Rapid aneuploidy detection with multiplex ligation-dependent probe amplification: a prospective study of 4000 amniotic fluid samples

• Published in: European journal of human genetics : EJHG 2009-01, Vol.17 (1), p.112-121
• Main Authors: Van Opstal, Diane, Boter, Marjan, de Jong, Danielle, van den Berg, Cardi, Brüggenwirth, Hennie T, Wildschut, Hajo I J, de Klein, Annelies, Galjaard, Robert-Jan H
• Format: Article
• Language: English
• Subjects: Amniocentesis; Amniocentesis - methods; Amniotic Fluid; Aneuploidy; Automation; Bioinformatics; Biological and medical sciences; Biomedical and Life Sciences; Biomedicine; Blood; Chromosome aberrations; Chromosomes; Contamination; Cytogenetics; Deoxyribonucleic acid; Diagnosis; DNA; False Negative Reactions; Female; Fetuses; Fluorescence in situ hybridization; Fundamental and applied biological sciences. Psychology; Gene Expression; General aspects. Genetic counseling; Genetic Testing - methods; Genetics of eukaryotes. Biological and molecular evolution; Human Genetics; Humans; Hybridization; In Situ Hybridization, Fluorescence; Karyotyping; Male; Medical genetics; Medical sciences; Molecular and cellular biology; Molecular Probe Techniques; Mosaicism; Nucleic Acid Amplification Techniques - methods; Polymerase chain reaction; Pregnancy; Prenatal development; Sensitivity and Specificity; Trisomy - diagnosis
• ISSN: 1018-4813; 1476-5438
• DOI: 10.1038/ejhg.2008.161

The introduction of prenatal screening requires rapid high-throughput diagnosis of common aneuploidies. Multiplex ligation-dependent probe amplification (MLPA) allows for quick, easily automated multiplex testing of these aneuploidies in one polymerase chain reaction. We performed a large prospective study using MLPA on 4000 amniotic fluid (AF) samples including all indications and compared its value to karyotyping and fluorescence in situ hybridization (FISH). MLPA can reliably determine common aneuploidies with 100% sensitivity and 100% specificity. Moreover, some mosaic cases and structural chromosome aberrations were detected as well. In cases of a male fetus, triploidies can be detected by an aberrant pattern of probe signals, which mimics maternal cell contamination (MCC). Macroscopic blood contamination was encountered in 3.2% of the AF samples. In 20% of these samples, an MLPA pattern was found consistent with MCC, although there were no false negatives of the most common aneuploidies. As the vast majority of inconclusive results (1.7%) is due to potential MCC, we designed a protocol in which we determine whether MLPA can be performed on blood-contaminated AF samples by testing if blood is of fetal origin. Then, the number of inconclusive results could be theoretically reduced to 0.05%. We propose an alternative interpretation of relative probe signals for rapid aneuploidy diagnosis (RAD). We discuss the value of MLPA for the detection of (submicroscopic) structural chromosome anomalies. MLPA is a reliable method that can replace FISH and could be used as a stand-alone test for RAD instead of karyotyping.