All obtained DNA sequence results were checked through the NCBI-Blast database.
Variation Profile Results
The base alterations detected in the DNA sequence of the samples were single nucleotide changes, and these changes did not result in an amino acid change in the codon, according to the DNA sequence results of Cry1 gene of all samples of the Nannospalax species analyzed in the study. In the Cry1 gene, two missense variations were discovered. The two variations, p.L370* (CTA > CTG) and p.N312* (AAC > AAT), were shown to be conserved among all species (Fig. 1).
The variations listed below did not result in an amino acid change in the codon, and all the changes observed in the nucleotide sequence data of the Cry2 gene are single nucleotide changes. The Cry2 gene has six missense variations. Amino acids are found in positions p.R181* (CGC > CGT), p.A184* (GCC > GCT), p.P192* (CCC > CCT), p.L202* (TTG > TTA), p.S220* (AGT > AGC), and p.E225* (GAG > GAA) are conserved in all species (Fig. 2–3).
In the Cry2 gene, codon 184 was encoded as Alanine for the Nannospalax galili species, while it was encoded as Valine in Nannospalax species, resulting in the p.A184V (GCC > GTC) variant. While p.V197G detected in Cry2 was Valine for Nannospalax galili species, it was encoded as Glycine instead of Valine in codon 197. The amino acid in the same codon evolved as Glycine in Homo sapiens, Rattus norvegicus and Heterocephalus glaber species. Similarly, in another example of the Cry2 gene, codon 197 was encoded as Valine in Nannospalax galili species, while it was encoded as Phenylalanine in Nannospalax species, p.V197F (GTT > TTT). In the codon of the same gene, two different variations were detected in different samples. In Homo sapiens Cry2, p.G187F and p.A174V variants were identified as shown in Fig. 2.
On the other hand, there were no base differences in the Reference Bmal1 genome with the Nannospalax species in this study. The results of the NCBI BLAST DNA scan indicated that the Reference genome and our samples were 100% identical. As a result, no variations in this gene have been found.
Although there are single nucleotide alterations at codons 6 (GAA > GAG), 25 (GTC > GTT), 27 (TCC > TCT), 57 (TCA > TCG), 58 (AAC > AAT), 63 (AGG > AGA), 79 (AAG > AAA), 81 (TCG > TCA), and 85 (GAG > GAA) of the Per1 gene, amino acids were conserved in all species. These were indicated as p.E6*, p.G7R, p.V25*, p.S27*, p.N57*, p.S57*, p.N58*, p.R63R*, p.K79*, p.S81* and p.E85*, respectively (Fig. 4–6). In addition, a nucleotide change occurred in the 5'-untranslated (5'-UTR) region of the Per1 gene, as illustrated in Fig. 7.
Seven different variations were observed in Per2 gene. Although there were single nucleotide alterations in Per2 codons 202 (TTG > TTA), 220 (AGT > AGC), and 247 (CCC > CCG), amino acids remain unchanged in all species as p.L202*, p.S220*, p.247P*. While p.N206D was Asparagine in Nannospalax galili, it was coded as Aspartic acid in this study instead of Asparagine in codon 206 (AAC > GAC) (Fig. 8–9).
A single nucleotide change at codon 221, p.D221N (GAT > AAT), evolved as Aspartic Acid in the Nannospalax galili species but as Asparagine in the Nannospalax species studied here.
In addition, p.M250V (ATG > GTG) amino acid was evolved in Nannospalax sp. and Homo sapiens. In the reference sequence Nannospalax galili at codon 252 (TGC > AGC), the amino acid encoded as Cysteine changed as Serin in all species, but in Heterocephalus glaber (p.C252S) it had changes as Glycine. Furthermore, while the p.P247* protein was maintained in Nannospalax unaltered, it was encoded as Leucine (p.L250P) in Homo sapiens.
Pathogenicity Results
A qualitative evaluation ("probably damaging", "possibly damaging", "benign", or "unknown") and a probability score were derived for each amino acid alteration. The PolyPhen-2 score indicates the possibility that this amino acid change can lead to harm, with values close to 1 suggesting that it is highly likely to be harmful (Adzhubei et al. 2013). It was determined that the p.D224N, p.L250P, p.M253V variants on the Per2 human protein. The scoring of the possible results of these variations using PolyPhen-2 has been confirmed to be benign (Fig. 11).
p.G187F and p.A174V variants were discovered in the human homolog of the Nannospalax Cry2 protein. The p.G187F variant of the human Cry2 protein has been found to be potentially dangerous, while the pA174V variant has been found to be benign (Fig. 12).
The functional effects of single-point variations or mutations in the amino acid sequence were calculated using the SNAP2 tool. The prediction is more accurate and reliable when the score is greater; on the other hand, when the score is lower, the variant is more likely to be neutral (Hecht et al. 2015).
The SNAP2 algorithm was used to see if the human variations of Per2 proteins detected in the study were pathogenic. p.D224N, p.L250P, and p.M253V variants on humans had a score of -43, -32, and − 44, respectively, and their estimated pathogenic effect values were "neutral" (Fig.s 13–15).
The in-silico SNAP2 results of Per1 protein showed that the p.G7R variant had a score of 65 and a positive predicted pathogenic effect score (Fig. 16).
Also, the Cry2 protein SNAP2 analysis results showed that the variants p.A174V and p.G187F had the scores of 20 and 2 positive predicted pathogenic effect scores (Fig. 17).
Protein-protein interaction analysis Results
STRING analysis was used to identify the functional connections of the proteins Per1, Per2, Cry1, Cry2, and Bmal1 in cell processes. The analysis results show the number of interactions, whereas the nodes show the proteins. The node rank is an estimate of the typical number of interactions per node. The clustering coefficient displays the average node density on the map. The STRING analyses performed here include all nodes and interactions for all proteins, which are provided in Table 2. A low PPE-enrichment p-value suggests that the observed edges are important and that the nodes are not random.
Table 2
Numerical results of the STRING analysis.
Protein name
|
Number of edges
|
Node number
|
Average node degree
|
Average interaction number (n)
|
Expected number of edges
|
PPE
p-value
|
Cry1
|
11
|
54
|
9.82
|
0.982
|
11
|
< 1.0e-16
|
Cry2
|
11
|
54
|
9.82
|
0.982
|
11
|
< 1.0e-16
|
Bmal1
|
11
|
43
|
7.82
|
0.888
|
13
|
3.69e-11
|
Per1
|
11
|
54
|
9.82
|
0.982
|
10
|
< 1.0e-16
|
Per2
|
11
|
55
|
10
|
1
|
11
|
< 1.0e-16
|
(n = Average Number of Interactions per Node; PPE (protein-protein interactions)) (https://string-db.org).
The Per2 protein exhibits protein-protein interaction (n = 1) more frequently than other proteins, according to the STRING analysis. The proteins Per1, Cry1, and Cry2 all have the same typical mean number of interactions (n = 0.982) and they are extensive (Table 2). The protein Per1 interacts with the following other proteins: Per2, Per3, Csnk1e, Csnk1d, Bhlhe41, Cry2, Cry1, Clock, and XP_008820661.1. Cry2 protein interacts with the proteins Nr1d1, Arntl1 (Bmal1), Bhlhe41, Fbxl3, Clock, Per2, Csnk1d, Csnk1e, and Npas2 proteins. Protein Per2 interacts with Clock, Arntl, Bhlhe41, Csnk1d, Btrc, Cry1, Per1, Cry2, Csnk1e, and Npas2. Cry1 protein interacts with Arntl (Bmal1), Bhlhe41, Fbxl3, Cry2, Clock, Csnk1d, Csnk1e, Npas2, and Per2 proteins. Since there were not any nucleotide differences between reference genome N. galili and Nannospalax species in Turkey for Bmal1 gene, the protein sequence remained the same. This means the visualized figure in Fig. 18 shows the original protein-protein interactions of Bmal1 protein.