A mutation in the neurofibromatosis type 2 tumor-suppressor gene, giving rise to widely different clinical phenotypes in two unrelated individuals.

We have sought mutations in the recently identified neurofibromatosis type 2 (NF2) tumor-suppressor gene in a large panel of NF2 patients, using PCR-based SSCP and heteroduplex analysis, followed by cloning and sequencing of appropriate PCR products. Two unrelated NF2 patients were found to have identical nonsense mutations caused by a C-to-T transition in a CpG dinucleotide that is a potential mutational hot spot in the NF2 tumor-suppressor gene. Unexpectedly, the two individuals had widely different clinical phenotypes, representing the severe Wishart and mild Gardner clinical subtypes. Analysis of DNA samples from different tissues of the mildly affected patient suggests that he is a somatic mosaic for the mutation.     

Novel and recurrent rearrangements in the CFTR gene: clinical and laboratory implications for cystic fibrosis screening

Because standard techniques used to detect mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene do not detect single or multiple exonic rearrangements, the importance of such rearrangements may be underestimated. Using an in-house developed, single-tube, semi-quantitative fluorescent PCR (SQF PCR) assay, we analyzed 36 DNA samples submitted for extensive CFTR sequencing and identified ten samples with rearrangements. Of 36 patients with classic CF, 10 (28%) harbored various deletions in the CFTR gene, accounting for 14% of CF chromosomes. A deletion encompassing the CFTR promoter and exons 1 and 2 was detected in a sample from one proband, and in the maternal DNA as well. In another family, a deletion of the promoter and exon 1 was detected in three siblings. In both of these cases, the families were African American and the 3120+1G>A splice site mutation was also identified. These promoter deletions have not been previously described. In a third case, a deletion of exons 17a, 17b, and 18 was identified in a Caucasian female and the same mutation was detected in the paternal DNA. In the other seven cases, we identified the following deletions: exons 2 and 3 (n=2); exons 4, 5, and 6a; exons 17a and 17b; exons 22 and 23; and exons 22, 23, and 24 (n=2). In our series, the frequency of CFTR rearrangements in classic CF patients, when only one mutation was identified by extensive DNA sequencing, was >60% (10/16). Screening for exon deletions and duplications in the CFTR gene would be beneficial in classic CF cases, especially when only one mutation is identified by standard methodologies. An erratum to this article can be found at     

Three Novel <Emphasis Type="Italic">NF1</Emphasis> Gene Mutations in a Cohort of Bulgarian Neurofibromatoses Patients

Neurofibromatosis (NF) is a clinically heterogeneous autosomal dominant disorder. Three distinct forms have been identified: neurofibromatosis type 1 (NF1), type 2 (NF2) and schwannomatosis. In the present study, we report clinical and genetic findings in the NF1 and NF2 genes in a cohort of 27 Bulgarian patients, with 18 cases (67%) genetically verified. Both NF1 and NF2 genes were screened by Sanger sequencing on DNA samples. The Sanger negative samples were screened by Multiplex Ligation-dependent Probe Amplification (MLPA) for deletions and duplications. The results from genetic testing revealed three novel mutations and fifteen previously reported ones (13 in the NF1 gene and 2 in the NF2 gene). The novel variants in the NF1 gene are a splice site mutation c.4725-1G>A, a small deletion of five bases c.823delATCTT, p.Leu275ValfsTer14, and a single base duplication c.6547dupC, p.Arg2183ProfsTer11. The novel splice site mutation is manifested by multiple “café au lait” macules and neurofibromas. Both novel out of frame mutations were found in patients with multiple “café au lait” spots and focal epilepsy. A segmental neurofibromatosis (SNF1) is restricted to one or more body segments. Here we present a case with SNF1 caused by a somatic deletion of exons 1 to 12 of the NF1 gene which is manifested by multiple neurofibromas in the right hand. Two nonsense mutations are found in the NF2 gene. Our study adds three novel mutations to the NF1 mutation spectra and contributes to the clinical-genetic NF1-characterization. Here we report strikingly different phenotypic spectra caused by the same mutation in a single family. Our findings contribute to the genotype- phenotype correlations which are difficult to establish, due to the extremely complex NF phenotype being a combination of clinical features.     

Molecular description of three macro-deletions and an Alu-Alu recombination-mediated duplication in the HPRT gene in four patients with Lesch-Nyhan disease

Mutations in the HPRT gene cause a spectrum of diseases that ranges from hyperuricemia alone to hyperuricemia with profound neurological and behavioral dysfunction. The extreme phenotype is termed Lesch-Nyhan syndrome. In 271 cases in which the germinal HPRT mutation has been characterized, 218 different mutations have been found. Of these, 34 (13%) are large- (macro-) deletions of one exon or greater and four (2%) are partial gene duplications. The deletion breakpoint junctions have been defined for only three of the 34 macro-deletions. The molecular basis of two of the four duplications has been defined. We report here the breakpoint junctions for three new deletion mutations, encompassing exons 4-8 (20033bp), exons 4 and 5 (13307bp) and exons 5 and 6 (9454bp), respectively. The deletion breakpoints were defined by a combination of long polymerase chain reaction (PCR) amplifications, and conventional PCR and DNA sequencing. All three deletions are the result of non-homologous recombinations. A fourth mutation, a duplication of exons 2 and 3, is the result of an Alu-mediated homologous recombination between identical 19bp sequences in introns 3 and 1. In toto, two of three germinal HPRT duplication mutations appear to have been caused by Alu-mediated homologous recombination, while only one of six deletion mutations appears to have resulted from this type of recombination mechanism. The other five deletion mutations resulted from non-homologous recombination. With this admittedly limited number of characterized macro-mutations, Alu-mediated unequal homologous recombinations account for at least 8% (3 of 38) of the macro-alterations and 1% (3 of 271) of the total HPRT germinal mutations.     

Detection of somatic mutations by high-resolution DNA melting (HRM) analysis in multiple cancers

Identification of somatic mutations in cancer is a major goal for understanding and monitoring the events related to cancer initiation and progression. High resolution melting (HRM) curve analysis represents a fast, post-PCR high-throughput method for scanning somatic sequence alterations in target genes. The aim of this study was to assess the sensitivity and specificity of HRM analysis for tumor mutation screening in a range of tumor samples, which included 216 frozen pediatric small rounded blue-cell tumors as well as 180 paraffin-embedded tumors from breast, endometrial and ovarian cancers (60 of each). HRM analysis was performed in exons of the following candidate genes known to harbor established commonly observed mutations: PIK3CA, ERBB2, KRAS, TP53, EGFR, BRAF, GATA3, and FGFR3. Bi-directional sequencing analysis was used to determine the accuracy of the HRM analysis. For the 39 mutations observed in frozen samples, the sensitivity and specificity of HRM analysis were 97% and 87%, respectively. There were 67 mutation/variants in the paraffin-embedded samples, and the sensitivity and specificity for the HRM analysis were 88% and 80%, respectively. Paraffin-embedded samples require higher quantity of purified DNA for high performance. In summary, HRM analysis is a promising moderate-throughput screening test for mutations among known candidate genomic regions. Although the overall accuracy appears to be better in frozen specimens, somatic alterations were detected in DNA extracted from paraffin-embedded samples.     

SNP identification,haplotype analysis,and parental origin of mutations in TSC2

Inactivating mutations in the TSC2 gene, consisting of 41coding exons in 40 kb on 16p13, cause the hamartoma syndrome tuberous sclerosis. During TSC2 mutational analysis we identified ten SNPs that occur within or close to exon boundaries at minor allele frequencies greater than 5%. We determined the haplotypes for six of these SNPs and the microsatellite marker kg8 in the 3' region of TSC2 in a set of 40 parent-child trios. The most common haplotypes accounted for 53%, 11%, 6%, and 5% of chromosomes. Thirty-eight TSC2 mutation-bearing haplotypes had a similar distribution, indicating that there was no haplotype that predisposed to mutation in this region of TSC2. Family analysis was possible in 12 sporadic cases, and indicated that the mother was the parent of origin in 7 cases (3 point mutations, 2 small deletions, 2 large deletions), while the father was in 5 cases (2 point mutations, 3 small deletions). We conclude that TSC2 mutations occur at substantial frequency on both the maternally and paternally derived TSC2 alleles, in contrast to many other genetic diseases including NF1. The observations have implications for genetic counseling in TSC.     

Sensitive and specific detection of K-ras mutations in colon tumors by short oligonucleotide mass analysis

Short oligonucleotide mass analysis (SOMA) is a technique by which small sequences of mutated and wild-type DNA, produced by PCR amplification and restriction digestion, are characterized by HPLC-electrospray ionization tandem mass spectrometry. We have adapted the method to specifically detect two common point mutations at codon 12 of the c-K-ras gene. Mutations in DNA from 121 colon tumor samples were identified by SOMA and validated by comparison with sequencing. SOMA correctly identified 26 samples containing the 12GAT mutation and four samples containing the 12AGT mutation. Sequencing did not reveal mutant DNA in three samples out of the 26 samples shown by SOMA to contain the 12GAT mutation. In these three samples, the presence of mutant DNA was confirmed by SOMA analysis after selective PCR amplification in the presence of BstN1 restriction enzyme. Additional mutations in codons 12 and 13 were revealed by sequencing in 24 additional samples, and their presence did not interfere with the correct identification of G to A or G to T mutations in codon 12. These results provide the basis for a sensitive and specific method to detect c-K-ras codon 12-mutated DNA at levels below 10–12% of wild-type DNA.     

Screening for EGFR and KRAS mutations in endobronchial ultrasound derived transbronchial needle aspirates in non-small cell lung cancer using COLD-PCR

EGFR mutations correlate with improved clinical outcome whereas KRAS mutations are associated with lack of response to tyrosine kinase inhibitors in patients with non-small cell lung cancer (NSCLC). Endobronchial ultrasound (EBUS)-transbronchial needle aspiration (TBNA) is being increasingly used in the management of NSCLC. Co-amplification at lower denaturation temperature (COLD)-polymerase chain reaction (PCR) (COLD-PCR) is a sensitive assay for the detection of genetic mutations in solid tumours. This study assessed the feasibility of using COLD-PCR to screen for EGFR and KRAS mutations in cytology samples obtained by EBUS-TBNA in routine clinical practice. Samples obtained from NSCLC patients undergoing EBUS-TBNA were evaluated according to our standard clinical protocols. DNA extracted from these samples was subjected to COLD-PCR to amplify exons 18-21 of EGFR and exons two and three of KRAS followed by direct sequencing. Mutation analysis was performed in 131 of 132 (99.3%) NSCLC patients (70F/62M) with confirmed lymph node metastases (94/132 (71.2%) adenocarcinoma; 17/132 (12.8%) squamous cell; 2/132 (0.15%) large cell neuroendocrine; 1/132 (0.07%) large cell carcinoma; 18/132 (13.6%) NSCL-not otherwise specified (NOS)). Molecular analysis of all EGFR and KRAS target sequences was achieved in 126 of 132 (95.5%) and 130 of 132 (98.4%) of cases respectively. EGFR mutations were identified in 13 (10.5%) of fully evaluated cases (11 in adenocarcinoma and two in NSCLC-NOS) including two novel mutations. KRAS mutations were identified in 23 (17.5%) of fully analysed patient samples (18 adenocarcinoma and five NSCLC-NOS). We conclude that EBUS-TBNA of lymph nodes infiltrated by NSCLC can provide sufficient tumour material for EGFR and KRAS mutation analysis in most patients, and that COLD-PCR and sequencing is a robust screening assay for EGFR and KRAS mutation analysis in this clinical context.     

Mutation analysis of Taiwanese Wilson disease patients

Wilson disease (WD) is an autosomal recessive disorder of copper metabolism, which is caused by mutation in copper-transporting ATPase (ATP7B). In the present study, we report a molecular diagnosis method to screen the WD chromosome in patients or in heterozygotic carriers in Taiwan. Exons 8, 11, 12, 13, 16, 17, and 18 of ATP7B are selected for the screening of mutations. The most common mutation, Arg778Leu or Arg778Gln, was first screened by PCR-RFLP then we combined single-stranded conformation polymorphism (SSCP) analysis followed by direct DNA sequencing on the DNA fragments with mobility shift on SSCP analysis. The diagnostic rate was compared with standard ATP7B whole gene sequencing analysis. Ten different mutations were identified among 29 WD patients; among them four were novel (Ala1168Pro, Thr1178Ala, Ala1193Pro, and Pro1273Gln). The false positive rates were tested against 100 normal individuals and listed as follows: exon 8: 5%; exon 11: 4%; exon 12: 6%; exon 13: 5%; exon 16: 5%; exon 17: 3%; exon 18: 4%. The Arg778Leu mutation exhibited the highest allelic frequency (43.1%). The detection rate of WD chromosomes is 65.52%, which is as sensitive as whole gene sequencing scanning. According to our results, WD chromosomes in Taiwan are predominantely located at exons 8, 11, 12, 13, 16, 17, and 18. The standard sequencing analysis on the entire gene is time consuming. We recommend screening these 7 exons first on those individuals who have a higher risk in having WD, before whole gene and promoter sequencing analysis in Taiwan.     

NF2 mutation screening by denaturing high-performance liquid chromatography and high-resolution melting analysis

Neurofibromatosis type 2 (NF2) is an autosomal-dominant disorder caused by mutations in the NF2 gene and predisposing to the development of nervous system. Identification of germline mutations is essential to provide appropriate genetic counseling in NF2 patients, but it represents an extremely challenging task because the vast majority of mutations are unique and spread over the entire coding sequence. Moreover, about 30% of de novo patients are indeed mosaic, and direct sequencing can undetect mutated alleles present in a minority of cells. As most screening techniques do not meet the requirements for efficient NF2 testing, we have developed a semi-automated denaturing high-performance liquid chromatography (DHPLC) method for point mutation detection combined with a multiplex ligation-dependent probe amplification approach to screen for gene rearrangements. In addition, we have evaluated high-resolution melting analysis (HRMA) as an exon scanning procedure to identify point mutations in the NF2 gene. The results obtained in 92 NF2 patients expand the NF2 mutational spectrum and indicate DHPLC and HRMA as good systems to screen for point mutations in diseases with a heterogeneous spectrum of alterations.     

Loss-of-function mutations in PTPN11 cause metachondromatosis, but not Ollier disease or Maffucci syndrome

Metachondromatosis (MC) is a rare, autosomal dominant, incompletely penetrant combined exostosis and enchondromatosis tumor syndrome. MC is clinically distinct from other multiple exostosis or multiple enchondromatosis syndromes and is unlinked to EXT1 and EXT2, the genes responsible for autosomal dominant multiple osteochondromas (MO). To identify a gene for MC, we performed linkage analysis with high-density SNP arrays in a single family, used a targeted array to capture exons and promoter sequences from the linked interval in 16 participants from 11 MC families, and sequenced the captured DNA using high-throughput parallel sequencing technologies. DNA capture and parallel sequencing identified heterozygous putative loss-of-function mutations in PTPN11 in 4 of the 11 families. Sanger sequence analysis of PTPN11 coding regions in a total of 17 MC families identified mutations in 10 of them (5 frameshift, 2 nonsense, and 3 splice-site mutations). Copy number analysis of sequencing reads from a second targeted capture that included the entire PTPN11 gene identified an additional family with a 15 kb deletion spanning exon 7 of PTPN11. Microdissected MC lesions from two patients with PTPN11 mutations demonstrated loss-of-heterozygosity for the wild-type allele. We next sequenced PTPN11 in DNA samples from 54 patients with the multiple enchondromatosis disorders Ollier disease or Maffucci syndrome, but found no coding sequence PTPN11 mutations. We conclude that heterozygous loss-of-function mutations in PTPN11 are a frequent cause of MC, that lesions in patients with MC appear to arise following a "second hit," that MC may be locus heterogeneous since 1 familial and 5 sporadically occurring cases lacked obvious disease-causing PTPN11 mutations, and that PTPN11 mutations are not a common cause of Ollier disease or Maffucci syndrome.     

Recurrence of a nonsense mutation in the NF1 gene causing classical neurofibromatosis type 1

Summary The gene responsible for von Recklinghausen neurofibromatosis (NF1) has recently been identified, and several point mutations and deletions have been described. The availability of intron-exon boundaries of several exons of the NF1 gene facilitates the search for mutations in affected patients. We have analysed 38 patients for mutations in exon 4 of the NF1 gene, and found one patient with a CT transition at base position 1087 of the cDNA, changing an arginine codon to a stop codon, at amino acid position 365. Sequencing of other members of the family, including both parents, did not show the mutation, confirming that this mutation is responsible for this sporadic NF1 case. As the mutation described here was previously identified in an independent case by others, this case represents a recurrence of this mutation and suggests that codon 365 might be a hot spot for mutations in the NF1 gene. Thus, a specific search for this mutation should be performed when studying NF1 sporadic or familiar cases for genetic analysis.     

Papillary renal cell carcinoma: analysis of germline mutations in the MET proto-oncogene in a clinic-based population

Approximately 10% of all renal cell carcinomas (RCCs) present a distinctive papillary histology. Familial papillary RCC (PRCC) has been described, but the majority of cases appear to be sporadic. Recently, germline mutations in the MET proto-oncogene on chromosome 7 have been identified in families with hereditary PRCC. We evaluated 59 patients with PRCC for the frequency of MET germline mutations to determine the value of genetic screening of this patient population. Between 1976 and 1997, 165 patients were identified with PRCC by retrospective chart review. Fifty-nine of 133 surviving patients agreed to provide a family history, a blood specimen, and informed consent for genetic research. DNA was isolated from peripheral blood leukocytes. Denaturing high-performance liquid chromatography (DHPLC) followed by genomic sequencing was performed on eight exons of the MET proto-oncogene, including exons 5-7 of the extracellular domain, exon 14, and exons 16-19 of the tyrosine kinase domain. The 59 patients in this study included 49 men and 10 women with a mean age at diagnosis of 61 years. Bilateral and/or multifocal disease was present in 13 cases (22%). No germline mutations were detected in the studied exons of the MET proto-oncogene (exons previously reported to contain deleterious mutations in familial PRCC). No pathological MET proto-oncogene germline mutations were identified in 59 patients with PRCC. The germline mutation rate in this clinic-based population of individuals with PRCC approaches 0% (CI = 0-6.18). MET proto-oncogene germline mutation screening does not appear to be clinically indicated in patients with PRCC without additional evidence for a genetic predisposition (positive family history, unusual age at onset, bilateral disease).     

LD-PCR coupled to long-read direct sequencing: an approach for mutation detection in genes with compact genomic structures

A number of techniques have been developed as primary screens to scan for DNA sequence variants, including denaturing gradient gel electrophoresis, denaturing high-performance liquid chromatography, single-strand conformation polymorphism and heteroduplex analysis. Variant alleles detected by these assays are subsequently characterised by DNA sequencing. Sequencing itself is rarely used as a primary screen because of labour intensity, cost, and, upon automation, occasional inaccuracy in identifying heterozygous sites. We have previously developed an approach based on coupling long-distance PCR (LD-PCR) to long-read direct sequencing to allow the detection of mutations in the approximately 1.1 kb exon 3 of MECP2. Our use of dye-labelled primers generated high-quality bi-directional sequence runs > 650 bp and allowed easy discrimination of heterozygous bases. We now describe the application of this approach to the detection of mutations in a considerably larger 6.35 kb LD-PCR fragment spanning 10 exons (exons 32-41) of the structurally complex, but genomically compact, TSC2 gene. In a blind analysis, 15/15 previously characterised mutations were successfully identified using seven overlapping bi-directional sequencing reactions. Our approach of long-read sequencing of long-distance PCR products may allow rapid sequencing of multiple exons of compact genes and may be appropriate as a highly sensitive primary screen for mutations.     

Application of Combined Long Amplicon Sequencing (CoLAS) for Genetic Analysis of Neurofibromatosis Type 1: A Pilot Study

Elaborate analyses of the status of gene mutations in neurofibromatosis type 1 (NF1) are still difficult nowadays due to the large gene sizes, broad mutation spectrum, and the various effects of mutations on mRNA splicing. These problems cannot be solved simply by sequencing the entire coding region using next-generation sequencing (NGS). We recently developed a new strategy, named combined long amplicon sequencing (CoLAS), which is a method for simultaneously analysing the whole genomic DNA region and, also, the full-length cDNA of the disease-causative gene with long-range PCR-based NGS. In this study, CoLAS was specifically arranged for NF1 genetic analysis, then applied to 20 patients (five previously reported and 15 newly recruited patients, including suspicious cases) for optimising the method and to verify its efficacy and benefits. Among new cases, CoLAS detected not only 10 mutations, including three unreported mutations and one mosaic mutation, but also various splicing abnormalities and allelic expression ratios quantitatively. In addition, heterozygous mapping by polymorphisms, including introns, showed copy number monitoring of the entire NF1 gene region was possible in the majority of patients tested. Moreover, it was shown that, when a chromosomal level microdeletion was suspected from heterozygous mapping, it could be detected directly by breakpoint-specific long PCR. In conclusion, CoLAS not simply detect the causative mutation but accurately elucidated the entire structure of the NF1 gene, its mRNA expression, and also the splicing status, which reinforces its high usefulness in the gene analysis of NF1.     

Mutations in the hypoxanthine guanine phosphoribosyltransferase gene (HPRT1) in Asian HPRT deficient families

Inherited mutation of hypoxanthine guanine phosphoribosyltransferase, (HPRT) gives rise to Lesch-Nyhan syndrome or HPRT-related gout. We have identified 34 mutations in 28 Japanese, 7 Korean, and 1 Indian families with the patients manifesting different clinical phenotypes, including two rare cases in female subjects, by the analysis of all nine exons of HPRT from the genomic DNA and reverse transcribed mRNA using PCR technique coupled with direct sequencing.     

Genomic structure and identification of novel mutations in usherin, the gene responsible for Usher syndrome type IIa

Usher syndrome type IIa (USHIIa) is an autosomal recessive disorder characterized by moderate to severe sensorineural hearing loss and progressive retinitis pigmentosa. This disorder maps to human chromosome 1q41. Recently, mutations in USHIIa patients were identified in a novel gene isolated from this chromosomal region. The USH2A gene encodes a protein with a predicted molecular weight of 171.5 kD and possesses laminin epidermal growth factor as well as fibronectin type III domains. These domains are observed in other protein components of the basal lamina and extracellular matrixes; they may also be observed in cell-adhesion molecules. The intron/exon organization of the gene whose protein we name "Usherin" was determined by direct sequencing of PCR products and cloned genomic DNA with cDNA-specific primers. The gene is encoded by 21 exons and spans a minimum of 105 kb. A mutation search of 57 independent USHIIa probands was performed with a combination of direct sequencing and heteroduplex analysis of PCR-amplified exons. Fifteen new mutations were found. Of 114 independent USH2A alleles, 58 harbored probable pathologic mutations. Ten cases of USHIIa were true homozygotes and 10 were compound heterozygotes; 18 heterozygotes with only one identifiable mutation were observed. Sixty-five percent (38/58) of cases had at least one mutation, and 51% (58/114) of the total number of possible mutations were identified. The allele 2299delG (previously reported as 2314delG) was the most frequent mutant allele observed (16%; 31/192). Three new missense mutations (C319Y, N346H, and C419F) were discovered; all were restricted to the previously unreported laminin domain VI region of Usherin. The possible significance of this domain, known to be necessary for laminin network assembly, is discussed in the context of domain VI mutations from other proteins.     

KRAS and BRAF mutation analysis can be reliably performed on aspirated cytological specimens of metastatic colorectal carcinoma

N. K. B. Pang, M. E. Nga, S. Y. Chin, T.‐M. Ismail, G. L. Lim, R. Soong and M. Salto‐Tellez
KRAS and BRAF mutation analysis can be reliably performed on aspirated cytological specimens of metastatic colorectal carcinoma Background: Sanger sequencing is one of several reliable methods in use to detect KRAS and BRAF mutations to facilitate clinical patient selection for anti‐epidermal growth factor receptor (EGFR) monoclonal antibody therapy in unresectable metastatic colorectal adenocarcinoma (CRC). Most analyses are made on pretreatment biopsy or resection specimens. There is a scarcity of published studies on the suitability of cytological samples for KRAS testing in this setting. Methods: DNA extraction was attempted on 11 search‐retrieved paired cases of histological resections or excisions of CRC and their corresponding cytological samples (representing metastases) and tested for KRAS mutations in exon 2 and 3, as well as BRAF exon 15 mutations by Sanger sequencing. Only KRAS wild‐type cases were subjected to BRAF analysis because this is the setting with true diagnostic value, as these mutations are mutually exclusive. Results: Of the 11 paired cases analysed, only eight histology cases showed satisfactory DNA quality for sequencing. Thus, only eight of the corresponding cytology cases were analysed. Seven of the eight cases tested showed the same KRAS genotype on both the aspirated cytology specimen of metastatic carcinoma and the primary tumour (histological specimen), from which we derive an overall concordance rate of 87.5%. The single discordant case was likely to be a true difference as it was demonstrated again on repeat testing of both samples. No BRAF mutations were detected on the four KRAS wild‐type cases. Conclusion: A range of cytological samples are suitable for KRAS and BRAF mutation testing, be it from previously stained preparations or cell blocks. These samples would be highly valuable in cases where cytological samples are the only material available for mutation testing.     

Evaluation of denaturing high performance liquid chromatography (DHPLC) for the mutational analysis of the neurofibromatosis type 1 (<Emphasis Type="Italic">NF1</Emphasis>) gene

The identification of mutations in the NF1 gene causing type 1 neurofibromatosis (NF1) has presented a considerable challenge because of the large size of the gene, the lack of significant mutational clustering, the diversity of the underlying pathological lesions and the presence of NF1 pseudogenes. Denaturing high performance liquid chromatography (DHPLC), a high throughput, non-hazardous and largely automated heteroduplex-based technique, is in many ways ideally suited to mutation detection in this condition. DHPLC was therefore optimised for the rapid screening of the 60 exons and splice junctions of the NF1 gene in patients with NF1. The sensitivity of DHPLC was evaluated in a retrospective study of a cohort of 111 unrelated NF1 patients with known germline mutations; 97% of mutations were detected. In a subsequent prospective analysis of 50 unrelated NF1 patients, germline mutations were identified in 34 individuals (68%), 22 of these alterations being novel. This represents the highest rate of mutation detection so far reported for the NF1 gene with a single screening technique and genomic DNA as a target.