Exploring the utility of whole‐exome sequencing as a diagnostic tool in a child with atypical episodic muscle weakness

The advent of whole‐exome next‐generation sequencing (WES) has been pivotal for the molecular characterization of Mendelian disease; however, the clinical applicability of WES has remained relatively unexplored. We describe our exploration of WES as a diagnostic tool in a 3½‐year old female patient with a 2‐year history of episodic muscle weakness and paroxysmal dystonia who presented following a previous extensive but unrevealing diagnostic work‐up. WES was performed on the proband and her two parents. Parental exome data was used to filter potential de novo genomic events in the proband and suspected variants were confirmed using di‐deoxy sequencing. WES revealed a de novo non‐synonymous mutation in exon 21 of the calcium channel gene CACNA1S that has been previously reported in a single patient as a rare cause of atypical hypokalemic periodic paralysis. This was unexpected, as the proband's original differential diagnosis had included hypokalemic periodic paralysis, but clinical and laboratory features were equivocal, and standard clinical molecular testing for hypokalemic periodic paralysis and related disorders was negative. This report highlights the potential diagnostic utility of WES in clinical practice, with implications for the approach to similar diagnostic dilemmas in the future.     

An alternative extrinsic pathway of human blood coagulation

To study the interrelationships of the major human coagulation pathways, factor X activation in normal and various deficient human plasmas was evaluated when clotting was triggered by dilute rabbit or human thromboplastin. Various dilutions of thromboplastin were added to plasma samples containing 3H-labeled factor X, and the time course of factor X activation was determined. At a 1/250 dilution of rabbit brain thromboplastin the rate of factor X activation in factor VIII or factor IX deficient plasma was only 10% of the activation rate seen for normal or factor XI deficient plasma. Reconstitution of the deficient plasmas with factors VIII or IX, respectively, restored normal factor X activation. Similar results were obtained when various dilutions of human thromboplastin replaced the rabbit thromboplastin. From these experiments, it is inferred that normal activation of factor X in plasma due to dilute thromboplastin requires factors VII, IX and VIII. An alternative extrinsic pathway that involves factors VII, IX, and VIII may be a major physiologic extrinsic pathway, and this pathway may help to explain the clinical observations of bleeding diatheses in patients deficient in factors IX or VIII.     

Interleukin-4 and interleukin-5 map to human chromosome 5 in a region encoding growth factors and receptors and are deleted in myeloid leukemias with a del(5q)

Interleukin-4 (IL-4) is a potent mediator of growth and differentiation of cells of several hematopoietic lineages. Interleukin-5 (IL-5) is a lineage-specific hematopoietic growth factor that stimulates the production of eosinophils and eosinophil colonies from normal human bone marrow cells. By using somatic cell hybrids and in situ chromosomal hybridization, we localized the IL-4 and IL-5 genes to human chromosome 5 at bands q23-31, a chromosomal region that is frequently deleted [del(5q)] in patients with myeloid disorders. By in situ hybridization, the IL-4 and IL-5 genes were found to be deleted in the 5q- chromosome of four patients with refractory anemia (RA) or therapy-related acute nonlymphocytic leukemia (t-ANLL), who had a del(5q). Thus a small segment of chromosome 5 contains IL-4, IL-5, IL- 3, and GM-CSF as well as other genes such as CD14 and EGR1. Our findings that each of these genes was deleted in the 5q- chromosome suggest that loss of function of one or more of these genes may play an important role in the pathogenesis of hematologic disorders associated with a del(5q).     

Piecemeal degranulation of mast cells in the inflammatory eyelid lesions of interleukin-4 transgenic mice. Evidence of mast cell histamine release in vivo by diamine oxidase-gold enzyme-affinity ultrastructural cytochemistry

We used light and electron microscopy to analyze the eyelid inflammation that develops in transgenic mice that overexpress interleukin-4 (IL-4; Tepper et al, Cell 62:457, 1990). Analysis of alkaline Giemsa-stained plastic sections examined by light microscopy (Dvorak et al, J Exp Med 132:558, 1970), as well as by routine transmission electron microscopy, indicated that the mast cells in the inflammatory eyelid lesions were undergoing piecemeal degranulation, a form of secretion in which the cells' cytoplasmic granules exhibit characteristic morphologic changes that are thought to be associated with the prolonged, vesicle-mediated release of the granules' constituents. Moreover, by using a newly reported enzyme affinity-gold method, which stains histamine based on binding to diamine oxidase-gold (Dvorak et al, J Histochem Cytochem 41:787, 1993), we show that these activated mast cells had released much of their histamine content. The eyelid lesions also exhibited increased numbers of mast cells; interstitial fibrosis, particularly around cutaneous nerves and blood vessels; activated fibroblasts; focal axonal damage; venules with endothelial cells containing numerous vesiculo-vacuolar organelles; and infiltrates of neutrophils and eosinophils. Our findings illustrate that overexpression of the IL-4 gene in vivo can result in eyelid lesions associated with piecemeal degranulation of mast cells, as well as tissue fibrosis and a variety of other pathologic changes. These results also represent the first direct morphologic evidence for histamine secretion by mast cells in vivo.