A Preliminary in Silico Study on Experimental Animal Model for Hamamy Syndrome: A Rare Human Genetic Disorder

Objective : Hamamy syndrome is a recently discovered rare genetic disorder caused by a missense mutation of Iroquois (IRX5) gene. IRX-5 transcription factor orchestrates cell movement in the developing fetus which underlies head and gonad formation. It also modulates multiple organs during embryogenesis like heart, bone, blood, facial appearances. Study animals play an essential role in understanding the pathogenesis of diseases and in pre-clinical studies of drug development. Hence our study was aimed at conducting comparative genomic analysis to find suitable animal models with similar sequence for IRX-5 gene that can be used for further studies. In our preliminary study we used Bioinformatics software CLC sequence viewer 6.7 to study three parameters; multiple sequence alignment, phylogenetic relationships and protein analysis of the IRX-5 gene. Results : Comparative genomic studies of the human IRX-5 showed high degree of homology with respect to IRX-5 of P. troglodytes(99.6%), M.mulatta (99%), B.taurus (98.3%), C. lupus (97.7%), M.musculus (93.4%), R.norvegicus (94.6) and lower homologyto D.rerio(71.3%). These results further revealed a closer phylogenetic relationship of Homo sapiens IRX-5 with P. troglodytes, M.mulatta B. Taurus, C. lupus, than with M.musculus , R.norvegicus and D.rerio . Abbreviations: HGP- Human Genome Project; NCBI- National Center for Biotechnology Information; BLAST- Basic Local Alignment Search Tool; EURORDIS- European Organization for Rare Diseases; FDA- Food and Drug Administration Agency; MSA-Multiple Sequence Alignment; OMIM- Online Mendelian Inheritance in Man; OD- Orphan Disease A Preliminary Study on Experimental Animal Model for Hamamy Syndrome: A Rare Human Genetic Disorder.

two Jordanian brothers born to double first cousin parents in 2007 [7] later in Turkey with two brothers born to a consanguineous marriage [8][9]. Hamamy syndrome is caused by a missense mutation of the IRX5(Iroquois) gene which results in the formation of defective proteins that are necessary for the morphogenesis of the heart, brain, face, bone and gonads. It also contributes to the morphogenesis of secondary organs such as dental, lachrymal, salivary and thyroid structures [10]. The Iroquois (IRX) gene is a homeobox gene i.e., class of gene involved in the regulatory functions of embryonic development [6][7][8] and encodes six transcription factors which are conserved across species. The six IRX genes have been organized into two-three gene complexes i.e., IRXA which contains IRX-1, IRX-2, IRX-4 genes and IRXB which contains IRX-3, IRX-5 and IRX-6 genes [11]. The IRX-5 gene located on the long arm of chromosome 16 is involved in modulating the migration of progenitor cells in the branchial arches and gonads [8].
Clinically, Hamamy Syndrome is characterized by craniofacial dysmorphisms, including severe tele canthus, myopia, hypoplasia of the lacrimal-salivary apparatus, frontonasal anomalies, protruding ears and sensory-neural hearing impairment. Skeletal anomalies with repeated fractures of long bones, hypodontia, microcytic hypochromic anemia and congenital heart defects with intraventricular conduction delay have also been documented in all affected individuals [9]. To encourage studies focusing on the development of products to treat ODs, the Food and Drug Administration Agency (FDA) passed the 1983 Orphan Drug Act.
Within the first 25 years of passing this act in the US, 326 products which target more than 200 rare diseases have been approved.
Although this is a good start, until now therapy is available for only 5% of all the known ODs. The small market size and thus revenue of ODs, lack of a suitable animal model, lack of adequate clinical data, unknown or unclear etiology, problems in finding sufficient number of patients with the disease and scarcity of funding have been some of the problems cited into the lack of progress in the development of therapy for ODs [1][2][3][4].
Although animal models have been playing an important role in understanding human disease and therapy for common diseases, [1,[12][13] for rare genetic diseases the results weren't that much satisfactory. This is because researchers have been using animals mostly based on traditional view that is based on anatomical, physiological and pathological similarities to human and this approach usually ignores the core of all similarities i.e., genetic similarity. Hence many of the expected results did not go as they hoped. So, finding a suitable animal model is one crucial factor in the development of therapy for rare diseases [2]. The availability of an animal model provides excellent opportunities for pre-clinical studies of human genetic disorder. Comparative genomics is the direct comparison of complete genetic material of one organism against that of another to gain a better understanding of how species evolved and to determine the function of genes and noncoding regions in genomes and considered as an effective approach for identifying the genetic factors responsible for diseases and in the development of prevention and treatment strategies [14]. By identifying and studying gene ortholog across species, researchers will be able to accurately translate and apply experimental data from animal experiments to humans. These technologies can be used to create, de novo, appropriate animal model. Hence our study was aimed at finding suitable animal models which have similar IRX-5 gene for further studies on Hamamy syndrome pathogenesis.

1.
Homo sapiens IRX-5 sequence similarity search: Protein sequences of IRX-5which closely matches with Homo sapiens IRX-5were identified using BLAST toolsupported through NCBI, USA. BLAST tool is designed to identify potential homologues for a given sequenceusing a set of algorithms can be used toanalyse programs available and each of them serves for specific purpose [15].
usually presented as rows, so that aligned sequences will appear in successive columns. Whereas in case of text formats, aligned columns containing identical or similar characters are designated with specific conservation symbols. For protein sequence alignments, color sare often used to indicate amino acid properties to assist in deciding the conservation of a given amino acid substitution. In multiple sequence alignment, the last row in each column often represents the consensus sequence. In some cases, consensus sequence is also represented in graphical format with a sequence logo, where the magnitude of each nucleotide or amino acid letter relates to its extent of conservation.

b.
Phylogeny: [16] The ancestral relationships between living organisms can be denoted by using an evolutionary tree i.e., the tree of life. The tree of life characterizes the phylogeny of organisms i.e., the description of organismal roots as they change through time. By exploring the phylogeny of organisms, we can easily find out any differences that exists among plants, animals and microorganisms.
Phylogenetic and sequence alignment are interrelated fields due to the need of evaluating sequence relatedness. Phylogenetic study makes extensive use of sequence alignments in building and understanding of phylogenetic trees, which further used to determine the evolutionary relationships between the homologous genes presented in the genomes of divergent species. The degree to which sequences in a question set to differ is qualitatively linked to the sequence evolutionary distance from one another. For instance, high sequence identity indicates that the sequences in question are comparatively young and also have most recent common ancestor, while low identity suggests that the separation is more ancient.

Results and Discussion
In current scenario of medical research, comparative genomics applications has improved the outcome of genetic maps in human and other model organisms and detected many rare human disease genes in other mammalian models [17].  Table 1. is shown in Figure 2.

Conclusion
Although much has been achieved in the field of medical research through the use of traditional methods of animal model finding, but their widespread use is limited due to some inherent faults that prevent its relevance to clinical medicine [18]. As it is very crucial to use animal models, because testing of experimental hypothesis directly in human subjects is not always possible due to various ethical or logistical reasons. But with the help of comparative genomics, it is feasible to predict suitable animal models with highest precision through the detection of relationship in terms of structural and functional aspects of human genome and also the pathophysiological aspects of human disease with other animal models available. Ultimately, comparative genomics is becoming one of the most promising scientific fields today, based on its application in various field of study, we anticipate more and more exciting technological advances and important biological discoveries in the near future.

Ethics Approval and Consent to Participate
This preliminary study got Asmara College of Health Sciences (ACHS) Ethical committee clearance and also granted permission to participate in the study.

Consent for Publication
All the Authors have agreed to publish this article.

Availability of Data and Material
The data used in this study is available from the corresponding author upon reasonable request.