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Many mutation scanning techniques are capable of locating mutations in DNA fragments much larger than the average exon. We have developed a system called Meta-PCR that can maximize the length of sequence scanned by these techniques, improving their productivity and realizing their full potential. Meta-PCR is a simple, versatile, and powerful method for generating chimeric DNA molecules. Currently, up to five PCR amplifiable fragments can be combined to form a single linear amplimer. The Meta-PCR reaction is self-assembling and takes place in two coupled stages carried out in a single reaction vessel. The order of fragments is reproducible and determined by primer design. We have developed two Meta-PCR assays, one comprising exons 6-10 of the Neurofibromatosis type 2 (NF2) gene and the second exons 8-12 of the human mismatch repair gene, hMLH1. We verified by direct sequencing that the order and sequence of the component exons in the Meta-PCR products is as predicted. Meta-PCR products from seven previously ascertained heterozygotes for NF2 mutations were directly sequenced. All seven mutations were clearly visible as mixed bases at the expected nucleotide, confirming that Meta-PCR faithfully reproduces the original sample genotype. We have evaluated the downstream use of the NF2 Meta-PCR products in fluorescent solid-phase chemical cleavage of mismatches (CCM). Meta-PCR products from eleven NF2 mutant heterozygotes were screened retrospectively for piperidine cleavage after hydroxylamine or potassium permanganate modification of mismatched bases. Ten of the 11 mutants were detected by visible cleavage. One mutation predicted to be cleaved after potassium permanganate modification was not detected. However, we were able to attribute this false negative to a failure in the CCM method. Meta-PCR is likely to be useful to clinical molecular diagnostic laboratories, helping them to fulfill demand for rapid and accurate screening for point mutations in large multi-exon genes.

• Two recent international documents on the regulation of genetic testing services are reviewed with respect to their impact on quality assurance in molecular genetic diagnostics, i.e. the Additional Protocol concerning Genetic Testing for Health Purposes (Council of Europe, ETS No. 203), as an addendum to the “Convention on Human Rights and Biomedicine” (ETS No. 164) released by the Council of Europe (CE) and the OECD Guidelines for Quality Assurance in Molecular Genetic Testing.

Costello syndrome (CS) is characterised by postnatal growth retardation, relative macrocephaly, distinctive facies, loose, hyperpigmented skin with deep palmar and plantar creases, and developmental delay.1–4 Cutaneous papillomata, which when present are the hallmark of this syndrome, develop in childhood. Cardiac anomalies, either structural defects, hypertrophic cardiomyopathy, or tachyarrhythmia, are present in 60% of cases.5 The cause of CS is unknown. Segregation analysis and recognition of advanced paternal age support autosomal dominant inheritance, with most cases being the result of de novo mutations.6 For uncommon conditions such as CS, which are generally sporadic, gene identification often relies upon clinical observation or rare cases with chromosomal rearrangements. A single translocation has been described in CS, t(1;22)(q25;q11).7 Recently, malignant tumours have been reported in a significant number of patients with CS, suggesting that a predisposition to malignancy is part of the condition. Bladder carcinoma has been reported twice.8,9 Three patients developed ganglioneuroblastomas.10,11 There have been single reports of epithelioma12 and vestibular schwannoma.13 However, rhabdomyosarcoma (RMS) has been reported in 10 patients.14–18 In seven of these cases, the tumour histology was embryonal; one was pleomorphic, one of unknown subtype,18 and the other alveolar.15 Based on the published cases of CS, with and without tumours, a tumour frequency as high as 17% has been suggested.18 We hypothesise that the increased malignancy risk in CS is the result of involvement of a tumour suppressor gene in the causation of CS, either by deletion or mutation. We suggest that the predisposition to malignancy occurs when a second mutation in the tumour suppressor gene occurs. Demonstration of loss of heterozygosity (LOH) is a proven technique for localisation of tumour suppressor genes. We report here the outcome of LOH and candidate gene studies …

Familial melanoma predisposition is associated with germline mutations at the CDKN2A/ARF locus in up to 40% of families. The exact role of the two proteins encoded by this complex locus in this predisposition is unclear. Most mutations affect either CDKN2A only or products of both genes. Recently a deletion affecting ARF-specific exon 1beta was reported in a family with melanoma and neural tumours. However, the possibility of this deletion also altering the CDKN2A transcript could not be excluded. More convincingly, a 16 base pair insertion in exon 1beta has been reported in an individual with multiple melanomas suggesting a direct role for ARF in melanoma predisposition. We report here a splice mutation in exon 1beta in a family with melanoma that results in ARF haploinsufficiency. The mutation was observed in a mother and daughter with melanoma. A sibling of the mother with breast cancer also had this mutation. Analysis of the melanoma from one individual revealed a 62 bp deletion in exon 3 of the wildtype allele and loss of the mutant allele; these somatic changes would affect both CDKN2A and ARF. These somatic events suggest that concomitant inactivation of both ARF and CDKN2A may be necessary for melanoma development and that mutations in ARF and CDKN2A possibly confer different levels of susceptibility to melanoma, with the former associated with lesser predisposition. In this situation, the events follow a 'three-hit' model as observed in tumours from FAP patients with an attenuated phenotype. Overall, the data suggest a direct role for ARF haploinsufficiency in melanoma predisposition and co-operation between ARF and CDKN2A in tumour formation, consistent with recent observations in Cdkn2a-specific knockout mice.

The analysis of a patient's tumor mutation status has become increasingly important. Pharmaceuticals such as gefitinib require that a mutation test is performed prior to treatment to help predict a positive outcome to the patient before the drug is administered. However, obtaining a suitable tumor sample for analysis is not always possible and therefore some patients that may benefit will not receive the drug. The use of easily obtainable samples such as plasma or serum would be ideal if tumor derived material could be readily extracted and contained the same predictive mutations as the source tumor. We have evaluated circulating free (cf) DNA from plasma, and to a lesser extent serum, from lung cancer patients for EGFR and KRAS mutations using allele specific PCR tests (ARMSTM). The majority of the samples had no matched tumor sample available so the mutation detection rate was compared to the expected frequency of mutations in this tumor and population type. EGFR mutations were detected in 21 of 237 plasma cf DNA samples (8.9%) (expected: 10-15%). KRAS mutations were detected in 11 of 168 plasma cf DNA samples (6.6%) (expected: 17%). Where analysis was performed on the matched cf DNA extracted from serum the mutation detection rate was lower than in plasma. Interestingly the EGFR mutation frequency was higher than KRAS in the plasma samples, although the KRAS mutation frequency was expected to be higher than EGFR mutation frequency in the tumors. This could be due to the frequent amplification of mutant EGFR in lung tumors resulting in more target copies in the cf DNA, the EGFR assays could be more sensitive or perhaps the nature of EGFR mutation containing tumors is different making them shed more DNA or survive longer in the blood. In conclusion, cf DNA derived from plasma is a useful surrogate for mutation detection when no tumor sample is available, but the mutation detection rate is not equal for different mutations in different genes.

A library of 50,000 recombinants representative of the human X chromosome has been constructed. Human X chromosomes were physically separated using a fluorescence-activated cell sorter. The DNA was purified from the chromosomes, digested to completion with the restriction enzyme EcoRI and cloned into the phage lambda gtWES.lambda B. The X-derived nature of the recombinants was confirmed by hybridization to rodent/human cell line DNA containing only the human X chromosome. Such libraries will be particularly useful for the investigation of genetic diseases such as Duchenne muscular dystrophy, where the basic defect has not been elucidated, and of neoplasia, where several specific chromosomal anomalies, particularly for the leukaemias, have been identified.

Chorionic villi can be obtained by direct transcervical aspiration at 9 to 10 weeks' gestation and used for analysis of fetal DNA. However, for the method to be reliable, there must be no detectable contamination by maternal DNA. To investigate the question of contamination, we compared the DNA of chorionic villi from five fetuses with that obtained from maternal lymphocytes, using the restriction endonuclease Taql and specific DNA probes for a pair of alleles on the X chromosome. The alleles yield fragments of different lengths when digested with Taql (length polymorphism), which can be demonstrated by electrophoresis and hybridization with the radioactive DNA probes. If the pattern obtained with the chorionic DNA is different from that obtained with the maternal DNA, contamination is not present. In two cases the fetal DNA of the chorionic villi was shown to be uncontaminated by maternal tissue. In one of these cases the mother was heterozygous and the fetus was homozygous; in the other the mother was homozygous and the fetus was heterozygous. In three other cases no definitive conclusions could be drawn, because the genotypes of the fetus and mother were identical. We conclude that chorionic villi at 9 to 10 weeks' gestation are a source of fetal DNA that can be used for gene analysis, with no detectable contamination by maternal DNA.