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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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Array-MLPA analysis of normal controls (15 normal females and 15 normal males). The average copy numbers on chromosomes 13, 18, 21, X and Y were shown in A, B, C, D and E, respectively. The normal distributions of copy number in the control individuals were shown with two grey lines. Error bars represented the corresponding standard deviation (SD) of the copy numbers for the MLPA probes covering each chromosome.
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Figure 1: Array-MLPA analysis of normal controls (15 normal females and 15 normal males). The average copy numbers on chromosomes 13, 18, 21, X and Y were shown in A, B, C, D and E, respectively. The normal distributions of copy number in the control individuals were shown with two grey lines. Error bars represented the corresponding standard deviation (SD) of the copy numbers for the MLPA probes covering each chromosome.

Mentions: Array-MLPA is not a length-based discrimination method, which can overcome the main drawback of MLPA. The sensitivity and specificity of array-MLPA is based on the universal flow-through array hybridization and data analysis. To determine the reliability of array-MLPA, 30 normal control individuals (15 males and 15 females) were first tested with replicate measurements. The mean standard deviation across the 30 control individuals was 0.024, indicating the low inter-individual variability on the array-MLPA test. All probes designed on chromosomes 13, 18, 21 and X of female samples had an average relative signal of about 1.0, whereas the average signal of chromosomes X and Y in males was approximately 0.5. The copy numbers of almost all the controls were located within the normal distributions ( two grey lines in Figure 1). The intra-assay standard deviations of the probe signals on chromosome 13, 18, 21, X and Y ranged from 0.018 to 0.031. Examples of the copy numbers on array-MLPA for a normal female and a normal male are shown in Figure 2. The average gene copy numbers on the five chromosomes in the normal controls are listed in Additional file 3, Table S2. Overall, 89.4% of the relative probe signals in the normal controls were within the normal range (0.85 to 1.15).


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)

Array-MLPA analysis of normal controls (15 normal females and 15 normal males). The average copy numbers on chromosomes 13, 18, 21, X and Y were shown in A, B, C, D and E, respectively. The normal distributions of copy number in the control individuals were shown with two grey lines. Error bars represented the corresponding standard deviation (SD) of the copy numbers for the MLPA probes covering each chromosome.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Array-MLPA analysis of normal controls (15 normal females and 15 normal males). The average copy numbers on chromosomes 13, 18, 21, X and Y were shown in A, B, C, D and E, respectively. The normal distributions of copy number in the control individuals were shown with two grey lines. Error bars represented the corresponding standard deviation (SD) of the copy numbers for the MLPA probes covering each chromosome.
Mentions: Array-MLPA is not a length-based discrimination method, which can overcome the main drawback of MLPA. The sensitivity and specificity of array-MLPA is based on the universal flow-through array hybridization and data analysis. To determine the reliability of array-MLPA, 30 normal control individuals (15 males and 15 females) were first tested with replicate measurements. The mean standard deviation across the 30 control individuals was 0.024, indicating the low inter-individual variability on the array-MLPA test. All probes designed on chromosomes 13, 18, 21 and X of female samples had an average relative signal of about 1.0, whereas the average signal of chromosomes X and Y in males was approximately 0.5. The copy numbers of almost all the controls were located within the normal distributions ( two grey lines in Figure 1). The intra-assay standard deviations of the probe signals on chromosome 13, 18, 21, X and Y ranged from 0.018 to 0.031. Examples of the copy numbers on array-MLPA for a normal female and a normal male are shown in Figure 2. The average gene copy numbers on the five chromosomes in the normal controls are listed in Additional file 3, Table S2. Overall, 89.4% of the relative probe signals in the normal controls were within the normal range (0.85 to 1.15).

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