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

Abstract

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.

Figure 1

This is a part of the MLPA analysis report (Genemarker v1.51) of a normal female. In the upper part, all 37 probes in the SALSA MLPA P095 aneuploidy kit are shown in the second column with their length in base pairs in the third column. The last column represents the relative probe signal of each probe. Normal values are defined between 0.7 and 1.3 and are shown in a white background. If the relative probe signal is aberrant, it has a gray background. In this case, a normal male is set as a control, hence the X and Y probes are aberrant; there are no relative probe signals for Y and the relative probe signals for X are nearly 2. The lower part of the figure shows the length in base pairs on the X axes and the relative probe signal on the Y axes. Normal relative probe signals are between the grey lines (0.7 and 1.3) and are depicted in green. Aberrant relative probe signals are depicted in red.

Figure 2

(a) Part of an MLPA analysis report (Genemarker v1.51) of a trisomy 21 case: when normal samples of the opposite sex as that of the fetus are used as controls only 4 of 8 probes are >1.3. (b) Part of an MLPA analysis report (Genemarker v1.51) of the same trisomy 21 case as in (a) when normal samples of the same sex as that of the fetus are used as controls 8 of 8 probes are >1.3. (c) Part of an MLPA analysis report (Genemarker v1.51) of the XYY/XY mosaic: the relative probe signals of all Y probes are > 1.3. (d) Part of an MLPA analysis report (Genemarker v1.51) of the mosaic trisomy 18: the relative probe signals of 6 of 8 chromosome 18 probes are >cutoff values (see Table 3). (e) Part of an MLPA analysis report (Genemarker v1.51) of a partial deletion of chromosome 21, which showed to be an unbalanced complex translocation of an inverted chromosome 4 with chromosome 21. Normal females were set as controls.