Antimicrobial Effect and Cytotoxic Evaluation of Mg-Doped Hydroxyapatite Functionalized with Au-Nano Rods.

Hydroxyapatite (HA) is the main inorganic mineral that constitutes bone matrix and represents the most used biomaterial for bone regeneration. Over the years, it has been demonstrated that HA exhibits good biocompatibility, osteoconductivity, and osteoinductivity both in vitro and in vivo, and can be prepared by synthetic and natural sources via easy fabrication strategies. However, its low antibacterial property and its fragile nature restricts its usage for bone graft applications. In this study we functionalized a MgHA scaffold with gold nanorods (AuNRs) and evaluated its antibacterial effect against S. aureus and E. coli in both suspension and adhesion and its cytotoxicity over time (1 to 24 days). Results show that the AuNRs nano-functionalization improves the antibacterial activity with 100% bacterial reduction after 24 h. The toxicity study, however, indicates a 4.38-fold cell number decrease at 24 days. Although further optimization on nano-functionalization process are needed for cytotoxicity, these data indicated that Au-NRs nano-functionalization is a very promising method for improving the antibacterial properties of HA.

Effect of mechanical strain on the collagen VI pericellular matrix in anterior cruciate ligament fibroblasts.

Cell-extracellular matrix interaction plays a major role in maintaining the structural integrity of connective tissues and sensing changes in the biomechanical environment of cells. Collagen VI is a widely expressed non-fibrillar collagen, which regulates tissues homeostasis. The objective of the present investigation was to extend our understanding of the role of collagen VI in human ACL. This study shows that collagen VI is associated both in vivo and in vitro to the cell membrane of knee ACL fibroblasts, contributing to the constitution of a microfibrillar pericellular matrix. In cultured cells the localization of collagen VI at the cell surface correlated with the expression of NG2 proteoglycan, a major collagen VI receptor. The treatment of ACL fibroblasts with anti-NG2 antibody abolished the localization of collagen VI indicating that collagen VI pericellular matrix organization in ACL fibroblasts is mainly mediated by NG2 proteoglycan. In vitro mechanical strain injury dramatically reduced the NG2 proteoglycan protein level, impaired the association of collagen VI to the cell surface, and promoted cell cycle withdrawal. Our data suggest that the injury-induced alteration of specific cell-ECM interactions may lead to a defective fibroblast self-renewal and contribute to the poor regenerative ability of ACL fibroblasts.

Metal-on-metal hip prostheses: correlation between debris in the synovial fluid and levels of cobalt and chromium ions in the bloodstream.

PURPOSE: Hip prostheses with metal-on-metal (MoM) coupling can release cobalt-chromium particles and ions. The aim of this work is to verify the correlation between particles in the synovial fluid and circulating ions. METHODS: Forty patients were enrolled; particles from synovial fluid were analysed by SEM­EDX (Scanning Electron Microscopy-Energy Dispersion X-rays analysis) and levels of circulating Co and Cr were assayed by ICP-MS (inductively-coupled plasma mass spectrometry). RESULTS: In 16 cases we did not find any particles in the synovial fluid and the Co level in whole blood was 0.05­4.42 ppb; in seven with few particles the blood level was 2.2­15.6 ppb; in six cases with several particles the level was 5.0­54.3 ppb; finally, in 11 cases we isolated not only Co-Cr particles, but also Cr particles with low or absent Co and in these patients the circulating level of Co was 23.8­109.6 ppb. Co in serumand Cr level both whole blood and serum have shown a similar trend to Co; the correlation between all these values and the corresponding particles is statistically significant in all cases. CONCLUSION: Co and Cr both in serum and whole blood represents a systemic representation of the particle release at local level and can therefore be used to confirm a diagnosis and monitor the wear process of MoM articular prostheses.

Characterization of a Novel Species of Legionella Isolated from a Healthcare Facility: Legionella resiliens sp. nov.

Two Legionella-like isolates, 8cVS16T and 9fVS26, were isolated from a water distribution system (WDS) in a healthcare facility. Cells were Gram- and Ziehl Neelsen-stain-negative, rod-shaped, motile, and exhibited a blue-white fluorescence under Wood's lamp at 365 nm. The strains grew in a range of 32-37 °C on BCYE with L-cysteine (Cys+), GVPC, and MWY agar medium, with a positive reaction for oxidase, catalase, and gelatinase. The dominant fatty acids were summed features 3 (C16:1ω7c/C16:1ω6c) (27.7%), C16:0 iso (17.5%), and C16:0 (16.3%), and Q13 as the major ubiquinone. The mip and rpoB gene sequences showed a similarity of 96.7% and 92.4%, with L. anisa (ATCC 35292T). The whole genomes sequencing (WGS) performed displayed a GC content of 38.21 mol% for both. The digital DNA-DNA hybridization (dDDH) analysis demonstrated the separation of the two strains from the phylogenetically most related L. anisa (ATCC 35292T), with ≤43% DNA-DNA relatedness. The Average Nucleotide Identity (ANI) between the two strains and L. anisa (ATCC 35292T) was 90.74%, confirming that the two isolates represent a novel species of the genus Legionella. The name proposed for this species is Legionella resiliens sp. nov., with 8cVS16T (=DSM 114356T = CCUG 76627T) as the type strain.

Collagen VI Deficiency Impairs Tendon Fibroblasts Mechanoresponse in Ullrich Congenital Muscular Dystrophy.

The pericellular matrix (PCM) is a specialized extracellular matrix that surrounds cells. Interactions with the PCM enable the cells to sense and respond to mechanical signals, triggering a proper adaptive response. Collagen VI is a component of muscle and tendon PCM. Mutations in collagen VI genes cause a distinctive group of inherited skeletal muscle diseases, and Ullrich congenital muscular dystrophy (UCMD) is the most severe form. In addition to muscle weakness, UCMD patients show structural and functional changes of the tendon PCM. In this study, we investigated whether PCM alterations due to collagen VI mutations affect the response of tendon fibroblasts to mechanical stimulation. By taking advantage of human tendon cultures obtained from unaffected donors and from UCMD patients, we analyzed the morphological and functional properties of cellular mechanosensors. We found that the length of the primary cilia of UCMD cells was longer than that of controls. Unlike controls, in UCMD cells, both cilia prevalence and length were not recovered after mechanical stimulation. Accordingly, under the same experimental conditions, the activation of the Hedgehog signaling pathway, which is related to cilia activity, was impaired in UCMD cells. Finally, UCMD tendon cells exposed to mechanical stimuli showed altered focal adhesions, as well as impaired activation of Akt, ERK1/2, p38MAPK, and mechanoresponsive genes downstream of YAP. By exploring the response to mechanical stimulation, for the first time, our findings uncover novel unreported mechanistic aspects of the physiopathology of UCMD-derived tendon fibroblasts and point at a role for collagen VI in the modulation of mechanotransduction in tendons.

Ceramic debris in hip prosthesis: correlation between synovial fluid and joint capsule.

Detection of ceramic particles in synovial fluids allows early diagnosis of ceramic damage, but there is no evidence of a relationship between ceramic debris in the articular space and in the joint capsule. The aim of the present study is to verify if the particles isolated in the synovial fluid are comparable with those stored in the capsular tissue. Twenty-one patients were enrolled. Both synovial fluid and capsular samples were collected during revision surgery and ceramic particles were isolated and analyzed by scanning electron microscopy and energy-dispersive X-ray microanalysis. It resulted a significant correlation between the samples couples (18 out of 21). This study confirms that the synovial fluid analysis can give a clear definition of the presence of particles in the joint capsule.

Immunoelectron Microscopy Methods.

"Immunoelectron microscopy" defines a group of techniques developed for visualizing where components of cells or tissues are localized, by means of a transmission electron microscope (TEM) at a subcellular resolution. The method is based on antigen recognition by primary antibodies and subsequent visualization of recognized structures by means of electron-opaque gold granules, which are easily visible in TEM images. The potentially high resolution of this method relies on the very small size of the colloidal gold label, which consists of granules ranging from 1 to 60 nm in diameter, mostly used in the 5-15 nm sizes.

Altered chromatin organization and SUN2 localization in mandibuloacral dysplasia are rescued by drug treatment.

Mandibuloacral dysplasia type A (MADA) is a rare laminopathy characterized by growth retardation, craniofacial anomalies, bone resorption at specific sites including clavicles, phalanges and mandibula, mottled cutaneous pigmentation, skin rigidity, partial lipodystrophy, and insulin resistance. The disorder is caused by recessive mutations of the LMNA gene encoding for A-type lamins. The molecular feature of MADA consists in the accumulation of the unprocessed lamin A precursor, which is detected at the nuclear rim and in intranuclear aggregates. Here, we report the characterization of prelamin A post-translational modifications in MADA cells that induce alterations in the chromatin arrangement and dislocation of nuclear envelope-associated proteins involved in correct nucleo-cytoskeleton relationships. We show that protein post-translational modifications change depending on the passage number, suggesting the onset of a feedback mechanism. Moreover, we show that treatment of MADA cells with the farnesyltransferase inhibitors is effective in the recovery of the chromatin phenotype, altered in MADA, provided that the cells are at low passage number, while at high passage number, the treatment results ineffective. Moreover, the distribution of the lamin A interaction partner SUN2, a constituent of the nuclear envelope, is altered by MADA mutations, as argued by the formation of a highly disorganized lattice. Treatment with statins partially rescues proper SUN2 organization, indicating that its alteration is caused by farnesylated prelamin A accumulation. Given the major role of SUN1 and SUN2 in the nucleo-cytoskeleton interactions and in regulation of nuclear positioning in differentiating cells, we hypothesise that mechanisms regulating nuclear membrane-centrosome interplay and nuclear movement may be affected in MADA fibroblasts.

3D ECM-rich environment sustains the identity of naive human iPSCs.

The establishment of in vitro naive human pluripotent stem cell cultures opened new perspectives for the study of early events in human development. The role of several transcription factors and signaling pathways have been characterized during maintenance of human naive pluripotency. However, little is known about the role exerted by the extracellular matrix (ECM) and its three-dimensional (3D) organization. Here, using an unbiased and integrated approach combining microfluidic cultures with transcriptional, proteomic, and secretome analyses, we found that naive, but not primed, hiPSC colonies are characterized by a self-organized ECM-rich microenvironment. Based on this, we developed a 3D culture system that supports robust long-term feeder-free self-renewal of naive hiPSCs and also allows direct and timely developmental morphogenesis simply by modulating the signaling environment. Our study opens new perspectives for future applications of naive hiPSCs to study critical stages of human development in 3D starting from a single cell.

Recessive COL6A2 C-globular missense mutations in Ullrich congenital muscular dystrophy: role of the C2a splice variant.

Ullrich congenital muscular dystrophy (UCMD) is a disabling and life-threatening disorder resulting from either recessive or dominant mutations in genes encoding collagen VI. Although the majority of the recessive UCMD cases have frameshift or nonsense mutations in COL6A1, COL6A2, or COL6A3, recessive structural mutations in the COL6A2 C-globular region are emerging also. However, the underlying molecular mechanisms have remained elusive. Here we identified a homozygous COL6A2 E624K mutation (C1 subdomain) and a homozygous COL6A2 R876S mutation (C2 subdomain) in two UCMD patients. The consequences of the mutations were investigated using fibroblasts from patients and cells stably transfected with the mutant constructs. In contrast to expectations based on the clinical severity of these two patients, secretion and assembly of collagen VI were moderately affected by the E624K mutation but severely impaired by the R876S substitution. The E624K substitution altered the electrostatic potential of the region surrounding the metal ion-dependent adhesion site, resulting in a collagen VI network containing thick fibrils and spots with densely packed microfibrils. The R876S mutation prevented the chain from assembling into triple-helical collagen VI molecules. The minute amount of collagen VI secreted by the R876S fibroblasts was solely composed of a faster migrating chain corresponding to the C2a splice variant with an alternative C2 subdomain. In transfected cells, the C2a splice variant was able to assemble into short microfibrils. Together, the results suggest that the C2a splice variant may functionally compensate for the loss of the normal COL6A2 chain when mutations occur in the C2 subdomain.

Dual-Functional Nano-Functionalized Titanium Scaffolds to Inhibit Bacterial Growth and Enhance Osteointegration.

Implantable biomaterials play a key role for the success of orthopedic surgery procedures. However, infections remain one of the most damaging post-operative complications that lead to the implant failure. Recently, several approaches have been proposed to avoid or manage implant-associated infections. Among these, an appropriate surface functionalization to confer intrinsic antibacterial properties preserving the osteo-integration ability represents an appealing strategy for the development of innovative implant materials. Titanium and its alloys are the most used materials for manufacturing of both articular and bone skull prostheses as well as dental implants. However, to date there is still a significant clinical need to improve their bioactivity, osseointegration and antibacterial activity. In this study, titanium biomimetic scaffolds are prepared by nano-functionalization with TiO2 (Ti_TiO2) and γFe2O3 (Ti_γFe2O3). Both cytocompatibility and antibacterial activity have been evaluated. Data show that both nano-functionalized scaffolds exhibit a good antibacterial activity towards Staphylococcus aureus, reducing colony number to 99.4% (Ti_TiO2) and 99.9% (Ti_γFe2O3), respectively. In addition, an increase of both human adipose-derived mesenchymal stem cells (hADSCs) cell proliferation (up to 4.3-fold for Ti_TiO2 and 3.7-fold for Ti_γFe2O3) and differentiation has been observed. These data suggest that these nano-functionalized titanium substrates represent promising prototypes for new antimicrobial and osteoconductive biomaterials to be used in the orthopedic field to reconstruct significant bone defect.

A new Ag-nanostructured hydroxyapatite porous scaffold: Antibacterial effect and cytotoxicity study.

We report a new chemical method for the functionalization of Mg-hydroxyapatite (Mg-HA) scaffold with Ag nanoparticles (Ag NPs) integrating in one step both the synthesis of the Ag NPs and their nano-structuring into the HA matrix (Ag-Mg-HA scaffold). This method exploits a green photochemical synthesis and allows the direct growth of Ag NPs on the Mg-HA surface. The surface structure of Ag-Mg-HA scaffold, investigated by scanning electron microscopy, shows no significant changes in the morphology upon Ag NPs incorporation. The presence of Ag was confirmed by EDX analysis. TEM and spectroscopic investigations show Ag NPs spherical shaped with a mean diameter of about 20 nm exhibiting the typical plasmon absorption band with maximum at 420 nm. The antibacterial properties of Ag-Mg-HA scaffolds were tested against two bacterial strains, Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The results show excellent antibacterial properties achieving up to 99% and 100% reduction of colonies for both bacteria cultures after 24 h of incubation and 100% of reduction after 48 h of incubation. The cytotoxicity of Ag-Mg-HA was also in deep investigated assessing both cell proliferation and differentiation using hADSCs (human Adipose Derived Stem Cells) and testing data point at 0, 7, 14 and 24 days. The results show cytotoxic effect with cell proliferation decreasing up to 90% at 24 days and osteogenic differentiation inhibition. The observed cytotoxicity can be probable ascribed to the oxidative stress by ROS. Indeed, considering the effectiveness of the nanofunctionalization method and the excellent antibacterial properties showed by the Ag-Mg-HA scaffold, future works will be devoted to create nanofunctionalized scaffold satisfying both antimicrobial and osteo-regenerative properties.

Interleukin-6 neutralization ameliorates symptoms in prematurely aged mice.

Hutchinson-Gilford progeria syndrome (HGPS) causes premature aging in children, with adipose tissue, skin and bone deterioration, and cardiovascular impairment. In HGPS cells and mouse models, high levels of interleukin-6, an inflammatory cytokine linked to aging processes, have been detected. Here, we show that inhibition of interleukin-6 activity by tocilizumab, a neutralizing antibody raised against interleukin-6 receptors, counteracts progeroid features in both HGPS fibroblasts and LmnaG609G/G609G progeroid mice. Tocilizumab treatment limits the accumulation of progerin, the toxic protein produced in HGPS cells, rescues nuclear envelope and chromatin abnormalities, and attenuates the hyperactivated DNA damage response. In vivo administration of tocilizumab reduces aortic lesions and adipose tissue dystrophy, delays the onset of lipodystrophy and kyphosis, avoids motor impairment, and preserves a good quality of life in progeroid mice. This work identifies tocilizumab as a valuable tool in HGPS therapy and, speculatively, in the treatment of a variety of aging-related disorders.

Emerin-prelamin A interplay in human fibroblasts.

BACKGROUND INFORMATION: Emerin is a nuclear envelope protein that contributes to nuclear architecture, chromatin structure, and gene expression through its interaction with various nuclear proteins. In particular, emerin is molecularly connected with the nuclear lamina, a protein meshwork composed of lamins and lamin-binding proteins underlying the inner nuclear membrane. Among nuclear lamina components, lamin A is a major emerin partner. Lamin A, encoded by the LMNA gene (lamin A/C gene), is produced as a precursor protein (prelamin A) that is post-transcriptionally modified at its C-terminal region where the CaaX motif triggers a sequence of modifications, including farnesylation, carboxymethylation, and proteolytic cleavage by ZMPSTE 24 (zinc metalloproteinase Ste24) metalloproteinase. Impairment of the lamin A maturation pathway causing lamin A precursor accumulation is linked to the development of rare diseases such as familial partial lipodystrophy, MADA (mandibuloacral dysplasia), the Werner syndrome, Hutchinson-Gilford progeria syndrome and RD (restrictive dermopathy). RESULTS: In the present study, we show that emerin and different prelamin A forms influence each other's localization. We show that the accumulation of non-farnesylated as well as farnesylated carboxymethylated lamin A precursors in human fibroblasts modifies emerin localization. On the contrary, emerin absence at the inner nuclear membrane leads to unprocessed (non-farnesylated) prelamin A aberrant localization only. Moreover, we observe that the restoration of emerin expression in emerin-null cells induces the recovery of non-farnesylated prelamin A localization. CONCLUSION: These results indicate that emerin-prelamin A interplay influences nuclear organization. This finding may be relevant to the understanding of laminopathies.

Emerin Phosphorylation during the Early Phase of the Oxidative Stress Response Influences Emerin-BAF Interaction and BAF Nuclear Localization.

Reactive Oxygen Species (ROS) are reactive molecules required for the maintenance of physiological functions. Oxidative stress arises when ROS production exceeds the cellular ability to eliminate such molecules. In this study, we showed that oxidative stress induces post-translational modification of the inner nuclear membrane protein emerin. In particular, emerin is phosphorylated at the early stages of the oxidative stress response, while protein phosphorylation is abolished upon recovery from stress. A finely tuned balance between emerin phosphorylation and O-GlcNAcylation seems to govern this dynamic and modulates emerin-BAF interaction and BAF nucleoplasmic localization during the oxidative stress response. Interestingly, emerin post-translational modifications, similar to those observed during the stress response, are detected in cells bearing LMNA gene mutations and are characterized by a free radical generating environment. On the other hand, under oxidative stress conditions, a delay in DNA damage repair and cell cycle progression is found in cells from Emery-Dreifuss Muscular Dystrophy type 1, which do not express emerin. These results suggest a role of the emerin-BAF protein platform in the DNA damage response aimed at counteracting the detrimental effects of elevated levels of ROS.

Tendon Extracellular Matrix Remodeling and Defective Cell Polarization in the Presence of Collagen VI Mutations.

Mutations in collagen VI genes cause two major clinical myopathies, Bethlem myopathy (BM) and Ullrich congenital muscular dystrophy (UCMD), and the rarer myosclerosis myopathy. In addition to congenital muscle weakness, patients affected by collagen VI-related myopathies show axial and proximal joint contractures, and distal joint hypermobility, which suggest the involvement of tendon function. To gain further insight into the role of collagen VI in human tendon structure and function, we performed ultrastructural, biochemical, and RT-PCR analysis on tendon biopsies and on cell cultures derived from two patients affected with BM and UCMD. In vitro studies revealed striking alterations in the collagen VI network, associated with disruption of the collagen VI-NG2 (Collagen VI-neural/glial antigen 2) axis and defects in cell polarization and migration. The organization of extracellular matrix (ECM) components, as regards collagens I and XII, was also affected, along with an increase in the active form of metalloproteinase 2 (MMP2). In agreement with the in vitro alterations, tendon biopsies from collagen VI-related myopathy patients displayed striking changes in collagen fibril morphology and cell death. These data point to a critical role of collagen VI in tendon matrix organization and cell behavior. The remodeling of the tendon matrix may contribute to the muscle dysfunction observed in BM and UCMD patients.

Lamin A involvement in ageing processes.

Lamin A, a main constituent of the nuclear lamina, is the major splicing product of the LMNA gene, which also encodes lamin C, lamin A delta 10 and lamin C2. Involvement of lamin A in the ageing process became clear after the discovery that a group of progeroid syndromes, currently referred to as progeroid laminopathies, are caused by mutations in LMNA gene. Progeroid laminopathies include Hutchinson-Gilford Progeria, Mandibuloacral Dysplasia, Atypical Progeria and atypical-Werner syndrome, disabling and life-threatening diseases with accelerated ageing, bone resorption, lipodystrophy, skin abnormalities and cardiovascular disorders. Defects in lamin A post-translational maturation occur in progeroid syndromes and accumulated prelamin A affects ageing-related processes, such as mTOR signaling, epigenetic modifications, stress response, inflammation, microRNA activation and mechanosignaling. In this review, we briefly describe the role of these pathways in physiological ageing and go in deep into lamin A-dependent mechanisms that accelerate the ageing process. Finally, we propose that lamin A acts as a sensor of cell intrinsic and environmental stress through transient prelamin A accumulation, which triggers stress response mechanisms. Exacerbation of lamin A sensor activity due to stably elevated prelamin A levels contributes to the onset of a permanent stress response condition, which triggers accelerated ageing.

Long term breeding of the Lmna G609G progeric mouse: Characterization of homozygous and heterozygous models.

The transgenic LmnaG609G progeric mouse represents an outstanding animal model for studying the human Hutchinson-Gilford Progeria Syndrome (HGPS) caused by a mutation in the LMNA gene, coding for the nuclear envelope protein Lamin A/C, and, as an important, more general scope, for studying the complex process governing physiological aging in humans. Here we give a comprehensive description of the peculiarities related to the breeding of LmnaG609G mice over a prolonged period of time, and of many features observed in a large colony for a 2-years period. We describe the breeding and housing conditions underlining the possible interference of the genetic background on the phenotype expression. This information represents a useful tool when planning and interpreting studies on the LmnaG609G mouse model, complementing any specific data already reported in the literature about this model since its production. It is also particularly relevant for the heterozygous mouse, which mirrors the genotype of the human pathology however requires an extended time to manifest symptoms and to be carefully studied.

Prelamin A is involved in early steps of muscle differentiation.

Lamin A is a nuclear lamina constituent implicated in a number of human disorders including Emery-Dreifuss muscular dystrophy. Since increasing evidence suggests a role of the lamin A precursor in nuclear functions, we investigated the processing of prelamin A during differentiation of C2C12 mouse myoblasts. We show that both protein levels and cellular localization of prelamin A are modulated during myoblast activation. Similar changes of lamin A-binding proteins emerin and LAP2alpha were observed. Furthermore, prelamin A was found in a complex with LAP2alpha in differentiating myoblasts. Prelamin A accumulation in cycling myoblasts by expressing unprocessable mutants affected LAP2alpha and PCNA amount and increased caveolin 3 mRNA and protein levels, while accumulation of prelamin A in differentiated muscle cells following treatment with a farnesyl transferase inhibitor appeared to inhibit caveolin 3 expression. Our data provide evidence for a critical role of the lamin A precursor in the early steps of muscle cell differentiation.

Report of a novel ATP7A mutation causing distal motor neuropathy.

We describe a novel ATP7A gene mutation associated with distal motor neuropathy, mild connective tissue abnormalities and autonomic disturbances. Next-generation sequencing analysis of a lower-motor neuron diseases gene panel was performed in two sibs presenting with distal motor neuropathy plus an autonomic dysfunction, which main manifestations were retrograde ejaculation, diarrhea and hyperhydrosis. Probands underwent dysmorphological, neurological, electrophysiological as well as biochemical evaluations and somatic and autonomic innervation studies on skin biopsies. A novel missense mutation (p.A991D) was identified in the X-linked ATP7A gene, segregating in both brothers and inherited from their healthy mother. Biochemical studies on patients' blood samples showed reduced serum copper and ceruloplasmin levels. Clinical and neurophysiological evaluation documented dysautonomic signs. Quantitative evaluation of skin innervation disclosed a small fiber neuropathy with prevalent autonomic involvement. Mutations in the ATP7A gene, encoding for a copper-transporting ATPase, have been associated with the severe infantile neurodegenerative Menkes disease and in its milder variant, the Occipital Horn Syndrome. Only two ATP7A mutations were previously reported as causing, a pure axonal distal motor neuropathy (dHMN-SMAX3). The phenotype we report represents a further example of this rare genotype-phenotype correlation and highlights the possible occurrence in SMAX3 of autonomic disturbances, as described for Menkes disease and Occipital Horn Syndrome.