Molecular Human Reproduction, Vol. 9, No. 7, 421-427,
July 2003
© 2003 European Society of Human Reproduction and Embryology
Article |
Specific detection of deleted and non-deleted dystrophin exons together with gender assignment in preimplantation genetic diagnosis of Duchenne muscular dystrophy
Submitted on January 15, 2003; accepted on March 6, 2003
1 Laboratoire de Génétique Moléculaire, Centre Hospitalo-Universitaire (CHU) and Institut Universitaire de Recherche Clinique (IURC), 641 Avenue du Doyen Gaston Giraud, 34093 Montpellier cedex 5, 2 Service de Gynécologie Obstétrique and 3 Service de Génétique Médicale, Hôpital Arnaud de Villeneuve, 371 Avenue du Doyen Gaston Giraud, 34295 Montpellier cedex 5, France 4 To whom correspondance should be addressed. e-mail: girardet{at}igh.cnrs.fr
 |
ABSTRACT |
|---|
 Top ABSTRACT IntroductionMaterials and methodsResultsDiscussionREFERENCES |
|---|
We have developed a preimplantation genetic diagnosis (PGD) strategy for Duchenne muscular dystrophy (DMD) allowing the simultaneous amplification of four exons (6, 8, 28 and 32) of the dystrophin gene together with ZFX/ZFY genes for gender determination. Preliminary experiments were carried out on 215 single lymphocytes from male and female individuals. Amplification rates ranged from 90.2% for exon 6 to 96.7% for exons 8 and 32. At least four of the five sequences were successfully amplified in 95.8% of single cells, and sexing was possible in 98.5%. This 5-plex assay was found to be robust enough to be used in a PGD clinical procedure and was therefore applied to a family whose female partner was a heterozygous carrier of a large deletion extending from exon 21 to exon 34 of the dystrophin gene. We have thus analysed two exons located in the deleted region of the gene, two non-deleted exons used as intrasample controls, and ZFX/ZFY genes. Cleavage stage embryo biopsy followed by PCR resulted in transfer of three unaffected embryos. The advantage of the present approach is to identify and subsequently transfer unaffected male embryos in addition to female embryos, and is now applicable to all families displaying a deletion involving at least one of these exons.
Key words: deletion/Duchenne muscular dystrophy/preimplantation genetic diagnosis/sex determination
|
Introduction |
|---|
|
TopABSTRACTIntroductionMaterials and methodsResultsDiscussionREFERENCES |
|---|
Duchenne muscular dystrophy (DMD; OMIM accession number: 310200) is the most common inherited neuromuscular disorder, affecting about 1 in 3500 newborn males (Emery, 1991). The disease is characterized by progressive muscle degeneration resulting in death in the second decade of life. DMD is an X-linked disease caused by mutations in the dystrophin gene located at Xp21 locus (Koenig et al., 1987). This gene is the largest human gene identified, consisting of 79 exons spanning
2.5 Mb of genomic DNA (Roberts et al., 1993). It encodes dystrophin, a 427 kDa membrane-associated protein (Hoffman et al., 1987), which, through its interaction with other proteins, participates in the linkage of the extracellular matrix to the cytoplasmic cytoskeleton (Ervasti et al., 1991). Dystrophin is either absent or greatly reduced from the muscles of DMD patients (Hoffman et al., 1988). About 60% of DMD patients exhibit large deletions of one or more exon(s) clustered in a major and a minor hotspot region at the proximal and central portion of the gene respectively (Koenig et al., 1987). These deletions can be detected by hybridization with cDNA probes (Darras and Francke, 1988) or more commonly by multiplex PCR amplification of selected exons (Chamberlain et al., 1988; Beggs et al., 1990) allowing the detection of 90–95% of the deletions in male patients. Approximately 5% of mutations are partial gene duplications detected by Southern blot analysis (Den Dunnen et al., 1989) or by multiplex amplifiable probe hybridization (White et al., 2002). Substitutions, small insertions or deletions of one or more nucleotide(s) are responsible for the deficiency in 30–35% of the cases (Tuffery et al., 1998) and are much more difficult to identify because they are heterogeneous and randomly distributed throughout the extremely large dystrophin gene.
No efficient treatment has been developed so far for DMD. Genetic counselling and molecular diagnosis that allow the identification of carriers are therefore the only solutions offered to DMD families. Carrier detection and DNA-based prenatal diagnosis using either chorionic villi or amniotic fluid may be performed when the disease-causing mutation has been detected in an affected individual or when informative linked markers have been identified in the family (Kim et al., 2002). However, the use of these highly polymorphic markers in a linkage-based genetic diagnosis is limited by the high new mutation rate—30% of DMD cases are sporadic as the result of de-novo mutations in the dystrophin gene—the high intragenic recombination frequency (10%) (Abbs et al., 1990) and the frequent absence of a suitable family structure.
Preimplantation genetic diagnosis (PGD) offers an alternative to prenatal diagnosis for couples at high risk of transmitting inherited disorders to their children. The procedure involves biopsy and DNA analysis of one or two blastomeres from cleavage stage embryos obtained as a result of IVF treatment (Handyside et al., 1990). Only unaffected embryos are transferred to the mother’s uterus, thereby avoiding the eventual termination of pregnancy when fetuses are diagnosed as affected following prenatal diagnosis.
Clinical applications of PGD for DMD have been reported by several groups using different approaches. For X-linked recessive genetic diseases such as DMD, when no specific PCR test is available, gender determination may be performed to select clinically unaffected, carrier or non-carrier females, thereby avoiding the birth of potentially afflicted males. In PGD procedures, this approach has already been used both by fluorescence in-situ hybridization (FISH) (Staessen et al., 1999) and PCR techniques (Handyside et al., 1990); nevertheless, this diagnostic strategy displays the disadvantage that all male embryos are discarded, although half of them are not affected. The approach proposed by Hussey et al. (1999) relies on the single cell duplex amplification of a specific DMD exon (17, 19, 44, 45 and 48), together with the SRY gene for gender assignment, and is therefore suitable for families displaying a deletion involving at least one of these exons. Ray et al. (2001) have recently reported a single cell protocol for DMD diagnosis allowing the simultaneous analysis of the frequently deleted exons 8, 19, 45, 47 and 51 of the DMD gene together with the amelogenin gene, applicable to the detection of 70% of all dystrophin gene deletions. However, there are no reports to date of these two latter techniques being applied clinically. Alternative techniques including linkage analysis using two polymorphic markers (Lee et al., 1998) and preamplification of the whole genome coupled with subsequent DNA analysis of selected dystrophin exons (Kristjansson et al., 1994) have also been developed. These alternative techniques were not extensively applied or applied at all to the study of human blastomeres in clinical PGD procedures. The first specific PGD for DMD has been performed for a family carrier of a deletion encompassing exon 17 of the dystrophin gene, through the study of this exon in single blastomeres (Liu et al., 1995).
We describe here a single cell multiplex PCR protocol allowing the analysis of four exons of the dystrophin gene, together with ZFX/ZFY genes for sex determination of embryos. This technically improved approach was used for the first time in a PGD procedure for a family displaying a large deletion in the DMD gene, giving the ability to analyse two exons located in the deleted region of the gene, together with two non-deleted exons used as intrasample controls, and ZFX/ZFY genes.
|
Materials and methods |
|---|
|
TopABSTRACTIntroductionMaterials and methodsResultsDiscussionREFERENCES |
|---|
Family description
A 34 year old woman and her 38 year old husband with no fertility problems presented to our unit requesting information about PGD for DMD.
One of the wife’s brothers had died of DMD at the age of 14 years and her youngest brother was affected. Molecular analysis of an affected brother’s dystrophin gene was initially performed by multiplex PCR and revealed a 14 exon deletion extending from exon 21 to exon 34 of the dystrophin gene.
The woman’s serum creatine kinase levels were previously found to be markedly raised, giving evidence of her carrier status. Two prior consecutive pregnancies were investigated by chorionic villus sampling analysis in the first trimester of pregnancy. In both instances, as the disease-causative mutation was not yet identified, a sex diagnosis obtained by PCR and confirmed by karyotypes revealed that fetuses were girls and pregnancies were continued.
After genetic counselling of the couple with regard to their 50% risk of having an affected boy, they gave informed consent to perform PGD. This study was approved by the institutional ethical comittee and by the local multidisciplinary network licensed for prenatal diagnosis, in accordance with French law.
Sampling of single lymphocytes
Venous blood samples (10 ml) from male and female individuals, including both parents, were collected into heparinized tubes. Blood was then diluted 2-fold with Dulbecco’s phosphate-buffered saline (PBS) (Gibco BRL, France), layered over Ficoll-Paque gradient (Sigma, France) and centrifuged at 1000 g for 20 min. The interface containing the mononuclear cell fraction was collected and cells were washed three times with PBS. The cells were then resuspended in 1 ml Roswell Park Memorial Institute (RPMI) medium (Invitrogen, France) containing 10% dimethylsulphoxide (Sigma) and frozen at –80°C until use. For isolation of single lymphocytes, an aliquot of the cell suspension was thawed and 1 µl was added to 4 ml RPMI supplemented with 10% fetal calf serum (Eurobio, France) in a culture plate. Single lymphocytes were aspirated using hand-drawn microcapillaries under an inverted microscope and transferred to 200 µl PCR tubes containing 3 µl of alkaline lysis buffer (ALB; 200 mmol/l KOH, 50 mmol/l dithiothreitol) (Cui et al., 1989). Aliquots from the last washing droplets were taken to serve as blanks to track contamination. Moreover, additional tubes containing ALB were also processed in the same conditions as were single lymphocytes to check absence of contamination in the lysis buffer, PCR mixes and microtubes. Samples were lysed by incubation at 65°C for 10 min, then either used immediately for PCR or stored at –20°C until further processing.
No blood sample from an affected boy of the family was available for this PGD test optimization.
ICSI and embryo biopsy
Ovarian stimulation, ICSI procedure and blastomere biopsy were performed as previously described (Girardet et al., 2003). Briefly, following ovarian stimulation, oocytes were retrieved by vaginal ultrasound-guided puncture 36 h after hCG administration. Mature metaphase II oocytes were microinjected with a single sperm in order to avoid sperm contamination, then fertilization was assessed 16–18 h after injection. Sequential media SM1/SM2 (Medicult, Denmark) were used for embryo culture. Further development of the normally fertilized oocytes was evaluated on days 2 and 3. Blastomere biopsy was performed on the early morning of day 3, using a laser for zona pellucida dissection. One blastomere (embryos ≤6 blastomeres) or two blastomeres (embryos ≥7 blastomeres) were aspirated and released into the surrounding medium, then embryos were immediately transferred back into S2 medium and incubated until selection and transfer. Biopsied blastomeres were visually checked for the presence of a nucleus and transferred to microtubes containing 3 µl of ALB. A 1 µl aliquot of each embryo culture medium was transferred to a separate tube containing 3 µl of ALB and examined by PCR to assess the incidence of contamination with exogenous DNA. Moreover, for each embryo biopsied, a sample of media from each drop in which the blastomeres had been washed was placed in a PCR tube containing ALB. We also included several negative control tubes containing ALB that remained opened either in the area of embryo micromanipulation (biopsy laboratory) or in the laminar flow hood where PCR mixes were prepared (pre-PCR laboratory). All these tubes were processed as for the blastomeres. Samples were lysed at 65°C for 10 min then immediately used for amplification.
Single cell PCR
For the PGD, since the female patient was known to be heterozygous for a deletion of exons 21–34, a multiplex PCR strategy was chosen and designed in order to study exons 6 and 8 located outside the deleted region, exons 28 and 32 involved in the deletion, and ZFX/ZFY genes for gender determination.
The first round multiplex PCR was followed by five specific nested PCR allowing the amplification of exons 6, 8, 28 and 32 of the dystrophin gene, as well as ZFX/ZFY genes. Some of the primers have been previously described (Chamberlain et al., 1988; Beggs et al., 1990; Abbs and Bobrow, 1992; Chong et al., 1993), while others had to be specially designed based on sequences of the human dystrophin gene (http://www.dmd.nl/md.html), in order to perform two rounds of PCR (Table I). The first round PCR was performed in a volume of 30 µl containing 3 µl of neutralization buffer (900 mmol/l Tris–HCl pH 8.3, 300 mmol/l KCl, 200 mmol/l HCl), 3 µl of potassium-free amplification buffer [100 mmol/l Tris–HCl pH 8.3, 15 mmol/l MgCl2, 0.1% (w/v) gelatin], 2 mmol/l dNTP (Amersham Biosciences, France), 0.25 µmol/l of each outer primer (Table I), and 1 IU Taq DNA polymerase (Applera, France). The program used was 4 min at 96°C, 25 cycles of 20 s at 96°C, 20 s at 62°C, and 40 s at 72°C, then 10 min at 72°C. Three microlitres of the first round PCR products were then reamplified in a second PCR in five separate reactions. All PCR reactions contained 3 µl of reaction buffer [500 mmol/l KCl, 100 mmol/l Tris–HCl pH 8.3, 15 mmol/l MgCl2, 0.1% (w/v) gelatin], 2 mmol/l dNTP, nested primers according to the locus analysed (Table I), and 1 IU Taq DNA polymerase, in a total volume of 30 µl. The PCR cycling conditions are described in Table I. All reactions were cycled in Gene Amp 9700 and Gene Amp 2400 PCR machines (Applied Biosystems).
|
During the PGD cycle, microtubes containing single lymphocytes from male and female individuals were run simultaneously with biopsied blastomeres as positive controls for DNA amplification and gender assignment.
In all experiments, stringent precautions were taken to prevent contamination: preparation of reagents and PCR mixes was carried out in a positive pressure room with all materials and supplies restricted to this activity, and analysis of PCR products was performed in a laboratory separated from the pre-PCR room in order to exclude carry-over DNA contamination. Moreover, the blastomere biopsy area was physically distinct from the molecular genetics laboratory.
Analysis of PCR products
Following the nested PCR, 10 µl of amplification products were electrophoresed on 1.5% agarose gels (Eurobio, France). Deletion was diagnosed when the three bands corresponding to exons 6, 8 and ZFX/ZFY genes were present, and the two bands corresponding to exons 28 and 32 were absent from the studied blastomeres when compared with non-deleted controls.
Sex determination was performed by mixing 15 µl of the ZFX/ZFY second-round PCR products with 10 IU of the restriction enzyme HaeIII (Ozyme, France) and 3 µl of the supplied buffer, in a total volume of 30 µl. After incubation at 37°C for 2.5 h, the digestion products were electrophoresed on 3% low melting agarose gels (Eurobio) containing ethidium bromide, for 40 min at 120 V, then visualized by UV transillumination.
Reanalysis of untransferred embryos
The day following the PGD clinical procedure, the remaining cells of untransferred cleavage stage embryos were removed from the zona pellucida, collected into microtubes containing lysis buffer, and exposed to PCR analysis to confirm the initial diagnosis.
|
Results |
|---|
|
TopABSTRACTIntroductionMaterials and methodsResultsDiscussionREFERENCES |
|---|
Lymphocyte testing
In order to set up a multiplex PCR assay that amplified exons 6, 8, 28 and 32 of the DMD gene as well as ZFX/ZFY genes, we first determined the best conditions to perform reproducible PCR for each sequence: PCR conditions were separately set up for each locus on single lymphocytes, then additional primer sets were included into the PCR reaction mixes until obtaining satisfactory results for the five sequences. PCR success was indicated by the presence of a single, specific and correctly sized band of reasonable intensity for each sequence when analysed on agarose gels.
The efficiency and accuracy of the multiplex PCR was evaluated in preclinical experiments on 215 single lymphocytes isolated from male (91 lymphocytes) and female individuals (124 lymphocytes). A summary of the amplification results is shown in Table II. Amplification rates ranged from 90.2% for exon 6 (194/215) to 96.7% for exons 8 and 32 (208/215). Successful amplification of the five sequences was achieved in 87.4% (188/215) of single lymphocytes and 95.8% (206/215) were amplified for at least four of the five loci analysed. Out of the 18 single cells that were amplified for four loci, failure of amplification happened 14 times for exon 6, twice for exon 28, once for exon 32 and once for ZFX/ZFY genes. No amplification signal for any sequence was observed in 1.9% (4/215) of single lymphocytes, probably because individual cells were not successfully transferred into the reaction tubes. All 20 blank controls, corresponding to the medium washing drops, included in the single cell test series were negative, and none of the tubes containing only the lysis buffer displayed amplification signals on agarose gels. All these data indicate absence of contamination.
|
Gender assignment was determined following HaeIII digestion of ZFX/ZFY nested PCR products; for XX cells, the 344 bp second round PCR product was cut into two fragments of 300 and 44 bp whereas XY cells yielded four fragments of 300, 216, 84 and 44 bp because of an additional HaeIII restriction site on the Y chromosome. An accurate predicted digestion pattern (two correctly sized bands for XX cells and four correctly sized bands for XY cells) was obtained in 96.1% (199/207) of the cells. However, sexing was still possible in a slightly larger proportion of single cells (204/207, i.e. 98.5%): when the three bands of 216, 84 and 44 bp are visualized and the X-specific band of 300 bp is missing, it can be concluded that the cell is from an XY individual, as all lymphocytes should have at least one X chromosome. Similarly, the visualization of three bands of 300, 216 and 44 bp or 300, 84 and 44 bp indicates that the cell is XY even though a typical band related to the XY pattern is missing. A misdiagnosis (male diagnosed as female due to the loss of the Y allele) was observed in three cells (1.4%), but no contamination with a Y sequence was detected in XX cells that always displayed only two digested bands of 300 and 44 bp. The analysis of single lymphocytes from the male individual that gave a positive amplification signal provided information on the degree of allele drop-out (ADO) at the ZFX/ZFY locus that occurred in four out of 89 (4.5%) male lymphocytes: once for X chromosome and three times for Y chromosome as shown by the absence of the corresponding fragments.
PGD cycle
During the PGD cycle, a total of 20 oocytes was collected after stimulation. Eighteen of these were at the metaphase II stage and were injected using the ICSI technique. Eleven (61.1%) showed normal fertilization as indicated by the presence of two pronuclei 16 h after ICSI, resulting in 10 embryos on day 3 post-insemination. Two embryos were too poor quality to be biopsied, one of them being largely fragmented. Eight embryos were developing normally and found to be of good quality as they reached the 4-cell stage on day 2 and the 6–9-cell stage in the morning of day 3. These eight embryos were biopsied according to their morphology and developmental stage (biopsy of one or two blastomeres). In the cohort of biopsied embryos, blastomeres from two embryos (E4 and E10) showed a tendency to adhere to each other making the biopsy procedure slightly longer.
Table III summarizes the ICSI-PGD cycle performed for the couple. A total of 12 blastomeres from eight embryos were analysed. Of these, 11 showed amplification signals (91.7%). Figure 1 illustrates the amplification products of the dystrophin exons and X and Y sequences in two biopsied blastomeres from two different embryos. One blastomere (14.2) lysed during the biopsy procedure but was transferred into a microtube and gave PCR signals. Five embryos were unambigously unaffected (E5, E11, E14, E17, E19) and two displayed a deletion for exons 28 and 32 (E10 and E12). One blastomere did not amplify for exon 8 and ZFX/ZFY genes (E16) but showed amplification signals for exons 28 and 32. Therefore, this embryo is most probably unaffected. All single lymphocytes used as positive controls during the PGD procedure were efficiently amplified, providing results for the five sequences. None of the negative controls tested showed amplification signals of any products on agarose gels, consistent with absence of exogenous DNA contamination.
|
|
Following HaeIII digestion of ZFX/ZFY second round PCR products, a gender diagnosis was obtained for five embryos, while ambiguous results consisting of faint digested bands were visualized for three embryos thus not allowing an accurate discrimination between X and Y sequences. The combination of both exon analysis and sex determination revealed two affected male embryos (E10, E12), a normal male embryo (E19), two female embryos (E14, E17), as well as two non-affected embryos (E5 and E11) whose sex diagnosis could not be established, and one probably non-affected embryo (E16) without sex diagnosis result. Figure 2 shows electrophoresis profiles as obtained on single blastomeres along with male and female single lymphocytes, after digestion by HaeIII restriction enzyme. Embryos with the best morphology and evidence of cleavage post-biopsy were selected for transfer. According to these criteria, three unaffected embryos (E14, E17, E19) were transferred back to the patient in the evening of day 3, but hCG levels on day 10 after embryo transfer were not consistent with implantation.
|
The results obtained in the clinical cycle were confirmed when blastomeres were disaggregated from untransferred embryos. No discrepancies were observed between the analysis performed on the single blastomeres and the reanalysis carried out on the whole embryos. Moreover, as expected, the embryo for which no amplification product was visualized for exon 8 and ZFX/ZFY genes (E16) was healthy, and the three embryos for which no sex diagnosis could be obtained (E5, E11 and E16) were actually female, male and female embryos respectively.
|
Discussion |
|---|
|
TopABSTRACTIntroductionMaterials and methodsResultsDiscussionREFERENCES |
|---|
We have developed a PGD protocol for DMD, allowing the analysis of four dystrophin exons combined to ZFX/ZFY genes. This procedure was applied to a family displaying a deletion involving 14 exons of the dystrophin gene (exons 21–34). Although various strategies have been reported for PGD of DMD, we describe here the first PGD clinical case allowing the simultaneous analysis of both deleted and non-deleted dystrophin exons together with sex-specific sequences, whose obvious advantage is to identify and subsequently transfer female as well as non-affected male embryos from the cohort of embryos under investigation. In the present protocol, the study of ZFX/ZFY genes provides a double-check for the results obtained following analysis of the four dystrophin exons.
For the setting up of this PGD assay, it was decided to study two exons located outside the deletion to obtain an internal control of the amplification reaction, which, in addition to the analysis of two exons involved in the deleted region, provides secure information on the diagnostic obtained. Thus, several sets of primers were designed and tested to amplify DMD exons at the single cell level (exons 3, 4, 6, 8, 19, 27, 28, 29, 31, 32), first in singleplex reactions, then combined with other primer sets (data not shown). As a result of these preliminary experiments, the optimum amplification efficiency and accuracy were achieved with exons 6, 8, 28, 32 and ZFX/ZFY when used in a multiplex reaction. In a preclinical setting, we then tested this single-cell multiplex PCR protocol on 215 single lymphocytes from male and female individuals. Of the 215 single cells tested, 87.4% amplified all four DMD exons and ZFX/ZFY genes. Amplification efficiency for exon 6 was found to be slightly lower than for other loci. When amplified independently (singleplex reaction), exon 6 provided a high amplification rate that was slightly decreased when used in a multiplex reaction (data not shown). This phenomenon is probably related to suboptimal PCR conditions when primers for exon 6 are combined with the four other primer sets in the first round PCR. In the assay presently described, we studied exons 6 and 8 that were located outside the deletion. Although the amplification rate for exon 6 is lower than for the other sequences, it could not lead to the non-transfer of a healthy embryo as exon 8 is used as a second positive control of amplification. For future diagnoses involving specifically deletions encompassing exon 6, we will try to improve the amplification efficiency for this exon by designing other primers or by modifying the PCR mix composition. A total of 96.7% lymphocytes amplified for either exon 6 or exon 8, in addition to either exon 28 or exon 32, and ZFX/ZFY. This procedure ensures that simultaneous reliable data are obtained from at least one deleted exon and one non-deleted exon. No single lymphocyte failed to amplify for exons 28 and 32 while amplifying for the three other loci. This result predicts a very low risk of misdiagnosis leading to the non-transfer of healthy embryos. Sex determination was accomplished by simultaneous nested amplification of the homologous but non-allelic ZFX and ZFY sequences on the short arms of X and Y chromosomes respectively, by using shared primers, followed by HaeIII digestion of PCR products (Chong et al., 1993). As a result, sexing was possible in 98.5% of single lymphocytes. Two other groups have reported similar results for sex determination in single cells using other strategies, including amplification of the SRY gene (Hussey et al., 1999), and analysis of the amelogenin gene on chromosome X and its Y pseudogene (Ray et al., 2001). However, amplification failure of a Y chromosome-specific repetitive DNA sequence occurred in a clinical procedure and was the first report of PGD misdiagnosis (Handyside and Delhanty, 1993); consequently, current PCR sexing protocols use primers designed to simultaneously amplify both the X and Y chromosomes. Moreover, deletions of Y chromosome-specific amelogenin gene have been observed in a few male patients while amplifying for other Y-specific markers (Steinlechner et al., 2002; Thangaraj et al., 2002). In our experience, although a digestion step is required that undoubtedly makes the diagnostic procedure longer, the ZFX/ZFY system was found to be efficient and appropriate for sexing single cells. The total time for analysis remains compatible with the transfer of embryos within the same day of biopsy. The assay we set up was therefore considered to be robust enough to proceed to clinical application.
The couple underwent a cycle of treatment during which eight preimplantation embryos were available for biopsy. A total of 12 blastomeres was biopsied, 11 of which provided signals following amplification, deletions being easily identified as missing bands for exons 28 and 32, compared with the positive controls. Among the eight embryos that were retrieved and tested, results of both DMD exons and sex assignment were available for five embryos: two were diagnosed as affected males, one was a normal male, and two were female. Sex diagnosis failed for the three other embryos as weak digested bands were obtained following ZFX/ZFY amplification and HaeIII digestion. These three embryos displayed a ZFX/ZFY PCR product signal quite weak compared to products from the five other embryos. Furthermore, because of the time-constraints related to PGD with the transfer of unaffected embryos to the maternal uterus within the day of biopsy, we had to decrease the incubation time of PCR products with HaeIII compared with lymphocytes. Both parameters may explain the absence of a reliable sex diagnosis for these three embryos. Even though two of them were not affected (E5 and E11), the third being most probably not affected (E16), the priority in the embryo transfer was given to the normal, non-deleted embryos whose gender assignment was clearly determined, in order to exclude any probability of misdiagnosis. A total of three embryos was therefore transferred to the mother’s uterus on the evening of day 3 post-fertilization, but unfortunately, no pregnancy ensued. In the cohort of embryos studied, the ratio affected (2/8) versus non-affected (6/8) is in accordance with the transmission of an X-linked recessive genetic disease. No discrepancies were observed between two biopsied blastomeres from the same embryo. In addition, embryos that displayed a deletion for exons 28 and 32 were subsequently always diagnosed as male embryos following HaeIII digestion.
Using the multiplex PCR procedure described here, an affected embryo would be misdiagnosed as normal only if a contaminant was introduced into the PCR tube. The risk is difficult to quantify as no contamination has been detected in all blank samples tested, neither during the PGD test optimization on single lymphocytes, nor during the PGD cycle on human blastomeres. Nevertheless, the accuracy of this diagnostic strategy remains highly dependent upon the absence of DNA contamination throughout the PGD procedure and therefore requires stringent precautions to avoid any source of exogenous contamination. This strategy, based on the absence of amplification signals, has been used during the first PGD clinical application by amplifying DNA sequences found only on the Y chromosome, in order to determine sex of embryos for the avoidance of X-linked disorders (Handyside et al., 1990). A blastomere showing no PCR amplification products was indicative of a female embryo. A few specific strategies for PGD, based upon identification of a deleted sequence, were reported afterwards, mainly for DMD (Kristjansson et al., 1994; Liu et al., 1995) but also for ataxia telangectasia (Hellani et al., 2002).
During the development of this multiplex PCR assay, we tested the method described by Ray et al. (2001) who performed a double-nested multiplex PCR to co-amplify dystrophin exons with X and Y amelogenin genes. In our hands, two rounds of PCR with separate nested primers were found to be more reliable than the double-nested amplification strategy. The ability to adjust the PCR conditions in the second round for each of the different loci independently provided the flexibility to develop the assay with greater sensitivity. However, considering the 215 single lymphocytes that were tested in preliminary experiments, 1290 amplification reactions had to be performed instead of the 430 PCR that would have been otherwise necessary.
In most preimplantation diagnoses performed for monogenic diseases, one or two polymorphic markers are usually studied in addition to the causative mutation, as described for phenylketonuria (Verlinsky et al., 2001) and neurofibromatosis type 2 (Abou-Sleiman et al., 2002) among others. In DMD, such a strategy may also be used to follow the transmission of the mutation in informative families and therefore to confirm a result. However, to assign a valid dystrophin haplotype in order to maximize the chance of detecting recombination events which would otherwise lead to inaccurate predictions, at least three informative markers are needed, one located in the coding region and two flanking markers, one 5' and the other 3' of the gene. Development of the multiplex PCR that would be required under single cell conditions would be highly technically challenging and time-consuming, although it could, at least in theory, be circumvented by preamplification of the whole genome that allows subsequent analysis of multiple sequences (Zhang et al., 1992; Snabes et al., 1994). However, the overall time requirement of the assay remains high and is not compatible with time-constraints related to PGD. Nevertheless, in this kind of analysis where a deletion is diagnosed through the absence of an amplification product, the most important danger is contamination. As reported by others, the study of a highly polymorphic marker located either within the dystrophin gene or on another chromosome may detect the presence of extra-, non-parental alleles thus revealing when contamination by extraneous DNA has occurred (Kuliev et al., 1998; Piyamongkol et al., 2001). A mathematical model was recently developed to assess the risk of misdiagnosis in PGD which includes various parameters such as contamination, amplification efficiency and chromosomal abnormalities among others (Lewis et al., 2001).
In summary, we have described a 5-plex assay allowing the amplification of four exons of the dystrophin gene, in conjunction with ZFX/ZFY analysis for sex determination. This strategy has been applied to a family requesting preimplantation diagnosis for DMD, as the mother was a carrier of a large dystrophin gene deletion of exons 21–34. This assay is now available for couples displaying a deletion of at least one of the exons presently studied. Indeed, exons 6 and 8 are located within the proximal deletion hot spot region of the DMD gene, ranging from exon 2 to exon 20 of the dystrophin gene. Deletion of exons 6 and/or 8 accounts for 16.5% of the deleted exons in DMD patients according to the data from the Leiden muscular dystrophy pages. The advantage of the procedure described here over the use of sex diagnosis techniques for PGD of DMD is that it allows the transfer of non-affected male embryos together with female embryos.
|
Acknowledgements |
|---|
We particularly wish to thank Mr Guy Vergnes and Pr Jacques Bringer for their encouragement and constant support. This work was supported by grants from l’Association Française contre les Myopathies (AFM), Vaincre la Mucoviscidose (VLM), and the Clinical Research Department of the CHU of Montpellier. We are grateful to the CHU of Montpellier for supporting PGD activity and providing the necessary facilities.
|
REFERENCES |
|---|
|
TopABSTRACTIntroductionMaterials and methodsResultsDiscussionREFERENCES |
|---|
Abbs, S. and Bobrow, M. (1992) Analysis of quantitative PCR for the diagnosis of deletion and duplication carriers in the dystrophin gene. J. Med. Genet., 29, 191–196.
Abbs, S., Roberts, R.G., Mathew, C.G., Bentley, D.R. and Bobrow, M. (1990) Accurate assessment of intragenic recombination frequency within the Duchenne muscular dystrophy gene. Genomics, 7, 602–606.[CrossRef][Web of Science][Medline]
Abou-Sleiman, P.M., Apessos, A., Harper, J.C., Serhal, P., Winston, R.M. and Delhanty, J.D. (2002) First application of preimplantation genetic diagnosis to neurofibromatosis type 2 (NF2). Prenat. Diagn., 22, 519–524.[CrossRef][Web of Science][Medline]
Beggs, A.H., Koenig, M., Boyce, F.M. and Kunkel, L.M. (1990) Detection of 98% of DMD/BMD gene deletions by polymerase chain reaction. Hum. Genet., 86, 45–48.[Web of Science][Medline]
Chamberlain, J.S., Gibbs, R.A., Ranier, J.E., Nguyen, P.N. and Caskey, C.T. (1988) Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Res., 16, 11141–11156.
Chong, S.S., Kristjansson, K., Cota, J., Handyside, A.H. and Hughes, M.R. (1993) Preimplantation prevention of X-linked disease: reliable and rapid sex determination of single human cells by restriction analysis of simultaneously amplified ZFX and ZFY sequences. Hum. Mol. Genet., 2, 1187–1191.
Cui, X.F., Li, H.H., Goradia, T.M., Lange, K., Kazazian, H.H.J., Galas, D. and Arnheim, N. (1989) Single-sperm typing: determination of genetic distance between the G gamma-globin and parathyroid hormone loci by using the polymerase chain reaction and allele-specific oligomers. Proc. Natl Acad. Sci. USA, 86, 9389–9393.
Darras, B.T. and Francke, U. (1988) Normal human genomic restriction-fragment patterns and polymorphisms revealed by hybridization with the entire dystrophin cDNA. Am. J. Hum. Genet., 43, 612–619.[Web of Science][Medline]
Den Dunnen, J.T., Grootscholten, P.M., Bakker, E., Blonden, L.A., Ginjaar, H.B., Wapenaar, M.C., van Paassen, H.M., van Broeckhoven, C., Pearson, P.L. and van Ommen, G.J. (1989) Topography of the Duchenne muscular dystrophy (DMD) gene: FIGE and cDNA analysis of 194 cases reveals 115 deletions and 13 duplications. Am. J. Hum. Genet., 45, 835–847.[Web of Science][Medline]
Emery, A.E. (1991) Population frequencies of inherited neuromuscular diseases—a world survey. Neuromusc. Disord., 1, 19–29.[CrossRef][Medline]
Ervasti, J.M. and Campbell, K.P. (1991) Membrane organization of the dystrophin-glycoprotein complex. Cell, 66, 1121–1131.[CrossRef][Web of Science][Medline]
Girardet, A., Hamamah, S., Anahory, T., Déchaud, H., Sarda, P., Hédon, B., Demaille, J. and Claustres, M. (2003) First preimplantation genetic diagnosis of hereditary retinoblastoma using informative microsatellite markers. Mol. Hum. Reprod., 9, 111–116.
Handyside, A.H. and Delhanty, J.D.A. (1993) Cleavage stage biopsy of human embryos and diagnosis of X-linked recessive disease. In Edwards, R.G. (ed.), Preimplantation Diagnosis of Human Genetic Disease. Cambridge University Press, Cambridge, pp. 239–270.
Handyside, A.H., Kontogianni, E.H., Hardy, K. and Winston, R.M. (1990) Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification. Nature, 344, 768–770.[CrossRef][Medline]
Hellani, A., Lauge, A., Ozand, P., Jaroudi, K. and Coskun, S. (2002) Pregnancy after preimplantation genetic diagnosis for ataxia telangiectasia. Mol. Hum. Reprod., 8, 785–788.
Hoffman, E.P., Brown, R.H. Jr and Kunkel, L.M. (1987) Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell, 51, 919–928.[CrossRef][Web of Science][Medline]
Hoffman, E.P., Fischbeck, K.H., Brown, R.H., Johnson, M., Medori, R., Loike, J.D., Harris, J.B., Waterston, R., Brooke, M., Specht, L. et al. (1988) Characterization of dystrophin in muscle-biopsy specimens from patients with Duchenne’s or Becker’s muscular dystrophy. N. Engl. J. Med., 318, 1363–1368.[Abstract]
Hussey, N.D., Donggui, H., Froiland, D.A., Hussey, D.J., Haan, E.A., Matthews, C.D. and Craig, J.E. (1999) Analysis of five Duchenne muscular dystrophy exons and gender determination using conventional duplex polymerase chain reaction on single cells. Mol. Hum. Reprod., 5, 1089–1094.
Kim, U.K., Chae, J.J., Lee, S.H., Lee, C.C. and Namkoong, Y. (2002) Molecular diagnosis of Duchenne/Becker muscular dystrophy by polymerase chain reaction and microsatellite analysis. Mol. Cells, 13, 385–388.[Web of Science][Medline]
Koenig, M., Hoffman, E.P., Bertelson, C.J., Monaco, A.P., Feener, C. and Kunkel, L.M. (1987) Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell, 50, 509–517.[CrossRef][Web of Science][Medline]
Kristjansson, K., Chong, S.S., Van den Veyver, I.B., Subramanian, S., Snabes, M.C. and Hughes, M.R. (1994) Preimplantation single cell analyses of dystrophin gene deletions using whole genome amplification. Nature Genet., 6, 19–23.[CrossRef][Web of Science][Medline]
Kuliev, A., Rechitsky, S., Verlinsky, O., Ivakhnenko, V., Evsikov, S., Wolf, G., Angastiniotis, M., Georghiou, D., Kukharenko, V., Strom, C. and Verlinsky, Y. (1998) Preimplantation diagnosis of thalassemias. J. Assist. Reprod. Genet., 15, 219–225.[CrossRef][Web of Science][Medline]
Lee, S.H., Kwak, I.P., Cha, K.E., Park, S.E., Kim, N.K. and Cha, K.Y. (1998) Preimplantation diagnosis of non-deletion Duchenne muscular dystrophy (DMD) by linkage polymerase chain reaction analysis. Mol. Hum. Reprod., 4, 345–349.
Lewis, C.M., Pinel, T., Whittaker, J.C. and Handyside, A.H. (2001) Controlling misdiagnosis errors in preimplantation genetic diagnosis: a comprehensive model encompassing extrinsic and intrinsic sources of error. Hum. Reprod., 16, 43–50.
Liu, J., Lissens, W., Van Broeckhoven, C., Lofgren, A., Camus, M., Liebaers, I. and Van Steirteghem, A. (1995) Normal pregnancy after preimplantation DNA diagnosis of a dystrophin gene deletion. Prenat. Diagn., 15, 351–358.[Web of Science][Medline]
Piyamongkol, W., Harper, J.C., Sherlock, J.K., Doshi, A., Serhal, P.F., Delhanty, J.D. and Wells, D. (2001) A successful strategy for preimplantation genetic diagnosis of myotonic dystrophy using multiplex fluorescent PCR. Prenat. Diagn., 21, 223–232.[CrossRef][Web of Science][Medline]
Ray, P.F., Vekemans, M. and Munnich, A. (2001) Single cell multiplex PCR amplification of five dystrophin gene exons combined with gender determination. Mol. Hum. Reprod., 7, 489–494.
Roberts, R.G., Coffey, A.J., Bobrow, M. and Bentley, D.R. (1993) Exon structure of the human dystrophin gene. Genomics, 16, 536–538.[CrossRef][Web of Science][Medline]
Snabes, M.C., Chong, S.S., Subramanian, S.B., Kristjansson, K., DiSepio, D. and Hughes, M.R. (1994) Preimplantation single-cell analysis of multiple genetic loci by whole-genome amplification. Proc. Natl Acad. Sci. USA, 91, 6181–6185.
Staessen, C., Van Assche, E., Joris, H., Bonduelle, M., Vandervorst, M., Liebaers, I. and Van Steirteghem, A. (1999) Clinical experience of sex determination by fluorescent in-situ hybridization for preimplantation genetic diagnosis. Mol. Hum. Reprod., 5, 382–389.
Steinlechner, M., Berger, B., Niederstatter, H. and Parson, W. (2002) Rare failures in the amelogenin sex test. Int. J. Legal Med., 116, 117–120.[CrossRef][Web of Science][Medline]
Thangaraj, K., Reddy, A.G. and Singh, L. (2002) Is the amelogenin gene reliable for gender identification in forensic casework and prenatal diagnosis? Int. J. Legal Med., 116, 121–123.[CrossRef][Web of Science][Medline]
Tuffery, S., Chambert, S., Bareil, C., Sarda, P., Coubes, C., Echenne, B., Demaille, J. and Claustres, M. (1998) Mutation analysis of the dystrophin gene in Southern French DMD or BMD families: from Southern blot to protein truncation test. Hum. Genet., 102, 334–342.[CrossRef][Web of Science][Medline]
Verlinsky, Y., Rechitsky, S., Verlinsky, O., Strom, C. and Kuliev, A. (2001) Preimplantation testing for phenylketonuria. Fertil. Steril., 76, 346–349.[CrossRef][Web of Science][Medline]
White, S., Kalf, M., Liu, Q., Villerius, M., Engelsma, D., Kriek, M., Vollebregt, E., Bakker, B., van Ommen, G.J., Breuning, M.H. and den Dunnen, J.T. (2002) Comprehensive detection of genomic duplications and deletions in the DMD gene, by use of multiplex amplifiable probe hybridization. Am. J. Hum. Genet., 71, 365–374.[CrossRef][Web of Science][Medline]
Zhang, L., Cui, X., Schmitt, K., Hubert, R., Navidi, W. and Arnheim, N. (1992) Whole genome amplification from a single cell: implications for genetic analysis. Proc. Natl Acad. Sci. USA, 89, 5847–5851.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  H. Malmgren, I. White, S. Johansson, L. Levkov, E. Iwarsson, M. Fridstrom, and E. Blennow PGD for dystrophin gene deletions using fluorescence in situ hybridization Mol. Hum. Reprod., May 1, 2006; 12(5): 353 - 356. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Fiorentino, A. Biricik, A. Nuccitelli, R. De Palma, S. Kahraman, M. Iacobelli, V. Trengia, D. Caserta, M.A. Bonu, A. Borini, et al. Strategies and clinical outcome of 250 cycles of Preimplantation Genetic Diagnosis for single gene disorders Hum. Reprod., March 1, 2006; 21(3): 670 - 684. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||