Susan Arbuckle

Expanding the clinical, pathological and MRI phenotype of DNM2-related centronuclear myopathy

Mutations in dynamin-2 (DNM2) cause autosomal dominant centronuclear myopathy (CNM). We report a series of 12 patients from eight families with CNM in whom we have identified a number of novel features that expand the reported clinicopathological phenotype. We identified two novel and five recurrent missense mutations in DNM2. Early clues to the diagnosis include relative weakness of neck flexors, external ophthalmoplegia and ptosis, although these are not present in all patients. Pes cavus was present in two patients, and in another two members of one family there was mild slowing of nerve conduction velocities. Whole-body MRI examination in two children and one adult revealed a similar pattern of involvement of selective muscles in head (lateral pterygoids), neck (extensors), trunk (paraspinal) and upper limbs (deep muscles of forearm). Findings in lower limbs and pelvic region were similar to that previously reported in adults with DNM2 mutations. Two patients presented with dystrophic changes as the predominant pathological feature on muscle biopsies; one of whom had a moderately raised creatine kinase, and both patients were initially diagnosed as congenital muscular dystrophy. DNM2 mutation analysis should be considered in patients with a suggestive clinical phenotype despite atypical histopathology, and MRI findings can be used to guide genetic testing. Subtle neuropathic features in some patients suggest an overlap with the DNM2 neuropathy phenotype. Missense mutations in the C-terminal region of the PH domain appear to be associated with a more severe clinical phenotype evident from infancy.

DGKE Variants Cause a Glomerular Microangiopathy That Mimics Membranoproliferative GN

Renal microangiopathies and membranoproliferative GN (MPGN) can manifest similar clinical presentations and histology, suggesting the possibility of a common underlying mechanism in some cases. Here, we performed homozygosity mapping and whole exome sequencing in a Turkish consanguineous family and identified DGKE gene variants as the cause of a membranoproliferative-like glomerular microangiopathy. Furthermore, we identified two additional DGKE variants in a cohort of 142 unrelated patients diagnosed with membranoproliferative GN. This gene encodes the diacylglycerol kinase DGKε, which is an intracellular lipid kinase that phosphorylates diacylglycerol to phosphatidic acid. Immunofluorescence confocal microscopy demonstrated that mouse and rat Dgkε colocalizes with the podocyte marker WT1 but not with the endothelial marker CD31. Patch-clamp experiments in human embryonic kidney (HEK293) cells showed that DGKε variants affect the intracellular concentration of diacylglycerol. Taken together, these results not only identify a genetic cause of a glomerular microangiopathy but also suggest that the phosphatidylinositol cycle, which requires DGKE, is critical to the normal function of podocytes.

Small noncoding differentially methylated copy-number variants, including lncRNA genes, cause a lethal lung developmental disorder

An unanticipated and tremendous amount of the noncoding sequence of the human genome is transcribed. Long noncoding RNAs (lncRNAs) constitute a significant fraction of non-protein-coding transcripts; however, their functions remain enigmatic. We demonstrate that deletions of a small noncoding differentially methylated region at 16q24.1, including lncRNA genes, cause a lethal lung developmental disorder, alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV), with parent-of-origin effects. We identify overlapping deletions 250 kb upstream of FOXF1 in nine patients with ACD/MPV that arose de novo specifically on the maternally inherited chromosome and delete lung-specific lncRNA genes. These deletions define a distant cis-regulatory region that harbors, besides lncRNA genes, also a differentially methylated CpG island, binds GLI2 depending on the methylation status of this CpG island, and physically interacts with and up-regulates the FOXF1 promoter. We suggest that lung-transcribed 16q24.1 lncRNAs may contribute to long-range regulation of FOXF1 by GLI2 and other transcription factors. Perturbation of lncRNA-mediated chromatin interactions may, in general, be responsible for position effect phenomena and potentially cause many disorders of human development.

Assessment of Cooling on an Acute Scald Burn Injury in a Porcine Model

The current Australian and New Zealand Burn Association (ANZBA) recommended Burns First Aid Treatment is place the burn under cool running water for 20 minutes. Wet towels and water spray also have been used frequently. No scientific data exist to compare the effectiveness of these methods of cooling. This study sought to determine experimental evidence for current Burns First Aid Treatment recommendations and the optimal mode of cooling. Four partial-thickness scald burn injuries were induced in 10 piglets each. First aid was then applied for 20 minutes via cool running water, wet towels, or water spray, with no treatment as a control. At day 1 and day 9, biopsies and clinical photographs were assessed in a blinded manner. The control group showed worsening or no change of depth over the course of 9 days. The outcomes with wet towels and water spray were variable. Cool running water consistently demonstrated improvement in wound recovery over the course of 9 days (P < .05). This study demonstrated that cool running water appeared the most effective first aid for an acute scald burn wound in a porcine model compared with wet towels and water spray.

Human Cytomegalovirus Infection Is Detected Frequently in Stillbirths and Is Associated With Fetal Thrombotic Vasculopathy

BACKGROUND Human cytomegalovirus (CMV) is the most common congenital infection in developed countries and is a known cause of intrauterine fetal death. We examined CMV infection in stillbirths and the relationship with histopathological findings at autopsy. METHODS We collected liver, kidney, and placenta specimens from 130 stillbirths. CMV DNA and protein were detected using polymerase chain reaction and immunohistochemistry, along with routine autopsy of stillborn infants. RESULTS Overall, CMV DNA was detected in 15% of singleton, >20-week stillborn infants. CMV DNA was detected in kidney (9%), liver (11%), and placenta (5%) specimens, with 75% of infections confirmed by immunohistochemistry. Fetal thrombotic vasculopathy was the only histopathological abnormality associated with CMV infection (in 60% CMV-infected vs 28% uninfected stillbirths P = .010). CONCLUSIONS Stillbirth has multiple etiologies. However, the detection of CMV DNA in 15% of fetal tissues or placentae suggests a strong association between CMV infection in pregnancy and stillbirth. Molecular testing during postmortem investigation has an important role to determine the contribution of CMV infection.

High-molecular-weight tropomyosins localize to the contractile rings of dividing CNS cells but are absent from malignant pediatric and adult CNS tumors

Tropomyosin has been implicated in the control of actin filament dynamics during cell migration, morphogenesis, and cytokinesis. In order to gain insight into the role of tropomyosins in cell division, we examined their expression in developing and neoplastic brain tissue. We found that the high‐molecular‐weight tropomyosins are downregulated at birth, which correlates with glial cell differentiation and withdrawal of most cells from the cell cycle. Expression of these isoforms was restricted to proliferative areas in the embryonic brain and was absent from the adult, where the majority of cells are quiescent. However, they were induced under conditions where glial cells became proliferative in response to injury. During cytokinesis, these tropomyosin isoforms were associated with the contractile ring. We also investigated tropomyosin expression in neoplastic CNS tissues. Low‐grade astrocytic tumors expressed high‐molecular‐weight tropomyosins, while highly malignant CNS tumors of diverse origin did not (P ≤ 0.001). Furthermore, high‐molecular‐weight tropomyosins were absent from the contractile ring in highly malignant astrocytoma cells. Our findings suggest a role for high‐molecular‐weight tropomyosins in astrocyte cytokinesis, although highly malignant CNS tumors are still able to undergo cell division in their absence. Additionally, the correlation between high‐molecular‐weight tropomyosin expression and tumor grade suggests that tropomyosins are potentially useful as indicators of CNS tumor grade. GLIA 42:25–35, 2003.

Optimal duration of cooling for an acute scald contact burn injury in a porcine model.

The Australian and New Zealand Burn Association recommend 20 minutes of cold running tap water as burn first aid. Scientific evidence for the optimal duration of treatment is limited. Our aim was to establish the optimal duration of cooling using cold running tap water to treat the acute burn. Partial thickness contact scald burns were induced at five sites in each of 17 pigs. Treatments with cold running tap water for 5, 10, 20, and 30 minutes were randomly allocated to different sites together with an untreated control site. In the running water 5 and 10 minute treatments intradermal temperatures rose by 1°C per minute when cooling was stopped, compared with 0.5°C per minute for 20 and 30 minutes duration. No differences in the surface area of each burn were noted between the five treatments on day 9. Histological analysis of burn depth on days 1 and 9 revealed that a higher proportion of burns treated for 20 and 30 minutes showed improvement compared with those treated for 5 and 10 minutes only. This difference reached statistical significance (P < .05) only in the cold running water for 20 minutes treatment arm. There was a statistically significant (P < .05) improvement in burn depth in a porcine acute scald burn injury model when the burn was treated with cold running tap water for 20 minutes as opposed to the other treatment durations. This study supports the current burn first aid treatment recommendations for the optimal duration of cooling an acute scald burn.

Mutations of GPR126 Are Responsible for Severe Arthrogryposis Multiplex Congenita

Arthrogryposis multiplex congenita is defined by the presence of contractures across two or more major joints and results from reduced or absent fetal movement. Here, we present three consanguineous families affected by lethal arthrogryposis multiplex congenita. By whole-exome or targeted exome sequencing, it was shown that the probands each harbored a different homozygous mutation (one missense, one nonsense, and one frameshift mutation) in GPR126. GPR126 encodes G-protein-coupled receptor 126, which has been shown to be essential for myelination of axons in the peripheral nervous system in fish and mice. A previous study reported that Gpr126(-/-) mice have a lethal arthrogryposis phenotype. We have shown that the peripheral nerves in affected individuals from one family lack myelin basic protein, suggesting that this disease in affected individuals is due to defective myelination of the peripheral axons during fetal development. Previous work has suggested that autoproteolytic cleavage is important for activating GPR126 signaling, and our biochemical assays indicated that the missense substitution (p.Val769Glu [c.2306T>A]) impairs autoproteolytic cleavage of GPR126. Our data indicate that GPR126 is critical for myelination of peripheral nerves in humans. This study adds to the literature implicating defective axoglial function as a key cause of severe arthrogryposis multiplex congenita and suggests that GPR126 mutations should be investigated in individuals affected by this disorder.

Human cytomegalovirus-induces cytokine changes in the placenta with implications for adverse pregnancy outcomes.

Human cytomegalovirus (CMV) infection of the developing fetus can result in adverse pregnancy outcomes including death in utero. Fetal injury results from direct viral cytopathic damage to the CMV-infected fetus, although evidence suggests CMV placental infection may indirectly cause injury to the fetus, possibly via immune dysregulation with placental dysfunction. This study investigated the effects of CMV infection on expression of the chemokine MCP-1 (CCL2) and cytokine TNF-α in placentae from naturally infected stillborn babies, and compared these changes with those found in placental villous explant histocultures acutely infected with CMV ex vivo. Tissue cytokine protein levels were assessed using quantitative immunohistochemistry. CMV-infected placentae from stillborn babies had significantly elevated MCP-1 and TNF-α levels compared with uninfected placentae (p = 0.001 and p = 0.007), which was not observed in placentae infected with other microorganisms (p = 0.62 and p = 0.71) (n = 7 per group). Modelling acute clinical infection using ex vivo placental explant histocultures showed infection with CMV laboratory strain AD169 (0.2 pfu/ml) caused significantly elevated expression of MCP-1 and TNF-α compared with uninfected explants (p = 0.0003 and p<0.0001) (n = 25 per group). Explant infection with wild-type Merlin at a tenfold lower multiplicity of infection (0.02 pfu/ml), caused a significant positive correlation between increased explant infection and upregulation of MCP-1 and TNF-α expression (p = 0.0001 and p = 0.017). Cytokine dysregulation has been associated with adverse outcomes of pregnancy, and can negatively affect placental development and function. These novel findings demonstrate CMV infection modulates the placental immune environment in vivo and in a multicellular ex vivo model, suggesting CMV-induced cytokine modulation as a potential initiator and/or exacerbator of placental and fetal injury.

Low prevalence of RAS-RAF-activating mutations in Spitz melanocytic nevi compared with other melanocytic lesions.

Abstract:  Melanocytic lesions, including Spitz nevi (SN), common benign nevi (CBN) and cutaneous metastatic melanoma (CMM), were analyzed for activating mutations in NRAS, HRAS and BRAF oncogenes, which induce cellular proliferation via the MAP kinase pathway. One of 22 (4.5%) SN tested showed an HRAS G61L mutation. Another lesion, a ‘halo’ SN, showed a BRAF V600E (T1796A) mutation. BRAF V600E mutations were found in two thirds (20/31) of CBN, while a further 19% (6/31) showed NRAS codon 61 mutations. One third of CMM (10/30) had various BRAF mutations of codon 600, and a further 6% (2/31) showed NRAS codon 61 mutations. Seventeen SN tested for loss of heterozygosity (LOH) at 9p and 10q regions, known to be frequently deleted in melanoma, showed LOH at the 9p loci D9S942 and IFNA. A further lesion was found with low‐level microsatellite instability at one locus, D10S214. The low rate of RAS‐RAF mutations (2/22, 9.1%) observed in SN suggests that these lesions harbor as yet undetected activating mutations in other components of the RAS‐RAF‐MEK‐ERK‐MAPK pathway. Germline DNA from members of 111 multiple‐case melanoma families, representing a range of known (CDKN2A) and unknown predisposing gene defects, was analyzed for germline BRAF mutations, but none was found.