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Severe Combined Immunodeficiency Disorder due to a Novel Mutation in Recombination Activation Gene 2: About 2 Cases

Severe Combined Immunodeficiency Disorder due to a Novel Mutation in Recombination Activation... Hindawi Case Reports in Immunology Volume 2021, Article ID 8819368, 5 pages https://doi.org/10.1155/2021/8819368 Case Report Severe Combined Immunodeficiency Disorder due to a Novel Mutation in Recombination Activation Gene 2: About 2 Cases 1,2 1,3 2 3,4 Ibtihal Benhsaien , Fatima Ailal , Khadija Elazhary , Jalila El bakkouri, 2 1,3 Abdallah Badou , and Ahmed Aziz Bousfiha Clinical Immunology Unit, Infectious Disease Department; Children Hospital, IBN Rochd University Hospital, Casablanca, Morocco Cellular and Molecular Pathology Laboratory, Faculty of Medicine and Pharmacy of Casablanca, Hassan II University, Casablanca, Morocco ClinicalImmunology,AutoimmunityandInflammationLaboratory(LICIA),FacultyofMedicineandPharmacyofCasablanca, Hassan II University, Casablanca, Morocco Immunology Laboratory, IBN Rochd University Hospital, Casablanca, Morocco Correspondence should be addressed to Ibtihal Benhsaien; ibtihalbenhsaien@gmail.com Received 19 April 2020; Revised 7 December 2020; Accepted 26 December 2020; Published 7 January 2021 Academic Editor: Alessandro Plebani Copyright © 2021 Ibtihal Benhsaien et al. 'is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Severe combined immunodeficiency (SCID) comprises a heterogeneous group of inherited immunologic disorders with profound defects in cellular and humoral immunity. SCID is the most severe PID and constitutes a pediatric emergency. Affected children are highly susceptible to bacterial, viral, fungal, and opportunistic infections with life-threatening in the absence of hematopoietic stem cell transplantation. We report here two cases of SCID. 'e first case is a girl diagnosed with SCID at birth based on her family history and lymphocyte subpopulation typing. 'e second case is a 4-month-old boy with a history of recurrent op- portunistic infections, BCGitis, and failure to thrive, and the immunology workup confirms a SCID phenotype. 'e genetic study in the two cases revealed a novel mutation in the RAG2 gene, c.826G>A (p.Gly276Ser), in a homozygous state. 'e novel mutation in the RAG2 gene identified in our study may help the early diagnosis of SCID. causing SCID [4]. Depending on the ethnicity, it is estimated 1. Introduction that RAG1/2 mutations account for nearly 50% of patients Severe combined immunodeficiencies (SCIDs) are a group with T-B-NK+SCID [5]. Recombination-activating genes of inherited disorders responsible for severe dysfunctions of (RAG1/2) contribute to the VDJ (variable, diversity, and the immune system that lead to the absence or dysfunction joining) recombination which leads to the generation of diverse antigen receptors [6]. Null mutations cause severe of the Tand B cells derived from the thymus gland and bone marrow, thus affecting both cellular and humoral adaptive combined immunodeficiency (SCID), with the absence of immunity [1]. 'e incidence and prevalence of SCID vary in both B and Tcells and preserved natural killer (NK) cells (T- different parts of the world and are reported to be higher in B-NK+) [7]. countries with a high rate of consanguinity. In Saudi Arabia, By contrast, hypomorphic RAG mutations that allow the prevalence reported is 2,906 live births [2], which is residual expression and function of the mutant protein, (20×) higher than the incidence reported from USA NBS enabling partial T and B lymphocyte development, may programs [3]. 'is is the most severe disorder among pri- cause a spectrum of phenotypes with prominent immune mary immunodeficiency diseases threatening children’s life. dysregulations, as observed in patients with Omenn syn- Actually, seventeen molecular defects are recognized as drome [8], leaky SCID with a predominance of T-cell 2 Case Reports in Immunology receptor (TCR) cδ T cells [9, 10], and combined immu- efficiency and total read counts and quality. Following data nodeficiency with granulomatous disease and/or autoim- analysis, annotation of single-nucleotide variants, insertions, munity [11]. deletions, and splice site alterations was performed by the Ion Reporter Server System (Life Technologies) and Inge- nuity Variant Analysis (Qiagen). 2. Materials and Methods 2.1. Blood Sample Collection. Whole blood was collected in 2.5. Sanger Sequencing. 'e variant of interest was con- EDTA-precoated test tubes. firmed by Sanger sequencing of amplified PCR products. 2.2. Flow Cytometry. Red blood cells were removed from 3. Results whole blood using an RBC lysis buffer. Cells were then stained with fluorochrome-conjugated anti-human CD3, 3.1. Clinical Presentation CD4, CD8, CD19, CD16, and CD56 antibodies. Cells were collected with a FACSCanto II flow cytometer (a FACS- 3.1.1. Case 1. 'e patient was a newborn female from third- Calibur from BD Biosciences, San Jose, CA) and analyzed degree consanguineous marriage (Figure 1). She had a fa- with the FACSDiva software. milial history of death of siblings. 'e first brother had recurrent respiratory infections with chronic diarrhea and died at the age of 4 months without any diagnosis. 'e 2.3.GeneticStudy. Genomic DNA was isolated from 300µL second brother was admitted to the intensive care unit for of peripheral blood using Maxwell 16 Blood DNA Purifi- severe respiratory infection at six months of age and died cation Kit (Promega, Madison, WI, USA) according to the three days after admission. 'e laboratory workup showed manufacturer’s protocol. Both parents of the patient gave lymphopenia, and a SCID was suspected. Considering her their written and informed consent for the genetic study. family history, our patient was not vaccinated and an im- 10ng of DNA was used to amplify the coding region munological workup was done at the age of 20 days using Ion AmpliSeq Inherited Disease Panel (Ion Torrent, (Table 1). 'ermo Fisher Scientific) according to the manufacturer’s 'erefore, the diagnosis of a SCID T-B-NK+ was protocol. established. She was put on trimethoprim-sulfamethoxazole 'e sample was reduced to a final concentration at∼100 as a prophylaxis and on immunoglobulin replacement given pM. Emulsion PCR was performed using the Ion PGM Hi-Q every three weeks. She was free from infection until the age OT2 KIT (Ion Torrent , 'ermo Fisher Scientific) of one and a half months, when she developed oral thrush, according to the manufacturer’s protocol. Following am- treated by fluconazole. At the age of 2 months, she developed plification and recovery, an Ion Sphere Quality Control Kit an axillary lymphadenitis which was treated successfully (Ion Torrent , 'ermo Fisher) was used to evaluate the with ceftazidime and amikacine, given for ten days. At three sample’s quality according to the manufacturer’s protocol. months of age, the infant underwent successful hap- Next, the enrichment was completed by selectively binding loidentical hematopoietic stem cell transplantation with the ISPs containing amplified library fragments to strepta- good outcome. Four months posttransplant, there was a vidin-coated magnetic beads, removing empty ISPs through good immunoglobulin production with a 100% stable washing steps, and denaturing the sample strands to allow lymphoid chimerism. On the last follow-up, the patient was the collection of the positive template ISPs. 5 years old, infection-free with no signs of GVHD, and Sequencing primer and polymerase were added to the completely vaccinated. final enriched spheres ISPs prior to loading into an Ion 318 Chip according to the Ion PGM Hi-Q Sequencing Kit (Ion Torrent , 'ermo Fisher Scientific). 3.1.2. Case 2. 'e patient was a four-month-old boy from Sequencing was carried out on the Personal Genome first-degree consanguineous marriage (Figure 1). He pre- Material (Ion Torrent , 'ermo Fisher Scientific). For a sented by the age of two months chronic diarrhea, recurrent sequence variant to be considered authentic, sequencing respiratory tract infections, oral thrush, locoregional coverage of 250 was used as a minimum requirement in this BCGitis, and failure to thrive. 'e laboratory workup study. Ion AmpliSeq Inherited Disease Panel targets the showed a profound lymphopenia and an absent thymus following PID genes: IL2RG for X-linked SCIDs, RAG1 for shadow in the chest X-ray. A diagnosis of SCID was sus- severe combined immunodeficiency, RAG2 for severe pected, and an immunological workup was done (Table 2). combined immunodeficiency, BTK for agammaglobulin- 'erefore, the diagnosis of a SCID was established. He emia X-linked type 1, WAS for Wiskott–Aldrich syndrome, was on antituberculosis to treat the BCGitis (rifampicine, and AIRE for autoimmune polyendocrine syndrome. isonhiazid, and ethambutol) and antifungal (fluconazole). He received also trimethoprim-sulfamethoxazole as a pro- phylaxis and immunoglobulin replacement every three 2.4. DataAnalysis. Sequence data were processed using the weeks. Each infectious episode was treated actively. Due to a Torrent Suite software v 5.0.4 (Ion Torrent , 'ermo Fisher lack of hematopoietic stem cell transplantation, he died by Scientific) to align reads to the genome reference (HG19) the age of 1 year and a half. and to generate run metrics, including chip loading Case Reports in Immunology 3 Family tree case 1 Case 1 Family tree case 2 Case 2 Figure 1: Pedigrees of families with RAG2 mutation: case 1 and case 2. Squares: male subjects; circles: female subjects; black filled symbols: patients with mutation; grey filled symbols: mutation carriers; crossed-out symbols: deceased subjects. Table 1: Immunological workup. (1) HIV serology �negative 3 3 3 3 (2) CBC: Hb:18.3g/dl (8.00–17.6g/dl), PNN: 1976c/mm (1,000–9,500c/mm ), lymphocytes: 832c/mm (2,000–17,000c/mm ), and Plts: 3 3 383,000c/mm (170,000–500,000c/mm ) (3) Immunoglobulin dosage: IgG: 6.20g/l, IgM: 0.20g/l, and IgA: 0.17g/l 3 3 3 3 3 (4) Lymphocyte subpopulation: lymphocytes: 520c/mm , CD3: 26c/mm (2,500–5,900c/mm ), CD4: 10c/mm (1,400–4,300c/mm ), 3 3 3 3 3 3 CD8: 16c/mm (500–1,700c/mm ), CD19: 1c/mm (300–3,000c/mm ), and CD16-56: 493c/mm (160–950c/mm ) Note: this table shows laboratory results at the time of admission. All normal ranges cited here are adapted to the age [12, 13]. 3.2. Genetic Study. 'e mutation was detected with the analyses: SIFT, polyphen-2, and Grantham indicated that complete RAG2 gene sequencing using the Personal Ge- this variant is highly damaging with a score of 0.0, 1.0, and nome Material and the Ion Reporter System and Ingenuity 56.0, respectively; this was also predicated to damage in the Software and then confirmed by direct target Sanger se- software PopViz (Figure 2), with the MAF score of −7 and the CADD score of 26.6. 'is variant c.826G>A quencing for each patient. 'e two patients carried a variant in the RAG2 gene, (p.Gly276Ser) is considered a new mutation that could cause c.826G>A (p.Gly276Ser), in a homozygous state. Moreover, SCID. To date, this variant is not described in the Exome for the first case, familial segregation was realized and Aggregation Consortium, Exome Sequencing Project, or the exhibited the same variant in the RAG2 gene, c.826G>A 1000 Genomes Browser. To the best of our knowledge, this is (p.Gly276Ser), in a heterozygous state in both parents. It is the first study, which has revealed such a variant. It is located in a highly conserved nucleotide and amino acid classified as a variant of uncertain significance (class 3) position, with small physicochemical differences between according to the recommendations of the American College the exchanged amino acids (Alamut v.2.7.1). Software of Medical Genetics and Genomics (ACMG). 4 Case Reports in Immunology Table 2: Immunological workup. (1) HIV serology �negative 3 3 3 3 (2) CBC: Hb: 11g/dl (8.6–13.7g/dl), PNN: 1,500c/mm (1,500–6,900c/mm ), lymphocytes: 680c/mm (3,900–9,000c/mm ), and Plts: 3 3 114,300c/mm (175,000–500,000c/mm ) (3) Immunoglobulin dosage: IgG: 0.4g/l (2.9–5.5g/l), IgM: 0.04g/l (0.3–0.85g/l), IgA: 0.07g/l (0.2–0.62g/l), and IgE: 0.11 UI/l 3 3 3 3 3 (4) Lymphocyte subpopulation: lymphocytes: 680c/mm (3,900–9,000c/mm ), CD3: 62c/mm (2,500–5,600c/mm ), CD4: 10c/mm 3 3 3 3 (1,800–4,000c/mm ), CD8: 30c/mm3 (590–1,600c/mm ), CD19: 10c/mm (430–3,000c/mm ), and CD16-56: 600c/mm3 (170–830c/ mm ) Note: this table shows laboratory results at the time of admission. All normal ranges cited here are adapted to the age [12, 13]. Plot of CADD vs MAF RAG2 patients expressed a typical clinical SCID phenotype with opportunistic infections and especially in the second case which was very symptomatic. 'e two patients had a pro- found lymphopenia with a very low number of CD3 Tcells. 'e software analyses (SIFT, polyphen-2, Grantham, and PopViz) indicate that this novel mutation c.826G>A in 30 RAG 2 gene is deleterious and then probably responsible for SCID. 'is was also supported by the familial segregation which exhibited the same variants in the heterozygous state. 5. Conclusion We report in this article a newly discovered mutation in exon 2 of the RAG2 gene c.826G>A in a homozygous state. 'ese results indicate that the AmpliSeq libraries, PGM and the Ion Reporter Sever System- based NGS approach is extremely efficient, fast, and cheap. 'is approach could lead MSC cutoff 0.001 to a better patients management and genetic counseling for relatives at risk. –7 –6 –5 –4 –3 –2 –1 0 MAF: 10^X Conflicts of Interest Frameshi Stop gained 'e authors declare that they have no commercial rela- Inframe Del/Ins Synonymous Intronic UTR tionship or potential conflicts of interest related to the Missense User mutation submission. Figure 2: CADD vs. MAF plot of RAG2 by PopViz. 'e vertical Authors’ Contributions and horizontal axes show the combined annotation-dependent depletion (CADD) and the minor allele frequency (MAF) scores, I. Benhsaien performed analysis on the sample, interpreted respectively. 'e red dot is the c.826G>A variant and the other dots are the other variants for pathogenic RAG2 mutations data, and wrote the manuscript. F. Ailal was responsible for (frameshift mutations, inframe Del/Ins mutations, intronic mu- the management of the patient and data interpretation. tation, missense mutations, etc.) reported. 'e MAF score is −7 and J. Elbakkouri performed immunological analysis. the CADD score is 26.6, which indicates that the new mutation K. Elazhary interpreted data. A. Badou performed study c.826G>A is probably deleterious. design, data interpretation, and manuscript evaluation. A. A. Bousfiha conceived the study, helped to evaluate the man- uscript, and edited the manuscript. 4. Discussion 'e RAG1 and RAG2 proteins constitute a recombinase complex, which starts the VDJ somatic recombination by References cleaving double-stranded DNA at special sequences, known [1] E. Cirillo, G. Giardino, V. Gallo et al., “Severe combined as recombination signal sequences (RSSs), flanking the immunodeficiency—an update,” Annals of the New York variable , diversity , and joining gene exons encoding the Academy of Sciences, vol. 1356, no. 1, 2015. genes of immunoglobulins (Ig) and T-cell receptors. 'is [2] H. Almousa, G. Al-Dakheel, A. Jabr et al., “High incidence of somatic recombination generates a diverse repertoire of severe combined immunodeficiency disease in Saudi Arabia B cells and Tcells [14, 15]. Mutations in RAG1/2 genes lead detected through combined Tcell receptor excision circle and to loss or reduction of V(D) J recombination, consequently a next generation sequencing of newborn dried blood spots,” blockade in B- and T-cell development, which is referred to Frontiers in Immunology, vol. 9, no. 782, 2018. as severe combined immunodeficiency (SCID). Our two [3] A. Kwan, R. S. Abraham, R. Currier et al., “Newborn screening for severe combined immunodeficiency in 11 screening CADD score Case Reports in Immunology 5 programs in the United States,” JAMA, vol. 312, no. 7, pp. 729–738, 2014. [4] S. G. Tangye, W. Al-Herz, A. Bousfiha et al., “Human inborn errors of immunity: 2019 update on the classification from the international union of immunological societies expert com- mittee,” Journal of Clinical Immunology, vol. 40, no. 1, p. 24, [5] T. Niehues, R. Perez-Becker, and C. Schuetz, “More than just SCID-'ephenotypic range of combinedimmunodeficiencies associated with mutations in the recombinase activating genes (RAG) 1 and 2,” Clinical Immunology, vol. 135, no. 2, pp. 183–192, 2010. [6] J. L. Casanova, H. Chapel, M. E. Conley et al., “International union of immunological societies primary immunodeficiency diseases classification committee. primary immunodeficiency diseases: an update from the international union of immu- nological societies primary immunodeficiency diseases clas- sification committee,” Journal of Allergy and Clinical Immunology, vol. 120, pp. 776–794, 2007. [7] S. S. Meshaal, R. E. El Hawary, D. S. Abd Elaziz et al., “Phenotypical heterogeneity in RAG-deficient patients from a highly consanguineous population,” Clinical & Experimental Immunology, vol. 195, no. 2, pp. 202–212, 2019. [8] A. Villa, S. Santagata, F. Bozzi et al., “Partial V(D)J recom- bination activity leads to Omenn syndrome,” Cell, vol. 93, no. 5, pp. 885–896, 1998. [9] S. Ehl, K. Schwarz, A. Enders et al., “A variant of SCID with specific immune responses and predominance of T cells,” Journal of Clinical Investigation, vol. 115, no. 11, pp. 3140– 3148, 2005. [10] J.-P. De Villartay, A. Lim, H. Al-Mousa et al., “A novel im- munodeficiency associated with hypomorphic RAG1 muta- tions and CMV infection,” Journal of Clinical Investigation, vol. 115, no. 11, pp. 3291–3299, 2005. [11] C. Schuetz, K. Huck, S. Gudowius et al., “An immunodefi- ciency disease with RAG mutations and granulomas,” New England Journal of Medicine, vol. 358, no. 19, pp. 2030–2038, [12] S. J. Staffa, J. D. Joerger, E. Henry, R. D. Christensen, C. Brugnara, and D. Zurakowski, “Pediatric hematology normal ranges derived from pediatric primary care patients,” American Journal of Hematology, vol. 95, no. 10, 2020. [13] W. T. Shearer, H. M. Rosenblatt, R. S. Gelman et al., “Lymphocyte subsets in healthy children from birth through 18 years of age,” Journal of Allergy and Clinical Immunology, vol. 112, no. 5, pp. 973–980, 2003. [14] D. G. Schatz and P. C. Swanson, “V(D)J recombination: mechanisms of initiation,” Annual Review of Genetics, vol. 45, no. 1, pp. 167–202, 2011. [15] C. B. Geier, A. Piller, A. Linder, K. M. Sauerwein, M. M. Eibl, and H. M. Wolf, “Leaky RAG defciency in adult patients with impaired antibody production against bacterial polysaccha- ride antigens,” PLoS ONE, vol. 10, no. 7, Article ID e0133220, http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Case Reports in Immunology Hindawi Publishing Corporation

Severe Combined Immunodeficiency Disorder due to a Novel Mutation in Recombination Activation Gene 2: About 2 Cases

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Copyright © 2021 Ibtihal Benhsaien et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Abstract

Hindawi Case Reports in Immunology Volume 2021, Article ID 8819368, 5 pages https://doi.org/10.1155/2021/8819368 Case Report Severe Combined Immunodeficiency Disorder due to a Novel Mutation in Recombination Activation Gene 2: About 2 Cases 1,2 1,3 2 3,4 Ibtihal Benhsaien , Fatima Ailal , Khadija Elazhary , Jalila El bakkouri, 2 1,3 Abdallah Badou , and Ahmed Aziz Bousfiha Clinical Immunology Unit, Infectious Disease Department; Children Hospital, IBN Rochd University Hospital, Casablanca, Morocco Cellular and Molecular Pathology Laboratory, Faculty of Medicine and Pharmacy of Casablanca, Hassan II University, Casablanca, Morocco ClinicalImmunology,AutoimmunityandInflammationLaboratory(LICIA),FacultyofMedicineandPharmacyofCasablanca, Hassan II University, Casablanca, Morocco Immunology Laboratory, IBN Rochd University Hospital, Casablanca, Morocco Correspondence should be addressed to Ibtihal Benhsaien; ibtihalbenhsaien@gmail.com Received 19 April 2020; Revised 7 December 2020; Accepted 26 December 2020; Published 7 January 2021 Academic Editor: Alessandro Plebani Copyright © 2021 Ibtihal Benhsaien et al. 'is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Severe combined immunodeficiency (SCID) comprises a heterogeneous group of inherited immunologic disorders with profound defects in cellular and humoral immunity. SCID is the most severe PID and constitutes a pediatric emergency. Affected children are highly susceptible to bacterial, viral, fungal, and opportunistic infections with life-threatening in the absence of hematopoietic stem cell transplantation. We report here two cases of SCID. 'e first case is a girl diagnosed with SCID at birth based on her family history and lymphocyte subpopulation typing. 'e second case is a 4-month-old boy with a history of recurrent op- portunistic infections, BCGitis, and failure to thrive, and the immunology workup confirms a SCID phenotype. 'e genetic study in the two cases revealed a novel mutation in the RAG2 gene, c.826G>A (p.Gly276Ser), in a homozygous state. 'e novel mutation in the RAG2 gene identified in our study may help the early diagnosis of SCID. causing SCID [4]. Depending on the ethnicity, it is estimated 1. Introduction that RAG1/2 mutations account for nearly 50% of patients Severe combined immunodeficiencies (SCIDs) are a group with T-B-NK+SCID [5]. Recombination-activating genes of inherited disorders responsible for severe dysfunctions of (RAG1/2) contribute to the VDJ (variable, diversity, and the immune system that lead to the absence or dysfunction joining) recombination which leads to the generation of diverse antigen receptors [6]. Null mutations cause severe of the Tand B cells derived from the thymus gland and bone marrow, thus affecting both cellular and humoral adaptive combined immunodeficiency (SCID), with the absence of immunity [1]. 'e incidence and prevalence of SCID vary in both B and Tcells and preserved natural killer (NK) cells (T- different parts of the world and are reported to be higher in B-NK+) [7]. countries with a high rate of consanguinity. In Saudi Arabia, By contrast, hypomorphic RAG mutations that allow the prevalence reported is 2,906 live births [2], which is residual expression and function of the mutant protein, (20×) higher than the incidence reported from USA NBS enabling partial T and B lymphocyte development, may programs [3]. 'is is the most severe disorder among pri- cause a spectrum of phenotypes with prominent immune mary immunodeficiency diseases threatening children’s life. dysregulations, as observed in patients with Omenn syn- Actually, seventeen molecular defects are recognized as drome [8], leaky SCID with a predominance of T-cell 2 Case Reports in Immunology receptor (TCR) cδ T cells [9, 10], and combined immu- efficiency and total read counts and quality. Following data nodeficiency with granulomatous disease and/or autoim- analysis, annotation of single-nucleotide variants, insertions, munity [11]. deletions, and splice site alterations was performed by the Ion Reporter Server System (Life Technologies) and Inge- nuity Variant Analysis (Qiagen). 2. Materials and Methods 2.1. Blood Sample Collection. Whole blood was collected in 2.5. Sanger Sequencing. 'e variant of interest was con- EDTA-precoated test tubes. firmed by Sanger sequencing of amplified PCR products. 2.2. Flow Cytometry. Red blood cells were removed from 3. Results whole blood using an RBC lysis buffer. Cells were then stained with fluorochrome-conjugated anti-human CD3, 3.1. Clinical Presentation CD4, CD8, CD19, CD16, and CD56 antibodies. Cells were collected with a FACSCanto II flow cytometer (a FACS- 3.1.1. Case 1. 'e patient was a newborn female from third- Calibur from BD Biosciences, San Jose, CA) and analyzed degree consanguineous marriage (Figure 1). She had a fa- with the FACSDiva software. milial history of death of siblings. 'e first brother had recurrent respiratory infections with chronic diarrhea and died at the age of 4 months without any diagnosis. 'e 2.3.GeneticStudy. Genomic DNA was isolated from 300µL second brother was admitted to the intensive care unit for of peripheral blood using Maxwell 16 Blood DNA Purifi- severe respiratory infection at six months of age and died cation Kit (Promega, Madison, WI, USA) according to the three days after admission. 'e laboratory workup showed manufacturer’s protocol. Both parents of the patient gave lymphopenia, and a SCID was suspected. Considering her their written and informed consent for the genetic study. family history, our patient was not vaccinated and an im- 10ng of DNA was used to amplify the coding region munological workup was done at the age of 20 days using Ion AmpliSeq Inherited Disease Panel (Ion Torrent, (Table 1). 'ermo Fisher Scientific) according to the manufacturer’s 'erefore, the diagnosis of a SCID T-B-NK+ was protocol. established. She was put on trimethoprim-sulfamethoxazole 'e sample was reduced to a final concentration at∼100 as a prophylaxis and on immunoglobulin replacement given pM. Emulsion PCR was performed using the Ion PGM Hi-Q every three weeks. She was free from infection until the age OT2 KIT (Ion Torrent , 'ermo Fisher Scientific) of one and a half months, when she developed oral thrush, according to the manufacturer’s protocol. Following am- treated by fluconazole. At the age of 2 months, she developed plification and recovery, an Ion Sphere Quality Control Kit an axillary lymphadenitis which was treated successfully (Ion Torrent , 'ermo Fisher) was used to evaluate the with ceftazidime and amikacine, given for ten days. At three sample’s quality according to the manufacturer’s protocol. months of age, the infant underwent successful hap- Next, the enrichment was completed by selectively binding loidentical hematopoietic stem cell transplantation with the ISPs containing amplified library fragments to strepta- good outcome. Four months posttransplant, there was a vidin-coated magnetic beads, removing empty ISPs through good immunoglobulin production with a 100% stable washing steps, and denaturing the sample strands to allow lymphoid chimerism. On the last follow-up, the patient was the collection of the positive template ISPs. 5 years old, infection-free with no signs of GVHD, and Sequencing primer and polymerase were added to the completely vaccinated. final enriched spheres ISPs prior to loading into an Ion 318 Chip according to the Ion PGM Hi-Q Sequencing Kit (Ion Torrent , 'ermo Fisher Scientific). 3.1.2. Case 2. 'e patient was a four-month-old boy from Sequencing was carried out on the Personal Genome first-degree consanguineous marriage (Figure 1). He pre- Material (Ion Torrent , 'ermo Fisher Scientific). For a sented by the age of two months chronic diarrhea, recurrent sequence variant to be considered authentic, sequencing respiratory tract infections, oral thrush, locoregional coverage of 250 was used as a minimum requirement in this BCGitis, and failure to thrive. 'e laboratory workup study. Ion AmpliSeq Inherited Disease Panel targets the showed a profound lymphopenia and an absent thymus following PID genes: IL2RG for X-linked SCIDs, RAG1 for shadow in the chest X-ray. A diagnosis of SCID was sus- severe combined immunodeficiency, RAG2 for severe pected, and an immunological workup was done (Table 2). combined immunodeficiency, BTK for agammaglobulin- 'erefore, the diagnosis of a SCID was established. He emia X-linked type 1, WAS for Wiskott–Aldrich syndrome, was on antituberculosis to treat the BCGitis (rifampicine, and AIRE for autoimmune polyendocrine syndrome. isonhiazid, and ethambutol) and antifungal (fluconazole). He received also trimethoprim-sulfamethoxazole as a pro- phylaxis and immunoglobulin replacement every three 2.4. DataAnalysis. Sequence data were processed using the weeks. Each infectious episode was treated actively. Due to a Torrent Suite software v 5.0.4 (Ion Torrent , 'ermo Fisher lack of hematopoietic stem cell transplantation, he died by Scientific) to align reads to the genome reference (HG19) the age of 1 year and a half. and to generate run metrics, including chip loading Case Reports in Immunology 3 Family tree case 1 Case 1 Family tree case 2 Case 2 Figure 1: Pedigrees of families with RAG2 mutation: case 1 and case 2. Squares: male subjects; circles: female subjects; black filled symbols: patients with mutation; grey filled symbols: mutation carriers; crossed-out symbols: deceased subjects. Table 1: Immunological workup. (1) HIV serology �negative 3 3 3 3 (2) CBC: Hb:18.3g/dl (8.00–17.6g/dl), PNN: 1976c/mm (1,000–9,500c/mm ), lymphocytes: 832c/mm (2,000–17,000c/mm ), and Plts: 3 3 383,000c/mm (170,000–500,000c/mm ) (3) Immunoglobulin dosage: IgG: 6.20g/l, IgM: 0.20g/l, and IgA: 0.17g/l 3 3 3 3 3 (4) Lymphocyte subpopulation: lymphocytes: 520c/mm , CD3: 26c/mm (2,500–5,900c/mm ), CD4: 10c/mm (1,400–4,300c/mm ), 3 3 3 3 3 3 CD8: 16c/mm (500–1,700c/mm ), CD19: 1c/mm (300–3,000c/mm ), and CD16-56: 493c/mm (160–950c/mm ) Note: this table shows laboratory results at the time of admission. All normal ranges cited here are adapted to the age [12, 13]. 3.2. Genetic Study. 'e mutation was detected with the analyses: SIFT, polyphen-2, and Grantham indicated that complete RAG2 gene sequencing using the Personal Ge- this variant is highly damaging with a score of 0.0, 1.0, and nome Material and the Ion Reporter System and Ingenuity 56.0, respectively; this was also predicated to damage in the Software and then confirmed by direct target Sanger se- software PopViz (Figure 2), with the MAF score of −7 and the CADD score of 26.6. 'is variant c.826G>A quencing for each patient. 'e two patients carried a variant in the RAG2 gene, (p.Gly276Ser) is considered a new mutation that could cause c.826G>A (p.Gly276Ser), in a homozygous state. Moreover, SCID. To date, this variant is not described in the Exome for the first case, familial segregation was realized and Aggregation Consortium, Exome Sequencing Project, or the exhibited the same variant in the RAG2 gene, c.826G>A 1000 Genomes Browser. To the best of our knowledge, this is (p.Gly276Ser), in a heterozygous state in both parents. It is the first study, which has revealed such a variant. It is located in a highly conserved nucleotide and amino acid classified as a variant of uncertain significance (class 3) position, with small physicochemical differences between according to the recommendations of the American College the exchanged amino acids (Alamut v.2.7.1). Software of Medical Genetics and Genomics (ACMG). 4 Case Reports in Immunology Table 2: Immunological workup. (1) HIV serology �negative 3 3 3 3 (2) CBC: Hb: 11g/dl (8.6–13.7g/dl), PNN: 1,500c/mm (1,500–6,900c/mm ), lymphocytes: 680c/mm (3,900–9,000c/mm ), and Plts: 3 3 114,300c/mm (175,000–500,000c/mm ) (3) Immunoglobulin dosage: IgG: 0.4g/l (2.9–5.5g/l), IgM: 0.04g/l (0.3–0.85g/l), IgA: 0.07g/l (0.2–0.62g/l), and IgE: 0.11 UI/l 3 3 3 3 3 (4) Lymphocyte subpopulation: lymphocytes: 680c/mm (3,900–9,000c/mm ), CD3: 62c/mm (2,500–5,600c/mm ), CD4: 10c/mm 3 3 3 3 (1,800–4,000c/mm ), CD8: 30c/mm3 (590–1,600c/mm ), CD19: 10c/mm (430–3,000c/mm ), and CD16-56: 600c/mm3 (170–830c/ mm ) Note: this table shows laboratory results at the time of admission. All normal ranges cited here are adapted to the age [12, 13]. Plot of CADD vs MAF RAG2 patients expressed a typical clinical SCID phenotype with opportunistic infections and especially in the second case which was very symptomatic. 'e two patients had a pro- found lymphopenia with a very low number of CD3 Tcells. 'e software analyses (SIFT, polyphen-2, Grantham, and PopViz) indicate that this novel mutation c.826G>A in 30 RAG 2 gene is deleterious and then probably responsible for SCID. 'is was also supported by the familial segregation which exhibited the same variants in the heterozygous state. 5. Conclusion We report in this article a newly discovered mutation in exon 2 of the RAG2 gene c.826G>A in a homozygous state. 'ese results indicate that the AmpliSeq libraries, PGM and the Ion Reporter Sever System- based NGS approach is extremely efficient, fast, and cheap. 'is approach could lead MSC cutoff 0.001 to a better patients management and genetic counseling for relatives at risk. –7 –6 –5 –4 –3 –2 –1 0 MAF: 10^X Conflicts of Interest Frameshi Stop gained 'e authors declare that they have no commercial rela- Inframe Del/Ins Synonymous Intronic UTR tionship or potential conflicts of interest related to the Missense User mutation submission. Figure 2: CADD vs. MAF plot of RAG2 by PopViz. 'e vertical Authors’ Contributions and horizontal axes show the combined annotation-dependent depletion (CADD) and the minor allele frequency (MAF) scores, I. Benhsaien performed analysis on the sample, interpreted respectively. 'e red dot is the c.826G>A variant and the other dots are the other variants for pathogenic RAG2 mutations data, and wrote the manuscript. F. Ailal was responsible for (frameshift mutations, inframe Del/Ins mutations, intronic mu- the management of the patient and data interpretation. tation, missense mutations, etc.) reported. 'e MAF score is −7 and J. Elbakkouri performed immunological analysis. the CADD score is 26.6, which indicates that the new mutation K. Elazhary interpreted data. A. Badou performed study c.826G>A is probably deleterious. design, data interpretation, and manuscript evaluation. A. A. Bousfiha conceived the study, helped to evaluate the man- uscript, and edited the manuscript. 4. 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Published: Jan 7, 2021

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