X连锁迟发性脊椎骨骺发育不良家系TRAPPC2基因缺失突变的高通量测序分析

doi:10.3969/j.issn.1674-8115.2024.03.015

上海交通大学学报（医学版） ›› 2024, Vol. 44 ›› Issue (3): 407-411.doi: 10.3969/j.issn.1674-8115.2024.03.015

• 短篇论著 • 上一篇

X连锁迟发性脊椎骨骺发育不良家系TRAPPC2基因缺失突变的高通量测序分析

刘宇¹(), 王环环², 肖冰¹, 唐利芳³()

^1.上海交通大学医学院附属新华医院儿科医学研究所，上海 200092
^2.上海市儿童医院，上海交通大学医学院附属儿童医院医学遗传研究所，上海 200040
^3.复旦大学附属中山医院青浦分院儿科，上海 201700

收稿日期:2023-05-06 接受日期:2023-12-21 出版日期:2024-03-28 发布日期:2024-04-29
通讯作者: 唐利芳 E-mail:liuyu13817354143@163.com;tanglifang1119@126.com
作者简介:刘宇（1971—），女，主管技师，本科；电子信箱：liuyu13817354143@163.com。
基金资助:
上海市自然科学基金(23ZR1452700);上海市卫生健康委员会面上项目(202140103)

High-throughput sequencing analysis of deletion mutation of TRAPPC2 in an X-linked spondyloepiphyseal dysplasia tarda pedigree

LIU Yu¹(), WANG Huanhuan², XIAO Bing¹, TANG Lifang³()

^1.Institute for Pediatric Research, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China
^2.Institute of Medical Genetics, Shanghai Children′s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200040, China
^3.Department of Pediatrics, Qingpu Branch of Zhongshan Hospital, Fudan University, Shanghai 201700, China

Received:2023-05-06 Accepted:2023-12-21 Online:2024-03-28 Published:2024-04-29
Contact: TANG Lifang E-mail:liuyu13817354143@163.com;tanglifang1119@126.com
Supported by:
Natural Science Foundation of Shanghai(23ZR1452700);Project of Shanghai Municipal Health Commission(202140103)

摘要/Abstract

摘要：

目的·研究一个X连锁迟发性脊椎骨骺发育不良（spondyloepiphyseal dysplasia tarda，SEDT）家系的致病基因及突变类型。方法·提取一个SEDT家系6名成员外周血基因组DNA。应用Clearseq遗传性疾病试剂盒靶向捕获先证者基因组样本中与罕见遗传性疾病相关的致病区域，并进行高通量测序，过滤去除高频突变。采用外显子组隐马尔科夫模型（exome hidden Markov model，XHMM）分析拷贝数变异（copy number variant，CNV），并进一步对6名家系成员基因缺失片段的拷贝数进行实时定量PCR分析。结果·高通量测序分析结果显示，先证者X染色体存在2.5 kb缺失（chrX：13 732 385~13 734 927），该区域覆盖转运蛋白复合体亚单位2（transport protein particle complex subunit 2，TRAPPC2）基因的第4~6个外显子。定量PCR结果证实先证者及其表哥均存在该缺失，先证者母亲为杂合缺失，先证者父亲、姐姐和表型正常的舅舅拷贝数均正常。结论·TRAPPC2基因第4~6个外显子片段的缺失为SEDT的致病性突变；同时高通量测序分析中运用XHMM算法可检测到致病基因多个外显子的缺失。

关键词: 迟发性脊椎骨骺发育不良, 高通量测序, 转运蛋白复合体亚单位2基因

Abstract:

Objective ·To explore the pathogenic gene and the mutation type of a family with X-linked spondyloepiphyseal dysplasia tarda (SEDT). Methods ·Genomic DNA was extracted from the peripheral blood of 6 members of a SEDT family. Clearseq hereditary disease kit was applied to target pathogenic regions related to the rare hereditary diseases in the genomic sample of the proband, and then high-throughput sequencing and deletion of high-frequency variants were preformed. Copy number variant (CNV) was analyzed by using exome-hidden Markov model (XHMM). Real-time quantitative PCR was performed to further analyze the copy numbers of the gene deletion fragment in the 6 family members. Results ·High-throughput sequencing results showed that 2.5 kb fragment deletion existed in the chromosome X (chrX: 13 732 385?13 734 927) of the proband, which covered exon 4?6 of the transport protein particle complex subunit 2 (TRAPPC2) gene. The quantitative PCR results confirmed the proband and his male cousin carried the deficiency. The proband′s mother had a heterozygous deficiency, and the proband′s father, sister and the uncle with normal phenotypes all had normal copy numbers. Conclusion ·The fragment deletion of exon 4?6 of TRAPPC2 gene is the pathogenic mutation of SEDT, and the XHMM algorithm in high-throughput sequencing analysis can detect the deletion of multiple exons in pathogenic genes.

Key words: spondyloepiphyseal dysplasia tarda (SEDT), high-throughput sequencing, transport protein particle complex subunit 2 (TRAPPC2) gene

中图分类号:

R394.3

刘宇, 王环环, 肖冰, 唐利芳. X连锁迟发性脊椎骨骺发育不良家系TRAPPC2基因缺失突变的高通量测序分析[J]. 上海交通大学学报（医学版）, 2024, 44(3): 407-411.

LIU Yu, WANG Huanhuan, XIAO Bing, TANG Lifang. High-throughput sequencing analysis of deletion mutation of TRAPPC2 in an X-linked spondyloepiphyseal dysplasia tarda pedigree[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2024, 44(3): 407-411.

图/表 3

图1 X连锁SEDT患者家系图

Fig 1 Family diagram of the patient with X-linked SEDT

表1 TRAPPC2 基因缺失片段RT-qPCR引物序列

Tab 1 Primer sequences of TRAPPC2 gene deletion fragment

Name	Sequence (5′→3′)	Product length/bp	Annealing temperature/℃
Exon4Q	Forward: CAAACTGTTTTTTCCCCACAAGA	275	62
	Reverse: TAAGAAGGTAAGGATATGCCCCG
Exon5Q	Forward: GGTTGCTTTTTGTTTTTCATTTCTAA	327	60
	Reverse: TTCCTAAATACATATTCACCTGGCTG
Exon6Q	Forward: GATTTGTCAGCTTTTTTGCTTCACTAA	94	62
	Reverse: CACTTGATCGAATAGGAGAATTGGGTT

图2 家系成员 TRAPPC2 基因第4~6个外显子的RT-qPCR结果Note：Ⅱ- 4—the mother′s brother of the proband; Ⅱ-9—the mother of the proband; Ⅱ-10—the father of the proband; Ⅲ-5—the male cousin of the proband; Ⅲ-10—the sister of the proband; Ⅲ-11—the proband; NC—normal male control.

Fig 2 RT-qPCR results of exon 4?6 of TRAPPC2 gene in family members

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X连锁迟发性脊椎骨骺发育不良家系TRAPPC2基因缺失突变的高通量测序分析

High-throughput sequencing analysis of deletion mutation of TRAPPC2 in an X-linked spondyloepiphyseal dysplasia tarda pedigree

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