1 Center for Human Genetics and the Departments of Pediatrics and 2 Urology, Boston University School of Medicine and Boston Medical Center, Boston, MA, USA
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Abstract |
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Key words: congenital bilateral absence of the vas deferens/cystic fibrosis/genetic testing/mass spectrometry/mutation detection
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Introduction |
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In 1971, Holsclaw et al. showed that bilateral vassal agenesis was present in almost all male CF patients (Holsclaw et al., 1971). Our studies (Anguiano et al., 1992
) and those of others (Dumur et al., 1990
; Osborne et al., 1993
; Culard et al., 1994
) showed that otherwise healthy men with CBAVD frequently had cystic fibrosis transmembrane regulator (CFTR) gene mutations. This observation led to our suggestion that CBAVD was a primarily genital form of CF (Anguiano et al., 1992
). Some CFTR gene mutations associated with CBAVD are uncommon in CF patients. In CBAVD, compound heterozygosity is common, there being mostly one severe and one mild mutation or two mild mutations (Chillon et al., 1995
; Costes et al., 1995
; Zielenski et al., 1995
; Dork et al., 1997
; Kanavakis et al., 1998
). The 5-thymidine variant of the polythymidine tract (IVS8-5T) in the splice acceptor site of intron 8 is one of the mild mutations that were discovered to be present with high frequency in CBAVD patients (Chillon et al., 1995
; Costes et al., 1995
; Zielenski et al., 1995
; Bienvenu et al., 1997
). This variant has been demonstrated to cause a high level of exon 9 skipping, leading to a non-functional CFTR protein (Chu et al., 1993
; Bienvenu et al., 1997
). The variable level of expression of this 5T allele explains the incomplete penetrance of this allele, resulting in CBAVD and a mild form of CF (Mak et al., 1997
; Rave-Harel et al., 1997
; Cuppens et al., 1998
).
Routine, but limited, mutation analysis for CBAVD men has revealed only a minority in which two distinct mutations were detected. Given the frequency of CF mutations, especially in the Caucasian population (1 in 25), and the common request by CBAVD men to sire their own offspring by using surgical sperm aspiration in conjunction with ICSI, we sought to determine whether a more extensive mutation analysis might prove to be of benefit for genetic counselling and risk estimation, as well as for subsequent prenatal diagnosis. We studied a cohort of men with clinically diagnosed CBAVD using matrix-assisted laser desorption ionizationtime of flight (MALDI-TOF) mass spectrometry to analyse an approximately hierarchical set of the most common 100 CF mutations.
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Materials and methods |
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Methods
25 mutation panel (CF25):
Genomic DNA was extracted from peripheral blood from each patient. The polymerase chain reaction (PCR) was performed on genomic DNA by the standard method. The PCR-amplified products from each patient were digested with specific restriction enzymes prior to gel electrophoresis. The mutations in the 25 mutation panel were: F508, G542X, N1303K, G551D, W1282X, 17171G
A, R553X, 621+1G
T, R1162X, 2183AA
G, R117H,
I507, R560T, 3849+10kbC
T, S549N, S549I, S549R, R1283M, R1283K, R553G, R560K, R117L, 1774delCT, 1811+1G
C, and 400661del14.
ACMG 25 mutation panel (ACMG25):
The following mutations are the recommended core mutations for general population CF carrier screening by American College of Medical Genetics (ACMG) (Grody, et al 2001):
F508, G542X, N1303K, G551D, W1282X, 17171G
A, R553X, 621+1G
T, R1162X, R117H,
I507, 1898+1G
A, G85E, R347P, A455E, R560T, R334W, 3849+10kbC
T, 3659delC, 1078delT, 2789+5G
A, 711+1G
T, 2184delA, 3120+1G
A and I148T. These mutations are included in the CF100 mutation panel below.
100 mutation panel (CF100).
DNA extraction and PCR amplification were carried out the same way as in the routine CF25 mutation panel, except that the reverse primer had a universal sequence tail, and a third primer of the same universal sequence was also added to the reaction. The third primer was also 5' end-labelled with biotin which attaches to the streptavidin coated magnetic beads. The exons of the CFTR gene amplified were 3, 4, 5, 6a, 7, 9, 10, 11, 12, 13, 14b, 15, 16, 17b, 19, 20, 21 and their immediate flanking intronic sequences as well as intron 19. PCR primers were selected to cover whole exons, except for 13 and 17b, where only partial exons were amplified. Three or more exons were co-amplified in a multiplex PCR, followed by a multiplex primer extension of the oligonucleotides at the mutation sites. The extended diagnostic products were then measured by MALDI-TOF mass spectrometry. The CF100 panel includes the most common mutations and the 5T allele as well as other less common mutations (Table I) (Cystic Fibrosis Genetic Analysis Consortium, 1994
; Zielenski and Tsui, 1995
; Estivill et al., 1997
). This assay was validated in blinded analyses of all the mutations in the CF25 panel plus the 5T, 100% concordance being achieved in repeated assays.
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MALDI-TOF.
This is a technology that enables the mass of large biomolecules, like DNA, to be measured directly (Hillenkamp et al., 1991; Little et al., 1997
). DNA is combined with a small organic compound (the matrix) that is able to absorb energy from a laser beam. The DNA and matrix solution is dried onto the surface of a Sequenom SpectroCHIP microarray to produce a crystalline dispersion. Once the sample is introduced into the mass spectrometer and air is removed, a laser pulse causes a spontaneous volatilization and ionization of matrix and associated DNA fragments. These gas-phase ions are accelerated through a voltage potential and their TOF is recorded. The TOF results are then converted into mass values that are, in turn, translated into diagnostic results. As an example, a MALDI-TOF mass spectrum provides the diagnostic results in Figure 1
.
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Data analysis.
CBAVD patients samples were subjected to our new CF100 mutation panel and assay results compared with our earlier routine CF25 mutation panels, as well as the recently announced ACMG25 mutation panel as if it were tested separately. Results were also analysed including the 5T allele for the CF25 and ACMG25 panels (Tables II and III).
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Results |
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CFTR genotypes in CBAVD patients
In CF25 mutation analysis, 49/92 (53.3%) of the patients had at least one mutation identified, including three (3.3%) with two mutations and 46 (50.0%) with one mutation (Table III). The ACMG25 panel, if performed, would have identified 50/92 (54.3%) patients with at least one mutation, including a new heterozygote and a second mutation on a previously considered heterozygote by CF25 analysis. Further analysis of the 5T allele yielded an increase in the total number of patients with at least one mutation by 20.4% (from 49 to 59 patients) for the CF25 + 5T and 20.0% (from 50 to 60 patients) for the ACMG25 + 5T. However, the number of the patients detected with two mutant alleles was 6.8- and 8.6-fold greater than with the ACMG25 and CF25 panels without the 5T allele. CF100 analysis resulted in even greater increases of 26.5% (from 49 to 62 patients) and 24.0% (from 50 to 62 patients), when compared with the CF25 and ACMG25 panels respectively, in the total number of patients with at least one mutation. The number of the patients detected with two mutant alleles increased 8- and 11-fold respectively compared with the ACMG25 and CF25 panels. Here, our routine CF25 and the ACMG25 panels would miss 4/92 (4.3%) patients with two mutations and 89/92 (8.79.8%) patients with one mutation. In addition, 2526/33 (7679%) compound heterozygotes (two mutations) would be incorrectly identified as heterozygotes (Table III
).
Through CF100 mutation analysis, 33/92 (35.9%) of the CBAVD patients were found to have two mutations and 29/92 (31.5%) had one mutation, and in 30/92 (32.6%) no mutation was detected. Compound heterozygosity accounts for 94% (31/33) of the patients with two mutations. Two homozygotes for 5T and D1270N were also detected. The majority of the compound heterozygotes had genotypes of one severe and one mild mutation (Table III). The most common was the genotype of
F508 and 5T (16 patients). Two relatively frequent compound heterozygotes were F508/R117H (3/33) and W1282X/5T (4/33). Unilateral renal agenesis was noted in 4/92 patients, one of whom had the
F508 mutation.
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Discussion |
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The distribution of mutation genotypes in CBAVD was clearly different from that seen in the CF population. Of our 33 patients with two mutations, 30/33 (90.9%) of the patients were compound heterozygotes of severe/mild and 3/33 (9.1%) had mild/mild mutations. In CF, the common mutation genotypes are homozygotes or compound heterozygotes with severe mutations such as F508 and others (i.e. nonsense or frameshift mutations). Compound heterozygotes and homozygotes of severe/severe mutations were not found in our cohort of CBAVD men. These results are consistent with those reported previously (Chillon et al., 1995
; Mercier et al., 1995
; Dork et al., 1997
; Mak et al., 1999
; Claustres et al., 2000
), reflecting the general observation of compound heterozygosity with severe/mild or mild/mild mutations in CBAVD patients. In a large French CBAVD study (Claustres et al., 2000
), the authors found that in CF, 87.77% of patients carried two severe CF mutations, whereas only 11.33% had a severe mutation in trans with a mild mutation. In contrast, none of their CBAVD patients had two severe mutations, all having severe/mild or mild/mild mutation combinations. The high frequency of mild or very mild CFTR mutations in patients with CBAVD led us and others to the hypothesis that the vas deferens is one of the tissues most susceptible to the effect of changes in CFTR activity and that CBAVD is a primarily genital form of CF (Anguiano et al., 1992
; Oates and Amos, 1993
; Mercier et al., 1995
). The one unilateral renal agenesis patient with CBAVD and a
F508 mutation may be a chance finding for a phenotype due to a different aetiology and pathogenesis (McCallum et al., 2001
).
The compound heterozygote F508/R117H, previously reported to occur commonly in CBAVD patients (Dork et al., 1997
), was also a frequent genotype (3/33) in this study. It was noted that R117H occurred on two chromosomal backgrounds, one carrying a 5T allele in CF patients and the other carrying a 7T allele in CBAVD patients, when the other chromosome carries a severe mutation such as
F508 (Kiesewetter et al., 1993
). A strong linkage between
F508 and the 9T allele in cis on a chromosome has been reported in several studies (Chu et al., 1993
; Kiesewetter et al., 1993
; Cuppens et al., 1994
). It is interesting that, in this study, 7T/9T was the only haplotype found in all three CBAVD patients with
F508/R1I7H. Although it was not confirmed, our results indicate that
F508 and R117H were on chromosome backgrounds of 9T and 7T respectively, because at least one 9T allele was seen in each one of the 41 patients having a
F508 mutation, a 7T in each of the four patients with R117H mutation. Therefore, our results tend to lend further support to the concept of
F508 mutation originally arising on a single chromosome background, possibly with 9T. The association of R117H in cis with a 5T allele results in CF when patients carry in trans
F508 or one of the severe mutations, and probably represents a general theme that a splicing mutation such as 5T, in cis with a mild mutation, can aggravate that mild mutation. This observation could have clinical implications in genetic counselling of patients with mild mutations, especially for those compound heterozygotes with severe/mild mutations.
L206W is another mild mutation reported with relatively high frequency in both CF and CBAVD patients (Chillon et al., 1995; Mak et al., 1999
; Claustres et al., 2000
) and which we noted in three compound heterozygotes, two with
F508 and one with 5T. It appears that L206W is associated in cis with the 9T allele since both
F508/L206W patients have a homozygous 9T background, while 5T/L206W exists in a 5T/9T background.
The 5T allele is the second most frequent mutation (33/95) in this study, after the F508 which had a frequency of 39/95. The frequent 5T allele was previously proposed to be a partially penetrant and leaky splicing mutation (Chillon et al., 1995
; Costes et al., 1995
; Zielenski et al., 1995
). Homozygotes for 5T in our one CBAVD patient and in others add weight to the argument that the 5T allele is a disease-causing mutation resulting in mild CF or CBAVD (Cuppens et al., 1998
) and probably the most common one of the CFTR gene, since it has a 5% allele frequency in most investigated populations (Kiesewetter et al., 1993
; Chillon et al., 1995
). Partial penetrance could be a feature not only of the 5T allele but also of other missense mutations or other variants (polymorphisms).
CFTR gene mutation analysis is recommended for the female partners of men with CBAVD planning surgical sperm aspiration coupled with ICSI (Johnson, 1998; Lawler and Gearhart, 1998
). Negative results for the female partner by testing for only the major CF mutations still leave residual risk of having affected offspring. Therefore, the most extensive and least expensive test that contains both the major and less common/mild mutations is strongly recommended. Our CF100 mutation analysis helps diminish this risk. One of the residual difficulties in counselling the family with CBAVD is the lack of a clear genotypephenotype correlation, which precludes prediction of a specific phenotype.
In summary, we have developed an accurate assay for 100 CF mutations including not only the mutations found in classical CF but also mild/less common mutations that have been found in both CF and CBAVD patients. We have further demonstrated the utility of mass spectrometry (MALDI-TOF) as a diagnostic tool with its inherent qualities of high accuracy and ease for automation. When compared with the two routine, but limited, CF mutation panels, our CF100 panel detected 4.3% more homozygotes and compound heterozygotes and 8.0% more heterozygotes. It also identified a second mutation in >50% of those considered heterozygotes under CF25 and ACMG25 analyses. Excluding IVS8-5T, CF100 mutation analysis would identify one newly recognized mutation for every seven carriers found under the ACMG25 panel analysis. This panel provides significantly greater opportunities for mutation detection especially for men with CBAVD, their partners, the partners of known CF carriers, and for those with mild CF with rarer mutations.
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Notes |
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References |
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Submitted on January 10, 2002; accepted on April 8, 2002.