The size of mammals varies from mice to whales and can vary markedly from species to species within the same genus. Mus minutoides is recognized as one of the smallest mammals in the world. An adult typically weighs approximately 3 g and is approximately 30 mm long. It belongs to the same genus as Mus musculus , a well-known laboratory animal, but is tenfold lighter (Willan and Meester, 1978) . Despite the physiological similarities shared by the two species, the molecular mechanisms responsible for the notable differences in body size observed between these two species and across a wide range of mammals remain poorly understood.

The Gh-Igf1 axis plays a role in regulating body and organ size (Vasques et al. , 2019). It has been extensively studied in mice, humans, dogs, and cows, focusing on its function and mutations (Godowski et al. , 1989; Chen et al. , 1991; Lupu et al. , 2001; Iio et al. , 2020). The growth hormone receptor gene ( Ghr ), a key component of the Gh-Igf1 axis, is expressed primarily in the liver and is responsible for stimulating insulin-like growth factor 1 (Igf1) production in response to growth hormone (Gh) secreted by the anterior pituitary gland (Vasques et al. , 2019). Igf1 is crucial in promoting growth by stimulating cell division and metabolism throughout the body (Wang et al. , 2004; Yakar et al. , 2018). Laron syndrome (LS) is a form of human dwarfism associated with the Gh-Igf1 axis (Godowski et al. , 1989; Schaefer et al. , 1994; Iwatani et al. , 1997; Janecka et al. , 2016). It is caused by various genetic mutations, such as exon loss or mutations, in Ghr . Several reports have documented dwarfism resulting from defective Igf1 secretion due to Ghr dysfunction (Rosenbloom and Guevara-Aguirre, 1998; Werner et al. , 2020). Ghr knockout experiments in mice and pigs have successfully replicated the LS phenotype (Zhou et al. , 1997; Cui et al. , 2015), emphasizing the crucial role of Ghr in determining mammalian body size. Based on these findings, we propose that Ghr may be involved in the molecular mechanisms underlying dwarfism in M. minutoides .

M. minutoides and M. musculus formed the most closely related cluster within the order Rodentia, family Muridae ( Figure 1A ). In contrast, Homo sapiens and Sus scrofa formed a distinct cluster separate from the Rodentia group. Additionally, a detailed alignment analysis was conducted using CLUSTALW. The predicted length of the M. minutoides Ghr protein was 650 amino acids, which is equivalent to the length of M. musculus Ghr. However, 48 amino acid residues (7.38%) differed between M. minutoides and M. musculus Ghr ( Figure 1B ). To assess the functional implications of the amino acid differences in M. minutoides Ghr, we utilized VaProS (http://p4d-info.nig.ac.jp/vapros/). Four algorithms were employed, namely PolyPhen2, SIFT, PROVEAN, and PANTHER (cutoffs = 0.5, 0.05, -2.5, and -3, respectively). Amino acid substitutions that scored above the cutoff for PolyPhen2 or below the cutoff for SIFT, PROVEAN, and PANTHER were considered to have a negative effect. As a result, it was found that in all four algorithms, no significant impact on the protein's function was observed for 30 amino acid residues, while 16 amino acid residues were predicted to have the potential for functional impairment ( Figure 1B ). Insertions and deletions (A449_S450insS and N465del) could not be evaluated by these algorithms, as they are specifically designed to assess only amino acid substitutions.

Furthermore, an alignment analysis was performed on six animal species, including Xenopus tropicalis , which was used as an outgroup for the phylogenetic analysis. We identified seven regions that exhibited high conservation among the species but showed variations in amino acid residues between M. minutoides and M. musculus (red letters in Figure 1B and red square in Figure 1C). Among the compared Ghr sequences, the amino acid residues Y204H, K213I, H479D, and T642A were identical in M. musculus , Rattus norvegicus , S. scrofa , and H. sapiens but not in M. minutoides ( Figure 1C ). These findings indicate their significance in mammals. In contrast, amino acid residues Y121H, P469L, and S487N differed exclusively in M. minutoides . These unique amino acid residues in M. minutoides (X. tropicalis displayed identical residues to those of the other mammals) may contribute to the M. minutoides -specific phenotype. Specifically, P469L was predicted to be a "deleterious mutation" by all four algorithms in the variation effect analysis using VaProS. This suggests that amino acid residue changes in M. minutoides may significantly affect Ghr protein function. In the context of structure prediction using AlphaFold2, no significant differences between the structures of the Ghr proteins in M. minutoides and M. musculus were observed ( Figure 1D ). However, proline, the 469th and 458th amino acid in M. musculus and H. sapiens , respectively, is located in the intracellular domain of the Ghr protein and is crucial for transmitting signals received from Gh into the cell (Smith et al. , 1989; Lin et al. , 2018). Also, Rowland et al. (2005) reported that the amino acid sequence between positions 391 and 569 of the Ghr protein plays an important role in intracellular signal transduction. In most animals, this proline residue is highly conserved, suggesting the conservation of Ghr's intrinsic function across these species. Therefore, the alteration of M. minutoides Ghr from proline to leucine (P469L) may significantly modify signal transduction from the receptor to the cell. Furthermore, given that the intracellular domain encompasses a significant portion of the identified amino acid substitutions in M. minutoides , it is plausible that the combined effect of these 25 substitutions, including P469L, H479D, S487N, and T642A, may collectively impact its functionality. Our preliminary data showed that Igf1 expression in M. minutoides was significantly lower than in M. musculus . Taken together, these findings suggest that the altered amino acid sequence of M. minutoides Ghr may hinder intracellular signaling, resulting in decreased Igf1 expression, and thus contribute to dwarfing in M. minutoides .

In conclusion, our study has shed light on novel aspects of Ghr, a crucial component of the Gh-Igf1 axis that plays a significant role in mammalian growth. Although previous studies have examined protein function through amino acid substitutions and deletions in various mammalian Ghr proteins (Goujon et al. , 1994; Wang et al. , 1995; Vairamani et al. , 2017), investigations of various mutations are lacking. We believe that investigating specific functional alterations in Ghr resulting from these mutations using genome editing technology will enhance our understanding of the mechanisms governing mammalian body size determination.

This study investigated the characteristics of the Ghr gene sequence and explored the relationship between these characteristics and dwarfism in M. minutoides . All animal experiments were approved by the Yamaguchi University Animal Use and Care Committee (approval number:291). Total RNA was extracted from the livers of M. minutoides using ISOGEN II (NIPPON GENE, Tokyo, Japan). The extracted RNA was reverse transcribed into cDNA using the QuantiTect Reverse Transcription Kit (QIAGEN, Hilden, Germany). Subsequently, primers were designed based on the highly conserved regions among M. musculus , Mus pahari , and Mus spretus using the coding sequences in the Ghr gene database. The primer sequences are listed below. PCR amplification was performed using M. minutoides cDNA as the template with BIOTAQ DNA polymerase (NIPPON Genetics, Tokyo, Japan) under the following conditions: initial denaturation at 94°C for 3 min, followed by 35 cycles of denaturation at 94°C for 30 sec, annealing at 64°C for 45 sec, and extension at 72°C for 45 sec, with a final extension at 72°C for 1 min. After electrophoresis, the target DNA bands were excised from the agarose gel, and the DNA fragments were extracted using the FastGene Gel/PCR Extraction Kit (NIPPON Genetics). DNA sequencing of the extracted DNA was outsourced to the Yamaguchi University Center for Gene Research. Additionally, the nucleotide sequence was determined using three individuals of M. minutoides , and the sequence alignment confirmed their identity (Accession number for the CDS of M. minutoides Ghr in DDBJ is LC623619). A phylogenetic analysis was conducted using MEGA X software (Kumar et al. , 2018) based on the amino acid sequence predicted from the determined nucleotide sequence of M. minutoides . Additionally, the prediction of the protein's structure was performed using the putative amino acid sequence with AlphaFold2 (Jumper et al., 2021; Varadi et al., 2022) .