A  promoter  sequence upstream of TMEM33 is GXP_4428588. This 1069 base pair promoter sequence spans 41936535-41937603 on human chromosome 4. [ 6 ]
The promoter sequence overlaps with the 5' untranslated region, first coding exon, and first  intron  of the TMEM33 gene. Many different  transcription factors  are known to bind to this region. [ 6 ]
The most likely to bind transcription factors are expressed most in  connective tissue  (i.e. blood, adipose tissue, and bone), the  immune system , and  nervous system . [ 6 ]


Eleven different mRNA transcript variants of TMEM33 exist, 9 alternatively spliced variants and 2 unspliced forms. [ 7 ]
Only 5 variants (a, b, c, d, and e) encode proteins. Transcripts a, b, and c encode the same 247 amino acid long protein because each have the same 7 typical coding  exons . These three variants only vary in 3' UTR length. [ 7 ]
Transcripts a, b, and c have a 744 base pair long coding range and a particularly long 3’  UTR  that is 6000 base pairs long. [ 8 ]


In  Homo sapiens , TMEM33 protein has 5 different coding mRNA variants that encode 3 different protein  isoforms . [ 10 ]
The largest and most common human TMEM33 protein is 247 amino acid long protein with 3  transmembrane domains . [ 11 ]
The only known TMEM33 ortholog with four transmembrane domains is Tts1 in  Schizosaccharomyces pombe . [ 12 ]
The human protein has a predicted  molecular weight  of 28 kDa [ 13 ]
and an  isoelectric point  of 9.88. [ 14 ]
TMEM33 has a significantly high net positive charge and quantity of hydrophobic residues. In particular,  leucine  that makes up 17.8% of the human TMEM33 protein. [ 15 ]
TMEM33 contains a conserved domain in the protein super family UPF0121, in Homo sapiens this region spans from amino acids 1 to 246. [ 11 ]


Programs and experiments that analyze proteins predict various post-translational modifications of TMEM33. There is an experimentally determined  acetylation  point is at  alanine ,  amino acid  residue 2 in humans. [ 11 ]  Human TMEM33 has  phosphorylation  predicted on  serine  residues 197 and 198 and  threonine  residues 5, 127, and 193. [ 16 ]
The primary  kinases  that are predicted to act on TMEM33 are  Protein kinase C  (PKC) and  PKA . [ 17 ]
TMEM33 is predicted to have an  O-glycosylation  site at human amino acid residues 4 and 5 [ 18 ]


The exact secondary structure of TMEM33 is unknown, but it is predicted to be composed primarily of  alpha-helices  and coiled domains. [ 19 ]
The  tertiary structure  is unknown.


According to the sequence motifs of the Homo Sapiens, TMEM33 it is predicted to be at the  endoplasmic reticulum  (ER) 48%,  mitochondria  35%, and  nucleus  13%. [ 12 ]
TMEM33  orthologs  in  vertebrates ,  invertebrates , and  fungi  were also predicted to localize to these three cellular locations: the ER first and the mitochondria second, and some orthologs a third location was predicted and it was the nucleus. [ 12 ]
Homo Sapiens TMEM33 localized with ER membrane and NE exogenously. [ 20 ]
The fission yeast TMEM33 ortholog, Tts1, was reported to localize to the ER and to the NE ( nuclear envelope ). [ 21 ]


A TMEM33 ortholog is known to be in over 150 organisms and no known  paralogs  exist. [ 1 ]


TMEM33 is conserved throughout all animals, similarity to human TMEM33 is >80% for all vertebrates and >60% for all invertebrates. [ 22 ]
TMEM33 has very distant orthologs that have UPF0121 in fungus, yeast, and plants. [ 22 ]


Using human proteins, an  affinity chromatography  ran on TMEM33 showed that the protein bound to  reticulon  4C, 1A,  2B,  3C, and Arl6IP1 in vitro. [ 20 ]
TMEM33 was found to interact with  ubiquitin C , ubiquitin specific peptidase 19 (USP19), 40S ribosomal protein S14 ( RPS14 ),  replication protein A  (RPA1, RPA2, RPA3), transitional endoplasmic reticulum ATPase (VCP), and RNA polymerase III initiation factor ( BRF2 ) using affinity capture experiments with those proteins as bait. [ 23 ]
 Coimmunoprecipitation  experiment found  two-pore channel  (TPC), an ion channel, and TMEM33 bind. [ 24 ]


TMEM33 localizes to the endoplasmic reticulum (ER) membrane and the nuclear envelope, and therefore must function in these regions. [ 20 ]
TMEM33 exogenously suppresses reticulon 4C function, which is a protein that induces the formation of the tubular structure of the ER. [ 20 ]
Therefore, TMEM33 is thought to regulate tubular ER structure through modulation of reticulon activity. Tts1, TMEM33 ortholog in S. pombe, has been found to be involved in dictating ER curvature as well. [ 21 ]
In the nucleus, knock-out studies indicate Tts1 has a role in directing the spindle pole bodies and nuclear core complexes in the nuclear envelope during mitosis. [ 21 ]


Transmembrane protein 61 ( TMEM61)  is a protein that is encoded by the  TMEM61  gene in humans. It is located on the first chromosome in humans [ 5 ]  and is highly expressed in the intestinal regions predominantly the kidney, adrenal gland and pituitary tissues. [ 6 ]  The protein, unlike other transmembrane protein in the region does not promote cancer growth. [ 7 ]  However, the  TMEM61  protein when inhibited by secondary factors restricts normal activity in the kidney. [ 8 ]  The human protein shares many Orthologs and has been prevalent on Earth for millions of years.


There are no known aliases of  TMEM61 . The human protein can be identified with any tool that uses UniProt by Q8N0U2. [ 9 ]


TMEM61  is located on the plus strand of the human chromosome 1 at the locus 1 p32.3. [ 5 ]  The gene is 11, 661 base pairs long, it ranges from position 54,980,628 to 54,992,288 on chromosome 1.  TMEM61  lies between LOC124904184 and BSND. [ 10 ]


NCBI RefSeq contains seven mRNA transcript variants for  TMEM61.  Transcription variants X1, X2, and X2 both are splices of the original protein, but all three isoforms have their own variants. None of the variants share similar exon boundaries, domain or disordered regions.


There are six known Isoforms of the  TMEM61  protein, Isoform X1 is encoded by transcript variant X1, and Isoform X2 with variant X2 and so on. There are two different X2 isoforms, but both have the same amino acid sequence, both the X2 have five less amino acids in the start of the protein, which differs from isoform X1 with same protein sequence and size as the original protein.


The Isoform 1 of the TMEM61 protein is made up of 210 amino acids. [ 12 ]  The protein has a predicted molecular weight of about 22.2 KDa and a theoretical isoelectric point of about 4.54. [ 13 ]  In terms of amino acid composition,  TMEM61  is relatively rich in both the hydrophobic Proline and hydrophilic Serine. The protein is relatively poor in both hydrophilic Asparagine and Lysine. It is also poor in both hydrophobic  Isoleucine and Phenylalanine. [ 14 ]  The protein indicates acid components from it addition of Arginine and Lysine subtracted to the addition of Glutamic Acid and Aspartic Acid. [ 14 ]


TMEM61  Isoform 1 contains two transmembrane domains one of encompasses a DUF domain.  TMEM61  also contains a MTP domain, unlike the transmembrane domain this domains located in the Golgi Apparatus and involves spanning transportation. All four domain regions had low value scores except the second TMEM domain was not able to be scored.


The Ali2D, and I-TASSER models predicted that the secondary structure of  TMEM61  has both alpha helices and beta strands.


No confident model for tertiary structure for TMEM61.


While the modification are few, phosphorylation will not result in a change oil the amino acid for  TMEM61,  this is a result of the lack of glycosylation that takes place in the sequence. Results are represented by graph on bottom right.


Immunofluorescent standing experiments have detected the  TMEM61  protein in the endocrine tissues, kidney and Urinary bladder, and proximal digestive tract. The experiment also found slight expression in the brain tissues. [ 17 ]


According to HumanAtlas, Geoprofile, and NCBI,  TMEM61  is highly expressed in the Kidney,  Pituitary gland ,  Salivary gland ,  Adrenal , and brain tissues in a decreasing order. [ 17 ] [ 10 ] [ 18 ]


In situ hybridization staining a mouse embryo discovered high levels of  TMEM61  in Kidneys and found no other tissues to express the protein. [ 17 ]


TMEM61  was found to be very abundant in the human body in comparison to other proteins. [ 19 ]


Western blotting showed an over expression of lysate in mammalian, in this case rabbit. [ 20 ]


The staining of the human pancreas shows cytoplasmic positivity in exocrine cells. [ 20 ]


The IntAct, String, and BioGrid database found eight relevant interacting protein to the  TMEM61 . [ 21 ] [ 22 ] [ 23 ]  Other TMEM protein such as TMEM124 are closely monitored together for the cancer expression both in the same region but both did not promote cancer growth.


InTACT


TMEM61  has orthologs in mammals, reptiles, aves, amphibians, and fish. A table of orthologs is beside to the right. There is no known paralog of  TMEM61 .


West African lungfish  is the furthest-from-human known organism to express  TMEM61  approximately 408 million years ago. [ 24 ]  The expression of  TMEM61  protein throughout its closely related orthologs all indicate high expression in the Kidney.Based on a molecular clock analysis, the protein sequence of  TMEM61  has on average evolved faster than Cytochrome C but slower than Fibrinogen alpha.


TMEM61  was anticipated to be associated with the formation of  brain tumors  but was later debunked as there was low levels expressed, however the test did indicate its location to be in the mitochondrial neural membrane region. [ 7 ] [ 25 ]  The  TMEM61  has been hoped to promote cancer or tumor growth but there has been no clinical research that proves this idea.


The information obtained about the  TMEM61  does show expression on the kidney beyond it human organism [ clarification needed ]  and the studies show MIF limiting the expression of  TMEM61 . [ 8 ]


The  Aquaporin-11  deficiency, closing or breaking of water channels limits the protein expression in the membrane and restricts  TMEM61  expression and inhibits kidney function. [ 8 ]


Very close to other TMEM protein such as TMEM124 was closely monitored for the cancer expression both in the same region but both did not promote cancer growth. PMP22, YAP1.


TMEM156  is a  gene  that encodes the  transmembrane protein  156 (TMEM156) in  Homo sapiens .  It has the clone name of FLJ23235. [ 1 ]


TMEM156 is located on the short arm of chromosome 4. It is found at position 4p14. It has four known  transcripts , only two of which have proteins. [ 2 ]


Cytogenetic band: 4p14 [ 2 ]


The image above shows chromosome four and the various gene locations on it. TMEM156 can be seen at the thin red band that has been placed at p14.


TMEM156 is 66,499 bases and 296  amino acids  in length. [ 4 ]  TMEM156 spans from 38,966,744 to 39,032,922 bp. [ 5 ]


TMEM156 has one known  splice variant , TMEM156-003. It has one transmembrane region and is 179 amino acids in length resulting in a lower molecular mass of 20.9 kDa. [ 6 ]  In addition the isoelectric point is 7.0633 and the gene has four transcripts. [ 7 ]


There is one known  isoform  of TMEM156. This particular isoform uses an alternate in-frame  splice site  in the 3' coding region. The resulting isoform is shorter than the original. [ 9 ]


The TMEM156 protein is 296 amino acids in length. It has a  molecular weight  of 34.323 kDa and an  isoelectric point  of 7.98. [ 10 ]  The protein interacts with the membrane three times as seen in the figure below.


TMEM156 has a secondary structure composed of primarily  α-helices . Phyre was used to create this structure that can be viewed on the right hand side of this article. It is evident that the predicted protein structure is vastly composed of α-helices. The N terminus is located at the top of the image and the C terminus is at the bottom. [ 12 ]


This structure was predicted by analyzing the amino acid sequence using I-TASSER. The final result can be seen below. [ 13 ]


Glycosylation  at Asn 45 and Asn 156 along with  N-glycosylation  seen in the portion of the protein that is found in the  cytoplasm . [ 14 ]


The k-NN tool suggests the location of TMEM156 in the endoplasmic reticulum of the cell with 44.4% certainty. The following locations were all predicted with 11.1% certainty: vacuolar, vesicles of secretory system, extracellular, plasma membrane, and mitochondrial. [ 15 ]


TMEM156 is expressed in several tissues including  ascites ,  bone marrow ,  salivary glands , and  vascular  to name a few. This gene is not  ubiquitously  expressed, but is still evident in many tissues. This gene is predominately expressed in adults but there is a bit of expression in  fetuses . [ 16 ]


Transmembrane protein 156 has one known paralog. It also has various  orthologs  within  eukaryotes . The table below compares an overarching sample of the known orthologs and one paralog. [ 18 ]  The specific lineage of TMEM156 is:  Eukaryota ;  Metazoa ;  Chordata ;  Craniata ;  Vertebrata ;  Euteleostomi ;  Mammalia ;  Eutheria ;  Euarchontoglires ;  Primates ;  Haplorrhini ;  Catarrhini ;  Homindae ;  Homo . [ 1 ]


NM_001258276 NM_001258277 NM_001258278 NM_052913


NM_029881 NM_001358814


NP_001245205 NP_001245206 NP_001245207 NP_443145


NP_084157 NP_001345743


Transmembrane protein 200A  is a  protein  that in humans is encoded by the TMEM200A  gene . [ 5 ]


Tonsoku-like, DNA repair protein  is a  protein  that in humans is encoded by the TONSL  gene . [ 5 ]


The protein encoded by this gene is thought to be a negative regulator of  NF-kappa-B  mediated transcription. The encoded protein may bind NF-kappa-B complexes and trap them in the  cytoplasm , preventing them from entering the nucleus and interacting with the  DNA .  Phosphorylation  of this protein targets it for degradation by the ubiquitination pathway, which frees the NF-kappa-B complexes to enter the nucleus.


NM_014831 NM_001329998


NP_001316927 NP_055646


Tetratricopeptide repeat and ankyrin repeat containing 1  is a  protein  that in humans is encoded by the TRANK1  gene . [ 5 ]


Through a Genome-wide association study, TRANK1 has been relationed with the  Bipolar disorder . [ 6 ]


NM_001079512 NM_001318873


NM_001013778 NM_001359069


NP_001072980 NP_001305802


Trans-Golgi network vesicle protein 23 A ( TVP23A ) is a  protein  coded for the TVP23A  gene , formerly known as FAM18A. [ 5 ]  TVP23A is located on chromosome 16. [ 6 ]   It is known to have human paralogs,  TVP23B  and TVP23C, as well as orthologs in many different species, notably yeast, mice, and chickens. [ 7 ]  The general consensus on the TVP23A protein indicate that it has some function in the late  Golgi apparatus  and is involved in retrograde transport from endosomes back into the Golgi apparatus. The nature of this transport is still unknown. [ 8 ]


TVP23A is located at cytogenic band 16p13.13, on the negative strand of  Chromosome 16 . [ 6 ]


TVP23A stands for  T rans-Golgi network  V esicle  P rotein  23A  TVP23A, is the current name for the protein.  Aliases of TVP23A include FAM18A, and rarely YDR084C. [ 5 ]


There are two known  isoforms  of TVP23A, variant one and variant two, with variant one being the more common variant in humans. [ 6 ]


TVP23A is a member of the pfam superfamily containing the  domain of unknown function  846 (DUF846). [ 9 ]   TVP23A has a predicted molecular weight of 24.1 kilodaltons, an isoelectric point of 6.5, and relatively high amounts of  tryptophan  and  phenylalanine . [ 10 ]  The secondary structure of TVP23A consists primarily of  alpha helices  composing 4 transmembrane domains. [ 11 ]  There is not much information on the tertiary structure of TVP23A or its homologs.  iTASSER [ 12 ]  was used to generate a prediction for the folding pattern of TVP23A, which supports the presence of multiple helix structures.


TVP23A is ubiquitously expressed in all human tissues. [ 13 ]  There is evidence of higher expression in the brain tissue of mice. [ 14 ]  The promoter for TVP23A is GXP_91266, spanning 1403 base pairs located on the negative strand of chromosome 16. [ 15 ]


The hypothesized function of TVP23A is a transmembrane protein involved in retrograde transport of  vesicles  from early  endosomes  into the late Golgi apparatus. [ 16 ]  TVP23A interactions with  SNARE  TVI1 were found to be required for retrograde transport.
 [ 17 ]


TVP23A has been found to interact with four different proteins via Yeast  two hybrid arrays . [ 18 ]   Two of these proteins, YIPF1 and YIPF2, are believed to be Golgi transport proteins.


TVP23A is a DUF846 containing protein, which is homologous throughout TVP-type proteins.  This domain contains the 4 transmembrane domains of TVP23A [ 19 ]


TVP23A has two paralogs,  TVP23B  and TVP23C.  TVP23B and TVP23C are 96% similar to each other, and both are located on  chromosome 17 . [ 20 ]  Due to the locations of these three genes, and their identities to each other, it is probable that ancestral TVP23 underwent  duplication  and  translocation , giving rise to TVP23A on chromosome 16 and TVP23B/C on chromosome 17, which then underwent a second duplication to form TVP23B and TVP23C.


TVP23A has been found in all multicellular eukaryotes, including fungi.  This gene has not been found in bacteria. [ 21 ]


Transfer genes  or  tra genes  (also  transfer operons  or  tra operons ), are some genes necessary for non-sexual  transfer of genetic material  in both  gram-positive  and  gram-negative bacteria . The tra locus includes the  pilin  gene and regulatory genes, which together form pili on the cell surface, polymeric proteins that can attach themselves to the surface of F-bacteria and initiate the conjugation. The existence of the tra region of a  plasmid  genome was first discovered in 1979 by David H. Figurski and Donald R. Helinski [ 1 ]   In the course of their work, Figurski and Helinski also discovered a second key fact about the tra region – that it can act in  trans  to the mobilization marker which it affects. [ 1 ]


This finding suggested that there were two basic aspects necessary for a plasmid to move from one cell to another:


The tra genes encode proteins which are useful for the propagation of the plasmid from the host cell to a compatible donor cell or maintenance of the plasmid.  Not all transfer operons are the same.  Some genes are only found in a few species or a single  genus  of bacteria while others (such as  traL ) are found in very similar forms in many bacterial species.  Many of the transfer systems are incompatible.  For example,  oriT  and  bom  are two origins of transfer which interact with different sets of transfer genes.  A plasmid with a mob site (like many found in  Rhodococcus  species) cannot be transferred via transfer genes which normally interact with the oriT site (which is common in  E. coli ) [ 3 ]


Each of the individual genes in the tra  operon  codes for a different protein product.  These products may perform a number of tasks including interaction with one another to perform mating pair functions and regulation of different regions of the tra operon itself, [ 6 ]  or conjugative DNA metabolism and surface exclusion. [ 4 ]   Also, note that some proteins perform multiple functions or are associated closely with proteins which have non-similar functions.


T cell receptor gamma variable 1 (non-functional)  is a  protein  that in humans is encoded by the TRGV1  gene .
 [ 1 ]


NM_001145418 NM_015281 NM_001393403 NM_001393404 NM_001393405


NM_001267622 NM_024477


Tetratricopeptide repeat  domain 28  is a  protein  that in  humans  is encoded by the TTC28  gene . [ 5 ]


TCL1 upstream neural differentiation-associated RNA  is a  long noncoding RNA  that in humans is produced from the TUNAR  gene . [ 2 ]


1UVZ ,  1W4V ,  1W89


Thioredoxin, mitochondrial  also known as  thioredoxin-2  is a  protein  that in humans is encoded by the  TXN2   gene  on chromosome 22. [ 4 ] [ 5 ] [ 6 ]  This nuclear gene encodes a  mitochondrial  member of the  thioredoxin  family, a group of small multifunctional  redox -active proteins. The encoded protein may play important roles in the regulation of the mitochondrial  membrane potential  and in protection against  oxidant -induced  apoptosis . [ 4 ]


As a  thioredoxin , TXN2 is a 12-kDa protein characterized by the redox  active site  Trp-Cys-Gly-Pro-Cys. In its  oxidized  (inactive) form, the two cysteines form a  disulfide bond . This bond is then  reduced  by  thioredoxin reductase  and  NADPH  to a dithiol, which serves as a disulfide  reductase . In contrast to  TXN1 , TXN2 contains a putative  N-terminal  mitochondrial  targeting sequence , responsible for its mitochondria  localization , and lacks structural cysteines. [ 7 ] [ 8 ]  Two mRNA transcripts of the  TXN2  gene differ by ~330  bp  in the length of the  3′-untranslated region , and both are believed to exist in vivo. [ 8 ]


This nuclear gene encodes a  mitochondrial  member of the  thioredoxin  family, a group of small multifunctional  redox -active proteins. [ 4 ]  The encoded protein is ubiquitously expressed in all  prokaryotic  and  eukaryotic  organisms, but demonstrates especially high expression in tissues with heavy metabolic activity, including the  stomach ,  testis ,  ovary ,  liver ,  heart ,  neurons , and  adrenal gland . [ 7 ] [ 8 ]  It may play important roles in the regulation of the mitochondrial  membrane potential  and in protection against  oxidant -induced  apoptosis . [ 4 ] [ 7 ]  Specifically, the ability of TXN2 to reduce disulfide bonds enables the protein to regulate mitochondrial redox and, thus, the production of  reactive oxygen species  (ROS). By extension, downregulation of TXN2 can lead to increased ROS generation and cell death. [ 7 ]  The antiapoptotic function of TXN2 is attributed to its involvement in GSH-dependent mechanisms to scavenge ROS, or its interaction with, and thus regulation of, thiols in the  mitochondrial permeability transition pore  component  adenine nucleotide translocator  (ANT). [ 8 ]


Overexpression of TXN2 was shown to have  attenuated   hypoxia -induced  HIF-1alpha  accumulation, which is in direct opposition of the  cytosolic  TXN1, which enhanced HIF-1alpha levels. [ 9 ]  Moreover, although both TXN2 and TXN1 are able to  reduce   insulin , TXN2 does not depend on the oxidative status of the protein for this activity, a quality which may contribute to their difference in function. [ 7 ]


It has been demonstrated that genetic polymorphisms in the TXN2 gene may be associated with the risk of  spina bifida . [ 10 ]


TXN2 is known to inhibit transforming growth factor  (TGF)-β -stimulated ROS generation independent of  Smad  signaling. TGF-β is a pro- oncogenic   cytokine  that induces  epithelial–mesenchymal transition  (EMT), which is a crucial event in  metastatic  progression. In particular, TXN2 inhibits TGF-β-mediated induction of  HMGA2 , a central EMT mediator, and  fibronectin , an EMT marker. [ 11 ]


TXN2 is shown to  interact  with  ANT . [ 8 ]


UBB+1  is shorthand for  Ubiquitin -B+1, a frameshifted  mutant  arising from the  Ubiquitin B  gene. [ 1 ]  UBB+1 is thought to arise from molecular misreading, a poorly understood process.  Molecular misreading introduces  dinucleotide deletions (e.g. ΔGA, ΔGU) into  mRNA  transcripts. These deletions are not present in genomic  DNA . UBB+1 has been observed in the hallmarks of  Alzheimer's disease , as well as other tauopathies and in polyglutamine diseases (e.g. Huntington's disease) but not in  synucleinopathies  (e.g. Parkinson's disease). Since its discovery it has been shown  in vitro  and  in vivo  that UBB+1 inhibits the proteasome and gives rise to downstream effects (e.g. a behavioral phenotype; impaired contextual memory). In non-neuronal cells UBB+1 also accumulates suggesting a functional role in non-neuronal diseases. UBB+1 can be truncated by yeast's  ubiquitin hydrolase 1  (YUH1) [ 2 ]  and ubiquitin C-terminal hydrolase L3  UCHL3  even though the glycine at position 76 has been substituted for a tyrosine. [ 3 ] [ 4 ]


1WH0 ,  4X3G


NM_001351099 NM_001351100 NM_001351101 NM_001351102 NM_001351103 NM_001351104 NM_001351105 NM_001351106 NM_001351107 NM_001351108 NM_001389594 NM_001389595 NM_001389596 NM_001389597 NM_001389598 NM_001389599 NM_001389600 NM_001389601 NM_001389602 NM_001389603 NM_001389604 NM_001389605 NM_001389606 NM_001389607 NM_001389608 NM_001400288 NM_001400290 NM_001400292 NM_001400293 NM_001400294 NM_001400295 NM_001400296 NM_001400297 NM_001400298 NM_001400299


NM_001168371 NM_001168372 NM_001168373 NM_027804 NM_145407


NP_001338028 NP_001338029 NP_001338030 NP_001338031 NP_001338032 NP_001338033 NP_001338034 NP_001338035 NP_001338036 NP_001338037


NP_001161843 NP_001161844 NP_001161845 NP_082080 NP_663382


Ubiquitin specific peptidase 19  is a  protein  that in humans is encoded by the USP19  gene .
 [ 5 ]


A  variant of uncertain  ( or unknown )  significance  ( VUS ) is a genetic variant that has been identified through  genetic testing  but whose significance to the function or health of an organism is not known. [ 1 ]  Two related terms are "gene of uncertain significance" (GUS), which refers to a gene that has been identified through  genome sequencing  but whose connection to a human disease has not been established, and "insignificant mutation", referring to a gene variant that has no impact on the health or function of an organism. The term "variant' is favored in clinical practice over " mutation " because it can be used to describe an allele more precisely (i.e. without inherently connoting pathogenicity). When the variant has no impact on health, it is called a "benign variant". When it is associated with a disease, it is called a "pathogenic variant". A "pharmacogenomic variant" has an effect only when an individual takes a particular drug and therefore is neither benign nor pathogenic. [ 1 ]


A VUS is most commonly encountered by people when they get the results of a lab test looking for a  mutation  in a particular gene. For example, many people know that mutations in the  BRCA1  gene are involved in the development of  breast cancer  because of the publicity surrounding  Angelina Jolie's  preventative treatment. [ 2 ]  Few people are aware of the immense number of other genetic variants in and around  BRCA1  and other genes that may predispose to hereditary breast and ovarian cancer. A recent study of the genes  ATM ,  BRCA1 ,  BRCA2 ,  CDH1 ,  CHEK2 ,  PALB2  and  TP53  found 15,311 DNA sequence variants in only 102 patients. [ 3 ]  Many of those 15,311 variants have no significant  phenotypic  effect. That is, a difference can be seen in the DNA sequence, but the differences have no effect on the growth or health of the person. [ 3 ]


Identifying variants that are significant or likely to be significant is a difficult task that may require expert human and  in silico  analysis, laboratory experiments and even  information theory . [ 3 ]  In spite of those efforts, many people may be worried about their particular VUS, even though it has not been determined to be significant or likely to be significant. Most discovered VUSs will not be investigated in a peer-reviewed research paper, as this effort is usually reserved for likely pathogenic variants. [ citation needed ]


In 2001, an initial draft of the human genome was published by the  International Human Genome Sequencing Consortium . [ 4 ]  With the development of  next-generation sequencing , the cost of sequencing has plummeted and the number of human  genomes  and  exomes  sequenced each year is increasing dramatically. [ 5 ]  As of 2017 [update] , the cost of a quality whole genome sequence is $1,000 or less. [ 6 ]  If the ratio of approximately 20 DNA sequence variants per gene [ 3 ]  holds over the entire genome (with approximately 20,000 genes) that means that every person who elects to have their genome sequenced will be provided with almost half a million Variants of Unknown Significance. To assist people to understand the meaning of all these variants, classification is a first step. [ citation needed ]


In 2015, the  American College of Medical Genetics and Genomics  (ACMG), the  Association for Molecular Pathology  (AMP), and the  College of American Pathologists  (CAP) published a third revision of their guidelines on variant interpretation in  Mendelian disorders . [ 1 ]  The publication recommended the following categories:  pathogenic ,  likely pathogenic ,  uncertain significance ,  likely benign , and  benign . This guideline is one of many resources published by the ACMG in hopes of improving standardization of variant interpretation and reporting. [ 1 ] [ 7 ] [ 8 ] [ 9 ] [ 10 ]


As of 2020 [update] , there continues to be limited involvement from federal agencies to regulate the clinical validity (accuracy) and utility (risks and benefits) of genetic testing. [ 11 ] [ 12 ] [ 13 ]  Variant interpretation and classification is notably subjective, as laboratories developed their own criteria prior to the ACMG-AMP guidelines. [ 8 ] [ 9 ] [ 14 ]  This subjectiveness is further problematic when there is evidence that variant significance changes over time. [ 13 ]  Due to the lack of consistency in official guidelines, the genomics community is left struggling to efficiently categorize genetic variants. [ 9 ] [ 15 ] [ 16 ]


This category is reserved for variants that are well-documented to cause disease. Pathogenicity should be reported with respect to a specific condition and inheritance pattern (e.g., autosomal recessive or dominant). A pathogenic variant meets stringent criteria such as evidence from well-established functional studies or being identified in multiple unrelated individuals with the disease. [ 1 ]


Variants in this category show strong evidence of being disease-causing but lack definitive proof. The term “likely pathogenic” indicates that the data supports a high likelihood (greater than 90%) that the variant is pathogenic. [ 1 ]


This category is for variants where there is unknown or conflicting clinical significance. Additional evidence is needed in order to determine whether or not the variant is causative for a particular disease. [ 1 ]  As additional data and research emerges over time, variants of unknown significance may get moved to other categories, frequently towards the benign or likely benign categories. [ 17 ]


This category is used for variants that are unlikely to be causative of disease, typically supported by population frequency data or functional studies suggesting no deleterious impact. A “likely benign” classification implies more than 90% certainty that the variant is not causative for a disease. [ 1 ]


This category is for variants that are not disease-causing. These variants are usually observed at high frequencies in population databases and have strong evidence against a pathogenic effect, including predictive computational models and experimental data showing no effect on the encoded protein. [ 1 ]


Less than 5% of the human genome encodes proteins, and the rest is associated with  non-coding RNA  molecules,  regulatory DNA sequences ,  LINEs ,  SINEs ,  introns , and sequences for which as yet  no function  has been determined. [ 4 ]  Thus, only a small fraction of the almost half-million VUS's that are expected to be identified by whole genome sequencing can be categorized into the 5 categories above, leaving the patient nearly as uninformed about their variants as they would have been without this information. [ citation needed ]


Most of the base sequences regulating  gene expression  are found outside of protein-coding sequences, either within introns or outside of genes in  intergenic regions . Changes in those regulatory regions can lead to dysfunction of a gene(s) and produce phenotypic effects that can be relevant to health and function. [ 18 ]


An example of a variant in an intergenic  enhancer  is one that is associated with blond hair color in northern Europeans. The variant in an enhancer of the  KITLG  gene causes only a 20% change in gene expression, yet causes hair lightening. [ 18 ] [ 19 ]


An example of an intronic VUS controlling gene expression is the  SNP  found in an intron of the  FTO gene . The FTO gene encodes the fat mass and obesity-associated protein, and the SNP (or VUS) found in its intron was shown by  genome-wide association studies  to be associated with an increased risk for  obesity  and  diabetes . The initial assumption was that this mutation was misregulating FTO to cause the disease risk. However, it was later shown that the intronic variant was in fact regulating the distant  IRX3  gene and not the FTO gene. [ 20 ]  That is just one example of how difficult it can be to determine the significance of a VUS even when many research labs are focused on it, and it illustrates that clinicians cannot reliably interpret genetic results that have not been fully clarified by prior research. [ citation needed ]


The number of VUS reports makes it impossible to mention all such reports. To give a flavor for some applications in one field, it is perhaps of most interest to focus on breast cancer. Remember, this is only a fraction of the information available world-wide about VUS reports related to breast cancer, and as always, your results may vary. [ citation needed ]


In a 2009 US study of over 200 women who received BRCA VUS reports and were surveyed for one year thereafter, distress over the result persisted for the year. [ 21 ]


A 2012 survey of patient outcomes in the Netherlands found that, after genetic counseling for BRCA VUS, patients perceived themselves to have different cancer risks than what had been explained to them by genetic counselors, and that this misperception influenced decisions about radical medical procedures. [ 22 ]


In a 2015 study in the UK, where BRCA VUSs occur in 10-20% of tests, 39% of breast cancer specialists taking part in the study did not know how to explain a VUS report to a patient with no family history, and 71% were unsure about the clinical implications of the test reports. [ 23 ]


Vasa  is an  RNA  binding protein with an ATP-dependent RNA  helicase  that is a member of the DEAD box family of proteins. The vasa gene is essential for  germ cell  development and was first identified in  Drosophila melanogaster , [ 1 ]  but has since been found to be conserved in a variety of vertebrates and invertebrates including humans. [ 2 ] [ 3 ]  The Vasa protein is found primarily in germ cells in embryos and adults, [ 2 ]  where it is involved in germ cell determination and function, as well as in multipotent stem cells, where its exact function is unknown. [ 3 ]


The Vasa gene is a member of the  DEAD box family  of RNA  helicases  in  Drosophila melanogaster. [ 1 ]  Its human ortholog, Ddx4, is located on human chromosome 5q. It is syntenic to mouse chromosome 13, where the mouse vasa gene is located. [ 2 ]  The gene is conserved in many invertebrates and vertebrate species such as  Caenorhabditis elegans ,  Xenopus ,  Zebrafish ,  flatworms ,  echinoderms ,  molluscs ,  nematodes , mice and rats as an important part of germ line maintenance and function. [ 2 ] [ 3 ]


All vertebrate species and  Drosophila  have only one vasa  ortholog . However,  C. elegans  has four Vasa genes, of which only one (GLH-1) is essential. [ 4 ] [ 5 ]


All DEAD box genes, including Vasa, have 9 conserved sequence motifs. [ 6 ]  The Vasa gene family evolved from a duplication event followed by acquiring certain domains. [ 7 ]  Early in the evolution of multicellular animals, the duplication of PL10 related DEAD-box gene occurred. [ 8 ]  This resulted in animals having both Vasa and PL10 genes, but plants and fungi only have PL10 genes and no Vasa. [ 7 ]  After the duplication event, the N-terminal region acquired Zn-knuckle domains which are now conserved in invertebrates. Vertebrates and insects both have lost the Zn-knuckle domains. The number of these domains vary between different species Vasa genes. An important property of Zn-knuckles, which can be categorized as classical  zinc fingers , [ 9 ]  is that they are able to bind to single and double stranded DNA or RNA. [ 10 ]  The presence of Zn-knuckles in invertebrates and absence in vertebrates may be an indication of differences in target binding sites. Their presence may be important to functions outside germ line development. An exception to this theory is the presence of Zn-knuckles in all four  C. elegans  Vasa genes, which are restricted to functions in the germ line.  [ 11 ]


The protein product in humans has 724 amino acids, a molecular mass of 79 kDa and 8 conserved domains in all DEAD-box proteins that is involved in RNA helicase activity. Domain V contains the DEAD motif. [ 12 ]  As with other Vasa related proteins, human Vasa has a N terminus rich in glycine and RGG motif repeats that function in RNA binding. [ 13 ]


Vasa is regulated at the transcript and protein level. Developing embryos and adults regulate Vasa expression to cell and tissue specific locations. In  Drosophila ,  zygotic  transcription of Vasa occurs at pole cells, and stays germ-line specific throughout the life of the organism. [ 14 ]


The Vasa promoter is regulated through methylation. In cells were Vasa is transcribed successfully, the promoter is hypomethylated and in all other cells it is methylated. [ 14 ]  When Vasa is hypermethylated in testes, spermatogenesis defects may occur. [ 15 ]


Post-transcriptionally Vasa has several splice forms in different animals. [ 16 ] [ 17 ]  In  Parhyale hawaiensis , Vasa transcript is uniformly distributed in the embryo and is localized depending on the stabilization of the 3’UTR ( Untranslated Region  to the germ line cells. [ 18 ]  Translation can be inhibited by cis regulatory elements in the transcript's 5' and 3' UTRs. They may inhibit translation by forming secondary RNA structures or binding trans-acting factors. Vasa expression localization is directed by repressing these translation inhibitory pathways. [ 19 ]


Post-translationally, in  Drosophila , Vasa protein is localized to the pole plasm during embryonic development. Many other proteins in  Drosophila  are also localized to the poles. For example,  oskar  protein was found to localize to pole plasm and may be involved in anchoring Vasa to polar granules in the posterior pole of the oocyte. [ 20 ]  Another enzyme, fat facets, may further stabilize Vasa in the pole plasm. [ 21 ]  Other post-translational modification includes phosphorylation of the Vasa ortholog in  C. elegans , [ 22 ]  and arginine methylation in a conserved region of mice,  Xenopus  and  Drosophila  Vasa genes.  [ 23 ]


One of main function of Vasa protein is in germ cell determination and function. [ 2 ]  It uses ATP dependent RNA helicase catalytic activity to regulate the translation of multiple mRNAs. [ 24 ]  Vasa unwinds the duplex RNA by binding and bending short stretches of the duplex in a non-processive manner. [ 25 ] [ 26 ]  The conserved domain may act as chaperones by unwinding RNA secondary structures and refolding properly. [ 27 ]  pre-mRNA splicing, ribosome biogenesis, nuclear export, translational regulation and degradation. [ 6 ]


Vasa was found to bind RNA in a sequence-specific manner. In the  Drosophila  embryos, Vasa binds the Uracil rich motif of the mei-P26 UTR. A mutation in Vasa reduced the interaction of between Mei-P26 and initiation factor elF58 which in turn significantly reduced translation of the gene. [ 28 ]


Recent evidence in invertebrates suggests that Vasa has a role in multipotent stem cells, but the exact function is unknown. [ 3 ]


A null mutation causes female sterility due to severe defects in oogenesis [ 29 ]  but males are fertile.


Homozygous mutations for partial loss of function allows eggs to be fertilized but embryos lack germ cells. [ 8 ]


Mutations in Vasa homolog,  Mvh , cause defects in spermatogenesis but females are fertile. Male sterility may be due to deficiencies in germ cell proliferation and differentiation (the mouse homolog of Droso.). Female fertility may be due to functional redundancy by other DEAD-box family members. Null mutation still allows primordial germ cells to form but have severe defects. [ 30 ]


Although there are no studies done on Vasa mutations in humans, it is likely that it would cause sterility. [ 2 ]


These sex-specific phenotypes in mice and  Drosophila  mutants suggest that Vasa either regulated differently or has different target functions in the two germ line types. [ 3 ]


Vasa expression is restricted to tissue specific cells. Until recently it was thought that Vasa protein can only be found in gametes and is undetectable in somatic cells. [ 2 ]  Within germ cells, Vasa is expressed in the cytoplasm. During embryogenesis, Vasa is expressed in migratory primordial germ cells (PGCs) at the gonadal ridge in both males and females. This specificity allows Vasa to be used as a highly specific marker for germ cells. [ 2 ]  In a patient with  Sertoli cell syndrome , no Vasa signal was detected from testicular biopsy. [ 2 ]  However, recent studies show that Vasa functions in other cells as well. [ 3 ]


A study on  Macrostomum lignano  found Vasa expression in multipotent  neoblast  stem cells in addition to germ cells. [ 16 ]  However,  RNAi knockdown  revealed that either Vasa is non-essential in this organism or is made functionally redundant by other Vasa-like genes. Similar results were found on studies of the colonial ascidian  Botryllus primigenus , [ 31 ]   oysters , [ 32 ]   teleosts , [ 33 ]   clawed frog , [ 34 ]  the parasitic wasp, [ 35 ]  and the crustacean  Parhyale hawaiensis . [ 18 ]


Vasa expression has been observed in epithelial ovarian cancer cells. It was found to deter the DNA damage-induced G2 checkpoint by downregulating the expression of another gene. [ 36 ]  Vasa is also present in chicken embryonic stem cells where it induces expression of germ line genes. This function still supports the most important role of Vasa in germ line development. [ 37 ]  In  Cnidarians , Vasa has a role in nerve cells and gland cells. [ 38 ]  Other examples include Vasa in multipotent stem cell cluster of  Polyascus polygenea  buds and stolon, [ 39 ]  Vasa in auxiliary cells of oyster ovaries, [ 40 ]  Vasa in non-germ-line lineages in the snail  Ilyanassa , [ 41 ]  Vasa in progenitor mesodermal posterior growth zone of the polychaete annelid  Platynereis dumerilii , [ 17 ]  and Vasa present in non-genetical segments during  Oligochaete  development. [ 42 ]  But no reports of vasa expressed outside of germ line cells in  vertebrates  or  insects . [ 3 ]


In  Drosophila ,  vasa  expression is seen in germ cells, specifically the germline stem cells (GSC's) of female ovaries and in the early stages of spermatogenesis in the male testis.


Due to the localization of  vasa ,  immunohistochemistry  staining can be done with vasa  antibodies . For example,  vasa  antibody staining is specific for germ cells in the  D. melanogaster  germarium.


This protein is localized to the cytoplasm of fetal germ cells and to the cytoplasm of developing oocytes in mammals.


Pax-6 , member of the  Pax  gene class, is responsible for carrying the genetic information that will encode  Pax-6  (protein) which dictates the development of the  olfactory epithelium , eyes and  central nervous system  in vertebrates. Pax-6 is expressed as a  transcription factor  when neural  ectoderm  receives a combination of weak  sonic hedgehog  and a strong  TGF-Beta  signaling gradients. Expression is first seen in the forebrain, hindbrain, head ectoderm and spinal cord followed by later expression in midbrain. Expression in the head ectoderm will give rise to the  nasal placodes  and to the eye placodes. The nasal placodes will give rise to the olfactory epithelium while eye placodes give rise to the lens and cornea. Expression in the different brain regions is orchestrated with the combinatorial expression of other transcription factors to give rise to the central nervous system. [ 1 ]  Experiments in mice demonstrate that a deficiency in Pax-6 leads to decrease in brain size, brain structure abnormality leading to Autism, lack of iris formation or a thin cornea. Knockout experiments produced eyeless phenotypes reinforcing the gene's role in eye development. [ 2 ]  Advancing research in this area may lead us to better understanding of the complexity seen in neural development and maybe one day be able to grow eye tissue in vitro.


References:


The  W box  is a  deoxyribonucleic acid  (DNA)  cis-regulatory element  sequence, (T)TGAC(C/T), which is recognized by the family of  WRKY transcription factors . [ 1 ] [ 2 ]


Functionality and conservation of the W-box element across plant species has been shown by gel shift experiments, random binding site selection, yeast one-hybrid screens and co-transfection assays performed with many different WRKY proteins.   In silico -based studies together with functional studies of plant promoters have identified clusters of W-boxes in stress-inducible promoters. The binding of WRKY proteins to W-boxes is a feature of both biotic and abiotic stress responses, together with other plant processes such as germination. [ 3 ]   It has also been shown that multiple W-boxes have a synergistic effect on transcription.


Almost all WRKY transcription factors bind preferentially to W-boxes, and since their discovery, this has raised the question as to how they show specificity for the promoters of their target genes. [ 2 ]  Ciolkowski et al. (2008) showed that although the W-box core is required, adjacent sequences also play a role in determining binding-site preference. [ 4 ]  Recent evidence suggests that the TGAC core is more degenerate, composed of a  guanine   adenine   cytosine  (GAC) core, and the upstream thymine and downstream  pyrimidine  flanking sequences help dictate recognition by specific WRKY factors. [ 5 ]   Basic residues of the WRKY protein domain also are believed to recognize the phosphate backbone of the  cis -element. [ 5 ]


Recently, Yamasaki et al. have determined the solution structure of the C-terminal WRKY domain of Arabidopsis WRKY4 in complex with the W-box DNA by NMR. [ 6 ]  They found that a four-stranded β-sheet enters the major groove of DNA in a structure they called the β-wedge, where the sheet is nearly perpendicular to the DNA helical ais. As predicted amino acids in the conserved WRKYGQK signature motif contact the W-box DNA.


Waardenburg syndrome type 1  is a congenital disorder that caused by a mutation in the  PAX3 gene  that results in abnormal development in the  neural crest  during early development. Type 1 results in early graying and white forelock and a notable distance between the eyes, noted as  dystopia canthorum . Common symptoms of the disease also includes non-progressive hearing loss in majority of patients with type 1. Patients can display complete or partial  heterochromia  and hypoplastic blue irides and congenital leukoderma. [ 1 ]


Type 1 of the Waardenburg syndrome's notable feature is  dystopia canthorum . Along with this feature, some patients' eyelids are fused medially, resulting in medial sclerae. Inferior lachrymal is moved laterally, along with punctae opposite of the cornea. [ 2 ]  Other features include high and broad nasal root and also nasi hypoplasia. A squared jaw is reported in some patients. Others present with  spina bifida  due to the mutation in the neural crest during early development. Other features include the white forelock and graying that occurs in most patients prior to reaching age thirty. In extremely rare cases, the forelock may even be colored red rather than white. The coloring of the forelock may differ from patient to patient. It can appear at birth, or later in the life of those affected and size may vary. It is usually observed in the midline of the hair, but it can appear in other locations as well. [ 1 ]  Associated with the hair forelock, skin pigmentation can also appear in different parts of the body and limbs (suspected to be caused by the mutation in KIT). In parts of the body of some patients, congenital leukoderma is observed. [ 1 ]


Similar to other types of the syndrome, type 1 displays  heterochromia  in some patients, sometimes complete or partial. If it happens to be partial, the differently colored iris is separated from the radial segment of the eye. Deficient  iris stroma  along with hypoplastic blue arises have also been found in some patients with type 1. [ citation needed ]  Another one of the symptoms is hearing loss, presenting in about 69% of cases. [ 2 ]  The types of hearing loss reported in patients differ over a great spectrum. Some of which could be bilateral or  unilateral ,  sensorineural  sometimes a combination of different ones in each ear. The most common loss types reported for type 1 are profound and bilateral.  Waardenburg type 1 is estimated to cause 3% of overall congenital deaf children. [ 1 ]


Type 1 Waardenburg syndrome is a  dominant autosomal  disease, with extremely high  penetrance  of nearly 85%. [ 1 ]  Type 1, as well as type 3, are linked to a mutation in the  PAX3 gene  located in chromosome 2q35. This gene is responsible for encoding a transaction factor named Drosophila paired (noted as prd) resulting in essential proteins. These proteins are responsible for multiple functions in early human development such as stem cell pluripotency, as well as inhibition of differentiation when needed and cell-lineage specification, migration, and proliferation in the cells . These functions are essential in properly developing the central nervous system, somites, skeletal muscles, and neural crest-derived cells that are present throughout various cell types in the body. PAX3 controls the neural crest development through regulation of c-RET,  TGF-b2 , and  WNT1  which are essential controllers of migration and differentiation. [ 3 ]


PAX3 in combination with other transcription factors such as MITF and TRP1 can control melanocyte development. This gene also plays a major role in muscle development as it regulated  myoD  and  myf-5 , essential transcription factors in muscle development. [ 4 ]


The structure of the gene and protein are well understood in research, PAX3 consists of 10 exons and results in major proteins that has 479 amino acids. It has also been found that a majority of the mutations that occur on the PAX3 gene is located on the 2-6 exons.  Heterozygous  mutation is the basis of the majority of type 1 Waardenburg syndrome mutations. [ 3 ]


Type 1 Waardenburg syndrome is sometimes misdiagnosed as type 2, therefore when measuring  dystopia canthorum  if present in a certain patient has been set in strict guidelines. There is a biometric index used as well formula to create a discriminant analysis. These measurements are based on three indices, based on the distances of the inner canthal, interpupillary, and outer canthal. [ 5 ]  This testing of the dystopia canthorum is referred to as the W index. Other features and dysfunctions are used for diagnoses such as hearing loss or pigmentation of the skin and the notable forelock that appears in the hair. A  diagnosis can also be provided through  genetic testing  and identification of the PAX3 mutation in the patient's genes. Gene analysis can take on a variety of tests such as single-gene testing (focus on PAX3), a multigene panel (PAX3 along with other genes), and a more comprehensive genomic testing when available. [ 6 ]


A list of major criteria was created to assess patients for type 1 Waardenburg syndrome. The list includes similar features as listed above such as congenital sensorineural hearing loss, white forelock, and hair pigmentation, pigmentation abnormality of the iris, dystopia canthorum, and related family members who exhibit the disease. [ 7 ]


There is no direct treatment for the patients with Waardenburg syndrome type 1, however, there are multiple ways in which the symptoms are managed. There are some options for hearing loss aid depending on the type faced by the patient. In previous cases, cochlea implants were successful to aid the hearing loss. [ 1 ]  There is also some genetic screening available that can assess whether children can inherit the mutation in the PAX3 gene, but not an overall prediction on the manifestation of the disease in the future generations. [ 1 ]  Pregnant women who are at risk for children suffering from this disease are recommended Folic acid supplementation to assist with the neural crest development. [ 1 ]  Further counseling is available with life decisions such as pregnancies and starting families. Since type 1 is a dominant autosomal disease, any offsprings of those affected are likely to also have the disease.


Waardenburg syndrome  was first described by  Petrus J Waardenburg  in 1951 in the  American Journal of Human Genetics . It is now commonly thought that the description refers to Waardenburg syndrome type 1. The description focused on the distinguishing feature of  dystopia canthorum , the wide distance between the eyes observed in over 80% of patients with type 1. [ 1 ] [ 2 ]  Variations in type 1 and the difference from type 2 were later described by Arias in 1971. In 1991, Da-Silva in focused on Brazilian children who exhibited the physical characteristics of type 1 followed by Winship and Beighton in 1992 with 68 children to describe the onset and manifestation of type 1. [ 8 ]  Research in 2016 resulted in connecting the hearing loss and other physical features to the syndrome and provided more accurate diagnosis. [ 9 ]


NM_175575 NM_001330341


NP_001317270 NP_783165


WAP, follistatin/kazal, immunoglobulin, kunitz and netrin domain containing 2  is a  protein  that in humans is encoded by the WFIKKN2  gene . [ 5 ]


The  WFIKKN1  protein contains a WAP domain,  follistatin  domain,  immunoglobulin domain , two tandem  Kunitz domains , and an  NTR  domain. This gene encodes a WFIKKN1-related protein which has the same domain organization as the WFIKKN1 protein. The WAP-type, follistatin type, Kunitz-type, and NTR-type protease inhibitory domains may control the action of multiple types of proteases. [provided by RefSeq, Jul 2008]. ##Evidence-Data-START## Transcript exon combination :: AY358142.1, AK127743.1 [ECO:0000332] RNAseq introns :: single sample supports all introns ERS025083, ERS025084 [ECO:0000348] ##Evidence-Data-END##


This article on a  gene  on human  chromosome 17  is a  stub . You can help Wikipedia by  expanding it .


In cellular biology, the  Wnt signaling pathways  are a group of  signal transduction  pathways which begin with  proteins  that  pass signals  into a cell through  cell surface receptors . The name Wnt, pronounced "wint", is a  portmanteau  created from the names Wingless and Int-1. [ 1 ]  Wnt signaling pathways use either nearby cell-cell communication ( paracrine ) or same-cell communication ( autocrine ). They are highly evolutionarily  conserved  in animals, which means they are similar across animal species from fruit flies to humans. [ 2 ] [ 3 ]


Three Wnt signaling pathways have been characterized: the  canonical Wnt pathway , the  noncanonical planar cell polarity pathway , and the  noncanonical Wnt/calcium pathway . All three pathways are activated by the binding of a Wnt-protein  ligand  to a  Frizzled  family  receptor , which passes the biological signal to the  Dishevelled protein  inside the cell. The canonical Wnt pathway leads to regulation of  gene   transcription , and is thought to be negatively regulated in part by the  SPATS1  gene. [ 4 ]  The noncanonical  planar cell polarity  pathway regulates the  cytoskeleton  that is responsible for the shape of the cell. The noncanonical Wnt/calcium pathway regulates  calcium  inside the cell.


Wnt signaling was first identified for its role in  carcinogenesis , then for its function in  embryonic development . The embryonic processes it controls include  body axis  patterning,  cell fate  specification,  cell proliferation  and  cell migration . These processes are necessary for proper formation of important tissues including bone, heart and muscle. Its role in  embryonic development  was discovered when genetic mutations in Wnt pathway proteins produced abnormal  fruit fly   embryos . Later research found that the genes responsible for these abnormalities also influenced breast cancer development in mice. Wnt signaling also controls  tissue regeneration  in adult bone marrow, skin and intestine. [ 5 ]


This pathway's clinical importance was demonstrated by  mutations  that lead to various diseases, including  breast  and  prostate cancer ,  glioblastoma ,  type II diabetes  and others. [ 6 ] [ 7 ]  In recent years, researchers reported first successful use of Wnt pathway inhibitors in mouse models of disease. [ 8 ]


The discovery of Wnt signaling was influenced by research on  oncogenic  (cancer-causing)  retroviruses . In 1982,  Roel Nusse  and  Harold Varmus  infected mice with  mouse mammary tumor virus  in order to mutate mouse genes to see which mutated genes could cause breast tumors. They identified a new mouse proto-oncogene that they named int1 (integration 1). [ 3 ] [ 9 ]


Int1 is highly conserved across multiple species, including humans and  Drosophila . Its presence in  D. melanogaster  led researchers to discover in 1987 that the int1 gene in  Drosophila  was actually the already known and characterized  Drosophila  gene known as Wingless (Wg). [ 3 ]  Since previous research by  Christiane Nüsslein-Volhard  and  Eric Wieschaus  (which won them the  Nobel Prize  in Physiology or Medicine in 1995) had already established the function of Wg as a  segment polarity gene  involved in the formation of the body axis during  embryonic development , researchers determined that the mammalian int1 discovered in mice is also involved in embryonic development. [ 10 ]


Continued research led to the discovery of further int1-related genes; however, because those genes were not identified in the same manner as int1, the int gene  nomenclature  was inadequate. Thus, the int/Wingless family became the Wnt family and int1 became Wnt1. The name Wnt is a  portmanteau  of int and Wg and stands for "Wingless-related integration site". [ 3 ]


Wnt comprises a diverse family of secreted  lipid -modified signaling  glycoproteins  that are 350–400  amino acids  in length. [ 11 ]  The lipid modification of all Wnts is  palmitoleoylation  of a single totally conserved cysteine residue. [ 12 ]  Palmitoleoylation is necessary because it is required for Wnt to bind to its carrier protein Wntless (WLS) so it can be transported to the  plasma membrane  for secretion [ 13 ]  and it allows the Wnt protein to bind its receptor Frizzled  [ 14 ] [ 15 ]  Wnt proteins also undergo  glycosylation , which attaches a  carbohydrate  in order to ensure proper secretion. [ 16 ]  In Wnt signaling, these proteins act as  ligands  to activate the different Wnt pathways via paracrine and autocrine routes. [ 2 ] [ 7 ]


These proteins are highly conserved across species. [ 3 ]  They can be found in mice, humans,  Xenopus ,  zebrafish ,  Drosophila  and many others. [ 17 ]


Wnt signaling begins when a Wnt protein binds to the N-terminal extra-cellular cysteine-rich domain of a  Frizzled  (Fz) family receptor. [ 19 ]  These receptors span the  plasma membrane  seven times and constitute a distinct family of  G-protein coupled receptors  (GPCRs). [ 20 ]  However, to facilitate Wnt signaling,  co-receptors  may be required alongside the interaction between the Wnt protein and Fz receptor. Examples include  lipoprotein receptor-related protein  ( LRP )-5/6,  receptor tyrosine kinase  (RTK), and  ROR2 . [ 7 ]  Upon activation of the receptor, a signal is sent to the  phosphoprotein   Dishevelled  (Dsh), which is located in the  cytoplasm . This signal is transmitted via a direct interaction between Fz and Dsh. Dsh proteins are present in all organisms and they all share the following highly conserved  protein domains : an amino-terminal DIX domain, a central  PDZ domain , and a carboxy-terminal  DEP domain . These different domains are important because after Dsh, the Wnt signal can branch off into multiple pathways and each pathway interacts with a different combination of the three domains. [ 21 ]


The three best characterized Wnt signaling pathways are the canonical Wnt pathway, the noncanonical planar cell polarity pathway, and the noncanonical Wnt/calcium pathway. As their names suggest, these pathways belong to one of two categories: canonical or noncanonical. The difference between the categories is that a canonical pathway involves the protein  beta-catenin  (β-catenin) while a noncanonical pathway operates independently of it. [ 19 ]


The canonical Wnt pathway (or Wnt/ β-catenin  pathway) is the Wnt pathway that causes an accumulation of  β-catenin  in the cytoplasm and its eventual translocation into the  nucleus  to act as a transcriptional  coactivator  of  transcription factors  that belong to the  TCF/LEF family . Without Wnt, β-catenin would not accumulate in the cytoplasm since a destruction complex would normally degrade it. This destruction complex includes the following proteins:  Axin ,  adenomatosis polyposis coli  (APC),  protein phosphatase 2A  (PP2A),  glycogen synthase kinase 3  (GSK3) and  casein kinase 1 α (CK1α). [ 22 ] [ 23 ]  It degrades β-catenin by targeting it for  ubiquitination , which subsequently sends it to the  proteasome  to be digested. [ 19 ] [ 24 ]  However, as soon as Wnt binds Fz and  LRP5 / 6 , the destruction complex function becomes disrupted. This is due to Wnt causing the translocation of the negative Wnt regulator, Axin, and the destruction complex to the plasma membrane.  Phosphorylation  by other proteins in the destruction complex subsequently binds Axin to the cytoplasmic tail of LRP5/6. Axin becomes de-phosphorylated and its stability and levels decrease. Dsh then becomes activated via phosphorylation and its DIX and PDZ domains inhibit the GSK3 activity of the destruction complex. This allows β-catenin to accumulate and localize to the nucleus and subsequently induce a cellular response via gene transduction alongside the TCF/LEF (T-cell factor/lymphoid enhancing factor) [ 25 ]  transcription factors. [ 24 ]   β-catenin  recruits other transcriptional coactivators, such as  BCL9 ,  Pygopus [ 26 ]  and Parafibromin/Hyrax. [ 27 ]  The complexity of the transcriptional complex assembled by  β-catenin  is beginning to emerge thanks to new high-throughput  proteomics  studies. [ 28 ]  However, a unified theory of how β‐catenin drives target gene expression is still missing, and tissue-specific players might assist β‐catenin to define its target genes. [ 29 ]  The extensivity of the  β-catenin  interacting proteins complicates our understanding: β-catenin may be directly phosphorylated at Ser552 by Akt, which causes its disassociation from cell-cell contacts and accumulation in cytosol, thereafter 14-3-3ζ interacts with β-catenin (pSer552) and enhances its nuclear translocation. [ 30 ]   BCL9  and  Pygopus  have been reported, in fact, to possess several  β-catenin -independent functions (therefore, likely, Wnt signaling-independent). [ 31 ] [ 32 ] [ 33 ]


The noncanonical planar cell polarity (PCP) pathway does not involve β-catenin. It does not use LRP-5/6 as its co-receptor and is thought to use  NRH1 ,  Ryk ,  PTK7  or  ROR2 . The PCP pathway is activated via the binding of Wnt to Fz and its co-receptor. The receptor then recruits  Dsh , which uses its PDZ and DIX domains to form a complex with Dishevelled-associated activator of  morphogenesis  1 ( DAAM1 ). Daam1 then activates the small  G-protein   Rho  through a  guanine  exchange factor. Rho activates  Rho-associated kinase  (ROCK), which is one of the major regulators of the  cytoskeleton . Dsh also forms a complex with  rac1  and mediates  profilin  binding to  actin . Rac1 activates  JNK  and can also lead to  actin   polymerization .  Profilin  binding to actin can result in restructuring of the cytoskeleton and  gastrulation . [ 7 ] [ 34 ]


The noncanonical Wnt/calcium pathway also does not involve  β-catenin . Its role is to help regulate calcium release from the  endoplasmic reticulum  (ER) in order to control intracellular calcium levels. Like other Wnt pathways, upon ligand binding, the activated Fz receptor directly interacts with Dsh and activates specific Dsh-protein domains. The domains involved in Wnt/calcium signaling are the PDZ and DEP domains. [ 7 ]  However, unlike other Wnt pathways, the Fz receptor directly interfaces with a trimeric G-protein. This co-stimulation of Dsh and the G-protein can lead to the activation of either  PLC  or cGMP-specific  PDE . If PLC is activated, the plasma membrane component  PIP2  is cleaved into  DAG  and  IP3 . When IP3 binds its receptor on the ER, calcium is released. Increased concentrations of calcium and DAG can activate  Cdc42  through  PKC . Cdc42 is an important regulator of ventral patterning. Increased calcium also activates  calcineurin  and  CaMKII . CaMKII induces activation of the transcription factor  NFAT , which regulates cell adhesion, migration and tissue separation. [ 7 ]  Calcineurin activates TAK1 and  NLK  kinase, which can interfere with TCF/β-Catenin signaling in the canonical Wnt pathway. [ 35 ]  However, if PDE is activated, calcium release from the ER is inhibited. PDE mediates this through the inhibition of PKG, which subsequently causes the inhibition of calcium release. [ 7 ]


The binary distinction of canonical and non-canonical Wnt signaling pathways has come under scrutiny and an integrated, convergent Wnt pathway has been proposed. [ 36 ]  Some evidence for this was found for one Wnt ligand (Wnt5A). [ 37 ]  Evidence for a convergent Wnt signaling pathway that shows integrated activation of Wnt/Ca2+ and Wnt/ β-catenin  signaling, for multiple Wnt ligands, was described in mammalian cell lines. [ 38 ]


Wnt signaling also regulates a number of other signaling pathways that have not been as extensively elucidated. One such pathway includes the interaction between Wnt and  GSK3 . During cell growth, Wnt can inhibit GSK3 in order to activate  mTOR  in the absence of β-catenin. However, Wnt can also serve as a negative regulator of mTOR via activation of the  tumor suppressor   TSC2 , which is upregulated via Dsh and GSK3 interaction. [ 39 ]  During  myogenesis , Wnt uses  PA  and  CREB  to activate  MyoD  and  Myf5  genes. [ 40 ]  Wnt also acts in conjunction with  Ryk  and  Src  to allow for regulation of neuron repulsion during  axonal guidance . Wnt regulates  gastrulation  when  CK1  serves as an inhibitor of  Rap1-ATPase  in order to modulate the cytoskeleton during gastrulation. Further regulation of gastrulation is achieved when Wnt uses ROR2 along with the  CDC42  and  JNK  pathway to regulate the expression of  PAPC . Dsh can also interact with aPKC,  Pa3 ,  Par6  and  LGl  in order to control cell polarity and  microtubule  cytoskeleton development. While these pathways overlap with components associated with PCP and Wnt/Calcium signaling, they are considered distinct pathways because they produce different responses. [ 7 ]


In order to ensure proper functioning, Wnt signaling is constantly regulated at several points along its signaling pathways. [ 41 ]  For example, Wnt proteins are  palmitoylated . The protein  porcupine  mediates this process, which means that it helps regulate when the Wnt ligand is secreted by determining when it is fully formed. Secretion is further controlled with proteins such as  GPR177  (wntless) and  evenness interrupted  and complexes such as the  retromer  complex. [ 7 ] [ 24 ]


Upon  secretion , the ligand can be prevented from reaching its receptor through the binding of proteins such as the stabilizers  Dally  and  glypican 3  (GPC3), which inhibit diffusion. In cancer cells, both the heparan sulfate chains [ 42 ] [ 43 ]  and the core protein [ 44 ] [ 45 ]  of GPC3 are involved in regulating Wnt binding and activation for cell proliferation. [ 46 ] [ 47 ]  Wnt recognizes a heparan sulfate structure on GPC3, which contains IdoA2S and GlcNS6S, and the 3-O-sulfation in GlcNS6S3S enhances the binding of Wnt to the heparan sulfate glypican. [ 48 ]  A cysteine-rich domain at the N-lobe of GPC3 has been identified to form a Wnt-binding hydrophobic groove including phenylalanine-41 that interacts with Wnt. [ 45 ] [ 49 ]  Blocking the Wnt binding domain using a nanobody called HN3 can inhibit Wnt activation. [ 45 ]


At the Fz receptor, the binding of proteins other than Wnt can antagonize signaling. Specific  antagonists  include  Dickkopf  (Dkk),  Wnt inhibitory factor 1  (WIF-1), [ 50 ] [ 51 ]   secreted Frizzled-related proteins  (SFRP),  Cerberus ,  Frzb ,  Wise ,  SOST , and  Naked cuticle . These constitute inhibitors of Wnt signaling. However, other molecules also act as activators.  Norrin  and  R-Spondin2  activate Wnt signaling in the absence of Wnt ligand.


Interactions between Wnt signaling pathways also regulate Wnt signaling. As previously mentioned, the Wnt/calcium pathway can inhibit TCF/β-catenin, preventing canonical Wnt pathway signaling. [ 7 ] [ 24 ]   Prostaglandin E2  (PGE2) is an essential activator of the canonical Wnt signaling pathway. Interaction of  PGE2  with its receptors E2/E4 stabilizes β-catenin through cAMP/PKA mediated phosphorylation. The synthesis of PGE2 is necessary for Wnt signaling mediated processes such as tissue regeneration and control of stem cell population in zebrafish and mouse. [ 5 ]  Intriguingly, the unstructured regions of several oversized  intrinsically disordered proteins  play crucial roles in regulating Wnt signaling. [ 52 ]


Wnt signaling plays a critical role in embryonic development. It operates in both  vertebrates  and  invertebrates , including humans, frogs, zebrafish,  C. elegans ,  Drosophila  and others. It was first found in the segment polarity of Drosophila, where it helps to establish anterior and posterior polarities. It is implicated in other  developmental processes . As its function in  Drosophila  suggests, it plays a key role in  body axis  formation, particularly the formation of the  anteroposterior  and  dorsoventral  axes. It is involved in the induction of  cell differentiation  to prompt formation of important organs such as  lungs  and  ovaries . Wnt further ensures the development of these tissues through proper regulation of  cell proliferation  and  migration . Wnt signaling functions can be divided into axis patterning, cell fate specification, cell proliferation and cell migration. [ 53 ]


In early embryo development, the formation of the primary body axes is a crucial step in establishing the organism's overall body plan. The axes include the anteroposterior axis, dorsoventral axis, and right-left axis. Wnt signaling is implicated in the formation of the anteroposterior and dorsoventral (DV) axes. Wnt signaling activity in anterior-posterior development can be seen in mammals, fish and frogs. In mammals, the  primitive streak  and other surrounding tissues produce the morphogenic compounds Wnts,  BMPs ,  FGFs ,  Nodal  and  retinoic acid  to establish the posterior region during late  gastrula . These proteins form concentration gradients. Areas of highest concentration establish the posterior region while areas of lowest concentration indicate the anterior region. In fish and frogs, β-catenin produced by canonical Wnt signaling causes the formation of organizing centers, which, alongside BMPs, elicit posterior formation. Wnt involvement in DV axis formation can be seen in the activity of the formation of the  Spemann organizer , which establishes the dorsal region. Canonical Wnt signaling β-catenin production induces the formation of this organizer via the activation of the genes twin and siamois. [ 36 ] [ 53 ]  Similarly, in avian gastrulation, cells of the  Koller's sickle  express different mesodermal marker genes that allow for the differential movement of cells during the formation of the primitive streak. Wnt signaling activated by FGFs is responsible for this movement. [ 54 ] [ 55 ]


Wnt signaling is also involved in the axis formation of specific body parts and organ systems later in development. In vertebrates,  sonic hedgehog  (Shh) and Wnt morphogenetic signaling gradients establish the dorsoventral axis of the  central nervous system  during  neural tube  axial patterning. High Wnt signaling establishes the dorsal region while high Shh signaling indicates the ventral region. [ 56 ]  Wnt is involved in the DV formation of the central nervous system through its involvement in  axon guidance . Wnt proteins guide the axons of the  spinal cord  in an anterior-posterior direction. [ 57 ]  Wnt is also involved in the formation of the limb DV axis. Specifically, Wnt7a helps produce the dorsal patterning of the developing limb. [ 36 ] [ 53 ]


In the  embryonic differentiation waves  model of development Wnt plays a critical role as part a signalling complex in competent cells ready to differentiate. Wnt reacts to the activity of the cytoskeleton, stabilizing the initial change created by a passing wave of contraction or expansion and simultaneously signals the nucleus through the use of its different signalling pathways as to which wave the individual cell has participated in. Wnt activity thereby amplifies mechanical signalling that occurs during development. [ 58 ] [ 59 ]


Cell fate specification or  cell differentiation  is a process where undifferentiated cells can become a more specialized cell type. Wnt signaling induces differentiation of  pluripotent stem cells  into  mesoderm  and  endoderm   progenitor cells . [ 60 ]  These progenitor cells further differentiate into cell types such as endothelial, cardiac and vascular smooth muscle lineages. [ 61 ]  Wnt signaling induces blood formation from stem cells. Specifically, Wnt3 leads to mesoderm committed cells with  hematopoietic  potential. [ 62 ]  Wnt1 antagonizes neural differentiation and is a major factor in self-renewal of neural stem cells. This allows for regeneration of nervous system cells, which is further evidence of a role in promoting neural stem cell proliferation. [ 60 ]  Wnt signaling is involved in  germ cell  determination,  gut  tissue specification,  hair follicle  development, lung tissue development, trunk  neural crest cell  differentiation,  nephron  development, ovary development and  sex determination . [ 53 ]  Wnt signaling also antagonizes heart formation, and Wnt inhibition was shown to be a critical inducer of heart tissue during development, [ 63 ] [ 64 ] [ 65 ]  and small molecule Wnt inhibitors are routinely used to produce cardiomyocytes from pluripotent stem cells. [ 66 ] [ 67 ]


In order to have the mass differentiation of cells needed to form the specified cell tissues of different organisms, proliferation and growth of  embryonic stem cells  must take place. This process is mediated through canonical Wnt signaling, which increases nuclear and cytoplasmic β-catenin. Increased β-catenin can initiate transcriptional activation of proteins such as  cyclin D1  and  c-myc , which control the  G1  to  S phase  transition in the  cell cycle . Entry into the S phase causes  DNA replication  and ultimately  mitosis , which are responsible for cell proliferation. [ 68 ]  This proliferation increase is directly paired with cell differentiation because as the stem cells proliferate, they also differentiate. This allows for overall growth and development of specific tissue systems during embryonic development. This is apparent in systems such as the circulatory system where Wnt3a leads to proliferation and expansion of hematopoietic stem cells needed for red blood cell formation. [ 69 ]


The biochemistry of  cancer stem cells  is subtly different from that of other tumor cells. These so-called Wnt-addicted cells hijack and depend on constant stimulation of the Wnt pathway to promote their uncontrolled growth, survival and migration. In  cancer , Wnt signaling can become independent of regular stimuli, through mutations in downstream oncogenes and tumor suppressor genes that become permanently activated even though the normal receptor has not received a signal. β-catenin binds to transcription factors such as the protein  TCF4  and in combination the molecules activate the necessary genes. LF3 strongly inhibits this binding  in vitro,  in cell lines and reduced tumor growth in mouse models. It prevented replication and reduced their ability to migrate, all without affecting healthy cells. No cancer stem cells remained after treatment. The discovery was the product of " rational drug design ", involving AlphaScreens and ELISA technologies. [ 70 ]


Cell migration during embryonic development allows for the establishment of body axes, tissue formation, limb induction and several other processes. Wnt signaling helps mediate this process, particularly during convergent extension. Signaling from both the Wnt PCP pathway and canonical Wnt pathway is required for proper convergent extension during gastrulation. Convergent extension is further regulated by the Wnt/calcium pathway, which blocks convergent extension when activated. Wnt signaling also induces cell migration in later stages of development through the control of the migration behavior of  neuroblasts ,  neural crest  cells,  myocytes , and tracheal cells. [ 71 ]


Wnt signaling is involved in another key migration process known as the  epithelial-mesenchymal transition  (EMT). This process allows epithelial cells to transform into mesenchymal cells so that they are no longer held in place at the  laminin . It involves cadherin down-regulation so that cells can detach from laminin and migrate. Wnt signaling is an inducer of EMT, particularly in mammary development. [ 72 ]


Insulin  is a  peptide hormone  involved in  glucose   homeostasis  within certain organisms. Specifically, it leads to upregulation of  glucose transporters  in the cell membrane in order to increase glucose uptake from the  bloodstream . This process is partially mediated by activation of Wnt/β-catenin signaling, which can increase a cell's insulin sensitivity. In particular, Wnt10b is a Wnt protein that increases this sensitivity in skeletal muscle cells. [ 73 ]


Since its initial discovery, Wnt signaling has had an association with  cancer . When Wnt1 was discovered, it was first identified as a proto- oncogene  in a  mouse model  for breast cancer. The fact that Wnt1 is a  homolog  of Wg shows that it is involved in embryonic development, which often calls for rapid cell division and migration. Misregulation of these processes can lead to tumor development via excess cell proliferation. [ 3 ]


Canonical Wnt pathway activity is involved in the development of  benign  and  malignant  breast tumors. The role of Wnt pathway in tumor chemoresistance has been also well documented, as well as its role in the maintenance of a distinct subpopulation of cancer-initiating cells. [ 74 ]  Its presence is revealed by elevated levels of β-catenin in the nucleus and/or cytoplasm, which can be detected with  immunohistochemical staining  and  Western blotting . Increased  β-catenin  expression is correlated with poor prognosis in breast cancer patients. This accumulation may be due to factors such as mutations in  β-catenin , deficiencies in the β-catenin destruction complex, most frequently by mutations in structurally disordered regions of  APC , overexpression of Wnt ligands, loss of inhibitors and/or decreased activity of regulatory pathways (such as the Wnt/calcium pathway). [ 52 ] [ 75 ] [ 76 ]  Breast tumors can  metastasize  due to Wnt involvement in EMT. Research looking at metastasis of basal-like breast cancer to the lungs showed that repression of Wnt/β-catenin signaling can prevent EMT, which can inhibit metastasis. [ 77 ]


Wnt signaling has been implicated in the development of other cancers as well as in  desmoid fibromatosis . [ 78 ]  Changes in  CTNNB1  expression, which is the gene that encodes β-catenin, can be measured in breast,  colorectal ,  melanoma ,  prostate ,  lung , and other cancers. Increased expression of Wnt ligand-proteins such as Wnt1, Wnt2 and Wnt7A were observed in the development of  glioblastoma ,  oesophageal cancer  and  ovarian cancer  respectively. Other proteins that cause multiple cancer types in the absence of proper functioning include ROR1, ROR2,  SFRP4 , Wnt5A, WIF1 and those of the TCF/LEF family. [ 79 ]  Wnt signaling is further implicated in the pathogenesis of bone metastasis from breast and prostate cancer with studies suggesting discrete on and off states. Wnt is down-regulated during the dormancy stage by autocrine  DKK1  to avoid immune surveillance, [ 80 ]  as well as during the dissemination stages by intracellular  Dact1. [ 81 ]  Meanwhile Wnt is activated during the early outgrowth phase by  E-selectin . [ 82 ]


The link between PGE2 and Wnt suggests that a chronic inflammation-related increase of PGE2 may lead to activation of the Wnt pathway in different tissues, resulting in  carcinogenesis . [ 5 ]


Diabetes mellitus type 2  is a common disease that causes reduced insulin secretion and increased  insulin resistance  in the periphery. It results in increased blood glucose levels, or  hyperglycemia , which can be fatal if untreated. Since Wnt signaling is involved in insulin sensitivity, malfunctioning of its pathway could be involved. Overexpression of Wnt5b, for instance, may increase susceptibility due to its role in  adipogenesis , since  obesity  and type II diabetes have high  comorbidity . [ 83 ]  Wnt signaling is a strong activator of  mitochondrial biogenesis . This leads to increased production of  reactive oxygen species  (ROS) known to cause DNA and cellular damage. [ 84 ]  This ROS-induced damage is significant because it can cause acute hepatic insulin resistance, or injury-induced insulin resistance. [ 85 ]  Mutations in Wnt signaling-associated transcription factors, such as  TCF7L2 , are linked to increased susceptibility. [ 86 ]


2C63 ,  2C74


14-3-3 protein eta  also referred to as 14-3-3η is a  protein  that in humans is encoded by the  YWHAH   gene .


This gene product belongs to the  14-3-3  family of proteins that are normally intracellular in nature and help to mediate  signal transduction  by binding to  phosphoserine -containing proteins. This highly conserved protein family is found in both plants and mammals, and this protein is 99% identical to the mouse, rat and bovine  orthologs . This gene contains a 7 bp repeat sequence in its  5' UTR , and changes in the number of this repeat has been associated with  early-onset schizophrenia . [ 5 ]


YWHAH has been shown to  interact  with:


14-3-3n is normally intracellular. Two main mechanisms resulting in the release of 14-3-3η into the extracellular environment have been reported:


14-3-3 proteins are components of small extracellular vesicles that are secreted by most, if not all cells.  Tumor necrosis factor  alpha stimulation of macrophages, but not IL-6, promotes the secretion of 14-3-3η into the extracellular space through a TNF alpha-dependent necroptotic mechanism.


A 2021 systematic literature review published by authors from the University Hospitals of Morecambe Bay NHS Foundation Trust in the United Kingdom conclude the following about the 14-3-3n biomarker:


Exogenous 14-3-3η stimulation has been reported to stimulate various cell types including macrophages, monocytes, and fibroblast-like synoviocytes activating key cell signalling cascades including:


Stimulation of cells by extracellular 14-3-3η has been reported to increase key factors relevant to the pathophysiology of rheumatoid arthritis, including:


Exogenous stimulation of human fibroblast-like synoviocytes, in a dose dependent manner, resulted in the development of invadosomes. [ 20 ]  Silencing of 14-3-3n via shRNA resulted in decreased invadosome formation. Invadosomes are finger-like projections that bud from the cell membrane and are directly associated with increasing the migratory or invasive potential of cells. In RA, this might account for how the disease travels to neighboring joints.


Taken together, extracellular 14-3-3η upregulates pro-inflammatory factors directly that are targeted with small molecules and/or biological  Disease-modifying antirheumatic drug  (DMARDs), including TNF, IL-6 and Janus kinases. The table below defines the medicines by target.


Golimumab


Etanercept


Certolizumab


Inflixamab


Inflectra


Sarilumab


Upadacitinib


Baricitinib


Ruxolitinib


Fedratinib


The body of independent evidence supporting the clinical utility of 14-3-3η continues to expand. As of 2022, there have been over 50 peer-reviewed citations and 200 conference proceedings describing the clinical value of the 14-3-3η biomarker.  [ 21 ]


Diagnostic Value of 14-3-3η Marker.  As shown in the table above, a number of studies have evaluated the specificity, sensitivity and value of 14-3-3η in combination with rheumatoid factor (RF) and/or the anti-cyclic citrullinated protein (ACPA, anti-CCP). According to Abdelhafiz et al. meta-analysis of all studies reported a pooled sensitivity of 73% (95% CI: 71 - 75) and a pooled specificity of 88% (95% CI: 87 - 90). In an Early Undifferentiated Polyarthritis (EUPA) cohort from the University of Sherbrooke where 14-3-3n were assessed in 331 subjects, the diagnostic accuracy of RF, CCP and RF and/or CCP was increased by: 24.0%, 36.8% and 15.0% respectively.  [ 22 ]


Prognostic Value of the 14-3-3η Marker.  An analysis of 331 patients from the EUPA cohort with five (5) years of longitudinal follow-up demonstrated that baseline 14-3-3η positivity, at the diagnostic cut-off of > 0.19 ng/ml, was associated with more radiographic progression over the five (5) years. Sustained and/or elevated 14-3-3η positivity over the course of the five (5) years of follow-up was associated with a higher likelihood of radiographic progression. Serial decreases in 14-3-3η of 0.76 ng/ml or reversion to a negative test during follow-up was associated with less subsequent radiographic progression.  [ 22 ]


Serial Changes in 14-3-3η Marker with Effective Disease Management.  14-3-3η has been described as a modifiable marker, with post-treatment levels having been reported to decrease, stay the same, or in some cases increase despite patients being treated with different classes of therapy including small molecule and biological DMARDs like Methotrexate, Adalimumab, Tocilizumab, Tofacitinib and Upadacitinib.  [ 23 ]   [ 24 ]   Post-treatment decreases and/or reversion to a negative 14-3-3η test is typically associated with better patient outcomes, whereas sustained positivity and/or increases in the 14-3-3η marker longitudinally is often associated with worse outcomes.


Further research is required to determine if certain therapies have a greater impact on the modifiability of 14-3-3η. Current data points towards more of a proximal or interactive effect between 14-3-3η and TNF alpha.


Targeting Drug Free Remission . With the advent of more effective RA treatments and more aggressive patient management strategies, drug free remission is now a possibility however, an important consideration when weaning a patient off of a DMARD is the concomitant risk of flaring. In a cohort of 331 patients from the EUPA cohort, Carrier et al. demonstrated that 14-3-3η positivity at the time of remission was associated with increased likelihood of radiographic progression.  [ 22 ]  Whilst further research is required to investigate how 14-3-3η informs risk of flaring and radiographic progression in patients that have achieved remission, the current data indicates that patients in clinical remission with a positive 14-3-3η test should be considered as higher risk for disease progression.


Detection of 14-3-3η Pre-Disease or in Other Arthritides.  As earlier detection of RA is often associated with better clinical outcomes, evaluating how 14-3-3η informs risk of disease onset in asymptomatic patients is and will continue to be of paramount importance to investigate. In a study by Hitchon et al, the authors demonstrated that 14-3-3η levels were detectable in 1st degree relatives of  patients diagnosed with RA, and that levels of 14-3-3η increased in 1st degree relatives at the time of transition to RA. The authors suggested that 14-3-3η was an imminent marker of RA transition. Additional studies in a larger subset of patients is required to fully evaluate when, why and how 14-3-3η levels increase in at-risk patients. [ 25 ]


14-3-3η has also been demonstrated to be detectable in lupus patients diagnosed with secondary Sjogren’s, albeit at lower levels than RA patients.


NM_024630 NM_153746


NP_078906 NP_714968


Zinc finger, DHHC-type containing 14  is a  protein  that in humans is encoded by the ZDHHC14  gene . [ 5 ]


NM_009566 NM_001310531


NP_001297460 NP_033592


ZFP92 zinc finger protein  is a  protein  that in humans is encoded by the ZFP92  gene . [ 5 ]


NM_001135146 NM_001135147 NM_001135148 NM_022154


NM_001135149 NM_001135150 NM_026228


NP_001128618 NP_001128619 NP_001128620 NP_071437


NP_001128621 NP_001128622 NP_080504


Zinc transporter ZIP8  is a  cation / bicarbonate   symporter   protein  which in humans is encoded by the  SLC39A8   gene . [ 5 ] [ 6 ] [ 7 ]


This  transmembrane protein  is responsible for the  influx  of  zinc ,  manganese ,  iron , and  cadmium . [ 8 ]  ZIP8 is distributed among the  embryo ,  placenta , and  yolk sac  during development. [ 9 ]  Within the embryo, the concentration of ZIP8 is highest during the developmental period of different organ systems, specifically the heart where is it localized in the  endothelial cells .  Cardiac development  is a zinc-dependent event. Beginning around mouse E8.0, the heart is in a tubular form with an outer  myocardium  layer and an inner  endocardium  layer, separated by cardiac jelly. [ 10 ]  As development continues, trabeculation, the  protrusion  of  cardiomyocytes  into the cardiac jelly, begins and facilitates nutrient and oxygen exchange prior to the establishment of  coronary vessels . Simultaneous with coronary circulation development, the  trabeculae  then collapse into the ventricular wall in a process known as  compaction .  Cardiomyocyte  differentiation,  proliferation , and trabeculae patterning is regulated through  Notch 1   signaling , which is upregulated by the ECM.  ADAMTS   1 ,  5 ,  7 , 15, and 19 are zinc  metalloenzymes  responsible for degrading the ECM prior to compaction. [ 11 ]  Many studies have analyzed the effects of Slc39a8-/- on fetal heart development and have shown a decrease in  zinc influx  leading to an increase in  cardiomyocyte proliferation  through  BMP10 , hypertrabeculation through the upregulation of Notch1, and ventricular non-compaction due to the persistence of the ECM.


Zinc-finger nucleases  ( ZFNs ) are artificial  restriction enzymes  generated by fusing a  zinc finger   DNA-binding domain  to a  DNA-cleavage domain .  Zinc finger domains can be engineered to target specific desired  DNA  sequences and this enables zinc-finger nucleases to target unique sequences within complex  genomes .  By taking advantage of endogenous DNA repair machinery, these reagents can be used to precisely alter the genomes of higher organisms. Alongside  CRISPR/Cas9  and  TALEN , ZFN is a prominent tool in the field of  genome editing .


It was initially created by researcher  Srinivasan Chandrasegaran .


The DNA-binding domains of individual ZFNs typically contain between three and six individual  zinc finger  repeats and can each recognize between 9 and 18 basepairs.  If the zinc finger domains perfectly recognize a 3 basepair DNA sequence, they can generate a 3-finger array that can recognize a 9 basepair target site.  Other procedures can utilize either 1-finger or 2-finger modules to generate zinc-finger arrays with six or more individual zinc fingers.  The main drawback with this procedure is the specificities of individual zinc fingers can overlap and can depend on the context of the surrounding zinc fingers and DNA.  Without methods to account for this "context dependence", the standard modular assembly procedure often fails. [ 1 ]


Numerous selection methods have been used to generate zinc-finger arrays capable of targeting desired sequences.  Initial selection efforts utilized  phage display  to select proteins that bound a given DNA target from a large pool of partially randomized zinc-finger arrays.  More recent efforts have utilized yeast one-hybrid systems, bacterial one-hybrid and two-hybrid systems, and mammalian cells.  A promising new method to select novel zinc-finger arrays utilizes a bacterial two-hybrid system and has been dubbed "OPEN" by its creators. [ 2 ]  This system combines pre-selected pools of individual zinc fingers that were each selected to bind a given triplet and then utilizes a second round of selection to obtain 3-finger arrays capable of binding a desired 9-bp sequence.  This system was developed by the Zinc-Finger Consortium as an alternative to commercial sources of engineered zinc-finger arrays.


(see:  Zinc finger chimera  for more info on zinc finger selection techniques)


The non-specific cleavage domain from the type IIs  restriction endonuclease   FokI  is typically used as the cleavage domain in ZFNs. [ 4 ]
This cleavage domain must dimerize in order to cleave DNA [ 5 ]
and thus a pair of ZFNs are required to target non-palindromic DNA sites. Standard ZFNs fuse the cleavage domain to the C-terminus of each zinc finger domain. To let the two cleavage domains dimerize and cleave DNA, the two individual ZFNs must bind opposite strands of DNA with their C-termini a certain distance apart.  The most commonly used linker sequences between the zinc finger domain and the cleavage domain requires the 5′ edge of each binding site to be separated by 5 to 7 bp. [ 6 ]


Several different  protein engineering  techniques have been employed to improve both the activity and specificity of the nuclease domain used in ZFNs.   Directed evolution  has been employed to generate a FokI variant with enhanced cleavage activity that the authors dubbed "Sharkey". [ 7 ]  Structure-based design has also been employed to improve the cleavage specificity of FokI by modifying the dimerization interface so that only the intended heterodimeric species are active. [ 8 ] [ 9 ] [ 10 ] [ 11 ]


Zinc finger nucleases are useful to manipulate the genomes of many plants and animals including  arabidopsis , [ 12 ] [ 13 ]   tobacco , [ 14 ] [ 15 ]   soybean , [ 16 ]   corn , [ 17 ]   Drosophila melanogaster , [ 18 ]   C. elegans , [ 19 ]   Platynereis dumerilii , [ 20 ]   sea urchin , [ 21 ]
 silkworm , [ 22 ]
 zebrafish , [ 23 ]   frogs , [ 24 ]   mice , [ 25 ]   rats , [ 26 ]   rabbits , [ 27 ]   pigs , [ 28 ]   cattle , [ 29 ]  and
various types of mammalian cells. [ 30 ]  Zinc finger nucleases have also been used in a mouse model of  haemophilia [ 31 ]  and a clinical trial found CD4+ human T-cells with the  CCR5  gene disrupted by zinc finger nucleases to be safe as a potential treatment for  HIV/AIDS . [ 32 ]  ZFNs are also used to create a new generation of genetic disease models called  isogenic human disease models .


ZFNs can be used to disable dominant mutations in heterozygous individuals by producing double-strand breaks (DSBs) in the DNA (see  Genetic recombination ) in the mutant allele, which will, in the absence of a homologous template, be repaired by  non-homologous end-joining  (NHEJ). NHEJ repairs DSBs by joining the two ends together and usually produces no mutations, provided that the cut is clean and uncomplicated. In some instances, however, the repair is imperfect, resulting in deletion or insertion of base-pairs, producing  frame-shift  and preventing the production of the harmful protein. [ 33 ]  Multiple pairs of ZFNs can also be used to completely remove entire large segments of genomic sequence. [ 34 ]
To monitor the editing activity, a PCR of the target area amplifies both alleles and, if one contains an insertion, deletion, or mutation, it results in a heteroduplex single-strand bubble that cleavage assays can easily detect.
ZFNs have also been used to modify disease-causing alleles in triplet repeat disorders. Expanded CAG/CTG repeat tracts are the genetic basis for more than a dozen inherited neurological disorders including Huntington's disease, myotonic dystrophy, and several spinocerebellar ataxias. It has been demonstrated in human cells that ZFNs can direct double-strand breaks (DSBs) to CAG repeats and shrink the repeat from long pathological lengths to short, less toxic lengths. [ 35 ]


Recently, a group of researchers have successfully applied the ZFN technology to genetically modify the gol pigment gene and the ntl gene in zebrafish embryo. Specific zinc-finger motifs were engineered to recognize distinct DNA sequences.  The ZFN-encoding mRNA was injected into one-cell embryos and a high percentage of animals carried the desired mutations and phenotypes. Their research work demonstrated that ZFNs can specifically and efficiently create heritable mutant alleles at loci of interest in the germ line, and ZFN-induced alleles can be propagated in subsequent generations.


Similar research of using ZFNs to create specific mutations in zebrafish embryo has also been carried out by other research groups. The kdr gene in zebra fish encodes for the vascular endothelial growth factor-2 receptor. Mutagenic lesions at this target site was induced using ZFN technique by a group of researchers in US. They suggested that the ZFN technique allows straightforward generation of a targeted allelic series of mutants; it does not rely on the existence of species-specific embryonic stem cell lines and is applicable to other vertebrates, especially those whose embryos are easily available; finally, it is also feasible to achieve targeted knock-ins in zebrafish, therefore it is possible to create human disease models that are heretofore inaccessible.


ZFNs are also used to rewrite the sequence of an allele by invoking the  homologous recombination  (HR) machinery to repair the DSB using the supplied DNA fragment as a template. The HR machinery searches for homology between the damaged chromosome and the extra-chromosomal fragment and copies the sequence of the fragment between the two broken ends of the chromosome, regardless of whether the fragment contains the original sequence. If the subject is homozygous for the target allele, the efficiency of the technique is reduced since the undamaged copy of the allele may be used as a template for repair instead of the supplied fragment.