Marta Podralska

Recurrent APC gene mutations in Polish FAP families

The molecular diagnostics of genetically conditioned disorders is based on the identification of the mutations in the predisposing genes. Hereditary cancer disorders of the gastrointestinal tracts are caused by mutations of the tumour suppressor genes or the DNA repair genes. Occurrence of recurrent mutation allows improvement of molecular diagnostics. The mutation spectrum in the genes causing hereditary forms of colorectal cancers in the Polish population was previously described. In the present work an estimation of the frequency of the recurrent mutations of the APC gene was performed. Eight types of mutations occurred in 19.4% of our FAP families and these constitute 43% of all Polish diagnosed families.

Thyroid cancer in two siblings with FAP syndrome and APC mutation

Familial adenomatous polyposis (FAP) is characterized by the presence of numerous polyps that line the mucosa of the large intestine and, in some cases, with other extraintestinal manifestations such as other gastrointestinal adenomas, osteomas and desmoid tumours. In rare cases, duodenal or thyroid carcinomas accompany this syndrome. The thyroid cancer (TC) cases account for 1–2% of FAP patients, and the risk of development of papillary carcinoma in a female polyposis patient is 100–160 times higher than in the background population. So far, there were described only eight FAP families, with more than one family member suffering from TC. Thyroid carcinoma developing in FAP patients tends to share some common characteristics, e.g. early onset, multicentricity, high frequency of papillary histology and favourable prognosis. FAP is associated with germline mutation in the APC gene inherited in an autosomal dominant manner. In most cases, mutations of this gene are small deletions or insertions occurring in exon 15. The most frequent mutations found in Polish patients with this disease are 3927-3931delAAAGA and 3183-3187delACAAA, both in exon 15. In some reports, germline mutations in codons 1061 and 1309 were frequent in FAP patients with TC. In the present study, we report the FAP kindreds harbouring germline APC gene mutation in intron 3 at splice site junction. In the reported family, classical adenomatous polyposis coexists with thyroid cancer in two sisters. Patient 1, a 28-year-old female patient, was referred to the Regional Oncological Centre of Gdansk in 1999 because of abdominal symptoms and family history of cancer. The patient’s father was affected with FAP, and he died at the age of 43. She was diagnosed with FAP and recommended for surgery on the basis of colonoscopic finding of multicentric polyps. Procto-colectomia restorativa was performed. Histopathological review of the resected colon demonstrated numerous adenomatous polyps measuring up to 1 cm in diameter. The polyps showed moderate and severe dysplastic changes. An upper endoscopy provided at age of 28 revealed the presence of two small polyps in the duodenum, but at the age of 30, multiple duodenal polyposis was recognised with moderate and occasionally severe dysplastic changes (ampullectomy). At the age of 30, the patient underwent ultrasound examination of the thyroid gland, and fine needle aspiration of nodule 5 mm in diameter was performed. Histological analysis did not reveal any signs of malignancy. At the age of 35, a multinodular goitre was diagnosed, and total thyroidectomy was performed. Histological analysis revealed a small focus of well-differentiated papillary thyroid microcarcinoma (2.5 mm in diameter). The patient was treated with iodine-131 followed by levothyroxine therapy. Patient 2, the twin sister of patient 1, underwent surgery because of papillary thyroid cancer at the age of 23. Colonoscopy at the age of 28 revealed only a few polyps in the colon, but 5 years later, she required subtotal colectomy Int J Colorectal Dis (2008) 23:331–332 DOI 10.1007/s00384-007-0365-0

Ten new ATM alterations in Polish patients with ataxia‐telangiectasia

Inherited biallelic mutations of the ATM gene are responsible for the development of ataxia telangiectasia (AT). The objective of the present study was to conduct molecular analysis of the ATM gene in a cohort of 24 Polish patients with ataxia‐telangiectasia with aim being to provide an updated mutational spectrum in Polish AT patients. As a result of molecular analysis, the status of recurrent mutation was confirmed and ten new ATM variants were detected. Application of MLPA analysis allowed the detection of large genomic deletion. Previously, this type of mutation had never been seen in our population. Finally, in silico analysis was carried out for newly detected ATM alterations. In addition, functional analysis was performed to evaluate the effects of intronic variants: c.3402+30_3402+32delATC.

MYH Gene Status in Polish FAP Patients without APC Gene Mutations

Familial Adenomatous Polyposis (FAP) is an inheritable predisposition for the occurrence of numerous polyps in the large intestine. In about 50% of all patients, the occurrence of the disease is conditioned by heterozygotic mutations of the APC gene. Screening for genetic factors in persons without mutations in the APC gene led to the identification of homozygotic mutations of the MYH gene as the cause of the appearance of the polyposis form which is characterized by recessive heritability and a milder course than in the case of the classic form of the disease. The authors examined 90 persons from the DNA bank of patients with FAP from the Institute of Human Genetics of the Polish Academy of Sciences in Poznań in whom no mutations in the APC gene were detected. Two of the most frequent mutations of the MYH gene (Y165C and G382D) were found to be heterozygous in 13% of patients and no other mutations in this gene coding sequence were observed. In the group with heterozygotic occurrence of the mutation in the MYH gene, the disease phenotype was not milder in comparison with the entire examined group and the mean age of the disease manifestation was even lower. This observation allows one to conclude that the employed methods of mutation screening were correct and, in the case of the examined group, the mutation ratio of the MYH gene does not precondition the occurrence of the disease, but it cannot be excluded that it may modify its phenotype. The obtained results indicate that the criteria applied during the process of FAP qualification are more rigorous than those applied in other countries.

FAM13A as a Novel Hypoxia-Induced Gene in Non-Small Cell Lung Cancer

Several genome-wide association studies (GWASs), have identified that FAM13A and IREB2 loci are associated with lung cancer, but the mechanisms by which these genes contribute to lung diseases susceptibility, especially in hypoxia context, are unknown. Hypoxia has been identified as a major negative factor for tumor progression in clinical observation. It has been suggested, that lower oxygen tension, may modulate the IREB2 and FAM13A activity. However, the role of these genes in hypoxia response has not been explained. To precise the role of these genes in hypoxia response, we analyzed the FAM13A and IREB2 expression, in lung cancer cells in vitro and lung cancer tissue fragments cultured ex vivo. Three cell lines: non-small cell lung cancer (A549, CORL-105), human lung fibroblasts (HL) and 37 lung cancer tissue fragments were analyzed. The expression of IREB2, FAM13A and HIF1α after sustained 72 hours of hypoxia versus normal oxygen concentration were analyzed by TaqMan® Gene Expression Assays and Western Blot. The expression of FAM13A was significantly up-regulated by hypoxia in two lung cancer cell lines (A549, CORL-105, P<0.001), both at the level of protein and mRNA, and in lung cancer tissue fragments (P=0.0004). The IREB2 was down-regulated after hypoxia in A549 cancer cells (P<0.001). Conclusions: We found that FAM13A overexpression in human lung cancer cell lines overlapped with hypoxia effect on lung cancer tissues. FAM13A is strongly induced by hypoxia and may be identified as a novel hypoxia-induced gene in non-small cell lung cancer.

DNA bank for Polish patients with predispositions to occurrence of colorectal polyposis

Intestinal polyposis syndromes comprise of a group of diseases conditioned by the occurrence of hereditary mutations. In the Polish DNA bank of hereditary predisposition to polyposis we collected DNA samples derived from persons from families with a diagnosed adenomatous polyposis including familial adenomatous polyposis coli together with its recessive form, Turcot’s syndrome, inherited mixed polyposis as well as persons with recognised hamartomatous polyposis containing juvenile polyposis, Peutz-Jeghers syndrome, Cowden syndrome and Proteus syndrome. Investigations were conducted on DNA isolated from the peripheral blood cells. The search for mutations in APC, MUTYH, PTEN, BMPR1A, SMAD4 and STK11 genes preconditioning the occurrence of individual diseases was performed employing PCR-SSCP, PCR-HD, DHPLC as well as RFLP techniques and DNA sequencing. At the present time, the DNA Bank comprises the total of 1097 DNA samples derived from 449 families with intestinal polyposis of which 945 samples come from persons in whose families Familial Adenomatous Polyposis (FAP) occurred. In addition, the collected data also contain material derived from 25 families with Peutz-Jeghers syndrome and from 20 families with juvenile polyposis as well as single cases with the Cowden syndrome, Proteus syndrome and desmoid tumors. The performed molecular investigations allowed identification of mutations from 44 to 50% studied families. With regard to the quantity of the material collected for analyses and the efficacy level of the employed molecular methods, the obtained results are in keeping with the results found in the literature from the field of genetics and medicine and do not differ from world standards. The collection of data and materials for investigations in the case of rare diseases allows qualitative, organisational and economic optimisation of the performed investigations.

First Polish Cowden syndrome patient with confirmed PTEN gene mutation

Cowden syndrome is a rare hereditary disease. Incidence of the disease is conditioned by occurrence of mutations in the PTEN gene. The disease has a frequency of 1/120,000 newborn and it predisposes to the occurrence of hamartoma polyps in the gastrointestinal tract, skin tumours, as well as tumours of the breast, ovary and thyroid. Here we describe the case of a Polish patient diagnosed with Cowden syndrome with an identified mutation in the PTEN gene. The disease course of the patient is described and discussed along with other cases of carriers of substitution 68T>A in the PTEN gene.

Overexpression of miR-652-5p in new onset type 1 diabetes

Introduction. MicroRNAs (miRNAs) are small noncoding RNA regulating gene expression at the posttranscriptional level. miRNAs have emerged as an important regulators of central and peripheral immune tolerance, therefore study the RNA molecules in the context of type 1 diabetes (T1D) pathogenesis is an important issue. The aim of this study was to investigate miR-652-5p expression level in the new onset T1D and an impact on ADAR and MARCH5, potential target genes. Material and methods. The miR-652-5p expression was investigated in the peripheral blood mononuclear cell of newly diagnosed T1D pediatric patients (n = 28) and age-matched controls (n = 28) by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). miRNA targets were analyzed by luciferase reporter assays. Results. Expression analysis revealed upregulation of miR-652-5p in T1D group compared to non-diabetic controls (p Conclusion. Our study revealed miR-652-5p as potential marker of new onset type 1 diabetes.

Allel RET+3 : T a medullary thyroid cancer

In our studies, we compared the frequency of the occurrence of the RET+3 : T allele in our group of 48 medullary thyroid cancer (MTC)patients with the frequency of occurrence of the allele in the Polish population. The frequency of the occurrence of the heterozygote variant of the RET+3 : T for the Polish population reached almost 12% (18/152) of heterozygotes, but in the group of patients with MTC, we did not find even a single RET+3 : T allele. The frequency difference is statistically significant and in the Fishers Exact Test, the two-sided P value is 0.0080. This observation allows assuming that the occurrence of the RET+3 : T in the heterozygotic state may lead to the inhibition of the disease phenotype in the cases of the medullary thyroid carcinoma.

Participation of miRNA in conditioning the incidence and modification of the course of FAP

In July 2008, the report considering identification of miRNA associated with regulation of APC protein expression level was published. Two types of MIRNA; miR135a and miR-135b, which are encoded by three genes, were described. Those genes are located on chromosomes 3, 12 and 1. miR135a and miR-135b interact with sequences 3 `UTR of mRNA of gene APC on the basis of incomplete complementarity and lead to its degradation. The changes in the sequences encoding miR135a and miR-135b may lead to a change in their affinity to the 3 `UTR of mRNA of the gene APC and enhancing its degradation. It can result in a reduced number of functional APC protein. Also, changes in the sequence 3 `UTR of mRNA can change the affinity of the sites recognized by miRNA or induce new sites recognized by the miRNA and thus will induce rapid degradation of mRNA. Both these situations would provide a similar effect such as observed in case of the mutations leading to premature termination of protein translation. It could not be excluded that the mutations in the 3 `UTR and miRNA can also modify the disease course if co-occurring with mutations in the APC gene coding sequence.