T cell-inflamed gene expression profile is associated with favorable disease-specific survival in non-hypermutated microsatellite-stable colorectal cancer patients.

BACKGROUND: The anti-tumor immune response plays a key role in colorectal cancer (CRC) progression and survival. The T cell-inflamed gene expression profile (GEP) is a biomarker predicting response to checkpoint inhibitor immunotherapy across immunogenic cancer types, but the prognostic value in CRC is unknown. We evaluated associations with disease-specific survival, somatic mutations, and examined its differentially expressed genes and pathways among 84 sporadic CRC patients from the Seattle Colon Cancer Family Registry. METHODS: Gene expression profiling was performed using Nanostring's nCounter PanCancer IO 360 panel. Somatic mutations were identified by a targeted DNA sequencing panel. RESULTS: The T cell-inflamed GEP was positively associated with tumor mutation burden and microsatellite instability high (MSI-H). Higher T cell-inflamed GEP had favorable CRC-specific survival (hazard ratio [HR] per standard deviation unit = 0.50, p = 0.004) regardless of hypermutation or MSI status. Analysis of recurrently mutated genes having at least 10 mutation carriers, suggested that the T cell-inflamed GEP is positively associated with RYR1, and negatively associated with APC. However, these associations were attenuated after adjusting for hypermutation or MSI status. We also found that expression of genes RPL23, EPCAM, AREG and ITGA6, and the Wnt signaling pathway was negatively associated with the T cell-inflamed GEP, which might indicate immune-inhibitory mechanisms. CONCLUSIONS: Our results show that the T cell-inflamed GEP is a prognostic biomarker in non-hypermutated microsatellite-stable CRC. This also suggests that patient stratification for immunotherapy within this CRC subgroup should be explored further. Moreover, reported immune-inhibitory gene expression signals may suggest targets for therapeutic combination with immunotherapy.

Ultrastructure abnormalities of collagen and elastin in Arab patients with arterial tortuosity syndrome.

Arterial tortuosity syndrome (ATS) is a rare autosomal recessive disease characterized by elongation and tortuosity of the large- and medium-sized arteries. ATS patients display features that are also found in Ehlers-Danlos syndrome (EDS) patients. ATS is caused by pathogenic mutations in the SLC2A10 gene, which encodes for the glucose transporter, GLUT10. This study aimed at examining the ultrastructure of skin for abnormalities that can explain the loose skin and arterial phenotypes of Arab patients with the p.S81R mutation in SLC2A10. Forty-eight patients with SLC2A10 mutation were recruited for this study. Skin biopsy specimens from three children with ATS and a healthy child were examined by electron microscopy to determine the ultrastructure of collagen and elastin. Histopathologic staining of sections from tissue biopsy specimens was also performed. Large spaces were observed among the collagen fibrils in the skin biopsy specimens obtained from ATS patients, suggesting disorganization of the collagen structures. Furthermore, elastin fiber contents and their thickness are reduced in the skin. In small muscular arteries in the skin from ATS patients, discontinuous internal elastic lamina, lack of myofilaments, and disorganized medial smooth muscle cells with vacuolated cytoplasm are present. The disorganization of collagen fibrils and reduced elastin contents in the skin may explain the loose skin phenotype of ATS patients similar to the EDS patients. The lack of elastin in small muscular arteries may have contributed to the development of arterial tortuosity in these patients.

Novel and recurrent germline mutations in the VHL gene in 5 Arab patients with Von Hippel-Lindau disease.

Inherited germline mutations in the VHL gene cause predisposition to Von Hippel-Lindau (VHL) disease. Patients exhibit benign and cancerous lesions in multiple tissues, including hemangioblastomas, clear cell renal cell carcinoma, cysts in kidneys and pancreas, and pheochromocytomas. Although pathogenic germline mutations in the VHL gene have been widely described in different populations, only a single mutation was previously reported in a family from mixed Arab-Persian ethnicity. Here, we present five Arab patients with two new and two recurrent germline mutations in the VHL gene. These mutations include three in-frame deletions and a missense mutation. Infrequent in-frame deletions in previously described patients from other populations, as well as the presence of new mutations, suggests a distinct spectrum of VHL gene mutations in Arab patients. While pulmonary manifestation has been described rarely in VHL disease, we have identified two patients with a recurrent p.Phe76del in-frame deletion exhibiting multiple nodules in lungs. We also describe a first-ever in-frame deletion in the VHL gene in a patient with VHL type 2C disease, exhibiting bilateral pheochromocytoma. Overall, the study provides an insight into the genotype-phenotype relationship of VHL disease in Arab patients and provides a comparison with previously described patients from other ethnicities.

Hereditary 1,25-dihydroxyvitamin D-resistant rickets (HVDRR): clinical heterogeneity and long-term efficacious management of eight patients from four unrelated Arab families with a loss of function VDR mutation.

BACKGROUND: Vitamin D regulates the concentrations of calcium and phosphate in blood and promotes the growth and remodeling of bones. The circulating active form of vitamin D, 1,25-dihydroxyvitamin D, binds to the vitamin D receptor (VDR), which heterodimerizes with the retinoid X receptor to regulate the expression of target genes. Inactivating mutations in the VDR gene cause hereditary vitamin D-resistant rickets (HVDRR), a rare disorder characterized by an early onset of rickets, growth retardation, skeletal deformities, hypocalcemia, hypophosphatemia and secondary hyperparathyroidism, and in some cases alopecia. METHODS: We describe eight new HVDRR patients from four unrelated consanguineous families. The VDR gene was sequenced to identify mutations. The management of patients over a period of up to 11 years following the initial diagnosis is assessed. RESULTS: Although all patients exhibit main features of HVDRR and carry the same c.885C>A (p.Y295*) loss of function mutation in the VDR gene, there was heterogeneity of the manifestations of HVDRR-associated phenotypes and developmental milestones. These eight patients were successfully treated over a period of 11 years. All clinical symptoms were improved except alopecia. CONCLUSIONS: The study concludes that VDR sequencing and laboratory tests are essential to confirm HVDRR and to assess the effectiveness of the treatment.

A genome-wide association study for colorectal cancer identifies a risk locus in 14q23.1.

Over 50 loci associated with colorectal cancer (CRC) have been uncovered by genome-wide association studies (GWAS). Identifying additional loci has the potential to help elucidate aspects of the underlying biological processes leading to better understanding of the pathogenesis of the disease. We re-evaluated a GWAS by excluding controls that have family history of CRC or personal history of colorectal polyps, as we hypothesized that their inclusion reduces power to detect associations. This is supported empirically and through simulations. Two-phase GWAS analysis was performed in a total of 16,517 cases and 14,487 controls. We identified rs17094983, a SNP associated with risk of CRC [p = 2.5 × 10(-10); odds ratio estimated by re-including all controls (OR) = 0.87, 95% confidence interval (CI) 0.83-0.91; minor allele frequency (MAF) = 13%]. Results were replicated in samples of African descent (1894 cases and 4703 controls; p = 0.01; OR = 0.86, 95% CI 0.77-0.97; MAF = 16 %). Gene expression data in 195 colon adenocarcinomas and 59 normal colon tissues from two different studies revealed that this locus has genotypes that are associated with RTN1 (Reticulon 1) expression (p = 0.001), a protein-coding gene involved in survival and proliferation of cancer cells which is highly expressed in normal colon tissues but has significantly reduced expression in tumor cells (p = 1.3 × 10(-8)).

The effect of 5-fluorouracil/leucovorin chemotherapy on CpG methylation, or the confounding role of leukocyte heterogeneity: An illustration.

Blood-based epigenome-wide association studies that aim at comparing CpG methylation between colorectal cancer (CRC) patients and controls can lead to the discovery of diagnostic or prognostic biomarkers. Numerous confounders can lead to spurious associations. We aimed to see if 5-fluorouracil (5-FU)/leucovorin chemotherapy administered to cases prior to the collection of their blood has an effect on methylation. 304 patients who received treatment and 273 who did not were profiled on the HumanMethylation450 array. Association tests were adjusted for confounders, including proxies for leukocyte cell counts. There were substantial methylation differences between these two groups that vanished once the leukocyte heterogeneity was accounted for. We observed a significant decrease of T cells in the treatment group (CD4+: p=10(-6); CD8+: p=0.036) and significant increase of NK cells (p=0.05) and monocytes (p=0.0006). 5-FU/leucovorin has no effect on global and local blood-based methylation profiles, other than through differences in the leukocyte compositions that the treatment induced.

Long-range epigenetic regulation is conferred by genetic variation located at thousands of independent loci.

The interplay between genetic and epigenetic variation is only partially understood. One form of epigenetic variation is methylation at CpG sites, which can be measured as methylation quantitative trait loci (meQTL). Here we report that in a panel of lymphocytes from 1,748 individuals, methylation levels at 1,919 CpG sites are correlated with at least one distal (trans) single-nucleotide polymorphism (SNP) (P<3.2 × 10(-13); FDR<5%). These trans-meQTLs include 1,657 SNP-CpG pairs from different chromosomes and 262 pairs from the same chromosome that are >1 Mb apart. Over 90% of these pairs are replicated (FDR<5%) in at least one of two independent data sets. Genomic loci harbouring trans-meQTLs are significantly enriched (P<0.001) for long non-coding transcripts (2.2-fold), known epigenetic regulators (2.3-fold), piwi-interacting RNA clusters (3.6-fold) and curated transcription factors (4.1-fold), including zinc-finger proteins (8.75-fold). Long-range epigenetic networks uncovered by this approach may be relevant to normal and disease states.

Clinical, neuroimaging, and genetic features of L-2-hydroxyglutaric aciduria in Arab kindreds.

BACKGROUND AND OBJECTIVES: L-2-hydroxyglutaric aciduria is a neurometabolic disorder with autosomal recessive mode of inheritance in which patients exhibit elevated L-2-hydroxyglutaric acid in body fluids, central nervous system manifestations, and increased risk of brain tumor formation. Mutations in L2HGDH gene have been described in L-2-hydroxyglutaric aciduria patients of different ethnicities. The present study was conducted to perform a detailed clinical, imaging and genetic analysis. DESIGN AND SETTINGS: A cross-sectional clinical genetic study of 16 L-2-hydroxyglutaric aciduria patients from 4 Arab consanguineous families examined at the metabolic clinic of the hospital. PATIENTS AND METHODS: Genomic DNA was isolated from the blood of 12 patients and 10 unaffected family members, and the L2HGDH gene was sequenced. DNA sequences were compared to the L2HGDH reference sequence from GenBank. RESULTS: All patients exhibit characteristic clinical, biochemical, and imaging features of L-2-hydroxyglutaric aciduria, and 4 patients exhibited increased incidence of brain tumors. The sequencing of the L2HGDH gene revealed the c.1015delA, c.1319C > A, and c.169G > A mutations in these patients. These mutations encode for the p.Arg339AspfsX351, p.Ser440Tyr, and p.Gly57Arg changes in the L2HGDH protein, respectively. The c.169G > A mutation, which was shown to have a common origin in Italian and Portuguese patients, was also discovered in Arab patients. Finding of the homozygous c.159T SNP associated with the c.169G > A mutation in Arab patients points to an independent origin of this mutation in Arab population. CONCLUSION: The detailed description of clinical manifestations and L2HGDH mutation in this study is useful for diagnosis of L-2-hydroxyglutaric aciduria in Arab patients. While reoccurrence of an L2HGDH mutation in L-2-hydroxyglutaric aciduria patients of different ethnicity is extremely rare, the c.169G mutation has an independent origin in Arab patients. It is likely that this mutation may also be present in patients of other ethnicities.

Identification of genes expressed by immune cells of the colon that are regulated by colorectal cancer-associated variants.

A locus on human chromosome 11q23 tagged by marker rs3802842 was associated with colorectal cancer (CRC) in a genome-wide association study; this finding has been replicated in case-control studies worldwide. In order to identify biologic factors at this locus that are related to the etiopathology of CRC, we used microarray-based target selection methods, coupled to next-generation sequencing, to study 103 kb at the 11q23 locus. We genotyped 369 putative variants from 1,030 patients with CRC (cases) and 1,061 individuals without CRC (controls) from the Ontario Familial Colorectal Cancer Registry. Two previously uncharacterized genes, COLCA1 and COLCA2, were found to be co-regulated genes that are transcribed from opposite strands. Expression levels of COLCA1 and COLCA2 transcripts correlate with rs3802842 genotypes. In colon tissues, COLCA1 co-localizes with crystalloid granules of eosinophils and granular organelles of mast cells, neutrophils, macrophages, dendritic cells and differentiated myeloid-derived cell lines. COLCA2 is present in the cytoplasm of normal epithelial, immune and other cell lineages, as well as tumor cells. Tissue microarray analysis demonstrates the association of rs3802842 with lymphocyte density in the lamina propria (p = 0.014) and levels of COLCA1 in the lamina propria (p = 0.00016) and COLCA2 (tumor cells, p = 0.0041 and lamina propria, p = 6 × 10(-5)). In conclusion, genetic, expression and immunohistochemical data implicate COLCA1 and COLCA2 in the pathogenesis of colon cancer. Histologic analyses indicate the involvement of immune pathways.

Growth differentiation factor 5 regulates cardiac repair after myocardial infarction.

OBJECTIVES: The aim of this study was to examine the function of the bone morphogenic protein growth differentiation factor 5 (Gdf5) in a mouse model of myocardial infarction (MI). BACKGROUND: The Gdf5 has been implicated in skeletal development, but a potential role in the heart had not been studied. METHODS: The Gdf5-knockout (KO) and wild-type (WT) mice were subjected to permanent left anterior descending coronary artery (LAD) ligation. Cardiac pathology, function, gene expression levels, and signaling pathways downstream of Gdf5 were examined. Effects of recombinant Gdf5 (rGdf5) were tested in primary cardiac cell cultures. RESULTS: The WT mice showed increased cardiac Gdf5 levels after MI, with increased expression in peri-infarct cardiomyocytes and myofibroblasts. At 1 and 7 days after MI, no differences were observed in ischemic or infarct areas between WT and Gdf5-KO mice. However, by 28 days after MI, Gdf5-KO mice exhibited increased infarct scar expansion and thinning with decreased arteriolar density compared with WT. The Gdf5-KO hearts also displayed increased left ventricular dilation, with decreased contractility after MI. At 4 days after MI, Gdf5-KO mice exhibited increased cardiomyocyte apoptosis and decreased expression of anti-apoptotic genes Bcl2 and Bcl-xL compared with WT. Unexpectedly, Gdf5-KO hearts displayed increased Smad 1/5/8 phosphorylation but decreased p38-mitogen-activated protein kinase (MAPK) phosphorylation versus WT. The latter was associated with increased collagen gene (Col1a1, Col3a1) expression and fibrosis. In cultures, rGdf5 induced p38-MAPK phosphorylation in cardiac fibroblasts and Smad-dependent increases in Bcl2 and Bcl-xL in cardiomyocytes. CONCLUSIONS: Increased expression of Gdf5 after MI limits infarct scar expansion in vivo. These effects might be mediated by Gdf5-induced p38-MAPK signaling in fibroblasts and Gdf5-driven Smad-dependent pro-survival signaling in cardiomyocytes.

A novel HAX1 gene mutation in severe congenital neutropenia (SCN) associated with neurological manifestations.

Autosomal recessive severe congenital neutropenia (SCN) results from a maturation arrest of granulopoiesis at the level of promyelocytes and apoptosis of myeloid cells. In SCN patients, mutations have been described in the HAX1 gene. Most of the SCN patients who carry nonsense mutations that are common to both transcript variants of the HAX1 gene also exhibit neurological deficits. This study describes an SCN patient with neurological manifestations including daily episodes of atonic seizures, learning disabilities, and developmental delay. Sequencing of the HAX1 gene of this SCN patient identified a novel nonsense c.463_464insC homozygous mutation in exon 3, which is common to both transcript variants of the gene. This mutation encodes for a p.Gln155ProfsX14 change and causes premature truncation of the HAX1 protein. Neutrophils isolated from the patient exhibited spontaneous apoptosis and loss of inner mitochondrial membrane potential, which were further enhanced upon treatment with hydrogen peroxide. This study adds to the spectrum of novel HAX1 gene mutations and disease manifestations in ethnically distinct SCN patients. Our report describes the only nonsense mutation in the HAX1 gene present in SCN patients of Arab origin.

A common polymorphism in the cannabinoid receptor 1 (CNR1) gene is associated with antipsychotic-induced weight gain in Schizophrenia.

Antipsychotic-induced weight gain has emerged as a serious complication in the treatment of patients with atypical antipsychotic drugs. The cannabinoid receptor 1 (CNR1) is expressed centrally in the hypothalamic region and associated with appetite and satiety, as well as peripherally. An antagonist of CNR1 (rimonabant) has been effective in causing weight loss in obese patients indicating that CNR1 might be important in antipsychotic-induced weight gain. Twenty tag SNPs were analyzed in 183 patients who underwent treatment (with either clozapine, olanzapine, haloperidol, or risperidone) for chronic schizophrenia were evaluated for antipsychotic-induced weight gain for up to 14 weeks. The polymorphism rs806378 was nominally associated with weight gain in patients of European ancestry treated with clozapine or olanzapine. 'T' allele carriers (CT+TT) gained more weight (5.96%), than the CC carriers (2.76%, p=0.008, FDR q-value=0.12). This translated into approximately 2.2 kg more weight gain in patients carrying the T allele than the patients homozygous for the CC genotype (CC vs CT+TT, 2.21+/-4.51 vs 4.33+/-3.89 kg; p=0.022). This was reflected in the allelic analysis (C vs T allele, 3.84 vs 5.83%, p=0.035). We conducted electrophoretic mobility shift assays which showed that the presence of the T allele created a binding site for arylhydrocarbon receptor translocator (ARNT), a member of the basic helix-loop-helix/Per-Arnt-Sim protein family. In this study, we provide evidence that the CNR1 gene may be associated with antipsychotic-induced weight gain in chronic schizophrenia patients. However, these observations were made in a relatively small patient population; therefore these results need to be replicated in larger sample sets.

Novel and recurrent mutations in the C1NH gene of Arab patients affected with hereditary angioedema.

BACKGROUND: Autosomal dominant hereditary angioedema (HAE) results in episodes of subcutaneous edema in any body part and/or submucosal edema of the upper respiratory or gastrointestinal tracts. This disorder is caused by mutations in the C1NH gene, many of which have been described primarily in European patients. However, the genetic cause of HAE in Middle Eastern Arab patients has not yet been determined. METHODS: Four unrelated Arab families, in which 15 patients were diagnosed with HAE, were studied. DNA from 13 patients was analyzed for mutations in the C1NH gene by DNA sequencing. RESULTS: Three novel and 2 recurrent mutations were identified in the C1NH gene of HAE patients. In family 1, the patient was heterozygous for a novel c.856C>T and a recurrent c.1361T>A missense mutation encoding for p.Arg264Cys and p.Val432Glu, respectively. In patients from family 2, a novel c.509C>T missense mutation encoding for a p.Ser148Phe was identified. In patients from family 3, a novel c.1142delC nonsense mutation encoding for a p.Ala359AlafsX15 was discovered. In family 4, a recurrent c.1397G>A missense mutation encoding for a p.Arg444His was present. CONCLUSION: This is the first ever report of C1NH gene mutations in Middle Eastern Arab patients. Our study suggests that, despite the numerous existing mutations in the C1NH gene, there are novel and recurrent mutations in HAE patients of non-European origin. We conclude that the spectrum of C1NH gene mutations in HAE patients is wider due to the likely presence of novel and recurrent mutations in patients of other ethnicities.

Novel FBP1 gene mutations in Arab patients with fructose-1,6-bisphosphatase deficiency.

UNLABELLED: Deficiency of fructose-1,6-bisphosphatase (FBP) results in impaired gluconeogenesis, which is characterized by episodes of hyperventilation, apnea, hypoglycemia, and metabolic and lactic acidosis. This autosomal recessive disorder is caused by mutations in the FBP1 gene, which encodes for fructose-1,6-bisphosphatase 1 (FBP1). Although FBP1 gene mutations have been described in FBP-deficient individuals of various ethnicities, there has been limited investigation into the genetics of this disorder in Arab patients. This study employed five consanguineous Arab families, in which 17 patients were clinically diagnosed with FBP deficiency. Seven patients and six carrier parents were analyzed for mutations in the FBP1 gene. DNA sequencing of the FBP1 gene identified two novel mutations in these families. A novel six nucleotide repetitive insertion, c114_119dupCTGCAC, was identified in patients from three families. This mutation encodes for a duplication of two amino acids (p.Cys39_Thr40dup) in the N-terminal domain of FBP1. A novel nonsense c.841G>T mutation encoding for a p.Glu281X truncation in the active site of FBP1 was discovered in patients from two families. The newly identified mutations in the FBP1 gene are predicted to produce FBP1 deficiency. These mutations are the only known genetic causes of FBP deficiency in Arab patients. The p.Cys39_Thr40dup is the first reported amino acid duplication in FBP deficiency patients. CONCLUSION: This study provides a strong rationale for genetic testing of FBP deficient patients of Arab ethnicity for recurrent or novel mutations in the FBP1 gene.

A novel missense and a recurrent mutation in SLC2A10 gene of patients affected with arterial tortuosity syndrome.

Arterial tortuosity syndrome is an autosomal recessive disorder characterized by severe tortuosity of greater and systemic arteries in affected individuals. In addition, patients display connective tissue features which include hyperextensible skin, hypermobility of joints and characteristic facial features. This syndrome is caused by mutation in SLC2A10 gene which encodes for the facilitative glucose transporter, GLUT10. We describe seven patients of two unrelated Saudi Arabian families who display tortuosity, dilatation and stenosis of arteries, pulmonary hypertension and other cardiovascular manifestations. These patients exhibit characteristic connective tissue phenotypes and distinctive facial features. In the single patient of Family 1, sequencing of the candidate gene, SLC2A10, identified a novel missense c.313C>T mutation encoding a p.Arg105Cys substitution in the second extracellular domain of GLUT10. The Arg105 in GLUT10 is highly conserved across species and its replacement with cysteine is predicted to be pathogenic. In the second family, all of the six affected individuals carry recurrent c.243C>G missense mutation encoding a p.Ser81Arg change in the third transmembrane domain of GLUT10. The present study suggests that there exists an intra- and inter-familial phenotypic variability in arterial tortuosity patients carrying identical or different mutations in SLC2A10 gene. While skin hyperextensibility, small joint hypermobility, and facial features are similarly expressed in these patients, there is a range of other phenotypes which include arterial tortuosity and associated complications, and abnormalities of other organs.

A novel non-sense mutation in the SLC2A10 gene of an arterial tortuosity syndrome patient of Kurdish origin.

Arterial tortuosity syndrome (ATS) is a rare autosomal recessive disorder in which patients display tortuosity of arteries in addition to hyperextensible skin, joint laxity, and other connective tissue features. This syndrome is caused by mutations in the SLC2A10 gene. In this article we describe an ATS girl of Kurdish origin who, in addition to arterial tortuosity and connective tissue features, displays stomach displacement within the thorax and bilateral hip dislocation. Clinical details of this patient have been reported previously. Sequencing of the SLC2A10 gene identified a novel homozygous non-sense c.756C>A mutation in this patient's DNA. This mutation in the SLC2A10 gene replaces a cysteine encoding codon with a stop signal. This is believed to cause a premature truncation of GLUT10 protein in this patient. We conclude that patients of Kurdish origin who display arterial tortuosity associated with skin hyperextensibility, joint hypermobility, and characteristic facial features may carry mutations in the SLC2A10 gene.

Grebe-type chondrodysplasia: a novel missense mutation in a conserved cysteine of the growth differentiation factor 5.

Grebe-type chondrodysplasia is a congenital skeletal disorder that is characterized by markedly shortened limbs and very short digits. This defect has an autosomal recessive mode of inheritance and results from mutations in the growth differentiation factor 5 (GDF5) gene. Here, we report three affected children in a consanguineous family who display typical features of Grebe-type chondrodysplasia. Sequencing of the GDF5 genes of the affected children identified a novel c.1285T>C mutation encoding a p.Cys429Arg substitution. The Cys429 of human GDF5 belongs to a group of seven cysteines, which are highly conserved across species and among the various members of the transforming factor-beta (TGF-beta) super family of proteins. These cysteines are essential for the structure, processing, and activity of these proteins. Therefore, it is possible that the p.Cys429Arg change in the GDF5 has produced an inactive protein, resulting in a Grebe-type chondrodysplasia phenotype in the affected children. The absence of skeletal abnormalities in the carrier parents suggests that the p.Cys429Arg change did not produce a dominant negative effect or haploinsufficiency in these individuals. This finding differs from the previous report of skeletal abnormalities in heterozygous individuals of Grebe-type chondrodysplasia families.

Defective glycosylation of decorin and biglycan, altered collagen structure, and abnormal phenotype of the skin fibroblasts of an Ehlers-Danlos syndrome patient carrying the novel Arg270Cys substitution in galactosyltransferase I (beta4GalT-7).

The Ehlers-Danlos syndrome (EDS) is a heterogeneous group of connective tissue disorders affecting skin and joint function. Molecular defects in extracellular matrix proteins, including collagen (type I, III, and V) and tenascin X are associated with different forms of EDS. Compound heterozygous mutations in the B4GALT7 gene, resulting in aberrant glycosylation of the dermatan sulfate proteoglycan decorin, had been described in a single patient affected with the progeroid form of EDS. We have studied the molecular phenotype of decorin, biglycan, and collagen type I containing fibrils in skin fibroblasts of a patient carrying the novel homozygous C808T point mutation in the B4GALT7 gene, which causes an Arg270Cys substitution in beta4GalT-7. Compared to control fibroblasts, galactosyltransferase activity in beta4GalT-7(Arg270Cys) cells was approximately three times reduced over a temperature range of 25-41 degrees C. Pulse-chase experiments and confocal microscopy demonstrated that synthesis and secretion of decorin were normal in beta4GalT-7(Arg270Cys) cells. However, about 50% of decorin were synthesized as a protein core in addition to its proteoglycan form. Biglycan was found in a monoglycanated form in addition to its mature form. Glycosaminoglycan chains were of the dermatan/chondroitin sulfate type both in beta4GalT-7(Arg270Cys) and control cells, and epimerization was reduced for decorin and biglycan. Compared to control cells, beta4GalT-7(Arg270Cys) cells showed altered, highly spread or stretched phenotypes and decreased proliferation rates. At the ultrastructural level, an intracellular accumulation of multiple secondary lysosomes and degenerative vacuoles was seen in beta4GalT-7(Arg270Cys) cells. Furthermore, the collagen suprastructures were altered in the beta4GalT-7(Arg270Cys) cells. The reduced beta4GalT-7 activity resulting in defective glycosylation of decorin and biglycan may be responsible for the complex molecular pathology in beta4GalT-7 deficient EDS patients, given the role of these proteoglycans in bone formation, collagen fibrillogenesis, and skeletal muscle development.

Gene transfer of prostaglandin synthase maintains patency of the newborn lamb arterial duct.

In congenital heart disease with left- or right-sided obstruction, prostaglandin E (PGE)1 or PGE2 is infused to maintain ductus arteriosus (DA) patency. We hypothesized that transfection of the DA with PGE synthase would lead to a greater production of PGE2 in situ and, hence, patency of the DA. The cDNA for human prostaglandin synthase was sequenced and ligated into a eukaryotic expression vector. The negative control was created by ligating the cDNA encoding the bacterial protein chloramphenicol acetyltransferase into the same plasmid. Transfection (600 microg DNA) was achieved in lambs within the first 24 h of life using the hemagglutinating virus of Japan (HVJ)-liposome transfection method with a custom-made, basket-weave-perforated catheter. Echocardiography was performed to assess DA patency until the time of sacrifice. To confirm expression of the transgene, PGE2 concentration was measured in organ culture of the DA by immunoassay and by Western immunoblotting of homogenized DA tissue. Patency of the DA was demonstrated by color Doppler in all the lambs (7/7) in which the PGE synthase was delivered, whereas functional closure was seen in the control group (6/6). The PGE2 concentration in the culture medium of the explanted DA in the treatment group was 3-fold higher than that of the control groups. Western immunoblotting confirmed the presence of PGE synthase in the treatment group. Gene transfer of PGE synthase to the DA is feasible and will maintain patency for at least 1 wk.

Elafin-overexpressing mice have improved cardiac function after myocardial infarction.

Elevated serine elastase activity after myocardial infarction can contribute to remodeling associated with left ventricular dilatation and dysfunction. We therefore assessed the effects of overexpressing the selective serine elastase inhibitor elafin in transgenic mice in which a myocardial infarction was caused by ligation of the left anterior descending coronary artery (LAD). Elevated serine elastase activity was observed in nontransgenic littermates as early as 6 h after LAD ligation and persisted at 4 and 7 days but not in sham-operated or elafin-overexpressing transgenic mice. Myeloperoxidase activity (index of inflammatory cells) and matrix metalloproteinase 2 were also increased but only at 4 and 7 days and only in nontransgenic mice (P < 0.05 for both comparisons), and this increase correlated with inflammatory cell infiltration. Echocardiographic study at 4 days revealed indexes of diastolic dysfunction in nontransgenic versus elafin-overexpressing mice (P < 0.05). Morphometric and biochemical analyses at 28 days indicated impairment in cardiac performance, with greater scar thinning and infarct expansion in nontransgenic versus elafin transgenic littermates (P < 0.05 for all comparisons). Thus serine elastase inhibition appears to suppress inflammation, cardiac dilatation, and dysfunction after myocardial infarct.