FANCD2 Western blot as a diagnostic tool for Brazilian patients with Fanconi anemia

Abstract

Fanconi anemia is a rare hereditary disease showing genetic heterogeneity due to a variety of mutations in genes involved in DNA repair pathways, which may lead to different clinical manifestations. Phenotypic variability makes diagnosis difficult based only on clinical manifestations, therefore laboratory tests are necessary. New advances in molecular pathogenesis of this disease led researchers to develop a diagnostic test based on Western blot for FANCD2. The objective of the present study was to determine the efficacy of this method for the diagnosis of 84 Brazilian patients with Fanconi anemia, all of whom tested positive for the diepoxybutane test, and 98 healthy controls. The FANCD2 monoubiquitinated isoform (FANCDS+/FANCD2L-) was not detected in 77 patients (91.7%). In 2 patients (2.4%), there was an absence of both the monoubiquitinated and the non-ubiquitinated proteins (FANCD2S-/FANCD2L-) and 5 patients (5.9%) had both isoforms (FANCD2S+/FANCD2L+). This last phenotype suggests downstream subtypes or mosaicism. All controls were diepoxybutane negative and were also negative on the FANCD2 Western blot. The Western blot for FANCD2 presented a sensitivity of 94% (79/84) and specificity of 100% (98/98). This method was confirmed as an efficient approach to screen Brazilian patients with deleterious mutations on FANCD2 (FANCD2S-/FANCD2L-) or other upstream genes of the FA/BRCA pathway (FANCDS+/FANCD2L-), to confirm the chromosome breakage test and to classify patients according to the level of FA/BRCA pathway defects. However, patients showing both FANCD2 isoforms (FANCD2S+/FANCD2L+) require additional studies to confirm mutations on downstream Fanconi anemia genes or the presence of mosaicism.

Fanconi anemia: Diagnostic methods and the applicability of laboratory genetics

“…Based on the information provided by E1 and E2 and analyzed in graph 1, it is possible to see that the Western blot technique appears as one of the most adopted methods in the period from 2005 to 2009 and that, according to Pilonetto et al (2009), the results using this method involving molecular biology corroborate global data in which core complex complementation groups present great genetic variety for FA, including mutations in any of the genes that encode the central complex of proteins (FANCA , B, C, E, F, G, L and M). And they may also belong to other complementation groups to be identified and may be associated with the central complex of the FANCD2 protein.…”

Conserved function of Drosophila Fancd2 monoubiquitination in response to double-strand DNA breaks

“…Low levels of Fancd2 ubiquitination can be a diagnostic indicator of FA ( Pilonetto et al 2009 ; Liu et al 2013 ) and correlate with shorter time of disease-free survival in the context of sporadic breast cancer ( Feng and Jin 2019 ). Monoubiquitination of vertebrate Fancd2 is sufficient for targeting to chromatin in response to DNA damaging agents such as mitomycin C ( Matsushita et al 2005 ).…”

Anemia de Fanconi: Métodos diagnósticos e a aplicabilidade da genética laboratorial

“…A partir das informações fornecidas por E1 e E2 e analisadas pelo Gráfico 1 é possível constatar que a técnica de Western blot surge como um dos métodos mais adotados no período de 2005 a 2009 e que segundo Pilonetto et al (2009), os resultados por esse método envolvendo a biologia molecular corroboram com os dados mundiais em que grupos de complementação de núcleo complexo apresentam grande variedade genética para a AF que incluem mutações em qualquer um dos genes que codificam o complexo central das proteínas (FANCA, B, C, E, F, G, L e M). E também podem pertencer a outros grupos de complementação a serem identificados podendo estarem associados com o complexo central da proteína FANCD2.…”