High‐throughput analysis of chromosome abnormality in spontaneous miscarriage using an MLPA subtelomere assay with an ancillary FISH test for polyploidy

Bruno, Damien L.; Burgess, Trent; Ren, Hua; Nouri, Sara; Pertile, Mark D.; Francis, David; Norris, Fiona; Kenney, Bronwyn K.; Schouten, Jan P.; Choo, K.H. Andy; Slater, Howard R.

doi:10.1002/ajmg.a.31552

Cited by 48 publications

(54 citation statements)

References 14 publications

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“…However, a fetus with triploidy could not be diagnosed by MLPA but was determined by direct-FISH analysis in a 24h duration test. The inability to detectpolyploidy is one of the main limitations of the MLPA assay and this has also been reported previously (9)(10)(11)(12)(13). If the sensitivity and specifity of the probes were evaluated individually, our experiences showed that most of the probes had 100% sensitivity.…”

Section: Discussionsupporting

confidence: 79%

Performance of MLPA as a screening method for aneuploidy in uncultured amniocytes

Yurdakul¹,

Durak²,

Müslümanoğlu³

et al. 2010

J Turkish German Gynecol Assoc

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Objective: To test whether the Multiplex Ligation-dependent ProbeAmplification (MLPA) technique can be used as a screening test for rapid diagnosis of aneuploidies in uncultured amniocentesis. Material and Methods:In this prospective blind study, MLPA with chromosomes 13,18,21,X and Y specific probe mixes was performed in 500 amniotic fluid samples. Chromosome copy numbers were determined by analyzing size and peak area for each MLPA probe. Results were compared with those of karyotyping/FISH. Results:Conclusive test results were obtained in 98% of the samples, whereas 10 were inconclusive. In all conclusive tests, the MLPA results were concordant with that of cytogenetic and/or FISH analyses. There were no false-positive results. A case with 69,XXX triploidy could not be diagnosed by MLPA. In total, 28 aneuploidies were diagnosed. There were no false-positive results. The performance of each probe was determined. Conclusion:MLPA is a rapid, simple and reliable assay for aneuploidy screening in uncultured amniocytes. (J Turkish-German Gynecol Assoc 2010; 11: 199-203) Key words: MLPA, prenatal screening, common aneuploidies, uncultured amniocytes Received: 5 November, 2010 Accepted: 14 November, 2010 Amaç: MLPA tekniği ile prenatal dönem anöploidi tanısı yeni, alternatif bir metoddur. Çalışmamızda bu yeni tekniğin prenatal tanı testi olarak rutinde kullanılabilirliğinin sınanması, tekniğin sensivite, spesivite ve test başarısızlık oranlarının saptanması ve bu yeni yöntemin rutinde kullanılan diğer anöploidi tanı yöntemlerine göre avantaj ve dezavantajlarının belirlenmesi amaçlanmıştır. Gereç ve Yöntemler:Toplam 500 hastanın amniyon sıvısında MLPA tekniği ile 13., 18., 21., X ve Y kromozomları için doz tayini yapılmıştır. MLPA tekniği ile saptanan sonuçlar, bu hastalara ait diğer rutin yön-temlerle saptanan sonuçlar ile karşılaştırılmıştır. Bulgular:Tekniğin anöploidi tanısınındaki sensivitesi %100, spesivitesi %100 ve test başarısızlık oranı %4 olarak saptanmıştır. 69,XXX karyotipli örneğimizde MLPA tekniği ile doğru sonuç alınamamıştır.Sonuç: MLPA te kniği ile prenatal tanıda anöpoidi tayininin pratik, hız-lı ve güvenilir şekilde yapılabileceği düşünülmüştür. (J Turkish-German Gynecol Assoc 2010; 11: 199-203) Abstract ÖzetOriginal Investigation 199

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Section: Discussionsupporting

confidence: 79%

Performance of MLPA as a screening method for aneuploidy in uncultured amniocytes

Yurdakul¹,

Durak²,

Müslümanoğlu³

et al. 2010

J Turkish German Gynecol Assoc

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“…Combining the results of this study with previous studies by Diego-Alvarez et al 5 and Bruno et al, 6 the sensitivity of MLPA is estimated to be 87.9%, with a specificity of 90.2%. In our study, we identified seven structural variants (2.46% with 99% confidence interval of 0.09 -4.83), highlighting an important need for adequate counseling or diagnostic reporting practices if MLPA is performed alone.…”

supporting

confidence: 79%

“…Molecular methods such as multiplex ligation-dependent probe amplification (MLPA) offer an alternative approach for high throughput, cost-efficient chromosomal aneuploidy screening, as sufficient probes can be used to enable a quantitative survey of the entire chromosomal complement and identify all but the most telomeric unbalanced chromosomal translocations. The application and assessment of MLPA as a diagnostic method in this setting has previously been reported in two publications-in 2007 by Diego-Alvarez et al 5 and in 2006 by Bruno et al 6 In this study, we describe the use of MLPA as a screening method for aneuploidy in POC in the largest cohort described to date and provide comparisons with respect to the sensitivity, specificity, and clinical utility of the technique in comparison with classical cytogenetic analysis.…”

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A comparison of molecular and cytogenetic techniques for the diagnosis of pregnancy loss

Caramins

Saville

Shakeshaft

et al. 2011

Genetics in Medicine

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Purpose: To evaluate the sensitivity, specificity, advantages, and limitations of multiplex ligation-dependent probe amplification compared with conventional karyotype analysis in the investigation of contributing factors to recurrent pregnancy loss. Method: A cohort of 284 patients underwent side-by-side analysis of products of conception by both conventional karyotyping and multiplex ligation-dependent probe amplification with direct comparison of results. Results: Multiplex ligation-dependent probe amplification was shown to enable a diagnosis for an additional 47 (16.5%) patients compared with conventional karyotype analysis. However, this advantage was offset by some disadvantages of the method, including a high false-positive rate (8/104; 7.7%), as demonstrated by single-arm probe abnormalities of uncertain clinical significance, as well as the inability to characterize structural rearrangements, such as Robertsonian translocations, which comprised 2.46% of samples (99% confidence interval ϭ 0.09 -4.83), and ploidy changes. The calculated performance characteristics of multiplex ligation-dependent probe amplification in this cohort yielded a sensitivity of 86.9% and specificity of 92.4%. Conclusions: The advantages of now widely accepted molecular methodologies, such as lower failure rates, faster turnaround times, and lower cost, must be complemented by adequate counseling, family follow-up, and specific diagnostic reporting practices. It is particularly important to specifically address the important limitations of the methodology, including the inability to characterize balanced structural rearrangements and ploidy changes, especially if multiplex ligation-dependent probe amplification is to be performed alone. Genet Med 2011:13(1):46 -51. Key Words: recurrent pregnancy loss, products of conception, genetic diagnosis, cytogenetics, multiplex ligation-dependant probe amplificationA proximately 15-20% of recognized pregnancies end in a spontaneous abortion before 20 weeks gestation. The burden of pregnancy loss is, however, not uniformly distributed. As a result of the increase in the risk of trisomic pregnancies with advancing maternal age, Ͼ40% of pregnancies in women older than 40 years end in spontaneous abortion. Recurrent pregnancy loss (RPL) is also frequent, affecting approximately 5% of couples. The major factors contributing to pregnancy loss are chromosomal in origin with approximately 50% of products of conception (POC) demonstrating either aneuploid or polyploid karyotypes. 1,2

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“…The use of subtelomeric MLPA in POC specimens should identify all trisomies and unbalanced translocations but will miss polyploidy, 47 unless an concurrent FISH panel is also evaluated. 48 Additionally, array CGH removes the analytic ambiguity of poor chromosome morphology and provides a greater genomic resolution than both chromosome and FISH analysis. However, difficulties in interpretation of array CGH occur due to the detection of polymorphisms or variations of unknown clinical significance, in addition to the missed identification of balanced rearrangements and polyploid states.…”

Section: Genetic Testing Algorithm For Poc Samplesmentioning

confidence: 99%

Reflex fluorescent in situ hybridization testing for unsuccessful product of conception cultures: A retrospective analysis of 5555 samples attempted by conventional cytogenetics and fluorescent in situ hybridization

Shearer

Thorland

Carlson

et al. 2011

Genetics in Medicine

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Introduction:The use of chromosome analysis on products of conception from spontaneous abortions is recommended to identify a genetic etiology. However, 20% of products of conception cultures are unsuccessful due to microbial contamination or lack of viable dividing cells. Our laboratory implemented a reflex fluorescent in situ hybridization (FISH) assay to detect numeric chromosome abnormalities for unsuccessful cultures. Materials and Methods: All products of conception samples were simultaneously processed for both chromosome analysis and FISH analysis. If the chromosome analysis was unsuccessful, interphase FISH was performed for chromosomes 13,16,18,21,22, X, and Y. To assess the performance of the FISH assay, a 3-year retrospective comparative analysis of the FISH results versus chromosome results was performed. Results: Of 5555 total specimens, 4189 (75%) represented chorionic villi/fetal tissue and 1366 (25%) represented tissue of unidentified origin. Of the 1189 tissues of unidentified origin with chromosome or FISH results, 1096 (92%) were XX, indicating that the majority of these tissues are likely maternal in origin. Of the 3361 successful chromosome studies on the chorionic villi/fetal tissue specimens, 1734 (52%) samples had a chromosome abnormality. Of the 762 successful FISH studies on chorionic villi/fetal tissue specimens that were unsuccessful by chromosome studies, 181 (25%) had an abnormal result with the targeted FISH panel. Overall, the FISH panel detected approximately 70% of the chromosome abnormalities in products of conception detectable by karyotype. When the FISH panel results were combined with chromosome analysis for the 4189 chorionic villi/fetal tissue specimens, the overall abnormality rate is 47%. Conclusions: Our reflex FISH assay proved useful for the detection of common chromosome aneuploidies in products of conception samples that failed conventional chromosome analysis. Because of its limited view of the genome, cautious interpretation of FISH results is required for all samples, in particular, trisomy of an acrocentric chromosome, which may represent a Robertsonian translocation. An algorithmic approach to the genetic evaluation of products of conception specimens, with the potential for initial evaluation by a FISH panel, may be warranted. Genet Med 2011:13(6):545-552.

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High‐throughput analysis of chromosome abnormality in spontaneous miscarriage using an MLPA subtelomere assay with an ancillary FISH test for polyploidy

Cited by 48 publications

References 14 publications

Performance of MLPA as a screening method for aneuploidy in uncultured amniocytes

Performance of MLPA as a screening method for aneuploidy in uncultured amniocytes

A comparison of molecular and cytogenetic techniques for the diagnosis of pregnancy loss

Reflex fluorescent in situ hybridization testing for unsuccessful product of conception cultures: A retrospective analysis of 5555 samples attempted by conventional cytogenetics and fluorescent in situ hybridization

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