AMMECR1: a single point mutation causes developmental delay, midface hypoplasia and elliptocytosis

Background: Deletions in the Xq22.3–Xq23 region, inclusive of COL4A5, have been associated with a contiguous gene deletion syndrome characterised by Alport syndrome with intellectual disability (Mental retardation), Midface hypoplasia and Elliptocytosis (AMME). The extrarenal biological and clinical significance of neighbouring genes to the Alport locus has been largely speculative. We sought to discover a genetic cause for two half-brothers presenting with nephrocalcinosis, early speech and language delay and midface hypoplasia with submucous cleft palate and bifid uvula.Methods: Whole exome sequencing was undertaken on maternal half-siblings. In-house genomic analysis included extraction of all shared variants on the X chromosome in keeping with X-linked inheritance. Patient-specific mutants were transfected into three cell lines and microscopically visualised to assess the nuclear expression pattern of the mutant protein.Results: In the affected half-brothers, we identified a hemizygous novel non-synonymous variant of unknown significance in AMMECR1 (c.G530A; p.G177D), a gene residing in the AMME disease locus. Transfected cell lines with the p.G177D mutation showed aberrant nuclear localisation patterns when compared with the wild type. Blood films revealed the presence of elliptocytes in the older brother.Conclusions: Our study shows that a single missense mutation in AMMECR1 causes a phenotype of midface hypoplasia, mild intellectual disability and the presence of elliptocytes, previously reported as part of a contiguous gene deletion syndrome. Functional analysis confirms mutant-specific protein dysfunction. We conclude that AMMECR1 is a critical gene in the pathogenesis of AMME, causing midface hypoplasia and elliptocytosis and contributing to early speech and language delay, infantile hypotonia and hearing loss, and may play a role in dysmorphism, nephrocalcinosis and submucous cleft palate.<br/

Early Cretaceous vegetation and climate change at high latitude: Palynological evidence from Isachsen Formation, Arctic Canada

Quantitative palynology of the marginal marine and deltaic-fluvial Isachsen Formation of the Sverdrup Basin, Canadian Arctic, provides insight into high latitude climate during much of the Early Cretaceous (Valanginian to early Aptian). Detrended Correspondence Analysis of main pollen and spore taxa is used to derive three ecological groupings influenced by moisture and disturbance based on the botanical affinities of palynomorphs: 1) a mixed coniferous assemblage containing both lowland and upland components; 2) a conifer-filicopsid community that likely grew in dynamic lowland habitats; and, 3) a mature dry lowland community composed of Cheirolepidiaceans. Stratigraphic changes in the relative abundance of pollen and spore taxa reflect climate variability in this polar region during the ~20 Mya history of the Isachsen Formation. The late Valanginian was relatively cool and moist and promoted lowland conifer-filicopsid communities. Warming in the Hauterivian resulted in the expansion coniferous communities in well-drained or arid hinterlands. A return to relatively cool and moist conditions in the Barremian resulted in the expansion of mixed lowland communities. This work demonstrates the utility of a multivariate statistical approach to palynology to provide insight into the composition and dynamics of ecosystems and climate of high latitude regions during the Early Cretaceous

Reprogramming of primary human fetal fibroblasts towards cardiomyoctes

The mammalian heart is primarily composed of fibroblasts, cardiomyocytes, endothelial and smooth muscle cells. Cardiomyocytes show little regenerative capabilities following damage (e.g. myocardial infarction, MI) and subsequently cardiac fibroblasts migrate to the site of injury and secrete extracellular matrix proteins to form scar tissue which supports the damaged myocardium. Thus, there is a therapeutic need for cell-based therapies for the generation of functional cardiomyocytes. One method to achieve this is to directly reprogram fibroblasts into cardiomyocytes, which has previously been achieved in mice through overexpression of the transcription factors: Gata4, Mef2c and Tbx5. The present study has hypothesised that fibroblast cells, isolated from human fetal heart and skin, can be reprogrammed into cardiomyocytes by overexpressing GATA4, MEF2C and TBX5 (GMT).Initial experiments aimed to characterise the phenotype of cardiac cell types within the human fetal heart using protein markers. Flow cytometry data revealed that the human fetal heart is composed of approximately 75-80% cardiomyocytes and 20-25% non-myocytes. The results also showed that Thy-1 and vimentin, considered to be fibroblast markers, localise in a proportion of cells that also express sarcomeric proteins (cardiomyocyte markers), confirming that they are not specific markers of fibroblasts. Isolation of fibroblasts from primary human fetal tissue was achieved through explant migration and phenotyped by RT-PCR, immunocytochemistry and flow cytometry. These cells were transfected with two vectors that allowed bicistronic expression of GATA4 with GFP and MEF2C with TBX5. Three transfection methods were compared: JetPEI, FuGENE6 and nucleofection. Nucleofection was found to be the best method of vector delivery into primary fibroblasts and enabled the selection of G418- resistant cells that were viable for 8 weeks.Flow cytometry analysis showed upregulation of NKX2.5, cardiac troponin I (TnI) and α-actinin in skin fibroblasts transfected with GMT and treated with G418, however, expression was not sustained beyond two weeks, implying that these cells were not stably expressing GMT. Treatment with the signalling molecule TGFβ-1 increased the percentage of cells expressing NKX2.5, TnI and α-actinin. This study has demonstrated the viability of a non-viral system for the delivery of GMT into primary human fetal fibroblasts and has shown that overexpression of GMT in these cells appears to initiate the early stages of direct cardiomyocyte reprogramming

Defining cardiac cell populations and relative cellular composition of the early fetal human heart

While the adult human heart is primarily composed of cardiomyocytes, fibroblasts, endothelial and smooth muscle cells, the cellular composition during early development remains largely unknown. Reliable identification of fetal cardiac cell types using protein markers is critical to understand cardiac development and delineate the cellular composition of the developing human heart. This is the first study to use immunohistochemistry (IHC), flow cytometry and RT-PCR analyses to investigate the expression and specificity of commonly used cardiac cell markers in the early human fetal heart (8–12 post-conception weeks). The expression of previously reported protein markers for the detection of cardiomyocytes (Myosin Heavy Chain (MHC) and cardiac troponin I (cTnI), fibroblasts (DDR2, THY1, Vimentin), endothelial cells (CD31) and smooth muscle cells (α-SMA) were assessed. Two distinct populations of cTnI positive cells were identified through flow cytometry, with MHC positive cardiomyocytes showing high cTnI expression (cTnI High) while MHC negative non-myocytes showed lower cTnI expression (cTnI Low). cTnI expression in non-myocytes was further confirmed by IHC and RT-PCR analyses, suggesting troponins are not cardiomyocyte-specific and may play distinct roles in non-muscle cells during early development. Vimentin (VIM) was expressed in cultured ventricular fibroblast populations and flow cytometry revealed VIM High and VIM Low cell populations in the fetal heart. MHC positive cardiomyocytes were VIM Low whilst CD31 positive endothelial cells were VIM High. Using markers investigated within this study, we characterised fetal human cardiac populations and estimate that 75–80% of fetal cardiac cells are cardiomyocytes and are MHC +/cTnI High/VIM Low, whilst non-myocytes comprise 20–25% of total cells and are MHC -/cTnI Low/VIM High, with CD31 + endothelial cells comprising ~9% of this population. These findings show distinct differences from those reported for adult heart. </p

In vitro stem cell modelling demonstrates a proof-of-concept for excess functional mutant TIMP3 as the cause of Sorsby Fundus Dystrophy

Sorsby Fundus Dystrophy (SFD) is a rare autosomal dominant disease of the macula that leads to bilateral loss of central vision and is caused by mutations in the TIMP3 gene. However, the mechanisms by which TIMP3 mutations cause SFD are poorly understood. Here, we generated human induced pluripotent stem cell-derived retinal pigmented epithelial (hiPSC-RPE) cells from three SFD patients carrying TIMP3 p.(Ser204Cys) and three non-affected controls to study disease related structural and functional differences in the RPE. SFD-hiPSC-RPE exhibited characteristic RPE structure and physiology but showed significantly reduced transepithelial electrical resistance associated with enriched expression of cytoskeletal remodelling proteins. SFD-hiPSC-RPE exhibited basolateral accumulation of TIMP3 monomers, despite no change in TIMP3 gene expression. TIMP3 dimers were observed in both SFD and control hiPSC-RPE, suggesting mutant TIMP3 dimerization does not drive SFD pathology. Furthermore, mutant TIMP3 retained matrix metalloproteinase activity. Proteomic profiling showed increased expression of extracellular matrix proteins, endothelial cell interactions and angiogenesis-related pathways in SFD-hiPSC-RPE. By contrast, there were no changes in VEGF secretion. However, SFD-iPSC-RPE secreted higher levels of monocyte chemoattractant protein 1, platelet-derived growth factor, and angiogenin. Our findings provide a proof-of-concept that SFD patient-derived hiPSC-RPE mimic mature RPE cells and support the hypothesis that excess accumulation of mutant TIMP3, rather than an absence or deficiency of functional TIMP3, drives ECM and angiogenesis related changes in SFD

Expression and localisation of thymosin beta-4 in the developing human early fetal heart

BackgroundThe objective of this study was to investigate the expression and localisation of thymosin β4 (Tβ4) in the developing human heart. Tβ4 is a cardioprotective protein which may have therapeutic potential. While Tβ4 is an endogenously produced protein with known importance during development, its role within the developing human heart is not fully understood. Elucidating the localisation of Tβ4 within the developing heart will help in understanding its role during cardiac development and is crucial for understanding its potential for cardioprotection and repair in the adult heart.MethodsExpression of Tβ4 mRNA in the early fetal human heart was assessed by PCR using both ventricular and atrial tissue. Fluorescence immunohistochemistry was used to assess the localisation of Tβ4 in sections of early fetal human heart. Co-staining with CD31, an endothelial cell marker, and with myosin heavy chain, a cardiomyocyte marker, was used to determine whether Tβ4 is localised to these cell types within the early fetal human heart.ResultsTβ4 mRNA was found to be expressed in both the atria and the ventricles of the early fetal human heart. Tβ4 protein was found to be primarily localised to CD31-expressing endothelial cells and the endocardium as well as being present in the epicardium. Tβ4-associated fluorescence was greater in the compact layer of the myocardial wall and the interventricular septum than in the trabecular layer of the myocardium.ConclusionsThe data presented illustrates expression of Tβ4 in the developing human heart and demonstrates for the first time that Tβ4 in the human heart is primarily localised to endothelial cells of the cardiac microvasculature and coronary vessels as-well as to the endothelial-like cells of the endocardium and to the epicardium

The diverse roles of TIMP-3: Insights into degenerative diseases of the senescent retina and brain

Tissue inhibitor of metalloproteinase-3 (TIMP-3) is a component of the extracellular environment, where it mediates diverse processes including matrix regulation/turnover, inflammation and angiogenesis. Rare TIMP-3 risk alleles and mutations are directly linked with retinopathies such as age-related macular degeneration (AMD) and Sorsby fundus dystrophy, and potentially, through indirect mechanisms, with Alzheimer's disease. Insights into TIMP-3 activities may be gleaned from studying Sorsby-linked mutations. However, recent findings do not fully support the prevailing hypothesis that a gain of function through the dimerisation of mutated TIMP-3 is responsible for retinopathy. Findings from Alzheimer's patients suggest a hitherto poorly studied relationship between TIMP-3 and the Alzheimer's-linked amyloid-beta (Ab) proteins that warrant further scrutiny. This may also have implications for understanding AMD as aged/diseased retinae contain high levels of Ab. Findings from TIMP-3 knockout and mutant knock-in mice have not led to new treatments, particularly as the latter does not satisfactorily recapitulate the Sorsby phenotype. However, recent advances in stem cell and in vitro approaches offer novel insights into understanding TIMP-3 pathology in the retina-brain axis, which has so far not been collectively examined. We propose that TIMP-3 activities could extend beyond its hitherto supposed functions to cause age-related changes and disease in these organs

S6 Fig -

DDR2 dual immunohistochemistry of fetal human heart tissue: (A) DDR2 and MHC expression at the boundary (dotted line) of a blood vessel wall and the myocardium. (B) DDR2 and α-SMA expression in a cardiac blood vessel (C) DDR2 and CD31 expression in a cardiac blood vessel. Myo = myocardium. AO/PA = aorta/pulmonary artery. DAPI was used as a counter stain for cell nuclei. (TIF)</p

Fig 7 -

Fig 7 - </p

Complete list of flow cytometry data showing tissue age, percentage of positive cells recorded for each marker and number of samples analysed.

(DOCX)</p