Inherited skin diseases can be difficult to assess clinically and often diagnosis relies on multiple laboratory investigations. Traditionally, examination of skin biopsies is followed by biochemical testing and Sanger sequencing of genomic DNA. This approach is labour-intensive, costly and time-consuming. The advent of next-generation sequencing (NGS) methods provides an alternative or complementary approach to making highly accurate diagnoses, but is not without its own challenges.
by J. Lee, Dr A. Salam, Dr T. Takeichi and Prof. J. A. McGrath
Background
The identification of pathogenic mutations in monogenic diseases represents one of the major challenges, and fundamental goals, of early 21st Century human genetics. Most genetic diseases are rare, clinically heterogeneous, and difficult to diagnose – a task made more challenging by disparity in genotype–phenotype correlations, inter- and intra-familial variability, and well as mosaic patterns of disease. It is these hurdles that have led to the advent of Next-Generation DNA Sequencing (NGS); a group of technologies that can improve the speed, accuracy, and cost-efficiency of genetic sequencing, while simultaneously mapping normal variation, and thus furthering our understanding of human genetics in both health and disease. Inherited skin diseases encompass a collection of over 500 clinical entities – with variable structural or inflammatory manifestations that can also affect hair, nails, teeth and certain mucosal surfaces [1]. Individually these disorders are uncommon, but collectively they generate a significant health burden and many diagnostic conundrums.
Traditional approaches to the diagnosis of inherited skin diseases
For patients with inherited skin disorders, the traditional approach to diagnosis is to document a comprehensive patient history, including recording accurate family pedigrees, and noting any consanguinity. The clinician will then go on to perform a physical examination, take clinical photographs, and order laboratory investigations, which often include a skin biopsy. Light microscopy is usually uninformative, and the skin may need to be examined by transmission electron microscopy and immunohistochemistry. Additional blood or urine samples may be need for further diagnostic biochemical studies. Changes in skin structure or protein expression may provide clues to candidate genes, for which polymerase chain reaction primers can be designed and used for Sanger sequencing of genomic DNA. This ‘candidate gene’ approach has proved very useful for several autosomal recessive inherited skin diseases, but is typically unhelpful in most dominant diseases or in those with more subtle changes in skin morphology. Cue the advent of NGS technologies and a different approach to diagnostics, where the challenge in genetic discovery shifts away from the generation of data, to the filtering of relevant data [2, 3].
The impact of NGS
NGS encompasses a number of new technologies that vary in their sequencing protocols, thus determining the type of data produced. The approaches taken vary in template preparation, sequencing and imaging, genome alignment and assembly methods. The methodology is therefore also known as high throughput or massively parallel sequencing due to the ability of NGS to process large volumes of genetic data in a short time, in stark contrast to individual gene screening with Sanger sequencing. Whole-genome sequencing (WGS) and whole-exome sequencing (WES) are the two most commonly used NGS techniques. WGS has the ability to sequence an individual’s entire genome, but at the expense of speed and cost. In contrast, WES uses an array to capture protein-coding regions of the human genome, encompassing ~21,000 genes, which make up less than 2% of the genome. Compared with the 2–3 million variants generated by WGS, the data from WES typically reveals around 25,000 variants. Nevertheless, WES is a more economical option than WGS because ~85% of the pathogenic mutations in monogenic diseases are predicted to be in exons. The plethora of data then has to be filtered, with any potentially disease variant with evidence for causality established (Fig. 1). This process often involves the filtering of variants through databases of previously identified sequences, and cross referencing with known biological or genetic databases, for which considerable bioinformatics support is required: a single WES run can generate one terabyte of data.
Whole-exome sequencing: the possible advantages
The challenge
The key questions for WES in the diagnosis of inherited skin diseases are as follows. (1) Are the new technologies better than what already exist for diagnosing known diseases? (2) Can the new technologies be helpful in resolving unknown diagnoses or discovering new clinical entities? (3) Can the new technologies be introduced into clinical work and overcome any practical obstacles? Emerging data indicate a resounding yes to the first two questions, although the third remains work in progress [4].
Breadth of cover
WES encompasses most of the coding regions of the genome, whereas Sanger sequencing targets a predetermined gene, or part of a gene, between specially designed primers. WES is also efficient for sequencing large genes, such as COL7A1, which encodes type VII collagen. This gene, which is mutated in the blistering disease, dystrophic epidermolysis bullosa, is composed of 118 exons. Conventional Sanger sequencing approaches are based on designing ~72 primer pairs to amplify the COL7A1 exons and flanking introns. Thus the Sanger sequencing approach is therefore laborious and expensive, particularly as COL7A1 contains few recurrent mutations and the gene needs to be screened in its entirety to identify pathogenic mutations.
Genetic diagnosis
WES has emerged as an invaluable tool where a patient’s clinical diagnosis is unclear or erroneous. In this situation, Sanger sequencing of multiple candidate genes is destined to failure and to exhaust both time and resources. WES, on the other hand, can identify known variants in order to make a genetic diagnosis that was not initially considered, as has been demonstrated for subtypes of epidermolysis bullosa, and other inherited skin diseases [5, 6]. Indeed, WES has been used to accurately diagnose inherited skin diseases without any a priori clinical information [7]. The rationale is that more accurate and timely diagnoses offered by WES will allow for earlier targeted therapy and ultimately improved patient care.
Genetic discovery
The value of WES in genetic discovery is evident in the number of inherited skin diseases whose original genetic basis has been informed by WES. Recent examples include the discovery of inherited skin and bowel inflammation resulting from mutations in ADAM17 and EGFR [8, 9]. Given the protean nature of inherited skin diseases, many mutations cannot be anticipated based on clinical phenotype and initial investigations, leaving no candidate gene targets for Sanger sequencing. One pertinent example of the completely unexpected candidate gene is the identification of mutations in EXPH5 [10], which encodes a GTPase effector protein, exophilin-5, in a form of intra-epidermal epidermolysis bullosa – a disease that usually arises as a genetic disorder of keratin. WES is therefore superior to Sanger sequencing in the diagnosis of both novel and genetically heterogeneous conditions.
Cost efficiency
The cost of DNA sequencing has reduced by around 100,000-fold over the last 20 years. Although the technique remains relatively expensive at present (~£900 per sample at King’s College London, 2014 prices), further cost reductions are expected that will soon make WES a more economically viable option than Sanger sequencing, for all but a few disorders in which there are recurrent mutations in a small number of small genes. Even at current costs, however, WES already has advantages over Sanger sequencing for some genes, such as COL7A1, for which the cost of Sanger sequencing is ~£1000 (or greater) in the small number of laboratories that undertake sequencing of this gene.
Considering the patient
The diagnosis of many inherited skin disorders often relies on invasive investigations such as sampling a small piece of skin (punch or ellipse biopsy) (Fig. 2). The procedure involves injection of local anesthetic, which can be painful, and the wound usually heals with a small but evident scar. Occasionally, skin biopsy sites can be complicated by bleeding or infection. WES can be performed using DNA extracted from blood, saliva or tissue samples, and although Sanger sequencing can also be performed on similar templates, for many patients, a skin biopsy would have been necessary to determine the gene(s) for sequencing. Thus WES typically offers a less-invasive approach for the patient.
Variant mapping
Aside from discovering genes and pinpointing mutations in inherited skin diseases, WES also generates a huge amount of other data that can be used to map genetic variation. In the longer term, the dissection of bioinformatics data will lead to a better understanding of the implications of certain variants, refining genotype–phenotype correlation, thus providing insight into individual prognosis, and allowing stratified or personalized medicine and therapeutics.
Whole-exome sequencing: the possible disadvantages
Data analysis
The large quantity of sequencing data generated by WES is potentially also a disadvantage. Before WES can be used in routine clinical practice, fast and efficient filtering techniques must exist to allow clinicians and non-geneticists to interpret WES data and to extract the relevant information in order to manage their patient’s needs. But the plethora of data generated by WES also provides considerably more information beyond the pathogenic mutation itself, including several co-incidental potentially damaging mutations (known as ‘incidental findings’) that are completely unconnected to the primary disease being investigated. What should diagnosticians do with this information? Does it make a difference if the implications are clinically actionable or not? There are clearly several unresolved issues.
Accuracy of data
Given the volume of data produced by WES, it is inevitable that some false positive variants are identified. Most laboratories therefore still elect to confirm mutations via an alternative sequencing platform, generally Sanger sequencing, which is therefore a significant barrier to the routine use of WES in diagnostics. From a technical perspective, NGS methods still need to be improved to cover important regulatory elements such as promoters and enhancers, and poorly annotated parts of the genome. Moreover, if WES is to become a routine diagnostic technique, standardized operating procedures and protocols must be created and implemented. For inherited skin disease diagnostics there would also need to be a realignment of technical wet lab skills (skin microscopy) in favour of computer database and in silico work.
Time to diagnosis
Perhaps the biggest challenge for WES, however, lies in the time it takes to process and analyse a case. For many inherited skin diseases, a rapid diagnosis is often very important to optimize clinical management, for example in neonates with suspected epidermolysis bullosa. The diagnostic approach using skin biopsy assessment followed by Sanger sequencing of candidate genes (implicated by skin biopsy) allows for possible diagnoses to be made within 2 to 3 days. In contrast, the quickest time that WES could be completed (at present) would be a minimum of 5 days, although in practice WES often takes considerably longer to complete and analyse. New platforms to shorten WES protocols are in development, but only when more rapid sample analysis is feasible in a diagnostic lab setting can one really begin to think about wholesale change of diagnostic practice.
Conclusion
Since 2011, WES has proven to be a valuable asset in the diagnosis and discovery of inherited skin diseases. But the adoption of WES into clinical diagnostics diagnosis is still being refined and piloted. WES techniques are constantly being improved to become more accurate, quicker and cost-effective, while enrichment methodologies and sequencing technology become more reproducible and standardized. This progress may allow WES to function independently as the stand alone diagnostic and discovery tool in genetics, negating the need for Sanger sequencing to confirm WES findings. However, as our understanding of the role of non-coding DNA in molecular biology grows, and as WGS is further refined, WES is at risk of being superseded by newer NGS techniques both for genetic discovery diagnostics and prognostics. Innovation looms, but ever it was in molecular genetics.
References
1. Leech SN, Moss C. Br J Dermatol. 2007; 156: 1115–1148.
2. Metzker ML. Genome Res. 2005; 15: 1767–1776.
3. Metzker ML. Nat Rev Genet. 2010; 11: 31–46.
4. Cho RJ, et al. J Invest Dermatol. 2012; 132(E1): E27–28.
5. Takeichi T, et al. Br J Dermatol. 2014; doi: 10.1111/bjd.13190. [Epub ahead of print]
6. Salam A, et al. Matrix Biol. 2013; 33: 35–40.
7. Takeichi T, et al. Exp Dermatol. 2013; 22: 825–831.
8. Blaydon DC, et al. N Engl J Med. 2011; 365: 1502–1508.
9. Campbell P, et al. J Invest Dermatol. 2014; doi: 10.1038/jid.2014.164. [Epub ahead of print].
10. McGrath JA, et al. Am J Hum Genet. 2012; 91: 1115–1121.
The authors
John Lee, Amr Salam BSc, MBChB, MRCP(UK), Takuya Takeichi MD PhD, John A McGrath* MD FRCP
St John’s Institute of Dermatology, King’s College London (Guy’s Campus), London, UK.
*Corresponding author
E-mail: john.mccgrath@kcl.ac.uk
Next generation sequencing and metagenomics – fighting the scourge of antibiotic resistance
, /in Featured Articles /by 3wmediaThe emergence of drug-resistant bacteria is one of the biggest public health challenges today. In April 2014, the World Health Organization (WHO) warned that a “post-antibiotic era – in which common infections and minor injuries can kill” is far from “an apocalyptic fantasy” and has instead become “a very real possibility.” Two months later, the newly formed World Alliance Against Antibiotic Resistance (with over 700 members in 55 countries) launched an urgent appeal to label antibiotic resistance “a grave global threat, and asking that antibiotics be declared a cultural heritage deserving legal protection.”
The ‘horror’ of carbapenem resistance
The march of bacterial resistance seems unremitting, even as the pipeline of new antibiotics is drying up. What is especially worrying for public health authorities, however, is a new “horror”. This refers to the growth of bacterial strains resistant to carbapenem – the ‘last resort’ antibiotic for unresponsive patients.
In several countries, carbapenem does not work in more than half of the people with Klebsiella pneumoniae, a major cause of hospital-acquired respiratory tract infections.
During the month of June 2014 alone, the US saw its first case of carbapenem-resistant (CR) Pseudomonas aeruginosa. In Canada, routine testing of raw squid in Saskatoon revealed a bacterial strain resistant to carbapenem. The case was the first of its kind in a food store, and demonstrates an enhancement of exposure risk “from a relatively small slice of the public” – such as travellers to risk zones or those recently hospitalized – to a much larger sector, according to Joseph Rubin, an assistant professor at the University of Saskatchewan.
Resistant genes are the real challenge
The Canadian case also illustrates the real, long-term challenge facing microbiologists. The bacterium in question, Pseudomonas fluorescens, is not risky for people with healthy immune systems. However, it carries a gene to produce carbapenemase, the enzyme inducing resistance to carbapenem.
Indeed, the real problem is less the spread of antibiotic-resistant bacteria than of antibiotic-resistant genes. Bacteria swap “small bits of DNA that carry genes like those for carbapenem resistance” and then “quickly pass it on to other species through gene swapping.”
A ticking time bomb
The implications are stark, and pose Scylla and Charybdis scenarios for public health authorities.
For example, third generation cephalosporins have been shown to fail in treating gonorrhea in much of Europe, Australia and Japan. Given that over 1 million people are infected globally with gonorrhea, every day, this is clearly a ticking time bomb.
Nevertheless, the use of carbapenems seems ill-advised. As the Canadian Medical Association Journal noted in 2011, this is because “gonorrhea readily shares its antibiotic resistance genes”, and using carbapenems “would invariably result in increased resistance … in other microbes. This life-saving antimicrobial would then have been ‘wasted’ on non–life-threatening gonococcal infections.”
Genomics and molecular methods
The above challenges necessitate a sophisticated arsenal of tools in the microbiology lab. Fortunately, breakthroughs in biotechnology, especially DNA sequencing and genomics, have shown some paths to the future.
In 1995, clinical microbiology witnessed the launch of the genomic era when the first bacterial genome of
Haemophilus influenzae was sequenced. So far, “over 1,000 bacterial genomes and 3,000 viral genomes, including representatives of all significant human pathogens,” have been sequenced – leading in turn to “unprecedented advances in pathogen diagnosis and genotyping and in the detection of virulence and antibiotic resistance.”
Sophisticated genomic tools based on molecular array techniques have been key to this success. Backed by high-speed computing, automated platforms and bioinformatics software, they provide labs with a host of new capabilities.
Molecular methods principally address a major limitation with previous phenotypic methods: the long period (weeks or even months) required to isolate some slow-growing bacteria. Indeed, the effort on the Haemophilus influenzae genome in 1995 had taken over a year, and although the Sanger sequencing used for this had long been considered a ‘gold standard’ , it soon faced “inherent limitations in throughput, scalability, speed and resolution.”
NGS offers exponential leap in sequencing
One of the most promising new molecular techniques is next-generation sequencing (NGS), which is also referred to as second-generation sequencing or SGS.
In terms of its basic principle, NGS parallels the capillary electrophoresis (CE) used in Sanger sequencing, with DNA fragments identified by signals as they are resynthesized from a template strand. However, while CE is limited to one or a few DNA fragments, NGS uses massively parallel processes to cover millions, and sequence several human genomes in a single run within days. This allows for identification of DNA base pairs covering whole genomes, and compare genetic differences down to resolution of a single base pair.
The latest NGS systems can generate over 300 Gb per flow cell, discover SNPs and chromosomal rearrangements, conduct transcriptome analysis, generate expression profiles, detect splice variants and quantify protein-DNA interactions.
Predicting antibiotic resistance, rapidly
NGS has already been used as a frontline weapon to identify bacterial pathogens and conduct epidemiological typing to define transmission pathways and support outbreak investigations – including cholera in Haiti in 2010 and and E. coli O104:H4 in Germany in 2011.
The technique is also seen as a way to predict antibiotic resistance, complementing phenotypic tests with the high-speed investigation of anomalies in results. It also resolves some of the biggest hurdles confounding traditional techniques, which require isolating resistance from environmental samples by PCR amplification or the cloning of cultured bacteria.
Both techniques, however, ignore large reservoirs of potential antibiotic resistance. PCR is incapable of broad-spectrum screening and is generally limited to known resistance genes. A bigger problem is that several bacteria are simply not culturable. This is an especially major challenge. Given that most antibiotics are produced by environmental microorganisms, most antibiotic resistance genes are likely to also have emerged outside a clinical setting.
Along with the new field of metagenomics, NGS has been harnessed to directly address these limitations.
The promise of metagenomics
Metagenomics was developed in the late 1990s for the function-based analysis of mixed environmental DNA species – and the existence of genes or genetic variations causing resistance. Metagenomics was directly aimed to address limitations in culturing and PCR amplification.
In its early years, metagenomics was principally targeted at recovery of novel biomolecules from environmental samples. The emergence of NGS, however, opened up wholly new frontiers – above all, to allow a fraction of the DNA in the sample to be isolated and sequenced, without cloning.
Metagenomics has been used to identify a range of antibiotic resistance genes, including tetracycline, aminoglycosides, bleomycin and β-lactamase.
The fight against resistant bacteria is likely to intensify in the years to come. In May 2014, ‘Cell Biology’ published the results of “the largest metagenomic search for antibiotic resistance genes in the DNA sequences of microbial communities from around the globe.” The findings illustrate the scope of the challenge: “bacteria carrying those vexing genes turn up everywhere in nature that scientists look for them.”
Challenges ahead
NGS is no doubt going to play a major role in this process. In spite of its novelty, it has already proven to be user-friendly for clinical laboratories. Last year, researchers showed it to be capable of integrating whole genome sequencing into the routine, daily workflow of a laboratory and rapidly resolve complex bacterial populations. NGS succeeded in identifying all ‘mixed-sample’ organisms taken from primary isolation plates. These included strains with scarce reads, notwithstanding an extremely low depth of coverage across its genome.
However, there is still some way to go. Key issues include the need to establish a reference genome database to archive, access and exchange the massive amount of data which is still to be generated. Some experts have called for the database to be open and accessible to the global scientific community.
A related challenge is that of interdisciplinary skills and collaboration. The analysis of NGS data requires a variety of specialists, namely “clinical and biomedical informaticians, computational biologists, molecular pathologists, programmers, statisticians, biologists, as well as clinicians.” Given that laboratories or other single institutions are unlikely to have all these skills in-house, an open database would again be the best means to find collaborative solutions.
Limits to Moore’s Law
The greatest driver of NGS adoption will be cost. The Human Genome Project cost around USD 3 billion. NGS can reduce this to “a few thousand dollars”, and achieve it much faster.
So far, NGS seems to be one of a handful of technologies challenging Moore’s Law, which describes a long-term trend of computing power doubling every two years. Compared to $95.2 million in costs per genome estimated in autumn 2001, the figure has since fallen by over 25,000 times. The decline has however not been uniform. Typically, costs had been dropping at an average of 70% a year between 2001 and 2007. In 2008, however, they fell massively, to $342,500 per genome in October compared to $7.1 million the previous year. This was wholly due to the move from Sanger-based to NGS sequencing technologies.
Costs per genome were estimated to be just over $4,000 in January 2014.
NGS, of course, has a variety of uses. It is seen as a tool to revolutionize molecular biology, molecular epidemiology and predict the evolution of bacteria. However, the fight against the menace of antibiotic resistance is likely to be one of its highest profile uses in the years to come.
NGS for inherited skin diseases
, /in Featured Articles /by 3wmediaInherited skin diseases can be difficult to assess clinically and often diagnosis relies on multiple laboratory investigations. Traditionally, examination of skin biopsies is followed by biochemical testing and Sanger sequencing of genomic DNA. This approach is labour-intensive, costly and time-consuming. The advent of next-generation sequencing (NGS) methods provides an alternative or complementary approach to making highly accurate diagnoses, but is not without its own challenges.
by J. Lee, Dr A. Salam, Dr T. Takeichi and Prof. J. A. McGrath
Background
The identification of pathogenic mutations in monogenic diseases represents one of the major challenges, and fundamental goals, of early 21st Century human genetics. Most genetic diseases are rare, clinically heterogeneous, and difficult to diagnose – a task made more challenging by disparity in genotype–phenotype correlations, inter- and intra-familial variability, and well as mosaic patterns of disease. It is these hurdles that have led to the advent of Next-Generation DNA Sequencing (NGS); a group of technologies that can improve the speed, accuracy, and cost-efficiency of genetic sequencing, while simultaneously mapping normal variation, and thus furthering our understanding of human genetics in both health and disease. Inherited skin diseases encompass a collection of over 500 clinical entities – with variable structural or inflammatory manifestations that can also affect hair, nails, teeth and certain mucosal surfaces [1]. Individually these disorders are uncommon, but collectively they generate a significant health burden and many diagnostic conundrums.
Traditional approaches to the diagnosis of inherited skin diseases
For patients with inherited skin disorders, the traditional approach to diagnosis is to document a comprehensive patient history, including recording accurate family pedigrees, and noting any consanguinity. The clinician will then go on to perform a physical examination, take clinical photographs, and order laboratory investigations, which often include a skin biopsy. Light microscopy is usually uninformative, and the skin may need to be examined by transmission electron microscopy and immunohistochemistry. Additional blood or urine samples may be need for further diagnostic biochemical studies. Changes in skin structure or protein expression may provide clues to candidate genes, for which polymerase chain reaction primers can be designed and used for Sanger sequencing of genomic DNA. This ‘candidate gene’ approach has proved very useful for several autosomal recessive inherited skin diseases, but is typically unhelpful in most dominant diseases or in those with more subtle changes in skin morphology. Cue the advent of NGS technologies and a different approach to diagnostics, where the challenge in genetic discovery shifts away from the generation of data, to the filtering of relevant data [2, 3].
The impact of NGS
NGS encompasses a number of new technologies that vary in their sequencing protocols, thus determining the type of data produced. The approaches taken vary in template preparation, sequencing and imaging, genome alignment and assembly methods. The methodology is therefore also known as high throughput or massively parallel sequencing due to the ability of NGS to process large volumes of genetic data in a short time, in stark contrast to individual gene screening with Sanger sequencing. Whole-genome sequencing (WGS) and whole-exome sequencing (WES) are the two most commonly used NGS techniques. WGS has the ability to sequence an individual’s entire genome, but at the expense of speed and cost. In contrast, WES uses an array to capture protein-coding regions of the human genome, encompassing ~21,000 genes, which make up less than 2% of the genome. Compared with the 2–3 million variants generated by WGS, the data from WES typically reveals around 25,000 variants. Nevertheless, WES is a more economical option than WGS because ~85% of the pathogenic mutations in monogenic diseases are predicted to be in exons. The plethora of data then has to be filtered, with any potentially disease variant with evidence for causality established (Fig. 1). This process often involves the filtering of variants through databases of previously identified sequences, and cross referencing with known biological or genetic databases, for which considerable bioinformatics support is required: a single WES run can generate one terabyte of data.
Whole-exome sequencing: the possible advantages
The challenge
The key questions for WES in the diagnosis of inherited skin diseases are as follows. (1) Are the new technologies better than what already exist for diagnosing known diseases? (2) Can the new technologies be helpful in resolving unknown diagnoses or discovering new clinical entities? (3) Can the new technologies be introduced into clinical work and overcome any practical obstacles? Emerging data indicate a resounding yes to the first two questions, although the third remains work in progress [4].
Breadth of cover
WES encompasses most of the coding regions of the genome, whereas Sanger sequencing targets a predetermined gene, or part of a gene, between specially designed primers. WES is also efficient for sequencing large genes, such as COL7A1, which encodes type VII collagen. This gene, which is mutated in the blistering disease, dystrophic epidermolysis bullosa, is composed of 118 exons. Conventional Sanger sequencing approaches are based on designing ~72 primer pairs to amplify the COL7A1 exons and flanking introns. Thus the Sanger sequencing approach is therefore laborious and expensive, particularly as COL7A1 contains few recurrent mutations and the gene needs to be screened in its entirety to identify pathogenic mutations.
Genetic diagnosis
WES has emerged as an invaluable tool where a patient’s clinical diagnosis is unclear or erroneous. In this situation, Sanger sequencing of multiple candidate genes is destined to failure and to exhaust both time and resources. WES, on the other hand, can identify known variants in order to make a genetic diagnosis that was not initially considered, as has been demonstrated for subtypes of epidermolysis bullosa, and other inherited skin diseases [5, 6]. Indeed, WES has been used to accurately diagnose inherited skin diseases without any a priori clinical information [7]. The rationale is that more accurate and timely diagnoses offered by WES will allow for earlier targeted therapy and ultimately improved patient care.
Genetic discovery
The value of WES in genetic discovery is evident in the number of inherited skin diseases whose original genetic basis has been informed by WES. Recent examples include the discovery of inherited skin and bowel inflammation resulting from mutations in ADAM17 and EGFR [8, 9]. Given the protean nature of inherited skin diseases, many mutations cannot be anticipated based on clinical phenotype and initial investigations, leaving no candidate gene targets for Sanger sequencing. One pertinent example of the completely unexpected candidate gene is the identification of mutations in EXPH5 [10], which encodes a GTPase effector protein, exophilin-5, in a form of intra-epidermal epidermolysis bullosa – a disease that usually arises as a genetic disorder of keratin. WES is therefore superior to Sanger sequencing in the diagnosis of both novel and genetically heterogeneous conditions.
Cost efficiency
The cost of DNA sequencing has reduced by around 100,000-fold over the last 20 years. Although the technique remains relatively expensive at present (~£900 per sample at King’s College London, 2014 prices), further cost reductions are expected that will soon make WES a more economically viable option than Sanger sequencing, for all but a few disorders in which there are recurrent mutations in a small number of small genes. Even at current costs, however, WES already has advantages over Sanger sequencing for some genes, such as COL7A1, for which the cost of Sanger sequencing is ~£1000 (or greater) in the small number of laboratories that undertake sequencing of this gene.
Considering the patient
The diagnosis of many inherited skin disorders often relies on invasive investigations such as sampling a small piece of skin (punch or ellipse biopsy) (Fig. 2). The procedure involves injection of local anesthetic, which can be painful, and the wound usually heals with a small but evident scar. Occasionally, skin biopsy sites can be complicated by bleeding or infection. WES can be performed using DNA extracted from blood, saliva or tissue samples, and although Sanger sequencing can also be performed on similar templates, for many patients, a skin biopsy would have been necessary to determine the gene(s) for sequencing. Thus WES typically offers a less-invasive approach for the patient.
Variant mapping
Aside from discovering genes and pinpointing mutations in inherited skin diseases, WES also generates a huge amount of other data that can be used to map genetic variation. In the longer term, the dissection of bioinformatics data will lead to a better understanding of the implications of certain variants, refining genotype–phenotype correlation, thus providing insight into individual prognosis, and allowing stratified or personalized medicine and therapeutics.
Whole-exome sequencing: the possible disadvantages
Data analysis
The large quantity of sequencing data generated by WES is potentially also a disadvantage. Before WES can be used in routine clinical practice, fast and efficient filtering techniques must exist to allow clinicians and non-geneticists to interpret WES data and to extract the relevant information in order to manage their patient’s needs. But the plethora of data generated by WES also provides considerably more information beyond the pathogenic mutation itself, including several co-incidental potentially damaging mutations (known as ‘incidental findings’) that are completely unconnected to the primary disease being investigated. What should diagnosticians do with this information? Does it make a difference if the implications are clinically actionable or not? There are clearly several unresolved issues.
Accuracy of data
Given the volume of data produced by WES, it is inevitable that some false positive variants are identified. Most laboratories therefore still elect to confirm mutations via an alternative sequencing platform, generally Sanger sequencing, which is therefore a significant barrier to the routine use of WES in diagnostics. From a technical perspective, NGS methods still need to be improved to cover important regulatory elements such as promoters and enhancers, and poorly annotated parts of the genome. Moreover, if WES is to become a routine diagnostic technique, standardized operating procedures and protocols must be created and implemented. For inherited skin disease diagnostics there would also need to be a realignment of technical wet lab skills (skin microscopy) in favour of computer database and in silico work.
Time to diagnosis
Perhaps the biggest challenge for WES, however, lies in the time it takes to process and analyse a case. For many inherited skin diseases, a rapid diagnosis is often very important to optimize clinical management, for example in neonates with suspected epidermolysis bullosa. The diagnostic approach using skin biopsy assessment followed by Sanger sequencing of candidate genes (implicated by skin biopsy) allows for possible diagnoses to be made within 2 to 3 days. In contrast, the quickest time that WES could be completed (at present) would be a minimum of 5 days, although in practice WES often takes considerably longer to complete and analyse. New platforms to shorten WES protocols are in development, but only when more rapid sample analysis is feasible in a diagnostic lab setting can one really begin to think about wholesale change of diagnostic practice.
Conclusion
Since 2011, WES has proven to be a valuable asset in the diagnosis and discovery of inherited skin diseases. But the adoption of WES into clinical diagnostics diagnosis is still being refined and piloted. WES techniques are constantly being improved to become more accurate, quicker and cost-effective, while enrichment methodologies and sequencing technology become more reproducible and standardized. This progress may allow WES to function independently as the stand alone diagnostic and discovery tool in genetics, negating the need for Sanger sequencing to confirm WES findings. However, as our understanding of the role of non-coding DNA in molecular biology grows, and as WGS is further refined, WES is at risk of being superseded by newer NGS techniques both for genetic discovery diagnostics and prognostics. Innovation looms, but ever it was in molecular genetics.
References
1. Leech SN, Moss C. Br J Dermatol. 2007; 156: 1115–1148.
2. Metzker ML. Genome Res. 2005; 15: 1767–1776.
3. Metzker ML. Nat Rev Genet. 2010; 11: 31–46.
4. Cho RJ, et al. J Invest Dermatol. 2012; 132(E1): E27–28.
5. Takeichi T, et al. Br J Dermatol. 2014; doi: 10.1111/bjd.13190. [Epub ahead of print]
6. Salam A, et al. Matrix Biol. 2013; 33: 35–40.
7. Takeichi T, et al. Exp Dermatol. 2013; 22: 825–831.
8. Blaydon DC, et al. N Engl J Med. 2011; 365: 1502–1508.
9. Campbell P, et al. J Invest Dermatol. 2014; doi: 10.1038/jid.2014.164. [Epub ahead of print].
10. McGrath JA, et al. Am J Hum Genet. 2012; 91: 1115–1121.
The authors
John Lee, Amr Salam BSc, MBChB, MRCP(UK), Takuya Takeichi MD PhD, John A McGrath* MD FRCP
St John’s Institute of Dermatology, King’s College London (Guy’s Campus), London, UK.
*Corresponding author
E-mail: john.mccgrath@kcl.ac.uk
Assessment of tumour markers on the Maglumi 2000 Chemiluminescence Immunoassay System
, /in Featured Articles /by 3wmediaTumour markers have been widely used in clinical settings for early cancer detection, diagnosis, prognosis and recurrence surveillance. Due to the growing usage, it is of vital importance to assess the performance of common tumour markers on in-vitro diagnosis instruments. In this study, the most commonly used tumour markers have been selected to evaluate the performance of the SNIBE Maglumi 2000 chemiluminescence immunoassay system by comparing with our reference methods.
by Dr Xiao Hu, Dr Sheng Kang, Zhiyun Duan and Professor Guichen Zhang
Background
Tumour markers are substances that rise abnormally in the body when cancer is present. They are useful indicators for cancer risk determination, screening, diagnosis, prognosis, post-treatment surveillance and recurrence monitoring [1]. Alpha-fetoprotein (AFP) is a well established marker in liver cancer diagnosis and post-treatment monitoring [2]. Another well studied tumour marker, prostate-specific antigen (PSA), is recommended for the screening of prostate cancer with men over 50 years old [3]. Carcinoembryonic antigen (CEA) is particularly used as a tumour marker for bowel cancer. It measures the response to treatment and monitors whether the disease has revisited [4]. Elevated serum ferritin has been found in patients with pancreatic cancer, breast cancer, colon cancer, non small cell lung cancer, hepatocellular carcinoma and Hodgkin’s lymphoma [5]. Cancer antigen 125 (CA 125) is a marker commonly used for following up patients with ovarian cancer after treatment [6], while cancer antigen 15-3 (CA 15-3) is widely used for breast cancer management [7]. Cancer antigen 19-9 (CA 19-9) is the best validated marker for pancreatic cancer post-treatment evaluation [8]. Cytokeratin 19 fragment (CYFRA 21-1) and squamous cell carcinoma antigen (SCCA) are useful markers for lung cancer diagnosis in combination with other markers [9] [10]. This study has evaluated the performance of ten tumour markers on the SNIBE Maglumi 2000 chemiluminescence immunoassay system.
Precision
According to the principle and method of the CLSI EP5-A2 guideline [11], we made some adjustments to evaluate the precision of ten tumour markers. Intra-assay precision was evaluated on three different levels of serum samples. Each sample was repeatedly measured for 20 times in the same run to calculate the coefficient of variation (CV%). Inter-assay precision was assessed by repeatedly measuring three different levels of samples for 10 days with the same batch of kit. Samples were run in duplicates, two runs per day with at least 3 hours time interval to calculate the coefficient of variation. The results are displayed as mean value and CV%. Table 1 lists the precision results of ten tumour markers. The CVs of the intra- and inter-assays were less than 4.12% and 6.67% respectively (Table 1).
Method comparison
Serum samples from patients with benign diseases to various cancers were offered by the clinical laboratory of our hospital. The patient names were coded with confidentiality. The samples were measured by our reference system and the SNIBE Maglumi 2000 system to form correlation dot plots. Concordance between SNIBE Maglumi 2000 and reference systems for each tumour marker was analysed. For each tested marker, the number of serum samples is ranged from 166 to 460.
By comparing with our reference methods, good correlations were shown between the SNIBE Maglumi 2000 and the ROCHE Cobas e601 or the ABBOTT Architect i2000. The slopes for all markers were between 0.853 and 1.361 while the intercepts ranges from -2.515 to +5.138 (Figure 1A-J). Total PSA has the highest correlation between the SNIBE Maglumi 2000 and the ROCHE Cobas e601 while the lowest relevance (R2=0.981) was seen in CA 19-9 between the SNIBE Maglumi 2000 and the ROCHE Cobas e601 (Figure 1). The total coincidence rate is between 93.7% (Figure 1I) and 99.6% (Figure 1B).
Conclusion
In this study, we have evaluated the performance of the SNIBE Maglumi 2000 chemiluminescence immunoassay system via ten tumour markers. The intra-assay precision and inter-assay precision for all markers examined here are highly acceptable. By comparing with our reference methods, a high correlation has been shown for all markers tested with the SNIBE Maglumi 2000 system. The total coincidence rate is within the acceptable range for all markers examined. To conclude, the SNIBE Maglumi 2000 system is reliable for the measurement of tumour markers in clinical use.
References
1. Sturgeon CM, Hoffman BR, Chan DW, et al. National academy of clinical biochemistry laboratory medicine practice guidelines for use of tumour markers in clinical practice: Quality requirements. Clin Chem. 2008; 54 (8): e1-e10.
2. Manini MA, Sangiovanni A, Fornari F, et al. Clinical and economical impact of 2010 AASLD guidelines for the diagnosis of hepatocellular carcinoma. J Hepatol. 2014; 60 (5): p995-1001.
3. Qaseem A, Barry MJ, Denberg TD, et al. Screening for prostate cancer: a guidance statement from the clinical guidelines committee of the American College of Physicians. Ann Intern Med. 2013; 158 (10): 761-769.
4. Labianca R, Nordlinger B, Beretta GD, et al. Primary colon cancer: ESMO Clinical Practice Guidelines for diagnosis, adjuvant treatment and follow-up. Ann Oncol. 2010; 21 (S5): v70-v77.
5. Alkhateeb AA, Connor JR. The significance of ferritin in cancer: Anti-oxidation, inflammation and tumorigenesis. BBA. 2013; 1836 (2): 245-254.
6. Forstner R, Sala E, Kinkel K, et al. ESUR guidelines: ovarian cancer staging and follow-up. Eur Radiol. 2010; 20: 2773-2780.
7. Sandri MT, Salvatici M, Botteri E, et al. Prognostic role of CA15.3 in 7942 patients with operable breast cancer. Breast Cancer Res Treat. 2012; 132:317–326.
8. Duffy MJ, Sturgeon C, Lamerz R, et al. Tumor markers in pancreatic cancer: a European Group on Tumor Markers (EGTM) status report. Ann Oncol. 2010; 21: 441-447.
9. Molina R, Auge JM, Escudero JM, et al. Mucins CA 125, CA 19.9, CA 15.3 and TAG-72.3 as tumour markers in patients with lung cancer: comparison with CYFRA 21-1, CEA, SCC and NSE. Tumour Biol. 2008; 29:371–380.
10. Chu XY, Hou XB, Song WA, et al. Diagnostic values of SCC, CEA, Cyfra21-1 and NSE for lung cancer in patients with suspicious pulmonary masses: A single center analysis. Cancer Biol Ther. 2011; 11(12): 995-1000.
11. Tholen DW, Kallner A, Kennedy JW, et al. Evaluation of precision performance of quantitative measurement methods; approved guidelines-second edition, EP5 A2. 2004; 24(25).
The authors
Xiao Hu* MD, Sheng Kang PhD, Zhiyun Duan MSc, Dept of Clinical Laboratory, Shenzhen Sixth People’s Hospital, Shenzhen, Guangdong 518052, China
Guichen Zhang Professor, PhD, MD, Medical College, Shenzhen University, Shenzhen, Guangdong 518052 China
(*Corresponding author: xiao121386@163.com)
Mass spectrometry: the gold standard in clinical routine
, /in Featured Articles /by 3wmediaThe application of mass spectrometry has evolved considerably since its first use and mass spectrometric methods were initially introduced in laboratory medicine approximately 40 years ago [1]. The very recent popularity of clinical mass spectrometry can be attributed to the high specificity, accuracy and reliability due to the direct analysis of ions without the risk of cross reactivity as described for antibody detection in immunoassays [2] as well as the ability to detect multi-analytes in a single run. Initially, GC-MS was used for biological analysis, however, this method requires volatile analytes, demanding extensive extraction and derivatization steps for nonvolatile and thermally unstable compounds typically found in clinical analysis. This is not particularly attractive in a clinical setting, in contrast to LC-MS/MS which offers the advantages of mass spectrometry analysis in combination with a simpler sample preparation technique.
by Dr Nihâl Yüksekdag, Dr Marc Egelhofer and Dr Richard Lukacin
One such example is the analysis of methylmalonic acid (MMA), an important biomarker for the identification of vitamin B12 deficiency which, if left untreated, can lead in the long term to permanent neurological damage and/or to hematological and gastroenterological diseases. The sole determination of holoTC, the active form of vitamin B12, does not have the same diagnostic significance as the combined measurement of holoTC and MMA, as the MMA concentration shows a possible vitamin B12 deficiency even before the actual vitamin level decreases [3]. Traditionally, the reference method for this parameter in plasma/serum is GC-MS which, as mentioned above, requires an extremely complex sample preparation that can take several hours [4]. In contrast to this, the sample preparation for LC-MS/MS from Chromsystems is much easier, and, with just a few minutes processing time, considerably faster, while requiring only one quarter of the sample material (see table 1).
Furthermore, data from plasma and urine MMA determinations by the reference GC-MS method and the new LC-MS/MS technology show a strong correlation and excellent agreement (Fig. 1). Therefore, the described LC-MS/MS technique represents a fast, reliable and robust method for routine analysis, achieving a higher throughput and higher efficiency.
Sample preparation as a pivotal step
The correct analytical procedure from extraction and sample preparation, through to the chromatography and MS setup is a prerequisite to achieve optimal results by mass spectrometry, and to fulfil the requirements in clinical diagnostics. The development of an appropriate sample preparation procedure can be complicated and time-consuming, requiring considerable work in order to sensibly embed it in the overall analytical procedure. The ultimate goal is the enrichment of the molecule of interest by a simultaneous elimination of compounds that cause ion suppression or enhancement effects. Moreover, components from plastic, chemicals like salts or particularly from the human matrix (whole blood, serum, plasma, urine), potentially co-eluting from the LC system can compete with the analytes during the ionization process. This leads to a change in compound ionization, and consequently alters the MS signal at the detector [5]. This process is called “ion suppression” and Bonfiglio et al [6] systematically analysed these effects and have found not surprisingly that they are dependent on the sample preparation technique used as well as the compound to be analysed. More polar analytes also showed stronger effects than less polar ones. Short-term variations in ionization can also compromise the accuracy of analyses, if the method is not sufficiently robust. If these variations have a differential impact on the target analyte and internal standard, the overall analysis is affected [7]. The authors also concluded the need for calibration material to be as similar as possible to the sample matrix. In addition, the choice of an appropriate internal standard helps to reduce matrix effects; whenever possible, an isotopically labelled version of the analyte is the ideal choice.
Depending on sample specimens and analyte characteristics, sample preparation techniques can encompass liquid-liquid extraction, solid phase extraction or protein precipitation and are also crucial for the removal of materials that may contaminate the column, trap-column or the analytical system.
Considering all of these factors, successful method development where all parameters work well within at least acceptable levels of CVs, recovery and appropriate limits of quantification (LOQs) can be very challenging. Furthermore, full establishment of a method that is comprehensively validated in the laboratory is a laborious process. The use of commercially available kits, like the one mentioned above for MMA, which have gone through numerous optimization, verification and validation processes from sample preparation through to MS analysis represents a secure, robust and time-saving alternative for clinical laboratories.
Multi-analyte determination
The capability of LC-MS/MS systems for the analysis of several compounds in a single run sounds efficient and relevant, e.g. for the simultaneous analysis of drugs and their metabolites, but may not be as easy as it seems. Every single analyte in a patient sample may possess different chemical and physical properties that affect its recovery in the sample preparation procedure. Consequently, some compounds may be extracted more efficiently than others. Therefore, it can be a highly complex task with a significant amount of work to develop a general sample preparation procedure for quantification of numerous drugs and metabolites, with many of them being analysed in a single run (see Fig. 2), aimed at simplifying the laboratory workflow.
Automation for a higher throughput
One of the major challenges clinical laboratories have been facing is the simplification and acceleration of sample preparation for LC-MS/MS. By using an automated workflow potential pipetting errors can be minimized and, in parallel, the throughput can be drastically increased. This is relevant, for example, to large transplant centres that analyse a high number of patient samples for immunosuppressive drugs, but nevertheless need to achieve fast and reliable results by LC-MS/MS. To date, there is only one system on the market (MassSTAR) that allows a fully automated CE-IVD workflow for immunosuppressants including sample tracking, LIMS connectivity and clotting detection. The automated method offers a time saving of approximately 80% compared to manual preparation. A comparison between manual and automated sample preparation and measurement techniques for the four immunosuppressants cyclosporine A, everolimus, sirolimus and tacrolimus showed very high correlations (Fig. 3). Automated and manual preparation procedures therefore produce almost the same results, with automation reducing the time needed for sample extraction while also increasing sample throughput. These automation options are also provided by Chromsystems for other parameters, such as vitamin D3/D2, the immunosuppressant mycophenolic acid and antiepileptics, for which comparable correlations between the manual and the automated methods have also been shown.
A gold standard in routine
LC-MS/MS is a valuable technique that is often used in reference methods for a wide range of parameters. Its main drivers for growth in clinical laboratories are the limitations of immunoassays for low molecular weight compounds, the easier workflows and higher throughput [8]. However, there are certain downfalls that need to be addressed with one of the most, or even the most critical factor in clinical mass spectrometry being the application of an appropriate sample preparation procedure that is robust as well as reliably fulfilling analytical requirements. A number of proven and CE-IVD approved LC-MS/MS kits for sample preparation from Chromsystems are available and simplify the workflow in the laboratory. Furthermore, automation is also possible for a range of parameters, reducing hands-on time and increasing throughput for those laboratories with the need for higher throughput.
References
1. Vogeser M, Kirchhoff F (2011) Progress in automation of LC-MS in laboratory medicine. Clin Biochem 44(1): 4-13.
2. Korecka M, Shaw L, (2009) Review of the newest HPLC methods with mass spectrometry detection for determination of immunosuppressive drugs in clinical practice. Ann. Transplant 14(2): 61-72.
3. Obeid R, (2014) Methylmalonic acid – a biomarker for vitamin B12 deficiency. DIALOG 1/2014.
4. Obeid R, Geisel J, Kirchhoff F, Bernhardt K, Ranke D, Lukačin R. (2014) External validation of a novel commercially available mass spectrometry kit for MMA in serum/plasma and urine. Poster presented at the congress of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) WorldLab, Istanbul, Turkey.
5. Schneider H, Steimer W. (2006) Tandem mass spectrometry in drug monitoring: experience and pitfalls in application. J Lab Med 30(6): 428-437.
6. Bonfiglio R, King RC, Olah TV, Merkle K. (1999) The effects of sample preparation methods on the variability of the electrospray ionization response for model drug compounds. Rapid Commun Mass Spectrom 13(12): 1175-1185.
7. Vogeser M, Seger C. (2010) Pitfalls associated with the use of liquid chromatography-tandem mass spectrometry in the clinical laboratory. Clin Chem 56(8): 1234-1244.
8. Grebe S, Singh R. (2011) LC-MS/MS in the Clinical Laboratory – Where to From Here? Clin Biochem Rev 32: 5-31.
The authors
Nihâl Yüksekdağ PhD, Marc Egelhofer PhD*, and Richard Lukačin PhD.
Chromsystems Instruments & Chemicals GmbH, Am Haag 12, 82166 Gräfelfing, Germany
*Corresponding author, egelhofer@chromsystems.de
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