From e4682a36496913e3ed40449b3e5ad180595d72ae Mon Sep 17 00:00:00 2001 From: Pjotr Prins Date: Fri, 19 Feb 2021 12:41:04 +0000 Subject: BLOG --- .../Wang_WIlliams_Rat_Brain_Proteome_For_Blog.md | 411 +++++++++++++++++++++ blog/2021/proteome/media/image1.png | Bin 0 -> 13412 bytes blog/2021/proteome/media/image10.png | Bin 0 -> 243443 bytes blog/2021/proteome/media/image11.png | Bin 0 -> 145211 bytes blog/2021/proteome/media/image12.png | Bin 0 -> 41221 bytes blog/2021/proteome/media/image13.png | Bin 0 -> 117129 bytes blog/2021/proteome/media/image2.png | Bin 0 -> 195319 bytes blog/2021/proteome/media/image3.png | Bin 0 -> 278356 bytes blog/2021/proteome/media/image4.png | Bin 0 -> 184772 bytes blog/2021/proteome/media/image5.png | Bin 0 -> 169900 bytes blog/2021/proteome/media/image6.png | Bin 0 -> 40876 bytes blog/2021/proteome/media/image7.png | Bin 0 -> 28535 bytes blog/2021/proteome/media/image8.png | Bin 0 -> 87973 bytes blog/2021/proteome/media/image9.png | Bin 0 -> 92049 bytes 14 files changed, 411 insertions(+) create mode 100644 blog/2021/proteome/Wang_WIlliams_Rat_Brain_Proteome_For_Blog.md create mode 100644 blog/2021/proteome/media/image1.png create mode 100644 blog/2021/proteome/media/image10.png create mode 100644 blog/2021/proteome/media/image11.png create mode 100644 blog/2021/proteome/media/image12.png create mode 100644 blog/2021/proteome/media/image13.png create mode 100644 blog/2021/proteome/media/image2.png create mode 100644 blog/2021/proteome/media/image3.png create mode 100644 blog/2021/proteome/media/image4.png create mode 100644 blog/2021/proteome/media/image5.png create mode 100644 blog/2021/proteome/media/image6.png create mode 100644 blog/2021/proteome/media/image7.png create mode 100644 blog/2021/proteome/media/image8.png create mode 100644 blog/2021/proteome/media/image9.png (limited to 'blog') diff --git a/blog/2021/proteome/Wang_WIlliams_Rat_Brain_Proteome_For_Blog.md b/blog/2021/proteome/Wang_WIlliams_Rat_Brain_Proteome_For_Blog.md new file mode 100644 index 0000000..80898e0 --- /dev/null +++ b/blog/2021/proteome/Wang_WIlliams_Rat_Brain_Proteome_For_Blog.md @@ -0,0 +1,411 @@ +From: RWWilliams, rwilliams\@uthsc.edu, labwilliams\@gmail + +To: Many (and now about ready for a blog) + +Date: 14Feb2021 + +Version: Extended cut of 15Feb2021 v2 + +Dear colleagues (particularly those interested in alcohol and drug +addiction, brain proteomics, dopamine, causal modeling), + +You could either go tobogganing today or tomorrow OR learn a bit about a +cool new rat brain proteomics-genetics data set generated by Xusheng +Wang (University of North Dakota) and colleagues, including Jumin Peng +at St. Jude Children\'s Research Hospital, and Michal Pravenec at the +Czech Academy of Science. This is an open, but not yet final, +quantitative proteomics data for the whole brain for 21 strains of rat +(male and female isogenic littermates) from the HXB/BXH family---part of +the Hybrid Rat Diversity Panel. + +For those of you on this list at NIH, first, thank you for all of your +support over more than 20 years that has made this Valentine\'s primer +possible---starting with the NIH Human Brain Project and continuing now +with a NIDA P30. I hope you will be impressed not only by the data that +Xusheng generated, but by the FAIR-ness and ease of analysis of highly +valuable smart quadratic data. + +What the heck is *smart quadratic data*? Please see an extended +discussion on this topic (verging on a rant in sections) given at the +University of Virginia in the Data Science program, 20Nov2020: + *** *** + +> **Footnote on this presentation**: Do you wonder why translation fails +> from animal models to human clinical care? 1-N-der? NIH has funded *n +> = 1* biomedical experimental research for about 70 years without +> blinking. Anyone who read Roger J. Williams\'s *Biochemical +> Individuality* when it came out in 1956 should have known better +> (***https://en.wikipedia.org/wiki/Roger_J._Williams***). My excuse +> is that I was four years old, but James Shannon was 52 years old. +> Elias Zerhouni was 5 years old, Francis Collins was 6 years old, and +> Eric Lander was about --9 months old. But it is now 2021 and in this +> glorious \"post-genomics\" era of highly accurate personalized health +> care and prevention (not) we should grow up and embrace the diversity +> and complexity of living systems. *Necessary and sufficient*---only in +> your reductive dreams. Want more of this: see *Herding Cats---the +> Sociology of Data Integration*, 2009: PMID: 20228863 +> https://doi.org/10.3389/neuro.01.016.2009 + +Many of you have given talks with a hierarchy of traits. I remember a +lovely talk that Nora Volkow gave to the Human Brain Project teams in +about 2005---from gene variants at the bottom to variable outcome +measures at the top---susceptible vs resistant, fast vs slow +metabolizer, will relapse, won\'t replase. I would say we have made only +modest progress at true holistic integration, and few biomedical +researcher know much about causal quantitative modeling. We absolutely +need the proteome tier to model addiction, and we need proteomes from +dozen of brain regions and hundreds is not thousands of individuals to +model risk and make reasonable predictions. Otherwise we are just +flapping our hands and lips. The work by Xusheng and others shows that +we are finally ready to come out of the proteomics \"winter\". The +technolgy is mature; batch effect is well controlled; cost is about the +same as Affymetrix arrays were in 2005. Several new proteomic data sets +in GeneNetwork prove it, but only Xusheng\'s data is directly relevant +to addiction. + +End of context; on with the topic at hand: + +One small molecule of great fame---dopamine---and its modulation, +variation, and contribution to addiction + +![](media/image1.png) + +QUESTION:  + +> **What proteins related to dopamine and its many roles in behavior are +> strongly modulated by DNA variants, and can we determine what gene +> variants are related both to dopamine function and substance use +> disorders. ** +> +> The Red Hot Chili Peppers ask this question in *This is the Place.* +> +> \"[Can I isolate your gene? Can I kiss your +> dopamine?](https://genius.com/Red-hot-chili-peppers-this-is-the-place-lyrics#note-1422525) +> +> \... +> +> A master piece of DNA caught in a flashing ray\" +> +> (The lyrics are* *on the horror of drug addiction. The lead, Anthony +> Kiedis, has relapsed several times. The PG version of the +> song: ***  ***and the +> typical RHCP shirtless version for the cool +> kids:*** *** + +**Step 1. **To answer the BIG Question, we are going to review all +genes/proteins in NCBI **Gene Reference into Function**---RIF for +short---that are related in some way to *dopamine*.  + +There are two ways to do this: + +1\. Link +to [***https://www.genenetwork.org***](https://www.genenetwork.org) and +set up the **Select and search **screen to look as shown below: + +![](media/image2.png) + +Note that in the **Combined** field above, I have entered the string + +> **RIF=dopamine   LRS=(15 999)**  + +This string will retrieve all proteins in the Hybrid Rat Diversity Panel +(the HXB/BXD family in this specific case) that are expressed reasonably +well (just over 8,000 proteins and over 200,000 peptide fragments) in +the whole brain.   + +The second part of the search string (LRS\...) finds all proteins that +have strong linkage---a likelihood ratio statistic score of at least 15. +This is equivalent to a LOD score of 3.3, and this is a value that is +often close to the genome-wide significance level. The other value, 999, +is just a high upper limit. + +The second way to find these proteins is a bit easier---just paste this +URL into your browser: + +> [***https://genenetwork.org/search?species=rat&group=HXBBXH&type=Whole+Brain+Proteome&dataset=UND\_NIDA\_HXB-BXH\_WBPr\_log2z8\_0221&search\_terms\_or=&search\_terms\_and=RIF%3Ddopamine+LRS%3D%2815+999%29&FormID=searchResult***](https://genenetwork.org/search?species=rat&group=HXBBXH&type=Whole+Brain+Proteome&dataset=UND_NIDA_HXB-BXH_WBPr_log2z8_0221&search_terms_or=&search_terms_and=RIF%3Ddopamine+LRS%3D%2815+999%29&FormID=searchResult) + +(This link can be shared, and will work *in perpetuity throughout the +known universe; *I phrase I steal from the Walt Disney Company legal +department with trepidation.)   + +**Step 2**. At this point if you are following along, you should have a +list of 115 proteins that are abundantly expressed in brain AND are +linked to *dopamine* AND that have reasonable genetic linkage in the HXB +family to a particular genome coordinate (usually a SNP). The **Search +Results** table should look like the screenshot below.  + +![](media/image3.png) + +I have highlighted the row 8---the ARNTL protein---a major transcription +factor involved in circadian rhythms that is upregulated by DRD2 +signaling (PMID: 16606840 in PNAS, 2006) + +**Step 3.** To begin to answer the second question---is there a major +modulator of multiple dopamine-associated proteins---we need to re-sort +this table using the column labeled **Peak Location**. In this +screenshot below I have scrolled over to the right to display the **Peak +Location** column after having performing the sort. All of these +proteins map to Chr 1 at about 43.7 megabases (Mb). + +![](media/image4.png) + +We see ARNTL again and eight other proteins that are genetically +downstream of one or many DNA variants located on the proximal part of +chromosome 1 (Chr 1). The **Peak LOD** scores range between 4.1 and 7.1. + +If you scroll down this list (and you should), you will find another +region of the rat genome that is highly linked with dopamine-associated +proteins---Chr 19 at about 60 Mb. But before we head to Chr 19, let\'s +continue to work with this proximal part of Chr 1 and try to figure out +why the variation in expression of this band of nine proteins map to +this part of the rat genome. Step 3 below is a long step---my +apologysorry. Perhaps time for a coffee break. + +**Step 3** involves mapping one or more of these nine proteins. I will +pick SYT7 since it has the highest expression (9 log2 units of +expression) and the second highest LOD score (6.8). + +You can either click on the UNIPROT identifier---**Q62747 **in the +window, or you can just paste this URL command into a browser: + +> [***https://genenetwork.org/show\_trait?trait\_id=Q62747&dataset=UND\_NIDA\_HXB-BXH\_WBPr\_log2z8\_0221***](https://genenetwork.org/show_trait?trait_id=Q62747&dataset=UND_NIDA_HXB-BXH_WBPr_log2z8_0221) + +If all goes well, your browser will display this content (and much more +too): + +![](media/image5.png) + + +Before we map SYT7 protein expression, you may be curious to know how +this protein has been linked to dopamine.  + +The answer is one click away. Tap on the **GeneWiki** button, +highlighted below in grey. + +![](media/image6.png) + +A **GeneWiki** window will open, and RIF number 18 explains the +association with *dopamine* and also links to a 2011 paper (PMID +21576241) on somatodendritic dopamine release and the involvement of +synaptotagmin 7 (SYT7).\ +\ +Again we pause briefly for \"data due diligence\". In the **Statistics** +**histogram** window you will note that the distribution of SYT7 protein +levels in 21 strains has a hint of bimodality---that is a good thing. + + + +There are no outliers, so we can map these logged protein expression +data \"as given\" without further normalization. + +We can now finally proceed to the actual mapping of variation in protein +expression---using for the first time infinite marker maps for +chromosome of all of the HXB/BXH family, and using the +updated GEMMA linear mixed model mapping function in GeneNetwork. + +Open the **Mapping Tools** window + +![](media/image8.png) + +In the screenshot above I have mapped variation in SYT7 protein level +using the new **Genotypes file: Experimental (smoothed)** + +These are genotypes based on whole genome sequencing of the HXB/BXH +family using linked-read 10X Chromium DNA libraries at a mean sequence +coverage of just over 45X. Libraries were prepared at HudsonAlpha and +sequenced on an Illumina Novaseq across the street from NIH at *The +American Genome Center* (TAGC, thanks Michal, Melinda, Hao, Clifton, +Jonathan, David, Hakan, Tristan, Victor, Jun, many others\....). + +The Manhattan plot of variation in SYT7 protein expression should look +like this:  + +![](media/image9.png) + +Beneath the Manahattan plot there is a **Mapping Statistics** table that +provides estimates a SNP coordinates (Rnor6 assembly) calculated by +GEMMA with --logP values and additive effects (log2 scale). + +![](media/image10.png) + +A --logP value of 5.27 is good---normally at or above genome-wide +threshold of significance. (This assertion does need more support, and +we are testing thresholds using using other mapping methods, including +R/qtl\'s and WebQTL\'s standard interval mapping methods, and using +permutation tests.) + +**Step 4.** What is the approximate confidence interval of the SYT7 +protein expression quantitative trait locus (QTL) on Chr 1? To answer +this question we need to sort the **Mapping Statistics** by +the **Position** column. Once sorted, we have to decide how wide a +confidence interval is appropriate given the density of DNA variants, +gene density, and --logP values. Karl Broman and others recommend a drop +in the --logP linkage statistic of about 1.5 on either side of the peak, +or plateau in this case. For the QTL map of SYT7 the confidence interval +encompasses an stretch of DNA from about 35 megabases (Mb) to 43 Mb. + +Normally, in an interval this large, we would just hit the pause button +and spend more time increasing the sample size (in progress already by +Xusheng Wang and colleagues). But for the sake of this Valentine\'s day +email, I am going to forge ahead to get to the box of chocolates and +that essential dopamine kiss in nucleus accumbens that is so rewarding.\ +\ +**Step 5**. What genes are located along this part of Chr 1?  + +To answer this question, click on the chromosome number, **1** in this +case. + +This will generate a chromosome-specific view; part shown below. + + + +The QTL peak is a \"non-recombinant\" plateau that extends from 35.5 to +45 Mb---confirming visually what we had already determined from the +--logP values.  The blue blocks along the top are gene \"models\" and +the lighter blue dots are the linkage values at different SNP locations. +You can zoom to a map with specific start- and end-coordinates.  + +You can keep zooming in on a specific region of a chromosome by clicking +on the pink horizontal bar alonge the top. Here is the plateau region of +the SYT7 protein expression QTL. + +![](media/image12.png) + +As you can tell from the screenshot, there are lots of genes---real and +putative---that call this part of Chr 1 home. + +Underneath each map an **Interval Analyst** table of all genes and +pseudogenes in a specific interval. In this case, there are about 130 +gene, of which 32+ are protein-coding.  + +Let me list them out throught to about 44 Mb. + +ADAMTS16  + +ICE1  + +MED10  + +UBE2QL1  + +NSUN2  + +SRD5A1  + +PAPD7  + +ADCY2  + +FASTKD3  + +MTRR  + +ZFP874B  + +ZFP748  + +PPP1R14C  + +IYD  + +PLEKHG1  + +MTHFD1L  + +AKAP12  + +ZBTB2  + +RMND1  + +ARNTL1  + +ESR1  + +SYNE1  + +MYCT1  + +VIP  + +CCDC170  + +FBXO5  + +MTRF1L  + +RGS17  + +OPRM1  + +IPCEF1  + +CNKSR3  + +Anything catch your eye? Actually, lots to catch the eye +here---perhaps too much.  + +The gene/\'protein that most of you will catch is **OPRM1**---the mu +opioid receptor.  + +Variants in this gene and locus are definitely controllers of morphine +response---particularly so in the BXD mouse family (Paige Lemen, Hao +Chen, Guy Mittleman, and Price Dickson have a strong abstract on this at +the upcoming 2021 NIDA Genetics meeting). Also true in *Homo sapiens* +based on initial GWAS analysis.\ +\ +**Step 6**. How do we evaluate the strength of these candidates as +controller of some subset of the nine proteins with variable expression +that map to this region? + +Simple---clip out all of those positional candidate genes and paste them +into the search **Get Any** window of GeneNetwork. It should look like +this: + + ![](media/image13.png) + +About 12 of these proteins have reasonably high expression in the rat +brain, and three of these also are associated with reasonably strong +cis-acting modulation---FASTKD3, PPP1R14C, and MTRR. That means that DNA +variant in or around these genes modulate both mRNA expression but much +more importantly, also the protein level. + +You can review these three candidates at your leisure.  + +PPP1R14C (aka KEPI)---see PMID: 11812771 + +MTRR: not much related to CNS function---mainly cancer and development + +FASKD3: not much CNS but key in mitochondrial function + +Ok, time to go out and sled. + +Any one that made it this far---bravo---you have persistence. + +Any questions about the proteomics to Xusheng Wang. + +Any questions about the genotypes and HXB sequence to Hao Chen. + +Any questions about mapping to Pjotr Prins and me. + +Any questions about GeneNetwork user interface to me. + +> [[Can I isolate your gene? Can I kiss your +> dopamine?]{.underline}](https://genius.com/Red-hot-chili-peppers-this-is-the-place-lyrics#note-1422525)\.... +> +> A perfect piece of DNA caught in a flashing ray +> +> A master piece of DNA caught in a flashing ray + +Thanks RHCP for thinking of us NIDA- and NIAAA-funded genetics +researchers. + +\--  + +Rob + +ps. You may want to know about OPRM1 as a great position and biological +candidate gene---is it causal. Unfortunately expression is not +consistently high in this proteomics analysis and we will have to look +at bit harder to find peptide fragments for this protein. 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