Molecular diagnostics

Specialist using "Qiasymphony", an automation platform for molecular diagnostic tests

Molecular diagnostics is a collection of techniques used to analyse biological markers in the genome and proteomethe individual's genetic code and how their cells express their genes as proteinsby applying molecular biology to medical testing. The technique is used to diagnose and monitor disease, detect risk, and decide which therapies will work best for individual patients.[1][2](foreword)

By analysing the specifics of the patient and their disease, molecular diagnostics offers the prospect of personalised medicine.[3]

These tests are useful in a range of medical specialisms, including infectious disease, oncology, human leukocyte antigen typing (which investigates and predicts immune function), coagulation, and pharmacogenomicsthe genetic prediction of which drugs will work best.[4](v-vii) They overlap with clinical chemistry (medical tests on bodily fluids).


The field of molecular biology grew in the late twentieth century, as did its clinical application. In 1980, Yuet Wai Kan et al. suggested a prenatal genetic test for Thalassemia that did not rely upon DNA sequencingthen in its infancybut on restriction enzymes that cut DNA where they recognised specific short sequences, creating different lengths of DNA strand depending on which allele (genetic variation) the fetus possessed.[5] In the 1980s, the phrase was used in the names of companies such as Molecular Diagnostics Incorporated[6] and Bethseda Research Laboraties Molecular Diagnostics.[7][8]

During the 1990s, the identification of newly discovered genes and new techniques for DNA sequencing led to the appearance of a distinct field of molecular and genomic laboratory medicine; in 1995, the Association for Molecular Pathology (AMP) was formed to give it structure. In 1999, the AMP co-founded The Journal of Medical Diagnostics.[9] Informa Healthcare launched Expert Reviews in Medical Diagnostics in 2001.[1] From 2002 onwards, the HapMap Project aggregated information on the one-letter genetic differences that recur in the human populationthe single nucleotide polymorphismsand their relationship with disease.[2](ch 37) In 2012, molecular diagnostic techniques for Thalassemia use genetic hybridization tests to identify the specific single nucleotide polymorphism causing an individual's disease.[10]

As the commercial application of molecular diagnostics has become more important, so has the debate about patenting of the genetic discoveries at its heart. In 1998, the European Union's Directive 98/44/ECclarified that patents on DNA sequences were allowable.[11] In 2010 in the US, AMP sued Myriad Genetics to challenge the latter's patents regarding two genes, BRCA1, BRCA2, which are associated with breast cancer. In 2013, the U.S. Supreme Court partially agreed, ruling that a naturally occurring gene sequence could not be patented.[12][13]


The Affymetrix 5.0, a microarray chip

Development from research tools

The industrialisation of molecular biology assay tools has made it practical to use them in clinics.[2](foreword) Miniaturisation into a single handheld device can bring medical diagnostics into the clinic and into the office or home.[2](foreword) The clinical laboratory requires high standards of reliability; diagnostics may require accreditation or fall under medical device regulations.[14] As of 2011, some US clinical laboratories nevertheless used assays sold for "research use only".[15]

Laboratory processes need to adhere to regulations, for example Clinical Laboratory Improvement Amendments, Health Insurance Portability and Accountability Act, Good Laboratory Practice, and Food and Drug Administration specifications in the United States. Laboratory Information Management Systems help by tracking these processes.[16] Regulation applies to both staff and supplies. As of 2012, twelve US states require molecular pathologists to be licensed; several boards such as the American Board of Medical Genetics and the American Board of Pathology certify technologists, supervisors, and laboratory directors.[17]

Automation and sample barcoding maximise throughput and reduce the possibility of error or contamination during manual handling and results reporting. Single devices to do the assay from beginning to end are now available.[14]


Main articles: Assay and Biological assay

Molecular diagnostics uses in vitro biological assays such as PCR-ELISA or Fluorescence in situ hybridization.[18][19] The assay detects a molecule, often in low concentrations, that is a marker of disease or risk in a sample taken from a patient. Preservation of the sample before analysis is critical. Manual handling should be minimised.[20] The fragile RNA molecule poses certain challenges. As part of the cellular process of expressing genes as proteins, it offers a measure of gene expression but it is vulnerable to hydrolysis and breakdown by ever-present RNAse enzymes. Samples can be snap-frozen in liquid nitrogen or incubated in preservation agents.[2](ch 39)

Because molecular diagnostics methods can detect sensitive markers, these tests are less intrusive than a traditional biopsy. For example, because cell-free nucleic acids exist in human plasma, a simple blood sample can be enough to sample genetic information from tumours, transplants or an unborn fetus.[2](ch 45) Many, but not all, molecular diagnostics methods based on nucleic acids detection use polymerase chain reaction (PCR) to vastly increase the number of nucleic acid molecules, thereby amplifying the target sequence(s) in the patient sample.[2](foreword) The detection of the marker might use real time PCR, direct sequencing,[2](ch 17) or microarray chipsprefabricated chips that test many markers at once.[2](ch 24) The same principle applies to the proteome and the genome. High-throughput protein arrays can use complementary DNA or antibodies to bind and hence can detect many different proteins in parallel.[21]


A microarray chip contains complementary DNA (cDNA) to many sequences of interest. The cDNA fluoresces when it hybridises with a matching DNA fragment in the sample.


Conventional prenatal tests for chromosomal abnormalities such as Down Syndrome rely on analysing the number and appearance of the chromosomesthe karyotype. Molecular diagnostics tests such as microarray comparative genomic hybridisation test a sample of DNA instead, and because of cell-free DNA in plasma, could be less invasive, but as of 2013 it is still an adjunct to the conventional tests.[22]


Main article: Pharmacogenomics

Some of a patient's single nucleotide polymorphismsslight differences in their DNAcan help predict how quickly they will metabolise particular drugs; this is called pharmacogenomics.[23] For example, the enzyme CYP2C19 metabolises several drugs, such as the anti-clotting agent Clopidogrel, into their active forms. Some patients possess polymorphisms in specific places on the 2C19 gene that make poor metabolisers of those drugs; physicians can test for these polymorphisms and find out whether the drugs will be fully effective for that patient.[24] Advances in molecular biology have helped show that some syndromes that were previously classed as a single disease are actually multiple subtypes with entirely different causes and treatments. Molecular diagnostics can help diagnose the subtypefor example of infections and cancersor the genetic analysis of a disease with an inherited component, such as Silver-Russell syndrome.[1][25]

Infectious disease

See also: Pathogenomics

Molecular diagnostics are used to identify infectious diseases such as chlamydia,[26] influenza virus[27] and tuberculosis;[28] or specific strains such as H1N1 virus.[29] Genetic identification can be swift; for example a loop-mediated isothermal amplification test diagnoses the malaria parasite and is rugged enough for developing countries.[30] But despite these advances in genome analysis, in 2013 infections are still more often identified by other meanstheir proteome, bacteriophage, or chromatographic profile.[31] Molecular diagnostics are also used to understand the specific strain of the pathogenfor example by detecting which drug resistance genes it possessesand hence which therapies to avoid.[31]

Disease risk management

A patient's genome may include an inherited or random mutation which affects the probability of developing a disease in the future.[23] For example, Lynch syndrome is a genetic disease that predisposes patients to colorectal and other cancers; early detection can lead to close monitoring that improves the patient's chances of a good outcome.[32] Cardiovascular risk is indicated by biological markers and screening can measure the risk that a child will be born with a genetic disease such as Cystic fibrosis.[33] Genetic testing is ethically complex: patients may not want the stress of knowing their risk.[34] In countries without universal healthcare, a known risk may raise insurance premiums.[35]


Cancer is a change in the cellular processes that cause a tumour to grow out of control.[23] Cancerous cells sometimes have mutations in oncogenes, such as KRAS and CTNNB1 (β-catenin).[36] Analysing the molecular signature of cancerous cellsthe DNA and its levels of expression via messenger RNAenables physicians to characterise the cancer and to choose the best therapy for their patients.[23] As of 2010, assays that incorporate an array of antibodies against specific protein marker molecules are an emerging technology; there are hopes for these multiplex assays that could measure many markers at once.[37] Other potential future biomarkers include micro RNA molecules, which cancerous cells express more of than healthy ones.[38]

See also


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