好气啊

感觉像是小时候语文考试的作文，不到最后半小时，绝对是写不出来的。

今晚12点是deadline，只能靠它了！！！！

LASIK手术，全称是“准分子激光原位角膜磨镶术”（laser in situ keratomileusis, LASIK），手术对象是人的角膜，就是人们常说的近视眼手术。

理想状态下，眼睛把入射光聚焦到视网膜上，我们就能看清楚东西了。但是近视的人的焦点在视网膜之前，通过配戴近视眼镜——凹透镜能使光线发散，将焦点后移到视网膜上，达到矫正近视的作用。

手术矫正近视则是通过改变眼球的屈光结构来调整光线的聚焦。角膜提供了整个眼睛屈光力的约2/3，LASIK手术正是通过改变角膜屈光率来达到矫正近视的目的的。

搜索LASIK手术与基因关系

其实我本身近视度数不高，还不到400度，只是佩戴眼镜的方式不对，而且学习工作又是每天对着电脑，总是觉得哪里不太舒服。听说这个近视眼手术并不是每个人都适合做，跟人的先天性的基因有关系，正好我测了自己的全基因组，就查一查分析一下我是否适合做这个手术。

首先是google关键词LASIK gene，发现还真有一下广告是 Gene Testing for LASIK Surgery Candidates，里面有科普到，如果有人不适合近视眼手术，通常是因为他属于一个叫做granular corneal dystrophy (GCD)的遗传情况。需要100美元的检测费用，而且是包含在近视眼手术里面的。

这种情况通常是TGFΒI基因突变引起的，有两种GCD，第一种是经典的，由12号外显子突变引起，第二种是Avellino Corneal Dystrophy (ACD)，因为首次被发现在意大利的一个叫做Avellino的小镇，由第4号外显子突变引起。当然，GCD情况比较稀有，目前做了42万近视眼手术的人群中只有390例。2006年的文章 TGFBI gene mutations in corneal dystrophies 就已经报道了超过30个突变位点，还有一个网站专门提供能做各种各样基因检测的公司名录：https://www.genetests.org/disorders/?disid=15602

OMIN数据库搜索

直接搜索关键词 granular corneal dystrophy，可以得到下面几个相关性比较强的条目：

• # 121900. CORNEAL DYSTROPHY, GROENOUW TYPE I; CDGG1
• # 607541. CORNEAL DYSTROPHY, AVELLINO TYPE; CDA
• # 608470. CORNEAL DYSTROPHY, REIS-BUCKLERS TYPE; CDRB
• # 122200. CORNEAL DYSTROPHY, LATTICE TYPE I; LCD1

也许是我对这个数据库的理解不对， 它貌似只能把性状关联到基因，可是我已经知道了这些性状相关基因是TGFB1，就懒得去看了，我关心的是具体的位点。

SNPedia数据库搜索

还是直接搜索关键词 granular corneal dystrophy，能搜索到这个条目 https://www.snpedia.com/index.php/Corneal_dystrophy 发现居然有超过20种的corneal dystrophy了，实在是太可怕了。涉及到的基因也不再仅仅是 TGFBI ，下面我就摘抄一些关于TGFB1基因的一些突变位点如下：

我的基因情况

太多的陌生名词了，我只关注那个Avellino corneal dystrophy ，意大利的小镇。跟这个症状相关的位点是https://www.snpedia.com/index.php/Rs121909211 ， 参考基因组在该位点是G，如果突变了就是有风险。查看了一下我的全基因组测序结果得到的vcf文件，果然木有这个突变，这么稀有的概率，我想我应该没有这么好的运气吧。因为没有医学背景，也没有仔细去查看每个单词的意思，其实也是有granular相关的单词，因为那个GROENOUW TYPE I其实就是granular，就是rs121909208 ，同理我也查看了一下，还是没有。

省了100美元的基因检测费用

可以去做近视手术啦，虽然我不一定会去。

参考：

http://www.vistareye.com/blog/detail/2015/06/02/new-evolution-in-gene-testing-for-lasik-surgery-candidates.html

http://blogs.plos.org/dnascience/2014/07/31/gene-test-predicts-blindness-lasik/

http://www.ncbi.nlm.nih.gov/pubmed/16683255

历史目录：

直播我的基因组分析-目录-持续更新

【直播】我的基因组81：看看我的vcf文件的vaf分布情况

【直播】我的基因组82：如何对maf格式的突变文件统计vaf

批量IGV截图【直播】我的基因组83

从WGS测序得到的VCF文件里面提取位于外显子区域的【直播】我的基因组84

安装snpEFF工具并对VCF文件进行注释【直播】我的基因组85

基因组重测序的unmapped reads assembly探究 【直播】我的基因组86

探究某个基因的外显子覆盖度情况【直播】我的基因组87

刚才发现了一个更好的方案：用 R blogdown 搭建个人博客或科研网站

他已经介绍的非常详细了，我就不多说了，大家直接去他博客看哈！

早在2015年的83个药物对14个DIPG细胞系的筛选实验中，就发现哪怕是效果最好的multi-HDAC inhibitor panobinostat也会被某些DIPG细胞系产生耐药性。（http://www.biotrainee.com/thread-1599-1-1.html）

预示着需要联合用药，或者采取其它策略。
有据可考的有效例子是干扰 RNA polymerase II (RNAPII) transcription 转录活性
这时候需要引入两个基因的背景知识。
BRD4 a key activator of RNAPII transcription at active chromatin marks
CDK7, a member of the cyclin-dependent kinase family involved in regulation of RNAPII phosphorylation, controlling transcriptional initiation, pausing, and elongation
而且它们的抑制剂是：
THZ1 is a highly specific CDK7 inhibitor effective
BET (bromodomain and extra-terminal) protein inhibition, targeting family member BRD4
JQ1也被广泛使用来抑制 bromodomain activity

因为 transcriptional dysregulation in DIPG is chiefly driven by the H3K27M mutation
提出假说：we hypothesized that DIPG may be vulnerable to transcriptional disruption.

实验材料是8个DIPG细胞系，七个是K27M，一个是WT：
Eight patient-derived DIPG cultures and one pediatric cortical glioblastoma culture (SU-pcGBM2) were used in this study;
seven of the eight DIPGs exhibit the H3K27M mutation and one is histone wild-type (WT)
(H3.3K27M: SU-DIPG-VI, SU-DIPG-XIII-P, SU-DIPG-XVII, SU-DIPG-XXV, SF7761, and JHH-DIPG1; H3.1K27M: SU-DIPG-IV; H3WT and MYCN amplified: VUMC-DIPG-10;).
SU-pcGBM2 is histone-3 WT and exhibits a TP53 mutation and EGFR amplification

结果，经过了JQ1处理后，WT的DIPG的受影响最小。

然后测试了两个目前临床批准的 bromodomain inhibitor drugs ，分别是 iBET762 和  OTX015
然后用targeting BRD4 的shRNA处理，看看变化，结果knockdown后，DIPG严重被破坏了。
同样用THZ1来抑制DIPG细胞系体内的CDK7，Disruption of RNAPII transcription through CDK7 inhibition thus appears to potently disrupt DIPG cell viability.
所以可以用HDAC inhibition together with JQ1 or THZ1  联合治疗。
这样，即使有些DIPG细胞系对HDAC inhibition 耐受了，这些细胞系仍然可以被bromodomain or CDK7 inhibition来杀死。
而且作者对panobinostat, JQ1, and THZ1 这3种处理都做了RNA-seq。(14个数据，第6/8细胞系的3种药物单独处理+联合处理，还有对照)
然后有做了它们3个的ChIP-seq来看看它们的target genes的overlap情况咋样。(8个数据，第6/8细胞系的3个药物处理的H3K27ac，共有一个INPUT)
数据都在：https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE94259

首先是药物处理的表达数据差异分析结果是：
THZ1-treated cells showed preferential disruption of genes related to transcription and gene regulation,
such as ETS1, ELF4, MGA, SOX10, and HES5.
In contrast, both JQ1 and panobinostat disrupted key regulators of nervous system development,
including NTRK3, LINGO1, ASCL1, SYT4, SYT17, MYT1, MYRF, and SALL3.
In addition, both drugs disrupted genes that enriched for synapse organization and structure,
with one panobinostat target being NLGN3, a key mechanism mediating neuronal activity-regulated glioma growth

最后对3个药物处理的H3K27ac数据一直在分析super enhancer，我表示完全蒙圈了，搞不懂写的些什么鬼画符。

其次，药物是：
Using this DIPG culture panel, we screened 83 drugs selected by pediatric neurooncologists as either promising targeted agents for cancer or traditional chemotherapeutic agents in use for pediatric brain tumor therapy.
结果是：We found sensitivity to a limited number of drugs (14/83 compounds demonstrated activity against three or more DIPG cultures

最有效的是：multi-HDAC inhibitor panobinostat

筛查结果如下：

(a) Heat map demonstrating DIPG cell line sensitivity to each of the 83 agents tested in a chemical screen of 14-patient derived DIPG cell cultures. The values shown are the absolute IC50 divided by the maximum dose. All drugs had a maximum dose of 10 μM except for drugs 26, 32, 40, 59 and 61, which had a maximum dose of 100 μM.
Values are shown as gradations of red to white: red, submicromolar IC50 values; white, IC50 greater than the maximum dose for that drug (i.e., 10 μM or 100 μM); pink, the range in between. Gray boxes indicate drugs not included in the screen for that cell line. Numbers corresponding to the drug names in the key below the heat map are listed above, and culture IDs are listed at right. The histone status of each culture used in the screen is indicated by green (wild type, WT), yellow (H3.3K27M, H3F3A-K27M) or blue (H3.1K27M, HIST1H3B-K27M); see also Supplementary Table 1. Recurrent 'hits' are visible as a column of red or pink.

(b) Dose-response curves of patient-derived DIPG lines (SU-DIPG-IV, SU-DIPG-VI, SU-DIPG-XIII, JHH-DIPG1 or SF7761 (ref. 3)) treated with the indicated drugs (x-axis) at 0.001, 0.01, 0.1, 1 or 10 μM or with 0.1% DMSO (vehicle control). n = 3 wells per condition. Cell viabilities were assessed at 72 h. Data are expressed relative to control. A pediatric cortical glioblastoma cell line (SU-pcGBM-2; histone WT) was treated in parallel for comparison in a subset. Data are shown as mean ± s.d.

(c) Panobinostat time course: DIPG cells were treated with panobinostat at the indicated concentrations (25–500 nM) or with control. n = 4 wells per condition. Cell viabilities were assessed at 0, 24, 48 and 72 h after treatment. Data are shown as mean ± s.d. **P < 0.01, ***P < 0.001; two-tailed t-test; results are shown for the lowest concentration that revealed a significant difference at 48 or 72 h.

筛查完了还需要反复实验解释得到的最有效的multi-HDAC inhibitor panobinostat 的效果的原理：

Expression of proliferation-associated genes MKI67 and CCND1 in DIPG cells decreased with time after panobinostat exposure

short hairpin RNA (shRNA)-mediated knockdown of HDAC1 or HDAC2 in the DIPG cells decreased cell viability

Western blot analyses of cells expressing the H3.3K27M mutation demonstrated a dose-dependent increase in H3 acetylation and H3K27 trimethylation following panobinostat treatment

最后说明：suggesting that the drug produced partial rescue of the H3K27M-induced global hypotrimethylation phenotype.

但是这个号称最有效的药物其实也是会被耐受的：
Highlighting the need for effective combination therapies, DIPG cells that survived chronic panobinostat exposure demonstrated resistance when re-challenged with the drug
所以作者认为应该进行联合治疗。
就跟前人工作比较一下：
首先前人发现;A recent, elegant study demonstrated utility of the histone demethylase inhibitor GSK-J4 for treating DIPG
那么我们就试验一下呗；
GSK-J4 decreased cell viability in H3K27M DIPG cell cultures
Panobinostat synergized with GSK-J4 in H3.3K27M mutant DIPG cells

哦，忘记介绍一个背景知识了：
The finding that the leading DIPG therapeutic candidate is an epigenetic modifying agent is congruent with the discovery that the majority (~80%) of DIPG tumors harbor K27M mutations in the histone H3 genes HIST1H3B or H3F3A

同时还有17 MAY 2013一篇science文章提到了K27M mutant影响了PRC2活性。

we performed IHC for H3K27me3 in a large cohort of pHGGs with known H3F3A mutation status (n = 104).

Strikingly, all K27M mutant pHGGs (n = 21) showed a strong reduction of overall H3K27me3 levels.

这篇文章就这一个目的，抢这个热点。

这篇文章的数据只有表达芯片数据可以下载了！

又增加了22个表达芯片数据！

Microarray expression data of 22 tumor samples are available in National Center for Biotechnology Information’s Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo) through GEO Series accession numbers GSE36245, GSE34824, and GSE49822.

H3K27me3 ChIP-Seq and WGBS data are available through European Genome-phenome Archive accession number EGAS00001000578. 这些数据需要申请才能下载，我就不多说了！

我们知道H3蛋白是由多个基因编码的，而H3F3A这个基因的K27M突变居然使得细胞整体的H3K27me3水平下降，简直不可思议。

因为H3F3A这个基因翻译的H3蛋白只是占了细胞H3蛋白的极少比例，虽然H3F3A这个基因的K27M突变肯定使得它自己的H3K27me3不能成功，但是它为什么能影响其它H3蛋白，的确是非常奇怪的事情。

所以非常值得探究。但是作者只是介绍了这个发现而已。并没有深入探究。

We identified six epigenetic and biological GBM subgroups displaying distinct global DNA methylation patterns, which harbor unique hotspot mutations, DNA copy-number alterations, and transcriptomic patterns.

分组如下：

主要是做甲基化(GSE36278)

We investigated a cohort of GBMs from children (n = 59) and adult patients (n = 77) for genome-wide DNA methylation patterns using the Illumina 450k methylation array.

Consensus clustering using the 8,000 most variant probes across the data set robustly identified six distinct DNA methylation clusters.

Based on correlations with mutational status, DNA copy-number aberrations, and gene expression signatures, as outlined below, we have labeled these subgroups “IDH,” “K27,” “G34,” “RTK I (PDGFRA),” “Mesenchymal,” and “RTK II (Classic).”

还根据肿瘤部位，一下临床marker，还有生存情况来论证它分组的正确性。

样本量是：

Primary tumor samples for methylation (n = 136; Table S1), mutation (n = 460; Table S2), and gene expression (n = 69) analysis and all clinical data were collected at the DKFZ (Heidelberg, Germany) and at McGill University (Montreal, Canada).

测序用的是杂交捕获PCR纯化测序方法。

表达量增加了46个样本！

数据地址是;

The complete CpG methylation values are accessible through GEO Series accession number GSE36278.

The complete gene expression values are accessible through GEO Series accession numbers GSE36245 and, as part of a previously reported series, GSE34824 (Schwartzentruber et al., 2012).

To decipher the molecular pathogenesis of paediatric GBM, we undertook a comprehensive mutation analysis in protein-coding genes by performing whole-exome sequencing (WES) on 48 well-characterized paediatric GBMs, including 6 patients for whom we had matched non-tumour (germline) DNA.

只有6个患者有NT配对样本，用来找somatic的mutation，结果发现其中4个患者就有H3F3A的突变，但是H3F3A本身是非常保守的，所以这个现象值得研究。

to our knowledge no human disorders have specifically been associated with mutations in histones, including H3.3

所以才扩大了WES测序样本数量。

(数据在 https://www.ebi.ac.uk/ega/studies/EGAS00001000226   )

结果：A total of 15 samples had heterozygous H3.3 mutations (9 K27M, 5 G34R, 1 G34V) and 14 samples had a mutation in ATRX

但是早先的TCGA计划中的GBM只测序了600个基因，不包括我们感兴趣的基因，而且公共数据里面的22个成人GBM里面 没有发现H3F3A相关突变。

所以把自己医院的所有样本(784 gliomas)都测了个遍，总结如下：

因为H3F3A突变了，同时需要探究

ATRX, which encodes a member of a transcription/chromatin remodelling complex required for the incorporation of H3.3 at pericentric heterochromatin and at telomeres, as well as at several transcription factor binding sites

DAXX (because the gene product heterodimerizes with ATRX and participates in H3.3 recruitment to DNA

同时也探究了27个样本的mRNA表达水平

showed a clear mutation-specific expression pattern when comparing both between K27 and G34 mutants and with H3.3 wild-type GBMs, including DLX2, SFRP2, FZD7 and MYT1

数据在; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE34824

RNA from 27 primary tumor samples was subject to QC by Agilent BioAnalyser analysis and then hybridised to an Affymetrix U133 Plus2 gene expression array

同时也探究了拷贝数：

DNA from 31 of the 48 paediatric GBM tumours analysed by whole-exome sequencing was hybridized to Illumina Human Omni 2.5M Single Nucleotide Polymorphism (SNP) arrays

但是这个数据无法找到。

生信各种组学群 554052300

癌症信息学 375389720

bioconductor中文社区 535223278

• 软件方面的包（包括各种芯片数据处理，NGS数据处理，差异分析等等！）
• 注释方面的包(第二类是一系列的基因组注释包，主要是各种ID的转换，kegg或者GO这样的功能注释，还有其它基因信息注释，转录本，外显子起始终止等等)
• 实验数据的包(每一个实验数据包都是一篇优秀的生物信息学分析文章，分析方法，思路都是值得学习的！)
• S4对象的讲解(这个是综合性质的讲解，因为bioconductor系列的包的基础就是一系列对象及函数，需要细致的讲解)
• 分析流程的讲解（共有6个基础流程加上8个进阶流程）
• 其它学习资源收集与翻译(学习过程收集到的资源分享一下)

j基因检测 555689949

可视化 555631759