Analyzing the First Drafts of the Human Proteome

This letter analyzes two large-scale proteomics studies published in the same issue of <i>Nature</i>. At the time of the release, both studies were portrayed as draft maps of the human proteome and great advances in the field. As with the initial publication of the human genome, these papers have broad appeal and will no doubt lead to a great deal of further analysis by the scientific community. However, we were intrigued by the number of protein-coding genes detected by the two studies, numbers that far exceeded what has been reported for the multinational Human Proteome Project effort. We carried out a simple quality test on the data using the olfactory receptor family. A high-quality proteomics experiment that does not specifically analyze nasal tissues should not expect to detect many peptides for olfactory receptors. Neither of the studies carried out experiments on nasal tissues, yet we found peptide evidence for more than 100 olfactory receptors in the two studies. These results suggest that the two studies are substantially overestimating the number of protein coding genes they identify. We conclude that the experimental data from these two studies should be used with caution

Correction to “Analyzing the First Drafts of the Human Proteome”

Correction to “Analyzing the First Drafts of the Human Proteome

Analysis of Paired Primary-Metastatic Hormone-Receptor Positive Breast Tumors (HRPBC) Uncovers Potential Novel Drivers of Hormonal Resistance

<div><p>We sought to identify genetic variants associated with disease relapse and failure to hormonal treatment in hormone-receptor positive breast cancer (HRPBC). We analyzed a series of HRPBC with distant relapse, by sequencing pairs (n = 11) of tumors (primary and metastases) at >800X. Comparative genomic hybridization was performed as well. Top hits, based on the frequency of alteration and severity of the changes, were tested in the TCGA series. Genes determining the most parsimonious prognostic signature were studied for their functional role <i>in vitro</i>, by performing cell growth assays in hormonal-deprivation conditions, a setting that mimics treatment with aromatase inhibitors. Severe alterations were recurrently found in 18 genes in the pairs. However, only <i>MYC</i>, <i>DNAH5</i>, <i>CSFR1</i>, <i>EPHA7</i>, <i>ARID1B</i>, and <i>KMT2C</i> preserved an independent prognosis impact and/or showed a significantly different incidence of alterations between relapsed and non-relapsed cases in the TCGA series. The signature composed of <i>MYC</i>, <i>KMT2C</i>, and <i>EPHA7</i> best discriminated the clinical course, (overall survival 90,7 vs. 144,5 months; p = 0.0001). Having an alteration in any of the genes of the signature implied a hazard ratio of death of 3.25 (p<0.0001), and early relapse during the adjuvant hormonal treatment. The presence of the D348N mutation in <i>KMT2C</i> and/or the T666I mutation in the kinase domain of <i>EPHA7</i> conferred hormonal resistance <i>in vitro</i>. Novel inactivating mutations in <i>KMT2C</i> and <i>EPHA7</i>, which confer hormonal resistance, are linked to adverse clinical course in HRPBC.</p></div

External testing—Cases valid for analysis from the TCGA series.

<p>Male or nonreported patients were excluded; patients negative for both hormone receptors and/or HER-2–positive, equivocal, unavailable, and/or falling in the HER-2–enriched cluster were excluded as well. We excluded from the relapse analysis patients without a follow-up status data; in addition, 17 additional patients were excluded from the survival analysis due to death by a non–tumor-related cause (non-relapsed).</p

Allelic Expansion of Variants Causing Severe Functional Protein Alterations from the Primary to the Metastatic Lesions.

<p>Allelic Expansion of Variants Causing Severe Functional Protein Alterations from the Primary to the Metastatic Lesions.</p

Clinical Characteristics and Detected Deleterious Mutations in Each Patient Pair.

<p>Clinical Characteristics and Detected Deleterious Mutations in Each Patient Pair.</p

Variant call heat map.

<p>Each of the 1071 unique variants identified are depicted in the heatmap as "present" (numbered cell) or "absent" (empty) in each primary or metastatic tumor. With the exception of pair M, each tumor was more similar to its pair than to any other case, regardless of being primary (pink-colored cases) or metastatic (yellow-colored cases), or metastatizing into the same organs or not (green: bone; red: lung; blue: peritoneum).</p

Kaplan-meier curves for patients positive versus negative for the signature.

<p><b>(A)</b> Overall survival segregating patients by being positive (alteration in either MYC, EPHA7 and/or KMT2C) or negative (lack of alterations in the three genes) for the most parsimonious gene signature, showing the complete follow-up (250 months) of the TCGA HRPBC cohort. Log-rank test p<0.001. <b>(B)</b> Overall survival of the TCGA HRPBC cohort split by the positivity or negative of the three-gene signature during the first 50 months (log-rank p<0.001), months 50 to 100 (log-rank test p<0.283) <b>(C)</b> and beyond 100 months (log-rank p = 0.074) <b>(D)</b>. It can be observed how the majority of the deaths in the three-gene positive patients occur during the first 50 months, when they are receiving adjuvant hormonal treatment. "Signature-positive" means having an alteration with functional impact (mutation, amplification/deletion, mRNA expression up- or down-regulation) in either of the three genes according to the TCGA data; "Signature-negative" means lack of alterations in the three genes.</p

KMT2C and EPHA7 mutations are related with hormonal resistance.

<p><b>(A)</b> Left panel: Cell growth (luminescence units, normalized to the growth of the parental clone in complete medium) in complete medium (FBS) or after 96 hours of estrogen deprivation (DCC-FBS). For each cell line, the parental clone and the clone isolated after 1 year of culture maintenance in DCC FBS are shown. Right panel: baseline response to estrogen withdrawal for non-<i>KMT2C</i>-mutant cells. <b>(B)</b> Three-dimensional structure of the kinase domain of EPHA7. The relevant residues are represented in “stick”-format. The catalytic residues are represented in red; the ADP-interacting residues, in cyan; and residue 666 is in purple. In the wild-type protein lysine-665 next to residue 666 makes a salt bridge with the side chain of glutamate-682 that coordinates the beta-phosphate of ADP/ATP. The side chain of wild-type threonine-666 is in direct contact with the main chain of glutamate-646. This region is highly conserved among tyrosine kinases; in particular, the threonine residue is conserved in all ephrin receptors (except 8 and 10). The mutation to isoleucine in 666 would change the hydrophobicity of that residue and would mean that it moves away from glutamate-646. This is likely to lead to a change in the orientation of lysine-665, which in turn may break the salt bridge, affect the coordination of the ADP/ATP, and impair the catalytic activity. <b>(C)</b> Left panel: CAMA1 cells were stably infected with a TRIPZ lentiviral vector carrying an inducible shRNAmir against <i>EPHA7</i> gene. shEPHA7 induction restored cell growth inhibition induced by DCC (82.5%). Right panel: CAMA1 cells transiently transfected with empty vector (EV), WT- or T666I-mutant EPHA7-containing vector. The growth arrest was rescued by T666I vector (8.2%) but not the WT vector (32.3%) compared with EV (59.7%). Luminiscence values normalized as in <b>(A)</b>. *p<0.05, **p<0.005, ***p<0.001.</p

Additional file 2: of Extreme genomic erosion after recurrent demographic bottlenecks in the highly endangered Iberian lynx

Supplemental datasheets. Datasheet S1: Summary of the annotation of the human and felid degradomes. Datasheet S2: List of orphan genes with expression levels in different organs. Datasheet S3: List of potential chromosomal rearrangements with respect to domestic cat. Datasheet S4: Gene Ontology terms enriched in proteins that duplicated at specific points in the lynx, cat, and tiger phylogeny. Datasheet S5: List of putative pseudogenes. Datasheet S6: Partitions identified by Saguaro as yielding alternative topologies from a whole genome alignment of Felidae. Datasheet S7: List of genes with signatures of positive selection. Datasheet S8: List of Gene Ontology terms significantly enriched in windows of high and low diversity in Iberian lynx populations or of high of low difference with respect to Eurasian. (XLSX 340 kb