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Original Article Indian Pediatrics 2003; 40:612-619 |
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Clinical Profile and Frequency of Delta F508 Mutation in Indian Children with Cystic Fibrosis |
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From the Departments of Pediatrics, Microbiology and Biostatistics, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110 029, India. Correspondence to: Dr. S. K. Kabra, Additional
Professor, Department of Pediatrics, AIIMS, New Delhi 110 029, India.
Manuscript received: November 28, 2002, Initial review completed: February 5, 2003, Revision accepted: February 14, 2003.
Cystic fibrosis (CF) is the most common life limiting recessive genetic disorder in Caucasians with an incidence of approxi-mately 1 in 2500 children born in the United Kingdom. It is less common in African Americans (1 in 15000) and in Asian Americans (1:31000)(1) . It also affects other ethnic groups such as black population with an incidence of 1 in 17,000 and the native American population with an approximate incidence of 1 in 80,000(2,3). CF was thought to be extremely rare in India. However a review of all the reports from Indian subcontinent suggests that CF is probably far more common in people of India/Indian origin than previously thought but is underdiagnosed or missed in the majority of cases(4). The precise incidence of CF among Indians is unknown. As a result of the widespread belief that CF does not occur in Indians, the disease is rarely suspected and even if it is suspected the diagnosis is not confirmed due to the poor availability of facilities for diagnosis. Published reports suggest that genetic and clinical profile of Indian children with CF may be different(5-7). There are no large studies describing clinical and genetic profile of CF in Indian children. In this communication we present experience of CF at a tertiary care hospital in north India. Subjects and Methods The study was conducted at Pediatric Chest Clinic of All India Institute of Medical Sciences, New Delhi. Children presenting with meconium ileus, recurrent/persistent pneumonia or malabsorption were subjected to sweat chloride testing by indigenously prepared and validated method(8). If sweat chloride was more than 60 mEq/L, it was repeated after a week. Sweat chloride of more than 60 mEq/L in a child on two occasions with clinical features of CF was diagnosed as a case of CF. If sweat chloride was between 40-60 mEq/L on repeated occasions with suggestive clinical presentation, they were subjected to CF mutation analysis and presence of at least one mutation was required to label the child as suffering from CF. If sweat chloride was <40 mEq/L, it was considered as normal. All children diagnosed as suffering from CF were subjected to detailed history and physical examination. All children were scored on a scale of 5-25 for their nutritional status, physical activity, pulmonary findings and radiological findings(9). A composite score indicating severity of disease was calculated by summing up all four scores. A score of >80 suggested mild disease and score of <40 suggested an advanced disease. Baseline investigations included cough swab/sputum culture and chest radiograph. Blood sugar levels, liver and renal function tests, ultrasonography of abdomen, chest CT scan, blood gas analysis and blood electrolytes were done whenever indicated. For detection of common mutation (delta F 508 mutation) 2-5 mL blood was collected in EDTA vacutainer. DNA was extracted by Phenol-chloroform method(10). Mutation was identified by amplification refractory mutation system (ARMS) technique(11) in the first 20 samples and later on, by amplification of 50 bp of DNA in the region of 3bp deletion by PCR and the product was analysed on a 12% polyacrylamide gel(12). All children were managed with vitamin supplements, enzyme replacement, chest physiotherapy and antibiotics courses. They were followed up every 4-8 weeks in the Pediatric Chest Clinic. Results From July 1995 to June 2002, a total of 3500 new cases were registered in Pediatric Chest Clinic. During this period, a total of 1200 children were subjected to sweat chloride test and 120 (3.5%) children were diagnosed as CF. Demographic Profile Place of residence: Patients came from various parts of India, particularly north India. There were 71 (59%) children from Delhi, 8 (7%) from Haryana, 8 (7%) from UP, 7 (6%) from Bihar, 7 (6%) from J & K, 5 (4%) from West Bengal, 3 (3%) from Madhya Pradesh, 2 (2%) each from Orissa, Rajasthan, Uttaranchal, 1 (1%) each from Punjab, Kerala, Tamilnadu and Maharashtra. One child was residing in Bhutan. Patients place of origin could be traced to almost all states in North India and some to south India (Table I). Table I Place of Origin of Patients’ Parents
Consanguinity was present in 18 (15%). History of definite CF in family was present in 2 (2%). History suggestive of CF was present in 41 (34%) patients. Average age at diagnosis was 54 months (95% CI 3-154), median age at diagnosis was 30 months. Average age of onset of symptoms was 10.85 months (95% CI 0.1-60 months). Median age of onset of symptoms was 3 months. Eighty (67%) were boys and 40 were girls. Clinical Profile Details of clinical profile are shown in Table II. Out of 120 patients, 118 (98%) had recurrent or persistent pneumonia at the time of presentation, 108 (90%) gave history of failure to thrive, 96 (80%) had malabsorption, and rectal prolapse was present in 16 (13%). Sixteen (13%) gave history of dehydration in past, and 10 (8%) gave history of meconium ileus. History of salt craving, salty taste on kissing and skin rashes was present in 5 patients each. Table II Clinical Characteristics of Children with Cystic Fibrosis
Physical examination revealed severe malnutrition in 50 (42%) patients (IAP classification). Other manifestations included Vitamin A deficiency (Bitot’s spots) in 10 (8%), vitamin D deficiency (rickets) in 8 (6%), clubbing in 80 (67%) and hepatomegaly in 20 (15%) patients. At the time of diagnosis, 2 girls were >12 years and 8 boys were more than 14 years of age. These girls did not have clinical evidence of secondary sex characters, 6 boys between 14-16 years had not developed pubertal changes while 2 boys 24 years and 25 years respectively had developed full sexual maturation. Nasal polyposis was present in 5 (5%) of patients. Examination of chest revealed evidence of hyperinflation in 100 (83%), kyphosis in 20 (17%), crepitations in 110 (92%), wheezing in 40 (25%) and bronchial breathing in 20 (17%) patients (Table II). Average clinical scores were 51 (95% CI 20-80). Score of >80 was present in only one patient. Forty eight (40%) patients had evidence of advanced disease with a CF score of <40. Average sweat chloride values were 91 mEq/L. Sweat test could not be performed in 4 patients. In each of these four patients two mutations were identified. Hypochloremic alkalosis was documented in 16 (13%) patients. At the time of presentation, Pseudomonas spp was cultured from respiratory secretions of 51 (42%), Staphylococcus spp in 18 (15%), Klebsiella spp in 8 (7%) and Hemophilus influenzae in 2 (2%) patients. Delta F 508 mutation could be identified in 45 (19%) chromosomes out of 240 tested. Nineteen (16%) patients were homozygous and 7 (6%) were heterozygous for the mutation. Frequency of delta F508 mutation was higher in patients originating from Pakistan. Out of total of 23 patients whose origin was traced to Pakistan, 13 (56%) patients were positive for delta F508 mutation. While the same figure for patients with CF originating from India was 13 (12%) out of 97 patients. Discussion We registered 120 children over 7 years at our hospital. This was about 3.5% of total children attending Pediatric Chest Clinic (PCC). The incidence of CF in India is not known. In the absence of proper registration/referral system, we cannot draw a conclusion about the magnitude of problem of CF in India. The incidence in migrant Indian populations in the USA has been estimated to be 1 in 40000(13); same in UK has been estimated between 1 in 10000 to 1 in 12000(5,14). In a study of 1000 cord blood tested for carrier state of delta F 508 mutation, 4 samples were found positive for the mutation, giving a carrier rate of 0.4%. From this data indirect calculation of disease frequency was 1 in 40000 with very wide confidence interval due to small sample size(15). In present study, majority (67%) of the patients were from Delhi; however, the parents originated from various states of India. From the results of present study a precise incidence of CF cannot be measured but it can be said that there were patients from almost all the states of North India. Diagnosis of CF in our patients was based on demonstration of elevated sweat chloride (>60 mEq/L) on two occasions in presence of suggestive clinical features in 116 patients and demonstration of two mutations in 4 patients (in these patients, sweat chloride test was not done). An international consensus statement also suggests similar guidelines for making a diagnosis of CF(16). High chlorides in sweat may be seen in many conditions including malnutrition, hypothyroidism, pancreatitis, Addison disease, fucosidosis etc.(9). Though in majority of our patients diagnosis of CF was based on elevated sweat chloride values, chances of false diagnosis are very remote as all of them had typical clinical features of CF. The average age at diagnosis was 54 months (median 30 months) in our patients. This is much higher than the average age of 6 months at the time of diagnosis of CF in USA, suggesting a delay in diagnosis. Even in Americas, median age of diagnosis among Indian Americans was 12 months compared with 6 months among Caucasian American children(13). It also reflects a low index of suspicion for the disease among Indians even in western countries(13). The average age of onset of symptoms was 10 months (median 3 months). A wide gap between onset of symptoms and diagnosis suggests need for awareness about the occurrence of CF in Indian children. In a study of 18 children from Chandigarh the average age at diagnosis was 4.78 years and duration of symptoms were 4.05 years, suggesting delayed diagnosis(17). An early diagnosis and intervention may improve the outcome of the patients(18). The clinical features in our patients were similar to that classically described(9,19). In our patients the average clinical scores were much lower suggesting already advanced disease at the time of diagnosis, probably related to delayed diagnosis. As a result of delayed diagnosis, there was delay in specific interventions resulting in severe malnutrition in almost 42% of the patients. Severe mal-nutrition has been seen as one of the determinant of early mortality in CF patients(20). A small proportion of our patients had salt craving, salty taste on kissing, dehydration without significant external losses and hypochloremic alkalosis. A syndrome of chronic salt depletion with alkalosis (Pseudobarrter syndrome) has been described in children with CF in high ambient temperatures(21). Whether this finding can be used as diagnostic criteria in India needs further study. Fifty per cent of our patients were already colonized with Pseudomonas at the time of diagnosis. CF patients initially get colonized with Staphylococcus and with treatment it gets replaced with Pseudomonas(9). Majority of our patients had recurrent or persistent pneumonia at time of presentation. The median lag period between onset of symptoms and diagnosis was 27 months. During this period they received multiple courses of antibiotics either as inpatients or out patients. They were selecting out resistant bacteria, resistant to commonly used antibiotics for respiratory tract infections resulting in very high colonization rate with Pseudomonas. In our patients frequency of delta F508 mutation was 19% that is much lower than that (70-80%) reported in Caucasian population. Other studies performed on small number of Indian patients or patients of Indian origin report frequency of delta F 508 between 19-44%(6,13,22). Reports from other Asian countries suggest relatively lesser proportion of patients with delta F 508 mutations(23). Till date about 1000 mutations have been described and profile of mutations vary between different geographic regions. In the present study, delta F 508 mutation was observed in 56% patients who originated from Pakistan, while figure for patients originating from India was 12%. A recent report from Pakistan describing mutations in 15 conse-cutive patients reported delta F 508 mutation in 60% of the patients(24). It is suggested that there is need for identification of prevalent mutations in different ethnic population. This may help in prenatal diagnosis of CF. Acknowledgement We would like to thank International Cystic Fibrosis (Mucoviscidosis) Association for help in establishing cystic fibrosis services at AIIMS. We also thank Ms Sheji Chacko, Ms K N Sreedevi, and Ms Josy Mathew for helping in nursing care of these patients. Contributors: SKK collected and analyzed the data, prepared manuscript and will act as guarantor for the paper, MK did genetic studies and helped in manuscript preparation, RL helped in analysis of data and preparation of manuscript, SS and MG helped in genetic studies, RM helped in statistical analysis of data, AK and GA helped in bacteriological studies, VK helped in collection of data. All authors approved the final manuscript. Competing interests: None. Funding: Partly funded by International Cystic Fibrosis (Mucoviscidosis) Association.
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