K-ras point mutations in routinely processed tissues: non-radioactive screening by single strand conformational polymorphism analysis.

AIMS--To develop a non-radioactive method to screen routinely fixed, paraffin wax embedded specimens for the occurrence of point mutations; to evaluate the single strand conformational polymorphism (SSCP) analysis technique for the detection of K-ras point mutations as a result of electrophoretic mobility shifts. METHODS--DNA was extracted from archival specimens of colon cancer and from established colon cancer cell lines with known point mutations. A K-ras gene fragment containing codons 12 and 13 of exon 1 was amplified with the polymerase chain reaction (PCR). Denatured DNA fragments were run on 10% polyacrylamide gels under non-denaturing conditions. After electrophoresis DNA was blotted and the single stranded DNA was detected using a digoxigenin labelled ras probe. The nature of the detected point mutations was identified and confirmed by sequencing and hybridisation with oligonucleotides using 32P labelling. RESULTS--Wild type and aberrant alleles were detected caused by mobility shifts after electrophoresis of the PCR products. Commonly occurring mutations in the K-ras gene--in the first two positions of codon 12--could easily be detected in DNA from archival paraffin wax embedded colon cancer tissue. In all the colon tumour samples studied wild type gene alleles were also found, presumably derived from normal cells in the specimen. CONCLUSIONS--The SSCP method permits rapid non-radioactive screening of adenomas or carcinomas for the occurrence of point mutations in the K-ras gene. But if a mutation is detected by an electrophoretic mobility shift, its identification requires confirmation by sequencing or oligonucleotide hybridisation.     

Non-invasive screening method for Fabry disease by measuring globotriaosylceramide in whole urine samples using tandem mass spectrometry

Fabry disease is an X-linked sphingolipidosis due to a deficiency of alpha-galactosidase A, which leads to the accumulation of globotriaosylceramide (GL-3) in several organs. When recombinant human alpha-galactosidase A is intravenously administered repeatedly before the patient develops permanent tissue damage, there is evidence that the accumulation of GL-3 is decreased in some organs and that the clinical symptoms are alleviated in some patients. However, Fabry disease is rare and many patients are not diagnosed until adulthood after irreversible tissue damage has occurred. Our group has developed a simple and non-invasive screening method for Fabry disease that measures total GL-3 in whole urine samples by tandem mass spectrometry. Using this method, we found that the concentration of GL-3 in whole urine sample from hemizygous patients, including pre-symptomatic young children with classic type Fabry disease, was significantly higher than that in controls. The mean concentration of GL-3 in urine from heterozygotes with symptoms was significantly higher than control concentrations, but GL-3 levels in the urine from 2 out of 8 heterozygotes of classic type Fabry disease were within control levels. An asymptomatic 14-year old hemizygote in the family of a cardiac variant did not have elevated urinary GL-3. Therefore, screening for the classic type and probably renal variant of Fabry disease is possible by measuring urinary GL-3, using our method. The early diagnosis of cardiac variant hemizygotes and some heterozygotes with all types of Fabry disease will not be possible using our method. We propose that this procedure can be used as a reliable, non-invasive, simple method for general and high-risk population screening for hemizygotic patients with the classic type and probably renal variant of Fabry disease.     

Molecular basis of fabry disease: Mutations and polymorphisms in the human α-galactosidase A gene

Fabry disease, an X-linked inborn error of glycosphingolipid catabolism, results from mutations in the α-galactosidase A gene at Xq22.1. Studies of the mutations in unrelated Fabry families have identified a variety of lesions indicating the molecular genetic heterogeneity underlying the disease. Forty-nine different mutations have been described including five partial gene deletions, one partial gene duplication, nine small deletions and insertions, three splice junction consensus site alterations, and 31 coding region single base substitutions. Most mutations resulted in the classical disease phenotype; however, five missense mutations were detected in atypical hemizygotes who were asymptomatic or had symptoms confined to the heart, including N215S, which was described in three unrelated atypical males. Most mutations were confined to a single pedigree with the exception of N215S, R227Q, R227X, R342Q, and R342X, which were each found in several unrelated families. Five of the 14 coding region CpG dinucleotides were sites of point mutations including the CpGs in codons 227 and 342, which were each mutated in both orientations. The identification of the mutation in a given Fabry family permits precise prenatal diagnosis and heterozygote detection of other family members with this X-linked recessive disease. Studies of additional Fabry families will provide information on the nature and frequency of the mutations causing this disease as well as potential insights into the structure/ function relationships of this lysosomal hydrolase. © 1994 Wiley-Liss, Inc.     

Missense mutation in exon 11 (Codon 378) of the presenilin-1 gene in a French family with early-onset Alzheimer's disease and transmission study by mismatch enhanced allele specific amplification. Mutations in brief no. 141. Online. besancon@rockefeller1.univ.lyon1.fr

Mutations in the presenilin-1 (PS1) gene account for the majority of familial early-onset Alzheimer's disease (EOAD) cases. We screened the coding part of the PS1 gene for the present of mutations in a French family with EOAD, using single strand conformation polymorphism (SSCP) analysis. Patients in the pedigree showed a missense mutation in exon 11 of the PS1 gene involving a transition of G to A, altering glycine to glutamate at codon 378. The cosegregation of the mutation with EOAD in the family was studied by allele specific amplification, enhanced by the introduction of a mismatch at the penultimate position near the 3' primer end. The mutation has not been described before and is located within the third large cytoplasmic loop and may lead to the appearance of a short additional a-helix.     

PCR detection of HIV proviral DNA (gag) in the brains of patients with AIDS: comparison between results using fresh frozen and paraffin wax embedded specimens.

AIMS--To adapt the polymerase chain reaction (PCR) technique of HIV detection to paraffin wax embedded brain tissue and to compare the results with those obtained using frozen tissue. METHODS--HIV antigen and HIV proviral DNA were detected in specimens of frontal lobe using immunohistochemistry and PCR, respectively. DNA was extracted from fresh tissue using standard methods whereas the technique for extracting DNA from paraffin wax embedded tissue was partly modified. RESULTS--Twenty cases were examined. HIV DNA was detected in 16 cases in frozen specimens. Of these, 15 were also positive when paraffin wax embedded material was analysed. CONCLUSIONS--This study shows that HIV proviral DNA can be detected in formalin fixed, paraffin wax embedded brain tissue by PCR. The results obtained from paraffin wax embedded specimens showed a similar degree of reliability to those from fresh frozen brain. Factors such as fixative, fixation time, and delay in performing post mortem examinations did not seem to influence PCR amplification as positive results were obtained with specimens left in fixative for up to eight months, as well as in cases where post mortem examinations had been delayed for up to four days.     

Identification of three novel RB1 mutations in Brazilian patients with retinoblastoma by "exon by exon" PCR mediated SSCP analysis

AIMS: To carry out a retrospective study, screening for mutations of the entire coding region of RB1 and adjacent intronic regions in patients with retinoblastoma. METHODS: Mutation screening in DNA extracts of formalin fixed, paraffin wax embedded tissues of 28 patients using combined "exon by exon" polymerase chain reaction mediated single strand conformational polymorphism analysis, followed by DNA sequencing. RESULTS: Eleven mutations were found in 10 patients. Ten mutations consisted of single base substitutions; 10 were localised in exonic regions (eight nonsense, one missense, and one frameshift) and another one in the intron-exon splicing region. Three novel mutations were identified: a 2 bp insertion in exon 2 (g.5506-5507insAG, R73fsX77), a G to A transition affecting the last invariant nucleotide of intron 13 (g.76429G>A), and a T to C transition in exon 20 (g.156795T>C, L688P). In addition, eight C to T transitions, resulting in stop codons, were found in five different CGA codons (g.64348C>T, g.76430C>T, g.78238C>T, g.78250C>T, and g.150037C>T). Although specific mutation hotspots have not been identified in the literature, eight of the 11 mutations occurred in CGA codons and seven fell within the E1A binding domains (codons 393-572 and 646-772), whereas five were of both types-in CGA codons within E1A binding domains. CONCLUSIONS: CGA codons and E1A binding domains are apparently more frequent mutational targets and should be initially screened in patients with retinoblastoma. Paraffin wax embedded samples proved to be valuable sources of DNA for retrospective studies, providing useful information for genetic counselling.     

Direct multiplex amplification of DNA from a formalin fixed, paraffin wax embedded tissue section.

The extraction of DNA from formalin fixed, paraffin wax embedded tissue can be problematical, with long protocols producing low yields. This report describes a very simple and useful method for amplifying DNA from formalin fixed, paraffin wax embedded tissue without the need for prior DNA extraction. This method allows direct polymerase chain reaction (PCR) based molecular analysis of fixed tissue. It is an invaluable method if clinical biopsy specimens are to be investigated, because extraction of uncontaminated DNA from such small samples can be very difficult or even impossible. It will also facilitate the study of intratumour heterogeneity, with the analysis of multiple small areas from within a single tumour section. In addition, this method can be used for other samples where only a few tests are to be carried out and a stock of DNA is not required, thus shortening the analysis time.     

Chloroquine cardiotoxicity: clinicopathologic features in three patients and comparison with three patients with Fabry disease

BACKGROUND: Microscopic features of chloroquine cardiotoxicity are similar to those of Fabry disease. The purpose of the study was to compare clinicopathologic findings in both disorders. METHODS: Patients with a diagnosis of chloroquine cardiotoxicity or Fabry disease were identified who had undergone endomyocardial biopsy or autopsy at Mayo Clinic Rochester (1976-2000). Clinical information was collected from medical records and letters from referring physicians. Light and electron microscopy were performed in all cases. RESULTS: Three patients (two women, one man) with chloroquine cardiotoxicity ranged in age from 53 to 73 years. Chloroquine was given for rheumatoid arthritis in two and systemic lupus erythematosus in one. Three patients (two men, one woman) with Fabry disease ranged in age from 58 to 76 years. Two had angiokeratomas, but only one had a previous diagnosis of Fabry disease. All six patients presented with dyspnea. Light microscopy from all six revealed myocyte enlargement due to perinuclear vacuolization. By transmission electron microscopy, all six showed abundant myelinoid figures within involved myocytes. Curvilinear bodies were observed in two patients with chloroquine cardiotoxicity and in none with Fabry disease. CONCLUSIONS: Patients with cardiac dysfunction due to chloroquine cardiotoxicity or Fabry disease have similar ages, presenting clinical symptoms, cardiac light microscopy and sarcoplasmic myelinoid bodies ultrastructurally. Patients with Fabry disease may not have a personal or family history of the disease. Similarly, a history of chloroquine usage may not be known to the pathologist. In these settings, the presence of curvilinear bodies ultrastructurally is useful for the diagnosis of chloroquine cardiotoxicity.     

PCR analysis in archival postmortem tissues.

BACKGROUND: Formalin fixed and paraffin wax embedded tissues of necropsy origin are an important source for molecular analysis especially in rare diseases, neuropathology, or molecular epidemiology studies. Because of DNA degradation, only short sequences can be amplified from this type of tissue, very often less than 100 bases. This poses problems because studies on polymorphism and mutations occurring in large genes often require the analysis of long sequences. METHODS: The development of a simple treatment to obtain longer fragments of DNA for the analysis of archival postmortem paraffin wax embedded tissues. RESULTS: It was possible to amplify longer sequences ranging up to 300 bases from postmortem tissues, with no modification to the usual DNA extraction procedures. To obtain longer stretches of DNA, a pre-PCR restoration treatment was required, by filling single strand breaks, followed by a vigorous denaturation step. CONCLUSIONS: The development of this simple treatment allowed the analysis of longer fragments of DNA obtained from archival postmortem paraffin wax embedded tissues.     

Immunohistochemical analysis of CDw52 antigen expression in non-Hodgkin's lymphomas.

AIM--To determine the antigen expression of CDw52 using Campath-1 antibodies in a series of non-Hodgkin's lymphomas (NHLs). METHODS--Tissue sections of lymphoma were stained immunohistochemically using rat Campath-1G and humanised Campath-1H with avidin-biotin-peroxidase complex techniques. Fifty-two fresh frozen lymphomas and a further 26 paraffin wax embedded sections were studied. RESULTS--Thirty-seven out of 41 B cell lymphomas were positive with Campath-1H in frozen sections (low grade, 24 of 24; high grade, 13 of 17) as were three out of five T cell lymphomas. Reed-Sternberg cells in six cases of Hodgkin's disease did not react. Eleven out of 16 high grade B cell lymphomas also stained positively with Campath-1G in paraffin wax sections as did five out of 10 T cell lymphomas. CONCLUSIONS--The Campath-1 antibodies showed that CDw52 antigen expression was present in all cases of low grade B cell NHL examined. Immunohistochemical staining in high grade B cell NHL and in T cell NHL was variable. These findings may be relevant to patient selection when considering treatment with Campath-1 antibodies.     

Remarkable variability in renal disease in a large Slovenian family with Fabry disease

Following the diagnosis of Fabry disease in a 45-year-old male, in 31 family members alpha-galactosidase A (alpha-Gal) activity in leucocytes was measured and mutation analysis of the alpha-Gal gene was performed. In the proband, the unique mutation A10523G/N272S in exon 6 was found, which was subsequently detected in seven males (of which one twin) and 10 female subjects. All males showed decreased to absent alpha-Gal A activity in leucocytes, but three out of 10 female subjects had alpha-Gal A activities within normal range. Although all male patients had symptoms of classical Fabry disease, such as acroparesthesias, hypohydrosis and heat-intolerance, there was considerable variability in organ involvement, especially in deterioration of renal function. Detailed studies of large families with Fabry disease may give insight into factors that influence the phenotype of this disorder.     

Identification of four novel mutations in five unrelated Korean families with Fabry disease

Fabry disease is a X-linked recessively inherited metabolic disorder, which results from the deficient activity of the lysosomal hydrolase alpha-galactosidase A leading to the systemic deposition of glycosphingolipids with terminal alpha-galactosyl moieties. Single-strand conformation polymorphism (SSCP) analysis was performed, followed by DNA sequencing of PCR amplified exons of the human alpha-galactosidase A gene in 5 unrelated Korean patients with classic Fabry disease. Five different mutations were identified; two nonsense mutations (Y86X and R342X), one missense mutation (D266N), and two small deletions (296del2 and 802del4). Except for R342X mutation, four were novel mutations (Y86X, D266N, 296del2, 802del4). A T to G transversion at nucleotide position 5157 in exon 2 caused a tyrosine-to-stop substitution at codon 86. A G to A transition at position 10287 in exon 5 substituted an asparagine for an aspartate at codon 266. Mutation 296del2 in exon 2 resulted in a frame shift with a stop signal at the 22th codon downstream from the mutation, whereas mutation 802del4 resulted in a stop codon at the site of 4 bp deletion. In addition, the 802del4 was found to be a de novo mutation. This is the first report on mutation analysis of the human alpha-galactosidase A gene in Korean patients with Fabry disease.     

马凡综合征微纤维蛋白1基因突变检测及单倍型连锁分析

目的：检测中国人马凡综合征（Marfan syndrome,MFS)患者微纤维蛋白1（fibillin-1,FBN1)基因的突变及对马凡综合征患者的家系成员进行症状前诊断。方法：应用聚合酶链反应－单链构象多态性技术和测序方法，对汉族9个家系中共17个MFS患者进行基因突变检测；运用FBN1基因内4个内含子中的可变串联重复序列构建染色体单倍型，进行家系单倍型连锁分析和基因诊断。结果：发现MFS（A）家系Ⅱ1患者有单链构象改变，测序证实为位于FBN1基因第25号外显子3243－3256核苷酸之间有I个13bp的小片段缺失，为新位点基因移码突变，其序列为gcctctgcaccca;单倍型连锁分析发现MFS（B）家系Ⅲ1是1个无症状期患者。结论：中国人FBN1基因突变可以引起马凡综合征，应用突变检测与单倍型连锁分析方法能为马凡综合征基因诊断提供依据。     

一个异染性脑白质营养不良家系ARSA基因突变分析

目的分析并确定一个异染性脑白质营养不良（metachromatic leukodystrophy，MLD）家系芳基硫酸酯酶A（arylsulftase A，ARSA）的基因（ARSA）突变及遗传特征。方法收集先证者及其家系成员临床资料，采用聚合酶链反应和DNA直接测序方法进行ARSA突变检测，确定基因突变的位点，分析基因型与表型的关系。结果该家系先证者的ARSA基因同时存在第2外显子G251A（R84Q）与G296T（G99V）两个杂合突变，具有相似表型的胞弟与先证者的测序结果完全一致，先证者之父母分别携带ARSA基因第2外显子G251A（R84Q）与G296T（G99V）的杂合突变，表型正常的先证者之姐未发现这些突变。结论该家系中两位患儿均为ARSA基因复合杂合突变致病，其G251A（R84Q）突变来自父亲，G296T（G99V）突变来自母亲，其父母均为表型正常的基因突变携带者。     

Sources of DNA for detecting B cell monoclonality using PCR.

AIMS--To evaluate the polymerase chain reaction (PCR) demonstration of clonal immunoglobulin heavy chain gene rearrangements using routinely prepared, unstained, and stained formalin fixed, paraffin wax embedded tissue samples. METHODS--Extracts from (a) fresh frozen tissue samples, (b) unstained, and (c) haematoxylin and eosin stained formalin fixed, paraffin wax embedded 5 microns tissue sections from 42 cases of low grade B cell lymphoma, all shown to be monoclonal by Southern blot analysis, were analysed using PCR. Two regions of the variable segment of the immunoglobulin heavy chain gene were amplified (framework 2 to joining region [Fr2/JH] and framework 3 to joining region [Fr3/JH]). Twelve samples of reactive lymphoid tissue were studied as controls. Products from each case were directly compared on polyacrylamide gels. RESULTS--Using both primer combinations, monoclonality was detected in 38 of 42 (90%) cases using fresh material, 37 of 42 (88%) using unstained paraffin wax embedded samples, and in 35 of 42 (83%) cases using haematoxylin and eosin stained sections. No false positive results attributable to fixation, processing, or staining were identified, although the efficiency of amplification using the Fr2/JH primers was significantly reduced. CONCLUSIONS--PCR determination of B cell clonality using paraffin wax embedded material is sufficiently sensitive and reliable for use as a routine diagnostic adjunct to conventional morphological and immunocytochemical assessment of lymphoproliferative disease.     

Improved current methods for amplification of DNA from routinely processed liver tissue by PCR.

With both a classic DNA preparation protocol (including removal of paraffin wax and protein digestion) and a DNA extraction protocol with Chelex 100, the hepatitis B virus genome was searched for using the polymerase chain reaction (PCR) in 30 samples of paraffin wax embedded liver tissue from patients with chronic hepatitis. The classic protocol was more sensitive than the rapid Chelex 100 procedure (10 v six positive samples). A third protocol, including removal of paraffin wax, protein digestion, and Chelex 100 treatment of the digestion solution before PCR, was more sensitive than the others (16 positive samples). It is concluded that it could therefore be helpful for PCR analysis of paraffin wax embedded liver tissue.     

New approaches for genotyping paraffin wax embedded breast tissue from patients with cancer: the Iowa women's health study

BACKGROUND: The use of paraffin wax embedded tissue samples as a source of DNA for genotype analysis has been limited because of difficulties in DNA extraction and single nucleotide polymorphism (SNP) analysis.AIMS: To test the feasibility of applying the combination of a commonly used DNA isolation procedure, PureGene, and a high throughput SNP analysis method, the polymerase chain reaction (PCR)-INVADER assay, to genotype several types of paraffin wax embedded breast tissues. METHODS: Twenty formalin fixed, paraffin wax blocks were obtained from five participants in the Iowa women's health study. Each participant provided several types of tissue including normal lymph node, normal nipple/areola tissue, inflammatory/fibrotic breast tissue, or normal breast tissue, and tumour tissue. RESULTS: Good quality DNA (260/280 ratio >1.6) was obtained from all tissues. Normal lymph nodes yielded the largest amount of DNA (97.1 mug). DNA obtained from the samples was tested for a germline C1183T polymorphism in the MnSOD gene by three methods-PCR-RFLP (restriction fragment length polymorphism), INVADER assay, and PCR-INVADER assay. Of the 20 samples, PCR-RFLP genotyped 16, the PCR-INVADER assay 18, and the INVADER assay two. This methodology was then used to analyse five additional genotypes and confirmed the general applicability of the method. CONCLUSIONS: This study demonstrated the feasibility of (1) using several paraffin wax embedded breast tissues as a source of DNA for germline genetic analysis, with lymph nodes providing the highest yield, and (2) using the combination of a common extraction method with a high throughput SNP analysis method, the PCR-INVADER assay.     

Tissue diagnosis of intestinal microsporidiosis using a fluorescent stain with Uvitex 2B.

AIMS--To detect intestinal microsporidiosis in paraffin wax embedded biopsy specimens using a fluorescence technique incorporating optical brighteners. METHODS--Eight HIV infected patients with confirmed intestinal microsporidiosis (six with Enterocytozoon bieneusi, one with Encephalitozoon intestinalis and one with Encephalitozoon cuniculi infection) and 10 without infection were studied. Tissue sections of paraffin wax embedded duodenal biopsy specimens were stained with 1% Uvitex 2B, coded and analysed independently by two investigators. RESULTS--In all eight cases with confirmed intestinal microsporidian infection, spores could be detected easily in tissue sections using the fluorescence technique. Spores or other elements consistent with microsporidiosis were not found in the 10 patients without infection. CONCLUSION--Staining of tissue sections from paraffin wax embedded intestinal biopsy specimens with stains incorporating Uvitex 2B is a rapid and easy technique for the diagnosis of intestinal microsporidiosis.     

Genetic analysis of hydatidiform moles in paraffin wax embedded tissue using rapid,sequence specific PCR-based HLA class II typing.

AIMS: To determine the applicability of rapid, sequence specific polymerase chain reaction (PCR)-based HLA class II genotyping for the distinction of complete from partial hydatidiform moles (HM) using DNA extracted from formalin fixed and paraffin wax embedded tissue. METHODS: Nine HM were studied. DNA was extracted from formalin fixed and paraffin wax embedded tissue after mechanical separation of decidual and molar components. HLA class II DRB (DRB1, -3, -4, and -5) and DQB1 genotyping was performed using a parallel series of PCR reactions, each of which contained sequence specific primers designed to amplify different HLA DRB and DQB1 alleles or allele groups (PCR-SSP analysis). In each case the HLA DRB and DQB1 genotypes identified within the decidua and HM were compared. RESULTS: Within the decidual tissue, HLA DRB genotypes were assignable in all nine cases, and HLA DQB1 genotypes were identified in seven cases. Within the molar tissue, HLA DRB genotypes were assignable in seven cases, and at least one HLA DQB1 allele was identified in seven cases. Interpretation based on HLA class II genotyping was therefore possible in two cases classified on histological appearances as complete HM, in four classified as partial HM, and in one HM of uncertain type. Different HLA DRB and DQB1 haplotypes were identified within the decidual and molar tissue from both complete HM, consistent with a solely paternal origin and supporting the histological diagnosis. HLA DRB and DQB1 alleles common to the decidual and molar tissue were present within the four partial HM and the HM of histologically uncertain type, consistent with combined maternal and paternal genetic input to these HM, supporting the histological diagnosis in four cases and suggesting that the histologically equivocal case was also a partial HM. CONCLUSION: PCR-SSP HLA class II DRB and DQB1 typing is reliably applicable to DNA extracted from formalin fixed and paraffin wax embedded tissue. Therefore, in a suitably equipped HLA typing laboratory, this technique provides a useful adjunct to histological examination for differentiation of complete from partial HM.