Spondylocheiro dysplastic form of the Ehlers-Danlos syndrome--an autosomal-recessive entity caused by mutations in the zinc transporter gene SLC39A13.

We present clinical, radiological, biochemical, and genetic findings on six patients from two consanguineous families that show EDS-like features and radiological findings of a mild skeletal dysplasia. The EDS-like findings comprise hyperelastic, thin, and bruisable skin, hypermobility of the small joints with a tendency to contractures, protuberant eyes with bluish sclerae, hands with finely wrinkled palms, atrophy of the thenar muscles, and tapering fingers. The skeletal dysplasia comprises platyspondyly with moderate short stature, osteopenia, and widened metaphyses. Patients have an increased ratio of total urinary pyridinolines, lysyl pyridinoline/hydroxylysyl pyridinoline (LP/HP), of approximately 1 as opposed to approximately 6 in EDS VI or approximately 0.2 in controls. Lysyl and prolyl residues of collagens were underhydroxylated despite normal lysyl hydroxylase and prolyl 4-hydroxylase activities; underhydroxylation was a generalized process as shown by mass spectrometry of the alpha1(I)- and alpha2(I)-chain-derived peptides of collagen type I and involved at least collagen types I and II. A genome-wide SNP scan and sequence analyses identified in all patients a homozygous c.483_491 del9 SLC39A13 mutation that encodes for a membrane-bound zinc transporter SLC39A13. We hypothesize that an increased Zn(2+) content inside the endoplasmic reticulum competes with Fe(2+), a cofactor that is necessary for hydroxylation of lysyl and prolyl residues, and thus explains the biochemical findings. These data suggest an entity that we have designated "spondylocheiro dysplastic form of EDS (SCD-EDS)" to indicate a generalized skeletal dysplasia involving mainly the spine (spondylo) and striking clinical abnormalities of the hands (cheiro) in addition to the EDS-like features.

Differential diagnosis of muscular hypotonia in infants: the kyphoscoliotic type of Ehlers-Danlos syndrome (EDS VI).

The kyphoscoliotic type of Ehlers-Danlos syndrome (EDS VI) (OMIM 225400) is an inherited connective tissue disorder characterized by hypotonia and kyphoscoliosis at birth, joint hypermobility, and skin hyperelasticity and fragility. Biochemically, it is characterized by a deficiency of collagen lysyl hydroxylase (EC 1.14.11.4) due to mutations in PLOD1. This deficiency results in underhydroxylation of collagen lysyl residues and, hence, an abnormal pattern of lysyl pyridinoline (LP) and hydroxylysyl pyridinoline (HP) crosslinks excreted in the urine. Because of hypotonia and delay in gross motor development, a neuromuscular disease is usually suspected, and in most cases the diagnosis is considered only very late, after performing an invasive neuromuscular work-up with normal results. We report a 12-month-old boy with kyphoscoliosis and delayed gross motor development, in whom the differential diagnosis of kyphoscoliotic type of Ehlers-Danlos syndrome (EDS VI) was initially suspected and successively confirmed by the abnormal urinary ratio of total pyridinolines (LP to HP), and by mutation analysis. We advocate the analysis of urinary pyridinolines in all infants with severe hypotonia which is highly specific and sensitive, quick and inexpensive.

Recurrent attacks of status epilepticus as predominant symptom in 3-methylcrotonyl-CoA carboxylase deficiency.

A patient with isolated 3-methylcrotonyl-CoA carboxylase (MCC) deficiency with an unusual clinical presentation is described. The patient presented with clusters of seizures with two or three months disease free interval in the first year of life which then evolved into attacks of status epilepticus after the age of 12 months. MCC deficiency was suspected because of elevated C5-OH-carnitine in tandem mass spectrometry and elevated 3-hydroxy-isovaleric acid in urine organic acid analysis. Deficiency of MCC was confirmed in cultured fibroblasts and mutation analysis revealed a novel mutation in MCCB, p.S39F. Attacks of status epilepticus as a predominant symptom have not been described before in isolated MCC deficiency.

Potential misdiagnosis of 3-methylcrotonyl-coenzyme A carboxylase deficiency associated with absent or trace urinary 3-methylcrotonylglycine.

We report 2 patients with isolated 3-methylcrotonyl-coenzyme A carboxylase deficiency whose urine was devoid of, or contained only trace, 3-methylcrotonylglycine, the pathognomonic marker for this disorder. The first patient, a girl with trisomy 21, was detected through newborn screening with an elevated 5 carbon hydroxycarnitine species level, and the second patient came to clinical attention at the age of 5 months because of failure to thrive and developmental delay. Investigation of urinary organic acids revealed an elevated 3-hydroxyisovaleric acid level but no demonstrable 3-methylcrotonylglycine in both patients. Enzyme studies in cultured fibroblasts confirmed isolated 3-methylcrotonyl-coenzyme A carboxylase deficiency with residual activities of 5% to 7% and 12% of the median control value, respectively. Incorporation of 14C-isovaleric acid into intact fibroblasts was essentially normal, showing that the overall pathway was at least partially functional and potentially explaining the absence of 3-methylcrotonylglycine in urine. Mutation analysis of the MCCA and MCCB genes revealed that both patients were compound heterozygous for a missense mutation, MCCB-c.1015G-->A (p.V339M), and a second mutation that leads to undetectable MCCB messenger (poly A+) RNA. Absent or trace 3-methylcrotonylglycine levels in urine raises the potential for misdiagnosis in the clinical biochemical genetics laboratory based solely on urine organic acid analysis using combined gas chromatography-mass spectrometry.

Transcriptional and post-transcriptional regulation of preproendothelin-1 by plaque-prone hemodynamics.

OBJECTIVE: Plaque-prone areas are exposed to a particular hemodynamic environment characterized by a low mean shear stress value and a cyclic reversal flow. This mechanical environment, also termed oscillatory shear stress (OSS), induces the expression of several pro-atherogenic genes in the endothelial cells including the preproendothelin-1 (ppET-1) gene. The present paper investigates the molecular mechanisms of this induction. METHODS AND RESULTS: Several deletional mutants of ppET-1 gene promoter were cloned upstream of a luciferase gene and transiently transfected in bovine arterial endothelial cells that were further exposed to plaque-prone hemodynamics. After 24h of flow exposure, analysis of the transfected cells showed that a proximal promoter of 156 base pairs length retained OSS responsiveness. Mutation of an activator protein-1 (AP-1) binding site present in this minimal promoter completely abolished its activation by OSS. Consistently, electrophoresis mobility shift assay revealed a sustained activation of AP-1 transcription factor in endothelial cells exposed to OSS. In addition to the transcriptional activation, we demonstrated that OSS also induces a stabilization of ppET-1mRNA through the 3'-untranslated region (3'-UTR) of this gene. Fluvastatin, a drug known to improve endothelial function, was shown to prevent OSS up-regulation of the ppET-1 gene expression. Under this flow condition, fluvastatin affects ppET-1 gene expression via inhibition of its promoter activity without affecting ppET-1mRNA stability. CONCLUSIONS: The present study demonstrate that plaque-prone hemodynamic induces ppET-1 gene expression by both transcriptional and post-transcriptional mechanisms via an activation of AP-1 transcriptional factor and stabilization of mRNA. The transcriptional up-regulation of ppET-1 was shown to be fluvastatin sensitive.

Plaque-prone hemodynamics impair endothelial function in pig carotid arteries.

Hemodynamic forces play an active role in vascular pathologies, particularly in relation to the localization of atherosclerotic lesions. It has been established that low shear stress combined with cyclic reversal of flow direction (oscillatory shear stress) affects the endothelial cells and may lead to an initiation of plaque development. The aim of the study was to analyze the effect of hemodynamic conditions in arterial segments perfused in vitro in the absence of other stimuli. Left common porcine carotid segments were mounted into an ex vivo arterial support system and perfused for 3 days under unidirectional high and low shear stress (6 +/- 3 and 0.3 +/- 0.1 dyn/cm(2)) and oscillatory shear stress (0.3 +/- 3 dyn/cm(2)). Bradykinin-induced vasorelaxation was drastically decreased in arteries exposed to oscillatory shear stress compared with unidirectional shear stress. Impaired nitric oxide-mediated vasodilation was correlated to changes in both endothelial nitric oxide synthase (eNOS) gene expression and activation in response to bradykinin treatment. This study determined the flow-mediated effects on native tissue perfused with physiologically relevant flows and supports the hypothesis that oscillatory shear stress is a determinant factor in early stages of atherosclerosis. Indeed, oscillatory shear stress induces an endothelial dysfunction, whereas unidirectional shear stress preserves the function of endothelial cells. Endothelial dysfunction is directly mediated by a downregulation of eNOS gene expression and activation; consequently, a decrease of nitric oxide production and/or bioavailability occurs.

Cap G, a gelsolin family protein modulating protective effects of unidirectional shear stress.

Atherosclerosis is a progressive and complex pathophysiological process occurring in large arteries. Although it is of multifactorial origin, the disease develops at preferential sites along the vasculature in regions experiencing specific hemodynamic conditions that are predisposed to endothelial dysfunction. The exact mechanisms allowing endothelial cells to discriminate between plaque-free and plaque-prone flows remain to be explored. To investigate such mechanisms, we performed a proteomic analysis on endothelial cells exposed in vitro to these two-flow patterns. A few spots on the two-dimensional gel had an intensity that was differentially regulated by plaque-free versus plaque-prone flows. One of them was further investigated and identified as macrophage-capping protein (Cap G), a member of the gelsolin protein superfamily. A 2-fold increase of Cap G protein and a 5-fold increase of Cap G mRNA were observed in cells exposed to a plaque-free flow as compared with static cultures. This increase was not observed in cells exposed to plaque-prone flow. Plaque-free flow induced a corresponding increase in nuclear and cytoskeletal-associated Cap G. Finally, overexpression of Cap G in transfection assays increased the motility potential of endothelial cells. These observations together with the known functions of Cap G suggest that Cap G may contribute to the protective effect exerted by plaque-free flow on endothelial cells. On the contrary, in cells exposed to a plaque-prone flow, no induction of Cap G expression could be observed.

ET-1 and NOS III gene expression regulation by plaque-free and plaque-prone hemodynamic conditions.

Both plaque-free and plaque-prone hemodynamic environments induce an increase in the oxidative state of endothelial cells (ECs), whereas differential gene expression regulation was described in cells exposed to these conditions. In order to investigate the role of the increased oxidative state in flow-regulation of gene expression, we first exposed EC to non-pulsed unidirectional shear stress. These conditions only slightly increases ECs oxidative state and endothelin-1 (ET-1) mRNA expression, whereas endothelial nitric oxide synthase (NOS III) mRNA level were significantly up-regulated. On the contrary, both ET-1 and NOS III gene expression were significantly induced in EC exposed to pulsed-unidirectional flow (plaque-free). Only ET-1 gene expression was up-regulated by oscillatory flow (plaque-prone). Moreover, use of an antioxidant only partially inhibited NOS III gene up-regulation by unidirectional flow, whereas it completely abrogated ET-1 gene up-regulation by unidirectional and oscillatory flows. Thus suggesting that mechanical forces regulate gene expression in ECs both via oxidative stress-dependent and -independent mechanisms.