Correspondence Laurent Pascal1 Odile Beyne-Rauzy2,3 Sabine Brechignac4,3 Sylvestre Marechaux1 Dominique Vassilieff5,3 Olivier Ernst6 Celine Berthon7,3 Emmanuel Gyan8,3 Marie-Pierre Gourin9,3 Franc¸ois Dreyfus5,3 Pierre Fenaux4,3 Christian Rose1,3
Toulouse-Purpan, Toulouse, 3GFM, Groupe Francophone des Myelodysplasies, Hopital Avicenne, Paris (AP-HP), 4H^opital Avicenne, Assistance Publique des H^opitaux de Paris (AP-HP), 5H^opital Cochin, AP-HP, Paris, 6Radiologie, H^opital Huriez, CHU de Lille, 7Hematologie, CHU de Lille, Lille, , 8Hematologie, H^opital Bretonneau, CHU de Tours, Tours, and 9CHU de Limoges, Limoge, France E-mail:
[email protected]
Keywords: MRI, transfusion, MDS First published online 9 May 2013 doi: 10.1111/bjh.12368
1
Service d’Hematologie et de cardiologie H^opital Saint Vincent de Paul, Universite Catholique de Lille, Lille, 2Service de medecine interne, CHU
References Anderson, L.J., Holden, S., Davis, B., Prescott, E., Charrier, C.C., Bunce, N.H., Firmin, D.N., Wonke, B., Porter, J., Walker, J.M. & Pennell, D.J. (2001) Cardiovascular T2-star (T2*) magnetic resonance for the early diagnosis of myocardial iron overload. European Heart Journal, 22, 2171–2179. Chacko, J., Pennell, D.J., Tanner, M.A., Hamblin, T.J., Wonke, B., Levy, T., Thomas, P.W. & Killick, S.B. (2007) Myocardial iron loading by magnetic resonance imaging T2* in good prognostic myelodysplastic syndrome patients on long-term blood transfusions. British Journal of Haematology, 138, 587–593. Di Tucci, A.A., Matta, G., Deplano, S., Gabbas, A., Depau, C., Derudas, D., Caocci, G., Agus, A. & Angelucci, E. (2008) Myocardial iron overload assessment by T2* magnetic resonance imaging in adult transfusion dependent patients with acquired anemias. Haematologica, 93, 1385–1388.
Ezekowitz, J.A., McAlister, F.A. & Armstrong, P.W. (2003) Anemia is common in heart failure and is associated with poor outcomes: insights from a cohort of 12 065 patients with new-onset heart failure. Circulation, 107, 223–225. Gandon, Y., Olivie, D., Guyader, D., Aube, C., Oberti, F., Sebille, V. & Deugnier, Y. (2004) Non-invasive assessment of hepatic iron stores by MRI. Lancet, 363, 357–362. Konen, E., Ghoti, H., Goitein, O., Winder, A., Kushnir, T., Eshet, Y. & Rachmilewitz, E. (2007) No evidence for myocardial iron overload in multitransfused patients with myelodysplastic syndrome using cardiac magnetic resonance T2 technique. American Journal of Hematology, 82, 1013–1016. Malcovati, L., Porta, M.G.D., Pascutto, C., Invernizzi, R., Boni, M., Travaglino, E., Passamonti, F., Arcaini, L., Maffioli, M., Bernasconi, P., vvvvLazzarino, M. & Cazzola, M. (2005) Prognostic factors and life expectancy in myelodysplastic syndromes classified according to WHO
criteria: a basis for clinical decision making. Journal of Clinical Oncology, 23, 7594–7603. Malcovati, L., Della Porta, M.G., Strupp, C., Ambaglio, I., Kuendgen, A., Nachtkamp, K., Travaglino, E., Invernizzi, R., Pascutto, C., Lazzarino, M., Germing, U. & Cazzola, M. (2011) Impact of the degree of anemia on the outcome of patients with myelodysplastic syndrome and its integration into the WHO classification-based Prognostic Scoring System (WPSS). Haematologica, 96, 1433–1440. Pennell, D.J. (2005) T2* magnetic resonance and myocardial iron in thalassemia. Annals of the New York Academy of Sciences, 1054, 373–378. Roy, N.B.A., Myerson, S., Schuh, A.H., Bignell, P., Patel, R., Wainscoat, J.S., McGowan, S., Marchi, E., Atoyebi, W., Littlewood, T., Chacko, J., Vyas, P. & Killick, S.P. (2011) Cardiac iron overload in transfusion-dependent patients with myelodysplastic syndromes. British Journal of Haematology, 154, 521–524.
Novel deep intronic and missense UNC13D mutations in familial haemophagocytic lymphohistiocytosis type 3
Familial haemophagocytic lymphohistiocytosis (FHL) is a hyperinflammatory disorder that typically presents in infancy (Filipovich, 2011; Janka, 2012) with an estimated incidence of 1 in 50 000 live births (Henter et al, 1991). Chemo- and immunotherapy can control the disease, but haematopoietic stem cell transplantation (HSCT) is the only cure. FHL is associated with autosomal recessive mutations in PRF1, UNC13D, STX11 and STXBP2 (Pachlopnik Schmid et al, 2010). In this study, we report two novel mutations in UNC13D, one of which is located in intron 1 of the gene, adjacent to a previously reported intronic mutation (c.118-308C > T) causative ª 2013 John Wiley & Sons Ltd British Journal of Haematology, 2013, 162, 413–427
of FHL3 in a number of patients world-wide (Meeths et al, 2011; Seo et al, 2013). The index patient (II-3) was a previously healthy 8-year old boy, the third child of non-consanguineous Chinese parents from Singapore. Patients and methods are described in detail in the Supporting Information (Data S1). To determine whether the patient, who rapidly developed haemophagocytic lymphohistiocytosis (HLH), might represent a case of FHL, the phenotype of cytotoxic lymphocytes was examined. This was done on the patient, the parents (father I-1; mother I-2), two brothers (II-1 and II-2), as well as transport- and local 415
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(A)
(B)
Fig 1. Cytotoxic T cell and NK cell degranulation. Peripheral blood mononuclear cells (PBMCs) from one local Swedish control, one Singaporean transport control, as well as Singaporean family members were rested overnight (A) or stimulated with interleukin 2 (IL2) for 36 h (B). PBMCs were mixed with P815 target cells and either anti-CD3 or anti-CD16 monclonal antibodies, as indicated, at 37°C for 3 h. Thereafter, cells were surface-stained with fluorochrome-conjugated antibodies to CD3, CD4, CD8, CD56, CD57, and CD107a. Cytotoxic T cells were gated as CD3+CD4 CD8+CD57+ cells, while NK cells as CD3 CD56dim cells. Bar charts show the relative percentage of surface CD107apositive cells after subtraction of background degranulation of PBMCs with P815 target cells alone.
controls 2 months after the clinical diagnosis of HLH. In this setting, where samples were transported far afield for functional analyses, conventional natural killer (NK) cell cytotoxicity and degranulation assays using K562 target cells (Bryceson et al, 2012) were difficult to interpret. However, assays quantifying degranulation triggered by engagement of the T cell receptor (CD3) on cytotoxic T cells or the Fc receptor (CD16) on NK cells were more robust (Chiang et al, 2013). Whereas parental and transport controls displayed normal degranulation, the patient (II-3) and remarkably also the middle brother (II-2) displayed defective cytotoxic T cell and NK cell-mediated degranulation in response to anti-CD3 or anti-CD16, respectively (Fig 1A). Upon stimulation with interleukin 2, NK cell and T cell degranulation increased in the two affected brothers (Fig 1B). Thus, the results demonstrate how new read-outs of cytotoxic lymphocyte degranulation excel in the identification of patients with defective cytotoxic lymphocyte function. Based on the functional results, the FHL genes required for lytic granule exocytosis (UNC13D, STX11, and STXBP2) were bi-directionally sequenced in genomic DNA from the patient. A novel heterozygote UNC13D c.1388A > C; p.Gln463Pro missense mutation in the second last base of exon 15 was identified (Fig 2A, B). Furthermore, sequencing of intron 1 of 416
UNC13D revealed a heterozygous c.118-307G > A mutation (Fig 2A, B), located to an evolutionary conserved region. The oldest brother (II-1) was wild-type for the exon 15 mutation but carried the intron 1 mutation. Importantly, both mutations were also identified in brother II-2, with defective degranulation and cytotoxicity. Neither of the mutations was identified in the Single Nucleotide Polymorphism database, 1000 genomes or in any of the 96 healthy blood donors used as controls. The p.Gln463Pro amino acid change was not predicted to be damaging with the mutation prediction software Alamut. With the same prediction software, the donor splice site affected by the c.1388A > C mutation was predicted to be functional, albeit with reduced affinity. Analysis of cDNA from the family members carrying the heterozygous mutations revealed normal size and sequence. Notably, in the two affected brothers the mutated c.1388C allele was predominantly amplified, suggesting that transcription of the other allele carrying the intronic c.118-307G > A mutation might be impaired. Thus, allele-specific quantitative real-time polymerase chain reaction (PCR) was performed on the family members carrying this mutation. In exon 11 of UNC13D, a common synonymous polymorphism (c.888G > C) was used to discriminate between the mutated (linked to the c.888G allele) and wild-type allele (linked to the c.888C allele) in carriers of the c.118-307G > A mutation. For comparison, allele-specific transcription was quantified in healthy controls that did not carry the c.118307G > A mutation but were heterozygous for the c.888G > C polymorphism. In the heterozygous carriers (I-2, II-1, II-2 and II-3) of the c.118-307G > A mutation, transcription of the allele carrying the linked c.888G polymorphism was significantly reduced, representing 10% of total UNC13D transcripts (P = 0021) (Fig 2C). These data indicate that the c.118-307G > A mutation impairs UNC13D transcription, possibly by disrupting a transcription factor binding-site or enhancer element. The very low abundance of the transcript with the c.118-307G > A mutation implied that predominantly the transcript with the p.Gln463Pro missense mutation would be translated. To compare expression of mutated and wild-type UNC13D (Munc13-4), Western blot analysis was performed. Whereas UNC13D was clearly present in the parents and brother II-1, its expression was severely reduced in LAK cells from the brother with compound heterozygous mutations in UNC13D (Fig 2D), indicating that the mutated UNC13D was degraded. Expression of STXBP2 (Munc18-2), the protein encoded by STXBP2, was however not affected by the UNC13D mutations. Although the glutamine residue at position 463 is weakly conserved among species (Figure S1), the amino acid change to proline appears to reduce the protein stability. Introduction of proline residues in proteins is often damaging due to imposed structural constraints of the pyrrolidine ring (Pakula & Sauer, 1989). The combination of defective ª 2013 John Wiley & Sons Ltd British Journal of Haematology, 2013, 162, 413–427
Correspondence
(A)
(B)
(C)
(D)
Fig 2. Genetic findings. (A) Pedigree of the Singaporean family with FHL3 and identified mutations (c.118-307G > A and c.1388A > C) and polymorphism (c.888G > C) in UNC13D. Filled symbols denote phenotypically affected family members. Haplotype bars illustrate the phase and inheritance of the genetic findings. The arrow indicates the index patient. (B) Sequence chromatograms illustrating the identified mutations in UNC13D in the index patient as well as the normal sequences in a healthy control. (C) Allele-specific quantitative real-time polymerase chain reaction (PCR) of the UNC13D intron 1 mutation. Allele-specific expression analysis of UNC13D was assessed in peripheral blood mononuclear cells from four healthy controls and the four heterozygous carriers of the UNC13D intron 1 mutation, c.118-307G > A, using real-time polymerase chain reaction (standard curve method). Statistical significance was analysed using the 2-tailed Mann–Whitney U test (*P < 005). (D) UNC13D expression in the family with compound heterozygous UNC13D mutations. LAK cells from a healthy transport control, the parents (I-1 and I-2), the oldest brother (II-1) and the genetically affected middle brother (II-2) were lysed and protein content was analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis and Western blotting. Rabbit polyclonal antibodies for detection of UNC13D and STXBP2, and mouse monoclonal antibodies for ACTB (actin) were used.
transcription from one UNC13D allele with reduced protein stability encoded by the other allele is a probable cause of the defective cytotoxic lymphocyte function in the two brothers in this study. At the time of writing, the affected brother (II-2) is 13 years old, Epstein-Barr virus (EBV) IgG-seropositive, IgM-seronegative, EBV-PCR negative, and still asymptomatic. The possible causes of this variable phenotypic expression within the family are not clear. Environmental factors or modifier genes could be involved. Our results imply that a thorough functional evaluation of HLH patients as well as sibling is of great importance since they may be genetically affected and thus have a risk of developing disease late but with rapid-onset and high mortality. There are several arguments in favour of transplanting genetically affected asymptomatic individuals immediately, rather than waiting until they present with disease (Machaczka et al, 2012). However, this is a difficult decision. If affected individuals are not directly scheduled for HSCT, they should be monitored carefully and the identification of a suitable potential donor is of value. This study highlights the necessity of also examining non-coding evolutionary conserved sequences for identification ª 2013 John Wiley & Sons Ltd British Journal of Haematology, 2013, 162, 413–427
of mutations in inherited haematological disorders, in order to give more patients a correct genetic diagnosis.
Acknowledgements The authors thank the patient and his family for participation. Nina J€antti and Monika Mastafa are acknowledged for technical assistance. This work was supported by the Swedish Research Council, Swedish Cancer Foundation, Swedish Children’s Cancer Foundation, The Society for Child Care, Shizu Matsumura’s Donation, Ake Olsson Foundation for Haematological Research, Histiocytosis Association, Jeansson’s Foundation, Ake Wiberg’s Foundation, the Karolinska Institute Research Foundation, and the Stockholm County Council (ALF project).
Author contributions ME, SCCC, HS and MM performed the experiments and data analysis. MYC, SNM and SYS recruited patients and samples. The project was designed and supervised by YTB, JIH and MN. ME and YTB wrote the initial draft and all the 417
Correspondence authors contributed to the review and approval of the final manuscript.
Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden, 4Haematology/Oncology Service, Department of Paediatric Subspecialties, KK Women’s and Children’s Hospital, Singapore and 5Broegelmann Research Laboratory,
Conflict of interest
The Gades Institute, University of Bergen, Bergen, Norway E-mail:
[email protected]
The authors declare no conflict of interest. Miriam Entesarian1,2 Samuel C. C. Chiang3 Heinrich Schlums3 Marie Meeths1,2 Mei-Yoke Chan4 Soe-Nwe Mya4 Shui-Yen Soh4 Magnus Nordenskj€ old2 1 Jan-Inge Henter Yenan T. Bryceson3,5
Keywords: familial haemophagocytic lymphohistiocytosis, NK cells, T cells, UNC13D (Munc13-4), deep intronic mutation First published online 14 May 2013 doi: 10.1111/bjh.12371
Supporting Information
1
Childhood Cancer Research Unit, Department of Women’s and
Children’s Health, Karolinska Institutet, Karolinska University Hospital Solna, 2Clinical Genetics Unit, Department of Molecular Medicine and Surgery, and Centre for Molecular Medicine, Karolinska Institutet, Karolinska University Hospital Solna, 3Centre for Infectious Medicine,
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Additional Supporting Information may be found in the online version of this article: Data S1. Patients and methods. Fig S1. Alignment from Alamut of amino acid conservation across species at the site of the c.1388A > C; p.Gln463Pro mutation.
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