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 (473 bp long) has two MnO sites, Haplotype Analysis one of which is destroyed by the W1282X mutation. Digestion of normal DNA (N) reveals three bands: 178, 172, and 123 bp in Restriction-enzyme digestion of genomic DNA was length. DNA digestion of homozygous for the W1282X mutation performed as recommended by the supplier (New En- (M) reveals two bands: 301 and 172 bp in length. DNA digestion gland Biolabs). Agarose gel electrophoresis and blot of heterozygotes for the mutation (H) reveals four bands: 301, 178, hybridization analysis were carried out essentially ac- 172, and 123 bp. DNA markers (Kb) are also shown. cording to methods described elsewhere (Tsui et al. 1985). patients screened, 16 (15 Ashkenazi and 1 Arab) were Statistical Analysis homozygous for W1282X, 22 (20 Ashkenazi, 1 Se- The Student unpaired t-test was used to compare phardic, and 1 from an unclassified origin) were het- differences in group means ( ± SD in all tables) for erozygous for W1282X and AF508, 2 (Ashkenazi) continuous variables. x2 Analysis was used to compare patients were heterozygous for W1282X and G542X, differences between groups. and 11 patients were heterozygous for W1282X and an as yet unidentified mutation. DNA marker haplotype analysis for informative families showed that Results chromosomes carrying the W1282X mutation were associated with haplotype B at the H2.3A/ TaqI and Identification of Mutations El1PstI loci. These DNA markers are the same as PCR-amplified DNA samples from 25 CF patients XV2C/TaqI/ and KM19/PstI (Kerem et al. 1989c). carrying 39 chromosomes with unidentified mutations Another DNA alternation in exon 21 was found in 4 were subjected to sequence analysis. This included di- Ashkenazi and 2 Arab CF chromosomes (table 1). rect DNA sequencing of genomic DNA regions con- This was a G--C substitution at nucleotide position taining exons 9-12 and 19-21, all of which partici- 4041, changing the 1303 codon to Lysin (N1303K) pate in encoding the two nucleotide-binding folds. and corresponding to a mutation described elsewhere One DNA alternation in exon 20 was found in 10 (Osborne et al. 1991). Hence, together with AF508, individuals; this was a G--A substitution at nucleotide G542X, N1303K, and 1717-1G--A, 92% of the CF position 3978, changing the Trp1282 codon to a stop chromosomes could be identified in Jewish patients of codon (W1282X) and corresponding to a mutation Ashkenazi origin. described elsewhere (Vidaud et al. 1990). This mutation destroys an MnlI recognition site (fig. 1) and can Genotype-Phenotype Correlation be detected by PCR amplification of exon 20 followed The AF508 mutation has already been shown to be by MnlI digestion. Of the remaining chromosomes associated with severe clinical presentation (Kerem et carrying unidentified CF mutations, 63 were found to al. 1990a). Therefore, for assessment of the severity carry the W1282X mutation; 57 (90%) of these 63 of the W1282X mutation, it would have been appro- were of Ashkenazi origin. Thus, the W1282X muta- priate to compare patients homozygous for the tion is the most common mutation (60%) in the Ash- W1282X mutation with patients homozygous for the kenazi Jewish patient population (table 1). Among the AF508 mutation and with patients heterozygous for Association of Nonsense Mutation with Severe CF 225

Table I Analysis of Mutation Frequencies Identified in 97 Israeli CF Patients

JEWISH PATIENTS ARAB Ashkenazim Sepharadim Unclassified PATIENTS

No. of chromosomes sampled.... 95 51 8 40 W1282X (no. [%]) ...... 57 (60) 1 (2) 3 (38) 2 (5) AF508 (no. [%])...... 21 (23) 18 (35) 2 (25) 10 (25) G542X (no. [%])...... 4 (4) 2 (4)0 2 (5) N1303K (no. [%]) ...... 4 (4) 0 0 2 (5) 1717-1G-A (no. [%]) ...... 1 (1) 0 0 1 (3) S5491 (no. [%])...... 0 0 0 2 (5) S549R (no. [%])...... 0 1 (2) 2 (25) 2 (5)

Total mutations (no. [%]) ...... 87 (92) 22 (43) 7 (88) 21 (53)

both mutations. As the AF508 frequency in the pulmonary-function decline with age was observed in screened population is low, only a small number of both groups. Since pulmonary-function tests were per- patients homozygous for AF508 were found (n = 6). formed only in patients above 6 years of age, numbers Therefore, the clinical data for the patients homozy- were too small for least-squares regression analysis. gous for the W1282X mutation were compared with We therefore used as a reference the previously re- those for the heterozygous patients carrying both the ported least-squares regression line for patients homo- AF508 mutation and the W1282X mutation. zygote for AFS08 (Kerem et al. 1990a). All the patients from both genotypes were classified The FEV1 of both groups of our patients, though as pancreatic insufficient. Three patients homozygous variable with age, was symmetrically distributed on for the W1282X mutation died (at ages 0.5, 2, and both sides of the reference regression line. This indi- 12 years), and three patients heterozygous for both cates similar severity of pulmonary function in pa- AF508 and W1282X died (at ages 0.25, 5, and 23 tients either homozygous for W1282X or heterozy- years). As shown in table 2, there were no significant gous for the W1282X and AF508 mutations, as differences between the two patient groups, in all the compared with patients homozygous for the AF508 studied parameters. Because of the strong dependence mutation. of pulmonary function on age, values of FEVI were Two patients were heterozygous for two different plotted against patients' age. As shown in figure 2, a termination mutations, W1282X and G542X. One

Table 2 Comparison of Clinical Characteristics and Pulmonary Function: CF Patients Homozygous for W1282X Mutation versus CF Patients Heterozygous for W1282X and AF508 Mutations W1282X/W1282X W1282X/AF508

No. of patients sampled ...... 16 22

No. (%) with meconium ileus ...... 6 (37) 6 (27) Age at diagnosis (years) ...... 9 ± 2.6 .6 ± .8

Sweat chloride (mmol\liter) ...... 113 ± 12 109 ± 11 Current age (years) ...... 9.3 ± 7.5 7.5± 11.6 Current weight (%-ile) ...... 17 ± 17 28 ± 21 Current height (%-ile) ...... 29 ± 29 27 ± 24 FEV, (% predicted) ...... 64 ± 27(n = 11) 69 26(n = 12) FEV2575 (% predicted) ...... 32 ± 20(n = 11) 52 ± 36(n = 12) NOTE. -None of the variables were statistically significant either by x2 analysis (for meconium ileus) or unpaired Student t-test (for all other variables). 226 Shoshani et al.

120 r o W1282X\W1282X Kartner et al. 1991); however, its role in chloride se- * W1282X\AF508 cretion is still unknown. Therefore, identification of 100 F 0 additional mutations and characterization of geno- 0 0 * may provide further insights 0 type-phenotype relations 80 k into the CFTR function. W1282X is a termination mutation. Termination mutations in human genes SU 0 * * have been associated with decreased levels of mRNA 3T 60 F o (Baserga and Benz 1988; Daar and Maquat 1988; 0 o 0 0 Globerman et al. 1988; Lim et al. 1989), instability of 40 F 0 0 the truncated protein (Adams and Coleman 1990), 0 0 and diseases that are more severe than those caused 20 F by missense mutations (Gitschier et al. 1986; White 1989). The present study shows that patients homozy- Il 0 gous for W1282X had disease severity similar to that 0 5 1 0 1 5 20 25 30 35 of patients heterozygous for W1282X and AF508. AGE (YEARS) Comparison of the studied variables from our two Figure 2 Age-adjusted FEV1 values. The line is the least- groups of patients to previously published data from squares regression line for patients homozygous for the AF508 mu- patients homozygous for the AF508 mutation, shown tation who have been reported elsewhere. to be associated with a severe CF presentation (Kerem et al. 1990a), showed similar disease severity. Thus the termination mutation W1282X appears to be asso- patient, age 16 years, had pancreatic insufficiency, me- ciated with presentation of severe disease. conium ileus, an FEV1 63% of the predicted value, Pulmonary function in the present study was vari- and an FEF2575 21 % ofthe predicted value, suggesting able in each genotype, similar to the variability found a severe disease presentation. The other patient, age in other centers among patients homozygous for 5 years, had pancreatic insufficiency but was too AF508 (Kerem et al. 1990a; Santis et al. 1990). In young for pulmonary-function tests. both groups of our patients, FEV1, although variable with age, was symmetrically distributed on both sides ofthe least-squares regression line previously obtained Discussion for patients homozygous for AF508. The variability in The term "Ashkenazim" designates Jews settled in pulmonary function indicates that additional factors northwestern Europe since the sixth century. It is used might affect the severity or progression of the pulmo- in contradistinction to the Sepharadim, Jews who nary disease in CF. Recently, it has been reported that originated in Spain. Since the 15th century, there have two CF patients with termination mutations in both of been waves ofemigration ofAshkenazi Jews to eastern their chromosomes had pancreatic insufficiency with Europe, North America, Israel, and several other mild pulmonary disease (Cutting et al. 1990a). Since countries (Roth 1971). In the present study, we have pulmonary function in patients carrying severe CF mu- described the most common CF mutation among Ash- tations (i.e., AF508 and W1282X) have high variabil- kenazi Jews, W1282X, accounting for 60% of the CF ity, it is difficult to conclude whether the milder form chromosomes in the studied group. All the chromo- of pulmonary disease shown in these two patients is somes carrying this mutation were found to be associ- genetically determined. Future studies with CFTR an- ated with a single haplotype, suggesting that they were tibodies in cells from patients carrying two termina- all derived from the same origin. The W1282X mutation mutations may shed new light on the structure- tion has been found to be rare in other, non-Jewish function relationships of the CFTR protein. different patient groups; in a worldwide survey, only 1.6% ofthe CF chromosomes were found to carry this mutation (Cystic Fibrosis Genetic Analysis Consor- Acknowledgments tium, unpublished data). The authors wish to thank Dr. Peter Durie for performing Recent transfection studies suggest that the CF gene the immunoreactive trypsinogen test, Mary Corey for her product (i.e., CFTR) probably functions as a cAMP- help in the statistical analysis, and Dr. Hermona Sorek for regulated chloride channel (Anderson et al. 1991; helpful discussions. The research was supported by grants Association of Nonsense Mutation with Severe CF 227 from the March of Dimes-Birth Defects Foundation (USA) sensus sequence mutation in the cystic fibrosis gene. Hum and the Thyssen Foundation (Germany). Genet 85:450-453 Highsmith WE Jr, Strong T, Burch N, Smith T, Silverman References LM, Collins FS, Boucher R, et al (1990) Identification of a splicing error of exon 14b giving rise to a frameshift Adams JG III, Coleman MB (1990) Structural hemoglobin mutation in a consanguineous family with mild cystic fi- variants that produce the phenotype of thalassaemia. brosis. Pediatr Pulmonol 5 [Suppl]: 11A Semin Hematol 27:229-238 Tannuzzi MC, Stern RC, Collins FS, Catherine TH, Hidaka Anderson MP, Rich DP, Gregory RJ, Smith AE, Welsh MJ N, Strong T, Becker L, et al (1991) Two frameshift muta- (1991) Generation of CAMP-activated chloride current tions in the cystic fibrosis gene. AmJ Hum Genet 48:227- by expression of CFTR. Science 251:670-682 231 Baserga SJ, Benz EJ Jr (1988) Nonsense mutations in the Ivaschenco TE, White MB, Dean M, Baranov VS (1991) A human beta-globin gene affect mRNA metabolism. Proc deletion of two nucleotides in exon 10 of the CFTR gene Natl Acad Sci USA 85:2056-2060 in a soviet family with cystic fibrosis causing early infant Boat TF, Welsh MJ, Beaudet AL (1989) Cystic fibrosis. In: death. Genomics 10:298-299 Scriver CR, Beaudet AL, Sly WS, Valle D (eds) The meta- Kartner N, Hanrahan JW, Jensen TJ, Naismith AL, Sun S, bolic basis of inherited disease, 6th ed. MacGraw-Hill, Ackerley CA, Reyes EF, et al (1991) Expression of the New York, pp 2649-2680 cystic fibrosis gene in non epithelial invertebrate cells pro- Cutting GR, Kasch LM, Rosenstein BJ, Tsui L-C, Kazazian duces a regulated anion conductance. Cell 64:681-691 HH Jr, Antonarakis SE (1990a) Two cystic fibrosis pa- Kerem B, Buchanan JA, Durie P, Corey ML, Levison H, tients with mild pulmonary disease and nonsense muta- Rommens JM, Buchwald M, et al (1989a) DNA marker tions in each CFTR gene. N Engl J Med 323:1517-1522 haplotype association with pancreatic sufficiency in cystic Cutting GR, Kasch LM, Rosenstein BJ, Zielenski J, Tsui fibrosis. Am J Hum Genet 44:827-834 L-C, Antonarakis SE, Kazazian HH Jr (1990b) A cluster Kerem E, Corey M, Kerem B, Durie P, Tsui LC, Levison H of cystic fibrosis mutations in the first nucleotide-binding (1989b) Clinical and genetic comparisons of patients with fold of the cystic fibrosis conductance regulator protein. cystic fibrosis, with or without meconium ileus. J Pediatr Nature 346:366-369 114:767-773 Cystic Fibrosis Genetic Analysis Consortium (1990) World- Kerem E, Corey M, Kerem B, Rommens JM, Markiewicz wide survey ofthe AF508 mutation-report from the Cys- D, Levison H, Tsui L-C, et al (1990a) The relationship tic Fibrosis Genetic Analysis Consortium (1990) Am J between genotype and phenotype in cystic fibrosis - anal- Hum Genet 47:354-359 ysis of the most common mutation (AF508). N Engi J Daar 10, Maquat LE (1988) Premature translation termina- Med 323:1517-1522 tion mediates triose phosphate isomerase mRNA degrada- Kerem B, Rommens JM, Buchanan JA, Markiewicz D, Cox tion. Mol Cell Biol 8:802-813 TK, Chakravarti A, Buchwald M, et al (1989c) Identifi- Dean M, White M, Amos J, Gerrard B, Stewart C, Khaw cation of the cystic fibrosis gene: genetic analysis. Science K-T, Leppert M (1990) Multiple mutations in highly con- 245:1073-1080 served residues are found in mildly affected cystic fibrosis Kerem B, Zielenski J, Markiewicz D, Bozon D, Gazit E, patients. Cell 61:863-870 YahavJ, Kennedy D, et al (1990b) Identification ofmuta- Durie PR, Forstner GG, Gaskin KJ, Moore DJ, Cleghorn tions in regions corresponding to the two putative nucleo- GJ, Wong SS, Corey ML (1986) Age related alterations tide (ATP)-binding folds of the cystic fibrosis gene. Proc in immunoreactive pancreatic cationic trypsinogen in sera Natl Acad Sci USA 87:8447-8451 from cystic fibrosis patients with and without pancreatic Kobayashi K, Knowles MR, Boucher RC, O'Brien WE, insufficiency. Pediatr Res 20:209-213 Beaudet AL (1990) Benign missense variations in the cystic Gasparini P, Nunes V, Savola A, Dognini M, Morral N, fibrosis gene. Am J Hum Genet 47:611-615 Gaona A, Bonizzato A, et al (1991) The search for south Lemna WK, Feldman GL, Kerem B, Fernbach SD, Zevko- European cystic fibrosis mutations: Identification of two vich EP, O'Brion WE, Collins FS, et al (1990) Mutation new mutations, four variants, and intronic sequences. Ge- analysis for heterozygote detection and prenatal diagnosis nomics 10: 193-200 of cystic fibrosis. N Engl J Med 322:291-296 GitschierJ, Wood WI, Shuman MA, Lawn RM (1986) Iden- Lerer I, Cohen S, Chemke M, Sanilevich A, Rivlin J, Golan tification of a missense mutation in the factor VIII gene of A, Yahav J, et al (1990) The frequency of the AF508 a mild hemophiliac. Science 232:1415-1416 mutation on cystic fibrosis chromosomes in Israeli fami- Globerman H. Amor M, Parker KL, New MI, White PC lies: correlation to CF haplotypes in Jewish communities (1988) Nonsense mutation causing steroid 21-hydroxy- and Arabs. Hum Genet 416:85 lase deficiency. J Clin Invest 82:139-144 Lim S, MullinsJJ, Chen CM, Gross KW, Maquat LE (1989) Guillermit H. Fanem P. Ferec C (1990) A 3' splice site con- Novel metabolism of several beta zero-thalassaemia beta- 228 Shoshani et al.

globin mRNA in the erythroid tissues of transgenic mice. and weight velocity: British children, 1965. Arch Dis EMBO J 8:2613-2619 Child 41:613-635 Osborne L, Knight R, Santis G, Hodson M (1991) A muta- Tsui L-C, Buchwald M, Barker D, Braman RG, Knowlton tion in the second nucleotide binding fold of the cystic J, Schumm H, Eiberg J et al (1985) Cystic fibrosis locus fibrosis gene. Am J Hum Genet 48:608-612 defined by a genetically linked polymorphism DNA Polgar G, Promadhat V (1971) Pulmonary function in chil- marker. Science 230:1045-1057 dren: techniques and standards. WB Saunders, Philadel- van der Kamer JH, ten Bokkel Huinick H, Weyers HA phia (1949) Rapid method for the determination of fat in fae- Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, ces. J Biol Chem 177:347-355 Grzelczak Z, Zielenski J, et al (1989) Identification of Vidaud M, Fanen P, MartinJ, Ghanem N, Nicolas S, Goos- the cystic fibrosis gene: cloning and characterization of sens M (1990) Three mutations in the CFTR gene in complementary DNA. Science 245:1066-1073 French cystic fibrosis patients: identification by denatur- Rommens JM, Iannuzzi MC, Kerem B, Drumm ML, ing gradient gel electrophoresis. Hum Genet 85:446-449 Melmer G, Dean M, Rozmahel R, et al (1989) Identifica- White PC (1989) Analysis of mutations causing steroid tion of the cystic fibrosis gene: chromosome walking and 21-dydroxylase deficiency. Endocr Res 15:239-256 jumping. Science 245:1059-1065 White MB, Amos J, Hsu JMC, Gerrard B, Finn P, Dean M Rommens J, Kerem B, Greer W, Chang P, Tsui L-C, Ray P (1990) A frame shift mutation in the cystic fibrosis gene. (1990) Rapid nonradioactive detection ofthe major cystic Nature 344:665-667 fibrosis mutation. Am J Hum Genet 46:395-396 White MB, Kruger LJ, Holsclaw DS, Gerrard BC, Stewart Roth C (1971) Ashkenaz. Encyclopedia Judaica. KeterJeru- C, Quittell L, Dolganov G, et al (1991) Detection of three salem 3:719-721 rare frameshift mutations in the cystic fibrosis gene in an Saiki RK, Gelfand SH, Stoffel S, Scharf SJ, Higuchi R, Horn African-American (CF444delA), an Italian (CF2522insC), GT, Mullis KB, et al (1988) Primer-directed enzymatic and a Soviet (CF3821delT). Genomics 10:266-269 amplification ofDNA with a thermostable DNA polymer- Winship PR (1989) An improved method for directly se- ase. Science 239:487-491 quencing PCR amplified material using dimethyl sulph- Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich oxide. Nucleic Acids Res 17:1266-1269 HA, Arnheim N (1985) Enzymatic amplification of beta Zielenski J, Bozon D, Kerem B, Markiewicz D, Rommens globin genomic sequences and restriction site analysis for JM, Tsui L-C (199la) Identification ofmutations in exons diagnosis of sickle cell anemia. Science 230:1350-1354 1 through 8 of the cystic fibrosis transmembrane conduc- Santis G, Osborne L, Knight RA, Hodson ME (1990) tance regulator (CFTR) gene. Genomics 10:229-235 Linked marker haplotypes and the AF508 mutation in Zielenski J, Rozmahel R, Bozon D, Kerem B, Grzelczak Z, adults with mild pulmonary disease and cystic fibrosis. Riordan JR, Rommens JM, et al (1991b) Genomic DNA Lancet 335:1426-1429 sequences of the cystic fibrosis transmembrane conduc- Tanner JM, Whitehouse RH, Takaishi M (1966) Standards tance regulator. Genomics 10:214-228 from birth to maturity for height, weight, height velocity