Methods:
Blood samples were taken from the daughter, father and mother . Blood were either deposited on paper for exome analysis (Centogene) and extracted using Max-QiampDNA extraction kit (Qiagen, Hidden, Germany) according to the instructions of manufacture. DNA was quantified using Qubit (Thermo Fisher Scientific). SGCE primers for exon 5 were designed using (Primer 3 software ver 4.1.0) (F: 5’CCTCTGATGAGCCTTGGATT3’, and R:(5‘ TTCACAGACCAGGAACTTGAGA3’). PCR were conducted using 100 ng DNA using platinium Hot-start PCR2X masterMix on Veriti thermal cycler (Thermo Fisher Scientific) using cycling conditions (95°C 7 min; 95°C 30s, 58°C 40s 72°C 1 min for35 cycles; 72°C for 15 min ) . PCR amplification product was controlled on 2% agarose gel.
For sanger sequencing, the PCR product was purified using exosap method and sequenced using forward and reverse primers using Bigdye Terminator Kit v3.1 cycle sequencing kit (Applied Biosystems, Foster city, CA, USA) and runned on 3500 ABI sequencer (Applied Biosystems, Foster city, CA, USA). Sequence analysis was performed using SeqScape software v2.5 (Ref Seq consensus sequence: NM_003919)