Transversion In Codon Research Articles

Genetic repair of a human induced pluripotent cell line from patient with Dutch-type cerebral amyloid angiopathy

Dutch-type cerebral amyloid angiopathy (D-CAA), also known as hereditary cerebral haemorrhage with amyloidosis-Dutch type (HCHWA-D), is an autosomal dominant disorder caused by a G to C transversion in codon 693 of the amyloid precursor protein (APP) that results in a Gln-to-Glu amino acid substitution. CRISPR-Cas9 editing was used for genetic correction of the mutation in a human induced pluripotent stem cell (hiPSC-) line established previously. The isogenic hiPSCs generated showed typical pluripotent stem cell morphology, expressed all markers of undifferentiated state, displayed a normal karyotype and had the capacity to differentiate into the three germ layers.

Molecular Characterization of Microrna Interference and Aristolochic Acid Intoxication Found in Upper Tract Urothelial Carcinoma in Patients with Balkan Endemic Nephropathy: A Systematic Review of the Current Literature.

The term "aristolochic acid nephropathy" (AAN) is used to include any form of toxic interstitial nephropathy that is caused either by ingestion of plants containing aristolochic acids (AA) or by the environmental contaminants in food such as in Balkan endemic nephropathy (BEN). Aristolochic acid (AA) intoxication is strongly associated with the development of upper tract urothelial carcinoma (UTUC); however, the underlying molecular mechanism remains to be defined. MicroRNAs (miRNA) regulate several biological processes, including cell proliferation, differentiation, and metabolism, acting as oncogenes or tumor suppressors. A unique miRNA expression profile suggested that miRNAs could function as regulators in UTUC developmental processes. This review aimed to summarize data available in the literature about underlying molecular mechanisms leading to the expression of miRNAs in AA-UTUC patients with BEN. Strong correlation in AA-UTUC has a distinctive gene alteration pattern, AL-DNA adducts, and a unique tumor protein (TP53) mutational spectrum AAG to TAG (A: T→T: A) transversion in codon 139 (Lys → Stop) of exon 5 activates the p53 tumor suppressor protein. Further, p53 protein is responsible not only for the expression of miRNAs but also acts as a target molecule for miRNAs and plays a crucial function in the AA-UTUC pathogenicity through activation of cyclin-dependent kinase (CyclinD1) and cyclin protein kinase 6(CDK6) to support cell cycle arrest. This study, proposed a molecular mechanism that represented a possible unique relationship between AA intoxication, miRNAs expression, and the progression of UTUC in patients with BEN.

Oxidative DNA damage drives carcinogenesis in MUTYH-associated-polyposis by specific mutations of mitochondrial and MAPK genes

MUTYH is a DNA-base-excision-repair gene implicated in the activation of nuclear and mitochondrial cell-death pathways. MUTYH germline mutations cause an inherited polyposis, MUTYH-associated-polyposis, characterized by multiple adenomas and increased susceptibility to colorectal cancer. Since this carcinogenesis remains partially unknown, we searched for nuclear and mitochondrial gene alterations that may drive the tumorigenic process. Ninety-six adenomas and 7 carcinomas from 12 MUTYH-associated-polyposis and 13 classical/attenuated adenomatous polyposis patients were investigated by sequencing and pyrosequencing for the presence of mutations in KRAS, BRAF, MT-CO1/MT-CO2 and MT-TD genes. KRAS mutations were identified in 24% MUTYH-associated-polyposis vs 15% classical/attenuated familial polyposis adenomas; mutated MUTYH-associated-polyposis adenomas exhibited only c.34G>T transversions in codon 12, an alteration typically associated with oxidative DNA damage, or mutations in codon 13; neither of these mutations was found in classical/attenuated familial polyposis adenomas (P<0.001). Mutated MUTYH-associated-polyposis carcinomas showed KRAS c.34G>T transversions, prevalently occurring with BRAFV600E; none of the classical/attenuated familial polyposis carcinomas displayed these alterations. Comparing mitochondrial DNA from lymphocytes and adenomas of the same individuals, we detected variants in 82% MUTYH-associated-polyposis vs 38% classical/attenuated familial polyposis patients (P=0.040). MT-CO1/MT-CO2 missense mutations, which cause aminoacid changes, were only found in MUTYH-associated-polyposis lesions and were significantly associated with KRAS mutations (P=0.0085). We provide evidence that MUTYH-associated-polyposis carcinogenesis is characterized by the occurrence of specific mutations in both KRAS and phylogenetically conserved genes of mitochondrial DNA which are involved in controlling oxidative phosphorylation; this implies the existence of a colorectal tumorigenesis in which changes in mitochondrial functions cooperate with RAS-induced malignant transformation.

Combined point mutations in codon 12 and 13 of KRAS oncogene in prostate carcinomas

Prostate cancer is a common malignancy that develops by structural mutation(s) and/or other genetic alterations in specific genes.The G to T transversions in codon 12 and C to T transitions in codon 13 of KRAS proto-oncogene are predominant point mutations that occur in about 20% of different cancers in human. In the current study it was aimed to investigate the prevalence and predictive significance of KRAS mutations in patients with prostate carcinomas. In a total of 30 fresh tumoural tissue specimens were investigated in patients with prostate carcinoma. All tumoural specimens were histo-pathologically diagnosed and genotyped for codon 12, 13 KRAS point mutations by reverse hybridisation and direct sequencing methods. KRAS mutations were found in 12 (40%) samples with 29 samples deriving from adenocarcinomas and 1 sample was small cell prostate carcinoma. In 1 (3.44%) sample codon 12 was found to be mutated and in 2 (6.8%) samples codon 13 and in 9 (31%) samples combined codon 12 and 13 were found to be mutated particularly in higher grade of tumoural tissues. Our study, based on representative collection of human prostate tumours, indicates that combined mutations in codons 12 and 13 KRAS are relatively infrequent and most commonly occur in prostate carcinomas.

Novel mutations of endothelin-B receptor gene in Pakistani patients with Waardenburg syndrome

Mutations in EDNRB gene have been reported to cause Waardenburg-Shah syndrome (WS4) in humans. We investigated 17 patients with WS4 for identification of mutations in EDNRB gene using PCR and direct sequencing technique. Four genomic mutations were detected in four patients; a G to C transversion in codon 335 (S335C) in exon 5 and a transition of T to C in codon (S361L) in exon 5, a transition of A to G in codon 277 (L277L) in exon 4, a non coding transversion of T to A at -30 nucleotide position of exon 5. None of these mutations were found in controls. One of the patients harbored two novel mutations (S335C, S361L) in exon 5 and one in Intronic region (-30exon5 A>G). All of the mutations were homozygous and novel except the mutation observed in exon 4. In this study, we have identified 3 novel mutations in EDNRB gene associated with WS4 in Pakistani patients.

Identification of two novel missense mutations in the KAL1 gene in Han Chinese subjects with Kallmann Syndrome

To identify mutations in the KAL1, the KAL2, and PROKR2/PROK2 genes and to characterize phenotypic features in 5 Chinese subjects with Kallmann Syndrome (KS) and 6 subjects with normosmic hypogonadotrophic hypogonadism (NHH) in Taiwan. Five unrelated males (age range 22-52 yr) with clinical manifestations of KS and 6 unrelated males (age range 24-47 yr) with NHH were analyzed. In addition, 5 relatives of KS subjects were also evaluated. Genomic DNA extraction, PCR, and DNA sequence analyses were performed using standard procedures. The 1st patient had a single missense mutation in his copy of the KAL1 gene, a T→G transversion in codon 134 that results in replacement of cysteine by gly cine. The 2nd affected subject had a single missense mutation in the KAL1 gene, a T→C transition in codon 163 that results in replacement of cysteine by arginine. The 3rd case was hemizygous for a nonsense mutation in codon 424 of exon 9 (c.CGA→TGA) of the KAL1 gene. This mutation predicts a markedly truncated protein. Two of the mutations (p.C134G and p.C163R) we identified in the KAL1 gene are novel. We identified 3 mutations, including 2 novel mutations, in the KAL1 gene in patients with KS in Taiwan. These data extend the variety of KAL1 gene mutations in KS and further define the role of the KAL1 protein in olfactory bulb development.

Sequence-Specific Conversion of βA- to βS-Globin by Small Fragment Homologous Replacement In Hematopoietic Stem/Progenitor Cells.

AbstractAbstract 3754 Introduction:An ultimate goal of gene therapy is the development of effective strategies to correct mutant genomic sequences in pathologic cells. To that end, studies have been undertaken to evaluate the therapeutic potential of an oligo/polynucleotide-based sequence-specific gene modification strategy, small fragment homologous replacement (SFHR) in the correction of the mutation giving rise to sickle cell anemia. Small DNA fragments (SDFs) comprising the sickle cell anemia mutation (an A>T transversion in codon 6) and flanking DNA sequences in the human b-globin gene were introduced into Hematopoietic Stem/Progenitor Cells (HSPCs). The studies presented indicated modification at the level of DNA, RNA, and protein when cells were differentiated into erythrocytes. Methods:In this study, SFHR was used to convert A>T in codon 6 of the b-globin gene in CD34+/CD38-/Lin- HSPCs isolated from full term umbilical cord blood as a proof of principle. HSPCs were transfected with a defined number of a 559-bp SDF using the Amaxa electroporation (nucleofection) system. After growing the transfected cells in stem cell media containing EPO for different time intervals up to 32 days, RNA was extracted and DNase I-treated before further analysis. Erythrocytes were also analyzed using antibodies that differentiate between wild-type hemoglobin A (HBA) and sickle cell hemoglobin S (HBS). Results:RFLP analysis of a 430-bp PCR product generated from mRNA-derived cDNA with the DdeI enzyme indicated conversion of bA- to bS-globin. Sequencing of the 430-bp amplicon showed the A > T conversion. Analysis of the transfected wild-type HSPC-derived erythrocytes with HBA and HBS specific antibodies demonstrated the presence of a subpopulation of cells expressing HBS. These data are consistent with previous studies showing both conversion of bS- to bA-globin in SC1 cells and bA- to bS-globin in HSPCs after electroporation and microinjection of SDF, respectively. Conclusion:These studies represent a critical next step in developing SFHR as a therapy for sickle cell disease, in that they demonstrate long-term SFHR-mediated modification of b-globin following mass transfection by electroporation of HSPCs. Disclosures:No relevant conflicts of interest to declare.

Mutational analysis of CDKN2A gene in a group of 390 larynx cancer patients

CDKN2A gene belongs to the genes involved in cell cycle regulation. When is absent or inactivated by mutation or promoter hypermethylation a cell may undertake an uncontrolled proliferation. Inactivation of CDKN2A gene is observed in many human malignancies, including larynx cancer. In this study we investigated mutations in exon 1 and exon 2 of CDKN2A gene in a large group of 390 laryngeal cancers. We found 40 different alterations (17%) and nearly half of them was not described previously. Out of these alterations two transversions in codon 108: c.322G>C (Asp108His) and c.322G>T (Asp108Tyr) as well as a G>A transition in codon 110 (Trp110X) were found more frequently (altogether: 7 cases in codon 108 and 10 cases in codon 110). This result, concerning the location of these codons in the ankyrin repeat structures, may suggest that these two codons may be critical hot-spots in larynx carcinogenesis.

Targeted deletion of the genes encoding NTH1 and NEIL1 DNA N-glycosylases reveals the existence of novel carcinogenic oxidative damage to DNA

We have generated a strain of mice lacking two DNA N-glycosylases of base excision repair (BER), NTH1 and NEIL1, homologs of bacterial Nth (endonuclease three) and Nei (endonuclease eight). Although these enzymes remove several oxidized bases from DNA, they do not remove the well-known carcinogenic oxidation product of guanine: 7,8-dihydro-8-oxoguanine (8-OH-Gua), which is removed by another DNA N-glycosylase, OGG1. The Nth1 −/− Neil1 −/− mice developed pulmonary and hepatocellular tumors in much higher incidence than either of the single knockouts, Nth1 −/− and Neil1 −/− . The pulmonary tumors contained, exclusively, activating GGT → GAT transitions in codon 12 of K-ras of their DNA. Such transitions contrast sharply with the activating GGT → GTT transversions in codon 12 of K-ras of the pathologically similar pulmonary tumors, which arose in mice lacking OGG1 and a second DNA N-glycosylase, MUTY. To characterize the biochemical phenotype of the knockout mice, the content of oxidative DNA base damage was analyzed from three tissues isolated from control, single and double knockout mice. The content of 8-OH-Gua was indistinguishable among all genotypes. In contrast, the content of 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) derived from adenine and guanine, respectively, were increased in some but not all tissues of Neil1 −/− and Neil1 −/− Nth1 −/− mice. The high incidence of tumors in our Nth1 −/− Neil1 −/− mice together with the nature of the activating mutation in the K-ras gene of their pulmonary tumors, reveal for the first time, the existence of mutagenic and carcinogenic oxidative damage to DNA which is not 8-OH-Gua.

AA amyloidosis associated with a mutated serum amyloid A4 protein

AA amyloidosis invariably has been associated with fibrillar deposits of the acute phase high-density lipoprotein serum amyloid A isotypes SAA1 and SAA2. We now report the first case in a patient with no antecedent history of a chronic inflammatory or neoplastic process whose pathologic renal deposits were comprised of a mutated form of the constitutively expressed serum amyloid A4 (SAA4) protein. Analyses by tandem mass spectrometry of amyloid extracted from kidney biopsies revealed a component identical in sequence to the N-terminal portion of SAA4, except for the substitution of glycine for tryptophan at position 22 (W22G). Sequencing of genomic DNA using SAA4-specific primers showed a TGG to GGG transversion in codon 22 that accounted for the observed modification. Confirmation of the SAA4 nature of the amyloid was obtained immunohistochemically. Notably, only wild-type SAA4 was detected by mass spectrometry in the patient's serum and its concentration was within normal limits. Given the substitution of an amino acid lacking a side chain for a bulky residue, we posit that the W22G alteration would profoundly affect SAA4 stability, rendering it amyloidogenic. Our studies provide the first evidence for a novel type of AA amyloidosis in which the fibrils were formed from a mutated SAA4 protein.

Aflatoxin B 1-induced TP53 mutational pattern in normal human cells using the FASAY ( Functional Analysis of Separated Alleles in Yeast)

Mutations in the TP53 gene are the most common alterations in human tumours. In hepatocellular carcinoma (HCC) related to exposure to aflatoxin B 1, a specific G > T transversion in codon 249 is classically described as a hot spot. However, AFB 1 is suspected to be a potent carcinogen in tissues other than the liver. By using the FASAY functional assay in yeast, the present study aimed at depicting the mutational pattern of TP53 in normal human fibroblasts after in vitro exposure to AFB 1. Molecular analysis of mutants revealed that codon 245 was the main hot spot, whereas no mutations were found in codon 249. The locations of mutations within GG and GC/CG sequences are well in accordance with AFB 1-adduct location data. In our assay with normal human fibroblasts, AFB 1 mainly induced G > A transitions, followed by G > T and A > G mutations. This suggests that G > T transversions at codon 249 were likely the result of a selection bias in human HCC rather than a true fingerprint of AFB 1 adducts. Indeed, a comparison of the mutation pattern with that found in human HCC excluding codon 249 reveals that the two spectra are quite similar. Furthermore, the similarity between our in vitro spectrum with that identified in AFB 1-induced lung tumours in mice suggests that AFB 1 may be a potent lung carcinogen in humans.

Sickle cell disease caused by Hb S/Québec‐CHORI: Treatment with hydroxyurea and response

Sickle hemoglobin (Hb S;betaGlu 6 Val) is due to an A>T transversion in codon 6 of the beta-globin gene. Other variant hemoglobins mimic Hb A, S, or C on newborn screening and clinical laboratory diagnostic tools, thus making their correct identification potentially difficult. Sickling disorders can result in individuals who are compound heterozygous for beta-globin mutations (e.g., Hb SC, HbSO(Arab)). The authors report a second case of HbS/Québec-CHORI, a severe compound heterozygous sickling disorder and their experience managing this patient with hydroxyurea.

Small Fragment Homologous Replacement-Mediated Modification of Genomicβ-Globin Sequences in Human Hematopoietic Stem/Progenitor Cells

An ultimate goal of gene therapy is the development of a means to correct mutant genomic sequences in the cells that give rise to pathology. A number of oligonucleotide-based gene-targeting strategies have been developed to achieve this goal. One approach, small fragment homologous replacement (SFHR), has previously demonstrated disease-specific genotypic and phenotypic modification after introduction of small DNA fragments (SDFs) into somatic cells. To validate whether the gene responsible for sickle cell anemia (beta-globin) can be modified by SFHR, a series of studies were undertaken to introduce sickle globin sequences at the appropriate locus of human hematopoietic stem/progenitor cells (HSPCs). The characteristic A two head right arrow T transversion in codon 6 of the beta-globin gene was indicated by restriction fragment length polymorphic (RFLP) analysis of polymerase chain reaction (PCR) products generated by amplification of DNA and RNA. At the time of harvest, it was determined that the cells generally contained </=1 fragment per cell. Control studies mixing genomic DNA from nontransfected cells with varying amounts of the targeting SDFs did not indicate any PCR amplification artifacts due to the presence of residual SDF during amplification. RNA was analyzed after DNase treatment, thus eliminating the potential for SDF contamination. Stable SFHRmediated conversion of normal (beta (A)) to sickle (beta (S)) globin was detected at frequencies up to 13% in cells harvested 30-45 days posttransfection. The minimum conversion efficiency ranged from 0.2 to 3%, assuming modification of at least one cell per experiment showing conversion. Conversion of sickle (beta (S)) to normal (beta (A)) globin was detected up to 10 days posttransfection in lymphoblastoid cells from a sickle cell patient. These studies suggest that SFHR may be effective for ex vivo gene therapy of sickle cells in a patient's HSPCs before autologous transplantation.

Use of genotypic selection to detect P53 codon 273 CGT>CTT transversion: Application to an occupationally exposed population

CGT>CTT transversion in codon 273 of the P53 tumor-suppressor gene is one of the major mutations detected in human tumors. Within an epidemiological framework, we investigated the use of a genotypic selection method to measure this point mutation. The allele-specific polymerase chain reaction (AS-PCR) that was developed was able to detect 10 mutant copies of the gene among a total of 5×10 5 wild-type copies. We used this assay to detect CGT>CTT transversions in buccal cell DNA of production workers ( n=76) from a viscose factory exposed to carbon disulfide (amongst other pollutants) and in the DNA of non-exposed office workers ( n=67). The mutation appeared more frequently in the exposed than in the non-exposed worker who were smokers. The results of the study indicate that occupational exposure results in a significant increase in P53 CGT>CTT transversions and more especially identified occupational exposure in combination with smoking as a significant risk factor for the mutation. We conclude that AS-PCR of the P53 273rd codon transversions is a suitable technique for studying the effects of occupational exposure.

Mucopolysaccharidosis type IV: N-Acetylgalactosamine-6-sulfatase mutations in Tunisian patients

Mucopolysaccharidosis type IVA (MPS IVA; OMIM #253000) or Morquio A syndrome is an autosomal recessive inborn error resulting from the deficient activity of the lysosomal enzyme, N-acetylgalactosamine-6-sulfatase (GALNS), and the progressive lysosomal accumulation of sulfated glycosaminoglycans. Clinically, the severe form of this lysosomal storage disease is characterized by a characteristic severe bone dysplasia and normal intelligence. To date, a variety of mutations have been associated with the severe MPS IVA phenotype. Here, we report the GALNS mutations in six severe MPS IVA patients from four unrelated Tunisian families. For mutation detection, each of the 14 exons and adjacent intron–exon junctions of the GALNS gene were sequenced after PCR-amplification from genomic DNA. Two novel mutations were identified: a G to A transition in the conserved 5′ donor splice site of intron 1 (GACgt → GACat: designated IVS1 +1g→a) and a G to C transversion in codon 66 of exon 2 predicting a glycine to arginine substitution (G66R). The IVS1 +1g→a mutation was homozygous in five similarly affected patients from three presumably unrelated families, but haplotype analysis suggested a common ancestor. The affected patient in the fourth family was homozygous for the G66R mutation. These are the first GALNS mutations causing severe MPS IVA disease identified in Tunisia. These molecular findings provide genotype/phenotype correlations, and permit accurate carrier detection, prenatal diagnosis, and counseling for MPS IVA disease in Tunisia where first cousin consanguineous mating remains frequent.

Combined small‐cell carcinoma of the stomach: p53 and K‐ras gene mutational analysis supports a monoclonal origin of three histological components

Primary small-cell carcinoma (SmCC) is extremely rare in stomach. We reported an autopsy case of combined gastric SmCC with p53 and K-ras mutational analysis. Histologically, the tumour was composed of well-differentiated adenocarcinoma surrounding the central dominant SmCC component with scattered nests of squamous cell carcinoma. Immunohistochemically, all the neoplastic components revealed strong expression for p53 protein. Based on these findings, we hypothesized that these histologically different components originated from a same progenitor cell that possessed p53 mutation. Using Laser-capture microdissection technique and mutational analysis, we identified the same point mutations of p53 gene (A-->G transversion in codon 239) and K-ras gene (G-->A transversion in codon 13) in all the neoplastic components, but not in the adjacent normal gastric epithelium. Our results strongly support a hypothesis that the combined SmCC of stomach in this case was of monoclonal origin.

A Novel Sickle Hemoglobin: Hemoglobin S-South End

Sickle hemoglobin (Hb S; beta Glu6Val) is due to an A<T transversion in codon 6 of the beta-globin gene. Several Hb S variants have both the Hb S mutation plus another mutation in the same beta-globin gene. Some of these variant hemoglobins can lead to sickle cell disease even in the simple heterozygote. Moreover, some variant hemoglobins mimic Hb A, S, or C on one or several clinical laboratory diagnostic tools, thus making their correct identification potentially problematic. The authors report a novel Hb S variant hemoglobin, Hb S-South End (beta Glu6Val, GAG>GTG; beta Lys132Asn, AAA>AAC). When present alone, the beta Lys132Asn mutation has low oxygen affinity. Therefore, this mutation may enhance the polymerization of the Hb S variant. Furthermore, the variant hemoglobin mimics Hb A on high-pressure liquid chromatography, and its identity is not easily diagnosed. A succinct review of variant sickle hemoglobins is also presented.

Aberrant splicing induced by missense mutations in BRCA1: clues from a humanized mouse model.

Numerous missense mutations in human BRCA1 gene have been linked to predisposition to breast cancer. However, the functional significance of the majority of these mutations remains unknown. We have examined the molecular basis for three such cancer-causing mutations. The first mutation, a T-->G transversion in codon 64, is predicted to change a conserved cysteine residue to glycine in the RING finger domain of the 1863 amino acid BRCA1 protein. Using a humanized mouse model we demonstrate that this missense mutation actually results in a functionally null protein. This striking result occurs because the single base alteration generates a new 5' splice site in exon 5 and also disrupts a putative exonic splicing enhancer motif. Consequently, the normal splice donor site is disrupted and an internal cryptic splice site is activated. This results in a 22-nucleotide deletion and the aberrant transcript is predicted to encode a severely truncated protein consisting of only 63 amino acids. To identify other missense mutations in BRCA1 that may result in aberrant splicing, we screened various mutations using the Genscan program. We demonstrate that at least two other missense mutations in codons 1495 and 1823 result in aberrant splicing due to the possible disruption of cis-acting splicing regulatory elements. In conclusion, our study demonstrates for the first time the application of a humanized mouse model for functional analysis of human mutations in mice and also shows the need for a careful examination of the functional consequences of single base alterations and single nucleotide polymorphisms identified in human disease-causing genes.

Absence of the Kilifi mutation in the rhinovirus-binding domain of ICAM-1 in a Caucasian population.

Human rhinoviruses (HRV), responsible for approximately 60% of the common colds, are divided into two groups, according to their receptor specificity. The major group of HRVs gains access to human cells by binding to the intercellular adhesion molecule-1 (ICAM-1), whereas HRVs of the minor group use members of the low-density lipoprotein receptor (LDLR) family for cell entry. Previous studies confirmed that the HRV-binding region of ICAM-1 is located in the amino-terminal immunoglobulin-like (Ig) domain 1, which is encoded by exon 2 of the ICAM-1 gene. An A --> T transversion in codon 29 of ICAM-1 exon 2 causes a lysine to methionine substitution (K29M), and was found at a high frequency (33.2%) in Kilifi (Kenya), as well as in other African populations. In this study we examined whether polymorphisms in exon 2 of ICAM-1 could be detected in a Caucasian population, assuming that these could be of importance in HRV binding. DNA from 100 healthy, unrelated, Belgian volunteers was obtained through a noninvasive swish-and-spit method. Using a primer set in the adjacent intron sequences, the full-length ICAM-1 exon 2 was amplified by polymerase chain reaction (PCR), followed by direct sequencing of the PCR product. No polymorphisms could be demonstrated in exon 2 of the ICAM-1 gene among all 100 tested individuals. The rhinovirus-binding Ig domain 1 of ICAM-1 seems to be a highly conserved region in the Caucasian population.

Infrequent p53 Gene Mutations and Lack of p53 Protein Expression in Clear Cell Sarcoma of the Kidney: Immunohistochemical Study and Mutation Analysis of p53 in Renal Tumors of Unfavorable Prognosis

A high prevalence of p53 gene mutation and protein expression has been found in the anaplastic variant of Wilms’ tumor (WT), known to be associated with poor outcome. However, there are very few studies of p53 alterations in the other two rare and highly malignant renal tumors in childhood, in other words, clear cell sarcoma of the kidney (CCSK) and malignant rhabdoid tumor of the kidney (MRTK). Overexpression of p53 protein has been detected in eight CCSKs in one study, and in two in another, yet no molecular correlation with p53 gene mutations has been carried out. Our study is the first molecular analysis concerning p53 in CCSK. We investigated eight cases of CCSK and one case of MRTK for p53 protein expression by immunohistochemical staining. All were analyzed for p53 mutations in the region of exons 4 to 8 by polymerase chain reaction–single-strand conformational polymorphism (PCR-SSCP) method and DNA sequencing analysis. By histological study, no CCSK showed anaplastic features. None expressed p53 protein, but two harbored p53 mutations. One was in exon 5, with a base pair insertion between codons 162 to 163 causing frameshift alteration in amino acid. Another was a silent CTC → CTT transversion in codon 289 of exon 8. The case of MRTK did not show any alterations of p53 protein or gene. Our result indicates that p53 alterations are infrequent in CCSK and do not seem to be primary genetic events in the pathogenesis of CCSK.