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Clinical characteristics and mutation analysis of X-linked severe combined immunodeficiency in China 
Clinical characteristics and mutation analysis of X-linked severe combined immunodeficiency in China
  Cui Zhang, Zhi-Yong Zhang, Jun-Feng Wu, Xue-Mei Tang, Xi-Qiang Yang, Li-Ping Jiang, Xiao-Dong Zhao
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Clinical characteristics and mutation analysis of X-linked severe combined immunodeficiency in China

Cui Zhang, Zhi-Yong Zhang, Jun-Feng Wu, Xue-Mei Tang, Xi-Qiang Yang, Li-Ping Jiang, Xiao-Dong Zhao

Chongqing, China

Author Affiliations: Division of Immunology, Children's Hospital of Chongqing Medical University, Chongqing 400014, China (Zhang C, Zhang ZY, Wu JF, Tang XM, Yang XQ, Jiang LP, Zhao XD)

Corresponding Author: Xiao-Dong Zhao, Division of Immunology, Children's Hospital of Chongqing Medical University, Chongqing 400014, China (Tel: +86 23 6362 2554; Fax: +86 23 6360 2136; Email: zhaoxd530@yahoo.com.cn)

doi: 10.1007/s12519-011-0330-4

Background: X-linked severe combined immuno-deficiency (X-SCID) is a rare, life-threatening immune disorder, caused by mutations of the gene for the ¶√-chain (¶√c) of the interleukin-2 receptor, IL2RG. We analyzed the clinical, immunologic, and molecular characteristics of children with X-SCID, attempting to improve the diagnosis and treatment of X-SCID in China.

Methods: X-SCID was suspected in male infants with recurrent or persistent infections. Eleven male infants from ten unrelated Chinese families were included. The IL2RG gene was amplified and sequenced, followed by mutation analysis in these children and their female relatives. X-linked short tandem repeat (X-STR) typing was done to define the maternal lymphocyte engraftment.

Results: The 11 children exhibited recurrent infections and 10 of them had lymphopenia. B cells were present in all patients, T cells were markedly reduced in 10, and NK cells were markedly reduced in 9. Nine IL2RG gene mutations were identified in the 11 children, with 5 novel mutations. One patient was found to have the maternal lymphocyte engraftment.

Conclusion: The clinical presentations and immunologic characteristics of the X-SCID patients were accordingly quite uniform despite the heterogeneity of mutations locating almost in the entire ¶√c gene.

Key words: clinical characteristics; IL2RG gene; mutation; severe combined immunodeficiency; X-linked trait

World J Pediatr 2013;9(1):42-47


Severe combined immunodeficiency (SCID) is a group of rare single gene disorders characterized by profound cellular and humoral immune dysfunction.[1,2] SCID has some underlying genetic defects, and all forms of SCID manifest as a common clinical characteristic, such as extreme susceptibility to infections, which may lead to death in the first few months of life unless immunologic reconstitution is achieved. The treatment and prevention of these infections could prolong the survival of patients but could not cure the disorder.[3] Approximately half of the SCID patients are diagnosed with X-linked SCID (X-SCID) which is caused by mutations in the interleukin-2 receptor common ¶√-chain (IL2RG, ¶√c) gene encoding the ¶√c of the IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 receptors.[4] Immunologically, X-SCID patients are characterized by the absence of or markedly diminished T cells and natural killer (NK) cells together with normal to slightly increased B cells.[5]

Transplantation of hematopoietic stem cells (HSCs) from a HLA-identical sibling or matched unrelated donor is the first choice for the treatment of SCID. Unfortunately, HLA-matched relatives are often not available as donors. T cell-depleted haploidentical bone marrow transplantation has been well established as a curative procedure for these patients.[3,6] In addition, gene therapy may represent a valid alternative for patients with X-SCID. Early diagnosis and treatment with bone marrow transplantation markedly improve the long-term outcomes.[7]

On January 1, 2008, the Newborn Screening Program in Wisconsin, USA became the first in the world to routinely test all newborns for SCID. Real-time quantitative polymerase chain reaction assay is able to measure T-cell receptor excision circles (TRECs) which form during the maturation of normal T-cells. A lack or very low number of TRECs is consistent with T-cell lymphopenia.[8] In the present study, we analyzed the clinical, immunologic, and molecular characteristics of 11 children with X-SCID from 10 unrelated Chinese families in an attempt to improve the diagnosis and treatment of X-SCID in China.



X-SCID was suspected in male infants with recurrent or persistent infections. From 2000 to 2010, 11 male infants from 10 unrelated Chinese families were included. The clinical presentations included the infection history and family history. Specific laboratory examinations were also carried out. Based on these findings, the DNA sequencing of the IL2RG gene was performed. All patients were born to non-consanguineous parents. Patient 4 was the maternal cousin of patient 5. The study was approved by the ethical committee and valid informed consent was obtained from the parents.

IL2RG gene mutation analysis

After informed consent was obtained, 3 mL of fresh whole blood was collected from each child and their parents for DNA sequencing and immunologic analysis. Total RNA was isolated from peripheral blood mononuclear cells (PBMCs) using Total RNA Purification kit (Genemega, San Diego, CA, USA) and submitted to RT-PCR using a RT-PCR kit (Takara, Otsu, Japan). Genomic DNA was extracted from whole blood using the QIAamp DNA Blood mini kit (QiagenGmbH, Hilden, Germany).

Eight exons of the IL2RG gene and surrounding genomic sequences were amplified using primers previously described.[9] For patient 8, the DNA was amplified with the forward primer of exon 4 and reverse primer of exon 6. The cDNA of patient 11 was amplified by PCR with the primers: 5'-CGCCATGTTGAAGCCATCAT-3' (forward) and 5'-GTTCAGGTTTCAGGCTTTAG-3' (reverse).[10]

The PCR products were extracted from agarose gel with QIAquick PCR Purification kit (Qiagen, Hilden Germany) and sequenced using the same primers in PCR on an automated ABI PRISM 3100 Genetic Analyzer (PE Applied Biosystems). Mutant sequences were confirmed by forward and reverse sequencing of independently amplified templates from the index patient and carrier relatives.

Prenatal diagnosis

Four couples at risk for having a fetus with X-SCID were recruited into the study for fetal DNA evaluation. In brief, 20 mL of amniotic fluid was obtained between 18 and 20 weeks of gestation by amniocentesis. Genomic DNA was extracted from amniotic fluid cells, and then PCR amplification and sequencing were performed at the mutation regions on fetal DNA.

X-linked short tandem repeat (X-STR) typing

The DXS6804 and DXS7423 loci were amplified with the following primers.[11]


             Reverse  5'-GGCATGTGGTTGCTA TAACC-3'


               Reverse    5'-GTCTTCCTGTCATCTCC CAAC-3'

The PCR products of 8 patients, their mothers, positive control and negative control were analyzed by capillary electrophoresis in an Applied Biosystems 3730xl Genetic Analyzer. GeneMapper 4.0 analysis software was used to determine fragments size.


Clinical presentations

The mean ages of the patients at onset and diagnosis were 111 days (range: 20-182 days) and 154 days (range: 39-279 days), respectively, with a duration of 33 days between the onset of symptoms and diagnosis. The most common presentation was recurrent fever (n=10), followed by pneumonia (n=9), prolonged diarrhea (n=7), oral candidiasis (n=3) and impetigo (n=2). The microorganisms causing overt infections in these patients included bacillus calmette-guerin (BCG) infection (n=3), Candida albicans (n=3), fungi (n=2), cytomegalovirus (n=2), Haemophilus influenzae (n=2), coxsackie (n=1), pseudomonas (n=1), parainfluenza virus 3 (n=1), influenza viruses type A (n=1), Uckermann citrobacter (n=1), and Moraxelle catarrhalis (n=1). All patients received routine immunizations before the onset of symptoms, including BCG (patients 3 and 8 with recurrent ulceration of the BCG scar; patient 1 with diffuse BCG infection in the liver, spleen, lungs, and skin). Five patients had a family history that their related male family members died of recurrent infections in infancy. None of the patients received hematopoietic stem cell transplantation (HSCT), and the mean age of these patients at death was 276 days (range: 158-395 days). The main clinical presentations of the patients at diagnosis are summarized in Table 1.

Immunologic characteristics

Lymphopenia was a common finding (range: 550-2070 cells/¶ŐL) in all children except patient 5 (4950 cells/¶ŐL). The serum immunoglobulin levels were abnormal in all patients. Of the 11 children, 6 had hypo-IgG, 4 normal IgG and 1 hyper-IgG levels; 6 had hypo-IgA and 5 normal IgA levels; 7 had hypo-IgM and 4 normal IgM levels. The IgE levels were normal in all patients. B cells were present in all patients; T cells were markedly reduced in 10, and NK cells were markedly reduced in 9. Patient 5 had a normal number of T cells. Patients 7 and 9 had normal number of NK cells, and the absolute number was 577 cells/¶ŐL and 498 cells/¶ŐL, respectively. The main immunological features of the 11 patients at diagnosis are shown in Table 2.[12,13]

Mutation analysis

The IL2RG gene was detected in the 11 children, and 9 mutations were found in the IL2RG gene. Mutation types included missense mutation (n=6), nonsense mutation (n=3), large deletion mutation (n=1), and disruption of RNA splicing mutation (n=1). Seven mutations were identified in exon 5, and 5 mutations had not been reported (c.445C>T, Q149X; c.548T>A, L183X; c.711G>A, W237X; c. 595-430_757+140del723, E199RfsX218; c.854G>C, E253RfsX261) (Fig.). Direct sequence analysis of the products of RT-PCR from patient 11 revealed that exon 6 was deleted from the mRNA (Fig.). All the mothers were carriers of X-SCID except the mother of patient 2. Because patient 2 and his mother were lost to follow-up, and the blood sample of the mother was absent. Besides, another 4 carriers were identified in the present study. The prenatal diagnosis of 4 couples showed that one fetus was affected. The mutations and their locations of the 11 children are shown in Table 3.

X-STR typing in 8 patients and their mothers

STR typing of the DSX6804 and DXS7423 loci is shown in Table 4. Patient 5 and his mother both had two alleles (allele 12 and 14) in the DSX6804 locus. The other 7 patients had only one allele as the male positive control. Thus only patient 5 had maternal lymphocyte engraftment.


Our study demonstrated that the patients had typical characteristics of X-SCID including extreme susceptibility to infection, leading to death in infancy. The clinical presentations of 5 patients with novel mutations were similar to the other 6 patients with well known mutations, and the clinical presentations of these 11 children were similar to those in two large surveys.[1,14]

In our study, 3 patients had BCG infection. It was reported that the risk for BCG infection is increased in patients with primary immunodeficiency.[15] BCG might be a major cause of infection and an obstacle for future immune reconstitution. In China, BCG is regularly given to newborns at the first month of their life without screening for primary immunodeficiencies. In the present study, 5 of the 11 patients had a clear family history of vaccination but received contraindicated BCG vaccine. All living vaccines besides BCG vaccine are contraindicated in combined and cellular immunodeficiencies. Thus, the family history of deaths of children and vaccine-related complications should be inquired before the regular BCG vaccination. Moreover, primary immunodeficiencies should be suspected when patients had BCG infection.

Studies have revealed that the absolute lymphocyte count is the most useful test in screening diagnosis, because lymphopenia can be found in almost all SCID patients at birth.[16,17] In our study, lymphopenia was a common sign in 10 patients. But patient 5 had normal lymphocyte count and normal number of T cells with the maternal lymphocyte engraftment by the X-STR typing. However, no overt clinical manifestations of graft-versus-host disease (GVHD) was found in this patient, which was consistent with the result reported elsewhere.[14,18]

In the typical phenotypes of X-SCID, the number of T cells and NK cells is usually very low or absent and B cells are generally present but nonfunctional.[5] In our study, however, one patient had normal T cell count and two had normal NK cell count. Buckley et al[1] reported that 6 of 49 patients with ¶√c-deficiency had a normal or elevated number of NK cells. Thus, the diagnosis of X-SCID should be based on examinations other than just immunologic tests, and identification of mutations is necessary.

In the present study, 7 of 11 mutations were identified in exon 5 and the relative frequency of mutations was higher than that in other exons. The types and locations of mutations were unevenly distributed, consistent with the results of a previous report.[9] Five novel mutations were identified in the present study and 3 of them produced a premature stop codon. Studies have demonstrated that the mutations leading to a nonsense codon in the extracellular domain (1-5 exon) of the IL2RG gene are invariably associated with undetectable mRNA by Northern blot, presumably due to nonsense-mediated RNA decay[19-21] and lack of the surface expression of ¶√c chain.[9,22,23] The skipping of exon 6 in the ¶√c mRNA of patient 11 was found to be attributed to the 3'-terminal nucleotide of the outskipped exon 6 but not its splice acceptor site. Furthermore, two similar splicing mutations, G to T and G to A substitution at the same position, have been found to have the same consequences in splicing of transcripts.[10,24,25] In our study, three sites, cDNA670, cDNA677 and cDNA854, are located within cytosine-guanine (CpG) dinucleotides and are mutational hotspots in the ¶√c gene along with other hotspots involving CpG nucleotides.[10,26,27] The three mutational hotspots shown here account for 45.5% of the 11 mutations detected in our study.

In conclusion, the clinical presentations and immunologic characteristics of XSCID patients were accordingly quite uniform despite the heterogeneity of mutations locating almost in the entire ¶√c gene. Furhter understandings of the symptoms and mutations of the IL2RG gene in depth are necessary for the medical staff in clinical diagnosis of X-SCID.


We are grateful to all children and their family members participating in this study. We appreciate Qiang-Lin Duan from Tongji Hospital for critical reading of the manuscript.

Funding: This work was financially supported by a grant from the Chongqing Outstanding Youth Fund (CSCT, 2008BA5040).

Ethical approval: This study was approved by the regional committee for medical research ethics. Informed consent was obtained from the study participants.

Competing interest: No benefits in any form have been received or will be received from any commercial party related directly or indirectly to the subject of this article.

Contributors: Zhang C wrote the main body of the article under the supervision of Zhao XD. All authors contributed to the design and interpretation of the study and to further drafts. Zhao XD is the guarantor.


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Received February 17, 2011Accepted after revision June 14, 2011

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