Abstract
A subgroup of patients with 22q11·2 microdeletion and partial DiGeorge syndrome (pDGS) appears to be susceptible to non-cardiac mortality (NCM) despite sufficient overall CD4+ T cells. To detect these patients, 20 newborns with 22q11·2 microdeletion and congenital heart disease were followed prospectively for 6 years. Besides detailed clinical assessment, longitudinal monitoring of naive CD4+ and cytotoxic CD3+CD8+ T cells (CTL) was performed. To monitor thymic activity, we analysed naive platelet endothelial cell adhesion molecule-1 (CD31+) expressing CD45RA+RO−CD4+ cells containing high numbers of T cell receptor excision circle (TREC)-bearing lymphocytes and compared them with normal values of healthy children (n = 75). Comparing two age periods, low overall CD4+ and naive CD4+ T cell numbers were observed in 65%/75%, respectively, of patients in period A (< 1 year) declining to 22%/50%, respectively, of patients in period B (> 1/< 7 years). The percentage of patients with low CTLs (< P10) remained robust until school age (period A: 60%; period B: 50%). Low numbers of CTLs were associated with abnormally low naive CD45RA+RO−CD4+ T cells. A high-risk (HR) group (n = 11) and a standard-risk (SR) (n = 9) group were identified. HR patients were characterized by low numbers of both naive CD4+ and CTLs and were prone to lethal infectious and lymphoproliferative complications (NCM: four of 11; cardiac mortality: one of 11) while SR patients were not (NCM: none of nine; cardiac mortality: two of nine). Naive CD31+CD45RA+RO−CD4+, naive CD45RA+RO−CD4+ T cells as well as TRECs/106 mononuclear cells were abnormally low in HR and normal in SR patients. Longitudinal monitoring of naive CD4+ and cytotoxic T cells may help to discriminate pDGS patients at increased risk for NCM.
Keywords: chromosome 22q11·2 microdeletion, DiGeorge syndrome, immunodeficiency, naive CD4− T cells, recent thymic emigrants (TREC)
Introduction
The chromosome 22q11·2 microdeletion is associated with DiGeorge (DGS), Sedlackova, Takao and velocardiofacial syndrome comprising congenital heart disease (CHD), abnormal facies, thymic hypoplasia, cleft palate and hypoparathyroidism [1,2]. The prevalence of the chromosome 22q11·2 microdeletion is at least 1 in 4000 live births [3–5].
The majority of patients exhibit features of partial DGS (pDGS) with low but sufficient overall T cells because of hypoplastic or atypically located thymus glands, whereas athymic patients with complete DGS (cDGS) and very low T cell numbers (1–2% CD3+) are exceedingly rare (incidence 1:1 000 000), with a very poor prognosis without thymic transplant [5–13].
In mainly retrospective or cross-sectional analyses, pDGS patients were shown to have higher T cell percentages and broader ranges of lymphoproliferative responses, resulting in a much better overall prognosis than those with cDGS [2,5–9]. However, a subgroup of as-yet immunologically undefined pDGS patients appears to be at higher risk for relevant infectious and autoimmune complications [7,8]. In this prospective 6-year study, we investigated infants with an early diagnosis of pDGS and microdeletion by longitudinal monitoring of lymphocyte subsets with particular regard to non-cardiac mortality (NCM). A subgroup of infants with pDGS and low numbers of both naive (CD31+) CD45RA+RO−CD4+ and cytotoxic CD3+CD8+ T cells was at increased risk of developing lethal infectious and lymphoproliferative complications.
Materials and methods
Patients
Between 1995 and 1999, all consecutively born newborns with conotruncal anomalies and suspicious clinical signs were investigated for the presence of the 22q11·2 microdeletion. After approval by the institutional review board and with parental consent, 20 infants with microdeletion were followed prospectively every 3–6 months until 2005. Major clinical events and complications were documented. Immunological examinations were performed only at periods without overt infection or post-interventional complications. Referring to the study by Sullivan [13], the period of infancy (0–12 months = period A) and the period thereafter (> 12 months and < 7 years = period B) were analysed separately.
Cytogenetic analysis
Metaphase chromosome preparations were performed from phytohaemagglutinin (PHA)-stimulated lymphocyte cultures (if available, also from both parents) and performed according to standard procedures. Chromosome and fluorescence in situ hybridization (FISH) analyses were performed in samples from patients and their parents.
Flow cytometry
The following lymphocyte subsets were measured with a fluorescence activated cell sorter (FACS)Calibur device (Becton Dickinson, Heidelberg, Germany): CD3+, CD3+CD4+, CD45RA+RO−CD4+, CD31+ (platelet endothelial cell adhesion molecule-1) CD45RA+RO−CD4+, CD45RA−RO+CD4+, cytotoxic CD3+CD8+ T cells, CD19+ B cells and CD16+CD56+ natural killer cells. A cohort of Caucasian children (n = 807) served as healthy controls [14].
At least four representative flow cytometric measurements from each patient were analysed during both periods A and B and at the end of age 6 years. FACS results were considered to be abnormal if the highest single value was < P10. The highest single flow cytometric value of each individual patient was used for comparative analysis. The ratios between naive CD45RA+RO− and memory CD45RO+RA−CD4+ lymphocytes were determined at the end of both observation periods. A ratio < 1 was considered abnormal.
T cell receptor excision circle analysis with reverse transcription–polymerase chain reaction and flow cytometry of CD31+CD4 T cells
In seven patients, T cell receptor excision circle (TREC) containing T cells were measured to estimate thymic activity [15,16]. TREC analysis was performed with DNA extracted from Ficoll-separated peripheral blood mononuclear cells (PBMC) using the Taqman-polymerase chain reaction technique. TRECs were compared with values of previously published healthy age-matched controls (< 10 years of age: > 10 000 TREC/106 PBMC) [16]. TRECs were compared with absolute numbers of naive CD45RA+RO− and CD31+CD45RA+RO−CD4+ T cells. Seventy-five healthy children (≤ 16 years) were investigated at our centre and served as controls for absolute numbers of CD31+CD45RA+RO−CD4+ T cells.
Lymphocyte proliferation tests
Proliferative responses to PHA, Staphylococcal enterotoxin B (SEB) and tetanus toxoid (TT) were determined by incorporation of [3H]-thymidine after standard protocols (normal > 50 000 d.p.m. (dissociations per minute) for mitogens and > 10 000 d.p.m. for TT; normal stimulation index > 50 for mitogens and > 10 for TT). One stimulation test was performed in each observation period.
Immunoglobulin and protective antibody measurement
Humoral immune responses were tested 4–8 weeks after administration of at least three regular vaccinations with diphtheria (DT) and TT, and conjugated vaccine against Haemophilus influenzae type b (HIB) in period A and after a first booster injection in period B. Serum concentrations of immunoglobulins and immunoglobulin G (IgG) subclasses were measured by rate nephelometry and related to age in percentile curves. IgG subclasses were measured at least twice after the second birthday. Specific antibodies against TT, DT (protective > 100 U/l) and HIB (protective > 0·15 µg/ml) were measured repeatedly, at least twice per observation period, using an enzyme-linked immunosorbent assay.
Clinical evaluation and outcome
Infectious, autoimmune and non-infectious complications requiring hospitalization as well as cardiac and developmental outcome were documented.
Results
Cytogenetic and FISH investigations
All 20 patients were shown to have a de novo 22q11·2 microdeletion. Parental origin could be studied in 14 patients. Eight had deletions of maternal and six of paternal origin. No heterozygous parent could be identified. No parental DNA was available in six patients. No correlation was found between origin of microdeletion and clinical outcome.
Clinical evaluation at first diagnosis
All 20 infants (10 male and 10 female) had proven CHD, 16 had no detectable thymus tissue (diagnosed by open heart operation in seven, by chest X-ray in two and by ultrasound in two patients). No patient with cDGS was observed (Table 1).
Table 1.
Overview of clinical features of 20 patients with chromosome 22q11·2 deletion syndrome (1995–2005).
| Congenital heart disease | Other anomalies | |
|---|---|---|
| 1 | VSD | No |
| 2 | Truncus arteriosus communis, LPA atresia, MAPCA, VSD, right aortic arch | Vascular bronchus compression, HPT |
| 3 | Tetralogy of Fallot, ASD | Unbalanced translocation 16/22 |
| 4 | PA/VSD, MAPCA, ASD | Vascular brochus compression by arteria lusoria |
| 5 | VSD, right aortic arch | Vascular tracheal compression, inguinal hernia, HPT, velopharyngeal dysfunction |
| 6 | PA/VSD, MAPCA | Clumb feet, thumb anomalies, submucous cleft, inguinal hernia, tracheomalacia, bilateral megaureters |
| 7 | Interrupted aortic arch type B | Strabism divergens, GER, HPT, trachomalacia |
| 8 | Tetralogy of Fallot, MAPCAs, LPA stenosis | Arteria lusoria |
| 9 | Interrupted aortic arch type B | GER, tracheomalacia, megaureter, nephrolithiasis, strabismus divergens |
| 10 | PA/VSD, MAPCA | Stabismus divergens alternans, oesophageal stenosis, tracheomalacia |
| 11 | Interrupted aortic arch type B | No |
| 12 | Tetralogy of Fallot, single left coronary artery, ASD II, right aortic arch | Talipes equinovarus, GER |
| 13 | Interrupted aortic arch type B, severe LPA stenosis | No |
| 14 | PA/VSD, MAPCA | Velopharyneal dysfunction, camptodactyly |
| 15 | Interrupted aortic arch type B | GER |
| 16 | PA/VSD | Corneal clouding, unilateral renal agenesis, GER |
| 17 | PA/VSD, MAPCA, right aortic arch | Velopharyngeal dysfunction, submucous cleft |
| 18 | VSD, ASD | No |
| 19 | VSD, ASD, right aortic arch | Choanal stenosis, laryngeal synechia, GER, megaureter, velopharyngeal dysfunction |
| 20 | VSD, PDA | Renal agenesis, vertebral anomaly, choledochal cyst, pancreas anulare |
ASD, atrial septal defect; GER, gastro-oesophageal reflux; HPT, hypoparathyroidism; LPA, left pulmonary artery; MAPCA, major aortopulmonary collateral arteries; PA, pulmonary atresia; VSD, ventricular septal defect.
Analysis of lymphocyte subsets by flow cytometry
The comparison of two age periods revealed that the majority of overall CD4+ and naive CD45RA+RO−CD4+ T cell counts were abnormally low in the first year of life (n = 13 of 20 and n = 15 of 20 respectively), whereas the proportion below the normal range was less in period B (n = five of 18 and n = nine of 18 respectively). All patients with low naive CD4+ T cell counts in period B also had low naive CD4+ T cell counts in period A. The percentage of patients with low number of CTLs showed a less pronounced decrease within the same time-periods (A: 12 of 20 versus B: nine of 18) (Fig. 1a, Table 2a).
Fig. 1.
Changes of naive/memory CD4+ and cytotoxic CD8+ T cells from period A to period B. (a) White circles: naive CD45RA+RO−CD4+ T cells of standard-risk (SR) patients; black circles: naive CD45RA+RO−CD4+ T cells of high-risk (HR) patients; while triangles: memory CD45RA−RO+CD4+ T cells of SR patients; black triangles: memory CD45RA−RO+CD4+ T cells of HR patients. Grey areas represent normal age-related ranges according to data from Shearer et al.[14]. (b) White rhombi: CD3+ T cells of SR patients; black rhombi: CD3+ T cells of HR patients; white triangles: CD3+CD8+ cytotoxic T cells of SR patients; black triangles: CD3+CD8+ cytotoxic T cells of HR patients. Grey areas represent normal age-related ranges according to data from Shearer et al.[14].
Table 2a.
Profile of T cell subsets in 20 patients with chromosome 22q11·2 deletion syndrome.
| Time-period | Age 0–12 months (A) | Age 12–72 months (B) |
|---|---|---|
| Percentage of patients with CD4+T cells < P10 | 65 (13/20) | 28 (5/18) |
| Range of CD4+ (cells/µl)* | 500– 2825 (NR 1400–4300) | 354– 2833 (NR 700–3400) |
| Percentage of patients with CD45RA+CD4+ T cells < P10 | 75 (15/20) | 50 (9/18) |
| Range of CD45RA+CD4+ (cells/µl)* | 390– 2492 (NR 1100–3700) | 180– 2381 (NR 430–2900) |
| Percentage of patients with CD8+ T cells < P10 | 60 (12/20) | 50 (9/18) |
| Range of CD3+CD8+ (cells/µl)* | 403– 1450 (500–1700) | 305 – 1198 (NR 490–2000) |
| Percentage of patients with CD45RA+CD4+/CD45RO+CD4+ ratios < 1 | 0 | 33 (6/18) |
Range of the highest single value of each individual patient within periods A and B compared with the normal age-related range (NR) (according to data from Shearer et al.[14]); bold, abnormal.
Until the end of period B or death, all patients with abnormally low naive CD4+ T cells also had decreased CTLs. Such patients were defined as retrospectively high risk (HR), whereas patients with transiently reduced or normal numbers of naive CD45RA+RO−CD4+ or cytotoxic T cells within the same observation periods were defined as standard-risk (SR) patients. According to this definition, 11 patients with HR features could be identified (Table 2b) (patients 2, 6, 4, 7, 8, 9, 10, 13, 14, 15 and 20). At the end of period B or prior to death, the ratios between CD45RA+RO− and CD45RO+RA−CD4+ T cells were < 1 in six of 18 and > 1 in 12 of 18 patients respectively (Tables 2a, c). All six patients with inverted ratios (patients 2, 8, 9, 13, 15 and 20) belonged to the HR group (Fig. 1a, Table 2b).
Table 2b.
Differences between standard-risk (SR) and high-risk (HR) partial DiGeorge syndrome (pDGS) patients.
| Patients with pDGS | SR | HR§¶ |
|---|---|---|
| Number of patients | 9 | 11 |
| Range of CD4+T cells (cells/µl) | 1060–2825 (NR 1400–4300)† | 940–1340§ (NR 1400–4300)† |
| 871–2833 (NR 700–3400)† | 500–1340¶ (NR 1400–4300)† | |
| 354–1900¶ (NR 700–3400)† | ||
| Number of patients with CD4+T cells < P10 | 0 | 5 |
| Range of CD45RA+CD4+ T cells (cells/µl) | 820–2492 (NR 1100–3700)† | 635–1031§ (NR 1100–3700)† |
| 652–2381 (NR 430–2900)† | 390–1031¶ (NR 1100–3700)† | |
| 180–420¶ (NR 430–2900)† | ||
| Number of patients with CD45RA+CD4+ T cells < P10 | 0 | 11 |
| Range of CD8+ T cells (CTLs) (cells/µl) | 447–1442 (NR 500–1700)† | 331–484§ (NR 500–1700)† |
| 624–1198 (NR 490–2000)† | 280–1450¶ (NR 500–1700)† | |
| 305–488¶ (NR 490–2000)† | ||
| Number of patients with CD3+CD8+ T cells < P10 | 0 | 11 |
| Number of patients with CD45RA+CD4+/CD45RO+CD4+ ratios < 1 | 0 | 6 |
| Number of patients with MAPCAs | 1 | 6 |
Range of the highest single value of each individual patient of both risk-groups compared with the normal age-related normal range (NR) in period A†and period B‡ (according to data from Shearer et al.[14]);
HR patients 6 and 10 (both died in period A at the age of 4 and 12 months respectively).
hr patients 2, 4, 7, 8, 9, 13, 14, 15 and 20 (patients 2 and 14 died in period B). MAPCAs, major aortopulmonary collateral arteries; bold, abnormal.
Table 2c.
CD45RA+RO−CD4+, platelet endothelial cell adhesion molecule (PECAM)-1+ (CD31+) CD45RA+RO−CD4+ T cells and T cell receptor excision circles (TRECs)/106 PBMC in seven partial DiGeorge syndrome (pDGS) patients.
| HR | TRECs/106 MNC† | CD45RA+RO−CD4+ (cells/µl) | CD31+CD45RA+RO−CD4+ (cells/µl) | CD45RA+/CD45RO+CD4+ ratio |
|---|---|---|---|---|
| 2* | b.d.t. | 570 (NR > 1100)‡ | 553 (NR > 730)§ | < 1 |
| 4 | 60 | 429 (NR > 430)‡ | 298 (NR > 360)§ | < 1 |
| 8 | b.d.t. | 270–275 (NR > 430)‡ | 165 (NR > 360)§ | < 1 |
| 9 | 640 | 627 (NR > 1100)‡ | 572 (NR > 730)§ | < 1 |
| 13 | b.d.t. | 223–312 (NR > 430)‡ | 182 (NR > 360)§ | < 1 |
| SR | ||||
| 11 | 10 600 | 864 (NR > 430)‡ | 798 (NR > 360)§ | ≥ 1 |
| 18 | 13 000 | 846 (NR > 430)‡ | 702 (NR > 360)§ | ≥ 1 |
Deceased patient.
Normal TREC content in healthy infants and children < 10 years of age (> 10 000 TREC/106 PBMC, according to Zhang et al.[16] and Lavi et al.[17]);
> P10 of CD45RA+RO−CD4+ cells/µl (according to data from Shearer et al.[14]);
> P10 of CD31+(PECAM-1+)CD45RA+RO−CD4+ T cells in healthy infants and children between 1 and 15 years of age (n = 75); b.d.t., below detection threshold; bold, abnormal; HR, high-risk patients; PBMC, peripheral blood mononuclear cells; SR, standard-risk patients.
Analysis of TRECs and CD31+ naive CD45RA+RO−CD4+ lymphocytes
The P10-90 percentiles of naive CD31+CD45RA+RO−CD4+ T cells of 75 healthy paediatric controls are depicted in Fig. 2, whereas threshold P10 values (730 cells/µl; < 1 year of age and 320 cells/µl; < 7 years of age) are shown in Table 2c.
Fig. 2.
CD31+CD45RA+RO−CD4+ T cells in HR and SR patients. Normal percentiles (P10, P50, P90) for naive CD31+CD45RA+RO−CD4+ T cells (n = 75 healthy controls). CD31+CD45RA+RO−CD4+ T cells of two standard-risk (SR) patients with normal T cell receptor excision circle (TREC) levels (10 600/106 peripheral blood mononuclear cells, patient 11 and 13 000 copies/106 peripheral blood mononuclear cells, patient 18) (white quadrangles) and of five high-risk (HR) patients with significantly reduced (640 copies/106 MNC, patient 9 and 60 copies/106 peripheral blood mononuclear cells, patient 4) or TREC levels below the detection threshold (b.d.t.) (patients 2, 8 and 13) (black quadrangles) (for patient identification see also Table 2c).
TRECs were below the detection threshold in three patients (patients 2, 8 and 13), significantly low in two patients (patients 4 and 9) and normal in two patients (patients 11 and 18) (Table 2c) [16,17]. HR patients (patients 2, 4, 8, 9 and 13) with either abnormally low or undetectable TRECs were shown to have abnormally low numbers of both naive CD45RA+RO−CD4+ and CD31+CD4RA+RO−CD4+ lymphocytes, whereas SR patients (patients 11 and 18) with normal TRECs had normal numbers of both naive CD4 populations. Inverted CD45RA+/CD45RO+ ratios of CD4+ T cells were observed only in HR patients with very low or absent TRECs, while being normal in two SR patients with normal TREC numbers (Table 2c).
Lymphocyte proliferation tests
PHA-induced lymphocyte stimulation tests were normal in all patients, whereas SEB-induced lymphocyte stimulation tests were normal in 18 and abnormal in two patients (patients 6 and 20 of the HR subgroup). TT-induced lymphocyte stimulation tests were normal in 10 of 16 patients, whereas six remained below threshold (SR patients 3 and 5 and HR patients 2, 8, 13 and 20).
Humoral immunity
In period A, IgG concentrations were < P10 in one of 20 and > P90 in four of 20 patients. The IgA and the IgM values were < P10 in nine of 20 and in 10 of 20 patients respectively. In period B, IgG values were < P10 in none of 17 and > P90 in five of 17 patients. IgA and IgM values were < P10 in three of 17 and one of 17 respectively. Anti-TT/DT/HIB antibody titres were normal in all patients after normal childhood immunization. IgG subclasses were normal in 15 analysed patients.
Infectious and other complications
Non-lethal infectious and non-infectious complications occurred in both SR and HR groups (Table 3). At autopsy of patient 2, lymphoproliferative disease (LPD) with polyclonal T cell infiltrations of lymph nodes, liver, spleen, lungs and kidneys without skin or gut involvement was demonstrated. Epstein–Barr virus (EBV) in-situ hybridization was positive in lung tissue and lymph nodes. Secondary follicles in lymphatic tissues as well as thymus tissue were undetectable. Preceding findings were a monoclonal IgM-gammopathy, severe thrombocytopenia with giant splenomegaly, urinary cytomegalovirus (CMV) excretion and elevated EBV–DNA copies in plasma.
Table 3.
Overview of clinical outcome at last follow-up in 20 partial DiGeorge syndrome (pDGS) patients with 22q11·2 microdeletion (1995–2005).
| HR | Complications | Developmental outcome | Outcome |
|---|---|---|---|
| 2 | Recurrent bronchitis; stomatitis aphthosa, otitis, chylothorax, LPD (EBV), monoclonal gammopathy (IgM), Escherichia coli-sepsis after partial splenectomy of a giant splenomegaly | Remedial kindergarten (speech therapy) | Death at 58 months (because of atypical LPD) |
| 4 | Cholecystitis, ascites, pneumonia, sepsis, tracheitis, transient thromboctopenia | Normal kindergarten (speech therapy) | Alive/well |
| 6 | Chylothorax, cystitis, chronic granulomatous tracheitis, nephrocalcinosis, pneumonia | Severe developmental/motor delay | Death at 4 months (Stenotrophomonas maltophilia pneumonia) |
| 7 | Chylothorax | Normal school | Alive/well |
| 8 | Tracheitis, pneumonia, sepsis | Remedial kindergarten (speech therapy) | Alive, LPA stenosis |
| 9 | Recurrent bronchitis; tracheitis, pneumonia, RSV-bronchiolitis, bacterial sepsis | Mild atactic cerebral palsy, mild speech delay, remedial kindergarten | Alive (mild aortic insufficiency, chronic pneumopathy) |
| 10 | ARDS, tracheitis, bacterial sepsis (Pseudomonas), PSWI | Severe developmental/motor delay | Death at 12 months (Pseudomonas sepsis) |
| 13 | Gastroenteritis, Hashimoto thyroiditis | Normal school | Alive/well |
| 14 | Recurrent bronchitis; tracheitis, otitis media | Normal kindergarten (remedial support), moderate developmental delay | Death at 72 months (HF) |
| 15 | Enteroviral meningitis | Normal kindergarten (remedial support) | Alive (residual moderate aortic stenosis) |
| 20 | Cholangitis, pancreatitis, RSV bronchiolitis, bacterial sepsis, autoimmune neutropenia and thrombocytopenia | Severe developmental/motor delay | Death at 25 months (RSV bronchiolitis/Pseudomonas cepacia pneumonia) |
| SR | |||
| 1 | No | Normal kindergarten | Alive/well |
| 3 | Pneumonia, otitis media, laryngotracheitis | Normal school | Alive/well |
| 5 | Sepsis, ascites, cystitis, tracheitis, pyelonephritis, pneumonia (Pseudomonas) | Normal kindergarten (speech therapy) | Alive/well |
| 11 | Chylothorax, NEC, pneumonia, bacterial sepsis (Staphylococcus aureus) | Slight developmental delay, mild speech delay, normal kindergarten | Alive (severe subvalvular aortic stenosis) |
| 12 | Recurrent bronchitis; gastroenteritis, tracheitis, pneumonia, RSV-bronchiolitis, bacterial sepsis, tracheitis, PSWI | Severe developmental/motor delay | Death at 20 months (HF) |
| 16 | Ascites, chylothorax, gastroenteritis, tracheitis | Severe developmental/motoric delay | Death at 21 months (HF) |
| 17 | Recurrent bronchitis | Normal kindergarten (speech therapy) | Alive, cyanotic |
| 18 | No | Remedial kindergarten | Alive/well |
| 19 | Urinary tract infection, otitis media, RSV-bronchiolitis | Normal kindergarten (speech therapy) | Alive/well |
Bold: all deceased patients; bold and italic: patients deceased because of non-cardiac complications. Patients with autoimmunity (all within the high-risk group): patient 13 with Hashimoto thyroiditis; patient 20 with autoimmune neutropenia/thrombocytopenia; patient 2 with monoclonal gammopathy of immunoglobulin M (IgM)-type and polyclonal lymphoproliferative disease (LPD). ARDS, acute respiratory distress syndrome; EBV, Epstein–Barr virus; GER, gastroesophageal reflux; HF, heart failure; HPT, hypoparathyroidism; HR, high-risk patients; NEC, necrotizing enterocolitis; PSWI, post-sternotomy wound infection; RHF, right heart failure; RSV, respiratory syncytial virus; SR, standard-risk patients.
Outcome at last follow-up
The developmental and cardiac outcomes are depicted in Table 3. All but two patients underwent at least one surgical cardiac intervention. Seven children died at the age of 4, 14, 20, 21, 25, 58 and 72 months leading to an overall morality of 35% (seven of 20).
Four HR patients (patients 2, 6, 10 and 20) died for non-cardiac reasons: polyclonal LPD proven by autopsy; Stenotrophomonas maltophilia pneumonia; Pseudomonas pneumonia/sepsis; autoimmune neutropenia/thrombocytopenia, respiratory syncitial virus-bronchiolitis and P. cepacia pneumonia. One HR patient (patient 14) and two SR patients (patients 12 and 16) died from cardiac causes only (Table 3). Four of seven patients with major aortopulmonary collateral arteries (MAPCA), pulmonary atresia (PA) and ventricle septum defect (VSD) died from non-cardiac causes (patients 2, 6, 10 and 14). Six HR patients and one SR patient had MAPCA/PA/VSD (Table 2b).
Discussion
The overall mortality in our cohort was 35% (seven of 20) with four deaths within the first 2 years of life and one death after 72 months of age, indicating a fairly HR for lethal outcome before school age [2,18]. Reports on causes and rates of mortality in children with pDGS and microdeletion are rare and usually retrospective, presenting mixed cohorts of patients of different age with and without CHD and mainly without specific analysis of thymic activity [2,18–21]. The overall mortality in the report by Ryan et al. analysing retrospectively the largest cohort of pDGS with microdeletion so far (n = 558) was 8%, with all except one of the deaths resulting from CHD [2].
The proportion of patients with CHD within Ryan's and another large cohort (n = 100) published by Oskarsdottir ranged between 64% and 75% respectively, whereas the rate of CHD was much higher (92%) if microdeletion was diagnosed within the first year of life [18]. Because all patients of our study were newborns with symptomatic CHD, we cannot exclude a biased selection of more severely affected patients.
In a 5-year study analysing 350 children with conotruncal heart disease after heart surgery, patients with 22q11·2 microdeletion (n = 27) were shown to have a higher overall mortality rate (25·9 versus 10·7%) than patients with similar contruncal cardiac disease without deletion [19]. If patients with PA/VSD/MAPCA with and without microdeletion were analysed separately after heart surgery, patients with microdeletion and PA/VSD/MAPCA were shown to have a significantly inferior outcome compared with those with similar cardiac disease without microdeletion (six of 15 versus none of 22). The main causes for mortality were serious infectious complications related mainly to low CD4 T cells [20].
In our series, four of seven patients (patients 2, 6, 10 and 14) with MAPCA/PA/VSD died (57%). The presence of MAPCA/PA/VSD was associated with vascular bronchus compression, tracheomalacia and low numbers for both naive CD4+ and cytotoxic CD3+CD8+ T cells (Table 1). As the 10-year mortality rate of patients with microdeletion suffering from CHD without MAPCA/PA/VSD is as low as 3·7%, the presence of MAPCA/PA/VSD may indeed represent a morphological risk factor for mortality because of pulmonary complications [21]. Whether there is an association between the presence of MAPCA/PA/VSD and persistent low thymic activity is currently unknown. In our series, six HR patients and only one SR patient were shown to have MAPCA/PA/VSD (Table 2b).
During infancy, the majority of our patients were shown to have abnormally low numbers of both overall and naive CD45RA+RO−CD4+ T cells, indicating abnormal thymic migration and or thymic hypoplasia. Longitudinal immunological analysis until the age of 72 months revealed that – despite normalization of overall CD4+ T cells in numerous patients – naive CD45RA+RO−CD4+ T cells as well as CTLs remained < P10 in 50% of patients [14]. It is important to recognize that until 2003 representative normal values for naive and memory CD4+ T cells had not been available for all relevant age stages [22,23]. Gennery et al. reporting on cross-sectionally analysed pDGS patients with proven microdeletion showed that 20 of 25 had recurrent bacterial and viral infections in the presence of low CTLs, but only six of 25 had additional low overall CD4+ T cells [7]. A survey published by Sullivan et al. analysing 19 patients with microdeletion during their first year of life showed that CTLs were affected more significantly than the total CD4+ T cell compartment [13]. Reduced CTL counts as well as impaired T cell receptor diversity of CTLs were associated with serious courses of viral disease, e.g. chicken pox or CMV, in previously reported pDGS patients [13,18,24,25].
In our cohort, 11 patients were shown to have abnormally low numbers of CD45RA+RO−CD4+ as well as low cytotoxic CD3+CD8+ T cells and were therefore defined as HR patients. In this HR group, four patients (patients 2, 6, 10 and 20) died because of infectious or lymphoproliferative complications, indicating that these complications may occur preferentially in a subgroup of pDGS patients with persistent impaired thymic activity and therefore restricted T cell receptor repertoire (Tables 2b, c and 3) [22,26–29].
According to Pieliero et al., peripheral blood T cell numbers in pDGS patients are preserved by peripheral homeostatic mechanisms to compensate for low thymic output [26]. Reduced numbers of TRECs may lead to accelerated conversion of CD45RA+ to CD45RO+CD4+ T cells, reduced diverse T cell repertoire diversity and a more extensive replicative history of naive T cells [26,27].
Although the numbers of patients available for TREC analysis were low, both absent and very low TRECs were associated clearly with abnormally low numbers of both naive CD45RA+RO−CD4+ and CD31+CD45RA+RO−CD4+ T cells as well as inverted CD45RA+/RO+ ratios (Fig. 2, Table 2c) in HR patients, whereas SR patients exhibited normal numbers of TRECs and both naive CD4 subsets (Table 2c). As the presence of CD31 differentiates between two populations of naive CD45RA+RO−CD4+ T cells with different replicative history, T cell receptor diversity and TREC content [30–32], measurement of CD31+CD45RA+RO−CD4+ T cells might help detecting pDGS patients with persistent impaired thymic activity even earlier.
Pieliero et al. also hypothesized that impairment of proliferative responses to specific antigens may arise in a subgroup of DGS patients concomitant with a progressive loss of telomere length of T cells which may lead age-dependently to increased susceptibility towards infection and autoimmunity [26]. In a cross-sectional study Lavi et al., analysing 39 pDGS patients of various ages, showed that TRECs < 10 000 copies/106 PBMC correlated with abnormally low mean lymphocyte transformation tests towards antigens, e.g. Candida and Staphylococcal protein A, and to a lesser degree with naive CD4 or cytotoxic T cell counts [17].
In our experience, PHA-induced lymphocyte proliferation tests were mainly normal, confirming numerous previous reports [5,6,8,17,25]. However, SEB-induced lymphocyte proliferation tests turned out to be abnormal in two patients who finally died because of non-cardiac reasons (patients 6 and 20), while six patients had abnormal TT-induced lymphocyte proliferation tests despite vaccination (patients 2, 3, 5, 8, 13 and 20). Despite the fact that all patients produced detectable humoral anti-TT responses, two HR patients with abnormal TT-induced lymphocyte proliferation died because of infectious and autoimmune complications (patients 2 and 20; data not shown).
The causes for autoimmunity and LPD in pDGS patients are, in fact, currently unknown [7,8,18,25]. Gennery et al. reported on 10 of 30 pDGS patients with autoimmune symptoms with or without autoantibodies and showed associated low immunoglobulins or poor antibody responses after specific vaccination with protein and polysaccharide antigens [7]. All patients of our cohort with proven autoimmunity or LPD belonged to the HR group (Table 3); however, we were unable to detect relevant deficiencies of specific antibody responses to protein antigens in this group. We have no data on responses to polysaccharide vaccination.
A novel observation within our series was the development of polyclonal LPD (patient 2; Table 3) with a clinical picture different from previously published LPDs described in cDGS patients [10,11]. Secondary T cell losses after post-interventional chylothorax, removal of residual thymic tissue by repetitive cardiac surgery or multiple infections with lymphotropic viruses (Table 3) may be contributing factors to explain the development of LPD in the presence of sufficient overall T cells; however, definitive answers remain elusive. It is noteworthy that Kanaya et al. reported on one pDGS patient with microdeletion dying of haemophagocytic lymphohistiocytosis at the age of 26 months, a complication that may resemble LPD if autopsy is not performed [29].
In summary, nine of 18 of early diagnosed newborns with pDGS, CHD and proven microdeletion of our series exhibited persistent low numbers of both naive (CD31+) CD45RA+RO−CD4+ lymphocytes and CTLs until the age of 72 months. Two additional patients with similar immunological features died during infancy because of NCM. Patients with this specific immunological profile were prone to developing serious infectious, autoimmune or lymphoproliferative complications. Standard T cell enumeration of overall CD4 T cells, lymphocyte proliferation tests towards mitogens or detection of humoral immune responses failed to identify these patients in time. We, as others, believe that loss of immune surveillance because of persistent low thymic output, low TRECs and concomitant compromised T cell receptor diversity is the most likely explanation for such complications in pDGS patients, although non-immunological morphological factors may play a role [5,22,26,27,33–38].
Therefore, longitudinal monitoring of naive (CD31+) CD45RA+RO−CD4+ T cells, CTLs or TRECs will be helpful to detect these HR patients earlier [10,17,25,27,29–30,38]. Not all pDGS patients with such immunological features may essentially develop lethal infectious or autoimmune diseases; other factors, e.g. additional T cell losses because of post-interventional chylothorax or the presence of MAPCA may also contribute to NCM.
Our findings should be investigated in a prospective longitudinal study with more early diagnosed pDGS infants. This future study could prove whether or not an HR subgroup of pDGS patients has to be considered eligible for more aggressive treatment of immunodeficiency, such as thymic tissue, haematopoietic stem cell or mature T cell transplantation [11,39].
Acknowledgments
The authors would like to thank the technical staff of our immunologic laboratory for excellent technical assistance: Mrs Maja Rutishauser, Corinne Wenk, Jannie Nievergelt, Cindy Nützli and Irene Nauta.
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