Clinical, Immunological, and Molecular Analysis in a Large Cohort of Patients with X-Linked Agammaglobulinemia: An Italian Multicenter Study

Clinical Immunology Vol. 104, No. 3, September, pp. 221–230, 2002 doi:10.1006/clim.2002.5241

Clinical, Immunological, and Molecular Analysis in a Large Cohort of Patients with X-Linked Agammaglobulinemia: An Italian Multicenter Study Alessandro Plebani, 1* Annarosa Soresina, 1 Roberto Rondelli, 2 Giorgio M. Amato, 3 Chiara Azzari, 4 Fabio Cardinale, 5 Gianantonio Cazzola, 6 Rita Consolini, 7 Domenico De Mattia, 8 Grazia Dell’Erba, 9 Marzia Duse, 1 Maurilia Fiorini, 10 Silvana Martino, 11 Baldassarre Martire, 8 Massimo Masi, 2 Virginia Monafo, 12 Viviana Moschese, 13 Luigi D. Notarangelo, 1 Paola Orlandi, 13 Pietro Panei, 14 Andrea Pession, 2 Maria C. Pietrogrande, 15 Claudio Pignata, 16 Isabella Quinti, 17 Vanda Ragno, 18 Paolo Rossi, 13 Antonella Sciotto, 19 Achille Stabile, 20 and the Italian Pediatric Group for XLA-AIEOP 1 Dipartimento di Pediatria, Universita` di Brescia; 2Dipartimento di Pediatria, Universita` di Bologna; 3Dipartimento di Pediatria, Universita` di Palermo; 4Dipartimento di Pediatria, Universita` di Firenze; 5Istituto di Pediatria Clinica e Sociale, 8Dipartimento di Biomedicina dell’Eta` Evolutiva, Universita` di Bari; 6Centro Fibrosi Cistica, Ospedale di Verona; 7Dipartimento di Pediatria, Universita` di Pisa; 9Divisione di Pediatria, Ospedale “Maggiore” di Bologna; 10Istituto di Medicina Molecolare “Angelo Nocivelli,” Universita` di Brescia; 11 Dipartimento di Pediatria, Universita` di Torino; 12Dipartimento di Pediatria, Universita` di Pavia; 13Dipartimento di Pediatria, Universita` di Tor Vergata di Roma; 14Istituto Superiore di Sanita`, Roma; 15Dipartimento di Pediatria, Universita` di Milano; 16 Dipartimento di Pediatria, Universita` di Napoli; 17Istituto di Clinica Medica, 18Dipartimento di Pediatria, Universita` “La Sapienza” di Roma; 19Dipartimento di Pediatria, Universita` di Catania; 20Clinica Pediatrica Universita` Cattolica del S.Cuore, Roma

A questionnaire-based retrospective clinical and immunological survey was conducted in 73 males with a definite diagnosis of X-linked agammaglobulinemia based on BTK sequence analysis. Forty-four were sporadic and 29 familial cases. At December 2000, the patients’ ages ranged from 2 to 33 years; mean age at diagnosis and mean duration of follow-up were 3.5 and 10 years respectively. After the mid-1980s all but 2 were on intravenous immunoglobulin (IVIG) substitution therapy, with residual IgG >500 mg/dl in 94% of the patients at the time of enrollment. Respiratory infections were the most frequent manifestation both prior to diagnosis and over follow-up. Chronic lung disease (CLD) was present in 24 patients, in 15 already at diagnosis and in 9 more by 2000. The cumulative risk to present at diagnosis with CLD increased from 0.17 to 0.40 and 0.78 when the diagnosis was made at the ages of 5, 10, and 15 years respectively. For the 9 patients who developed CLD during follow-up, the duration of follow-up, rather than age at diagnosis; previous administration of intramuscular immunoglobulin; and residual IgG levels had a significant effect on the development of CLD. Chronic sinusitis was present in 35 patients (48%), in 15 already at diagnosis and in 20 by 2000. Sistemic infections such as sepsis and meningitis/meningoencephalitis decreased over * To whom correspondence and reprint requests should be addressed at Clinica Pediatrica, Universita` di Brescia, Spedali Civili, 25123 Brescia, Italy. Fax: ⫹39 30 3388099. E-mail: plebani@ master.cci.unibs.it.

follow-up, probably due to optimal protection provided by high circulating IgG levels reached with IVIG. © 2002 Elsevier Science (USA) Key Words: X-linked agammaglobulinemia; infections; intravenous immunoglobulin; BTK mutation.

INTRODUCTION

X-linked agammaglobulinemia (XLA) is a rare genetic disorder of B-cell maturation characterized by absence of mature B cells, very low serum levels of all immunolglobulin isotypes, and lack of specific antibody production. Mutations in the gene coding for a tyrosine kinase (BTK, Bruton tyrosine kinase) have been identified as responsible for XLA (1, 2) but the exact role of this kinase in B-cell development has not yet been established. After the decline of passively transferred maternal antibodies, affected boys usually present with protracted or recurrent bacterial infections to prevent which lifelong immunoglobulin replacement is indicated (3–5). Because T cells, which protect against intracellular microorganisms, develop and function normally in these patients, resistance to fungi and viruses is unimpaired with the notable exceptions of enteroviruses that may cause life-threatening meningoencephalitis (6, 7). Until now, only a few large multicenter studies (3–5, 8) have been carried out with the aim of defining the natural history of XLA. We report here clinical and

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molecular data from a multicenter retrospective survey of 73 male patients with a definitive diagnosis of XLA. MATERIALS AND METHODS

In 1994, 39 Italian centers belonging to the Immunodeficiency Working Group of the Italian Association of Pediatric Hemathology and Oncology (AIEOP) were invited to submit to the study all males being followed with a presumed diagnosis of XLA based on hypogammaglobulinemia and ⬍1% circulating B cells. A detailed individual questionnaire to be compiled on enrollment included the patient’s personal data, family pedigree, date of diagnosis, immunologic data and clinical manifestations up to enrollment, as well as information on the route and dosage of immunoglobulin replacement from diagnosis to enrollment. An annual questionnaire which included all relevant clinical features, immunological data, and dosage of immunoglobulin replacement was compiled for each year from enrollment to 2000. A senior clinician was responsible for the compilation of all questionnaires at each center. For each patient, the percentages of circulating T and B lymphocytes were evaluated at the time of enrollment by FACS analysis using anti-CD3, -CD6/-CD8, and -CD19 or -CD20 monoclonal antibodies. One hundred four male patients with a presumed diagnosis of XLA were collected and BTK mutation analysis has been performed so far in 88/104. This allowed us to make a definite diagnosis of XLA on 73 of them who are included in the present study. BTK mutation analysis was performed on genomic DNA either by sequencing the abnormal SSCP-PCR products obtained with primers flanking each of the 19 exons, including splice sites (manuscript in preparation), or by NIRCA, as previously described (9). An ABI Prism Big Dye Terminator Cycle Sequencing system was used for direct sequencing (Perkin Elinus, Chatsworth, CA). Statistical Analysis All information was stored, controlled, and analyzed by VENUS, an integrated system of software facilities running on an IBM mainframe at the North-East Italian Interuniversity Computing Center (CINECA). Cumulative risks of developing CLD or chronic sinusitis were calculated by the Kaplan–Meier method. Fisher’s exact test was used to compare differences in percentages for categorical variables, whereas Student’s t test was adopted to compare means for continuous variables. All P values are two-sided and values less than 0.05 were considered statistically significant. The SAS package (SAS Institute, Cary, NC) was used for the analysis of the data.

RESULTS

Characteristics of Patients Seventy-three male patients with a definitive diagnosis of XLA based on BTK mutation analysis and belonging to 18 of 39 centers that had been contacted were included in the study (Table 1). Family history was negative for XLA in 44 (60.3%) and positive in the remaining 29 (39.7%), who belonged to 19 families, 10 of whom had affected males in multiple generations (for a total of 15 of the patients included in the study), while 9 families had multiple affected males in the same sibship only (for a total of 14 patients). All patients had a normal percentage of CD3-positive cells. At diagnosis, 44 of 73 patients had serum IgG levels below 200 mg/dl, a value formerly considered a threshold limit for XLA (10). Of the remaining 29 patients, 26 had serum IgG levels higher than 200 mg/dl, but ⬎2 SD below the normal mean value for age, whereas 3 (Patients 21, 57, and 59) had serum IgG within the normal range. Moreover, normal IgA and IgM levels were detected in 3 (Patients 5, 51, and 53) and in 5 (Patients 27, 37, 50, 51, and 70) patients respectively. All of these patients had low serum IgG; in all of them, serum IgA and IgM decreased to very low levels during follow-up. In December 2000, the patients’ mean age was 14 years (median 12 years; range: 2–33 years). The diagnosis of agammaglobulinemia was made at a mean age of 3.5 years (median 3 years; range: 4 months–17 years), and the mean age at onset of symptoms was 2 years (median 1; range: 2 months–11 years). In order to assess the impact of awareness of the disease on the ability to establish a correct diagnosis early in life, we considered the five families in which more children were affected in the same sibship; one of them included two twins and therefore was analyzed no further. Within the four remaining families, the mean age at diagnosis for the first vs subsequent affected children was 4.7 and 1.7 years respectively. A reverse association was observed between year of birth and age at diagnosis (Fig. 1). Since the mid1980s, patients have been treated with IVIG at a dosage of 400 – 600 mg/kg for 21–28 days. In 66 of them the trough serum IgG level at the time of inclusion in the study was ⬎500 mg/dl, and in the remaining 4 ranged from 400 to 500 mg/dl. The mean duration of IVIG replacement therapy was 9 years (median 6 years; range: 4 –23 years), and the cumulated years of IVIG therapy were 585. Thirteen patients (18%) (Patients 26, 35, 36, 51, 64, 4, 29, 13, 65, 44, 46, 47, and 14) had initially received intramuscular or subcutaneous replacement for periods of time ranging from 1 to 15 years (mean 3.85 years). Three patients (Patients 8, 21, and 72), with a current age of 16, 13, and 4 years respectively, who

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TABLE 1 Characteristics of the Patients at Diagnosis and Duration of IVIG Replacement Therapy

Patient

Age at diagnosis (months)

1 2 3

72 4 28

4

41

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57

31 7 93 49 12 94 30 30 27 88 29 32 21 11 60 24 138 37 51 16 55 33 18 17 182 21 132 38 98 33 67 151 31 50 167 8 25 215 6 6 7 28 17 109 98 10 22 57 54 74 39 157 53

Family history 0 0 Brother of 4, nephew of 36, cousin of 45 Brother of 3, nephew of 36, cousin of 45 ⫹ 0 ⫹ 0 0 0 0 Nephew of 68 0 0 0 0 0 ⫹ ⫹ 0 0 0 Brother of 24 Brother of 23 ⫹ 0 Nephew of 47 0 0 0 0 0 ⫹ 0 0 Uncle of 3 and 4 0 0 0 0 0 0 Twin of 44 Twin of 43 Cousin of 3 and 4 0 Uncle of 27 0 0 0 Brother of 52 Brother of 51 0 0 ⫹ ⫹ 0

IgG (mg/dl)

IgA (mg/dl)

IgM (mg/dl)

B cells (%)

BTK gene mutation

Duration IVIG therapy (mos)

75 161 360

7 0 0

46 10 0

0 1 1

R520X K19X G594E

35 186 203

330

0

0

1

G594E

189

32 87 196 120 5 180 179 179 6 370 15 155 202 20 255 47 824 7 525 210 106 225 66 7 327 80 190 108 100 22 14 160 362 12 162 50 202 285 280 240 50 57 235 250 523 257 385 355 219 64 25 8 891

50 6 36 0 ⬍5 3 7 6 1 0 16 8 2 0 0 7 1 5 4 7 6 ⬍5 7 1 1 16 0 ⬍6 ⬍5 0 3 0 23 ⬍6 ⬍6 3 6 ⬍7 0.6 0.6 12 2 0 0 21 7 54 29 46 2 0 6 ⬍6

17 5 26 5 ⬍5 15 28 22 18 39 14 26 22 10 0 7 35 41 8 16 18 7 56 7 41 15 19 38 ⬍5 0 1 9 73 11 27 8 25 ⬍6 0.9 0.9 35 11 0 0 28 55 62 20 4 9 0 4 23

0 0 0 0 0 0 0 0 0 2 0 2 2 0 1 0 0 0 0.6 0.2 0 0.3 0 ⬍1 1 0 1 1 0 0.8 0 1 0 ⬍1 0.5 1 0 0 0 0 0 1 0 0 ⬍1 0 2 1 0 0 0.5 0 1

Splice donor defect; IVS17 R615S G302R G594R R525P R28H R641C Y344X F493X Y112H R255X R255X Q260X G594R R288W R255X G541D V568delT R28H R28H G302delGGA R525P C506Y T33P R288Q R520Q Q293X L175fsX193 Splice donor defect; IVS8 G54fsX56 F635insTTTTAG G594E Q363fsX398 R255X Splice donor defect; IVS14 E589K E90fsX120 R28C L512P L512P G594E R490delGCGC C506Y K300fsH325 Y39S L498V R288W R288W Splice acceptor defect; IVS16 C527delTG L32W S247fsX276 V113F

38 63 250 22 175 52 62 148 289 189 149 168 162 32 79 27 71 74 93 86 66 59 41 154 199 125 285 225 140 190 8 61 135 150 46 118 58 58 147 108 214 178 30 60 77 76 157 211 202 22 67

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TABLE 1—Continued

Patient

Age at diagnosis (months)

58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73

39 14 49 17 39 20 45 155 18 10 64 47 13 79 44 18

Family history 0 0 0 ⫹ ⫹ 0 0 ⫹ 0 0 Uncle of 12 Brother Brother Brother Brother

of of of of

0 71 70 73 72

IgG (mg/dl)

IgA (mg/dl)

IgM (mg/dl)

B cells (%)

102 679 163 349 334 50 59 307 63 10 20 41 224 118 251 86

⬍6 ⬍6 13 9 ⬍6 ⬍6 0 10 7 ⬍6 0 0 29 2 21 26

6 10 14 27 26 13 0 0 12 12 5 24 50 24 7 23

1 1 ⬍1 1 1 1 1 0 1 0 0 0 0.6 0.5 2 1

refused replacement therapy have so far remained in apparently good health with scarse use of antibiotics; in 1996, a 23-year-old patient (Patient 31) with chronic lung disease from diagnosis refused further replacement therapy. BTK Mutations Different types of BTK gene abnormalities (Table 1) were detected in the 63 families: 55.6% were missense mutations, 14.3% premature stop codons, 15.9% small deletions, 6.3% small insertions, and 7.9% were splice site mutations (manuscript in preparation). Clinical Manifestations before Diagnosis The frequency and type of infections reported up to the time of diagnosis are shown in Table 2. Respiratory tract infections (RTIs), by far the most frequent finding, were recorded in 68.5% (50/73) of the patients; in 11 of them the infections were limited to the upper respiratory tract, while in 39 they involved both upper and lower respiratory tract (URTI/LRTI). The mean age at diagnosis of the patients belonging to the former group was significantly lower (P ⫽ 0.03) than that of patients belonging to the latter (2.38 ⫾ 1.16 years vs 5.03 ⫾ 4.39 years). At diagnosis, 15 of 39 (38.5%) patients with LRTI had CLD (presence of recurrent or chronic symptoms of bronchitis associated with abnormal chest or CT scan radiographs such as peribronchial thickening, segmental atelectasis, and bronchiectasis). The mean age at diagnosis of these patients was higher, although not significantly different, than that of the 24 patients without CLD [7.8 years (range: 1.7–18 years) vs 4.8

BTK gene mutation R133fsX175 L512Q Splice donor defect; IVS15 R28H G612X K185fsX193 M509V R288Q S578Y F583fsX586 Y344X C154G R544G R544G W124C W124C

Duration IVIG therapy (mos) 202 176 214 42 36 73 163 192 65 30 252 23 42 45 20

years (range: 1.2–15 years) P ⫽ 0.06]. Statistical analysis showed that the risk of developing CLD increased with age at diagnosis; namely the cumulative risk of CLD increased from 0.17 to 0.40 and to 0.78 when the diagnosis was made at the ages of 5, 10, and 15 years, respectively (Fig. 2A). The contribution of the delayed diagnosis to the development of CLD is more evident when we consider the patients born before and after 1980. Among the 19 patients with LRTI born before 1980, 10 had CLD at diagnosis, whereas this complication was present in only 5 of 20 patients with LRTI born after 1980 (P ⫽ 0.001). Within the 19 patients born before 1980, the mean age at diagnosis was higher in the 10 with CLD at diagnosis as compared to that of the remaining 9 without CLD [mean age 8.5 years (range: 3–17 years) vs mean age 4.0 (range: 1– 8 years), respectively]. Likewise within the 20 patients born after 1980 and suffering from LRTI, the 5 with CLD at diagnosis had a higher mean age at diagnosis as compared to the remaining 15 without CLD [mean age 5.5 years (range: 2–13 years) vs mean age 3.4 years (range: 1.3–11 years)]. The year 1980 was chosen to separate the two populations based on the fact that intravenous immunoglobulins were introduced in Italy around that year. Chronic sinusitis diagnosed on clinical grounds (presence of two of the three major signs: rhinorrea, postnasal drip, and cough for at least 3 months) or on CT scan was present in 15 of 73 patients, 7 of whom also had CLD. The mean age at diagnosis was significantly higher in the group of patients with as compared to the group without chronic sinusitis [mean age 7 years (range: 10 months–18 years) vs 3.5 years (range: 4 months–15 years) P ⫽ 0.001].

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FIG. 1. Relationship between year of birth and age at diagnosis of XLA.

The other clinical manifestations reported prior to diagnosis were mostly of an infectious nature and often found in patients who also had RTI. Skin infections (boils or impetigo) were reported in 19 patients (27%). Three patients had suppurative superficial lymphadenitis (secondary to impetigo in one), and 2 had deep abscesses (1 hepatic and 1 perirectal), which were complications of sepsis and gastroenteritis, respectively. Multiple episodes of acute diarrhea occurred in 9 patients (13%). Two of them had chronic diarrhea; their stool cultures were repeteadly negative and histologic data of intestinal biopsies were not available. Severe infections, sepsis, and meningitis were reported prior to diagnosis in 4 and 3 patients, respectively. Sepsis developed in infancy in 3 of 4 cases, Pseudomonas was isolated in 2 of 4 cases and Streptococcus pneumoniae in 1 of 4. Neisseria meningitidis was isolated from the TABLE 2 Clinical Features up to Diagnosis Presenting manifestations

N patients (%)

Respiratory tract infections Upper tract Upper and lower tract Cutaneous infections Gastrointestinal infections Arthritis Sepsis Meningitis Lymphadenitis Abscesses Vaccine-associated paralytic polio Neutropenia Urinary tract infections

50 (68.5%) 11 (15%) 39 (53%) 19 (27%) 9 (13%) 7 (10%) 4 (6%) 3 (4%) 3 (4%) 2 (3%) 2 (3%) 1 (1%) 1 (1%)

FIG. 2. Cumulative risk of developing CLD in relation to age at diagnosis (A) and to duration of follow-up (B).

CSF of 1 of 3 patients with meningitis. A diagnosis of vaccine-associated paralytic poliomyelitis (VAPP) was made in 2 patients who had received the first three doses of oral polio vaccine; in both cases, VAPP developed after the third dose of vaccine. Arthritis was the presenting symptom of XLA in 7 patients; in all cases, it resolved with immunoglobulin substitution therapy. Growth retardation was present in 6 patients, all of whom (including the two with chronic diarrhea) had LRTI. One patient (Patient 70) was asymptomatic and the diagnosis of XLA was made at the age of 13 months because his brother was known to be affected by XLA. Clinical Manifestations during Follow-up Mean duration of follow-up at December 2000 was 10 years (median 7 years; range: 1–29 years). Infections

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remained the most relevant clinical feature even after IVIG replacement was started. Detailed data on the annual incidence of different types of infections requiring hospitalization are reported in Table 3. CLD already present at diagnosis in 15 patients developed in 9 more by 2000. Thirty-seven episodes of severe pneumonia requiring hospitalization were recorded in 15 patients; 26 (70%) episodes occurred in 7 of the 24 patients with CLD and the remaining 11 episodes in 8 of the remaining 49 patients without CLD up to 2000. Of the 9 patients who developed CLD during follow-up, 2 were hospitalized for severe pneumonia, and 2 others had one or more episodes of pneumonia which did not require hospitalization, whereas the remaining 5 suffered from recurrent bronchitis with no overt episodes of pneumonia. The mean age at diagnosis in the 9 patients who developed CLD during follow-up was not significantly different from that of the 49 remaining patients who did not develop CLD up to 2000 [2.6 ⫾ 2.3 years (range: 4 months–7 years) vs 2.8 ⫾ 2.2 years (range: 6 months– 11.5 years)]. However, mean follow-up time of the former (actual mean age of 17 years) was significantly longer (16.9 ⫾ 8.2 years; range: 2.5–36 years; median 15 years) than that of the latter (7.2 ⫾ 5.2 years; range: 8 months–21 years; median 5 years) (P ⬍ 0.0001), who had an actual mean age of 10 years. Statistical analysis evaluating the contribution of four variables (age at diagnosis, previous administration of intramuscular immunoglobulins, serum IgG levels, and duration of follow up) to the development of CLD showed that duration of follow up was the only variable with a significant effect on the development of CLD. The probability of developing CLD after 25 years of follow-up reached 90%. The relationship between CLD development and the duration of follow-up is shown in Fig. 2B. Chronic sinusitis diagnosed on clinical grounds and confirmed by CT scan, when available, developed during follow-up in 20 patients. Again, mean duration of follow-up in this group of patients was significantly higher (P ⫽ 0.008) than that of 38 patients without chronic sinusitis up to 2000 [12.6 ⫾ 5.7 years (range: 1–26 years) vs 6.5 ⫾ 6.2 years (range 1–24 years)]. The mean age of patients with chronic sinusitis was 13.7 years (range: 4.6 –28 years), whereas it was 9.6 years (range: 1.5–32 years) for XLA patients without chronic sinusitis. Ten of the 20 patients who developed chronic sinusitis during follow-up had CLD as well, which was present from diagnosis in 4 and with onset by 2000 in the remaining 6. Statistical analysis comparing the 20 patients who developed chronic sinusitis during follow-up with the 38 patients without this complication up to 2000 showed that duration of follow-up (but not age at diagnosis, previous administration of intramuscular immunoglobulins, or residual serum IgG levels)

TABLE 3 Infections Requiring Hospitalization during IVIG Therapy

Pneumonia Gastrointestinal infections Septicemia Encephalitis Osteomyelitis Other infections

N infections

N patients

Annual infection incidence a

37 3 1 1 1 8

15 3 1 1 1 6

0.06 0.005 0.002 0.002 0.002 0.014

a Number of infections/number years accumulated by 73 patients during a 585 accumulated years of IVIG therapy.

had a significant effect on the development of this complication (data not shown). Multiple episodes of gastroenteritis were observed in 14 patients, including the 2 with chronic diarrhea at diagnosis; however, only three episodes required hospitalization. Results of 4 positive stool cultures from 4 patients yielded Salmonella in 2, Campylobacter in 1, and Giardia in the remaining patient. During follow-up body weight remained ⬍3rd percentile in only 2, both with chronic diarrhea. A single episode each of sepsis (Patient 30); meningoencephalitis (Patient 3), which responded to high-dose intravenous immunoglobulins; and osteomyelitis (Patient 59) were recorded in three patients, all on intravenous replacement. Notably, the annual incidence of sepsis and meningoencephalitis was lower during substitution therapy than before (0.002 vs 0.01, P ⬍ 0.05; 0.002 vs 0.012, P ⬍ 0.1 per patient per year, respectively). Other acute infectious episodes requiring hospitalization were recorded in 6 patients and included pyodermitis, otitis with otorrhea, mononucleosis, myositis, bacterial orchitis, and febrile bronchitis. Recurrent bacterial conjuntivitis was reported in six patients (8%). Results of eight positive cultures from four patients were available: Haemophilus influenzae was isolated from all four patients; Group A beta hemolytic streptococcus and Branhamella catarrhalis were each isolated from two patients. A severe idiopathic cerebral atrophy was observed in one patient and transient arthritis was recorded in four patients. One patient died from acquired myocardiopathy of unknown origin at the age of 19 years. Positivity for HCV mRNA was detected in 1 of 73 sera tested. This patient had been on immunoglobulin replacement (intramuscular then intravenous) for many years and had also received several plasma transfusions. DISCUSSION

This is the first extensive survey of XLA carried out in Italy with the aim of gathering quantitative data on

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the clinical presentation and long-term complications associated with this disease. To this purpose, a detailed individual questionnaire to be compiled on enrollment in 1994, and each year from enrollment to 2000 for male patients with a presumed diagnosis of XLA based on hypogammaglobulinemia and ⬍1% circulating B cells, was obtained from 18 of the 39 centers belonging to the Immunodeficiency Working Group of AIEOP. One hundred four patients with a presumed diagnosis of XLA were enrolled and BTK gene sequence analysis performed in 88 of 104 patients allowed us to make a definite diagnosis of XLA in 73 patients, who are included in the present study. Three of the 73 patients had normal serum IgG levels at diagnosis (atypical form of XLA). BTK sequence analysis in these 3 patients showed glycine-to-asparticacid substitution at residue 541, valine-to-phenylalanine substitution at residue 113, and leucine-to-glutamine substitution at residue 512, respectively; none of these mutations were found in any of the other 70 patients with low levels of all 3 immunoglobulin isotypes. In addition these mutations were not reported in any of the three recent large surveys of XLA patients (8, 11, 12). This suggests that these missense mutations may predispose to a milder immunological phenotype. However, it is well known that other background genetic or epigenetic factors may influence the immunological phenotype, as the same mutation may lead to different phenotypic expression of the disease, ranging from panhypogammaglobulinemia to normal levels of one or more isotypes (13–19). In any case, in most of the “atypical” cases reported, circulating B cells were less than 1%, suggesting that the percentage of circulating B cells, rather than the extent of the immunoglobulin defect, is a better indicator of XLA (15, 17, 19). On the other hand, an immunological phenotype compatible with selective IgA deficiency has been reported in one patient with normal number of circulating B cells who shared the same BTK mutation with his brother, who was affected by the classical XLA phenotype (20). Moreover, recently one patient with normal immunoglobulin levels and a selective antipolysaccharide antibody deficiency has been diagnosed as affected with XLA on the basis of BTK sequence analysis (21). As a whole, our data confirm that the immunological spectrum of XLA is broader than originally thought. This has led in 1999 the joint ESID (European Society for Immune Deficiencies)/PAGID (Pan-American Group for Immune Deficiencies) Committee to refine the diagnostic criteria for XLA (22). RTIs were the most common clinical manifestation recorded prior to diagnosis in our series of patients. At diagnosis, isolated URTIs were recorded in 11 patients (15%), whereas URTIs and LRTIs were observed in 39 (53%). Fifteen patients (38.5%) of the latter group had already developed CLD at diagnosis. The mean age at

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diagnosis of these 15 patients was higher than that of the 24 patients without CLD at diagnosis (7.8 years vs 4.8 years). Likewise, the risk to develop chronic sinusitis was dependent on the age at diagnosis, since the mean age at diagnosis of the 15 patients with this complication was significantly higher than that of the remaining 57 without sinusitis. Almost 50% of the patients with chronic sinusitis has CLD as well, suggesting that persistent postnasal drip may increase the risk to develop LRTI. These findings are in agreement with others (8, 23– 25) and emphasize the crucial importance of early diagnosis to permit prompt introduction of adequate immunoglobulin replacement in order to prevent or decrease severity of pulmonary infections, which remain a main cause of death in XLA. However, a clear trend toward earlier diagnosis is apparent both from our data and from those of others (3). This is very likely due to the widespread assessement of immunoglobulin serum levels in recent years as well as to the pediatricians’ increasing awareness of this primary immunodeficiency, first described in 1952 (26). Respiratory tract infections remained the most prominent clinical problem observed also during follow-up despite immunoglobulin substitution treatment. Of particular significance, chronic lung disease is a major complication of XLA. In our series, CLD was already present at diagnosis in 15 of 73 patients and developed during follow-up in 9 more. Interestingly, the subgroup of patients who developed CLD during follow-up did not differ for age at diagnosis or trough immunoglobulin serum levels from the subgroup who did not develop CLD. However, the duration of follow-up was significantly longer in the former. The overall probability of developing CLD reached about 80% after 17 years of follow-up. Different explanations may be offered to justify the role of prolonged follow-up as a risk factor for CLD. First, patients with a longer follow-up are more likely to have received inappropriate immunoglobulin substitution therapy (such as intramuscular treatment, low-dose IVIG, or plasma infusions) early during treatment. While the groups who did and did not develop CLD during follow-up did not differ in the proportion of patients who received intramuscular injections, we did not evaluate possible differences in the number of doses of im immunoglobulin received, nor in the total dose administered. Alternatively, it is possible that prolonged follow-up is associated with a higher risk of developing CLD because of inability of immunoglobulin substitution to reach the mucosal surfaces and thus provide full protection. Finally, it is possible that presently unknown modifier genes may also be involved. Whatever the mechanism, these results are partly in keeping with those recently reported by Kainulainen et al. (27) and by Quartier et al. (8) showing that progression of pulmonary abnor-

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malities may occur in hypogammaglobulinemic patients despite appropriate immunoglobulin replacement therapy. Similar considerations apply to the development of chronic sinusitis which is also associated with a long duration of follow-up. On the whole these data suggest that, despite early diagnosis and appropriate immunoglobulin replacement, CLD remains the most severe long-term complication in XLA patients. In almost all of our patients, trough serum IgG levels ranged from 500 to 800 mg/dl when determined on serum samples obtained in 1994, and all of the patients had been on IVIG replacement since the mid-1980s. Residual IgG levels of this order have generally been considered to be protective (28). The recent finding (8) of a significantly lower incidence of serious bacterial infections with residual serum IgG ⬎800 mg/dl as compared to 500 mg/dl suggest that a threshold of 500 mg/dl may be insufficient, in some patients, to provide adequate protection from pulmonary infections. Thus, at least in patients with recurrent pulmonary bacterial infections, prevention of lung complications should be attempted by increasing the dosage of IVIG to attain serum IgG levels ⬎800 mg/dl and/or by long-term antibiotic prophylaxis together with chest physiotherapy. However, although highdose IVIG have been well documented to decrease the frequency of bacterial infections (8, 23–25), it is not yet clear whether residual serum IgG higher than those currently considered protective (⬎500 mg/dl) will prevent mucosal infections, in particular recurrent bronchitis and pneumonia. Chronic sinusitis is another common long-term complication in XLA patients. Its treatment is still problematic. Medical treatment, based on systemic antibiotics combined with topical decongestants and steroids, is generally unsatisfactory. Moreover, surgery of the sinuses can at best provide only temporary improvement (8, 29). Treatment with high-dose IVIG (8, 23, 29) would not be expected to contribute significantly to the prevention of sinus infections, not only because of their scarse availability at mucosal surfaces but also because protection at mucosal surfaces is normally provided by secretory IgA and IgM antibodies, which are also lacking in XLA. As the usually recommended treatment of chronic sinusitis has proven unsatisfactory, a polydisciplinary approach is needed to define an optimal regimen treatment. Joint involvement was mild and less frequent in our patients than in those reported in two other large surveys (3, 4). This might be attributed to the higher residual IgG levels (⬎500 mg/dl) reached with IVIG in our study. In fact, a recent study has demonstrated that recurrence of arthritis is effectively prevented by high serum IgG levels (8). Sepsis and meningitis/meningoencephalitis decreased in frequency in our patients during follow-up. Further-

more, both before and after immunoglobulin replacement the frequency of these complications was much lower than that reported in previous studies (3, 4) in which replacement was mainly by intramuscular route, but was similar to what reported in a recent study in which IVIG were used (8). Again, higher residual IgG levels, impossible to reach by intramuscular route, explain the lower frequency of systemic complications observed more recently by us and others (8). The estimated risk of VAPP in XLA, based on its occurrence in 2 of 69 patients who received three doses of oral polio vaccine, is as high as 1 case/100 doses and thus much higher than in normal children (1 case/ 2.400.000 doses) (30). Reduction, though not elimination, of this risk is to be expected in Italy as a consequence of the recent change in the national vaccination policy that prescribes administration of inactivated polio vaccine for the first two doses. Nevertheless, until the risk of VAPP is eliminated, a detailed family history aimed at ruling out XLA, must be obtained in order to minimize the occurrence of this serious complication. In our survey, no patient developed malignancies. The real incidence of malignancies in XLA is still undefined. The frequencies reported in two earlier large surveys (3, 4) were 2 of 96 and 0 of 44, respectively, while in another study 3 of 52 adults with XLA developed colorectal cancer, a prevalence 30-fold greater than expected (31). As these studies were all published prior to the description of BTK mutations in XLA, it is possible that they had included cases of common variable immunodeficiency, which, compared to XLA, is associated with a greater risk of malignancy. Furthermore, the differential diagnosis between these conditions is sometimes difficult and was even more so before genetic diagnosis of XLA became possible (32, 33). No malignancies have been reported in a recent retrospective survey of 31 agammaglobulinemic patients, with a definitive diagnosis of XLA in 27 (8). Screening for HCV RNA was positive in 1 of 73 patients, a frequency much lower than that of 3 of 7 XLA patients recently reported (34). It was not possible to trace back the source of infection in our patient since the positivity has been documented only long after he had started immunoglobulin replacement and had received plasma transfusion; in the cited study infection was due to contaminated lots of immunoglobulins in 2 patients and to plasma transfusion in the other one. The exclusion of anti-HCV-positive donors from the plasma pools used to prepare IVIG toghether with the incorporation of additional viral-inactivation steps and routine screening of all batches by PCR should further reduce the risk of viral transmission through immunoglobulins. The mortality rate in the present study is approximately similar to that reported in a recent survey (1.4% vs 3.2%) (8) and much lower than that reported

X-LINKED AGAMMAGLOBULINEMIA

in the two earlier large surveys (3, 4) that showed a mortality rate of 17% (38% of deaths were due to CLD) and 25% (50% of deaths were due to disseminated viral infections). This could well be due to the presence of a higher proportion of older patients and patients with more severe complications in both earlier studies. The difference in mortality rates between our and the two earlier studies (3, 4) is certainly in part attributable to a positive bias in the selection of our patients: during patient enrollment only living patients were included in the study; thus our mortality rate refers only to a 6-year period (1994 –2000). Further follow-up of these patients and prospective analysis of the clinical course of newly diagnosed XLA patients should provide information useful for improving prophylactic measures and reducing the frequency of major complications, thus permitting a better quality of life and prolonged survival.

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14. ACKNOWLEDGMENTS This study was partially supported by grants from Immune Deficiency Foundation; Telethon, Grant E668 to L.D.N.; Murst Cofinanziamento 1999 to A.P.; European Union (Grant QLG1-CT-200101536 to A.P.); Associazione Immunodeficienze Primitive (AIP); and Societa` Italiana di Allergologia e Immunologia Pediatrica (SIAIP). We thank all the patients and families for their generous cooperation in this study.

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