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NextgenerationsequencingrevealedDNAligaseIVdeficiencyina“developmentallynormal”patientwithmassive...

ArticleinClinicalImmunology·January2016

DOI:10.1016/j.clim.2016.01.002

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Letter to the Editor

Next generation sequencing revealed DNAligase IV deficiency in a “developmentallynormal” patient with massive brain Epstein–Barr virus-positive diffuse large B-cell lym-phoma

Keywords:LIG4 mutationEpstein–Barr virus-positive diffuse large B-cell lymphoma

DNA ligase IV deficient patients usually present with microcephaly,low birth weight, growth and/or mental retardation, dysmorphic facialfindings, variable immunodeficiency, pancytopenia, and radiosensitivi-ty due to impaired repair of DNA double-strand breaks by non-homologous end-joining [1,2]. Non-Hodgkin's lymphoma is known tobe the most frequent malignancy associated with immunodeficiency.Diffuse large B-cell lymphoma (DLBCL) is considered to be the mostcommon type frequently associated with Epstein–Barr virus (EBV)and having a predilection for extranodal sites [3]. We report a case ofthe female patient with DNA ligase IV deficiency syndrome, who hadno clinical features of that syndrome (Fig. 1A.) and developed EBV-

associated DLBCL with the right lung involvement and a massive braintumor lesion at the age of 2 years old. Next generation sequencingrevealed c.2736 + 3delC, c.8 CNT (p.A3V) and c.26CNT (p.T9I) in LIG4gene and additional polymorphisms in ATM, NOD2 and NLRP3 genes.

A healthy female was born at term (weight 3050 g., height 50 сm,circumference of the head 34 cm) from a non-consanguineousmarriage,and was vaccinated BCG without any clinical complications. She had ahistory of primary EBV infection at the age of 11 months with persistedEBV in blood proven by PCR. She had stomatitis with ulceration andperforation of the hard palate. Six months later she developed encepha-litis with facial and oculomotor nerve paresis, generalized convulsions,hemiplegia of the right side of the body. PCR of cerebrospinal fluidwas positive for EBV. Brain CT revealed multiple foci of calcification(Fig. 1B,C,D). The patient received short courses of antiviral treatmentas well as intravenous immunoglobulin and dexamethasone with ashort-term clinical response.

At the age of 2 years and 3months, she had another episode of activeEBV infection with fever and cough followed by a rapid deterioration ofthe neurological status. Chest CT scan showed foci in the right lung(Fig. 2A,B,C). Brain CT revealed negative dynamics with tumor massesin the white matter in the left brain stem with moderate contrastenhancement and decreased density and differentiation of gray andwhite matter. Lung and brain biopsy was performed, and the diagnosis

Clinical Immunology 163 (2016) 108–110

Fig. 1. Patient at the age of 2 years and 10monthswithout dysmorphic facialfindings andmicrocephaly, (A)written informed consentwas obtained fromparents for publishing photo andclinical data Brain CT scan of the patient at the age of 1 year and 7months revealsmultiple focal calcification in the right hemisphere of the brain (B), in the left (C), in both (D), probably asa manifestation of viral encephalitis.

http://dx.doi.org/10.1016/j.clim.2016.01.0021521-6616/© 2016 Elsevier Inc. All rights reserved.

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of DLBCL, EBV-positive was confirmed. Immunoblastic tumor cellsstained positive for CD20 and EBV-encoded RNAs (EBERs) but negativefor CD30, CD15, ALK, EMA, CD10, CD3 by immunohistochemistry(Fig. 2D,E,F).

Flow cytometry analysis of her peripheral blood mononuclear cellsshowed decreased number of T cells (CD3+29.2% (307 cells/μL, normalrange for age (NRA): 2000–4500 cells/μL), CD4+ 13.7% (144 cells/μL),CD8+ 3.4% (36 cells/μL)), elevated percentage of B cells (CD19+57.5% (604 cells/μL)) with normal B memory distribution and normalpercentage of NK 9.8% (103 cells/μL). Abnormalities in humoral immuni-ty were revealed as well as low IgA level and titer of isohemagglutinins(IgG 14.02 g/L (after intravenous immunoglobulin replacement), IgM1.44 g/L (NRA: 0.19–1.46 g/L), IgA 0.08 g/L (NRA: 0.2–1 g/L); titer ofisohemagglutinins was 1:2 (blood group II (+)), NRA: 1:16–1:128).Due to a small number of CD8+ cell, Zap-70 expression was checkedby flow cytometry. No differences between the patient's and thecontrol's data were found.

Recent thymic emigrants (RTE) were extremely reduced, CD4 +CD45RA + CD31+ 2.56% (NRA: N35%) and T cell receptor clonalityrevealed absence of TCRVb9, TCRVb20, TCRVb18, TCRVb5.2 and a high ex-pression of TCRVb14. The patient's fibroblasts were sensitive for ionizingradiation checked by gamma-H2AX expression by confocal microscopy(NRA n = 6: 24 h. no ionization radiation (IR)/yes IR 2.5 ± 0.3/6 ± 0.5foci per cell nucleus, the patient 4.1/9.8).

Abnormalities in the DNA reparation machinery gave the reasons toassumeDNA recombination defects (RAG1, RAG2, Artemis,DNA PKcs andothers) [1]. Next generation sequencingwas performed, and it revealedc.2736 + 3delC, c.8 CNT (p.A3V) and c.26CNT (p.T9I) in LIG4 gene. Asmall deletion and p.A3V (PolyPhen-2 software gave a slightly higherthe score of damaging 0.38, substitution was predicted to be benign)were inherited from her mother and the point mutation (p.T9I)(PolyPhen-2, Score of damaging = 0.52, the mutation was predictedto be possible damaging) was identified in her father. The presence oftwomissense substitutions inherited from both parents with additionaldeletion from mother was the reason for establishing the geneticdiagnosis for the patient. Additional polymorphisms in ATM genec.146CNG, p.S49C, NOD2 gene c.2653GNC, p.A885P and in NLRP3 genec.410GNA, p.R137H were found.

She was treated with NHL-BFM-based chemotherapy and re-ceived 4 courses of high-dose methotrexate (5 g/m2) and a courseof high-dose cytarabine (12 g/m2) combined with dexamethasone.She received rituximab as well, both intravenously (six doses) andintrathecally (two doses). During the chemotherapy, the patientreceived antiviral treatment (acyclovir, ganciclovir and foscarnet).A partial response was achieved, but a rapid brain tumor growth oc-curred after the fifth course of chemotherapy, while EBV DNA by PCRremained positive both in blood and cerebrospinal fluid (Fig. 2G,H,I).She died seven months after DLBCL was diagnosed due to lymphoma

Fig. 2. Chest CT scan of the patient's lungs at the age of 2 years and 3 months at the time of lymphoma diagnosis shows two homogeneous soft tissue infiltrations in the right upper lobeparamediastinally (25 × 30 × 30mm in diameter) and subpleural nodule (10 × 10× 15mm) in the lower lobe, and two smaller foci (4–5mm) in the upper and lower lobes. Additionally,twoperivascularmasses (2mm) are detected in both lungs (A – frontal; B, C – sagittal sections) Histopathology of the lung tumor showing a diffuse proliferation of large atypical lymphoidcellsmediumand large size with eosinophilic cytoplasm, large nuclei with prominent central nucleolus (DH&E 10×, E H&E 20×). Tumor cells are positive for CD20, (F – 20×), detected byimmunohistochemistry Brain CT scan of the patient at the age of 2 years and 8 months (G, H, I) shows nonhomogeneous-solid “granulomas” with moderate contrast enhancement,progression of diffuse leukomalacia, focal calcification of the left hemisphere of the brain, the rise of internal hydrocephalus and signs of toxic encephalopathy.

109Letter to the Editor

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progression and severe infectious complications. An autopsy was notperformed.

Confirmation carriers of mutations in the parents gave a possibilityto carry out prenatal diagnosis in this family. At the twelfth week ofpregnancy, chorionic villi were taken from themother, and prenatal di-agnosis was performed. Only the paternal mutation p.T9I was detectedin fetal DNA. At the 39th week, a healthy female was born with normallymphocyte subsets, RTE, TRECs and the thymus size.

Twenty-eight cases of identified mutations in LIG4 gene have beenpreviously reported [5–10]. They were divided into seven distinct cate-gories: 1) leukemia; 2) DNA ligase IV deficiency syndrome; 3) Dubowitssyndrome; 4) Omenn syndrome; 5) radiosensitive severe combinedimmunodeficiency [5]; 6) Seckel syndrome with AML/MDS [8];7) slowly-progressing SCID [10]. Patients with LIG4 gene had increasedincidence of hematologicalmalignancies [1,2,4–9]. Additionally, 8 out of28 had hematologicalmalignancies, two of themEBV-associated or neg-ative lymphomas [6,7]. Twenty-seven out of 28 had facial abnormalities[4–8]. The main differences of our case included lack of classic clinicalstigmata, the presence of B cells in the peripheral blood and an earlyage of lymphoma manifestation in comparison with other patientswho were described. The only similar case of DNA ligase IV deficiencyto be reported was a “developmentally normal” 14-year-old boy (withpresumably normal growth) who exhibited severe radiosensitivityduring treatment for leukemia [9].

We present a patient with a combination of clinical and immunolog-ic data, whichwere not previously reported as DNA ligase IV deficiency.Next generation sequencing helped to identify a genetic basis of atypicalsevere combined immunodeficiency complicated with a massive brainand lung EBV-positive DLBCL and to reveal previously unreportedphenotype due to mutations in LIG4 gene.

Conflict of Interest

The authors declare that they have no conflict of interest.

References

[1] M.A. Slatter, A.R. Gennery, Primary immunodeficiencies associated with DNA-repairdisorders, Expert Rev. Mol. Med. 12 (2010), e9.

[2] D.A. Chistiakov, Ligase IV syndrome, Adv. Exp. Med. Biol. 685 (2010) 175–185.[3] H. Tran, J. Nourse, S. Hall, M. Green, L. Griffiths, M.K. Gandhi, Immunodeficiency-

associated lymphomas, Blood Rev. 22 (2008) 261–281.

[4] J.E. Murray, L.S. Bicknell, G. Yigit, A.L. Duker, M. van Kogelenberg, S. Haghayegh,et al., Extreme growth failure is a common presentation of ligase IV deficiency,Hum. Mutat. 35 (2014) 76–85.

[5] H.I. Jspeert, A. Warris, M. van der Flier, I. Reisli, S. Keles, S. Chishimba, et al., Clinicalspectrum of LIG4 deficiency is broadened with severe dysmaturity, primordialdwarfism, and neurological abnormalities, Hum. Mutat. 34 (2013) 1611–1614.

[6] N. Toita, N. Hatano, S. Ono, M. Yamada, R. Kobayashi, I. Kobayashi, et al., Epstein–Barr virus-associated B-cell lymphoma in a patient with DNA ligase IV (LIG4)syndrome, Am. J. Med. Genet. A 143A (2007) 742–745.

[7] C.M. Bacon, S.J. Wilkinson, G.P. Spickett, D. Barge, H.H. Lucraft, G. Jackson, et al.,Epstein–Barr virus-independent diffuse large B cell lymphoma in DNA ligase 4deficiency, J. Allergy Clin. Immunol. 131 (2013) 1237–1239.

[8] M.Y. Zhang, S.B. Keel, T. Walsh, M.K. Lee, S. Gulsuner, A.C. Watts, et al., Genomicanalysisof bone marrow failure and myelodysplastic syndromes reveals phenotypicand diagnostic complexity, Haematologica 100 (2015) 42–48.

[9] E. Riballo, S.E. Critchlow, S.H. Teo, A.J. Doherty, A. Priestley, B. Broughton, et al.,Identification of a defect in DNA ligase IV in a radiosensitive leukaemia patient,Curr. Biol. 9 (1999) 699–702.

[10] S. Tamura, K. Higuchi, M. Tamaki, C. Inoue, R. Awazawa, N. Mitsuki, Y. Nakazawa,et al., Novel compound heterozygous DNA ligase IV mutations in an adolescentwith a slowly-progressing radiosensitive severe combined immunodeficiency,Clin. Immunol. 160 (2015) 255–260, http://dx.doi.org/10.1016/j.clim.2015.07.004.

Svetlana O. SharapovaResearch department, Belarusian Research Center for Pediatric Oncology,

Hematology and Immunology, Minsk region, BelarusCorresponding author at: Research department, Immunology

Laboratory, Belarusian Research Center for Pediatric Oncology,Hematology and Immunology, Minsk region, settlement of Borovliani

223053, Belarus.E-mail address: sharapovasv@gmail.com.

Elizabeth Yenhui ChangDepartment of Pathology and Laboratory Medicine All Children's Hospital

Johns Hopkins Medicine, St. Petersburg, FL, USA

Irina E. GuryanovaInna V. Proleskovskaya

Alina S. FedorovaElena A. RutskayaOlga V. Aleinikova

Research department, Belarusian Research Center for Pediatric Oncology,Hematology and Immunology, Minsk region, Belarus

5 August 2015Available online 13 January 2016

110 Letter to the Editor

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