Medicina molecular trisomia 21

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DOI: 10.1002/pd.2922

ORIGINAL ARTICLE

Selective analysis of cell-free DNA in maternal blood for evaluation of fetal trisomy
Andrew B. Sparks1†, Eric T. Wang1†, Craig A. Struble1, Wade Barrett1, Renee Stokowski1, Celeste McBride1, Jacob Zahn1, Kevin Lee1, Naiping Shen1, Jigna Doshi1, Michel Sun1, Jill Garrison1, Jay Sandler1, Desiree Hollemon1, Patrick Pattee1, Aoy Tomita-Mitchell2, MichaelMitchell2, John Stuelpnagel1, Ken Song1* and Arnold Oliphant1
1 2

Aria Diagnostics, Inc., 5945 Optical Court, San Jose, CA 95138, USA. Medical College of Wisconsin, Milwaukee, WI 53226, USA. *Correspondence to: Ken Song. E-mail: ksong@ariadx.com † Authors contributed equally to the work.

ABSTRACT
Objective To develop a novel prenatal assay based on selective analysis of cell-free DNA inmaternal blood for evaluation of fetal Trisomy 21 (T21) and Trisomy 18 (T18). Methods Two hundred ninety-eight pregnancies, including 39 T21 and seven T18 confirmed fetal aneuploidies, were analyzed using a novel, highly multiplexed assay, termed digital analysis of selected regions (DANSR™). Cell-free DNA from maternal blood samples was analyzed using DANSR assays for loci on chromosomes 21 and 18.Products from 96 separate patients were pooled and sequenced together. A standard Z-test of chromosomal proportions was used to distinguish aneuploid samples from average-risk pregnancy samples. DANSR aneuploidy discrimination was evaluated at various sequence depths. Results At the lowest sequencing depth, corresponding to 204 000 sequencing counts per sample, average-risk cases
wheredistinguished from T21 and T18 cases, with Z statistics for all cases exceeding 3.6. Increasing the sequencing depth to 410 000 counts per sample substantially improved separation of aneuploid and average-risk cases. A further increase to 620 000 counts per sample resulted in only marginal improvement. This depth of sequencing represents less than 5% of that required by massively parallel shotgunsequencing approaches.

Conclusion Digital analysis of selected regions enables highly accurate, cost efficient, and scalable noninvasive fetal aneuploidy assessment. © 2012 John Wiley & Sons, Ltd.
Funding sources: None Conflicts of interest: None declared

INTRODUCTION
Prenatal testing for fetal chromosomal aneuploidies is commonly practiced and endorsed by professional medical organizations.1,2Prenatal testing currently encompasses both screening and diagnostic modalities. Screening involves analysis of serum markers and/or ultrasound interpretation of fetal measurements such as nuchal translucency, but has suboptimal sensitivity and specificity.1 Diagnostic testing includes invasive procedures such as chorionic villus sampling (CVS) or amniocentesis, and although these tests are highlyaccurate, they come at significant health risks to the fetus and mother, including the potential loss of a healthy fetus.3 For the past several decades, numerous efforts have been pursued to develop a maternal blood test with improved accuracy
Prenatal Diagnosis 2012, 32, 3–9

for the detection of major fetal aneuploidies. Such blood tests could improve current screening practices for fetalaneuploidy. Initial efforts targeting isolation and analysis of circulating fetal cells in the maternal bloodstream have not proven successful, because of the challenges in detecting sufficient fetal cell numbers in circulation.4–6 By contrast, analysis of cell-free DNA (cfDNA) in maternal circulation has shown promise for evaluation of fetal aneuploidy. Several groups have demonstrated the use of massivelyparallel DNA shotgun sequencing (MPSS) to assay for fetal Trisomy 21 (T21) from cfDNA in maternal blood.7–10 More recently, several pilot studies have shown the ability of MPSS to assay for Trisomy 18 (T18) and Trisomy 13 (T13) from cfDNA in maternal blood, although with more variable results.10,11 Whereas MPSS has demonstrated robust technical performance, its cost and complexity, including...
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