**6. Future perspective**

CRISPR/Cas9-based therapy, are been used as a candidate to be administered systemically, via intravenous infusion, for precision editing of a gene in target tissue in humans [85]. Similarly using this technology, gene therapy was developed to treat the rare neurodegenerative condition, Dopamine Transporter Deficiency Syndrome (DTDS) using a personalized approach with a view to counter the exact genetic fault present in a patient's neurons [85]. Using a novel approach where, skin cells from patients, turned into pluripotent stem cells in the laboratory with the aim to get neuronal cells with the disease-causing mutation. A vector carrying adenoassociated virus gene therapy was created to target the neurological fault and its efficacy was tested in both neuronal human cell lines and a mouse model, with the corresponding loss of function mutations in *SLC6A3* [85]*.* This research ingenuity/ approach has provided, promising results leading to some clinical trials that may put an end to this cruel disease.

DTDS is an area of unmet medical needs and the disease is also known as infantile parkinsonism-dystonia, due to it having neurodegenerative and movement symptoms similar to Parkinson's disease [85]. It is a very rare inherited condition known to affect around 50 children around the world. Although this might be due to under-diagnosis by clinicians bearing the symptoms are similar to other inherited movement disorders e.g., cerebral palsy [85].

Environmental factors are implicated in the formation of ROS affecting human health by directing epigenetics signature of the genome, such could also drive the addition of methyl group (▬CH3) to some nucleotides neighboring guanosine (CpG islands) of the genome. These are areas where drug repurposing becomes essential as they could target methylation processes which are amongst, inherent biochemical/epigenetics machinery of cells, containing necessary pathways that allow environmental agents to induce mutations. Bearing these epigenetic signatures play a significant role in genomic balance, they play a leading role in several diseases hence are the essential target for drug repurposing.

Many diseases present some inherent opportunities via epigenetics markers that required intelligent manipulation of phyto-compounds to access new therapy that is efficient and easily accessible. Phytochemicals are known to play vital roles in preventing oxidative stress with concomitant damages [2, 85]. At the cellular and molecular level, they inactivate Reactive Oxygen Species (ROS). And under specific low concentration, inhibit or delay oxidative processes by interrupting the radical

chain reaction of lipid peroxidation [2, 86]. Bioactive components with anti-oxidative capacity naturally present in food are of great interest due to their beneficial effects on human health as they offer protection against oxidative deterioration.

DNA processes such as replication, transcription, recombination, and repair are, known to be facilitated by several factors covered in this chapter and others such as supercoiling that help facilitate both the packaging of DNA and many fundamental genetic processes that enabled the enzymatic manipulation of DNA. Aberrant RBP-RNA interactions are now known to promote disease progression, as much as mutations in TFs. RBP's role in disease was initially understudied because of their systematic evaluation was limited by, lack of sensitive and efficient assays for phenotypic interrogation of individual RBPs.

There is profound evidence that suggests, consumption of food rich in phytochemicals may progressively reduce the risk of different diseases by modulating immune-inflammatory markers [87]. Using the combination of disparate molecular/biophysical tools we recently [88], compared the binding affinity of artesunate and azadirachitin to gephyrin E this is towards enabling insights into natural bioactive compounds useful for rational drug design, essential in the race to manage myriad of disease phenotypes. The results from our research and others are necessary as they, may provide, the impetus for more studies into bioactive components of plant origin towards the effective management of different disease phenotypes.

#### **6.1 Next-generation sequence in disease diagnosis**

Next-generation sequencing (NGS), is a massively parallel and a high-throughput DNA sequencing technology that enables the fast generation of data on thousands to millions of base pairs of DNA from an individual patient by sequencing large numbers of genes in a single reaction [89]. NGS can sequence millions of DNA fragments in a massively parallel fashion, instead of sequencing a single DNA fragment one at a time, as observed in traditional capillary electrophoresis sequencing. The general workflow of NGS includes four main steps:

I.library preparation,

II. cluster generation,

III. sequencing, and

IV.data analysis.

Sequence reads are produced from fragment libraries, a pool of adaptor-ligated and enriched DNA fragments. One advantage is that a small quantity of DNA, from a patient, is needed to produce a library.

In step 1, patient DNA is randomly fragmented by different methods and then prepared for sequencing by ligating specific adaptor oligonucleotides to both ends of each DNA fragment. Adapter-ligated fragments are further enriched with specific oligonucleotides designed for the target genes included in the NGS panel and are then amplified by polymerase chain reaction (PCR). The prepared library is loaded into a flow cell for cluster generation and subsequent sequencing.

During sequencing, short read lengths (35–250 bp, depending on the platform) sequences that are produced are then aligned to a reference genome with bioinformatics software [89].

*Recent Progress in Drug Repurposing Using Protein Variants and Amino Acids in Disease… DOI: http://dx.doi.org/10.5772/intechopen.102571*

During data analysis, variant calling can be achieved by various standard and in-house analysis pipelines. All detected variants are checked against standard databases (e.g., dbSNP137, 1000 Genomes Project, Exome Variant Server, ExAC Browser, OMIM catalog, ClinVar, Human Gene Mutation Database) to enable interpretation of the pathogenicity of a given variant.

Next-generation sequencing panels are now commonly used in clinical diagnosis to identify genetic causes of various monogenic disease groups, such as epilepsy [90], intellectual disability [91, 92], neurodevelopmental disorders [93], neurometabolic disorders [94], amongst others.

The use of NGS in clinical laboratories is increasing, with application in the diagnosis of immune disorders, infectious diseases, human hereditary disorders, in non-invasive prenatal diagnosis, and recently, in the therapeutic decision making for somatic cancers [95, 96].

Today two different NGS technologies are mainly used in clinical laboratories: Ion Torrent and Illumina systems [97].

The Ion Torrent exploited the emulsion PCR using native dNTP chemistry that releases hydrogen ions during base incorporation by DNA polymerase and a modified silicon chip detecting the pH modification [98], while Illumina technology is based on the existing Solexa *sequencing by synthesis* chemistry with the use of very small flow-cells, reduced imaging time and fast sequencing process [97].

#### **6.2 Usefulness of NGS**

NGS approaches will remain useful because:

1.It is highly accurate and cost-effective.

2.It has a wide application for use in clinically heterogeneous inherited disorders, resulting in an increase in the number of reported disease-causing genes.

NGS is appealing when there is a genetic contribution in heterogeneous and complex diseases, such as in cardiomyopathies, in cardiac arrhythmias, in connective tissue disorders, in mental retardation or autism, and where a large number of genes are involved in a large phenotypic syndrome [99, 100]. In these cases, NGS approaches allow us to test a large number of genes simultaneously in a costeffective manner [101].

Two options of NGS are currently available [101]:


Targeted sequencing is applicable for genetic disorders, such as non-syndromic deafness [98], common diseases, such as hypertension and diabetes [102], or in traditional cytogenetic and Mendelian disorder diagnosis [103]. The main limitation of targeted sequencing is the rigidity of testing only a selected number of genes. Since the genetic field is rapidly evolving, new genes may be associated with a clinical phenotype, and as such redesigning and revalidation of the panel is needed [101].

The WES application could be applicable for the identification of genes responsible for the dominant Freeman-Sheldon syndrome, the recessive Miller syndrome, and the dominant Schinzel-Giedion syndrome [104]. The shortcoming of WES is

that about 10% of targeted bases sequenced in WES do not get the 20 read depth [105], required for clinical confidence and interpretation, and approximately only 85% of genes associated with human diseases into the principle database (OMIM) receive the adequate coverage [106].

## **6.3 Challenges of NGS in disease diagnosis**

In the NGS process, one limiting step is the complexity of genetic variation interpretation in whole-exome, due to the presence of thousands of rare single nucleotide variations without pathogenic effect. Moreover, in the majority of human diseases, the pathological phenotype may be caused by a pathogenic rare mutation with a strong effect or it may be caused by a co-presence of multiple genetic variations [107].

Another important challenge of the use of the NGS approach in clinical diagnostic is the management of the amount of data generated [108]. Indeed generation, analysis, and also storage of NGS data require sophisticated bioinformatics infrastructure [109], which could be capital intensive.

A skilled bio(chem)-informatics staff is needed to manage and analyze NGS data, therefore increasing the impact of computing infrastructure and manpower on costs of NGS applications in clinical diagnostics [110, 111].
