Clinical Immunology (2014) 154, 100–104
available at www.sciencedirect.com
Clinical Immunology www.elsevier.com/locate/yclim
BRIEF COMMUNICATION
NK and B cell deficiency in a MPS type II family with novel mutation in the IDS gene Leuridan Cavalcante Torres a,c,⁎, Diogo Cordeiro de Queiroz Soares a,b , Leslie Domenici Kulikowski d , Jose Francisco Franco b , Chong Ae Kim b a
Translational Research Laboratory Prof. C. A. Hart, Instituto de Medicina Integral Prof. Fernando Figueira (IMIP), Recife, Brazil b Medical Genetics Unit, Hospital das Clínicas, Faculdade de Medicina, Universidade de São Paulo (USP), São Paulo, Brazil c Medical Investigation Laboratory (LIM 36), Hospital das Clínicas, Faculdade de Medicina, Universidade de São Paulo (USP), São Paulo, Brazil d Department of Pathology, Citogenomics Laboratory (LIM 03), Universidade de São Paulo (USP), São Paulo, Brazil
Received 2 July 2013; accepted with revision 4 July 2014 KEYWORDS Mucopolysaccharidoses; NK cells; B cells; Immunodeficiency; Autoimmunity
Abstract The mucopolysaccharidoses (MPSs) are a group of rare, inherited lysosomal storage disorders that are clinically characterized by abnormalities in multiple organ systems and reduced life expectancy. Whereas the lysosome is essential to the functioning of the immune system, some authors suggest that the MPS patients have abnormalities in the immune system similar to the patients with primary immunodeficiency. In this study, we evaluated 8 male MPS type II patients of the same family with novel mutation in the IDS gene. We found in this MPS family a quantitative deficiency of NK and B cells with normal values of IgG, IgM and IgA serum antibodies and normal response to polysaccharide antigens. Interestingly, abnormalities found in these patients were not observed in other MPS patients, suggesting that the type of mutation found in the IDS gene can be implicated in the immunodeficiency. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Hunter syndrome, or mucopolysaccharidosis type II (MPS II, OMIM 607014), is a rare X-linked recessive disorder caused by a deficiency of the lysosomal enzyme iduronate-2-sulfatase ⁎ Corresponding author at: Translational Research Laboratory Prof. C. A. Hart, Instituto de Medicina Integral Prof. Fernando Figueira (IMIP), Rua dos Coelhos, 300, Recife, PE, 50070-550, Brazil. Fax: +55 81 2122 4703. E-mail address: [email protected] (L.C. Torres).
http://dx.doi.org/10.1016/j.clim.2014.07.001 1521-6616/© 2014 Elsevier Inc. All rights reserved.
(IDS), leading to the progressive accumulation of glycosaminoglycans (GAGs) in several organs. It affects approximately 1 in 170,000 male newborns, whereas female carriers rarely manifest symptoms [1]. The phenotypic spectrum of MPS II varies, with two typical clinical forms. The severe form is responsible for approximately 75% of all MPS II patients, with the signs and symptoms emerging between 18 months and 4 years of age. It is characterized mostly by facial dysmorphism, hepatosplenomegaly, hernias, short stature, stiff joints and contractures, cardiac valve disease and obstructive respiratory
NK and B cell deficiency in a MPS type II family with novel mutation in the IDS gene Table 1
101
Immunophenotyping of T, B and NK cells of 8 male family with MPS type II.
Patient Total T cells (P) lymphocytes (CD3*)
T CD4* (CD3*CD4*)
P1 P2 P3 P4 P5 P6 P7 P8
944 (476–1136) 554 (248–724) 2,1 798 (476–1136) 625 (248–724) 1,5 1278 (476–1136) 852 (248–724) 1,9 1193 (618–1348) 795 (390–1024) 2,5 849 (476–1136) 1082 (248–724) 1,1 329 (618–1348) 536 (390–1024) 0,8 1470 (780–2086) 1064 (453–1700) 1,9 1203 (780–2086) 802 (453–1700) 1,6
1612 1581 1440 2368 2272 1008 2849 2255
1499 (844–1943) 1422 (844–1943) 2131 (844–1943) 1989 (1280–2413) 1931 (844–1943) 866 (1280–2413) 2535 (1515–3701) 2006 (1515–3701)
T CD8* (CD3*CD8*)
Ratio B cells CD4*/CD8* (CD19*)
NK cells (CD3*/CD16*/CD56*)
217 (138–544) 106 (134–545) 221 (138–544) 79 (134–545) 72 (138–544) 34 (134–545) 288 (471–1031) 106 (127–515) 304 (138–544) 52 (134–545) 140 (471–1031) 16 (127–515) 370 (631–1283) 130 (135–601) 252 (631–1283) 22 (135–601)
Absolute values between parentheses indicate the reference values for the Brazilian population, by age, as described by Moraes-Pinto et al. [37].
complications, hearing loss, chronic diarrhea, and communicating hydrocephalus. On the contrary, the attenuated form presents a slower progression of the disease, usually marked by fairly normal intelligence, hepatosplenomegaly, joint contractures and conductive and sensorineural hearing loss [2,3]. Almost 350 mutations in the IDS gene have been described. The majority of these are missense mutations and lead to impaired degradation of GAGs by lysosomes and progressive accumulation of these metabolites [2]. Lysosome is critical for normal function of the immune system and the classical view of the lysosomal compartment as a digestive organelle has now expanded, with the lysosome involved in the control of cell-surface receptor-mediated signal transduction [4,5]. Moreover, the catabolism of macromolecules in the lysosome is essential for the correct function of several immune system functions including antigen processing and presentation [6,7], cytokine secretion [8], phagocytosis [9,10], and secretion of molecules [11,12]. Natural killer (NK) cells are considered to characterize an arm of the innate immune system, as their effector functions are mediated by repertoire of germline-encoded receptors that do not undergo somatic recombination [13,14]. However, NK cells express several costimulatory ligands including OX40L and CD40L (CD 154), which permit them to provide a costimulatory signal to B cells or T cells [15–17]. Thereby, NK cells participate in early defense against intracellular microbial infections and several types of tumors and may also be implicated in autoimmunity and hypersensitivity reactions [18–20]. Given the above, and considering the complexity attributed to MPS for being a chronic, systemic and progressive, in which life expectancy is correlated with the severity of clinical symptoms, whose heart, recurrent respiratory infections and upper airway obstruction are the main causes of death [2], the aim of this investigation was to evaluate the cellular immune response in a Brazilian family with 8 individuals affected by MPS type II.
2. Methods 2.1. Patients We studied 8 male individuals of the same family with MPS type II. The clinical history of the patients and a family tree were previously described by our group in this journal [21].
The evaluated patients includes brothers and cousins with the ages varied from 3 to 27 years (mean of 21 years), all of them reporting recurrent sinopulmonary infections, mild coarse faces, hepatosplenomegaly, hernias, short stature, stiff joints and contractures, cardiac valve disease and communicating hydrocephalus. The patients doesn't require of enzyme-replacement therapy because they have attenuated Form of Hunter Syndrome (MPS II).
2.2. Immunophenotyping Six-color flow cytometric immunophenotyping of peripheral blood was performed on a FACS LSRII FORTESSA (BD Biosciences). The following monoclonal antibodies were used: anti-CD3, anti-CD4, anti-CD8, anti-CD19, anti-CD16, anti-CD56, anti-CD314 (NKG2D). The cells were analyzed with the most appropriate lymphocyte gate using the combination of forward and side scatters and the obtained data were analyzed using FACS Diva software (BD Biosciences).
2.3. Molecular analysis Genomic DNA was extracted from peripheral blood using blood genomic Spin Mini Prep kit (GE Healthcare, USA) and amplified using 9 primer pairs flanking all exons and exon/ intron boundaries. The primers used in this study for exons 1 to 9 were described by Kato et al. [22]. Amplicons were purified by column-purification kit GFX PCR DNA (Amersham Pharmacia Biotech, USA) and sequenced in a MegaBACE 1000 DNA Sequencer (Amersham Pharmacia Biotech, USA). Sequencing analysis was performed using the Chromaspro version 1.34 (Technelysium Pty Ltd., Australia) and was compared with the sequence contained in the NCBI database [23].
2.4. Western blot analysis The membrane was incubated overnight at 4 °C with goat antibody against IDS (0.3 μg/ml, AF2449, R&D Systems Inc., Minneapolis, MN, USA) after treatment with 5% skim milk/ Tris-buffered saline for blocking nonspecific reactions. The immune complexes were detected with peroxidase-labeled horse anti-goat IgG (1:1000, Vector Laboratories Inc., Burlingame, CA, USA) and Immobilon Western HRP Substrate (EMD Millipore, Billerica, MA, USA).
102
Figure 1
L.C. Torres et al.
Relative values of NK cells (CD3−/CD16+/CD56+) of a healthy control and 8 male family with MPS type II by flow cytometry.
NK and B cell deficiency in a MPS type II family with novel mutation in the IDS gene
Figure 2 Iduronate 2-sulfatase protein expression by Western Blot analysis of 7 patients with MPS II family. CTL (control).
3. Results 3.1. Immunological investigation We observed that the majority of patients presented normal absolute number for TCD8+ and TCD4+ lymphocytes, except patient 6 that showed decreased values of TCD4+ with an inversion of CD4/CD8 ratio. Low absolute number of B cells with normal levels of IgG, IgM and IgA and an appropriate response to polysaccharides antigens in five patients (p3, p4, p6, p7 and p8) (Table 1). We found reduced absolute numbers of NK cells with normal expression of stimulatory receptors NKG2D when compared to reference values for the Brazilian population (Fig. 1).
4. Discussion In this study, we describe a family of 8 patients whose molecular analysis revealed a novel mutation in the IDS gene, confirming the diagnosis of MPS type II. It usually cannot be determined whether a certain mutation will cause severe or mild MPS type II. However, missense mutations frequently result in decreased expression of IDS enzyme activity and comprise the majority of gene mutations in MPS type II, unlike mutations that result in complete absence of the enzyme activity, such as total rearrangements or partial deletions that seem to be associated with the severe form of the disease in up to 25% of cases of MPS type II [2]. Western Blot analysis of Iduronate-2-sulfatase (IDS) protein (76 kDa) was performed, according to Araya et al. [24], and detect that all patients express some degree of IDS protein, but wasn't observed a correlation between the protein expression and the patient's phenotype (Fig. 2). In immunological evaluation, we observed absolute and relative NK cells deficiency in all MPS patients and 5 of them present B cell deficiency also, without antibody defects. NK cells are lymphocytes of the innate immune system, do not express TCR or immunoglobulin genes, but in its place use a variety of germline-encoded receptors to induce their functions, which include cytokine production, perforindependent cytotoxicity and expression of co-stimulatory molecules. In this capacity, NK cells participate in antiviral and anticancer responses and can contribute to adaptive immunity [25,26]. Early studies suggested that NK cells, like B cells and myeloid-lineage cells, develop primarily in the bone marrow. NK precursor (NKP) cells derived from hematopoietic stem cells (HSCs) give rise to immature NK (iNK) and then mature NK cells. Several transcription factors, such as Ikaros, Ets-1, PU.1, T-bet and E4BP4 are required for NK cell maturation. Among these, Ets-1, Ikaros and PU.1 are critical for production of NKPs, and the other factors become implicated after cells are committed to the NK cell lineage.
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However, these factors are not necessarily specific to the NK lineage, because their individual deletions sometimes cause defects in other hematopoietic cell lineages [27–29]. The transcription factor Ikaros (also known as LyF-1) was initially described for its ability to bind and activate the enhancer of the CD3δ gene, an early and definitive marker of T cell differentiation [30,31]. However, gene-targeting studies evidenced the fundamental role of Ikaros in hematopoiesis, in particular in T and B lymphocytes, natural killer (NK) and dendritic cells, and stem cells [32–34]. Interestingly, 8 patients with MPS type II of the same family presented immunologic alterations not found in MPS type II patients with other types of mutations in the IDS gene, suggesting that mutation found in this family of MPS is directly associated with low levels of NK cells and B cell on peripheral blood. Similarly, Ng et al. [35] reported a case of a woman that present profound deficiency of NK and B cells, associated with recurrent infections. Based on the clinical and laboratory presentation of this case, the authors considered the many similarities between the clinical case and the Ikaros-null knockout mouse and raise the possibility of a mutation in Ikaros as the cause of an immune defect, including NK and B cell deficiency and emphasizing the importance of a further investigation, since mutation or alternative splicing of Ikaros may cause the findings observed [35]. Given these results, we believe that the IDS gene can act as a transcriptional coactivator of some proteins, such as Ikaros, and that novel mutation in the IDS gene presented by this family may interfere somehow in the expression of Ikaros, compromising the development and maturation of B and NK cells. Thus, further studies are needed to better understand these immune abnormalities. Additionally, it is known that NK cells are distributed on multiple organs, being more frequent in lungs and liver and less frequent in lymph node and thymus, where NK cells are almost undetectable. Therefore, we believe that one of the possibilities for the predisposition of these patients, specifically to recurrent respiratory infections, may be related to the reduced distribution of NK cells in the lungs [36]. In this study, we evaluated 8 male MPS type II patients of the same family with novel missense mutation (p.A77D) in the IDS gene. We found in this MPS family a quantitative deficiency of NK and B cells with normal values of IgG, IgM and IgA serum antibodies and normal response to polysaccharide antigens. Interestingly, these abnormalities found in these patients were not observed in other MPS patients, suggesting that the type of mutation found in the IDS gene can be implicated in the immunodeficiency.
Conflict of interest statement None of the authors has any potential financial conflict of interest related to this manuscript.
Acknowledgments The authors thank the patients, their families and grant No. 2010/52694-8 of the São Paulo Research Foundation (FAPESP).
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References [1] J. Muenzer, The mucopolysaccharidoses: a heterogeneous group of disorders with variable pediatric presentations, J. Pediatr. 144 (5 Suppl.) (2004) S27–S34. [2] R. Martin, M. Beck, C. Eng, et al., Recognition and diagnosis of mucopolysaccharidosis II (Hunter syndrome), Pediatrics 121 (2) (2008) e377–e386 (Review). [3] J. Muenzer, M. Beck, C.M. Eng, et al., Multidisciplinary management of Hunter syndrome, Pediatrics 124 (6) (2009) e1228–e1239 (Epub 2009. Review). [4] W. Kolch, Coordinating ERK/MAPK signalling through scaffolds and inhibitors, Nat. Rev. Mol. Cell Biol. 6 (2005) 827–837. [5] A. Mor, M.R. Philips, Compartmentalized Ras/MAPK signaling, Annu. Rev. Immunol. 24 (2006) 771–800. [6] L.C. Hsing, A.Y. Rudensky, The lysosomal cysteine proteases in MHC class II antigen presentation, Immunol. Rev. 207 (2005) 229–241. [7] P. Li, J.L. Gregg, N. Wang, et al., Compartmentalization of class II antigen presentation: contribution of cytoplasmic and endosomal processing, Immunol. Rev. 207 (2005) 206–217. [8] S. Radoja, A.B. Frey, S. Vukmanovic, T-cell receptor signaling events triggering granule exocytosis, Crit. Rev. Immunol. 26 (2006) 265–290. [9] D. Schmid, C. Munz, Immune surveillance of intracellular pathogens via autophagy, Cell Death Differ. 12 (Suppl. 2) (2005) 1519–1527. [10] D. Schmid, J. Dengjel, O. Schoor, S. Stevanovic, C. Munz, Autophagy in innate and adaptive immunity against intracellular pathogens, J. Mol. Med. 84 (2006) 194–202. [11] E.J. Blott, G.M. GriYths, Secretory lysosomes, Nat. Rev. Mol. Cell Biol. 3 (2002) 122–131. [12] O.J. Holt, F. Gallo, G.M. GriYths, Regulating secretory lysosomes, J. Biochem. (Tokyo) 140 (2006) 7–12. [13] Y.T. Bryceson, E.O. Long, Line of attack: NK cell specificity and integration of signals, Curr. Opin. Immunol. 20 (2008) 344–352. [14] L.L. Lanier, Up on the tightrope: natural killer cell activation and inhibition, Nat. Immunol. 9 (2008) 495–502. [15] C.A. Biron, More things in heaven and earth: defining innate and adaptive immunity, Nat. Immunol. 11 (2010) 1080–1082. [16] A. Zingoni, T. Sornasse, B.G. Cocks, Y. Tanaka, A. Santoni, L.L. Lanier, Cross-talk between activated human NK cells and CD4+ T cells via OX40-OX40 ligand interactions, J. Immunol. 173 (2004) 3716–3724. [17] I.R. Blanca, E.W. Bere, H.A. Young, J.R. Ortaldo, Human B cell activation by autologous NK cells is regulated by CD40-CD40 ligand interaction: role of memory B cells and CD5+ B cells, J. Immunol. (2001) 6132–6139. [18] E. Vivier, E. Tomasello, M. Baratin, T. Walzer, S. Ugolini, Functions of natural killer cells, Nat. Immunol. 9 (2008) 503–510.
L.C. Torres et al. [19] M. Flodstrom-Tullberg, Y.T. Bryceson, F.D. Shi, P. Hoglund, H. G. Ljunggren, Natural killer cells in human autoimmunity, Curr. Opin. Immunol. 21 (2009) 634–640. [20] D. Von Bubnoff, E. Andres, F. Hentges, T. Bieber, T. Michel, J. Zimmer, Natural killer cells in atopic and autoimmune diseases of the skin, J. Allergy Clin. Immunol. 125 (2010) 60–68. [21] C.R.D.C. Quaio, H. Grinberg, M.L.C. Vieira, et al., Report of a large Brazilian family with a very attenuated form of Hunter syndrome (MPS II), JIMD Rep. 4 (2012) 125–128. [22] T. Kato, Z. Kato, I. Kuratsubo, et al., Mutational and structural analysis of Japanese patients with mucopolysaccharidosis type II, J. Hum. Genet. 50 (2005) 395–402. [23] NCBI — NationalCenter for Biotechnology Information, Accessed at http://www.ncbi.nlm.nih.gov. [24] K. Araya, N. Sakai, I. Mohri, K. Kagitani-Shimono, T. Okinaga, Y. Hashii, H. Ohta, I. Nakamichi, K. Aozasa, M. Taniike, K. Ozono, Localized donor cells in brain of a Hunter disease patient after cord blood stem cell transplantation, Mol. Genet. Metab. 98 (3) (Nov. 2009) 255–263. [25] J.S. Orange, Z.K. Ballas, Natural killer cells in human health and disease, Clin. Immunol. 118 (2006) 1–10. [26] A. Cerwenka, L.L. Lanier, Ligands for natural killer cell receptors: redundancy or specificity, Immunol. Rev. 181 (2001) 158–169. [27] J.P. Di Santo, Natural killer cell developmental pathways: a question of balance, Annu. Rev. Immunol. 24 (2006) 257–286. [28] J.C. Sun, L.L. Lanier, NK cell development, homeostasis and function: parallels with CD8+ T cells, Nat. Rev. Immunol. 11 (2011) 645–657. [29] D.G. Hesslein, L.L. Lanier, Transcriptional control of natural killer cell development and function, Adv. Immunol. 109 (2011) 45–85. [30] K. Georgopoulos, B.A. Morgan, D.D. Moore, Functionally distinct isoforms of the CRE-BP DNA binding protein mediate activity of a T cell receptor enhancer, Mol. Cell. Biol. 12 (1992) 747–757. [31] K. Georgopoulos, D.D. Moore, B. Derfler, Ikaros, an early lymphoid-specific transcription factor and a putative mediator for T cell commitment, Science 258 (1992) 808–812. [32] H. Clevers, M. Oosterwegel, K. Georgopoulos, Transcription factors in early T cell development, Immunol. Today 14 (1993) 591–596. [33] K. Georgopoulos, M. Bigby, J.H. Wang, et al., The Ikaros gene is required for the development of all lymphoid lineages, Cell 79 (1994) 143–156. [34] P. Kirstetter, M. Thomas, A. Dierich, P. Kastner, S. Chan, Ikaros is critical for B cell differentiation and function, Eur. J. Immunol. 32 (2002) 720–730. [35] S. Ng, C. Fanta, M. Okam, A.S. Bhatt, NK-cell and B-cell deficiency with a thymic mass, N. Engl. J. Med. 364 (6) (2011) 586–588. [36] C. Grégoire, L. Chasson, C. Luci, et al., The trafficking of natural killer cells, Immunol. Rev. 220 (2007 Dec) 169–182. [37] M.I. Moraes-Pinto, Valores de referência de linfócitos/mm3 em população brasileira saudável, Braz.Group Prim, Immunodefic. Disord. 3 (2006) 1–4.
available at www.sciencedirect.com
Clinical Immunology www.elsevier.com/locate/yclim
BRIEF COMMUNICATION
NK and B cell deficiency in a MPS type II family with novel mutation in the IDS gene Leuridan Cavalcante Torres a,c,⁎, Diogo Cordeiro de Queiroz Soares a,b , Leslie Domenici Kulikowski d , Jose Francisco Franco b , Chong Ae Kim b a
Translational Research Laboratory Prof. C. A. Hart, Instituto de Medicina Integral Prof. Fernando Figueira (IMIP), Recife, Brazil b Medical Genetics Unit, Hospital das Clínicas, Faculdade de Medicina, Universidade de São Paulo (USP), São Paulo, Brazil c Medical Investigation Laboratory (LIM 36), Hospital das Clínicas, Faculdade de Medicina, Universidade de São Paulo (USP), São Paulo, Brazil d Department of Pathology, Citogenomics Laboratory (LIM 03), Universidade de São Paulo (USP), São Paulo, Brazil
Received 2 July 2013; accepted with revision 4 July 2014 KEYWORDS Mucopolysaccharidoses; NK cells; B cells; Immunodeficiency; Autoimmunity
Abstract The mucopolysaccharidoses (MPSs) are a group of rare, inherited lysosomal storage disorders that are clinically characterized by abnormalities in multiple organ systems and reduced life expectancy. Whereas the lysosome is essential to the functioning of the immune system, some authors suggest that the MPS patients have abnormalities in the immune system similar to the patients with primary immunodeficiency. In this study, we evaluated 8 male MPS type II patients of the same family with novel mutation in the IDS gene. We found in this MPS family a quantitative deficiency of NK and B cells with normal values of IgG, IgM and IgA serum antibodies and normal response to polysaccharide antigens. Interestingly, abnormalities found in these patients were not observed in other MPS patients, suggesting that the type of mutation found in the IDS gene can be implicated in the immunodeficiency. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Hunter syndrome, or mucopolysaccharidosis type II (MPS II, OMIM 607014), is a rare X-linked recessive disorder caused by a deficiency of the lysosomal enzyme iduronate-2-sulfatase ⁎ Corresponding author at: Translational Research Laboratory Prof. C. A. Hart, Instituto de Medicina Integral Prof. Fernando Figueira (IMIP), Rua dos Coelhos, 300, Recife, PE, 50070-550, Brazil. Fax: +55 81 2122 4703. E-mail address: [email protected] (L.C. Torres).
http://dx.doi.org/10.1016/j.clim.2014.07.001 1521-6616/© 2014 Elsevier Inc. All rights reserved.
(IDS), leading to the progressive accumulation of glycosaminoglycans (GAGs) in several organs. It affects approximately 1 in 170,000 male newborns, whereas female carriers rarely manifest symptoms [1]. The phenotypic spectrum of MPS II varies, with two typical clinical forms. The severe form is responsible for approximately 75% of all MPS II patients, with the signs and symptoms emerging between 18 months and 4 years of age. It is characterized mostly by facial dysmorphism, hepatosplenomegaly, hernias, short stature, stiff joints and contractures, cardiac valve disease and obstructive respiratory
NK and B cell deficiency in a MPS type II family with novel mutation in the IDS gene Table 1
101
Immunophenotyping of T, B and NK cells of 8 male family with MPS type II.
Patient Total T cells (P) lymphocytes (CD3*)
T CD4* (CD3*CD4*)
P1 P2 P3 P4 P5 P6 P7 P8
944 (476–1136) 554 (248–724) 2,1 798 (476–1136) 625 (248–724) 1,5 1278 (476–1136) 852 (248–724) 1,9 1193 (618–1348) 795 (390–1024) 2,5 849 (476–1136) 1082 (248–724) 1,1 329 (618–1348) 536 (390–1024) 0,8 1470 (780–2086) 1064 (453–1700) 1,9 1203 (780–2086) 802 (453–1700) 1,6
1612 1581 1440 2368 2272 1008 2849 2255
1499 (844–1943) 1422 (844–1943) 2131 (844–1943) 1989 (1280–2413) 1931 (844–1943) 866 (1280–2413) 2535 (1515–3701) 2006 (1515–3701)
T CD8* (CD3*CD8*)
Ratio B cells CD4*/CD8* (CD19*)
NK cells (CD3*/CD16*/CD56*)
217 (138–544) 106 (134–545) 221 (138–544) 79 (134–545) 72 (138–544) 34 (134–545) 288 (471–1031) 106 (127–515) 304 (138–544) 52 (134–545) 140 (471–1031) 16 (127–515) 370 (631–1283) 130 (135–601) 252 (631–1283) 22 (135–601)
Absolute values between parentheses indicate the reference values for the Brazilian population, by age, as described by Moraes-Pinto et al. [37].
complications, hearing loss, chronic diarrhea, and communicating hydrocephalus. On the contrary, the attenuated form presents a slower progression of the disease, usually marked by fairly normal intelligence, hepatosplenomegaly, joint contractures and conductive and sensorineural hearing loss [2,3]. Almost 350 mutations in the IDS gene have been described. The majority of these are missense mutations and lead to impaired degradation of GAGs by lysosomes and progressive accumulation of these metabolites [2]. Lysosome is critical for normal function of the immune system and the classical view of the lysosomal compartment as a digestive organelle has now expanded, with the lysosome involved in the control of cell-surface receptor-mediated signal transduction [4,5]. Moreover, the catabolism of macromolecules in the lysosome is essential for the correct function of several immune system functions including antigen processing and presentation [6,7], cytokine secretion [8], phagocytosis [9,10], and secretion of molecules [11,12]. Natural killer (NK) cells are considered to characterize an arm of the innate immune system, as their effector functions are mediated by repertoire of germline-encoded receptors that do not undergo somatic recombination [13,14]. However, NK cells express several costimulatory ligands including OX40L and CD40L (CD 154), which permit them to provide a costimulatory signal to B cells or T cells [15–17]. Thereby, NK cells participate in early defense against intracellular microbial infections and several types of tumors and may also be implicated in autoimmunity and hypersensitivity reactions [18–20]. Given the above, and considering the complexity attributed to MPS for being a chronic, systemic and progressive, in which life expectancy is correlated with the severity of clinical symptoms, whose heart, recurrent respiratory infections and upper airway obstruction are the main causes of death [2], the aim of this investigation was to evaluate the cellular immune response in a Brazilian family with 8 individuals affected by MPS type II.
2. Methods 2.1. Patients We studied 8 male individuals of the same family with MPS type II. The clinical history of the patients and a family tree were previously described by our group in this journal [21].
The evaluated patients includes brothers and cousins with the ages varied from 3 to 27 years (mean of 21 years), all of them reporting recurrent sinopulmonary infections, mild coarse faces, hepatosplenomegaly, hernias, short stature, stiff joints and contractures, cardiac valve disease and communicating hydrocephalus. The patients doesn't require of enzyme-replacement therapy because they have attenuated Form of Hunter Syndrome (MPS II).
2.2. Immunophenotyping Six-color flow cytometric immunophenotyping of peripheral blood was performed on a FACS LSRII FORTESSA (BD Biosciences). The following monoclonal antibodies were used: anti-CD3, anti-CD4, anti-CD8, anti-CD19, anti-CD16, anti-CD56, anti-CD314 (NKG2D). The cells were analyzed with the most appropriate lymphocyte gate using the combination of forward and side scatters and the obtained data were analyzed using FACS Diva software (BD Biosciences).
2.3. Molecular analysis Genomic DNA was extracted from peripheral blood using blood genomic Spin Mini Prep kit (GE Healthcare, USA) and amplified using 9 primer pairs flanking all exons and exon/ intron boundaries. The primers used in this study for exons 1 to 9 were described by Kato et al. [22]. Amplicons were purified by column-purification kit GFX PCR DNA (Amersham Pharmacia Biotech, USA) and sequenced in a MegaBACE 1000 DNA Sequencer (Amersham Pharmacia Biotech, USA). Sequencing analysis was performed using the Chromaspro version 1.34 (Technelysium Pty Ltd., Australia) and was compared with the sequence contained in the NCBI database [23].
2.4. Western blot analysis The membrane was incubated overnight at 4 °C with goat antibody against IDS (0.3 μg/ml, AF2449, R&D Systems Inc., Minneapolis, MN, USA) after treatment with 5% skim milk/ Tris-buffered saline for blocking nonspecific reactions. The immune complexes were detected with peroxidase-labeled horse anti-goat IgG (1:1000, Vector Laboratories Inc., Burlingame, CA, USA) and Immobilon Western HRP Substrate (EMD Millipore, Billerica, MA, USA).
102
Figure 1
L.C. Torres et al.
Relative values of NK cells (CD3−/CD16+/CD56+) of a healthy control and 8 male family with MPS type II by flow cytometry.
NK and B cell deficiency in a MPS type II family with novel mutation in the IDS gene
Figure 2 Iduronate 2-sulfatase protein expression by Western Blot analysis of 7 patients with MPS II family. CTL (control).
3. Results 3.1. Immunological investigation We observed that the majority of patients presented normal absolute number for TCD8+ and TCD4+ lymphocytes, except patient 6 that showed decreased values of TCD4+ with an inversion of CD4/CD8 ratio. Low absolute number of B cells with normal levels of IgG, IgM and IgA and an appropriate response to polysaccharides antigens in five patients (p3, p4, p6, p7 and p8) (Table 1). We found reduced absolute numbers of NK cells with normal expression of stimulatory receptors NKG2D when compared to reference values for the Brazilian population (Fig. 1).
4. Discussion In this study, we describe a family of 8 patients whose molecular analysis revealed a novel mutation in the IDS gene, confirming the diagnosis of MPS type II. It usually cannot be determined whether a certain mutation will cause severe or mild MPS type II. However, missense mutations frequently result in decreased expression of IDS enzyme activity and comprise the majority of gene mutations in MPS type II, unlike mutations that result in complete absence of the enzyme activity, such as total rearrangements or partial deletions that seem to be associated with the severe form of the disease in up to 25% of cases of MPS type II [2]. Western Blot analysis of Iduronate-2-sulfatase (IDS) protein (76 kDa) was performed, according to Araya et al. [24], and detect that all patients express some degree of IDS protein, but wasn't observed a correlation between the protein expression and the patient's phenotype (Fig. 2). In immunological evaluation, we observed absolute and relative NK cells deficiency in all MPS patients and 5 of them present B cell deficiency also, without antibody defects. NK cells are lymphocytes of the innate immune system, do not express TCR or immunoglobulin genes, but in its place use a variety of germline-encoded receptors to induce their functions, which include cytokine production, perforindependent cytotoxicity and expression of co-stimulatory molecules. In this capacity, NK cells participate in antiviral and anticancer responses and can contribute to adaptive immunity [25,26]. Early studies suggested that NK cells, like B cells and myeloid-lineage cells, develop primarily in the bone marrow. NK precursor (NKP) cells derived from hematopoietic stem cells (HSCs) give rise to immature NK (iNK) and then mature NK cells. Several transcription factors, such as Ikaros, Ets-1, PU.1, T-bet and E4BP4 are required for NK cell maturation. Among these, Ets-1, Ikaros and PU.1 are critical for production of NKPs, and the other factors become implicated after cells are committed to the NK cell lineage.
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However, these factors are not necessarily specific to the NK lineage, because their individual deletions sometimes cause defects in other hematopoietic cell lineages [27–29]. The transcription factor Ikaros (also known as LyF-1) was initially described for its ability to bind and activate the enhancer of the CD3δ gene, an early and definitive marker of T cell differentiation [30,31]. However, gene-targeting studies evidenced the fundamental role of Ikaros in hematopoiesis, in particular in T and B lymphocytes, natural killer (NK) and dendritic cells, and stem cells [32–34]. Interestingly, 8 patients with MPS type II of the same family presented immunologic alterations not found in MPS type II patients with other types of mutations in the IDS gene, suggesting that mutation found in this family of MPS is directly associated with low levels of NK cells and B cell on peripheral blood. Similarly, Ng et al. [35] reported a case of a woman that present profound deficiency of NK and B cells, associated with recurrent infections. Based on the clinical and laboratory presentation of this case, the authors considered the many similarities between the clinical case and the Ikaros-null knockout mouse and raise the possibility of a mutation in Ikaros as the cause of an immune defect, including NK and B cell deficiency and emphasizing the importance of a further investigation, since mutation or alternative splicing of Ikaros may cause the findings observed [35]. Given these results, we believe that the IDS gene can act as a transcriptional coactivator of some proteins, such as Ikaros, and that novel mutation in the IDS gene presented by this family may interfere somehow in the expression of Ikaros, compromising the development and maturation of B and NK cells. Thus, further studies are needed to better understand these immune abnormalities. Additionally, it is known that NK cells are distributed on multiple organs, being more frequent in lungs and liver and less frequent in lymph node and thymus, where NK cells are almost undetectable. Therefore, we believe that one of the possibilities for the predisposition of these patients, specifically to recurrent respiratory infections, may be related to the reduced distribution of NK cells in the lungs [36]. In this study, we evaluated 8 male MPS type II patients of the same family with novel missense mutation (p.A77D) in the IDS gene. We found in this MPS family a quantitative deficiency of NK and B cells with normal values of IgG, IgM and IgA serum antibodies and normal response to polysaccharide antigens. Interestingly, these abnormalities found in these patients were not observed in other MPS patients, suggesting that the type of mutation found in the IDS gene can be implicated in the immunodeficiency.
Conflict of interest statement None of the authors has any potential financial conflict of interest related to this manuscript.
Acknowledgments The authors thank the patients, their families and grant No. 2010/52694-8 of the São Paulo Research Foundation (FAPESP).
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