From the Double Helix to the Personal Genomes

Abstract

In 1954, the seminal short paper in the Nature from James Watson and Francis Crick proposed the double helix model of deoxyribose nucleic acid. This amateur-looking concept changed the way molecular biology is now perceived. We are now celebrating the 70 years of the phenomenon discovery of the modern science that has revolutionized life sciences with a huge impact on many other related scientific disciplines. Fifty years later, the Human Genome Project offered the nearly complete draft sequence of the human genome that is now fully sequenced in the 70^th year of the double helix. Technological advances have led to a rapid reduction in the cost of whole genome sequencing. It is now possible for anyone to get the personal genome sequenced for <US$500! The investigative field of human genetics has evolved into promising field of clinical genomics and genomic medicine. By far the major impact of human genome sequencing is evident from precision medicine and healthcare. Most common and rare disorders of unknown etiology can now be diagnosed and deciphered with precision using one of the next-generation genome sequencing techniques. Wide-ranging precision genome diagnostic opportunities now exist along with precision and personalized therapeutic options. In addition, precise personalized preventive healthcare is now possible for healthy people with a family history of monogenic/Mendelian disease or common complex medical conditions. The technology permits us to have the whole genome sequenced in a new born baby or anyone else of any age to assist with long-term preventive health and life style planning and necessary precautions.

INTRODUCTION

Charles Darwin, the famous biologist of generations, referred to hereditary factors in his seminal book “On the Origin of Species.” However, it took several years before biologists and others working on different aspects of life sciences could properly conceptualize the shape and location of these so-called hereditary factors. Gregor Mendel’s seminal simple garden pea cross-over experiments and wealth of observations probably offered the first physical evidence for Darwin’s theory. Following a series of deliberations and research, most scientists agreed on the nucleus as the possible location of the heredity. Biological chemists were convinced that nucleic acids should have the key role in heredity. As soon as the term “gene” became the common reference for hereditary factors, intense work began on the precise location, structure, and function of genes. The focus was largely on nucleic acids-the ribose nucleic acid (RNA) and the deoxyribose nucleic acid (DNA). Historical archives describe several models of structure and function that were proposed, investigated, and eventually discarded.

This article provides an overview of the genes, genetics, and present-day genomics, all fundamentally linked to the structure of DNA. The focus is largely on its impact on clinical and healthcare applications with particular reference to personal genomics.

THE WATSON-CRICK DEOXYRIBOSE NUCLEIC ACID MODEL

In the early stages of research and development, the preferred model for the DNA was considered to be the triple helical molecule. It was almost impossible to agree on the basic requirement of the gene splitting into two halves and then reuniting again at conception.

The molecular biology research inevitably suffered during the second World war. The momentum in this field started rising again soon after the War. The team at King’s College, London (England), led by the French biologist Dr. Rosalind Franklin, began to study the bio-physical characteristics of the DNA molecule. Dr. Franklin, an expert in X-ray crystallography, developed very impressive pictures of the bovine calf muscle DNA. Whilst she argued the double-strand structure of the DNA, she could not convince fellow colleagues of its biological significance. The X-ray films and photographs were left unattended for some time. Her work, deciphering the structure of the DNA stopped. However, her experimental X-ray crystallography pictures prompted James Watson and Francis crick to develop the model. Unfortunately, Dr. Franklin failed to receive the credit. Many of her enthusiasts and admirers call her the “Forgotten Queen of the DNA”!^[1]

Both James Watson and Francis Crick, working as junior research fellows in the Cavendish Laboratory, at the University of Cambridge, had an entirely different line of research but soon became close friends arguing over basic molecular biology. However, they had a lot of disagreement on the structure of cell nucleus, nature of genes, and differing views on genetics. Nevertheless, they found a common ground to unravel the mystery of the nucleic acids. They agreed to include four nucleotides – adenine, thymine, cytosine, and guanine. The preliminary prototype was not acceptable to many scientists. The main gap was the “unclear and nonfunctional linking of the nucleotides.” Further work and minor adjustments to the nucleotides links were made. The revised model should be final and the decision was made to report to the scientific world [Figure 2]. A very short report was drafted and the Director of the Cavendish Laboratory traveled to London to discuss it with the Editor of Nature. The editor instantly agreed but suggested to invite the King’s College Laboratory Chief, Frederick Hugh Maurice Wilkins, to produce a report on their work as the only evidence to complement the hypothetical double helix model of the DNA. Both reports were published in the Nature in April 1953. Both scientists humbly accepted that “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.”

Figure 1: The Double Helix model of the DNA-note spiral arrangement of adenine-thymine (A-T) and cytosine-guanine (C-G) nucleotides or bases along the double helical sugar phosphate backbone

Figure 2: The triple-nucleotide genetic code along with amino acid selection for the peptide molecule

The double helix concept and model of the DNA molecule was initially not appreciated but was later accepted following the confirmation and replication by several biology laboratories from worldwide [Figure 1]. All biology students universally are now expected to know the four bases (nucleotides), arranged in A-T and C-G pairs, held together with the sugar-phosphate double helix. The true era of real genetics began with this discovery! The 1962 Nobel Prize in Medicine and Physiology was jointly conferred on James Watson, Francis Crick, and Maurice Hugh Frederick Wilkins “for their discoveries concerning the molecular structure of nucleic acids and its significance for information transfer in living material.” The omission of Dr. Rosalind Franklin’s name was noted by several people. Unfortunately, at the time of the Nobel Prize award decision in 1962, she had passed away in 1958 at the age of 37! Her omission remains a subject of discontent and deep resentment among many scientists, particularly the Women scientists all over the world. Brenda Maddox 2002 described this historical error in her book “Rosalind Franklin-The Dark Lady of DNA”. Even today, after 70 years, this episode remains a contentious subject and quoted as an example of criminal and unethical act in the scientific research. The Nobel Awards committee later accepted this gap but defended that the Nobel Prize was not conferred posthumously.

THE GENETIC CODE

Following the acceptance and applications of the double helix DNA structure, the focus turned toward understanding the arrangements of nucleotides or bases. It was unclear how the position of base pairs, A-T and C-G, might determine the function, particularly in the construction of the gene and subsequent process of transcription in the peptide molecule synthesis. This led to many technological advances and several laboratories started working in this novel field of molecular biology research.

In early 1960, the group at the University of Wisconsin, led by Dr. Har Gobind Khorana, developed a set of three nucleotides while analyzing the E. Coli gene. This triple nucleotide set was later identified specifically to a particular amino acid involved in the synthesis of the peptide molecule. At the same time, two other NIH scientists, Nirenberg and Matthaei, also produced similar results. Thus, the triple-nucleotide genetic code was invented [Figure 2].^[2,3] The genetic code laboratory techniques have been replicated in many laboratories with identical results.^[4] Following the genetic code, Dr. Khorana pioneered the technique of synthesis of the gene that laid the foundation of functional genomics.^[5]

FROM CHROMOSOMES TO DEOXYRIBOSE NUCLEIC ACID DIAGNOSIS

While the search for the structure of DNA was actively pursued, cell biologists made significant advances in sorting out the role and structure of chromosomes. This nomenclature simply implied “colorful threads” like components located in the cell nucleus. It was soon confirmed that this appearance was only seen in the eukaryotic cell since the nuclear material was dispersed in the cytoplasm of the prokaryotic cell. It was also noticed that the chromosome unit, the chromatid, was held in place around histones as tight helical fiber. As soon as the double helical structure of the DNA was accepted, the chromatid was shown as the multiply super coiled arrangement of nucleotides [Figure 3].

Figure 3: Organization of the genome-from single base pair (A-T Or C-G) to multiple super coiled chromatids of a eukaryotic chromosome

In the early twentieth century, there was lot of interest in principles of heredity or inheritance. It was fuelled by the rediscovery of Mendelian laws of inheritance and the successes of cytologists elucidating chromosomes. Thus, two major concepts of genetics, particularly heredity, became the main area of focus for biologists and related life sciences scientists. There were many reports on the possible numbers and constitutions of human chromosomes. Earlier work by Theophilus Painter in 1923 showed possible 48 diploid numbers in humans.^[6] It was based on the analysis of paraffin block embedded testicular tissue obtained from an institutionalized male. Other reports did not replicate this finding. It was later agreed that the institutionalized male probably has a numerical chromosomal condition. Later, Albert Levan, working in the same area demonstrated 46 chromosomes. This finding was finally confirmed in 1956 jointly with Tjio and Levan.^[7] The classic paper included summary data from 261 unique chromosome spreads obtained from 22 different cell cultures of fetal lung tissue. All of the cultures were used within a few days after the tissue was obtained, thus minimizing the possibility of long-term culture-induced errors of chromosome number. The results were both clear and replicable. Tjio and Levan confessed that they were surprised to find that the chromosome number 46 predominated in the tissue cultures from all four embryos. However, the numbers did not match in a few singe cases. Several scientists and laboratories were able to replicate their findings. Thus, towards the end of 1960, the double helix model of the DNA and 46 human diploid chromosome numbers were confirmed.

Confirmation of the diploid human chromosomes was greeted by the golden era of human cytogenetics. The cytogenetic analysis became an essential genetic tool investigating a range of human disorders, notably children with mental retardation. Amongst the initial reports, the report of extra chromosome 21 chromosome in the Dr. Down’s case of “Mongolian idiocy” led to the confirmation of Down syndrome as numerical chromosome aberration. Similar reports followed and we now have several distinct human conditions with deviation from 46 chromosomes, referred to as aneuploidies, such as trisomy 18 (Edwards syndrome), trisomy 13 (Patau syndrome), and monosomy of the X chromosome (Turner syndrome).

Following the elucidation of numerical chromosomal syndromes, the focus shifted on children with physical and developmental difficulties who were otherwise shown to have normal 46 diploid numbers. As soon as the quality of cytogenetic preparations improved and with the advent of G-banding technique, it became possible to analysis each chromosome with better resolution. This remarkable development led to the classification and nomenclature of chromosomes.^[8] In several cases, structural chromosome aberrations were shown, for example, ring X chromosome in a severely retarded girl with Ullrich-Turner syndrome.^[9] Several reports led the better understanding of structural chromosome changes-deletion, duplication, and inversion (paracentric or pericentric). Whilst most of these resulted from errors in the post-zygotic mitotic cell division, a small number were shown to result from meiotic errors during the gamete formation in a normal parent with a chromosome rearrangement or translocation. The normal and healthy parent apparently had a balanced chromosome rearrangement involving two chromosomes. The meiotic error could result in either loss (deletion) or gain (duplication), for instance, it was shown that a small number of Down syndrome had an additional part of the chromosome 21 resulting from meiotic recombination involving chromosomes 14 and 21, the phenomenon of the Robertsonian translocation.^[10] There are now several other similar reports involving short arm (p), long arm (q), telomeres (tel), and centromeres. In most cases, the structural chromosome aberrations are named on the basis of precise location, such as 4pdel and 5pdel. However, some syndromes are eponymously named after the person or patient of the initial report, for example, Wolf–Hirschhorn (4pdel), cri du chat (5pdel), Williams–Beuran (7pdel), Langer–Giedion (8qdel), Smith–Magenis (17pdel), and Alagille (20pdel). Rapid improvements in the cytogenetic techniques, particularly the prometaphase banding, contributed to the elucidation of several microdeletion syndromes. The European chromosome abnormalities database includes all numerical and structural chromosome aberrations syndromes.^[11]

With the success of G-banding and pro-metaphase banding, the cytogenetic applications in biological research and clinical medicine reached to a very high level. Many other techniques for much better visualization of the whole or parts of the individual chromosome were introduced, tried, and discarded. Amongst these attempts, the technique of fluorescence in Situ Hybridization (FISH) looked more promising and very attractive. It allowed the laboratory scientist to paint and demonstrate the structure of a specific part of the chromosome. FISH is essentially a molecular technique that allows the localization of a specific DNA sequence in a small part or the entire chromosome in a cell.^[12] It is utilized in chromosomal localization, gene mapping, diagnose of genetic diseases, and identification of chromosomal abnormalities. It may also be used to study comparisons amongst the inter-species chromosome or gene arrangements.^[13] FISH involves binding the DNA of all probes attached to a fluorescent molecule with a specific sequence of sample single strand DNA.^[14]) The final product can be visualized under the fluorescent microscope. In addition to the specific DNA sequence of interest, a standard probe is used to identify the normal chromosome; for instance the normal 22 chromosome is highlighted to demonstrate the deleted chromosome 22 in a patient with the22qdeletion syndrome or Di George syndrome^[15]). While the FISH technique is now replaced with other sophisticated molecular methods, it remains of interest in clinical genetics and genetic counseling as pictorial evidence for clinic and patient records.

Perhaps the best evidence of the DNA double helix is provided by the technique of genomic hybridization in which two separated single strands of similar DNA sequences can be joined together, however of different cellular origin. Arrays of DNA sequences in a specific part of chromosomes can be used to identify precise chromosomal location. The revolutionary technique of array comparative genomic hybridization (cCGH), also referred to as the microarray chromosome analysis, has replaced both conventional and molecular cytogenetics as the first line of genetic investigation.^[14] However, there are exceptions where this method might not be appropriate in certain situations such as chromosome rearrangements, chromosomal mosaicism, and complex structural aberrations involving multiple chromosomes.

The technique of aCGH or chromosome microarray analysis involves using a single DNA strand from the patient sample or any other source of interest. The single-strand DNA is hybridized with reference DNA strands obtained from the library of normal DNA strands. The microarray slide is digitally visualized. Any deviation or gaps in the DNA hybridization can be seen using the computational software. The deviations are typically loss (deletion) or gain (duplication) [Figure 4].^[16]

Figure 4: Diagrammatic of the chromosome microarray analysis (CMA) in prenatal diagnosis (Courtesy-Hong Kong J Gynaecol Obstet Midwifery 2019;19(2):114-22 | https://doi.org/10.12809/hkjgom.19.2.08)

MENDELIAN GENETICS AND GENOMICS

The status of the Austrian Monk, Reverend Gregor Mendel, is probably higher than most biologists and geneticists of several generations. His seminal experiments on various garden pea plants and seeds laid the foundation of key principles of heredity. His observations, methodical randomization and superb mathematical analysis remain unchallenged and replicated in several different approaches. Mendel’s laws of inheritance set the stage for modern genetics and provided evidence for the existence of genes and related elements governing heredity and traits. In contrast to Darwin and Wilkins theory of natural selection for evolution, Gregor Mendel’s work provides firm evidence of traits selection based on the existence and segregation of recessive and dominant alleles. An interested reader can find many sources for details on Mendel’s experiments and impact on the modern biology.^[17]

The core Mendelian genetics is related to three laws of inheritance:^[18]

1. Law of Dominance – The first law of inheritance implies that the hybrid offspring will only inherit the dominant trait in the phenotype. The alleles that are suppressed are called the recessive traits while the alleles that determine the trait are known as the dominant traits
2. Law of Independent Assortment – The second law of inheritance states that a pair of traits segregates independently of another pair during gamete formation. Thus, different traits get equal opportunity to occur together
3. Law of Segregation – The third law of inheritance states that during the production of gametes, two copies of each hereditary factor segregate so that offspring acquire one factor from each parent. Thus allele, an alternative form of the gene, pairs segregate during the formation of gamete and re-unite randomly during fertilization.

Based on the Mendel’s laws of inheritance, the analysis of phenotype traits and the segregation pattern in families led to the concept of recessive and dominant diseases. In the recessive inheritance, single copy of the allele (heterozygote) is not considered sufficient to result in a specific trait unless joined by another similar allele (homozygote) or another allele of the same gene (compound heterozygote). In contrast, the dominant single copy of the allele (heterozygote) is sufficient to result in the phenotype trait. Following the distinction between autosomes and X chromosomes, terms such as autosomal recessive, autosomal dominant, X-linked recessive, and X-linked dominant were introduced and firmly established as non-chromosomal patterns of inheritance.)

It soon became apparent that in several instances, phenotype traits did not conform to the Mendelian patterns.^[19] The concept of polygenic inheritance combined with nongenetic or acquired environmental factors emerged. This formed the basis for multifactorial/polygenic inheritance, specifically for isolated congenital anomalies and the most common complex diseases.^[20] Following the cell biological confirmation of mitochondrial genes, the matrilineal mitochondrial inheritance was recognized. The mitochondrial genetic diseases either result from mutations or polymorphisms within the mitochondrial genome or associated with several nuclear genes.^[21,22] The recognition of uniparental disomy, gene methylation changes, and genomic loci regulating the gene function laid the foundation of epigenetics and epigenomics.^[23] The concept of nontraditional inheritance is based on the evidence for added influence on phenotype traits related to epigenetic factors and structural genomic alterations (single nucleotide polymorphisms [SNPs], insertion-deletions (InDels), trinucleotide (triplet) repeats, and copy number variations (CNVs).^[24]

DIAGNOSTIC GENETICS AND GENOMICS

Compared to the cell biology and cytogenetic techniques, methods for DNA analysis developed at slow place and faced many obstacles. Initial hurdles of DNA extraction from various sources were soon overcome. The main success was achieved with the advent of restriction enzymes that were sensitive to specific base-pair sites and managed to produce variable-sized DNA fragments spread across all chromosomal loci. Some of these happened to be located within the gene and thus accepted as a useful biomarker. These restriction enzymes were harnessed from a wide range of microbial sources. For many years the technique of restriction fragment length polymorphisms (RFLPs) occupied the space in DNA diagnostics. Many other laboratory techniques followed, such as denaturing gradient gel electrophoresis, single strand conformation polymorphisms, variable number tandem repeats, mini or micro satellite polymorphisms, trinucleotide repeats, and may others.^[25] These were used in specific genetic diagnosis and as well as sequencing. The technique of Prof. Ed Southern allowed scientists to save DNA fragments from these methods, popularly referred to as Southern blotting. This technique allowed scientists to carry out laborious large-scale linkage studies for gene mapping, in particular the positional cloning approach.^[26] Needless to add, these techniques also found a place in forensic science with the precision personal identification tool of the DNA fingerprinting pioneered by Sir Alec Jaffrey in Leicester, England.

Undoubtedly, by far the most significant advancement in the DNA laboratory analysis came from the invention of polymerase chain reaction or PCR. In 1985, the California scientist, Kary Mullis, invented this method in which a small amount of DNA can be copied in large quantities over a short period. By applying heat, the double-stranded DNA molecule is separated and the DNA building blocks that have been added are bonded to each strand. With the help of the enzyme DNA polymerase, new DNA chains are formed and the process can then be repeated. PCR has been of major importance in medical research, medical diagnostics, and forensic science. The PCR method allowed harnessing large amount of DNA segments of choice thus facilitating specific disease gene analysis or DNA sequence in selected sections of the genome.^[27] The 1993 Chemistry Nobel Prize was conferred on Kary Mullis for his ground breaking work.

Specific gene analysis

Following the successes of cytogenetics, the field of molecular genetics made huge progress from mid-1980s onwards. Major advancements in the molecular genetic methods were rapidly utilized in specific gene analysis and multiple gene analysis. The PCR alone made this possible, specifically the technique of multiplex-PCR. By far the best example of multiplex-PCR application is the precision genetic diagnosis, specifically in prenatal diagnosis.^[28,29] In addition, this method is used in the diagnosis and managing acute microbial infections.^[30]

Most diagnostic molecular genetic laboratories use multiplex-PCR as an essential tool. There were two specific areas – variable phenotype spectrum (clinical heterogeneity) associated with single gene or multiple genes closely related to disease molecular mechanisms (molecular heterogeneity). Another challenge was numerous alleles associated with single gene (allelic heterogeneity). The initial focus was naturally on common Mendelian genetic diseases, particularly common in certain ethnic communities, for example, inherited hemoglobinopathies; beta-thalassemia in the Mediterranean and north western Indian subcontinent and sickle cell disease in western Africa], cystic fibrosis (north Europeans), G6PD (Arabs and Middle East), Tay-Sachs disease (Jewish people), and others.^[31] Gene-specific databases soon emerged with information on different mutations (alleles) correlated with the gene product and clinical manifestations.

In addition to common autosomal recessive disorders, the precision genetic counseling required molecular diagnosis in other Mendelian disorders. The advent of dystrophin gene in the X-linked debilitating disease Duchenne muscular dystrophy (DMD), allelic to Becker muscular dystrophy, led to ground breaking and bench mark clinical genetic practice managing inherited muscle diseases. In some cases, further research has led to specific therapy, specifically the anti-sense oligonucleotide model for the incurable DMD.^[32,33]

Gene panel testing

Extensive research in Mendelian genetic diseases unravels several examples where similar phenotypes were in fact part of the variable clinical spectrum with overlapping clinical manifestations. Many novel genes were discovered with closely related molecular mechanisms. The concept of “gene-molecule family” was universally agreed, for example, malformation-neurodevelopmental delay syndromes related to RAS-MAPK-MTOR pathway (RASopathies), arterial-aortic dilatation syndromes due to TGFß pathway genes (TGFbetapathies), and transcription factors related diseases (Transcriptionpathies) and many more.^[34] The technique for combining several closely related genes or even unrelated genes allowed the construction of many multi-gene DNA diagnostic kits.^[35] Some of the commonly used kits include neuromuscular diseases, epilepsy syndromes, familial/inherited cancer (breast/ovarian; colorectal cancer; familial dementia; inherited cardiomyopathies. Whilst both limited gene and multigene DNA diagnosis made a huge impact in the clinical genetic practice, the focus rapidly moved to genome sequencing methods.

Genome sequencing

The PCR technique allowed the analysis and studying any fragment size or amount of DNA. It also became possible to amplify DNA from different sections or sources simultaneously referred to as the multiplex PCR. By far the best application is evident from the enhanced efficiency of sequencing, both the Sanger and next-generation sequencing (NGS) techniques. An interested reader is advised to refer to details of these methods in other resources due to limited available space in this article.^[25] Essentially, the Sanger sequencing, a lengthy and labor-intensive process, allows each nucleotide (A-T and C-G) displayed individually compared to the full chart of nucleotides put together. Interpretation of the Sanger sequencing is relatively straightforward whilst the NGS requires sophisticated computational analysis. Both methods are now possible using the automated systems.

In contrast to the Sanger sequencing method, the NGS is designed for rapid automated sequencing with data directly fed into the computer for bioinformatic analysis. The benefits and challenges of both DNA sequencing methods are important in practice [Figure 5]. The laboratory part is relatively less time-consuming and thus efficient. However, the sequencing data analysis requires the size of the computing system for large-scale data and software programming for specific and efficient analysis. In addition, the sequencing data needs to be analyzed in the context of the phenotype and the source population ethnicity. It is a rapidly evolving system that has taken the center stage in both genomic research and diagnostic applications. It is now the universal practice to use the Sanger sequencing for the validation of sequence variants identified by the NGS method.^[25]

Figure 5: Comparison between the Sanger sequencing and the Next generation sequencing methods (https://www. illumina. Com /science/technology/next-g eneration-sequencing/ngs-vs- sanger-sequencing. html

In diagnostic genomics, there are three major applications of NGS-targeted exome sequencing basis on the clinical phenotype (clinical exome sequencing-CES) covering exons of around 5000–6000 genes of interest, whole exome sequencing (WES) covering exons or coding region of all 20,000 genes and the whole genome sequencing (WGS) covering exons and introns of all genes and all other genomic material between genes, including the evolutionary conserved genomic elements. While both CES and WES are established in clinical diagnostic genomics, the scope and role of WGS is widening to many horizons.^[36] Apart from identification of pathogenic mutations or variants in established or likely monogenic (Mendelian) disease, WES can also detect rare variants of unknown or likely significance.^[37] Recent reports advocate strongly, the importance of WGS in public health domains, specifically new born screening (NBS).^[38] It is a new territory that has raised several ethical and logistic questions. Nevertheless, the argument has shifted more towards its use in NBS and acute pediatrics, particularly with the prospect of rapid WGS method.^[39,40]

Genome variants

By far the most important task following the genome sequencing is the genome variant interpretation, particularly missense mutations or sequence variation occurring within the gene including the intronic splice site. There are many different variant interpretation approaches in relation to monogenic disorders, specifically rare genetic diseases. The classification system proposed by the working group of the American College of Medical Genetics and Genomics is widely accepted and used by most diagnostic genetic and genomic laboratories.^[41] This system includes the 5-tiered scheme to classify a genetic variant as pathogenic, likely pathogenic, a variant of uncertain significance (VUS), likely benign, or benign. Each category of the variant requires evidence that could be very strong, strong, supporting and moderate.

In the context of genome wide polymorphisms (variants), the challenge is to distinguish pathogenic (disease causing) from nonpathogenic variants (benign or likely benign). This would depend on the frequency in a given population or distinct ethnic community. It is recognized that this is by far most relevant to tribal and indigenous populations in Africa, Australia, India, and South East Asia and Far East/Asia Pacific regions. In all cases, the spectrum of variants includes polymorphisms not associated with disease, polymorphisms possibly associated with disease, mutations or variants not disease causing, mutations or variants probably disease causing, and finally disease causing mutations or variants. The fundamental basis of this or any classification system remains careful case selection with in-depth phenotyping (deep phenotyping) and all other supporting evidence. The system proposed by the Human Phenotype Ontology provides the necessary framework for deep phenotyping, an essential component for the variant interpretation, specifically VUS.^[42]

THE HUMAN GENOME PROJECT-THE GENOME ERA

Amongst major joint institutional, industrial, charity and state funded scientific projects, the huma genome project (HGP) stands out probably the largest human effort with the sole aim of sequencing the human genome. It is often compared with the mammoth Manhattan project for the atomic bomb. The concept and the scientific commitment for mapping and sequencing the human genome dates back to 1990s.^[43] The first draft of human genome sequence appeared in 2003 simultaneously from NIH and Craig Venter’s laboratories.^[44] The final complete sequence of the human genome was published in 2022.^[45] The multi-billion dollar HGP initiative completed the huge task in record time and within the allocated resources. Incidentally, the completion of HGP coincided with the 50 years of the Double helix, both major landmarks in the history of genetics. Many people thought it was the beginning of the genome era! However, Dr. Francis Collins, the Director of NHGRI-NIH and the Lead Scientist of HGP, argued that genome era did not began with HGP. This happened to be another project in succession with the Mendel’s laws of inheritance, discovery of the DNA double helix, the genetic code and many others. He believes that there will never be the postgenome era as it will continue for considerably long period. The initial publication was not the complete human genome sequence as around 2% of the genome could not be done, as mostly this included repetitive DNA sequences confined to the telomeric regions. It took almost 20 years for achieving the 100% mark for the human genome sequencing.^[45] Undoubtedly, all sequencing efforts and outcomes massively contributed to the realization of the precision and personalized medicine.^[46]

GENOMICS AND MULTI-OMICS

The term genome was coined by Hans Winkler in 1920, a German biologist. It was essentially to convey gene and chromosome in single word. The word gene was later added to omics, the Greek word for the compilation of knowledge. According to the NHGRI-NIH, the genome is the entire set of DNA instructions found in a cell. In humans, the genome consists of 23 pairs of chromosomes located in the cell’s nucleus, as well as a small chromosome in the cell’s mitochondria. A genome contains all the information needed for an individual to develop and function. In simple words, the science of genomics is in reality continuation of the science of genetics. Professor Victor McKusick promoted genomics along with many of his contemporaries.

The genomics is one of several biological sciences ending with the omics suffix, for example proteomics, metabolomics, microbiomics, glycomics, lipidomics, phenomics and many more.^[47] In addition to genomics, the three other OMIC sciences (transcriptomics, proteomics, and metabolomics) are joined together with systems biology.^[47] All these four omic sciences constitute the large field of systems biology including many other integrated multi-OMICS disciplines [Figure 6].^[48–50]

Figure 6: Integrative approaches for correlation of disease relevant target tissues and the Multi-omics data

COMPLEX GENOMICS

The bulk of genetic and genomic diseases do not conform to distinct categories of mode of inheritance (chromosomal, Mendelian, and mitochondrial). These belong to the large group of medical and few surgical conditions causally related to hundreds and thousands of genome wide variations closely interacting with range of apparently non-genetic or acquired life-long factors. The list of these acquired or postconceptional factors is too long but can be summarised as per stage of life-prenatal (maternal drugs, infection, toxins), perinatal (exposure to hypoxia, infection, birth trauma) and postnatal (infection, nutrition, trauma), infancy/childhood (infection, nutrition, trauma, and psycho-social deprivation), adolescent/young adult (infection, trauma, autoimmune diseases, and psycho-social), late adult/elderly (chronic medical diseases, drug-alcohol abuse, and life-style related).

Most clinical manifestations can be accommodated within the broad spectrum, however, could be age or gender specific. In some instances, the age of onset, severity and ultimate prognosis could be correlated with the variance and quantity of genomic factors. By far the best, however crude, evidence is derived from the three generation family history highlighting the common phenotypic features segregating from both maternal and paternal lineages. Not surprisingly, a clinician often comes across extremely complex multi-system manifestations that might be difficult to explain, such as the metabolic syndrome.^[51,52] Clearly, it might be logical to carry out the whole genome sequence in this patient and build the molecular landscape based on the genome profile. However, this would require a comprehensive genome database with fully annotated variant information on all aspects of genomic constitution and function.^[53,54]

Heritability

The term heritability refers to the proportion of phenotype variation attributed to genetic or genomic factors. By far the best evidence for heritability is based on large scale twin studies comparing the outcome in monozygotic (identical) and dizygotic (non-identical) twins [Table 1].^[55] This approach was used for several years and used in offering advice on the life-long risk and avoidance of acquired factors to mitigate the impact of inherited genetic or genomic factors. Real genomic evidence emerged with the advent and generous use of newer laboratory techniques that allowed the use of different elements of the genome (HapMap; CNVs; SNPs) in carrying out quantitative analysis. The genome wide studies led to major transformation in understanding molecular mechanisms underpinning the complex medical diseases. Genomic approaches have made major inroads to the understanding of complex diseases, particularly the uncovering the missing heritability.

Table 1: The concordance data and heritability estimates in common medical and psychiatric diseases^{[ 55 ]}

HERITABILITY ESTIMATES FOR COMMON COMPLEX TRAITS BASED ON MZ AND DZ TWIN STUDIES DATA

Genome wide studies

Advances in genome analysis offered scientists the opportunity to study causal associations of genomic regions or loci with different phenotypic traits, specifically assigned with common medical diseases. This approach is not aimed at specific diagnosis, like chromosomal or Mendelian disease, but to assemble genomic variance data closely matching with the phenotypic variance or traits. The genome wide association study (GWAS) collects data to find out the common variants in a number of individuals, both with and without a common trait (e.g., a disease), across the genome, using genome wide SNP arrays. A typical GWAS analyze common variants that do not generally identify causal variants.^[56] These common variants tag a region of linkage disequilibrium (LD) containing causal variant(s).

Recently, in addition to GWAS, several studies have reported similar approaches to integrate multi-level data derived from closely related OMICS sciences, specifically transcriptomics, proteomics, metabolomics and phenomics [Figure 7]. By far the most important component of the omics, wide association study is the phenomics focused on quantitative traits loci.^[57]

Figure 7: The genomic approach to the preventive healthcare

Polygenic scores

In any complex multi-factorial/polygenic disease, there are three basic elements-genomic constitution, physical characteristics and environmental factors. Out of these, genomic profile is static, the physical characteristics are probably modifiable (within limits), and largely modifiable and to some extent, avoidable environmental factors. The heritability data were generated based on very crude analysis and probabilities. This approach could never convince the clinician to apply in clinical medicine. Researchers interested in complex genomics have developed a new quantitative system, the polygenic score (PRS), that takes into account all the variables causally associated with the phenotypic variance.^[58] This approach convinced clinicians in many disciplines who regularly manage patients and advise families affected with a complex polygenic disease. Investigators in complex genomics have produced impressive and enormous data within relatively short space of time. Perhaps the best example of PRS clinical application is the coronary artery disease-CAD.^[59] The genomic data generated are statistically separated with individual risk allele and compared with controls. Multiple PRS figures are thus developed and tested for association using the linkage disequilibrium model. The association data is assigned to classify clinical cases into high, moderate and average risk categories [Table 2]. It is likely that the future management protocol for complex diseases would use PRS in risk assessment and advising accordingly, specifically on prevention.^[60]

Table 2: Outcomes of polygenic risk scores in coronary artery disease^{[ 61 ]}

GENOMIC MEDICINE AND HEALTHCARE

Undoubtedly, the biggest and by far the most important translation and application of the novel genomic advances has been the revolutionary genomic medicine and healthcare. This development is notably talked about in the context of precision and personalized healthcare. To this, other dimensions such as participation and prevention are also added. However, the core elements, diagnosis-treatment-prevention, remain unchanged. Unlike any other medicine system, the genomic led precision medicine focuses on demography, environment, natural history and phenotype of the disease and specific-targeted pharmacotherapy.^[61] The core triad of precision medicine includes diagnosis, treatment, and prevention.

In addition to conventional genetic laboratory techniques, the major tools for genomic medicine practice include microarray chromosome analysis (MCA), specific genetic testing, multi-gene panel testing and the next generation genome sequencing. Other genomic laboratory techniques of RNA sequencing, the mitochondrial genome sequencing, and epigenomic analysis are applied in selected cases. The holistic individual metabolic turnover, referred to as the metabolomics, can be assessed using emerging new proteomic analysis methods. The model of the step-wise stratification using multiple OMIC methods is now accepted for the precision and personalized medicine.^[62]

The path from the laboratory to the clinic or “bench to bedside” requires multi-faceted research and development (RandD) strategy for successful genomic medicine and healthcare program. Main areas for RandD include discovery of diagnostic molecular (genomic) profiles, identification of novel genetic or genomic associations and their mechanisms, construction of polygenic risk scores, biomarker discovery, specific pharmacogenomic markers, molecular or genomic profile-based patient cohort build up and finally drug repurposing through discovery of novel factors targeted by existing drugs for other indications. Some of these new applications have moved into preventive genomic healthcare targeted at healthy people with real or perceived genetic risks.^[63]

PERSONAL GENOMICS

The emerging field of personal genomics is the targeted preventive healthcare based on the individual genetic risks.^[64] The genetic risks are determined based on the allele frequency of single or multiple genes with the recognized disease causing potential. The targeted people could be the single person, family, ethnic community or the specific population.^[65] The selection of targeted cohort is commonly based on the family history, physical characteristics, and conventional radiologic, biochemical, and immunological investigations. However, increasingly, many people now volunteer for genetic/genomic profiling irrespective of the family history or ethnic specific health risks. Some people are simply concerned for their personalized health risks. In all cases, the genomic profiling is the key in personal genomics to select person(s) at risk including the level (age and severity) of disease development.^[66]

There are five distinct categories of people seeking personal health-care advice based on genetic or genomic risk status.

• Class 1: First degree relative of an index patient with a chromosomal or monogenic (Mendelian) disease
• Class 2: Extended family member with history of a chromosomal or monogenic (Mendelian) disease
• Class 3: Member of the family with a common or complex disease
• Class 4: Member of the ethnic population group with increased prevalence of monogenic/Mendelian disease(s)
• Class 5: Any one from the population concerned for health risk(s) including the new born.

Note-the person seeking personal genomic information or advice should be deemed healthy (asymptomatic) at the time of genome sequencing or selective genomic testing.

Class 1-First degree relative of a patient with confirmed diagnosis and/or known disease causing mutation of a recognizable chromosomal or monogenic (Mendelian) disease

This is by far the most common group of people seeking personal genetic or genomic advice or information. The individual is concerned for his or health risks related to the disease in the sister, brother or including parents. Most of these people are referred to the clinical genetic or genetic counseling service.

In the chromosomal context, a typical scenario would be parents of a child with distinct facial profile, growth delay and range of neurodevelopmental difficulties. The child’s G-banded karyotype confirmed unbalanced chromosome rearrangement involving two acrocentric chromosomes.^[13–22] The parental karyotype analysis showed one parent with the classic Robertsonian translocation. The parent would be clinically normal. In practice, this is seen in Down’s syndrome without trisomy 21. In this case, the extra chromosome material belonging to the long arm of chromosome 21 results from meiotic rearrangement. Clinically, most such cases are indistinguishable from the common trisomy 21 Down syndrome. This phenomenon is seen in around 5% cases. The parents are offered genetic counseling including detailed discussion on their reproductive choices. In most cases, early prenatal testing is preferred with chorion villous biopsy at 11+ weeks. The affected pregnancy would qualify for option of medical termination.

In the context of a recognizable Mendelian disease or familial disorder, the prior genetic risk to the first degree relative could be quantified based on the inheritance pattern. The prior probability is used in making prediction either for risk of recurrence or even the risk of developing the disease itself. In the case of an autosomal recessive disease, such as the spinal muscular atrophy (SMA), the prior risk of a sibling would be 2 in 3 for being the carrier. This could be used in calculating SMA risk in any future child using the population frequency of carrier applicable to his or her spouse or partner. However, if the sibling were shown to carry normal allele, then the risk would be substantially reduced. Similar approach could also be used for advising a young sister of an affected brother with an X-linked disorder such as DMD. Her prior 50% genetic risk for being the carrier would be considerably reduced if she had not inherited the same dystrophin gene mutation, except for very small residual risk related to gonadal mosaicism. On the other hand, on confirmation of inheriting the same mutation, her risk would rise to 100%. She could then be counseled accordingly with discussion on her reproductive options.

In the above two situations, the risk of developing symptoms and signs of the genetic disease in the carrier person are extremely small except for exceptional biological reasons. In autosomal dominant Mendelian disease, the first degree relative carries 50% risk for inheriting the similar gene change of mutation. There is very high life-time risk of developing the disease, however mild or severe, and early or late onset. There are instances where the person might escape from being clinically affected, although could have subclinical signs detectable on special investigations, such as minimal changes in the brain in a variable autosomal dominant neurodegenerative disease. This is often referred to as incomplete penetrance. Nevertheless, confirmation of the disease causing gene mutation would be extremely helpful to other first degree relatives for accurate risk prediction, and if at all possible, applying any form of prevention or lowering the impact of the disease using drugs and devices. In the case of familial cancers, such as BRCA associated breast and ovarian cancers, the healthy unaffected lady with confirmed disease causing BRCA mutation could be offered many preventable options including the risk reducing surgery involving mastectomy with or without oophorectomy.

Class 2: Extended family member with history of a chromosomal or monogenic (Mendelian) disease

The prior genetic risk to a second or third degree family member with the common ancestral parents would depend on the mode of inheritance as outlined in the previous category. This scenario is increasingly coming to the genetic or genomic clinic as more and more people are aware equipped with information from the media, internet and social interactions. As far as possible, it is extremely important to have the confirmation of the disease in the index family member along with the outcome of genetic testing results. The prior genetic risk could be accurately modified with the help of specific targeted genetic testing. However, in the event of nonavailability of the genetic information, it might be possible to carry out genetic testing based on the diagnosis or the natural history in the index family member. The clinical geneticist or even genetic counselor would be in a good position to advice if the genetic testing should involve the multi-gene panel or targeted CES. In the case of recessive conditions, it is the normal practice to offer testing in the spouse or partner based on specific disease causing gene change or mutation. The outcome of combined approach would allow clinical geneticist, genetic counselor or even a fetal medicine professional to discuss most appropriate reproductive options including interventions during the pregnancy as well.

In the case of an autosomal dominant disorder, such as familial cancer or untreatable chronic progressive neurodegenerative condition, the gene carrier or heterozygote, would be at increased life-time risk for developing the disease as per the natural history of the condition. In addition to the genetic counseling, the person might also benefit from other specialist input, such as serial X-ray mammography, breast MRI scan, and ovarian ultrasound examination. Most large tertiary hospitals have the provision of multi-disciplinary team care. This is an excellent example of specific targeted genetic or genomic management.

Class 3: Member of the family with a common or complex disease

Most of the non-communicable diseases have complex or mixed etiology. These are common and constitute the bulk of morbidity and mortality globally irrespective of geographic location or ethnic population origin. It has been recognized for long time that there are several hundreds and thousands of gene alleles or polymorphic variants spread across the genome either closely associated or contributing to the causation of the disease phenotype through one or more molecular mechanisms. The term multi-factorial/polygenic inheritance is used to emphasize distinct genetic disease category. Several examples include isolated congenital anomalies such as ventricular septal defect, arial septal defect, pyloric stenosis, congenital dislocation of hips, and many more. Among the life long and progressive diseases, conditions such as psoriasis, bronchial asthma, coronary artery disease, essential hypertension, rheumatoid arthritis, bipolar depression, and schizophrenia. The genetic factors are generally perceived on the basis of family history, association with selected life style or socio-cultural practices and higher than expected prevalence among the monozygotic twins compared to dizygotic or non-identical twins. The proportion of the phenotype attributed to hereditary factors is referred to as the heritability index. In general, higher heritability for a complex disease could be used for advising increased risk, possibly modifiable by adopting new life style, dietary habits, enhanced physical activity, or occupation.

There are several families or cluster of closely knit communities, where some of the common or complex diseases are more prevalent compared to the general population. New advances and techniques for genomic analysis have helped to collect huge amounts of data on genomic variants supposedly closely associated with some of the common and complex diseases. Genomic variants could be SNPs, CNVs, cluster of dispersed InDels, gene-specific exonic or intronic variants, and many more evolutionary conserved genomic areas. Typically, GWAS produce spike-like peaks for the most significant loci with genome variants, displayed as the Manhattan plot.^[67]

Genome-wide association studies have helped to produce useful data for some the complex diseases. In this context, in a simplified manner, common diseases result from environment and genome variants of medium to small effect [Figure 8]. In exceptional cases, few common diseases may result from high-effect variants. Most Mendelian diseases are associated with high-effect rare alleles. It is not clear what is the role of low-frequency variants with intermediate effects.^[68]

Figure 8: Variable frequency of rare to common disease alleles in a given population-The basis of personal genomics

Despite huge data accumulated from numerous GWA studies, clinical applications are limited. Nevertheless, the enthusiasm has not diminished and efforts are continued to find a much more robust and translational tool for medical and healthcare applications. The PRS system is a good example in this context that has offered a new dimension in risk assessment and evaluation of the family history with the sole purpose of precise and personalized genomic healthcare interventions in complex disorders. However, there are many technical and logistic difficulties faced by proponents of PGS. Current practice followed and recommended by genomic physicians, is to base the advice on a pragmatic approach relying on the family history, degree of relationship with affected family members, severity and age of onset in a younger affected family member, and selection using conventional serologic (proteomic), immunological and inflammatory bio-markers. At present, clinical risk assessment and effective preventive measures based on personalized genomic profiling are not considered to be of any value due to limited predictive power and robust case selection.

Class 4: Member of the ethnic population group with increased prevalence of monogenic/Mendelian disease(s)

There are several common and rare genetic disorders encountered more frequently in certain ethnic population groups. There are several factors for increased prevalence including isolated existence, preferred socio-cultural cohabitation (dietary habits, life style, and common faith), and endogamy or consanguineous promoting non-assortative spouse or partner selection. Most frequent genetic conditions are recessively inherited; examples include beta-thalassemia (Greek; Cypriots, Turkish, Iranians, and peoples in western parts of the Indian subcontinent), cystic fibrosis (north Europeans), G6PD (Arabic and Persian speaking people), and Tay-Sachs and related metabolic diseases (Jewish people). The natural history and causative genes or alleles are documented in several studies. This valuable resource is harnessed for risk assessment and advising on prevention in prospective parents belonging to the ethnic community groups. In some countries, there are laws prohibiting marriage amongst the known carriers, for example, Iran and Cyprus for beta-thalassemia.

Preconception counseling is now increasingly offered to prospective parents from these ethnic communities or anyone else with the family history of the unknown genetic condition. This is often combined with carrier identification using multi-gene panel for several common and rare recessively inherited diseases. Most couples contemplating having a new baby opt for preconception carrier testing to allow themselves informed family planning.

Class 5: Any person from the population concerned for health risk(s) including the new born

The last category for personalized genomic healthcare includes healthy people, including the new born, from any population group irrespective of the geographic location. In most cases, there is no real reason to seek advice for health risks or any form of prevention. There is an upsurge of genomic laboratories who now offer health risks assessment based on personalised genomic profiling. This practice of “direct to consumer” genetic testing (DTC-Genetics/Genomics) is actively promoted and considered lucrative with a projected multi-billion dollar market.^[69] Several such private enterprises exist that offer DTC for specific diseases, such as breast/ovarian cancer, colorectal cancer, prostate cancer, coronary heart disease, type 2 diabetes mellitus and many more. There are packages available for a broad range of unrelated incurable late-onset diseases including Alzheimer’s dementia, Parkinson’s disease, and many more. In some cases, genomic profiling and interpretation are carried out for practically every possible disease without any clear evidence of genetic or genomic association. Except for few laboratories, most do not have any pre- or post-testing information or counseling support available. There is increasing concern for the DTC genetic or genomic profiling since it might result in unnecessary or unwarranted genomic information or “genomicisation” with the danger of creating a healthy cohort of “worried well” people.^[70] Recent reports of new born genomic profiling is viewed with great caution. Despite few claims of positive outcomes, many pediatricians and other clinicians remain hugely sceptical. This is undoubtedly the most contentious approach that requires a thorough public debate on ethical, legal, and social issues (ELSI) for the large-scale genomic profiling in any person, specifically in the newborn.^[40]

SUMMARY AND CONCLUSIONS

All scientific discoveries, innovations, translations, and applications are exciting to experience to all humans. Many such novel advances might not stand the test of time and simply disappear. Nevertheless, enthusiasm and unpalatable desire lead to further new discoveries and the cycle is continually repeated. The science of genetics and genomics is no exception. Perhaps, this field is more relevant due to its direct impact on living creatures, particularly humans in the context of society and environment. This chapter takes the reader, probably a young enthusiastic student or new fellow, through major hypotheses, experiments, discoveries, landmarks, and applications. The broad subject of medical and preventive healthcare is reflected against the mirror of genetics and genomics. The need for indigenous population-specific genome databases is required for delivering effective, fair, ethical, and legally justified genomic healthcare to many people, particularly those in ethnic minority communities. Major global genomic organizations are actively pursuing the proposal for setting up the Pangenome, the digital global genome database available to all irrespective of political or economic systems.^[71]

There is no doubt about the Genomic or OMIC power in making precision diagnoses, advising on the outcomes, and assisting individuals and families for making informed choices for basic living in relation to a particular genetic condition. Clearly, many unexpected and unwanted ethical and social issues were encountered and dealt with. However, the situation changed with the scope and prospects of predictive healthcare based on the individual genetic or genomic profile. This natural desire received further boost with the advent of highly sensitive personalized genome sequencing methods. Inevitably, commercial exploitation of novel genomic developments led to “direct to consumer” testing opportunities along with possible prevention using drugs and other measures, often without any credible scientifically validated evidence. Most health-care providers are sceptical about the new emerging field of personal genomics and seek advice and views of experts in medical ethics, health economics, health sociology, and the medical law. There is a call for efficient and effective regulation and governance using the current or new statutory instruments.

Conflicts of interest

There are no conflicts of interest.

Funding

Nil.