Filaggrin mutations are genetic modifying factors exacerbating X-linked ichthyosis

Mutations inactivating the STS gene cause X-linked ichthyosis (XLI), whereas null mutations in the FLG gene cause ichthyosis vulgaris. Two brothers presented with XLI. One had a typical fine scaling, and the other was much more severely affected. Both patients carried STS missense mutation T165I. Furthermore, the more severely affected patient also carried heterozygous FLG mutation R501X, which was absent from his mildly affected brother. These data suggest that disrupting epidermal differentiation via different pathways can increase phenotypic severity. Owing to the high population frequency of FLG mutations, filaggrin is a possible genetic modifier in other genodermatoses.     

四种遗传性鱼鳞病基因型与临床表型的相关性分析

目的对四种鱼鳞病(寻常型鱼鳞病;X性-联鱼鳞病;板层状鱼鳞病;大疱性鱼鳞病)的致病基因进行精细定位,并分析其基因型与临床表型的关系。方法对四种鱼鳞病各1例患者进行临床表型分析以及外周血DNA直接测序检测鱼鳞病FLG基因、STS基因、TGM1基因和K1,K10角蛋白基因的突变位点。结果①寻常型鱼鳞病患者在FLG基因的外显子5的第278位有G-T突变,613位有G-A突变。②板层状鱼鳞病患者TGM1基因外显子3的第504位碱基有C-T突变,使第142位氨基酸由精氨酸(R)转变为半胱氨酸(C),即R142C错义突变;外显子7的第1122位碱基有C-T突变,使348位氨基酸由精氨酸(R)突变为终止密码(R348X),导致其编码的蛋白缺失了C端的470个氨基酸。③X-性联鱼鳞病患者STS基因完全缺失。④大疱性鱼鳞病患者外显子5的第242碱基存在A-C突变,外显子6的第599位碱基均存在A-G突变,导致K1蛋白第633位氨基酸由赖氨酸(Lys)变为精氨酸(Arg)。结论鱼鳞病患者临床表型的不同与致病基因的突变位点密切相关。     

Oculocutaneous albinism type 3: a Japanese girl with novel mutations in TYRP1 gene

Background

Oculocutaneous albinism (OCA) type 3 caused by mutations of the TYRP1 gene is an autosomal recessive disorder of pigmentation characterized by reduced biosynthesis of melanin pigment in the skin, hair, and eye. The clinical phenotype has been reported as mild in Caucasian OCA3 patients.

Objective

We had the opportunity to examine a Japanese girl with OCA3 and investigated activity of TYRP1 protein derived from the mutant allele detected in the patient.

Methods

Mutation search for OCA responsible genes was done. A mutant allele with a missense mutation was analyzed using melanocyte cultures (b cells) established from a mouse model of OCA3.

Results

Compound heterozygous mutations, p.C30R and p.367fsX384, were detected in the Japanese girl. Then we revealed that the missense mutation, p.C30R, was functionally incapable of melanin synthesis with in vitro experiments.

Conclusion

This is the first report of the occurrence of OCA3 in Japanese population.     

X-性连锁鱼鳞病一家系类固醇硫酸酯酶基因研究

目的 检测一个中国汉族人X-性连锁鱼鳞病家系的类固醇硫酸酯酶(STS)基因突变情况.方法 收集1个X-性连锁鱼鳞病家系的临床资料,提取外周血DNA,通过PCR扩增外周血基因组DNA类固醇硫酸酯酶基因的第1和第10外显子,以表型正常家系成员及50例健康人为正常对照.结果 家系内全部患者均存在STS基因的完全缺失,即10个外显子均缺失,家系中正常人及对照者未发现上述缺失.结论 STS基因的完全缺失可能为导致该家系临床表型的主要原因.

Abstract：

Objective To detect the steroid sulfatase (STS) gene mutation in a Chinese pedigree with X-linked ichthyosis (XLI). Methods Genomic DNA was extracted from the peripheral blood of 3 affected patients and unaffected members in this family and 50 unrelated healthy volunteers followed by the amplification of the exon 1 and exon 10 of STS gene by PCR. Results Complete deletion of the exon 1 to 10 of STS gene was detected in all the patients in this pedigree with XLI, while no mutation was found in this gene in unaffected members of this family or normal human controls. Conclusion The complete deletion of STS gene is likely to be the main cause of the phenotype of XLI in this family.     

Pressure ulcers in Jordan: a point prevalence study

Background

Pressure ulcers are a common problem among hospitalised patients. Several prevalence studies have been conducted internationally but there is a paucity of research on pressure ulcer in the Arab world in general and in Jordan specifically.

Purpose

The aim of this study was to quantify the prevalence rate of pressure ulcers in Jordan, and to compare these figures with other studies conducted using the same methods.

Design

A cross-sectional survey design.

Participants

All inpatients older than eighteen in both university and general hospitals in Jordan. Patients in the emergency, day care and maternity wards were excluded.

Instruments

European Pressure Ulcer Advisory Panel (EUPAP) data collection form.

Methods

The survey was conducted by examining patients’ skin. Pressure ulcers were classified according to the EPUAP grading system. Risk of pressure ulcer development was assessed using the Braden scale. Data were collected on preventive measures recorded in the clinical setting.

Results

The overall prevalence rate was 12% (All percentages are rounded to the nearest digit) (7% when Grade I excluded). The sacrum and heel were the most common affected sites. Grade one was the most common grade (44%). Only 17% of the patients at risk received adequate prevention.

Conclusion

The pressure ulcer prevalence rate in Jordan is lower than that published in most studies utilising the same methodology. Despite this relatively low prevalence very few patients at risk received adequate prevention, and there is therefore a need to raise the awareness for pressure ulcer prevention in Jordan. Furthermore, differences in age and frailty in the Jordanian sample could explain the low prevalence.     

EC-SOD induces apoptosis through COX-2 and galectin-7 in the epidermis

Background

Extracellular superoxide dismutase (EC-SOD) is an anti-oxidant enzyme found in the extracellular matrix of tissues, and plays an important role in the prevention of many diseases caused by oxidative stress. However, other functions of EC-SOD in epidermis are not well known.

Objective

We investigated the functions of EC-SOD in epidermis using keratinocyte cell line and EC-SOD transgenic mice.

Methods

Expression of galectin-7 in pEC-SOD transfected cells or skin of EC-SOD transgenic mice was detected by western blot analysis. The percentage of apoptotic cells was determined by propidium iodide staining and subsequent FACS analysis. COX-2 siRNA or scrambled siRNA was transfected into HaCaT cells and western blot analysis was performed to detect pro-apoptotic protein levels.

Results

The epidermis of EC-SOD transgenic mice was thinner than wild type mice. In addition, we showed that the thin epidermis of EC-SOD transgenic mice results from the apoptosis of epidermal cells. To elucidate which molecules are involved in EC-SOD-induced apoptosis, we utilized two-dimensional electrophoresis; the results showed that the epidermis of EC-SOD transgenic mice produces more galectin-7, a pro-apoptotic factor, than the wild type. Furthermore, we showed that the transfection of EC-SOD-expressing plasmids induces the production of galectin-7, and pro-apoptotic proteins in keratinocytes. This suggests that EC-SOD induces apoptosis through increased galectin-7 expression. Finally, we demonstrated that EC-SOD-induced galectin-7 results from the production of COX-2.

Conclusion

Our results imply that EC-SOD plays a role not only as a reactive oxygen species scavenger, but also as a pro-apoptotic factor via COX-2/galectin-7 pathways in the epidermis.     

Implementation of an optimized strategy for genetic testing of the Chinese patients with oculocutaneous albinism

Background

Oculocutaneous albinism (OCA) is a relatively common inherited disorder in all populations worldwide. The mutational spectra of OCA are population-specific.

Objective

Based on our previous molecular epidemiological studies, we have implemented an optimized strategy for the genetic testing of Chinese OCA patients.

Methods

Genomic DNA was extracted from the blood samples of 52 clinically diagnosed OCA patients and 100 unaffected subjects. The amplified DNA segments were screened for mutations of TYR, OCA2, TYRP1, SLC45A2 and HPS1 by direct sequencing. To exclude the previously unidentified alleles (PUAs) from polymorphisms, samples from 100 unaffected controls were sequenced for the same regions of variations.

Results

Among the 52 OCA patients, 26 (50.0%) were found mutations on TYR gene, 8 (15.4%) on OCA2, 12 (23.1%) on SLC45A2, 2 (3.8%) on HPS1, and 4 (7.7%) patients uncharacterized. We identified 18 PUAs in these patients, 2 in TYR, 7 in OCA2, 8 in SLC45A2, and 1 in HPS1.

Conclusion

The optimized method to screen the OCA mutations is efficiently implemented in the routine genetic testing of Chinese OCA patients accompanied with genetic counseling.     

Expression of antimicrobial peptides in diabetic foot ulcer

Background

Foot ulcers are one of the main diabetes complications due to its high frequency and difficulty of complete healing. There are several factors that participate in diabetic ulcers development and limited information exists about the role of antimicrobial peptides (AMP) in its pathogenesis.

Objective

The aim of this study was to analyze the expression pattern of the main AMPs: Human Neutrophil Peptide (HNP)-1, Human β-defensin (HBD)-1, HBD-2, HBD-3, HBD-4 and cathelicidin LL-37 in biopsies from diabetic foot ulcers (DFU).

Methods

20 biopsies from DFU grade 3 according to Wagner's classification and 20 biopsies from healthy donors were obtained. Real time PCR, immunohistochemistry and primary cell cultures were performed.

Results

β-Defensins were overexpressed in DFU, whereas LL-37 has low or none expression in comparison with healthy skin. When primary cell culture from these biopsies were performed and infected with Staphylococcus aureus, epidermal cell from diabetic ulcers showed lower LL-37 expression compared with cell cultures from healthy donors skin.

Conclusion

These results suggest that though most AMPs are expressed in DFU, this production is not appropriate to promote wound healing and contain secondary infections.     

The hsp27kD heat shock protein and p38-MAPK signaling are required for regular epidermal differentiation

Background

In human epidermal keratinocytes the expression of hsp27 is closely related to differentiation in vitro and in situ.

Objective

We aimed to gain further insight into the role of hsp27 in epidermal differentiation by specific inhibition through siRNA and inhibition of p38-MAPK, the key enzyme of hsp27 phosphorylation.

Methods

Normal human keratinocytes (KC) and organotypic skin cultures (SE-skin equivalents) were used. Expression and phosphorylation of hsp27 was inhibited in these models by siRNA and SB203580, a specific inhibitor of p38-MAPK, respectively. Modification of morphology and expression of hsp27 and other differentiation associated proteins was investigated by immunofluorescence, western blot, and RT-PCR.

Results

Inhibition of p38-MAPK resulted in a downregulation of hsp27 in KC and SE. Additionally, in the presence of SB203580 Ca²⁺ induced expression of pro-filaggrin and loricrin was inhibited at the protein level and expression of filaggrin, keratin 10, and transglutaminase 1 at the mRNA level. Addition of SB203580 to SE, as well as hsp27 knockdown in this model resulted in identical patterns of irregular differentiation, disturbance of epidermal layers, and delayed expression of K10.

Conclusion

These results provide evidence that the expression of hsp27 and its phosphorylation by p38-MAPK are required for keratinocyte differentiation and for the formation of a regularly stratified epidermis.     

Cutaneous manipulation of vascular growth factors leads to alterations in immunocytes, blood vessels and nerves: Evidence for a cutaneous neurovascular unit

Background

Skin cells produce soluble factors which influence keratinocyte proliferation, angiogenesis, nerve innervation and immunocyte response.

Objective

To test the hypothesis that epidermal-dermal interactions influence neural outgrowth, vascular survival, immunocyte recruitment and keratinocyte proliferation.

Methods

We genetically manipulated the epidermis to express excess vascular endothelial growth factor (VEGF) and/or angiopoietin-1 (Ang1) and then examined the epidermal and dermal phenotypes. We compared these findings with those occurring following overexpression of the Ang1 receptor Tie2 in endothelial cells or keratinocytes.

Results

Keratinocyte-overexpression of Ang1 resulted in increased epidermal thickness compared to control littermates. Keratinocyte-specific overexpression of Ang1 or VEGF increased dermal angiogenesis compared to control animals and combined Ang1-VEGF lead to further increases. Cutaneous leukocyte examination revealed increases in CD4⁺ T cell infiltration in mice with keratinocyte-specific overexpression of Ang1, VEGF and Ang1-VEGF combined; in contrast only keratinocyte-specific Ang1 overexpression increased cutaneous F4/80⁺ macrophage numbers. Interestingly, combined keratinocyte-derived Ang1-VEGF overexpression reduced significantly the number of F4/80⁺ and Cd11c⁺ cells compared to mice overexpressing epidermal Ang1 alone. Endothelial cell-specific Tie2 overexpression increased dermal angiogenesis but failed to influence the epidermal and immune cell phenotypes. Keratinocyte-specific Tie2 expressing mice had the highest levels of CD4⁺, CD8⁺ and CD11c⁺ cell numbers and acanthosis compared to all animals. Finally, increases in the number of cutaneous nerves were found in all transgenic mice compared to littermate controls.

Conclusion

These findings demonstrate that change to one system (vascular or epidermal) results in change to other cutaneous systems and suggest that individual molecules can exert effects on multiple systems.     

Analysis of the VCX3A, VCX2 and VCX3B genes shows that VCX3A gene deletion is not sufficient to result in mental retardation in X-linked ichthyosis

BACKGROUND: X-linked ichthyosis (XLI), an inborn error of metabolism, is due to steroid sulphatase (STS) deficiency. Most patients with XLI harbour complete deletion of the STS gene and flanking sequences. The presence of low copy number repeats on either side of the STS gene seems to have a major role in the high frequency of these deletions. Some patients with XLI with terminal deletions of Xp22.3 involving marker DXS1139 and the STS gene show mental retardation (MR); VCX3A is the only gene located on this critical region. OBJECTIVES: To analyse the VCX3A, VCX, VCX2 and VCX3B genes in 80 unrelated Mexican patients with XLI with normal intelligence. METHODS: STS activity was measured in the leucocytes using 7-[3H]-dehydroepiandrosterone sulphate as a substrate. Amplification of the regions from telomeric DXS89 to centromeric DXS1134 including both extremes of the STS and the VCX3A, VCX, VCX2 and VCX3B genes was performed using polymerase chain reaction. RESULTS: No STS activity was detected in the patients with XLI (0.00 pmol mg(-1) protein h(-1)). We observed two different deletion patterns: the first group included 62 patients with deletion of VCX3A and VCX genes. The second group included 18 patients with breakpoints at several regions on either side of the STS gene not including the VCX3A gene. CONCLUSIONS: These data indicate that more complex mechanisms, apart from possible VCX3A gene participation, are occurring in the genesis of MR in XLI, at least in the sample of Mexican patients analysed.     

Deletion of distal promoter of VCXA in a patient with X-linked ichthyosis associated with borderline mental retardation

BACKGROUND: X-linked ichthyosis (XLI) is caused by deficiency of steroid sulfatase (STS) activity. About 90% XLI patients have large deletions involving the entire STS gene and flanking regions. Recently, VCXA, which is located approximately 0.7Mb telomeric to the STS gene, was reported as a candidate gene for mental retardation (MR) in patients with XLI. OBJECTIVE: To delineate the X-chromosomal deletion of a XLI patient with borderline mental retardation. METHODS: We carried out FISH analysis to show that the whole STS gene is deleted, and PCR analysis for fine-scale deletion mapping. RESULTS: The deleted segment is approximately 1.6Mb in size, and includes the entire STS and VCXB1 genes. VCXA itself is intact, but its promoter is deleted. CONCLUSION: A deletion that includes the VCXA promoter is associated with borderline mental retardation in a patient with XLI.     

X性联锁遗传鱼鳞病一家系的STS基因研究

目的 研究一 X性联锁遗传鱼鳞病(XLI)家系基因突变,探讨基因突变与临床表现的关系,为进一步开展基因诊断和基因治疗奠定基础。方法 应用PCR方法扩增家系中的先证者及其母亲及与该家系无关的50例正常人外周血基因组DNA STS基因的第一外显子和第十外显子。以角蛋白hHb6为引物,作内对照。结果 家系中先证者的STS基因全部缺失,而先证者之母和与该家系无关的50例正常人未发现缺失。先证者及先证者之母的内对照引物PCR扩增后都有产物。结论 该XLI家系存在STS基因缺失,该缺失引发出XLI特有的皮肤病变。     

X‐linked ichthyosis: Clinical and molecular findings in 35 Italian patients

Recessive X‐linked ichthyosis (XLI), the second most common ichthyosis, is caused by mutations in the STS gene encoding the steroid sulfatase enzyme. A complete deletion of the STS gene is found in 85%‐90% of cases. Rarely, larger deletions involving contiguous genes are detected in syndromic patients. We report the clinical and molecular genetic findings in a series of 35 consecutive Italian male patients. All patients underwent molecular testing by MLPA or aCGH, followed, in case of negative results, by next‐generation sequencing analysis. Neuropsychiatric, ophthalmological and paediatric evaluations were also performed. Our survey showed a frequent presence of disease manifestations at birth (42.8%). Fold and palmoplantar surfaces were involved in 18 (51%) and 7 (20%) patients, respectively. Fourteen patients (42%) presented neuropsychiatric symptoms, including attention‐deficit hyperactivity disorder and motor disabilities. In addition, two patients with mental retardation were shown to be affected by a contiguous gene syndrome. Twenty‐seven patients had a complete STS deletion, one a partial deletion and 7 carried missense mutations, two of which previously unreported. In addition, a de novo STS deletion was identified in a sporadic case. The frequent presence of palmoplantar and fold involvement in XLI should be taken into account when considering the differential diagnosis with ichthyosis vulgaris. Our findings also underline the relevance of involving the neuropsychiatrist in the multidisciplinary management of XLI. Finally, we report for the first time a de novo mutation which shows that STS deletion can also occur in oogenesis.     

rAAV2-mediated restoration of LEKTI in LEKTI-deficient cells from Netherton patients

Background

Netherton syndrome (NS, MIM 256500) is a potential live threatening autosomal-recessive skin disorder clinically characterized by the trias of congenital erythroderma, hair shaft anomalies and atopic diathesis. It is caused by mutations in the gene SPINK5 resulting in a deficiency of its processed protein named lympho-epithelial Kazal-type related inhibitor (LEKTI). LEKTI controls the activity of several serine proteases in the skin that are involved in terminal differentiation. Loss of LEKTI results in protease hyperactivity, increased degradation of intercellular junctions, reduced stratum corneum adhesion and impaired skin barrier function. Today NS can only be treated symptomatically.

Objective

Does gene transfer offer a therapeutic option for NS in the future?

Methods

A recombinant adeno-associated virus type 2 vector was constructed containing the full length cDNA (rAAV2/C-SPINK5) of functional human LEKTI. Infectious virus particles were used for transfection of LEKTI-deficient-keratinocytes of NS patients in vitro.

Results

Gene transfer of SPINK5 in NS-keratinocytes led to a five-fold increase in mRNA expression of SPINK5 reaching almost 75% of normal value. The functionality of the expressed LEKTI was proven in a hydrolytic activity assay demonstrating that the activity of LEKTI after gene transfer increased closely to the level seen in keratinocytes of healthy individuals.

Conclusion

The results provide first evidence that gene transfer of SPINK5 results in increased LEKTI activity in NS-keratinocytes, thus offering a rational to further pursue such a gene therapy approach for NS.     

Quantification of activated and total caspase-14 with newly developed ELISA systems in normal and atopic skin

Background

Activation of caspase-14 occurs during terminal differentiation of keratinocytes and may play a role in filaggrin degradation. Therefore, down-regulation of caspase-14 may lead to impaired barrier function.

Objective

To compare the levels of active and total caspase-14 in healthy subjects in various age groups and in patients with atopic dermatitis (AD), using two enzyme-linked immunoassay (ELISA) systems.

Methods

We established four clones of monoclonal antibodies to caspase-14 and used clone 3 as the immobilizing antibody. A cleavage site-directed antibody, h14D146 [4] was used for specific quantification of active caspase-14 in extracts of tape-stripped corneocytes. Total caspase-14 was measured with a commercial antibody, H-99.

Results

The amount of caspase-14 remained constant (ca. 0.1% of extractable proteins) in healthy males from their twenties to their fifties. Caspase-14 was mostly in active form (71-94%) in these extracts. In contrast, caspase-14 level and active caspase-14 ratio were significantly decreased in females in their fifties and sixties. Contents of free amino acids were decreased in females in their sixties, and transepidermal water loss was increased in females in their forties and sixties. In patients with AD, active caspase-14 was markedly down-regulated compared to age-matched controls in both lesional (7.5%) and non-lesional skin (10.6%). Staining of active caspase-14 was considerably weaker in non-lesional skin and was hardly detectable in lesional skin with parakeratosis.

Conclusion

Our new ELISA systems are effective tools to quantify activation of caspase-14. Our results indicate a role of caspase-14 in epidermal barrier function.