Introduction to Molecular Diagnostics

Ianis G. Matsoukas PhD

School of Sport and Biomedical Sciences,

The University of Bolton,
QS32 Queens Specialist Building,
Bolton BL4 7DX.

x3409
E-mail: I.Matsoukas@bolton.ac.uk
 Outline
• Introduction to essential concepts in molecular diagnostics that impact on the identification of
markers of human diseases;
• Distinguish the different types of clinical genetic testing currently used in molecular
diagnostics laboratory globally;
• Explain the steps of developing a molecular genetic test from research laboratory-based
findings;
• Evaluate and judge the international standards behind the principles of analytical validity,
clinical validity, and clinical utility when using genetic testing techniques;
• Describe some examples of the different molecular diagnostic platforms and techniques,
including high throughput sequencing.
 History of Diseases Diagnostics
 The ability of a health care practitioner to efficiently deliver effective care depends upon their
ability to accurately identify the cause of the patient’s problem, i.e., make the diagnosis.
 Over time, our understanding of the mechanistic basis of disease has increased and furthered our
ability to make more accurate and specific diagnoses.
 Early healers needed only to feel the patient’s skin to diagnose (fever), but with further
understanding of different illnesses and their symptoms, doctors gained the ability to distinguish
between yellow fever, scarlet fever, bubonic fever and other types of infections.
 It was the advent of modern microscopy and histopathology, or cellular pathology, in the
nineteenth century, however, which enabled a tremendous leap forward in our understanding of
the biology of disease at a cellular level.
 Today, cell cultures, additional antibiotic sensitivity testing, and genetic information from molecular
diagnostics provide critical additional information.
 Molecular Diagnostics

Molecular diagnostics is the use of the molecular biology techniques to:

1. expand scientific knowledge of natural history of diseases,

2. identify individuals who are at risk of acquiring specific diseases,
3. diagnose infectious and other human diseases at the molecular level.
 Molecular Diagnostics Impact
 Monitor diseases more accurately:
 Allows for early treatment and better patient care.

 Determine most appropriate treatment:

 Reduces or eliminates unnecessary treatment.
 Reduces or eliminates inadequate treatment.
 Greater cost effectiveness.

 Reduce patient morbidity and mortality.

 Types of clinical genetic testing
1. Newborn screening
 Newborn screening is used just after birth to identify genetic disorders that can be treated early in life.
2. Diagnostic testing
 Diagnostic testing is used to identify or rule out a specific genetic or chromosomal condition. In many cases,
genetic testing is used to confirm a diagnosis when a particular condition is suspected based on physical signs
and symptoms.
3. Prenatal testing
 Prenatal testing is used to detect changes in a fetus's genes or chromosomes before birth. This type of testing
is offered during pregnancy if there is an increased risk that the baby will have a genetic or chromosomal
disorder.
4. Carrier testing
 Carrier testing is used to identify people who carry one copy of a gene mutation that, when present in two
copies, causes a genetic disorder.
 Types of clinical genetic testing
5. Preimplantation testing
 Preimplantation testing, also called preimplantation genetic diagnosis (PGD), is a specialized technique that can
reduce the risk of having a child with a particular genetic or chromosomal disorder. It is used to detect genetic
changes in embryos that were created using assisted reproductive techniques such as in-vitro fertilization.
6. Predictive and presymptomatic testing
 Predictive and presymptomatic types of testing are used to detect gene mutations associated with disorders
that appear after birth, often later in life. These tests can be helpful to people who have a family member with
a genetic disorder, but who have no features of the disorder themselves at the time of testing.
7. Forensic testing
 Forensic testing uses DNA sequences to identify an individual for legal purposes. Unlike the tests described
above, forensic testing is not used to detect gene mutations associated with disease. This type of testing can
identify crime or catastrophe victims, rule out or implicate a crime suspect, or establish biological relationships
between people (for example, paternity test).
 Molecular Biomarkers: Definition & Types
 In 1998, the National Institutes of Health Biomarkers Definitions Working Group defined a biomarker as:
Characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic
processes, or pharmacologic responses to a therapeutic intervention.
 Identification of novel molecular markers will facilitate the development of useful molecular assays for detection,
diagnosis, predication of the diseaseoutcomes.
 There are three key types of biomarkers employed for clinical decision-making including:
 diagnostic,
 prognostic, and
 predictive biomarkers.
 Diagnostic biomarkers facilitate identification of a cancer type or subtype.
 Prognostic biomarkers aid clinicians in determining the risk of relapse or disease progression after therapy,
wherein patients with high risk are selected for aggressive screening or adjuvant therapy to prevent recurrence.
 Clinicians utilize predictive biomarkers to select one therapy over others, based on associations between
biomarker results and likelihood of response to certain therapies. In practice, predictive biomarkers often
identify the molecular targets of relevance to targeted anticancer drugs.
 Biomarker Discovery:
Biomarker Development Process
 Genetic Tests: Analytical Validity & Clinical Validity
AnalyticalValidity
 Analytical validity is the test’s ability to accurately detect and measure the biomarker of
interest (i.e., protein, DNA, RNA).
• Are the test results repeatable when performed under identical conditions?
• Are the test results reproducible when the test is performed in different conditions?
• Is the test sensitive enough to detect biomarker levels as they occur in a real-life setting?

ClinicalValidity
 Clinical validity asks the question:
 is the biomarker being tested associated with a disease or outcome or the response to a treatment?
 What is the clinical performance of the test, including its sensitivity (ability to identify those with the
disease), specificity (ability to identify those without the disease), positive predictive value, and negative
predictive value?
 Genetic Tests: Clinical Utility
Clinical Utility
 Clinical utility is perhaps one of you’re the most important when considerations determining whether or not
to order or cover a genetic test.
 While the meaning of the term has some variability depending on the context or source, there is a largely
agreed-upon definition. Four factors are generally considered when evaluating the clinical utility of a genetic
test:
1. Patient outcomes – do the results of the test improve health outcomes (e.g., reduce mortality or
morbidity) or other outcomes that are important to patients such as quality of life?
2. Diagnostic thinking – does the test confirm or change a diagnosis? Does it determine the genetic etiology
for a condition or does it clarify the prognosis?
3. Decision-making guidance – will the test results determine the appropriate dietary, physiological, medical
(including pharmaceutical), and/or surgical intervention?
4. Familial and societal impacts – does the test identify at-risk family members, high-risk race/ethnicities, and
the impact on health systems and/or populations?
 Timelines of major Molecular:
Biology Discoveries

Source: Chapter 1: Human Heredity by Cummings Thompson Learning Brooks/ Cole

 Genomic Variations and Disease Association
 A particular gene whose progression of base pairs is typical of the population at large is often termed the
―wild type or ―normal‖ gene sequence.
 Gene variants that are deviations from the wild type may or may not be clinically significant and
extensive human research is often needed to determine their relevance.
 A gene variant present in more than 1% of the population is called a polymorphism and may be considered a
normal variant.
 When specific mutations, or sets of mutations, are known to be biomarkers associated with a disease or
condition, molecular diagnostic tests can examine a patient’s genes to determine whether those mutations are
present.
 These tests may look only for those certain gene variants, or map the entire sequence of a targeted portion
of DNA to detect all mutations in the sequence.
Molecular Diagnostics Techniques & Platforms
 A number of techniques are employed in modern diagnostics to detect
and quantify specific DNA or RNA sequences, as well as proteins.

 The following are a few examples of these techniques:

1. Polymerase Chain Reaction (PCR)
2. Real-Time qPCR
3. In Situ Hybridization (ISH or FISH)
4. DNA Chips and Microarrays
5. Mass spectrometry (MS)
6. Sanger Sequencing (CE)
7. Next Generation Sequencing (NGS)
 Molecular Diagnostics Techniques & Platforms
1. Polymerase Chain Reaction (PCR)
 One of the essential methods
underlying many molecular
diagnostics is amplification, the
process of making copies of a specific
DNA or RNA sequence found in a
sample (e.g., blood or tumor).
 There are a number of different
amplification technologies, but
Polymerase Chain Reaction (PCR)
testing is the most widely used and is
considered a work horse in molecular
diagnostics.
 There are different PCR types (article on
moodle).
 Molecular Diagnostics Techniques & Platforms
2. Real-Time qPCR
 Real-time qPCR utilizes the PCR reaction to detect targets of
interest.

 Gene-specific primers are used and the target is detected either by

the incorporation of a double-stranded DNA (dsDNA)-specific dye
or by the release of a TaqMan FRET (fluorescence resonance energy
transfer) probe through polymerase 5′−3′ exonuclease activity.

 Developed in the early 1990s, qPCR is widely used in both clinical

and research settings for genotyping, gene expression analysis, CNV
assays and pathogen detection.

 qPCR is extremely rapid and robust, which is beneficial for point-of-

care applications. Its high sensitivity and specificity make it the gold
standard for clinical gene detection with several US FDA-approved
tests.

 The number of simultaneous targets that can be detected is in the

hundreds rather than the thousands for microarrays and NGS.

 This method also requires primers and/or probes designed for

specific targets.
 Molecular Diagnostics Techniques & Platforms
3. In Situ Hybridization (ISH or FISH)
 A technique that unzips DNA or RNA in the
sample and uses a so- called probe—a labeled
DNA or RNA strand that hybridizes with the
target, complementary sequence on an unzipped
strand and thereby identifies and quantifies the
target sequence in the sample.
 FISH stands for Fluorescent In Situ
Hybridization due to its use of a fluorescent
probe that facilitates automated reading of the
results.
 This is distinct from immunohistochemistry,
which usually localizes proteins in tissue sections.
 Molecular Diagnostics Techniques & Platforms
4. Array technologies A. cDNA Micoarray C. Array-CGH protocol flowchart

1. RNA or cDNA (the expression of a large number of

genes).

2. SNP arrays identify common polymorphisms

associated with disease and phenotypes and GWAS.

3. Array-CGH (Comparative genomic hybridization) is

a molecular cytogenetic method for analysing copy
number variations (CNVs) relative to ploidy level in
the DNA of a test sample compared to a reference
sample, without the need for culturing cells. The aim
of this technique is to quickly and efficiently compare
two genomic DNA samples arising from two sources, B. Micoarray data analysis
which are most often closely related, because it is
suspected that they contain differences in terms of Green =
either gains or losses of either whole chromosomes Down-regulated
or subchromosomal regions (a portion of a whole
chromosome). Red = Up-regulated Garnis et al. 2004 Mol Cancer, 3:9.

Yellow= No change
Can identify structural variation, copy number
variants (CNVs) [lower-resolution arrays].
 Molecular Diagnostics Techniques & Platforms
5. Mass spectrometry (MS)
 A technology that determines the molecular
mass of a charged particle by measuring its
mass-to- charge (m/z) ratio.
 This technology is used to find and analyze
protein based biomarkers and is broadly
classified into gel-based and gel- free
techniques.
 Molecular Diagnostics Techniques & Platforms
6. Sanger Sequencing
 Molecular Diagnostics Techniques & Platforms
1st, 2nd & 3rd Sequencing Generation
 Molecular Diagnostics Techniques & Platforms
7. NGS
 A technique used to map out the sequence
of the nucleotides that comprise a strand of
DNS.
 Next Generation Sequencing (NGS)
technology was specifically designed to
simultaneously evaluate variation in several
genes.
 This technology can also be used to detect
different types of geneticalterations.

Escalona M, Rocha S, Posada D. A comparison of tools for the simulation of genomic

next-generation sequencing data. Nature Reviews Genetics,17, 459–469, (2016). doi:10.1038/nrg.2016.57.
Molecular Diagnostics Techniques & Platforms
A Comparison of NGS Platforms
 NGS Applications

• Whole Genome Sequencing (WGS)

• Whole Exome Sequencing (WES)

• Target Re-sequencing

• RNA-Seq

• Chip-Seq

• SNP/Indel/Structural Variation Discovery

Bioinformatics Analysis: Next Lecture
 Successful Molecular Diagnostics Examples
1. Screening/Diagnostic Test Example: Cystic Fibrosis
 One of the most common mutations causing CF is the delta F508 deletion mutation which can be detected
using a number of molecular techniques.
 These tests use DNA amplification methods to amplify key portions of the Cystic Fibrosis Transmembrane
Conductance Regulator (CFTR) gene and look for the mutations that cause CF.

Cytogenetic Location: 7q31.2

https://ghr.nlm.nih.gov/gene/CFTR
 Successful Molecular Diagnostics Examples
2.Therapeutic Decision-Making Example: KRAS
 An example of a molecular diagnostic that improves therapeutic decision-making is K-Ras testing. K- Ras is a
protein, encoded by the KRAS gene, that plays a critical role in cell division, cell differentiation, and the self-
destruction of cells. KRAS mutations that produce an abnormal, overactive K-Ras protein are found in
pancreatic, colorectal, lung and other cancers.
 Detecting a K-RAS mutation is used to determine patient suitability for certain therapies in colorectal and
lung cancer.
 Anti-EGFR (epidermal growth factor receptor) drugs (Erbitux (cetuximab) and Vectibix (panitumumab)) are
key therapies for those cancers, but patients whose cancers contain KRAS typically fail to respond to these
anti-EGFR therapies.
 Therefore, Erbitux and Vectibix labels recommend against prescribing the drugs for patients with certain KRAS
mutations.
 KRAS mutation testing is typically performed for patients with metastatic colorectal cancer or metastatic lung
cancer via direct DNA sequencing or PCR and other amplification-based methods.
 Successful Molecular Diagnostics Examples
3.Therapeutic Monitoring Example:HIV
 Many molecular diagnostics improve monitoring and management of disease.
 Some of the most common monitoring molecular tests are tests for HIV viral load in patients affected with
HIV.
 HIV viral load tests are amplification-based tests that measure the amount of HIV RNA to determine how
many copies of the virus are present (measured in copies per milliliter of blood). Keeping viral levels as low as
possible is key to staying healthy with HIV.
 We have made great strides in the care of HIV patients in the three decades since HIV was first detected,
essentially transitioning it into a chronic disease in many developed countries.
 The HIV viral load test has become an essential tool as patients cycle through different antiretroviral therapies
and develop resistance to certain therapies.
 Periodic HIV viral load tests indicate whether or not the disease is progressing, either because an untreated
patient’s immune system can no longer suppress replication of the virus, or because the therapy for a patient in
treatment is no longer working.
 Successful Molecular Diagnostics Examples
4. Molecular diagnosis of retinoblastoma
 Retinoblastoma (RB) is the most common malignant childhood tumor of the eye and results from inactivation
of both alleles of the RB1 gene.
 Nowadays RB genetic diagnosis requires classical chromosome investigations, Multiplex Ligation-dependent
Probe Amplification analysis (MLPA) and Sanger sequencing.
 Nevertheless, these techniques show some limitations.
 NGS and RB1-custom aCGH have demonstrated to be an effective combined approach in order to
optimize the overall diagnostic procedures of RB.
 Custom aCGH is able to accurately detect genomic rearrangements allowing the characterization of their
extension.
 NGS is extremely accurate in detecting single nucleotide variants, relatively simple to perform, cost effective,
efficient, sensitive and accurate.
Grotta et al. BMC Cancer (2015) 15:841. DOI 10.1186/s12885-015-1854-0.
Array-CGH profiles of large deletions
in patients with URB & TRB

Grotta et al. BMC Cancer (2015) 15:841. DOI 10.1186/s12885-015-1854-0.

Epidermal Growth Factor Receptor gene
(EGFR): EGFR L747P Mutation
 L747P (2239 - 2240 TT>CC) in
exon 19 is a rare EGFR mutation
that appears to lead to
Gefitinib resistanceand might
accelerate liver metastases.

 A CT scan revealed that the

number and relative volume of
the liver metastases had
increased after treatment.
 Sequencing results for EGFR mutation
L747P (2239-2240 TT>CC) on exon 19

EGFR mutation L747P led to gefitinib resistance and accelerated liver metastases in a Chinese patient with lung
adenocarcinoma https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4555769/
 Quality control: Complex challenges
for molecular diagnostics*
 There is a need for sufficient protocols in place to assure the accuracy laboratory genetic test results that could be
achieved through solid quality assurance program including quality control (QC) samples, a validated laboratory test, quality
control monitoring, and error prevention protocols.
 One example of a well thought-out approach is the Tang et al., 2012.

Although the article focuses on RNA profiling using large scale arrays, the principles for quality assurance can
be applied to multiplex DNA sequencing and DNA array testing.
The Tang paper describes the successful adaptation of traditional QC principles to minimize failure risk in a
highly complex test protocol.
However, in light of increasing pressure on healthcare budgets and limited personnel, it is understandable that
the laboratory may ask if equivalent assurance of quality can be provided with a simpler approach.
 In view of the risks and costs of delivering poor quality results, careful consideration should be given to the possible
effects from QC shortcuts and simplifications.

*Tang W, et al. Quality assurance of RNA expression profiling in clinical laboratories. J Mol Diagn. 2012;14(1)1-11.
 Technical issues with NGS implementation
 Bioinformatics methods for sequence alignment keep undergoing rapid improvements
– Need to update bioinformatics pipeline at frequent intervals

 Structural genetic variation:

– Optimal algorithms for detection of copy number variation remain unclear.

 Several regions with inadequate coverage

– Backup Sanger sequencing/alternative methodology necessary for several exons in
the context of inherited disorders – Sensitivity to detect somatic mutations will not
be the same in all the analyzed.
 Molecular Diagnostics: Future Perspectives
 New technologies are making it possible to gather more information to more fully characterize the disease
state, the best treatment alternatives for a patient, or in the case of infectious disease, rapidly identify a
specific pathogen out of many that could be responsible for a particular infection (e.g., respiratory viruses).
 Approaches that may address this need for increasing amounts of data include multi-marker or multiplexed
approaches (methods that can test for multiple biomarkers in a single assay) and gene sequencing that can
detect multiple defects associated with a particular subset of patients with a disease.
 Practical application of these methods can be challenging, due to complexity of the assays involved, but these
challenges are being met by integration of molecular methods with advanced information technology (IT) and
data analytic methods.
 The fast pace of technological development in molecular diagnostics, and the rapidly growing body of research
regarding biomarkers, challenge regulatory bodies to stay current.
 Molecular Diagnostics: Future Perspectives
 Clear understanding of regulatory expectations for multiplex tests, companion diagnostics and next
generation sequencing as well as the increasing investments necessary to generate the clinical evidence
required for approval or clearance will be integral.
 The ability and capacity to capture, store and analyze the tremendous and rapidly increasing amount of
diagnostics data, particularly with gene sequencing tests, is an ongoing challenge. IT systems must have enough
power to process and analyze these huge volumes of information. In addition, another key challenge is the
need for data standards that would speed interpretation of these complex genomic data sets and make the
results relevant for specific patients. This task is further complicated by the fact that the patient’s medical
record is often in many unconnected places, whether in multiple out-patient offices, multiple hospitals, or
multiple pharmacy systems.
 Ethical questions are also being raised about the disclosure and use of genetic information.
 Genetic markers are being identified that can signal the propensity of a patient to develop a disease over their
lifetime and the question has been raised of how, if at all, such information should be disclosed, particularly in
cases where prevention or treatments are not yet available. Policy makers continue to grapple with these
challenges.
 Outline of the Lecture
• Introduction to essential concepts in molecular diagnostics that impact on the identification of
markers of human diseases;
• Distinguish the different types of clinical genetic testing currently used in molecular
diagnostics laboratory globally;
• Explain the steps of developing a molecular genetic test from research laboratory-based
findings;
• Evaluate and judge the international standards behind the principles of analytical validity,
clinical validity, and clinical utility when using genetic testing techniques;
• Describe some examples of the different molecular diagnostic platforms and techniques,
including high throughput sequencing.
Introduction to Molecular Diagnostics
Ianis G. Matsoukas PhD

School of Sport and Biomedical Sciences,

The University of Bolton,
QS32 Queens Specialist Building,
Bolton BL4 7DX.

x3409
E-mail: I.Matsoukas@bolton.ac.uk