Unique genetic features of the naked mole-rat’s THADA gene

Thyroid Adenoma Associated (THADA) is a protein-coding gene that maps to chromosomal band 2p21 and first has been described as a target of recurrent translocation partner in thyroid tumors. Many genome-wide association studies have revealed an association between THADA and two frequent human diseases, i.e. type 2 diabetes and polycystic ovary syndrome. Nevertheless, the function of its protein is not been completely understood. However, recent evidence suggests that in a Drosophila model THADA can act as a sarco/endoplasmic reticulum Ca2+-ATPase (SERCA)-interacting protein which uncouples SERCA from this function. Once being uncoupled, SERCA produces an increased amount of heat without transporting calcium thus triggering nonshivering thermogenesis. This data prompted us to compare human THADA with that of 65 other eutherian mammals. This includes a comparison of THADA of a variety of eutherian mammals with that of the naked-mole rat (Heterocephalus glaber) which is known to display unique features of thermoregulation compared to other mammals. Our analysis revealed five positions where only the naked-mole rat presented differences. These latter positions included four single amino acid substitutions and one unique deletion of six or seven amino acids, respectively, between residues 858 and 859. In future studies these changes will be analyzed further in detail for their functional relevance.


Introduction
and polycystic ovary syndrome (PCOS) (e.g. Zhao et al., 2012, Eriksen et al., 2012, Wang et al., 2012, Cui et al., 2013, Sun et al., 2014, Day et al., 2015, Cui et al., 2015, Xia et al., 2019, Park et al., 2019, Bakhashab et al., 2019, Tian et al., 2020, Vishnubotla et al., 2020. Again, due to a lack of knowledge, for a long time it was impossible to tie these associations with any type of causal relationship. Nevertheless, both associations turned out to be highly reproducible and apparently did not depend on ethnicity (Goodarzi et al., 2012). Nevertheless, unlike sarcolipin THADA does not seem to be inducible by cold, pointing to a more complex network. The Drosophila experiments suggest that THADA is not only a very "old" gene in terms of evolution but also has served functions in thermogenesis very early.
These functions may explain the association between THADA and T2DM as well as with PCOS and suggest a causal link. The anti-obesity effect of THADA may be mediated through enhanced thermogenesis or reduced ER (endoplasmic reticulum) calcium. Of note, in mice changes of calcium levels in the endoplasmic reticulum during pancreatic maturation were correlated with the expression of THADA (West et al., 2021). Overall, an in depth characterization of the metabolic effects of the THADA-driven network will not only aim at a better understanding of its function in general but might also provide new therapeutic targets for the treatment of both conditions associated with THADA by GWAS (Chatterjee et al., 2017).
THADA as well as its protein were found to be highly conserved among mammals and even among vertebrates (Drieschner et al., 2007, Soller et al., 2008. To better understand functional relationships between THADA and thermogenesis, this study aims at the characterization of Thada of the naked-mole rat (NMR Thada) (Heterocephalus glaber) which, when compared to other rodents and mammals in general, is expressing many unusual traits including its limited ability to maintain a stable body temperature and, at least in part together with other bathyergids, appears to have a different thyroid hormone physiology. While some evidence has been presented that NMR are even non-ageing mammals i.e. lack an increase of likelihood of mortality with chronological age (Ruby et al., 2018)

Materials and methods
THADA is a well conserved protein among mammalian species. For protein alignment of multiple sequences the NCBI blast tool has been used (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins).

Results
In a first approach, THADA AS-sequences of a small set of eutherian (placental) mammals have been compared with that of the naked-mole rat by multiple sequence alignment. For this first analysis we referred to NMR Thada sequence XP_004839417.1 (NCBI reference sequence) and compared it to a first set of 16 species including: Bos taurus (European cattle), Callithrix jacchus (common marmoset), Canis lupus familiaris THADA was unambiguously identified in all these species. Furthermore, this search confirmed the high level of identity between human THADA and that of the other mammalian species investigated ranging between 99.1 % (gorilla and chimpanzee) and 78.3 % (mouse) (Tab.1).
Between human and NMR-Thada, an identity of 85.1% was observed.
A total of six amino acid residues of NMR-Thada turned out to be unique among orthologs present in these 16 other mammalian species with no variation of each of the corresponding amino acid seen in the other mammals analyzed. In addition, NMR-Thada differed from all other investigated mammalian species by a unique deletion site of six or seven amino acids,

Discussion and further experimental approach
Human THADA and its gene have been identified nearly twenty years ago by positional cloning of a breakpoint region of chromosome 2 recurrently involved in chromosomal alterations in thyroid tumors (Bol et al., 2001, Rippe et al., 2003. In genome-wide association studies, the SNPs of the THADA gene have been found to be associated with T2DM as well as with PCOS and SNPs in THADA belong to the most strongly positively selected SNPs during the evolution of the recent human species (Green et al., 2010). Nevertheless, surprisingly little is known about THADA function. Some recent data, however, suggest that, as a SERCA uncoupler, THADA is involved in the control of thermogenesis and energy production.
Compared to other eutherian mammals, naked-mole rats show an extraordinary biology e.g. In Drosophila, THADA knockout leads to obese hyperphagic flies with reduced energy production and enhanced cold-sensitivity (Muraru et al., 2017). These effects could be reversed by transfer of intact human THADA. As a next experimental step an in depth characterization of the NMR-unique changes of THADA as determined herein seems reasonable. It e.g. remains to be determined if transfection of NMR´s Thada and Thada with single changes as present in NMR can also reverse these effects. In order to analyze the possible biological significance of the NMR-unique mutations NMR-derived fibroblasts can also be transfected using human THADA to study the effects on the transcriptome.