Barker, U Alabama Birm, USA Kant, U Penn, USA

Beaudet, Baylor, USA Kitzis, CHU-Paris, France

Boué, Paris, France Klinger, Integ Genet, USA

Bowcock, Stanford, USA Knight, London, England

Cao, U Cagliari, Italy Lissens, Vrije U Brussels

Carbonara, Torino, Italy Loukopoulos, Athens, Greece

Cassiman, U Leuven, Belgium Lucotte, College de France

Claustres, Montpellier, France Malcolm, ICH-London, England

Collins, U Michigan, USA Malik, Basler-Basel, Switzerland

Cutting, Johs Hopkins, USA Mao, Collab Res, USA

Dallapiccola, Roma, USA McIntosh, WGH-Edinburgh, Scotland

Dean, NCI Frederick, USA Morel, Lyon, France

De Arce, Dublin, N Ireland Morgan, McGill, Canada

Edwards, Oxford, England Naylor, UT San Antonio, USA

Elles, St Mary's-Manchester, England Olek, U Bonn, West Germany

Erlich, Cetus, USA Orr, U Minnesota, USA

Estivill, Barcellona, Spain Pignatti, U Verona, Italy

Ferec, Brest, France Ramsay, SAMIR, South Africa

Ferrari, Milano, Italy Richards, GeneScreen, USA

Godet, Villeurbanna, France Romeo, Gaslini-Genoa, Italy

Goossens, Creteil, France Rozen, Montreal Children, Canada

Graham, Belfast, N Ireland Scheffer,UGottingen,TheNetherlands

Gruenert, UCSF, USA Schmidtke, IHG, Berlin

Halley, Rotterdam, The Netherlands Schwartz, U Copenhagen, Denmark

Harris, Guy's-London, England Super, Royal Manchester, England

Highsmith, NC Mem Hosp, USA Thibodeau, Rochester, USA

Horst, Münster, West Germany Tsui, Toronto, Canada

Jaume-Roig, Son Dureta, Spain Tümmler, Hannova, West Germany

Kalaydjieva, Sofia, Bulgaria Williamson,St Mary'sLondon,England



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NEWSLETTER #5, February 9, 1990


1. The DNA sequences of the introns flanking exon 9 are attached. Please note that there is an intron in the reported exon 6; this exon is now split into 6a and 6b. The latter information is supplied by Kathy Klinger et al. (see attached).

2. Four (4) additional mutations have been found in exon 11 and 1 in exon 9. One of the exon 11 mutations is reported by Garry Cutting and the rest are from the Toronto group.

a. Garry Cutting reports that the alanine at amino acid position 559 is changed to threonine (A559T*). G559 is a highly conservative residue within the ATP-binding fold. The corresponding nucleotide change is G->A at position 1807.

*The previously proposed arrow in the short descriptive symbols for amino acid changes (see last newsletter) is now deleted in order to avoid editorial problems in the future, as suggested by Art Beaudet.

b. The glycine codon (GGA) at amino acid position 542 is changed to a stop codon (TGA) (G542X). The single chromosome carrying this mutation is of Ashkenazic Jewish origin (family A) and has the B haplotype (XV2C allele 1; KM.19 allele 2). The mutant sequence can be detected by hybridization analysis with allele-specific oligonucleotides (ASOs) on genomic DNA amplified by PCR with the 11i-5 and 11i-3 oligonucleotide primers. The normal ASO is 5'-ACCTTCTCCAAGAACT-3' and the mutant ASO, 5'-ACCTTCTCAAAGAACT-3'. The oligonucleotide hybridization condition is as described in Kerem et al. (1989) and the washing conditions are twice in 5xSSC for 10 min each at room temperature followed by twice in 2XSSC for 30 min each at 45[[ring]]C. The mutation is not detected in 52 other non-[[Delta]]F508 CF chromosomes, 11 of which are of Jewish origin (3 have a B haplotype), nor in 13 normal chromosomes.

c. The highly conserved serine residue at position 549 is changed to arginine (S549R); the codon change is AGT->AGG. The CF chromosome with this mutation is carried by a non-Ashkenazic Jewish patient in Moroco (family B). The chromosome also has the B haplotype. Detection of this mutation may be achieved by ASO hybridization or allele-specific PCR. In the ASO hybridization procedure, the genomic DNA sequence is first amplified by PCR with the 11i-5 and 11i-3 oligonucleotides; the ASO for the normal sequence is 5'-ACACTGAGTGGAGGTC-3' and that for the mutant is 5'-ACACTGAGGGGAGGTC. The oligonucelotide hybridization condition is as described by Kerem et al. (1989) and the washings are done twice in 5xSSC for 10 min each at room temperature followed by twice in 2xSSC for 30 min each at 56[[ring]]C. In the allele-specific PCR amplification, the oligonucleotide primer for the normal sequence is 5'-TGCTCGTTGACCTCCA-3', that for the mutant is 5'-TGCTCGTTGACCTCCC-3' and that for the common, outside sequence is 11i-5. The reaction is performed with 500 ng of genomic DNA, 100 ng of each of the oligoncleotides and 0.5 unit of Taq polymerase. The DNA template is first denatured by heating at 94[[ring]]C for 6 min, followed by 30 cycles of 94[[ring]] for 30 sec, 55[[ring]] for 30 sec and 72[[ring]] for 60 sec. The reaction is completed by a 6 min heating at 72[[ring]] for 7 min. This S549R mutation is not present in 52 other non-[[Delta]]F508 CF chromosomes, 11 of which are of Jewish origin (3 have a B haplotype), nor in 13 normal chromosomes.

d. The arginine (AAG) at amino acid position 560 is changed to threonine (AAC). The individual carrying this mutation (R560T) is from family #32 and the chromosome is marked by haplotype IIIb. The mutation creates a MaeII site which cleaves the PCR product of exon 11 (generated with primers 11i-5 and 11i-3) into 2 fragments of 214 and 204 bp in size. None of the 36 non-[[Delta]]F508 CF chromosomes (7 of which have haplotype IIIb) or 23 normal chromosomes (16 have haplotype IIIb) carries this sequence alteration. The R560T mutation is also not present on 8 CF chromosomes with the [[Delta]]F508 mutation.

e. A mutation in exon 9 is detected at amino acid position 455- the alanine (GCG) is changed to glutamic acid (GAG) (A455E). The 2 CF chromosomes carrying this mutation are from patients of a French-Canadian origin (families #27 and #53) and they belong to haplotype group Ib. The mutation is detectable by allele-specific oligonucleotide (ASO) hybridization with PCR-amplified genomic DNA sequence. The PCR primers are 9i-5 (5'-TAATGGATCATGGGCCATGT-3') and 9i-3 (5'-ACAGTGTTGAATGTGGTGCA-3'). The ASOs are 5'-GTTGTTGGCGGTTGCT-3' for the normal allele and 5'-GTTGTTGGAGGTTGCT-3' for the mutant. The oligonucleotide hybridization condition is as described in Kerem et al. (1989) at 37[[ring]]C and the washings are done twice with 5xSSC for 10 min each at room temperature followed by twice with 2xSSC for 30 min each at 52[[ring]]C. Although the alalnine at position 455 (Ala455) is not present in all ATP-binding folds across species, it is present in all known members of the P-glycoprotein family. Further, A455E is believed to be a mutation rather than a sequence polymorphism because the change is not found in other chromosomes with group I or other haplotypes; the ones examined are 62 CF chromosomes in group I, including those with [[Delta]]F508 (haplotype Ia), 22 CF chromosomes of other haplotypes, 11 normal chromosomes with the same group I haplotype and 29 other normal chromosomes.

3. There was an error in the sequence reported for the 5' flanking intron of exon 11. The sequence should read GGAGATGCAATG instead of GGAGATCAATG (we missed a "G" which was clearly there). Thanks to Iain McIntosh for noting this error. We apologize for any inconvenience that might have caused.

4. A report from Pier Franco Pignatti on the 4th European Community Meeting on the molecular genetics of cystic fibrosis, carrier testing and prenatal diagnosis, held in Verona on 19-20 January.