18个trio家系突变研究

文章是：Trio-based exome sequencing arrests de novo mutations in early-onset high myopia

高度近视的遗传病因几十年来一直不清楚。早发性高度近视（EOHM）定义为在6岁以下的时候屈光度低于或等于-6屈光度，这个年龄段的儿童不太可能接受高教育压力，我们研究了早发性高度近视（EOHM）的遗传因素。使用trio（两个非患病父母和一个患病孩子）全外显子测序分析致病突变。我们在16个先证者中鉴定了遗传自父母的复合杂合突变或未知功能的新生突变。有趣的是，在EOHM患者中确定的新发突变的概率明显增加。在新鉴定的候选基因中，在一个EOHM先证者中鉴定出BSG新发突变。在1,040名患者扩大筛选，在BSG基因上发现另外四个突变。然后，我们构建了Bsg突变小鼠，以进一步阐明该基因的功能影响，并观察到典型的近视表型，包括延长的轴向长度。在EOHM中使用基于一家三口trio外显子研究，我们解释了没有近视父母的EOHM患者的新发突变的突出作用。疾病基因BSG的发现为近视发展及其病因提供了新的见解，这扩大了我们目前对高度近视的认识，可能对未来的治疗和预防有所帮助。

本文的科研思路是对16个trio家系进行全外显子分析，通过隐性遗传模式分析发现了6个已知的致病基因，通过新发突变发现了17个候选致病基因，但是因为样本量少没有重叠的新发突变基因，接下来用14种生信软件分析这些位点的危害性，发现BSG基因的危害性比较高，并且BSG的功能与高度近视比较相关，然后在1000多例散发病例中只验证这个BSG基因，又发现了四个可疑的致病位点（悲催的是没有父母样本，不知道是不是新发突变，哪怕只有一个证明是新发突变也是很强的证据），没有办法，只能构建敲除小鼠看看基因表达与表型情况，结果还是很不错的，最后生信分析一下这个基因与之前已知致病基因有没有蛋白左右作用。

Raw sequencing reads were filtered using the FastQC program to remove reads with a sequencing quality lower than 20, and the 3′/5′ adapter sequences on each read were trimmed using the Cutadapt program . The clean reads were mapped to human reference genome (hg19/GRCH37) by the BWA software, and PCR duplications were removed by the Sequence Alignment/Map tools (SAMtools). Local realignments of reads, quality recalibration, and variant calling (VCF format) were performed by the Genome Analysis Toolkit (GATK). De novo and rare inherited mutations (including SNVs and small indels) in each trio were detected by the mirTrios program based on the VCF files produced by GATK. Annotation of the detected variants with respect to the consequences of their mutation on known genes (gene name, functional effect, amino acid change, etc.), mutation effect predicted by multiple computational methods (SIFT, Polyphen2, MutationTaster, etc.), and population allele frequency (from 1000 Genomes, EVS, and ExAC database) were performed using the ANNOVAR software. The sequencing data have been deposited in the figshare database (DOI: 10.6084/m9.figshare.3497660; https://figshare.com/s/0eab58f90f3b1b20a181).

A typical screenshot of mirTrios:

mirTrios was developed for identification and comprehensive analysis the de novo and rare inherited variants with one or multiple trios samples based on high-throughput sequencing data starting from a VCF file (version 4). It uses reference gene definitions and hg19 genomic coordinates for annotation.

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