Rita Gardella

Mutations in the facilitative glucose transporter GLUT10 alter angiogenesis and cause arterial tortuosity syndrome

Arterial tortuosity syndrome (ATS) is an autosomal recessive disorder characterized by tortuosity, elongation, stenosis and aneurysm formation in the major arteries owing to disruption of elastic fibers in the medial layer of the arterial wall. Previously, we used homozygosity mapping to map a candidate locus in a 4.1-Mb region on chromosome 20q13.1 (ref. 2). Here, we narrowed the candidate region to 1.2 Mb containing seven genes. Mutations in one of these genes, SLC2A10, encoding the facilitative glucose transporter GLUT10, were identified in six ATS families. GLUT10 deficiency is associated with upregulation of the TGFβ pathway in the arterial wall, a finding also observed in Loeys-Dietz syndrome, in which aortic aneurysms associate with arterial tortuosity. The identification of a glucose transporter gene responsible for altered arterial morphogenesis is notable in light of the previously suggested link between GLUT10 and type 2 diabetes. Our data could provide new insight on the mechanisms causing microangiopathic changes associated with diabetes and suggest that therapeutic compounds intervening with TGFβ signaling represent a new treatment strategy.

Homozygosity mapping of a gene for arterial tortuosity syndrome to chromosome 20q13

Background: Arterial tortuosity syndrome (ATS) is an uncommon connective tissue disorder of unknown aetiology. The most prominent feature is tortuosity of the large arteries, but lengthening, stenosis, and aneurysm formation are also frequent. Methods: We performed a genomewide screen by homozygosity mapping of three consanguineous multiplex families, two from Morocco, and one from Italy, which included 11 ATS patients. The two families from Morocco may possibly have a common ancestor. Results: We mapped the ATS gene to chromosome 20q13. Recombinations within an extended haplotype of 11 microsatellite markers localised the ATS gene between markers D20S836 and D20S109, an interval of 9.5 cM. Conclusions: Cloning and completing functional and structural analysis of the ATS gene may provide new insights into the molecular mechanisms of elastogenesis.

Exclusion of candidate genes in a family with arterial tortuosity syndrome

Arterial tortuosity syndrome (ATS) is a rare hereditary disorder with variable clinical presentation including tortuosity and elongation of the major arteries, often associated with pulmonary artery stenosis, pulmonary hypertension, and skin and joint laxity, suggestive of a connective tissue disorder. ATS is transmitted in an autosomal recessive mode, but the causal gene is unknown. We report an Italian pedigree with three inbred families in which five patients show signs of ATS. In particular, four adult patients present arterial tortuosity and elongation of the main arteries. Two of these patients, with the most severe degree of arterial tortuosity, also show severe peripheral stenosis of the main pulmonary artery. The fifth young patient shows a severe pulmonary valve stenosis in the absence of arterial tortuosity. All patients show signs of Ehlers–Danlos syndrome (EDS): soft skin with abundant subcutaneous tissue and joint laxity, hernias, and disorganization of the extracellular matrix (ECM) of fibronectin (FN) and of actin microfilaments in cultured skin fibroblasts. Linkage analysis of the genes involved in EDS and other connective tissue disorders, excluded COL1A1, COL1A2, COL2A1, COL3A1, COL5A1, COL5A2, COL5A3, COL6A1, COL6A2, ADAMTS2, ELN, FN1, TNXA, and TNXB as candidate genes in the family under study, thus indicating that ATS is a distinct clinical and molecular entity.

Two novel SLC2A10/GLUT10 mutations in a patient with arterial tortuosity syndrome†

Bruno Drera, Andrea Guala, Nicoletta Zoppi, Rita Gardella, Piergiorgio Franceschini, Sergio Barlati, and Marina Colombi* Division of Biology and Genetics, Department of Biomedical Sciences and Biotechnology, University of Brescia, Brescia, Italy Ambulatorio di Genetica Clinica, Ospedale SS. Pietro e Paolo, Borgosesia, Italy Dipartimento di Scienze Pediatriche e dell’Adolescenza, Servizio di Genetica Clinica, Università degli Studi di Torino, Torino, Italy

Different phenotypes in recessive dystrophic epidermolysis bullosa patients sharing the same mutation in compound heterozygosity with two novel mutations in the type VII collagen gene

Background Dystrophic epidermolysis bullosa (DEB) is a bullous skin disease caused by mutations in the type VII collagen gene (COL7A1).

Three homozygous PTC mutations in the collagen type VII gene of patients affected by recessive dystrophic epidermolysis bullosa: analysis of transcript levels in dermal fibroblasts.

The Hallopeau‐Siemens variant of recessive dystrophic epidermolysis bullosa (HS‐RDEB) is a severe inherited skin disease characterized by the absence of collagen type VII (COLVII) and anchoring fibrils (AF), caused by mutations in collagen type VII gene (COL7A1). Mutations leading to the formation of premature termination codons (PTCs) of translation are the characteristic genetic lesions in HS‐RDEB patients; many PTC mutations have been found to be associated with a marked reduction or complete absence of COLVII mRNA. In this article, we report homozygosity for three different mutations in the COL7A1 of HS‐RDEB patients. One mutation, the R2685X, falling in exon 109, is a novel mutation, whereas the other two, the 425A→G falling in exon 3 and the 497insA in exon 4, have been previously identified in compound heterozygosity with different mutations in other unrelated RDEB patients. Haplotype analysis in three Italian families carrying the 497insA mutation suggested a common origin of this mutation and indicated that this is an ancestral Italian mutation. All these mutations generate PTCs and are associated with the absence of COLVII expression, as detected by immunofluorescence analysis of the patients skin. Evaluation of the levels of the mutated COLVII mRNAs in cultured skin fibroblasts of the patients and of their parents showed that all the mutated transcripts were expressed at consistent levels. Therefore, our results indicate that a marked mRNA reduction is not a constant feature associated with PTC mutations in COL7A1. Hum Mutat 13:439–452, 1999.

Exclusion of stromelysin-1, stromelysin-2, interstitial collagenase and fibronectin genes as the mutant loci in a family with recessive epidermolysis bullosa dystrophica and a form of cerebellar ataxia

SummaryThe interstitial collagenase gene (CLG), one of the main candidates in severe generalized recessive epidermolysis bullosa dystrophica (SGREBD), is closely linked to the stromelysin-1 (STMY1) and stromelysin-2 (STMY2) genes. These three loci map on chromosome 11 (q21–q22.3), where they constitute a cluster of genes coding for metalloproteinases involved in the degradation of the extracellular matrix (ECM). A recessive form of cerebellar ataxia of post-puberal onset (CLA1) has also been assigned to chromosome 11 (q14–q21). Since useful restriction fragment length polymorphisms (RFLPs) for the CLG gene are not available, we have studied the inheritance of the marker TaqI RFLP of the STMY1 gene in a North Italian family with a child affected by SGREBD, and his two sisters showing cerebellar ataxia (CA) of post-puberal onset. We have also studied the MspI RFLP of the fibronectin gene (FN1), which is located on chromosome 2q34–q36, and which codes for non-collagenous matrix proteins. Since we did not observe the segregation of the pathological phenotypes with STMY1 and FN1 RFLPs, we excluded the involvement of these genes in both the SGREBD and CA present in this family. The exclusion of the STMY1 gene indicates that the mutation causing SGREBD cannot be located in the CLG and/or STMY2 genes because of their proximity to the STMY1 locus. These data also indicate that the CA form here reported is not attributable to alterations in regions close to the collagenase cluster on chromosome 11.

EFFECT OF DEXAMETHASONE ON THE ASSEMBLY OF THE MATRIX OF FIBRONECTIN AND ON ITS RECEPTORS ORGANIZATION IN EHLERS-DANLOS SYNDROME SKIN FIBROBLASTS

The effect of dexamethasone (DEX) on the expression of fibronectin (FN), proα1(I) collagen (Col1), integrin α2, α5and β1subunits mRNAs, were studied by quantitative in situ hybridization (ISH) with radiolabelled probes in relationship with the organization of the extracellular matrix (ECM) of FN in human skin fibroblasts. In particular, two fibroblast strains were analysed, one derived from a control donor, typically organizing a rich ECM of FN, and the other from a patient affected by Ehlers‐Danlos syndrome (EDS), which did not assemble the FN‐ECM.

A ‐96C→T mutation in the promoter of the collagen type VII gene (COL7A1) abolishing transcription in a patient affected by recessive dystrophic epidermolysis bullosa

Hereditary dystrophic epidermolysis bullosa (DEB) refers to a group of clinically heterogeneous skin blistering diseases due to mutations in the collagen type VII gene (COL7A1). We report two novel mutations found in a patient affected by the most severe form of DEB, the recessive Hallopeau‐Siemens variant (HS‐RDEB): the R1978X nonsense mutation, in exon 72, and the ‐96C→T transition, in the promoter region. The allele specific analysis of the transcripts from skin fibroblasts of the patient showed that the ‐96C→T mutation is associated to the absence of collagen type VII (COLVII) mRNA. This mutation, the first one ever identified in the promoter of COL7A1, falls in an Sp1 motif, localized between nucleotides ‐96 and ‐91. Gel shift analysis indicated that the ‐96/‐91 sequence is a functional Sp1 site and that the ‐96C→T transition inhibits the binding of the trascription factor. These data indicate that the ‐96/‐91 sequence is a crucial Sp1 site whose integrity is necessary for COL7A1 expression. The compound heterozygosity for the ‐96C→T null mutation and for the R1978X mutation leads to the absence of COLVII at skin level and to the severe phenotype of the HS‐RDEB patient. Hum Mutat 16:275, 2000.

Phenotypic correction of the defective fibronectin extracellular matrix of Ehlers-Danlos syndrome fibroblasts

In vitro cultured skin fibroblasts derived from Ehlers-Danlos Syndrome (EDS) type I to VIII patients lack fibronectin-containing extracellular matrix (FN-ECM) which can be restored when EDS cells are cocultivated over a feeder of control fibroblasts. Further analysis, focused on EDS types III and IV cells, showed that partial matrix correction in EDS type III cells can be obtained by their cultivation over a feeder of EDS type IV fibroblasts, but not vice versa. An apparently normal FN-ECM can be restored in EDS types III and IV cells also by the addition of cellular--but not plasma--FN. These biological features might be used for a better understanding of ECM assembly and for the characterization of the different EDS cell types.