MIRAGE syndrome (myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital (gonadal) phenotypes, and enteropathy) (OMIM: 617053) is a well-established complex multisystem disorder caused by pathogenic gain-of-function variants in the gene SAMD9. ^{1 , 2} Changes in this gene were first described in 2016 and to date more than 100 affected individuals have been reported. We have recently undertaken a meta-analysis of all published SAMD9-associated variants and shown that the range of clinical phenotypes is very variable. ³ Increasingly, children with MIRAGE syndrome are diagnosed who do not have adrenal insufficiency, and a large proportion of children and young people with SAMD9 variants present with myelodysplastic syndrome (MDS) alone. ^{3 , 4}

SAMD9 has been extensively shown to be a growth repressor in in vitro cellular models, explaining the typical phenotypic growth restriction and tissue hypoplasia observed in children with this condition. However, the molecular mechanism or mechanisms by which SAMD9 affects cell growth and proliferation are still unknown. Two studies have reported characteristic enlarged endosomes in skin fibroblasts derived from MIRAGE patients, including “giant” endosomes in some cells. ^{1 , 5} These authors proposed that endosome dysfunction results in reduced recycling of epidermal growth factor receptor (EGFR), with consequent decreased cell growth and proliferation. ¹ We have also observed somewhat larger vesicles in our patients’ fibroblasts using transmission electron microscopy (TEM) imaging and Rab5a and Rab7a as early- and late-endosomal markers respectively, in live fibroblast cultures. ² Massively enlarged or giant endosomes were not seen. Of note, these TEM studies were only reviewed at low power and in limited sections, and more detailed high-power imaging was not undertaken.

TEM remains an extremely powerful approach for visualising and analysing intracellular structures in biological samples. This technique is extensively used in clinical diagnosis as well as research settings, and therefore represents an invaluable analytical tool. In this study, we have used TEM to systematically analyse ultrastructural morphology of skin fibroblasts, which had been previously derived from MIRAGE patients, ² to investigate any potential characteristic features that could help elucidate the molecular role of SAMD9.

All three patient samples showed similar ultrastructural features which were distinct from the control sample. Given the qualitative, observational nature of TEM, the major consistent changes are described below.

A general increase in endosome number and activity was observed in the patients’ fibroblasts ( Figure 1). Characteristic features of increased endosomal activity included pinocytic vesicles, early endosomes showing chains and clumps of vesicles, as well as vesicle budding ( Figure 1). Several late endosomes had multivesicular bodies (MVB) ( Figure 2). Evidence of increased endosomal activity was also demonstrated by enlarged empty endosomes and large (over 1.0 μm diameter), single membrane bound lysosomes, which were mainly empty with a rim of electron dense material ( Figure 2). No giant endosomes were seen.

All patient-derived cells displayed a regular array of organelles ( Figure 3). Mitochondria did not show any significant pathological features except swelling and internal cristae disruption in a few cells. Endoplasmic reticulum was prominent in several fibroblasts and was mainly of the rough endoplasmic type (rER). A moderate distention of the cisternae was observed, with granular electron dense material. Dilatation of smooth endoplasmic reticulum (sER) was also present, indicated by an irregular outline with granular material within, but without any obvious ribosomes lining the membranes ( Figure 3, Patient 1, top left).

No major differences in cell nuclei between control and patient samples were observed ( Figure 4). Several features were common in both control and patient cells, including a convoluted nuclear envelope with a marginated chromatin pattern, nucleoli often prominent in size and occasionally multiple nucleoli present in one cell.

In this study, we have used TEM to examine ultrastructural morphology of fibroblasts derived from MIRAGE patients. Although our early specimens were variable, ² we have now obtained and systematically analysed more images from our original fibroblast samples. All samples were well preserved with ample numbers of cells for analysis. The three patients’ samples showed essentially similar features and were distinct from the control sample, demonstrating that carrying SAMD9 variants resulted in differences in intracellular structures.

Evidence of increased endosomal activity was present in all patients’ samples. This was reflected by an increase in pinocytosis and budding in early endosomes, and the presence of large endosomes and lysosomes. Endosomes can be broadly classified as early endosomes, late endosomes and recycling endosomes, which will fuse with lysosomes for degradation of waste matter. Lysosomes are single membrane bound organelles filled with hydrolytic enzymes capable of degrading many types of biomolecules, cellular organelles and micro-organisms. ⁶ Thus, lysosomes typically appear as sphere-shaped sacks with electron dense contents. Taken together, consistent markers of increased endosome activity in patients’ samples were seen.

We also observed a dilation of both rER and sER. The main function of rER is the synthesis and modification of proteins that need to be delivered to organelles within the cell or secreted from the cell. sER is associated with the synthesis of lipids such as cholesterol and phospholipids, which are essential for the formation of cellular membranes. sER also plays a role in glycogen metabolism. ⁷ Glycogen content and intermediate filaments were at varying levels in all samples and no excess lipid was detected.

Mitochondria were easily detected in TEM images, as they are bounded by a double membrane and the inner membrane forms the characteristic infolding of lamellar cristae and matrix space. Their major role is the provision of energy by the production of ATP and phosphorylation of ADP to regulate cellular metabolism. ⁸ We detected bloated mitochondria in a few cells; however, this is quite a common feature and could indicate a sign of cellular stress, such as a delay in fibroblast preservation.

Cell nuclei did not show any major differences between control and patients’ samples and appeared with a convoluted nuclear envelope. A convoluted nuclear membrane provides an increased area of contact between the nucleus and the cytoplasm and may suggest heightened metabolic activity. ⁹

TEM is widely used in clinical settings to diagnose specific conditions, such as lysosomal storage disorders, glomerular diseases and metabolic and congenital myopathies, ^{10 – 12} however, over the past years its use has been valuable in research settings too. Indeed, TEM has been used to further investigate the detailed structures of cell organelles to validate potential novel findings discovered through research studies. For example, TEM has been used to: a) assess ciliary ultrastructural defects in ciliary disorders ¹³ ; b) identify stored material in lysosomal storage disorders, including neuronal ceroid lipofuscinoses and Batten disease ¹⁴ ; and c) isolate highly infectious and contagious microorganisms, primarily viruses, to enable production of vaccines in microbial diseases, e.g. SARS-CoV-2/coronavirus. ¹⁵ However, there is no standard protocol for TEM and different preparation methods can be used, which can often affect the observed results.

The most widely used method is “traditional ultrathin-section EM”, ¹⁶ which in brief consists of a series of steps (fixation, wash, dehydration, embedding) to preserve the specimen before sectioning and staining. This has been proven to maintain cellular integrity and sample structure, without leading to cellular artefacts. This approach is, therefore, potentially more reliable. In our original study, cultured fibroblasts were first pelleted and then fixed in 2.5% glutaraldehyde followed by 1% osmium tetroxide. They were then processed into agar 100 resin, sectioned (90 nm) and stained with uranyl acetate and lead citrate. In contrast, in other studies reporting giant endosomes in MIRAGE patients, ^{1 , 5} fibroblasts were seeded on a chamber slide and then fixed with 2.5% glutaraldehyde followed by 1.0% osmium tetroxide. They were then embedded in Epon, sectioned (70 nm) and stained with uranyl acetate and lead citrate. The use of different methodologies, especially the initial step of cell pelleting compared to growing cells directly on chamber slides, might therefore account for some of the variability in the appearance of the vesicles and endosomes in different, independent studies.

Whilst SAMD9-associated conditions provide a fascinating model for the dynamic evolution of genetic disease, the exact mechanism by which SAMD9 regulates cell growth and mediates a cellular effect is yet to be fully established. The paralogous gene SAMD9L has been shown to regulate endosomal fusion through degradation of EGFRs. ¹⁷ Several patients with MIRAGE syndrome had characteristic enlarged endosomes, ^{1 , 5} therefore SAMD9 could potentially be involved in the regulation of endocytosis of receptors acting as an endosome fusion facilitator, ¹ or in lysosomal activation. ¹⁸ In our original study, we showed some large vesicles in our patient fibroblasts, but these were quite heterogenous. We also carried out live cell imaging of early and late endosomes and the changes in early endosomes from live imaging were only subtle. ² In the present study, all three MIRAGE patients’ fibroblasts exhibited increased endosomal activity compared to controls, as well as large lysosomes. These data could, therefore, still support the role of SAMD9 in recycling of EGFRs.

SAMD9 has also been associated with the viral host defence mechanism, especially poxviruses, ^{19 – 21} and tumour suppression. ^{17 , 22} More recently, it has been shown to have nucleic acid binding capacity, important for antiviral and antiproliferative functions. ²³ As more insight is obtained on the biological function or functions of SAMD9, detailed data from patient-derived materials, and associated cellular ultrastructural changes, could provide more evidence for underlying disease mechanisms.

This study has several limitations. TEM is a powerful tool, however its assessment remains subjective, as it mainly relies on the experience of the individual analysing the images. Qualitative evaluation can be difficult since the cells are in a three dimensions and TEM is allowing visualisation of a very thin slice through the cell. The number of individuals with MIRAGE syndrome studied to date is relatively small, so data are limited. It would be of interest to study cellular ultrastructure from individuals with other gain-of-function or loss-of function changes in SAMD9, who do not have classic MIRAGE syndrome features, to see if similar changes are seen there too.

In conclusion, we have observed evidence of increased endosomal activity in patients-derived fibroblasts carrying SAMD9 variants compared to controls. These findings provide more data for ultrastructural morphology of SAMD9-associated conditions and supports the original findings of enlarged vesicles in patients’ fibroblasts. However, more insight into the pathogenic mechanisms of SAMD9 disruption is needed. Indeed, induced pluripotent stem cell lines (iPSC) have been generated from fibroblasts from two MIRAGE patients. ^{2 , 24} These represent a potentially important resource to further investigate the disease mechanisms of MIRAGE syndrome and model the underlying molecular basis, at least in vitro. Understanding the molecular function of SAMD9 is extremely important to help us develop personalised management and effective therapies for individuals with SAMD9-associated variants.