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The standard-of-care (SOC) diagnostic prenatal testing includes a combination of cytogenetic methods such as karyotyping, fluorescence in situ hybridization (FISH), and chromosomal microarray (CMA) using either direct or cultured amniocytes or chorionic villi sampling (CVS). However, each technology has its limitations: karyotyping has a low resolution (>5Mb), FISH is targeted, and CMA does not detect balanced structural variants (SVs) or decipher complex rearrangements in the genome. These limitations necessitate the use of multiple tests, either simultaneously or sequentially to reach a genetic diagnosis. This long-standing prenatal testing workflow demonstrates the need for an alternative technology that can provide high-resolution results in a cost and time-effective manner. Optical genome mapping (OGM) is an emerging technology that has demonstrated its ability to detect all classes of SVs, including copy number variations (CNVs) and balanced abnormalities in a single assay, but has not been evaluated in the prenatal setting. This retrospective validation study analyzed 114 samples (including replicates), representing 94 unique and well-characterized samples that were received in our laboratory for traditional cytogenetic analysis with karyotyping, FISH, and/or CMA. Samples comprised 84 cultured amniocytes, and 10 phenotypically normal and cytogenetically negative controls. Six samples were run in triplicate to evaluate intra-run, inter-run, and inter-instrument reproducibility. Clinically relevant SVs and CNVs were reported using the Bionano Access software with standardized and built-in filtration criteria and phenotype-specific analysis. OGM was 100% concordant in identifying the 101 aberrations that included 29 interstitial/terminal deletions, 28 duplications, 26 aneuploidies, 6 absence of heterozygosity (AOH), 3 triploid genomes, 4 Isochromosomes, 1 translocation, and revealed the identity of 3 marker chromosomes, and 1 chromosome with additional material not determined by karyotyping. Additionally, OGM detected 64 additional clinically reportable SVs in 43 samples. OGM demonstrated high technical and analytical robustness and a limit of detection of 5% allele fraction for interstitial deletions and duplications, and 10% allele fraction for translocation and aneuploidy. This study demonstrates that OGM has the potential to identify unique genomic abnormalities such as CNVs, AOHs, and several classes of SVs including complex structural rearrangements. OGM has a standardized laboratory workflow and reporting solution that can be adopted in routine clinical laboratories and demonstrates the potential to replace the current SOC methods for prenatal diagnostic testing. We recommend its use as a first-tier genetic diagnostic test in a prenatal setting.

Rett (RTT) syndrome, a neurodevelopmental disorder caused by pathogenic variation in the MECP2 gene, is characterized by developmental regression, loss of purposeful hand movements, stereotypic hand movements, abnormal gait, and loss of spoken language. Due to the X-linked inheritance pattern, RTT is typically limited to females. Recent studies revealed somatic mosaicism in MECP2 in male patients with RTT-like phenotypes. While detecting mosaic variation using Sanger sequencing is theoretically possible for mosaicism over ~15%-20%, several variables, including efficiency of PCR, background noise, and/or human error, contribute to a low detection rate using this technology. Mosaic variants in two males were detected by next generation sequencing (NGS; Case 1) and by Sanger re-sequencing (Case 2). Both had targeted digital PCR (dPCR) to confirm the variants. In this report, we present two males with classic RTT syndrome in whom we identified pathogenic variation in the MECP2 gene in the mosaic state (c.730C T (p.Gln244*) in Patient 1 and c.397C T (p.Arg133Cys) in Patient 2). In addition, estimates and measures of mosaic variant fraction were surprisingly similar between Sanger sequencing, NGS, and dPCR. The mosaic state of these variants contributed to a lengthy diagnostic odyssey for these patients. While NGS and even Sanger sequencing may be viable methods of detecting mosaic variation in DNA or RNA samples, applying targeted dPCR to supplement these sequencing technologies would provide confirmation of somatic mosaicism and mosaic fraction.

An expanding range of genetic syndromes are characterized by genome-wide disruptions in DNA methylation profiles referred to as episignatures. Episignatures are distinct, highly sensitive and specific biomarkers that have recently been applied in clinical diagnosis of genetic syndromes. Episignatures are contained within the broader disorder-specific genome-wide DNA methylation changes which can share significant overlap amongst different conditions. In this study we performed functional genomic assessment and comparison of disorder-specific and overlapping genome-wide DNA methylation changes related to 65 genetic syndromes with previously described episignatures. We demonstrate evidence of disorder-specific and recurring genome-wide differentially methylated probes (DMPs) and regions (DMRs). The overall distribution of DMPs and DMRs across the majority of the neurodevelopmental genetic syndromes analyzed showed substantial enrichment in gene promoters and CpG islands, and under-representation of the more variable intergenic regions. Analysis showed significant enrichment of the DMPs and DMRs in gene pathways and processes related to neurodevelopment, including neurogenesis, synaptic signaling and synaptic transmission. This study expands beyond the diagnostic utility of DNA methylation episignatures by demonstrating correlation between the function of the mutated genes and the consequent genomic DNA methylation profiles as a key functional element in the molecular etiology of genetic neurodevelopmental disorders. This article is protected by copyright. All rights reserved.

Conventional cytogenetic analysis of products of conception (POC) is of limited utility because of failed cultures, as well as microbial and maternal cell contamination (MCC). Optical genome mapping (OGM) is an emerging technology that has the potential to replace conventional cytogenetic methods. The use of OGM precludes the requirement for culturing (and related microbial contamination). However, a high percentage of MCC impedes a definitive diagnosis, which can be addressed by an additional pre-analytical quality control step that includes histological assessment of H&E stained slides from formalin-fixed paraffin embedded (FFPE) tissue with macro-dissection for chorionic villi to enrich fetal tissue component for single nucleotide polymorphism microarray (SNPM) analysis. To improve the diagnostic yield, an integrated workflow was devised that included MCC characterization of POC tissue, followed by OGM for MCC-negative cases or SNPM with histological assessment for MCC-positive cases. A result was obtained in 93% (29/31) of cases with a diagnostic yield of 45.1% (14/31) with the proposed workflow, compared to 9.6% (3/31) and 6.4% (2/31) with routine workflow, respectively. The integrated workflow with these technologies demonstrates the clinical utility and higher diagnostic yield in evaluating POC specimens.

Conventional cytogenetic analysis of products of conception (POC) is of limited utility because of failed cultures, microbial and maternal cell contamination (MCC). Optical genome mapping (OGM) is an emerging technology that has the potential to replace conventional cytogenetic methods. The use of OGM precludes the requirement for culturing (and related microbial contamination). However, a high percentage of MCC impedes a definitive diagnosis, which can be addressed by an additional pre-analytical quality control step that includes histological assessment of H&E stained slides from FFPE tissue with macro-dissection for Chorionic villi to enrich fetal tissue component for Single nucleotide polymorphism (SNP) microarray analysis. An internal audit of POC cases subjected to karyotype-only analysis showed a low yield on clinically actionable information that contributed to patient care. To improve the diagnostic yield, an integrated workflow was devised that included MCC characterization of POC tissue, followed by OGM for MCC negative cases or SNPM with histological assessment for MCC positive cases. A result was obtained in 93% (29/31) cases with a diagnostic yield of 45.1% (14/31) with proposed workflow compared to 9.6% (3/31) and 6.4% (2/31) with routine workflow, respectively. The integrated workflow with these technologies demonstrates the clinical utility and higher diagnostic yield in evaluating POC specimens.

It is well documented that electrospun tissue engineering scaffolds can be fabricated with variable degrees of fiber alignment to produce scaffolds with anisotropic mechanical properties. Several attempts have been made to quantify the degree of fiber alignment within an electrospun scaffold using image-based methods. However, these methods are limited by the inability to produce a quantitative measure of alignment that can be used to make comparisons across publications. Therefore, we have developed a new approach to quantifying the alignment present within a scaffold from scanning electron microscopic (SEM) images. The alignment is determined by using the Sobel approximation of the image gradient to determine the distribution of gradient angles with an image. This data was fit to a Von Mises distribution to find the dispersion parameter κ, which was used as a quantitative measure of fiber alignment. We fabricated four groups of electrospun polycaprolactone (PCL) + Gelatin scaffolds with alignments ranging from κ = 1.9 (aligned) to κ = 0.25 (random) and tested our alignment quantification method on these scaffolds. It was found that our alignment quantification method could distinguish between scaffolds of different alignments more accurately than two other published methods. Additionally, the alignment parameter κ was found to be a good predictor the mechanical anisotropy of our electrospun scaffolds. The ability to quantify fiber alignment within and make direct comparisons of scaffold fiber alignment across publications can reduce ambiguity between published results where cells are cultured on "highly aligned" fibrous scaffolds. This could have important implications for characterizing mechanics and cellular behavior on aligned tissue engineering scaffolds. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1680-1686, 2016.

Early decompressive fasciotomy is essential in the prevention of the sequelae of compartment syndrome. Many techniques have been described for the closure of the fasciotomy wound, and controversy exists as to which method is the best. Primary closure is often impossible secondary to tissue retraction and edema. Split-thickness skin grafting leaves a thin, insensate, and often aesthetically unpleasing result. Gradual mechanical dermal apposition has been used with increasing frequency, and has been shown to be effective in the closure of fasciotomies, but often takes 7–10 days for closure. We present our experience with the STAR, a mechanical method of fasciotomy wound closure that is effective in 2–4 days, and is extremely simple to use.