Prenatal diagnosis of xeroderma pigmentosum and trichothiodystrophy in 76 pregnancies at risk

Kleijer, Wim J.; Sterre, Marianne L. T. van der; Garritsen, Victor H.; Raams, Anja; Jaspers, Nicolaas G. J.

doi:10.1002/pd.1849

Cited by 15 publications

(9 citation statements)

References 14 publications

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“…UVC treatment elicited UDS activity as shown by an increase in nuclear fluorescence, after conjugation of fluorescent azide to incorporated EdU (Figure 1A and B). As reported in previous publications employing 3 H-thymidine labeling and autoradiography (15,16), strong signals from scheduled DNA synthesis in S-phase cells can be distinguished from much weaker cell-cycle independent unscheduled EdU incorporation, resulting from repair synthesis (Figure 1B). Additionally, the fluorescent-azide coupling reaction seems specific to incorporated EdU, as we barely detected any non-specific cytoplasmic staining or DNA-replication-independent nuclear signals (Figure 1B).…”

Section: Resultssupporting

confidence: 80%

“…For this purpose, in a practical UDS-based XP diagnosis, normal fibroblasts have been preloaded with latex-beads in the cytoplasm and co-cultured with patient-derived fibroblasts. The ‘bead-labeled-cells’ provide an internal standard that can be detected under phase contrast microscopy which eliminates sample-to-sample staining variability (15). We tested if this technique is also compatible with the EdU assay.…”

Section: Resultsmentioning

confidence: 99%

“…For inducing DNA damage 254-nm UVC-irradiation is commonly used and cells are then incubated in the presence of either radiolabeled thymidine or a nucleoside analog. The most frequently used methods in XP-diagnostic laboratories are: (i) 3 H-thymidine incorporation, followed by evaluation of grain counting after autoradiography of tissue-culture coverslips—this technique is accurate and compatible with most immunostaining-based methods, but the procedures are labor-intensive and time-consuming (15,16). (ii) Bromodeoxyuridine (BrdU) incorporation, followed by alkaline denaturation of DNA, and immunofluorescent detection by anti-BrdU antibody—this is a convenient variant of method (i); replacement of 3 H-thymidine by BrdU diminishes the time needed for the assay, however it has reduced sensitivity (∼50% reduction of UDS activity is not detectable) (18).…”

Section: Introductionmentioning

confidence: 99%

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A rapid non-radioactive technique for measurement of repair synthesis in primary human fibroblasts by incorporation of ethynyl deoxyuridine (EdU)

Limsirichaikul

Niimi

Fawcett

et al. 2008

Nucleic Acids Research

107

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Xeroderma pigmentosum (XP) is an autosomal recessive genetic disorder. Afflicted patients show extreme sun-sensitivity and skin cancer predisposition. XP is in most cases associated with deficient nucleotide excision repair (NER), which is the process responsible for removing photolesions from DNA. Measuring NER activity by nucleotide incorporation into repair patches, termed ‘unscheduled DNA synthesis (UDS)’, is one of the most commonly used assays for XP-diagnosis and NER research. We have established a rapid and accurate procedure for measuring UDS by replacement of thymidine with 5-ethynyl-2'-deoxyuridine (EdU). EdU incorporated into repair patches can be directly conjugated to fluorescent azide derivatives, thereby obviating the need for either radiolabeled thymidine or denaturation and antibody detection of incorporated bromodeoxyuridine (BrdU). We demonstrate that the EdU incorporation assay is compatible with conventional techniques such as immunofluorescent staining and labeling of cells with micro-latex beads. Importantly, we can complete the entire UDS assay within half a day from preparation of the assay coverslips; this technique may prove useful as a method for XP diagnosis.

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Section: Resultssupporting

confidence: 80%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A rapid non-radioactive technique for measurement of repair synthesis in primary human fibroblasts by incorporation of ethynyl deoxyuridine (EdU)

Limsirichaikul

Niimi

Fawcett

et al. 2008

Nucleic Acids Research

107

View full text Add to dashboard Cite

show abstract

“…The notable exception is inactivation of the DNA damageinducible gene 14-3-3 σ (50), which may be related to the effects on the NER pathway that we have observed. The highest NER capacity observed in our TSI samples was 74% of normal [NER capacities of 50% of normal and lower are considered to be diagnostic for XP (4,51)]. If 0.70 times the average of the NER capacity of the breast reduction samples is established as a cutoff (Fig.…”

Section: Discussionmentioning

confidence: 99%

Nucleotide excision repair deficiency is intrinsic in sporadic stage I breast cancer

Latimer

Johnson

Kelly

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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The molecular etiology of breast cancer has proven to be remarkably complex. Most individual oncogenes are disregulated in only approximately 30% of breast tumors, indicating that either very few molecular alterations are common to the majority of breast cancers, or that they have not yet been identified. In striking contrast, we now show that 19 of 19 stage I breast tumors tested with the functional unscheduled DNA synthesis assay exhibited a significant deficiency of DNA nucleotide excision repair (NER) capacity relative to normal epithelial tissue from disease-free controls (n = 23). Loss of DNA repair capacity, including the complex, damage-comprehensive NER pathway, results in genomic instability, a hallmark of carcinogenesis. By microarray analysis, mRNA expression levels for 20 canonical NER genes were reduced in representative tumor samples versus normal. Significant reductions were observed in 19 of these genes analyzed by the more sensitive method of RNase protection. These results were confirmed at the protein level for five NER gene products. Taken together, these data suggest that NER deficiency may play an important role in the etiology of sporadic breast cancer, and that early-stage breast cancer may be intrinsically susceptible to genotoxic chemotherapeutic agents, such as cis-platinum, whose damage is remediated by NER. In addition, reduced NER capacity, or reduced expression of NER genes, could provide a basis for the development of biomarkers for the identification of tumorigenic breast epithelium.human primary breast cell explant culture | human breast tumor explant culture | breast epithelial attached epispher | hypermutability

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“…NER was measured by using the clinically applied functional UDS assay, a technique requiring attached, live, and actively proliferating cells for its application (Kleijer et al 2007).…”

Section: Primary Culture Of Ovarian Epitheliummentioning

confidence: 99%

Cell-type-specific level of DNA nucleotide excision repair in primary human mammary and ovarian epithelial cell cultures

et al. 2008

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DNA repair, a fundamental function of cellular metabolism, has long been presumed to be constitutive and equivalent in all cells. However, we have previously shown that normal levels of nucleotide excision repair (NER) can vary by 20-fold in a tissue-specific pattern. We have now successfully established primary cultures of normal ovarian tissue from seven women by using a novel culture system originally developed for breast epithelial cells. Epithelial cells in these cultures aggregated to form three-dimensional structures called "attached ovarian epispheres". The availability of these actively proliferating cell cultures allowed us to measure NER functionally and quantitatively by the unscheduled DNA synthesis (UDS) assay, a clinical test used to diagnose constitutive deficiencies in NER capacity. We determined that ovarian epithelial cells manifested an intermediate level of NER capacity in humans, viz., only 25% of that of foreskin fibroblasts, but still 2.5-fold higher than that of peripheral blood lymphocytes. This level of DNA repair capacity was indistinguishable from that of normal breast epithelial cells, suggesting that it might be characteristic of the epithelial cell type. Similar levels of NER activity were observed in cultures established from a disease-free known carrier of a BRCA1 truncation mutation, consistent with previous normal results shown in breast epithelium and blood lymphocytes. These results establish that at least three "normal" levels of such DNA repair occur in human tissues, and that NER capacity is epigenetically regulated during cell differentiation and development.

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Prenatal diagnosis of xeroderma pigmentosum and trichothiodystrophy in 76 pregnancies at risk

Abstract: Reliable prenatal diagnosis for XP and TTD can be made by the demonstration of clearly reduced UV-induced DNA repair synthesis due to defective global genome nucleotide excision repair.

Cited by 15 publications

References 14 publications

A rapid non-radioactive technique for measurement of repair synthesis in primary human fibroblasts by incorporation of ethynyl deoxyuridine (EdU)

A rapid non-radioactive technique for measurement of repair synthesis in primary human fibroblasts by incorporation of ethynyl deoxyuridine (EdU)

Nucleotide excision repair deficiency is intrinsic in sporadic stage I breast cancer

Cell-type-specific level of DNA nucleotide excision repair in primary human mammary and ovarian epithelial cell cultures

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