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2. Rapid screening of CF mutations and polymorphisms with a reverse line blot assay
Supplement / The Netherlands Journal of Medicine 54 (1999) S3 –S84 In Nov 1998 ERCF contained data on 431 patients with FEV1 values (% predicted) separated by 2 yrs who had received Pulmozyme (P) for $ 2 yrs since enrolment (T group), and 2,125 patients with similar FEV1 records who had never received P (NT group). The mean change in FEV1 over 2 yrs was 2 0.7 (T) vs 2 2.2 (NT) (males 1 0.6 and 2 1.8, females 2 2.1 and 2 2.6 respectively). The greatest difference was in patients 6– , 13 yrs (T 5 1 1.8%, NT 5 2 1.3%), and in those denying daily cough (T 5 1 1.5%, NT 5 2 3.1%) or sputum production (T 5 1 3.3%, NT 5 2 2.8%). Patients who performed airway clearance (T 5 no change in FEV1 , NT 5 2 2.1%) differed from those who did not (T 5 2 7.2%, NT 5 2 2.3%). Patients without Pseudomonas changed 1 4% (T) and 2 1.7% (NT). Data on exacerbations (EX) from 493 patients who had 1 yr without P followed by $ 1 yr with P (T group) were compared with those who had no P throughout (NT group). In the 2nd yr the mean change in EX frequency from yr 1 was 2 0.2 (T) vs 1 0.1 (NT), i.e. 3 fewer EX / 10 treated patients / yr. Patients aged 6 to , 13 yrs appeared to benefit most from P, with a net reduction of 4 EX / 10 patients / yr. These observations are consistent with the known benefits of P. Younger patients and those with mild disease appear to benefit most.
E2.4. Influence of screening on medical status of CF patients. Data from the ERCF. J. Navarro, O. Mouterde, H.K. Harms, M.E. Hodson, C. Koch, G. Mastella, M. Rainisio, S. McKenzie on behalf of ERCF. 1684 of 13684 (12%) patients in ERCF by end 1998 were diagnosed by neonatal screening (S population). 95% CIs of means were compared between S and non-S patients in three age groups. 0 – , 6 years: S 5 744 / 3669 (20%): weight for age percentile was better (37 vs 34) and oral nutritional supplements less common (26 vs 39%) in S. Respiratory symptoms except crepitations (13 vs 18%) and clubbing (7 vs 12%) were equally prevalent. Prevalence of Ps. aeruginosa was lower (26 vs 32%), H. influenzae higher (45 vs 37%) and S. aureus similar. Fewer S patients received inhaled bronchodilators (35 vs 47%), inhaled corticosteroids (18 vs 23%) or non-oral antibiotics (28 vs 34%). Respiratory function was not analysed. 6 – , 13 years: S 5 518 / 3771 (14%): nutritional status was better (weight for age percentile 36 vs 32; 35 vs 45% received oral supplements). There was no difference in pulmonary symptoms except clubbing (33 vs 40%) and hyperinflation (24 vs 30%). Bacteriology and respiratory function were similar. 13 – , 18 years: S 5 244 / 2332 (10%): no difference in any characteristic. Conclusion: Nutritional status was better in S patients aged , 13 years, supporting recent work by Farrell. Respiratory status (based on symptoms, prevalence of Ps. aeruginosa and use of treatments) seemed to be better in S patients , 6 years old.
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ABSTRACTS I. CFTR 1. Expression of wild-type and DF508 CFTR in cells expressing different levels of Hsc70 and Hsp70 chaperones. C.M. Farinha 1,2 , P.C. Alves 2 , D. Penque 2 , M.D. Amaral 1,2 . 1 Dept. ´ ´ ˆ Quımica e Bioquımica , Faculdade de Ciencias , Universidade ´ Lisboa, Portugal, 2 Centro de Genetica Humana, Instituto Nacion´ Dr. Ricardo Jorge, Lisboa, Portugal. al Saude Polypeptides in the Hsp70 major stress protein family are known to act as molecular chaperones. Hsp70 has been described to be involved in the cellular ‘quality control’ responsible for retention of DF508-CFTR within the endoplasmic reticulum. We compared expression of Hsc70 and Hsp70 under heat-shock (438C) in non-transfected CHO cells (K1) and in CHO cells stably expressing wt-CFTR or DF508-CFTR, by immunoblot with specific antibodies. Results show that: 1) neither of these cells express Hsp70 unless exposed to stress; 2) equivalent levels of Hsc70 are present in all three types of non-stressed cells; 3) in K1 cells Hsc70 is unchanged after a 18h post-shock recovery period and Hsp70 is significantly induced; 4) in wt- and DF508 CFTR cells Hsc70 is reduced and Hsp70 is less induced over the same period and these changes occur simultaneously with CFTR destabilization of either wt- (bands B and C) or DF-CFTR (band B). This work was partially financed by PRAXIS XXI research grant P/ SAU / 55 and CMF is a recipient of a PRAXIS XXI doctoral fellowship. 2. Rapid screening of CF mutations and polymorphisms with a reverse line blot assay. Klaus R. Huber 1,3 , Rhea Nersesian 2 , 1 1 2 1 Kurt Bauer , Sabina Andra¨ , Randall Saiki . Ludwig Boltzmann Institute for Moleculargenetic Laboratory Diagnostics, Donauspital, Vienna, Austria. 2 Roche Molecular Systems, Inc., Alameda, CA, USA. In a cohort of 100 patients that were referred to our laboratory for analyses of CFTR gene mutations, a comparison between an established and a new reverse line probe assay was performed. The new assay format includes 16 mutations that are found most commonly plus polymorphism analyses at the locus 508 in exon 10 and at the poly-T tract in intron 8. In all, the new assay detected 97% of the mutations that we found in the 100 patients by methods that included reverse line blots, DGGE, and sequencing. The added feasibility to probe the intron 8 polymorphism make possible the routine analyses of patients referred from in vitro fertility clinics. Diagnosis of hereditary human disease to date requires arduous techniques and intense manual handling. Because of this, clinical laboratories must rely on test systems that screen for a panel of the most frequent mutations that cause disease in a high percentage of patients, in order to minimize the need for further elaborate protocols. Our results show the general suitability of the test kit for a rapid screening of the most common CF mutations.
E2.4. Influence of screening on medical status of CF patients. Data from the ERCF. J. Navarro, O. Mouterde, H.K. Harms, M.E. Hodson, C. Koch, G. Mastella, M. Rainisio, S. McKenzie on behalf of ERCF. 1684 of 13684 (12%) patients in ERCF by end 1998 were diagnosed by neonatal screening (S population). 95% CIs of means were compared between S and non-S patients in three age groups. 0 – , 6 years: S 5 744 / 3669 (20%): weight for age percentile was better (37 vs 34) and oral nutritional supplements less common (26 vs 39%) in S. Respiratory symptoms except crepitations (13 vs 18%) and clubbing (7 vs 12%) were equally prevalent. Prevalence of Ps. aeruginosa was lower (26 vs 32%), H. influenzae higher (45 vs 37%) and S. aureus similar. Fewer S patients received inhaled bronchodilators (35 vs 47%), inhaled corticosteroids (18 vs 23%) or non-oral antibiotics (28 vs 34%). Respiratory function was not analysed. 6 – , 13 years: S 5 518 / 3771 (14%): nutritional status was better (weight for age percentile 36 vs 32; 35 vs 45% received oral supplements). There was no difference in pulmonary symptoms except clubbing (33 vs 40%) and hyperinflation (24 vs 30%). Bacteriology and respiratory function were similar. 13 – , 18 years: S 5 244 / 2332 (10%): no difference in any characteristic. Conclusion: Nutritional status was better in S patients aged , 13 years, supporting recent work by Farrell. Respiratory status (based on symptoms, prevalence of Ps. aeruginosa and use of treatments) seemed to be better in S patients , 6 years old.
S23
ABSTRACTS I. CFTR 1. Expression of wild-type and DF508 CFTR in cells expressing different levels of Hsc70 and Hsp70 chaperones. C.M. Farinha 1,2 , P.C. Alves 2 , D. Penque 2 , M.D. Amaral 1,2 . 1 Dept. ´ ´ ˆ Quımica e Bioquımica , Faculdade de Ciencias , Universidade ´ Lisboa, Portugal, 2 Centro de Genetica Humana, Instituto Nacion´ Dr. Ricardo Jorge, Lisboa, Portugal. al Saude Polypeptides in the Hsp70 major stress protein family are known to act as molecular chaperones. Hsp70 has been described to be involved in the cellular ‘quality control’ responsible for retention of DF508-CFTR within the endoplasmic reticulum. We compared expression of Hsc70 and Hsp70 under heat-shock (438C) in non-transfected CHO cells (K1) and in CHO cells stably expressing wt-CFTR or DF508-CFTR, by immunoblot with specific antibodies. Results show that: 1) neither of these cells express Hsp70 unless exposed to stress; 2) equivalent levels of Hsc70 are present in all three types of non-stressed cells; 3) in K1 cells Hsc70 is unchanged after a 18h post-shock recovery period and Hsp70 is significantly induced; 4) in wt- and DF508 CFTR cells Hsc70 is reduced and Hsp70 is less induced over the same period and these changes occur simultaneously with CFTR destabilization of either wt- (bands B and C) or DF-CFTR (band B). This work was partially financed by PRAXIS XXI research grant P/ SAU / 55 and CMF is a recipient of a PRAXIS XXI doctoral fellowship. 2. Rapid screening of CF mutations and polymorphisms with a reverse line blot assay. Klaus R. Huber 1,3 , Rhea Nersesian 2 , 1 1 2 1 Kurt Bauer , Sabina Andra¨ , Randall Saiki . Ludwig Boltzmann Institute for Moleculargenetic Laboratory Diagnostics, Donauspital, Vienna, Austria. 2 Roche Molecular Systems, Inc., Alameda, CA, USA. In a cohort of 100 patients that were referred to our laboratory for analyses of CFTR gene mutations, a comparison between an established and a new reverse line probe assay was performed. The new assay format includes 16 mutations that are found most commonly plus polymorphism analyses at the locus 508 in exon 10 and at the poly-T tract in intron 8. In all, the new assay detected 97% of the mutations that we found in the 100 patients by methods that included reverse line blots, DGGE, and sequencing. The added feasibility to probe the intron 8 polymorphism make possible the routine analyses of patients referred from in vitro fertility clinics. Diagnosis of hereditary human disease to date requires arduous techniques and intense manual handling. Because of this, clinical laboratories must rely on test systems that screen for a panel of the most frequent mutations that cause disease in a high percentage of patients, in order to minimize the need for further elaborate protocols. Our results show the general suitability of the test kit for a rapid screening of the most common CF mutations.