Expanded molecular detection of MPL codon p.W515 and p.S505N mutations in myeloproliferative neoplasms.

BACKGROUND: Patients negative for the JAK2 p.V617F somatic variant are frequently reflexed to testing for MPL exon 10 variants. Detection of these variants via multiplexed allele-specific PCR followed by fragment analysis has been previously published. The present study builds on this concept by improving the detection of the p.W515A variant, adding a second allele-specific primer to detect the p.W515R variant, and incorporating an improved primer for p.S505N detection. METHODS: The W515 amplification employs 5'-labeled allele-specific forward primers to detect p.W515K, p.W515L, p.W515R, and p.W515A. The p.S505N amplification includes an allele-specific reverse primer with a tail extension. Fragments were subject to capillary electrophoresis on an ABI 3500 Genetic Analyzer and analyzed using GeneMapper 6.0 (Thermo Fisher Scientific). RESULTS: Thirty MPL-negative and 13 MPL-positive samples previously tested by a reference laboratory were tested with the MPL LDT. Results were 100% concordant. The MPL LDT has a limit of detection of at least 5% VAF for the p.W515 variants and 10% VAF for the p.S505N variant. CONCLUSION: Current MPL assays are predominantly focused on p.W515L/K and p.S505N mutations. We have engineered an MPL test for detecting p.W515A/L/K/R and p.S505N variants, thereby increasing the diagnostic yield with little additional expense or technician time.

Development and Validation of Two RT-qPCR Diagnostic Assays for Detecting Severe Acute Respiratory Syndrome Coronavirus 2 Genomic Targets across Two Specimen Types.

Following the outbreak and subsequent pandemic of coronavirus disease 2019 (COVID-19), clinical diagnostic laboratories worldwide sought accurate and reliable testing methodologies. However, many laboratories were and still are hindered by a number of factors, including an unprecedented demand for testing, reagent and laboratory supply shortages and availability of qualified staff. To respond to these concerns, two separate laboratory-developed tests were validated for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using two different specimen types. In addition, these assays target different genomic regions of SARS-CoV-2, allowing for viral detection and mitigating genetic variation. Lower limit of detection and clinical evaluation studies showed detection of SARS-CoV-2 at 500 cp/mL with nasopharyngeal and saliva samples. These multiplexed RT-qPCR assays, although based on modified CDC, New York State Department of Health, and World Health Organization Emergency Use Authorization tests, allow for higher throughput and rapid turnaround time, benefiting patients, clinicians, and communities as a whole. These cost-effective tests also use readily obtainable reagents, circumventing commercial assay supply chain issues. The laboratory-developed tests described here have improved patient care and are highly adaptable should the need arise at other clinical diagnostic laboratories. Furthermore, the foundation and design of these assays may be modified in the future for detection of COVID-19 variants or other RNA-based viral detection tests.

Clustered mutations in the GRIK2 kainate receptor subunit gene underlie diverse neurodevelopmental disorders.

Kainate receptors (KARs) are glutamate-gated cation channels with diverse roles in the central nervous system. Bi-allelic loss of function of the KAR-encoding gene GRIK2 causes a nonsyndromic neurodevelopmental disorder (NDD) with intellectual disability and developmental delay as core features. The extent to which mono-allelic variants in GRIK2 also underlie NDDs is less understood because only a single individual has been reported previously. Here, we describe an additional eleven individuals with heterozygous de novo variants in GRIK2 causative for neurodevelopmental deficits that include intellectual disability. Five children harbored recurrent de novo variants (three encoding p.Thr660Lys and two p.Thr660Arg), and four children and one adult were homozygous for a previously reported variant (c.1969G>A [p.Ala657Thr]). Individuals with shared variants had some overlapping behavioral and neurological dysfunction, suggesting that the GRIK2 variants are likely pathogenic. Analogous mutations introduced into recombinant GluK2 KAR subunits at sites within the M3 transmembrane domain (encoding p.Ala657Thr, p.Thr660Lys, and p.Thr660Arg) and the M3-S2 linker domain (encoding p.Ile668Thr) had complex effects on functional properties and membrane localization of homomeric and heteromeric KARs. Both p.Thr660Lys and p.Thr660Arg mutant KARs exhibited markedly slowed gating kinetics, similar to p.Ala657Thr-containing receptors. Moreover, we observed emerging genotype-phenotype correlations, including the presence of severe epilepsy in individuals with the p.Thr660Lys variant and hypomyelination in individuals with either the p.Thr660Lys or p.Thr660Arg variant. Collectively, these results demonstrate that human GRIK2 variants predicted to alter channel function are causative for early childhood development disorders and further emphasize the importance of clarifying the role of KARs in early nervous system development.

Heat shock 70-kDa protein 5 (Hspa5) is essential for pronephros formation by mediating retinoic acid signaling.

Heat shock 70-kDa protein 5 (Hspa5), also known as binding immunoglobulin protein (Bip) or glucose-regulated protein 78 (Grp78), belongs to the heat shock protein 70 kDa family. As a multifunctional protein, it participates in protein folding and calcium homeostasis and serves as an essential regulator of the endoplasmic reticulum (ER) stress response. It has also been implicated in signal transduction by acting as a receptor or co-receptor residing at the plasma membrane. Its function during embryonic development, however, remains largely elusive. In this study, we used morpholino antisense oligonucleotides (MOs) to knock down Hspa5 activity in Xenopus embryos. In Hspa5 morphants, pronephros formation was strongly inhibited with the reduction of pronephric marker genes Lim homeobox protein 1 (lhx1), pax2, and ß1 subunit of Na/K-ATPase (atp1b1). Pronephros tissue was induced in vitro by treating animal caps with all-trans-retinoic acid and activin. Depletion of Hspa5 in animal caps, however, blocked the induction of pronephros as well as reduced the expression of retinoic acid (RA)-responsive genes, suggesting that knockdown of Hspa5 attenuated RA signaling. Knockdown of Hspa5 in animal caps resulted in decreased expression of lhx1, a transcription factor directly regulated by RA signaling and essential for pronephros specification. Co-injection of Hspa5MO with lhx1 mRNA partially rescued the phenotype induced by Hspa5MO. These results suggest that the RA-Lhx1 signaling cascade is involved in Hspa5MO-induced pronephros malformation. This study shows that Hspa5, a key regulator of the unfolded protein response, plays an essential role in pronephros formation, which is mediated in part through RA signaling during early embryonic development.

barx1 is necessary for ectomesenchyme proliferation and osteochondroprogenitor condensation in the zebrafish pharyngeal arches.

Barx1 modulates cellular adhesion molecule expression and participates in specification of tooth-types, but little is understood of its role in patterning the pharyngeal arches. We examined barx1 expression during zebrafish craniofacial development and performed a functional analysis using antisense morpholino oligonucleotides. Barx1 is expressed in the rhombencephalic neural crest, the pharyngeal arches, the pectoral fin buds and the gut in contrast to its paralogue barx2, which is most prominently expressed in the arch epithelium. Additionally, barx1 transient expression was observed in the posterior lateral line ganglia and developing trunk/tail. We show that Barx1 is necessary for proliferation of the arch osteochondrogenic progenitors, and that morphants exhibit diminished and dysmorphic arch cartilage elements due to reductions in chondrocyte differentiation and condensation. Attenuation of Barx1 results in lost arch expression of osteochondrogenic markers col2a1, runx2a and chondromodulin, as well as odontogenic marker dlx2b. Further, loss of barx1 positively influenced gdf5 and chordin, markers of jaw joint patterning. FGF signaling is required for maintaining barx1 expression, and that ectopic BMP4 induces expression of barx1 in the intermediate region of the second pharyngeal arch. Together, these results indicate an essential role for barx1 at early stages of chondrogenesis within the developing zebrafish viscerocranium.

Zebrafish dlx2a contributes to hindbrain neural crest survival, is necessary for differentiation of sensory ganglia and functions with dlx1a in maturation of the arch cartilage elements.

The Dlx genes are expressed in a coordinate manner, establishing proximal-distal polarity within the pharyngeal arches. In zebrafish, dlx2a is expressed in the migrating cranial neural crest that contributes to the pharyngeal arches. Expression of dlx2a in the arches is subsequently followed by overlapping expression of the physically linked dlx1a gene, and of other paralogues that include dlx5a/dlx6a and dlx3b/dlx4b. To investigate the patterning and establishment of arch proximodistal polarity in zebrafish, we characterized the function of dlx2a and dlx1a, using antisense morpholino oligonucleotides (MOs). We show that embryos injected with dlx1a and dlx2a MOs exhibit reduced and dysmorphic arch cartilage elements. The combined loss of dlx1a and dlx2a causes severe arch cartilage dysmorphology, revealing a role for these genes in maturation and patterning of arch chondrogenesis. Knockdown of dlx2a affects migrating neural crest cells as evidenced by reduced expression of crestin, and sox9a transcripts, in addition to increased levels of apoptosis. During pharyngogenesis, loss of dlx2a results in aberrant barx1 expression and the absence of goosecoid transcripts in the dorsal region of the ceratohyal arch. Defects in the differentiation of ectomesenchymal derivatives, including sensory ganglia and cartilage elements, indicate a role for dlx2a in specification and maintenance of cranial neural crest.

Isolation and expression of zebrafish zinc-finger transcription factor gene tsh1.

We report the expression patterns of tsh1, a zebrafish homologue of the Drosophila homeotic gene teashirt. Expression of tsh1 is first detected at the 2-somite stage (10h post-fertilization, hpf) at the anterior end of the spinal cord. Expression expands toward the posterior spinal cord, and by the prim-5 stage (24 hpf) tsh1 transcripts are detected throughout spinal cord. Between the 14- and 25-somite stage (16-24 hpf), spinal cord expression shows a clear anterior boundary at the rostral margin of rhombomere 7. Around the prim-25 stage (36 hpf), while the spinal expression of tsh1 decreases, new expression is detected in the pectoral fin buds and dorsal forebrain. By the long-pec stage (48 hpf), spinal cord expression is undetectable, but strong expression is observed in the rhombencephalon, telencephalon, tectum opticum, midbrain-hindbrain boundary, in the first pharyngeal arch and in the eyes. This expression persists at least until the larval stages. Retinoic acid signaling influences tsh1 expression. Zebrafish tsh1 expression was induced in the anterior neural tube in embryos treated briefly with exogenous retinoic acid. Furthermore, tsh1 expression was down-regulated in the spinal cord in the zebrafish neckless mutant in which RA signaling is disrupted due to a missense mutation in the gene encoding retinaldehyde dehydrogenase type 2.

Intergenic enhancers with distinct activities regulate Dlx gene expression in the mesenchyme of the branchial arches.

The vertebrate Dlx genes, generally organized as tail-to-tail bigene clusters, are expressed in the branchial arch epithelium and mesenchyme with nested proximodistal expression implicating a code that underlies the fates of jaws. Little is known of the regulatory architecture that is responsible for Dlx gene expression in developing arches. We have identified two distinct cis-acting regulatory sequences, I12a and I56i, in the intergenic regions of the Dlx1/2 and Dlx5/6 clusters that act as enhancers in the arch mesenchyme. LacZ transgene expression containing I12a is restricted to a subset of Dlx-expressing ectomesenchyme in the first arch. The I56i enhancer is active in a broader domain in the first arch mesenchyme. Expression of transgenes containing either the I12a or the I56i enhancers is dependent on the presence of epithelium between the onset of their expression at E9-10 until independence at E11. Both enhancers positively respond to FGF8 and FGF9; however, the responses of the reporter transgenes were limited to their normal domain of expression. BMP4 had a negative effect on expression of both transgenes and counteracted the effects of FGF8. Furthermore, bosentan, a pharmacological inhibitor of Endothelin-1 signaling caused a loss of I56i-lacZ expression in the most distal aspects of the expression domain, corresponding to the area of Dlx-6 expression previously shown to be under the control of Endothelin-1. Thus, the combinatorial branchial arch expression of Dlx genes is achieved through interactions between signaling pathways and intrinsic cellular factors. I56i drives the entire expression of Dlx5/6 in the first arch and contains necessary sequences for regulation by at least three separate pathways, whereas I12a only replicates a small domain of endogenous expression, regulated in part by BMP-4 and FGF-8.