FGFR3 and PIK3CA mutations are involved in the molecular pathogenesis of solar lentigo

Background  Solar lentigines (SL) are frequent benign skin lesions appearing on sun-exposed areas especially in elderly people and therefore represent a hallmark of (photo)aged skin. It has been proposed that SL may subsequently evolve into adenoid seborrhoeic keratosis (SK). However, little is known about the genetic basis of SL. In human SK, FGFR3 and PIK3CA mutations have recently been identified.
Objectives  To analyse SL for potential FGFR3 and PIK3CA mutations.
Methods  We screened 30 SL for FGFR3 mutations using a SNaPshot^® multiplex assay. For PIK3CA mutations we used direct sequencing of exon 9 and a SNaPshot^® assay for the H1047R hotspot mutation (exon 20). Because psoralen plus ultraviolet A (PUVA) lentigines show the V600E BRAF hotspot mutation, we additionally investigated this mutation in SL by allele-specific polymerase chain reaction.
Results  FGFR3 mutations were detected in five of 30 (17%) SL and PIK3CA mutations in two of 28 (7%) SL. None of 28 SL available for BRAF analysis revealed the V600E mutation.
Conclusions  Our results suggest that FGFR3 and PIK3CA mutations are involved in the pathogenesis of SL. The occurrence of these mutations in both SL and SK suggests a common genetic basis. Our findings furthermore substantiate previous speculations that UV exposure may be a causative factor for FGFR3 and PIK3CA mutations in human skin.     

FGFR3 and PIK3CA mutations in stucco keratosis and dermatosis papulosa nigra

Background Stucco keratosis (STK) and dermatosis papulosa nigra (DPN) are referred to as variants of seborrhoeic keratosis. However, the genetic alterations involved in the pathogenesis of these benign tumours are unknown. Objectives Because FGFR3 and PIK3CA mutations have been reported to be involved in the pathogenesis of seborrhoeic keratosis, we analysed whether these mutations are also present in STK and DPN. Methods A SNaPshot^® multiplex assay was used for analysis of 11 previously described FGFR3 hotspot mutations. PIK3CA mutations were analysed by a SNaPshot^® assay covering five PIK3CA hotspot mutations. Results Five STK and two DPN samples were analysed. Three of five STK samples revealed a PIK3CA mutation (E542K, E545K), but no FGFR3 mutation was found. In contrast, both DPN samples harboured an FGFR3 mutation (R248C, S249C) but no PIK3CA mutation. Control tissues available for three samples did not show PIK3CA or FGFR3 mutations, excluding germline mutations and indicating a strong genotype–phenotype correlation between the mutation and the lesion. Conclusions These results indicate that FGFR3 and PIK3CA mutations are involved in the pathogenesis of STK and DPN. The molecular genetic findings furthermore support the concept that both skin lesions are specific variants of seborrhoeic keratosis, sharing a common genetic background.     

High frequency of FGFR3 mutations in adenoid seborrheic keratoses

FGFR3 germline mutations cause autosomal dominant skeletal disorders including achondroplasia, thanatophoric dysplasia, severe achondroplasia with developmental delay and acanthosis nigricans, and Crouzon syndrome. Somatic mutations of FGFR3 have been identified in bladder cancer, multiple myeloma, and other neoplasms. FGFR3 mutations have also been detected in 40% of seborrheic keratoses (SKs) of the hyperkeratotic and acanthotic subtype, which are very common benign skin tumors. Using a multiplex SNaPshot assay that covers 11 activating FGFR3 mutations, we investigated a series of 27 SKs of the adenoid subtype. Mutations were detected in 23 of 27 (85%) adenoid SKs. R248C mutations were the most frequent mutation type. In two SKs, the A393E mutation was found, which has not been described in acanthotic and hyperkeratotic SKs so far. Three adenoid SKs displayed two simultaneous FGFR3 mutations. Adenoid SKs seem to be characterized by a higher frequency of FGFR3 mutations than hyperkeratotic and acanthotic SKs. The mechanism for the high rate of somatic FGFR3 mutations in these benign skin tumors remains elusive, but UV light exposure may play a potential role, especially in the R248C mutations.     

FGFR3, PIK3CA and RAS mutations in benign lichenoid keratosis

Background Benign lichenoid keratoses (BLKs) are solitary skin lesions which have been proposed to represent a regressive form of pre‐existent epidermal tumours such as solar lentigo or seborrhoeic keratosis. However, the genetic basis of BLK is unknown. Objectives FGFR3, PIK3CA and RAS mutations have been shown to be involved in the pathogenesis of seborrhoeic keratosis and solar lentigo. We thus investigated whether these mutations are also present in BLK. Methods After manual microdissection and DNA isolation, 52 BLKs were screened for FGFR3, PIK3CA and RAS hotspot mutations using SNaPshot^® multiplex assays. Results We identified 6/52 (12%) FGFR3 mutations, 10/52 (19%) PIK3CA mutations, 6/52 (12%) HRAS mutations and 2/52 (4%) KRAS mutations. FGFR3 and RAS mutations were mutually exclusive. One BLK showed a simultaneous PIK3CA and HRAS mutation. In nine BLKs with a mutation, nonlesional control tissue from the epidermal margin and the dermal lymphocytic infiltrate were wild‐type, indicating that these mutations are somatic. To demonstrate that these findings are specific, 10 samples of lichen planus were analysed without evidence for FGFR3, PIK3CA or RAS mutations. Conclusions Our results indicate that FGFR3, PIK3CA and RAS mutations are present in approximately 50% of BLKs. These findings support the concept on the molecular genetic level that at least a proportion of BLKs represents regressive variants resulting from former benign epidermal tumours such as seborrhoeic keratosis and solar lentigo.     

Activation of the PI3K/AKT signalling pathway in non‐melanoma skin cancer is not mediated by oncogenic PIK3CA and AKT1 hotspot mutations

Please cite this paper as: Activation of the PI3K/AKT signalling pathway in non‐melanoma skin cancer is not mediated by oncogenic PIK3CA and AKT1 hotspot mutations. Experimental Dermatology 2010; 19 : e222–e227. Abstract: Non‐melanoma skin cancer represents the most frequent human cancer entity. Activation of the PI3K/AKT signalling pathway has been reported both in squamous cell carcinoma (SCC) of the skin and in basal cell carcinoma (BCC). In many cancers, including SCC of the head and neck, the oesophagus and the penis, activation of this pathway is mediated by oncogenic PIK3CA and AKT1 mutations. We therefore screened 61 non‐melanoma skin cancer samples (30 SCC and 31 BCC) for the presence of activating PIK3CA and AKT1 mutations. PIK3CA hotspot mutations were analysed using a highly sensitive SNaPshot assay, and exon 4 of AKT1 was sequenced directly. In addition, immunohistochemistry was performed for phosphorylated AKT protein. Immunohistochemical expression of pAkt was observed both in SCC and in BCC samples, indicating an activation of the PI3K/AKT pathway. Although SCC showed higher expression levels than BCC, this difference was not significant. However, none of the 61 non‐melanoma skin cancer samples revealed any PIK3CA and AKT1 hotspot mutations at the investigated loci. We conclude that PIK3CA and AKT1 hotspot mutations do not contribute to the activation of the PI3K/AKT signalling pathway in non‐melanoma skin cancer. The distinct PIK3CA mutation spectrum between SCC of the skin and SCC of other tissues may reflect the different carcinogens which are involved into the mutagenesis of these cancers. PIK3CA and AKT1 hotspot mutations are obviously not caused by UV light exposure, the main risk factor in non‐melanoma skin cancer.     

Spectrum of FGFR3 mutations in multiple intraindividual seborrheic keratoses

Somatic FGFR3 mutations have recently been identified in seborrheic keratoses (SK). Affected individuals often show a large number of SK, but their spectrum of FGFR3 mutations has not been investigated yet. We analyzed 78 SK of four patients using a SNaPshot multiplex assay. FGFR3 mutations were detected in 46 of 78 SK (59%). The mutation rates of the patients ranged from 26 to 89%. Each patient showed at least four different mutated loci. FGFR3 mutations appear to be common genetic alterations in multiple SK with a varying interindividual mutation frequency but without specific intraindividual hot spots.     

Malignant melanoma in patients with hereditary nonpolyposis colorectal cancer

Background  Malignant melanoma (MM) is the most aggressive skin cancer. Most MMs are sporadic, and in this setting an association with mismatch repair (MMR) gene mutations, typical of hereditary nonpolyposis colorectal cancer (HNPCC) tumours, has been proposed.
Objectives  To characterize clinically and/or by molecular biology the patients with MM belonging to a cohort of 60 kindreds with HNPCC.
Methods  Patients with HNPCC with a diagnosis of MM were studied by immunohistochemistry (IHC) on tumour tissue using antibodies to MLH1, MSH2, p16, β-catenin and E-cadherin, and by direct sequencing of MMR genes on germline DNA, and BRAF and NRAS on somatic DNA extracted from MM.
Results  Nine cutaneous MMs were detected in the tumour spectrum of eight families with HNPCC. The median age at diagnosis was 46 years. In one HNPCC family the diagnosis of MM was made in two first-degree relatives fitting the clinical definition of familial melanoma. IHC and sequencing analysis showed an MSH2 mutation in one patient with MM.
Conclusions  Dermatological surveillance should be recommended to families in which MM is diagnosed in at least one member, especially at a young age. The combination of MMR gene mutations and abnormalities of p16 or other molecular pathways is needed to induce melanocytic carcinogenesis in a familial setting as well as in sporadic MM.     

CHILD syndrome in 3 generations: the importance of mild or minimal skin lesions

BACKGROUND: CHILD syndrome (congenital hemidysplasia with ichthyosiform nevus and limb defects, Online Mendelian Inheritance in Man 308050) is an X-linked dominant trait with lethality for male embryos. The disorder is caused by mutations in NSDHL (Online Mendelian Inheritance in Man 300275), a gene playing an important role in the cholesterol biosynthetic pathway. Most reports deal with sporadic cases, and only 5 cases of mother-to-daughter transmission have been documented. We present here a family with mild features of CHILD syndrome in 3 generations. Molecular analysis was used to confirm the diagnosis. OBSERVATIONS: We studied 14 members of a family with CHILD syndrome. The 23-year-old proposita, her mother, 2 aunts, and her grandmother presented with mild or minimal skin lesions that had been present since infancy. Analysis of the NSDHL gene showed missense mutation c.370G-->A in these 5 patients. This mutation was absent in the 9 clinically unaffected family members tested. CONCLUSIONS: In this family, we recognized CHILD syndrome with mild or minimal features in 3 generations because we were able to verify our clinical diagnosis by means of molecular analysis. We assume that many cases that so far have been considered sporadic may in fact be familial when a meticulous physical examination of female family members is combined with molecular testing.     

A novel splice-site mutation in the CDH3 gene in hypotrichosis with juvenile macular dystrophy

Background.  Hypotrichosis with juvenile macular dystrophy (HJMD) is a rare autosomal recessive disorder characterized by sparse hair on the scalp and early blindness. Mutations in the CDH3 gene have been reported to underlie HJMD.
Aim.  To identify a gene responsible for HJMD in a large, four-generation Pakistani family.
Methods.  Genotyping of 13 members of the family, including 6 affected and 7 unaffected members, was carried out using polymorphic microsatellite markers closely linked to the CDH3 on chromosome 16q22.1. To screen for mutations in the CDH3 gene, all of its exons and splice junctions were amplified using PCR from genomic DNA and sequenced directly, using an automated DNA sequencer.
Results.  Microsatellite analysis showed linkage of the family to the CDH3 gene on chromosome 16q22.1. Sequence analysis of the CDH3 gene revealed a novel splice-site mutation (IVS10–1 G→T), leading to probable skipping of exon 11 and a shift in the reading frame.
Conclusion.  The mutation identified here represents the first mutation in the CDH3 gene causing HJMD in a Pakistani population.     

Deregulation of the tumour suppressor genes p14ARF, p15INK4b, p16INK4a and p53 in basal cell carcinoma

Background  Basal cell carcinoma (BCC) is a locally aggressive slowly growing tumour that rarely metastasizes and is mostly seen in older members of the population.
Objectives  To determine the involvement of the tumour suppressor genes p14^ARF, p15^INK4b, p16^INK4a and p53 in BCC.
Methods  We investigated the integrity of the CDKN2A locus in 15 BCC samples by analysing the presence of allelic imbalance/loss of heterozygosity (LOH). Moreover, we studied the mRNA expression levels of the tumour suppressor genes p14^ARF, p15^INK4b, p16^INK4a and p53 in the BCC samples and compared them with mRNA levels in the corresponding normal tissue. The presence of mutations was examined by sequencing for exons 1a and 2 of p16^INK4a.
Results  We found LOH in one BCC sample for the marker D9S1748. A polymorphism (G442A) of exon 2 was detected in three cases. p14^ARF, p15^INK4b and p53 presented high expression levels, whereas p16^INK4a exhibited low mRNA levels compared with the corresponding normal tissue. Significant correlations were detected among the genes studied.
Conclusions  Our results demonstrate a different expression profile between p16^INK4a and p14^ARF, p15^INK4b and p53 in BCC. Moreover, we found a low percentage of LOH and of a polymorphic sequence variant (Ala148Thr) for the CDKN2A locus.     

Analysis of the CDKN2A and CDK4 genes and HLA-DR and HLA-DQ alleles in two Spanish familial melanoma kindreds

Some confusion exists in the literature about which criteria should be used to define familial melanoma. This could explain the different reported frequencies of mutations in predisposing genes, mostly CDKN2A, in these patients. This study evaluated the human leucocyte antigen (HLA) class II genotype and the presence of mutations in CDKN2A and CDK4 genes in 2 families with very different clinical features. The family with a germinal mutation in exon 2 of CDKN2A (Gly101Try) presented the following clinical features: 3 first-degree affected members, 1 of whom had 2 melanomas, and all the melanomas appearing before 35 years of age. In contrast, the second family did not present any mutation in the studied genes and included 2 first-degree affected members diagnosed at over 45 years of age. Neither family showed an association with HLA genotype. Other genes are also involved in familial melanoma but, when the CDKN2A gene is affected, some clinical features seem to be uniform.     

BRAF and KRAS mutations in sporadic glomus tumors

Glomus tumors are rare soft tissue neoplasms resembling the normal glomus body, which is a specialized form of arteriovenous anastomosis that regulates heat. The molecular genetics of sporadic glomus tumors has not been studied. We genotyped tumors from 28 patients (16 female patients and 12 male patients) ranging from 13 to 77 years and correlated the results with the tumor site (15 finger/1 hand/4 arm/7 leg/1 eyelid), Ki-67 index, and clinical follow-up. Tumor DNA from paraffin-embedded tissue was screened by multiplex polymerase chain reaction and mass spectroscopy, using a panel covering 370 mutations across 30 genes, including AKT1, BRAF, CTNNB1, EGFR, ERBB2, FGFR1/2/3, HRAS, KIT, KRAS, MEK1/2, NRAS, PDGFRA, and PIK3CA. A BRAF V600E mutation was identified in 3 cases, all of which occurred in proximal locations (upper shin, thigh, and upper arm). Two of the patients with BRAF-mutated tumors were quite young (21 and 13 years) and one of the BRAF-mutated tumors recurred in 3 years. A KRAS G12A mutation was found in tumor removed from the finger. Ki-67 index did not correlate with genotype. To our knowledge, this is the first report of oncogenic mutations in sporadic glomus tumors.     

A novel mutation of CYLD in a Chinese family with multiple familial trichoepithelioma

Background Trichoepithelioma is a benign cutaneous tumour that originates from hair follicles and occurs either as a sporadic non-familial or a multiple-familial type. Recently, several mutations in the cylindromatosis (CYLD) gene have been reported in multiple familial trichoepithelioma (MFT). Objectives To report a Chinese family of MFT and to explore the genetic mutation. Methods A Chinese pedigree of typical MFT was subjected to mutation detection in CYLD. Direct sequencing of all PCR products of the whole coding regions of CYLD gene was performed to identify the mutation. Results The c.1178-1179delCA (p.T393fs) mutation was found in CYLD gene in the affected members, but not in the healthy individuals in the family. Conclusion Our study found a novel mutation in exon 10 of CYLD gene.     

Activation of fibroblast growth factor receptor 3 and oncogene-induced senescence in skin tumours

Background  The activation of oncogenes is an important step in tumorigenesis, and recently, oncogene-induced senescence (OIS) was proposed as a critical barrier against malignant transformation in normal primary cells.
Objectives  The aim of this study was to examine the activation of fibroblast growth factor receptor 3 (FGFR3) as an oncogene product and OIS in human skin tumours.
Methods  We investigated the activation of FGFR3 and OIS by mutation and immunohistochemical analysis in skin tumours, including seborrhoeic keratosis, actinic keratosis (AK), Bowen's disease (BD), basal cell carcinoma (BCC) and squamous cell carcinoma (SCC).
Results  Activated point mutations of FGFR3 were identified in four of 22 cases (18%) of seborrhoeic keratosis, but no mutation was detected in the other skin tumours. Twenty-seven of 31 cases (87%) of seborrhoeic keratosis showed moderately to strongly positive expression of the FGFR3 protein, but almost all the other skin tumours were negative. On the other hand, almost all the seborrhoeic keratoses showed negative immunoreactivity for antiphoshohistone H2AX (γ-H2AX) as a marker of OIS, but 17 of 22 cases (77%) of AK were moderately to strongly positive. Immunoreactivity for γ-H2AX was significantly greater in AK than in seborrhoeic keratosis, BD, BCC and SCC.
Conclusions  The activation of FGFR3 might be a common feature in the tumorigenesis in seborrhoeic keratosis, although the activation does not induce a typical oncogenic signal in keratinocytes. In addition, OIS due to some oncogenic signals rather than activation of FGFR3 might be involved in the early skin carcinogenesis related to chronic ultraviolet radiation exposure.     

Paternal germline mosaicism in Herlitz junctional epidermolysis bullosa

We studied a single patient with the lethal (Herlitz) type of junctional epidermolysis bullosa (H-JEB). Screening for mutations in the LAMB3 gene in the patient revealed the previously described hotspot mutation R635X and a novel one basepair deletion in exon 10. The single basepair deletion 1094delA could be detected in the clinically unaffected mother, while the nonsense mutation R635X could not be found in the peripheral blood DNA of either parent. After excluding non-paternity by microsatellite analysis using random markers on chromosomes 3, 8 and 18, we determined that the mutation R635X in the proband was most likely the result of a de novo event or alternatively, germline mosaicism. The parents requested prenatal diagnosis for a second pregnancy, and while the maternal mutation 1094delA could not be detected in DNA from the fetus, unexpectedly, the mutation R635X was present in the chorionic villus DNA. These findings were most consistent with paternal germline mosaicism for the recessive mutation R635X. The results have had a significant impact on the genetic counseling in this family. To our knowledge, this study represents the first documented case of germline mosaicism in junctional epidermolysis bullosa, and serves as a reminder that germline mosaicism should be considered in cases in which a 'new' mutation is found in the offspring of a clinically and/or genetically unaffected parent.     

毛囊角化病一家系及三例散发患者ATP2A2基因突变研究

目的：对毛囊角化病（Darier's disease, DD）一家系及三例散发患者进行ATP2A2基因的突变分析。方法：收集先证者及其家系成员、散发病例的临床资料和外周血,采用PCR技术扩增ATP2A2基因所有编码区及侧翼序列,用Sanger法测序检测潜在的突变,选取与患者无亲缘关系的100例健康人作为对照,同时对已报道的ATP2A2基因突变进行文献回顾。结果：家系中三例患者均检测出ATP2A2基因第5号外显子c.380 G>A(p.G127D)新发错义突变;散发患者S1检测出第13号外显子C.1676G>A(p.R559Q)错义突变,散发患者S2检测出第14号外显子c. 2001C>T(p.D667D)同义突变,散发患者S3未检测出突变。结论：本研究中共发现三个突变,其中c.380G>A(p.G127D)在中国人群中首次报道,拓展了ATP2A2的基因突变谱。     

Familial acanthosis nigricans due to K650T FGFR3 mutation

BACKGROUND: Acanthosis nigricans is a feature of several syndromes caused by activating mutations of the fibroblast growth factor receptor 3 gene (FGFR3), including Crouzon syndrome with acanthosis nigricans, thanatophoric dysplasia, and severe achondroplasia with developmental delay and acanthosis nigricans (SADDAN syndrome). OBSERVATIONS: We describe a healthy 4-year-old African American girl with generalized acanthosis nigricans since infancy. Her father had a history of acanthosis nigricans since childhood, in addition to Crohn disease, obesity, and adult-onset diabetes mellitus. A pedigree with numerous affected family members was constructed. Other than slightly short stature, no associated anomalies were found, including dysmorphic features or skeletal or neurologic defects. Genetic testing revealed a previously undescribed, heterozygous lysine to threonine mutation at codon 650 of the FGFR3 gene in the 4 affected family members who were tested. CONCLUSION: Extensive acanthosis nigricans in early childhood, especially with a family history of acanthosis nigricans, may warrant testing for FGFR3 mutations.     

Molecular basis of non-lethal junctional epidermolysis bullosa: identification of a 38 basepair insertion and a splice site mutation in exon 14 of the LAMB3 gene

Abstract: Epidermolysis bullosa (EB) is a group of genodermatoses characterized by fragility and easy blistering of the skin. In the junctional forms of EB (JEB), blisters occur at the level of the lamina lucida, and specific mutations have been detected in the genes encoding different components of the hemidesmosomal-anchoring filament complex. In the non-lethal form of JEB (NL-JEB), mutations in genes encoding two of the polypeptide chains of the anchoring filament protein laminin 5 have recently been described. In this study, we searched for mutations in a family using PCR amplification of exon 14 of LAMB3 , the laminin 5 β₃ chain gene, followed by heteroduplex analysis and automated sequencing of the PCR products. We detected a novel combination of mutations in this family, consisting of an out-of frame insertion on one allele, and a splice site mutation on the other allele, representing the first report of a large insertion in LAMB3 , together with a splice site mutation inherited in trans , which result in the NL-JEB phenotype.     

Acanthosis nigricans and hypochondroplasia in a child with a K650Q mutation in FGFR3

Acanthosis nigricans has been described in several autosomal dominant skeletal dysplasia syndromes due to germline FGFR3 mutations, but rarely specifically in patients with hypochondroplasia. We report a child who presented with extensive acanthosis nigricans, short stature, and radiographic evidence of hypochondroplasia. Genetic analysis revealed a heterozygous K650Q mutation in FGFR3.