Compendium of proteins containing segments that exhibit zero-tolerance to amino acid variation in humans.

Genetic missense tolerance ratio (MTR) analysis systematically evaluates all possible segments in a given protein-encoding transcript found in the human population. This method scores each segment for the number of observed missense variants versus the number of silent mutations in that same segment. An MTR score of 0 indicates that no missense mutations are observed within a given segment. This is indicative of evolutionary purifying selection, which excludes mutations in that segment from the general human population. Here, we conducted MTR analysis on each of the roughly 20,000 protein-encoding human genes. It was seen that there are 257 genes with at least one 31-residue encoding segment with MTR = 0 (1.3% of all human genes). The proteins encoded by these 257 genes were tabulated along with information regarding the sequence location of each intolerant segment, the likely function of the protein, and so forth. The most functionally-enriched family among these proteins is a collection of several dozen proteins that are directly involved in RNA splicing. Some of the other proteins with zero-tolerance segments have thus far escaped significant characterization. Indeed, while a number of these proteins have previously been genetically linked to human disorders, many have not. We hypothesize that this compendium of human proteins with zero-tolerance segments can be used to complement disease mutation data as a pointer to genes and proteins that are associated with interesting and underexplored human biology.

missense tolerance ratio

INTRODUCTION

Genetic intolerance analysis has emerged as a powerful tool for studying protein evolution, structure–function relationships, and linkage of proteins to disease. , , , , , , , , , Here we examine an extreme form of protein sequential intolerance by identifying human proteins that contain segments in which genetic variation is completely disallowed by evolution.

Petrovsky and coworkers introduced an approach to measure the “missense tolerance ratios” (MTR) for segments of human protein‐encoding genes. , For a given gene, this method is based on analyzing the >105 sequences for that gene in the gnomAD database and comparing the number of missense mutations present in each 31 amino acids segment versus the number of observed silent missense mutations in that same segment. Coding genes with segments that exhibit fewer amino acid‐encoding mutations than expected based on the observed number of silent mutations for that segment are deemed to be genetically intolerant. Intolerance indicates that amino acid‐encoding missense mutations within that gene segment are evolutionarily excluded from the human gene pool by “purifying selection.” Because of the limited number of currently available sequences in gnomAD, statistically meaningful single codon MTR ratios cannot yet be determined. However, analysis of 93‐base segments encoding 31 amino acids usually yields robust statistics. A segmental MTR score of 1.0 indicates that the sequence of the analyzed gene segment is under no purifying selective pressure, whereas an MTR score of 0 means that the introduction of even a single amino acid‐varying missense mutation into a segment is not seen in gnomAD, indicative of stringent purifying selection for variations associated with that segment. Mutations occurring in an intolerant segment of a protein can result in reduced evolutionary fitness through any one of a variety of potential mechanisms, such as triggering the loss of that protein's native function or inducing the formation of toxic aggregates, as we have reviewed elsewhere.

Previous studies have explored the relationship of MTR analysis to specific proteins, particularly with respect to the use of segmental intolerance analysis to predict or illuminate the linkage of proteins to human disease. , , , , , , This highlights the fact that proteins containing intolerant segments are sometimes subjected to known disease mutations in other parts of the protein and, more rarely, even within the intolerant segment. The latter instance occurs when a disease mutation is observed in a patient‐derived database such as ClinVar that is too rare to be seen in the sample of the global (mostly healthy) human population represented by the current gnomAD collection of sequences.

Our objective in this paper differs from the previous work. Here we sought to systematically identify all protein‐encoding human genes that contain one or more MTR = 0 (“zero‐tolerance”) segments. The resulting list is the main deliverable of this paper. It is hoped that this list will serve as a useful resource for the research community in identifying proteins that contain segments in which mutations result in such catastrophic consequences that they are filtered out of the human population. Evidently, these proteins are profoundly important and/or perilous, such that their study in some cases may yield groundbreaking insight into human biology and molecular pathophysiology. We also reported a few selected observations that can be made regarding the 257 proteins that contain at least one zero‐tolerance segment. One important finding is that proteins involved in RNA splicing are the most common group of proteins that contain absolutely intolerant segments. Another important finding is that there are many proteins that contain at least one intolerant segment, but for which there exist no known disease or ClinVar pathogenic mutations to date. We hypothesized that some of these proteins must be essential to human reproduction and/or development, despite, in many cases, having escaped much prior attention or recognition.

RESULTS AND DISCUSSION

Human proteins with zero‐tolerance segments

We observed 257 proteins—ca. 1.3% of all human proteins—that contain at least one amino acid segment at least 31 residues long (or an N‐ or C‐terminal segment at least 16 residues long) in which amino acid variations appear not to be evolutionarily tolerated (MTR score = 0), as determined by MTR analysis of the human gene sequences in the gnomAD database. These proteins are listed in Table 1, ordered alphabetically by their corresponding gene symbol. For each entry, a variety of supporting information is included, such as the location of the intolerant segment(s) in the protein, the function of the protein, and whether it is a membrane protein. Many of the proteins contain multiple zero‐tolerance segments and some of these segments extend well beyond 31 residues. Figure 1a shows a histogram that summarizes the distribution of all possible 31 amino acids segment MTR scores within the 257 proteins. Even within these proteins, only 1.8% of all segments exhibited an MTR score at or near zero. Figure 1b shows the distribution of the whole‐protein median MTR score for each protein, where it is seen that the level of genetic tolerance within these proteins is typically not low, with a median score of 0.75 and a mean of 0.71 ± 0.26. These data complement results reported in a column of Table 1 in which the median MTR score is presented for all segments within each protein.

TABLE 1

Human proteins that contain at least one zero‐tolerance segment

Gene symbol, UniProt ID, transcript ID	Encoded protein	Function	GO pathway or process	Protein length	Transmembrane?	Intolerant segment(s) (UniProt numbering)	Median MTR score for entire protein	No. of ClinVar variants in intolerant segment(s)	No. of ClinVar variants in the whole protein
ABL1 P00519‐1 ENST00000318560	Tyrosine‐protein kinase ABL1	NR tyrosine kinase that is linked to cell growth and survival, as well as chromatin remodeling. Regulates CDC42 signal transduction.	GO:0009790; embryo development	1,130	No	393–423 c	0.821	None	Many
ACTB P60709‐1 b ENST00000675515	Actin, cytoplasmic 1	Actin component	GO:0030029; actin filament‐based process	375	No	53–91, 124–185, 247–278 b , (based on ENST00000331789)	0.1245	9	Many
ACTC1 P68032‐1 ENST00000290378	Actin, alpha cardiac muscle 1	Actin	GO:0060048; cardiac muscle contraction	377	No	105–151	0.435	4	Many
ACTL6B O94805‐1 ENST00000160382	Actin‐like protein 6B	Transcriptional activation and repression of select genes by chromatin remodeling. Role in neuronal development.	GO:0016573; histone acetylation	426	No	1–19	0.726	None	2
ACTR2 P61160‐1 ENST00000260641	Actin‐related protein 2	ATP binding component of Arp23 complex.	GO:0007010; cytoskeleton organization	394	No	1–16	0.654	None	None
AGO2 Q9UKV8‐1 ENST00000220592	Protein argonaute‐2	Essential for RNAi. May inhibit translation.	GO:1905114; cell surface receptor signaling pathway involved in cell–cell signaling	859	No	446–485	0.554	None	None
AP2M1 Q96CW1‐1 ENST00000292807	AP‐2 complex subunit mu	Component of AP‐2. Adaptor protein that plays a role in trafficking.	GO:1905114; cell surface receptor signaling pathway involved in cell–cell signaling	435	No	403–447 c	0.4645	None	None
AR P10275‐1 ENST00000374690	Androgen receptor	Steroid hormone receptor that can affect proliferation and differentiation.	GO:0009790; embryo development	920	No	891–932	0.849	None	None
ARF1 P84077‐1 ENST00000541182	ADP‐ribosylation factor 1	GTP binding protein involved in protein trafficking.	GO:0032880; regulation of protein localization	181	No	16–59	0.448	1	3
ARF5 P84085‐1 ENST00000000233	ADP‐ribosylation factor 5	GTP‐binding protein involved in protein trafficking.	GO:0006886; intracellular protein transport	180	No	41–74	0.603	None	None
ARIH1 Q9Y4X5‐1 ENST00000379887	E3 ubiquitin‐protein ligase ARIH1	E3 ubiquitin ligase. Interacts with cullin‐RING ubiquitin ligase complexes.	GO:0000209; protein polyubiquitination	557	No	336–369, 450–483	0.5155	None	None
ATF2 P15336‐1 ENST00000264110	Cyclic AMP‐dependent transcription factor ATF‐2	Transcriptional activator that involves anti‐apoptosis, cell growth, and DNA damage response. Can impair mitochondrial membrane potential.	GO:0045930; negative regulation of mitotic cell cycle	505	No	365–395	0.848	None	2
ATP1A1 P05023‐1 ENST00000295598	Sodium/potassium‐transporting ATPase subunit alpha‐1	Sodium potassium pump	GO:0030001; metal ion transport	1,023	Yes	604–637	0.537	None	8
ATP1A3 P13637‐2 d ENST00000543770	Sodium/potassium‐transporting ATPase subunit alpha‐3	Sodium potassium pump	GO:0030001; metal ion transport	1,024	Yes	355–398 d	0.494	5	Many
ATP2B1 P20020‐3 ENST00000428670	Plasma membrane calcium‐transporting ATPase 1	Calcium transporter	GO:0030001; metal ion transport	1,220	Yes	421–451	0.712	None	None
ATP6V0C P27449‐1 ENST00000330398	V‐type proton ATPase 16 kDa proteolipid subunit	Proton‐conducting pore forming subunit of the membrane integral V0 complex of vacuolar ATPase responsible for acidifying a variety of intracellular compartments in eukaryotic cells.	GO:0030001; metal ion transport	155	Yes	133–170	0.3715	None	None
ATRX P46100‐1 ENST00000373344	Transcriptional regulator ATRX	Involved in transcriptional regulation and chromatin remodeling. May be involved in telomere maintenance.	GO:0065004; protein–DNA complex assembly	2,492	No	1,782–1,814, 2,095–2,142, 2,159–2,213	0.837	3	Many
BCL11B Q9C0K0‐1 ENST00000357195	B‐cell lymphoma/leukemia 11B	Key regulator of differentiation and survival of T‐lymphocytes. Required for CCR7 and CCR9 receptors.	GO:0000904; cell morphogenesis involved in differentiation	894	No	789–822	0.675	1	4
BRD4 O60885‐1 ENST00000263377	Bromodomain‐containing protein 4	Chromatin reader protein that binds acetylated histones and plays a role in epigenetics.	GO:0031056; regulation of histone modification	1,362	No	508–542	0.76	None	1
BRD8 Q9H0E9‐1 ENST00000254900	Bromodomain‐containing protein 8	May act as a coactivator during transcriptional activation by hormone‐activated nuclear receptors. Component of NuA4 histone acetyltransferase.	GO:0016573; histone acetylation	1,235	No	704–736	0.891	None	None
CACNA1A O00555‐8 ENST00000360228	Voltage‐dependent P/Q‐type calcium channel subunit alpha‐1A	Voltage dependent calcium channel	GO:0030001; metal ion transport	2,506	Yes	287–325	0.762	1	Many
CACNA1C Q13936‐11 d ENST00000347598	Voltage‐dependent L‐type calcium channel subunit alpha‐1C	Calcium channel	GO:0030001; metal ion transport	2,186	Yes	731–764 d	0.710	1	Many
CACNA1E Q15878‐1 ENST00000367573	Voltage‐dependent R‐type calcium channel subunit alpha‐1E	Voltage gated calcium channel	GO:0030001; metal ion transport	2,313	Yes	1,648–1,679	0.786	None	17
CALM1 P0DP23‐1 ENST00000356978	Calmodulin‐1	Modulates the function of numerous proteins in a calcium dependent manner. Involved in centrosome cycle and cytokinesis.	GO:1905114; cell surface receptor signaling pathway involved in cell–cell signaling	149	No	110–142	0.3975	None	12
CALM2 P0DP24‐1 ENST00000272298	Calmodulin‐2	Controls a large number of enzymes and, with CCP110 and centrin, is involved in the centrosome cycle and progression through cytokinesis.	GO:0055074; calcium ion homeostasis	149	No	78–118	0.4675	3	12
CAMK2A Q9UQM7‐1 ENST00000348628	Calcium/calmodulin‐dependent protein kinase type II subunit alpha	Kinase that is activated by calcium or calmodulin	GO:0030001; metal ion transport; GO:1905114	478	No	111–163	0.515	None	9
CAND1 Q86VP6‐1 ENST00000545606	Cullin‐associated NEDD8‐dissociated protein 1	Key assembly factor of SCF ubiquitin ligase	GO:0010265; SCF complex assembly	1,230		46–76	0.786	None	1
CASK O14936‐1 ENST00000378163	Peripheral plasma membrane protein CASK	Neuronal development protein trafficking	GO:0030001; metal ion transport	926	No	73–103	0.652	None	Many
CDC42 P60953‐2 ENST00000400259	Cell division control protein 42 homolog	Epithelial polarization, attachment of spindle to microtubules. Cell migration. Present in neuronal cells.	GO:1905114; cell surface receptor signaling pathway involved in cell–cell signaling	191	No	28–109	0.2035	5	11
CDC73 Q6P1J9‐1 ENST00000367435	Parafibromin	RNA pol II recruitment (PAF1 interaction). Recruits E2 ligases to histones.	GO:0050684; regulation of mRNA processing; GO:1905114	531	No	133–173	0.721	None	Many
CDK11B P21127‐1 ENST00000407249	Cyclin‐dependent kinase 11B	Cyclin dependent kinase involved in many roles. Pre‐mRNA splicing.	GO:0050684; regulation of mRNA processing	795	No	733–801	0.7135	None	None
CELF2 O95319‐1 ENST00000416382	CUGBP Elav‐like family member 2	RNA splicing	GO:0050684; regulation of mRNA processing	508	No	413–449	0.552	None	None
CHD2 O14647‐1 ENST00000394196	Chromodomain‐helicase‐DNA‐binding protein 2	DNA binding helicase. Promotes deposition of histone H3.3.	GO:0032508; DNA duplex unwinding	1,828	No	484–519	0.743	1	Many
CHD4 Q14839‐1 ENST00000544040	Chromodomain‐helicase‐DNA‐binding protein 4	Part of NuRD complex and remodels chromatin	GO:0043044; ATP‐dependent chromatin remodeling	1,912	No	1,110–1,160, 1,165–1,212	0.672	2	17
CLASRP Q8N2M8‐1 ENST00000391953	CLK4‐associating serine/arginine rich protein	Probably functions as an alternative splice regulator.	GO:0008380; RNA splicing	674	No	1–32	0.8205	None	None
CLCN4 P51793‐1 ENST00000380833	H(+)/Cl(−) exchange transporter 4	Hydrogen chloride outward rectifying exchanger	GO:0006811; ion transport	760	Yes	519–549	0.584	1	Many
CLTC Q00610‐1 ENST00000269122	Clatherin heavy chain 1	Central protein of clathrin coated pits. Key role in endocytosis.	GO:0030001; metal ion transport	1,675	No	1,302–1,336	0.660	None	5
CNOT6L Q96LI5‐1 ENST00000504123	CCR4‐NOT transcription complex subunit 6‐like	Has poly(A) exoribonuclease activity. Catalytic component of the CCR4‐NOT complex.	GO:0006402; mRNA catabolic process	555	No	404–434	0.7255	None	None
CPSF4 O95639‐1 ENST00000292476	Cleavage and polyadenylation specificity factor subunit 4	Pre‐mRNA processing. Poly‐A cap	GO:0050684; regulation of mRNA processing	269	No	68–115	0.585	None	None
CREB1 P16220‐2 ENST00000430624	Cyclic AMP‐responsive element‐binding protein 1	Phosphorylation‐dependent transcription factor. Binds to CRE and is enhanced by TORC coactivators. Circadian rhythm and differentiation of adipose tissue.	GO:0007623; circadian rhythm	327	No	271–315 c	0.668	None	1
CREBL2 O60519‐1 ENST00000228865	cAMP‐responsive element‐binding protein‐like 2	May play a role in cell cycle. Transcriptional activity involved in adipose differentiation.	GO:0006351; transcription, DNA‐templated	120	No	20–54	0.836	None	None
CSNK2B P67870‐1 ENST00000375882	Casein kinase II subunit beta	Regulatory subunit of casein kinase 2, a normally constitutively active kinase. Participates in Wnt signaling.	GO:1905114; cell surface receptor signaling pathway involved in cell–cell signaling	215	No	1–19	0.5695	1	3
CSTF2 P33240‐1 ENST00000372972	Cleavage stimulation factor subunit 2	Required for polyadenylation and pre‐mRNA cleavage	GO:0006379; mRNA cleavage	577	No	555–577 c	0.8155	None	None
CTCF P49711‐1 ENST00000264010	Transcriptional repressor CTCF	Involved in transcriptional regulation by binding to chromatin insulators. Plays a role in CENPE recruitment during mitosis.	GO:0071824; protein–DNA complex subunit organization	727	No	279–324	0.6235	None	16
CUL1 Q13616‐1 ENST00000325222	Cullin‐1	Core component of cullin‐RING‐based SCF E3 ubiquitin ligase in ubiquitination of proteins involved in cell cycle progression.	GO:1905114; cell surface receptor signaling pathway involved in cell–cell signaling	776	No	532–566	0.484	None	None
CUL4B Q13620‐2 ENST00000371322	Cullin‐4B	Core component of cullin‐RING‐based E3 ubiquitin ligase	GO:0016567; protein ubiquitination	913	No	709–742 c	0.631	None	Many
DDX3X O00571‐2 d ENST00000457138	ATP‐dependent RNA helicase DDX3X	ATP‐dependent helicase. Binds RNA G4s. Transcription regulation. Required for ATF4 mRNA translation. Mediates virus replication.	GO:1905114; cell surface receptor signaling pathway involved in cell–cell signaling	646	No	476–507 d	0.603	1	Many
DENND1A Q8TEH3‐1 ENST00000373624	DENN domain‐containing protein 1A	Guanine nucleotide exchange factor regulating clathrin endocytosis through RAB35 activation	GO:0046907; intracellular transport	1,009	No	1–18	0.875	None	None
DHX15 O43143‐1 ENST00000336812	Pre‐mRNA‐splicing factor ATP‐dependent RNA helicase DHX15	Pre‐mRNA processing factor involved in disassembly of spliceosomes.	GO:0006397; mRNA processing	795	No	462–509, 532–573	0.501	None	None
DHX9 Q08211‐1 ENST00000367549	ATP‐dependent RNA helicase A	Helicase activity. Some mRNA splicing activity.	GO:0050684; regulation of mRNA processing	1,270	No	708–757	0.666	None	None
DKC1 O60832‐1 ENST00000369550	H/ACA ribonucleoprotein complex subunit DKC1	Catalyzes uridine to psuedouridine in RNA	GO:0006396; RNA processing	514	No	88–138, 167–207, 218–256, 371–404	0.584	2	Many
DLG3 Q92796‐1 ENST00000374360	Disks large homolog 3	Role in learning, through NMDA receptor signaling	GO:2000310; regulation of NMDA receptor activity	817	No	522–572	0.800	None	14
DUSP8 Q13202‐1 ENST00000397374	Dual specificity protein phosphatase 8	Phosphatase that regulates MAPK activity	GO:0009966; regulation of signal transduction	625	No	610–625	0.780	None	None
EHBP1 Q8NDI1‐1 ENST00000263991	EH domain‐binding protein 1	May play a role in actin reorganization.	GO:0033036; macromolecule localization	1,231	No	1–21	0.931	None	None
EHMT2 Q96KQ7‐1 ENST00000375537	Histone‐lysine N‐methyltransferase EHMT2	Histone methyltransferase that mono or di‐methylates H3K9	GO:0016570; histone modification	1,210	No	1,070–1,108	0.823	None	None
EIF1AX P47813‐1 ENST00000379607	Eukaryotic translation initiation factor 1A, X‐chromosomal	Seems to be required for maximal protein biosynthesis.	GO:0006413; translational initiation	144	No	5–45, 56–128	0.2105	None	None
EIF1AY O14602‐1 ENST00000361365	Eukaryotic translation initiation factor 1A, Y‐chromosomal	Seems to be required for maximal protein biosynthesis rate.	GO:0006413; translational initiation	144	No	124–144	0.5075	None	None
EIF2S2 P20042‐1 ENST00000374980	Eukaryotic translation initiation factor 2 subunit 2	Initiation of translation	GO:0009790; embryo development	333	No	226–285, 318–333	0.645	None	None
EIF2S3 P41091‐1 ENST00000253039	Eukaryotic translation initiation factor 2 subunit 3	Subunit of eIF‐2 involved in early steps of protein synthesis.	GO:0006413; translational initiation	472	No	150–204, 429–461	0.478	None	6
EIF3A Q14152‐1 ENST00000369144	Eukaryotic translation initiation factor 3 subunit A	Subunit of the eIF‐3 complex. Required for protein synthesis. Targets a subset of mRNA involved in cell proliferation.	GO:0006413; translation initiation	1,382	No	1–18	0.887	None	None
EIF4A3 P38919‐1 b ENST00000649764	Eukaryotic initiation factor 4A‐III	ATP dependent helicase. Pre‐mRNA splicing. Core component of exon junction complex. Involved in craniofacial development.	GO:0009790; embryo development	411	No	204–234 b (based on ENST00000269349)	0.4735	None	1
ERH P84090‐1 ENST00000557016	Enhancer of rudimentary homolog	May have a role in cell cycle	GO:0007049; cell cycle	104	No	30–61	0.325	None	None
ETF1 P62495‐1 ENST00000360541	Eukaryotic peptide chain release factor subunit 1	Directs termination of nascent peptide synthesis in response to stop codons. Component of SURF complex.	GO:0002184; cytoplasmic translational termination	437	No	55–87, 324–355	0.490	None	None
F8 P00451‐1 ENST00000360256	Coagulation factor VIII	Factor VIII, along with calcium and phospholipid, acts as a cofactor for F9/factor IXa, when it converts F10/factor X to the activated form, factor Xa.	GO:0016491; oxidoreductase activity	2,351	No	95–131	0.893	4	Many
FGD1 P98174‐1 ENST00000375135	FYVE, RhoGEF, and PH domain‐containing protein 1	Activates CDC42. Plays a role in cytoskeleton and cell shape	GO:0007010; cytoskeleton organization	961	No	575–616, 739–769	0.7615	1	Many
FMR1 Q06787‐1 ENST00000370475	Synaptic functional regulator FMR1	mRNA regulation. Maybe DNA repair in neuronal cells.	GO:0050684; regulation of mRNA processing	632	No	69–99	0.858	None	16
FOXG1 P55316‐1 ENST00000313071	Forkhead box protein G1	Transcription repression factor important for neurogenesis.	GO:0007420; brain development	489	No	175–209, 217–247	0.564	2	Many
FOXJ3 Q9UPW0‐1 ENST00000372572	Forkhead box protein J3	Transcriptional activator of MEF2C. Plays an important role in spermatogenesis.	GO:0010468; regulation of gene expression	622	No	100–139	0.848	None	None
GABPA Q06546‐1 ENST00000354828	GA‐binding protein alpha chain	Transcription factor capable of interacting with purine rich repeats.	GO:0009790; embryo development	454	No	376–406	0.698	None	None
GABRA2 P47869‐1 ENST00000514090	Gamma‐aminobutyric acid receptor subunit alpha‐2	Ligand gated chloride channel that is a component of the receptor for GABA.	GO:0099536; synaptic signaling	451	Yes	279–316	0.6545	None	None
GABRA3 P34903‐1 ENST00000370314	Gamma‐aminobutyric acid receptor subunit alpha‐3	GABA receptor	GO:0099536; synaptic signaling	492	Yes	306–338	0.688	None	None
GABRB2 P63137‐1 ENST00000274547	Gamma‐aminobutyric acid receptor subunit beta‐2	Ligand‐gated chloride channel component of the GABA receptor.	GO:0099536; synaptic signaling	512	Yes	151–190	0.618	None	Many
GDF11 O95390‐1 ENST00000257868	Growth/differentiation factor 11	Secreted signal involved in development.	GO:0045664; regulation of neuron differentiation	407	No	1–21	0.728	None	None
GJB1 P08034‐1 ENST00000374029	Gap junction beta‐1 protein	Forms gap junctions	GO:0007267; cell–cell signaling	283	Yes	50–89	0.713	3	Many
GLRA2 P23416‐1 ENST00000218075	Glycine receptor subunit alpha‐2	Glycine ligand gated chloride channel. Also triggered by taurine and beta‐alanine.	GO:1905114; cell surface receptor signaling pathway involved in cell–cell signaling	452	Yes	261–315	0.698	None	None
GNAL P38405‐1 ENST00000535121	Guanine nucleotide‐binding protein G(olf) subunit alpha	G protein that may be involved in olfactory and visual transduction.	GO:0019932; second‐messenger‐mediated signaling	381	No	35–66, 140–171, 178–215 c	0.615	1	6
GNAQ P50148‐1 ENST00000286548	Guanine nucleotide‐binding protein G(q) subunit alpha	G protein involved in many transmembrane signaling pathways. Is important for B cell selection and chemotaxis of neutrophils and dendritic cells.	GO:0019932; second‐messenger‐mediated signaling	359	No	171–202	0.575	None	4
GNAS Q5JWF2‐1 ENST00000371100	Guanine nucleotide‐binding protein G(s) subunit alpha isoforms short	G protein that is activated by GPCRs including beta‐adrenergic receptors, stimulates Ras signaling.	NA	1,037	No	899–959	0.8455	None	17
GNG3 P63215‐1 ENST00000294117	Guanine nucleotide‐binding protein G(I)/G(S)/G(O) subunit gamma‐3	G protein subunit and required for GTPase activity.	GO:0055074; calcium ion homeostasis	75	No	58–75	0.819	None	1
GOLGA8G Q08AF8‐1 b ENST00000526619	Putative golgin subfamily A member 8F/8G	Possibly a psuedogene	GO:0000226; microtubule cytoskeleton organization	430	No	389–422 b (based on ENST00000525590)	0.9045	None	None
GORASP2 Q9H8Y8‐1 ENST00000234160	Golgi reassembly‐stacking protein 2	Role in assembly and membrane stacking of the Golgi cisternae. May regulate intracellular transport. Required for normal acrosome formation in spermiogenesis. Mediates ER‐stress and induced unconventional trafficking of core‐glycosylated CFTR to cell membrane.	GO:0045184; establishment of protein localization	454	No	1–16	0.896	None	None
GRIA2 P42262‐1 ENST00000264426	Glutamate receptor 2	Receptor for glutamate that functions as an ion channel in the CNS.	GO:0099536; synaptic signaling	833	Yes	521–554	0.6745	None	1
GRIA3 P42263‐1 b ENST00000622768	Glutamate receptor 3	Glutamate gated ion channel	GO:0007215; glutamate receptor signaling pathway	894	Yes	487–543, 602–638, 745–777, 879–894 b (based on ENST00000371256)	0.608	2	Many
GRIN1 Q05586‐1 ENST00000371561	Glutamate receptor ionotropic, NMDA 1	NMDA subunit that binds glutamate	GO:0050684; regulation of mRNA processing	938	Yes	549–581 c	0.544	3	Many
GRIN2A Q12879‐1 ENST00000396573	Glutamate receptor ionotropic, NMDA 2A	Ligand‐gated ion channel	GO:0030001; metal ion transport	1,464	Yes	631–670	0.799	5	Many
GRIN2B Q13224‐1 ENST00000609686	Glutamate receptor ionotropic, NMDA 2B	Component of NMDA receptor complex	GO:1905114; cell surface receptor signaling pathway involved in cell–cell signaling	1,484	Yes	504–567, 662–700, 744–774	0.659	5	Many
GSPT1 P15170‐1 ENST00000563468	Eukaryotic peptide chain release factor GTP‐binding subunit ERF3A	Translation termination	GO:0002184; cytoplasmic translational termination	499	No	170–204 c	0.739	None	None
HCFC1 P51610‐1 ENST00000310441	Host cell factor 1	Control of cell cycle from G1 to S. Coactivator of GABP2.	GO:0009790; embryo development	2035	No	143–173, 207–240, 1,600–1,631, 1,976–2,007	0.648	1	Many
HMGN4 O00479‐1 ENST00000377575	High mobility group nucleosome‐binding domain‐containing protein 4	Chromatin binding	GO:0031492; nucleosomal DNA binding	90	No	1–17	0.978	None	None
HNF1B P35680‐1 b ENST00000617811	Hepatocyte nuclear factor 1‐beta	Transcription factor. Binds to FPC element in PLAU gene. Organ development.	GO:0009790; embryo development	557	No	281–313 b (based on ENST00000225893)	0.8195	None	Many
HNRNPC P07910‐1 ENST00000554455	Heterogeneous nuclear ribonucleoproteins C1/C2	Binds pre‐mRNA and nucleates the assembly of 40S hnRNP particles. May play a role in spliceosome assembly and pre‐mRNA splicing.	GO:0043487; regulation of RNA stability	306	No	39–83	0.67	None	None
HNRNPD Q14103‐1 ENST00000313899	Heterogeneous nuclear ribonucleoprotein D0	Binds with high affinity to RNA with AU‐rich elements. Functions as transcription factor.	GO:0006401; RNA catabolic process	355	No	108–148	0.924	None	None
HNRNPH2 P55795‐1 ENST00000316594	Heterogeneous nuclear ribonucleoprotein H2	Component of hnRNP which processes pre‐mRNAs.	GO:0008380; RNA splicing	449	No	78–111, 151–190	0.504	None	5
HNRNPK P61978‐2 d ENST00000351839	Heterogeneous nuclear ribonucleoprotein K	mRNA processing (one of major pre‐mRNA binding proteins). DNA binding. TP53 coactivator.	GO:0050684; regulation of mRNA processing	463	No	441–463 a (based on ENST00000376263)	0.6985	None	8
HSD17B10 Q99714‐1 ENST00000168216	3‐hydroxyacyl‐CoA dehydrogenase type‐2	Mitochondrial dehydrogenase involved in pathways of fatty acid, branched‐chain amino acid and steroid metabolism.	GO:1901575; organic substance catabolic process	261	No	145–181	0.628	None	15
HUWE1 Q7Z6Z7‐1 ENST00000342160	E3 ubiquitin‐protein ligase HUWE1	E3 ubiquitin ligase	GO:0000209; protein polyubiquitination	4,374	No	499–529, 547–578, 3,006–3,042, 3,917–3,952, 4,358–4,390	0.728	1	Many
INTS6 Q9UL03‐1 ENST00000311234	Integrator complex subunit 6	Component of integrator complex. Involved in U1 and U2 transcription.	GO:0006366; transcription by RNA polymerase II	887	No	76–107	0.7715	None	None
IRAK1 P51617‐1 ENST00000369980	Interleukin‐1 receptor‐associated kinase 1	Serine/threonine kinase that plays a critical role in initiating the innate immune system.	GO:0002218; activation of innate immune response	712	No	26–58	0.801	None	1
KAT7 O95251‐1 ENST00000259021	Histone acetyltransferase KAT7	Catalytic component of HBO1 histone acetyltransferase complexes.	GO:0016573; histone acetylation	611	No	405–443	0.678	None	None
KCNA3 P22001‐1 ENST00000369769	Potassium voltage‐gated channel subfamily A member 3	Voltage gated potassium channel	GO:0030001; metal ion transport	575	Yes	359–392	0.865	None	1
KCNB1 Q14721‐1 ENST00000371741	Potassium voltage‐gated channel subfamily B member 1	Voltage‐gated potassium channels that can form heterotetrameric channels with other potassium channels.	GO:0030001; metal ion transport	858	Yes	82–113, 326‐357, 369–413	0.664	3	Many
KCNC2 Q96PR1‐1 ENST00000549446	Potassium voltage‐gated channel subfamily C member 2	Voltage gated potassium channel. Also acts in various signaling pathways such as NO signaling.	GO:0030001; metal ion transport	638	Yes	370–401	0.720	None	None
KCND3 Q9UK17‐1 ENST00000315987	Potassium voltage‐gated channel subfamily D member 3	Voltage gated inactivated A‐type potassium channel. May contribute to current in heart or neuron.	GO:0030001; metal ion transport	655	Yes	298–332, 364–407	0.7405	3	Many
KCNH7 Q9NS40‐1 ENST00000332142	Potassium voltage‐gated channel subfamily H member 7	Voltage gated potassium channel	GO:0030001; metal ion transport	1,196	Yes	612–642	0.834	None	1
KCNJ3 P48549‐1 ENST00000295101	G protein‐activated inward rectifier potassium channel 1	Inward rectifier potassium channel controlled by G proteins and playing a crucial role in regulating heartbeat.	GO:0030001; metal ion transport	501	Yes	161–194	0.6365	None	None
KCNMA1 Q12791‐1 ENST00000286628	Calcium‐activated potassium channel subunit alpha‐1	Export of potassium triggered by changes in cytosolic calcium or magnesium. Regulates smooth muscles, hair cells in cochlea, transmitter release, and innate immunity.	GO:0030001; metal ion transport	1,236	Yes	554–598, 1,009–1,039	0.681	None	Many
KCNQ2 O43526‐1 ENST00000359125	Potassium voltage‐gated channel subfamily KQT member 2	Heterotetramerizes with KCNQ3 to form a voltage gated channel important for regulation of neuronal excitability.	GO:0030001; metal ion transport	872	Yes	197–237	0.721	5	Many
KDM2A Q9Y2K7‐1 ENST00000529006	Lysine‐specific demethylase 2A	Histone demethylase that preferentially demethylates H3K36. Regulates circadian clock.	GO:0007623; circadian rhythm	1,162	No	275–311, 585–627	0.704	None	None
KDM3B Q7LBC6‐1 ENST00000314358	Lysine‐specific demethylase 3B	Histone demethylase that specifically demethylates histone H3.	GO:0016570; histone modification	1,761	No	1,678–1714, 1716–1748	0.786	None	None
KIF11 P52732‐1 ENST00000260731	Kinesin‐like protein KIF11	Motor protein required for establishing a bipolar spindle during mitosis. Also involved in Golgi‐to‐cell surface trafficking.	GO:0007051; spindle organization	1,056	No	259–299	0.840	None	13
KIF1A Q12756‐1 ENST00000498729	Kinesin‐like protein KIF1A	Motor for anterograde axonal transport of synaptic vesicle precursors. Interacts with CALM1. Required for neuronal dense core vesicles transport to dendritic spines and axons.	GO:0030705; cytoskeleton‐dependent intracellular transport	1,791	No	1,465–1,498	0.779	1	Many
KIF5A Q12840‐1 ENST00000455537	Kinesin heavy chain isoform 5A	Kinesin transport of neurofilament proteins	GO:0030705; cytoskeleton‐dependent intracellular transport	1,032	No	230–264	0.788	1	Many
KMT2C Q8NEZ4‐1 ENST00000262189	Histone‐lysine N‐methyltransferase 2C	Histone methyltransferase to H3K4. Chromatin remodeling.	GO:0016571; histone methylation	4,911	No	349–379	0.923	None	Many
KPNB1 Q14974‐1 ENST00000290158	Importin subunit beta‐1	Binds to nuclear localization signals and imports proteins into the nucleus.	GO:0051169; nuclear transport	876	No	706–745	0.580	None	None
LPA P08519‐1 ENST00000316300	Apolipoprotein(a)	Main constituent of lipoprotein(a). Serine protease activity. Inhibits plasminogen activator 1.	GO:0006508; proteolysis	4,584	No	99–130	0.935	None	None
LUC7L3 O95232‐1 ENST00000505658	Luc7‐like protein 3	Binds cAMP regulatory element DNA sequence. May play a role in RNA splicing	GO:0008380; RNA splicing	432	No	185–225	0.808	None	None
MAMLD1 Q13495‐4 a ENST00000426613	Mastermind‐like domain‐containing protein 1	Transactivates HES3 independent of NOTCH	GO:0006357; regulation of transcription by RNA polymerase II	749	No	713–747 a (based on ENST00000432680)	0.916	None	None
MAPRE2 Q15555‐1 ENST00000300249	Microtubule‐associated protein RP/EB family member 2	May be involved in microtubule polymerization by anchoring at centrosome.	GO:0051493; regulation of cytoskeleton organization	327	No	45–87, 131–162	0.5785	1	3
MED12 Q93074‐1 ENST00000374080	Mediator of RNA polymerase II transcription subunit 12	Component of mediator complex. Involved in the regulation of nearly all RNA pol‐II dependent genes. May specifically regulate transcription of targets of Wnt signaling pathway and SHH signaling.	GO:1905114; cell surface receptor signaling pathway involved in cell–cell signaling	2,177	No	1,138–1,169	0.693	1	Many
MED14 O60244‐1 ENST00000324817	Mediator of RNA polymerase II transcription subunit 14	Component of the mediator complex, needed for nearly all RNA pol II dependent genes.	GO:0006366; transcription by RNA polymerase II	1,454	No	1,277–1,308	0.845	None	None
MEF2C Q06413‐1 ENST00000437473	Myocyte‐specific enhancer factor 2C	Transcription activator that binds specifically to MEF2 element in many muscle‐specific genes. Controls cardiac morphogenesis and myogenesis. Plays a role in hippocampal learning. Important for immune cells.	GO:1905114; cell surface receptor signaling pathway involved in cell–cell signaling	473	No	1–36	0.6485	6	Many
METTL14 Q9HCE5‐1 ENST00000388822	N6‐adenosine‐methyltransferase non‐catalytic subunit	Component of methyltransferase complex that methylates at the N6 position of some mRNAs and regulates circadian rhythm, differentiation of embryonic stem cells and cortical neurogenesis.	GO:0032259; methylation	456	No	104–135	0.801	None	None
MMP16 P51512‐1 ENST00000286614	Matrix metalloproteinase‐16	Endopeptidase that degrades components of extracellular matrix. Matrix remodeling of blood vessels.	GO:0009790; embryo development	607	Yes	199–233	0.7975	None	None
MOB4 Q9Y3A3‐1 ENST00000323303	MOB‐like protein phocein	May play a role in membrane trafficking, specifically membrane budding.	GO:0046872; metal ion binding	225	No	123–153	0.7225	None	None
MRC1 P22897‐1 b ENST00000569591	Macrophage mannose receptor 1	Mediates endocytosis of glycoproteins.	GO:0044419; interspecies interaction between organisms	1,456	Yes	168–213, 411–471b (based on ENST00000239761)	0.778	None	1
MYB P10242‐1 ENST00000367814	Transcriptional activator Myb	Transcriptional activator. DNA‐binding to YAAC[GT]G. Plays an important role in the control of proliferation and differentiation of hematopoietic progenitor cells.	GO:0006338; chromatin remodeling	640	No	118–157	0.841	None	None
NAA10 P41227‐1 ENST00000464845	N‐alpha‐acetyltransferase 10	Acetyltransferase, particularly the first amino acid following removal of methionine.	GO:0006473; protein acetylation	235	No	17–47, 53–95, 104–148	0.4335	6	21
NAA15 Q9BXJ9‐1 ENST00000296543	N‐alpha‐acetyltransferase 15, NatA auxiliary subunit	Auxiliary subunit of N‐terminal acetyltransferase activity. May be important for vascular, hematopoietic and neuronal growth and development. Required to control retinal neovascularization.	GO:0006473; protein acetylation	866	No	97–137	0.795	None	None
NEDD8 Q15843‐1 ENST00000250495	NEDD8	Plays an important role in cell cycle control and embryogenesis via its conjugation to target proteins. Ubiquitin‐like	GO:0043687; post‐translational protein modification	81	No	16–46	0.4175	None	None
NIPBL Q6KC79‐1 ENST00000282516	Nipped‐B‐like protein	Loading of cohesion complex onto chromatin.	GO:0009790; embryo development	2,804	No	2,073–2,107	0.816	3	Many
NONO Q15233‐1 ENST00000276079	Non‐POU domain‐containing octamer‐binding protein	Plays a variety of roles in nuclear processes.	GO:0006281; DNA repair	471	No	174–216, 221–265	0.600	None	5
NR4A2 P43354‐1 ENST00000339562	Nuclear receptor subfamily 4 group A member 2	Transcriptional regulator for differentiation of neurons during development. Crucial for expression of SLC6A3, SLC18A2, TH, and DRD2.	GO:1905114; cell surface receptor signaling pathway involved in cell–cell signaling	598	No	261–293, 318–348	0.825	None	None
NRBP1 Q9UHY1–1 a ENST00000379852	Nuclear receptor‐binding protein	May play a role in trafficking between ER and the Golgi through interaction with rho‐type GTPases.	GO:0006810; transport	535	No	199–232 a (based on ENST00000379863)	0.671	None	None
NSMF Q6X4W1‐1 ENST00000371475	NMDA receptor synaptonuclear signaling and neuronal migration factor	Part of CREB shut off pathway. Couples NMDA‐sensitive glutamate receptor and triggers long lasting changes to dendrites and synapses.	GO:0048814; regulation of dendrite morphogenesis	530	No	1–19	0.804	None	None
NUDT11 Q96G61‐1 ENST00000375992	Diphosphoinositol polyphosphate phosphohydrolase 3‐beta	Cleaves a beta‐phosphate from the diphosphate groups in PP‐InsP5	GO:0009058; biosynthetic process	164	No	1–20	0.522	None	None
NUDT21 O43809‐1 ENST00000300291	Cleavage and polyadenylation specificity factor subunit 5	Component of cleavage factor Im. Involved in mRNA processing.	GO:0050684; regulation of mRNA processing	227	No	170–215	0.5065	None	None
OGT O15294‐1 ENST00000373719	UDP‐N‐acetylglucosamine‐peptide N‐acetylglucosaminyltransferase 110 kDa subunit	Glycosylates other proteins.	GO:0006493; protein O‐linked glycosylation	1,046	No	21–52, 54–84, 212–248, 349–382, 384–452, 499–538	0.542	1	5
OR4F17 Q8NGA8‐1 ENST00000585993	Olfactory receptor 4F17	Predicted olfactory receptor	GO:0007165; signal transduction	305	Yes	1–22	0.938	None	None
OTUD5 Q96G74‐1 ENST00000156084	OTU domain‐containing protein 5	Deubiquitining functioning as a negative regulator of immune system.	GO:0016579; protein deubiquitination	571	No	171–201, 343–411	0.5085	None	1
PAK2 Q13177‐1 ENST00000327134	Serine/threonine‐protein kinase PAK 2	Serine/threonine kinase. Involved in cytoskeleton regulation, cell motility, cell cycle progression apoptosis, or proliferation. Downstream of CDC42 and RAC1.	GO:0031098; stress‐activated protein kinase signaling cascade	524	No	362–397	0.719	None	None
PAK3 O75914‐1 ENST00000372010	Serine/threonine‐protein kinase PAK 3	Serine/threonine kinase that affects cytoskeleton regulation, cell migration, and cell cycle. Acts downstream of CDC42.	GO:0006468; protein phosphorylation	559	No	68–99, 290–335, 413–455, 458–489 c	0.645	1	15
PBX1 P40424‐1 ENST00000420696	Pre‐B‐cell leukemia transcription factor 1	Binds DNA in junction with HOX proteins. Spleen development.	GO:0009790; embryo development	430	No	275–306	0.632	None	4
PCBP2 Q15366‐1 ENST00000439930	Poly(rC)‐binding protein 2	Single strand nucleotide binding protein that preferentially binds to dC. Acts as adaptor between MAVS and E3 ITCH	GO:0043161; proteasome‐mediated ubiquitin‐dependent protein catabolic process	365	No	90–120	0.4825	None	None
PCYT1B Q9Y5K3‐1 ENST00000379144	Choline‐phosphate cytidylyltransferase B	Rate‐limiting step in the CDP‐choline pathway for phosphatidylcholine biosynthesis	GO:0009058; biosynthetic process	369	No	220–268	0.675	None	None
PHF5A Q7RTV0‐1 ENST00000216252	PHD finger‐like domain‐containing protein 5A	Involved in PAF1 complex in transcriptional elongation. Involved in pre‐mRNA splicing and deposition of certain histones.	GO:0006397; mRNA processing	110	No	1–18, 85–110	0.324	None	None
PIK3CA P42336‐1 ENST00000263967	Phosphatidylinositol 4,5‐bisphosphate 3‐kinase catalytic subunit alpha isoform	Subunit of PI3K	GO:0009749; response to glucose	1,068	No	927–975, 1,010–1,041	0.691	None	Many
PLS3 P13797‐1 ENST00000355899	Plastin‐3	Actin bundling proteins found in microvilli, stereocilia, filopodia and may play a role in bone development.	GO:0007010; cytoskeleton organization	630	No	456–503	0.766	None	None
POLR2A P24928‐1 b NA	DNA‐directed RNA polymerase II subunit RPB1	Forms RNA polymerase active center with another catalytic subunit.	GO:0006366; transcription by RNA polymerase II	1,970	No	476–506 b (based on ENST00000572844)	0.667	None	None
POLR2B P30876‐1 ENST00000381227	DNA‐directed RNA polymerase II subunit RPB2	DNA dependent RNA polymerase catalyzing transcription.	GO:0006366; transcription by RNA polymerase II	1,174	No	490–522, 524–557, 746–778, 979–1,026, 1,072–1,115	0.627	None	None
POU3F2 P20265‐1 ENST00000328345	Histone‐lysine N‐methyltransferase EHMT2	Histone methyltransferase that mono or di‐methylates Lys‐9.	GO:0006479; protein methylation	443	No	278–314	0.667	None	None
POU3F3 P20264‐1 ENST00000361360	POU domain, class 3, transcription factor 3	Transcription factor that acts synergistically with SOX11 and SOX4. Role in neuronal development.	GO:0030900; forebrain development	500	No	317–352	0.641	None	1
PPP1CB P62140‐1 ENST00000395366	Serine/threonine‐protein phosphatase PP1‐beta catalytic subunit	Protein phosphatase that forms complexes with over 200 regulatory proteins. Glycogen metabolism, muscle contractility, protein synthesis, chromatin structure, and cell cycle progression.	GO:0000278; mitotic cell cycle	327	No	51–113	0.3295	2	11
PPP2CA P67775‐1 ENST00000481195	Serine/threonine‐protein phosphatase 2A catalytic subunit alpha isoform	Major phosphatase for microtubule‐associated proteins.	GO:1904528; Positive regulation of microtubule binding	309	No	142–181	0.392	None	None
PPP3R1 P63098‐1 ENST00000234310	Calcineurin subunit B type 1	Regulatory subunit of calcineurin, a calcium‐dependent, calmodulin stimulated protein phosphatase. Confers calcium sensitivity.	GO:1905114; cell surface receptor signaling pathway involved in cell–cell signaling	170	No	62–96	0.464	None	None
PRPF4B Q13523‐1 ENST00000337659	Serine/threonine‐protein kinase PRP4 homolog	Has a role in pre‐mRNA splicing. Phosphorylates SF2/ASF.	GO:0006468; protein phosphorylation	1,007	No	811–841	0.761	None	None
PRPF8 Q6P2Q9‐1 ENST00000572621	Pre‐mRNA‐processing‐splicing factor 8	Core component of spliceosome.	GO:0000398; mRNA splicing, via spliceosome	2,335	No	505–537, 764–800, 837–879, 1,494–1,539, 1,811–1,848, 1,888–1,919	0.527	None	21
PRPS1 P60891‐1 ENST00000372435	Ribose‐phosphate pyrophosphokinase 1	Essential for nucleotide synthesis.	GO:0019438; aromatic compound biosynthetic process	318	No	1–30, 84–121, 123–162, 168–199	0.446	7	Many
PSMC1 P62191‐1 ENST00000261303	26S proteasome regulatory subunit 4	26S proteosome subunit	GO:1905114; cell surface receptor signaling pathway involved in cell–cell signaling	440	No	291–332	0.652	None	None
PSMC2 P35998‐1 ENST00000435765	26S proteasome regulatory subunit 7	Component of 26S proteosome	GO:1905114; cell surface receptor signaling pathway involved in cell–cell signaling	433	No	284–318	0.644	None	None
PSMC5 P62195‐1 ENST00000310144	26S proteasome regulatory subunit 8	26S proteosome subunit	GO:1905114; cell surface receptor signaling pathway involved in cell–cell signaling	406	No	112–144,158–188	0.563	None	None
PSMD14 O00487‐1 ENST00000409682	26S proteasome non‐ATPase regulatory subunit 14	26S proteosome subunit. Metalloprotease that specifically cleaves “Lys‐63” linked polyubiquitin chains. Plays a role in DSBs and in recombination repair by promoting RAD51 loading.	GO:1905114; cell surface receptor signaling pathway involved in cell–cell signaling	310	No	65–97	0.515	None	None
PUF60 Q9UHX1‐1 ENST00000526683	Poly(U)‐binding‐splicing factor PUF60	DNA and RNA binding, involved in several nuclear processes such as pre‐mRNA splicing, apoptosis, and transcription regulation. Binds to poly(U) RNA.	GO:0000398; mRNA splicing, via spliceosome	559	No	90–164	0.5175	1	5
PURA Q00577‐1 ENST00000331327	Transcriptional activator protein Pur‐alpha	Probable transcription activator that binds to purine rich single strand of PUR element upstream od MYC gene	GO:0032508; DNA duplex unwinding	322	No	54–92	0.464	2	Many
RAB2A P61019‐1 ENST00000262646	Ras‐related protein Rab‐2A	Required for transport from ER to Golgi	GO:0046907; intracellular transport	212	No	8–46	0.563	None	None
RAC1 P63000‐1 ENST00000348035	Ras‐related C3 botulinum toxin substrate 1	GTPase that cycles between GTP active and GDP inactive and plays a role in secretory processes, phagocytosis of apoptotic cells, epithelial cell polarization, neurons adhesion, migration and differentiation, and growth‐factor induced formation of membrane ruffles.	GO:1905114; cell surface receptor signaling pathway involved in cell–cell signaling	192	No	141–171 c	0.236	None	9
RAN P62826‐1 ENST00000543796	GTP‐binding nuclear protein Ran	GTPase involved in nucleocytoplasmic import/export. Required for normal progression through mitosis.	GO:0071426; ribonucleoprotein complex export from nucleus	216	No	12–50, 118–187	0.179	None	None
RBBP4 Q09028‐1 ENST00000373493	Histone‐binding protein RBBP4	Core histone binding subunit. Chromatin remodeling. Component of CAF‐1, HDAC, NuRD, PRC2, and NURF.	GO:0043044; ATP‐dependent chromatin remodeling	425	No	12–63, 228–260, 294–333, 335–384	0.3305	None	None
RBBP5 Q15291‐1 ENST00000264515	Retinoblastoma‐binding protein 5	Plays crucial role in differentiation potential in embryonic stem cells. Gene regulation. Stimulates histone methyltransferases.	GO:0016569; covalent chromatin modification	538	No	1–18	0.716	None	None
RBBP7 Q16576‐1 ENST00000380087	Histone‐binding protein RBBP7	Core histone binding subunit that may target histone remodeling factors. Component of some histone remodeling complexes.	GO:0006338; chromatin remodeling	425	No	122–156 c	0.5405	None	None
RBM10 P98175‐1 ENST00000377604	RNA‐binding protein 10	mRNA processing	GO:0050684; regulation of mRNA processing	930	No	332–375	0.7125	None	4
RBM22 Q9NW64‐1 ENST00000199814	Pre‐mRNA‐splicing factor RBM22	Required for pre‐mRNA splicing as component of the activated spliceosome.	GO:0000398; mRNA splicing, via spliceosome	420	No	20–50	0.798	None	None
RBM3 P98179‐1 ENST00000376759	RNA‐binding protein 3	RNA binding	GO:0050684; regulation of mRNA processing	157	No	1–20	0.853	None	None
RBM39 Q14498‐1 ENST00000253363	RNA‐binding protein 39	Acts as pre‐mRNA splicing factor.	GO:0008380; RNA splicing	530	No	373–403	0.655	None	None
RBMX2 Q9Y388‐1 ENST00000305536	RNA‐binding motif protein, X‐linked 2	Involved in pre‐mRNA splicing as component of spliceosome.	GO:0008380; RNA splicing	322	No	1–17	0.907	None	1
RBMY1A1 P0DJD3‐1 ENST00000382707	RNA‐binding motif protein, Y chromosome, family 1 member A1	mRNA binding	GO:0050684; regulation of mRNA processing	496	No	99–130	0.934	None	None
RHOA P61586‐1 ENST00000418115	Transforming protein RhoA	GTPase involved in cytoskeleton organization. Regulates KCNA2. Can be activated by CaMKII.	GO:1905114; cell surface receptor signaling pathway involved in cell–cell signaling	193	No	1–77	0.268	8	9
RHOB P62745‐1 ENST00000272233	Rho‐related GTP‐binding protein RhoB	Mediates apoptosis in neoplastically transformed cells after DNA damage. Myosin contractile ring formation during cell cycle cytokinesis.	GO:0000278; mitotic cell cycle	196	No	18–48	0.566	None	None
RPL10 P27635‐1 ENST00000424325	60S ribosomal protein L10	Component of large ribosomal subunit. May play a role in embryonic brain development.	GO:0009790; embryo development	214	No	48–78	0.405	1	8
RPL36A P83881‐1 ENST00000553110	60S ribosomal protein L36a	Ribosomal protein	GO:0002181; cytoplasmic translation	106	No	81–106 c	0.580	None	None
RPS28 P62857‐1 ENST00000600659	40S ribosomal protein S28	NA	GO:0006413; translational initiation	69	No	54–69	0.5585	None	1
RPS6KA3 P51812‐1 ENST00000379565	Ribosomal protein S6 kinase alpha‐3	Serine/threonine kinase downstream of ERK. Regulates translation. Modulates mTOR signaling. Role in other pathways.	GO:0006468; protein phosphorylation	740	No	114–150, 457–494, 559–595, 681–711	0.5825	1	Many
RRAGA Q7L523‐1 ENST00000380527	Ras‐related GTP‐binding protein A	Guanine nucleotide binding protein that plays an important role in MTORC1 signaling for amino acid availability. May lead to cell death through TNF‐α signaling.	GO:0043200; response to amino acid	313	No	16–46	0.5725	None	None
RRM2 P31350‐1 ENST00000304567	Ribonucleoside‐diphosphate reductase subunit M2	Provides the precursors necessary for DNA synthesis.	GO:0019438; aromatic compound biosynthetic process	389	No	345–378 c	0.769	None	None
RTF1 Q92541‐1 ENST00000389629	RNA polymerase‐associated protein RTF1 homolog	Component of Paf1 complex. Implicated in regulation of development of embryonic stem cell pluripotency. Required for Wnt and Hox genes.	GO:1905114; cell surface receptor signaling pathway involved in cell–cell signaling	710	No	688–710	0.672	None	None
RYR2 Q92736‐1 ENST00000366574	Ryanodine receptor 2	Mediates calcium release from the sarcoplasmic reticulum and plays a critical role in cardiac muscle contraction.	GO:0030001; metal ion transport	4,967	Yes	4,856–4,889	0.841	1	Many
SAT1 P21673‐1 ENST00000379270	Diamine acetyltransferase 1	Acetylation of small molecule polyamines (e.g., spermidine)	GO:0009058; biosynthetic process	171	No	148–171	0.533	None	None
SCN2A Q99250‐1 ENST00000375437	Sodium channel protein type 2 subunit alpha (NaV1.2)	Voltage dependent release of sodium permeability. Implicated in hippocampal replay occurring with sharp wave ripples.	GO:0030001; metal ion transport	2,005	Yes	404–434, 854–885	0.7285	1	Many
SCN8A Q9UQD0‐1 ENST00000354534	Sodium channel protein type 8 subunit alpha (NaV1.6)	Voltage dependent sodium ion channel	GO:0030001; metal ion transport	1,980	Yes	396–439, 837–875, 910–961, 1,287–1,323, 1,449–1,499, 1,639–1,671, 1,680–1716,1746–1776	0.639	Many	Many
SF1 Q15637‐1 ENST00000377390	Splicing factor 1	Required for first step in ATP dependent spliceosome assembly	GO:0000387; spliceosomal snRNP assembly	639	No	223–258 c	0.751	None	None
SF3A2 Q15428‐1 ENST00000221494	Splicing factor 3A subunit 2	Involved in pre‐mRNA splicing as a component of the SF3A complex.	GO:0006376; mRNA splice site selection	464	No	42–72	0.714	None	None
SF3B1 O75533‐1 ENST00000335508	Splicing factor 3B subunit 1	Pre‐mRNA splicing as part of SF3B complex.	GO:0000245; spliceosomal complex assembly	1,304	No	537–573, 816–848, 962–1,002, 1,005–1,062, 1,133–1,170, 1,189–1,236	0.567	None	18
SF3B4 Q15427‐1 ENST00000271628	Splicing factor 3B subunit 4	mRNA splicing	GO:0050684; regulation of mRNA processing	424	No	1–23	0.642	1	4
SIN3A Q96ST3‐1 ENST00000394947	Paired amphipathic helix protein Sin3a	Transcriptional repressor. Regulates cell cycle progression. Required for cortical neuron differentiation and callosal axon elongation.	GO:0009790; embryo development	1,273	No	112–156	0.778	None	4
SLC25A5 P05141‐1 ENST00000317881	ADP/ATP translocase 2	ADP:ATP antiporter that mediates ATP synthesis in the mitochondria.	GO:0055085; transmembrane transport	298	Yes	283–298	0.714	None	None
SLC9A6 Q92581‐1 ENST00000370698	Sodium/hydrogen exchanger 6	Exchange of protons for sodium and potassium across endosomes. Contributes to calcium homeostasis.	GO:0030001; metal ion transport	699	Yes	334–371 c	0.758	None	Many
SMARCA2 P51531‐1 ENST00000382203	Probable global transcription activator SNF2L2	Component of SWI/SNF complex which carries out chromatin remodeling. Also belongs to the neural progenitors‐specific chromatin remodeling complex (npBAF complex) and the neuron‐specific chromatin remodeling complex (nBAF complex).	GO:0006338; chromatin remodeling	1,590	No	933–969	0.634	1	Many
SMARCA4 P51532‐1 ENST00000344626	Transcription activator BRG1	Involved in chromatin remodeling, part of SWI/SNF complex	GO:0006338; chromatin remodeling	1,647	No	754–789, 879–912, 955–987, 1,035–1,067	0.559	1	Many
SMARCA5 O60264‐1 ENST00000283131	SWI/SNF‐related matrix‐associated actin‐dependent regulator of chromatin subfamily A member 5	Helicase that has ATP‐dependent nucleosome‐remodeling activity. Component of ISWI. Binds to histones	GO:0043044; ATP‐dependent chromatin remodeling	1,052	No	290–321	0.651	None	None
SMARCE1 Q969G3‐1 ENST00000348513	SWI/SNF‐related matrix‐associated actin‐dependent regulator of chromatin subfamily E member 1	Chromatin remodeling to activate or repress genes. Component of SWI/SNF.	GO:0006338; chromatin remodeling	411	No	54–109	0.785	5	Many
SMC1A Q14683‐1 ENST00000322213	Structural maintenance of chromosomes protein 1A	Central component of the cohesion complex, which is essential for cohesion of sister chromatids after DNA replication. Involved in DNA repair.	GO:0007059; chromosome segregation	1,233	No	36–73, 290–321, 636–670, 1,103–1,153	0.5205	4	Many
SNAI2 O43623‐1 ENST00000020945	Zinc finger protein SNAI2	Transcriptional repressor. Involved in neural development.	GO:0031056; regulation of histone modification	268	No	202–241	0.838	None	2
SNRPC P09234‐1 ENST00000244520	U1 small nuclear ribonucleoprotein C	Component of U1 snRNP spliceosome	GO:0008380; RNA splicing	159	No	9–47	0.689	None	None
SNX12 Q9UMY4‐2 ENST00000374274	Sorting nexin‐12	May be involved in intra‐cellular trafficking	GO:0051049; regulation of transport	162	No	6–37	0.691	None	None
SP3 Q02447‐1 ENST00000310015	Transcription factor Sp3	Transcription factor that can act as an activator or repressor depending on isoform or PTM. Binds to GT and GC boxes. Cell cycle regulation, hormone induction, and house‐keeping.	GO:0009790; embryo development	781	No	625–658	0.8405	None	None
SPIN1 Q9Y657‐1 ENST00000375859	Spindlin‐1	Chromatin reader. Activator of Wnt. May play a role in cell‐cycle regulation during transition from gamete to embryo.	GO:1905114; cell surface receptor signaling pathway involved in cell–cell signaling	262	No	83–115, 240–262	0.616	None	None
SPOP O43791‐1 ENST00000393328	Speckle‐type POZ protein	Component of Cullin ring based BCR E3 ubiquitin ligase.	GO:0016567; protein ubiquitination	374	No	19–60, 62–124	0.453	6	15
SRF P11831‐1 ENST00000265354	Serum response factor	Transcription factor that binds to serum response element. Together with MRTFA is coupled to cytoskeletal expression and dynamics. Required for cardiac differentiation and maturation.	GO:0009790; embryo development	508	No	134–168	0.785	None	None
SRSF10 O75494‐1 ENST00000492112	Serine/arginine‐rich splicing factor 10	Pre‐mRNA splicing	GO:0050684; regulation of mRNA processing	262	No	1–44, 52–96	0.79	None	None
SRSF2 Q01130‐1 ENST00000392485	Serine/arginine‐rich splicing factor 2	Splicing of pre‐mRNA	GO:0050684; regulation of mRNA processing	221	No	1–17	0.603	None	None
SRSF3 P84103‐1 ENST00000373715	Serine/arginine‐rich splicing factor 3	RNA binding and pre‐mRNA cleavage	GO:0050684; regulation of mRNA processing	164	No	24–56	0.390	None	None
SRY Q05066‐1 ENST00000383070	Sex‐determining region Y protein	Transcriptional regulator that controls a genetic switch in male development.	GO:0030238; male sex determination	204	No	129–163	0.9095	None	16
STAG2 Q8N3U4‐1 ENST00000371160	Cohesin subunit SA‐2	Component of cohesin complex. Required for cohesion of sister chromatids after DNA replication.	GO:0007059; chromosome segregation	1,231	No	113–145	0.700	None	5
SUMO2 P61956‐1 ENST00000420826	Small ubiquitin‐related modifier 2	Ubiquitin like protein that can be attached to proteins on lysine residues.	GO:0018205; peptidyl‐lysine modification	95	No	17–54	0.231	None	None
SUZ12 Q15022‐1 ENST00000322652	Polycomb protein SUZ12	Polycomb group protein. Involved in histone methylation.	GO:0034968; histone lysine methylation	739	No	308–342	0.811	None	2
TAF1 P21675‐1 ENST00000423759	Transcription initiation factor TFIID subunit 1	Largest component and core scaffold of the TFIID basal transcription factor complex. Kinase and histone acetyltransferase activity.	GO:0016573; histone acetylation	1,872	No	1,328–1,370	0.738	2	25
TAOK1 Q7L7X3‐1 ENST00000261716	Serine/threonine‐protein kinase TAO1	Serine/threonine protein kinase involved in MAPK cascade, DNA damage response, and regulation of cytoskeleton stability	GO:0070507; regulation of microtubule cytoskeleton organization	1,001	No	181–215	0.742	None	None
TBC1D3H P0C7X1‐1 b ENST00000455054	TBC1 domain family member 3H	Acts as a GTPase activating protein for RAB5.	GO:0006886; intracellular protein transport	549	No	344–376 b (based on ENST00000455054)	0.986	None	None
TBL1XR1 Q9BZK7‐1 ENST00000430069	F‐box‐like/WD repeat‐containing protein TBL1XR1	Recruitment of ubiquitin/19S proteosome to nuclear receptor‐regulated transcription units. Probably acts as integral component of the N‐Cor corepressor complex.	GO:0009790; embryo development	514	No	261–294, 322–355	0.588	None	Many
TCF4 P15884‐1 ENST00000564999	Transcription factor 4	Transcription factor that binds to immunoglobulin enhancer. Involved in neuron differentiation.	GO:0006366; transcription by RNA polymerase II	667	No	552–587 c	0.732	1	Many
THOC2 Q8NI27‐1 ENST00000245838	THO complex subunit 2	Required for efficient export of poly‐A spliced mRNA. Component of TREX complex.	GO:0006397; mRNA processing	1,593	No	135–188, 698–731, 733–786, 1,059–1,100	0.653	1	17
TLK2 Q86UE8‐1 ENST00000326270	Serine/threonine‐protein kinase tousled‐like 2	Serine/threonine kinase involved in chromatin assembly	GO:1902275; regulation of chromatin organization	772	No	651–689	0.631	None	2
TOP1 P11387‐1 ENST00000361337	DNA topoisomerase 1	Topoisomerase. DNA repair/strain resolution.	GO:0009790; embryo development	765	No	476–512	0.700	None	2
TRA2B P62995‐1 ENST00000453386	Transformer‐2 protein homolog beta	mRNA splicing	GO:0050684; regulation of mRNA processing	288	No	104–138	0.757	None	None
TRIM24 O15164‐1 ENST00000343526	Transcription intermediary factor 1‐alpha	Transcriptional coactivator that interacts with numerous nuclear receptors and modulates transcription. Interacts with chromatin histone H3 modifications.	GO:0006351; transcription, DNA‐templated	1,050	No	818–858	0.854	None	None
TRPC5 Q9UL62‐1 ENST00000262839	Short transient receptor potential channel 5	Calcium channel. Causes neuron apoptosis.	GO:0030001; metal ion transport	973	Yes	290–323	0.716	None	None
TUBA1A Q71U36‐1 ENST00000301071	Tubulin alpha‐1A chain	Tubulin chain	GO:0000226; microtubule cytoskeleton organization	451	No	1–41, 43–172, 174–241, 243–286, 288–338, 340–399, 401–439	0.000	Many	Many
TUBA1B P68363‐1 ENST00000336023	Tubulin alpha‐1B chain	Tubulin chain	GO:0007017; microtubule‐based process	451	No	1–18, 81–117, 119–160, 185–231, 243–286, 341–374, 382–441	0.16	None	None
TUBB P07437‐1 ENST00000327892	Tubulin beta chain	Major component of microtubules	GO:0000226; microtubule cytoskeleton organization	444	No	49–120, 122–157, 190–240, 242–273, 297–330	0.171	5	14
TUBB2A Q13885‐1 ENST00000333628	Tubulin beta‐2A chain	Tubulin component.	GO:0000226; microtubule cytoskeleton organization	445	No	300–367	0.256	1	17
TUBB2B Q9BVA1‐1 ENST00000259818	Tubulin beta‐2B chain	Tubulin component. Implicated in neuronal migration.	GO:0009790; embryo development	445	No	283–356	0.256	2	Many
TUBB4B P68371‐1 ENST00000340384	Tubulin beta‐4B chain	Subunit of microtubules	GO:0007017; microtubule‐based process	445	No	163–198	0.257	None	None
U2AF1 Q01081‐1 ENST00000291552	Splicing factor U2AF 35 kDa subunit	Plays critical role in mRNA splicing.	GO:0008380; RNA splicing	240	No	1–32	0.446	2	4
U2AF2 P26368‐1 ENST00000308924	Splicing factor U2AF 65 kDa subunit	Pre‐mRNA splicing	GO:0050684; regulation of mRNA processing	475	No	188–220, 245–309	0.4625	None	None
U2SURP O15042‐1 ENST00000473835	U2 snRNP‐associated SURP motif‐containing protein	RNA binding	GO:0008380; RNA splicing	1,029	No	291–325, 585–621	0.7705	None	None
UBC P0CG48‐1 ENST00000536769	Polyubiquitin‐C	Ubiquiti	GO:1905114; cell surface receptor signaling pathway involved in cell–cell signaling	685	No	470–509	0.455	None	None
UBE2D3 P61077‐1 ENST00000453744	Ubiquitin‐conjugating enzyme E2 D3	E2 ubiquitin enzyme	GO:0006513; protein monoubiquitination	147	No	33–98, 130–147 c	0.178	None	None
UBE2E3 Q969T4‐1 ENST00000410062	Ubiquitin‐conjugating enzyme E2 E3	Accepts ubiquitin from E1 complex. Participates in regulation of transepithelial sodium transport in renal cells.	GO:0016567; protein ubiquitination	207	No	65–95	0.546	None	None
UBE2H P62256‐1 ENST00000355621	Ubiquitin‐conjugating enzyme E2 H	E2 ubiquitin ligase	GO:0000209; protein polyubiquitination	183	No	33–67	0.326	None	1
UBE2I P63279‐1 ENST00000355803	SUMO‐conjugating enzyme UBC9	Covalently attaches SUMO to target proteins.	GO:0018205; peptidyl‐lysine modification	158	No	62–98, 117–148	0.193	None	None
UBE2K P61086‐1 ENST00000261427	Ubiquitin‐conjugating enzyme E2 K	E2 ubiquitin ligase	GO:0000209; protein polyubiquitination	200	No	1–23	0.370	None	None
UHRF2 Q96PU4‐1 ENST00000276893	E3 ubiquitin‐protein ligase UHRF2	E3 ubiquitin ligase that plays important roles in DNA methylation, histone modifications, cell cycle, and DNA repair. Reads for 5‐hydroxymethylcytosine in DNA.	GO:0016567; protein ubiquitination	802	No	582–626	0.721	None	None
USP9Y O00507‐1 ENST00000338981	Probable ubiquitin carboxyl‐terminal hydrolase FAF‐Y	Probable deubiquitinase. Essential component of TGF‐Beta/BMP signaling.	GO:0016579; protein deubiquitination	2,555	No	1,326–1,381	0.951	None	None
UTY O14607‐1 ENST00000331397	Histone demethylase UTY	Male specific histone demethylase	GO:0016570; histone modification	1,347	No	124–158, 873–918, 1,094–1,138, 1,194–1,225 c	0.841	None	None
VAV1 P15498‐1 ENST00000602142	Proto‐oncogene vav	Couples to tyrosine kinase signals with rho/Rac GTPases, and leads to cell differentiation and/or proliferation.	GO:0010942; Positive regulation of cell death	845	No	365–401	0.778	None	3
WNK3 Q9BYP7‐1 ENST00000354646	Serine/threonine‐protein kinase WNK3	Serine/threonine kinase that plays an important role in electrolyte homeostasis.	GO:0043270; positive regulation of ion transport	1,800	No	332–364	0.915	None	2
XPR1 Q9UBH6‐1 ENST00000367590	Xenotropic and polytropic retrovirus receptor 1	Phosphate homeostasis. Phosphate export. Binds inositol polyphosphates.	GO:0006873; cellular ion homeostasis	696	Yes	112–149	0.790	1	5
YTHDC1 Q96MU7‐1 ENST00000344157	YTH domain‐containing protein 1	Pre‐mRNA splicing; mRNA export; involved in spermatogenesis.	GO:0050684; regulation of mRNA processing	727	No	361–395	0.872	None	None
YY1 P25490‐1 ENST00000262238	Transcriptional repressor protein YY1	Transcription factor that exhibits positive and negative control on large number of genes. Binds to CCGCCATNTT.	GO:0001558; regulation of cell growth	414	No	288–326, 338–410	0.526	4	5
ZBTB16 Q05516‐1 ENST00000335953	Zinc finger and BTB domain‐containing protein 16	Transcriptional repressor. May play a role in myeloid maturation.	GO:0009790; embryo development	673	No	576–622	0.7855	None	1
ZBTB20 Q9HC78‐1 ENST00000474710	Zinc finger and BTB domain‐containing protein 20	May be a transcription factor involved in hematopoiesis, oncogenesis and, immune response.	GO:0001678; cellular glucose homeostasis	741	No	574–618	0.7355	9	19
ZEB2 O60315‐1 ENST00000558170	Zinc finger E‐box‐binding homeobox 2	Transcriptional inhibitor of E‐cadherin and represses expression of MEOX2. Binds to CACCT in different promoters.	GO:0009790; embryo development	1,214	No	296–326, 1,064–1,094	0.786	1	Many
ZFX P17010‐1 ENST00000379177	Zinc finger X‐chromosomal protein	Probably a transcriptional activator.	GO:0006357; regulation of transcription by RNA polymerase II	805	No	414–444	0.720	None	None
ZFY P08048‐1 ENST00000383052	Zinc finger Y‐chromosomal protein	Probable transcription factor	GO:0006357; regulation of transcription by RNA polymerase II	801	No	654–691 c	0.828	None	None
ZMAT2 Q96NC0‐1 ENST00000274712	Zinc finger matrin‐type protein 2	Involved in pre‐mRNA splicing as a component of the spliceosome.	GO:0000398; mRNA splicing, via spliceosome	199	No	67–101	0.709	None	None
ZMYM3 Q14202‐1 ENST00000314425	Zinc finger MYM‐type protein 3	Plays a role in cell morphology and cytoskeletal organization.	GO:0007010; cytoskeleton organization	1,370	No	1,185–1,215	0.724	None	3
ZMYND8 Q9ULU4‐1 ENST00000311275	Protein kinase C‐binding protein 1	Transcriptional corepressor for KDM5D. Function seems to be histone recognition.	GO:0060284; regulation of cell development	1,186	No	1,033–1072 c	0.790	None	None
ZNF84 P51523‐1 ENST00000327668	Zinc finger protein 84	May be involved in transcription	GO:0006357; regulation of transcription by RNA polymerase II	738	No	486–527	0.885	None	None

The transcript containing the intolerant segment is not found in UniProt and is not the canonical transcript. The amino acid numbering provided for the zero‐tolerance segment is based on the sequence of the protein encoded by the indicated transcript.

The transcript containing the intolerant segment is not found in UniProt. The amino acid numbering provided for the zero‐tolerance segment is based on the sequence of the protein encoded by the indicated transcript.

UniProt numbering, which in this case is different from the numbering of the MTR‐designated canonical transcript in gnomAD.

UniProt transcript used that is not considered the canonical sequence by UniProt.

Abbreviations: ATP, adenosine triphosphate; ER, endoplasmic reticulum; GO, gene ontology; GPCR, G‐protein coupled receptor; GDP, guanosine diphosphate; GTP, guanosine triphosphate; MTR, missense tolerance ratio; PTM, post‐translational modification.

FIGURE 1

Histograms for intolerance within the 257 proteins containing a zero‐tolerance segment. (a) Distribution of MTR scores for all possible 31 residue segments. Segments with a score in the “at or near zero” bin represent 1.9% of all segments. The mean MTR score is 0.69 ± 0.26 and the median score is 0.73. (b) Distribution of median protein MTR scores based on analysis of all possible 31 amino acid segments within each protein. The mean of these medians is 0.71 ± 0.26. MTR, missense tolerance ratio

P60709‐1

P13637‐2

Q13936‐11

O00571‐2

P38919‐1

Q08AF8‐1

P42263‐1

P35680‐1

P61978‐2

Q13495‐4

P22897‐1

Q9UHY1–1

P24928‐1

P0C7X1‐1

In addition to the 257 proteins with certain zero‐tolerance segments, we also found 33 human proteins that have 31 or longer residue segments with an MTR score of 0, but for which the statistics associated with this score are uncertain because of an insufficient number of observed silent mutations with the intolerant segments. These 33 proteins are listed in Table S1 and will require additional data to determine whether the preliminary MTR = 0 score seen for at least one segment within each of these proteins is confirmed in a statistically robust manner. It may be significant that 7 of these 33 proteins have sites of known ClinVar variants, suggestive of high relevance to human health (Table S1).

Homology of human zero‐tolerance proteins to corresponding proteins from other mammals

For each of the 257 proteins containing one or more zero‐tolerance segments, we conducted BLASTP sequence homology searches for both the entire protein sequence and the zero‐tolerance segments. Results were analyzed for the 250 closest mammalian homologs. Figure 2 gives a representative sample of the results, while Figure S1 shows the results for all 257 proteins. For each protein the statistical distribution of sequence identity to the closest 250 mammalian homologs is presented for both full‐length protein sequence (black) and zero‐tolerance segment(s) (red).

FIGURE 2

Representative examples of sequence identity patterns for proteins containing zero‐tolerance segments, comparing both the whole‐protein (black plots) and the intolerant segmental (red plots) homology levels to the 250 nearest mammalian homologs following BLASTP searches of NCBI. GENE.1, GENE.2, and so forth indicate which non‐contiguous intolerant segment for that gene was searched. The distributions of sequence identities seen for the 250 closest homologs to each protein are presented as box‐and‐whiskers plots. The bold bar is the median, the wings of the bars are the quartiles and the whiskers are 1.5 times the inner quartile ranges. The dots are outliers that lie beyond the whiskers. The complete results for all 257 proteins with zero‐tolerance segments are presented in Figure S1

For most proteins, it is seen that the median of the distribution of % sequence homology is much higher for the zero‐tolerance segments than it is for the entire protein sequence. Indeed, for a great many zero‐tolerance segments, the degree of sequence identity to all 250 closest mammalian homologs is 100%. However, there are also a number of exceptions seen in Figures 2 and S1, where the median sequence homology observed for a given zero‐tolerance segment is significantly less than 100%. For example, the intolerant segment found in CTCF exhibits a lower median homology score to mammalian homologs than does the full protein sequence of that protein.

There are a variety of potential explanations for why any given zero‐tolerance segment is not absolutely conserved among mammalian homologs. Because currently there are only roughly 150 fully sequenced mammalian genomes, the fact that we are considering the data for the 250 nearest homologs implies that many of the homologs included in the analysis for a given protein are paralogs, not orthologs. Between paralogs, even functionally critical residues are sometimes expected to exhibit variation. Another and particularly intriguing possibility is that some less‐than‐100%‐conserved human zero‐tolerance segments play critical roles in establishing traits that are unique to humans. Only careful future studies of specific instances will provide convincing explanations for why some of the proteins shown in Figures 2 and S1 contain human zero‐tolerance segments that are less‐than‐100% conserved.

Likely protein basis for most evolutionary intolerance associated with protein‐encoding genes

While it cannot be ruled out for all entries in Table 1 that the mechanism responsible for evolutionary purifying selection involves changes in parent DNA or mRNA structure (see the previous review ), we hypothesize that for the vast majority of cases, evolutionary intolerance stems from the altered properties of the encoded mutant protein. This is here supported by two observations. (a) A number of the proteins listed in Table 1 are known to directly form complexes with other proteins appearing in this table, suggesting that disruption by a single mutation in a single subunit of critical multi‐protein complexes is a common mechanism of underlying zero‐tolerance. In some other cases, multiple proteins containing zero‐tolerance segments are seen to be on the same pathways, even if they do not actually form a complex. (b) There are many proteins appearing in Table 1 that are known to be central players in human biology and physiology—proteins that one might expect to contain intolerant segments. These include calmodulin, ubiquitin, SUMO, clathrin, various tubulin subunits, actin, and the rynanodine receptor. In light of these considerations, this paper focuses on the implications of genetic intolerance as it relates to the encoded proteins.

A case study of intolerance: The voltage‐gated potassium and sodium channels

Although a comprehensive structural study of all 257 proteins with zero‐tolerance regions is beyond the scope of the current work, we nonetheless subjectively perused several dozen of the proteins in Table 1. Results suggest that zero‐tolerance segments tend to occupy well‐structured regions of proteins, often including functionally‐critical sites. An example is provided by the six voltage‐gated potassium channels and two voltage‐gated sodium channels appearing in Table 1 (see list in Table S2). With only one exception, the 19 intolerant segments documented for these eight channels are contained within the part of the channels that spans the critical transmembrane S4 segment of the voltage sensor domain through the transmembrane S6 segment, the latter of which includes the channel gate and ends the pore domain (see Table S2). The structural elements in this span are all known to be critical to voltage‐gated sodium and potassium channel function, where S4 and the S4–S5 linker are central to channel regulation by the transmembrane electrical potential. The actual pore is comprised of S5, the selectivity filter, the pore helix, and S6. , The location of zero‐tolerant segments in these structural elements strongly implicates mutation‐induced alteration of channel function as the mechanistic basis for evolutionary intolerance associated with these segments. It is interesting that from channel to channel the exact location of the intolerant segments varies. For example, the single zero‐tolerance segment in KCNA3 spans the S4 segment and S4–S5 linker, which are key for voltage regulation, while the single zero‐tolerance segment in KCNH7 spans the pore helix, selectivity filter, and S6, which are critical for ion selectivity and flux. ,

The single zero‐tolerance segment that was not located within the functionally‐central S4 through S6 part of the channels is the 82–113 residues segment found in the KCNB1 potassium channel. This segment is located in its N‐terminal tetramerization (T) domain, a domain found in some, but not all voltage‐gated potassium channels. Mutagenesis studies of H105 located within this intolerant segment revealed that mutations at this site do not interfere with KCNB1 homotetramerization, but rather disrupt heterotetramerization with subunits of voltage‐gated KV6 potassium channel family members. This strongly suggests that the basis for zero‐tolerance in this segment is not disruption of the formation of homotetrameric KCNB1 channels but rather disruption of the formation of heterotetrameric KCNB1/Kv6 channels.

A final observation should be made about the sodium channel SCN2A. Unlike homotetrameric potassium channels, human voltage‐gated sodium channels combine all four subunits in a single long chain in which the four connected “pseudo‐subunits” are homologous, but are not identical in sequence, resulting in a fourfold semi‐symmetric channel. It is interesting that only two of the pseudo‐subunits of SCN2A contain zero‐tolerance segments, not all four. Some pseudo‐subunits in voltage‐dependent sodium channels are evidently more tolerant of mutations than others.

Previously overlooked proteins containing zero‐tolerance segments

While the zero‐tolerance proteins include a number of prominent proteins, on the opposite end of the spectrum are a number of genes/proteins listed in Table 1 that are almost completely uncharacterized. A February 2022 PubMed search on each of the following nine genes yielded, at most, only eight papers mentioning each: CLASRP, GOLGA8G, HMGN4, OR4F17, RBMX2, TBC1D3H, U2SURP, ZMAT2, and ZNF84. The presence of zero‐tolerance segments within these proteins suggests that at least some of them are associated with critical physiological functions and/or pathophysiology. While only further study will confirm this prediction, the MTR data seems compelling that such studies are merited. Here, we further highlight the case of OR4F17, which is a membrane protein and putative olfactory receptor. Only 36 of the zero‐tolerance proteins (13%) are integral membrane proteins, which include the aforementioned voltage‐gated channels (Table 1). This is despite the fact that membrane proteins represent roughly 20–30% of all human proteins and are the targets for more than 50% of all approved drugs. This highlights the fact that the factors that decide what represents a good target for drug development correlate only partially with the priorities of natural selection. Indeed, a particularly intriguing observation is that while the human G‐protein coupled receptor (GPCR) superfamily includes the targets for about one third of all approved drugs, OR4F17 is the only GPCR among the 257 proteins of Table 1 and is classified as one of the 500 human olfactory receptors. This raises the question that why an olfactory receptor would contain a zero‐tolerance segment. We suggest three competing hypotheses. First, it could be that mutations in the intolerant segment of this receptor (located at its N‐terminus) could result in a toxic gain‐of‐function effect such as promoting the formation of aggregates or amyloids by this protein. Another possibility is that OR4F17 is not actually an olfactory receptor but has a different and very important physiological function that is disrupted by mutations in its intolerant segment. A third possibility is that it is an olfactory receptor but has additional physiological functions. This would not be unprecedented. Only future experiments will determine which, if any, of these hypotheses are correct. However, this serves as another illustration of the power of intolerance analysis to direct attention to interesting biological questions.

Proteins involved in RNA splicing represent the largest group of proteins containing at least one intolerant segment

We sought preliminary insight into which pathways, networks, and protein complexes are most commonly represented among the 257 proteins with intolerant segments. Cytoscape stringApp with a confidence cutoff of 0.95 was used to determine high confidence interactors. This approach yielded protein interaction maps that group proteins based on broad molecular or cellular functional categories (Figure 3). The largest clusters of networked proteins are associated with central cellular processes such as chromatin remodeling, protein degradation, RNA splicing, the cytoskeleton, the cell cycle, and nucleic acids biochemistry.

FIGURE 3

Protein interaction network using Cytoscape stringApp based on an interactor cut‐off stringdb score ≥ 0.95. Not all proteins returned by this analysis (~150) are visualized here, as networks that consisted of two proteins were excluded from the visualization (with one exception). The clusters highlighted were manually assigned by identifying the general functions of proteins in the clustered area

Further analysis of the protein interaction‐mapping presented in Figure 3 using the stringApp network clustering with a granularity parameter of three was used to help identify sets of proteins that may participate in functional complexes (Figure 4). Major complexes include proteins of the spliceosome, translation, tubulin, and NMDA receptor.

FIGURE 4

Granulated protein interaction networks among proteins containing intolerant segments. We used a granularity parameter of 3 to form more discrete interaction nodes that may represent specific protein complexes. Proteins are labeled according to gene symbol. The darkness/thickness of the lines connecting nodes is indicative of Cytoscape stringApp experimental score, which is based on high‐throughput interaction mapping, where thicker darker lines reflect more confident interactions based on experiments. The networks shown are manually identified based of the general function of the cluster. Sub‐networks of six or fewer proteins are not shown

GO Panther's overrepresentation analysis was employed to identify biological processes that are overrepresented in the intolerant gene list as compared to the Homo sapiens reference. We filtered for processes that had less than 500 proteins in the reference list to avoid very general biological processes and instead focused on more specific pathways. In addition, the p value cut‐off was required to be less than 5 × 10−10. As can be seen in Figure 5a, the most overrepresented biological processes are all or in some way related to mRNA processing, particularly RNA splicing. The two other pathways noted were histone modification and regulation of membrane potential. For further confirmation, the list of proteins was analyzed using Enrichr. Consistent with Panther, it was seen that three different databases (Bioplanet, WikiPathway 2021 Human, and KEGG 2021 Human34423492) indicate that mRNA processing was the most significantly enriched pathway. Approximately 50 of the 290 proteins with zero‐tolerance segments were identified by Panther having the gene ontology (GO) term mRNA processing (see Table 1). When GO Panther's overrepresentation analysis results were filtered to retain results only for the proteins with a zero‐tolerance segment that is at least 41 residues long, the only two categories yielding p <5 × 10−10 were RNA splicing and mRNA processing (Figure 5b), further highlighting the robust enrichment of these protein functional categories in Table 1.

FIGURE 5

Panther overrepresentation GO biological process term analysis of biological pathways associated with proteins containing a zero‐tolerance segment. Only pathways with less than 500 proteins in the Homo sapiens reference list and p < 5 × 10−10 were considered. (a) The results for analysis of proteins in which the minimum size of the zero‐tolerance segment was 31 residues. (b) The results when the minimum length of the zero‐tolerant segment was 41 residues. GO, gene ontology

The gene enrichment analyses all point to RNA splicing as the biological process that is associated with the largest number of proteins that contain an intolerant segment. This may well reflect the importance of mRNA splicing in early human development (conception to birth), where this process enables proteins to be remodeled to suit varying roles during the developmental phases of human gestation. , , Failure to express the correct protein isoforms at the right time may be a particularly common mechanism of purifying selective pressure on the responsible gene variations.

Association of ClinVar variants with proteins having intolerant segments

For each protein with an MTR = 0 segment, we also examined whether there are ClinVar missense variants that encode amino acid changes in that protein and recorded this observation in Table 1. As of February 2022, we found that 127 of the 257 proteins contain at least one ClinVar missense mutation encoding an amino acid change in the protein. It is interesting that the other 130 of these proteins have no known or suspected disease mutations associated with them, highlighting the ability of intolerance analysis to detect proteins that evidently may be essential to human reproduction or gestational development but are not associated with known human genetic disorders. Currently, detection of disease variants is usually based on genetic sampling and analysis of people after they have been born, explaining why mutations in such essential genes may have escaped detection.

For the 127 intolerant proteins that are seen to be associated with ClinVar variations, we also examined whether any of the encoded amino acid changes are located within MTR = 0 segments. We found this to be the case for 68 proteins. For such proteins, while mutations are not observed within their MTR = 0 gene segments in any of the >105 sequences from mostly healthy people in the current gnomAD database, there are very rare variants that are detected in clinical patient populations, often sick children with de novo (non‐inherited) mutations. These very rare variants may cause or contribute to human disorders, but are not absolutely filtered out of the human population because they do not prevent birth.

CONCLUSION

This paper reports that 257 human proteins contain zero‐tolerance segments, as identified by MTR analysis. Some of these proteins were previously known to be associated with genetic disorders and some were not. While not all proteins containing zero‐tolerance segments can be functionally grouped with other such proteins, about half were found in one of a half dozen functionally‐related groups of protein, the largest of which (containing nearly 20% of all zero‐tolerance proteins) is associated with RNA splicing and related RNA biochemistry.

We hope that this report of 257 human proteins that contain zero‐tolerance segments will motivate studies of these proteins to establish exactly how and why mutations in intolerant segments within each protein result in purifying selection in the human population. This will require insight into the human physiological role(s) of each protein and also structural and structure–function data (see Perszyk et al. for a recent method that may support such efforts). For some of these proteins, such as the voltage‐gated potassium and sodium channels, there may already be enough information in the literature to rationalize the presence of zero‐tolerant segments. However, even for these channels, questions remain. For example, mutations in the zero‐tolerance segments of the sodium channels SCN2A and SCN8A are subjected to purifying selection even though these mutations would occur under heterozygous WT/mutant expression conditions and even though sodium channels, unlike potassium channels, form monomeric channels. Does this mean a 50% reduction in the function of SNC2A or SCN8A is sufficient to prevent human reproduction or terminate life before birth or is it instead the case that mutations in zero‐tolerant segments in these proteins induce some sort of toxic gain‐of‐function effect that compounds the impact of partial loss‐of‐function under WT/mutant heterozygous conditions? Future studies may be required to address such questions.

For proteins that have previously escaped significant notice, such as the putative olfactory receptor, OR4F17, observation of a zero tolerant segment suggests a critical and previously overlooked role for these proteins in human reproduction and/or health. Observation of zero‐tolerance segments in proteins may be particularly useful as a way of pointing investigators to proteins that are critical for human reproduction and/or pre‐birth development, but for which associated causative mutations have never been detected.

Finally, there are other interesting questions triggered by this work. These include the aforementioned question of why some zero‐tolerance segments in human protein are not 100% conserved among their nearest mammalian relatives. Another question is inspired by Figure 1a, where it is seen that there is a modest population of proteins that have not only a zero‐tolerance segment, but also contain segments with MTR values higher than 1.0, suggesting these latter segments are experiencing evolutionary pressure to rapidly mutate. Does this suggest that such proteins are critical to human reproduction and/or health, yet also are being pressured either to adapt to changes in the human environment, to further optimize a current function, or to acquire a new function or mode of regulation? We hope that addressing questions such as these will ultimately advance our understanding of the molecular biology of human health, reproduction, development, and disease.

MATERIALS AND METHODS

MTR analysis

An Excel file containing a well‐annotated list of all canonical human genes was provided by Prof. Anthony Capra of the University of California, San Francisco. From this list, we deleted all genes that encode various forms of non‐coding RNA, leaving a list of roughly 20,000 protein‐encoding genes. Each gene was then subjected to MTR analysis using the web‐mounted MTR‐Viewer server (http://biosig.unimelb.edu.au/mtr-viewer/). Version 2 of MTR analysis was run using the default window size of 31 residues. This program conducts MTR analysis in “sliding sequence” fashion for each possible 93 nucleotide segment in the coding gene transcript and returns a plot of the segmental MTR score versus the position of the amino acid in the middle of the encoded 31 residues segment. When a residue is within 16 residues of the protein's N‐ or C‐terminus, analysis is conducted, but in a truncated manner. For example, for residue 10 in any given protein, the reported MTR score will be for the gene segment that encodes residues 1–25. The MTR plots generated by the server for each protein were then manually inspected and the minimum MTR score observed for the analyzed gene/protein was recorded along with the corresponding residue number at the center of the analyzed segment. For proteins having multiple overlapping and/or non‐overlapping MTR = 0 segments, the locations of all such segments were recorded. MTR plots revealed that there were 257 human proteins that exhibited at least one statistically robust MTR = 0 segment.

The 257 genes and their encoded proteins that exhibited at least one statistically robust MTR = 0 segment are tabulated (Table 1) with both gene codes and UniProt identifiers (https://www.UniProt.org/). It was found that the canonical transcript for a given gene analyzed by the MTR version 2 program does not always correspond to the canonical protein sequence listed in UniProt, usually because the MTR‐analyzed transcript is a splice variant of the transcript that encodes the UniProt‐canonical protein. For such instances this is noted in Table 1 and, to avoid confusion, the reported amino acid sequence of the intolerant segment is provided using the residue numbering for the canonical UniProt sequence. There were a few cases where the intolerant segment was not found in the sequence of the UniProt‐listed splice variant form(s) of the protein. In these cases, a note is added to the table.

In conjunction with the primary MTR plot for each gene/protein, the output of the MTR‐Viewer server also includes a plot of the positions of any known ClinVar variants for the analyzed gene/protein. Along with tabulated MTR data for each protein entry we also included the total number of ClinVar variants in the protein and the number that are located within the MTR = 0 segment(s), if any.

Proteins with intolerant segments were also manually characterized based on their function. The GO terms , were tabulated and pathway analysis was also conducted, as described in the following sections. We also recorded whether each protein entry contains a transmembrane domain.

In addition to the 257 proteins with statically robust zero‐tolerance segments there were additional 33 proteins that contained MTR = 0 segments, but for which there were not enough observed silent mutations within these segments in gnomAD to ensure that MTR = 0 is statistically robust. These proteins are listed in Table S1, along with additional information regarding the location of the candidate intolerant segment(s) in each protein's sequence. These 33 proteins remain candidates as having zero‐tolerance segments, but more human sequences will be required to increase the number silent mutations to the point where statistically reliable MTR scores can be calculated.

Sequence homology searches

For each of the 257 protein sequences of Table 1 that contain one or more zero‐tolerance segments we ran BLASTP using the default search parameters against all available mammalian protein sequences. For each search we saved the output for the 250 closest mammalian homologs. We also ran BLASTP for each protein's zero‐tolerance segment(s). For each protein, the median % sequence identity for both the full‐length sequence and the zero‐tolerance segment(s) was determined along with related statistics (Figure 2 and S1).

BLASTP searches were also conducted for the 33 proteins of Table S1 that contain an MTR = 0 segment, but for which the result is not statistically definitive, as summarized in Figure S2.

Protein–protein interaction analysis

Protein networks for proteins with MTR = 0 segments were constructed using the Cytoscape stringApp (https://apps.cytoscape.org/apps/stringapp) with a confidence cutoff of 0.95 stringdb score. Next, a granulation value of 3 was applied to determine refined complexes. The thickness of the lines connecting protein pairs in the granulated Cytoscape networks was set based on the stringdb experiment scores.

Pathway analysis

Gene symbols for proteins with at least one MTR = 0 segment were input into GO Panther's statistical overrepresentation test to determine which biological pathways are overrepresented compared to the reference human gene list (http://www.pantherdb.org/). The processes considered for evaluation were those that encompass less than 500 genes in the human genome reference to filter for GO biological processes functions that are more specific and reduce non‐specific overarching GO terms. In addition to this criterion, the GO term must also have p < 5.0 x 10−10. Additionally, the gene list was input into Enrichr (https://maayanlab.cloud/Enrichr/) to determine the biological pathways involved.

AUTHOR CONTRIBUTIONS

Adam Sanders: Data curation (equal); formal analysis (equal); investigation (equal); writing – review and editing (equal). Jake Hermanson: Data curation (equal); formal analysis (equal); investigation (equal); visualization (equal); writing – review and editing (equal). David Samuels: Investigation (equal); writing – review and editing (equal). Lars Plate: Investigation (equal); methodology (equal); supervision (equal); writing – review and editing (equal). Charles Sanders: Conceptualization (equal); funding acquisition (equal); project administration (equal); supervision (equal); writing – original draft (lead); writing – review and editing (lead).

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Appendix S1. Supporting Information

Click here for additional data file.

Figure S1. % Sequence identities for human proteins containing zero‐tolerance segments to their 250 closest mammalian homologs. For each, the % sequence identity for the entire encoded protein sequence is given first (black), followed by the % sequence identity for each non‐contiguous intolerant segment found in that gene (red), where GENE.1, GENE.2, and so forth indicate which intolerant protein segment encoded by that gene was searched. The data is presented in the form of “box and whiskers” plots. The bold bar indicates the median, the wings of the bars are the quartiles, the whiskers are 1.5 times the inner quartile range, and the dots are outliers that lie beyond the whiskers. Zero‐tolerance protein segments for the following genes failed to yield a successful BLASTP search, which is why they do not appear in this figure: DUSP8.1, EIF1AY, GDF11, HCFC1 (third zero‐tolerance segment only), HMGN4, HUWE1 (fourth zero‐tolerance segment only), KCNC2, NSMF, RBMY1A1, RGPD2, RPS28, SCN8A (eighth zero‐tolerance segment only), and TERT. Some of the possible reasons the amino acid sequences for the zero‐tolerance segments failed to return BLASTP results are given in https://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastDocs&DOC_TYPE=FAQ#nohits

Figure S2. % Sequence identities for the 33 human proteins that contain currently‐statistically‐unconvincing zero‐tolerance segments to their 250 closest mammalian homologs. % Sequence identities for human proteins containing zero‐tolerance segments to their 250 closest mammalian homologs. For each gene, the % sequence identity for the entire encoded protein sequence is given first (black), followed by the % sequence identity for each non‐contiguous intolerant segment found in that gene (red), where GENE.1, GENE.2, and so forth indicate which intolerant protein segment encoded by that gene was searched. The data is presented in the form of “box and whiskers” plots. The bold bar indicates the median, the wings of the bars are the quartiles, the whiskers are 1.5 times the inner quartile range, and the dots are outliers that lie beyond the whiskers.

Table S1. Human proteins that contain at least one zero‐tolerance segment, but for which the statistics for these segments are uncertain.

Table S2. Locations of zero‐tolerance segments in voltage‐gated potassium channels.

Click here for additional data file.