Joost Schalkwijk

A genome-wide association study identifies new psoriasis susceptibility loci and an interaction between HLA-C and ERAP1

To identify new susceptibility loci for psoriasis, we undertook a genome-wide association study of 594,224 SNPs in 2,622 individuals with psoriasis and 5,667 controls. We identified associations at eight previously unreported genomic loci. Seven loci harbored genes with recognized immune functions (IL28RA, REL, IFIH1, ERAP1, TRAF3IP2, NFKBIA and TYK2). These associations were replicated in 9,079 European samples (six loci with a combined P < 5 × 10−8 and two loci with a combined P < 5 × 10−7). We also report compelling evidence for an interaction between the HLA-C and ERAP1 loci (combined P = 6.95 × 10−6). ERAP1 plays an important role in MHC class I peptide processing. ERAP1 variants only influenced psoriasis susceptibility in individuals carrying the HLA-C risk allele. Our findings implicate pathways that integrate epidermal barrier dysfunction with innate and adaptive immune dysregulation in psoriasis pathogenesis.

On the nature of hypertrophic scars and keloids: A review

On the Nature of Hypertrophic Scars and Keloids: A Review Frank Niessen;Paul Spauwen;Joost Schalkwijk;Moshe Kon; Plastic and Reconstructive Surgery

Psoriasis is associated with increased beta-defensin genomic copy number

Psoriasis is a common inflammatory skin disease with a strong genetic component. We analyzed the genomic copy number polymorphism of the β-defensin region on human chromosome 8 in 179 Dutch individuals with psoriasis and 272 controls and in 319 German individuals with psoriasis and 305 controls. Comparisons in both cohorts showed a significant association between higher genomic copy number for β-defensin genes and risk of psoriasis.

Deletion of the late cornified envelope LCE3B and LCE3C genes as a susceptibility factor for psoriasis.

Psoriasis is a common inflammatory skin disease with a prevalence of 2–3% in individuals of European ancestry. In a genome-wide search for copy number variants (CNV) using a sample pooling approach, we have identified a deletion comprising LCE3B and LCE3C, members of the late cornified envelope (LCE) gene cluster. The absence of LCE3B and LCE3C (LCE3C_LCE3B-del) is significantly associated (P = 1.38E–08) with risk of psoriasis in 2,831 samples from Spain, The Netherlands, Italy and the United States, and in a family-based study (P = 5.4E–04). LCE3C_LCE3B-del is tagged by rs4112788 (r 2 = 0.93), which is also strongly associated with psoriasis (P < 6.6E–09). LCE3C_LCE3B-del shows epistatic effects with the HLA-Cw6 allele on the development of psoriasis in Dutch samples and multiplicative effects in the other samples. LCE expression can be induced in normal epidermis by skin barrier disruption and is strongly expressed in psoriatic lesions, suggesting that compromised skin barrier function has a role in psoriasis susceptibility.

Haploinsufficiency of TNXB Is Associated with Hypermobility Type of Ehlers-Danlos Syndrome

To the Editor: Ehlers-Danlos syndrome (EDS) is a heterogeneous group of heritable connective-tissue disorders, generally affecting skin, joints, and blood vessels. The most recent classification recognizes six subtypes (Beighton et al. 1998), of which the hypermobility type (HT-EDS [formerly EDS type III] [MIM 130020]) is the most common. This type of EDS is similar to benign joint hypermobility syndrome (BJHS), and both are often considered to represent the same hyperlaxity syndrome, since no clear clinical distinction can be made (Grahame 1999). Although various causative genes have been found in all other types of EDS, the genetic basis of HT-EDS or BJHS remains unexplained (Steinmann et al. 2002). One family has been described that has a missense mutation in COL3A1 (Narcisi et al. 1994), resulting in a phenotype that resembles HT-EDS, without obvious vascular complications. Mutations in COL3A1 generally result in the severe vascular type of EDS (MIM 130050). To our knowledge, no other cases of COL3A1 mutations in HT-EDS have been reported. Recently, we showed that deficiency of the extracellular-matrix protein tenascin-X (TNX), encoded by the TNXB gene, causes a new type of recessively inherited EDS (Schalkwijk et al. 2001). Patients with complete deficiency of TNX showed marked joint hypermobility, skin hyperextensibility, and easy bruising. The absence of atrophic scars and recessive inheritance distinguishes TNX deficiency from the classical type of EDS. In our initial report (Schalkwijk et al. 2001), only a few heterozygous family members were available for examination. Here, we have examined all 20 heterozygous family members (individuals from families A–D in table 1) who were available for further study, regardless of clinical symptoms; in all of these individuals, we have found significantly reduced serum TNX levels (56% ± 6% vs. 100% ± 14% in the control population; P<.001, by Students t test) (fig. 1f), and, in 17 of them, we have confirmed heterozygosity for a truncating TNXB mutation (table 1). Clinical examination revealed generalized joint hypermobility in nine family members (45%), using the Beighton score (Beighton et al. 1973), for HT-EDS, or the Brighton criteria (Grahame et al. 2000), for BJHS (table 1 and fig. 1e). Skin hyperextensibility and easy bruising, frequently seen in the individuals with complete TNX deficiency, were absent. A number of patients with haploinsufficiency had recurring joint dislocations and chronic joint pain, as are seen in HT-EDS and BJHS. Only four family members carrying two normal TNXB alleles were available for study, of whom none had hypermobility. The local medical ethics committee (CMO Regio Arnhem-Nijmegen) approved the study protocol, and informed consent was obtained from all patients. Figure  1 TNXB haploinsufficiency and generalized joint hypermobility. a–d, Pedigrees of families A–D (also see table 1). e, Joint hypermobility in individual III9 from family A. f, Distribution of serum TNX levels in control population, population ... Table 1 Clinical and Molecular Findings in Individuals with TNXB Haploinsufficiency/Reduced Serum TNX Levels A striking finding is that 0 of the 6 males with haploinsufficiency fulfilled the clinical criteria for HT-EDS or BJHS, whereas 9 of 14 (64%) females were positive. This finding is in accordance with previous population-based studies that show a female preponderance in joint hypermobility syndromes (Larsson et al. 1987; Rikken-Bultman et al. 1997). In a control group of 30 unaffected females of the same age as the females with haploinsufficiency in the present study, we found no individuals with a Beighton score >4. This indicates that the prevalence of generalized joint hypermobility in a population of females with haploinsufficiency is significantly higher than in a control population (P<.001, by χ2 test). No sex differences in serum TNX levels in unaffected individuals and individuals with haploinsufficiency were found (not shown). Because our observations in families carrying previously described TNXB mutations suggested an association between TNXB haploinsufficiency and joint hypermobility, we wondered about the prevalence of TNXB haploinsufficiency in patients with HT-EDS. We measured serum TNX levels (by ELISA) in an unselected cohort of 80 patients with HT-EDS who were recruited through the Dutch organization for patients with EDS. All patients were diagnosed with HT-EDS by a medical specialist, and ∼90% were female. Although the mean serum TNX level was not different in the cohort with HT-EDS overall (99.4%±19.7%) (fig. 1f), six of these patients (7.5% [all female]) had serum TNX levels >2.5 SDs (65%) below the mean for unaffected individuals. On the basis of the normal distribution of serum TNX levels, only 0.6% of individuals would be expected to have such low serum TNX levels, which is significantly less than the frequency found in the population with HT-EDS described in the present study (P<.001, by Fishers exact test). Clinically, patients with reduced TNX levels showed hypermobile joints, often associated with joint subluxations and chronic musculoskeletal pain (table 1). The clinical findings in these patients differ from those with complete TNX deficiency. Patients with haploinsufficiency do not have skin hyperextensibility and lack the easy bruising seen in patients with TNX deficiency. In addition, TNXB haploinsufficiency is expected to be an autosomal dominant trait, which is in accordance with the observed mode of inheritance of HT-EDS and BJHS. On screening for the presence of a 30-kb deletion described previously (Burch et al. 1997; Schalkwijk et al. 2001), we found that this deletion was present in one of these six patients. The 30-kb deletion creates a fusion gene of TNXB and XA, a partial duplicate of TNXB. The XA gene has an internal deletion that truncates its ORF, rendering XA and the fusion gene nonfunctional (Gitelman et al. 1992). The deleted allele also lacks CYP21, so this individual is also a carrier for congenital adrenal hyperplasia. Subsequently, we PCR amplified and directly sequenced the coding regions and the intron-exon boundaries of TNXB in the other five patients presumed to have haploinsufficiency (for primers used, see Schalkwijk et al. 2001). One patient (individual F in table 1) was heterozygous for a 2-bp deletion, [AA56063] del, in exon 8, resulting in a premature stop codon at the position of amino acid 1231. In the other four patients, we were unable to identify mutations in TNXB. These patients may have mutations, in regulatory sequences or in exons of the TNXB gene, that have not yet been identified, or they may represent the extreme in normal variation of TNX expression. In conclusion, in the present study, we have reported a genetic defect associated with HT-EDS or BJHS. On the basis of the observed phenotype in patients with complete TNX deficiency and the high prevalence of generalized joint hypermobility in heterozygous females, this is likely to be a causative relationship. Reduced TNX expression could disturb deposition of collagen (Mao et al. 2002) and the elastic fiber network (Burch et al. 1997), as has been shown for complete TNX deficiency, resulting in increased laxity of ligaments and tendons. TNXB haploinsufficiency is dominantly inherited and appears to produce clinical findings primarily in women, consistent with clinical descriptions of HT-EDS. Although we identified inactivating TNX mutations in only 2.5% of this cohort with HT-EDS, 7.5% had serum TNX levels low enough to affect collagen metabolism. The present study demonstrates that TNXB haploinsufficiency is associated with HT-EDS and suggests that locus heterogeneity exists for this disorder, as it does for other types of EDS (Byers 1994).

Accurate, high-throughput typing of copy number variation using paralogue ratios from dispersed repeats

Recent work has demonstrated an unexpected prevalence of copy number variation in the human genome, and has highlighted the part this variation may play in predisposition to common phenotypes. Some important genes vary in number over a high range (e.g. DEFB4, which commonly varies between two and seven copies), and have posed formidable technical challenges for accurate copy number typing, so that there are no simple, cheap, high-throughput approaches suitable for large-scale screening. We have developed a simple comparative PCR method based on dispersed repeat sequences, using a single pair of precisely designed primers to amplify products simultaneously from both test and reference loci, which are subsequently distinguished and quantified via internal sequence differences. We have validated the method for the measurement of copy number at DEFB4 by comparison of results from >800 DNA samples with copy number measurements by MAPH/REDVR, MLPA and array-CGH. The new Paralogue Ratio Test (PRT) method can require as little as 10 ng genomic DNA, appears to be comparable in accuracy to the other methods, and for the first time provides a rapid, simple and inexpensive method for copy number analysis, suitable for application to typing thousands of samples in large case-control association studies.Recent work has demonstrated an unexpected prevalence of copy number variation in the human genome, and has highlighted the part this variation may play in predisposition to common phenotypes. Some important genes vary in number over a high range (e.g. DEFB4 , which commonly varies between two and seven copies), and have posed formidable technical challenges for accurate copy number typing, so that there are no simple, cheap, high-throughput approaches suitable for large-scale screening. We have developed a simple comparative PCR method based on dispersed repeat sequences, using a single pair of precisely designed primers to amplify products simultaneously from both test and reference loci, which are subsequently distinguished and quantified via internal sequence differences. We have validated the method for the measurement of copy number at DEFB4 by comparison of results from >800 DNA samples with copy number measurements by MAPH/REDVR, MLPA and array-CGH. The new Paralogue Ratio Test (PRT) method can require as little as 10 ng genomic DNA, appears to be comparable in accuracy to the other methods, and for the first time provides a rapid, simple and inexpensive method for copy number analysis, suitable for application to typing thousands of samples in large case-control association studies.

Constitutive and inducible expression of SKALP/elafin provides anti-elastase defense in human epithelia.

Skin-derived antileukoproteinase (SKALP), also known as elafin, is a serine proteinase inhibitor first discovered in keratinocytes from hyperproliferative human epidermis. In addition to the proteinase inhibiting domain which is directed against polymorphonuclear leukocyte (PMN) derived enzymes such as elastase and proteinase 3, SKALP contains multiple transglutaminase (TGase) substrate domains which enable crosslinking to extracellular and cell envelope proteins. Here we show that SKALP is constitutively expressed in several epithelia that are continuously subjected to inflammatory stimuli, such as the oral cavity and the vagina where it co-localizes with type 1 TGase. All epithelia from sterile body cavities are negative for SKALP. In general, stratified squamous epithelia are positive, whereas pseudostratified epithelia, simple/glandular epithelia and normal epidermis are negative. SKALP was found in fetal tissues of the oral cavity from 17 wk gestation onwards where it continued to be expressed up to adult life. Remarkably, in fetal epidermis SKALP was found from week 28 onwards, but was downregulated to undetectable levels in neonatal skin within three months, suggesting a role during pregnancy in feto-maternal interactions or in the early maturation phase of the epidermis. Immunoelectron microscopy revealed the presence of SKALP in secretory vesicles including the lamellar granules. In culture models for epidermal keratinocytes we found that expression of the endogenous SKALP gene provided protection against cell detachment caused by purified elastase or activated PMNs. Addition of exogenous recombinant SKALP fully protected the keratinocytes against PMN-dependent detachment whereas superoxide dismutase and catalase were only marginally effective. These findings strongly suggest that the constitutive expression of SKALP in squamous epithelia, and the inducible expression in epidermis participate in the control of epithelial integrity, by inhibiting PMN derived proteinases.

β-Defensin-2 Protein Is a Serum Biomarker for Disease Activity in Psoriasis and Reaches Biologically Relevant Concentrations in Lesional Skin

Background Previous studies have extensively documented antimicrobial and chemotactic activities of beta-defensins. Human beta-defensin-2 (hBD-2) is strongly expressed in lesional psoriatic epidermis, and recently we have shown that high beta-defensin genomic copy number is associated with psoriasis susceptibility. It is not known, however, if biologically and pathophysiologically relevant concentrations of hBD-2 protein are present in vivo, which could support an antimicrobial and proinflammatory role of beta-defensins in lesional psoriatic epidermis. Methodology/Principal Findings We found that systemic levels of hBD-2 showed a weak but significant correlation with beta defensin copy number in healthy controls but not in psoriasis patients with active disease. In psoriasis patients but not in atopic dermatitis patients, we found high systemic hBD-2 levels that strongly correlated with disease activity as assessed by the PASI score. Our findings suggest that systemic levels in psoriasis are largely determined by secretion from involved skin and not by genomic copy number. Modelling of the in vivo epidermal hBD-2 concentration based on the secretion rate in a reconstructed skin model for psoriatic epidermis provides evidence that epidermal hBD-2 levels in vivo are probably well above the concentrations required for in vitro antimicrobial and chemokine-like effects. Conclusions/Significance Serum hBD-2 appears to be a useful surrogate marker for disease activity in psoriasis. The discrepancy between hBD-2 levels in psoriasis and atopic dermatitis could explain the well known differences in infection rate between these two diseases.

Coal tar induces AHR-dependent skin barrier repair in atopic dermatitis

Topical application of coal tar is one of the oldest therapies for atopic dermatitis (AD), a T helper 2 (Th2) lymphocyte-mediated skin disease associated with loss-of-function mutations in the skin barrier gene, filaggrin (FLG). Despite its longstanding clinical use and efficacy, the molecular mechanism of coal tar therapy is unknown. Using organotypic skin models with primary keratinocytes from AD patients and controls, we found that coal tar activated the aryl hydrocarbon receptor (AHR), resulting in induction of epidermal differentiation. AHR knockdown by siRNA completely abrogated this effect. Coal tar restored filaggrin expression in FLG-haploinsufficient keratinocytes to wild-type levels, and counteracted Th2 cytokine-mediated downregulation of skin barrier proteins. In AD patients, coal tar completely restored expression of major skin barrier proteins, including filaggrin. Using organotypic skin models stimulated with Th2 cytokines IL-4 and IL-13, we found coal tar to diminish spongiosis, apoptosis, and CCL26 expression, all AD hallmarks. Coal tar interfered with Th2 cytokine signaling via dephosphorylation of STAT6, most likely due to AHR-regulated activation of the NRF2 antioxidative stress pathway. The therapeutic effect of AHR activation herein described opens a new avenue to reconsider AHR as a pharmacological target and could lead to the development of mechanism-based drugs for AD.

Neuromuscular involvement in various types of Ehlers-Danlos syndrome.

Ehlers–Danlos syndrome (EDS) is a clinically and genetically heterogeneous group of heritable connective tissue disorders characterized by joint hypermobility, skin hyperextensibility, and tissue fragility. Muscle involvement is plausible based on recently discovered interactions between muscle cells and extracellular matrix molecules; however, muscle symptoms are only sporadically reported. We designed a cross‐sectional study to find out whether neuromuscular features are part of EDS.