Mary K. Dunkel

Noninvasive prenatal diagnosis of a fetal microdeletion syndrome.

This proof-of-principle study shows that it is possible to detect a genetic microdeletion carried by a fetus through analysis of DNA in circulating maternal blood.

High resolution non-invasive detection of a fetal microdeletion using the GCREM algorithm.

The non‐invasive prenatal detection of fetal microdeletions becomes increasingly challenging as the size of the mutation decreases, with current practical lower limits in the range of a few megabases. Our goals were to explore the lower limits of microdeletion size detection via non‐invasive prenatal tests using Minimally Invasive Karyotyping (MINK) and introduce/evaluate a novel statistical approach we recently developed called the GC Content Random Effect Model (GCREM).

Gender influences the response to experimental silica-induced lung fibrosis in mice

Accumulating evidence suggests that gender can have a profound effect on incidence and severity of a variety of pulmonary diseases. To address the influence of gender on the development of silica-induced pulmonary fibrosis, we instilled 0.2 g/kg silica into male and female C57BL/6 mice and examined the fibrotic and inflammatory response at 14 days postexposure. Both silica-exposed male and female mice had significant increases in total lung hydroxyproline compared with saline controls. However, silica-exposed female mice had significantly less total lung hydroxyproline than silica-exposed male mice. This observation was confirmed by color thresholding image analysis. Interestingly, silica-exposed female mice had significantly more inflammatory cells, the majority of which were macrophages, as well as higher levels of the macrophage-specific chemokines MCP-1 and CCL9 in whole lung lavage compared with silica-exposed male mice. We also show that at baseline, estrogen receptor α (ERα) mRNA expression is lower in female mice than in males and that ERα mRNA expression is decreased by silica exposure. Finally, we show that the response of ovariectomized female mice to silica instillation is similar to that of male mice. These observations together show that gender influences the lung response to silica.

Innate Immune Activation by Inhaled Lipopolysaccharide, Independent of Oxidative Stress, Exacerbates Silica-Induced Pulmonary Fibrosis in Mice

Acute exacerbations of pulmonary fibrosis are characterized by rapid decrements in lung function. Environmental factors that may contribute to acute exacerbations remain poorly understood. We have previously demonstrated that exposure to inhaled lipopolysaccharide (LPS) induces expression of genes associated with fibrosis. To address whether exposure to LPS could exacerbate fibrosis, we exposed male C57BL/6 mice to crystalline silica, or vehicle, followed 28 days later by LPS or saline inhalation. We observed that mice receiving both silica and LPS had significantly more total inflammatory cells, more whole lung lavage MCP-1, MIP-2, KC and IL-1β, more evidence of oxidative stress and more total lung hydroxyproline than mice receiving either LPS alone, or silica alone. Blocking oxidative stress with N-acetylcysteine attenuated whole lung inflammation but had no effect on total lung hydroxyproline. These observations suggest that exposure to innate immune stimuli, such as LPS in the environment, may exacerbate stable pulmonary fibrosis via mechanisms that are independent of inflammation and oxidative stress.

Cell-free nucleic acids as non-invasive biomarkers of gynecological disorders, fetal aneuploidy and constitutional maternal chromosomal mosaicism

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., 2014, 2015). 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., 2015). 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.

Comparative evaluation of the Minimally-Invasive Karyotyping (MINK) algorithm for non-invasive prenatal testing

Minimally Invasive Karyotyping (MINK) was communicated in 2009 as a novel method for the non-invasive detection of fetal copy number anomalies in maternal plasma DNA. The original manuscript illustrated the potential of MINK using a model system in which fragmented genomic DNA obtained from a trisomy 21 male individual was mixed with that of his karyotypically normal mother at dilutions representing fetal fractions found in maternal plasma. Although it has been previously shown that MINK is able to non-invasively detect fetal microdeletions, its utility for aneuploidy detection in maternal plasma has not previously been demonstrated. The current study illustrates the ability of MINK to detect common aneuploidy in early gestation, compares its performance to other published third party methods (and related software packages) for prenatal aneuploidy detection and evaluates the performance of these methods across a range of sequencing read inputs. Plasma samples were obtained from 416 pregnant women between gestational weeks 8.1 and 34.4. Shotgun DNA sequencing was performed and data analyzed using MINK RAPIDR and WISECONDOR. MINK performed with greater accuracy than RAPIDR and WISECONDOR, correctly identifying 60 out of 61 true trisomy cases, and reporting only one false positive in 355 normal pregnancies. Significantly, MINK achieved accurate detection of trisomy 21 using just 2 million aligned input reads, whereas WISECONDOR required 6 million reads and RAPIDR did not achieve complete accuracy at any read input tested. In conclusion, we demonstrate that MINK provides an analysis pipeline for the detection of fetal aneuploidy in samples of maternal plasma DNA.

High Levels of Sample-to-Sample Variation Confound Data Analysis for Non-Invasive Prenatal Screening of Fetal Microdeletions.

Our goal was to test the hypothesis that inter-individual genomic copy number variation in control samples is a confounding factor in the non-invasive prenatal detection of fetal microdeletions via the sequence-based analysis of maternal plasma DNA. The database of genomic variants (DGV) was used to determine the “Genomic Variants Frequency” (GVF) for each 50kb region in the human genome. Whole genome sequencing of fifteen karyotypically normal maternal plasma and six CVS DNA controls samples was performed. The coefficient of variation of relative read counts (cv.RTC) for these samples was determined for each 50kb region. Maternal plasma from two pregnancies affected with a chromosome 5p microdeletion was also sequenced, and analyzed using the GCREM algorithm. We found strong correlation between high variance in read counts and GVF amongst controls. Consequently we were unable to confirm the presence of the microdeletion via sequencing of maternal plasma samples obtained from two sequential affected pregnancies. Caution should be exercised when performing NIPT for microdeletions. It is vital to develop our understanding of the factors that impact the sensitivity and specificity of these approaches. In particular, benign copy number variation amongst controls is a major confounder, and their effects should be corrected bioinformatically.