MOLECULAR

DIAGNOSTICS

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 ACKNOWLEDGEMENTS
Firstly, I would like to thank the school for letting me perform
such a wonderful research project, which helped me enrich my
knowledge.
I would also like to thank my class teacher, Mrs. Piyali Sen
Chatterjee, without whose help the project could not have been
completed
Lastly I would like to thank my parents, who have been an
integral part of this journey.
RAHUL ROY

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 CONTENTS

Topic Page Number

1.Introduction 3
2.General Details 6-8
3.Case Studies 9-15
4.Conclusion 16

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 INTRODUCTION
Molecular diagnostics is a collection of techniques used to
analyse biological markers in the genome and proteome—the
individual's genetic code and how their cells express their genes
as proteins—by 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

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 leucocyte antigen typing
(which investigates and predicts immune function), coagulation,
and pharmacogenomics—the genetic prediction of which drugs will work
best.[4](v-vii)They overlap with clinical chemistry (medical tests on bodily fluids).

BRIEF HISTORY:-
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 sequencing—then
in its infancy—but 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]

DNA microarrays, sometimes called "gene chips," allow researchers to see

the expression of hundreds or thousands of genes at one time. A DNA
microarray is a thin-sized chip with thousands of single-stranded DNA
fragments corresponding to various genes of interest that have been
inserted into "spots" in the microarray.

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A single microarray may contain 10,000 or more spots with each spot
containing pieces of DNA from a different gene. A single gene chip can
even hold representative fragments from the entire human genome.

Molecular diagnostics uses techniques such

as mass spectrometry and gene chips to
capture the expression patterns of genes and
proteins

From 2002 onwards, the HapMap Project aggregated information on the

one-letter genetic differences that recur in the human population—
the single nucleotide polymorphisms—and 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.

Nowadays, molecular diagnosis also allows for the identification of tumor

markers that are used in the detection, diagnosis, treatment, and
monitoring of some types of cancer.

Tumor markers are substances found in the blood, urine, or tissues

produced by tumor cells or other cells in the body in response to cancer or
certain benign conditions.

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 GENERAL DETAILS
TECHNIQUES:-
Development from research tools
The industrialisation of molecular biology assay tools has made it practical
to use them in clinics.[2](
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

Assays
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.

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.

PCR is currently the most widely used method for detection of DNA
sequences.[21] The detection of the marker might use real time PCR, direct
sequencing,[2](ch 17) , microarray chips—prefabricated chips that test many
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markers at once,[2](ch 24)or MALDI-TOF[22] 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

Applications:-

Prenatal
Conventional prenatal tests for chromosomal abnormalities such as Down
Syndrome rely on analysing the number and appearance of the chromosomes—
the karyotype. Molecular diagnostics tests such as microarray comparative genomic
hybridization test a sample of DNA instead, and because of cell-free DNA in plasma,
could be less invasive.

Infectious disease
Molecular diagnostics are used to identify infectious diseases such
as chlamydia,[29] influenza virus[30] and tuberculosis;[31] or specific strains
such as H1N1 virus.[32] Genetic identification can be swift; for example
a loop-mediated isothermal amplification test diagnoses the malaria
parasite.
Molecular diagnostics are also used to understand the specific strain of the
pathogen—for example by detecting which drug resistance genes it
possesses—and hence which therapies to avoid.

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. [26] 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

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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.

Cancer
Cancer is a change in the cellular processes that cause a tumour to grow
out of control.[26] Cancerous cells sometimes have mutations in oncogenes,
such as KRAS and CTNNB1(β-catenin).[39] Analysing the molecular
signature of cancerous cells—the DNA and its levels of expression
via messenger RNA—enables physicians to characterise the cancer and to
choose the best therapy for their patients.
To do the molecular diagnostic test for cancer, one of the significant issue is
the DNA sequence variation detection.

Tumor biopsy samples used for diagnostics always contain as little as 5%

of the target variant as compared to wildtype sequence. Also, for
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noninvasive applications from peripheral blood or urine, the DNA test must
be specific enough to detect mutations at variant allele frequencies of less
than 0.1%.[21]
Currently, by optimizing the traditional PCR, there’s a new invention,
amplification-refractory mutation system (ARMS) is a method for detecting
DNA sequence variants in cancer. The principle behind ARMS is that the
enzymatic extension activity of DNA polymerases is highly sensitive to
mismatches near the 3′ end of primer.

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 CASE STUDIES
CASE 1:-
Initial Diagnosis
This 42-year-old Indian woman was referred for diagnosis and therapy of an
ampullary adenoma. The patient had been in good health until 3 months earlier,
when she developed episodic right upper quadrant (RUQ) and back pain. On
gallbladder ultrasound, there was evidence of calcified gallstones with normal
gallbladder wall and bile duct. Laboratory tests revealed a mild elevation in alanine
amino-transferase (ALT) (48 U/liter).

MOLECULAR DIAGNOSTIC STUDIES:-

Loss of Heterozygocity at the APC locus.

The mutational alterations being tested for in this case manifest as allelic loss, also
known as loss of heterozygosity, generally regarded as a measure of tumor suppressor
gene deletion. Relative intensity of polymorphic electrophoretic bands is determined
from PCR amplified microsatellite tandem repeats situated in proximity to known
or putative tumor suppressor genes.

Mutation Analysis of the APC gene;-

A mutation scanning technique was used to

determine the presence of mutations in the
adenomatous polyposis coli gene (APC) gene.
A PCR-based assay was used to amplify all 15
exons of the APC gene and intron/exon
boundaries.

A single base pair change of a cytosine (C) to a Red inflamed polyps in colon tissue biopsy

thymine (T) was detected at nucleotide position 481 (481 C>T), predicted to result in
a change of a glutamine residue to a stop codon at codon position 161 (Q161X).
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The Q161X mutation identified in this patient is predicted to produce a truncated
protein and is interpreted as a disease-causing mutation. These findings are
consistent with the diagnosis of familial adenomatous polyposis (FAP)

Chromsome Banding PCR studies

AGE SEX Type of Diagnosis And

Ailment Treatment
42 years Female Familial Tissue biopsy, followed
old adenomatuos with PCR studies
polyposis. revealing FAP, due to
point mutation.
(Hereditary)
Surgery done, recovering
well.

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Case 2:-
Initial Diagnosis:-
A 30-year-old man developed liver cirrhosis and diabetes. Laboratory studies
showing high serum ferritin and high transferrin saturation led to the
hypothesis that iron toxicity to the liver and pancreas contributed to his
cirrhosis and diabetes. A blood specimen was tested for mutation in
the HFE gene that is responsible, at least in part, for hereditary hemochromatosis.

Disease Details

Skin Pigmentation

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MOLECUAR DIAGNOSTIC STUDIES:-
Polymerase chain reaction (PCR) followed by melt curve analysis was
performed, and a pathologist interpreted the findings as HFE C282Y
mutation without wild-type DNA at that locus.

Homozygous HFE gene mutation with the predicted amino acid

substitution predisposes to iron overload by overabsorption of iron from
the diet.

He was treated with therapeutic phlebotomy until his serum iron levels
returned to the normal range. He remains at risk for iron overload, and he
should be periodically monitored and managed accordingly.

A genetic counselor educated him about the increased risk of iron

overload faced by blood relatives if they, too, inherited two mutated
alleles of the HFE gene.

AGE SEX TYPE OF AILMENT DIAGNOSIS

AND
TREATMENT
30 MALE HEREDITARY PCR
HEMOCHROMATOSIS FOLLOWED BY
MELT CURVE
ANALYSIS
SHOWED
MUTATION IN
Hfe gene,
TREATED WITH
PHLEBOTOMY

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CASE 3:-

INITIAL DIAGNOSIS:-

A 78-year-old woman was referred for further investigation of a

bicytopenia. She had a history of rheumatoid arthritis and
Sjögren syndrome for which she was treated with methotrexate
and etanercept. She had no complaints, and during physical
examination, no lymphadenopathy, masses, or visceromegaly
was found.
A peripheral blood smear did not show blasts or dysplastic
features of the white blood cells. The patient had normal
ferritin, vitamin B12, and folic acid concentrations without
evidence of hemolysis.
MOLECULAR DIAGNOSTICS:-

Serum protein electrophoresis (SPE)4 and immunofixation

electrophoresis (IFE) with pentavalent antiserum were performed as M-
protein screening. These data suggested the presence of an IgG heavy
chain M protein (γ-HC).

Capillary electrophoresis (CE) combined with immunosubtraction (IS)

analysis (Fig. 1C) confirmed that the abnormal pattern was caused by a
γ-HC. IgG-IS (Fig. 1D) illustrated that the γ region consisted of
polyclonal IgG-κ and IgG-λ (green).

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The β2 region largely consisted of IgG not associated with light chains
(γ-HC, represented in red), and the remaining fraction (blue) was a
combination of polyclonal IgA, polyclonal IgG, and
nonimmunoglobulins.

Bone marrow analysis showed the presence of approximately 2%

plasma cells, of which about 30% were atypical or binucleated (Fig. 1E).
No other abnormalities were observed within the bone marrow. Using
flow cytometry, a small population of plasma cells was detected.

Capillary Electrophoresis

Bone marrow Aspirate

AGE SEX TYPE OF DIAGNOSIS AND

AILMENT TREATMENT
78 MALE BICYTOPENIA CAPILLAY
ELECTROPHORESI
S SHOWED
ABNORMAL HC
PROTEIN.
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CASE 4:-

74-year-old retired high school teacher

In July of 2013, the patient was diagnosed with Non Small Cell Lung
Carcinoma(NSCLC) after presenting to her PCP with dyspnea and
intermittent back and chest pain; cardiac workup was negative, and the
patient has no history of smoking

Initial CT scan showed a large mass in the right lower lobe and 2 small
lesions in the T9 and T10 vertebra, suspicious for metastatic disease

MOLECULAR DIAGNOSTICS:-
Patient screened for circulating tumor DNA in urine, which shows
T790M-positive disease.
Mutational analysis on the primary mass showed EGFR exon 19
deletion, consistent with NSCLC and no other actionable mutations.

Biopsy and histology of the primary mass

and metastatic lesion showed TTF-1+, p40 -,
p60 - consistent with bronchogenic
adenocarcinoma NSCLC.

She is initiated on systemic therapy with

erlotinib for metastatic disease.

After 5 cycles, the patient displays good

response, with clinical improvement and
radiologic improvement in primary and
metastatic lesions Xray showing NSCLC

Age Sex Type Of Ailment Diagnosis And Treatment

74 Female Non Small Cell Lung Mutational analysis on primary
Carcinoma mass EFGR 19 exon deletion,
Chemotherapy Treatment

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 CONCLUSION
The medical community has recognized the importance of
molecular diagnostics for several decades, and this field
is especially important to cancer care. Molecular
diagnostics have already improved cancer diagnosis and
treatment techniques, and research is continuing.

Molecular diagnostics analyzes how these genes and

proteins are interacting inside a cell. The focus is on
patterns--gene and protein activity patterns--in different
types of cancerous or precancerous cells.

Molecular diagnostics has two major hurdles to

overcome.

 The first and most formidable is identification and

analysis of relevant tumor markers.

 The second is developing portable, sensitive,

accurate instruments that can be used in the clinic.

Molecular diagnostics also impact cancer treatment.

Microarrays are being applied to pharmacogenomics-an
area of research that studies why certain drugs work in
combination with particular genetic expression patterns
but not with others-to design new drugs that target
cancer cells and do not affect normal ones.

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So, it can be concluded that molecular diagnostics plays a
very important role in modern medical sciences.

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