Gene Mutation Detection And Preliminary Analyses Of Genotype And Phenotype Of Diffuse Palmoplantar Keratoderma And Darier’s Disease

Background1. The Background of EPPKDiffuse palmoplantar keratoderma (DPPK), is one of the most common types of hereditary palmoplantar keratoderma. It is an autosomal dominant inherited disease and develops in all races. DPPK is of infancy onset with only rough skin on palmoplantar or diffuse plaques, clear-edge thickening like yellow callosum. Sometimes thickening can spread to the edges or extensor aspects of palmoplantars and feet accompanied with knuckle pads, thickened and opaque nails and camptodactyly. According to the pathological characteristics, DPPK can be divided into2subtypes (epidermolytic palmoplantar keratoderma called DEPPK or EPPK and diffuse non-epidermolytic palmoplantar keratoderma callled DNEPPK). EPPK (OMIM#144200) is an autosomal dominant inherited skin disorder originally reported by Vorner in1901. EPPK has the similar clinical manifestations of diffuse hyperkeratosis of epidermis with DNEPPK, but there are no epidermis cleavages and loosening keratinocytes in DNEPPK. Also EPPK is more common in races and clinical cases. The candidate gene of EPPK was located on chromosome17q21.1-q21.2by Reis through genetic linkage analysis of German patients with EPPK in1992. This site was the domain of type I keratin gene cluster and keratin9 (KRT9) gene was naturally considered as a candidate gene of EPPK. Keratin is the most common intermediate filament protein, including30kinds of molecules divided into type Ⅰ acidic protein and type Ⅱ neutral/alkaline protein. Keratin molecule is a major component of the intermediate filament in epidermal cells and epithelial cells. Therefore, intermediate filament assembly will be destroyed by keratin mutations, forming abnormal aggregates, which can increase the risk of cell rupture and cause some diseases. KRT9gene mutations are closely related to skin diseases, because KRT9is type Ⅰ acidic protein mostly presenting in palmoplantar skin. So far, it has been reported that25mutations of KRT9gene are associated with EPPK including missense mutations, deletions or insertions.2. The Background of Darier’s Disease(1)The clinical characteristics of the Darier’s disease (DD)DD is a rare autosomal dominant dyskeratosis skin disease. It usually begins at the age of10-20years old with the equal rate between male and female. Epidemiological investigation showed that the morbidity is of1/30000to1/100000. The clinical phenotypes are various and the distribution is symmetric and wide. Lesions occur mainly in the seborrheic areas, such as the forehead, scalp, chest, back. At the beginning, the lesions can be recognized as small, solid, normal skin papules. Then they gradually turn into plaques and warty accompanied with palmoplantar keratoderma(PPK) and typical nail damage. In some condition, it can involve mucous membranes such as the mucosae of oral pharynx, esophagus, larynx, rectal. The lesions are exacerbated in summer and relieved in winter. Histological examinations of DD show typically epidermal hyperkeratosis, focal dyskeratosis, acantholysis and cleavages between suprabasal keratinocytes. There are corps ronds and grains investigated among abnormal keratinized cells. The destruction of desmosome connection, protein filaments aggregation around the cell nucleus and vacuolization in cytoplast can be viewed by electron microscope.(2)The localization of pathogenic gene of DDDD was first reported by Darier and White in1889. In1993, pathogenic gene of DD was located by Bashir and Craddock in chromosome12q23-q24.1, the very gene site of ATP2A2encoding the sarcoplasmic/endoplasmic reticulum Ca2+-ATP enzyme2(SERCA2). SERCA2which was responsible for regulating the concentration of intracellular calcium ion and played a crucial role in the maintenance of cellular calcium signal transduction was a member of the P type calcium ATP enzyme family. ATP2A2gene encoded a defective SERCA2in patients with DD.(3)The characteristics, functions and mutations of ATP2A2geneThe length of ATP2A2gene is about76kb. It consists of21exons and encodes sarcoplasmic/endoplasmic reticulum Ca2+-ATP enzyme2(SERCA2). According to the different splice sites, there are three different transcripts, SERCA2a, SERCA2b and SERCA2c. Their distributions are of tissue specificity. SERCA2a mainly expresses in heart, brain and slow skeletal muscle contraction. SERCA2b mainly distributes in muscle, skin and other muscle tissues. SERCA2c can be detected in hematopoietic cells and mononuclear cells. The structure of SERCA2includes the cervical region, actuator domain, phosphorylation site, ATP binding region and10or11transmembrane helical regions (M1-10/11). M4, M5, M6, M8have the Ca2+-binding sites. The hinge region combines the ATP binding region and the membrane protein, forming a complete functional unit. SERCA2which is responsible for regulating the concentration of intracellular calcium ion and plays a crucial role in the maintenance of cellular calcium signal transduction is a member of the P type calcium ATP enzyme family. SERCA2maintains a low level of calcium in the cytoplasm by transporting calcium ion from cytoplasmic to the endoplasmic reticulum. Intracellular calcium plays an important role in regulation of cell differentiation and the connection of cells. Mutations in the ATP2A2gene cause functional disruption of SERCA2(the calcium pump), including a reduction of ATP affinity, a loss of calcium affinity, effects on the phosphorylation of ATP, blocking of dephosphorylation and uncoupling levels. The mutations of ATP2A2are scattered over the entire gene, and there is no mutational hotspots, although there are two mutation clusters, which are located in the amino terminal upstream stalk and S1, and in the carboxyl-terminal transmembrane regions. However, there are also some reports showing that mutations of ATP2A2associated with DD distribute in the middle region of the gene, especially phosphorylation and ATP binding region.ObjectiveTo detect the KRT9mutations in Chinese family with EPPK and analysis clinical features of EPPK and mutation screening results in order to obtain more information of genotype and phenotype. Review KRT9gene mutation cases reported by previous literature, attempting to find out the relationship between mutation sites and clinical phenotype.To detect pathogenic mutations of ATP2A2gene in the two sporadic cases of DD and analyze clinical features and mutation results in order to obtain more information of genotype and phenotype.Methods(1)Family investigation and Clinical data collection:We collected Clinical data of a Chinese family with EPPK and two patients with DD, then carried out family investigation.(2)Histopathological examination:We took biopsy tissues from the skin lesions of the proband’s left palm and the patient2with DD by operation. We then carried out pathological examination.(3)Genomic DNA extraction:We collected peripheral blood samples of the proband and her brother, the family members with EPPK, the two patients with DD and100normal healthy individuals. DNA was extracted from these samples.(4)PCR reaction:The hot spots of KRT9gene, the first exons including intron-exon boundaries, and21exons of the ATP2A2gene including intron-exon boundaries were amplified by polymerase chain reaction (PCR).(5)1%agarose gel electrophoresis:The DNA product was assessed by1%Agarose gel electrophoresis after PCR reaction. The DNA product can be sent to DNA sequencing when a single bright band was seen in the electrophoresis.(6)DNA sequencing:Gene mutations of the proband, the proband’s brother, the two patients with DD and normal healthy individuals were detected. Finally, we summarized the clinical characteristics and analyzed their mutation screening results.(7)We reviewed keratin9gene mutations in previous reports referring to Human Intermediate Filament Database(http://www.interfil.org/) and the United States National Center for Biotechnology Information (http://www.ncbi.nlm.nih. gov)Results(1) Clinical characteristics:The proband was a32-year-old female who presented with hyperkeratosis on the palms and soles from infancy. About half month after her birth, she began to develop erythema, scale on both palms and soles, and then it turned into diffuse yellow thickening and hyperkeratosis, with knuckle pad-like keratoses on the finger joints and camptodactyly. Some involved nails of her toes were thickened and opaque. Family investigation:There were a total of16people of4generations, including6patients. The minimum patient was1and a half years old and the mean age of onset was about half a month after birth. They had the similar clinical manifestations.(2) A missense mutation, c.470T>G, expected to transform the corresponding amino acid methionine into arginine (p.M157R), was found out in the two patients by the detection of mutation hot spots of KRT9gene. But the same mutation could not be found in the100unrelated healthy individuals.(3) So far, mutations of KRT9gene reported previously gathered around exon1and exon6. The most common mutation site was the487th nucleotide of cDNA. There were25mutations,19in the1A region and6in the2B domain. Besides the deletion of A in the500th nucleotide of cDNA resulting in the insertion of four nucleotides (c.500delAinsGGCT), which were expected to transform the corresponding amino acid tyrosine to tryptophan and leucine (p.Y167delinsWL), the main mutation pattern was missense mutation.. Genotype-phenotype analysis shows that according to the current literature, the cases with mutations in the163rd amino acids of KRT9gene display typical clinical manifestations and pathological features. In addition, the KRT1mutations could also lead to some clinical manifestations similar to palmoplantar keratoderma.(4)Clinical characteristics of the patients with DD Two patients taking part in our study had no family history of a similar disease. One was a25-year-old male from Hunan province with more than20years of medical history. The lesions of small, hemispheric, skin color or light red papules mainly presented on the skin of his forehead, oxter and groin. His condition was slight but recrudescent, which was exacerbated in summer sometimes with pruritus. He was treated with some oral medicines by himself with a partial response. He had a period of neuropsychiatric abnormalities history six years ago, but no nail, palmar or mucosal signs.The other was a46-year-old male from Hunan province with multiple lesions characterized by severe follicular hyperkeratotic papules, warty papules or plaques in the regions ranging from the forehead, cheek, neck, chest, both the front shoulders, abdomen and lumbosacral portion, in agreement with the typical features of DD. The lesions were also exacerbated in summer and relieved in winter. His condition was severe and recrudescent for20years.(5)Two novel missense mutations (c.2098A>G, c.742C>A) of ATP2A2gene were found out in the two sporadic cases, but cannot be detected in100healthy individuals.(6) Genotype-phenotype analyses Mutations of ATP2A2gene were different in the two DD cases. And the severity of the disease and their age were different, too. Literature analysis showed that phenotypes and age could be different in the same fanmily with the same mutation. Therefore, we could not find significant correlation between clinical phenotype and genotype.Conclusion(1)The missense mutation of KRT9detected in this pedigree with EPPK was not single nucleotide polymorphism (SNP) but a pathogenic mutation. The novel mutations found in the two sporadic cases are not the SNP of ATP2A2but pathogenic mutations and also the novel mutations in the wold.(2)Our mutation screening results further enriched the genotype and phenotype database of keratosic skin genetic diseases and also laid the foundation of future antenatal genetic counseling, prenatal diagnosis and gene therapy.