[Preimplantation genetic diagnosis and monogenic inherited eye diseases]. / Preimplantacní genetická diagnostika a dedicná onemocnení oka.

OBJECTIVE: Preimplantation genetic diagnosis (PGD) is an established application of genetic testing in the context of in vitro fertilization. PGD is an alternative method to prenatal diagnosis which aims to prevent the transmission of an inherited disorder to the progeny by implanting only embryos that do not carry genetic predisposition for a particular disease. The aim of this study is to provide an overview of eye disorders for which PGD has been carried out. METHODS: The European literature search focused on best practices, ethical issues, risks and results of PGD for inherited eye disorders. RESULTS: PGD is performed for a number of ocular disorders; a prerequisite for its application is however, the knowledge of a disease-causing mutation(s). The main advantage of this method is that the couple is not exposed to a decision of whether or not to undergo an abortion. Qualified counselling must be provided prior to the PGD in order to completely understand the risk of disability in any child conceived, consequences of disease manifestation, and advantages as well as limitations of this method. In the group of non-syndromic eye diseases and diseases in which ocular findings dominate, PGD has been performed in European countries for aniridia, choroideremia, congenital fibrosis of extraocular muscles, Leber congenital amaurosis, ocular albinism, retinitis pigmentosa, X-linked retinoschisis, Stargardt disease, blepharophimosis-ptosis-inverse epicanthus syndrome and retinoblastoma. Sexing for X-linked or mitochondrial diseases has been carried out for blue cone monochromatism, choroideremia, familial exudative vitreoretinopathy, Leber hereditary optic neuropathy, macular dystrophy (not further specified), Norrie disease, X-linked congenital stationary night blindness, X-linked retinoschisis and nystagmus (not further specified). CONCLUSION: In recent years, there has been an increase in potential to use PGD. The spectrum of diseases for this method has widened to include severe inherited eye diseases.Key words: preimplantation genetic diagnosis; monogenic eye diseases; in vitro fertilization.

[Implementation of arrays in first trimester prenatal diagnosis]. / Implementace array vysetrení do prenatální diagnostiky v I. trimestru.

OBJECTIVE: Array technology in chorionic villus sampling (CVS) - analysis of clinical benefit and a proposal of a more effective 1st trimester genetic testing policy. DESIGN: Retrospective study. SETTING: Gennet, Center of Medical Genetics and Reproductive Medicine, Prague. MATERIAL AND METHODS: Total of 913 CVS were performed at Gennet between 2010-2014. All 913 samples were tested by QF-PCR rapid test for aneuploidy of chromosomes 13, 18, 21, X and Y and karyotyping following standard long term culture. Microarray analysis (Illumina HumanCytoSNP12 v2.1) was performed on 179 samples with normal result from both - QF-PCR and karyotyping. RESULTS: At 229 samples the common chromosomal aneuploidy was detected using rapid QF-PCR (25% from 911 successful rapid tests). Conventional karyotyping revealed 239 unbalanced chromosome aberrations (27% from 897 successful cultivations). 227/239 (95%) positive karyotypes confirmed QF-PCR finding of common aneuploidies. 10 unbalanced chromosome aberrations were not covered by rapid QF-PCR test. Microarray analysis of samples with normal result from both- QF-PCR and karyotyping- revealed 13 clinically relevant chromosome aberrations (7.5%). CONCLUSION: New policy for chorionic villi testing at Gennet was established. Based on evaluation of the results of karyotyping, array and QF-PCR and analysis of published data we decided to replace karyotyping by microarray analysis in all cases of foetuses with normal results from QF-PCR. More effective detection of pathological and clinically relevant chromosome aberrations in examined foetuses is expected.

[When is it useful to look for TP53 germline gene mutations in families of oncology patients?]. / Kdy má smysl hledat v rodinách onkologických pacientu zárodecné mutace v genu TP53?

BACKGROUND: The Li-Fraumeni syndrome is a relatively rare familial cancer syndrome associated with germline mutations in the tumour-suppressor gene TP53. Members of affected families can suffer from a wide variety of tumours. Identification of a germline TP53 mutation is particularly important in families of patients affected by one of several characteristic types of tumours. METHODS AND RESULTS: The data used for the distribution analysis of mean age at the cancer diagnosis in TP53 mutation carriers and in the Czech population were extracted from a database of 176 families (469 cancers in 346 patients) with germline TP53 mutations and from Czech statistical data of cancer incidence in years 1994 to 1998 (UZIS). The comparison of the age distribution of the relative tumour incidence in these two groups clearly separated childhood adrenocortical sarcoma, rhabdomyosarcoma, osteosarcoma and brain tumour patients. In their families genetic counselling should be recommended. CONCLUSIONS: Patients with low age at diagnosis of these tumours, particularly patients with additional personal or family history of malignancy, should be considered as potential TP53 mutation carriers. It should be relevant not only for the treatment and follow-up of the patients but also for preventive measures aimed at other members of their families.

The expression of PAX5, p53 immunohistochemistry and p53 mutation analysis in superficial bladder carcinoma tissue. Correlation with pathological findings and clinical outcome.

OBJECTIVES: The expression pattern of PAX5 in the tissue of superficial bladder transitional cell carcinoma (TCC), its prognostic value and its correlation with p53 immunohistochemistry and p53 mutation analysis were evaluated. METHODS: Study comprised 61 patients with histologically confirmed superficial bladder TCC. Expression level of PAX5 mRNA was investigated using reverse transcriptase-polymerase chain reaction (RT-PCR) and determined semiquantitatively. The presence of p53 mutations was determined by SSCP and confirmed by direct sequencing. The p53 immunohistochemistry was performed with DO1 antibody and semiquantitatively evaluated using HSCORE (HS) method. As the control group for the evaluation of the PAX5 expression served 8 men with benign prostatic hyperplasia. RESULTS: PAX5 expression was found in 50 patients with bladder TCC but in no patient from the control group. Its quantity however correlated neither with the stage nor with the grade of the tumor. P53 mutation was confirmed only in 1 patient with pTaG2 tumor in exon 5 (deletion of proline 128). On the contrary, positive immunohistochemical staining of p53 was detected in most patients. Using the cutoff value of HS 200, 56.9% of patients showed p53 overexpression. Quantity of p53 immunochistochemical positivity did not correlate with the quantity of PAX5 expression. Using the cutoff values of HS 200 for p53 and of 0.2 for PAX5, 7 of 8 patients with future progression had p53 and 4 had PAX5 overexpression respectively. CONCLUSION: The expression of gene PAX5 is a frequent event in superficial TCC of the bladder.

The application of comparative genomic hybridization to previously karyotyped cervical cancer cell lines.

This investigation is concerned with the application of comparative genomic hybridization (CGH) to DNA from previously fully karyotyped cervical cancer cell lines using G-banding and fluorescence in situ hybridization (FISH) to compare the chromosome copy numbers observed in karyotypes with the profile shifts seen in CGH analysis. It has demonstrated that diploid DNA can be used as a reference to cohybridize with a test sample of any modal number because of the proportional representation of every chromosome arm and region in equal volumes of both test and reference DNAs. Profile shifts in the near-diploid line gave a clear indication of over and under-representation of either the whole or parts of chromosome arms. In near-tetraploid samples, profile shifts, either gain or loss due to copy number changes from four to five, five to six, or four to three were smaller and were not always seen; however, the points of profile shift would have allowed us to work out most of the breakpoints if karyotype information had not been available. The profiles, however, did not provide accurate information on the ploidy status; this would need to be measured by other means for the CGH data to be interpreted correctly. The 3q and 8q gain in all the squamous cell carcinoma cell lines appeared very clearly. Comparative genomic hybridization revealed a new breakpoint at 7q31 which was not detected originally on the karyotype in DE3. A breakpoint on 9q was reassigned on the basis of the profile shift from 9q13 to 9q22 in JE6. Clarification of the origin of a small fragment from chromosome 20 constantly present in JE6 showed it to be 20q22-qter.