Clinical application of non-invasive prenatal diagnosis of phenylketonuria based on haplotypes via paired-end molecular tags and weighting algorithm

Background Phenylketonuria (PKU) is a metabolic disease that can cause severe and irreversible brain damage without treatment. Methods Here we developed a non-invasive prenatal diagnosis (NIPD) technique based on haplotypes via paired-end molecular tags and weighting algorithm and applied it to the NIPD of PKU to evaluate its accuracy and feasibility in the early pregnancy. A custom-designed hybridization probes containing regions in phenylalanine hydroxylase (PAH) gene and its 1 Mb flanking region were used for target sequencing on genomic and maternal plasma DNA (7–13 weeks of gestation) to construct the parental haplotypes and the proband’s haplotype. Fetal haplotype was then inferred combined with the parental haplotypes and the proband’s haplotype. The presence of haplotypes linked to both the maternal and paternal mutant alleles indicated affected fetuses. The fetal genotypes were further validated by invasive prenatal diagnosis in a blinded fashion. Results This technique has been successfully applied in twenty-one cases. Six fetuses were diagnosed as patients carrying both of the mutated haplotypes inherited from their parents. Eleven fetuses were carriers of one heterozygous PAH variants, six of which were paternal and five of which were maternal. Four fetuses were absence of pathogenic alleles. All results were consistent with the prenatal diagnosis through amniotic fluid. Conclusions The results showed that our new technique applied to the genotyping of fetuses with high risk for PKU achieves an accurate detection at an early stage of pregnancy with low fetal fraction in cell free DNA.

Hi-C detects genomic structural variants in peripheral blood of pediatric leukemia patients

B-cell acute lymphoblastic leukemia (B-ALL) is often driven by chromosome translocations that result in recurrent and well-studied gene fusions. Currently, fluorescent in-situ hybridization probes are employed to detect candidate translocations in bone marrow samples from B-ALL patients. Recently Hi-C, a sequencing-based technique originally designed to reconstruct the three-dimensional architecture of the nuclear genome, was shown to effectively recognize structural variants. Here, we demonstrate that Hi-C can be used as a genome-wide assay to detect translocations and other structural variants of potential clinical interest. Structural variants were identified in both bone marrow and peripheral blood samples, including an ETV6-RUNX1 translocation present in one pediatric B-ALL patient. Our report provides proof-of-principle that Hi-C could be an effective strategy to globally detect driver structural variants in B-ALL peripheral blood specimens, reducing the need for invasive bone marrow biopsies and candidate-based clinical tests.

A transcriptome atlas of the mouse iris at single-cell resolution defines cell types and the genomic response to pupil dilation

The iris controls the level of retinal illumination by controlling pupil diameter. It is a site of diverse ophthalmologic diseases and it is a potential source of cells for ocular auto-transplantation. The present study provides foundational data on the mouse iris based on single nucleus RNA sequencing. More specifically, this work has (1) defined all of the major cell types in the mouse iris and ciliary body, (2) led to the discovery of two types of iris stromal cells and two types of iris sphincter cells, (3) revealed the differences in cell type-specific transcriptomes in the resting vs. dilated states, and (4) identified and validated antibody and in situ hybridization probes that can be used to visualize the major iris cell types. By immunostaining for specific iris cell types, we have observed and quantified distortions in nuclear morphology associated with iris dilation and clarified the neural crest contribution to the iris by showing that Wnt1-Cre-expressing progenitors contribute to nearly all iris cell types, whereas Sox10-Cre-expressing progenitors contribute only to stromal cells. This work should be useful as a point of reference for investigations of iris development, disease, and pharmacology, for the isolation and propagation of defined iris cell types, and for iris cell engineering and transplantation.

Hi-C detects genomic structural variants in peripheral blood of pediatric leukemia patients

ABSTRACTB-cell acute lymphoblastic leukemia (B-ALL) is often driven by chromosome translocations that result in recurrent and well-studied gene fusions. Currently, fluorescent in-situ hybridization probes are employed to detect candidate translocations in bone marrow samples from B-ALL patients. Recently Hi-C, a sequencing-based technique originally designed to reconstruct the three-dimensional architecture of the nuclear genome, was shown to effectively recognize structural variants. Here, we demonstrate that Hi-C can be used as a genome-wide assay to detect translocations and other structural variants of potential clinical interest. Structural variants were identified in both bone marrow and peripheral blood samples, including an ETV6-RUNX1 translocation present in one pediatric B-ALL patient. Our report provides proof-of-principle that Hi-C could be an effective strategy to globally detect driver structural variants in B-ALL peripheral blood specimens, reducing the need for invasive bone marrow biopsies and candidate-based clinical tests.

Performance Comparison of Multiplexed Fluorescent Resonance Emission Transfer Hybridization Probes Across Roche LightCycler® Real-Time PCR Systems for the Detection of Bartonella species

Introduction/Objective Molecular assays for Bartonella species are important in diagnosing infection and expediting patient treatment. Real time polymerase chain reaction (RT-PCR) using fluorescent resonance energy transfer (FRET) hybridization probes can be used to detect Bartonella species in blood and fresh/fixed tissue biopsies in RT-PCR instruments. Over time, new technologies and reagents are introduced and existing PCR primers and FRET probes must be re-validated on new platforms. This study aimed to compare the performance of a Bartonella RT-PCR assay using the sunsetting Roche LightCycler® 2.0 (Roche Diagnostics, Indianapolis, IN) and newer LightCycler® 480 RT- PCR instruments. Methods/Case Report DNA was extracted from 132 historically positive, whole organism spiked, and historically negative whole blood and formalin fixed paraffin embedded (FFPE) samples. Samples were run on the LightCycler® 2.0 using instrument specific LightCycler® FastStart DNA Master HybProbe enzyme and compared to results generated using the LightCycler® 480 and its instrument specific LightCycler® 480 Genotyping Master enzyme. During optimization, MgCl2 concentrations and thermocycling profiles were adjusted. Accuracy, specificity, inclusivity, and limit of detection studies were performed. Crossing point (Cp), melting temperature (Tm), fluorescent peak and fluorescent background values were compared between the two instruments. Results (if a Case Study enter NA) The agreement in accuracy between the LightCycler® 2.0 and the LightCycler® 480 was 100% for whole blood samples. For historically positive FFPE samples, LightCycler® 2.0 sensitivity and LightCycler® 480 sensitivity were 86% and 100%, respectively. Specificity and inclusivity of the assay were identical between the two instruments. The limit of detection in whole blood was 5-fold lower on the LightCycler® 480 (50 copies/µL) compared to the LightCycler® 2.0 (250 copies/µL). Mean Cp and fluorescent peak intensity values increased by 5.1% and 65-fold, respectively. Conclusion The study demonstrates similar performance and improved limit of detection for the Bartonella FRET hybridization probe RT-PCR assay on the LightCycler® 480 compared to the LightCycler® 2.0.

A transcriptome atlas of the mouse iris at single cell resolution defines cell types and the genomic response to pupil dilation

The iris controls the level of retinal illumination by controlling pupil diameter. It is a site of diverse ophthalmological diseases and it is a potential source of cells for ocular auto-transplantation. The present study provides foundational data on the mouse iris based on single nucleus RNA sequencing. More specifically, this work has (1) defined all of the major cell types in the mouse iris and ciliary body, (2) led to the discovery of two types of iris stromal cells and two types of iris sphincter cells, (3) revealed the differences in cell type-specific transcriptomes in the resting, constricted, and dilated states, and (4) identified and validated antibody and in situ hybridization probes that can be used to visualize the major iris cell types. By immunostaining for specific iris cell-types, we have observed and quantified distortions in nuclear morphology associated with iris dilation and clarified the neural crest contribution to the iris by showing that Wnt1-Cre-expressing progenitors contribute to nearly all iris cell types, whereas Sox10-Cre-expressing progenitors contribute only to stromal cells. This work should be useful as a point of reference for investigations of iris development, disease, and pharmacology, for the isolation and propagation of defined iris cell types, and for iris cell engineering and transplantation.

Molecular-level similarity search brings computing to DNA data storage

As global demand for digital storage capacity grows, storage technologies based on synthetic DNA have emerged as a dense and durable alternative to traditional media. Existing approaches leverage robust error correcting codes and precise molecular mechanisms to reliably retrieve specific files from large databases. Typically, files are retrieved using a pre-specified key, analogous to a filename. However, these approaches lack the ability to perform more complex computations over the stored data, such as similarity search: e.g., finding images that look similar to an image of interest without prior knowledge of their file names. Here we demonstrate a technique for executing similarity search over a DNA-based database of 1.6 million images. Queries are implemented as hybridization probes, and a key step in our approach was to learn an image-to-sequence encoding ensuring that queries preferentially bind to targets representing visually similar images. Experimental results show that our molecular implementation performs comparably to state-of-the-art in silico algorithms for similarity search.

Multiplex Real-Time PCR for genotyping Glutathione S-Transferase deletion polymorphisms v1

Glutathione S-transferases (GSTs) are enzymes that act in the conjugation of reactive metabolites to the reduced glutathione. The GSTM1 and GSTT1 deletion polymorphisms promote a detriment in the metabolic detoxification process of xenobiotics, due to present a non-functional null allele, causing oxidative stress, may be related to the pathophysiology of various diseases. Genetic association case-control studies usually determine whether a statistical association exists between the disease trait and the genetic marker, such as GST deletion polymorphism. Therefore, techniques to genotyping these polymorphisms through multiplex PCR are highly desired and of wider interest. This method allows for the detection of genotypes for genetic association studies in molecular pathology and it is more cost-effective than hybridization probes.

Comparative detection and enumeration strengths of quantitative real-time PCR & FISH for waterborne bacterial pathogens in municipal wastewater

This study comparatively evaluates the detection and enumeration strengths of Real-Time PCR (RT PCR) and FISH, for selected bacterial pathogens in municipal wastewater. Both assays were performed using three primer and probe sets complementary to the same chromosomal virulence gene sequences. Primer & probe specificity was confirmed with DNA & fixed cells from pure bacterial cultures as well as seeded wastewater samples. Detection limits calculated for the RT PCR assay were 25 to 3030 tir gene copies for Escherichia coli O157:H7 and 3 x 10⁴ to 293 x10⁷ invA gene copies for Salmonella enterica, using pure cultures and seeded wasewater samples, respectively. In spite of the confirmed specificity of the DNA hybridization probes with target nucleic acids, fluorescent signals from hybridized whole target cells were below the detection limit of the FISH assay, and consequently were not quantified. This research demonstrates both the utility of RT PCR in detecting bacterial pathogens and the need for further optimization with DNA-targeted FISH, using environmental samples.