HUMAN MUTATION 5:lOl-102 (1995)

MUTATION IN BRIEF

Mutation Gene causing cystic

+IG+T> in the c F Fibrosis

Joanna Crawford, Agatha Labrinidis, William F. Carey, Paul V. Nelson, John S. Harvey, and C. Phillip Morris* Department of Chemical Pathology, Women’s and Children’s Hospital, North Adelaide, South Australia 5006, Awtralia; Fax: 61 -8-204-71 00

Communicated by H. H. Dahl

Cystic fibrosis (CF) is the most common auto- soma1 recessive disease in Caucasian populations, with an incidence of approximately 1 in 2,500 live births and a carrier frequency of about 1 in 25 (Boat et al., 1989). The fundamental defect in CF appears to be dysfunctional regulation of chloride ion flux in exocrine gland epithelia, leading to abnormally thick mucus secretions. This is re- flected in the clinical features of the disorder which are dominated by lung disease, currently the major cause of morbidity in CF, and gradual d e g radation of the pancreas.

Cystic fibrosis is caused by mutations in the cys- tic fibrosis transmembrane conductance regulator (CFTR) gene. The CFTR gene spans approxi- mately 250 kb and is composed of 27 exons (Rom- mens et al., 1989; Riordan et al., 1989). To date more than 400 disease-causing mutations have been identified (CF Consortium, personal commu- nication); however, most of these are rare or are unique to specific families.

The 5-year-old patient described in this study was originally diagnosed with CF at 3 years of age, based on clinical observations and elevated sweat electrolyte levels. The patient is of Portuguese/ Chinese extraction and has pancreatic insuffi- ciency, which is being treated by enzyme replace- ment therapy and she has recurrent respiratory infections for which she is receiving aggressive an- tibiotic treatment. She has one older sister, who also has CF.

The patient was screened for the 15 most frequent CF mutations and was found to be nega- tive for all those tested. The prevalence and rela- tively high frequency of missense mutations affect- ing the first nucleotide binding fold of the CFTR protein led to polymerase chain reaction (PCR) amplification of the exons and intron boundaries of exons 10, 11, and 12. To detect possible se-

0 1995 WILEY-LISS, INC.

quence alterations, the amplified products were subjected to single-stranded conformational poly- morphism (SSCP) analysis as previously described (Harvey et al., 1993). The exon 12 PCR product showed the presence of a new band in addition to the two normal bands, indicating that the patient is heterozygous for a change in this exon (Fig. 1). Sequence analysis of the exon 12 PCR product revealed a heterozygous G-+T transversion in the highly conserved GT of the intron 12 splice donor site (5’ splice site), generating a 1,898 + 1G+T mutation. Allele specific oligonucleotides (ASO) were designed to detect the normal (5’-ATT- TGAAAGGTATGTTCTT-3’) and mutant (5’- AT T TG AAAGT TATGT TCT T-3 ’ ) sequences of 1,898+1G-+T in unaffected and CF patient populations. Exon 12 PCR products were amplified and A S 0 detection was performed as previously described (Harvey et al., 1993), except that the filters were washed at 54°C in 2 x SSC, 0.1% sodium dodecyl sulfate (SDS). In addition to the patient, her mother and sister were both found to be heterozygous for 1898 + 1G-T; however, the mutation was not present in any of the other 69 individuals who were screened for the mutation.

As this patient is 5 years old and already exhib- its many of the classic features of CF, it is reason. able to assume that the 1,898 + 1G-T mutation is associated with severe disease presentation. A large number of mutations affecting 5’ splice sites that lead to abnormal mRNA splicing, and thus disease presentation, have now been described in a variety of human genetic disorders (reviewed in Krawczak et al., 1992; Sakuraba et al., 1992). Of the 5’ splice mutations, 77% result in exon skip-

Received April 19, 1994 accepted May 3, 1994. *To whom reprint requestslcorrespondence should be ad-

dressed.

102 CRAWFORD ET AL.

FIGURE 1. SSCP detection of a base change in exon 12 of the CF gene. Lanes I , 3-8, amplified DNA from CF patients who do not contain a mutation in this region. Lane 2, contains amplified DNA from the patient with the 1,898 + 1G+T mu- tation. Arrow indicates a band with altered mobility. PCR primers used to amplify exon 12 were 12-j-5 (sense) 5’ - TCATCTACACTAGATGACCAGGAA-3’ and 12-j-3 (an- tisense) 5‘-AGGTAAAATGCAATCTATGATGGG-3’.

ping or a combination of exon skipping and use of cryptic sites (Sakuraba et al., 1992). Assuming the result of mutant 5’ splicing caused by 1,898+ lG+T correlates with that seen at these other donor splice sites, it is likely that this mutation, in conjunction with another as yet unidentified del- eterious allele, will generate loss of CFTR function and lead to the disease presentation recognised in the patient described in this study.

Interestingly, both the other possible sequence variants at this position, i.e., 1,898 + 1G-A (Strong et al., 1992) and 1,898+ lG+C (Cup- pens et al., 1992) have previously been described. Two patients who had the 1,898 + 1G+A muta- tion were reported to have pancreatic insufficiency and impaired pulmonary function (Strong et al., 1992). This supports our observation that changes at nucleotide 1,898 + 1 are associated with severe disease presentation. The description of a G+T mutation at 1,898 + 1 means all possible nucle-

otide changes have been recognised at this position and indicates a possible region of genetic instabil- ity, or at least mutation susceptibility, at this site.

ACKNOWLEDGMENTS

We are grateful to Dr. Bronte Gabb, Genetics Department, University of Adelaide, for his help- ful discussions and guidance during aspects of this work. This work was supported by the South Aus- tralian Health Commission.

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