Department of Clinical Genetics, Shengjing Hospital of China Medical University, Shenyang, 110004, Liaoning, China.
Center of Reproductive Medicine, Department of Obstetrics and Gynecology, Shengjing Hospital of China Medical University, Shenyang, 110004, Liaoning, China.
Reprod Biol Endocrinol. 2023 Mar 1;21(1):23. doi: 10.1186/s12958-023-01074-w.
Kallmann syndrome (KS) is a common type of idiopathic hypogonadotropic hypogonadism. To date, more than 30 genes including ANOS1 and FGFR1 have been identified in different genetic models of KS without affirmatory genotype-phenotype correlation, and novel mutations have been found.
A total of 35 unrelated patients with clinical features of disorder of sex development were recruited. Custom-panel sequencing or whole-exome sequencing was performed to detect the pathogenic mutations. Sanger sequencing was performed to verify single-nucleotide variants. Copy number variation-sequencing (CNV-seq) was performed to determine CNVs. The pathogenicity of the identified variant was predicted in silico. mRNA transcript analysis and minigene reporter assay were performed to test the effect of the mutation on splicing.
ANOS1 gene c.709 T > A and c.711 G > T were evaluated as pathogenic by several commonly used software, and c.1063-2 A > T was verified by transcriptional splicing assay. The c.1063-2 A > T mutation activated a cryptic splice acceptor site downstream of the original splice acceptor site and resulted in an aberrant splicing of the 24-basepair at the 5' end of exon 8, yielding a new transcript with c.1063-1086 deletion. FRFR1 gene c.1835delA was assessed as pathogenic according to the ACMG guideline. The CNV of del(8)(p12p11.22)chr8:g.36140000_38460000del was judged as pathogenic according to the ACMG & ClinGen technical standards.
Herein, we identified three novel ANOS1 mutations and two novel FGFR1 variations in Chinese KS families. In silico prediction and functional experiment evaluated the pathogenesis of ANOS1 mutations. FRFR1 c.1835delA mutation and del(8)(p12p11.22)chr8:g.36140000_38460000del were assessed as pathogenic variations. Therefore, our study expands the spectrum of mutations associated with KS and provides diagnostic evidence for patients who carry the same mutation in the future.
卡尔曼综合征（KS）是一种常见的特发性低促性腺激素性性腺功能减退症。迄今为止，在不同的 KS 遗传模型中已经发现了 30 多个基因，包括 ANOS1 和 FGFR1，但没有肯定的基因型-表型相关性，并且发现了新的突变。
共招募了 35 名具有性发育障碍临床特征的无亲缘关系患者。进行定制面板测序或全外显子组测序以检测致病突变。进行 Sanger 测序以验证单核苷酸变异。进行拷贝数变异测序（CNV-seq）以确定 CNV。使用计算机预测鉴定出的变异的致病性。进行 mRNA 转录分析和 minigene 报告基因检测以测试突变对剪接的影响。
通过几种常用软件评估 ANOS1 基因 c.709T > A 和 c.711G > T 为致病性，通过转录剪接检测验证 c.1063-2A > T。c.1063-2A > T 突变激活了原始剪接受体位点下游的隐蔽剪接受体位点，导致外显子 8 的 5'端的 24 个碱基发生异常剪接，产生新的转录本，c.1063-1086 缺失。根据 ACMG 指南评估 FRFR1 基因 c.1835delA 为致病性。根据 ACMG & ClinGen 技术标准，判断 del(8)(p12p11.22)chr8:g.36140000_38460000del 的 CNV 为致病性。
在此，我们在中国 KS 家族中鉴定了三个新的 ANOS1 突变和两个新的 FGFR1 变异。通过计算机预测和功能实验评估了 ANOS1 突变的发病机制。FRFR1 c.1835delA 突变和 del(8)(p12p11.22)chr8:g.36140000_38460000del 被评估为致病性变异。因此，我们的研究扩展了与 KS 相关的突变谱，并为未来携带相同突变的患者提供了诊断证据。
