Cezary Cybulski

Germline 657del5 mutation in the NBS1 gene in breast cancer patients

In this report the proportion of consecutive and familial breast cancer cases harboring the 657del5 of exon 6 of the NBS1 gene was determined to assess whether it is associated with the increased risk of breast cancer development. The study consisted of 3 groups of patients: a series of consecutive 150 patients with histologically confirmed breast cancer, diagnosed under the age of 50 in the city of Szczecin; a series of 80 breast cancer patients with a family history of breast cancer in their first‐degree relatives; and a series of 530 consecutive individuals without the diagnosis of breast cancer selected at random by family doctors from the city of Szczecin. Molecular examination included allele‐specific PCR assay for the common Slavic NBS1 mutation (657del5), LOH analysis using denucleotide CA repeat microsatellite markers, haplotype analysis and sequencing. The NBS1 founder mutation was detected in 2 of 150 (1.3%) consecutive breast cancer cases diagnosed under the age of 50 years; in 3 of 80 familial breast cancer cases (3.7%); and in 3 of 530 individuals (0.6%) from the general population. Examination of tumor DNA from patients with the NBS1 mutation (groups A and B) revealed loss of heterozygosity (LOH) in all cases. Additional haplotype analysis revealed that allelic loss affects specifically wild‐type alleles. The majority of probands with breast cancer and the NBS1 mutation had a positive family history of breast cancer in their first‐degree relatives. It appears that the 657del5 mutation in exon 6 of the NBS1 gene is responsible for the occurrence of a small but significant proportion of familial breast cancer patients.

A high frequency of BRCA2 gene mutations in Polish families with ovarian and stomach cancer

Germ-line mutations in the BRCA2 gene are associated with a wide range of cancer types, including the breast, ovary, pancreas, prostate and melanoma. In this study, we evaluated the importance of a family history of stomach cancer in predicting the presence of a BRCA2 mutation in Polish patients with ovarian cancer. A BRCA2 mutation was found in eight of 34 women with ovarian cancer and a family history of stomach cancer versus three of 75 women with ovarian cancer and a family history of ovarian cancer, but not of stomach cancer (odds ratio=7.4; 95% CI 1.8–30; P=0.004). The results of this study suggest that, in the Polish population, the constellation of ovarian and stomach cancer predicts the presence of a germ-line BRCA2 mutation and confirms that stomach cancer is part of the spectrum of BRCA2 mutations. It is expected that the penetrance of BRCA2 mutations for stomach cancer will vary from country to country, reflecting local environmental and lifestyle factors.

Germline MSH2 and MLH1 mutational spectrum including large rearrangements in HNPCC families from Poland (update study).

Germline mutations in the DNA mismatch repair genes MSH2 and MLH1 account for a significant proportion of hereditary non‐polyposis colorectal cancer (HNPCC) families. One approach by which development of an efficient DNA‐testing procedure can be implemented is to describe the nature and frequency of common mutations in particular ethnic groups. Two hundred and twenty‐six patients from families matching the Amsterdam II diagnostic criteria or suspected HNPCC criteria were screened for MSH2 and MLH1 germline mutations. Fifty different pathogenic mutations were found, 25 in MSH2 and 25 in MLH1. Twenty‐four of these had not previously been described in other populations. Among our 78 families with MSH2 or MLH1 mutations, 54 (69.2%) were affected by recurrent mutations including 38 found at least twice in our own series. Two of the most frequent alterations were a substitution of A to T at the splice donor site of intron 5 of MSH2 and a missense change (A681T) of MLH1 found in 10 and eight families, respectively. Among large deletions detected by the multiplex ligation‐dependent probe amplification assay, exon 9 deletions in the MSH2 gene were found in two families. Our results indicate that a screening protocol specific for the Polish population that is limited to the detection of all reported mutations will result in the identification of the majority of changes present in MLH1 and MSH2 genes in Polish HNPCC kindreds.

Optimization of experimental conditions for RNA-based sequencing of MLH1 and MSH2 genes

The most sensitive technique for the detection of germline mutations is exon by exon sequencing of the gene under investigation using genomic DNA as a template for analysis. This approach, however, has cost and sensitivity limitations that can, at least in part, be overcome by RNA‐based analysis. Germline mutations of MLH1 and MSH2 are the most frequent cause of the inherited susceptibility to colorectal and other epithelial cancers known as hereditary non‐polyposis colorectal cancer (HNPCC). We compared the analysis of the MLH1 and MSH2 genes using mRNA and genomic DNA as starting material from 21 HNPCC patients. All samples were investigated by RT‐PCR, sequencing of cDNA and simultaneous sequencing of genomic DNA. The cDNA was generated using specific primers complementary to the ends of MLH1 and MSH2 genes, respectively. Mutations in MLH1 and MSH2 were detected in 11 out of 21 unrelated patients. In 10 out of 11 cases, mutations were detected independently of the type of primers used for reverse transcription (RT). One novel missense mutation (K751R) in MLH1 was detected using this method. One nonsense mutation (E205X) in MSH2 was only detectable when RT was performed using MSH2 gene‐specific primers. Shorter PCR products indicative of alternatively spliced transcripts were not observed when MLH1 or MSH2 specific cDNA RT primers were employed to generate template, except in one case where exon skipping was observed for exons 9 and 10. In this report we demonstrate that primers specific for RT of MLH1 and MSH2 are crucial for increasing the sensitivity of cDNA analysis. DNA sequencing using RNA as a basis for template construction may be a valuable and economical alternative to genomic DNA sequencing. Hum Mutat 17:52–60, 2001.

Retinal function in the von Hippel-Lindau disease

Purpose: To assess the retinal function in patients with von Hippel-Lindau disease (VHL). Patients: Studies were undertaken in 12 patients (17 eyes) with detected VHL gene mutation and 12 normal healthy controls (17 eyes). Methods: Pattern ERG (PERG), standard flash electroretinogram (ERG) recordings were performed in accordance with the International Society for Clinical Electrophysiology of Vision (ISCEV) standards. Results: In VHL patients, electrophysiological statistically significant changes were found. In PERG examination, increased latency of P50 was found in the total VHL group (p<0.02) and in the VHL subgroup with retinal angiomas (p<0.04). In ERG examination, photopic b-wave latency was increased in the total VHL group (p<0.03) and also in the VHL subgroup without retinal angiomas (p<0.05). In OPs, latency increase of OP2, OP3 waves and reduced amplitude of OP3 wave in the total VHL group (OP2 latency, p<0.05; OP3 latency, p<0.01; OP3 amplitude, p<0.03) and in the VHL subgroup with retinal angiomas (OP2 latency, p<0.02; OP3 latency, p<0.008; OP3 amplitude, p<0.02) were obtained. Conclusions: It can be hypothesized that dysfunction of the inner retinal layer is present in individuals with VHL disease even in patients without retinal angiomas.

CHAPTER 22:Selenium and Cancer

Selenium is a trace element that is an essential component of many enzymes that play an important role in several major metabolic pathways and ameliorate environmental insult, including the antioxidant defense system, the immune system and functioning of the thyroid gland. There is a range of serum selenium levels associated, with the lowest rate of all causing mortality – the levels should be maintained at an optimal level and be neither too low nor too high. Selenium intake varies, largely based on the selenium content of food. The mean level of selenium in the population varies considerably between countries. Thus, for some countries selenium supplementation should be considered, whereas for others it could be contraindicated. Recent meta-analyses indicate unequivocally that selenium supplementation of people with low initial serum selenium levels decreases the incidence of cancer incidence by ∼35% and of cancer mortality by almost 50%. Additionally, it appears that in Se-deficient countries selenium levels may be a useful marker to select patients for cancer surveillance using for example computerized tomography for detection of the earliest stages of bronchial malignancy and colonoscopy for detection of the early stage colorectal cancer.