- Email: [email protected]
A Novel Deletion Mutation in Keratin 5 Causing the Removal of 5 Amino Acids and Elevated Mutant mRNA Levels in Dowling–Meara Epidermolysis Bullosa Simplex
124:5 MAY 2005
distribution in case and control groups, respectively, from HWE can have considerable consequences for conclusions made in all studies. Alarmed through the raised objection that we might have misinterpreted our data, we carefully considered Gyo¨rffy et al’s arguments. In our study, we focused on the genetic variability of seven loci that are considered to be related to a risk for psoriasis (Richter-Hintz et al, 2003). In the case of two loci, GSTM and NQOR, Gyo¨rffy et al found deviations from HWE and stated that our conclusions are not tenable. But tests for HWE are not available that can be applied to dominant-recessive markers with only two alleles, e.g., GST. The genotype distribution of the NQOR locus indeed deviated significantly from HWE, but the risk of mistyping genotypes can increase the probability of making type II errors (i.e., erroneously accepting deviations from HWE). Assuming that only two rare NQOR homozygotes are not classified correctly and using the Bonferroni correction for multiple testing, no significant deviation from HWE was found (p ¼ 0.028). Therefore, we conclude that Gyo¨rffy et al’s criticism based on their meta-analysis has no solid basis for all of the reported cases. Nevertheless, we agree with Gyo¨rffy et al’s statement that genotypic data of case–control studies must first be tested for deviations from HWE and results should be reported. Deviations from HWE can point to either a sampling bias, mistyping of genotypes, or spurious gene associations because of population stratification. If genotype frequencies of both groups do not deviate significantly from HWE, there are no serious concerns about the genetic structure of samples. If only the genotype distribution of the patient group shows deviation from HWE, this may provide additional support for an association of the marker locus with
LETTERS TO THE EDITOR
1083
the disease in question. If, however, the genotype frequencies found in the control group deviate from HWE, data must be reanalyzed carefully for all the reasons mentioned above. Finally, although it would be best to analyze first the control group genetic testing in clinical research has to fit into practical possibilities. Nevertheless, we want to stress that a case–control design can be an optimal choice for many human genetic studies when appropriate statistics and population genetic concepts are applied (Schulze and McMahon, 2002). Charlotte Esser and Ju¨rgen Tomiukw Institute for Environmental Medical Research (IUF), Du¨sseldorf, Germany; wInstitute of Human Genetics, University of Tu¨bingen, Tu¨bingen, Germany
DOI: 10.1111/j.0022-202X.2005.23709.x Manuscript received December 20, 2004; revised January 11, 2005; accepted for publication January 13, 2005 Address correspondence to: Dr Charlotte Esser, IUF, Auf’m, Hennekamp 50, 40225 Du¨sseldorf, Germany. Email: [email protected]
References Gyo¨rffy B, Kocsis I, Vasarhelyi B: Missed calculations and new conclusions: Recalculation of genotype distribution data published in Journal of Investigative Dermatology, 1998–2003. J Invest Dermatol 122:644–646, 2004 Richter-Hintz D, Thier R, Steinwachs S, et al: Allelic variants of drug metabolizing enzymes as risk factors in psoriasis. J Invest Dermatol 120:765–770, 2003 Schulze TG, McMahon FJ: Genetic association mapping at the crossroads: Which test and why? Overview and practical guidelines. Am J Med Genet 114:1–11, 2002
A Novel Deletion Mutation in Keratin 5 Causing the Removal of 5 Amino Acids and Elevated Mutant mRNA Levels in Dowling–Meara Epidermolysis Bullosa Simplex To the Editor: Epidermolysis bullosa simplex (EBS) is one of the three broad groups into which cases of Epidermolysis bullosa are classified on the basis of clinical, histological, and genetic evidence obtained from the affected patient (Pulkkinen et al, 1999). EBS is further classified into three subgroups, EBS Weber–Cockayne (EBS-WC), EBS Koebner (EBS-K), and EBS Dowling–Meara (EBS-DM), on the basis of the severity and distribution of the symptoms, the age of onset, and the ultrastructural investigation of the epidermis and basement membrane zone.
Abbreviations: EBS, epidermolysis bullosa simplex; GAPDH, glycerylaldehyde-3-phosphate; KRT5, keratin 5; KRT14, keratin 14; qPCR, quantitative polymerase chain reaction
The phenotypic manifestations of the different subtypes of EBS have been extensively reviewed elsewhere, as has the experimental evidence initially linking keratins 5 (KRT5) and 14 (KRT14) mutations to EBS (Fine et al, 1991; IshidaYamamato, 1991; Vassar, 1991; Fine et al, 2000). The characteristic bullae or blisters seen in EBS result from the lysis of unusually fragile basal keratinocytes as a result of mutations in the genes encoding KRT5 and KRT14. EBS-DM is considered the most severe form of EBS with widespread herpetiform blistering, frequent extracutaneous involvement, and evidence of tonofilament clumping at the electron microscope level in basal keratinocytes (Irvine et al, 1999; Fine et al, 2000). Here we report the identification of a novel mutation in the 2B helical region of KRT5 in a three-generation nonconsanguinous Australian family of caucasian extraction (Fig 1). The proband was the only member of the family to
1084 LETTERS TO THE EDITOR
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Figure 2 Schematic representation of novel DRNKLA identified in proband.
Figure 1 Clinical Features of mature affected proband. (A) Hyperkeratosis on sole, (B) blister on upper arm, and (C) pedigree of affected family. Filled symbol indicates proband.
exhibit clinical symptoms of EBS, presenting at birth with herpetiform blistering involving the limbs, trunk, and face. Primers and PCR conditions used for the ampification of KRT5 exons from genomic DNA extracted from peripheral blood leucocytes were as reported previously (Stephens et al, 1997). The study was approved by the institutional ethics committee of the Southeast Sydney Area Health Service and conducted according to the Declaration of Helsinki principles. All patients and family members signed informed consents. Two 3 mm punch biopsies were taken from the underarm skin of the proband in locations previously shown to be affected by blistering and immediately immersed in RNAlater transport solution (Qiagen, Hilden, Germany). Total cellular RNA was used as a template for cDNA production, from which the expression of the mutation was confirmed and quantified using quantitative polymerase chain reaction (qPCR). The novel mutation detected in exon 7 of KRT5 in this pedigree resulted in the deletion of five amino acid residues from the 2B domain of K5, 6 residues downstream of the 2B discontinuity (KRT5 429delRNKLA, K5 2B:69-73 del) (Fig 2). The phenotypic expression of EBS-DM in this case has shown significant amelioration in both severity and distribution with age (Fig 1). No other mutations were found in KRT14 or the remaining KRT5 exons. The analysis (by direct sequencing) of both the phenotypically normal parents excluded the mutation in the other family members. Analysis by gel electrophoresis separating the smaller allele containing the deletion from the wild-type allele excluded the mutation in 50 unaffected, unrelated controls, showing that this mutation is found only in the affected proband. Using the 15 nucleotide difference between the wild-type and mutant alleles, differential primers
were designed to selectively amplify either wild-type or mutant alleles. Utilizing qPCR, RNA samples derived from 3 mm skin punch biopsies showed that, normalized against the housekeeping gene GAPDH, the mutant transcript was present at a level 2.7-fold greater than that of the wild-type allele. qPCR calculations were performed using the DDCT method as previously described, and melting curve analysis was used to ensure the amplification of a single PCR product (Livak et al, 2001). The 2B region of the KRT5 protein is one of the four a helical segments that comprises the rod domain. Each of these four segments possesses a repeating heptad amino acid residue motif (a–b–c–d–e–f–g)n, where a and d are generally predicted to be positions occupied by apolar residues (North et al, 1994). The 2B region is thought to be highly important in facilitating the lateral alignment of keratin molecules as a prelude to the formation of the KRT5/KRT14 obligate heterodimers, which form the basis of the basal keratinocyte intermediate filaments. The 2B segment differs from the remaining three in that the heptad repeat breaks down and stutters in the middle of the segment (North et al, 1994). The phenotype of the proband expressing clinically diagnosable EBS-DM in this study has exhibited significant amelioration of severity with age. (Fig 1). Normal daily activities are now generally unimpeded by blister formation, which is now characterized by the less frequent occurrence of grouped blisters. In other forms of EBS, three previous non-missense keratin mutations have been reported to lead to a range of phenotypes: (1) a mutation in the 30 splice acceptor site of KRT14 exon 1 leading to the development of a highly truncated keratin protein within the 1B helical domain and severe recessive EBS, (2) a donor splice site mutation in KRT5 resulting in the removal of 22 amino acids from the H1 and 1A rod domains and the expression of EBS-DM, (3) a 3 nucleotide deletion in the 2B domain of KRT14, resulting in the development of EBS-WC (refer to www.interfil.org for a comprehensive summary of EBS mutations). This is a report of a non-missense mutation in the 2B domain of KRT5, which leads to the development of EBSDM. Interestingly, the deletion of a relatively large sequence
124:5 MAY 2005
of amino acid residues has, in this instance, led to the development of a surprisingly mild phenotype. This is in direct contrast to a number of cases in which a much smaller genetic lesion has led to the development of persistently severe EBS-DM (Cummins et al, 2001). The alignment of six individual-type 2 keratins (KRT 1, 3, 4, 5, 7, 8) showed that the RNKLA motif is poorly conserved across these 6 individual proteins, with only lysine (K) at codon 431 being absolutely conserved. A review of the literature shows that no EBS-causing mutations have been reported between codons 429 and 433 in KRT5. On the basis of this information, it is possible that this region does not play an important functional role, such that the mutation seen in this family may not cause significant disruption to normal intermediate filament assembly and function, at least later in life. Analysis of this region indicates that this region is able to accommodate the helical interruption within a small region of the protein (North et al, 1994). Even though the loss of five amino acids from an exon is a significant genetic lesion, it is possible in this case that the location of the lesion (in an area of low sequence conservation that has been shown to be able to accommodate other helical disruptions) and the form of the mutation (the mutation is in-frame and does not affect the downstream translation of the exon) contribute to reducing the effect of the deletion and result in the expression of a mild disease phenotype. The greater significance of the qPCR data in this case is unknown; however, the finding that the mutant allele is present (at the RNA level) in the cell at 2.7 times the level of the normal allele disproved our initial hypothesis that the disease phenotype was mild due to lack of mutant allele expression. Further investigation into the effect of the abnormal mutant allele mRNA level identified may assist in delineating the correlation between genotype and phenotype, which continues to increase in complexity as more novel EBS mutations of this nature are identified and reported. M. W. Kemp,z S. Klingberg,w L. Lloyd,w T. J. Molloy,z P. Marr,y Y. Wang,z G. A. C. Murrell,z and D. F. Murrell Department of Dermatology, The St George Hospital Campus, University of New South Wales, Sydney, NSW, Australia; wDepartment of
LETTERS TO THE EDITOR
1085
Chemical Pathology, Queensland Health Pathology Service, Royal Brisbane Hospital Campus, QLD, Australia; zOrthopaedic Research Institute, The St George Hospital Campus, University of New South Wales, Sydney, NSW, Australia; yDepartment of Anatomical Pathology, St George Hospital, Sydney, NSW, Australia
DOI: 10.1111/j.0022-202X.2005.23739.x Manuscript received August 2, 2004; revised December 17, 2004; accepted for publication January 12, 2005 Address correspondence to: De´de´e Murrell, MD, Department of Dermatology, The St George Hospital, Kogarah, Sydney, NSW 2217, Australia. Email: [email protected]
References Cummins RE, Klingberg S, Wesley J, Rogers M, Zhao Y, Murrell DF: Keratin 14 point mutations at codon 119 of helix 1A resulting in different epidermolysis bullosa simplex phenotypes. J Invest Dermatol 117: 1103–1107, 2001 Fine JD, Bauer EA, Briggaman RA, et al: Revised clinical and laboratory criteria for subtypes of inherited epidermolysis bullosa. J Am Acad Dermatol 24:119–135, 1991 Fine JD, Eady RAJ, Bauer EA, et al: Revised classification system for inherited epidermolysis bullosa; Report of the second international consensus meeting on diagnosis and classification of epidermolysis bullosa. J Am Acad Dermatol 42:1051–1066, 2000 Irvine AD, McLean WHI: Human keratin diseases: The increasing spectrum of disease and subtlety of the phenotype–genotype correlation. Br J Dermatol 140:815–828, 1999 Ishida-Yamamoto A, McGrath JA, Chapman SJ, et al: Epidermolysis bullosa simplex (Dowling–Meara type) is a genetic disease characterized by an abnormal keratin-filament network involving keratins K5 and K14. J Invest Dermatol 97:959–968, 1991 Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real time quantitative PCR and the 2DDCt Method. Methods 25:402–408, 2001 North ACT, Steinert PM, Parry DAD: Colied coil and link segments in keratin and other intermediate filament molecules: A computer modeling study. Proteins 20:174–184, 1994 Pulkkinnen L, Uitto J: Mutation analysis and molecular genetics of epidermolysis bullosa. Matrix Biol 18:29–42, 1999 Stephens K, Ehrlich P, Weaver M, Le R, Spencer A, Sybert VP: Primers for exon specific amplification of the KRT5 gene; identification of novel and recurrent mutations in epidermolysis bullosa simplex patients. J Invest Dermatol 108:349–353, 1997 Vassar R, Coulombe PA, Degenstein L, et al: Mutant keratin expression in transgenic mice causes marked abnormalities resembling a human genetic skin disease. Cell 64:365–380, 1991
Single Nucleotide Polymorphisms of the Fibroblast Growth Factor Receptor 2 Gene in Patients with Chronic Venous Insufficiency with Leg Ulcer To the Editor: Chronic, non-healing leg ulcers, frequently attributed to chronic venous insufficiency result in an enormous deAbbreviations: BEK, bacterially expressed kinase; CVI, chronic venous insufficiency; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; KGFR, keratinocyte growth factor receptor; SNP, single nucleotide polymorphism; UTR, untranslated region
crease in the quality of patients’ life. Several factors are suspected to play a role in the pathomechanism of prolonged wound healing in these patients, but the exact pathogenesis of the disease is still unknown (Stucker et al, 2003). In this study, we aimed to identify single nucleotide polymorphism (SNP) in the fibroblast growth factor receptor-2 (FGFR-2) gene that might contribute to the pathogenesis of prolonged wound healing in leg ulcer patients.
distribution in case and control groups, respectively, from HWE can have considerable consequences for conclusions made in all studies. Alarmed through the raised objection that we might have misinterpreted our data, we carefully considered Gyo¨rffy et al’s arguments. In our study, we focused on the genetic variability of seven loci that are considered to be related to a risk for psoriasis (Richter-Hintz et al, 2003). In the case of two loci, GSTM and NQOR, Gyo¨rffy et al found deviations from HWE and stated that our conclusions are not tenable. But tests for HWE are not available that can be applied to dominant-recessive markers with only two alleles, e.g., GST. The genotype distribution of the NQOR locus indeed deviated significantly from HWE, but the risk of mistyping genotypes can increase the probability of making type II errors (i.e., erroneously accepting deviations from HWE). Assuming that only two rare NQOR homozygotes are not classified correctly and using the Bonferroni correction for multiple testing, no significant deviation from HWE was found (p ¼ 0.028). Therefore, we conclude that Gyo¨rffy et al’s criticism based on their meta-analysis has no solid basis for all of the reported cases. Nevertheless, we agree with Gyo¨rffy et al’s statement that genotypic data of case–control studies must first be tested for deviations from HWE and results should be reported. Deviations from HWE can point to either a sampling bias, mistyping of genotypes, or spurious gene associations because of population stratification. If genotype frequencies of both groups do not deviate significantly from HWE, there are no serious concerns about the genetic structure of samples. If only the genotype distribution of the patient group shows deviation from HWE, this may provide additional support for an association of the marker locus with
LETTERS TO THE EDITOR
1083
the disease in question. If, however, the genotype frequencies found in the control group deviate from HWE, data must be reanalyzed carefully for all the reasons mentioned above. Finally, although it would be best to analyze first the control group genetic testing in clinical research has to fit into practical possibilities. Nevertheless, we want to stress that a case–control design can be an optimal choice for many human genetic studies when appropriate statistics and population genetic concepts are applied (Schulze and McMahon, 2002). Charlotte Esser and Ju¨rgen Tomiukw Institute for Environmental Medical Research (IUF), Du¨sseldorf, Germany; wInstitute of Human Genetics, University of Tu¨bingen, Tu¨bingen, Germany
DOI: 10.1111/j.0022-202X.2005.23709.x Manuscript received December 20, 2004; revised January 11, 2005; accepted for publication January 13, 2005 Address correspondence to: Dr Charlotte Esser, IUF, Auf’m, Hennekamp 50, 40225 Du¨sseldorf, Germany. Email: [email protected]
References Gyo¨rffy B, Kocsis I, Vasarhelyi B: Missed calculations and new conclusions: Recalculation of genotype distribution data published in Journal of Investigative Dermatology, 1998–2003. J Invest Dermatol 122:644–646, 2004 Richter-Hintz D, Thier R, Steinwachs S, et al: Allelic variants of drug metabolizing enzymes as risk factors in psoriasis. J Invest Dermatol 120:765–770, 2003 Schulze TG, McMahon FJ: Genetic association mapping at the crossroads: Which test and why? Overview and practical guidelines. Am J Med Genet 114:1–11, 2002
A Novel Deletion Mutation in Keratin 5 Causing the Removal of 5 Amino Acids and Elevated Mutant mRNA Levels in Dowling–Meara Epidermolysis Bullosa Simplex To the Editor: Epidermolysis bullosa simplex (EBS) is one of the three broad groups into which cases of Epidermolysis bullosa are classified on the basis of clinical, histological, and genetic evidence obtained from the affected patient (Pulkkinen et al, 1999). EBS is further classified into three subgroups, EBS Weber–Cockayne (EBS-WC), EBS Koebner (EBS-K), and EBS Dowling–Meara (EBS-DM), on the basis of the severity and distribution of the symptoms, the age of onset, and the ultrastructural investigation of the epidermis and basement membrane zone.
Abbreviations: EBS, epidermolysis bullosa simplex; GAPDH, glycerylaldehyde-3-phosphate; KRT5, keratin 5; KRT14, keratin 14; qPCR, quantitative polymerase chain reaction
The phenotypic manifestations of the different subtypes of EBS have been extensively reviewed elsewhere, as has the experimental evidence initially linking keratins 5 (KRT5) and 14 (KRT14) mutations to EBS (Fine et al, 1991; IshidaYamamato, 1991; Vassar, 1991; Fine et al, 2000). The characteristic bullae or blisters seen in EBS result from the lysis of unusually fragile basal keratinocytes as a result of mutations in the genes encoding KRT5 and KRT14. EBS-DM is considered the most severe form of EBS with widespread herpetiform blistering, frequent extracutaneous involvement, and evidence of tonofilament clumping at the electron microscope level in basal keratinocytes (Irvine et al, 1999; Fine et al, 2000). Here we report the identification of a novel mutation in the 2B helical region of KRT5 in a three-generation nonconsanguinous Australian family of caucasian extraction (Fig 1). The proband was the only member of the family to
1084 LETTERS TO THE EDITOR
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Figure 2 Schematic representation of novel DRNKLA identified in proband.
Figure 1 Clinical Features of mature affected proband. (A) Hyperkeratosis on sole, (B) blister on upper arm, and (C) pedigree of affected family. Filled symbol indicates proband.
exhibit clinical symptoms of EBS, presenting at birth with herpetiform blistering involving the limbs, trunk, and face. Primers and PCR conditions used for the ampification of KRT5 exons from genomic DNA extracted from peripheral blood leucocytes were as reported previously (Stephens et al, 1997). The study was approved by the institutional ethics committee of the Southeast Sydney Area Health Service and conducted according to the Declaration of Helsinki principles. All patients and family members signed informed consents. Two 3 mm punch biopsies were taken from the underarm skin of the proband in locations previously shown to be affected by blistering and immediately immersed in RNAlater transport solution (Qiagen, Hilden, Germany). Total cellular RNA was used as a template for cDNA production, from which the expression of the mutation was confirmed and quantified using quantitative polymerase chain reaction (qPCR). The novel mutation detected in exon 7 of KRT5 in this pedigree resulted in the deletion of five amino acid residues from the 2B domain of K5, 6 residues downstream of the 2B discontinuity (KRT5 429delRNKLA, K5 2B:69-73 del) (Fig 2). The phenotypic expression of EBS-DM in this case has shown significant amelioration in both severity and distribution with age (Fig 1). No other mutations were found in KRT14 or the remaining KRT5 exons. The analysis (by direct sequencing) of both the phenotypically normal parents excluded the mutation in the other family members. Analysis by gel electrophoresis separating the smaller allele containing the deletion from the wild-type allele excluded the mutation in 50 unaffected, unrelated controls, showing that this mutation is found only in the affected proband. Using the 15 nucleotide difference between the wild-type and mutant alleles, differential primers
were designed to selectively amplify either wild-type or mutant alleles. Utilizing qPCR, RNA samples derived from 3 mm skin punch biopsies showed that, normalized against the housekeeping gene GAPDH, the mutant transcript was present at a level 2.7-fold greater than that of the wild-type allele. qPCR calculations were performed using the DDCT method as previously described, and melting curve analysis was used to ensure the amplification of a single PCR product (Livak et al, 2001). The 2B region of the KRT5 protein is one of the four a helical segments that comprises the rod domain. Each of these four segments possesses a repeating heptad amino acid residue motif (a–b–c–d–e–f–g)n, where a and d are generally predicted to be positions occupied by apolar residues (North et al, 1994). The 2B region is thought to be highly important in facilitating the lateral alignment of keratin molecules as a prelude to the formation of the KRT5/KRT14 obligate heterodimers, which form the basis of the basal keratinocyte intermediate filaments. The 2B segment differs from the remaining three in that the heptad repeat breaks down and stutters in the middle of the segment (North et al, 1994). The phenotype of the proband expressing clinically diagnosable EBS-DM in this study has exhibited significant amelioration of severity with age. (Fig 1). Normal daily activities are now generally unimpeded by blister formation, which is now characterized by the less frequent occurrence of grouped blisters. In other forms of EBS, three previous non-missense keratin mutations have been reported to lead to a range of phenotypes: (1) a mutation in the 30 splice acceptor site of KRT14 exon 1 leading to the development of a highly truncated keratin protein within the 1B helical domain and severe recessive EBS, (2) a donor splice site mutation in KRT5 resulting in the removal of 22 amino acids from the H1 and 1A rod domains and the expression of EBS-DM, (3) a 3 nucleotide deletion in the 2B domain of KRT14, resulting in the development of EBS-WC (refer to www.interfil.org for a comprehensive summary of EBS mutations). This is a report of a non-missense mutation in the 2B domain of KRT5, which leads to the development of EBSDM. Interestingly, the deletion of a relatively large sequence
124:5 MAY 2005
of amino acid residues has, in this instance, led to the development of a surprisingly mild phenotype. This is in direct contrast to a number of cases in which a much smaller genetic lesion has led to the development of persistently severe EBS-DM (Cummins et al, 2001). The alignment of six individual-type 2 keratins (KRT 1, 3, 4, 5, 7, 8) showed that the RNKLA motif is poorly conserved across these 6 individual proteins, with only lysine (K) at codon 431 being absolutely conserved. A review of the literature shows that no EBS-causing mutations have been reported between codons 429 and 433 in KRT5. On the basis of this information, it is possible that this region does not play an important functional role, such that the mutation seen in this family may not cause significant disruption to normal intermediate filament assembly and function, at least later in life. Analysis of this region indicates that this region is able to accommodate the helical interruption within a small region of the protein (North et al, 1994). Even though the loss of five amino acids from an exon is a significant genetic lesion, it is possible in this case that the location of the lesion (in an area of low sequence conservation that has been shown to be able to accommodate other helical disruptions) and the form of the mutation (the mutation is in-frame and does not affect the downstream translation of the exon) contribute to reducing the effect of the deletion and result in the expression of a mild disease phenotype. The greater significance of the qPCR data in this case is unknown; however, the finding that the mutant allele is present (at the RNA level) in the cell at 2.7 times the level of the normal allele disproved our initial hypothesis that the disease phenotype was mild due to lack of mutant allele expression. Further investigation into the effect of the abnormal mutant allele mRNA level identified may assist in delineating the correlation between genotype and phenotype, which continues to increase in complexity as more novel EBS mutations of this nature are identified and reported. M. W. Kemp,z S. Klingberg,w L. Lloyd,w T. J. Molloy,z P. Marr,y Y. Wang,z G. A. C. Murrell,z and D. F. Murrell Department of Dermatology, The St George Hospital Campus, University of New South Wales, Sydney, NSW, Australia; wDepartment of
LETTERS TO THE EDITOR
1085
Chemical Pathology, Queensland Health Pathology Service, Royal Brisbane Hospital Campus, QLD, Australia; zOrthopaedic Research Institute, The St George Hospital Campus, University of New South Wales, Sydney, NSW, Australia; yDepartment of Anatomical Pathology, St George Hospital, Sydney, NSW, Australia
DOI: 10.1111/j.0022-202X.2005.23739.x Manuscript received August 2, 2004; revised December 17, 2004; accepted for publication January 12, 2005 Address correspondence to: De´de´e Murrell, MD, Department of Dermatology, The St George Hospital, Kogarah, Sydney, NSW 2217, Australia. Email: [email protected]
References Cummins RE, Klingberg S, Wesley J, Rogers M, Zhao Y, Murrell DF: Keratin 14 point mutations at codon 119 of helix 1A resulting in different epidermolysis bullosa simplex phenotypes. J Invest Dermatol 117: 1103–1107, 2001 Fine JD, Bauer EA, Briggaman RA, et al: Revised clinical and laboratory criteria for subtypes of inherited epidermolysis bullosa. J Am Acad Dermatol 24:119–135, 1991 Fine JD, Eady RAJ, Bauer EA, et al: Revised classification system for inherited epidermolysis bullosa; Report of the second international consensus meeting on diagnosis and classification of epidermolysis bullosa. J Am Acad Dermatol 42:1051–1066, 2000 Irvine AD, McLean WHI: Human keratin diseases: The increasing spectrum of disease and subtlety of the phenotype–genotype correlation. Br J Dermatol 140:815–828, 1999 Ishida-Yamamoto A, McGrath JA, Chapman SJ, et al: Epidermolysis bullosa simplex (Dowling–Meara type) is a genetic disease characterized by an abnormal keratin-filament network involving keratins K5 and K14. J Invest Dermatol 97:959–968, 1991 Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real time quantitative PCR and the 2DDCt Method. Methods 25:402–408, 2001 North ACT, Steinert PM, Parry DAD: Colied coil and link segments in keratin and other intermediate filament molecules: A computer modeling study. Proteins 20:174–184, 1994 Pulkkinnen L, Uitto J: Mutation analysis and molecular genetics of epidermolysis bullosa. Matrix Biol 18:29–42, 1999 Stephens K, Ehrlich P, Weaver M, Le R, Spencer A, Sybert VP: Primers for exon specific amplification of the KRT5 gene; identification of novel and recurrent mutations in epidermolysis bullosa simplex patients. J Invest Dermatol 108:349–353, 1997 Vassar R, Coulombe PA, Degenstein L, et al: Mutant keratin expression in transgenic mice causes marked abnormalities resembling a human genetic skin disease. Cell 64:365–380, 1991
Single Nucleotide Polymorphisms of the Fibroblast Growth Factor Receptor 2 Gene in Patients with Chronic Venous Insufficiency with Leg Ulcer To the Editor: Chronic, non-healing leg ulcers, frequently attributed to chronic venous insufficiency result in an enormous deAbbreviations: BEK, bacterially expressed kinase; CVI, chronic venous insufficiency; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; KGFR, keratinocyte growth factor receptor; SNP, single nucleotide polymorphism; UTR, untranslated region
crease in the quality of patients’ life. Several factors are suspected to play a role in the pathomechanism of prolonged wound healing in these patients, but the exact pathogenesis of the disease is still unknown (Stucker et al, 2003). In this study, we aimed to identify single nucleotide polymorphism (SNP) in the fibroblast growth factor receptor-2 (FGFR-2) gene that might contribute to the pathogenesis of prolonged wound healing in leg ulcer patients.