Trisomy of chromosome 21, 13, or 18 as well as sex chromosome aneuploidy are the main genetic diseases of newborns. So far, there is no effective treatment for these diseases. Therefore, it is crucial to develop a rapid, reliable and highly sensitive molecular diagnostic technique. In this study, a 4-well universal flow-through array based on a three-dimensional aluminum-oxide substrate was used to analyze MLPA amplification products (array-MLPA). The main advantages of array-MLPA over other techniques such as karyotype analysis, FISH and multiplex PCR, are its simplicity, speed, and analysis of 124 detector probes in parallel. Four different samples for 124 genetic markers can be tested all at once within 6 hours starting from fresh blood or amniotic fluid samples. The cost of the universal chip is limited to $50 per sample. An additional advantage of this method is the automated measurement and data analysis. Compared to AneuVysion FISH analysis, the array-MLPA system demonstrates at least 4-fold shorter assay time and at least 6-fold less assay cost. The reproducibility and accuracy of the array technology were demonstrated previously in studies on gene expression profiling [29, 30] and quantification of PMP22 gene copy number  and detection of DMD genomic rearrangements .
To further investigate the applicability of array-MLPA for rapid chromosomal aneuploidy screening, especially in fetuses at risk for Down's syndrome, we performed array-MLPA analysis on 161 peripheral blood and 12 amniotic fluid samples. Our results showed that 169 of 173 (97.7%) samples including all the amniotic fluid samples identified by array-MLPA were concordant with karyotype analysis. Our study demonstrates the successful application and strong potential of array-MLPA in clinical diagnosis and prenatal testing for rapid chromosomal aneuploidy screening. Further validation studies must be performed to ensure the clinical applicability of our array-MLPA assay.
To improve the reliability of detection, we designed two different probes per targeted gene, located in the 5' and 3' region of the same gene, and the average values of two probes were used to calculate the copy number of the gene. The data indicated that 89.4% of the relative probe signals from array-MLPA ranged from 0.85 to 1.15 in the normal controls. In the previous MLPA analysis , the normal region was usually set defined as 0.7 to 1.3. This indicates that array-MLPA is more accurate than the reported results with conventional MLPA for the determination of copy number.
It is well-known that intrinsic sensitivity limitations of MLPA make it unsuitable for reliable detection of mosaicism. It has become possible to detect the mosaicism of chromosomal aneuploidies by array-MLPA due to its higher accuracy. In this study, we identified sex chromosome mosaicism in two patients by array-MLPA. In one case (B10), the average copy number on chromosome X was 0.71 revealing approximately 50% mosaic rate of chromosome X. The result was concordant with that of conventional cytogenetic analysis. In another case (B11), a similar result was observed on array-MLPA. However, G-banded karyotype analysis indicated the chromosome karyotypes 45,X(70%)/47,XXX(30%). The result showed that there were polyploid cells in the patient B11. This suggests array-MLPA might have a bias in detection. Array-MLPA appeared to be more suitable for detecting mosaicism of monosomy than polyploidy.
In addition to X chromosome aneuploidies, unknown marker chromosomes in four cases (B5, B6, B8 and B9) were found by karyotype analysis, but G-banding did not distinguish their origins. Finally, we identified the regions of these unknown marker chromosomes by array-MLPA. Two of the four cases were confirmed by Y-chromosome-specific PCR. In another case (B7), abnormality on the Y chromosome was identified by both array-MLPA and G-banding analysis. In this study, we designed ten pairs of probes to cover the long and short arms of chromosome Y. The data obtained on array-MLPA and PCR analysis indicate the presence of Y chromosome-specific probe signals, especially TSPY probes. Our study demonstrates the power of array-MLPA for detecting small abnormalities on chromosomes.
However, four samples with chromosome translocations or inversions were not identified by array-MLPA since the recombination regions were not covered by the MLPA probes. Currently, we have only used one (Cy5) of the four available detection wavelengths for screening chromosomes 13, 18, 21, X, and Y. Using three additional sets of amplification primers, each labelled with a different fluorophore , it should be simple to expand the number of MLPA probes for other chromosomal abnormalities with potential clinical significance. Still, array-MLPA cannot completely replace conventional cytogenetic analysis. It is suitable to be used as a screening test in conjunction with karyotype analysis for diagnosis.