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Technical Briefs |
1
Service de Biochimie et de Génétique Moléculaire, INSERM U468, Hôpital Henri Mondor, AP-HP, 51 Avenue du Maréchal de Lattre de Tassigny, 94010 Créteil, France
2
Laboratoire de Génétique Moléculaire, Faculté des Sciences Pharmaceutiques et Biologiques, 4 Av de l’Observatoire, 75006 Paris, France
3
Service de Cytogénétique, Hôpital de Hautepierre, 67098 Strasbourg, France
a author for
correspondence: fax 33-1-4981-2842, e-mail girodon{at}im3.inserm.fr
Cystic fibrosis (CF; MIM No. 219700), the most common autosomal recessive disease in Caucasians (1), is caused by mutations in the gene encoding CF transmembrane conductance regulator (CFTR; MIM No. 602421) (2). The disease may be revealed by fetal bowel hyperechogenicity during routine ultrasonography in the second trimester of pregnancy, even when there is no family history of CF. However, fetal bowel hyperechogenicity is not specific for CF: when found at this stage of pregnancy, it corresponds to CF in 2–20% of cases (1)(3)(4)(5). Diagnostic investigations are based on screening for CF-causing mutations, fetal karyotyping, and screening for infections. Normal intestinal enzyme activities in amniotic fluid before 18 weeks of gestation reasonably rules out CF (6). However, most women are referred after this time, and it is necessary to screen for CF mutations in the parents and, if possible, fetus. To date, almost 900 mutations have been described throughout the CFTR gene, but very few deletions have been identified (7). Indeed, large deletions are not routinely screened for, given their rarity and the lack of suitable diagnostic tools. Conventional PCR-based methods usually detect deletions only when they are present in the homozygous state. As a result, the frequency of CFTR deletions is underestimated. Two relatively frequent large deletions have recently been described: 3120+1kbdel8.6kb was found in 13% of CF chromosomes in Israeli-Arab patients (8), and CFTRdele2,3 accounts for 1–6.4% of CF chromosomes in Slavic populations (9).
A couple was referred to our laboratory for fetal hyperechogenic bowel diagnosed during a routine ultrasound scan at 20 weeks of gestation. The couple, who were first cousins of Turkish extraction, had no family history of CF. Diagnostic investigations were requested after genetic counseling, with the couple’s informed consent. The fetal karyotype (amniotic cells) was normal, as were intestinal fetal enzyme activities.
Screening with an oligonucleotide ligation assay (CF-OLA; PE
Biosystems) for 31 CF mutations frequently found among Caucasians
showed a normal pattern in the parents, as in the control, whereas 3 of
the 29 expected peaks were missing in the fetus (Fig. 1A
). Because these three peaks corresponded to two different PCR
products spanning exon 19 of the CFTR gene, a deletion
spanning exon 19 was suspected at the homozygous site in the
fetus, rather than a mutation in the region recognized by either the
PCR-OLA primers or a CF-OLA probe, that prevented ligation of
the labeled products. Southern blotting was not suitable in this
emergency situation to confirm the deletion. In addition, not enough
fetal DNA was available, and in heterozygotes (the parents), only the
detection of a new junction fragment on Southern blot can point to a
gene rearrangement. Indeed, determination of the gene copy number after
hybridization to CFTR cDNA is hazardous because the
intensity of hybridization signals may vary with exon length.
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To firmly confirm the deletion and to rapidly establish the
prenatal diagnosis of CF, we used fluorescent
TaqManTM-based kinetic quantitative
PCR on an ABI PrismTM 7700 Sequence Detection
System (PE Biosystems). We determined the relative copy numbers of
CFTR exon 19 and the albumin gene (ALB), which
served as a reference internal control in each single reaction
(10), in DNA from amniocytes, and from white blood cells of
the parents and a control. The copy number of exon 19 was twofold lower
in each parent’s DNA than in the control DNA and was near zero in the
fetal DNA, confirming the presence of a deletion and strongly
suggesting CF in the fetus (Table 1
). However, given the late stage of gestation, the parents
decided to continue the pregnancy, and a girl was delivered at term.
The meconium was emitted spontaneously but was thick. The diagnosis of
CF was confirmed at 6 weeks of life by a positive sweat test.
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The deletion was restricted to exon 19; PCR products were obtained in
the fetus from exon 18 (not shown) and a fragment of intron 19,
including the site of the 3849+10kbC
T mutation (peak 16 in Fig. 1A
).
To identify the two breakpoints between exons 18 and 20, several
long-range PCR experiments were performed on genomic DNA from cultured
placental cells obtained after birth, using two forward primers located
in intron 18 and two reverse primers located in intron 19, all designed
from the sequence of human BAC clone 133K23 (GenBank Accession No.
AC000061). The expected PCR product obtained from control DNAs was 7762
bp long, whereas an additional 2483-bp fragment was obtained with the
parents’ DNA, corresponding to a 5279-bp deletion. Only the small
fragment was observed in placental DNA.
Direct sequencing of this product identified the deletion breakpoints.
The deletion, starting 15 kb after the last nucleotide of exon 18 and
spanning 5279 bp, was designated 3600+15kbdel5.3kb, or CFTRdele19. The
resulting putative CFTR protein presumably lacks 83 amino acids and,
thus, part of the second nucleotide-binding domain (NBD2), which is
involved in CFTR gating by adenosine triphosphate hydrolysis
(11) and appears critical for CFTR function. This deletion
could be the consequence of nonhomologous recombination, favored by the
possible topoisomerase I recognition sites [(G/C)(A/T)T]
(12) or PyTT sites (13) that lie in the close
vicinity of the breakpoints. Alternatively, it may result from a
replication slippage mechanism, favored by the direct repeated 4-bp
motif (AACT) and the sequence homology around the breakpoints (15 of 20
nucleotides 5' to the breakpoint in intron 18 were identical to 15 of
25 nucleotides 5' to the breakpoint in intron 19; Fig. 1B
). Similarly,
such a short motif has been found at the breakpoints of two other
CFTR gene deletions (8)(9).
We designed a duplex-PCR experiment to detect, in a single step, both the wild-type and the deleted alleles, and to determine the frequency of the deletion in 116 CF patients of various origins in whom one or two mutations remained to be identified (total of 161 CF chromosomes, including 6 from Turkish patients). Forty PCR cycles were run with primer annealing at 55 °C and 0.2 µmol/L each primer. Primers 5'CFdel19 (5'-TACTTGAGGATAGAACCAGGAT-3') and 3'CFdel19 (5'-GCAAATGAAGGTCTGTGAAACT-3'), which flank the deletion breakpoints, yielded a 248-bp fragment in the presence of the deletion, whereas control primers 5'CF19 (5'-GTGAAATTGTCTGCCATTCTT-3') and 3'CF19 (5'-AGGCTACTGGGATTCACTTA-3') amplified a 407-bp fragment that included exon 19. This assay precisely identifies the 3600+15kbdel5.3kb mutation and may be more applicable than long-range PCR in routine laboratories. No other CFTRdele19 alleles were found in the 161 CF chromosomes tested. The deletion should therefore be screened for in a wider population, especially in Turkish patients, to evaluate its frequency and, hence, the relevance of the molecular test. Nevertheless, prenatal diagnosis is now possible for the couple’s future pregnancies, if requested, as well as carrier testing of the family.
This case illustrates the limitations of routinely used CFTR gene screening methods and the value of testing the fetus with hyperechogenic bowel even in the absence of a detectable mutation in the parents. The diagnosis of the deletion of exon 19 here was rapidly confirmed by real-time quantitative PCR, which demonstrated one-half the copy number of exon 19 in each parent compared with a control, and no copy in the fetus. To our knowledge, this is the first report of prenatal CF diagnosis ascertained by this approach.
Only eight large CFTR gene deletions that remove coding
regions have been reported to date (7), probably because
conventional PCR-based techniques are designed to detect point
mutations and miss large deletions. In five cases, as in the case
reported here, deletions were suspected after failure of conventional
PCR amplification targeting particular exons because they were present
in CF patients in the homozygous state. In two others, hemizygosity was
suggested by the lack of heterozygosity for the 
F508 or Y1092X
mutation in one parent of an apparently homozygous CF child
(14)(15). This underlines the need to study the
family when CF patients appear homozygous for a mutation. Partial
hemizygosity may in other cases be shown by segregation studies of
CFTR intragenic microsatellites when the deletions encompass
such polymorphic sites (16), but this approach would
have failed in the case reported here. Nasal epithelial CFTR
transcript analysis may also point to a deletion, in the homozygous or
heterozygous state, as in the case of the very recently described
CFTRdele2,3 deletion (9).
Quantitative PCR on genomic DNA represents an interesting alternative to Southern blotting or pulse field gel electrophoresis for the diagnosis of deletions, especially in heterozygous patients who escape detection through routinely used PCR-based methods. This technique was recently shown to be particularly useful in ascertaining chromosome aneuploidy and gene amplification in cancers (10)(17). Carrier screening for Duchenne and Becker muscular dystrophy and spinal muscular atrophy, in which deletions are frequent and for which quantitative assays have already been developed (18)(19)(20)(21), should benefit from this technical improvement. However, large CFTR genomic deletions are quite rare in CF, although they are likely underestimated. No hot-spot or target site for deletions has been identified on the basis of the few deletions described to date. Therefore, several CFTR probes scattered throughout the gene would be necessary to screen for unknown CFTR gene deletions. Although this remains a technical challenge, the case reported here illustrates the interest to further develop a quantitative PCR-based approach for the diagnosis of unknown CFTR deletions, complementary to the detection of point defects.
Acknowledgments
This work was supported by the Association Française de Lutte contre la Mucoviscidose (AFLM). We thank Josiane Martin and Pascal Vivien for expert technical assistance.
Footnotes
1 these authors contributed equally to this work and should both be considered first authors 
References
The following articles in journals at HighWire Press have cited this article:
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  I. Schrijver, K. Rappahahn, L. Pique, M. Kharrazi, and L.-J. Wong Multiplex Ligation-Dependent Probe Amplification Identification of Whole Exon and Single Nucleotide Deletions in the CFTR Gene of Hispanic Individuals with Cystic Fibrosis J. Mol. Diagn., July 1, 2008; 10(4): 368 - 375. [Abstract] [Full Text] [PDF]
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F. M. Hantash, J. B. Redman, D. Goos, A. Kammesheidt, M. J. McGinniss, W. Sun, and C. M. Strom Characterization of a Recurrent Novel Large Duplication in the Cystic Fibrosis Transmembrane Conductance Regulator Gene J. Mol. Diagn., September 1, 2007; 9(4): 556 - 560. [Abstract] [Full Text] [PDF]
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 F Niel, J Martin, F Dastot-Le Moal, B Costes, B Boissier, V Delattre, M Goossens, and E Girodon Rapid detection of CFTR gene rearrangements impacts on genetic counselling in cystic fibrosis J. Med. Genet., November 1, 2004; 41(11): e118 - e118. [Full Text] [PDF]
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 C. Costa, J.-M. Jouannic, N. Stieltjes, J.-M. Costa, E. Girodon, and M. Goossens Quantitative Real-Time PCR Assay for Rapid Identification of Deletion Carriers in Hemophilia Clin. Chem., July 1, 2004; 50(7): 1269 - 1270. [Full Text] [PDF]
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V N Ninis, M O Kylync, M Kandemir, E Dathly, and A Tolun High frequency of T9 and CFTR mutations in children with idiopathic bronchiectasis J. Med. Genet., July 1, 2003; 40(7): 530 - 535. [Full Text] [PDF]
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