Laura Lucarini

Dominant and recessive COL6A1 mutations in Ullrich scleroatonic muscular dystrophy.

In this study, we characterized five Ullrich scleroatonic muscular dystrophy patients (two Italians, one Belgian, and two Turks) with a clinical phenotype showing different degrees of severity, all carrying mutations localized in COL6A1. We sequenced the three entire COL6 complementary DNA. Three of five patients have recessive mutations: two patients (P1and P3) have homozygous single‐nucleotide deletions, one in exon 9 and one in exon 22; one patient (P2) has a homozygous single‐nucleotide substitution leading to a premature termination codon in exon 31. The nonsense mutation of P2 also causes a partial skipping of exon 31 with the formation of a premature termination codon in exon 32 in 15% of the total COL6A1 messenger RNA. The remaining two patients carry a heterozygous glycine substitution in exons 9 and 10 inside the triple‐helix region; both are dominant mutations because the missense mutations are absent in the DNA of their respective parents. As for the three homozygous recessive mutations, the apparently healthy consanguineous parents all carry a heterozygous mutated allele. Here, for the first time, we report a genotype–phenotype correlation demonstrating that heterozygous glycine substitutions in the triple‐helix domain of COL6A1 are dominant and responsible for a milder Ullrich scleroatonic muscular dystrophy phenotype, and that recessive mutations in COL6A1 correlate with more severe clinical and biochemical Ullrich scleroatonic muscular dystrophy phenotypes. Ann Neurol 2005;58:400–410

Detection of common and private mutations in the COL6A1 gene of patients with Bethlem myopathy

Background: Dominant mutations in COL6A1, COL6A2, and COL6A3, the three genes encoding collagen type VI, a ubiquitous extracellular matrix protein, are associated with Bethlem myopathy (BM) and Ullrich scleroatonic muscular dystrophy. Methods: The authors devised a method to screen the entire coding sequence of the three genes by reverse transcriptase–PCR amplification of total RNA from skin fibroblasts and direct sequencing of the resulting 25 overlapping cDNA fragments covering 107 exons. Results: Four splicing and four missense mutations were identified in 16 patients with BM, six of which are novel mutations in COL6A1. Both common and private mutations are localized in the α1 (VI) chain between the regions corresponding to the 3′ end of the NH2-globular domain and the 5′ end of the triple helix, encoded by exons 3 through 14. Conclusions: The clustering of the mutations in a relatively narrow area of the three collagen type VI chains in patients with Bethlem myopathy (BM) suggests that mutations in different regions could result in different phenotypes or in no phenotype at all. Moreover, the detection of mutations in only 60% of the patients suggests the existence of at least another gene associated with BM. The authors propose the direct sequencing of COL6 cDNAs as the first mutation screening analysis in BM, given the high number of exon-skipping events.

FBN1 mutation screening of patients with Marfan syndrome and related disorders: detection of 46 novel FBN1 mutations

Fibrillin‐1 gene (FBN1) mutations cause Marfan syndrome (MFS), an inherited connective tissue disorder with autosomal dominant transmission. Major clinical manifestations affect cardiovascular and skeletal apparatuses and ocular and central nervous systems. We analyzed FBN1 gene in 99 patients referred to our Center for Marfan Syndrome and Related Disorders (University of Florence, Florence, Italy): 85 were affected by MFS and 14 by other fibrillinopathies type I. We identified mutations in 80 patients. Among the 77 different mutational events, 46 had not been previously reported. They are represented by 49 missense (61%), 1 silent (1%), 13 nonsense (16%), 6 donor splice site mutations (8%), 8 small deletions (10%), and 3 small duplications (4%). The majority of missense mutations were within the calcium‐binding epidermal growth factor‐like domains. We found preferential associations between The Cys‐missense mutations and ectopia lentis and premature termination codon mutations and skeletal manifestations. In contrast to what reported in literature, the cardiovascular system is severely affected also in patients carrying mutations in exons 1–10 and 59–65. In conclusion, we were able to detect FBN1 mutations in 88% of patients with MFS and in 36% of patients with other fibrillinopathies type I, confirming that FBN1 mutations are good predictors of classic MFS.

COL6A1 genomic deletions in Bethlem myopathy and Ullrich muscular dystrophy

We have identified highly similar heterozygous COL6A1 genomic deletions, spanning from intron 8 to exon 13 or intron 13, in two patients with Ullrich congenital muscular dystrophy and the milder Bethlem myopathy. The 5′ breakpoints of both deletions are located within a minisatellite in intron 8. The mutations cause in‐frame deletions of 66 and 84 amino acids in the amino terminus of the triple‐helical domain, leading to intracellular accumulation of mutant polypeptides and reduced extracellular collagen VI microfibrils. Our studies identify a deletion‐prone region in COL6A1 and suggest that similar mutations can lead to congenital muscle disorders of different clinical severity. Ann Neurol 2005

ACE and TGFBR1 genes interact in influencing the susceptibility to abdominal aortic aneurysm

A role of ACE I/D polymorphism in the pathogenesis of abdominal aortic aneurysm (AAA) has been demonstrated, possibly due to the effect of angiotensin II on vascular tissue remodelling. Angiotensin II exerts profibrogenic effects through the local induction of TGF-beta. Dysregulated TGF-beta signalling may result from mutations in TGFBR1 and TGFBR2 genes, thus resulting in degenerative changes in the vessel wall. We performed a case-control study in order to investigate the role of TGFBR1 9A6A polymorphism as predisposing factor to AAA per se, and in the presence of ACE DD and AT1R 1166 CC genotypes in 201 AAA patients (mean age+/-S.D., 71.5+/-6.9) referred to the Unit of Vascular Surgery of the University of Florence, compared with 252 healthy controls (mean age+/-S.D., 70.6+/-8.6). A significant difference in genotype distribution and allele frequency between patients and controls was found for ACE, but not for AT1R and TGFBR1 polymorphisms. At univariate analysis a significant association between ACE DD, but not AT1R CC and TGFBR1 6A allele, and the susceptibility to the disease was found [ACE DD OR=1.86 (95% CI 1.26-2.76), p=0.002]. After adjustment for age, gender, traditional cardiovascular risk factors, and CAD, PAD and CVD, ACE DD genotype still affected the susceptibility to AAA [OR=2.13 (95% CI 1.06-4.28), p=0.03], and the contemporary presence of ACE DD genotype and TGFBR1 6A allele, increased the predisposition to the disease [OR=5.09 (95% CI 1.44-18.02), p=0.01]. This study, which demonstrates an interaction between ACE and TGFBR1 genes in predisposing to AAA, may provide further information on the mechanisms contributing to AAA susceptibility, and offer a topic for future larger studies.

Cytokine gene expression and production by human LPS-stimulated mononuclear cells are inhibited by sulfated heparin-like semi-synthetic derivatives

Summary.  Background: The K5 polysaccharide obtained from Escherichia coli strain 010:K5:H4 is a polymer of the disaccharidic unit formed by D‐glucuronic acid and N‐acetylglucosamine. This structure is akin to N‐acetylheparosan, the precursory polymer of heparin and of heparan sulfate. This structural affinity with N‐acetylated heparin and with de‐sulfated heparin makes the K5 polysaccharide extremely useful for the preparation of sulfated heparin‐like semi‐synthetic derivatives. It has been demonstrated that heparins are able to inhibit tissue factor and cytokine production and expression by human monocytes. Objective: The aim of this study was to evaluate the effects of four different heparin‐like semi‐synthetic derivatives on inflammatory cytokine production and expression by human mononuclear cells. Results: The simultaneous addition of lipopolysaccharide (LPS; 0.2 and 10 µg mL−1) and the K5 polysaccharide did not inhibit interleukin (IL)‐1β, IL‐6 or tumor necrosis factor (TNF)‐α production by stimulated mononuclear cells. IL‐1β, IL‐6 and TNF‐α concentrations in supernatants of LPS‐stimulated mononuclear cells were not influenced by the addition of N,O‐sulfated K5 polysaccharide (K5‐N, OS) and epimerized N‐sulfated K5 polysaccharide (K5 NS epi) at 5 and 10 µg mL−1, whereas the addition of epimerized N,O‐sulfated K5 polysaccharide (K5‐N, OS epi) (5 and 10 µg mL−1) and O‐sulfated K5 polysaccharide (K5‐OS) (5 and 10 µg mL−1) to LPS‐stimulated cells caused a significant dose‐dependent inhibition of IL‐1β, IL‐6 and TNF‐α. All sulfated heparin‐like semi‐synthetic derivatives did not influence the IL‐10 production by LPS‐stimulated mononuclear cells. In LPS‐stimulated cells (0.2 and 10 µg mL−1), K5‐OS or K5‐N, OS epi at 5 and 10 µg mL−1 markedly decreased TNF‐α mRNA expression. Conclusions: These results indicate that the sulfated heparin‐like semi‐synthetic derivatives K5‐OS and K5‐N, OS epi are able to inhibit both expression and production of inflammatory cytokines, whereas they do not influence the anti‐inflammatory cytokine IL‐10, suggesting a potential role for these products as modulators of inflammatory reactions.

Structural insights on carbonic anhydrase inhibitory action, isoform selectivity, and potency of sulfonamides and coumarins incorporating arylsulfonylureido groups.

Sulfonamides and coumarins incorporating arylsulfonylureido tails were prepared and assayed as inhibitors of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). Some derivatives incorporating 3-pyridinesulfonamide and arylsulfonylureoido fragments were low nanomolar inhibitors of isoforms CA II and XII (upregulated or overexpressed in glaucoma) and showed effective in vivo intraocular pressure lowering effects in an animal model of the disease, which were several times better compared to those of the antiglaucoma drug dorzolamide. By means of X-ray crystallography of adducts of several sulfonamides with CA II, the effective inhibitory properties were rationalized at the molecular level. The coumarins were ineffective as hCA I and II inhibitors but showed low nanomolar activity for the inhibition of the tumor-associated isoforms hCA IX and XII. The presence of arylsulfonylureido tails in these CA inhibitors possessing quite different mechanisms of action led to highly effective and isoform-selective compounds targeting enzymes involved in severe pathologies such as glaucoma or cancer.

Histamine H4 receptor activation alleviates neuropathic pain through differential regulation of ERK, JNK, and P38 MAPK phosphorylation.

Abstract Histamine plays a complex role in pain modulation with opposite roles in nociception for histamine receptor subtypes 1, 2, and 3. The histamine H4 receptor (H4R) is expressed primarily on cells involved in inflammation and immune responses with a proinflammatory activity, but little is known about the role in nociception of neuronal H4R. To investigate the effects of neuronal H4R in pain transmission, the effects produced by the H4R agonist ST-1006 were detected in the spared nerve injury model of neuropathic pain. ST-1006 counteracted mechanical allodynia in neuropathic mice, an effect prevented by the H4R antagonist JNJ 10191584. In spared nerve injury mice, an early over-phosphorylation of ERK1 and ERK2 was observed in the dorsal root ganglia (DRG), spinal cord, and sciatic nerve. A progressive and long-lasting activation of JNK1 was observed in the sciatic nerve and, to a lesser extent, in the spinal cord and DRG. An increased p-P38 content was detected in the spinal cord and DRG, with no modification in the sciatic nerve. Administration of ST-1006 prevented phosphorylation of all 3 MAPK within DRG, and phosphorylation of ERK1, ERK2, and pJNK1 in the sciatic nerve. In the spinal cord, the H4R agonist prevented selectively the pERK2 increase with no effect on pJNK1 and p-P38 levels. Double immunofluorescence experiments showed a neuronal localization and site of action for H4R. These findings suggest a prevalent modulation of ERK activity after H4R stimulation and indicate the DRG as prominent site of action for H4R-mediated antineuropathic activity. Targeting neuronal H4R with selective agonists could have therapeutic potential for neuropathic pain treatment.

A homozygous COL6A2 intron mutation causes in-frame triple-helical deletion and nonsense-mediated mRNA decay in a patient with Ullrich congenital muscular dystrophy

Ullrich congenital muscular dystrophy (UCMD) is a severe disorder caused, in most cases, by a deficiency in collagen VI microfibrils. Recessive mutations in two of the three collagen VI genes, COL6A2 and COL6A3, have been identified in eight of the nine UCMD patients reported thus far. A heterozygous COL6A1 gene deletion, resulting in a mutant protein that exerts a dominant negative effect, has recently been described in a severely affected UCMD patient. Here we describe a patient in whom reverse transcription-PCR analysis of fibroblast RNA suggested a heterozygous in-frame deletion of exon 13 in the triple-helical domain of COL6A2, which is predicted to be dominantly acting. However, a homozygous A→G mutation at −10 of intron 12 was found in the genomic DNA. The intron mutation activated numerous cryptic splice acceptor sites, generating normal and exon 13-deleted COL6A2 mRNA, and multiple aberrant transcripts containing frameshifts that were degraded through a nonsense-mediated decay mechanism. Northern analysis indicated diminished COL6A2 mRNA expression as the primary pathogenic mechanism in this UCMD patient. Our results underscore the importance of multifaceted analyses in the accurate molecular diagnosis and interpretation of genotype-phenotype correlations of UCMD.

Evidence for oxidative stress in plasma of patients with Marfan syndrome

0.91–1.31 1.11 0.77–1.60 Metabolic syndrome — NCEP 1.16 0.99–1.35 1.56* 1.06–2.29 1.14 0.95–1.37 1.20 0.83–1.74 Abdominal obesity — IDF 1.27** 1.08–1.50 1.25 0.85–1.84 0.96 0.79–1.17 1.11 0.75–1.64 Abdominal obesity — NCEP 1.18* 1.01–1.39 1.26 0.86–1.85 1.20 0.99–1.44 0.95 0.65–1.39 Hypertension 1.35*** 1.14–1.59 1.21 0.82–1.80 1.15 0.94–1.41 1.02 0.69–1.53 Low high-density lipoprotein 0.92 0.74–1.14 1.98* 1.10–3.57 0.89 0.69–1.15 1.00 0.60–1.66 Elevated triglycerides 0.75 ** 0.61–0.92 0.92 0.57–1.47 0.93 0.73–1.17 1.31 0.83–2.06 Insulin resistance TG/HDL 0.87 0.75–1.02 1.55* 1.05–2.29 0.90 0.74–1.09 0.88 0.60–1.28 Hyperglycemia 1.34*** 1.14–1.57 1.38 0.95–2.01 1.13 0.94–1.36 1.00 0.69–1.46 Women Metabolic syndrome — IDF 1.52*** 1.34–1.75 1.44* 1.07–1.94 1.15* 1.00–1 .33 0.74 0.53–1.05 Metabolic syndrome — NCEP 1.54*** 1.35–1.77 1.35* 1.00–1.82 1.26** 1.09–1.45 0.86 0.61–1.22 Abdominal obesity — IDF 1.47*** 1.25–1.73 0.82 0.57–1.17 1.23* 1.03–1.46 0.73 0.51–1.06 Abdominal obesity — NCEP 1.56*** 1.36–1.78 1.00 0.82–1.22 1.34*** 1.16–1.56 0.88 0.63–1.23 Hypertension 2.15*** 1.84–2.52 1.23 0.90–1.68 1.19* 1.02–1.39 0.90 0.64–1.27 Low high-density lipoprotein 1.12 0.96–1.31 1.59* 1.11–2.27 0.98 0.83–1.16 1.01 0.68–1.50 Elevated triglycerides 1.08 0.89–1.30 0.94 0.62–1.42 1.01 0.82–1.24 1.32 0.83–2.11 Insulin resistance TG/HDL 1.15* 1.00–1.34 1.60** 1.14–2.26 1.04 0.89–1.22 0.93 0.63–1.37 Hyperglycemia 1.45*** 1.27–1.65 1.48** 1.11–1.98 1.16* 1.01–1.33 1.01 0.72–1.41