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Summer Research Fellowship Programme of India's Science Academies

Molecular characterization of severe combined immunodeficiency (SCID)

Ankita Jha

Ramaiah Institute of Technology, Bengaluru, Karnataka 560054

Dr. Manisha Madkaikar

Director, National Institute of Immunohaematology, 13th floor, New Multistoreyed Building, KEM Hospital Campus, Parel, Mumbai 400012

Abstract

SCID is the most severe form of primary immunodeficiency disease. It is characterised by abnormality in the function of immune system due to the genetic defects in the development of T cells. SCID is seen in infants and is fatal if left untreated in the first year of life. Based on IUIS classification, immunodeficiency affecting cellular and humoral immunity is categorized into different types of SCID depending on defective T, B and/or NK cells. It could be (T- B-NK+ SCID), (T-B-NK- SCID), (T-B+NK- SCID), (T-B+NK+ SCID). Currently, hematopoietic stem cell transplant (HSCT) is the treatment modality for SCID, however gene therapy trials are showing great promise and attempting to become an alternative to the bone marrow transplant. The type of SCID helps in determining the best treatment for which diagnosis has to be made on family history, clinical and immunological profile. It includes flow cytometric evaluation of T, B and NK cells, pSTAT5 on whole blood, protein expression of CD127, CD132, Human Leukocyte antigen-D related (HLA-DR) on lymphocytes, and concentration of T cell receptor excision circles (TRECs). A low lymphocyte count, low or absent TRECs and defects in lymphocyte subsets account for the diagnosis of SCID. Due to overlapping phenotypes, SCID involves a number of genes such as IL2RG, ADA, RAG1/RAG2, JAK3, PNP etc and its assessment becomes tiresome and time-consuming using conventional methods. Molecular characterization has helped in identifying genetic defects in a much simpler way by designing Targeted Gene panel. It consists of genes which have suspected associations with the disease and are ideal for analysing specific mutations. Genes are sequenced using Next Generation Sequencing (NGS), a massive parallel sequencing tool in which multiple DNA fragments can be sequenced at the same time. NGS panels assess multiple genes simultaneously and identify variations in a simpler and cost-effective manner. The identified mutations can be then confirmed by Sanger sequencing.​

Keywords: IUIS classification, flow cytometry, NGS gene panel

Abbreviations

Abbreviations
 IUISInternational Union of Immunological Societies
HSCTHematopoietic Stem Cell Transplant
 TRECT cell receptor excision circles  
NGS Next Generation Sequencing 
PCR Polymerase Chain Reaction 
 ADAAdenosine Deaminase  
 JAK3Janus Kimase 3 
dNTP Deoxyribonucleotide Triphosphate 
 WGSWhole Genome Sequencing 
 WESWhole Exome Sequencing 
 TGPTargeted Gene Panel 
DMSO Dimethyl Suphoxide 
PB Phosphate Buffer 
EB Elution Buffer 
 NCBINational Centre for Biotechnology Information 
CBC Complete blood cell  
 ALCAbsolute Lymphocyte Count 

INTRODUCTION

Background/Rationale

SCID is the most severe form of primary immunodeficiency. It is a rare genetic disorder characterised by impairment in development of lymphocytes affecting cellular and humoral adaptive immunity. The incidence of this disease is 1 in 58,000 births (​​ Neil A. Holtzman et al, 2014). ​​It is seen in infants where they are susceptible to various infections and if not given treatment in the first year of life it becomes fatal and leads to their death. The detection of SCID goes undetectable during birth and until about six months of age as they carry maternal antibodies, but it can be suspected because of recurrent, persistent infections. SCID can be recognized by severely low lymphocyte count, low or absent TRECs (T-cell receptor excision circles) and defects in lymphocyte subsets. Screening for SCID can be done by using quantitative real time PCR to measure the level of TRECs. IUIS (International Union of Immunological Societies) classifies typical SCID based on the analysis of CD3, CD19, CD16 on T, B and NK cells. CD3 count in typical SCID is lower than 300cells/microL (​​Shearer et al, 2014​)​. Presence of low or normal CD19 on B cells can further group SCID into T-B-NK+ SCID, T-B-NK- SCID, T-B+NK- SCID, T-B+NK+ SCID ( Bousfiha et al, 2017 ​​​​).

IUIS CLASSIFICATION_1.jpg
    IUIS CLASSIFICATION OF SCID

    Haematopoietic stem cell transplant provides the clinically approved cure for SCID. Gene therapy is also paving its way towards the treatment of SCID especially for patients with ADA-SCID and X-SCID. As SCID involves phenotypic overlaps, genes responsible for SCID can be identified by targeted Next Generation Sequencing (NGS) panel which allows recognition of particular type of SCID. NGS is a massive parallel DNA sequencing tool which can sequence the entire human genome in a single day unlike conventional sanger sequencing technology. Sanger sequencing tool allows a single DNA fragment to be sequenced at a time and makes the diagnosis of SCID patients complex and laborious. Targeted NGS panel focuses on the genes associated with the SCID and allows simultaneous high depth sequencing to find rare variants in a simpler and cost-effective manner. However, results of NGS need to be validated by sanger sequencing.

    Objectives

    This project involves the study of Severe combined immunodeficiency (SCID) and helps in achieving following objectives-

    • To understand the diagnostic approach for SCID.
    • To perform molecular characterization of T-B+NK- SCID.

    LITERATURE REVIEW

    Information

    SCID involves more than 30 genes, some of them are JAK3, RAG1/RAG2, IL-2RG, ADA, PNP. Mutations in any of these lead to SCID pathogenesis (​​Fausto Cossu, 2010​​)​. Mostly, mutations are seen in IL2RG gene under normal CD19 level. This gene encodes common gamma chain (Yc) protein which helps in the development and differentiation of T and B cells. Mutations that result in non-function gamma chain causes failure of the immune system with low T, NK cells and non-functional B cells. As IL2-receptor gamma or IL2R(Y) is located on X chromosome the defect associated with it is called X linked SCID. Another defect involving common gamma chain (Yc) that leads to SCID is due to the mutations in JAK3. It is a cytosolic tyrosine kinase that help in the signalling of cytokine receptors (IL-2, IL-4, IL-7, IL-9, IL-15, IL-21) which uses common gamma chain (Yc). JAK3 is mostly expressed in T cells and NK cells. So, the mutations inactivating JAK3 causes immunodeficiency of type T-B+NK- SCID (​​Marko Pesu et al, 2005​​). SCID also occurs due to Adenosine Deaminase (ADA) Deficiency. It is an autosomal recessive disorder caused by a defective enzyme, ADA. This defect causes accumulation of deoxyadenosine (dATP) which inhibits the enzyme ribonucleotide reductase. The reduction in ribonucleotide reductase impairs dNTP synthesis which is necessary for lymphocyte proliferation. In the absence of dNTPs, immune system gets compromised causing SCID.

    Treatment of SCID

    The first hematopoietic stem cell transplant (HSCT) for SCID was carried out in 1968 (​​KANEGANE et al, 2017​​). Since then it became the most common treatment for SCID patients using either a matched related or unrelated donor, or a half-matched donor, who would be either parent. Half-matched donor requires his/her marrow to be depleted of all mature T cells to avoid the occurrence of graft-versus-host disease (GVHD) (​​Chinen and H. Buckley, 2010​​). A popular case of bone marrow transplant was David Phillip Vetter, referred to as “The bubble boy” by the media. He was born in 1971 and immediately after his birth, he was placed in a plastic germ-free environment where he spent 12 years of his life. He had bone marrow transplant from his sister, an unmatched donor. While his body did not reject the transplant, he became ill with an unscreened virus Epstein-Barr present in his newly transplanted bone marrow from his sister. He eventually died after15 days of his transplant in 1984.

    the bubble boy.jpg
      David Vetter, a child born with SCID in 1971

      Gene therapy is an alternative to bone marrow transplant especially patients with ADA SCID and X-SCID. In 1990, four-year-old Ashanthi DeSilva who was born with ADA SCID underwent successful gene therapy. Researchers extracted some of her white blood cells, and used a retrovirus to insert a healthy adenosine deaminase (ADA) gene into them. These cells were then injected back into her body. Gene therapy gave Ashanti a supply of her own cells that could produce ADA and provided her with a functional immune system. Success of gene therapy has also been impaired with the development of leukaemia in some patients and so, several efforts are being made for improving gene therapy (​ Cavazzana-Calvo and  Fischer, 2007​​).

      Diagnostic Tool for SCID

      Next-generation DNA sequencing is a high-throughput and cost-efficient diagnostic technology. It provides three approaches which can rapidly identify variants in the gene responsible for a disease (​Seleman et al, 2017​ ). The most comprehensive NGS technique is whole genome sequencing (WGS) which sequences a patient’s entire genome and identifies variants in coding and non-coding regions. Then, there is Whole exome sequencing (WES) which sequences only the protein-coding regions. Targeted gene panel (TGP) sequencing is the third approach which sequences a specific cohort of genes and allows an increased depth of sequencing coverage.

      NGS.png
        VARIANTS IDENTIFIED BY WGS, WES, TGP

        After designing NGS panel and sequencing, NGS data is analysed using different bioinformatics tools. Variants are identified and can be classified as pathogenic, likely pathogenic, benign, likely benign, or a variant of uncertain significance (​​Seleman et al, 2017​​). Pathogenicity occurs when there is loss of altered protein expressions or function, when the location of the variant is in the coding region or because of a null variant present in a gene capable of causing human disease. Benign variants are those that have high minor allelic frequency and have no impact on health. Variants of uncertain significance are those that do no meet any criteria of other variants and its effect to human kind is not known yet. Later, functional validation of the disease is done by analysing protein expression or/and by using animal models (​Seleman et al, 2017​).

        ngs2_1.png
          STEPS FOR THE IDENTIFICATION AND VALIDATION OF DISEASE CAUSING VARIANTS

          Despite of the increase diagnostic yield of NGS compared to conventional technologies, many patients remain undiagnosed because of the deficits in the technology, data analysis and understanding of the disease. This faster and cheaper sequencing gives result of less accuracy due to its problems with copy number variants ( Zhao et al, 2013​ ). It is because of the inherent statistical methods that are used for assembly ( Cottrell, 2018​ ). Therefore, clinical applications have to be confirmed using Sanger sequencing method.

          METHODOLOGY

          Patient

          Female child of 2 months old born out of 2nd degree consanguineous marriage was suspected of SCID. Patient was presented with complaints of intermittent high-grade fever since birth and required multiple admissions for the same. A clinical proforma was filled which included the age, consanguinity, family history, clinical parameters like number of infections, site of infections, age of presentation, administration of vaccines and post live vaccine complications.

          Sample and Immunological Workup

          Peripheral Blood was collected in EDTA, Plain and Heparin vacutainers. A complete blood cell count was done in Sysmex XS-800i (Sysmex Co., Cobe, Japan) 5-part automated haematological analyser. Flow cytometric analysis was done for lymphocyte subsets and CD132 expression on B cells using specific cell surface markers (anti-CD19 allophycocyanin [APC]).

          Next Generation Sequencing

          The sample was referred for targeted next generation sequencing under primary immunodeficiency (PID) panel. Around 60 genes were covered, and the libraries were sequenced on Illumina sequencing platform (mean coverage >80 to 100X). List of genes given below-

          GENES COVERED IN PID PANEL 
          GENESCD8A MTHFD1 RBM8A TAZ 
             ADA  DCLRE1CMRE11A RFX5 TNFRSF4 
          AK2 FOXN1MAGT1 RFXANK TP63 
          ATM GATA2 MALT1 RFXAP TRAC 
          CD247 IKBKB NFKBIA RHOH TTC74 
          CD8A IKBKG NHEJ1  SEMA3E TTC74
           CD27IL21 NME1 SH2D1A WAS 
           CD3DIL21R ORAI1 SLC46A1 CTPS1 
           CD3EIL2RG PNP STAT5B LIG4 
          CECR1 IL7R PRKDC STIM1 RAG2 
           CHD7ITK PTPRC STK4 TAPBP 
           CIITAJAK3 RAC2 TAP1 
          CORO1A LCK RAG1 TAP2 

          The variants obtained from PID panel were then confirmed by Sanger sequencing.

          Sanger Validation

          Standardisation of polymerase chain reaction (PCR)

          Standardisation of PCR was done by taking the primers for the exon of the gene containing mutation and finding its annealing temperature. Two different temperatures were taken for a gradient PCR according to the melting temperature (Tm) of the primers. The annealing temperature should be around 5℃ less than the Tm and so the temperatures taken were 62℃ and 64℃. PCR used was “TAKARA PCR thermal cycler dice”, a gradient PCR. The whole process begins with reconstituting the stock primer which had a concentration of 100pM and needed to be converted into 50µl of 10pM. This was accomplished by adding 45µl of distilled water and 5ul of stock primer. Reagents used for the PCR are given in the table below. All the volumes are in Microlitres (µl).

          PCR PROTOCOL
           DEPC WaterBuffer dNTP Taq polymerase Mgcl2 DMSO Forward primer Reverse primer DNA 
          18.15  2.5 0.5 0.25 - -0.8 0.8 2 
          16.15 2.5 0.5 0.25 2 - 0.8 0.8 2
           15.15 2.5 0.5 0.25 21  0.80.8  2

          The protocol given in the 1st row in the table was used with a normal DNA sample. Correct annealing temperature shows a clear distinct band when run in agarose gel allowing the primer to be used for PCR with patient DNA at that particular annealing temperature. In case where there is a blurred band obtained, PCR enhancers -MgCl2, DMSO or in combination are added, 2µl each. MgCl2 acts as a cofactor for the enzyme Taq polymerase and increases productivity. But its higher concentration decreases the specificity so the concentration used here was 2mM. DMSO disrupts the secondary structure of DNA and facilitates the annealing of primer with template thus, increasing the specificity and yield of PCR.

          After finding the correct annealing temperature, reaction mixture was made with patient’s DNA and a normal PCR was performed. Reaction mixture with the DNA samples of father and mother were also prepared for PCR. The PCR used here was GeneAmp® PCR System9700 and the total volume of the reaction mixture was 25µl.

          PCR protocol.png
            PCR

            The amplicons were visualized by agarose gel electrophoresis.

            Agarose gel electrophoresis

            • 1.2% of agarose gel was prepared which requires 0.6-0.8 5g of agarose.
            • 50X tris acetate buffer was converted to 70 ml of 1X buffer to which 0.84g agarose was added. The solution was heated to 95℃ for 5-10 mins until the solution became clear.
            • Then 7µl ethidium bromide was added to visualise the DNA under UV light. Ethidium bromide is an intercalating agent which works by binding to the grooves of DNA double helix. It is a toxic mutagen and carcinogen in nature. This limitation can be overcome by adopting different alternatives such as crystal violet, gel red, SYBR safe which are less mutagenic than ethidium bromide.
            • Electrophoresis tank should be set up in which the gel should be poured and solidified. It was placed with comb to make wells for the samples to be loaded in it.
            • 3µl of sample loading dyes such as bromophenol blue and xylene cyanol was mixed with 7µl of samples and a total of 10ul was added to each well.
            • The gel was filled with buffer and connections were made at a voltage of 120 Volts for 22 mins.
            • After the run of gel was over, distinct bands were seen. They were seen as orange bands under UV transilluminator.

            Amplicons then had to be purified by PCR clean-up process (column purification method). This step is necessary to remove remaining primers, incomplete products or mis primed artifacts. Other components such as polymerase or buffer components should also be removed before it goes for sequencing PCR.

            Cleanup of PCR product using GeneAll cleaning kit

            • Clean up begins with binding buffer - PB buffer which helps in binding of DNA to silica membrane in column.
            • Five times the PB buffer was added to the sample and then the whole was transferred to SV column.
            • The column was centrifuged at 10,000 rpm for 1 min.
            • NW buffer of 650µl was added to the column. NW buffer is a washing buffer containing ethanol which removes unwanted impurities such as salts, proteins, nucleotides, dyes.
            • Residual ethanol was removed by centrifuging the column for 1 min.
            • The pass through the membrane present in collection tube was discarded and an empty centrifuge was given.
            • The SV column was then put in a new 1.5ml tube and the sample was allowed to get air dried for 15 mins.
            • Lastly, Elution buffer -EB buffer of 25µl was added on to the silica membrane and centrifuged for 1 min.
            • The pass through the membrane collected at the bottom of the tube was the purified product.
            • The purified product was ready for sequencing PCR.

            Sequencing PCR

            Sequencing PCR is done to know the order of nucleotides of a gene. Sequencing PCR is very similar to normal PCR. The differences are -it uses dideoxynucleotide triphosphates, only one primer and the process begin with PCR fragments. Here the reaction mixture was of 10µl and the primer needed to be converted from concentration of 10pM to 20µl solution of 0.3pM. This was obtained by adding 13µl of water and 7µl of 10pM primer. The composition of reaction mixture is given in table below.

            SEQUENCING PCR PROTOCOL
            DEPC water Bigdye  RR  Primer  Purified Product 
             4µl2µl 1µl 1µl2µl 
            seqPCR.png
              SEQUENCING PCR (25 CYCLES)

              Clean up of sequencing PCR product (by column purification method)

              • Ten times the NR buffer (binding buffer) was added to the sample, mixed and the whole was transferred to the column.
              • Centrifugation for 1 min at 10,000 rpm.
              • 650µl of NW (washing buffer) was added to the tube and centrifuged for 1 min.
              • The pass through the membrane present in collection tube was discarded and an empty centrifuge was given.
              • SV column was transferred to a new 1.5ml tube
              • Finally, 18µl HiDi formamide was added on to the silica membrane and is centrifuged for 1 min.
              • The pass through collected at the bottom of the tube was the purified product.

              Sequencing by 3730XL DNA analyser, applied biosystems

              The purified product after sequencing PCR stage was fed to sanger sequencer after a short denaturation step (5 mins) which converted double stranded DNA into single stranded DNA. Result was seen as a chromatogram file showing DNA sequence where mutations could be present.

              Bioinformatics analysis

              Nucleotide Blast of the obtained sequence was performed using bioinformatic tool NCBI BLAST. The variants identified and confirmed using prediction tools such as mutation taster.

              RESULTS AND DISCUSSION

              Patient Characteristics

              Clinical findings reveal that there was history of BCG given but no BCG scar found. CBC done on day 2 showed only 1% lymphocyte. Chest X ray showed no thymic shadow and no organisms were isolated. Patient was suffering with recurrent fever and responded poorly to IV antibiotics.

              Laboratory Findings

              Complete blood cell count and lymphocyte subset analysis done for the patient are given in the table below-

              IMMUNOLOGICAL FINDING
              WBC/µl NEUTROPHIL(%) LYMPHOCYTE(%) MONOCYTE(%) EOSINOPHIL(%)BASOPHIL(%) ALC/µl  B(%) Abs count/µl T(%) Abs count/µl   Th(%)Abs count/µl  Tc(%)  Abs count/µl NK(%) Abs count/µl 
               PATIENT21010 67 21 10 0.2 4412 86 3839 0  00 0 0 0 2 88 

              There was absent or severly low T, NK cell count and evaluation of CD132 expression on B cells indicated that the patient had (T-B+NK-) phenotype SCID.

              Flow cytometric analysis result was obtained and interpreted by lab professionals. As a trainee i had observed the approach to diagnose different types of SCID.

              Molecular Findings

              Depending on the immunophenotypic pattern, Next Generation Sequencing was done, and a homozygous missense variation was found in exon13 of JAK3 gene (chr19:g.17947959C>A; Depth: 46x). It results in the substitution of Cysteine for Glycine at codon 589(p. Gly589Cys). This defect is a homozygous autosomal recessive deficiency and the variant found was c.1765G>T, classified as likely pathogenic.

              Sanger Sequencing

              Polymerase chain reaction

              The annealing temperature came out to be 60℃ for the primer to be used for exon13 of JAK3 gene. The result was a clear band in agarose gel as shown below-

              PCR STANDARDISATION_1.jpg
                STANDARDISATION OF PCR AMPLIFICATION OF EXON 13 AT 60℃

                PCR of patient, father and mother’s sample was performed with standard protocol and the amplicons obtained were run in agarose gel which produced bands of high intensity and allowed us to proceed with the clean-up process, sequencing PCR, post sequencing PCR clean-up and sanger sequencing.

                PCR1_1.jpg
                  AMPLIFICATION OF DNA SAMPLE OF FATHER(LEFT), MOTHER(MIDDLE),PATIENT(RIGHT)

                  Aanlysis of variants

                  Nucleotide Blast was performed on the sequences of the patient, father and mother. Non-matched nucleotide was found and few adjacent nucleotides along with non-matched was copied in mutation Taster tool to find the nature of variant.

                  nucleotide blast.png
                    SUBSTITUTION OF T FOR G IN THE CODING REGION AT POSITION 589

                    Upon checking the variant in Mutation Taster, it was found to be disease causing mutation. The variant was c.1765G>T and aletration region was CDS at position 589. This mutation affects protein kinase1 domain which loses its function. It is a homozygous autosomal recessive deficiency where mother and father are both carrier showing heteropeaks in the chromatogram. Therefore this confirms the mutation obtained in Targeted Next generation sequencing.

                    CHROMATOGRAM_1.png
                      CHROMATOGRAM OF PATIENT, MOTHER AND FATHER (FATHER AND MOTHER SHOWING HETEROPEAKS)

                      CONCLUSION

                      In this research, we learnt about SCID and different approaches to diagnose it. Next generation sequencing has made screening process easier by including genes which are associated with the disease and providing specific results based on the mutations identified. HSCT and Gene therapy are paving their way towards the treatment of SCID. Targeted gene panel focuses on the genes responsible for the disease and the mutations identified in them are checked by sanger sequencing which later involves the use of bioinformatic tools to confirm it.

                      ACKNOWLEDGEMENTS

                      Firstly, I would like to extend my acknowledgments to Indian Academy of Sciences (IASc-INSA-NASI) for making me part of Summer Research Fellowship programme,2019.

                      I owe my heartiest gratitude to respected Dr. Manisha Madkaikar, Director, department of paediatric immunology and leukocyte biology, National institute of immunohematology, Mumbai for providing me this golden opportunity to work in her lab and extending all her critical supervision, advice, and constant encouragement.

                      I profoundly thank Dr. Priyadarshini Dey, Assistant professor, department of Biotechnology, Ramaiah Institute of Technology for providing my letter of recommendation to Indian Academy of Sciences.

                      I am deeply indebted to Dr. Priyanka M. Kambli and Ms. Sneha Sunil Sawant for providing me with all lab facilities, outstanding guidance and timely help despite of their busy schedule.

                      I am heartily thankful to all my seniors and co interns working in the lab for their cooperation and making friendly atmosphere for research work. I am highly obliged for their constant support and encouragement during my stay in Mumbai.

                      I also pay my sincere thanks to my beloved parents and friends who supported me throughout the fellowship and guided me with their valuable suggestions.

                      References

                      • Neil A. Holtzman, 2014, Newborn Screening for Severe Combined Immunodeficiency, JAMA, vol. 312, no. 7, pp. 701

                      • William T. Shearer, Elizabeth Dunn, Luigi D. Notarangelo, Christopher C. Dvorak, Jennifer M. Puck, Brent R. Logan, Linda M. Griffith, Donald B. Kohn, Richard J. O'Reilly, Thomas A. Fleisher, Sung-Yun Pai, Caridad A. Martinez, Rebecca H. Buckley, Morton J. Cowan, 2014, Establishing diagnostic criteria for severe combined immunodeficiency disease (SCID), leaky SCID, and Omenn syndrome: The Primary Immune Deficiency Treatment Consortium experience, Journal of Allergy and Clinical Immunology, vol. 133, no. 4, pp. 1092-1098

                      • Aziz Bousfiha, Leïla Jeddane, Capucine Picard, Fatima Ailal, H. Bobby Gaspar, Waleed Al-Herz, Talal Chatila, Yanick J. Crow, Charlotte Cunningham-Rundles, Amos Etzioni, Jose Luis Franco, Steven M. Holland, Christoph Klein, Tomohiro Morio, Hans D. Ochs, Eric Oksenhendler, Jennifer Puck, Mimi L. K. Tang, Stuart G. Tangye, Troy R. Torgerson, Jean-Laurent Casanova, Kathleen E. Sullivan, 2017, The 2017 IUIS Phenotypic Classification for Primary Immunodeficiencies, Journal of Clinical Immunology, vol. 38, no. 1, pp. 129-143

                      • Fausto Cossu, 2010, Genetics of SCID, Italian Journal of Pediatrics, vol. 36, no. 1, pp. 76

                      • Marko Pesu, Fabio Candotti, Matthew Husa, Sigrun R. Hofmann, Luigi D. Notarangelo, John J. O'Shea, 2005, Jak3, severe combined immunodeficiency, and a new class of immunosuppressive drugs, Immunological Reviews, vol. 203, no. 1, pp. 127-142

                      • Hirokazu KANEGANE, Kohsuke IMAI, Tomohiro MORIO, 2017, Severe combined immunodeficiency: From its discovery to the perspective, Japanese Journal of Clinical Immunology, vol. 40, no. 3, pp. 145-154

                      • Javier Chinen, Rebecca H. Buckley, 2010, Transplantation immunology: Solid organ and bone marrow, Journal of Allergy and Clinical Immunology, vol. 125, no. 2, pp. S324-S335

                      • Marina Cavazzana-Calvo, Alain Fischer, 2007, Gene therapy for severe combined immunodeficiency: are we there yet?, Journal of Clinical Investigation, vol. 117, no. 6, pp. 1456-1465

                      • Michael Seleman, Rodrigo Hoyos-Bachiloglu, Raif S. Geha, Janet Chou, 2017, Uses of Next-Generation Sequencing Technologies for the Diagnosis of Primary Immunodeficiencies, Frontiers in Immunology, vol. 8

                      • Min Zhao, Qingguo Wang, Quan Wang, Peilin Jia, Zhongming Zhao, 2013, Computational tools for copy number variation (CNV) detection using next-generation sequencing data: features and perspectives, BMC Bioinformatics, vol. 14, no. S11

                      • Paul Cottrell, 2018, Advantages and Drawbacks of Next Generation Sequencing, SSRN Electronic Journal

                      Source

                      • Fig 1: Aziz Bousfiha , Leïla Jeddane, Capucine Picard, Fatima Ailal, H. Bobby Gaspar,Waleed Al-Herz et al. The 2017 IUIS Phenotypic Classification for Primary Immunodeficiencies
                      • Fig 2: Name: Microbiology ID: e42bd376-624b-4c0f-972f-e0c57998e765@4.4 Language: English Summary: Subjects: Science and Technology Keywords: Print Style: License: Creative Commons Attribution License (by 4.0) Authors: OpenStax Microbiology Copyright Holders: OpenStax Microbiology Publishers: OpenStax Microbiology Latest Version: 4.4 First Publication Date: Oct 17, 2016 Latest Revision: Nov 11, 2016 Last Edited By: OpenStax Microbiology,https://cnx.org/contents/5CvTdmJL@4.4
                      • Fig 3: Uses of Next-Generation Sequencing Technologies for the Diagnosis of Primary immunodeficiencies,Division of Immunology, Michael Seleman , Rodrigo Hoyos-Bachiloglu , Raif S. Geha and Janet Chou,Boston Children’s Hospital, Boston, MA, United States
                      • Fig 4: Uses of Next-Generation Sequencing Technologies for the Diagnosis of Primary immunodeficiencies,Division of Immunology, Michael Seleman , Rodrigo Hoyos-Bachiloglu , Raif S. Geha and Janet Chou,Boston Children’s Hospital, Boston, MA, United States
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