Mutations in the filament aggregating protein (filaggrin) gene have recently been identified as the cause of the common genetic skin disorder ichthyosis vulgaris (IV), the most prevalent inherited ...disorder of keratinization. The main characteristics of IV are fine-scale on the arms and legs, palmar hyperlinearity, and keratosis pilaris. Here, we have studied six Irish families with IV for mutations in filaggrin. We have identified a new mutation, 3702delG, in addition to further instances of the reported mutations R501X and 2282del4, which are common in people of European origin. A case of a 2282del4 homozygote was also identified. Mutation 3702delG terminates protein translation in filaggrin repeat domain 3, whereas both recurrent mutations occur in repeat 1. These mutations are semidominant: heterozygotes have an intermediate phenotype most readily identified by palmar hyperlinearity and in some cases fine-scale and/or keratosis pilaris, whereas homozygotes or compound heterozygotes generally have more marked ichthyosis. Interestingly, the phenotypes of individuals homozygous for R501X, 2282del4, or compound heterozygous for R501X and 3702delG, were comparable, suggesting that mutations located centrally in the filaggrin repeats are also pathogenic.
Cutaneous squamous cell carcinoma (SCC) is the most prevalent invasive malignancy with metastatic potential. The epidermis is exposed to a variety of environmental DNA-damaging chemicals, principal ...among which are polyaromatic hydrocarbons (PAHs) ubiquitous in the environment, tobacco smoke, and broiled meats. Langerhans cells (LCs) comprise a network of dendritic cells situated adjacent to basal, suprabasal, and follicular infundibular keratinocytes that when mutated can give rise to SCC, and LC-intact mice are markedly more susceptible than LC-deficient mice to chemical carcinogenesis provoked by initiation with the model PAH, 7,12-dimethylbenzaanthracene (DMBA). LCs rapidly internalize and accumulate DMBA as numerous membrane-independent cytoplasmic foci. Repopulation of LC-deficient mice using fetal liver LC-precursors restores DMBA-induced tumor susceptibility. LC expression of p450 enzyme CYP1B1 is required for maximal rapid induction of DNA-damage within adjacent keratinocytes and their efficient neoplastic transformation; however, effects of tumor progression also attributable to the presence of LC were revealed as CYP1B1 independent. Thus, LCs make multifaceted contributions to cutaneous carcinogenesis, including via the handling and metabolism of chemical mutagens. Such findings suggest a cooperative carcinogenesis role for myeloid-derived cells resident within cancer susceptible epithelial tissues principally by influencing early events in malignant transformation.
To identify an allele-specific short interfering RNA (siRNA), against the common KRT12 mutation Arg135Thr in Meesmann epithelial corneal dystrophy (MECD) as a personalized approach to treatment.
...siRNAs against the K12 Arg135Thr mutation were evaluated using a dual luciferase reporter gene assay and the most potent and specific siRNAs were further screened by Western blot. Off-target effects on related keratins were assessed and immunological stimulation of TLR3 was evaluated by RT-PCR. A modified 5' rapid amplification of cDNA ends method was used to confirm siRNA-mediated mutant knockdown. Allele discrimination was confirmed by quantitative infrared immunoblotting.
The lead siRNA, with an IC(50) of thirty picomolar, showed no keratin off-target effects or activation of TLR3 in the concentration ranges tested. We confirmed siRNA-mediated knockdown by the presence of K12 mRNA fragments cleaved at the predicted site. A dual tag infrared immunoblot showed knockdown to be allele-specific, with 70% to 80% silencing of the mutant protein.
A potent allele-specific siRNA against the K12 Arg135Thr mutation was identified. In combination with efficient eyedrop formulation delivery, this would represent a personalized medicine approach, aimed at preventing the pathology associated with MECD and other ocular surface pathologies with dominant-negative or gain-of-function pathomechanisms.
Summary Background Pachyonychia congenita (PC) is a rare autosomal dominant keratin disorder, subdivided into two major variants, PC-1 and PC-2. Predominant characteristics include hypertrophic nail ...dystrophy, focal palmoplantar keratoderma and oral leukokeratosis. Multiple steatocystomas that develop during puberty are a useful feature distinguishing PC-2 from PC-1. At the molecular level it has been shown that mutations in keratin K6a or K16 cause PC-1 whereas those in K6b or K17 lead to PC-2. Objective To identify mutations in 22 families presenting with clinical symptoms of either PC-1/focal non-epidermolytic palmoplantar keratoderma (FNEPPK) or PC-2. Methods Mutation analysis was performed on genomic DNA from PC patients by direct sequencing. Results Here, we report four new missense and five known mutations in K6a; one new deletion and three previously identified missense mutations in K16; plus one known mutation in K17. Conclusion With one exception, all these heterozygous mutations are within the highly conserved helix boundary motif regions at either end of the keratin rod domain. In one sporadic case, a unique mutation in K16 resulting in deletion of 24 bp was found within the central rod domain, in a child with a phenotype predominantly consisting of focal plantar keratoderma. The identification of mutations in cases of PC is prerequisite for future development of gene-specific and/or mutation-specific therapies.
Meesmann epithelial corneal dystrophy (MECD) is an inherited eye disorder caused by dominant-negative mutations in either keratins K3 or K12, leading to mechanical fragility of the anterior corneal ...epithelium, the outermost covering of the eye. Typically, patients suffer from lifelong irritation of the eye and/or photophobia but rarely lose visual acuity; however, some individuals are severely affected, with corneal scarring requiring transplant surgery. At present no treatment exists which addresses the underlying pathology of corneal dystrophy. The aim of this study was to design and assess the efficacy and potency of an allele-specific siRNA approach as a future treatment for MECD.
We studied a family with a consistently severe phenotype where all affected persons were shown to carry heterozygous missense mutation Leu132Pro in the KRT12 gene. Using a cell-culture assay of keratin filament formation, mutation Leu132Pro was shown to be significantly more disruptive than the most common mutation, Arg135Thr, which is associated with typical, mild MECD. A siRNA sequence walk identified a number of potent inhibitors for the mutant allele, which had no appreciable effect on wild-type K12. The most specific and potent inhibitors were shown to completely block mutant K12 protein expression with negligible effect on wild-type K12 or other closely related keratins. Cells transfected with wild-type K12-EGFP construct show a predominantly normal keratin filament formation with only 5% aggregate formation, while transfection with mutant K12-EGFP construct resulted in a significantly higher percentage of keratin aggregates (41.75%; p<0.001 with 95% confidence limits). The lead siRNA inhibitor significantly rescued the ability to form keratin filaments (74.75% of the cells contained normal keratin filaments; p<0.001 with 95% confidence limits).
This study demonstrates that it is feasible to design highly potent siRNA against mutant alleles with single-nucleotide specificity for future treatment of MECD.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Background Polymorphisms in the serine protease inhibitor gene serine peptidase inhibitor Kazal type 5 (SPINK5) and the serine protease kallikrein-related peptidase 7 gene (KLK7) appear to confer ...risk to eczema in some cohorts, but these findings have not been widely replicated. These genes encode proteins thought to be involved in the regulation of posttranslation processing of filaggrin (FLG) , the strongest identified genetic risk factor for eczema to date. Objectives We sought to clarify the individual risk of eczema conferred by the SPINK5 polymorphism rs2303067 (Glu420Lys) and a previously described insertion in the 3′ untranslated region of KLK7 and to examine potential epistatic effects between these variants and FLG mutations. Methods Initially, we examined the effects of these polymorphisms and FLG in 486 unrelated patients from a German family-based study, an additional 287 German patients, and 418 unrelated Irish/English patients with eczema (n for 3 genes studied = 1191 vs 4544 control subjects). We then additionally studied the SPINK5 polymorphism and FLG mutations in 1583 patients with eczema from the Avon Longitudinal Study of Parents and Children cohort (sample size for 2 genes studied = 2774 vs 10,607 control subjects). Results No association was seen with the SPINK5 or KLK7 variants in the case-control analysis; however, a weaker effect was observed for the SPINK5 variant with maternal transmission in the family-based study. No interactions were seen between the polymorphisms in KLK7 , SPINK5 , and FLG. Conclusion The SPINK5 420LysSer mutation confers a risk of eczema when maternally inherited but is not a major eczema risk factor. The KLK7 insertion appears to confer no risk of eczema. We found no interaction between the SPINK5 risk allele or the putative KLK7 risk allele and FLG mutations.
Dowling–Degos disease (DDD) is an autosomal-dominant genodermatosis characterized by reticulate pigmentation of the flexures. By direct DNA sequencing, we have identified a frameshift mutation in ...exon 1 of KRT5 in the proband from an extended Spanish DDD kindred. Cloning of PCR products confirmed that this was a 2-bp deletion mutation, designated c.442delAG, leading to a premature termination codon in the V1 domain of the K5 polypeptide, designated p.S148fsX30. These data confirm that haploinsufficiency for K5 causes DDD and points to a prominent role for the keratin intermediate filament cytoskeleton within basal keratinocytes in epidermal pigment biology.
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.
The systemic complications of acute hematologic emergencies account for the high mortality rates seen during inpatient management. Perhaps the most challenging diagnostic entity among all hematologic ...emergencies is leukostasis. In acute myeloid leukemia (AML), myeloid blasts are often highly adherent to the endothelial vasculature, and high peripheral blood blast count in excess of 100,000 cells per microliter can predispose patients to pulmonary and neurologic complications, leading to rapid clinical deterioration even before a formal diagnosis of leukostasis is made. The mobilization of appropriate healthcare personnel in the inpatient setting at inopportune times sometimes poses a major barrier to the successful treatment of patients with leukostasis, and patients can pass away quickly. In this report, we describe clinico-radio-pathologic correlations of leukostasis using pre- and post-mortem analysis in a patient with AML with a FLT3-TKD mutation, and we describe the current literature on best management approaches based on recent evidence, including consideration of first-line FLT3 (CD135) inhibitors such as quizartinib.
The Genetic Basis of Pachyonychia Congenita Smith, Frances J.D.; Liao, Haihui; Cassidy, Andrew J. ...
The Journal of investigative dermatology symposium proceedings,
10/2005, Letnik:
10, Številka:
1
Journal Article
Recenzirano
Odprti dostop
In 1994, the molecular basis of pachyonychia congenita (PC) was elucidated. Four keratin genes are associated with the major subtypes of PC: K6a or K16 defects cause PC-1; and mutations in K6b or K17 ...cause PC-2. Mutations in keratins, the epithelial-specific intermediate filament proteins, result in aberrant cytoskeletal networks which present clinically as a variety of epithelial fragility phenotypes. To date, mutations in 20 keratin genes are associated with human disorders. Here, we review the genetic basis of PC and report 30 new PC mutations. Of these, 25 mutations were found in PC-1 families and five mutations were identified in PC-2 kindreds. All mutations identified were heterozygous amino acid substitutions or small in-frame deletion mutations with the exception of an unusual mutation in a sporadic case of PC-1. The latter carried a 117 bp duplication resulting in a 39 amino acid insertion in the 2B domain of K6a. Also of note was mutation L388P in K17, which is the first genetic defect identified in the helix termination motif of this protein. Understanding the genetic basis of these disorders allows better counseling for patients and paves the way for therapy development.
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