Journal of Immunology Research

Journal of Immunology Research / 2014 / Article
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Advances in Primary Immunodeficiencies: Challenges in Biology, Diagnosis, Management, and Treatment

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Research Article | Open Access

Volume 2014 |Article ID 683160 |

Lin-Lin Wang, Wei Zhou, Wei Zhao, Zhi-Qing Tian, Wei-Fan Wang, Xiao-Fang Wang, Tong-Xin Chen, "Clinical Features and Genetic Analysis of 20 Chinese Patients with X-Linked Hyper-IgM Syndrome", Journal of Immunology Research, vol. 2014, Article ID 683160, 12 pages, 2014.

Clinical Features and Genetic Analysis of 20 Chinese Patients with X-Linked Hyper-IgM Syndrome

Academic Editor: Catharina Schuetz
Received27 May 2014
Accepted31 Jul 2014
Published20 Aug 2014


X-linked hyper-IgM syndrome (XHIGM) is one type of primary immunodeficiency diseases, resulting from defects in the CD40 ligand/CD40 signaling pathways. We retrospectively analyzed the clinical and molecular features of 20 Chinese patients diagnosed and followed up in hospitals affiliated to Shanghai Jiao Tong University School of Medicine from 1999 to 2013. The median onset age of these patients was 8.5 months (range: 20 days–21 months). Half of them had positive family histories, with a shorter diagnosis lag. The most common symptoms were recurrent sinopulmonary infections (18 patients, 90%), neutropenia (14 patients, 70%), oral ulcer (13 patients, 65%), and protracted diarrhea (13 patients, 65%). Six patients had BCGitis. Six patients received hematopoietic stem cell transplantations and four of them had immune reconstructions and clinical remissions. Eighteen unique mutations in CD40L gene were identified in these 20 patients from 19 unrelated families, with 12 novel mutations. We compared with reported mutation results and used bioinformatics software to predict the effects of mutations on the target protein. These mutations reflected the heterogeneity of CD40L gene and expanded our understanding of XHIGM.

1. Introduction

X-linked hyper-IgM syndrome (XHIGM; HIGM1; OMIM: 308230) is one type of primary immunodeficiency diseases (PIDs), resulting from defects in the CD40 ligand/CD40 signaling pathways leading to impairment of immunoglobulin isotype switching in B cells and characterized by recurrent infections in association with markedly decreased serum IgG, IgA, and IgE levels but normal or elevated serum IgM levels [1]. Patients with XHIGM usually develop symptoms by the first or second year of life. Over 50% of patients have chronic or intermittent neutropenia, often associated with oral ulcers. Opportunistic infections, especially Cryptosporidium parvum and Pneumocystis jirovecii infections, are common and prominent clinical feature of the patients. Besides the susceptibility to recurrent bacterial and opportunistic infections, these patients are prone to autoimmune manifestations, especially hematologic abnormalities, arthritis, and inflammatory bowel disease. Furthermore, an unusual susceptibility to liver, pancreas, or biliary tract tumours has been reported [2].

CD40L is the disease-causing gene of XHIGM [37], located in Xq26.3-Xq27.1 and containing five exons and four intervening introns. The corresponding translated protein CD154 (also called CD40L, gp39, TNFSF5, or TRAP) contains 261 amino acids and is mainly expressed on activated mature T cells. Its receptor, CD40, is expressed on a variety of cells, such as B cells, monocytes/macrophage, dendritic cells, endothelial cells, and epithelial cells [8, 9]. CD40-CD154 interactions provide a costimulatory signal for T cell activation and can lead to B cell proliferation and immunoglobulin class switching, affecting both cellular and humoral immunity [10, 11]. Therefore, XHIGM belongs to a combined immunodeficiency disease category.

XHIGM is the most common subtype of hyper-IgM syndromes (affecting about 70% of the patients) [12]. More subtypes of hyper-IgM syndromes have been gradually revealed. Diseases resulting from mutations in AICDA (ID: 57379), CD40 (ID: 958), UNG (ID: 7374), NEMO (ID: 8517), or NFKBIA (ID: 4792) genes are all involved in the CD154-CD40- NF-B pathways and affected the class switch recombination (CSR), somatic hypermutation (SHM), or germinal center formation and form the subtypes of hyper-IgM syndromes. Mutations in the promoter of CD40L were also found [13]. The heterogeneity of hyper-IgM syndrome brings the challenge for diagnosis and accurate and reliable molecular testing methods are needed.

With diagnosed patients accumulated, people began to analyze the clinical features and mutation characteristics of these patients since 1992 [14]. A CD40L mutation database ( has been founded and updated [15]. Retrospective research results of XHIGM patients in Europe [16], USA [17], Japan [18], and Latin America [19] were published. However, little information about Chinese XHIGM patients has been reported. In this study, we collected clinical data of Chinese XHIGM patients diagnosed and followed up in our center, reviewed their clinical and immune features, therapy, and response, and investigated the mutation characteristics, intending to increase our knowledge of this disease and finally improve the quality of life in these patients.

2. Methods

2.1. Patients

Patients in present study were first diagnosed in hospitals affiliated to Shanghai Jiao Tong University School of Medicine or referred from other hospitals and followed up in Shanghai Children’s Medical Center (also affiliated to Shanghai Jiao Tong University School of Medicine) during 1999–2013. A total of 28 candidate patients with HIGM phenotype were recruited. The inclusion criteria were low sera IgG and IgA (2 standard deviations below normal value for age), normal or elevated serum IgM, and compatible infectious events. Patients with secondary immunodeficiency conditions such as HIV or congenital rubella infections, immunosuppressive drug use, or neoplasms were excluded. Then CD154 expression on active T cells was detected by flow cytometry and sequences of CD40L gene were analyzed. A total of 20 patients with XHIGM were finally identified.

2.2. Data Collection

An informed consent was obtained from each patient’s parent or guardian before enrollment in the study. Clinical data were collected from the patients’ medical records, including initial clinical manifestation, onset age, diagnosis age, parental consanguinity, family history of immunodeficiency, recurrent infections, autoimmune diseases or malignancy diseases, vaccination and allergic history, complication, laboratory tests (including immunoglobulin level and lymphocyte subpopulations), and treatments. A total of 5 mL venous blood was collected from patients for pathogen screening and gene sequencing.

2.3. Molecular and Genetic Diagnosis

Detection of CD154 expression on activated CD4+ T cells was performed by flow cytometry (FACScan, Becton Dickinson, USA) using specific fluorescent-labeled monoclonal antibodies according to the method described previously [20].

Genomic DNA was isolated from heparinized peripheral blood by using the DP319 kit (Tiangen Biotech Co. LTD., China). The individual exons of CD40L gene, including exon-intron boundaries, were amplified by polymerase chain reaction (PCR) as previously described [21]. The system for PCR reaction contained 13.5 μL ddH2O, 1 μL primer for each direction, and 8.5 μL PCR MasterMix (containing 0.1 UTaq/μL, 500 μM dNTP, 20 mM Tris-HCl (pH 8.3), 100 mM KCl, and 3 mM MgCl2 (Tiangen Biotech Co. LTD., China), with total volume 25 μL). Amplification conditions were 95°C for 10 minutes, then 40 cycles of 94°C for 30 seconds, 55°C for one minute, and 72°C for 30 seconds, followed by a final extension of 72°C for 2 minutes. The PCR products were electrophoresed in 1.5% agarose gel, purified, and sequenced. Mutations were investigated by alignment with standard sequence published in NCBI. We sequenced CD40L genes in 50 Chinese controls. Human SNP databases (dbSNP in NCBI and 1000 Genomes) were also searched to confirm the detected mutations.

2.4. Analysis Tools

Data were analyzed by SPSS statistical software (version 21.0, SPSS Inc., Chicago, IL). Median and range were used to present the characters of focused variable. Bioinformatics software PolyPhen-2 ( and MutationTaster ( were used to predict the effects of point mutations.

3. Results

3.1. Demographic Characteristics

From 1999 to 2013, 20 patients from 19 unrelated families (P2 and P3 are cousins) were diagnosed as XHIGM, considering the clinical manifestations, lab examination, family history, decreased CD154 expression, and the mutation of CD40L gene (Table 1). The median onset age of these patients is 8.5 months (range: 20 days–21 months). And the median of diagnosis lag is 50 months (range: 5 months–15 years). Half of the patients had positive family histories of previous sibling deaths at an early age and the diagnosis lag in them was much shorter than that in patients with negative family histories ( months versus months, ). Positive family history with early death in male members may help in diagnosis.

PatientsOnset age (m)Diagnosis age (m)Diagnosis lag (m)StatusFamily historyTime for IVIGHSCTOral ulcerOtitis mediaRecurrent sinopulmonary infectionsRecurrent diarrheaNeutropeniaOther significant eventsSpecial pathogens

P1188466L(+)7 y, IR(+)*(+)

P212483611 y(+)4 y, IR(+)*(+)(+)(+)Impetigo;
perianal abscess

P36126D(+)1 y, R(+)(+)(+)*(+)(+)Elevated liver enzyme;
perianal abscess;

P415205L(+)19 m, IR(+)(+)(+)Encephalitis*;
elevated liver enzyme;
Candida albicans; Pseudomonas aeruginosa

cerebral infarction;

P6261599 y(+)5 y, R(+)(+)(+)(+)(+)BCGitis* 
Pseudomonas aeruginosa

P7554497 y5 y, R(+)(+)(+)BCGitis*

P8412311927 y3 y, R(+)(+)(+)(+)*(+)Arthritis

P9124836L(+)4 y, IR(+)(+)(+)*(+)Chronic kidney disease;

P10960519 y2 y 5 m, R(+)(+)(+)*(+)Crohn’s disease

P111219218021 yNA(+)(+)(+)*Ulcerative colitis;
intestinal perforation;
elevated liver enzyme;
chronic kidney disease;

P12560557 y(+)2 y 3 m, IR(+)(+)(+)(+)(+)BCGitis*Mycobacterium tuberculosis

P13123624D3 y, R(+)(+)(+)*(+)(+)HepatosplenomegalyFungus

P14810597D20 m, IR(+)(+)(+)*(+)Tuberculous meningitisMycobacterium tuberculosis

P156777111 y7 y, IR(+)(+)(+)*Perianal abscess;
facial cellulitis;
red blood cell aplastic anemia

P160.676059.336 y3 y 4 m, R(+)(+)(+)*BCGitis;


P182149285 y(+)NA(+)(+)(+)*(+)

P19317142 y(+)NA(+)(+)(+)BCGitis*

P20641354 y3 y, R(+)(+)(+)(+)*(+)


Note: (a) L: lost in followup; D: dead; R: regularly; IR: irregularly; NA: not available; y: year; m: month; IVIG: intravenous immunoglobulin; HSCT: hematopoietic stem cell transplantation; VZV: varicella zoster virus; HBV: hepatitis B virus. (b) *initial symptom.

3.2. Clinical Manifestations

The most common symptoms of XHIGM patients were recurrent sinopulmonary infections (18 patients, 90%), neutropenia (14 patients, 70%), oral ulcer (13 patients, 65%), and protracted diarrhea (13 patients, 65%). Respiratory and gastrointestinal systems were more frequently affected. Skin (7 patients, 35%), bone and joint system (4 patients, 20%), central nervous system (3 patients, 15%), and urinary system (2 patients, 10%) were also affected (detailed in Table 1).

The median onset age of diarrhea was 12.0 months (range: 20 days to 20 months). Among them, two patients (P10, P11) were diagnosed with inflammatory bowel diseases (IBDs) under enteroscopes, and Cryptosporidium was isolated from the stool of P11. Fungus was isolated from the stool of P13. Pathogens in other patients were not identified.

Six patients had BCGitis (Table 2). The median onset time of adverse drug reactions was 2 months old (range: 50 days–7 months). Two of the patients received antituberculosis medicine treatments and recovered. The other four patients had severe local adverse events after BCG vaccinations, presenting ulceration >10 mm or suppurative lymphadenitis, and received surgeries to drain. Among them, P12 was reinfected by tubercle bacillus at 35 months old and recovered after antituberculosis drug therapy. Besides, another patient P14 suffered pulmonary tuberculosis at 8.5 years old, with restrictive pulmonary capacity and splenomegaly and lymphadenopathy. The patient received isoniazide and rifampicin treatment unregularly for nearly one year and developed tuberculous meningitis and died at 10 years old.


P62 mBCGitis (ipsilateral axillary lymphadenitis)Surgery

P72 mBCGitis (ipsilateral axillary lymphadenitis)Surgery

P1250 dBCGitis (ipsilateral axillary lymphadenectasis)Antituberculosis medicine (rifampicin, unregularly)
35 mPulmonary tuberculosisAntituberculosis medicine (isoniazide + rifampicin + VitB6), antibiotics

P167 mBCGitis (ipsilateral axillary lymphadenitis)Surgery

P172 mBCGitis (ipsilateral axillary lymphadenitis)Surgery

P193 mBCGitis (ipsilateral cervical lymphadenectasis)Antituberculosis medicine

P148.5 yPulmonary tuberculosis, restrictive pulmonary capacity, splenomegaly and lymphadenopathyAntituberculosis medicine (isoniazide + rifampicin, unregularly), antibiotics, IVIG, thymosin
10 yTuberculous meningitisAntituberculosis medicine (isoniazide + rifampicin, unregularly)

Note: IVIG: intravenous immunoglobulin.

Some special manifestations deserved extra attention. P5 was infected by measles and developed subacute sclerosing panencephalitis (SSPE). P11 had Cryptosporidium infection, with protracted diarrhea and elevated liver enzyme. Moreover, this patient developed chronic kidney disease, presenting oliguria, edema, acidosis, increased creatinine concentration, and decreased glomerular filtration rate (GFR), lasting for over four months. Renal biopsy showed tubulointerstitial lesions and the patient received dialysis. P9 also developed chronic kidney disease, with oliguria, albuminuria, hematuria, increased serum creatinine and urea nitrogen concentration, and anemia for over three months. He also exhibited electrolyte disturbance (hypocalcemia and hyperphosphatemia) and convulsion. Ultrasound examination revealed diffuse kidney impairments.

3.3. Hematologic Findings

Anemia was found in seven patients (Table 3). Among them, P10 and P11 had IBDs and the chronic bloody purulent stools might partly cause the anemia. P9 and P11 had chronic kidney disease which may lead to renal anemia. P15 had pure red blood cell aplastic anemia demonstrated by bone marrow biopsy. Neutropenia was the most common hematologic complication, occurring in 70% of the patients (14 patients), 11 of which complicated with oral ulcer.

Patients ()IgG
Lowest neutropenia (/L)Absolute lymphocyte (/L)Hb (g/L)PLT (*1000/L)CD3 (/L)CD4 (/L)CD8 (/L)CD19 (/L)CD4/CD8CD16/56 (/L)


Note: NA: not available.

The median serum IgM concentration was 3.15 g/L (range: 0.74–11.7 g/L). Seventeen patients had elevated IgM levels and three patients had normal IgM levels. Serum IgG levels were uniformly decreased. IgA levels were reduced in the majority of patients.

Lymphocyte counts were generally normal. Three patients (P5, P9, and P11) exhibit significantly decreased CD4/CD8 ratio. They all had severe clinical symptoms and the decreased ratio reflected the impaired cellular immunity. P5 had measles infection, leading to SSPE, and died within one year. P11 had Cryptosporidium infection, with prolonged elevated liver enzyme and impaired renal functions. P9 also had severe manifestation, complicated with chronic renal failure.

3.4. Mutation Analysis

Eighteen unique mutations were identified in 20 patients from 19 unrelated families (P2 and P3 were cousins and they had the same mutation. Besides, P12 and P16 had the same mutation). Among the 19 families, we found 7 deletions, 6 missense mutations, 2 nonsense mutations, 2 splice site mutations, and 2 insertions (Table 4). The mutations were distributed throughout the entire gene, affecting all the domain of the protein, and exon 5 was the most frequently affected region (10/19, 52.6%). Detailed descriptions and analysis were in the Discussion section.

Patients ()Genomic DNA mutation (NC_000023.10)cDNA mutation (NM_000074.2)Predicted effect on protein (NP_000065.1)Affected domainExon/intronCD40L expressionReferences

P1g.175delCc.175delCp.Gln35ArgfsX36TMExon 1NA
P2g.186delGc.186delGp.Ser39GlnfsX48TMExon 10.09%
P3g.186delGc.186delGp.Ser39GlnfsX48TMExon 10.31%
P4g.6196g>a (IVS2-1g>a)Skipping exon3Skipping exon3Intron 2NA
P5g.8181_8182insGTc.420_421insGTp.Asp117ValfsX128ECUExon 40.02%
P6*g.10952G>Cc.499G>Cp.Gly167ArgTNFHExon 50.27%[39]
P7*g.11160C>Ac.707C>Ap.Ser236XTNFHExon 50.75%[36]
P8g.11069_11072delCTCAc.616_619delCTCAp.Leu206GlufsX240TNFHExon 50.80%
P9*g.12090C>Ac.654C>Ap.C218XTNFHExon 5NA[18, 19, 43]
P10*g.10861a>g (IVS4-2a>g)c.482delTGTTACAGExon5 absentTNFHIntron 40.31%[37]
P11g.100C>Tc.100C>Tp.Pro10SerICExon 14.55%
P12*g.2091_2094delTAGAc.158_161delTAGAp.Ile53LysfsX65ECUExon 20.01%[18, 19, 44, 45]
P13g.11220T>Cc.767T>Cp.Phe256SerTNFHExon 50.53%
P14g.11067T>Cc.686T>Cp.Leu205SerTNFHExon 51.78%
P15g.173_189delcccagatgattgggtcac.173_189delcccagatgattgggtcap.Thr34CysfsX42TMExon 10.42%
P16*g.2091_2094delTAGAc.158_161delTAGAp.Ile53fsLysX65ECUExon 2NA[18, 19, 44, 45]
P17g.11075G>Cc.694G>Cp.Ala208ProTNFHExon 5NA
P18g.10986delGc.605delGp.Cys178PhefsX190TNFHExon 5NA
P19g.10882_10883insAc.501_502insAp.Gly144ArgfsX158TNFHExon 5NA
P20*g.10871T>Cc.490T>Cp.Trp140ArgTNFHExon 5NA[19, 38]

Note: *mutation that has been reported. IC: intracytoplasmic domain; TM: transmembrane region; ECU: extracellular unique domain; TNFH: tumor necrosis factor homology domain; NA: not available.

3.5. Therapy and Prognosis

Five patients had died (Table 1). P5 developed SSPE after measles virus infection at 15 years old and died within one year. P14 was infected by mycobacterium tuberculosis and died of tuberculous meningitis at 10 years old. P17 had severe diarrhea and pneumonia since 14 months and died at 2 years old. P3 and P13 received hematopoietic stem cell transplantations (HSCT) but suffered from severe infections and died at 22 months and 56 months, respectively. Three patients were lost in followups. The other twelve patients were alive till now, with the median age of 8 years old (range: 2–27 years old).

Fifteen patients (75%) received intravenous immunoglobulin (IVIG) treatment (Table 1). Eight patients could receive IVIG regularly (400 mg/kg/month) after diagnosis as HIGM. All of them were free of severe bacterial infections, although three of them still had recurrent oral ulcers. Seven patients could not receive full-dose IVIG regularly. Among them, one patient (P14) died of tuberculosis infection. Two patients still had recurrent bacterial infections (P2 had recurrent otitis media and perianal abscess. P12 had otitis media, pneumonia, and fever every half month). Three patients were lost in followup. The other five patients did not receive IVIG therapy, including the two patients who died from measles infection (P5) and severe diarrhea and pneumonia (P17).

Six patients received HSCTs and the median age at transplantation was 6 years old. Four patients received hematopoietic stem cells from HLA-identical unrelated donors and they had clinical remissions and were followed up from months to 6 years. The other two patients had HLA-half identical sibling donors and died from severe infections (Table 5).

PatientsAge at diagnosis (y)Age at HSCT (y)Pretransplantation statusDonorsComplicationsOutcomes

P312Oral ulcer, elevated liver enzyme, neutropeniaHaploidentical sibling VOD, paralytic ileus, pulmonary infection, DICDied

P656.5Gingivitis, neutropeniaHLA-identical unrelated donorGVHD Grade I,
ADH abnormal secretion.
9 y, clinical remission.
Oral ulcer.
Free of IVIG.

P810.2521Oral ulcerHLA-identical unrelated donorGVHD Grade III,
sepsis (Brucella),
CMV infection.
27 y, clinical remission. Pneumonia once a year within the first two years after HSCT. Free of IVIG.

P1056Oral and perianal ulcer, abdominal pain, diarrhea, fever, prolonged APTT, anemia HLA-identical unrelated donorGVHD Grade I,
Hemorrhagic shock, VOD, CMV infection, diarrhea.
9 y, clinical remission.
Free of IVIG.

P1334.5Oral ulcer, diarrhea, neutropeniaHaploidentical siblingSepsisDied

P203.44.6Oral ulcer, neutropeniaHLA-identical unrelated donorGVHD Grade II,
pulmonary infection.
4 y 10 m, clinical remission.

Note: HSCT: hematopoietic stem cell transplantation; VOD: hepatic venoocclusive disease; DIC: disseminated intravascular coagulation;
GVHD: graft-versus-host disease; ADH: antidiuretic hormone; IVIG: intravenous immunoglobulin.

4. Discussions

This study described the clinical features of Chinese XHIGM patients. Respiratory and gastrointestinal systems were more frequently affected. Skin, bone and joint system, central nervous system, and urinary system were also involved. In this study, two patients (P9 and P11) developed chronic kidney diseases. Cardiovascular disease, infections, and nonsteroidal anti-inflammatory drugs (NSAIDs) are accepted risk factors for causing chronic kidney disease [22]. P9 was born with congenital heart diseases (ventricular septal defect and patent ductus arteriosus). P11 had ulcerative colitis and received NSAIDs treatment. Both of the patients had severe recurrent infections because of immunodeficiency. These complications might partly contribute to the kidney involvement in these two patients. Few reports on XHIGM patients with chronic kidney diseases have been published till now. We should pay more attention to the urinary system function in further followups.

Opportunistic infections, especially Cryptosporidium parvum and Pneumocystis jirovecii infections, are common and prominent clinical feature of XHIGM patients [16, 17, 23]. Besides, infections caused by tubercle bacillus were detected [16, 17, 19]. A number of PIDs are reported to be susceptible to severe mycobacterial disease following vaccination with BCG, including severe combined immunodeficiency (SCID), chronic granulomatous disease (CGD), and Mendelian susceptibility to mycobacterial diseases (MSMD). Increased BCG infections are also reported in patients with hyperimmunoglobulin E syndrome (HIES) and XHIGM, with less prevalence and severity of BCG complications compared with the PIDs mentioned above [24]. In a European survey, only two of the 56 XHIGM patients present with tuberculosis [16]. However, in our study concerning 20 XHIGM patients, we found six patients had BCGitis during the first year of life. One of them was reinfected by tuberculosis at 35 months old. One more patient suffered pulmonary tuberculosis and tuberculous meningitis and died at 10 years old. In another cohort study focusing on Chinese patients, five (38%) of the 13 XHIGM patients had lymphoid tuberculosis after BCG vaccination [25], the relatively high incidence of which was very similar to ours. An Indian research group also reported that two patients with pulmonary tuberculosis and one patient with disseminated BCGiosis were found in the cohort study including seven XHIGM patients [26]. Global Tuberculosis Report 2013 by WHO pointed out that India and China had the largest number of tuberculosis cases, taking up to 26% and 12% of the global total, respectively [27]. In both China and India, BCG vaccine is given to every newborn after birth to prevent tuberculous meningitis and disseminated disease, while in Europe, where incidence and prevalence of tuberculosis are low, less than half of the European countries have nationally recommended BCG vaccination. The differences in tuberculosis prevalence and BCG coverage may partly contribute to the differences of mycobacteria complications in XHIGM patients.

Some recent discoveries may help to elucidate the susceptibility to mycobacterial infections in XHIGM patients. Hayashi’s results indicated that CD40-CD154 signaling might be an important step in host immune response against Mycobacterium avium infection [28]. Klug-Micu et al. revealed that activation of monocytes via CD40L resulted in a Vitamin D-dependent antimicrobial activity against Mycobacterium tuberculosis [29]. IL12/23-IFN-γ axis is involved in defending against BCG infection [24, 30]. Jain et al. [31] demonstrated that activated T cells from XHIGM patients produced markedly reduced levels of IFN-γ and failed to induce antigen-presenting cells to synthesize IL-12. We should continue to observe the susceptibility to tuberculous infections in XHIGM patients and be concerned about the immunity affected by tuberculous infection. Furthermore, we found two patients with varicella-zoster virus infections, one patient with measles virus infections, one patient with fungus infections, and one patient with Cryptosporidium infection, which all indicated the defects in cellular immunity.

Mutations of CD40L geneare highly heterogeneous [1820, 32]. CD40Lbase, which was last updated in 2011, contained 250 public entries. Besides, more mutations on CD40L gene were reported in other studies [19, 33]. Considering the published results, the common type of mutations in CD40L might be missense mutations, followed by deletion/insertion mutations, nonsense mutations, and splice site mutations (detailed in Table 6). In our results, missense mutations and deletion/insertion mutations were the main types, composing about 3/4 of the mutations.

ReportsYearNumber of mutationsMissense (%)Nonsense (%)Deletion (%)Insertion (%)Splice site (%)

This study20131931.610.536.810.510.5
Nonoyama et al. [18]19971330.823.123.1023.1
Seyama et al. [20]19982832.
Lee et al. [32]20056118.024.621.311.524.6
Winkelstein et al. [17]20035422.218.524.111.124.1
CD40Lbase  [46]201122131.723.517.210.416.3
Cabral-Marques et al. [19]20142634.626.911.57.711.5

Some possible mutation hotspots have been identified. Notarangelo et al. [15] pointed out that W140 represents a mutational hotspot. In Lee’s study [32], four possible hotspots were identified that were Thr254, IVS1+1 gt, IVS3+1 ga/t, and IVS4+1 gc. In the CD40Lbase, mutations were concentrated in some special positions; that is, mutations in C218, T254, and W140 have been identified in more than 10 patients, respectively, till now.

In this study, we identified two splice site mutations (P4: IVS2−1 ga and P10: IVS4−2 ag). Vargas-Hernández et al. [34] and García-Pérez et al. [35] separately reported a IVS3−1 ga mutation, resulting in exon 4 skipping and a protein with 21-amino acid truncation. Weller et al. [36] reported an IVS2−1 gt mutation, resulting in exon 3 skipping and forming a protein consisting of 107 amino acids. In our study, P4 mutated in 3′-acceptor splice sites, the same positions as the three cases, and it can be inferred that this mutation could also result in the skipping of exon 3. For the mutation in P10, it has been reported in 1994 [37]. Mutation in the acceptor site of intron 4 allowed utilization of a cryptic acceptor site and a few base pairs downstream in exon 5 and led to 8-nucleotides deletion in P10.

Six patients had missense mutations in this study. From data published in CD40Lbase (updated to 2011), the most frequent amino acid substitutions were Leu to Ser/Pro, Thr to Met, Gly to Arg, and Ala to Asp/Glu. Thusberg and Vihinen [38] reported that there were 10 invariant positions corresponding to the amino acids W140, L161, G167, Y169, Y172, L205, G226, G227, L231, and G257 in CD40L, according to sequence alignment and calculation. Disease causing mutations are typically located at conserved positions within a protein family, since these positions are usually essential for the structure and/or function of the protein [38]. In our study, the mutation in P6 (Gly167Arg) was previously reported in two Iranian patients [39]. Substitution from glycine to arginine introduced a larger side chain group and a positive charge, which would profoundly change structure and functions of the protein. The mutation in P20 (p.Trp140Arg) was reported as one of the “hotspots” and eleven patients in all had been reported to have this mutation until now. Trp140 is a large hydrophobic residue buried inside the protein while arginine had a large hydrophilic side chain with positive charge. The Trp140Arg substitution would obviously lead to destabilization of the structure.

Four missense mutations were novel. Mutation in P14 (p.Leu205Ser) changed the conserved hydrophobic residue into a hydrophilic residue. Proline was involved in the mutations in P11 (p.Pro10Ser) and P17 (p.Ala208Pro). Proline is the only amino acid forming a ring with the backbone and bends the main chain of the protein in a characteristic way. Proline is a known breaker of secondary structures. The substitution in the two patients would profoundly change structure and functions of the protein. A p.Ala208Asp mutation was identified in an XHIGM patient and reported in 2002 [40].

Effects of the four novel point mutations were analyzed by using bioinformatics software PolyPhen-2 [41] and MutationTaster [42]. PolyPhen-2 predicted probably damaging effects to the protein with a score over 0.99, respectively. MutationTaster predicts that mutations in P13, P14, and P17 were disease causing, with probability value over 0.98.

Two patients had nonsense mutations at codon 236 and codon 218, both of which were already reported [18, 36, 43]. Cys218 was involved in formation of a disulfide bridge and nonsense mutation (p.C218X) had been reported in at least twelve XHIGM patients and one carrier, usually associated with more severe clinical presentation.

Nine patients had insertion/deletion mutations, all changing the reading frames and leading to large changes of the protein structure. P12 and P16 were from unrelated families and they had the same mutation (g.2091_2094delTAGA, c.158_161delTAGA, p.Ile53fsX13). This mutation had been reported in at least another 6 unrelated patients and was the fifth common mutation position in the CD40Lbase. The 4 deleted nucleotides were very close to the acceptor splice site of intron 1 and were flanked by repeat sequences, suggesting that these mutations might be caused by slipped mispairing mechanisms. The frame shift deletion led to a premature stop codon within the extracellular unique domain. P2 and P3 were cousins and they had the same mutation (g.186delG). The deletion in exon 1 led to a premature stop codon in the transmembrane domain.

In conclusion, we retrospectively investigated the clinical, immunological, and molecular features of 20 Chinese XHIGM patients diagnosed and followed up in our center. We identified 18 unique mutations in CD40L in these patients, with 12 novel mutations. Various mutations reflected the heterogeneity of CD40L gene, and we should go further to investigate the relationship between genotype and phenotype.

Conflict of Interests

None of the authors has any potential financial conflict of interests related to this paper.

Authors’ Contribution

Lin-Lin Wang and Wei Zhou are co-first authors and had equal contributions to the study.


The authors express their gratitude to all the patients and their families for their kindness for permission and cooperation. The authors are also grateful to Koon-Wing Chan, Department of Paediatrics and Adolescent Medicine, The University of Hong Kong, for his kind advice on sequence analysis.


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