Human Reproduction Update Advance Access published March 12, 2015 Human Reproduction Update, Vol.0, No.0 pp. 1– 3, 2015
LETTER TO THE EDITOR
Cell-free nucleic acids as non-invasive biomarkers of gynecological disorders, fetal aneuploidy and constitutional maternal chromosomal mosaicism
& The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: [email protected]
Downloaded from http://humupd.oxfordjournals.org/ at KTH Royal Institute of Technology on August 12, 2015
Sir, We enjoyed reading the article entitled ‘Cell-free nucleic acids as noninvasive biomarkers of gynecological cancers, ovarian, endometrial and obstetric disorders and fetal aneuploidy’ by Traver et al. (2014). It is now widely accepted that massively parallel sequencing of maternal plasma DNA provides high sensitivity and specificity for non-invasive detection of fetal aneuploidy (Wang et al., 2014a, b). However, recent studies have raised concerns about high false-positive rates of such noninvasive prenatal testing (NIPT) assays reaching up to 7% for trisomy 21 (Wang et al., 2014a). One important consideration is the impact of the maternal genome as a possible cause of false-positive results, including gynecological conditions (Lau et al., 2013; Traver et al., 2014). There is also another important factor to consider; the presence of constitutional maternal chromosomal mosaicism. We analyzed a 28-year-old primigravida who was enrolled into our research study under an IRB-approved protocol due to abnormal fetal ultrasound showing cystic hydroma at 10 weeks of gestation. NIPT using plasma DNA sequencing, coupled with the ‘Minimally Invasive Karyotyping’ (MINK) analysis algorithm (Chu et al., 2009) was performed and returned a significant P-value consistent with a gain in copy number of chromosome 21, yet the fetal karyotype was normal as determined by chorionic villus sampling (CVS) followed by classical chromosome and FISH analyses. NIPT was initially interpreted as a false-positive finding. However, we suspected there might be a biological explanation and performed high-resolution copy number variation analyses of maternal
genomic DNA, CVS derived DNA and maternal plasma DNA. By comparing the affected maternal plasma and maternal genomic DNA libraries, respectively, against normal plasma and normal genomic DNA libraries, we identified a gain of 26% in maternal plasma and a gain of 28% in maternal genomic DNA involving the 21q11.2–q22.12 chromosome region (chr21:14 350 000– 32 650 000) (Fig. 1A and B). Similarly, microarray analysis on a pure maternal DNA sample revealed a gain in the chr21:14 420 615– 32 635 501 (hg19) region, which is suggestive of a mosaicism for an extranumerary abnormal chromosome 21 (Fig. 1C). FISH analysis on uncultured interphase cells from maternal peripheral blood (Fig. 1D and E) showed 28% of cells with an additional chromosome 21. Our data demonstrate that an apparent ‘false-positive’ NIPT result for trisomy 21 was due to maternal somatic mosaicism for an extranumerary chromosome 21. Recurrent trisomy 21 in offspring of young, apparently healthy parents has been explained in some families by maternal germline mosaicism, also reviewed by Taylor et al. (2014). We would like to raise an important issue on interpretation of positive NIPT findings and clinical significance of chromosomal mosaicism. Sequencing of the maternal plasma may reveal a maternal genomic abnormality which may elevate the risk of future pregnancies with trisomy 21, trisomy for an abnormal chromosome or 21q deletion. Despite the lack of current apparent health consequences of mosaic trisomy 21 for the mother, she may have elevated risk for hematopoietic malignancy and/or dementia and late onset conditions. Gonadal and somatic mosaicism for point mutations is a well-documented cause for a number of genetic diseases; however low-level mosaicism for aneuploidy or structurally abnormal chromosomes is likely also a common phenomenon, yet underestimated. We propose that all positive NIPT results should be followed by diagnostic fetal testing, parental DNA analysis, and genetic counseling.
2
Letter to the Editor
References Chu T, Bunce K, Hogge WA, Peters DG. Statistical model for whole genome sequencing and its application to minimally invasive diagnosis of fetal genetic disease. Bioinformatics 2009;25:1244– 1250. Lau TK, Jiang FM, Stevenson RJ, Lo TK, Chan LW, Chan MK, Lo PS, Wang W, Zhang HY, Chen F et al. Secondary findings from non-invasive prenatal testing for common fetal aneuploidies by whole genome sequencing as a clinical service. Prenat Diagn 2013; 33:602– 608.
Taylor TH, Gitlin SA, Patrick JL, Crain JL, Wilson JM, Griffin DK. The origin, mechanisms, incidence and clinical consequences of chromosomal mosaicism in humans. Hum Reprod Update 2014;20:571–581. Traver S, Assou S, Scalici E, Haouzi D, Al-Edani T, Belloc S, Hamamah S. Cell-free nucleic acids as non-invasive biomarkers of gynecological cancers, ovarian, endometrial and obstetric disorders and fetal aneuploidy. Hum Reprod Update 2014;20:905–923. Wang JC, Sahoo T, Schonberg S, Kopita KA, Ross L, Patek K, Strom CM. Discordant noninvasive prenatal testing and cytogenetic results: a study of 109 consecutive cases. Genet Med 2014a. doi: 10.1038/gim.2014.92 (epub ahead of print).
Downloaded from http://humupd.oxfordjournals.org/ at KTH Royal Institute of Technology on August 12, 2015
Figure 1 Maternal mosaicism for partial trisomy 21 detected via non-invasive prenatal testing for fetal aneuploidy of a karyotypically normal fetus. (A) Copy number plots of affected maternal (red) and normal maternal plasma control (n ¼ 7, blue) DNA samples. Y-axis represents normalized, and GC corrected, read counts. X-axis represents indices of mappable 50 kb regions in chr21. (B) Copy number plots of affected maternal leukocyte genomic DNA (green), a CVS genomic DNA sample from the same pregnancy (red), and confirmed normal CVS genomic DNA samples (n ¼ 6, blue). (C) Array-CGH log2 ratio plot of chromosome 21 of maternal genomic DNA, showing a gain in the 21q11.2– q22.11 chromosome region. Bottom, idiogram of chromosome 21. Oligonucleotide probes represented by colored dots on a log2 scale (Y-axis) and arranged on X-axis according to the Human Genome assembly (GRCh37/hg19) position from the centromere (left) to the telomere (right) of chromosome 21. Black dots indicate normal copy number for DNA probes with mean log2 of 0.0. Gain of copy number (blue dots) is detected for regions with a positive log2 ratio (above 0), while red dots with a negative log2 ratio (below 0) indicate loss of DNA copy number. Mean log2 ratio of +0.58 is expected for an extra copy gain in 100% of the cells. Mosaic gain of an 18.2 Mb segment (21q11.2 – q22.11) is detected by 768 consecutive oligonucleotide probes with an average log2 ratio of 0.203. Top, a position of probes for FISH analysis using clones RP11-143A3 (labeled red) and RP11-108H5 (labeled green) is shown. (D) Interphase FISH analysis demonstrates a normal chromosome 21 complement detected in 101/140 (72%) of cells studied. (E) FISH analysis reveals three sets of the red and green signals consistent with an extra abnormal chromosome 21 in 39/140 (28%) of cells scored. CGH, comparative, genomic hybridization; CVS, chorionic villus sampling.
3
Letter to the Editor
Wang Y, Chen Y, Tian F, Zhang J, Song Z, Wu Y, Han X, Hu W, Ma D, Cram D et al. Maternal mosaicism is a significant contributor to discordant sex chromosomal aneuploidies associated with noninvasive prenatal testing. Clin Chem 2014b; 60:251– 259.
Tianjiao Chu1,2, Suveyda Yeniterzi1,2, Svetlana A. Yatsenko1, Mary Dunkel2, Aleksandar Rajkovic1, W. Allen Hogge1,2 and David G. Peters1,2,* 1 Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh, Pittsburgh, PA, USA
2
Center for Fetal Medicine, Magee-Women’s Research Institute, Pittsburgh, PA, USA * Correspondence address. Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh, 204 Craft Avenue, Pittsburgh, PA 15213, USA. Tel: 1-412-641-2979, Fax: 1-412-641-6156; E-mail: [email protected] doi:10.1093/humupd/dmv015
Downloaded from http://humupd.oxfordjournals.org/ at KTH Royal Institute of Technology on August 12, 2015
LETTER TO THE EDITOR
Cell-free nucleic acids as non-invasive biomarkers of gynecological disorders, fetal aneuploidy and constitutional maternal chromosomal mosaicism
& The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: [email protected]
Downloaded from http://humupd.oxfordjournals.org/ at KTH Royal Institute of Technology on August 12, 2015
Sir, We enjoyed reading the article entitled ‘Cell-free nucleic acids as noninvasive biomarkers of gynecological cancers, ovarian, endometrial and obstetric disorders and fetal aneuploidy’ by Traver et al. (2014). It is now widely accepted that massively parallel sequencing of maternal plasma DNA provides high sensitivity and specificity for non-invasive detection of fetal aneuploidy (Wang et al., 2014a, b). However, recent studies have raised concerns about high false-positive rates of such noninvasive prenatal testing (NIPT) assays reaching up to 7% for trisomy 21 (Wang et al., 2014a). One important consideration is the impact of the maternal genome as a possible cause of false-positive results, including gynecological conditions (Lau et al., 2013; Traver et al., 2014). There is also another important factor to consider; the presence of constitutional maternal chromosomal mosaicism. We analyzed a 28-year-old primigravida who was enrolled into our research study under an IRB-approved protocol due to abnormal fetal ultrasound showing cystic hydroma at 10 weeks of gestation. NIPT using plasma DNA sequencing, coupled with the ‘Minimally Invasive Karyotyping’ (MINK) analysis algorithm (Chu et al., 2009) was performed and returned a significant P-value consistent with a gain in copy number of chromosome 21, yet the fetal karyotype was normal as determined by chorionic villus sampling (CVS) followed by classical chromosome and FISH analyses. NIPT was initially interpreted as a false-positive finding. However, we suspected there might be a biological explanation and performed high-resolution copy number variation analyses of maternal
genomic DNA, CVS derived DNA and maternal plasma DNA. By comparing the affected maternal plasma and maternal genomic DNA libraries, respectively, against normal plasma and normal genomic DNA libraries, we identified a gain of 26% in maternal plasma and a gain of 28% in maternal genomic DNA involving the 21q11.2–q22.12 chromosome region (chr21:14 350 000– 32 650 000) (Fig. 1A and B). Similarly, microarray analysis on a pure maternal DNA sample revealed a gain in the chr21:14 420 615– 32 635 501 (hg19) region, which is suggestive of a mosaicism for an extranumerary abnormal chromosome 21 (Fig. 1C). FISH analysis on uncultured interphase cells from maternal peripheral blood (Fig. 1D and E) showed 28% of cells with an additional chromosome 21. Our data demonstrate that an apparent ‘false-positive’ NIPT result for trisomy 21 was due to maternal somatic mosaicism for an extranumerary chromosome 21. Recurrent trisomy 21 in offspring of young, apparently healthy parents has been explained in some families by maternal germline mosaicism, also reviewed by Taylor et al. (2014). We would like to raise an important issue on interpretation of positive NIPT findings and clinical significance of chromosomal mosaicism. Sequencing of the maternal plasma may reveal a maternal genomic abnormality which may elevate the risk of future pregnancies with trisomy 21, trisomy for an abnormal chromosome or 21q deletion. Despite the lack of current apparent health consequences of mosaic trisomy 21 for the mother, she may have elevated risk for hematopoietic malignancy and/or dementia and late onset conditions. Gonadal and somatic mosaicism for point mutations is a well-documented cause for a number of genetic diseases; however low-level mosaicism for aneuploidy or structurally abnormal chromosomes is likely also a common phenomenon, yet underestimated. We propose that all positive NIPT results should be followed by diagnostic fetal testing, parental DNA analysis, and genetic counseling.
2
Letter to the Editor
References Chu T, Bunce K, Hogge WA, Peters DG. Statistical model for whole genome sequencing and its application to minimally invasive diagnosis of fetal genetic disease. Bioinformatics 2009;25:1244– 1250. Lau TK, Jiang FM, Stevenson RJ, Lo TK, Chan LW, Chan MK, Lo PS, Wang W, Zhang HY, Chen F et al. Secondary findings from non-invasive prenatal testing for common fetal aneuploidies by whole genome sequencing as a clinical service. Prenat Diagn 2013; 33:602– 608.
Taylor TH, Gitlin SA, Patrick JL, Crain JL, Wilson JM, Griffin DK. The origin, mechanisms, incidence and clinical consequences of chromosomal mosaicism in humans. Hum Reprod Update 2014;20:571–581. Traver S, Assou S, Scalici E, Haouzi D, Al-Edani T, Belloc S, Hamamah S. Cell-free nucleic acids as non-invasive biomarkers of gynecological cancers, ovarian, endometrial and obstetric disorders and fetal aneuploidy. Hum Reprod Update 2014;20:905–923. Wang JC, Sahoo T, Schonberg S, Kopita KA, Ross L, Patek K, Strom CM. Discordant noninvasive prenatal testing and cytogenetic results: a study of 109 consecutive cases. Genet Med 2014a. doi: 10.1038/gim.2014.92 (epub ahead of print).
Downloaded from http://humupd.oxfordjournals.org/ at KTH Royal Institute of Technology on August 12, 2015
Figure 1 Maternal mosaicism for partial trisomy 21 detected via non-invasive prenatal testing for fetal aneuploidy of a karyotypically normal fetus. (A) Copy number plots of affected maternal (red) and normal maternal plasma control (n ¼ 7, blue) DNA samples. Y-axis represents normalized, and GC corrected, read counts. X-axis represents indices of mappable 50 kb regions in chr21. (B) Copy number plots of affected maternal leukocyte genomic DNA (green), a CVS genomic DNA sample from the same pregnancy (red), and confirmed normal CVS genomic DNA samples (n ¼ 6, blue). (C) Array-CGH log2 ratio plot of chromosome 21 of maternal genomic DNA, showing a gain in the 21q11.2– q22.11 chromosome region. Bottom, idiogram of chromosome 21. Oligonucleotide probes represented by colored dots on a log2 scale (Y-axis) and arranged on X-axis according to the Human Genome assembly (GRCh37/hg19) position from the centromere (left) to the telomere (right) of chromosome 21. Black dots indicate normal copy number for DNA probes with mean log2 of 0.0. Gain of copy number (blue dots) is detected for regions with a positive log2 ratio (above 0), while red dots with a negative log2 ratio (below 0) indicate loss of DNA copy number. Mean log2 ratio of +0.58 is expected for an extra copy gain in 100% of the cells. Mosaic gain of an 18.2 Mb segment (21q11.2 – q22.11) is detected by 768 consecutive oligonucleotide probes with an average log2 ratio of 0.203. Top, a position of probes for FISH analysis using clones RP11-143A3 (labeled red) and RP11-108H5 (labeled green) is shown. (D) Interphase FISH analysis demonstrates a normal chromosome 21 complement detected in 101/140 (72%) of cells studied. (E) FISH analysis reveals three sets of the red and green signals consistent with an extra abnormal chromosome 21 in 39/140 (28%) of cells scored. CGH, comparative, genomic hybridization; CVS, chorionic villus sampling.
3
Letter to the Editor
Wang Y, Chen Y, Tian F, Zhang J, Song Z, Wu Y, Han X, Hu W, Ma D, Cram D et al. Maternal mosaicism is a significant contributor to discordant sex chromosomal aneuploidies associated with noninvasive prenatal testing. Clin Chem 2014b; 60:251– 259.
Tianjiao Chu1,2, Suveyda Yeniterzi1,2, Svetlana A. Yatsenko1, Mary Dunkel2, Aleksandar Rajkovic1, W. Allen Hogge1,2 and David G. Peters1,2,* 1 Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh, Pittsburgh, PA, USA
2
Center for Fetal Medicine, Magee-Women’s Research Institute, Pittsburgh, PA, USA * Correspondence address. Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh, 204 Craft Avenue, Pittsburgh, PA 15213, USA. Tel: 1-412-641-2979, Fax: 1-412-641-6156; E-mail: [email protected] doi:10.1093/humupd/dmv015
Downloaded from http://humupd.oxfordjournals.org/ at KTH Royal Institute of Technology on August 12, 2015
