RESUMEN
Purpose: The aim of this study was to identify the molecular genetic basis of hereditary retinal dystrophies (HRDs) in five unrelated Iranian families. Methods: Whole exome sequencing and Sanger sequencing were performed in all families. Variants were analyzed using various bioinformatics databases and software. Results: Based on the selected strategies, we identified potentially causative variants in five families with HRDs: the novel homozygous deletion mutation c.586_589delTTTG (p.F196Sfs*56) in the TTC8 gene of family A, the novel homozygous missense mutation c.2389T>C (p.S797P) in the CRB1 gene in family B, the novel homozygous frameshift mutation c.2707dupA (p.S903Kfs*66) in the LRP5 gene in family C, the novel homozygous splice mutation c.584-1G>T in the MERTK gene in family D, and the novel homozygous missense mutation c.1819G>C (p.G607R) rs61749412 in the ABCA4 gene of family E. Conclusions: This study highlights the presence of five novel variants associated with retinal dystrophies in selected Iranian families with hereditary blindness.
Asunto(s)
Secuenciación del Exoma , Enfermedades Hereditarias del Ojo/diagnóstico , Predisposición Genética a la Enfermedad , Distrofias Retinianas/diagnóstico , Transportadoras de Casetes de Unión a ATP/genética , Adulto , Pueblo Asiatico/genética , Niño , Proteínas del Citoesqueleto , Análisis Mutacional de ADN , Enfermedades Hereditarias del Ojo/genética , Proteínas del Ojo/genética , Femenino , Humanos , Lactante , Irán , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Masculino , Proteínas de la Membrana/genética , Biología Molecular , Proteínas del Tejido Nervioso/genética , Linaje , Proteínas/genética , Distrofias Retinianas/genética , Tirosina Quinasa c-Mer/genéticaRESUMEN
BACKGROUND: In vitro generation of germ cells introduces a novel approach to male infertility and provides an effective system in gene tracking studies, however many aspects of this process have remained unclear. We aimed to promote mouse embryonic stem cells (mESC) differentiation into germ cells and evaluate its effectiveness with tracking the expression of the Tsga10 during this process. METHODS: mESCs were differentiated into germ cells in the presence of Retinoic Acid. Based on developmental schedule of the postnatal testis, samples were taken on the 7th, 12th, and 25th days of the culture and were subjected to expression analysis of a panel of germ cell specific genes. Expression of Tsga10 in RNA and protein levels was then analyzed. RESULTS: Transition from mitosis to meiosis occurred between 7th and 12th days of mESC culture and post-meiotic gene expression did not occur until the 25th day of the culture. Results showed low level of Tsga10expression in undifferentiated stem cells. During transition from meiotic to post-meiotic phase, Tsga10 expression increased in 6.6 folds. This finding is in concordance with in vivo changes during transition from pre-pubertal to pubertal stage. Localization of processed and unprocessed forms of the related protein was similar to those in vivo as well. CONCLUSIONS: Expression pattern of Tsga10, as a gene with critical function in spermatogenesis, is similar during in vitro and in vivo germ cell generation. The results suggest that in vitro derived germ cells could be a trusted model to study genes behavior during spermatogenesis.
Asunto(s)
Regulación de la Expresión Génica/genética , Proteínas de Plasma Seminal/biosíntesis , Espermatogénesis/genética , Animales , Western Blotting , Proteínas del Citoesqueleto , Células Madre Embrionarias/fisiología , Regulación de la Expresión Génica/fisiología , Técnicas In Vitro/métodos , Masculino , Meiosis/genética , Meiosis/fisiología , Ratones , Ratones Endogámicos C57BL , Mitosis/genética , Mitosis/fisiología , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas de Plasma Seminal/genética , Espermatogénesis/fisiología , Testículo/citología , Testículo/metabolismo , Testículo/fisiologíaRESUMEN
OBJECTIVE(S): Sertoli cells support in vivo germ cell production; but, its exact mechanism has not been well understood. The present study was designed to analyze the effect of Sertoli cells in differentiation of mouse embryonic stem cells (mESCs) to germ cells. MATERIALS AND METHODS: A fusion construct composed of a Stra8 gene promoter and the coding region of enhanced green fluorescence protein was produced to select differentiated mESCs. To analyze sertoli cells' effect in differentiation process, mESCs were separated into two groups: the first group was cultured on gelatin with retinoic acid treatment and the second group was co-cultured with sertoli cell feeder without retinoic acid induction. Expressions of pre-meiotic (Stra8), meiotic (Dazl and Sycp3) and post-meiotic (Prm1) genes were evaluated at different differentiation stages (+7, +12 and +18 days of culture). RESULTS: In the first group, expressions of meiotic and post-meiotic genes started 12 and 18 days after induction with retinoic acid, respectively. In the second group, 7 days after co-culturing with Sertoli cells, expression of meiotic and post-meiotic genes was observed. CONCLUSION: These results show that differentiation process to germ cells is supported by Sertoli cells. Our findings provide a novel effective approach for generation of germ cell in vitro and studying the interaction of germ cells with their niche.