Unidade de Endocrinologia do Desenvolvimento, Laboratório de Hormônios e Genética Molecular/LIM42, Hospital das Clínicas, Disciplina de Endocrinologia, Faculdade de Medicina da Universidade de São Paulo, São Paulo, Brazil.
Division of Endocrinology, Diabetes, and Hypertension, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA.
Neuroendocrinology. 2018;107(2):127-132. doi: 10.1159/000490059. Epub 2018 May 15.
Loss-of-function mutations in the coding region of MKRN3, a maternally imprinted gene at chromosome 15q11.2, are a common cause of familial central precocious puberty (CPP). Whether MKRN3 alterations in regulatory regions can cause CPP has not been explored to date. We aimed to investigate potential pathogenic variants in the promoter region of MKRN3 in patients with idiopathic CPP.
PATIENTS/METHODS: A cohort of 110 patients with idiopathic CPP was studied. Family history of precocious sexual development was present in 25%. Mutations in the coding region of MKRN3 were excluded in all patients. Genomic DNA was extracted from peripheral blood leukocytes, and 1,100 nucleotides (nt) of the 5'-regulatory region of MKRN3 were amplified and sequenced. Luciferase assays were performed in GT1-7 cells transiently transfected with plasmids containing mutated and wild-type MKRN3 promoter.
We identified a rare heterozygous 4-nt deletion (c.-150_-147delTCAG; -38 to -41 nt upstream to the transcription start site) in the proximal promoter region of MKRN3 in a girl with CPP. In silico analysis predicted that this deletion would lead to the loss of a binding site for a downstream res-ponsive element antagonist modulator (DREAM), a potential transcription factor for MKRN3 and GNRH1 expression. Luciferase assays demonstrated a significant reduction of MKRN3 promoter activity in transfected cells with a c.-150_- 147delTCAG construct plasmid in both homozygous and heterozygous states when compared with cells transfected with the corresponding wild-type MKRN3 promoter region.
A rare genetic alteration in the regulatory region of MKRN3 causes CPP.
MKRN3 是位于 15q11.2 染色体上的母系印记基因，其编码区的功能丧失突变是家族性中枢性性早熟（CPP）的常见原因。到目前为止，还没有研究过调节区的 MKRN3 改变是否会导致 CPP。我们旨在研究特发性 CPP 患者 MKRN3 启动子区的潜在致病变异。
患者/方法：研究了 110 例特发性 CPP 患者。25%的患者有性早熟家族史。所有患者均排除 MKRN3 编码区的突变。从外周血白细胞中提取基因组 DNA，并扩增和测序 MKRN3 5'-调控区的 1100 个核苷酸（nt）。用含有突变和野生型 MKRN3 启动子的质粒瞬时转染 GT1-7 细胞，进行荧光素酶检测。
我们在一名 CPP 女孩中发现了 MKRN3 近端启动子区罕见的杂合 4-nt 缺失（c.-150_-147delTCAG；转录起始位点上游-38 至-41nt）。计算机分析预测，该缺失将导致一个下游反应元件拮抗剂调节剂（DREAM）结合位点丢失，DREAM 是 MKRN3 和 GNRH1 表达的潜在转录因子。荧光素酶检测显示，与转染相应野生型 MKRN3 启动子区域的细胞相比，c.-150_-147delTCAG 构建质粒转染的细胞中 MKRN3 启动子活性显著降低，无论是纯合状态还是杂合状态。
MKRN3 调节区的罕见遗传改变导致 CPP。
