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.