Beta 1 integrin activation mediates adhesive differences between trisomy 21 and non-trisomic fibroblasts on type VI collagen

Trisomy 21 (Down syndrome) is a common genetic condition with a high incidence of congenital heart defects (CHD), particularly those involving abnormal development of the embryonic atrioventricular (AV) canal. Type VI collagen (Col VI) is expressed in the developing AV canal extracellular matrix, and has been associated with trisomy 21 AV canal defects in human genetic studies. Although the molecular mechanisms linking Col VI and trisomy 21 AV canal defects are not well understood, a computer model predicts increased cell adhesiveness is responsible for these CHD. We compared integrin-mediated cell adhesive properties for skin fibroblasts isolated from trisomy 21 and non-trisomic individuals on Col VI, fibronectin (FN) and type I collagen (Col I). Cell lines demonstrate similar adhesion profiles to FN and Col I, but all trisomy 21 cells display increased adhesive capacity for Col VI compared to non-trisomic fibroblasts. Cell adhesion to type VI collagen was shown to be GRGDS independent, but beta(1) integrin family dependent. Function-blocking antibodies identified alpha(3)beta(1) as the predominant integrin mediating trisomy 21 and non-trisomic skin fibroblast adhesion on Col VI. Trisomy 21 and non-trisomic fibroblasts display similar expression levels for each of the integrin receptors examined. A beta(1) integrin-activating antibody augments non-trisomic cell adhesion on Col VI, but has no effect upon trisomy 21 fibroblasts. These results demonstrate that beta(1) integrin family members mediate trisomy 21 and non-trisomic skin fibroblast adhesion for Col VI. Altered activation state of the beta(1) integrin is a mechanism responsible for increased trisomy 21 cell adhesion on Col VI.     

Copper/zinc superoxide dismutase (SOD-1) activity in regular trisomy 21, trisomy 21 by translocation and mosaic trisomy 21

Cu/Zn superoxide dismutase (SOD-1) (E.C.1.15.1.1.) activity was estimated in children with regular trisomy 21-Down syndrome as well as in cases of translocation and mosaic trisomy 21, as identified by the GTG, CBG and RHG banding techniques. SOD-1 activity was found to be increased in all examined cases except trisomy 21 mosaicism. These findings provide further proof of the gene dosage theory and additional biochemical evidence for the triplicate existence of the SOD-1 gene localized on chromosome 21     

A child with complementary mosaic trisomy 8 and mosaic trisomy 21; clinical description of Warkany-Down syndrome and mechanism of origin

Aneuploidy mosaicism involving two complementary different autosomal trisomy cell lines is extremely rare. Although a mosaic double trisomy 8/trisomy 21 has been described in literature, this is the first report of Warkany (+8)–Down (+21) syndrome due to two complementary mosaic trisomy cell lines. The phenotype of the male patient with Warkany–Down syndrome includes upslanting palpebral fissures, hypertelorism, small low-set ears with unilateral aural stenosis, large and broad hands and feet with deep palmar and plantar creases, bilateral cryptorchidism, generalized mild hypotonia and transient neonatal thrombocytopenia. At the age of two years, his developmental quotient is around 50. His height, weight and head circumference are below the third centile. We speculate on the mechanism of origin of the complementary trisomy cell lines based on molecular cytogenetic studies that showed no evidence for a chimera.     

C-band polymorphism: Comparison between trisomy 21 cases and mentally retarded controls

C-banding was done prospectively on 50 Down syndrome (trisomy 21) cases and 50 mentally retarded controls. Heterochromatin was quantitated by measuring the lengths of heterochromatin blocks and comparing these segments to the length of the short arm of chromosome 16 for 1, 9 and 16 heterochromatin, and to the total length of the Y chromosome for the Y heterochromatin in the distal long arm. For the first 30 individuals in each group, there was no difference in the mean lengths of C-band blocks of the 1, 9, 16 and Y chromosomes. For the total sample, there also was no difference between the trisomy 21 cases and controls in the number or size of pericentric inversions involving the heterochromatin blocks of chromosomes 1 and 9. Assuming random segregation of the parental C-band polymorphisms, this study gives no evidence for an association between such polymorphisms of the 1, 9, 16 and Y chromosomes and nondisjunction of chromosome 21.     

Association between presenilin‐1 polymorphism and maternal meiosis II errors in Down syndrome

Several lines of evidence suggest a shared genetic susceptibility to Down syndrome (DS) and Alzheimer disease (AD). Rare forms of autosomal‐dominant AD are caused by mutations in the APP and presenilin genes (PS‐1 and PS‐2). The presenilin proteins have been localized to the nuclear membrane, kinetochores, and centrosomes, suggesting a function in chromosome segregation. A genetic association between a polymorphism in intron 8 of the PS‐1 gene and AD has been described in some series, and an increased risk of AD has been reported in mothers of DS probands. We therefore studied 168 probands with free trisomy 21 of known parental and meiotic origin and their parents from a population‐based material, by analyzing the intron 8 polymorphism in the PS‐1 gene. An increased frequency of allele 1 in mothers with a meiosis II error (70.8%) was found compared with mothers with a meiosis I error (52.7%, P < 0.01), with an excess of the 11 genotype in the meiosis II mothers. The frequency of allele 1 in mothers carrying apolipoprotein E (APOE) ϵ4 allele (68.0%) was higher than in mothers without ϵ4 (52.2%, P < 0.01). We hypothesize that the PS‐1 intronic polymorphism might be involved in chromosomal nondisjunction through an influence on the expression level of PS‐1 or due to linkage disequilibrium with biologically relevant polymorphisms in or outside the PS‐1 gene. Am. J. Med. Genet. 93:366–372, 2000. © 2000 Wiley‐Liss, Inc.     

Sphingosine‐1‐phosphate activates chemokine‐promoted myeloma cell adhesion and migration involving α4β1 integrin function

Myeloma cell adhesion dependent on α4β1 integrin is crucial for the progression of multiple myeloma (MM). The α4β1‐dependent myeloma cell adhesion is up‐regulated by the chemokine CXCL12, and pharmacological blockade of the CXCL12 receptor CXCR4 leads to defective myeloma cell homing to bone marrow (BM). Sphingosine‐1‐phosphate (S1P) regulates immune cell trafficking upon binding to G‐protein‐coupled receptors. Here we show that myeloma cells express S1P1, a receptor for S1P. We found that S1P up‐regulated the α4β1‐mediated myeloma cell adhesion and transendothelial migration stimulated by CXCL12. S1P promoted generation of high‐affinity α4β1 that efficiently bound the α4β1 ligand VCAM‐1, a finding that was associated with S1P‐triggered increase in talin‐β1 integrin association. Furthermore, S1P cooperated with CXCL12 for enhancement of α4β1‐dependent adhesion strengthening and spreading. CXCL12 and S1P activated the DOCK2‐Rac1 pathway, which was required for stimulation of myeloma cell adhesion involving α4β1. Moreover, in vivo analyses indicated that S1P contributes to optimizing the interactions of MM cells with the BM microvasculture and for their lodging inside the bone marrow. The regulation of α4β1‐dependent adhesion and migration of myeloma cells by CXCL12‐S1P combined activities might have important consequences for myeloma disease progression. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Mucopolysaccharidosis type VI: Structural and clinical implications of mutations in N‐acetylgalactosamine‐4‐sulfatase

Mucopolysaccharidosis type VI (MPS‐VI) is an autosomal recessive lysosomal storage disorder caused by the deficiency of N‐acetylgalactosamine‐4‐sulfatase (4S; or ARSB). Mutations in the 4S gene are responsible for 4S deficiency, which leads to the intralysosomal storage of partially degraded glycosaminoglycans, dermatan sulfate, and chondroitin 4‐sulfate. To date, a total of 45 clinically relevant mutations have been identified in the human 4S gene. Missense mutations are the largest group, with 31 identified mutations. Nonsense mutations and small insertions or deletions comprise the remainder, with seven mutations each. Six polymorphisms have also been reported: two amino acid substitutions and four silent transitions. Mapping of the missense mutations onto the 4S structure shows that they are distributed throughout the three subunits of the mature 4S polypeptide. Mutations have been identified in active site residues, in residues adjacent to the active site, in potential substrate binding residues, in residues exposed on the surface, and in residues buried within the protein core. Missense mutations have also been identified in disulfide crosslinks. Molecular modeling of MPS‐VI mutations onto the 4S structure suggests that the majority cause 4S deficiency via destabilization and the consequent reduction of 4S protein concentration. The vast majority of MPS‐VI mutant alleles are either unique to a patient or are present in a small number of patients. So far, no common mutations have been described. Therefore, screening of the general population for MPS‐VI alleles will be difficult. Hum Mutat 18:282–295, 2001. © 2001 Wiley‐Liss, Inc.     

Musculocontractural Ehlers‐Danlos Syndrome (former EDS type VIB) and adducted thumb clubfoot syndrome (ATCS) represent a single clinical entity caused by mutations in the dermatan‐4‐sulfotransferase 1 encoding CHST14 gene

We present clinical and molecular findings of three patients with an EDS VIB phenotype from two consanguineous families. The clinical findings of EDS kyphoscoliotic type (EDS type VIA and B) comprise kyphoscoliosis, muscular hypotonia, hyperextensible, thin and bruisable skin, atrophic scarring, joint hypermobility and variable ocular involvement. Distinct craniofacial abnormalities, joint contractures, wrinkled palms, and normal urinary pyridinoline ratios distinguish EDS VIB from EDS VIA. A genome‐wide SNP scan and sequence analyses identified a homozygous frameshift mutation (NM_130468.2:c.145delG, NP_569735.1:p.Val49*) in CHST14, encoding dermatan‐4‐sulfotransferase 1 (D4ST‐1), in two Turkish siblings. Subsequent sequence analysis of CHST14 identified a homozygous 20‐bp duplication (NM_130468.2:c.981_1000dup, NP_569735.1:p.Glu334Glyfs*107) in an Indian patient. Loss‐of‐function mutations in CHST14 were recently reported in adducted thumb–clubfoot syndrome (ATCS). Patients with ATCS present similar craniofacial and musculoskeletal features as the EDS VIB patients reported here, but lack the severe skin manifestations. By identifying an identical mutation in patients with EDS VIB and ATCS, we show that both conditions form a phenotypic continuum. Our findings confirm that the EDS‐variant associated with CHST14 mutations forms a clinical spectrum, which we propose to coin as “musculocontractural EDS” and which results from a defect in dermatan sulfate biosynthesis, perturbing collagen assembly. Hum Mutat 31:1–7, 2010. © 2010 Wiley‐Liss, Inc.     

Ultrastructural triple localization of laminin‐1, nidogen‐1, and collagen type IV helps elucidate basement membrane structure in vivo

The basement membrane models which have been proposed to date are generally based on biochemical data, mainly binding studies and artificially synthesized polymers in vitro. Basically these have led to models proposing two three‐dimensional laminin‐1 and collagen type IV networks interconnected by nidogen‐1. Whether they reflect the in vivo basement membrane structure is still not clear. We localized laminin‐1, nidogen‐1, and collagen type IV ultrastructurally in adult and fetal mouse kidney basement membranes with the help of immunogold‐histochemistry performing double and triple localization to try to elucidate the molecular organization of basement membranes in vivo. We found laminin‐1, nidogen‐1, and collagen type IV distributed over the entire basement membranes in adult and fetal kidneys. This contradicts earlier studies ascribing laminin‐1 to the lamina lucida and collagen type IV to the lamina densa. In addition, various basement membrane segments exhibited an organized labeling pattern for the BM components. Double‐labeling revealed co‐localization of laminin‐1 and nidogen‐1. We conclude that the combination of laminin‐1 with collagen type IV as double‐network basement membrane partially interconnected by nidogen‐1 is found already in the early fetal kidney in vivo. However, our data cannot exclude the possibility of other variants of basement membrane assemblages. This is also indicated by a changing structure even in individual segments of one basement membrane type which renders a more flexible basement membrane architecture plausible. Anat Rec 254:382–388, 1999. © 1999 Wiley‐Liss, Inc.     

Carbon monoxide‐treated dendritic cells decrease β1‐integrin induction on CD8+ T cells and protect from type 1 diabetes

Carbon monoxide (CO) treatment improves pathogenic outcome of autoimmune diseases by promoting tolerance. However, the mechanism behind this protective tolerance is not yet defined. Here, we show in a transgenic mouse model for autoimmune diabetes that ex vivo gaseous CO (gCO)‐treated DCs loaded with pancreatic β‐cell peptides protect mice from disease. This protection is peptide‐restricted, independent of IL‐10 secretion by DCs and of CD4⁺ T cells. Although no differences were observed in autoreactive CD8⁺ T‐cell function from gCO‐treated versus untreated DC‐immunized groups, gCO‐treated DCs strongly inhibited accumulation of autoreactive CD8⁺ T cells in the pancreas. Interestingly, induction of β1‐integrin was curtailed when CD8⁺ T cells were primed with gCO‐treated DCs, and the capacity of these CD8⁺ T cells to lyse isolated islet was dramatically impaired. Thus, immunotherapy using CO‐treated DCs appears to be an original strategy to control autoimmune disease.     

Toll‐like receptor 3 stimulation promotes Ro52/TRIM21 synthesis and nuclear redistribution in salivary gland epithelial cells,partially via type I interferon pathway

Up‐regulated expression of Ro52/tripartite motif‐containing protein 21 (TRIM21), Ro60/TROVE domain family, member 2 (TROVE2) and lupus LA protein/Sjögren's syndrome antigen B (La/SSB) autoantigens has been described in the salivary gland epithelial cells (SGEC) of patients with Sjögren's syndrome (SS). SGECs, the key regulators of autoimmune SS responses, express high levels of surface functional Toll‐like receptor (TLR)‐3, whereas Ro52/TRIM21 negatively regulates TLR‐3‐mediated inflammation. Herein, we investigated the effect of TLR‐3‐signalling on the expression of Ro52/TRIM21, as well as Ro60/TROVE2 and La/SSB autoantigens, by SGECs. The effect of TLR‐3 or TLR‐4 stimulation on autoantigen expression was evaluated by polyI:C or lipopolysaccharide (LPS) treatment, respectively, of SGEC lines (10 from SS patients, 12 from non‐SS controls) or HeLa cells, followed by analysis of mRNA and protein expression. PolyI:C, but not LPS, resulted in a two‐step induction of Ro52/TRIM21 mRNA expression by SGECs, a 12‐fold increment at 6 h followed by a 2·5‐fold increment at 24–48 h, whereas it induced a late two‐fold up‐regulation of Ro60/TROVE2 and La/SSB mRNAs at 48 h. Although protein expression levels were not affected significantly, the late up‐regulation of Ro52/TRIM21 mRNA was accompanied by protein redistribution, from nucleolar‐like pattern to multiple coarse dots spanning throughout the nucleus. These late phenomena were mediated significantly by interferon (IFN)‐β production, as attested by cognate secretion and specific inhibition experiments and associated with IFN regulatory factor (IRF)3 degradation. TLR‐3‐signalling had similar effects on SGECs obtained from SS patients and controls, whereas it did not affect the expression of these autoantigens in HeLa cells. TLR‐3 signalling regulates the expression of autoantigens by SGECs, implicating innate immunity pathways in their over‐expression in inflamed tissues and possibly in their exposure to the immune system.     

The PTPN22 1858T allele but not variants in the proximal promoter region of IL‐21 gene is associated with the susceptibility to type 1 diabetes and the presence of autoantibodies in a Brazilian cohort

Interleukin (IL)-21 and protein tyrosine phosphatase non-receptor 22 (PTPN22) regulate lymphocyte function and have been implicated in the pathogenesis of autoimmune diabetes. We sequenced the proximal promoter of the IL-21 gene for the first time and analysed the PTPN22 1858T polymorphism in type 1A diabetes (T1AD) patients and healthy controls (HC). We correlated the frequencies of islet and extra-pancreatic autoantibodies with genotypes from both loci. The case series comprised 612 T1AD patients and 792 HC. Genotyping of PTPN22 C1858T was performed on 434 T1AD patients and 689 HC. The −448 to +83 base pairs (bp) region of the IL-21 gene was sequenced in 309 Brazilian T1AD and 189 HC subjects. We also evaluated human leucocyte antigen (HLA) DR3/DR4 alleles. The frequencies of glutamic acid decarboxylase (GAD65), tyrosine phosphatase-like protein (IA)-2, anti-nuclear antibody (ANA), thyroid peroxidase (TPO), thyroglobulin (TG), thyrotrophin receptor autoantibody (TRAb), anti-smooth muscle (ASM) and 21-hydroxylase (21-OH) autoantibodies were higher in T1AD patients than in HC. The PTPN22 1858T allele was associated with an increased risk for developing T1AD [odds ratio (OR) = 1·94; P < 0·001], particularly in patients of European ancestry, and with a higher frequency of GAD65 and TG autoantibodies. HLA-DR3/DR4 alleles predominated in T1AD patients. A heterozygous allelic IL-21 gene variant (g.-241 T > A) was found in only one patient. In conclusion, only PTPN22 C1858T polymorphism and HLA-DR3 and/or DR4 alleles, but not allelic variants in the 5′-proximal region of the IL-21 gene were associated with T1AD risk. Patients with T1AD had increased frequencies of anti-islet-cell, anti-thyroid, anti-nuclear, anti-smooth muscle and anti-21-OH autoantibodies. The C1858T PTPN22 polymorphism was also associated with a higher frequency of GAD65 and TG autoantibodies.