Biol_72_1

Introduction

The evolution of human skeleton over millions of years has provided useful information about skeletal diversity, its origin, and the changes brought about by environment and other factors of evolution (Eckhardt, 1989; Kralick and Zemel, 2020; Kun et al., 2023). The research conducted dives into the evolution and genetic architecture of human skeleton, accentuating the intricate play of different evolutionary forces responsible for shaping the traits of the human skeleton as well as diversifying it (Kun et al., 2023). Additionally, the evidence from fossils indicates vital morphological modifications in the size of the torso, limbs, and body width, denoting that these transitions happened gradually over time, with gradual evolution over the span of 6 to 7 million years. Humans are the sole bipedal great ape’s characteristic of their distinct skeletal structure, which holds an upright posture. Bipedalism is facilitated by distinct anatomy of skeletal system, like short arms as compared to legs, a narrow trunk and pelvic region, and orientation nature of the vertebral column (Kun et al., 2023).

All these modifications to structural proportions started to emerge around the separation of the human and chimpanzee heritage and could possibly have promoted the usage of tools as well as altered the higher brain functions and cognitive evolution (Kun et al., 2023). Modern-day human skeleton is gracile described in relation to previously found hominin fossils and great ape remains. Evolutionary biologists classify these influences into neutral and non-neutral forces. Neutral processes implicate mutations that evoke unique diversity without straightforward benefits or impediments to individuals, adding up to random shifts within populations. On the other hand, non-neutral processes happen when mutations impact the individual’s fitness, affecting their capacity to adapt to environmental factors (Fournier-Level et al., 2011).

Genomic studies on human-specific skeletal traits

The genomic study of human-specific skeletal traits has made substantial advancements in recent years. A thorough scientific analysis uncovered that distinctive genetic loci concerned with human skeletal proportions were focused within areas of genome that have gone through evolutionary changes in humans, as well as in regulatory components of genes that display differential patterns between humans and our closest evolutionary lineages, the great apes (Kun et al., 2023). After studying the genes concerned with evolution of human skeletal morphology, a candidate gene has been specified. The DMD gene, seems to have exhibited some point mutations for positive selection. Duchenne muscular dystrophy (DMD) is a hereditary disease distinguished by progressive muscle deficiency due to mutations in the DMD gene, which encodes the protein dystrophin (Farrokhi, 2021). The DMD gene displays an elevated rate of unique mutations, contributing to the preponderance of the disorder across all populations (Fischer et al., 2002). This fascinating discovery indicates that hereditary modifications that happened in these pivotal genomic regions may have played a climactic function in navigating the development of the distinct skeletal morphology found in human lineage (Buckler et al., 2009).

Materials and Methods

Sequence data collection across primates

Based on the comprehensive literature review, the genes DMD, ZMIZ1, and FN1 have been identified to human skeletal development. ZMIZ1 (Zinc Finger MIZ-Type Containing 1) is a crucial transcriptional co-activator that plays a pivotal role in regulating gene expression. Fibronectin, encoded by the FN1 gene, is a crucial glycoprotein component of the extracellular matrix (ECM) that plays a vital role in various cellular processes essential for bone development and repair. After investigating these genes for human-specific mutations, it was discovered that ZMIZ1 did not exhibit any human-specific non-synonymous mutations. Consequently, DMD gene has been determined as a candidate for analyzing positive selection in human skeletal morphology. The GTEx portal (https://gtexportal.org/home/) authorizes the exploration of gene expression patterns, study of genetic variants, knowledge of tissue-specificity, analysis of diseases, collection of data from numerous references, and visualization of the results (Stanfill and Cao, 2021).

Selection of orthologs sequences

To further scrutinize the evolutionary relationships of the DMD gene, the orthologs protein sequences of human DMD and its paralogs (UTRN and SPTB) were retrieved from Ensembl utilizing the BLASTP (https://blast.ncbi.nlm.nih.gov/Blast.cgi) sequence alignment tool (Pervaiz and Abbasi, 2016).

Species that were employed in the study are Homo sapiens (humans), Macaca mulatta (macaque), Pongo abelii (orangutan), Pan troglodytes (chimpanzee), Pan paniscus (bonobo), and Saimiri boliviensis (squirrel monkey), Rattus norvegicus (rat), Mus musculus (mouse) Gorilla gorilla (gorilla), Equus caballus (horse), Ornithorhynchus anatinus (platypus), Gallus gallus (chiken), Loxdonta africana (elephant), Dasypus novemcinctus (armadillo), Sarcophilus hairrisii (tasmanian devil), Felis Catus (cat), Xenopustropicalis (Tropical clawed frog), Gasterosteus aculeatus (stickleback), and Denio rerio (zebra fish).

Node sequences

After creating the phylogenetic tree, which illustrates the branching pattern and transformation of species over time, downloading the hereditary sequences from the internal nodes of the tree is the next step. This is accomplished by employing the ExtAncSeqMEGA file, which collects hereditary information for these ancestral origins (https://sourceforge.net/projects/extancseqmega/). The commands used in research are as follows Figure 1:

The last step in the procedure was to align the sequences of the modern human and non-human primate species with the downloaded node sequences.

The human Duchenne muscular dystrophy (DMD) gene has two closely affiliated putative paralogs, as discerned by the paralog prediction tool in the Ensembl database (http://www.ensembl. Org). These are the SPTB (spectrin beta chain) and UTRN (utrophin) genes (Hubbard et al., 2002).

Comparative genomic analysis across primates

A comparative analysis was executed for the human DMD gene across different primate species, like chimpanzee, gorilla, macaque, marmoset, orangutan, and bonobo. The MEGA software (https://www.megasoftware.net/) was used to execute this comparative analysis with the prior goal of determining any human-specific mutations in this gene and then computing the Ka/Ks ratio, which is a criterion used to evaluate whether the gene has gone through positive selection during the process of evolution over time. By comparing the sequence of this gene across multiple primate species, the analysis aimed to reveal the molecular mechanisms and evolutionary influences that have led to the hereditary multiplicity and transformation of these organisms. In Figure 2 and 3, the interface of MEGA 11 software represented human-specific mutations in coding and protein sequences.

Calculation of Ka/Ks ratio

The synonymous (Ks) and non-synonymous (Ka) mutations were calculated to discern the Ka/Ks ratio, which is a valuable measurement for assessing the evolutionary stresses acting on a gene. The Ka/Ks ratio, where Ka is the rate of non-synonymous mutations (those that alter the amino acid sequence) and Ks is the rate of synonymous mutations (those that do not influence the amino acid sequence), assists in determining whether the gene has undergone positive selection.

Synonymous and non-synonymous mutation identification

MEGA11 was used to align the coding sequences of DMD gene using the CLUSTALW algorithm. This process allowed the sequences to be compared and their similarities and disparities to be specified and identify the synonymous and non-synonymous mutations.

Sliding window analysis of Ka/Ks ratio

Sliding window analysis of the Ka/Ks ratio (SWAKK) was employed to determine regions under positive selection in the coding sequences of the gene commonly found in humans and chimpanzees. In SWAKK server (https://ibl.mdanderson.Org/swakk/1dkaks.html), the coding sequences of gene of interest into overlapping windows of 10 codons (30 nucleotides), with each window shifted by 10 codons were inserted. To achieve a balance between localized evolutionary changes and statistical reliability in Ka/Ks estimation a window size of 10 codons was selected on SWAKK server. It helps to reduce the excessive noise and identify region-specific selection signals precisely.

The estimation of the Ka (non-synonymous substitution rate) and Ks (synonymous substitution rate) values for each window across a gene define the deviations in the selective stress influencing the gene. A Ka/Ks ratio greater than 1 indicates the presence of positive selection, which demonstrates that the gene has experienced adaptive transformations. On the contrary, a Ka/Ks ratio less than or equal to 1 suggests either neutral or purifying selection.

Plot data preparation

In order to yield the desired plot, the analysis used the Ka and Ks values that were previously acquired through the SWAKK (Sliding Window Analysis of Ka/Ks) method. This ensured that the data was structured in a symmetric way that could be easily plotted and depicted visually, facilitating additional analysis and interpretation of the Ka/Ks proportions across the various window positions.

Visualizing data with GNUPLOT

The structured and categorized data were then fed into the GNUPLOT software, which is a strong open-source data visualization tool (https://gnuplot.en.lo4d.com/windows). The precise commands and code that were used to create and depict this graphical illustration are as follows in Figure 4:

Selection scans

Statistical trials: Tajima’s D, Fu and Li’s Test

The neutrality tests, such as Tajima’s D and Fu and Li’s D, were used to evaluate the genetic variability patterns within coding sequences of gene across human populations and their primate counterparts. These analyses were conducted using the DnaSP6 software (http://www.ub.edu/dnasp/).

Determination of statistical significance

Statistical importance was demonstrated through the calculation of p-values. Conventionally, a p-value lower than 0.05 is considered statistically substantial, symbolizing significant variations from the neutral model’s expectations. Such divergences indicate the impact of factors beyond genetic drift, such as natural selection or demographic shifts, in shaping observed genetic deviation across human populations and primates (Wang et al., 2023). This understanding carries substantial significance for explaining genetic variation, susceptibility to diseases, and the evolutionary dynamics describing the genes.

Phylogenetic analysis

The evolutionary relationship among the target gene, as well as their putative paralogs and orthologs, were specified using phylogenetic tree that employed the Maximum Likelihood (ML) method (Yang and Evolution, 2007). ML tree was yielded based on the Whelan and Goldman (WAG) model, which is a well-established statistical prototype for emulating amino acid substitutions throughout evolutionary cycles.

Comparative protein domain annotation

A thorough study of relevant academic literature was done to determine distinct domains present within the gene of interest. Subsequent to this extensive literature review, the SMART (Simple Modular Architecture Research Tool) bioinformatics resource (http://smart.embl-heidelberg.de/) was employed to execute a more thorough analysis and depiction of these specified domains (Schultz et al., 1998).

Protein structural evolution analysis

Structure prediction using PHYRE2

A computational tool PHYRE2 was utilized to predict the structure of target gene shared among humans, chimpanzees, and their common ancestor. PHYRE2 employs a technique called homology modeling, which involves predicting the 3D structure of a DMD domain protein. The C-terminal domain of DMD gene was selected due to presence of mutation. The sequences of selected domains were then uploaded in PHYRE2 server. PHYRE2 provide a more comprehensive understanding of the structural characteristics of the target gene shared among the studied species.

Visualization and analysis with UCSF chimera

The predicted structures were then analyzed and compared by utilizing Chimera, a technological software program developed for visualizing and investigating complicated biomolecular structures (https://www.cgl.ucsf.edu/chimera/)(Sgro, 2017). The comparative study was done using chimera’s matchmaker tool.

Table 1: Utilization of online resources for current study.

Resources	URLs	Last accessed
Ensembl	https://www.ensembl.org/index.html	25-05-2024
Uniprot	https://www.uniprot.org/	02-06-2024
GTEx	https://gtexportal.org/home/	10-05-2024
CLUSTALW or MSA	https://megasoftware.net/dload_win_beta	15-05-2024
SWAKK	https://ibl.mdanderson.org/swakk/1dkaks.html	20-05-2024
GNUPlot	http://www.gnuplot.info/	18-04-2024
DnaSP	http://www.ub.edu/dnasp/	28-05-2024
SMART	http://smart.embl.de	04-06-2024
I-TASSER	https://zhanggroup.org/I-TASSER/	22-05-2024
PHYRE2	http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index	14-06-2024
UCSF Chimera	https://www.cgl.ucsf.edu/chimera/download.html	16-06-2024

Results and Discussion

Identification and analysis of species unique mutations and positive selection across primates

The study of Ka/Ks ratios in primates provides insights into how gene has evolved under the forces of natural selection. The marmoset, macaque, orangutan, gorilla, and bonobo heritage, as well as their common ancestral nodes, show lower Ka/Ks ratios as compared to humans. This suggests that these primate species have experienced fewer adaptive modifications at the protein level comparative to humans. The distinctive Ka/Ks ratios for the human and non-human primates, as well as their common ancestral nodes, are provided in Figure 6. Furthermore, Table 2 illustrates the places within the genome where these non-synonymous mutations have occurred, proposing additional insights into the evolutionary trajectories of these species.

SWAKK assessment of human and chimpanzee genome

This relative analysis of the coding sequences between humans and chimpanzees, enabled by the SWAKK server, bared patterns of conservation and deviation, eventually shedding light on the complex evolutionary affinity between these two primates.

Regions exhibiting positive selection

After executing a thorough analysis of the hereditary sequences, many distinctive regions were specified that

Table 2: Depicts non-synonymous substitutions and ka/ks ratios for human and non-human primates.

Species	Amino acid replacement	Positions	TM	No of Ka	No of Ks	Ka/ks
Human	A → D	219	20	12	8	1.5
Human	E → D	378
	N → Y	998
	N → S	1097
	S → L	2013
	N → K	2243
	I → V	2366
	R → Q	2492
	I → V	2614
	I → T	2636
	G → R	2672
	Q → R	2938
Bonobo	None	None	7	0	7	0
Marmoset	V → I	25	190	58	132	0.43
Marmoset	C → S	188	190	58	132	0.43
	F → Y	266
	Y → F	302
	T → A	309
	S → A	320
	Q → R	402
	Q → K	639
	T → A	673
	T → A	865
	R → K	929
	M → T	943
	A → T	951
	T → A	961
	D → E	972
	I →V	1015
	N → S	1049
	L → I	1079
	Q → K	1114
	K → T	1196
	V → A	1232
	V → I	1360
	K → R	1429
	M → T	1484
	M → T	1593
	D → N	1609
	I → V	1913
	T → M	1982
	M → T	1984
	P → S	2022
	Table continues on next page............
Species	Amino acid replacement	Positions	TM	No of Ka	No of Ks	Ka/ks
	D → N	2031
	S → I	2049
	I → V	2055
	T → K	2068
	R → H	2109
	T → I	2177
	L → P	2239
	K → R	2322
	Q → L	2326
	D → A	2333
	L → F	2334
	E → D	2336
	Q → H	2398
	R → H	2632
	H → Q	2675
	R → H	2721
	V → I	2752
	K → R	2762
	Q → R	2807
	T → A	2933
	I → V	2986
	K → R	2990
	E → D	2991
	N → S	2992
	S → G	2994
	A → S	3049
Chimpanzee	None	None	7	0	7	0
Macaque	E → K	477	90	23	67	0.34
	A → V	582
	K →R	585
	T → M	641
	A → V	787
	K → R	803
	Q → E	885
	A → T	897
	V → M	926
	A → V	985
	G → T	1028
	V → A	1038
	A → V	1148
	A → T	1395
	P → S	1711
	L → I	2082
	Q → R	2298
	L → F	2390
	Table continues on next column............
Species	Amino acid replacement	Positions	TM	No of Ka	No of Ks	Ka/ks
	A → T	2413
	D → G	2491
	R → Q	2681
	I → V	2763
	A → S	3224
Gorilla	R → C	489	21	2	19	0.10
Gorilla	Q → R	594	21	2	19	0.10
Orangutan	I → V	228	43	16	27	0.59
	S → T	330
	R → Q	396
	N → T	426
	Q → E	503
	V → L	516
	K → Q	535
	T → K	603
	T → A	631
	I → M	1116
	S → L	1706
	S → G	2180
	N → H	2190
	N → T	2300
	D → E	2323
	H → R	2389

exhibited a Ka/Ks ratio greater than 1. The marmoset, macaque, orangutan, gorilla, and bonobo heritage, as well as their common ancestral nodes, show lower Ka/Ks ratios as compared to humans. This suggests that these primate species have experienced fewer adaptive modifications at the protein level comparative to humans. Studies like those on HR, HAR1, and RUNX2, among others, highlight how specific genes underwent positive selection to promote human-specific traits, offering deeper understanding into the molecular mechanisms underlying human evolution. These pivotal genes have been determined as playing significant roles in the transition of the human skeletal structure, especially the evolution of bipedalism and the S-shaped backbone including Runt genes (Runx1, Runx2, Runx3), these genes express during cartilage construction and are critical for skeleton-genesis in vertebrates. Runt Genes, specifically Runx2 is involved in BMP signaling pathway. BMPs bind to receptors, phosphorylating SMAD1/5/8. The SMAD complex activates RUNX2, promoting osteoblast differentiation and bone formation. Genes in human accelerated regions (HARs), and loci of skeletal ratios were enormously enhanced in HARs, which are components of the genome that have developed rapidly in humans in relation to other species. Similar to these genes, the regions exhibiting positive selection depicted the possibility of changes in skeletal development that permit bipedalism.

Regions exhibiting purifying selection

In contrast, other genomic domains were also determined that displayed a Ka/Ks ratio less than 1. This pattern is characteristic of purifying selection, a procedure in which deleterious mutations are efficiently extracted from the population, conserving the integrity and functionality of vital genetic components.

GNUPLOT visualization of genomic evolutionary dynamics

The study utilized the GNUPLOT software, which is integrated into the SWAKK server, to visually depict the selective pressures exerted across various genomic regions. The resulting graphical depiction, as shown in Figure 7, delivers an explicit and thorough knowledge of the selective forces at play.

The distinct substitutions, where the encoded amino acid was transformed, are complicated and illustrated in Table 3.

The Table 3 depicts that since the variation of the human origin from chimpanzees, there have been 13 specific amino acid substitutes in proteins that are distinctive to humans. These amino acid alternates signify modifications that have arisen at the genetic level, which may have contributed to the evolution of specific functional or structural traits in humans compared to chimpanzees.

Table 3: Represents ancestral amino acid residues and substitutions in human and chimpanzee linages.

Regions	Position	Ancestral residue	Replacement in chimpanzee	Replacement in human
Region 1	219	A		D
Region 2	343 373 377	R E E	G D	D
Region 3	882	G		D
Region 4	997 1034 1096 1129	N S N T	C K	Y S
Region 5	2012	S		L
Region 6	2161 2242	T N	A	K
Region 7	2365	I		V
Region 8	2491 2613 2635 2671	R I I G		Q V T R
Region 9	2937	Q		R

It suggests that there have been five amino acid alterations that have ensued particularly within the chimpanzee lineage. This implies that both human and chimpanzee genomes have gone through distinct evolutionary modifications since their split, resulting in the observed disparities between the two species.

Evaluating genetic diversity and neutrality utilizing Tajima’s D and Fu and Li’s D and F tests

Analysis of Tajima’s Test for the DMD Gene

The Tajima’s Test analysis operated on the DMD gene provides insights into the selective pressures affecting this genetic locus. The outcomes suggest that the DMD gene is not going through neutral selection, implying that the observed genetic mutations are probably impacted on by other evolutionary means. The analysis was conducted on an alignment file comprising coding sequences, which covered a region of 14,014 areas.

This conclusion is illustrated in the study’s Figure 8, providing a pictorial manifestation of Tajima’s D result and its importance for the evolutionary dynamics of the DMD gene.

Analysis of Fu and Li’s test for the DMD gene

The outcomes of the Fu and Li’s tests for the DMD gene are illustrated in Figure 9 These statistical tests provide helpful insights into the evolutionary patterns of the DMD gene, demonstrating explicit evidence that it has not evolved under neutral selection. Several key findings support this conclusion:

Significant negative Fu and Li’s D and F values

The Fu and Li’s D* and F* statistics, which are employed to witness divergences from neutral evolution, exhibit highly substantial negative values of -1.77525 and -1.95457, respectively, with p-values smaller than 0.02. These negative values suggest an excess of rare genetic variants in the DMD gene sequence described in relation to what would be anticipated under a neutral evolutionary model.

High level of haplotype diversity

The population under investigation displays an incredibly high level of haplotype multiplicity, as evidenced by the haplotype diversity index (Hd) being analogous to 1.000. This conclusion indicates the existence of a significant number of unique haplotypes within the studied population. Such an increased extent of genetic diversity implies the gene being subjected to selective forces, rather than just going through random genetic drift.

Excessive number of singleton mutations

The analysis demonstrates a considerable excess of singleton mutations, with 822 out of the total 847 mutations being distinctive to single individuals. The existence of a substantial number of singleton mutations implies the population has encountered somewhat recent genetic changes, potentially steered by adaptive procedures.

Nucleotide diversity (Pi)

The high haplotype diversity, overabundance of singleton mutations, and the nucleotide variation observed in the data collectively indicate that the gene under investigation is subject to selective forces, potentially driven by recent adaptive circumstances, rather than being impacted exclusively by random genetic drift. These conclusions provide valuable understanding into the evolutionary dynamics and genetic arrangement of the studied population.

Analysis of phylogenetic tree

The UTRN (Utrophin) gene comprises a distinct clade below the DMD clade with multiple species. SPTB (Spectrin Beta) gene constructs another singular clade further below the UTRN clade. Since the UTRN clade is placed between the DMD clade and the SPTB clade, this implies that UTRN is more closely associated to DMD than SPTB. These results are visually portrayed in the phylogenetic tree displayed in Figure 10.

Functional annotation analysis of candidate gene

The Duchenne muscular dystrophy (DMD) protein is a complicated and multifunctional molecule that plays a critical role in different cellular functions. Using the SMART (Simple Modular Architecture Research Tool) bioinformatics mechanism, several different functional domains and areas are determined within the DMD protein. These determined domains are thought to be accountable for the protein’s diverse functional capabilities, as they likely contribute to its structural integrity, interactions with other molecules, and overall biological actions.

Identified functional regions and domains of DMD gene

Dystrophin (DMD) protein displays a diverse array of structural and functional regions that contribute to its vital biological functions. The specified domains and functional zones of the DMD protein include:

CH (calponin homology domain)

The calponin homology (CH) region is a structural motif found at the N-terminus of the DMD protein, mainly implicated in the organization and dynamics of the actin cytoskeleton. There are two CH domains in DMD protein. One is found at position (17-117) while the second CH domain is found at position (136-235). Depicted in yellow and orange color, these areas play a vital role in the binding and cross-linking of actin filaments, which is necessary for the construction of actin-based configurations, such as bundles and networks. The existence of a predicted CH domain has also been specified in the Cdc24 protein. Cdc24 is a pivotal regulator of cell division and polarization, indicating that the CH domain may be concerned with the spatial organization and dynamics of the actin cytoskeleton during these important cellular procedures.

SPEC (spectrin repeats)

These structural characteristics are important for the proper assembly and function of the cytoskeletal network, which provides mechanical support and organization to the cell’s interior. Moreover, twenty-two SPEC domains at the N-terminus of Human DMD were found between amino acid positions (1: 343-446, 2: 451-555, 3: 562-666, 4: 722-827, 5: 833-933, 6: 942-1044, 7: 1051-1153, 8: 1160-1262, 9: 1269-1366, 10: 1468-1567, 11: 1574-1675, 12: 1682-1779, 13: 1784-1873, 14: 1880-1970, 15: 1998-2100, 16: 2107-2207, 17: 2214-2317, 18: 2473-2576, 19: 2583- 2685, 20: 2692-2801, 21: 2808-2930, 22: 2973-3039).

WW (WWP or rsp5 domain)

Illustrated in green, the domain is distinguished by the existence of two positively conserved tryptophan (Trp or W) amino acid residues, and is thus often referred to as the WWP (Trp-Trp-Pro) or Rsp5 domain. The two tryptophan residues are thought to be vital for mediating these protein-protein interactions, accentuating the importance of the unique structural architecture of this domain. WWP domain is found at positions (3056-3088) portrayed in Figure 11.

EF-hand domains (Pfam Domain)

Depicted in Red and Maroon, the EF-hand domain adopts a distinctive helix-loop-helix tertiary configuration, identical to other EF-hand motifs found in different proteins. The preliminary role of this EF-hand-like domain seems to be the provision of particularity in the distinction and binding of beta-dystroglycan, a pivotal element of the dystrophin-glycoprotein complex. This interchange enables to promote the association between dystrophin and the dystroglycan complex.

ZnF (zinc finger domain)

Displayed in purple, the zinc-binding territory found in the dystrophin protein plays a critical part in its function. The distinct “ZZ” motif within the dystrophin configuration seems to have the ability to attach to the calcium-binding protein calmodulin. This ZZ-type zinc finger field is strategically placed between a WW domain (which is flanked by EF-hand) of the zinc-binding domain in dystrophin are vital for retaining the integrity of dystrophin between the intracellular and extracellular components of the muscle, and any disturbances in this domain or its interactions can have harsh outcomes for muscle function and fitness. The ZnF domain is found at positions of (3307-3352).

Functional characteristics of structural domains

N-terminal domain

The N-terminal domain of dystrophin is found from 1-240 amino acids. The N-terminal domain encodes the dystrophin protein, which binds to F-actin in the cytoskeleton of muscle cells.

C-terminal domain

The dystrophin protein is composed of 3685 amino acids. Particularly, the C-terminal domain spans roughly from 3240-3685 amino acids. The major role of C-terminal domain is to anchor dystrophin to the muscle cell membrane by attaching to β-dystroglycan, thereby retaining muscle cell integrity and strength during contraction and relaxation.

Comparative domain analysis across different species

Analyzing the domains across different species highlights considerable conservation with some deviation in species. The results are as follows:

Putative orthologs in human DMD

The proteins from different species, such as horse (3820 amino acids), chicken (3700 amino acids), mouse (3678 amino acids), and cat (3677 amino acids), display a favorably conserved domain configuration. These orthologous proteins, which have developed from a common ancestral gene, share an incredible resemblance in their structural organization, constituting key domains such as the CH (calponin homology), SPEC (spectrin repeat), WWP (WW domain-containing protein), EF-hand 2, EF-hand 3, and ZnF (zinc finger) domains. This implies that the arrangement and structural characteristics of zebra fish protein variants vary from other organisms, potentially indicating functional variation.

Putative paralogs in human DMD

UTRN (3433 amino acids) indicates a high degree of resemblance to DMD, especially in the conservation of the CH domains and spectrin repeats, implying functional redundancy and compensation in the absence of dystrophin. SPTB (2328 amino acids) has a shorter sequence with lesser spectrin repeats and absence of WW, EF-hand 2, EF-hand 3 and ZnF domains. It reflected functional deviation where spectrin might play more restricted roles described in relation to dystrophin and utrophin.

Assessing evolutionary conservation of protein structures

PHYRE2, a computational tool was utilized to specify the three-dimensional protein configurations of DMD protein in humans Figur 13, chimpanzees, and their common evolutionary predecessor displayed in Figure 12 and Figure 14. These protein configurations are visually demonstrated below.

Comparative analysis of human and ancestral protein structures

The investigation demonstrates that while the human DMD protein and its ancestral companion share well-aligned area, there are substantial divergences in other areas, as shown by the high overall RMSD and low Q-score in Figure 15. These findings suggest that although certain regions of the proteins are structurally similar, the overall structures diverge considerably due to differences in sequence length and domain composition Figure 16.This comprehensive analysis highlights the complexity of protein structure-function relationships, where sequence similarity does not always correlate with structural congruenceshown in Figure 17 and Figure 18.

Comparative analysis of ancestor and chimpanzee protein structures

Overall, the analysis reveals a high degree of sequence similarity between both proteins with significant structural differences in Figure 20. The conserved core regions exhibit strong structural similarity, evidenced by the low RMSD of 1.122 Å in pruned areas and 1.872 Å in fully aligned columns. However, the overall high RMSD of 132.767 Å for all pairs, the SDM value of 129.018, and the low Q-score indicated substantial structural variations beyond these well-aligned areas in Figure 19 The protein comparison suggests that, despite evolutionary conservation in key functional domains, there are species-specific structural adaptations in these proteins shown in Figure 21.

Conclusion

The rapidly progressing field of genomics has facilitated extensive bioinformatics analyses across various animal species, providing valuable insights into the evolutionary affinities of genes associated with distinct phenotypic characteristics and human skeletal diseases. A meticulous analysis of the DMD gene across primates has shed light on its evolutionary dynamics and functional significance. The presence of human-specific mutations and positive selection signals, as demonstrated by the Ka/Ks ratio, implies that this gene has experienced adaptive evolution and functional transformations within the human heritage. The calculated Ka/Ks value was found to be 1.5 in humans. Further research through SWAKK analysis, Tajima’s D, and Fu and Li’s tests has yielded a negative value of -1.71219 with a p-value less than 0.05, while Fu and Li’s test produced a negative value of -1.95457, negative values for both tests indicated deviation from neutral evolution, all of which lead to an excess of rare alleles and genetic diversity at specific loci. Phylogenetic estimation of the DMD gene and its paralogs, UTRN and SPTB, has demonstrated powerful evolutionary affinities across vertebrates. The maximum likelihood method has confirmed the close evolutionary affinity between DMD and UTRN.

Recommendations

The study of the DMD gene, which is responsible for the growth and function of muscles, and its evolutionary dynamics supplies a valuable prospect to extend our understanding across various scientific domains. Investigating the distinctive mutations and adaptive evolution of the DMD gene in human sheds light on its key function in muscular disorders and skeletal development, potentially directing the development of more targeted and useful therapies. Moreover, advanced bioinformatics can enable the extension of these analyses to other genes associated with skeletal disorders and morphological characteristics across a wider spectrum of species, facilitating a more thorough awareness of the genetic signatures of these traits. Beyond the significance for human health and biology, the findings from the evolution of the DMD gene can also shape conservation methods for endangered species, supporting the maintenance of hereditary diversity and improving the strength of these species in the face of environmental challenges. For further experimental validation, a site directed mutagenesis in vitro protein expression maybe done in future to analyze structural and functional effects of DMD substitutions. Additionally, the biological relevance may also be confirmed by doing the in-vivo studies in engineered model organisms (like mouse) carrying the human-specific mutations.

Acknowledgement

I acknowledge faculty and HOD of Biotechnology department, University of Sialkot for insightful suggestion regarding the research.

Novelty Statement

The present study uniquely integrates positive selection signatures, comparative domain architecture, and structural divergence analyses to highlight candidate mutations that may have contributed to human-specific skeletal adaptations. These findings provide a basis for experimental validation, advanced structural studies and functional analysis of other novel skeletal genes.

Author’s Contribution

SN designed the methodology and written the manuscript. FS collected the data and TS assisted in result interpretation.

ZI, UM and IA contributed to manuscript editing, validation of findings, and reference management.

All authors have read and approved the final version of the manuscript.

Generative AI and AI assisted technology statement

The authors declare that no generative AI and AI assisted technology was used in the creation of this manuscript.

Conflict of interest

The authors have declared no conflict of interest.

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