INTRODUCTION

Primary immunodeficiency diseases (PIDs) are a group of diseases that present as an unusual increased susceptibility to infections, characterized by 10 distinct warning signs (1). Since Bruton's first description of a patient with agammaglobulinemia and recurrent sinopulmonary infections in 1952 (2), about 100 different types of PIDs have been recognized (1, 3). For some patients, however, little distinct presentations and lack of convenient diagnostic approaches have resulted in inappropriate treatment for several years until critical health events and irreversible sequel were precipitated (3). Recently, advanced techniques of molecular diagnosis and exploring knowledge of immunology increase in the recognition rate of PIDs, subsequently revise an update categories (4).

Epidemiological studies have revealed wide geographical and racial variations in the incidence or prevalence and distribution patterns of PIDs. Most developed countries have their own clinical pictures and molecular basis of PIDs. However in Taiwan, we currently do not have sufficient comprehension in systemic approach to patients with PIDs. Extending our investigation from a single pediatric tertiary hospital to nationwide (5), the aim of this study was to speculate the distribution, infections, treatments and molecular analysis in Taiwanese patients with PIDs on the basis of an updated eight categories (4).

PATIENTS AND METHODS

Data Collection

Since July 1985 to June 2005, patients diagnosed as PIDs from a computer database search in Chang Gung Memorial Hospital (CG) were enrolled. Secondary immunodeficiencies were excluded as a previous study (5). Reported cases (6–15) and new patients in CG or referred from National Taiwan University Hospital (NT), Mackay Memorial Hospital (MK), Veterans Hospital (Ve), and Kaohsing University Hospital (Ks) were all collected. Patients with International Classification of Disease, Ninth Revision (ICD-9) number regarding PIDs were reclassified by an update eight categories, including “predominant antibody deficiencies,” “T- and B-cell immunodeficiency,” “congenital defects of phagocyte,” “other well-defined immunodeficiency syndromes,” “disease in immune deregulation,” “defects in innate immunity,” “auto inflammatory disorders,” or “complement deficiency.”

Analysis of Candidate Genes

After informed consent obtained from patients, 10–20 ml of venous blood was sent to our laboratory within 24–72 h. Extraction of RNA and synthesis of complementary DNA were performed as described previously (5). One or two pairs of oligonucleotide primers were selected for each gene to cover the entire coding region (5, 16, 17). The mutations identified from cDNA were confirmed by sequence analysis of genomic DNA. The individual exons, including the exon-intron boundaries, were amplified using designed primers (5, 16, 17). The 22q11.2 deletion responsible for DiGeorge syndrome (DGS) was detected by fluorescence in situ hybridization (FISH) (18). Expression of candidate molecules and/or proteins was evaluated using flow cytometry or immunostaining for anti-Btk antibody (mouse IgG1, provided by T. Futatani MD, and H.D. Ochs MD, University of Washington Medical Center, Seattle, WA), CD40L, IL-2 receptor common gamma chain (IL2RG or CD132), IL-12 receptor β1 (IL-12RB1), CD11b, CD18, human leukocyte antigen (HLA) class I (HLA-A, B, C) and class II (HLA-DR, DP, DQ; all purchased from Pharmingen, San Diego, CA), and Wiskott-Aldrich Syndrome Protein (WASP) antibodies (rabbit IgG1, a gift from Q. Zhu MD, University of Washington Medical Center, Seattle, WA) (5, 16–20).

RESULTS

Distribution in a Large Cohort of Patients with PIDs

The medical records of 124 patients from 120 unrelated families were collected from 5 tertiary medical centers. Fifty-three cases were from Chang Gung Memorial Hospital, a tertiary medical center responsible for 2,355,497 live births within the most recent 20 years (21), which corresponded to an estimated incidence of 2.2 per 100,000 live births. Together with patients from other hospitals, the distribution and clinical features of this cohort were summarized in Tables I and II. “Predominant antibody deficiencies” were the most common cases in 45 patients (36.3%). “T- and B-cell immunodeficiencies,” “congenital defects of phagocyte,” and “other well-defined immunodeficiency syndromes” were almost equal (approximate 20%) and the next common. “Immune deregulation” (Chediak-Higashi syndrome) and “complement 3 deficiency” were only found in one boy each (below 1%), respectively. None had “defects in innate immunity” or “auto inflammatory disorders.” Forty-two individuals (from 39 unrelated families) had a family history of PIDs. No antenatal diagnosis or consanguinity was traced.

Table I. Distributions of a Large Cohort of 124 Patients with PIDs and Disease Patterns
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Table II. Clinical Events in 124 Patients with PIDs
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Common Presentations, Infections and Mortality

One patient with Chediak-Higashi syndrome that was reclassified into “disease of immune deregulation” by an update categories, was conventionally classified into “congenital defects of phagocyte” (Table II) in this study. The age of onset ranged from the first day of life to 24-years-old, commencing below 3 years for 69.4% of all. Recurrent sinopulmonary infections (otitis media, sinusitis and/or pneumonia) were the most common presentations in all except “congenital defects of phagocyte” in this category severe skin infections and septicemia were more common. Splenomegaly and/or hepatomegaly in patients with “other well-defined immunodeficiency syndromes” were as common as recurrent sinopulmonary infections.

Fifty-two patients (33.9%) endured 68 episodes of identified septicemia, mainly Gram-negative bacteria in 46 episodes (Pseudomonas and Salmonella infections in 15 and 12; respectively as Table III).

Table III. Identified Pathogens in 68 Episodes of Septicemia from 52 Patients with Defective Cell Types
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The causes for mortality in 23 patients were infections (n=14; four associated with unsuccessful transplantation), malignancy (n=4; one related to EBV-induced lymphoproliferative disorders), complicated congenital heart diseases (n=3), veno-occlusive disease after transplantation (n=1), and disseminated intravascular coagulopathy (n=1) as Table IV.

Table IV. Mortality in 23 Patients with PIDs
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Treatments

Regular IVIG infusions were given for patients with hypogammaglobulinemia and recurrent sinopulmonary infections (Table V) (22, 23). Short-term IVIG treatment (<4 doses) was administered in one individual with CVID and four with selective immunoglobulin deficiency (IgG2 subclass in 1, IgG2 and IgG4 subclass in 2, and IgG3 and IgG4 in 1) during the period of recurrent infections. Prophylactic antibiotics were prescribed in cases with T-cell defects, phagocyte defects and both predominant B-cell deficiencies and bronchiectasis. Granulocyte-colony stimulating factor (G-CSF) was administered to three patients with severe neutropenia and recurrent infections. Interferon-gamma (IFN-γ) treated two patients with IFN-γ-associated immunodeficiency when one had intractable salmonella infection and another had severe mycobacteria infections. IFN-γ also treated seven CGD patients to decrease the rate of severe infections, however, one of them died of Enterobacter cloacae sepsis while waiting for transplant donors. Stem-cell transplantation succeeded in seven cases (SCID [n=4], WAS [n=2], CHS [n=1]) but failed in five (SCID [n=2], WAS [n=2], LAD [n=1]) as Table VI. Engraftment succeeded in two SCID patients who received related HLA-matched bone borrow neither myeloablation nor GvHD prophylaxis, and another SCID patient received unrelated four-matched cord-blood transplantation at 5 months of age because of none matched bone marrow available from parents, siblings or volunteers, and achieved IVIG-independent immune reconstruction at 6 months post transplant.

Table V. Current Treatment in 101 Living Patients with PIDs
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Table VI. Prognosis in 12 Patients with PIDs, (6 SCID, 4 WAS, 1 LAD and 1CHS) Receiving Stem Cell Transplantation
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Molecular Analysis

Molecular analysis was studied in 33 patients from 29 unrelated families (Table VII). We directly sequenced candidate genes in 24 patients and evaluated the expression level of candidate proteins in 22 patients, respectively as Table VII. As expected, the expressional level of translated proteins from causative genes were decreased or absent.

Table VII. The Survey of Molecular Evidence in 33 Agreed Patients (from 29 Unrelated Families)
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Twenty biologic parents received the carrier detection. All were carriers except for one with de novo mutated IL2RG (Trp 74 Gly).

DISCUSSION

The study from a large cohort of Taiwanese patients with PIDs shows that predominant antibody deficiencies (B cells) are the most common, similar to the worldwide reports (1). According to an update eight categories (4), the phenotypes and characterized laboratory findings are easily recognized in patients with SCID (12 cases) and HIGM (10 cases) [classified to “T- and B-cell immunodeficiencies”], WAS (10 cases) and DGS (9 cases) [“other well-defined immunodeficiency syndromes”], CGD (9 cases) and HIES (8 cases) [“congenital defects of phagocytes”]. These PIDs could be often diagnosed by well-experienced physicians referring simple laboratory tests. Thus, such three categories were quickly reminded and became the approximately second common cases. None patient with “defects in innate immunity” (for instant, anhidrotic ectodermal dysplasia with immunodeficiency [EDA-ID]) or “auto inflammatory disorders” (for example, familial Mediterranean fever or other period fever syndromes) may in part ascribe to racial variation, but the lower incidence rate (estimated at 2.2 per 100,000 live births), compared to 2.7 per 100,000 in Singapore (24) and 8.4 per 100,000 in Sweden (25), reflects an underestimation of the burden of patients with PIDs, especially for those genetically ill-defined patients with adult-onset cases of CVID (onset age over 24 years of age), a polysaccharide deficiency but normal immunoglobulins, and, extremely critical cases that did not survive before referral to medical centers. Moreover, patients with single-gene mutation constitute approximately 40.0% in other countries (1), but as high as 60.0 % in Taiwan (74/124 patients as Table I). The ratio also implies an underestimation in those patients with ill-defined genetic basis of PIDs.

In this cohort, clinical manifestations of recurrent sinopulmonary infections and septicemia are common in patients with PIDs. Similar infectious pathogens as other series, choosing empiric antibiotics to presumed pathogens of Strep. Pneumonia (in airway infections), pseudomonas and salmonella (in sepsis) are life saving until proven other else. Catalase-producing pathogens (e.g., Staph. aureus and Aspergillus spp) usually infect patients with phagocytic defects. Opportunistic infections (e.g., PCP and cytomegalovirus) often occur in patients with T-cell defects, containing “T- and B-cell” and “other well-defined immunodeficiency syndromes” by update categories (4). Compared to worldwide 1233 patients (63.0% of 1956 patients) with successful hematopoietic stem cell transplantation (HSCT) since 1968–1997 (26), only seven patients (58.3% of 12 patients) succeeded within the most recent 20 years. Thirteen cases deceased while waiting for suitable HSCT. Encouraging, a SCID patient with de novo IL2RG mutation received unrelated four-matched umbilical cord HSCT (UCSCT). One year later, he has 82% donor chimerism in the lymphocytes and recovery of T-cell function. UCSCT is an alternative source for patients with critical status of severe PIDs, including SCID, CGD, WAS, XLP, and DGS (27–32). Compared with bone marrow, the benefits of UCSCT in earlier recovery of immune function, lower GvHD risk, and lower viral transmission rate are proposed (33), but long-term prognosis will be not yet determined.

The fundamentally conservative nature of Taiwan culture released just 33 patients (from 29 unrelated families) for molecular analysis, revealing twelve unique mutations (14 patients from 12 families) from 20 identified mutations that are not located on hot spots (website://bioinf.uta.ti). The higher percentage of novel mutations (60.0%, 12/20) reflects distinct Taiwan geography.

For developing comprehensive molecular diagnosis in Taiwanese patients with PIDs, we continue elucidating genetic basis of CVID-the most popular disease in PIDs. CVID is a heterogeneous syndrome which “masks” or overlaps disorders in patients with mutations of the Btk, CD40L, SH2D1A/DHSP/SAP or ICOS genes (16, 17, 34). We analyzed these four possible candidate genes in 12 CVID patients (3/9 F/M). Subsequently, a mutated [Asp521Val] Btk gene in two cousins and [Lys96Stop] CD40L in a boy were identified in Table VII, initially diagnosed as CVID. None mutation was found in the SH2D1A/DHSP/SAP and ICOS genes. In most recent, taking emerging concept that the BAFF/Blys signaling (B-cell-activating factor of the tumor necrosis factor family) enhances B-cell survival, CD40L (T-cell) independent antibody isotype switching, and germinal center maintenance through three receptors mainly on the surface of B cell (35–37): BAFFR (BAFF receptor), TACI (transmembrane activator and calcium modulator and cyclophilin ligand interactor) and BCMA (B-cell maturation antigen). Such observation in gene knock-out mice led to the exploration of patients with mutations of BAFFR and TACI from the Caucasian CVID cohort by Grimbacher and Geha study groups (38–40). Meanwhile, mutations of CD19, caspase-8 and caspase-9 could have the CVID phenotype (41, 42). These new causative genes are the ongoing subjects to investigate CVID patients.

In conclusion, our experience reported here shows that predominate antibody deficiencies, found in many studies more 60% (1), is only 36.6%, more likely that it is the absence of adult-onset PIDs, especially for adult-onset CVID. Ethic factors may contribute to the higher unique mutations in the isolated Formosa Island, Taiwan. This review is to raise awareness in physicians rather than pediatricians, and keep tune for exploring knowledge to identify new candidate genes in PIDs. Clinically, high index of suspicion, well-control infection, regular IVIG and optimal HSCT will rescue more PIDs patients.