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)