ABSTRACT
Objective:To identify the causative gene and observe the phenotypic characteristics of a family with isolated microphthalmia-anophthalmia-coloboma (MAC).Methods:A retrospective clinical study. One patient (proband) and 3 family members of a family with MAC visited the Henan Eye Hospital from May 2019 to May 2022 were included in the study. The patient's medical history and family history were inquired in detail, and the best corrected visual acuity (BCVA), slit lamp microscope, fundus photography, optical coherence tomography (OCT), ophthalmological B mode ultrasound and axial length (AL) measurement were performed. The peripheral venous blood of the proband, his parents and brother was collected for Trio whole-exome sequencing and pathogenic gene screening. Fluorescence quantitative Polymerase chain reaction was used to verify the suspicious variations. The clinical features of the patient's ocular and systemic also were observed.Results:The proband, male, was 3 years old at the first visit. The horizontal pendular nystagmus was detected in both eyes. Vertical elliptical microcornea and keyhole-shaped iris colobomas were detected in both eyes. The objective refraction at first visit (3 years old) was -4.00 DS/-0.50 DC×105° (OD) and -3.50 DS/-1.25 DC×80° (OS). Refraction and BCVA at 6 years old: -6.50 DS/-2.00 DC×110°→0.05 (OD) and -6.00 DS/-1.50 DC×80°→0.2 (OS). The AL at 4 years and 10 months old was 24.62 mm (OD) and 23.92 mm (OS), respectively. The AL at 5 years and 7 months old was 25.24 mm (OD) and 24.36 mm (OS), respectively. Ultrasonography shows tissue defects in both eyes. Fundus photography showed the inferior choroidal coloboma involving optic disc. OCT showed the optic disc in both eyes was abnormal with colobomas around, and the retinal neurosensory layer in colobomas area was disordered and thin; the retinoschisis was visible in the left eye. The proband's parents and siblings have normal phenotypes. Whole exome sequencing reveals a denovo heterozygous deletion of YAP1 gene: YAP1, chr11: 10280247-102100671, NM_ 001130145, loss 1 (EXON: 6-9). The results of bioinformatics analysis were pathogenic variants. Parents and siblings were of the wild type. Conclusions:Loss of heterozygosity in exons 6-9 of YAP1 gene is the pathogenic variation in this family. It can cause abnormal development of anterior segment, chorioretinal colobomas, deepening of axial myopia, even severe macular colobomas and retinoschisis.
ABSTRACT
Objective: To establish a tetracycline-induced gene knockdown system and study the effect of YAP1 on the function of gastric cancer cells. Methods: We constructed pLKO.1-tetON-YAP1 knock-down lentivirus and detected the vector by enzyme digestion and sequencing. Gastric cancer cell lines SGC-7901 and MKN-28 were infected with lentivirus, and YAP1 knocked-down gastric cancer cell lines induced by DOX were established. The mRNA level of YAP1 was detected by RT-qPCR, and the protein level of YAP1 was detected by Western blotting. Cell proliferation was detected by plate cloning experiment, and cell migration was detected by scratch-healing assay and Transwell assay. Results: The results of double enzyme digestion showed two bands at 6 000 bp and 3 000 bp, and that the sequencing results were consistent with the designed shRNA sequence. In the DOX-induced group, the mRNA and protein levels of YAP1 in gastric cancer cells infected with pLKO.1-tetON-YAP1 lentivirus significantly decreased compared with those in non-induced group. In the plate cloning experiment, the number of clones in shYAP1 groups decreased significantly after DOX induction, but there was no significant change in the non-induced group. Scratch-healing assay and Transwell assay showed that after DOX induction, the cell migration ability of shYAP1 groups was inhibited, but without significant change in the non-induced group. Results: We have successfully established a tetracycline-induced lentivirus system, and knocked down YAP1 gene of gastric cancer cells with this system. The proliferation and migration of gastric cancer cells are inhibited by YAP1 in this tetracycline induced lentivirus system.