Pulmonary disease is a complication of Gaucher disease (GD), a lysosomal disorder due to the deficiency of glucocerebrosidase. Lung involvement was investigated through chest radiography, high-resolution computed tomography of the chest, pulmonary function tests (PFT), and oxygen saturation (SaO2 ) at 21% Fi O2 in 13 Italian GD patients, six homoallelic for the L444P mutation (Group A), seven with various genotypes (Group B). Echocardiography and transcutaneous oxygen tension measurement at room air and after breathing 100% oxygen were performed to exclude pulmonary hypertension and/or intrapulmonary shunts. A score index (SI) including lung involvement evaluated the severity of GD. In three Group A patients with respiratory symptoms and in an asymptomatic male interstitial involvement was demonstrated; one child died of aspiration pneumonia. Group B patients had no signs of lung damage; PFT were normal in all cases but one. SaO2 was normal in both groups. Pulmonary vascular disease was ruled out in three cases with respiratory symptoms. In Groups A and B the median SI were 22 and 13, respectively (p < 0.01). L444P homozygotes appear at major risk for developing pulmonary disease, even at earlier ages. A comprehensive evaluation of lung involvement is recommended primarily in these subjects.
Gaucher disease (GD) is a lysosomal storage disorder caused by a recessively inherited deficiency of glucocerebrosidase activity (1). The hallmark of GD is the presence in a variety of tissues of lipid-laden macrophages (Gaucher cells), the largest numbers being found in spleen, liver, bone marrow, lymph nodes, and the central nervous system.
Clinical expression of the disease is variable. A significant phenotypic variability resulting from genetic heterogeneity has been reported (2, 3). The substitution of serine for asparagine at amino acid residue 370 (the N370S mutation) is associated with type I disease (nonneuronopathic variant), whereas a substitution of proline for leucine at amino acid residue 444, especially in the homozygous state is associated with type II and type III diseases (acute and subacute neuronopathic variants, respectively). In patients homozygous for the D409H glucocerebrosidase mutation the phenotypic manifestations have been correlated with this specific and rare genotype (4).
Among clinical aspects of GD, lung involvement seems to be a complication described in all types: it was demonstrated in more than one-third of the subjects examined post mortem (5). However, serious pulmonary disease seems to occur preferably in children with a more severe course of GD (1). The goal of the present study was to investigate lung involvement in a group of GD patients with different genotypes.
We prospectively studied 13 patients with GD (median age 15 yr; range, 1 to 49.4 yr; 7 males, 6 females). The patients were born in two regions of southern Italy, Campania (ten) and Calabria (three). The diagnosis of GD had been made on the basis of clinical features and low activity of β-glucosidase in leukocytes (6).
Molecular analysis was carried out in all patients. Genomic DNA was extracted using standard methods. Mutation analysis was performed using polymerase chain reaction (PCR) amplification of appropriate fragments of genomic DNA. The primers were chosen to amplify the glucocerobrosidase gene selectively from the pseudogene. After amplification, the samples were examined for the characterization of the mutations. Mutation N370S was identified by nonisotopic single strand conformation polymorphism (SSCP) (7). Mutation L444P creates a NciI site; thus the appropriate PCR product underwent restriction enzyme digestion and was separated by electrophoresis on a 1.5% agarose gel. The rare mutations were characterized by direct sequencing combined with restriction fragmentation analysis of the samples showing an altered pattern by SSCP analysis. On the basis of the results of the genetic analysis patients were categorized into two groups: the first included six patients homoallelic for the L444P mutation (Group A, median age 9.1 yr; 5 males); the second group included the remaining seven subjects with various genotypes (Group B, median age 24.4 yr; 2 males).
A standardized pediatric questionnaire devised by the American Thoracic Society (8) and adapted to an Italian population was administered to obtain a personal and family history of respiratory disease and allergic symptoms. In all patients the existence of an atopic state was evaluated by skin-prick tests, using common allergen extracts, and by serum IgE determination.
Pulmonary studies were based on posteroanterior and lateral chest radiographs (CXR), high-resolution computed tomography (HRCT) of the lung, and pulmonary function tests (PFT). HRCT was done with a third-generation machine with 1.5 mm collimation and a 1.9-s scan time at 130 kV and 175 mA/s settings, and 10-mm intervals at full inspiration, with the patient in the supine decubitus position. The images were reconstructed using a bone algorithm for targeted fields. Results of imaging studies were analyzed by radiologists who were unaware of the patient's clinical status. Spirometry, lung volumes (by the N2 washout method), and diffusing capacity for carbon monoxide (by the single-breath method; Dl CO) were performed in cooperative patients (Model 2200; SensorMedics Inc., Yorba Linda, CA). Measurements of FVC, FEV1, FEV1/VC, FEF25–75, FEF75%, TLC, and FRC were obtained. All parameters, with the exception of FEV1/VC were expressed as percentages of the predicted values (9). Results for Dl CO, corrected for hemoglobin concentration and alveolar volume, were expressed as Krogh's constant and reported as percentages of predicted normal values (10). Pulse oximetry for the measurement of oxygen saturation (SaO2 ) at 21% Fi O2 was also performed in all subjects (OXImeter; Radiometer, Copenhagen, Denmark).
In patients with clinical respiratory symptoms suspected of having pulmonary hypertension we performed Doppler echocardiography to evaluate the systolic pulmonary artery pressure through the tricuspid regurgitation flow speed (Acuson 128 XP/5 Ultrasound System; Acuson Corp., Mountain View, CA). Finally, the measurement of transcutaneous oxygen tension (TcPo 2; MicroGas 7650, Kontron Instruments Ltd, Watford, UK) while breathing 21% Fi O2 and after administration of 100% oxygen for 15 min was obtained in subjects with respiratory symptoms in order to exclude intrapulmonary shunts.
The relative severity of GD was based on the severity score index (SI) which was a modification of the original score proposed by Zimran and colleagues (11). In addition to the age at diagnosis and to the involvement of various organs, the involvement of respiratory tract was considered as well. This was graded according to the following criteria: 0 point in the absence of lung disease, 1 point if only functional abnormalities were observed, 2 points if CXR and/or HRCT anomalies and/or pulmonary vascular abnormalities with or without functional defects were detected. A final value ⩽ 12 indicated mild GD disease, 13 ⩽ SI < 28 moderate GD disease and ⩾ 28 severe GD disease.
At the time of the study all patients but one were receiving infusion of modified human placental glucocerebrosidase (alglucerase; 120 U/kg/mo and 60 U/kg/mo in Group A and Group B, respectively).
The Mann-Whitney U test was performed to evaluate differences in the SI between Group A and Group B patients. Except for age and SI which were expressed as median, all values are presented as mean ± SD. Statistical significance was assumed at p < 0.05.
Informed consent was obtained from the patients and/or their families. The study protocol was approved by the hospital ethical committee.
Clinical data, the results of the CXR and HRCT evaluation, the score indices of the patients, and the genotypes are summarized in Table 1. Cases from 1 to 6 included Group A patients, while Group B subjects were from 7 to 13.
|Case No.||Sex/Age (yr)||Clinical Features||CXR Findings||HRCT Findings||Score Index||Genotype|
|Fatal respiratory||tial and alveolar|
|2||M||/5||Hepatosplenomegaly||Bronchial thick-||Localized interstitial||20||L444P/L444P|
|Recurrent respiratory||ening||interlobular thick-|
|Focal air trapping|
|Partial splenectomy||Reticulonodular||intralobular thick-|
|1 > A + 1|
|Dyspnea on exertion|
The HRCT scans were free of respiratory motion artifacts in all patients but three (Cases 1, 3, and 12), who were unable to cooperate by breath holding. None of the patients had signs of heart disease.
Within Group A three patients showed oculomotor apraxia (Cases 4, 5, and 6), while overt neurological signs and symptoms were not evident in the remaining three subjects. Therefore, according to a recent description of type III clinical variants (3), Cases 1, 2, and 3 could be classified as type IIIb patients and Cases 4, 5, and 6 as type IIIa patients. One patient died at the age of 19 mo of respiratory failure due to severe aspiration pneumonia (Case 1). A CXR performed at the age of 12 mo revealed diffuse interstitial and alveolar densities. Glucocerebrosidase treatment had been started 5 mo prior to the exitus. Recurrent respiratory infections were reported in two subjects (Cases 2 and 3). Three patients had never had respiratory symptoms (Cases 4, 5, and 6). HRCT and/or CXR evidence of pulmonary involvement was demonstrated in four of the six subjects. Figure 1 shows chest X-ray film and HRCT scan of Patient 4. Lung imaging studies were normal in two subjects (Cases 5 and 6).
Three patients underwent PFT. In Case 4 we observed mild reduction of FEF25–75 and FEF75% (58% and 61% of predicted, respectively), and of TLC (66% of predicted), whereas Case 5 showed only mild reduction of FVC (65% of predicted). Dl CO was normal in both subjects. PFT were normal in the third subject (Case 6).
In the six patients from Group A SaO2 at 21% Fi O2 was 95 ± 2% (range, 92 to 98%).
Six of the seven patients from Group B were asymptomatic and had no evidence of lung involvement on either CXR or HRCT (cases from 7 to 12). PFT of five cooperating subjects (cases from 7 to 11) showed that FVC was 97 ± 14%; FEV1 and FEV1/VC were 100 ± 12% and 88 ± 7%, respectively; FEF25–75 was 104 ± 16%; TLC and FRC were within normal range (95 ± 6% and 104 ± 10%, respectively); and Krogh's constant was 98 ± 5%. A 26-yr-old male (Case 13) had mild dyspnea on exertion and a history of episodic asthma; he had massive hepatomegaly and skin-prick tests were positive for Parietaria officinalis, Olea europea, and cat fur. This patient was a current, regular smoker (two cigarettes per day); he started smoking at the age of 14 yr. Enzyme replacement therapy had been started 1 yr prior to the study. Apart from mild dyspnea on exertion, he did not report respiratory symptoms at the time of the study. Pulmonary function evaluation disclosed only mild reduction of FEF25–75 and FEF75% (62% and 53% of predicted, respectively). CXR and HRCT of the chest were normal.
In the seven patients from Group B SaO2 at 21% Fi O2 was 96 ± 1% (range, 94 to 98%).
Doppler echocardiography was performed only in three of the four subjects who had had respiratory symptoms (Cases 2, 3, and 13) because another patient died of fatal aspiration pneumonia before the echocardiography could be performed (Case 1). Results showed in all cases a normal value of pulmonary artery pressure (less than 25 mm Hg). Measurement of TcPo 2 at Fi O2 21% and after administration of 100% oxygen was obtained only in one subject (Case 13) because the remaining three patients were unable to cooperate while breathing 100% oxygen. In this subject TcPo 2 was 71 mm Hg at 21% Fi O2 and increased to 500 mm Hg after 100% oxygen.
In the whole study population the median score index was 17 (range, 8 to 26). In Group A and Group B the median score indices were 22 (range, 17 to 26) and 13 (range, 8 to 17), respectively (p < 0.01). All patients but one from Group A (Case 6) had a pulmonary score > 0 because lung involvement was demonstrated at the imaging studies and/or at PFT. Among patients from Group B the pulmonary score was 0 in all subjects but one (Case 13).
Answers to the questionnaire showed that 3 of 13 patients (Cases 2, 3, and 13) had a family history of atopy (asthma, rhinitis, eczema), but only one of them (8% of the total) reported a family history of allergic asthma; the same patients (23% of the total) had had sporadic episodes of airway obstruction diagnosed as asthma or asthmatic bronchitis, but not in the year preceding the study. Atopy was excluded by skin-prick tests in all but two subjects. In addition to Case 13, Case 2 had positive skin tests to Dermatophagoides pteronyssinus and Dermatophagoides farinae. Serum IgE were normal in all subjects. Except for two subjects (Cases 7 and 13), none of the patients was a smoker at the time of the study.
Twelve of the patients are currently alive.
At the time of the study, Group A patients with primary lung involvement (Cases 1, 2, 3, and 4) had been receiving the treatment with alglucerase for a mean period of 1.9 ± 1.3 yr; the remaining two subjects without demonstrated primary lung disease (Cases 5 and 6) had started the enzymatic therapy 3 yr before the study. Within Group B all patients but one (Case 11) had been receiving the treatment with alglucerase for a mean period of 1.7 ± 0.5 yr.
In this study of patients with GD intrinsic pulmonary involvement appeared significantly more frequent in subjects homoallelic for the L444P mutation than in patients with other genotypes. The foremost feature of lung disease was the involvement of the lung interstitium, as shown on CXR and/or HRCT scans. One subject with hepatomegaly and kyphosis showed minimal functional impairment consisting of mild reduction of FVC in the absence of other signs of primary lung disease.
Gaucher patients with genotypes other than L444P/L444P did not show CXR and/or HRCT evidence of lung damage. Clinical symptoms and/or functional abnormalities were not detected in all cases but one. This subject had massive hepatomegaly and was a current smoker; he had had atopic asthma and mild dyspnea on exertion. With the exception of slight small airways functional abnormalities, no additional signs of lung parenchymal and/or vascular disease could be demonstrated in this patient.
In this study the age of the patients could be regarded as a bias factor for the presence of pulmonary disease because the two groups were not age-matched. However, in the group of homozygotes for the L444P mutation we found pulmonary signs or symptoms at a median age of 6 yr, even in subjects with no overt neurological disease. By contrast, GD patients with other genotypes did not show even minimal signs of lung involvement at a median age of 24 yr. In addition to this, the duration of the enzymatic treatment did not appear significantly different in the two groups. Finally, the patients homozygous for the L444P mutation appeared to have more severe disease, as shown by both higher severity score indices and pulmonary scores.
Clinical signs and symptoms of pulmonary disease have been reported in Gaucher patients (1). Most of the described cases with severe lung disease, characterized by recurrent pulmonary infections and progressive dyspnea culminating in fatal respiratory insufficiency, were children (12). Abnormalities on chest radiographs or CT scans have been described also in adults with type I disease (13, 14). Kerem and coworkers have recently demonstrated that type I patients with pulmonary function abnormalities have significantly more severe disease than those with normal pulmonary function (15). This study included mostly subjects with the common “mild” genotype N370S. Younger subjects with different genotypes, more severe disease, and possibly, more serious respiratory manifestations were not included in this analysis. Hence the authors could not come to the conclusion that a genotype–phenotype correlation with regard to lung disease exists in GD.
Lung involvement in GD is multifaceted, and several possible pathophysiological mechanisms account for it. Gaucher cells can fill the alveolar spaces and/or the inter- and intralobular septa, leading to air space and/or interstitial disease, respectively. Pulmonary vascular disease seems to be more common than suspected: pulmonary hypertension even in the absence of vascular plugging by Gaucher cells, and intrapulmonary shunts related to the hepatopulmonary syndrome have been reported (16, 17). Finally, hepatosplenomegaly and spinal deformities can progressively lead to small lung volumes and to changes of the pulmonary vascular bed, and secondary hypoventilation can eventually occur (18).
In this survey the functional impairment shown in three individuals from both subgroups consisted of mild obstructive and/or restrictive defects. Finally, pulmonary hypertension was ruled out in three cases with respiratory symptoms, while intrapulmonary shunts were excluded only in one of them (Case 13). However, because the remaining patients did not have either hypoxemia or additional features including finger clubbing, orthodeoxia, and platypnea, we exclude that the other patients reporting respiratory symptoms had intrapulmonary shunts.
Data from the questionnaire showed that the prevalence of a family history of asthma in GD patients is not different from the prevalence recently seen in a population of school children in central Italy (8% versus 8.3%, respectively), whereas the prevalence of asthma is higher in GD subjects than in the normal population (23% versus 15%, respectively) (19). All patients with GD and previous episodes of asthma were clinically stable at the time of the study and had no symptoms of acute respiratory tract disease. Therefore, on the basis of the results of the questionnaire, we conclude that the radiological signs of lung disease in our patients with L444P genotype are primarily due to GD.
In summary, in view of the large proportion in our study of GD patients homoallelic for the L444P mutation with demonstrated lung damage, and even though no clear evidence of a genotype–phenotype relationship can be demonstrated, we hypothesize that homozygosity for the mutation L444P is associated with a major risk of developing intrinsic pulmonary involvement. In these patients primary lung disease is also likely to occur at early ages and does not seem related to the duration of the enzymatic therapy.
We recommend that in GD a comprehensive evaluation of pulmonary disease is made as soon as possible even in the absence of clinical symptoms. It should primarily include imaging studies of the lung to diagnose air space and/or interstitial disease, and PFT to detect functional impairment. Finally, GD patients should be examined for pulmonary vascular disorders through noninvasive tests such as echocardiography to evaluate pulmonary hypertension and the administration of 100% oxygen to reveal even small amounts of intrapulmonary shunts (20).
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