RESUMO
Anophthalmia/microphthalmia (A/M) is a genetically heterogeneous birth defect for which the etiology is unknown in more than 50% of patients. We used exome sequencing with the ACE Exome(TM) (Personalis, Inc; 18 cases) and UCSF Genomics Core (21 cases) to sequence 28 patients with A/M and four patients with varied developmental eye defects. In the 28 patients with A/M, we identified de novo mutations in three patients (OTX2, p.(Gln91His), RARB, p.Arg387Cys and GDF6, p.Ala249Glu) and inherited mutations in STRA6 in two patients. In patients with developmental eye defects, a female with cataracts and cardiomyopathy had a de novo COL4A1 mutation, p.(Gly773Arg), expanding the phenotype associated with COL4A1 to include cardiomyopathy. A male with a chorioretinal defect, microcephaly, seizures and sensorineural deafness had two PNPT1 mutations, p.(Ala507Ser) and c.401-1G>A, and we describe eye defects associated with this gene for the first time. Exome sequencing was efficient for identifying mutations in pathogenic genes for which there is no clinical testing available and for identifying cases that expand phenotypic spectra, such as the PNPT1 and COL4A1-associated disorders described here.
Assuntos
Anoftalmia/genética , Anormalidades do Olho/genética , Microftalmia/genética , Mutação , Anoftalmia/metabolismo , Colágeno Tipo IV/genética , Análise Mutacional de DNA , Exoma , Exorribonucleases/genética , Feminino , Humanos , Lactente , Masculino , Proteínas de Membrana/genética , Microftalmia/metabolismo , Fatores de Transcrição Otx/genética , Receptores do Ácido Retinoico/genéticaRESUMO
The relationship between DNA crosslinks and cell death as a result of exposure to melphalan (MLN) was studied in the F1 variant of B16 melanoma cells. The formation of DNA crosslinks is believed to represent the lethal lesion following exposure of cells to bifunctional alkylating agents. The production of DNA crosslinks by MLN was determined by the recently described ethidium bromide fluorescence assay [De Jong et al., Int. J. Cancer 37, 557 (1986)]. A direct correlation between the percentage of DNA crosslinks (Ct) and cytotoxicity of melphalan has not been previously reported utilizing the fluorescence assay. The cytotoxicity of MLN and the production of DNA crosslinks by this drug were determined following a 1-hr incubation at 37 degrees. The concentrations of MLN necessary to reduce colony growth to 37% of control and 10% of control were 6.7 microM (EC37) and 26 microM (EC10) respectively. Utilizing the ethidium bromide fluorescence assay (EFA), the relationship between MLN concentration (x axis) and DNA crosslinks expressed as Ct (y axis) was best described by a power curve (y = 0.28 x 0.81; r = 0.985). The respective Ct values at the EC37 and EC10 of MLN were 1.3 and 3.8%. It appears that the sensitivity of the EFA is similar to the alkaline elution assay and, in addition, that the EFA is less technically difficult to employ with tumor cells obtained from patients.
