Diamond-Blackfan anaemia: genetic homogeneity for a gene on chromosome 19q13 restricted to 1.8 Mb.

Diamond-Blackfan anaemia (DBA; MIM#205900) is a rare disorder manifested as a pure red-cell aplasia in the neonatal period or in infancy. The clinical hallmark of DBA is a selective decrease in erythroid precursors and anaemia. Other lineages are usually normal and the peripheral white blood cell count is normal. In approximately one-third of cases, the disease is associated with a wide variety of congenital anomalies and malformations. Most cases are sporadic, but 10-20% of them follow a recessive or a dominant inheritance pattern. A female with DBA and a chromosomal translocation involving chromosome 19q was recently identified. We undertook a linkage analysis with chromosome 19 markers in multiplex DBA families of Swedish, French, Dutch, Arabic and Italian origin. Significant linkage to chromosome 19q13 was established for dominant and recessive inherited DBA with a peak lod score at D19S197 (Zmax = 7.08, theta = 0.00). Within this region, a submicroscopic de novo deletion of 3.3 Mb was identified in a patient with DBA. The deletion coincides with the translocation break-point and, together with key recombinations, restricts the DBA gene to a 1.8-Mb region. The results suggest that, despite its clinical heterogeneity, DBA is genetically homogeneous for a gene in 19q13.

Hematopoietic stem cell transplantation in severe congenital neutropenia.

BACKGROUND: Severe congenital neutropenia (SCN) is an immunodeficiency characterized by disturbed myelopoiesis and an absolute neutrophil count (ANC) <0.5 × 10(9)/L. SCN is also a premalignant condition; a significant proportion of patients develop myelodysplastic syndrome or leukemia (MDS/L). Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative treatment for SCN. PROCEDURE: Since 2004, eight HSCT have been performed in seven patients at our center. The indications were transformation to MDS/L (n = 2), granulocyte colony-stimulating factor receptor (CSF3R) mutation(s) (n = 2), granulocyte colony-stimulating factor (G-CSF) resistance (n = 2), and at the patient's own request (n = 1). RESULTS: The mean age at transplantation was 13 years (2.8-28 years) (mean follow-up 32 months, range 21-60). Three patients harbored ELANE mutations, three HAX1 mutations, and in one patient no causative mutation was identified. Two of the ELANE mutations were novel mutations. Three patients initially received myeloablative conditioning and four had reduced intensity conditioning (RIC). Three grafts were from HLA-identical siblings, three from matched unrelated donors and two were cord blood units. Engraftment occurred in all patients. Two of seven (29%) patients died; both had MDS/L and both were among the three that underwent myeloablative conditioning. One patient has chronic GVHD 2 years post-transplant. CONCLUSIONS: The role of HSCT should be explored further in patients with SCN. In particular, the influence of the conditioning regime needs to be evaluated in a larger cohort of patients.

Perforin gene defects in familial hemophagocytic lymphohistiocytosis.

Familial hemophagocytic lymphohistiocytosis (FHL) is a rare, rapidly fatal, autosomal recessive immune disorder characterized by uncontrolled activation of T cells and macrophages and overproduction of inflammatory cytokines. Linkage analyses indicate that FHL is genetically heterogeneous and linked to 9q21.3-22, 10q21-22, or another as yet undefined locus. Sequencing of the coding regions of the perforin gene of eight unrelated 10q21-22-linked FHL patients revealed homozygous nonsense mutations in four patients and missense mutations in the other four patients. Cultured lymphocytes from patients had defective cytotoxic activity, and immunostaining revealed little or no perforin in the granules. Thus, defects in perforin are responsible for 10q21-22-linked FHL. Perforin-based effector systems are, therefore, involved not only in the lysis of abnormal cells but also in the down-regulation of cellular immune activation.

Spectrum, and clinical and functional implications of UNC13D mutations in familial haemophagocytic lymphohistiocytosis.

OBJECTIVE: Familial haemophagocytic lymphohistiocytosis (FHL) is a fatal disorder of immune dysregulation with defective cytotoxic lymphocyte function. Disease-causing mutations have been identified in the genes encoding perforin (PRF1), syntaxin-11 (STX11), and Munc13-4 (UNC13D). We screened for UNC13D mutations and studied clinical and functional implications of such mutations in a well defined patient cohort. METHODS: Sequencing of UNC13D was performed in 38 FHL patients from 34 FHL families in which PRF1 and STX11 mutations had been excluded. RESULTS: We identified six different mutations affecting altogether 9/38 individuals (24%) in 6/34 (18%) unrelated PRF1/STX11-negative families. Four novel mutations were revealed; two homozygous nonsense mutations (R83X and W382X), one splice mutation (exon 28), and one missense mutation (R928P). In addition, two known mutations were identified (R214X and a deletion resulting in a frame-shift starting at codon 782). There was considerable variation in the age at diagnosis, ranging from time of birth to 14 years (median 69 days). Three of nine patients (33%) developed central nervous system (CNS) symptoms. Natural killer (NK) cell activity was impaired in all four patients studied. Defective cytotoxic lymphocyte degranulation was evident in the two patients investigated, more pronounced in the patient with onset during infancy than in the patient with adolescent onset. CONCLUSIONS: Biallelic UNC13D mutations were found in 18% of the PRF1/STX11-negative FHL families. Impairment of NK cell degranulation was less pronounced in a patient with adolescent onset. FHL should be considered not only in infants but also in adolescents, and possibly young adults, presenting with fever, splenomegaly, cytopenia, hyperferritinaemia, and/or CNS symptoms.

Genotype-phenotype study of familial haemophagocytic lymphohistiocytosis due to perforin mutations.

BACKGROUND: PRF1 gene mutations are associated with familial haemophagocytic lymphohistiocytosis type 2 (FHL2). Genotype-phenotype analysis, previously hampered by limited numbers of patients, was for the first time performed by data pooling from five large centres worldwide. PATIENTS AND METHODS: Members of the Histiocyte Society were asked to report cases of FHL2 on specific forms. Data were pooled in a common database and analysed. RESULTS: The 124 patients had 63 different mutations (including 15 novel mutations): 11 nonsense, 10 frameshift, 38 missense and 4 in-frame deletions. Some mutations were found more commonly: 1122 G-->A (W374X), associated with Turkish origin, in 32 patients; 50delT (L17fsX22) associated with African/African American origin, in 21 patients; and 1090-91delCT (L364fsX), in 7 Japanese patients. Flow cytometry showed that perforin expression was absent in 40, reduced in 6 and normal in 4 patients. Patients presented at a median age of 3 months (quartiles: 2, 3 and 13 months), always with fever, splenomegaly and thrombocytopenia. NK activity was absent in 36 (51%), 5% in 4 (6%), "reduced" in 2 (3%) (not reported, n = 54). Nonsense mutations were significantly associated with younger age at onset (p<0.001) and absent natural killer activity (p = 0.008). CONCLUSION: PRF1 mutations are spread over the functional domains. Specific mutations are strongly associated with Turkish, African American and Japanese ethnic groups. Later onset and residual cytotoxic function are observed in patients with at least one missense mutation.

Central nervous system involvement in severe congenital neutropenia: neurological and neuropsychological abnormalities associated with specific HAX1 mutations.

OBJECTIVES: Homozygous mutations in the HAX1 gene were recently identified in severe congenital neutropenia patients belonging to the original Kostmann family in northern Sweden. Our observations suggested that these patients also develop neurological and neuropsychological symptoms. METHODS: Detailed clinical studies and mutation analyses were performed in the surviving patients belonging to the Kostmann kindred and in two patients not related to this family, along with studies of HAX1 splice variant expression in normal human tissues. RESULTS: Five of six Kostmann family patients and one other patient from northern Sweden harboured homozygous HAX1 mutations (568C-->T, Q190X) and one carried a heterozygous ELA2 gene mutation. One Swedish patient of Kurdish extraction carried alternative homozygous HAX1 mutations (131G-->A, W44X). All the three patients with Q190X mutations who were alive and available for evaluation developed neurological disease with decreased cognitive function, and three of four patients who reached 10 years developed epilepsy. In contrast, the patients with the ELA2 and W44X HAX1 mutations, respectively, showed no obvious neurological abnormalities. Moreover, two alternative HAX1 splice variants were identified in normal human tissues, including the brain. Both transcripts contained exon 5, harbouring the Q190X mutation, whereas the 5' end of exon 2 containing the W44X mutation was spliced out from the second transcript. CONCLUSIONS: We describe neurological and neuropsychological abnormalities for the first time in Kostmann disease patients. These central nervous system symptoms appear to be associated with specific HAX1 mutations.

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 (> or = 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.

Cleavage of Bcl-2 is an early event in chemotherapy-induced apoptosis of human myeloid leukemia cells.

The proto-oncogene product Bcl-2 protects a wide variety of cell types from apoptosis via a hitherto unknown mechanism. Bcl-2 has been shown to function upstream of the death proteases (caspases) in some, but not all, occurrences of apoptotic cell death. Using the myeloid leukemic cell line P39 we report the chemotherapy-induced caspase-dependent cleavage of endogenous Bcl-2. Etoposide treatment of these cells triggered a time-dependent activation of type II and type III caspases and cleavage of Bcl-2 yielding a 23 kDa cleavage fragment. The emergence of this cleavage product was blocked by the general caspase inhibitor zVAD-fmk, as well as the type III caspase inhibitor IETD-fmk and the caspase-9-selective inhibitor LEHD-fmk, while the type II caspase inhibitor DEVD-fmk proved considerably less efficient. Bcl-2 cleavage preceded cleavage of the known caspase-3 substrate, poly(ADP-ribose) polymerase (PARP), as well as that of the caspase-6 substrate, lamin B, indicating that Bcl-2 cleavage is a relatively early event in the apoptosis cascade in this experimental model. While evidence for cleavage of Bcl-2 in several subcellular compartments of etoposide-treated cells was obtained, this cleavage was detected predominantly in the mitochondrial fraction, thus providing further support for the central role of mitochondria in apoptosis. Caspase-mediated cleavage following etoposide treatment of these myeloid leukemic cells may represent a means for the attenuation of Bcl-2 function upon apoptosis induction.

Hemophagocytic lymphohistiocytosis. Report of 122 children from the International Registry. FHL Study Group of the Histiocyte Society.

Hemophagocytic lymphohistiocytosis (HLH) is a rare, often fatal, disease of early infancy. The diagnosis of HLH is frequently delayed or made at autopsy because no genetic or biologic marker has been identified. To improve the classification and treatment of HLH, the Histiocyte Society has established an 'International Registry for HLH'. Data collected included family history, clinical and laboratory features at the onset of illness, and treatment outcome. Stringent diagnostic criteria (ie fever, splenomegaly, cytopenia, hypertriglyceridemia, and/or hypofibrinogenemia, and hemophagocytosis without evidence of malignancy) were used for patient selection. One hundred and twenty-two patients (61 males, 61 females) were enrolled from 17 centers in 11 countries. The rate of parental consanguinity was 24%. A positive family history was reported in 49% of cases including two pairs of affected male twins. The median age at disease onset was 2.9 months, with no difference between familial and sporadic cases. Age at onset was similar in affected sibs from 10 of 14 families, but in four up to 3-year differences were observed. Hemophagocytosis was present at diagnosis in 75%. An associated infection (usually by common viral pathogens) was reported in 50 of the 122 (41%) cases, of which 25 had familial disease. Natural killer activity was impaired in 36 of 37 patients studied. Chromosome analysis was normal in all tested patients. A decreased frequency of HLA-B7 and B8 alleles and increased frequency of HLA-B21 and DQ3 were observed. The estimated 5-year survival (SE) was 21% (18.7) for all patients. It was 66% (37.8) for patients who received allogeneic bone marrow transplant and 10.1% (9.6) for patients treated with chemotherapy alone (P=0.0001). None of the previously proposed prognostic indicators (age, associated infection, cerebrospinal fluid pleocytosis, family history) correlated with treatment outcome.

Bone marrow transplantation in two children with congenital amegakaryocytic thrombocytopenia.

Two thrombocytopenic infants with essentially normal initial bone marrow morphology were believed to have idiopathic thrombocytopenic purpura. However, they failed to respond to steroids and intravenous immunoglobulins and had a normal platelet recovery after transfusions. The diagnosis was revised to congenital amegakaryocytic thrombocytopenia after bone marrow biopsies, which revealed a marked paucity of megakaryocytes. Repeated biopsies disclosed gradually decreasing numbers of megakaryocytes and increasing marrow hypoplasia. At the ages of 42 and 22 months the children underwent allogeneic bone marrow transplants, one of them with an unrelated marrow donor. Both patients are well with good engraftment of donor marrow and normal peripheral blood counts, 31 and 12 months after BMT, respectively.

Allogeneic bone marrow transplantation for hemophagocytic lymphohistiocytosis in Sweden.

Six children (aged 9 months to 10 and 5/12 years) with hemophagocytic lymphohistiocytosis (HLH) have undergone allogeneic BMT in Sweden. In two of the children unrelated donors were used. Parents were available as donors in two of the cases and siblings in the other two. Conditioning before BMT consisted of etoposide, busulfan and cyclophosphamide with the addition of ATG in two cases and OKT 3 in one case. For post-transplant immunosuppression, i.v. methotrexate and cyclosporin A (CsA) were used in five cases, and in one child CsA was combined with methylprednisolone. Of the six children, four are alive and well 2 and 3/12 to 3 and 1/12 years after BMT. One child, who had an unrelated donor with one DR-antigen mismatch, died 30 days after BMT of fulminant grade IV GVHD. Another patients, seropositive for CMV, received marrow from an unrelated HLA-A, -B, -DR and -DP identical donor. After an initially uneventful course, CMV was isolated from her leukocytes. Seven months after BMT she developed a progressive obstructive chronic bronchiolitis and succumbed to respiratory insufficiency 14 months after the transplant. This study supports the view that BMT is the treatment of choice in HLH, particularly if an HLA-identical related donor is available.

Familial hemophagocytic lymphohistiocytosis. Primary hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis represents a spectrum of pathogenetically different diseases including the rapidly fatal autosomal recessive disease of familial hemophagocytic lymphohistiocytosis (FHL). The onset is usually during the first years of life with fever, cytopenia, and hepatosplenomegaly. Neurologic symptoms may supervene. Similar symptoms may occur in the infection-(virus-)associated or malignancy-associated hemophagocytic syndromes (IAHS/MAHS). Triggering infections can be found in all these diseases and do not allow for reliable differentiation. An international treatment protocol (HLH-94) has been developed for FHL, but immunomodulatory treatment may be justified in IAHS and MAHS as well, since they also have a high fatality rate.

Infection- and malignancy-associated hemophagocytic syndromes. Secondary hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis represents a spectrum of pathogenetically different diseases in which a T-cell induced, uncontrolled activation of phagocytosing macrophages may lead to fever, organomegaly, and pancytopenia. The underlying immunologic disturbance can either be genetically transmitted, like in FHL, or acquired, as in IAHS or MAHS. Triggering infections can be found in all these diseases and do not allow a reliable differentiation. An international treatment protocol has been developed for FHL. IAHS and MAHS also have a high fatality rate, justifying immunomodulatory treatment if the disease is progressive.

Familial hemophagocytic lymphohistiocytosis: too little cell death can seriously damage your health.

Familial hemophagocytic lymphohistiocytosis (FHL) is a rare and fatal disease of early childhood characterized by a non-malignant accumulation of activated T lymphocytes and histiocytes in the reticuloendothelial system. Moreover, immune system derangement, with prominent hypercytokinemia and low or absent cytotoxic T and natural killer (NK) cell activity, is a consistent feature of this autosomal recessive disorder. Recent work has demonstrated that the degree of spontaneous caspase activation in FHL lymphocytes is attenuated in vitro whereas Fas-mediated caspase activation and apoptosis induction remains unmitigated, and FHL can thus be distinguished from the related chronic disorder of immune regulation termed autoimmune lymphoproliferative syndrome or ALPS. However, subsequent studies have identified mutations in the gene encoding perforin, a cytotoxic granule constituent required for apoptotic killing of target cells, in a number of FHL patients. Hence, the underlying defect in FHL may be conceived of as a lack of apoptosis triggering within the immune system, rather than apoptosis resistance per se. These observations represent an important step in our understanding of the pathogenesis of FHL and also serve to emphasize the pivotal role of cellular (perforin-based) cytotoxicity in the regulation of immune homeostasis.

[Apoptosis required for maintenance of homeostasis: familial hemophagocytic lymphohistiocytosis caused by too little cell death]. / Lagom är bäst även när det gäller apoptos. Brist på apoptos orsak till familjär hemofagocyterande lymfohistiocytos.

Apoptosis or programmed cell death is essential for the maintenance of tissue homeostasis. Recent studies have implicated the dysregulation of apoptosis in a plethora of human diseases. Hence, perturbation of the death program intrinsic to every cell may result in essentially too little or too much apoptosis, which may in turn lead to proliferative or degenerative diseases respectively. In the present review, we discuss our current molecular understanding of the apoptotic process and its possible role in human disease, with particular emphasis on the rare and invariably fatal disease of early childhood, termed familial hemophagocytic lymphohistiocytosis, in which mutations in the perforin gene were recently identified.

[Familial hemophagocytic lymphohistiocytosis: diagnosis, treatment and pathophysiological mechanisms]. / Familjär hemofagocyterande lymfohistiocytos. Från dödligt sjuka barn till bot och immunologiskt genombrott.

Familial hemophagocytic lymphohistiocytosis (FHL) is an invariably fatal disease typically seen in infancy and early childhood, with a median survival without therapy of two months. It is characterized by prolonged fever, hepatosplenomegaly, cytopenia, and deficient NK-cell activity and T-cell cytotoxic capacity. Severe neurological symptoms as well as coagulation disorders and abnormalities in liver function and lipid status may also develop. Since the mid 1980's there has been a remarkable increase in our understanding of this disease. In a large-scale international collaborative effort mediated through the Histiocyte Society, diagnostic criteria and an international treatment protocol (HLH-94) based on immunochemotherapy and BMT have been developed. A large proportion of affected children can now be cured and, moreover, successful chemotherapy in utero of FHL has been achieved. It has been shown that the symptoms and signs are mediated through a pronounced hypercytokinemia. Previous suggestions that FHL may be caused by a deficiency in apoptosis were recently confirmed when perforin gene defects were described, which may well explain the disastrous lymphohistiocytic accumulation and subsequent T-cell activation.

[Parvovirus B19 infection--an insidious chameleon]. / Parvovirus B19-infektion--en lömsk kameleont.

Parvovirus B19 is a common source of infection with a seroprevalence of 60-70 per cent in the adult population. The most common manifestation is erythema infectiosum ('fifth disease'), with exanthem, fever and upper airway symptoms in children. The infection can give rise to a multifacetted clinical picture and is probably underdiagnosed, particularly in risk groups (individuals with haemolytic anaemia or immunosuppression, and fetuses). Serological diagnosis can now be complemented with the demonstration of viral DNA using the PCR (polymerase chain reaction) test in various body fluids, or tissue biopsy. Recent years have witnessed manifest increase in clinical knowledge of parvovirus B19-associated complications, and their diagnosis and treatment.

Hematopoietic stem cell transplantation in children with cancer and the risk of long-term psychological morbidity in the bereaved parents.

We have investigated whether hematopoietic stem cell transplantation (HSCT) before the death of children with cancer has a long-term effect on the physical and psychological well-being of the parents. A nationwide questionnaire was sent out to all bereaved parents in Sweden who had lost a child due to a malignancy from 1992 to 1997. Self-reported levels of anxiety, depression and quality of life as well as overall psychological and physical well-being in bereaved parents of children who underwent HSCT were compared with bereaved parents whose children did not receive a transplant. Bereaved parents whose children underwent HSCT had, according to a visual digital scale, an increased relative risk (RR) of long-term anxiety (RR 1.5; 95% confidence interval (CI) 1.0-2.1), poor psychological well-being (RR1.3; 95% CI 1.1-1.5), low quality of life (RR 1.4; 95% CI 1.2-1.7) and poor physical health (RR 1.3; 95% CI 1.1-1.5), whereas the State-Trait Anxiety Inventory and 'The Göteborg Quality of Life Instrument' were non-significantly increased (RR 1.3; 95% CI 0.8-2.3 and RR 1.7; 95% CI 0.9-3.3, respectively). The risks of these consequences were further augmented in case of multiple HSCT. We suggest that bereaved parents of children undergoing HSCT may be at greater risk of decreased psychological well-being than other bereaved parents of children with cancer.