) Granzyme B: a natural born killer.
Immunol. Rev.
 ,
193
,
31
–38.
18
Barbosa, M.D., Barrat, F.J., Tchernev, V.T., Nguyen, Q.A., Mishra, V.S., Colman, S.D., Pastural, E., Dufourcq-Lagelouse, R., Fischer, A., Holcombe, R.F. et al. (
1997
) Identification of mutations in two major mRNA isoforms of the Chediak–Higashi syndrome gene in human and mouse.
Hum. Mol. Genet.
 ,
6
,
1091
–1098.
19
Menasche, G., Pastural, E., Feldmann, J., Certain, S., Ersoy, F., Dupuis, S., Wulffraat, N., Bianchi, D., Fischer, A., Le Deist, F. et al. (
2000
) Mutations in RAB27A cause Griscelli syndrome associated with haemophagocytic syndrome.
Nat. Genet.
 ,
25
,
173
–176.
20
Stinchcombe, J., Bossi, G. and Griffiths, G.M. (
2004
) Linking albinism and immunity: the secrets of secretory lysosomes.
Science
 ,
305
,
55
–59.
21
Prekeris, R., Klumperman, J. and Scheller, R.H. (
2000
) Syntaxin 11 is an atypical SNARE abundant in the immune system.
Eur. J. Cell. Biol.
 ,
79
,
771
–780.
22
Ravichandran, V., Chawla, A. and Roche, P.A. (
1996
) Identification of a novel syntaxin- and synaptobrevin/VAMP-binding protein, SNAP-23, expressed in non-neuronal tissues.
J. Biol. Chem.
 ,
271
,
13300
–13303.
23
Valdez, A.C., Cabaniols, J.P., Brown, M.J. and Roche, P.A. (
1999
) Syntaxin 11 is associated with SNAP-23 on late endosomes and the trans-Golgi network.
J. Cell. Sci.
 ,
112
(Pt 6),
845
–54.
24
Henter, J.I., Elinder, G. and Ost, A. (
1991
) Diagnostic guidelines for hemophagocytic lymphohistiocytosis. The FHL Study Group of the Histiocyte Society.
Semin. Oncol.
 ,
18
,
29
–33.
25
Schneider, E.M., Lorenz, I., Walther, P. and Janka-Schaub, G.E. (
2003
) Natural killer deficiency: a minor or major factor in the manifestation of hemophagocytic lymphohistiocytosis?
J. Pediatr. Hematol. Oncol.
 ,
25
,
680
–683.
26
Chen, Y.A. and Scheller, R.H. (
2001
) SNARE-mediated membrane fusion.
Nat. Rev. Mol. Cell. Biol.
 ,
2
,
98
–106.
27
Teng, F.Y., Wang, Y. and Tang, B.L. (
2001
) The syntaxins.
Genome Biol.
 ,
2
, Reviews 3012.1–3012.
7
.
28
Tang, B.L., Low, D.Y. and Hong, W. (
1998
) Syntaxin 11: a member of the syntaxin family without a carboxyl terminal transmembrane domain.
Biochem. Biophys. Res. Commun.
 ,
245
,
627
–632.
29
Rothman, J.E. and Warren, G. (
1994
) Implications of the SNARE hypothesis for intracellular membrane topology and dynamics.
Curr. Biol.
 ,
4
,
220
–233.
30
Hay, J.C. (
2001
) SNARE complex structure and function.
Exp. Cell. Res.
 ,
271
,
10
–21.
31
Russell, J.H. and Ley, T.J. (
2002
) Lymphocyte-mediated cytotoxicity.
Annu. Rev. Immunol.
 ,
20
,
323
–370.
32
Fernandez, N.C., Lozier, A., Flament, C., Ricciardi-Castagnoli, P., Bellet, D., Suter, M., Perricaudet, M., Tursz, T., Maraskovsky, E. and Zitvogel, L. (
1999
) Dendritic cells directly trigger NK cell functions: cross-talk relevant in innate anti-tumor immune responses in vivo.
Nat. Med.
 ,
5
,
405
–411.
33
Yu, Y., Hagihara, M., Ando, K., Gansuvd, B., Matsuzawa, H., Tsuchiya, T., Ueda, Y., Inoue, H., Hotta, T. and Kato, S. (
2001
) Enhancement of human cord blood CD34+ cell-derived NK cell cytotoxicity by dendritic cells.
J. Immunol.
 ,
166
,
1590
–1600.
34
zur Stadt, U., Kabisch, H., Janka, G. and Schneider, E.M. (
2003
) Rapid LightCycler assay for identification of the perforin codon 374 Trp→ stop mutation in patients and families with hemophagocytic lymphohistiocytosis (HLH).
Med. Pediatr. Oncol.
 ,
41
,
26
–29.
35
zur Stadt, U., Pruggmayer, M., Jung, H., Henter, J.I., Schneider, M., Kabisch, H. and Janka, G. (
2002
) Prenatal diagnosis of perforin gene mutations in familial hemophagocytic lymphohistiocytosis (FHLH).
Prenat. Diagn.
 ,
22
,
80
–81.
36
Henter, J.I. and Elinder, G. (
1991
) Familial hemophagocytic lymphohistiocytosis. Clinical review based on the findings in seven children.
Acta Paediatr. Scand.
 ,
80
,
269
–277.
37
Broman, K.W., Murray, J.C., Sheffield, V.C., White, R.L. and Weber, J.L. (
1998
) Comprehensive human genetic maps: individual and sex-specific variation in recombination.
Am. J. Hum. Genet.
 ,
63
,
861
–869.
38
Lathrop, G.M., Lalouel, J.M., Julier, C. and Ott, J. (
1984
) Strategies for multilocus linkage analysis in humans.
Proc. Natl Acad. Sci. USA
 ,
81
,
3443
–3446.
39
Sobel, E. and Lange, K. (
1996
) Descent graphs in pedigree analysis: applications to haplotyping, location scores, and marker-sharing statistics.
Am. J. Hum. Genet.
 ,
58
,
1323
–1337.
40
zur Stadt, U., Eckert, C., Rischewski, J., Michael, K., Golta, S., Muller, M., Schneppenheim, R. and Kabisch, H. (
2003
) Identification and characterisation of clonal incomplete T-cell-receptor Vdelta2-Ddelta3/Ddelta2-Ddelta3 rearrangements by denaturing high-performance liquid chromatography and subsequent fragment collection: implications for minimal residual disease monitoring in childhood acute lymphoblastic leukemia.
J. Chromatogr. B Analy. Technol. Biomed. Life. Sci.
 ,
792
,
287
–298.
41
Kasper, B., Thole, H.H., Patterson, S.D. and Welte, K. (
1997
) Cytosolic proteins from neutrophilic granulocytes: a comparison between patients with severe chronic neutropenia and healthy donors.
Electrophoresis
 ,
18
,
142
–149.
42
Bradford, M.M. (
1976
) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem.
 ,
72
,
248
–254.
43
Laemmli, U.K. (
1970
) Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
 ,
227
,
680
–685.
44
Kasper, B., Tidow, N., Grothues, D. and Welte, K. (
2000
) Differential expression and regulation of GTPases (RhoA and Rac2) and GDIs (LyGDI and RhoGDI) in neutrophils from patients with severe congenital neutropenia.
Blood
 ,
95
,
2947
–2953.