Haplotype-based analysis resolves missing heritability in oculocutaneous albinism type 1B.

Oculocutaneous albinism (OCA) is a rare disorder of pigment production. Affected individuals have variably decreased global pigmentation and visual-developmental changes that lead to low vision. OCA is notable for significant missing heritability, particularly among individuals with residual pigmentation. Tyrosinase (TYR) is the rate-limiting enzyme in melanin pigment biosynthesis and mutations that decrease enzyme function are one of the most common causes of OCA. We present the analysis of high-depth short-read TYR sequencing data for a cohort of 352 OCA probands, ∼50% of whom were previously sequenced without yielding a definitive diagnostic result. Our analysis identified 66 TYR single-nucleotide variants (SNVs) and small insertion/deletions (indels), 3 structural variants, and a rare haplotype comprised of two common frequency variants (p.Ser192Tyr and p.Arg402Gln) in cis-orientation, present in 149/352 OCA probands. We further describe a detailed analysis of the disease-causing haplotype, p.[Ser192Tyr; Arg402Gln] ("cis-YQ"). Haplotype analysis suggests that the cis-YQ allele arose by recombination and that multiple cis-YQ haplotypes are segregating in OCA-affected individuals and control populations. The cis-YQ allele is the most common disease-causing allele in our cohort, representing 19.1% (57/298) of TYR pathogenic alleles in individuals with type 1 (TYR-associated) OCA. Finally, among the 66 TYR variants, we found several additional alleles defined by a cis-oriented combination of minor, potentially hypomorph-producing alleles at common variant sites plus a second, rare pathogenic variant. Together, these results suggest that identification of phased variants for the full TYR locus are required for an exhaustive assessment for potentially disease-causing alleles.

A custom capture sequence approach for oculocutaneous albinism identifies structural variant alleles at the OCA2 locus.

Oculocutaneous albinism (OCA) is a heritable disorder of pigment production that manifests as hypopigmentation and altered eye development. Exon sequencing of known OCA genes is unsuccessful in producing a complete molecular diagnosis for a significant number of affected individuals. We sequenced the DNA of individuals with OCA using short-read custom capture sequencing that targeted coding, intronic, and noncoding regulatory regions of known OCA genes, and genome-wide association study-associated pigmentation loci. We identified an OCA2 complex structural variant (CxSV), defined by a 143 kb inverted segment reintroduced in intron 1, upstream of the native location. The corresponding CxSV junctions were observed in 11/390 probands screened. The 143 kb CxSV presents in one family as a copy number variant duplication for the 143 kb region. In the remaining 10/11 families, the 143 kb CxSV acquired an additional 184 kb deletion across the same region, restoring exons 3-19 of OCA2 to a copy-number neutral state. Allele-associated haplotype analysis found rare SNVs rs374519281 and rs139696407 are linked with the 143 kb CxSV in both OCA2 alleles. For individuals in which customary molecular evaluation does not reveal a biallelic OCA diagnosis, we recommend preliminary screening for these haplotype-associated rare variants, followed by junction-specific validation for the OCA2 143 kb CxSV.

Automated Digital Quantification of Pulmonary Fibrosis in Human Histopathology Specimens.

Pulmonary fibrosis is characterized by abnormal interstitial extracellular matrix and cellular accumulations. Methods quantifying fibrosis severity in lung histopathology samples are semi-quantitative, subjective, and analyze only portions of sections. We sought to determine whether automated computerized imaging analysis shown to continuously measure fibrosis in mice could also be applied in human samples. A pilot study was conducted to analyze a small number of specimens from patients with Hermansky-Pudlak syndrome pulmonary fibrosis (HPSPF) or idiopathic pulmonary fibrosis (IPF). Digital images of entire lung histological serial sections stained with picrosirius red and alcian blue or anti-CD68 antibody were analyzed using dedicated software to automatically quantify fibrosis, collagen, and macrophage content. Automated fibrosis quantification based on parenchymal tissue density and fibrosis score measurements was compared to pulmonary function values or Ashcroft score. Automated fibrosis quantification of HPSPF lung explants was significantly higher than that of IPF lung explants or biopsies and was also significantly higher in IPF lung explants than in IPF biopsies. A high correlation coefficient was found between some automated quantification measurements and lung function values for the three sample groups. Automated quantification of collagen content in lung sections used for digital image analyses was similar in the three groups. CD68 immunolabeled cell measurements were significantly higher in HPSPF explants than in IPF biopsies. In conclusion, computerized image analysis provides access to accurate, reader-independent pulmonary fibrosis quantification in human histopathology samples. Fibrosis, collagen content, and immunostained cells can be automatically and individually quantified from serial sections. Robust automated digital image analysis of human lung samples enhances the available tools to quantify and study fibrotic lung disease.

Robust biofilm assay for quantification and high throughput screening applications.

Bacterial biofilms are populations of bacteria within a self-produced adherent extracellular matrix that are notoriously resistant to treatment. Existing methods for biofilm quantification are often limited in their dynamic range of detection (signal-to-background), throughput, and require modifications to the protocol depending on the bacterial species. To address these limitations, a broad utility, high-throughput (HTP) method was required. Using a fluorescent dye, FM1-43, we stained the biofilm, followed by solvent extraction and quantitation of biofilm employing a fluorescent plate reader. Utilizing eight different bacterial pathogens, we demonstrate that this method is widely applicable for biofilm quantification. Depending on the species, this biofilm assay offered a large dynamic range of 8-146 fold change compared to 2-22 fold for crystal violet staining under similar conditions. In addition to routine biofilm quantification using this new assay, as a proof-of-concept, 1200 compounds were screened against two different bacterial species to identify biofilm inhibitors. In our HTP screens we successfully identified compounds rifabutin and ethavarine as potential biofilm inhibitors of Burkholderia pseudomallei Bp82 and Acinetobacter baumannii biofilm production respectively. This newly validated biofilm assay is robust and can be readily adapted for antibiofilm screening campaigns and can supplant other less sensitive and low throughput methods.

Bioengineering of bacterial pathogens for noninvasive imaging and in vivo evaluation of therapeutics.

Critical bacterial pathogens of public health and biodefense concerns were engineered to constitutively express Escherichia coli enzyme thymidine kinase (TK) that allows for noninvasive nuclear imaging via phosphorylation and entrapment of radiolabeled nucleoside analog 1-(2'deoxy-2'-fluoro-ß-D-arabinofuranosyl)-5-iodouracil (FIAU). Expression of functional TK was established using a nucleoside analog Zidovudine that impeded the growth of tk-engineered bacteria. Significantly, no observable growth differences were detected for FIAU. High resolution mass spectrometry with Pseudomonas aeruginosa PAO1 and its tk variant (PAO1TK) confirmed FIAU phosphorylation and retention only in PAO1TK. In vitro gamma counting with wild-type PAO1, Acinetobacter baumannii and Burkholderia pseudomallei Bp82 and their tk derivatives with [18F]FIAU further confirmed that tk variants selectively incorporated the radiotracer, albeit with varying efficiencies. In vitro [18F]FIAU labeling coupled with in vivo Positron Emission Tomography/Computed Tomography (PET/CT) imaging of PAO1 and PAO1TK confirmed that only PAO1TK can be imaged in mice at sensitivities ≥107 bacteria per infection site. This was further verified by administering [18F]FIAU to animals infected with PAO1 and PAO1TK. Utility of tk-engineered P. aeruginosa in noninvasive PET/CT imaging for bacterial therapeutic evaluation in animals was demonstrated employing antibiotic ciprofloxacin, underscoring the immediate use of PAO1TK and potentially other engineered pathogens for evaluating experimental therapeutics.