Rapid genetic diagnosis and prenatal diagnosis of spinal muscular atrophy by denaturing high-performance liquid chromatography

Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder1 (1 in 6000 to 10 000 births) caused by mutations in the SMN1 gene at 5q13. More than 90%-98% of SMA patients show homozygous deletion of SMN1,2 which has proved to be useful in the diagnosis of SMA. But it is hampered because of the existence of a highly homologous gene, SMN23 Based on nucleotide mismatches between SMN1 and SMN2, the following two DNA tests are usually performed: single-strand conformational polymorphism (SSCP)3 and polymerase chain reaction (PCR) followed by a restriction enzyme digestion.4,5 In this study we developed a new method for rapid genetic diagnosis of SMA by denaturing high-performance liquid chromatography (DHPLC), which is based on different retention of homoduplexes and heteroduplexes in detecting the homozygous deletion of SMN1. Both genetic and prenatal diagnoses were performed successfully for a SMA family by DHPLC, which was confirmed as a rapid and effective technique for detecting the deletion of SMN1. METHODS Patients A total of 49 samples were taken from 5 SMA patients diagnosed by restriction enzyme digestion at the National Laboratory of Medical Genetics (deletion of exon 7 and 8 in 4 patients and deletion of exon 7 in 1 patient), 10 parents (obligate carriers) of the 5 patients, and 30 normal controls. Another couple (family 1) with an 8-year-old SMA-affected boy desired to carry out prenatal genetic diagnosis for their fetus (20-week gestation, Fig. 1). After informed consent was obtained, peripheral blood (I:1, I:2, and II:1) and amniotic fluid (II:2) were collected for extraction of genomic DNA by the phenolchloroform method.Fig. 1.: Pedigree of family 1. II: 1 is the proband of the family, his mother (I: 2) has another gestation. The amniotic fluid of the fetus II: 2 was analyzed at the 20th gestational week.DHPLC analysis The following primers were synthesized to amplify exon 7 of the SMN gene: SMN forward 5′-AGA CTA TCA ACT TAA TTT CTG ATCA-3′, and reverse 5′-GAT TCA CTT TCA TAA TGC TGG-3′. 541C960 and 541C1120 for exon 8 were synthesized as described.3 Twenty microliters of reaction mixture contained 30 ng genomic DNA in 10 × PCR buffer (Qiagen, Germany) with 15 mmol/L MgCl2, 5× Q-solution, 200 μmol/L dNTPs, 20 pmol/L primers, and 0.1 U HotStart Taq (Qiagen, Germany). PCR was carried out on a Perkin-Elmer thermal cycler. A 15-minute initial denaturation at 95°C was followed by 35 cycles (95°C for 30 seconds, 54°C and 58°C for exon 7 and 8, respectively for 30 seconds, 72°C for 30 seconds), and a final elongation at 72°C for 7 minutes, and finally ended with a holding period at 4°C. For heteroduplex analysis, the PCR amplification products were subject to an additional 5-minute denaturation at 95°C, followed by gradually reannealing from 95°C to 25°C over a period of 60 minutes. The prepared sample was then directly loaded into the autosampler of the automated DHPLC system, the WAVE® nucleic acid fragment analysis system, equipped with a DNASep1 cartridge (Transgenomic, USA). The samples were run under partially denaturing conditions at 53.6°C (exon 7) and 55.8°C (exon 8). The start- and end-points of the gradient were adjusted according to the size of the PCR products using an algorithm provided by navigator software. PCR-enzyme digestion analysis In order to compare with the DHPLC result, the samples from the SMA family were simultaneously examined by restriction enzyme digestion. The primers for PCR-enzyme digestion (both SMN1 and SMN2) were R111, X7-Dra, and 541C940, 541C1120, respectively4 The amplicons of exon 7 and exon 8 were digested by restriction enzyme DraI and DdeI respectively as described.5 DNA sequencing To confirm the accuracy of DHPLC, exon 7 amplicons of I:1, II:1, II:2, and two normal controls (C:1 with both heteroduplex and homoduplex peaks, C: 2 with homoduplex peak only) were sequenced. The amplified products were purified by exonuclease and then sequenced by the ABI Big Dye terminator cycle sequencing kit (ABI Biosystems, USA) according to the manufacture's instructions and run on an ABI 3100 sequencer. The sequencing results were analyzed with the DNASTAR® software program package. RESULTS DHPLC analysis The parents of SMA patients and 29 control samples displayed heteroduplex peaks in both exon 7 and 8. In Fig. 2A (chromatogram of exon 7), I:1, II:2 and one control (C:1) showed two homoduplexes and one heteroduplex of SMN1 and SMN2, which were clearly separable on the DHPLC chromatogram based on different retention time. For II:1 and a control individual (C:2), only one homoduplex peak was observed on the chromatogram. However, each homoduplex peak had its own retention time, which distinguished SMN1 (4.58 minutes) from SMN2 (4.39 minutes). The retention time of the heteroduplex peak (4.26 minutes) was shorter than that of homoduplex peak of SMN2 or SMN1. These findings revealed that II:1 lacking SMN1 but retaining SMN2, and C:2 bearing only SMN1. Similar DHPLC results were also found in exon 8 (pictures were not provided).Fig. 2.: A: chromatographic analysis of family 1 and two control samples. I: 1, II: 2, C: 1 with two homoduplex peaks and one heteroduplex peak; II: 1, C: 2 with one homoduplex peak only. B: Sequence analysis. a, II: 2 with SMN1/SMN2 genes; b, II: 1 with the SMN2 gene only; c, normal control C: 2 with the SMN1 gene only.Interestingly, one SMA patient was detected deletion of exon 7 only by PCR-enzyme digestion previously, but deletion of both exon 7 and 8 by DHPLC. DNA sequencing I: 1, II: 2 and C: 1 showed C/T heterozygous at position +6 of exon 7, suggesting that they carried both SMN1 and SMN2 genes (Fig. 2B-a, only provided the sequencing result of II:2). Nevertheless, II:1 (Fig. 2B-b) and C:2 (Fig. 2B-c) displayed only T and C, indicating that they carried only SMN2 and SMN1 genes, respectively. PCR-enzyme digestion of family 1 DraI digestion of exon 7, II:1 showed only one band of 163 bp, whereas I:1, I:2, II:2 and C:1 showed two bands of 187 bp and 163 bp;6 DdeI digestion of exon 8, II:1 showed only one band of 122 bp, but the others showed two bands of 187 bp and 122 bp (Fig. 3). The enzyme digestion result indicated that the proband was homozygous deletion of SMN1, which was consistent with the DHPLC result.Fig. 3.: Enzymatic digestion results of family 1. M: 100 bp DNA marker, B: blank. On the left of M is the result before and after DraI digest of exon 7. On the right of M is the result before and after DdeI digest of exon 8. Lanes 1 to 5 mean I: 1, I: 2, II: 1, II: 2 and C: 1, respectively.DISCUSSION Molecular diagnosis of SMA presents a special difficulty owing to the duplication and inversion of 500 kb in the region, which result in the presence of two homologous SMN genes differing only in 3 intronic and 2 exonic nucleotides.7 It has been demonstrated that the translationally silent, single-nucleotide C→T difference between SMN1 and SMN2 at position +6 of exon 7 disrupts a putative exonic splicing enhancer (ESE) site8 or a splice inhibitor site,9 resulting in alternative splicing of exon 7 of SMN2, in which exon 7 is skipped, and an unstable and inactive protein isoform, SMNΔ7, with a different carboxy terminus, causes the molecular defect responsible for SMA. Two methods have been used to detect the homozygous deletion of the SMN1 gene, but SSCP10 is considered to be time-consuming. The enzymatic digestion5 performed in our laboratory sometimes showed digestion failure or partial digestion, which may lead to misdiagnosis. DHPLC as a novel, nongel-based method is very sensitive to DNA sequence variation including the absence of the homozygous SMN gene in SMA patients.11,12 It is performed on a WAVE DNA fragment analysis system by a DNASep column containing nonporous alkylated polystyrenedivinylbenzene particles that are electrically neutral and hydrophobic. Triethylammonium acetate (TEAA) is a positively charged reagent that facilitates interaction between the stationary matrix and negatively charged DNA molecules. DNA fragments are eluted from the column by increasing the ratio of acetonitrile to TEAA. The eluted DNA from the column is then detected by scanning with a UV detector at 260 nm. The detection is based on differences in the retention time of perfectly matched homoduplexes and heteroduplexes containing one mismatched base pair. In this study, the previously identified homozygous deletions of SMN1 exon 7 and 8 were detected by DHPLC. In one SMA patient, only deletion of exon 7 of SMN1 was detected by PCR-enzyme digestion; however, he was detected with deletion of exon 7 and 8 by DHPLC. The difference between the results of the two methods might be caused by incomplete enzymatic digestion. In family 1, the proband was confirmed to be a SMA patient bearing only SMN2, whereas the fetus did not inherit the homozygous deletion. And the DHPLC results of family 1 were completely consistent with those of enzymatic digestion and sequencing analysis, indicating the sensitivity and preciseness of this method. About 9% of normal individuals showed homozygous deletion of the SMN2 gene,13 and in our study a normal control with only SMN1 was also found. Moreover, DHPLC analysis showed that the retention time of homoduplex SMN1 was different from that of homoduplex SMN2. Therefore, we could distinguish SMA patients with SMN2 only from normal individuals, despite the normal one carried SMN1 only or SMN1 and SMN2 both. In this study, only one heterozygous peak and two homozygous peaks were observed, which were not completely identical to the results reported previously,14 two heterozygous peaks formed by SMN1 and SMN2 could be clearly identified. The chromatographic difference between various groups may be resulted from different DNA polymerases used in PCR, or different acetonitrile concentrations in dilution buffer. However, this difference did not influence the results of analysis. In conclusion, a new DHPLC-based method for detection of homozygous deletion of the SMN1 gene is feasible in gene diagnosis and prenatal diagnosis of SMA patients and fetus at risk. In detecting the homozygous deletion of SMN1, compared with SSCP and PCR-enzyme digestion, DHPLC is rapid, accurate and sensitive. DHPLC can be used for genetic diagnosis and prenatal diagnosis of SMA patients.