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Next generation sequencing (NGS), also known as high-throughput sequencing involves different types of modern sequencing technologies such as Illumina, Roche 545, Ion torrent and SOLiD sequencing. These techniques sequence DNA and RNA much more rapidly and efficiently than first generation sequencing (Sanger) and has therefore revolutionised the study of genomics and molecular biology. This essay focuses on three different diseases, Cystic Fibrosis (CF), Huntington’s disease (HD) and Charcot-Marie-Tooth Disease (CMTD), and how they can be diagnosed using both non-NGS techniques and whole genome Illumina sequencing. Thereafter, the advantages and limitations of using RNA-sequencing in a patient with congenital muscular dystrophy will be discussed.
Cystic Fibrosis molecular cause
Cystic fibrosis is the most common autosomal recessive disease in Caucasians and affects around 1 in 2,500 individuals (1). It is caused by the mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR), and the most common mutation is the deletion of three-nucleotides causing a loss of a phenylalanine residue at the amino acid position 508 (ΔF508) (1).
The CFTR protein is a cyclic adenosine monophosphate (c-AMP) dependent channel that works as an electrostatic attractant by outlining intracellular and extracellular anions toward the positively charged transmembrane (TM) domains inside the channel (2). Two TMs form a chloride channel pore, allowing chloride and bicarbonate transportation (2). Normally, when CFTR is activated, the chloride ions are secreted out of the cell as epithelial sodium channels are inhibited. This leads to water leaving the cell through osmosis, providing fluid for epithelial tissue secretions (1).
In CF patients, the combination of loss of chloride secretion and sodium hyper-absorption via the epithelia sodium channel results in loss of airway surface secretions. This effects the overlying mucous becoming adherent to the airway cells and preventing effective ciliary activity (2). Thus, the inhaled bacteria are no longer cleared but instead set up low grade persistent infection where the associated inflammatory and immune responses lead to airway damage and airways obstruction due to the increased concentration of salt in sweat (2).
Huntington’s Disease molecular cause
Huntington’s disease is an autosomal dominant and late-onset neurodegenerative disorder that affects 5-10 out of 100,000 individuals (3). It is caused by a CAG trinucleotide repeat expansion (>35 repeats) in the IT15 gene that results in a long stretch of polyglutamine protein close to the amino terminus of the huntingtin gene (HTT) (4).
One of the hallmarks of HD is the formation of cytoplasmic aggregates and nuclear presences throughout the brain (4). Polyglutamine inclusions contain highly ordered amyloid fibres with high β-sheet content and low detergent solubility that sequester numerous other proteins, including factors that are important for transcription and protein quality control. This suggests that the presence of polyglutamine has an impact on the cellular function and contributes to a complex loss-of-function phenotype (4). This leads to an inherited neurological illness causing involuntary movements, severe emotional disturbance and cognitive decline (3).
Charcot-Marie-Tooth disease (CMTD) molecular cause
The hereditary motor and sensory neuropathy, also known as CMTD is the most common inherited neuromuscular disorder affecting at least 1 in 2,500 (5). The inheritance of CMT can be autosomal dominant, autosomal recessive, or X linked (5). Mutations leading to CMT are grouped into demyelinating, axonal and intermediate forms that are based on electrophysiological and pathological findings.
The demyelinating types are characterised by severely reduced motor nerve conduction velocities (MNCVs) and mainly by myelin abnormalities (5). The majority of people with CMTD show a predominantly demyelinating peripheral neuropathy and are classified as CMT1. CMT1A is a subtype that is associated with an autosomal dominant duplication on the peripheral myelin protein 22 gene (PMP22) and is expressed in the compact myelin of Schwann cells of the peripheral nervous system (6). Thus, CMT is mainly characterised by distal muscle weakness and atrophy leading to motor handicap (5).
Cystic Fibrosis diagnostic tests
As CF is a complex genetic disease in which mutations in the CFTR gene alter the function of the anion channel that leads to an increased concentration of salt in sweat. The sweat secretions can be stimulated either through cholinergic or β-adrenergic pathways. The cholinergic pathway is important for normal thermoregulation and is not affected in CF patients. However, the β-adrenergic pathway is either absent or markedly reduced in CF patients that can be measured using a non-NGS technique called sweat chloride concentration. Other non-NGS techniques include abnormalities of an ion transport in respiratory epithelia of patients with CF that are associated with a different pattern of nasal epithelia compared with normal epithelia. This test has either very little or no response to chloride free solutions (8).
The role of NGS in diagnosing CF is beneficial for detecting the bacterial pathogens (such as Pseudomonas aeruginosa) during an infection. This can be achieved by resequencing individual colonies and whole populations from single sputum samples from CF patients that is marked by acceleration in the decline of pulmonary function (9). Since CF has a high genetic heterogeneity (due to different types of mutations), it greatly affects the allele detection rate and overall frequency of mutant alleles of genetic tests such as whole genome Illumina sequencing. The algorithm of immunoreactive trypsinogen (IRT) analysis and next generation sequencing (NGS) contributes to an improved timeliness by having a high throughput. NGS also provides visibility into the CFTR gene for molecular diagnostic testing of CF and can be used to make informed family planning decisions and choose optimised treatments, leading to a better quality of life.
However, as Illumina sequencing produces shorter reads, there is still a need for more adequate read lengths of genes to make it an effective test for CF patients (11). Additionally, to limit costs and time, a gene panel of CF mutations would be more beneficial to have a role in mutational searches that can be performed for both CF diagnostics and screening purposes.
Huntington’s Disease diagnostic tests
The clinical diagnosis of HD using a non-NGS technique is based on the neurological evaluation with the manifestation of an obvious extrapyramidal movement disorder, and a positive genetic test for the HD CAG expansion or a confirmed family history of HD (12). The neuropathology indicates loss of medium gamma-amino butyric acid (GABAergic) spiny neurons, sparing of large cholinergic interneurons, and specific neuronal loss of the cerebral cortex (12). The morphometric analyses from MRI scans suggest marked atrophy in the striatum, thinning of the cortical ribbon and evidence of white matter volume loss (12).
A gold standard NGS technique for HD diagnosis is the DNA determination, showing at least 36 CAG-repeats on the huntingtin gene on chromosome 4 (13). This can be tested by using Southern blot for longer repeats that provides accurate size repeat expansions, but the detection rate is low. Completing a WGS is a more useful test for identifying repeat expansions as it is able to identify the genome coverage when detecting the disease compared to WES. The penetrance of HD is unpredicted as some patients may not manifest the phenotype until late in life and will therefore be included in the reference data (14). Thus, having the use of WGS of Illumina will avoid costly unnecessary tests such as MRIs, treatments (i.e. gamma globulin for inherited neuropathy) and additional referrals are strong reasons to increase the appropriate genetic testing used for HD patients (15).
Currently, the main technical limitation of NGS is the inadequate for disorders caused by expansion of oligonucleotide repeats and alterations in highly repetitive DNA regions such as in HD (16). Meaning that additional tests such as Southern blot are required to confirm the diagnosis.
Charcot-Marie-Tooth Disease diagnostic tests
A family history of CMT-like symptoms combined with signs of nerve damage from an individual’s physical exam (leg weakness, deep tendon reflexes), suggest CMT or another hereditary neuropathy (17). If the diagnosis is consistent with CMT, a neurologist will arrange a genetic testing (DNA blood test) to detect the most common genetic defects known to cause CMT and perform a nerve conduction velocity (NCV) that measures the strength and speed of electrical signals transmitted through the peripheral nerves (17). Delayed responses are a sign of demyelination and small responses are signs of axonopathy and is often used to distinguish between CMT types; CMT1 and CMT2 (17). Other non-NGS procedures may include electromyography (EMG) that measures the electrical signs in muscles (17).
Whole genome Illumina sequencing has shown to identify both known and novel genes associated with CMTD that has sometimes been missed using the Sanger sequencing in a diagnostic laboratory caused by small indels of the DNA (17). Thus, a gene panel of all known genes associated with CMT will thereafter be able to link with the patient’s phenotyping shown in the clinic and will increase the throughput, meaning more novel genes will be introduced to the panel (17). Using whole genome Illumina sequencing to detect CMT as a diagnosis has been beneficial to some extent as their degree of exome coverage is of insufficient leading to more genes needing to be covered. For example, having the CTMX, where the X chromosome and many GC rich regions are poorly covered by the WGS, a better option would be to have a disease-targeted sequencing. This will allow the analysis of multiple or more genes known to be related to a given phenotype (16).
The main disadvantage however is the interpretation of all the genes that have been screened, as most of them might not be related to CMT. This makes it more difficult to understand the novel gene and identify the potentially relevant mutation as there are no reliable functional tests to prove the pathogenic nature of a given mutation (17).
Next Generation Sequencing ethical, economical and technical factors
One of the most common reasons for referrals to a specialist clinic is the genetic evaluation of patients that wish to start a family (17). Some of these patients will undergo antenatal testing such as amniocentesis to determine the genotype of the embryo (17). While the requests are a minority, it highlights the importance of ensuring that a mutation is indeed pathogenic in an individual patient, but such a process is becoming increasingly complex with the advent of NGS technologies and the subsequent identification of novel genes (as some could be discovered as very rare) (17). It can also have an impact on the patient’s life (work, future living). These may include HD, where the penetrance is not obvious at the start, but drastically changes with time. This highlights some of the ethical dilemmas that both patients and healthcare professionals may face, considering such tests may determine what future the parents may decide for their child.
Clinicians are often faced with the question whether they should start a diagnostic workup with a disease-targeted test or directly with a genomic analysis (WES or WGS), for the sake of time, cost and efficiency (16). For instance, excellent results have been obtained with WES/WGS for investigation of seemingly genetic disorders that present atypical manifestations that are difficult to confirm using simple clinical or laboratory criteria or otherwise require extensive or costly evaluation. These are usually disorders with high clinical and genetic heterogeneity, such as intellectual disability and congenital malformations (16). The cost of implementation including equipment set up, routine sequencing costs for reagents and consumables as well as post-processing bioinformatics costs is an obvious, but significant factor (18). However, the significant requirements in computation resources and time would render such analyses unusable in a clinical environment (18).
RNA-sequencing and Congenital Muscular Dystrophy
Congenital Muscular Dystrophy (CMD) is a genetic condition that is caused by muscle weakness and wasting starting very early in life. This affects the skeletal muscles that are responsible for the body movements. A patient has completed a whole genome sequencing alongside some of its family members to identify a novel mutation. However, as there was a 10Mb interval identified with the disease but with no obvious single nucleotide observed in the exome region, an RNA-sequencing test can be useful to uncover multiple features of transcriptome and to facilitate the biological applications.
The main applications of RNA-seq analysis are novel gene identification, expression, and splicing analysis (19). This is because it has the ability to reveal unannotated protein- and microRNA-coding genes expressed in the cells without prior knowledge of the reference or sequence of interest (20). It also has the ability to quantify the expression of isoforms and unknown transcripts, and splice analyse exons to identify the functional gene and protein diversification in a disease as CMD (20).
Thus, a combination of long-read RNA sequencing and short-read RNA seq of the patient and the family members will enable characterisation of the splicing landscape of CMD by identifying the allele-specific expressions and disease-associated SNPs (19). In this case, single-cell RNA-seq analysis will be more advantageous as it will provide the expression profile of individual cells. Through gene clustering analyses, rare cell types within the cell population can be identified, thereby applying the study of the cellular heterogeneity and diversity in neuroscience. This will make it easier to identify uncommon RNA but also reveal copy number distribution of the whole mRNA population in individual cells that will be more helpful to understand the causal variant that affects the patient (19). RNA-seq is more sensitive in detecting genes with very low expression and more accurate in detecting expression of extremely abundant genes.
The challenges of RNA require comprehensive solutions including differential gene expression analysis and detection of fusion genes (19). RNA-seq is complicated by having multiple-step processes to identify and quantify all RNA species from the reads sequenced (19). Thus, quality assessment is the first step of the bioinformatics pipeline of RNA-seq and a step before analysis (19). It is also necessary to filter data, removing (trimming) low quality sequences or bases adaptors, contaminations or overrepresented sequences to ensure a coherent final result (19). Another problem in reads mapping is that the polymorphisms are especially common for the large and complex transcriptomes. This leads to sequence reads aligning to multiple locations of the genome resulting in unidentified region of interest that is causing the disease (19).
After getting the read counts, data normalisation is one of the most crucial steps of data processing that is essential to ensure accurate inference of gene expression and subsequent analyses thereof (19). However, there are multiple features of the RNA-seq data that can be taken into account including transcript size, GC content, sequencing depth sequencing error rate and insert size (19). RNA-seq has the benefit of delivering low background signal due to the fact that the DNA sequences are unambiguously mapped to unique regions of the genome and as a result the noise in the experiment is easily eliminated during the analysis (20). These limitations include solving big output files that require high level of volume storage memory, huge obtained data needing required powerful and strong tools and equipment like computation units to process and analyse data (20).
In conclusion, the aim of completing whole genome Illumina sequencing in patients in diagnostic laboratories is to have a higher throughput, more efficient, timely and cost-effective method for genetic diagnosis. This is to detect known and novel genes in an entire human genome for various diseases. Currently, the major limiting factor for genetic testing is the pace of discovery of genes potentially relevant to a phenotype and its interpretations. Furthermore, RNA-seq is another high-throughput, quantitative method allowing us to explore the transcriptome of an organism of interest. This has enabled us to potentially identify a variant call causing a patient in a family.
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