The Most Common Mutation in the Ashkenazi Jewish Cystic

Am. J. Hum. Genet. 50:222-228, 1992 Association of a Nonsense Mutation (WI 282X), the Most Common Mutation in the Ashkenazi Jewish Cystic Fibrosis Patients in Israel, with Presentation of Severe Disease Tzipora Shoshani,* Arie Augartent Ephraim Gazit,1'§ Nurit Bashan,* Yaakov Yahav4 Yosef Riviin,11 Asher Tal,# Hagit Seret,* Liora Yaar,§ Eitan Kerem,t and Bat-sheva Kerem* *Department of Genetics, Life Sciences Institute, and tDepartment of Pediatrics, Bikur Cholim Hospital Hadassah Medical School, Hebrew University, Jerusalem; tDepartment of Pediatrics and §Tissue Typing Laboratory, Chaim Sheba Medical Center, Sackler Medical School, Tel Aviv University, Tel Aviv; I"Department of Pediatrics, Carmel Hospital, Technion Medical School, Haifa; and #Department of Pediatrics, Soroka Medical Center, Negev University, Beer Sheba, Israel Summary Only about 30% of the cystic fibrosis chromosomes in the Israeli cystic fibrosis patient populations carry the major CF mutation (/F508). Since different Jewish ethnic groups tended to live as closed isolates until recent times, high frequencies of specific mutations are expected among the remainder cystic fibrosis chromosomes of these ethnic groups. Genetic factors appear to influence the severity of the disease. It is therefore expected that different mutations will be associated with either severe or mild phenotype. Direct genomic sequencing of exons included in the two nucleotide-binding folds of the putative CFTR protein was performed on 119 Israeli cystic fibrosis patients from 97 families. One sequence alteration which is expected to create a termination at residue 1282 (W1282X) was found in 63 chromosomes. Of 95 chromosomes, 57 (60%) are of Ashkenazi origin. Together with the AF508 (23% in this group), G542X, N1303K, and 1717-1G-*A mutations, the identification of 92% of cystic fibrosis chromosomes of Ashkenazi origin becomes possible. Patients homozygous for the W1282X mutation (n = 16) and patients heterozygous for the AF508 and W1282X mutations (n = 22) had similarly severe disease, reflected by pancreatic insufficiency, high incidence of meconium ileus (37% and 27%, respectively), early age at diagnosis, poor nutritional status, and variable pulmonary function. In conclusion, the W1282X mutation is the most common cystic fibrosis mutation in the Ashkenazi Jewish patient population in Israel. This nonsense mutation is associated with presentation of severe disease. Introduction frequencies have been found among CF black, Italian, The gene responsible for cystic fibrosis (CF) has re- Greek, Spanish, Turkish, and Jewish populations cently been identified (Riordan et al. 1989; Rommens (Cystic Fibrosis Genetic Analysis Consortium 1990). et al. 1989), and the major mutation, AF508, has been Numerous additional mutations have recently been defined (Kerem et al. 1989c). A worldwide survey of identified in the CF gene (Cutting et al. 1990a, 1990b; the AF508 frequency revealed that this mutation ac- Dean et al. 1990; Guillermit et al. 1990; Highsmith counts for approximately 70% of all CF chromo- et al. 1990; Kerem et al. 1990b; Kobayashi et al. 1990; somes. However, the frequency varies greatly between Vidaud et al. 1990; White et al. 1990, 1991; Gaspar- different ethnic and geographic populations. Lower ini et al. 1991; Iannuzzi et al. 1991; Ivaschenco et al. 1991; Osborne et al. 1991; Zielenski et al. 1991a). Each disease-related mutation marks a functionally Received July 10, 1991; revision received September 4, 1991. important region of the cystic fibrosis transmembrane Address for correspondence and reprints: Bat-sheva Kerem, conductance regulator (CFTR), providing informa- Ph.D., Department of Genetics, Life Sciences Institute, Hebrew tion on structure-function relationships ofthe protein. University, Jerusalem 91904, Israel. is an i 1992 by The American Society of Human Genetics. All rights reserved. Furthermore, this information essential require- 0002-9297/92/5001-0024$02.00 ment for both genetic counseling of CF families and 222 Association of Nonsense Mutation with Severe CF 223 population screening for CF carriers. In particular, representing the two majorJewish ethnic groups (Ash- identification of additional mutations becomes critical kenazim and Sepharadim) in Israel. Blood samples in populations in which the frequency of AF508 is from the patients, their parents, and available sibs low, e.g., the Jewish population. were collected. DNA was prepared essentially ac- The incidence ofCF in Israel is about 1 /5,000 (Boat cording to a method described elsewhere (Tsui et al. et al. 1989). The frequency of AF508, both among 1985). Patient diagnosis had been previously con- the Jewish and Arab CF patient populations in Israel firmed by typical clinical findings of pulmonary and/ and among theJewish population in North America, is or gastrointestinal disease (Boat et al. 1989) and/or approximately 30% (Cystic Fibrosis Genetic Analysis the presence of family history of CF, together with Consortium 1990; Lemna et al. 1990; Lerer et al. abnormal sweat chloride test. 1990). In a previous study of a small group of Israeli CF patients, four additional rare mutations (1717- Assessment of Disease Severity 1G--A, G542X, S5491, and S549R) were found The parameters described below were used in the (Kerem et al. 1990b). Since the different Jewish ethnic analysis of clinical phenotypes: Pancreatic function groups tended to live as closed isolates until recent was determined by either 3-d fecal fat collection or times, high frequencies of specific mutations are ex- plasma immunoreactive trypsinogen test (van der pected among the remainder of Jewish CF chromo- Kamer et al. 1949; Durie et al. 1986). Pulmonary somes within these populations. function was assessed in all CF patients over 6 years The CF disease is characterized by a wide variability of age. Forced vital capacity (FVC), forced expiratory of clinical expression. There is heterogeneity in mode volume in one second (FEV1), and forced expiratory ofpresentation, organ involvement, type of complica- flow rate in the middle half of expiration (FEF25-75) tions, disease severity, and life expectancy (Boat et al. were measured and expressed as a percentage of pre- 1989). A genotype-phenotype correlation in CF has dicted values for height and sex, by using previously been suggested on the basis of both haplotype analysis described standardized pulmonary equations (Polgar and the different distribution of the AF508 mutation and Promadhat 1971). Current height and weight per- among pancreatic-sufficient and -insufficient patients centiles were computed using the tables of Tanner (Kerem et al. 1989a, 1989c). More recently, in a study (Tanner et al. 1966). Meconium ileus was defined as of a large CF cohort, the AF508 mutation was shown failure to pass meconium within 24 h or longer after to be associated with a severe clinical presentation birth and as occurring in conjunction with clinical (Kerem et al. 1990a). In addition, several other muta- signs of acute intestinal obstruction and characteristic tions were associated with presentation of either se- radiological findings (Kerem et al. 1989b). Ages at vere or mild disease, although a small number of pa- diagnosis and at assessment, sex, and diagnostic sweat tients were studied (Kerem et al. 1990b). These studies chloride levels were from patient charts. stress the importance of results associating different mutations in the CF gene with either severe or mild Mutations Analysis phenotypes. Here we report both the identification DNA sequences spanning individual exons of the of the most common CF mutation in the Ashkenazi CF gene were amplified by PCR (Saiki et al. 1985, Jewish patient population in Israel and the results of 1988), with oligonucleotide primers (Kerem et al. its genotype-phenotype correlation. 1990b) located in the respective flanking introns ofthe CF gene (Zielenski et al. 1991b). Amplified exon 20 DNA fragments were digested with the MnnI restric- Patients and Methods tion endonuclease for 3 h at 370C and electrophoresed on 7% polyacrylamide gels. DNA from all the families Patients was tested for AF508 by heteroduplex formation be- The patient cohort involved in the present study tween the normal and the mutant sequences and by included 119 CF patients from 97 families who regu- subsequent polyacrylamide gel electrophoresis (Rom- larly attended the CF clinics at Chaim Sheba Medical mens et al. 1990). DNA samples from patients nonho- Centre, Tel Aviv; Bikur Cholim General Hospital, Je- mozygous for AF508 were included in a subsequent rusalem; Carmel Medical Centre, Haifa; and Soroka search for additional mutations, as described below. Medical Centre, Beer Sheva. This group of patients The G542X, S5491, S549R, N1303K, and 1717- comprised of20 Arab and 77Jewish families, the latter 1G--A mutations were detected by PCR and subse- 224 Shoshani et al. quent allele specific oligo nucleotide hybridization as N H H M M Kb described elsewhere (Kerem et al. 1990b; Osborne et al. 1991). DNA Sequence Determination 301 - Amplified genomic DNA fragments eluted from 5% polyacrylamide gels were extracted with phenol/ 178- chloroform and were subjected to the dideoxy-chain- 172 termination sequencing method essentially as described (Winship 1989), by using the U.S. Biochemi- cals SequenaseTm kit with either one ofthe PCR primers 123- or internal oligonucleotides as sequencing primers (Kerem et al. 1990b). Figure I Analysis of MnlI digestion of exon 20-amplified genomic DNA. The PCR product

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