key: cord-1029826-ougc319c
authors: Pattan, Vishwanath; Kashyap, Rahul; Bansal, Vikas; Candula, Narsimha; Koritala, Thoyaja; Surani, Salim
title: Genomics in medicine: A new era in medicine
date: 2021-09-20
journal: World J Methodol
DOI: 10.5662/wjm.v11.i5.231
sha: 5580b0fd089d9cee0921b43e3ac15f8045cf1907
doc_id: 1029826
cord_uid: ougc319c

The sequencing of complete human genome revolutionized the genomic medicine. However, the complex interplay of gene-environment-lifestyle and influence of non-coding genomic regions on human health remain largely unexplored. Genomic medicine has great potential for diagnoses or disease prediction, disease prevention and, targeted treatment. However, many of the promising tools of genomic medicine are still in their infancy and their application may be limited because of the limited knowledge we have that precludes its use in many clinical settings. In this review article, we have reviewed the evolution of genomic methodologies/tools, their limitations, and scope, for current and future clinical application.

Understanding the human genome has come a long way since the initial discovery of DNA structure by Watson and Crick in 1953 [1] . The genome study and reference used to be a very specialized area, but lately with the advent of the messenger based RNA vaccine have brought the concept of genetics even to the lay public. In the 1970s, the ability to manipulate DNA with recombinant DNA technology increased the horizon. Our understanding of medical genetics began with inheritance patterns of single-gene diseases. The database of Mendelian Inheritance in Man (MIM) was initiated in the early 1960s by McKusick [2] . As of January 5, 2021, 4368 genes were mapped to phenotype-causing mutations [3] . However, only a small portion of diseases have a monogenic cause. The majority of the common diseases are polygenic, and elucidation of their mechanism has remained elusive.

The human genome project, which was completed in 2003, revolutionized the understanding of the human genome and served as a turning point to fast forward the genomic methodologies. However, the clinical application of findings from these genomic studies is still in its infancy. This is largely because we still have not understood or made complete sense of the available information. That is, the sequence data have been difficult to correlate to functional outcomes, making it difficult to understand the genetic basis of diseases and the complex gene-lifestyle-environment influences or their interaction. Moreover, most of the initial focus of the research had been on coding regions of DNA which comprises approximately 2% of the DNA and the knowledge about specific implications of non-coding DNA regions (98% of DNA) are largely unknown [4, 5] .

Remarkably, the human genome and the closest related species chimpanzees differ in single nucleotide alterations by a mere 1.23% and in deletions, insertions, and copy number variations by 3% [6] . In humans, the genomes of any two individuals are about 99.9% identical. However, a mere 0.1% variation allows for changes in a massive number of nucleotides because the human genome has approximately 30 billion base pairs (3.3 × 10 9 ) [7] .

In this review, we will discuss the evolution in genomic methodology, limitations, and their scope for current and future clinical application.

After the initial DNA sequencing method by Maxam and Gilbert [8] in 1977, the chaintermination DNA sequencing method developed by Sanger et al [9] in 1977 was used for the next few decades. It relied on the template DNA strand and had limited capacity for sequencing gene panels. Subsequently, with commercial production of high throughput technologies or next-generation sequencing (NGS) revolutionized the DNA sequencing by 2007 [10] . Also called as massively parallel sequencing, NGS does parallel sequencing of millions of small DNA fragments. Each DNA fragment is fixed at a unique location on the solid support. While the sample of the patient's DNA which serves as a template in NGS is amplified and fragmented, the third-generation sequencing uses single DNA molecules rather than the amplified DNA as a template thus eliminating errors from DNA amplification processes. The NGS can be used for whole-genome sequencing, exome sequencing, or targeted gene panels comprising tens to hundreds of genes.

Single nucleotide polymorphism (SNP) is the variation in genetic sequence by a single nucleotide. It is the most common type of genetic variation in man [11] . It was detected in the 1980s using restriction enzymes [12] . With application of the microarray technology to SNPs, the scope of SNP in clinical practice has widened, especially in oncology. The first SNP array analysis was done in 1998 and the first application of SNP array analysis in cancer was done in 2000 [13] . SNP array analysis is used to determine loss of heterozygosity, allelic imbalance, genomic copy number changes, frequency of homozygous chromosome regions, uniparental disomy, DNA methylation alterations and linkage analysis of DNA polymorphisms in cancer cells [13, 14] .

Kary Banks Mullis successfully demonstrated polymerase chain reaction (PCR) in 1983 [15] . PCR is a cost-effective method that can amplify a single DNA exponentially [16] . It is a rapid, highly specific, and extremely sensitive method. PCR is being used in SNP genotyping, detection of rare sequences, insertion-deletion variants, and structural variants like copy-number variants. useful. In genome-wide association studies (GWAS), genotyping of hundreds or thousands of SNPs is done in cases and control populations and their association with heritability is analyzed. A combination of linkage and association methodologies helps to identify and characterize the wider range of disease-susceptibility variants [22] . Fluorescence in Situ Hybridization (FISH) was developed in 1987. It is a cytogenetic technique which uses fluorescent DNA probes which are designed to label precise chromosomal locations. The advantage of FISH over conventional cytogenetic metaphase karyotype analysis is lack of cell culture requirement. It can rapidly evaluate interphase nuclei in the fresh or paraffin-embedded sample [23] . However, the resolution of this technique is only as good as that of karyotype bands. Cloned DNA FISH probes of about 100 kb, called bacterial artificial chromosomes, are now available. FISH is being utilized more in making clinical diagnosis among Oncology due to its simplicity and reliability to evaluate the key biomarkers in various malignancies.

Comparative genomic hybridization (CGH) was developed in 1992. CGH can detect DNA copy number changes across the entire genome of a patient sample in a single experiment. It compares the hybridization signal intensity of a test sample (for example tumor sample) against a reference sample along the chromosomes[13].

The HapMap project was started in 2002 to develop a haplotype map of the human genome. It can also describe the common patterns of human genetic variation [24] . The 1000 Genomes Project comprised a total of 26 diverse population set in which wholegenome sequencing was performed. It also used deep exome sequencing and dense microarray genotyping to give a comprehensive description of common human genetic variation[25].

It involves modification of the genome at a precise, prespecified locus using programmable nucleases. Examples of some of the programmable nucleases include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPRassociated) system. These programmable nucleases are designed to impart site-specific double-strand breaks (dsBs) in chromosomal DNA. The cell is therefore forced to use one of the endogenous DNA repair mechanisms -homologous recombination or homology-directed repair (HDR) and nonhomologous end-joining (NHEJ). This enables targeted genetic modifications during the repair process in the living cells (in vivo) (Table 1) In the year 2013, Cong et al [27] and Mali et al [28] showed successful genome editing in mammalian cells using the CRISPR system. In the last 5 years, we have seen a leap in the research interest (both animal and human) in CRISPR genomic editing.

While genome editing holds promise to correct the defective genome in vivo, therapies can also be designed to alter the gene expression without altering the genomic code. For example, anti-sense oligonucleotide can be used to alter the splice points of pre-mRNA to correct for a defective gene or suppress its expression. Examples of drugs which use splice modulation and approved by Food and Drug Administration (FDA) are Eteplirsen (exon skipping, approved for Duchenne muscular dystrophy) and nusinersen (exon inclusion, approved for spinal muscular atrophy) [29] . Table 1 summarizes the commonly used genomic tools, their working principle, advantages/applications and limitations (see Table 1 ). Table 2 summarizes the major genome/gene editing tools their working principle, advantages/applications and limitations. Table 3 summarizes gene-based therapies that are either FDA approved therapies or investigational therapies showing promise. Epigenetic modulators (e.g., DNA methylase) can also be fused with dCas9 to achieve controlled epigenetic modulations. Cas-9 NHEJ is simpler and efficient; Cas-9 HDR is more precise but lower efficiency than NHEJ. The mutant version of the Cas9 called Cas9 nickase can be used to minimize the risk of offtargets 

The newer genomic technology and tools have broadened the scope and pushed the time limits for development of new diagnostic kits, preventive strategies like vaccines, therapeutic strategies like gene modulation and gene therapy. A lot is yet to be studied in terms of the complex interaction of gene-environment-lifestyle-disease. Knowing the impact of genomics on disease pathophysiology and response to medications [30] . expands the scope of research and clinical application. While genome editing holds promise to correct the defective genome in vivo, therapies can also be designed to alter the gene expression without altering the genomic code (example exon skipping, or inclusion discussed above). The newer genomic editing tools have showed great potential and promise but they need to be studied extensively before clinical application. Also, uniform international ethical guidelines and guiding principles need to be established so that these genomic technologies are not misused. September 20, 2021

Volume 11 Issue 5 It is very important to include diverse populations and to represent minority population in the genomic studies, so that results could be generalized and more accurate diagnostic, predictive and therapeutic tools can be developed.

Genome Editing for Rare Genetic Diseases Without Double-Strand Breaks or Donor DNA

Search-and-replace genome editing without double-strand breaks or donor DNA

Use of Zinc-Finger Nucleases for Crop Improvement

Engineering and Application of Zinc Finger Proteins and TALEs for Biomedical Research

Combining Engineered Nucleases with Adeno-associated Viral Vectors for Therapeutic Gene Editing

Fact Sheet for Healthcare Providers Administering Vaccine (Vaccination Providers): emergency use authorization (EUA) of the Janssen COVID-19 vaccine

COVID-19 Vaccine Use Following Thorough Safety Review

Gruber WC; C4591001 Clinical Trial Group. Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine

Fact Sheet for Healthcare Providers Administering Vaccine (Vaccination Providers): Emergency use authorization (EUA) of the Pfizer-BioNTech COVID-19 vaccine to prevent coronavirus disease 2019 (COVID-19)

Pfizer-BioNTech COVID-19 Vaccine

Beigel JH; mRNA-1273 Study Group. An mRNA Vaccine against SARS-CoV-2 -Preliminary Report

Fact Sheet for Healthcare Providers Administering Vaccine (Vaccination Providers): Emergency use authorization (EUA) of the moderna COVID-19 vaccine to prevent coronavirus disease 2019 (COVID-19)

Moderna COVID-19 Vaccine

An Investigational RNAi Therapeutic Targeting Glycolate Oxidase Reduces Oxalate Production in Models of Primary Hyperoxaluria

Viltolarsen for the treatment of Duchenne muscular dystrophy

Advances in chimeric antigen receptor T cells

Golodirsen: First Approval

Givosiran: First Approval

Onasemnogene Abeparvovec: First Global Approval

Inotersen Treatment for Patients with Hereditary Transthyretin Amyloidosis

Safety and efficacy of axicabtagene ciloleucel in refractory large B-cell lymphomas

Tisagenlecleucel in Children and Young Adults with B-Cell Lymphoblastic Leukemia

Nusinersen for the treatment of spinal muscular atrophy

Eteplirsen in the treatment of Duchenne muscular dystrophy

Talimogene Laherparepvec (T-VEC) and Other Oncolytic Viruses for the Treatment of Melanoma

Pfizer and Sangamo Announce Updated Phase 1/2 Results Showing Sustained Factor VIII Activity Levels and No Bleeding Events or Factor Usage in 3e13 vg/kg Cohort Following giroctocogene fitelparvovec (SB-525) Gene Therapy

Clinical implications and outcomes of the ORION Phase III trials

Volanesorsen and Triglyceride Levels in Familial Chylomicronemia Syndrome

CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia