Hum Genet (1992) 90 : 474
h u m a n .. genet,cs 9 Springer-Verlag 1992
A rare D N A variant in exon 15 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene M. Chill6n, A. Palacio, V. Nunes, X. Estivili Molecular Genetics Department, Cancer Research Institute (IRO), Duran Reynals Hospital, L'Hospitalet de Llobregat. Campus Bellvitge, E-08907 Barcelona, Spain Received: 13 April 1992 / Revised: 6 July 1992
Abstract. W e d e s c r i b e a rare s i n g l e - s t r a n d c o n f o r m a t i o n p o l y m o r p h i s m that occurs at n u c l e o t i d e 3030 ( G o r A ) in exon 15 of the cystic fibrosis t r a n s m e m b r a n e c o n d u c t a n c e r e g u l a t o r gene.
Source~description. W e have amplified a 485-bp f r a g m e n t c o n t a i n i n g exon 15 of the cystic fibrosis t r a n s m e m b r a n e c o n d u c t a n c e r e g u l a t o r ( C F T R ) g e n e , using p r i m e r s 15i-5 (5' G T G C A T G C T C T T C T A A T G C A 3') a n d 15i-3 (5' AAGGCACATGCCTCTGTGCA 3 ' ) , which h a v e previously b e e n d e s c r i b e d ( Z i e l e n s k y et al. 1991). Variant. A n a b n o r m a l s i n g l e - s t r a n d c o n f o r m a t i o n polym o r p h i s m (SSCP) b a n d p a t t e r n of exon 15 was o b s e r v e d in two individuals o u t of 70 u n r e l a t e d C a u c a s i a n s analysed (Fig. I a). T h e v a r i a n t consists o f a G to A substitution at n u c l e o t i d e 3030 of the C F T R gene. Chromosomal localization. T h e C F T R gene m a p s to c h r o m o s o m e 7 (7q31) ( R i o r d a n et al. 1989). Other comments. T h e c o n d i t i o n s of the p o l y m e r a s e chain r e a c t i o n ( P C R ) w e r e : d e n a t u r a t i o n at 96~ for 30 s, a n n e a l i n g at 56~ for 30 s a n d e x t e n s i o n at 74~ for 4 0 s in 35 cycles. T h e r e a c t i o n mix c o n t a i n e d 0.1 lal ad C T P (32p 3000 C i / m m o l ) to label the f r a g m e n t s . T h e P C R p r o d u c t s w e r e d i g e s t e d with the DdeI restriction e n z y m e . S S C P e l e c t r o p h o r e s i s was p e r f o r m e d at r o o m t e m p e r a t u r e with 6% a c r y l a m i d e and 10% glycerol, at 1 0 W for 2 4 h . O n l y o n e individual s h o w e d an u n u s u a l p a t t e r n . W e r e - a m p l i f i e d the s a m p l e with the s a m e P C R c o n d i t i o n s and the D N A p r o d u c t was s e q u e n c e d a u t o m a t i c a l l y ( A B I 373A) using f l u o r e s c e n t t e r m i n a t o r s . T h e c h a n g e was a t t r i b u t a b l e to a G to A substitution in exon 15 (Fig. l b ) ; this substitution does not m o d i f y the a m i n o acid t h r e o n i n e , at p o s i t i o n 966 of the C F T R p r o Correspondence to: M. Chilldn
Fig. 1. a SSCP analysis of the exon 15 of the CFTR gene of 4 unrelated CF individuals. The extra band in the pattern corresponds to the 3030 (G or A) polymorphism, b G to A substitution in exon 15, visualised by automatic sequencing: this substitution does not modify the amino acid threonine at position 966 of the CFTR protein. A comparison between a normal control (3030 G) and the heterozygous individual who has a shared signal (N) with respect to the normal signal (G) tein. This v a r i a n t can also be r e c o g n i s e d by e n z y m a t i c digestion, b e c a u s e the n u c l e o t i d e c h a n g e ( A ) d e s t r o y s a MaeII ( A / C G T ) site.
Acknowledgements. This work was supported by the Fondo de Investigaciones Sanitarias de la Seguridad Social (90ED1254) and Institut Catala de la Salut. M.C. received a fellowship from the Spanish Ministry of Education and Science.
References Riordan J, Rommens J, Kerem B-S, Alon N, Rozmahel R. Grzelczak Z, Zielinsky J, Lok S, Plavsic N, Chou JL. Drumm ML, Iannuzzi MC, Collins FS, Tsui L-C (1989) Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245:1066 1073 Zielensky J, Rozmahel R, Bozon D, Kerem B, Grzelczak Z, Riordan J, Rommens J, Tsui L-C (199[) Genomic DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Genomics 10 : 214-228
h u m a n .. genet,cs 9 Springer-Verlag 1992
A rare D N A variant in exon 15 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene M. Chill6n, A. Palacio, V. Nunes, X. Estivili Molecular Genetics Department, Cancer Research Institute (IRO), Duran Reynals Hospital, L'Hospitalet de Llobregat. Campus Bellvitge, E-08907 Barcelona, Spain Received: 13 April 1992 / Revised: 6 July 1992
Abstract. W e d e s c r i b e a rare s i n g l e - s t r a n d c o n f o r m a t i o n p o l y m o r p h i s m that occurs at n u c l e o t i d e 3030 ( G o r A ) in exon 15 of the cystic fibrosis t r a n s m e m b r a n e c o n d u c t a n c e r e g u l a t o r gene.
Source~description. W e have amplified a 485-bp f r a g m e n t c o n t a i n i n g exon 15 of the cystic fibrosis t r a n s m e m b r a n e c o n d u c t a n c e r e g u l a t o r ( C F T R ) g e n e , using p r i m e r s 15i-5 (5' G T G C A T G C T C T T C T A A T G C A 3') a n d 15i-3 (5' AAGGCACATGCCTCTGTGCA 3 ' ) , which h a v e previously b e e n d e s c r i b e d ( Z i e l e n s k y et al. 1991). Variant. A n a b n o r m a l s i n g l e - s t r a n d c o n f o r m a t i o n polym o r p h i s m (SSCP) b a n d p a t t e r n of exon 15 was o b s e r v e d in two individuals o u t of 70 u n r e l a t e d C a u c a s i a n s analysed (Fig. I a). T h e v a r i a n t consists o f a G to A substitution at n u c l e o t i d e 3030 of the C F T R gene. Chromosomal localization. T h e C F T R gene m a p s to c h r o m o s o m e 7 (7q31) ( R i o r d a n et al. 1989). Other comments. T h e c o n d i t i o n s of the p o l y m e r a s e chain r e a c t i o n ( P C R ) w e r e : d e n a t u r a t i o n at 96~ for 30 s, a n n e a l i n g at 56~ for 30 s a n d e x t e n s i o n at 74~ for 4 0 s in 35 cycles. T h e r e a c t i o n mix c o n t a i n e d 0.1 lal ad C T P (32p 3000 C i / m m o l ) to label the f r a g m e n t s . T h e P C R p r o d u c t s w e r e d i g e s t e d with the DdeI restriction e n z y m e . S S C P e l e c t r o p h o r e s i s was p e r f o r m e d at r o o m t e m p e r a t u r e with 6% a c r y l a m i d e and 10% glycerol, at 1 0 W for 2 4 h . O n l y o n e individual s h o w e d an u n u s u a l p a t t e r n . W e r e - a m p l i f i e d the s a m p l e with the s a m e P C R c o n d i t i o n s and the D N A p r o d u c t was s e q u e n c e d a u t o m a t i c a l l y ( A B I 373A) using f l u o r e s c e n t t e r m i n a t o r s . T h e c h a n g e was a t t r i b u t a b l e to a G to A substitution in exon 15 (Fig. l b ) ; this substitution does not m o d i f y the a m i n o acid t h r e o n i n e , at p o s i t i o n 966 of the C F T R p r o Correspondence to: M. Chilldn
Fig. 1. a SSCP analysis of the exon 15 of the CFTR gene of 4 unrelated CF individuals. The extra band in the pattern corresponds to the 3030 (G or A) polymorphism, b G to A substitution in exon 15, visualised by automatic sequencing: this substitution does not modify the amino acid threonine at position 966 of the CFTR protein. A comparison between a normal control (3030 G) and the heterozygous individual who has a shared signal (N) with respect to the normal signal (G) tein. This v a r i a n t can also be r e c o g n i s e d by e n z y m a t i c digestion, b e c a u s e the n u c l e o t i d e c h a n g e ( A ) d e s t r o y s a MaeII ( A / C G T ) site.
Acknowledgements. This work was supported by the Fondo de Investigaciones Sanitarias de la Seguridad Social (90ED1254) and Institut Catala de la Salut. M.C. received a fellowship from the Spanish Ministry of Education and Science.
References Riordan J, Rommens J, Kerem B-S, Alon N, Rozmahel R. Grzelczak Z, Zielinsky J, Lok S, Plavsic N, Chou JL. Drumm ML, Iannuzzi MC, Collins FS, Tsui L-C (1989) Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245:1066 1073 Zielensky J, Rozmahel R, Bozon D, Kerem B, Grzelczak Z, Riordan J, Rommens J, Tsui L-C (199[) Genomic DNA sequence of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Genomics 10 : 214-228
