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J Med Genet 2003;40:907-912
Prenatal diagnosis of syndromes caused by chromosomal abnormality is a long established part of obstetric care in developed countries. In this area, there have been recent significant advances in the identification of high risk pregnancies using sophisticated serum analyte and ultrasound screening methods. (1) (2) For follow up diagnostic testing, karyotyping has provided the gold standard. This technology has remained essentially unchanged over 30 years, as no new technology has yet proven superior in terms of being able to detect such a wide range of abnormalities with the necessary precision. Nevertheless, molecular tests, such as fluorescent in situ hybridisation (FISH) (3) and short tandem repeat analysis, (4) are now in common practice for the diagnosis of specific abnormalities. These adjunctive tests importantly decrease turnaround times from 1-2 weeks to 1-2 days.
We assessed the performance of multiplex ligation dependent probe amplification (MLPA) as an alternative method for the detection of aneuploidy, which is by far the most common prenatal chromosome abnormality. This novel technique (5) detects sequence dosage differences in a semi-quantitative manner and has many potential applications in diagnostic molecular genetics and cytogenetics. For example, a recent report describes its use for detection of large genomic deletions and duplications in the BRCA1 gene. (6) This technology appears to have significant advantages in that it is extremely versatile in its applications, flexible in its target loci, highly automated, suitable for high throughput testing, efficient, and cost effective. Its application for aneuploidy detection has not been reported in a clinical setting. In order to assess the precision and robustness of the test, we conducted a prospective blind trial on 492 consecutive amniocentesis samples referred to our cytogenetics laboratory.
MATERIALS AND METHODS
This study was designed to blind test amniocentesis samples prospectively. The samples were collected over a 4 month period as they were referred to the cytogenetics laboratory of Genetic Health Services. The referral reasons covered the whole range from low risk anxiety to very high risk cases with known clinical abnormalities assessed by ultrasound and/or serum screening. With the exception of 30 blood contaminated samples, none was excluded from the study. Usually 15-20 ml of amniotic fluid was received and 1 ml was removed for MLPA testing. Tests were processed in batches of 16-96 samples. Data processing of the MLPA tests was performed without knowledge of the karyotype/FISH results.
Lysates were made by centrifuging 1 ml of amniotic fluid for 20 minutes at 3500 rpm in a benchtop centrifuge. The supernatant was discarded and the remaining cell pellet lysed with 25 [micro]l of 50 mmol/l NaOH and heated at 95[degrees]C for 15 minutes. Each lysate was neutralised with 5 [micro]l of 1 mol/l Tris-HCl buffer (pH 7.4, 25[degrees]C) giving a total volume of 30 [micro]l, of which 5 [micro]l was used in the MLPA reaction. When required, lysate DNA was concentrated by ethanol precipitation and redissolved in 5 [micro]l of TE buffer.
The MLPA-Trisomy test kit (P001) was obtained from MRC Holland, Amsterdam, the Netherlands. The principle of MLPA has been described in detail previously. (5) Briefly, for each specific genomic target, a set of two probes is designed to hybridise immediately adjacent to each other on the same target strand. Both probes consist of a short (22-43 nt) target specific sequence and a universal forward or reverse PCR primer binding site. In addition, one of the probes contains a so-called "stuffer" sequence. For each probe in the multiplex, the stuffer part has a specific length (19-364 nt) and sequence. After overnight hybridisation to the target DNA, each pair of adjacent probes is joined by a ligation reaction. Next, PCR is performed with a single fluorescent labelled primer pair, which ensures that the relative yield of each of …