Incidence of severe congenital neutropenia in Sweden and risk of evolution to myelodysplastic syndrome/leukaemia

Authors

  • Göran Carlsson,

    Corresponding author
    • Childhood Cancer Research Unit, Department of Women's and Children's Health, Karolinska Institutet, Karolinska University Hospital Solna, Stockholm, Sweden
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  • Anders Fasth,

    1. Department of Paediatrics, University of Gothenburg, Gothenburg, Sweden
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  • Elisabet Berglöf,

    1. Childhood Cancer Research Unit, Department of Women's and Children's Health, Karolinska Institutet, Karolinska University Hospital Solna, Stockholm, Sweden
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  • Kristina Lagerstedt-Robinson,

    1. Clinical Genetics Unit, Department of Molecular Medicine and Surgery, and Centre for Molecular Medicine, Karolinska Institutet, Karolinska University Hospital Solna, Stockholm, Sweden
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  • Magnus Nordenskjöld,

    1. Clinical Genetics Unit, Department of Molecular Medicine and Surgery, and Centre for Molecular Medicine, Karolinska Institutet, Karolinska University Hospital Solna, Stockholm, Sweden
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  • Jan Palmblad,

    1. Departments of Medicine and Haematology, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden
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  • Jan-Inge Henter,

    1. Childhood Cancer Research Unit, Department of Women's and Children's Health, Karolinska Institutet, Karolinska University Hospital Solna, Stockholm, Sweden
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    • These authors have contributed equally as senior authors.
  • Bengt Fadeel,

    1. Childhood Cancer Research Unit, Department of Women's and Children's Health, Karolinska Institutet, Karolinska University Hospital Solna, Stockholm, Sweden
    2. Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden
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    • These authors have contributed equally as senior authors.

Correspondence: Göran Carlsson, MD, PhD, Childhood Cancer Research Unit, Karolinska University Hospital, Q6:05, SE-171 76 Stockholm, Sweden.

E-mail: goran.carlsson@ki.se

Summary

Severe congenital neutropenia (SCN) is characterized by low blood neutrophil counts, early bacterial infections, and risk of leukaemia development. As yet, no population-based incidence estimates of SCN have been reported. Children less than 16 years of age with SCN were sought in Sweden during the 20-year period 1987–2006 by a questionnaire to all Swedish Departments of Paediatrics and by reviewing the Swedish Health and Welfare Statistical Databases. Thirty-two patients were diagnosed with congenital neutropenia during this period. All received treatment with recombinant granulocyte-colony stimulating factor (G-CSF). Twenty-one patients were diagnosed as SCN or probable SCN, corresponding to 1·0 per 100 000 live births. Nine (43%) had ELANE mutations, four (19%) HAX1 mutations and eight (38%) were children with disease of unknown genetic aetiology. Four out of 21 patients (19%) developed myelodysplastic syndrome/leukaemia and three (14%) died, all with leukaemia. The cumulative incidence of myelodysplastic syndrome/leukaemia was 31%. The observed incidence of SCN in this population-based study was higher than previously estimated, possibly because genetic testing now can identify SCN cases previously thought to be idiopathic or benign neutropenia. The risk of developing myelodysplastic syndrome/leukaemia is considerable. ELANE mutations are the most commonly identified genetic defects.

Severe congenital neutropenia (SCN) includes a heterogeneous group of rare disorders characterized by very low absolute neutrophil count (ANC) in the peripheral blood (<0·5 × 109/l), maturation arrest in the bone marrow of the myelopoiesis at the promyelocyte/myelocyte stage, and early onset of bacterial infections (Carlsson & Fasth, 2001). SCN was first described as an autosomal hereditary disorder by the Swedish paediatrician Rolf Kostmann (Kostmann, 1956). He described it as an autosomal recessive haematological disorder (Kostmann, 1956, 1975). We now know that most cases of SCN are autosomal dominant or sporadic, and the eponym Kostmann disease is now commonly used for patients with the recessive form of SCN (Carlsson et al, 2006a, 2007). During the last 10 years, mutations that cause SCN have been identified in several genes. The recessive form of the disease was shown to be the result of homozygous mutations in the HAX1 gene (Klein et al, 2007). Genetic analyses of autosomal dominant and sporadic cases of SCN have revealed mutations in the ELANE gene (formerly known as ELA2), encoding neutrophil elastase, in a majority of cases (Dale et al, 2000). Other, less frequently mutated genes include CDKN2A (p14), and G6PC3 in recessive SCN, GFI1 in autosomal dominant SCN and WAS in X-linked SCN (XLN) (Devriendt et al, 2001; Person et al, 2003; Bohn et al, 2007a; Boztug et al, 2009). The term SCN is also commonly used for patients with ANC persistently below 0·5 × 109/l, maturation arrest of myelopoiesis and severe bacterial infections early in life, even in the absence of mutations in any of the genes known to cause SCN. In addition, congenital neutropenia may also be associated with other disorders, such as Shwachman-Diamond Syndrome (SDS), glycogen storage disease, type 1b (GSD1b), Cohen and Barth syndromes (Johnston et al, 1997; Bohn et al, 2007b).

The frequency of the different gene defects in SCN appears to be related to the ethnic composition of the patient population. Hence, the reported frequency of ELANE mutations is highly variable, ranging from between 30% in a British population (Smith et al, 2009) and 56% in the North American Chronic Neutropenia Repository (Xia et al, 2009). For HAX1 mutations, the figures vary between 0% in North America and 4% in Britain, while such mutations are present in 12% of the patients with SCN in the European sub-registry of the Severe Chronic Neutropenia International Registry (SCNIR) (Zeidler et al, 2009). ELANE mutations account for 57% of the cases reported. Overall, about one third of the SCN patients have no known disease-causing mutation.

Severe congenital neutropenia is a premalignant disorder. Rosenberg et al (2010) reported a cumulative incidence of myelodysplastic syndrome/acute myeloid leukaemia (MDS/AML) of 22% in SCN patients enrolled in SCNIR, after 15 years on granulocyte-colony stimulating factor (G-CSF) therapy. The extent to which continuous G-CSF treatment may contribute to the evolution of secondary malignancies has been debated (Zeidler et al, 2000). Notably, patients with SDS and XLN may also develop MDS/leukaemia (Dale et al, 2006; Beel & Vandenberghe, 2009).

One group estimated the frequency of SCN as 1-2 per million individuals (Zeidler et al, 2009), while the incidence of SCN in a well-defined population has not been previously reported. Here, we studied the incidence of SCN in the Swedish population, and also report the SCN-causing mutations found and the incidence of MDS/leukaemia.

Methods

Children less than 16 years of age with SCN were sought in Sweden during the 20-year period between 1 January 1987 and 31 December 2006. The cohort was divided into two 10-year periods to determine whether there was a difference in the frequency of SCN during these time periods. During the total time period 2 092 401 children were born; 1 136 287 during 1987–1996 and 956 114 during 1997–2006 (National Board of Health & Welfare in Sweden, 2009). Two independent methods were used to identify children with SCN (Table 1). First, a questionnaire was sent to all Departments of Paediatrics in Sweden with a request to identify patients at their Department that had been diagnosed with SCN between 1987 and 2006. Patients with the following International Classification of Disease (ICD) code numbers in ICD-9 and ICD-10 were requested: 288, 288A, 288W, 270C, 271X, 277W 279N in ICD-9 and D70, D70·9, D70·9A, D70·9B, D70·9C, D70·9D, D70·9W, D72, E70·3A, E71·1C, E74·0 in ICD-10 (WHO, 1978, 2010). Secondly, we searched for the same code numbers in ICD-9 and ICD-10 in the Swedish Health and Welfare Statistical Databases, at the National Board of Health and Welfare in Sweden. A list of the ICD codes and diseases/diagnoses is presented as Supplemental Material. Inclusion criteria were presence of a mutation in the genes known to be associated with SCN (HAX1, ELANE, CDKN2A, G6PC3, WAS, GFI1). A probable SCN case was defined as a case with persistent ANC < 0·5 x 109/l, maturation arrest of myelopoiesis at the promyelocyte/myelocyte level and recurrent bacterial infections, but no identified mutation in the genes listed above. An application for ethical vetting was sent to The Regional Ethics Review Board in Stockholm, which advised that ethical vetting was not needed according to Swedish law applicable at that time.

Table 1. The diagnoses recognized in the study
Diagnoses1987–19961997–2006Total
  1. SCN, severe congenital neutropenia.

SCN-ELANE459
SCN-HAX1404
SCN-Unknown mutation358
Shwachman-Diamond syndrome235
Glycogen storage disease, type 1b202
Cohen syndrome011
Barth syndrome011
Griscelli syndrome type 2011
Pearson's anaemia011
Total151732

Results

Thirty-two patients were diagnosed with congenital neutropenia during the 20-year period 1987–2006 (Table 1). Twenty-one of these were diagnosed as SCN (n = 13) and probable SCN (n = 8), respectively (Table 2), giving a total incidence of 1·0 per 100 000 live births of verified or probable SCN. No major difference in incidence was found for the two 10-year periods; 11 patients were diagnosed between 1987 and 1996 and 10 patients diagnosed between 1997and 2006. Nine children (43%) had heterozygous ELANE mutations, four (19%) homozygous or compound heterozygous HAX1 mutations and eight (38%) had no known mutations, including two that were not tested for mutations. All HAX1 mutations were diagnosed during the first time period and they have all been reported previously (Carlsson et al, 2008, 2011). The male:female ratio was 2:1 (14 males, 7 females. In the second time period all patients with ELANE mutations were males. Five of the nine ELANE mutations were familial cases, with two pairs of siblings, and the other four were sporadic (Table 3). Two ELANE mutations were new mutations not previously reported; both were familial cases. Five patients were negative for mutations in ELANE and HAX1. One was also negative for mutations in GFI1 and CSF3R. We did not screen for other neutropenia-related gene mutations e.g. CDKN2A or G6PC3, as our patients did not correspond to the reported phenotypes. Mutational analysis could not be performed in two patients. One of these patients had no typical maturation arrest in the bone marrow but the clinical presentation was otherwise consistent with SCN. Among the other patients with congenital neutropenia, five patients had SDS, two patients had GSD1b, and one each was diagnosed with Barth syndrome, Cohen syndrome, Griscelli syndrome type 2 and Pearson syndrome, respectively.

Table 2. Data of the SCN patients in relation to the underlying genetic defect
 NumbersSex (female/male)Age at diagnosisMDS/leukaemiaHSCTDead
  1. SCN, severe congenital neutropenia; MDS, myelodysplastic syndrome; HSCT, haematopoietic stem cell transplantation.

1987–1996
SCN-ELANE42/20–3 months11 
SCN-HAX141/31–7 months131
Unknown mutations32/112 months–3½ years0  
SCN, total11     
1997–2006
SCN-ELANE50/50 months–6 years111
SCN-HAX10     
Unknown mutations52/31 months–2½ years111
SCN, total10     
Total, 1987–2006217/14 463
Table 3. Patient characteristics for patients with genetically verified (n = 13) and probable SCN (n = 8)
PatientAge (years)DiagnosisSexAge at diagnosisMaturation arrestMutationsG-CSF (μg/kg/d)MDS/LeukaemiaHSCTComments
  1. 1Karlsson et al (2007), 2Carlsson et al (2008), 3Carlsson et al (2011), 4Carlsson et al (2009), 5Lundén et al (2009).

  2. SCN, severe congenital neutropenia; G-CSF, granulocte colony-stimulating factor; MDS, myelodysplastic syndrome; HSCT, haematopoietic stem cell transplantation; F, female; M, male.

1987–1996
124SCNF3 monthsYesELANE: c.164G>C (p.Cys55Ser)15·5  Mother with ELANE mutation
223SCNF13 monthsYesHAX1: [c.568C>T]+[c.568C>T] (p.Gln190X)2,3. Neg ELANE3 YesHSCT due to CSF3R mutation
322SCNMNewbornYesELANE: c.164G>C (p.Cys55Ser)18  Brother to Patient 1
421SCNM18 monthsYesHAX1: [c.131G>A]+[c.131G>A] (p.Trp44X)2,3. Neg ELANE5 YesHSCT due to patients own wish
520Probable SCNM12 monthsYesNeg ELANE and HAX13·8   
619SCNM3 monthsYesELANE: c.259_261del (p.His87del)315YesYesSevere GVHD
718SCNM5 monthsYesHAX1: [c.568C>T]+[c.568C>T] (p.Gln190X)2,3. Neg ELANE6YesYesDied after HSCT
818SCNM3 monthsYesHAX1: [c.91del]+[c.568C>T] (p.Glu31LysfsX54 +  p.Gln190X)4. Neg ELANESporadic   
917SCNFNewbornYesELANE: c.367C>T (p.Leu123Phe)7·5  Mutation not previously reported. Mother with ELANE mutation
1016Probable SCNF3·5 yearsYesNeg ELANE and HAX113   
1115Probable SCNF3·5 yearsNoNot analysed1·2   
1997–2006
1213Probable SCNM1 monthYesNot analysed60Yes Died at 21 months of age with malignant histiocytosis/leukaemia. Father and grandfather have agranulocytosis
1313SCNM4 monthsYesELANE: [c.90T>G + c.194T>A] (p.Ile30Met and p.Val65Asp)5. Neg HAX112   
1413SCNMNewbornYesELANE: c.367C>T (p.Leu123Phe)5·3  Brother to Patient 9
1511Probable SCNM15 monthsYesNeg ELANE and HAX17   
1611SCNM2 monthsYesELANE: c.452G>C (p.Cys151Ser)350YesYesDied after HSCT
1710SCNM5 monthsYesELANE: c.458C>A (p.Ala153Asp)14·5   
189Probable SCNF1·5 yearsYesNeg ELANE and HAX13·2   
198SCNM6 yearsNoELANE: c.367-1G>C (p.?)Sporadic  Mutation not previously reported. Mother with ELANE mutation
207Probable SCNF13 monthsYesNeg ELANE, HAX1, GFI1 and CSF3R380 YesHSCT due to G-CSF resistance
215Probable SCNM2·5 yearsYesNeg ELANE and HAX130   

The time of diagnosis varied between birth and 6 years of age for patients with ELANE mutations, 3 to 7 months for HAX1 mutations, and 1 month to 3·5 years for children with unknown mutations. Four out of 21 patients with SCN (19%) developed MDS/leukaemia; two had ELANE mutations, one had HAX1 mutations, and one who was never tested for mutations. The time between diagnosis of SCN and development of MDS/leukaemia varied between 1·5 and 13 years. All patients were on G-CSF treatment. Three of these patients were considered to be relatively resistant to G-CSF, as they did not achieve normal ANC at doses of 50–80 μg G-CSF/kg per day. At 15 years of age, the cumulative incidence was 25% for MDS/leukaemia in the entire cohort of patients with SCN (n = 21) (Fig 1A). The cumulative incidence of MDS/leukaemia after 15 years on treatment with G-CSF in the 18 children regularly treated with G-CSF was 31% (Fig 1B). The corresponding figures at 10 years were 10% and 12%, respectively. Three patients (14%) have died, all with leukaemia. None of the patients died because of infections. Six patients have undergone haematopoietic stem cell transplantation (HSCT) (Carlsson et al, 2011).

Figure 1.

Cumulative incidence of MDS/leukaemia. (A) Cumulative incidence of MDS/leukaemia in 21 patients with SCN, related to age in years after birth. Last follow up date was set to 1st of July 2011. (B) Cumulative incidence of MDS/leukaemia in 18 patients with SCN and regular G-CSF treatment, related to years of G-CSF treatment. Note that three patients were excluded from the cohort of 21 patients: two patients received G-CSF treatment only sporadically and one patient only had G-CSF treatment for a short time and received haematopoietic stem cell transplantation at an early stage because she was G-CSF resistant. Last follow up date was set to 1st of July 2011.

No patient was lost to follow up.

Discussion

We report herein an incidence of SCN in Sweden of 1·0 per 100 000 live births, including patients with verified disease-causing mutations and patients with probable SCN. On average one child per year in Sweden has been diagnosed with SCN. Previously, the prevalence of SCN has been estimated to 1–2 patients per million inhabitants with equal gender distribution (Zeidler et al, 2009). This estimate implies that Sweden, with a population of about 9 million inhabitants, would have 9–18 patients currently alive with SCN. In fact, our study identified 21 patients born during a period of 20 years, and of these, 18 patients are currently alive corresponding to a minimal prevalence of two patients per million inhabitants. However, the current study does not provide information on patients alive and born prior to 1987, who would have contributed to even higher prevalence figures.

The present study was not designed to determine the incidence of all known SCN mutations. Notwithstanding, we can report the frequency of mutations in ELANE and HAX1, the two most common genetic SCN-causing defects, in this population-based study. Thus, while the overall proportion of patients with known mutations (62%) is comparable to previous reports, we found a higher proportion of patients harbouring HAX1 mutations as compared to other European and US surveys (Smith et al, 2009; Xia et al, 2009; Zeidler et al, 2009). This is probably due to the fact that patients from the original Kostmann family (n = 3) were included in our cohort. Notably, no patient with HAX1 mutations has been born since 1993, while the number of patients with ELANE mutations have increased somewhat. We also found a high proportion of males (14/21, 67%) while others have reported an equal gender distribution (Dale et al, 2000; Xia et al, 2009), but there is a slight male predominance in the SCNIR (Dale et al, 2006). The male predominance in our study could suggest X-linked inheritance; however, the phenotype for XLN does not fit to our patients and we did therefore not screen for mutations in WAS.

In our cohort, 4 out of 21 patients (19%) developed leukaemia, and the cumulative incidence of MDS/leukaemia after 15 years on G-CSF treatment was 31%, which is higher than reported by the SCNIR with a cumulative incidence of 22% MDS/AML after 15 years of G-CSF treatment (Rosenberg et al, 2010). The French Severe Chronic Neutropenia Registry reported a risk of MDS/AML of 10·8% at 20 years of age, but they did not relate this to G-CSF treatment (Donadieu et al, 2005). These differences could be a result of our smaller population, as an additional case of MDS/leukaemia would have a proportionally higher impact. Furthermore, a different genetic background of SCN could yield a higher risk of malignant transformation, although this remains to be understood. Finally, in a small country, the health care system may be more likely to detect a rare disorder and avoid not detecting an underlying mild neutropenia before it transforms to MDS/leukaemia.

Of the four patients that developed MDS/leukaemia in the present study, two had ELANE mutations, one harboured a HAX1 mutation, and one died before genetic diagnosis was possible. In total, 3/21 (14%) patients died, all with MDS/leukaemia. Hence, patients with SCN, irrespective of mutations, should be followed closely for leukaemia transformation.

The role of continuous G-CSF treatment for the malignant evolution is not fully understood. The commonly held view is that the growth factor, G-CSF, might stimulate an established malignant clone but that this treatment may not trigger the emergence of such a clone. Leukaemia has been reported in patients with SCN prior to the introduction of treatment with G-CSF, while MDS/leukaemia has not been reported in G-CSF-treated patients with cyclic neutropenia (Carlsson et al, 2006a, 2007). It has been suggested that the pathogenesis of SCN and its evolution to secondary malignancies may involve an underlying genomic instability and pharmacological doses of G-CSF could potentially stimulate the stepwise acquisition of genetic changes in bone marrow cells in these patients and proliferation of a potential malignant clone of cells (Carlsson et al, 2006b, 2011; Zeidler et al, 2009). In a very recent study, Beekman et al (2012) performed next-generation sequencing on serial haematopoietic samples of an SCN patient who developed AML 17 years after initiation of G-CSF therapy. The authors concluded that the sequential gain of two CSF3R mutations implicated abnormal G-CSF signalling as the driver of leukaemic transformation in this particular case.

In our study no patient has died of infections, while SCNIR reported a 10% septicaemia-related mortality (Rosenberg et al, 2010) and the French Severe Chronic Neutropenia Registry reported six septic deaths out of 101 patients with SCN, although none of these patients received G-CSF (Donadieu et al, 2005). These differences compared to our study might be due to our more uniform patient population and that more of our patients are on G-CSF. We also believe that the mortality of septic infections patients with SCN has decreased due to a higher awareness of neutropenic fever in this patient group as well as in patients with therapy related-neutropenia in patients treated for malignant disease.

Normally, the diagnosis of SCN is established during the first year of life as a result of severe bacterial infections. With the increasing availability of a molecular genetic diagnosis, we have diagnosed some patients at a later age with clinically milder symptoms, the oldest being 6 years at diagnosis. Previously, these children may have been diagnosed as idiopathic neutropenia. Therefore, we conclude that mutation analysis should also be considered for children older than 1 year with a suspicion of SCN despite having a clinically milder phenotype. In the present study, two patients displayed persistent severe neutropenia and recurrent bacterial infections yet did not fulfil the criteria of maturation arrest. Notably, one of these patients harboured an ELANE mutation. Therefore, SCN may have to be considered also in patients who do not present with all of the typical clinical and laboratory signs. It is also known that it is difficult to clearly define maturation arrest. In our opinion it is sometimes difficult to separate a left winged myelopoiesis and a maturation arrest.

In summary, we found an incidence of SCN in the Swedish population of 1·0 per 100 000 live births. This is the first published incidence study of SCN. We also observed a high risk of evolution to MDS/leukaemia, corresponding to about 30% after 15 years on G-CSF therapy. We noted that more patients with later onset and also less prominent disease are now being identified, probably as a result of the availability of molecular diagnosis for many, but not all, cases of SCN.

Acknowledgements

The authors would like to thank all colleagues at the Departments of Paediatrics in Sweden that have contributed to this study.

Funding

The study was supported by grants from the Swedish Children's Cancer Foundation (GC, JIH), Swedish Cancer Foundation (BF, MN, JIH), Swedish Research Council (BF, MN, JIH), and Stockholm County Council (ALF project) (project coordinator: BF).

Authorship and disclosures

GC and JIH initiated the study. GC, JIH, JP, MN and BF contributed to the study design. KLR and MN were responsible for the genetic analysis. AF participated in collecting data. EB participated in the statistical analysis. GC wrote the first draft of the manuscript. All authors contributed to data analysis and the final version of the manuscript. The authors have no conflicts of interest to declare.

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