Uniparental inheritance of microsatellite alleles of the cystic fibrosis gene

©1993 Oxford University Press

Human Molecular Genetics, 1993, Vol. 2, No. 6

677-681

Uniparental inheritance of microsatellite alleles of the cystic fibrosis gene (CFTR): identification of a 50 kilobase deletion Nuria Morral, Virginia Nunes, Teresa Casals, Nicolas Cobos1, Oscar Asensio2, Javier Dapena3 and Xavier Estivill* Molecular Genetics Department, Cancer Research Institute (IRO), Hospital Duran i Reynals, A.Castelldefels Km 2.7, 08907 L'Hospitalet de Llobregat, Barcelona, Catalunya, 1Cystic Fibrosis Unit, Hospital de la Vail d'Hebron, Barcelona, 2 Cystic Fibrosis Unit, Hospital Infantil de Sabadell, Barcelona and 3Pediatric Unit, Hospital Virgen del Rocfo, Sevilla, Spain Received February 23, 1993; Revised and Accepted April 7, 1993

ABSTRACT More than 250 mutations have been detected in the cystic fibrosis (CF) transmembrane regulator (CFTK) gene, most of which are single point mutations or small deletions or insertions of a few nudeotides. Here we report the first large deletion identified in the CFTR gene, which involves 50 kb in two stretches of DNA: one of 10 kb from exon 4 to exon 7, and another of 40 kb, spanning exons 11 to 18. Hie deletion has been detected via uniparental inheritance of CFTR microsatellite alleles (TVS17BTA and IVS17BCA) in 3 independent CF famines. Clinical status of the 3 CF patients, of which two have the AF508 mutation as the other CF allele, suggests that this mutation is responsible for a severe clinical phenotype, indistinguishable from homozygous AF508 patients. The deletion detected here suggests that other large, but less complex molecular defects could also exist in the CFTR gene. INTRODUCTION More than 250 cystic fibrosis (CF) mutations have been identified in the CFTR gene (ref. 1-3 and CF Genetic Analysis Consortium). A 3-bp deletion at codon 508 ( AF508 mutation) (3) accounts for approximately 70% of CF chromosomes in populations of Northern Europe and North America, being less frequent in the Mediterranean region, where less than 50% of CF chromosomes have the AF508 mutation (4-6). There is a high molecular heterogeneity in the CF Mediterranean population with a large proportion (30%) of CF chromosomes as yet uncharacterised (ref. 6 and unpublished). In CF families from this region genetic analysis is being performed by combining direct and indirect molecular methods. Three microsatellite markers (7), located in intronic regions of the CFTR gene [IVS8CA (8), IVS17BTA (9) and IVS17BCA (9,10)], together with mutation analysis provide complete informativity in more than 99% of CF families (11). We have analysed more than 500 Spanish CF families for mutations in the CFTR gene and have identified about 30 differemt mutations, which account for more than 70% of CF chromosomes (ref. 12 and unpublished). Microsatellite CFTR • To whom correspondence should be addressed

haplotypes are associated with specific CF mutations and dius allows the development of frameworks useful for diagnosis (12). In the analysis of CF families with microsatellite markers three families showed uniparental inheritance of alleles at the IVS17BTA and/or IVS17BCA microsatellites, suggesting that these loci were lacking one CF chromosome. Here we present the molecular and genetic characterisation of a large deletion in the CFTR gene, involving exons 4 to 7 and 11 to 18, which is responsible for CF in the three families. RESULTS Uniparental inheritance of IVS17BTA and IVS17BCA microsatellite alleles Three CF families showed abnormal inheritance of alleles at the IVS17BTA and/or IVS17BCA microsatellite loci (Fig. 1 and Table 1). In families B235 and B290 the CF individuals only inherited the paternal alleles, whereas in family B91 die CF child inherited only the maternal alleles. Paternity and maternity were confirmed in the three families by analysis with 5 microsatellite markers located on different chromosomes (D1S117, D6S89, D11S35, APOC2, and D21S168), for which allele frequencies have been extensively studied in the Spanish population (13), giving a probability of paternity and maternity of higher tiian 99%. Because in the diree families the CF patients inherited only one allele from one of the parents, it was suspected tiiat the anomalous pattern was due to a deletion in this genomic region. Extended analysis with other intragenic polymorphisms located in intron 12 (1898 + 152 T or A, near exon 12) (14) and intron 18 (3601 - 6 5 C or A, very close to exon 19) (15), showed that the deletion possibly included intron 12, but not the 3' end of intron 18. The 5' end of the deletion was further defined to be between intron 10 and exon 12 with the information obtained for the AF508 mutation in two families. Further analysis with intragenic and flanking markers (XV-2c, KM. 19, MP6d-9, G2, and J3.ll) (16—18) showed that the uniparental segregation of alleles was restricted to the CFTR region, suggesting a deletion that starts in the region between intron 10 and exon 12, and finishes at exon/intron 18 (Table 1).

678 Human Molecular Genetics, 1993, Vol. 2, No. 6 B F

g1

B2 3 5

M CF F M CF C C

B290

CF

F M CF

- 420

- 5 4

-370 32 - 3 1 - 30 IVS17BTA

-280

- 25

-230 kb B

_

_ 1 7 I V817BCA

B

S B

7

1 7 1 6

IVS 8CA

S B

16

Figure 2. Southern blot of a pulsed field gel electrophoresis of DNA from family B235 by using enzymes BssHU and Sail, hybridised with the cDNA probe pS7 (exons 19 to 24). The resolution range was from 50 to 600 kb. The 420-kb BssHR fragment, and the 370-kb band detected in the CF patient (CF) and his mother (M) indicates the loss of 50 kb in the CF chromosome in the region from exons 10 to 19. In the double digestion with BssHU and Sail an additional band of 230 kb apart from the 280-kb band was detected. Father (F) showed a normal pattern of digestion in both cases. Partial digestion of Sail was observed in the double digestion of Sail and BssHU. B, BssHU; S, Sail.

a)

F

M CF

F

M CF

E K on

- 1 3 -10 8

_ 1 1 Loci

R*p«ats

7 8

I. (

Figure 1. Multiplex PCR analysis of three microsateUites within the CFTR gene (TVS8CA, IVS17BTA, and IVS17BCA) in families B91, B235, and B29O. In family B235, absence of segregation of maternal alleles for IVS17BTA and IVS17BCA is shown in the CF child as well as in two sibs, who were also found to be carriers of the 50-kb deletion involving exons 4 to 7 and 11 to 18. F, father, M, mother; CF, cystic fibrosis.

A complex 50-kb deletion in the CFTR gene To define more precisely the length of the deletion, long range restriction mapping was performed in all index cases and their parents using pulsed field gel electrophoresis (PFGE). The DNA of normal, carrier and CF individuals was digested with the rare cutter restriction enzymes BssHU and Sail, and was hybridised with the cDNA probe pS7 (exons 19 to 24) (Fig. 2). A normal 420-kb and an extra 370-kb BssHR fragments were observed in the affected individuals and in their corresponding carrier parents. The three CF patients and the carrier parents showed normal 280-kb and 230-kb bands in double digestions of Sail and BssHU (Fig. 2). The difference in kb between the normal bands and the extra fragments is in agreement with a 50 kb deletion, the 370-kb & J H I I and 230-kb SaWBssHU bands corresponding to junction fragments. The same pattern of fragments was observed when filters were hybridised with probe pE8 (exons 6 to 9; data not shown). DNA from members of the 3 families was digested with £coRI, Hindm and Pstl and was hybridised to several genomic, and cDNA probes spanning the whole CFTR region. Probe pHP3 (exons 1 to 11) gave junction fragments of 7.8 and 7.0 kb in EcoKL and Pstl digestions, respectively, in the CF patients and

E c o Rl

b) F 8 J I

M

CF

F

M CF

9 8 - | 7 8 -

E c o Rl

P« t I

Figure 3. Identification of a junction fragment with probe pE8. (a) Southern blots of genomic DNA digested with EcoRl and Pstl were hybridised with probe pE8, which contains exons 6 to 9. This probe detected a junction fragment (j.f.) of 7.8 kb in the £coRI digestion, and a 7-kb in the Pstl digestion, (b) Hybridisation of the same Southern blot with a PCR product probe of exon 8, showing that it was the responsible for the extra fragment. Other extra bands corresponding to other regions of the genome were observed in the three lanes, and are homologous to exons 7 and 9 (J.Zidenski and L-C.Tsui, and A.Bosch, unpublished results). F, father; M, mother; CF, cystic fibrosis.

Human Molecular Genetics, 1993, Vol. 2, No. 6 679 TaMe 1. Haplotypes for flanking and intragenic CFTR markers in three CF families with abnormal microsatellite segregation Family Sample B235 Father Mother CF B290 Father

Haplotype XT

KP

8CA

F508

rvi2

17TA

17CA

1 *1 1 •1 1 1

1 2 1 1

F DF F F DF F

1 1

2 =

2 1

16 17 17 16 17 16

1 =

30 32 54 = 32 =

13 13 11 = 13 =

2

1 1 2 2 2 1

16 16 17 17 17 16

F F F DF DF F

1 = 2 1 1 =

31 = 53 31 31 =

16 = 11 13 13 =

2 *2

1 1 2 2

16 16 16 16

F F F F

1 = 2 1

25 = 7 7

13 = 17 17

1 2

1 2

16 16

F F

= 2

= 7

17

•1

Mother

2 •1

CF B91 Father

1 1 1 •1

Mother CF

[V18

G2

311

_ _ _ -

1 1 2 1 1 1

7) 1) 1) 2) 2) 1)

_ _ -

2 1 2 1 1 1

2 2 1 2

2 2 1 1 2 1

1 1 2 2 1 2

= , absence of alleles;CF, cystic fibrosis; F, normal phenylalanine at codon 508; DF, F5O8; *, CF chromosomes in parents; XT, X\-2dTaq\ (16); KP, KM A9/Pst\ (16); 8CA, FVS8CA (8); IV12, 1898+152 T-A/Bc/I (14); 17TA, IVS17BTA (9); 17CA, IVS17BCA (8, 9); IV18, 3601-65 C-A/tfmfl (15); G2, pG2/XbaI (18); 311, J3.ll (17); ( - ) assignment of alleles not possible.

carrier parents. Attempts to detect these fragments by using PCR probes containing exons 10, 11, or 12, which supposedly contained the 5' end of the deletion, failed. Surprisingly, hybridisation of EcoRI and Pstl digestions with probe pE8 (Fig. 3a), which includes exons 6 to 9, showed the 7.8 kb EcoRl and 7.0 kb Pstl junction fragments. These results suggested that the deletion covered two regions of the CFTR gene that were separated by a non-deleted genomic region. Hybridisation with a PCR probe of exon 8 produced the two extra EcoRl and Pstl fragments (Fig. 3b) (an additional fragment was also detected with PvuU), which were not found in 250 normal and 150 CF chromosomes analysed, confirming that they are junction fragments due to a break point and not polymorphisms. Individual PCR probes of exons 3 to 19 were obtained and hybridised to DNA from the parents and the CF patients. Half dosage was observed in the index cases and in their carrier parents for exons 4 to 7 and 11 to 18, indicating a deletion involving these exons (data not shown). Attempts to define the junction fragments at introns 3, 11, and 18, failed. This complex 50-kb deletion detected in three patients has been named CF50kbdel # 1. Single origin and severity of mutation CF50kbdel # 1 Mutation CF50kbdel # 1 has been found associated to the same XV-2c and KM. 19 (D7S23), MP6d-9 (D7S399), IVS8CA, IV18, G2, and J3.ll (D7S8) haplotype (8,15-18) in the three chromosomes, suggesting a common origin for this mutation (Table 1). Parents and grandparents of the three patients, carriers of mutation CF50kbdel # 1, are unrelated, but they all are from the same geographical region in the South of Spain, Andalucfa (Cdrdoba, Almeria and Cidiz). The CF50kbdel # 1 mutation has been found in three cases and has a frequency of about 0.3%. Two patients (CFB91 and CFB235) are CF50kbdel # 1/ AF508 heterozygous and have pancreatic and lung disease with different

levels of involvement. Patient CFB91 is a severely affected 8-year-old girl, diagnosed at 7 months of age due to a respiratory infection. Sweat chloride was 110 mEq/L, forced vital capacity (FVC) and forced expiratory volume in 1 sec (FEV1) were 40% of normal values and she has often been treated due to pulmonary infection. Height and weight were at percentiles of 37 and 17, respectively. Patient CFB235 is a moderately affected 14-yearold girl, diagnosed when she was 7 years old due to a respiratory infection. Sweat chloride was 100 mEq/L, FVC was 76% and FEV1 was 86% of normal values. Height and weight were at percentiles of 17 and 10, respectively. Clinical score of Shwachman-Kulczycki (19), for which 100 is the best score, was of 52 in patient CFB91 and 65 in patient CFB235. The third patient, a 2-year-old boy diagnosed due to a lung infection at the age of 10 months, is heterozygous for mutation CF50kbdel# 1 and an as yet unknown mutation. Height and weight were at percentiles of 25 and 7, respectively. He has never been hospitalized for treatment of pulmonary infection. Clinical score of Shwachman—Kulczycki was of 95. The patient also has pancreatic disease that needs treatment with enzymes. Comparison of the three patients with others of similar ages and period of diagnosis, homozygous for mutation AF5O8, did not reveal differences in the severity of the disease. DISCUSSION Mutation CF50kbdel # 1 is the largest deletion identified so far in the CFTR gene. The deletion has been found in 3 unrelated families, but it has been shown to have a common origin in patients from the South of Spain. The CF50kbdel # 1 mutation is expected to lead to a complete absence of CFTR or to an aberrant protein (Fig. 4). Clinical status of the 3 affected patients, of which two have the AF5O8 mutation as the other CF allele,

680 Human Molecular Genetics, 1993, Vol. 2, No. 6 IVS17BTA/CA

WS8CA IV12

-

IV18

-

-

02

-

POLYMORPHIC MARKERS

pHB pHP3

PLASMID PROBES

p87 pS12

PH1 E3 E4

E6 E8b ~ Eta

E7

E8 ~ ES

E10

E19

E12

PCR PROBES

E1S

E11

DDODDDDDODO 1

2

3

4

6 U

6b

7

8

B

TM1

10

11 12

13

14* 14b

NBF1

16

16 17* 17b

18

18

20 21 22 23

-31 24

NBF2

TM2

normal

DDODO

DDD 1

2

3

TM1

8

9

19

10

-3'

20 21 22 23 24

NBF1

NBF2

CF50kbdel#1 Figure 4. Characterization of mutation CFSOkbdel # 1. Schematic representation of the exons of the normal and CF50kbdel # 1 CFTR gene (empty boxes). Normal and the putative deleted parts of the protein (grey boxes) are shown. All the probes used to characterise the deletion are also shown. TM1 and TM2, transmembrane domains; NBF1 and NBF2, nucleotide binding fold domains; R, regulatory domain.

suggests that this mutation is responsible for a severe clinical phenotype, indistinguishable from homozygous AF5O8 patients. Identification of gene deletions has been widely reported for the 0-globin gene (20), the Human Factor VIII gene (21), and the LDL receptor gene (22). Sequence analysis of these deletions has shown that the major mechanism involved in their generation is non-homologous recombination, facilitated in many cases by Alu elements or other repetitive sequences (22). Preliminary experiments with cosmid clones spanning the non deleted exons in the CF50kbdel # 1 patients suggest that the region between the two deletions is not inverted (data non shown). It is possible that the mechanism involved in the formation of the CF50kbdel ft 1 mutation in the CFTR gene, could be illegitimate recombination between direct or inverted short repetitive elements (1 to 10 bp), located several kb apart. This mechanism has been shown to be implicated in the generation of several small deletions in disease genes (21). Further knowledge on the sequence of the junction fragments should provide information on the intricate mechanism responsible for the rearrangement leading to this complex deletion. Probably due to the fact that no cytogenetic abnormalities were detected in CF patients, and also to the limitation of the PCR technology, the possibility of large deletions involving part of the CFTR gene had been ignored, with only a small sample of 19 chromosomes having been reported to be tested for deletions

prior to the CFTR locus being discovered, using very distant probes (23). The three cases presented here may not be unique and other large deletions might be discovered in the CFTR gene. Because of the power of PCR, special attention should be focused on apparently 'homozygous' patients for a given mutation. The absence of a given exon could be read as homozygosity for a particular mutation in the same exon on the other chromosome (X.E., unpublished). The study of the respective parents should resolve these peculiar 'homozygous' cases. The analysis of CF families with unknown mutations, using cDNA clones and the search for unusual allele segregation using intragenic markers, should help to uncover part of the as yet unknown molecular mutation mechanisms in CF. Segregation analysis of microsatellite alleles might play an important role in the identification of other large molecular defects, as in the case of the DMD gene (24). The ability of microsatellite markers to detect allelic losses depends on the heterozygosity of each polymorphism and on the localisation of these markers within a given gene. MATERIALS AND METHODS CF families and mutation analysis More than 500 families were analysed for mutations and microsatellites in the CFTR gene (12, 25). Mutation analysis was performed using methodology previously described (26), direct sequencing, or SSCP analysis (27).

Human Molecular Genetics, 1993, Vol. 2, No. 6 681 Multiplex PCR amplification Microsatellites IVS8CA, IVS17BTA and IVS17BCA were analysed by simultaneous PCR amplification of the three loci as described previously (28). PFGE analysis Fresh human lymphocytes were embedded in agarose plugs and lysed. Afterwards the DNA was digested with the rare cutting restriction enzymes BssHn or fiuHH and Sail. The DNA fragments were separated by pulsed field gel electrophoresis (PFGE) (29) in 1.2% agarose gels with 0.5 XTBE buffer at 4°C. Separation of 50 to 600 kb was achieved after 15 hours at 300 Volts and pulse times of 15 seconds using a LKB (Pharmacia) Pulsaphor. Nylon filters were hybridized with probes pHl, pH6, and pS7. Southern blotting analysis DNA (8 jig) was digested with Eco RI, HimttH and Pstl and the fragments separated on a 0.8% agarose gel. Hybridisation and autoradiography were performed as described (30). cDNA and PCR probes Subclones pHP3, pHl, pH6, pE8, pS12 and pS7 were obtained from the fulllength CFTR cDNA pBQ6.2, kindly provided by J. Rommens and L.-C. Tsui. Subclones were introduced into the pBluescribe vector. pHP3 (1783 bp) contains exons 1 to 11; pHl (1231 bp) has exons 11 to 15; pH6 (395 bp) exons 15 to 17; pE8 (610 bp) contains exons 6 to 9; and pS12 (251 bp) contains exons 17 to 19, and pS7 (926 bp) has exons 19 to 24. Individual PCR probes of fragments containing exons 3, 4, 5, 6, 7, 8, 10, 11, 12, 18, and 19, and part of the respective flanking introns, were obtained by DNA amplification using primers previously described (31, 32). Conditions for PCR were as follows: 200 ng DNA; 1.5 mM MgCl2; 50 mM KC1; 10 mM Tris (pH 8.3); 30 pmol of each primer; and 2 U of Taq polymerase in a total volume of 100 fi}. The device was programmed to carry out 35 cycles of: 94°C for 30 sec, 62°C for 30 sec, and 74°C for 90 sec. PCR products were used as probes in Southern blot filters.

ACKNOWLEDGEMENTS We thank H.Kruyer for help with the manuscript, F.J.Gimenez for technical assistance, and the Spanish CF families for support. This work was supported by the Fondo de Investigaciones Sanitarias de la Seguridad Social (grant 90E1254 and 93/0202) (Spain) and the Institut Catala de la Salut (Catalonia).

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