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Rapid screening for chromosomal aneuploidies using array-MLPA.

Yan JB, Xu M, Xiong C, Zhou DW, Ren ZR, Huang Y, Mommersteeg M, van Beuningen R, Wang YT, Liao SX, Zeng F, Wu Y, Zeng YT - BMC Med. Genet. (2011)

Bottom Line: However, results are usually not available for 3-4 days or more.Furthermore, we detected two chromosome X monosomy mosaic cases in which the mosaism rates estimated by array-MLPA were basically consistent with the results from karyotyping.Our study demonstrates the successful application and strong potential of array-MLPA in clinical diagnosis and prenatal testing for rapid and sensitive chromosomal aneuploidy screening.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Medical Genetics, Children's Hospital of Shanghai, Shanghai Jiao Tong University, Shanghai, P.R. China.

ABSTRACT

Background: Chromosome abnormalities, especially trisomy of chromosome 21, 13, or 18 as well as sex chromosome aneuploidy, are a well-established cause of pregnancy loss. Cultured cell karyotype analysis and FISH have been considered reliable detectors of fetal abnormality. However, results are usually not available for 3-4 days or more. Multiplex ligation-dependent probe amplification (MLPA) has emerged as an alternative rapid technique for detection of chromosome aneuploidies. However, conventional MLPA does not allow for relative quantification of more than 50 different target sequences in one reaction and does not detect mosaic trisomy. A multiplexed MLPA with more sensitive detection would be useful for fetal genetic screening.

Methods: We developed a method of array-based MLPA to rapidly screen for common aneuploidies. We designed 116 universal tag-probes covering chromosomes 13, 18, 21, X, and Y, and 8 control autosomal genes. We performed MLPA and hybridized the products on a 4-well flow-through microarray system. We determined chromosome copy numbers by analyzing the relative signals of the chromosome-specific probes.

Results: In a blind study of 161 peripheral blood and 12 amniotic fluid samples previously karyotyped, 169 of 173 (97.7%) including all the amniotic fluid samples were correctly identified by array-MLPA. Furthermore, we detected two chromosome X monosomy mosaic cases in which the mosaism rates estimated by array-MLPA were basically consistent with the results from karyotyping. Additionally, we identified five Y chromosome abnormalities in which G-banding could not distinguish their origins for four of the five cases.

Conclusions: Our study demonstrates the successful application and strong potential of array-MLPA in clinical diagnosis and prenatal testing for rapid and sensitive chromosomal aneuploidy screening. Furthermore, we have developed a simple and rapid procedure for screening copy numbers on chromosomes 13, 18, 21, X, and Y using array-MLPA.

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The male fetus with trisomy 21 and XXY were analyzed with array-MLPA. The left figures showed the relative signal of each probe. The probes on chromosomes 13, 18, 21, X and Y were depicted in yellow, blue, green, purple and purplish red, respectively. Eight autosomal control probes were shown in grey. The right figures showed the average copy numbers on each chromosome. Error bars represented the corresponding SD of the copy numbers on the MLPA probes covering each chromosome. The copy number of each chromosome was listed in the tables.
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Figure 3: The male fetus with trisomy 21 and XXY were analyzed with array-MLPA. The left figures showed the relative signal of each probe. The probes on chromosomes 13, 18, 21, X and Y were depicted in yellow, blue, green, purple and purplish red, respectively. Eight autosomal control probes were shown in grey. The right figures showed the average copy numbers on each chromosome. Error bars represented the corresponding SD of the copy numbers on the MLPA probes covering each chromosome. The copy number of each chromosome was listed in the tables.

Mentions: Next, array-MLPA was performed on 161 peripheral blood and 12 amniotic fluid samples including the 30 normal controls described above. Karyotype analysis was independently carried out on these samples. The results obtained by array-MLPA and the corresponding G-banded karyotypes were presented in Table 2. Of 173 tested samples, 169 (97.7%) including 12 amniotic fluid samples were correctly identified by array-MLPA. Figure 3 showed the abnormal copy numbers on chromosome 21 or X for two fetus samples. One was a male fetus (A9) in which the average copy number of chromosome 21 reached 1.53 indicating trisomy 21 (Figure 3A), while another was a male fetus (A10) in which the copy number on the X chromosome was 0.85 ±0.02 suggesting XXY (Figure 3B). However, four abnormal karyotypes including 46,XX,-14,+t(14;21);(p11.2;p11.2); 46,XY,rec(14)(18qter→18q22::14qter)pat; 46,XY,inv(9)(p12q21) and 46,XY,t(1;7)(p36.3;p13) could not be detected by array-MLPA (Table 2). A comparison of the results from array-MLPA and karyotyping was summarized in Table 3.


Rapid screening for chromosomal aneuploidies using array-MLPA.

Yan JB, Xu M, Xiong C, Zhou DW, Ren ZR, Huang Y, Mommersteeg M, van Beuningen R, Wang YT, Liao SX, Zeng F, Wu Y, Zeng YT - BMC Med. Genet. (2011)

The male fetus with trisomy 21 and XXY were analyzed with array-MLPA. The left figures showed the relative signal of each probe. The probes on chromosomes 13, 18, 21, X and Y were depicted in yellow, blue, green, purple and purplish red, respectively. Eight autosomal control probes were shown in grey. The right figures showed the average copy numbers on each chromosome. Error bars represented the corresponding SD of the copy numbers on the MLPA probes covering each chromosome. The copy number of each chromosome was listed in the tables.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3111339&req=5

Figure 3: The male fetus with trisomy 21 and XXY were analyzed with array-MLPA. The left figures showed the relative signal of each probe. The probes on chromosomes 13, 18, 21, X and Y were depicted in yellow, blue, green, purple and purplish red, respectively. Eight autosomal control probes were shown in grey. The right figures showed the average copy numbers on each chromosome. Error bars represented the corresponding SD of the copy numbers on the MLPA probes covering each chromosome. The copy number of each chromosome was listed in the tables.
Mentions: Next, array-MLPA was performed on 161 peripheral blood and 12 amniotic fluid samples including the 30 normal controls described above. Karyotype analysis was independently carried out on these samples. The results obtained by array-MLPA and the corresponding G-banded karyotypes were presented in Table 2. Of 173 tested samples, 169 (97.7%) including 12 amniotic fluid samples were correctly identified by array-MLPA. Figure 3 showed the abnormal copy numbers on chromosome 21 or X for two fetus samples. One was a male fetus (A9) in which the average copy number of chromosome 21 reached 1.53 indicating trisomy 21 (Figure 3A), while another was a male fetus (A10) in which the copy number on the X chromosome was 0.85 ±0.02 suggesting XXY (Figure 3B). However, four abnormal karyotypes including 46,XX,-14,+t(14;21);(p11.2;p11.2); 46,XY,rec(14)(18qter→18q22::14qter)pat; 46,XY,inv(9)(p12q21) and 46,XY,t(1;7)(p36.3;p13) could not be detected by array-MLPA (Table 2). A comparison of the results from array-MLPA and karyotyping was summarized in Table 3.

Bottom Line: However, results are usually not available for 3-4 days or more.Furthermore, we detected two chromosome X monosomy mosaic cases in which the mosaism rates estimated by array-MLPA were basically consistent with the results from karyotyping.Our study demonstrates the successful application and strong potential of array-MLPA in clinical diagnosis and prenatal testing for rapid and sensitive chromosomal aneuploidy screening.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Medical Genetics, Children's Hospital of Shanghai, Shanghai Jiao Tong University, Shanghai, P.R. China.

ABSTRACT

Background: Chromosome abnormalities, especially trisomy of chromosome 21, 13, or 18 as well as sex chromosome aneuploidy, are a well-established cause of pregnancy loss. Cultured cell karyotype analysis and FISH have been considered reliable detectors of fetal abnormality. However, results are usually not available for 3-4 days or more. Multiplex ligation-dependent probe amplification (MLPA) has emerged as an alternative rapid technique for detection of chromosome aneuploidies. However, conventional MLPA does not allow for relative quantification of more than 50 different target sequences in one reaction and does not detect mosaic trisomy. A multiplexed MLPA with more sensitive detection would be useful for fetal genetic screening.

Methods: We developed a method of array-based MLPA to rapidly screen for common aneuploidies. We designed 116 universal tag-probes covering chromosomes 13, 18, 21, X, and Y, and 8 control autosomal genes. We performed MLPA and hybridized the products on a 4-well flow-through microarray system. We determined chromosome copy numbers by analyzing the relative signals of the chromosome-specific probes.

Results: In a blind study of 161 peripheral blood and 12 amniotic fluid samples previously karyotyped, 169 of 173 (97.7%) including all the amniotic fluid samples were correctly identified by array-MLPA. Furthermore, we detected two chromosome X monosomy mosaic cases in which the mosaism rates estimated by array-MLPA were basically consistent with the results from karyotyping. Additionally, we identified five Y chromosome abnormalities in which G-banding could not distinguish their origins for four of the five cases.

Conclusions: Our study demonstrates the successful application and strong potential of array-MLPA in clinical diagnosis and prenatal testing for rapid and sensitive chromosomal aneuploidy screening. Furthermore, we have developed a simple and rapid procedure for screening copy numbers on chromosomes 13, 18, 21, X, and Y using array-MLPA.

Show MeSH
Related in: MedlinePlus