The Human Genome Project (HGP), officially completed in April 2003, is thought to be the biggest revelation in the past decade with regards to medicine. However, this achievement is only the start of a new era in genomics as the aim to determine gene function and therapeutic answers to all ~27,000 genes of the human genome has just begun . In this review, the prospects of the HGP, in particular in eliminating genetic diseases are discussed, focusing on LQTS as well as the study of genome analysis, and how this may have an impact on both ethical and social issues.
The science of genes has been profoundly affected during the past decade due to the HGP which will soon begin to influence both medicine and society. Following years of vague explanations concerning cellular processes, the aim of understanding both human physiology and disease is impending. For many centuries linkage analysis was used in attempt to pinpoint the causes of genetic diseases within families, however this was very time-consuming and not so efficient. The dawn of the HGP has allowed a more extensive strategy in that the entire human genome would be sequenced for the benefit of mankind [1, 36].
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Since the completion of the HGP, the advances in DNA technology have allowed the tracing of genetic disorders to be promoted, in that the majority of genetic diseases are now thought to be a consequence of one or more gene mutations. Through the recent progression in molecular genetics, the vast bulk of causal mutations have been identified, which has allowed diagnostic testing to be greatly accelerated. Hence, the phenomenon of genotype-phenotype correlations in which molecular defects in the genetic make-up of an organism are related to their actual traits is now studied on a large-scale basis. However, many complications arise in this research area since there are numerous determinants with respect to cause and effect linkages, as well as the possibility of both environmental influences and the involvement of multiple genes .
Genome sequencing is an effective method for the cloning and large-scale detection of genes. Numerous approaches have been applied in the past in order to decipher large, complex genomes [4, 6]. This review discusses two alternative strategies: ordered-clone sequencing and whole-genome shotgun (WGS) sequencing which are summarised in figure 1 .
These two strategies both initially involve the assembly of genomic libraries. Once the genome has been broken down into short segments they are introduced into a vector, commonly plasmids or artificial chromosomes (e.g. BAC), and proliferated into microbes such as yeast or bacteria. Construction of genomic libraries involves restriction enzymes; DNA ligase is used to close the gaps between the resulting ssDNA fragments and the complementary vector. Upon insertion of the recombinant DNA, each of the recombinant molecules proliferates, producing clones of the original inserted fragment, creating a genomic library of clones. This library is consequently used for DNA sequence analysis of the inserted fragment, depending upon the genome-sequencing method implemented . Both the WGS sequencing concept and the ordered-clone approach have been used in sequencing the human genome .
In order to perform ordered-clone sequencing (figure 2), the strategy employed by the HGP, high molecular weight (HMW) DNA is partially digested and subjected to size-selection in order to be inserted into a large bacterial cloning vector, e.g. BACs. The overlapping BAC clones can be identified using high throughput fingerprinting e.g. via Hind III restriction patterns. To date, all known BAC libraries with large insert sizes, have been constructed from partial digests of megabase DNA through the use of restriction enzymes [4, 6]. The minimum tile set that represents the genome is selected from the BAC-based physical map, where these unique overlapping clones, via shotgun sequencing, serve as substrates for assembling the whole finished sequence [6, 7]. Once the suitable BAC clones have been selected, they are purified and cloned by randomly shearing them into appropriately sized fragments, i.e. 1-2 and 2-10 kb, and divided into subclones. These subclones can then be sequenced by various techniques including automated DNA sequencing [6, 8]. Subsequently, the data achieved through these sequencing reactions is analysed to construct contigs . This strategy has many advantages and disadvantages, as does the WGS sequencing method, as can be seen in table 1.
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On the other hand, the WGS sequencing method was the approach employed my Celera in order to attempt the sequencing of the human genome [2, 7]. In this strategy (figure 3), HMW gDNA is firstly purified and then cloned by randomly shearing them into plasmid vectors of varied insert sizes: typically 1, 5-10 and 150 kb, allowing the formation of three shotgun genomic libraries [2, 6]. As with the ordered-clone approach, the sequence overlaps gathered from the use of these genomic libraries are edited and used to build sequence contigs . In WGS sequencing, there are both small-insert clones (used to construct the sequence contigs and draft sequence), and large-insert clones which are fused to join single-copy contigs my means of paired-end reads into larger units, termed 'scaffolds' [3, 6]. Finally, genome finishing is implemented in order to eliminate any errors, as well as sealing the gaps between the scaffolds, resulting in an accurately-assembled representative contig [2, 3, 5]. However, there are other, newer techniques that have been implemented in genome sequencing such as pyrosequencing and Solexa sequencing.
Assigning Gene Function
As the post-genomic era is just around the corner, the prospect of utilising the genomic information from the HGP to progress from the gene sequence, to the functional protein and in turn to understanding its function in vivo draws closer . Functional genomics attempts to understand the relationship between genotype and phenotype through the interpretation of one's genome through reverse genetics, transcriptomics, comparative genomics and proteomics. This review will discuss comparative genomics in attempt to understand gene function.
Firstly, reverse genetics involves disturbing the DNA sequence of a gene in attempt to analyse its effect in vivo either through random mutagenesis, targeted mutagenesis or phenocopying . Moreover, transcriptomics involves exploiting information from all expressed transcripts within cells, tissues, or whole organisms e.g. exploiting the expression of particular genes in certain cell types under certain conditions using DNA microarrays. The tool of proteomics incorporates many techniques such as 2-D gel electrophoresis in order to study the proteome (the expressed proteins of an entire genome) within a system .
Now that the human genome has been completely sequenced, it can act as a reference for comparison with other species in attempt to fully understand human disease and physiology, in addition to knowledge of evolutionary divergence [32, 34]. This review discusses the use of mice models as an example in determining human gene function; however other models can be used such as chimpanzees.
The mouse is often used as a model for analysing gene function in humans since it has been extensively studied. Since the human-mouse divergence occurred around 75 million years ago, any mutations that might have developed will have affected the genome and hence, any mutual sequences between the two organisms almost certainly signify common functions. Furthermore, when carrying out comparative genomics, the initial stage is to identify homologous sequences (orthologs and paralogs). From studying the mouse genome, it is clear that it contains approximately the same amount of protein-coding genes as in the human genome, and that around 99 percent of genes in the mouse have homologous traits in Homo sapiens, and vice versa. This indicates that the proteins encoded for in both the human and mouse genomes are equivalent .
Moreover, not only do these two species share vast similarity in their protein-encoded genes, but are also alike in their genome composition. As shown in the example in figure 4, there are many domains of conserved synteny between the human and mouse chromosomes. However, there are obviously a few differences between the two genomes such as mice contain extra copies of genes that are involved in immunity for example, but these differences are somewhat trivial when observing the whole picture .
Long QT Syndrome
The completion of the HGP has helped accelerate the search for genetic factors primarily associated with genetically complex disorders . The main goal of the HGP is to pinpoint those genetic variations that cause individuals to be more susceptible to major killers such as cancer and cardiovascular diseases, with the aim of creating a genetic disease-free society . This review discusses the notion of long QT syndrome (LQTS), a complex genetic disease .
LQTS is characterised by abnormal patterns seen on the electrocardiogram (ECG) in which corrected QT (QTc) prolongation is observed (figure 5) . This disease is one of the major causes of sudden cardiac death owing to the formation of malignant arrhythmias termed torsade de pointes (TdP) . There are two forms of LQTS: congenital (an inherited condition subdivided depending on locus-specific mutations as seen in table 2) and acquired (due to precipitating factors such as diuretics). Moreover, there are two modes of congenital LQTS heredity: Romano-Ward syndrome (RWS) and Jervell and Lange-Neilsen syndrome (JLNS), the autosomal dominant and autosomal recessive forms, respectively. JLNS is much rarer and is also associated with deafness . RWS is thought to affect 1 in 10,000 individuals, whereas JLNS is significantly rarer, estimated to affect between 1-6 individuals in 6 million . Due to the HGP, it is clear that the majority of LQTS forms are the consequence of mutations in ion channels or their auxiliary subunits . To date, twelve mutations relating to LQTS have been identified, termed LQT1-12, (table 2) [11, 15].
The Role of Genetics in Acquired Arrhythmias
The HGP has shed a new light on the genetics of LQTS in that scientists are now aiming to establish the true role of predisposition in acquired arrhythmias. Through the HGP, it is now believed that a substantial amount of the susceptibility factors associated with acquired disorders are encoded by single nucleotide polymorphisms (SNPs) of the human genome. Not only are these modifying genes thought to be involved in complex genetic disorders such as LQTS, but are also assumed to be implicated in monogenetic disorders as well. Furthermore, it is clear that there are another set of genes which regulate both inherited and acquired disorders, in which the underlying disease determines their expression .
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The Prediction and Prevention of Arrhythmias
Currently, LQTS is usually diagnosed by a cardiologist through ECG recordings and certain exercise tests. Nonetheless, in those individuals with a known family history of LQTS, it may be possible to estimate the risk of developing the disease . In genetic disorders, there is often a gain- or loss-of-function in the resulting protein of the mutated gene .
Since the human genome has been sequenced, and some of the mutations linked to LQTS identified, tests are now being developed in order to establish the specific genotype of LQTS patients and in turn determine whether or not their first-degree relatives have or are susceptible to the disease (predictive genetic testing) [11, 21]. However, in the majority of cases in which there is a strong family history, it is apparent that the genetic component is a susceptibility factor since LQTS is a multifactorial disease in which there are both hereditary genetic predispositions and environmental influences e.g. exercise and gender . Genetic predisposition influences the phenotype of individuals along with other factors such as penetrance and expressivity .
Therefore, those environmental factors that cause individuals to become more susceptible to cardiac disorders can be avoided in order to implement preventative strategies. As a result, it is key that both genetic and environmental triggers of cardiac disorders are identified . Even though genetic testing is not as yet available for use in clinical practice, the use of the HGP in the future will almost certainly allow genetic testing to permit the diagnosis and early treatment of LQTS and hopefully its prevention .
At the moment, there is no cure for LQTS, but treatments can be used as seen in table 3. However, with the knowledge gained from the sequenced human genome, the future of LQTS treatment seems promising.
Congenital LQTS is best treated if the clinical treatment given to patients is genotype-specific, since it could possibly be used to plan and tailor treatment to specific patient's needs [10, 24]. For example, studies have shown that individuals with mutations in the LQT3 gene don't respond as well to beta-blockers, and instead may benefit from treatment such as mexiletine and lidocaine which block sodium channels in attempt to normalise the QT interval i.e. gene-specific treatments [9, 24]. A summary of the clinical features for LQT1-3 can be seen in table 4.
On the contrary, out of the estimated 27,000 genes of the human genome, it is now known that the discrepancies in genes between individuals is crucial, in that sometimes these differences can control one's susceptibility to particular genetic disorders . Therefore, establishing the genetic make-up and its associated interactions with particular medications is essential while heading into the age of personalised medicine .
Even though the detection of gene mutations is key in beginning to fully understand the genetics behind LQTS, understanding the epigenetics, for instance genomic imprinting, is just as significant. The imprinting of genes refers to whether gene expression is regulated maternally or paternally . Recently, the KCNQ1 gene has been mapped to chromosome 11p15.5 which shares synteny with mouse chromosome 7 - the Kcnq1 domain. Here, an array of six genes are located whose expression is genomically imprinted in some tissues, and which has been associated with Beckwith-Wiedemann syndrome [15, 19].
Moreover, the Kcnq1 gene is paternally imprinted in mice, and hence is maternally expressed during early fetal development [15, 30]. However, the paternal allele becomes methylated during embryogenesis; a similar imprinting process is thought to occur in humans. As can be seen from figure 6, the human KCNQ1OT1 promoter has been localised to intron 11 on chromosome 11 of the KCNQ1 gene .
This imprinting in the mouse may explain why LQTS still persists even when the coding sequence mutation isn't present in the KCNQ1 gene. Upon differentiation, it is crucial that the paternal allele becomes methylated to ensure that the truncated KCNQ1OT1 transcript is not generated. However, this methylation can be blocked during development by a single mutation that disrupts the CpG island of the Kcnq1 overlapping transcript 1 (Kcnq1ot1), resulting in gene silencing of the cardiac paternal allele, and hence LQTS symptoms. Therefore, epigenetic effects may play a major role in the pattern of inheritance in some families who suffer from LQTS .
Future of Genomics in Cardiovascular Arrhythmias
As more mutations associated with LQTS are discovered owing to the HGP, researchers are coming closer to understanding LQTS causes as well as more accurate diagnoses. This genetic research should hopefully allow doctors to directly treat the disease as opposed to treating the symptoms. Scientists are still awaiting the clinical practice of gene-specific treatments which will hopefully be able to replace the less effective beta-blockers currently used. A new line of research involves understanding the way defective genes influence the electrical properties of myocytes and hence, the cardiac action potential. This research may ultimately lead to the discovery of epigenetic processes of gene silencing or gene repair of the defective gene that is causative of LQTS. Information gained from genetic typing of genealogies will hopefully allow treatment to be matched to specific families in the near future .
Social and Ethical Issues
Not only is science extensively influenced by the HGP; society is also widely affected . The new and exciting field of genomics has bought new ides to life such as the discovery of mutations associated with genetic diseases as well as the development of more accurate therapies. However, with the prospect of 'engineering' a genetic disease-free society, ethical, legal, and social implications (ELSI) must be acknowledged [27, 28].
Genetic testing provides many benefits such as the detection of genetic conditions, but there are a number of uncertainties, for example prejudicial treatment of individuals regarding DNA-based tests, and misuse of confidential information may give rise to genetic discrimination by life and health cover insurers as well as employees . Moreover, this genetic testing is only an estimation and hence, those it deems susceptible may never actually develop the disease in question . Therefore, it is important to consider the psychological associations involved in being diagnosed with the disorder, and whether to 'label' those individuals who are genetically affected with the disease but show no physical signs, just purely on molecular aberrations .
Furthermore, there are several concerns associated with the privacy of genetic information because of genome projects, for instance informed consent to take DNA samples, patient confidentiality, and third-party access . Currently, many UK health insurance companies base their enquiries upon genetic screening tests in order to establish whether or not the individual has an increased chance of developing a certain genetic condition e.g. cardiovascular disorders . This also raises the issue of unreliable tests in which perfectly healthy people could be denied insurance cover purely based on the disease-linked alleles they carry [17, 29].
In the future, genetic information is most likely to affect pharmacogenetics, and even though most people will agree that there are few ethical concerns associated, it will ultimately broaden public awareness on the topic. The amount of research into personalising medicines for the minority of patients may be restricted since major pharmaceutical companies may focus on larger subsets of individuals in order to attempt to treat the maximum number of people. Therefore, those smaller subsets of patients will be at a significant disadvantage .
Therefore, a complete set of legislation laws for public health is required in order to see the full benefits of genome projects such as the HGP, because once genetic information ends up in the wrong hands, it is virtually impossible to prevent its disclosure [17, 29]. Hence, much thought went into the ELSI upon sequencing the human genome .
The HGP allowed the prospect of a post-genomic age, and will be crucial, hopefully in the near future, in understanding the genetics behind diseases such as LQTS . The genomic knowledge of LQTS from the HGP, has led to the possibility of personalised medicine . However, such genome projects will also bring along much controversy with respect to ethical issues, and hence will also influence some of the possible benefits of the HGP. Sequencing the human genome is only the first step in seeking therapies for genetic diseases - the next chapter in genomics is to be able to link sequence to function in hope of initially eradicating various Mendelian diseases .
Although the HGP has harvested many benefits to the field of genomics such as prenatal genetic testing and the management of some monogenic disorders, there are still several issues that need to be resolved. For example, large epidemiologic studies need to be implicated in order to see the true effect of certain genes in human genetic disorders; and genetic testing is too expensive to be used on a large-scale clinical basis. Along with others, these hindrances will postpone the rate at which the HGP promotes the breakthrough in molecular medicine that will lead us into the new genomic age .