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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References

In this study, we report the clinical and genetic features of Chinese patients with X-linked lymphoproliferative syndrome (XLP). Male patients with fulminant infectious mononucleosis (FIM), Epstein–Barr virus (EBV)-associated hemophagocytic lymphohistiocytosis (HLH) or persistent EBV viremia were enrolled in this study. Direct sequencing was used to detect SH2D1A/XIAP gene mutations. The patients' clinical features were assessed by retrieval of data from medical records. Twenty-one male patients with FIM, EBV-associated HLH or persistent EBV viremia were evaluated. Four patients had SH2D1A mutations, and one patient had an XIAP mutation. All five of these patients had symptoms of HLH and EBV infection. Among the five patients, the youngest one was only 1 month old at onset. One patient exhibited hypogammaglobulinemia. Of four patients evaluated for immunological function, all exhibited reduced CD4/CD8 ratios. Three patients had rapid disease progression and died. One patient received haematopoietic stem cell transplantation and is well. The overall clinical phenotypes of Chinese patients with XLP matched previous reports. For patients with severe EBV-associated HLH, our results indicate the need to examine the possibility of XLP.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References

X-linked lymphoproliferative syndrome (XLP) is a rare inherited immunodeficiency. Two genes associated with the development of XLP have been identified [1]. The first gene, SH2D1A, encodes signalling lymphocytic activation molecule (SLAM)-associated protein (SAP). The second gene is XIAP, also known as BIRC4, which encodes X-linked inhibitor of apoptosis protein. While they are located close together at Xq25, mutations in SH2D1A and XIAP seem to lead to forms of XLP with distinct molecular pathogenesis and clinical features.

XLP is characterized by extreme vulnerability to Epstein–Barr virus (EBV) infection. The major clinical phenotypes of XLP include fulminant infectious mononucleosis (FIM), EBV-associated hemophagocytic lymphohistiocytosis (HLH), lymphoproliferative disorder and dysgammaglobulinemia [2, 3]. Patients with XLP often manifest an array of these phenotypes over time. XLP survival rates are very poor, even with treatment, and the vast majority of patients die in childhood [4, 5]. Haematopoietic stem cell transplantation (HSCT) is the only curative therapy for XLP [6, 7]. Therefore, rapid, definitive diagnosis and immediate treatment are critical to improve the prognosis and survival of patients with XLP.

To date, only one Chinese case of XLP reported in a local journal [8]. We report here the clinical and genetic features of five additional Chinese patients diagnosed with XLP in our hospital over the past 2 years.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References

Patients

During the period from January 2010 to December 2012, male patients met one of the following three criteria were enrolled in the study. (1) Patients were diagnosed with FIM, according to the previous study [9]. (2) Based on the guideline of HLH-2004 [10], the patients were diagnosed with HLH, and with evidence of EBV infection. (3) Patients had active EBV infection lasting more than 6 months. After obtaining written informed consent, 5 ml of venous blood from patients and their parents was collected into heparin-containing syringes and used for immunological assays and sequencing. The study protocol was approved by the Ethics Committee of the Children's Hospital of Fudan University.

Routine evaluation of immunological function

Routine evaluation of immunological function included analysis of lymphocyte subsets and the detection of immunoglobulins G, A, M and E. As previously reported [11], lymphocyte subsets were analysed using a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Immunoglobulins G, A and M were detected by nephelometry. Immunoglobulin E was detected by UniCAP (Pharmacia, Uppsala, Sweden).

Gene sequencing

Genomic DNA was isolated from peripheral blood mononuclear cells using the RelaxGene Blood DNA System (Tiangen Biotech, Beijing, China) according to the manufacturer's instructions. Genomic DNA was amplified by PCR using synthetic oligonucleotide primers designed to amplify the SH2D1A and XIAP genes. The primer sequences are shown in Supplemental Table. After an initial denaturation step of 5 min at 95 °C, 35 cycles of amplification were performed as follows: 95 °C for 30 s, 60 °C for 30 s and 72 °C for 40 s. Final extension was performed at 72 °C for 7 min. PCR products were purified with Performa DTR Gel Filtration Cartridges and directly sequenced by ABI Prism BigDye terminators. Both strands were sequenced.

Clinical assessment

After patients were confirmed with SH2D1A or XIAP gene mutation, the patients' clinical events and laboratory features were assessed by retrieval of data from medical records.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References

During the study period, 21 male patients with FIM (n = 2), EBV-associated HLH (n = 13) or active EBV infection lasting more than 6 months (n = 6) were enrolled and completed SH2D1A and XIAP sequencing. Five patients with EBV-associated HLH were found to have SH2D1A or XIAP mutations. Therefore, we summarize the clinical and genetic features of these five patients below.

Clinical characteristics

Patient 1 was 4 years old at diagnosis. He initially received treatment in our hospital for fever. He tested positive for EBV-DNA and EBV-VCA IgM and exhibited low serum immunoglobulin G levels. He was administered acyclovir and IVIG, and his symptoms improved. One month later, he showed neutropenia, anaemia and thrombocytopenia. After the SH2D1A gene mutation was found, he received HSCT and is well.

Patient 2 is the youngest among the five patients, with his age of onset being only 1 month. He had fever, thrombocytopenia and liver dysfunction (ALT 95 IU/l, AST 83 IU/l). Atypical lymphocyte counts were elevated, accounting for 36% of peripheral blood leucocytes, while bone marrow was normal. His mother had negative EBV-VCA IgM and EBV-VCA IgG. Although he tested negative for EBV-DNA and EBV-VCA IgM, he was diagnosed with EBV infection. He was treated with acyclovir, IVIG and other symptomatic treatments. After 2 weeks of treatment, the patient showed symptoms of HLH and died from multiple system organ failure (MSOF) at 3 months of age.

Both Patient 3 and Patient 4 had rapid disease progression. Patient 3 was a 9-month-old boy. His disease progressed from onset to death in only 23 days. In the first 2 weeks of the course of the disease, he only had moderate fever. However, he then showed jaundice (TB 54.7 μm, DB 45.4 μm), liver dysfunction (ALT 297 IU/l, AST 380 IU/l) and high atypical lymphocyte counts (27%). He tested positive for EBV-DNA and EBV-VCA IgM. After treatment with acyclovir, IVIG and other symptomatic treatments for 7 days, he showed encephalitic symptoms (convulsions and coma) and symptoms of HLH. Two days later, the boy died from MSOF. Patient 4 was a 1-year, 5-month-old boy. He was transferred to our hospital after having a persistent fever for 20 days. As with Patient 3, he showed jaundice (TB 93.4 μm, DB 77.2 μm), liver dysfunction (ALT 763 IU/l, AST 864 IU/l) and high atypical lymphocyte counts. He also tested positive for EBV-DNA and EBV-VCA IgM. After treatment with acyclovir, IVIG and other symptomatic treatments for 4 days, he developed HLH symptoms. Two days later, he exhibited convulsions and died from MSOF.

Patient 5 was a 4-year-old boy. He had fever, rash and liver dysfunction (ALT 341 IU/l, AST 258 IU/l) and tested positive for EBV-VCA IgM. However, he tested negative for EBV-DNA. After 2 weeks of treatment with ganciclovir and other symptomatic treatments, symptoms improved. However, 1 month later, fever and rash reappeared. Moreover, he showed symptoms of HLH. At this time, the SH2D1A gene mutation was found. He is alive and waiting for HSCT.

Totally, none of the five patients had a family history of XLP or a history of recurrent infections. All of the five patients had EBV infection and presented with symptoms of HLH. They were treated according to the guideline of HLH-2004 [10]. Three patients died from MSOF. Routine evaluation of immunological function was completed on 4 of the 5 patients. All four of these patients had decreased CD4/CD8 ratios due to abnormal CD8+ T cell proliferation. Only one of these four patients showed hypogammaglobulinemia. Clinical characteristics, including immunological phenotypes of the five patients, are summarized in Tables 1 and 2 and Fig. 1.

Table 1. Clinical features of patients with X-linked lymphoproliferative syndrome
 Age at diagnosisClinical presentationEBV statusFamily historyOutcome
EBV-VCA IgMEBV-DNA (copies/μg)
  1. HLH, hemophagocytic lymphohistiocytosis; MSOF, multiple system organ failure; HSCT, Haematopoietic stem cell transplantation.

Patient 14 yearsHLH, hypogammaglobulinemiaNegative6.14 × 107NegativeAlive well after HSCT
Patient 21 monthHLH, liver dysfunction and developed into MSOFNegativeNegativeNegativeDead
Patient 39 monthsHLH, encephalitis, liver dysfunction and developed into MSOFPositive8.67 × 106NegativeDead
Patient 41 year and 5 monthsHLH, encephalitis, liver dysfunction and developed into MSOFPositive3.46 × 106NegativeDead
Patient 54 yearsHLH and liver dysfunctionPositive5.21 × 106NegativeAlive and waiting HSCT
Table 2. Immunological phenotypes of patients with X-linked lymphoproliferative syndrome.
 Patient 1Patient 2Patient 3Patient 4Patient 5Age-matched reference value
2 days – 11 months1–6 years
  1. ND, not determined.

CD3 (cells/μl)23953481ND1123925901800–33001500–2900
CD4 (cells/μl)9261480ND4500632900–23001000–2100
CD8 (cells/μl)13821936ND57771927700–1500700–1100
CD16/56 (cells/μl)64642ND561223300–800300–600
CD19 (cells/μl)45362ND443180700–1700500–1200
      1–3 months7–11 months1–2 years3–6 years
IgG (g/l)0.212.5ND22.57.282.75–7.503.50–8.905.52–11.464.95–12.74
IgA (g/l)0.190.23ND5.250.090.05–0.600.06–0.540.06–0.740.33–1.89
IgM (g/l)0.110.89ND11.70.110.10–0.700.36–1.200.60–2.120.65–2.01
IgE (Ku/l)<2<2ND<2<2<100<100<100<100
image

Figure 1. Disease development process of the five patients with X-linked lymphoproliferative syndrome. EBV: Epstein–Barr virus; HLH: Hemophagocytic lymphohistiocytosis; HSCT: Haematopoietic stem cell transplantation; MSOF: multiple system organ failure.

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Genetic characteristics

Four of the five patients had SH2D1A mutations, and one patient was found to have an XIAP mutation. Each of their mothers was heterozygotic for the same mutation, and their fathers had no SH2D1A or XIAP gene mutations. The mutations of Patients 3, 4 and 5 are reported in the previous studies [12-14]. The mutations of Patient 1 and Patient 2 were however not reported before and were not found in the 1000 genome database as polymorphisms (Table 3, Fig. 2).

Table 3. Genetic features of patients with X-linked lymphoproliferative syndrome
 Genetic defectMutation
TypeCDS level changeProtein level changeEffect
Patient 1SH2D1ASubstitutionc.293T>CP.L98PMissense defect
Patient 2XIAPSubstitutionc.1100A>GP.D367GSplice site mutation
Patient 3SH2D1ASubstitutionc.138-2A>G_Splice acceptor defect, Exon skipping
Patient 4SH2D1ASubstitutionc.161A>GP.Y54CMissense defect 
Patient 5SH2D1ASubstitutionc.138-2A>G_Splice acceptor defect, Exon skipping
image

Figure 2. Mutations identified in the SH2D1A or XIAP gene in the five patients. Arrows indicate the mutation points.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References

XLP is a rare but life-threatening disease. The estimated prevalence of XLP is 2–3 per 1 million males [15]. However, the frequency may be under-reported for a variety of reasons, including failure to properly diagnose the disorder. Due to the discovery of two causative genes, a definitive genetic diagnosis is now possible in many patients with XLP phenotypes. As the common clinical features of XLP are FIM, EBV-associated HLH and lymphoproliferative disorder [2, 3], we completed SH2D1A and XIAP gene sequencing in the patients with one or more of these symptoms in this study.

Most XLP patients appear healthy prior to contracting EBV [16]. However, following infection, patients often develop T and B cell lymphoproliferation and secondary HLH [16, 17]. Using gene sequencing, we diagnosed five patients with XLP of the 21 male patients in our study with FIM, EBV-associated HLH or persistent EBV viremia. The overall clinical phenotypes of the affected persons matched those previously reported. All of the five patients had symptoms of HLH and four tested positive for EBV-DNA. This finding indicated that EBV infection triggers HLH in patients with SH2D1A or XIAP deficiency. Although Patient 2 was EBV-DNA negative, we still consider HLH as triggered by EBV infection based on the elevated atypical lymphocyte counts. Previous study reported that about 13 XLP patients showed hypogammaglobulinemia [18]. In our study, 1 patient with SH2D1A deficiency had lower IgG, IgA and IgM levels, especially IgG. The results indicate that the patient had hypogammaglobulinemia. All four patients evaluated for immunological function showed a low CD4/CD8 ratio, which may be associated with EBV infection.

In patients with XLP, disease onset is usually at 2–5 years of age and is often triggered by EBV infection [16, 19]. Among the five patients in the study, the youngest one was only 1 month old at time of onset. It is different with the western world, maybe due to early encountering of the EBV infection. Although there is no precise epidemiological data of EBV infection, the age of onset is thought to vary widely, with developed countries having higher ages at primary infection, most likely due to better hygienic conditions and other socioeconomic and demographic factors including household size and population density [20]. The result indicates that patients with SH2D1A or XIAP deficiency can show XLP associated symptoms at a very young age.

Prior reports indicate that the prognosis for XLP is poor, with 70% of patients dying before the age of 10 and mortality nearing 96% for those with a history of EBV infection [2, 4, 5]. In our study, three patients had rapid disease progression and died. Only one patient received HSCT and is well. The prognosis observed in our study is therefore similar to previous studies.

In summary, we report the clinical and genetic features of five Chinese patients with SH2D1A/XIAP deficiency in this study. For patients with severe EBV-associated HLH, our results indicate the need to consider the possibility of XLP.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References

This work was supported by the National Natural Science Foundation of China (81172877, 81000260) and Shanghai Rising-Star Program (11QA1400700).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Conflict of interest
  9. References