Maternally inherited 133kb deletion of 14q32 causing Kagami-Ogata syndrome.

We present a case of a newborn female with multiple anomalies demonstrating that the causes of imprinting disorders rely not only on the parent-of-origin of the chromosomal aberrations, but also the scope of genes contained in the imprinted region. The newborn female presented with prenatal polyhydramnios, neonatal respiratory distress, distal contractures, abdominal hernia, bell-shaped thorax, and abnormal ribs. The neonate required mechanical ventilation due to apnea, underwent surgery for laryngomalacia, and showed development delay by age 11 months. Chromosomal microarray analysis identified a single copy number loss in chromosome region 14q32.2q32.31, containing genes that are differentially expressed based on parent-of-origin. Microarray analysis also confirmed the identical deletion in the patient's mother, who was reported to be normal. Additional molecular analyses determined the exact size and breakpoints of the deletion as well as methylation states in both the patient and her mother. The maternally transmitted deletion was responsible for Kagami-Ogata syndrome in the patient.

Evaluating melanocytic lesions with single nucleotide polymorphism (SNP) chromosomal microarray.

Histopathology is the gold standard for diagnosing melanocytic lesions; however, distinguishing benign versus malignant is not always clear histologically. Single nucleotide polymorphism (SNP) microarray analysis may help in making a definitive diagnosis. Here, we share our experience with the Oncoscan FFPE Assay and demonstrate its diagnostic utility in the context of ambiguous melanocytic lesions. Eleven archival melanocytic lesions, including three benign nevi, four melanomas, three BAP1-deficient Spitzoid nevi and one nevoid melanoma were selected for validation. SNP-array was performed according to the manufacturer's protocol, using the recommended 80ng of DNA; however, as little as 15ng was used if the extraction yield was lower. Concordance was assessed with H&E and various combinations of BAP1 and p16 immunohistochemical stains (IHC) and external reference laboratory chromosomal microarray results. After validation, the SNP array was utilized to make definitive diagnoses in four challenging cases. Oncoscan SNP array findings were in concordance with H&E, IHC, and reference laboratory chromosomal microarray testing. The SNP-based microarray can accurately detect copy number changes and aid in making a more definitive diagnosis of challenging melanocytic lesions. This can be accomplished using significantly less DNA than is required by other microarray technologies.

Development of HLA-B*57:01 Genotyping Real-Time PCR with Optimized Hydrolysis Probe Design.

HLA-B*57:01 genotyping before abacavir (ABC) administration is a standard of care to avoid ABC-driven hypersensitivity reactions. Several HLA-B*57:01 tests have been developed, each with advantages and disadvantages. Some have limited accuracy, require special instrumentation, and/or are labor intensive and expensive. We developed a novel hydrolysis probe-based real-time PCR method of HLA-B*57:01 genotyping. Primer and probes were designed based on published sequence variations in exon 3 of HLA-B that distinguish HLA-B*57:01 from ABC-insensitive alleles such as HLA-B*57:03 and HLA-B*58:01. We designed PCR primers to amplify HLA-B*57:01 along with closely related alleles, such as HLA-B*57:03, directly from genomic DNA. Most ABC-insensitive alleles, including HLA-B*58:01, would not produce any products in the PCR reaction. Our hydrolysis probes enable differentiation of HLA-B*57:01 from the other amplified, but ABC-insensitive, alleles. In addition to using real-time PCR, we used restriction enzymes to generate differential digestion patterns that led to the development of an HLA-B*57:01 PCR-restriction fragment length polymorphism marker. When used to genotype a set of 75 selected clinical samples, our real-time PCR assay demonstrated 100% accuracy in distinguishing between the HLA-B*57:01-positive and -negative alleles when results were compared to those of sequence-specific oligonucleotide probe typing and reference laboratory testing. Our newly developed test will allow clinical laboratories with real-time PCR capabilities to perform HLA-B*57:01 genotyping in a timely and economical manner.

Utilization of the oncoscan microarray assay in cancer diagnostics

Current strategies for cancer patient management include the use of genomic and proteomic test results to help guide therapeutic selection. The need for multi-target variant analysis is highlighted by the growing number of novel therapies to treat tumors with specific profiles and the increasing recognition that cancer is an extremely heterogeneous syndrome. Microarray analysis is a powerful genomic tool that provides genome-wide genetic information that is critical for guiding cancer treatments. Unlike constitutional applications of microarray analysis which are performed on whole blood samples, microarray analysis of solid tumors is challenging because tumor tissues are typically formalin fixed and paraffin embedded (FFPE). Genomic DNA extracted from FFPE tissues can also be fragmented into small pieces and yield much lower concentrations of DNA. We validated and implemented the Affymetrix OncoScan® FFPE assay to enable genome-wide analysis from these types of samples. The Affymetrix OncoScan® assay utilizes molecular inversion probes that generate multiplexed array hybridization targets from as short as 40 base-pairs of sequence and as low as approximately 80 ng of genomic DNA. OncoScan microarray analysis provides genomic information that includes structural variations, copy number variations and SNPs in a timely and a cost-effective manner from FFPE tumor tissues (AU)

A new alternative in plant retrograde signaling.

The reduced or oxidized state of plastoquinone in chloroplasts regulates splicing in the nucleus to control nuclear gene expression in response to changing environmental conditions.

Subset of heat-shock transcription factors required for the early response of Arabidopsis to excess light.

Sunlight provides energy for photosynthesis and is essential for nearly all life on earth. However, too much or too little light or rapidly fluctuating light conditions cause stress to plants. Rapid changes in the amount of light are perceived as a change in the reduced/oxidized (redox) state of photosynthetic electron transport components in chloroplasts. However, how this generates a signal that is relayed to changes in nuclear gene expression is not well understood. We modified redox state in the reference plant, Arabidopsis thaliana, using either excess light or low light plus the herbicide DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone), a well-known inhibitor of photosynthetic electron transport. Modification of redox state caused a change in expression of a common set of about 750 genes, many of which are known stress-responsive genes. Among the most highly enriched promoter elements in the induced gene set were heat-shock elements (HSEs), known motifs that change gene expression in response to high temperature in many systems. We show that HSEs from the promoter of the ASCORBATE PEROXIDASE 2 (APX2) gene were necessary and sufficient for APX2 expression in conditions of excess light, or under low light plus the herbicide. We tested APX2 expression phenotypes in overexpression and loss-of-function mutants of 15 Arabidopsis A-type heat-shock transcription factors (HSFs), and identified HSFA1D, HSFA2, and HSFA3 as key factors regulating APX2 expression in diverse stress conditions. Excess light regulates both the subcellular location of HSFA1D and its biochemical properties, making it a key early component of the excess light stress network of plants.

Linking photoreceptor excitation to changes in plant architecture.

Plants sense neighbor proximity as a decrease in the ratio of red to far-red light, which triggers a series of developmental responses. In Arabidopsis, phytochrome B (PHYB) is the major sensor of shade, but PHYB excitation has not been linked directly to a growth response. We show that the basic helix-loop-helix (bHLH) transcription factor PIF7 (phytochrome-interacting factor 7), an interactor of PHYB, accumulates in its dephosphorylated form in shade, allowing it to bind auxin biosynthetic genes and increase their expression. New auxin synthesized through a PIF7-regulated pathway is required for shade-induced growth, linking directly the perception of a light quality signal to a rapid growth response.

Arabidopsis thaliana PGR7 encodes a conserved chloroplast protein that is necessary for efficient photosynthetic electron transport.

A significant fraction of a plant's nuclear genome encodes chloroplast-targeted proteins, many of which are devoted to the assembly and function of the photosynthetic apparatus. Using digital video imaging of chlorophyll fluorescence, we isolated proton gradient regulation 7 (pgr7) as an Arabidopsis thaliana mutant with low nonphotochemical quenching of chlorophyll fluorescence (NPQ). In pgr7, the xanthophyll cycle and the PSBS gene product, previously identified NPQ factors, were still functional, but the efficiency of photosynthetic electron transport was lower than in the wild type. The pgr7 mutant was also smaller in size and had lower chlorophyll content than the wild type in optimal growth conditions. Positional cloning located the pgr7 mutation in the At3g21200 (PGR7) gene, which was predicted to encode a chloroplast protein of unknown function. Chloroplast targeting of PGR7 was confirmed by transient expression of a GFP fusion protein and by stable expression and subcellular localization of an epitope-tagged version of PGR7. Bioinformatic analyses revealed that the PGR7 protein has two domains that are conserved in plants, algae, and bacteria, and the N-terminal domain is predicted to bind a cofactor such as FMN. Thus, we identified PGR7 as a novel, conserved nuclear gene that is necessary for efficient photosynthetic electron transport in chloroplasts of Arabidopsis.

Mutations in Arabidopsis YCF20-like genes affect thermal dissipation of excess absorbed light energy.

Plants dissipate excess absorbed light energy as heat to protect themselves from photo-oxidative stress. The Arabidopsis thaliana npq6 mutant affected in thermal dissipation was identified by its partial defect in the induction of nonphotochemical quenching of chlorophyll fluorescence (NPQ) by excess light. Positional cloning revealed that npq6 contains a frameshift mutation caused by a single base-pair deletion in the At5g43050 gene, which encodes a member of the hypothetical chloroplast open reading frame 20 (YCF20) family of proteins with unknown function(s). The YCF20 protein family is mostly conserved in oxygenic photosynthetic organisms including cyanobacteria, eukaryotic algae, and plants. Amino acid sequence comparison identified two other genes in Arabidopsis that encode similar proteins to NPQ6: At1g65420 and At3g56830. These three Arabidopsis proteins have functional chloroplast-targeting transit peptides. Using reverse genetics, a mutant with a T-DNA insertion within the At1g65420 gene was identified and shown to exhibit a low NPQ phenotype similar to that of npq6; therefore, At1g65420 was named NPQ7. In contrast, a knockdown mutant in the At3g56830 gene with lower transcript levels showed wild-type levels of NPQ. The npq6 npq7 double mutant had an additive NPQ defect, indicating that the YCF20 family members in Arabidopsis have overlapping functions affecting thermal dissipation.

Quantitative genetic analysis of thermal dissipation in Arabidopsis.

Feedback deexcitation is a photosynthetic regulatory mechanism that can protect plants from high light stress by harmlessly dissipating excess absorbed light energy as heat. To understand the genetic basis for intraspecies differences in thermal dissipation capacity, we investigated natural variation in Arabidopsis (Arabidopsis thaliana). We determined the variation in the amount of thermal dissipation by measuring nonphotochemical quenching (NPQ) of chlorophyll fluorescence in Arabidopsis accessions of diverse origins. Ll-1 and Sf-2 were selected as high NPQ Arabidopsis accessions, and Columbia-0 (Col-0) and Wassilewskija-2 were selected as relatively low NPQ accessions. In spite of significant differences in NPQ, previously identified NPQ factors were indistinguishable between the high and the low NPQ accessions. Intermediate levels of NPQ in Ll-1 x Col-0 F1 and Sf-2 x Col-0 F1 compared to NPQ levels in their parental lines and continuous distribution of NPQ in F2 indicated that the variation in NPQ is under the control of multiple nuclear factors. To identify genetic factors responsible for the NPQ variation, we developed a polymorphic molecular marker set for Sf-2 x Col-0 at approximately 10-centimorgan intervals. From quantitative trait locus (QTL) mapping with undistorted genotype data and NPQ measurements in an F2 mapping population, we identified two high NPQ QTLs, HQE1 (high qE 1, for high energy-dependent quenching 1) and HQE2, on chromosomes 1 and 2, and the phenotype of HQE2 was validated by analysis of near isogenic lines. Neither QTL maps to a gene that had been identified previously in extensive forward genetics screens using induced mutants, suggesting that quantitative genetics can be used to find new genes affecting thermal dissipation.

Plastid-to-nucleus retrograde signaling.

Plant cells store genetic information in the genomes of three organelles: the nucleus, plastid, and mitochondrion. The nucleus controls most aspects of organelle gene expression, development, and function. In return, organelles send signals to the nucleus to control nuclear gene expression, a process called retrograde signaling. This review summarizes our current understanding of plastid-to-nucleus retrograde signaling, which involves multiple, partially redundant signaling pathways. The best studied is a pathway that is triggered by buildup of Mg-ProtoporphyrinIX, the first intermediate in the chlorophyll branch of the tetrapyrrole biosynthetic pathway. In addition, there is evidence for a plastid gene expression-dependent pathway, as well as a third pathway that is dependent on the redox state of photosynthetic electron transport components. Although genetic studies have identified several players involved in signal generation, very little is known of the signaling components or transcription factors that regulate the expression of hundreds of nuclear genes.

Is PsbS the site of non-photochemical quenching in photosynthesis?

The PsbS protein of photosystem II functions in the regulation of photosynthetic light harvesting. Along with a low thylakoid lumen pH and the presence of de-epoxidized xanthophylls, PsbS is necessary for photoprotective thermal dissipation (qE) of excess absorbed light energy in plants, measured as non-photochemical quenching of chlorophyll fluorescence. What is known about PsbS in relation to the hypothesis that this protein is the site of qE is reviewed here.