Mary Goetsch

Investigation of somatic NKX2-5 mutations in congenital heart disease

Background: Reports of somatic mutations found in hearts with cardiac septal defects have suggested that these mutations are aetiologic in pathologic cardiac development. However, the hearts in these reports had been fixed in formalin for over 22 years. Because of the profound implication of this finding, we attempted to replicate it using fresh frozen tissue obtained in the current era from 28 patients with septal defects who underwent cardiac surgery and who were enrolled in our congenital heart disease tissue bank. Methods: Our cohort included patients with atrial septal defects (ASD, n = 13), ventricular septal defects (VSD, n = 5), and atrioventricular canal defects (AVCD, n = 10). Cardiac tissue samples were collected both from diseased tissue located immediately adjacent to the defect and from anatomically normal tissue located at a site remote from the defect (right atrial appendage). Tissue samples were immediately frozen in liquid nitrogen and stored at −80°C. Genomic DNA was isolated and amplified using the same methodology described in the previously published reports. 42 pathologic cardiac tissue samples were sequenced. Results: One non-synonymous germline sequence variant was identified in one patient. Two synonymous germline sequence variants were identified in two separate patients. A common single nucleotide polymorphism (SNP) was identified in 16 patients. Based on the incidence of somatic mutations described in the previously published reports, our study was adequately powered to replicate the previous studies. No evidence of somatic mutations was found in this study. Conclusion: Somatic mutations in NKX2-5 do not represent an important aetiologic pathway in pathologic cardiac development in patients with cardiac septal defects.

Influence of Temperature during Transportation on Cell-Free DNA Analysis

Objective: To determine the effect of shipping blood in Streck blood collection tubes (BCT) prior to processing on cell-free DNA (cf-DNA) levels. Methods: Blood was collected in ethylenediaminetetraacetic acid (EDTA) and BCT tubes from 10 pregnant women carrying male fetuses. One set of each tube for each subject was processed to plasma immediately (standard cf-DNA protocol), whereas the other set was shipped by air courier and then processed. DNA was extracted and total and fetal DNA concentrations were measured by TaqMan multiplexed quantitative real-time PCR. Results: No significant difference was observed in total cf-DNA in plasma between immediately processed EDTA (control) and immediately processed BCT samples. Moreover, no significant change in total cf-DNA was detected in plasma of BCT samples shipped at room temperature. Significant differences in total cf-DNA leading to a significantly decreased fetal fraction were found in shipped EDTA samples and BCT samples shipped at 4°C. Discussion: BCT tubes are suitable for shipping whole blood prior to processing with respect to cf-DNA levels. However, care should be taken to ensure that samples are not exposed to extreme temperatures during shipment. This finding will benefit the development and clinical application of noninvasive methods of fetal diagnosis that utilize cf-DNA.

Non-Invasive Prenatal Detection of Trisomy 21 Using Tandem Single Nucleotide Polymorphisms

Background Screening tests for Trisomy 21 (T21), also known as Down syndrome, are routinely performed for the majority of pregnant women. However, current tests rely on either evaluating non-specific markers, which lead to false negative and false positive results, or on invasive tests, which while highly accurate, are expensive and carry a risk of fetal loss. We outline a novel, rapid, highly sensitive, and targeted approach to non-invasively detect fetal T21 using maternal plasma DNA. Methods and Findings Highly heterozygous tandem Single Nucleotide Polymorphism (SNP) sequences on chromosome 21 were analyzed using High-Fidelity PCR and Cycling Temperature Capillary Electrophoresis (CTCE). This approach was used to blindly analyze plasma DNA obtained from peripheral blood from 40 high risk pregnant women, in adherence to a Medical College of Wisconsin Institutional Review Board approved protocol. Tandem SNP sequences were informative when the mother was heterozygous and a third paternal haplotype was present, permitting a quantitative comparison between the maternally inherited haplotype and the paternally inherited haplotype to infer fetal chromosomal dosage by calculating a Haplotype Ratio (HR). 27 subjects were assessable; 13 subjects were not informative due to either low DNA yield or were not informative at the tandem SNP sequences examined. All results were confirmed by a procedure (amniocentesis/CVS) or at postnatal follow-up. Twenty subjects were identified as carrying a disomy 21 fetus (with two copies of chromosome 21) and seven subjects were identified as carrying a T21 fetus. The sensitivity and the specificity of the assay was 100% when HR values lying between 3/5 and 5/3 were used as a threshold for normal subjects. Conclusions In summary, a targeted approach, based on calculation of Haplotype Ratios from tandem SNP sequences combined with a sensitive and quantitative DNA measurement technology can be used to accurately detect fetal T21 in maternal plasma when sufficient fetal DNA is present in maternal plasma.

Impact of MYH6 variants in hypoplastic left heart syndrome

Hypoplastic left heart syndrome (HLHS) is a clinically and anatomically severe form of congenital heart disease (CHD). Although prior studies suggest that HLHS has a complex genetic inheritance, its etiology remains largely unknown. The goal of this study was to characterize a risk gene in HLHS and its effect on HLHS etiology and outcome. We performed next-generation sequencing on a multigenerational family with a high prevalence of CHD/HLHS, identifying a rare variant in the α-myosin heavy chain (MYH6) gene. A case-control study of 190 unrelated HLHS subjects was then performed and compared with the 1000 Genomes Project. Damaging MYH6 variants, including novel, missense, in-frame deletion, premature stop, de novo, and compound heterozygous variants, were significantly enriched in HLHS cases (P < 1 × 10−5). Clinical outcomes analysis showed reduced transplant-free survival in HLHS subjects with damaging MYH6 variants (P < 1 × 10−2). Transcriptome and protein expression analyses with cardiac tissue revealed differential expression of cardiac contractility genes, notably upregulation of the β-myosin heavy chain (MYH7) gene in subjects with MYH6 variants (P < 1 × 10−3). We subsequently used patient-specific induced pluripotent stem cells (iPSCs) to model HLHS in vitro. Early stages of in vitro cardiomyogenesis in iPSCs derived from two unrelated HLHS families mimicked the increased expression of MYH7 observed in vivo (P < 1 × 10−2), while revealing defective cardiomyogenic differentiation. Rare, damaging variants in MYH6 are enriched in HLHS, affect molecular expression of contractility genes, and are predictive of poor outcome. These findings indicate that the etiology of MYH6-associated HLHS can be informed using iPSCs and suggest utility in future clinical applications.

254 Highly Sensitive Transplant Rejection Surveillance Using Targeted Detection of Donor Specific Cell Free DNA

Provided herein are methods and computer-readable storage media related to cell-free DNA and uses thereof to determine risk of a condition, such as transplant rejection or cancer, in a subject.

Characterization of the pixB gene in Xenorhabdus nematophila and discovery of a new gene family

Xenorhabdus nematophila are Gram-negative bacteria that engage in mutualistic associations with entomopathogenic nematodes. To reproduce, the nematodes invade insects and release X. nematophila into the haemolymph where it functions as an insect pathogen. In complex medium, X. nematophila cells produce two distinct types of intracellular crystalline inclusions, one composed of the methionine-rich PixA protein and the other composed of the PixB protein. Here we show that PixB crystalline inclusions were neither apparent in X. nematophila cells grown in medium that mimics insect haemolymph (Graces medium) nor in cells grown directly in the insect haemocoel. The identified pixB gene was regulated by a conserved σ70 promoter while the pixA promoter was less well conserved. Expression of pixA and pixB under biological conditions was analysed using GFP promoter reporters. Microplate fluorescence detection and flow cytometry analyses revealed that pixB was expressed at high levels in Graces medium and in insect haemolymph and at lower levels in complex medium, while pixA was expressed at lower levels under all conditions. Although pixB was highly expressed in Graces medium, PixB crystalline inclusions were not present, suggesting that under biological conditions PixB production may be controlled post-transcriptionally. Although a pixB-minus strain was constructed, the function of PixB remains unresolved. The pixB gene was present in few Xenorhabdus species and pixB-type genes were identified in some Proteobacteria and Gram-positive species, while pixA was only present in Xenorhabdus species. Two conserved sequences were identified in PixB-type proteins that characterize this previously unrecognized gene family.