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Autoimmune lymphoproliferative syndrome in a patient with a new minimal deletion in the death domain of the FAS gene
Human Pathology (2008) 39, 137–141
www.elsevier.com/locate/humpath
Case study
Autoimmune lymphoproliferative syndrome in a patient with a new minimal deletion in the death domain of the FAS geneB Gabriela Gualco MD a,⁎, Anke van den Berg PhD b , Sicco Koopmans RA b , Lívia M. Bacchi MS c , Siderley S. Carneiro MD d , Everaldo Ruiz Jr MD e , Ana Paula Vecchi MD e , John K.C. Chan MD f a
Consultoria em Patologia, Rua Major Leonidas Cardoso 739, PO Box 151, CEP 18602-010, Botucatu, Sao Paulo, Brazil Department of Pathology and Laboratory Medicine, University Medical Center Groningen and University of Groningen, Groningen, The Netherlands c University of São Paulo Medical School, São Paulo, Brazil d Laboratório Atalaia e Hospital Materno Infantil, Goiânia, Goias, Brazil f Department of Pathology, Queen Elizabeth Hospital, Hong Kong b
Received 12 June 2007; revised 23 July 2007; accepted 26 July 2007
Keywords: Autoimmune lymphoproliferative syndrome; ALPS; Apoptosis; FAS gene deletion; CD95; Rosai-Dorfman
Summary We present a case of autoimmune lymphoproliferative syndrome (ALPS) caused by a previously undescribed minimal deletion in the death domain of the FAS gene. ALPS is an uncommon disease associated with an impaired Fas-mediated apoptosis. The patient presented with a history of splenomegaly since 4 months of age, associated with cervical lymphadenopathy, which improved with oral corticosteroid treatment. Relevant laboratory findings were the presence of anemia, thrombocytopenia, and positive direct and indirect Coombs tests. He was not an offspring of consanguineous parents. Two cervical lymph node biopsies were performed, at 4 years and at 6 years of age. In both lymph nodes, there was marked paracortical expansion by lymphocytes in variable stages of immunoblastic transformation and a very high cell proliferating index. Some clear cells were also present, raising the suspicion of malignant lymphoma. In one of the lymph nodes, there was also a focus rich in large histiocytes with round nuclei and emperipolesis, consistent with focal Rosai-Dorfman disease. Immunostaining showed numerous CD3+ cells, many of which were double-negative (CD4− CD8−) and expressed CD57, especially around the follicles. Molecular studies of the lymph node biopsy showed a point deletion (4–base pair deletion) in exon 9 of the FAS gene (930del TGCT), which results in 3 missense amino acids. © 2008 Elsevier Inc. All rights reserved.
1. Introduction ☆ This study was partially supported by National Institutes of Health (Bethesda, MD) grants NIH CA 070058 and NIH CA 082274. ⁎ Corresponding author. E-mail address: [email protected] (G. Gualco).
0046-8177/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2007.07.013
The autoimmune lymphoproliferative syndrome (ALPS) is an uncommon disease resulting from an impaired Fasmediated apoptosis. Since the original clinical description by
138
G. Gualco et al.
Fig. 1 Histologic and immunohistochemical findings of the cervical lymph node (second biopsy). A, The paracortex is markedly expanded by lymphoid cells with pale cytoplasm; there are hyperplastic as well as regressive follicles (hematoxylin and eosin [H&E], original magnification ×100). B, Paracortical area populated by lymphocytes, plasma cells, and immunoblasts with mitotic activity (H&E, magnification ×400). C, Lymph node showing expansion of paracortex by numerous CD3+ T cells (immunoperoxidase with hematoxylin counterstain, magnification ×100). D, In this area, the Ki-67 proliferative index is very high (immunoperoxidase with hematoxylin counterstain, magnification ×100).
Canale and Smith [1], followed by the characterization of an abnormal T-cell proliferation with lymphocyte apoptosis defect and the demonstration of FAS gene mutations [2-5], many cases have been described [6-8]. The main features of ALPS are as follows: early childhood onset, prominent nonmalignant lymphadenopathy, hepatosplenomegaly, autoimmune manifestations and blood count alterations such as expanded populations of CD4− CD8− (double-negative T cells) [4,9], and in vitro defects in lymphocytes apoptosis. In most patients, there is a genetic defect in FAS (APT1/ TNFRSF-6) gene, which encodes Fas protein (also called APO-1 or CD95), a cell-surface receptor that regulates lymphocyte survival by triggering programmed cell death or apoptosis. In a low percentage of ALPS cases, the genetic mutation involves CASPASE-8, CASPASE-10, or FAS LIGAND genes. There remain some ALPS patients with unidentified genetic defects [7,9]. Recently, Oliveira et al [10] reported a case with a germline mutation in NRAS.
By far, the most common gene mutations are missense point mutations in the intracellular death domain of the FAS gene [7,11]. Deletions involving the FAS gene are rarely described, with some affecting exon 9 [5]. This report describes a previously undescribed minimal deletion in the death domain of the FAS gene in a 6-year-old boy with ALPS.
2. Case report A 6-year-old white boy presented with a history of splenomegaly since he was 4 months old, associated with cervical lymphadenopathy. By the time he was 1 year old, he developed thrombocytopenia, which improved with prednisone treatment. At 3 years of age, he presented with autoimmune hemolytic anemia and hepatomegaly; there was
Autoimmune lymphoproliferative syndrome
139 San Francisco, CA); CD25 (4C9) and CD4 (1FG) (Novocastra, Newcastle, England); and CD57 (VP-C361) (Vector Laboratories, Burlingame, CA). The reaction was visualized using 3,3′-diaminobenzidine as chromogen. Immunostaining showed numerous CD3+ cells in the paracortical area (Fig. 1B). There was a population of CD4− CD8− cells, mostly around the follicles (ie, CD4+ cells and CD8+ cells together being less than CD3+ cells). There were sheets of CD57+ cells, especially around the follicles. Ki-67 cell proliferation index was very high in the interfollicular and paracortical areas (Fig. 1B insert). Sections from both lymph nodes were examined for the presence of the Epstein-Barr viral RNA by in situ hybridization using the EBER-1 probe [12] and were negative.
Fig. 2 Cervical lymph node (second biopsy): focus of RosaiDorfman disease revealing large histiocyte with emperipolesis, accompanied by many plasma cells (H&E, magnification ×400).
persistent splenomegaly and cervical lymphadenopathy. In a period of 6 years of life, the patient had several episodes of cervical lymphadenopathy, usually associated with upper airway infections, which improved with oral corticosteroid therapy. There was no history of cutaneous lesions, articular pain, or specific infectious disease. The boy was not an offspring of consanguineous parents, and there was no history of rheumatologic disease in the family. Relevant laboratory findings were the presence of anemia, leukopenia with lymphocytosis, thrombocytopenia, and positive direct and indirect Coombs tests. All serologic tests for specific infection diseases were negative. Tests for rheumatoid factor, anti–nuclear factor, anti–extractable nuclear antigens, and anti-DNA were negative.
2.1. Morphological, immunohistochemical, and Epstein-Barr virus in situ hybridization findings Two cervical lymph node biopsies were performed, at 4 years and at 6 years of age. The histologic findings of both lymph nodes looked similar. The architecture was distorted but not totally effaced. There was marked paracortical expansion by lymphocytes in variable stages of immunoblastic transformation (Fig. 1A and A insert). These lymphocytes were admixed with some medium-sized clear cells and plasma cells. The paracortical lymphoid cells demonstrated high mitotic activity. There were scattered reactive follicles in the cortex. In the second lymph node biopsy, there was also a pale focus rich in large histiocytes with round nuclei and emperipolesis, consistent with focal Rosai-Dorfman disease (Fig. 2). Immunohistochemical staining was performed using the avidin-biotin-peroxidase complex method with the following antibodies: CD20 (L26), CD30 (BerH2), Ki-67 (MIB1), BCL-2 (124), p24 (KaL-1), BCL-6 (PG-B6p), and CD8 (C8/144B) (Dako, Carpinteria, CA); CD3 (SP7) (Labvision,
3. Molecular pathology findings DNA was isolated from both formalin-fixed, paraffinembedded lymph nodes with PK1 buffer as described previously [13].
3.1. T-cell receptor gene rearrangement Clonality study for T-cell receptor (TCR) γ and TCRβ was performed by polymerase chain reaction (PCR) amplification using multiplex PCR according to the BIOMED-2 protocol [14]. No clonal TCRγ nor TCRβ rearrangement was detected.
3.2. Amplification of the FAS gene and mutation detection Ten sets of primers were used to amplify all 9 exons of the FAS gene [7,8]. PCR products were denatured for 10 minutes at 94°C, and heteroduplex molecules were formed for 50 minutes at 55°C. The resulting homo/heteroduplex molecules were analyzed on a denaturing gradient gel electrophoresis (DGGE). The gel was stained with ethidium bromide, and aberrant homoduplex bands were excised from the gel and eluted in distilled water. A volume of 1 μL was reamplified using the same set of primers. Direct sequence analysis was performed using both PCR primers on a MegaBace fluorescence sequencer and a dye terminator sequence kit (Blue Lion Biotech, Snoqualmie, WA). Sequence was compared with the sequence present in the GenBank (APO1 sequence accession no. x63717). Codons 1 to 16 represent the signal peptide, and the mature peptide consists of 319 amino acids (17-335) [8]. The same aberrant banding pattern was detected by DGGE in both biopsies (Fig. 3A). Sequence of the aberrant band showed a deletion of 4 base pairs (bp) in exon 9 (930del TGCT) of the FAS gene (10q24.1, GenBank accession no. M67454), which results in 3 missense amino acids and a premature stop at position 223, supporting the diagnosis of ALPS (Fig. 3B).
140
G. Gualco et al.
Fig. 3 (A) DGGE analysis showed the same aberrant band pattern in both samples (arrow). (B) The sequence showed 4-bp deletion in exon 9 of the FAS gene (box).
4. Discussion ALPS is the first human disease whose etiology has been attributed to a primary defect in apoptosis. Homeostasis through apoptosis is crucial to remain within the limited containment capacity of the lymphoid compartment to eliminate autoreactive lymphocytes and to prevent malignant transformation [3,5]. A defective apoptosis of lymphocytes through the Fas pathway occupies a central role in the pathogenesis of ALPS. The main manifestations of ALPS are lymphoid proliferation causing lymphadenopathies, hepatomegaly and splenomegaly, autoimmune phenomena, and increased risk for lymphomas. The morphological features of lymph node biopsies are often worrisome and not uncommonly lead to a misdiagnosis of malignant lymphoma. In fact, we received the first biopsy of this patient in consultation with the diagnosis of T-cell lymphoma. Taking into account the clinical picture (very young age) and the characteristic immunophenotype [9,15] of double-negative (CD4− CD8−) CD57+ CD3+ paracortical T cells, it is possible to make a presumptive diagnosis of ALPS.
Genetic studies of families with ALPS have identified a variety of causative mutations, permitting formulation of a genotype-based ALPS classification adopted by the National Institutes of Health [15]. ALPS types 0 and 1a are due to homozygous or heterozygous FAS mutations, type 1b a defect of the FAS ligand, type 2 a defect of caspase 10, and type 3 unknown genetic defects [10,11,14]. Our patient belongs to type 1a. More than 70 different FAS gene mutations have been described so far, either in the coding regions or in the splice sites, with most affecting the intracellular or death domain (exon 9). Most of them are point mutations, and only a few deletions have been described in exon 9 [5,16]. Our patient has a 4-bp deletion (930delTGCT), which results in I220fs, with 3 missense amino acids and a premature stop codon at amino acid position 223; the same alteration was detected in the 2 lymph node biopsies performed with a 2-year interval in between. To the best of our knowledge, this mutation has not been described before. There are 2 patients reported with closed stop codons 224 and 227 originated by 2-bp insertion and 8-bp deletion, respectively, [8,9], associated with a
Autoimmune lymphoproliferative syndrome Fas-induced apoptosis of 1% and 13% of control value [9]. ALPS patients with germline mutation of the intracellular domain of FAS have significantly increased risk (14- and 51-fold) of developing non-Hodgkin lymphoma and Hodgkin lymphoma, respectively [16]. Another interesting finding in our case was the presence of a focus of RosaiDorfman disease in one lymph node. Recently, Maric et al [17] reported an association between ALPS and RosaiDorfman disease. Both disorders share many clinical features including autoimmune phenomena. The presence of focal Rosai-Dorfman disease in 41% of patients with ALPS opens the possibility that a subpopulation of patients with Rosai-Dorfman disease might have a forme fruste form of ALPS [17].
Acknowledgments We thank Lucimara Chioato, PhD, and Elida B. Ojopi, PhD, for their help related to the TCR gene rearrangement study.
References [1] Canale VC, Smith CH. Chronic lymphadenopathy simulating malignant lymphoma. J Pediatr 1967;70:891-9. [2] Sneller MC, Straus SE, Jaffe ES, et al. A novel lymphoproliferative/ autoimmune syndrome resembling murine lpr/gld disease. J Clin Invest 1992;90:334-41. [3] Watanabe-Fukunaga R, Brannan CI, Copeland NG, Jenkins NA, Nagata S. Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature 1992;356:314-7. [4] Fisher GH, Rosenberg FJ, Straus SE, et al. Dominant interfering Fas gene mutations impair apoptosis in a human autoimmune lymphoproliferative syndrome. Cell 1995;81:935-46.
141 [5] Rieux-Laucat F, Le Deist F, Hivroz C, et al. Mutations in Fas associated with human lymphoproliferative syndrome and autoimmunity. Science 1995;268:1347-9. [6] Bettinardi A, Brugnoni D, Quiros-Roldan E, et al. Missense mutations in the Fas gene resulting in autoimmune lymphoproliferative syndrome: a molecular and immunological analysis. Blood 1997;89: 902-9. [7] van den Berg A, Tamminga R, de Jong D, Maggio E, Kamps W, Poppema S. FAS gene mutation in a case of autoimmune lymphoproliferative syndrome type IA with accumulation of gammadelta+ T cells. Am J Surg Pathol 2003;27:546-53. [8] van den Berg A, Maggio E, Diepstra A, de Jong D, van Krieken JS. Germline FAS gene mutation in a case of ALPS and NLP Hodgkin lymphoma. Blood 2002;99:1492-4. [9] Straus S, Sneller M, Leonardo M, Puck J, Strober W. An inherited disorder of lymphocyte apoptosis: the autoimmune lymphoproliferative syndrome. NIH conference. Ann Intern Med 1999;130:591-601. [10] Oliveira JB, Bidère N, Niemela J, et al. NRAS mutation causes a human lymphoproliferative syndrome. PNAS 2007;104:8953-8. [11] Del-Rey MJ, Manzanares J, Bosque A, et al. Autoimmune lymphoproliferative syndrome (ALPS) in a patient with a new germline Fas gene mutation. Immunobiology 2007;212:73-83. [12] Bacchi MM, Bacchi CE, Alvarenga M, Miranda R, Chen YY, Weiss LM. Burkitt's lymphoma in Brazil: strong association with EpsteinBarr virus. Mod Pathol 1996;9:63-7. [13] Limpens J, Beelen M, Stad R, et al. Detection of the t(14;18) translocation in frozen and formalin-fixed tissue. Diagn Mol Pathol 1993;2:99-107. [14] van Dongen JJM, Langerak AW, Bruggemann M, et al. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 concerted action BMH4-CT98-3936. Leukemia 2003;17:2257-317. [15] Lim MS, Straus SE, Dale JK, et al. Pathological findings in human autoimmune lymphoproliferative syndrome. Am J Pathol 1998;153: 1541-50. [16] Rao VK, Straus SE. Causes and consequences of the autoimmune lymphoproliferative syndrome. Hematology 2006;11:15-23. [17] Maric I, Pittaluga S, Dale J, et al. Histologic features of sinus histiocytosis with massive lymphadenopathy in patients with autoimmune lymphoproliferative syndrome. Am J Surg Pathol 2005;29: 903-11.
www.elsevier.com/locate/humpath
Case study
Autoimmune lymphoproliferative syndrome in a patient with a new minimal deletion in the death domain of the FAS geneB Gabriela Gualco MD a,⁎, Anke van den Berg PhD b , Sicco Koopmans RA b , Lívia M. Bacchi MS c , Siderley S. Carneiro MD d , Everaldo Ruiz Jr MD e , Ana Paula Vecchi MD e , John K.C. Chan MD f a
Consultoria em Patologia, Rua Major Leonidas Cardoso 739, PO Box 151, CEP 18602-010, Botucatu, Sao Paulo, Brazil Department of Pathology and Laboratory Medicine, University Medical Center Groningen and University of Groningen, Groningen, The Netherlands c University of São Paulo Medical School, São Paulo, Brazil d Laboratório Atalaia e Hospital Materno Infantil, Goiânia, Goias, Brazil f Department of Pathology, Queen Elizabeth Hospital, Hong Kong b
Received 12 June 2007; revised 23 July 2007; accepted 26 July 2007
Keywords: Autoimmune lymphoproliferative syndrome; ALPS; Apoptosis; FAS gene deletion; CD95; Rosai-Dorfman
Summary We present a case of autoimmune lymphoproliferative syndrome (ALPS) caused by a previously undescribed minimal deletion in the death domain of the FAS gene. ALPS is an uncommon disease associated with an impaired Fas-mediated apoptosis. The patient presented with a history of splenomegaly since 4 months of age, associated with cervical lymphadenopathy, which improved with oral corticosteroid treatment. Relevant laboratory findings were the presence of anemia, thrombocytopenia, and positive direct and indirect Coombs tests. He was not an offspring of consanguineous parents. Two cervical lymph node biopsies were performed, at 4 years and at 6 years of age. In both lymph nodes, there was marked paracortical expansion by lymphocytes in variable stages of immunoblastic transformation and a very high cell proliferating index. Some clear cells were also present, raising the suspicion of malignant lymphoma. In one of the lymph nodes, there was also a focus rich in large histiocytes with round nuclei and emperipolesis, consistent with focal Rosai-Dorfman disease. Immunostaining showed numerous CD3+ cells, many of which were double-negative (CD4− CD8−) and expressed CD57, especially around the follicles. Molecular studies of the lymph node biopsy showed a point deletion (4–base pair deletion) in exon 9 of the FAS gene (930del TGCT), which results in 3 missense amino acids. © 2008 Elsevier Inc. All rights reserved.
1. Introduction ☆ This study was partially supported by National Institutes of Health (Bethesda, MD) grants NIH CA 070058 and NIH CA 082274. ⁎ Corresponding author. E-mail address: [email protected] (G. Gualco).
0046-8177/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.humpath.2007.07.013
The autoimmune lymphoproliferative syndrome (ALPS) is an uncommon disease resulting from an impaired Fasmediated apoptosis. Since the original clinical description by
138
G. Gualco et al.
Fig. 1 Histologic and immunohistochemical findings of the cervical lymph node (second biopsy). A, The paracortex is markedly expanded by lymphoid cells with pale cytoplasm; there are hyperplastic as well as regressive follicles (hematoxylin and eosin [H&E], original magnification ×100). B, Paracortical area populated by lymphocytes, plasma cells, and immunoblasts with mitotic activity (H&E, magnification ×400). C, Lymph node showing expansion of paracortex by numerous CD3+ T cells (immunoperoxidase with hematoxylin counterstain, magnification ×100). D, In this area, the Ki-67 proliferative index is very high (immunoperoxidase with hematoxylin counterstain, magnification ×100).
Canale and Smith [1], followed by the characterization of an abnormal T-cell proliferation with lymphocyte apoptosis defect and the demonstration of FAS gene mutations [2-5], many cases have been described [6-8]. The main features of ALPS are as follows: early childhood onset, prominent nonmalignant lymphadenopathy, hepatosplenomegaly, autoimmune manifestations and blood count alterations such as expanded populations of CD4− CD8− (double-negative T cells) [4,9], and in vitro defects in lymphocytes apoptosis. In most patients, there is a genetic defect in FAS (APT1/ TNFRSF-6) gene, which encodes Fas protein (also called APO-1 or CD95), a cell-surface receptor that regulates lymphocyte survival by triggering programmed cell death or apoptosis. In a low percentage of ALPS cases, the genetic mutation involves CASPASE-8, CASPASE-10, or FAS LIGAND genes. There remain some ALPS patients with unidentified genetic defects [7,9]. Recently, Oliveira et al [10] reported a case with a germline mutation in NRAS.
By far, the most common gene mutations are missense point mutations in the intracellular death domain of the FAS gene [7,11]. Deletions involving the FAS gene are rarely described, with some affecting exon 9 [5]. This report describes a previously undescribed minimal deletion in the death domain of the FAS gene in a 6-year-old boy with ALPS.
2. Case report A 6-year-old white boy presented with a history of splenomegaly since he was 4 months old, associated with cervical lymphadenopathy. By the time he was 1 year old, he developed thrombocytopenia, which improved with prednisone treatment. At 3 years of age, he presented with autoimmune hemolytic anemia and hepatomegaly; there was
Autoimmune lymphoproliferative syndrome
139 San Francisco, CA); CD25 (4C9) and CD4 (1FG) (Novocastra, Newcastle, England); and CD57 (VP-C361) (Vector Laboratories, Burlingame, CA). The reaction was visualized using 3,3′-diaminobenzidine as chromogen. Immunostaining showed numerous CD3+ cells in the paracortical area (Fig. 1B). There was a population of CD4− CD8− cells, mostly around the follicles (ie, CD4+ cells and CD8+ cells together being less than CD3+ cells). There were sheets of CD57+ cells, especially around the follicles. Ki-67 cell proliferation index was very high in the interfollicular and paracortical areas (Fig. 1B insert). Sections from both lymph nodes were examined for the presence of the Epstein-Barr viral RNA by in situ hybridization using the EBER-1 probe [12] and were negative.
Fig. 2 Cervical lymph node (second biopsy): focus of RosaiDorfman disease revealing large histiocyte with emperipolesis, accompanied by many plasma cells (H&E, magnification ×400).
persistent splenomegaly and cervical lymphadenopathy. In a period of 6 years of life, the patient had several episodes of cervical lymphadenopathy, usually associated with upper airway infections, which improved with oral corticosteroid therapy. There was no history of cutaneous lesions, articular pain, or specific infectious disease. The boy was not an offspring of consanguineous parents, and there was no history of rheumatologic disease in the family. Relevant laboratory findings were the presence of anemia, leukopenia with lymphocytosis, thrombocytopenia, and positive direct and indirect Coombs tests. All serologic tests for specific infection diseases were negative. Tests for rheumatoid factor, anti–nuclear factor, anti–extractable nuclear antigens, and anti-DNA were negative.
2.1. Morphological, immunohistochemical, and Epstein-Barr virus in situ hybridization findings Two cervical lymph node biopsies were performed, at 4 years and at 6 years of age. The histologic findings of both lymph nodes looked similar. The architecture was distorted but not totally effaced. There was marked paracortical expansion by lymphocytes in variable stages of immunoblastic transformation (Fig. 1A and A insert). These lymphocytes were admixed with some medium-sized clear cells and plasma cells. The paracortical lymphoid cells demonstrated high mitotic activity. There were scattered reactive follicles in the cortex. In the second lymph node biopsy, there was also a pale focus rich in large histiocytes with round nuclei and emperipolesis, consistent with focal Rosai-Dorfman disease (Fig. 2). Immunohistochemical staining was performed using the avidin-biotin-peroxidase complex method with the following antibodies: CD20 (L26), CD30 (BerH2), Ki-67 (MIB1), BCL-2 (124), p24 (KaL-1), BCL-6 (PG-B6p), and CD8 (C8/144B) (Dako, Carpinteria, CA); CD3 (SP7) (Labvision,
3. Molecular pathology findings DNA was isolated from both formalin-fixed, paraffinembedded lymph nodes with PK1 buffer as described previously [13].
3.1. T-cell receptor gene rearrangement Clonality study for T-cell receptor (TCR) γ and TCRβ was performed by polymerase chain reaction (PCR) amplification using multiplex PCR according to the BIOMED-2 protocol [14]. No clonal TCRγ nor TCRβ rearrangement was detected.
3.2. Amplification of the FAS gene and mutation detection Ten sets of primers were used to amplify all 9 exons of the FAS gene [7,8]. PCR products were denatured for 10 minutes at 94°C, and heteroduplex molecules were formed for 50 minutes at 55°C. The resulting homo/heteroduplex molecules were analyzed on a denaturing gradient gel electrophoresis (DGGE). The gel was stained with ethidium bromide, and aberrant homoduplex bands were excised from the gel and eluted in distilled water. A volume of 1 μL was reamplified using the same set of primers. Direct sequence analysis was performed using both PCR primers on a MegaBace fluorescence sequencer and a dye terminator sequence kit (Blue Lion Biotech, Snoqualmie, WA). Sequence was compared with the sequence present in the GenBank (APO1 sequence accession no. x63717). Codons 1 to 16 represent the signal peptide, and the mature peptide consists of 319 amino acids (17-335) [8]. The same aberrant banding pattern was detected by DGGE in both biopsies (Fig. 3A). Sequence of the aberrant band showed a deletion of 4 base pairs (bp) in exon 9 (930del TGCT) of the FAS gene (10q24.1, GenBank accession no. M67454), which results in 3 missense amino acids and a premature stop at position 223, supporting the diagnosis of ALPS (Fig. 3B).
140
G. Gualco et al.
Fig. 3 (A) DGGE analysis showed the same aberrant band pattern in both samples (arrow). (B) The sequence showed 4-bp deletion in exon 9 of the FAS gene (box).
4. Discussion ALPS is the first human disease whose etiology has been attributed to a primary defect in apoptosis. Homeostasis through apoptosis is crucial to remain within the limited containment capacity of the lymphoid compartment to eliminate autoreactive lymphocytes and to prevent malignant transformation [3,5]. A defective apoptosis of lymphocytes through the Fas pathway occupies a central role in the pathogenesis of ALPS. The main manifestations of ALPS are lymphoid proliferation causing lymphadenopathies, hepatomegaly and splenomegaly, autoimmune phenomena, and increased risk for lymphomas. The morphological features of lymph node biopsies are often worrisome and not uncommonly lead to a misdiagnosis of malignant lymphoma. In fact, we received the first biopsy of this patient in consultation with the diagnosis of T-cell lymphoma. Taking into account the clinical picture (very young age) and the characteristic immunophenotype [9,15] of double-negative (CD4− CD8−) CD57+ CD3+ paracortical T cells, it is possible to make a presumptive diagnosis of ALPS.
Genetic studies of families with ALPS have identified a variety of causative mutations, permitting formulation of a genotype-based ALPS classification adopted by the National Institutes of Health [15]. ALPS types 0 and 1a are due to homozygous or heterozygous FAS mutations, type 1b a defect of the FAS ligand, type 2 a defect of caspase 10, and type 3 unknown genetic defects [10,11,14]. Our patient belongs to type 1a. More than 70 different FAS gene mutations have been described so far, either in the coding regions or in the splice sites, with most affecting the intracellular or death domain (exon 9). Most of them are point mutations, and only a few deletions have been described in exon 9 [5,16]. Our patient has a 4-bp deletion (930delTGCT), which results in I220fs, with 3 missense amino acids and a premature stop codon at amino acid position 223; the same alteration was detected in the 2 lymph node biopsies performed with a 2-year interval in between. To the best of our knowledge, this mutation has not been described before. There are 2 patients reported with closed stop codons 224 and 227 originated by 2-bp insertion and 8-bp deletion, respectively, [8,9], associated with a
Autoimmune lymphoproliferative syndrome Fas-induced apoptosis of 1% and 13% of control value [9]. ALPS patients with germline mutation of the intracellular domain of FAS have significantly increased risk (14- and 51-fold) of developing non-Hodgkin lymphoma and Hodgkin lymphoma, respectively [16]. Another interesting finding in our case was the presence of a focus of RosaiDorfman disease in one lymph node. Recently, Maric et al [17] reported an association between ALPS and RosaiDorfman disease. Both disorders share many clinical features including autoimmune phenomena. The presence of focal Rosai-Dorfman disease in 41% of patients with ALPS opens the possibility that a subpopulation of patients with Rosai-Dorfman disease might have a forme fruste form of ALPS [17].
Acknowledgments We thank Lucimara Chioato, PhD, and Elida B. Ojopi, PhD, for their help related to the TCR gene rearrangement study.
References [1] Canale VC, Smith CH. Chronic lymphadenopathy simulating malignant lymphoma. J Pediatr 1967;70:891-9. [2] Sneller MC, Straus SE, Jaffe ES, et al. A novel lymphoproliferative/ autoimmune syndrome resembling murine lpr/gld disease. J Clin Invest 1992;90:334-41. [3] Watanabe-Fukunaga R, Brannan CI, Copeland NG, Jenkins NA, Nagata S. Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature 1992;356:314-7. [4] Fisher GH, Rosenberg FJ, Straus SE, et al. Dominant interfering Fas gene mutations impair apoptosis in a human autoimmune lymphoproliferative syndrome. Cell 1995;81:935-46.
141 [5] Rieux-Laucat F, Le Deist F, Hivroz C, et al. Mutations in Fas associated with human lymphoproliferative syndrome and autoimmunity. Science 1995;268:1347-9. [6] Bettinardi A, Brugnoni D, Quiros-Roldan E, et al. Missense mutations in the Fas gene resulting in autoimmune lymphoproliferative syndrome: a molecular and immunological analysis. Blood 1997;89: 902-9. [7] van den Berg A, Tamminga R, de Jong D, Maggio E, Kamps W, Poppema S. FAS gene mutation in a case of autoimmune lymphoproliferative syndrome type IA with accumulation of gammadelta+ T cells. Am J Surg Pathol 2003;27:546-53. [8] van den Berg A, Maggio E, Diepstra A, de Jong D, van Krieken JS. Germline FAS gene mutation in a case of ALPS and NLP Hodgkin lymphoma. Blood 2002;99:1492-4. [9] Straus S, Sneller M, Leonardo M, Puck J, Strober W. An inherited disorder of lymphocyte apoptosis: the autoimmune lymphoproliferative syndrome. NIH conference. Ann Intern Med 1999;130:591-601. [10] Oliveira JB, Bidère N, Niemela J, et al. NRAS mutation causes a human lymphoproliferative syndrome. PNAS 2007;104:8953-8. [11] Del-Rey MJ, Manzanares J, Bosque A, et al. Autoimmune lymphoproliferative syndrome (ALPS) in a patient with a new germline Fas gene mutation. Immunobiology 2007;212:73-83. [12] Bacchi MM, Bacchi CE, Alvarenga M, Miranda R, Chen YY, Weiss LM. Burkitt's lymphoma in Brazil: strong association with EpsteinBarr virus. Mod Pathol 1996;9:63-7. [13] Limpens J, Beelen M, Stad R, et al. Detection of the t(14;18) translocation in frozen and formalin-fixed tissue. Diagn Mol Pathol 1993;2:99-107. [14] van Dongen JJM, Langerak AW, Bruggemann M, et al. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 concerted action BMH4-CT98-3936. Leukemia 2003;17:2257-317. [15] Lim MS, Straus SE, Dale JK, et al. Pathological findings in human autoimmune lymphoproliferative syndrome. Am J Pathol 1998;153: 1541-50. [16] Rao VK, Straus SE. Causes and consequences of the autoimmune lymphoproliferative syndrome. Hematology 2006;11:15-23. [17] Maric I, Pittaluga S, Dale J, et al. Histologic features of sinus histiocytosis with massive lymphadenopathy in patients with autoimmune lymphoproliferative syndrome. Am J Surg Pathol 2005;29: 903-11.