Methods
Clinical data and routine test results of this patient, admitted to the Department of Endocrinology in Beijing Children hospital, were collected, summarized, analyzed and discussed. In search for a monogenic cause of the early onset of the disease, gene sequencing was performed.
DNA extraction: 2mL of peripheral venous blood was collected (EDTA anticoagulant), and genomic DNA was extracted using the QIAamp whole blood DNA extraction kit (Qiagen, Germany).
Genetic examination and technical route of exon gene detection: Whole-exon sequencing analysis after informed consent from the patient’s parents was conducted. DNA was extracted from the peripheral blood of the subjects to construct the genome library. The biotin-labelled probe was hybridized with the library DNA under certain conditions, and the streptavidin-modified magnetic beads were covalently bound to the biotin-labelled probe to capture the target gene. Finally, magnetic beads carrying the target gene were adsorbed by magnetic frame, eluted, purified and enriched. The enriched target genes were sequenced by NextSeq 500 high-throughput sequencer (Illumina). The sequencing data were compared with the hG19 reference sequence of the human genome using BWA software, and the comparison files were sorted, filtered, and compared locally with multiple sequences to eliminate false positives. Sanger sequencing was used to verify gene variants associated with the clinical phenotype of the subject. Genetic analyses were completed by Beijing McGinow Technology Co., LTD.