Hum Genet (1992) 88:479-481
9 Springer-Verlag1992
Carrier detection and prenatal diagnosis of cystic fibrosis using an intragenic TA-repeat polymorphism Etienne Mornet 1, Corinne Chateau 1, Brigitte Simon-Bouy 1, Joelle Boue 1, Julian Zielenski 2, Lap Chee Tsui 2, and Andr~ Boue 1 1Centre d'Etudes de Biologie Prdnatale (CEBIOP), Chateau de Longchamp, Bois de Boulogne, F-75016 Paris, France 2The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada Received July 22, 1991 / Revised October 8,1991
Summary. W e have analysed the segregation of a TA-repeat p o l y m o r p h i s m in intron 17b of the cystic fibrosis t r a n s m e m b r a n e conductance regulator gene responsible for cystic fibrosis (CF) in 23 French CF families non-informative for the AF508 mutation (i.e. with at least one parent not carrying AF508) or closely linked D N A markers. A t least 13 different alleles ranging from 7 to 45 repeats were observed and the detected heterozygosity was 89%. O f the 23 families studied, 19 were fully informative for prenatal diagnosis or carrier detection, 3 were partially informative and one was not informative. In 6 families, prenatal diagnosis for CF or carrier detection in siblings of CF cases were p e r f o r m e d using this polymorphism.
t r a n s m e m b r a n e conductance regulator ( C F T R ) gene. T h e y have found 95% of heterozygosity in their population and consequently this m a r k e r is highly informative. We report here the screening of French CF families not informative for AF508 or linked D N A m a r k e r s with the T A repeat of this region.
Materials and methods Patients and CF families Twenty-three unrelated French families with at least one affected child (three of them with a related CF case in the sibling) were referred for genetic counselling or prenatal diagnosis. Diagnosis of cystic fibrosis was established by the usual clinical and laboratory criteria.
Introduction Polymerase chain reaction (PCR) amplification 1 Cystic fibrosis (CF) is the most c o m m o n severe autosomal recessive genetic disorder affecting E u r o p e a n and North A m e r i c a n populations with an incidence of about 1 in 2500. The three base-pair deletion (AF508) in exon 10 causes CF in a large proportion of patients ( K e r e m et al. 1989; E W G C F G 1990). A large n u m b e r of the remaining CF mutations in the French population lead to a low degree of informativity in prenatal or genetic diagnosis when CF mutations are directly tested after AF508 is shown not to be informative (Simon-Bouy et al. 1991). AF508 is said to be not informative when at least one parent in the couple at risk for CF does not carry this mutation. A solution for prenatal or genetic diagnosis of CF in siblings of CF patients is the use of intragenic polymorphisms that m a y be employed as genetic markers without any risk caused by extra-genic recombination. Dinucleotide repeats are particulary useful because they generally show a high degree of polymorphism. Zielenski et al. (1991) have recently described a T A - r e p e a t polym o r p h i s m downstream of exon 17b of the cystic fibrosis
Offprint requests to: E. Mornet
About 0.5 ixg genomic DNA were mixed with 4pmoles of the two oligonucleotide primers 17B-R1 and 17B-RE3 (Zielenski et al. 1991), annealed to the DNA template for polymerase extension at 72~ with 2 units Taq DNA polymerase and denatured at 93~ for further cycles. Primers were annealed at 55~ for 30 s and extension was performed at 72~ for I min. Amplification was checked by electrophoresis of a 10-~tl sample from the 100-~ reaction.
PCR amplification 2 A 5 ~tl sample of the reaction mixture was used as a DNA template for the second PCR amplification. Approximately 0.2pmoles of the TA-repeat primer 17TA-3 or the CA repeat 17CA-5 (Zielenski et al. 1991) were end-labelled with gamma 32p ATP and polynucleotide kinase, and mixed with the DNA matrix, dNTPs and PCR buffer. The same PCR cycle was used as in PCR 1.
Electrophoresis The extended, radiolabelled fragments were separated by electrophoresis on 7% denaturing polyacrylamide gel for 4 h and exposed overnight to X-ray film. The sizes of the different alleles were determined using control sequences.
480 Results and discussion
Table 1. Genotyping for the TA repeat detected in intron 17b in the 23 couples at risk for CF. ND, Not done
The 45 individuals forming 23 unrelated couples (90 chromosomes) showed 13 different alleles ranging from 7 to 45 T A repeats (Table 1). The frequencies of these alleles did not significantly differ from those previously reported in a sample of normal chromosomes, exept for the relatively high frequency of T A 45 (Zielenski et al. 1991). Five of the tested individuals were homozygous. Thus, the heterozygosity detected was 89%, and does not significantly differ from that in the report of Zielenski et al. (1991) (95%). In most of the cases, the families were completely informative making possible the determination of the CF status in siblings. An example (family 12) is shown in Fig. 1; the parents carry alleles 45/32 and 28/7, the patient 45/7 and an unaffected child 32/28. In three cases (families 5, 15, 16), one parent was homozygous and consequently the informativity was only 50% because the analysis could only be based on the transmission of alleles from the other parent: for example, in family 5, if the patient and a fetus do not carry the same maternal allele, the fetus is normal (heterozygote or homozygote, probability = 0.5) but if the patient and the fetus carry the same maternal allele, the fetus m a y be affected or heterozygous (probability 0.5) and no conclusion is possible. The two parents were homozygous for the same allele (30 repeats) in only one case (family 8), and the analysis was consequently non-informative. In the latter family, amplification of the C A repeat was performed and showed that the four m e m b e r s were homozygous for a fragment of undetermined size. Attempts to demonstrate consanguinity in this family failed. However, the 30-repeats allele is frequent in the tested population and the probability of finding two unrelated individuals homozygous for this allele is 0.6%.
Family
Father
Mother
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
30/29 36/28 30/ 7 30/28 7/ 7 30/31 45/ 7 30/30 45/30 45/30 32/ 8 45/32 32/31 30/ 7 31/31 45/31 36/ 7 30/31 29/31 31/36 31/39 30/34 45/30
30/ 7 30/ 7 30/31 34/29 31/30 30/31 30/ 7 30/30 30/24 31/ 7 36/32 28/ 7 36/30 36/31 45/31 31/31 29/30 45/29 45/30
31/ 7 32/33 ND 24/ 7
Application to prenatal diagnosis Families 1, 4 and 20 were referred for genetic counselling and prenatal diagnosis. These families were not completely informative with AF508 and linked D N A markers. In family 1, the father was heterozygous for AF508 but the m o t h e r did not carry this mutation. Analysis of the T A repeats showed that the parents carried genotypes 30/29 and 30/7, and the index case 30/29 (Fig. 1). The fetus carried AF508 and the CF allele from the mother. H e was consequently genotyped 30/29 and affected. In family 4, the father was heterozygous for AF508 and the m o t h e r did not carry this mutation. Analysis of the T A repeats showed that the parents carried genotypes 30/28 and 34/29; the index case and the fetus carried 34/30 showing that the fetus was consequently affected. In family 20, the parents did not carry AF508 but this family was informative for the intron 6 polymorphism (Chehab et al. 1991). Both T A repeats and intron 6 polym o r p h i s m showed that the fetus was not affected.
Application to carrier detection In three families (21, 22, 23), siblings of CF cases were referred for genetic counselling to determine whether
Fig. 1. Autoradiography of the TA repeats detected in 6 French CF families. An extra band corresponding to 18 repeats is always associated with allele 7 and consequently was not considered as an allele. The father in family 7 (lane 9) was analysed again, and genotyped as 45/7
they were carriers of CF. An example is shown in Fig. 2: the index case was genotyped 30/24 and his mother 24/7. In this family, the sister of the mother had a 50% a priori risk of being a carrier. She carried the genotype 29/7 and consequently did not carry a CF allele.
481
I 45/3O
30/24
I1-1 Fig. 2, Carrier detection in a sibling of a CF case using TA repeats polymorphism; 1-1 is obligatory carrier and the risk of her sister I-3 of being a carrier is 0.5. The segregation of the TA repeats alleles shows that she is not a carrier
In conclusion, we have s h o w n that the T A r e p e a t detected in intron 17b of the C F T R gene is useful for prenatal diagnosis and familial carrier detection. H o w e v e r , this m e t h o d c a n n o t be used w h e n the index case is d e a d or w h e n the family is not informative.
Acknowledgements. This work was supported by grants from Association Frangaise de Lutte contre la Mucoviscidose (E. M., C. C., B. S-B., J. B., A.B.), the National Institutes of Health (DK34944), the Canadian Cystic Fibrosis Foundation and the Cystic Fibrosis Foundation (J. Z., L. C. T.).
References T h e C F gene has n o w b e e n extensively analysed b y the 86 laboratories o f the Cystic Fibrosis G e n e t i c A n a l y sis C o n s o r t i u m . Because this gene is 250 kb long (with a c D N A of 6 kb), it was expected that it w o u l d exhibit m a r k e d intragenic p o l y m o r p h i s m s . Several p o l y m o r p h i c sites have b e e n described, including a d i m o r p h i c 4-bp repeat in the flanking intron of exon 6b ( C h e h a b et al. 1991), a G T r e p e a t in intron 8 (N. M o r r a l and X. Estivill, personal c o m m u n i c a t i o n ) , and the T A and C A repeats, in intron 17b (Zielenski et al. 1991). Because these polym o r p h i s m s are repeats, they show a very high d e g r e e of polymorphism and heterozygosity, which thus lead to considerable informativity in a family where other mutations or linked D N A m a r k e r s are unsuccessful. B e c a u s e these repeats are intragenic, they are reliable w h e n used as genetic m a r k e r s for prenatal diagnosis or carrier detection.
Chehab EF, Johnson J, Louie E, Goossens M, Kawasaki E, Erlich H (1991) A dimorphic 4-bp repeat in the cystic fibrosis gene is in absolute linkage disequilibrium with the AF508 mutation: implications for prenatal diagnosis and mutation origin. Am J Hum Genet 48 : 223-226 EWGCFG (1990) Gradient of distribution in Europe of the major CF mutation and of its associated haplotype. Hum Genet 85 : 436-441 Kerem B, Rommens JM, Buchanan D, Markiewicz TK, Cox A, Chakravarti M, Buchwald M, Tsui LC (1989) Identification of the cystic fibrosis gene: genetic analysis. Science 245:10731089 Simon-Bouy B, Mornet E, Serre JL, Taillandier A, Bou6 J, Bou6 A (1991) Nine mutations in the cystic fibrosis (CF) gene account for 80% of the CF chromosomes in French patients. Clin Genet 40 : 218-224 Zielenski J, Markiewicz D, Rininsland F, Rommens JR, Tsui LC (1991) A cluster of highly polymorphic dinucleotide repeats in intron 17b of the CFTR gene. Am J Hum Genet (in press)
9 Springer-Verlag1992
Carrier detection and prenatal diagnosis of cystic fibrosis using an intragenic TA-repeat polymorphism Etienne Mornet 1, Corinne Chateau 1, Brigitte Simon-Bouy 1, Joelle Boue 1, Julian Zielenski 2, Lap Chee Tsui 2, and Andr~ Boue 1 1Centre d'Etudes de Biologie Prdnatale (CEBIOP), Chateau de Longchamp, Bois de Boulogne, F-75016 Paris, France 2The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada Received July 22, 1991 / Revised October 8,1991
Summary. W e have analysed the segregation of a TA-repeat p o l y m o r p h i s m in intron 17b of the cystic fibrosis t r a n s m e m b r a n e conductance regulator gene responsible for cystic fibrosis (CF) in 23 French CF families non-informative for the AF508 mutation (i.e. with at least one parent not carrying AF508) or closely linked D N A markers. A t least 13 different alleles ranging from 7 to 45 repeats were observed and the detected heterozygosity was 89%. O f the 23 families studied, 19 were fully informative for prenatal diagnosis or carrier detection, 3 were partially informative and one was not informative. In 6 families, prenatal diagnosis for CF or carrier detection in siblings of CF cases were p e r f o r m e d using this polymorphism.
t r a n s m e m b r a n e conductance regulator ( C F T R ) gene. T h e y have found 95% of heterozygosity in their population and consequently this m a r k e r is highly informative. We report here the screening of French CF families not informative for AF508 or linked D N A m a r k e r s with the T A repeat of this region.
Materials and methods Patients and CF families Twenty-three unrelated French families with at least one affected child (three of them with a related CF case in the sibling) were referred for genetic counselling or prenatal diagnosis. Diagnosis of cystic fibrosis was established by the usual clinical and laboratory criteria.
Introduction Polymerase chain reaction (PCR) amplification 1 Cystic fibrosis (CF) is the most c o m m o n severe autosomal recessive genetic disorder affecting E u r o p e a n and North A m e r i c a n populations with an incidence of about 1 in 2500. The three base-pair deletion (AF508) in exon 10 causes CF in a large proportion of patients ( K e r e m et al. 1989; E W G C F G 1990). A large n u m b e r of the remaining CF mutations in the French population lead to a low degree of informativity in prenatal or genetic diagnosis when CF mutations are directly tested after AF508 is shown not to be informative (Simon-Bouy et al. 1991). AF508 is said to be not informative when at least one parent in the couple at risk for CF does not carry this mutation. A solution for prenatal or genetic diagnosis of CF in siblings of CF patients is the use of intragenic polymorphisms that m a y be employed as genetic markers without any risk caused by extra-genic recombination. Dinucleotide repeats are particulary useful because they generally show a high degree of polymorphism. Zielenski et al. (1991) have recently described a T A - r e p e a t polym o r p h i s m downstream of exon 17b of the cystic fibrosis
Offprint requests to: E. Mornet
About 0.5 ixg genomic DNA were mixed with 4pmoles of the two oligonucleotide primers 17B-R1 and 17B-RE3 (Zielenski et al. 1991), annealed to the DNA template for polymerase extension at 72~ with 2 units Taq DNA polymerase and denatured at 93~ for further cycles. Primers were annealed at 55~ for 30 s and extension was performed at 72~ for I min. Amplification was checked by electrophoresis of a 10-~tl sample from the 100-~ reaction.
PCR amplification 2 A 5 ~tl sample of the reaction mixture was used as a DNA template for the second PCR amplification. Approximately 0.2pmoles of the TA-repeat primer 17TA-3 or the CA repeat 17CA-5 (Zielenski et al. 1991) were end-labelled with gamma 32p ATP and polynucleotide kinase, and mixed with the DNA matrix, dNTPs and PCR buffer. The same PCR cycle was used as in PCR 1.
Electrophoresis The extended, radiolabelled fragments were separated by electrophoresis on 7% denaturing polyacrylamide gel for 4 h and exposed overnight to X-ray film. The sizes of the different alleles were determined using control sequences.
480 Results and discussion
Table 1. Genotyping for the TA repeat detected in intron 17b in the 23 couples at risk for CF. ND, Not done
The 45 individuals forming 23 unrelated couples (90 chromosomes) showed 13 different alleles ranging from 7 to 45 T A repeats (Table 1). The frequencies of these alleles did not significantly differ from those previously reported in a sample of normal chromosomes, exept for the relatively high frequency of T A 45 (Zielenski et al. 1991). Five of the tested individuals were homozygous. Thus, the heterozygosity detected was 89%, and does not significantly differ from that in the report of Zielenski et al. (1991) (95%). In most of the cases, the families were completely informative making possible the determination of the CF status in siblings. An example (family 12) is shown in Fig. 1; the parents carry alleles 45/32 and 28/7, the patient 45/7 and an unaffected child 32/28. In three cases (families 5, 15, 16), one parent was homozygous and consequently the informativity was only 50% because the analysis could only be based on the transmission of alleles from the other parent: for example, in family 5, if the patient and a fetus do not carry the same maternal allele, the fetus is normal (heterozygote or homozygote, probability = 0.5) but if the patient and the fetus carry the same maternal allele, the fetus m a y be affected or heterozygous (probability 0.5) and no conclusion is possible. The two parents were homozygous for the same allele (30 repeats) in only one case (family 8), and the analysis was consequently non-informative. In the latter family, amplification of the C A repeat was performed and showed that the four m e m b e r s were homozygous for a fragment of undetermined size. Attempts to demonstrate consanguinity in this family failed. However, the 30-repeats allele is frequent in the tested population and the probability of finding two unrelated individuals homozygous for this allele is 0.6%.
Family
Father
Mother
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23
30/29 36/28 30/ 7 30/28 7/ 7 30/31 45/ 7 30/30 45/30 45/30 32/ 8 45/32 32/31 30/ 7 31/31 45/31 36/ 7 30/31 29/31 31/36 31/39 30/34 45/30
30/ 7 30/ 7 30/31 34/29 31/30 30/31 30/ 7 30/30 30/24 31/ 7 36/32 28/ 7 36/30 36/31 45/31 31/31 29/30 45/29 45/30
31/ 7 32/33 ND 24/ 7
Application to prenatal diagnosis Families 1, 4 and 20 were referred for genetic counselling and prenatal diagnosis. These families were not completely informative with AF508 and linked D N A markers. In family 1, the father was heterozygous for AF508 but the m o t h e r did not carry this mutation. Analysis of the T A repeats showed that the parents carried genotypes 30/29 and 30/7, and the index case 30/29 (Fig. 1). The fetus carried AF508 and the CF allele from the mother. H e was consequently genotyped 30/29 and affected. In family 4, the father was heterozygous for AF508 and the m o t h e r did not carry this mutation. Analysis of the T A repeats showed that the parents carried genotypes 30/28 and 34/29; the index case and the fetus carried 34/30 showing that the fetus was consequently affected. In family 20, the parents did not carry AF508 but this family was informative for the intron 6 polymorphism (Chehab et al. 1991). Both T A repeats and intron 6 polym o r p h i s m showed that the fetus was not affected.
Application to carrier detection In three families (21, 22, 23), siblings of CF cases were referred for genetic counselling to determine whether
Fig. 1. Autoradiography of the TA repeats detected in 6 French CF families. An extra band corresponding to 18 repeats is always associated with allele 7 and consequently was not considered as an allele. The father in family 7 (lane 9) was analysed again, and genotyped as 45/7
they were carriers of CF. An example is shown in Fig. 2: the index case was genotyped 30/24 and his mother 24/7. In this family, the sister of the mother had a 50% a priori risk of being a carrier. She carried the genotype 29/7 and consequently did not carry a CF allele.
481
I 45/3O
30/24
I1-1 Fig. 2, Carrier detection in a sibling of a CF case using TA repeats polymorphism; 1-1 is obligatory carrier and the risk of her sister I-3 of being a carrier is 0.5. The segregation of the TA repeats alleles shows that she is not a carrier
In conclusion, we have s h o w n that the T A r e p e a t detected in intron 17b of the C F T R gene is useful for prenatal diagnosis and familial carrier detection. H o w e v e r , this m e t h o d c a n n o t be used w h e n the index case is d e a d or w h e n the family is not informative.
Acknowledgements. This work was supported by grants from Association Frangaise de Lutte contre la Mucoviscidose (E. M., C. C., B. S-B., J. B., A.B.), the National Institutes of Health (DK34944), the Canadian Cystic Fibrosis Foundation and the Cystic Fibrosis Foundation (J. Z., L. C. T.).
References T h e C F gene has n o w b e e n extensively analysed b y the 86 laboratories o f the Cystic Fibrosis G e n e t i c A n a l y sis C o n s o r t i u m . Because this gene is 250 kb long (with a c D N A of 6 kb), it was expected that it w o u l d exhibit m a r k e d intragenic p o l y m o r p h i s m s . Several p o l y m o r p h i c sites have b e e n described, including a d i m o r p h i c 4-bp repeat in the flanking intron of exon 6b ( C h e h a b et al. 1991), a G T r e p e a t in intron 8 (N. M o r r a l and X. Estivill, personal c o m m u n i c a t i o n ) , and the T A and C A repeats, in intron 17b (Zielenski et al. 1991). Because these polym o r p h i s m s are repeats, they show a very high d e g r e e of polymorphism and heterozygosity, which thus lead to considerable informativity in a family where other mutations or linked D N A m a r k e r s are unsuccessful. B e c a u s e these repeats are intragenic, they are reliable w h e n used as genetic m a r k e r s for prenatal diagnosis or carrier detection.
Chehab EF, Johnson J, Louie E, Goossens M, Kawasaki E, Erlich H (1991) A dimorphic 4-bp repeat in the cystic fibrosis gene is in absolute linkage disequilibrium with the AF508 mutation: implications for prenatal diagnosis and mutation origin. Am J Hum Genet 48 : 223-226 EWGCFG (1990) Gradient of distribution in Europe of the major CF mutation and of its associated haplotype. Hum Genet 85 : 436-441 Kerem B, Rommens JM, Buchanan D, Markiewicz TK, Cox A, Chakravarti M, Buchwald M, Tsui LC (1989) Identification of the cystic fibrosis gene: genetic analysis. Science 245:10731089 Simon-Bouy B, Mornet E, Serre JL, Taillandier A, Bou6 J, Bou6 A (1991) Nine mutations in the cystic fibrosis (CF) gene account for 80% of the CF chromosomes in French patients. Clin Genet 40 : 218-224 Zielenski J, Markiewicz D, Rininsland F, Rommens JR, Tsui LC (1991) A cluster of highly polymorphic dinucleotide repeats in intron 17b of the CFTR gene. Am J Hum Genet (in press)
