Mutation analysis and prenatal diagnosis of keratin 9 gene in a large Chinese family with epidermolytic palmoplantar keratoderma / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze potential mutation in keration 9 (KRT9) gene in a large Chinese family with epidermolytic palmoplantar keratoderma (EPPK) and to perform prenatal diagnosis on the fetus at 10th gestational week.</p><p><b>METHODS</b>Peripheral venous blood samples were obtained from 5 affected and 8 unaffected individuals of the family. Fifty unrelated healthy individuals were also recruited as controls. PCR was used to amplify exons 1 and 6 of KRT9 gene, and the products were sequenced directly. After the mutation was confirmed, prenatal diagnosis was performed on the fetus during the first trimester of pregnancy.</p><p><b>RESULTS</b>A heterozygous missense mutation c.482A to G in the KRT9 gene, which has led to substitution of Asparaginate by Serine at codon 161 (p.N161S), was detected in all patients but not in other individuals of the family and the 50 healthy controls. The fetus was found to have carried the p.N161S mutation too. Following selected abortion, analysis of fetal tissue was consistent with prenatal diagnosis.</p><p><b>CONCLUSION</b>The missense mutation c.482A to G (p.N161S), which has been shown previously to cause EPPK, is found in the KRT9 gene of patients in this family. Gene mutation analysis for prenatal diagnosis is efficient to facilitate detection of affected fetus in time.</p>

Differential protein expressions in breast cancer between drug sensitive tissues and drug resistant tissues / 中南大学学报(医学版)

OBJECTIVE@#To investigate the differential expression of the sensitive and resistant relative proteins in human breast cancer tissue.@*METHODS@#A drug sensitive group and a drug resistant group for chemotherapy in patients with breast cancer were selected through neoadjuvant. The differential protein expression in 2 groups was detected by proteomics techniques, and parts of differential proteins were identified by Western blot.@*RESULTS@#There were 13 differential proteins in the 2 groups, in which the expression of 3 proteins was up-regulated and 10 down-regulated. Seven proteins were identified by Western blot. The expression of keratin type I cytoskeletal 19 (KIC19), thymidine phosphorylase (TYPH) was upregulated, and the expression of heat shock protein 27 (HSP27), keratin type I cytoskeletal 9 (KIC9), collagen alpha-2(VI) (CO6A2), vimentin (VIME), and actin cytoplasmic 1 (ACTB) was down-regulated in the drug resistant group. There was significant difference between the 2 groups (P<0.01).@*CONCLUSION@#The expression of KIC19 and TYPH may be correlated with drug resistance in patients with breast cancer, and HSP27, KIC9, CO6A2, VIME, and ACTB may be correlated with drug sensitivity.

Mutation analysis of keratin 9 gene in a family with epidermolytic palmoplantar keratoderma / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze potential mutation of keration 9 gene (KRT9) in a Chinese family affected with epidermolytic palmoplantar keratoderma (EPPK) and to correlate genotype with the phenotype.</p><p><b>METHODS</b>Genomic DNA was extracted from peripheral blood samples of 12 patients and 13 healthy individuals from the family and 100 unrelated individuals. Polymerase chain reaction (PCR) was used to amplify exons 1 and 6 of KRT9 gene. PCR products were sequenced bidirectionally in order to identify potential mutations.</p><p><b>RESULTS</b>A heterozygous transversional mutation, 488G→A, was identified in exon 1 of KRT9 gene in all patients, which has resulted in substitution of a glutamine residue for arginine acid at position 163 (R163Q) of the KRT9 protein. The same mutation was not found in the 13 healthy members from the family and 100 unrelated individuals.</p><p><b>CONCLUSION</b>The 488G→A mutation of KRT9 gene is probably the cause of EPPK in this Chinese family.</p>

Mutation analysis of a Uighur family with epidermolytic palmoplantar keratoderma / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To map and identify the disease gene for the epidermolytic palmoplantar keratoderma (EPPK) in a Uighur family of China.</p><p><b>METHODS</b>Blood samples were collected and genomic DNA was extracted from 48 members of the Xinjiang Uighur family. Six microsatellite repeat sequences on chromosome region 17q12-q21 and 12q13 were selected based on the two known candidate genes KRT9 and KRT1. Two-point linkage analysis and haplotype analysis were performed. Exons and their flanking intronic sequence of the KRT9 gene were amplified by polymerase chain reaction (PCR) and sequenced.</p><p><b>RESULTS</b>Data from the marker D17S1787 suggested linkage and yielded a Lod score of 8.65 at theta=0 by using MLINK software. Genotypes and haplotypes were acquired. The disease gene of the EPPK family is located between markers 17/TG/36620115 and D17S846. Chromosome 12q13 region was excluded with the negative Lod score obtained in marker D12S96 (Lod=-infinity at theta=0). No pathogenic mutation was detected in the KRT9 gene.</p><p><b>CONCLUSION</b>The disease gene of the EPPK family is located on chromosome region 17q21.2. The keratin 9 gene might not be the disease gene.</p>

Serum proteomic analysis on metastasis-associated proteins of hepatocellular carcinoma / 南方医科大学学报

<p><b>OBJECTIVE</b>To screen the serum proteins associated with the metastasis of hepatocellular carcinoma (HCC) using a comparative proteomic approach.</p><p><b>METHODS</b>The serum samples of HCC patients with the same disease background were divided into metastatic (n=20) and non-metastatic (n=20) groups. The proteins extracted from the patients and 20 normal subjects, after depletion of the highly abundant proteins, underwent two-dimensional gel electrophoresis (2-DE). Comparative analyses of the 2-DE protein patterns between the 3 groups were conducted using a computerized image analysis system. The proteins with statistically significant differential expression between the metastatic and non-metastatic patients were identified by mass spectrometry. Western blotting was performed to examine the differential expression of the candidate proteins.</p><p><b>RESULTS</b>Four protein spots were identified by mass spectrometry among the 12 differentially expressed protein spots in the serum samples of HCC patients with intrahepatic metastasis, and confirmed by searching in MASCOT database. Of the 4 proteins, cytokeratin 9 (CK9) was up-regulated by 2 folds, and inter-alpha (globulin) inhibitor H4, complement factor H-related protein 1 precursor (FHR-1), and apolipoprotein E were down-regulated by 2 folds. CK9 was found to be specifically over-expressed in the metastatic group in comparison with the non-metastatic group, as confirmed by Western blotting.</p><p><b>CONCLUSION</b>The metastasis of HCC might be correlated to the specific variation of protein expression profiles. The overexpression of CK9 may play a crucial role in HCC metastasis, and can be used as a potential serum marker for predicting HCC metastasis.</p>

Mutation analysis of the keratin 9 gene in a pedigree with diffuse epidermolytic plamoplantar keratoderma / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To analyze the mutation of the keratin 9 gene (KRT9) in a pedigree with epidermolytic plamoplantar keratoderma (EPPK).</p><p><b>METHODS</b>Blood samples were obtained from 4 affected and 3 normal individuals in this family. Mutation screening was carried out by polymerase chain reaction (PCR) and direct DNA sequencing.</p><p><b>RESULTS</b>A heterozygous nucleotide C to T transition at position 484 in exon 1 of the KRT9 gene was detected in the 3 affected in this family, but was not found in normal individuals in the family and 100 unrelated individuals.</p><p><b>CONCLUSION</b>A missense mutation (484 C to T) in the KRT9 gene has been detected in this EPPK family, which is probably one of the molecular bases of the pathogenesis of the disease.</p>

The Proteomics Approaches to Find Bio-Markers in Serum of Locally Advanced Breast Cancer Patients / 한국유방암학회지

PURPOSE: Breast cancer continues to be a major cause of death, despite the advances in the study of many prognostic factors. Although many prognostic factors have been studied, none reliably predict the response to treatment. This uncertainty in the prognostic factors could be overcome by defining the changes, occurring in patients at either the gene or protein level. Herein, attempts were made to examine the protein repertoire of patients using Proteomics. MATERIALS AND METHODS: Using conventional Proteomics, the high resolution 2-D electrophoresis followed by computational image analysis(Melanie program) and protein identification with mass spectrometry (MALDI-TOF), the serum of locally advanced breast cancer patients (stage III) was analyzed, and attempts were made to define the differences between recurred (or metastasis) patients ,and disease free patients of more than 4-years duration after surgery. RESULTS: In the 2-D electrophoresis of serum, about 1,000 spots were gained in each gel, with the up and down expressed protein spots compared to the normal control protein map. Six of seven patients had Cytokeratin 9 in their peripheral blood. In the serum of recurred patients (one of two), no Haptoglobin-related proteins were detected. All five un-recurred patients had normal or elevated levl of serum Haptoglobin-related proteins. CONCLUSIONS: The reduction of Haptoglobin-related proteins indicated the humoral immuno-depression in recurred patients. These findings may suggest the continuation of proper humoral immunity was important in the prevention of cancer recurrences or metastasis after surgery, especially in locally advanced breast cancer patients, which may suggests the value of immunotherapy in breast cancer patients to obtain good results.

The Proteomics Approaches to Find Bio-Markers in Serum of Locally Advanced Breast Cancer Patients / 한국유방암학회지

PURPOSE: Breast cancer continues to be a major cause of death, despite the advances in the study of many prognostic factors. Although many prognostic factors have been studied, none reliably predict the response to treatment. This uncertainty in the prognostic factors could be overcome by defining the changes, occurring in patients at either the gene or protein level. Herein, attempts were made to examine the protein repertoire of patients using Proteomics. MATERIALS AND METHODS: Using conventional Proteomics, the high resolution 2-D electrophoresis followed by computational image analysis(Melanie program) and protein identification with mass spectrometry (MALDI-TOF), the serum of locally advanced breast cancer patients (stage III) was analyzed, and attempts were made to define the differences between recurred (or metastasis) patients ,and disease free patients of more than 4-years duration after surgery. RESULTS: In the 2-D electrophoresis of serum, about 1,000 spots were gained in each gel, with the up and down expressed protein spots compared to the normal control protein map. Six of seven patients had Cytokeratin 9 in their peripheral blood. In the serum of recurred patients (one of two), no Haptoglobin-related proteins were detected. All five un-recurred patients had normal or elevated levl of serum Haptoglobin-related proteins. CONCLUSIONS: The reduction of Haptoglobin-related proteins indicated the humoral immuno-depression in recurred patients. These findings may suggest the continuation of proper humoral immunity was important in the prevention of cancer recurrences or metastasis after surgery, especially in locally advanced breast cancer patients, which may suggests the value of immunotherapy in breast cancer patients to obtain good results.

PCR products with heterozygous mutations containing two types of heteroduplexes / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To confirm that PCR products with heterozygous mutations contain not only wide-type and mutant homoduplexes, but also two types of heteroduplexes.</p><p><b>METHODS</b>An insertion-deletion mutation in the exon 1 of KRT9 gene (497delAinsGGCT), which caused Chinese epidermolytic palmoplantar keratoderma (EPPK) was investigated by polymerase chain reaction (PCR), polyacrylamide gel electrophoresis (PAGE) and denaturing high-performance liquid chromatography(DHPLC).</p><p><b>RESULTS</b>Two heteroduplexes and two homoduplexes in the PCR product from the heterozygous mutation of the exon 1 of KRT9 (497delAinsGGCT) were detected.</p><p><b>CONCLUSION</b>PCR products from KRT9 gene with heterozygous mutations contain two types of heteroduplexes. It is without the need to perform heating and cooling PCR products obtained from heterozygous mutations in advance before the mutation screening steps such as denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), conformation-sensitive gel electrophoresis (CSGE), DHPLC and heteroduplex analysis (HA), etc.</p>

Present status of the molecular genetics in epidermolytic palmoplantar keratoderma / 中华医学遗传学杂志

In this article we reviewed the current researches on the molecular basis of epidermolytic palmoplantar keratoderma (EPPK) and the structure and function of the keratins with mutations that can cause inherited keratin disorders. Also summarized are seventeen mutations of keratin 9 in EPPK in different ethnic populations.

DNA-based Prenatal Diagnosis of Epidermolytic Palmoplantar Keratoderma in Two Pregnancies at Risk in One large Pedigree

BACKGROUND: Epidermolytic palmoplantar keratoderma (EPPK) is an autosomal dominant disease of cornification which presents as severe thickening of the palms and soles with prominent epidermolytic hyperkeratosis pathologically. Recent studies have shown that EPPK is caused by mutations in the keratin 9 (K9) gene which is expressed essentially only in the palms and soles. Previously, We have reported that patients in one large pedigree of EPPK have an R162W substitution in the K9 protein. In this pedigree, two women whose husbands are both EPPK patients had become pregnant. OBJECTIVE: Since both women were concerned about this genetic disorder, we have performed prenatal diagnosis by biopsy analysis of chorionic villi tissue. METHODS: Chorionic villi biopsies were performed at 12 weeks gestation. Since the skin lesions are strictly confined to the palms and soles of the babies, the prenatal diagnosis of EPPK by ultrastructural analysis of fetal skin biopsy or amniotic fluid cells is highly problematic. Polymerase chain reaction amplification of specific allele (PASA) assay and direct DNA sequencing analyses were performed whether the fetuses carried mutant allele of K9 gene. RESULTS: PASA assay and direct DNA sequencing analyses showed that one fetus was normal, but the other fetus carried the abnormal allele. Subsequently, the mother of the unaffected fetus delivered a normal child, but the mother of the affected fetus terminated the pregnancy. CONCLUSION: We describe the analysis of the K9 mutation in the two fetuses at risk for EPPK. We believe that this is the first report of prenatal diagnosis for EPPK. But, we have to think about the ethical problems before we decide to perform the prenatal diagnosis of any kind of skin diseases.

DNA - based Prenatal Diagnosis of Epidermolytic Palmoplantar Keratoderma / 대한산부인과학회잡지

OBJECTIVE: The purpose of this investigation was to establish the prenatal diagnosis for identifying the risk for epidermolytic palmoplantar keratoderma(EPPK) of a fetus by sequence analysis of fetal genomic DNA from chorionic villi. METHODS: Chorionic villus sampling under transvaginal sonography at 12 weeks of gestation from a woman at risk for a child in a EPPK-affected family was perfomed. Polymerase chain reaction amplification of specific allele (PASA) assay was carried out for the detection of mutation(R162W in keratin 9 [K9] gene) previously identified in this family. Direct DNA sequencing analysis of K9 gene was accomplished to confirm the mutation. RESULTS: We had found the point mutation, R162W of K9 gene, in affected family members and confirmed by PASA assay. Affected family members were shown to have PCR products reactive with both the mutant and wildtype specific primers. Because we could not find any expected products after PASA assay with the primers la(+)/KSmt(-) of the fetal DNA, we predicted that the fetus did not inherited the mutant allele and that the fetus could be unaffected. After PASA assay, we analyzed DNA sequences of two family members to confirm the mutation. A C-to-T substitution at bp 545 was detected in the father, instead the fetus did not have any mutant band at that base pair. CONCLUSION: The PASA assay and direct DNA sequencing analysis of K9 gene through chorionic villi sampling and extraction of genomic DNA had validity to early prenatal diagnosis whether fetus was affected in EPPK or not.