Subject Name: Genetic Engineering- II

Subject Code: MTI-605

Unit No:5
Unit Name: Molecular Diagnostics
Faculty Name : Dr. V. Suresh Gupta

Sub Topic Name Diagnosis of

-Cystic fibrosis,
-Thalassemia,
-Fragile X syndrome

Cystic fibrosis, Thalassemia, Fragile X syndrome

DIAGNOSIS OF CYSTIC FIBROSIS USING MULTIPLEX PCR

Cystic fibrosis, Thalassemia, Fragile X syndrome

Cystic Fibrosis
A chronic, progressive, and frequently fatal genetic disease of the
bodys mucus glands.
Affects the respiratory and digestive systems in children and young
adults.
An average person has a lifespan of 40 years with the right
treatment.
The name cystic fibrosis refers to the characteristic scarring
(fibrosis) and cyst formation within the pancreas, first recognized in
the 1930s.

Cystic fibrosis, Thalassemia, Fragile X syndrome

Defective CFTR gene

Cystic Fibrosis is caused by a defective CFTR [cystic fibrosis

transmembrane conductance regulator] gene which codes for
a Na+ and Cl- transporter found on the surface of epithelial
cells of lungs and other organs.
This results in thickened secretions in the airways, pancreatic
ducts, biliary tree, intestines and reproductive tract leading to
the clinical manifestations of the disease.
As many as 1300 mutations in the CFTR gene have been
observed worldwide.
The severity of the disease is directly related to the
characteristic effects of the particular mutations that have
been inherited by the individual sufferer.

Cystic fibrosis, Thalassemia, Fragile X syndrome

Cystic fibrosis, Thalassemia, Fragile X syndrome

Cystic fibrosis, Thalassemia, Fragile X syndrome

Signs and Symptoms

The hallmark signs and symptoms of cystic fibrosis are:

salty tasting skin

poor growth
poor weight gain despite a normal food intake
accumulation of thick, sticky mucus
frequent chest infections
coughing or shortness of breath.

Cystic fibrosis, Thalassemia, Fragile X syndrome

Test for Cystic Fibrosis
Sweat Test If a person shows symptoms of CF or if a baby has a
positive newborn screen for CF, a doctor may order a sweat test. This
simple, painless test is the best way to diagnose CF. It measures the
concentration of salt in a persons sweat. A high salt level indicates CF.
- Sweat tests should be done at a Cystic Fibrosis Foundation-accredited
care center where strict guidelines help ensure accurate results.
Newborn Screening Newborns screened for cystic fibrosis can benefit
from early diagnosis and treatment.
Genetic Carrier Testing More than 10 million Americans are
symptomless carriers of the defective CF gene. This blood test can help
detect carriers, who could pass CF onto their children. To have cystic
fibrosis, a child must inherit one copy of the defective CF gene from each
parent.

Cystic fibrosis, Thalassemia, Fragile X syndrome

Multiplex pcr
Multiplex PCR, is a modification of PCR in order to rapidly
detect deletions or duplications in a large gene.
This process amplifies genomic DNA samples using multiple primers and a
temperature-mediated DNA polymerase in a thermal cycler.
It consists of multiple primer sets within a single PCR mixture to
produce amplicons of varying sizes that are specific to different DNA
sequences.
By targeting multiple genes at once, additional information may be gained
from a single test-run that otherwise would require several times the reagents
and more time to perform.

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Cystic fibrosis, Thalassemia, Fragile X syndrome

DETECTION OF THALASSEMIA BY ARMS PCR

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Cystic fibrosis, Thalassemia, Fragile X syndrome

Thalassemias are forms of inherited autosomal blood disorders.
Caused by weakening and destruction of red blood cells.
Missing or mutation in the genes responsible for hemoglobin prodn results in
thalassemia.
Hemoglobin is a heterotetramer consisting of 2 subunits & 2 subunits.
Mutation affects either of the subunit resulting in defected RBC prodn.
Depending on the subunit affected thalassemia is classified accordingly:
Alpha thalassemia:
Caused due to mutations in alpha globin genes. Alpha globin is composed of
4 genes and mutations in either of these genes results into alpha
thalassemia.
Depending on the missing or mutation in these alpha globin genes alpha
thalassemia is further classified as alpha thalassemia (Major, Minor), Silent
thalassemia & hemoglobin H disease.

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Cystic fibrosis, Thalassemia, Fragile X syndrome

Beta thalassemia:
It is due to mutations or missing of gene or genes responsible for globin
protein.
Accordingly there are 3 types of beta thalassemia: Beta thalassemia minor (beta
thalassemia trait), major (Cooleys anemia) & intermedia.

DIAGNOSIS OF THALASSEMIA
4 different ways are used for diagnosis:
Full blood count
blood film
haematinic assay
electrophoresis
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Cystic fibrosis, Thalassemia, Fragile X syndrome

ARMS (Amplification Refractory Mutation System)

ARMS-PCR a PCR technique that depends on annealing of
oligonucleotide primers to specific target DNA, facilitating
amplification of unique seqs.
ARMS is ideally suited for the detection of point mutations & single
deletion/insertions and involves PCR for each of the allele.
ARMS-PCR has been termed as allele specific PCR (ASP) or PCR
amplification of specific allele (PASA).

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Cystic fibrosis, Thalassemia, Fragile X syndrome

WORKING OR PRINCIPLE OF ARMS
A standard ARMS PCR consists of 2 complementary reactions & utilizes 3
primers
one primer is constant & complementary to the templates in both the
reactions .
the other primers differ at their 3 terminals and are specific to either the
wild type DNA or the mutated seq.
ARMS is based on the principle that Taq polymerase lacks 3-5exonuclease
activity.
For polymerases to extend, the primers must be perfectly annealed to their
target sites at 3 end.
Thus a mismatch between the 3end of the PCR primer and the template will
result in greatly reduced amplification efficiency.

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Cystic fibrosis, Thalassemia, Fragile X syndrome

ARMS-PCR reaction

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Cystic fibrosis, Thalassemia, Fragile X syndrome

PCR products are separated by electrophoresis through an agarose gel
containing ethidium bromide.
ARMS primer can be designed to amplify a specific member of a multiallelic system while remaining refractory to amplification of another
allele that may differ by as little as a single base from the former.
If the sample is homozygous mutant or homozygous wild type,
amplification will only occur in only one of the tubes, if the sample is
heterozygous amplification will be seen in both tubes.
ARMS-PCR detects one mutation per reaction. To overcome this problem
multiplex-PCR (MARMS-PCR) has been developed.

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Cystic fibrosis, Thalassemia, Fragile X syndrome

ADVANTAGES & Disadvantages OF ARMS-PCR

ARMS is a rapid, inexpensive and simple
device to work with.
ARMS requires any conventional PCR
equipments although the method used
varies according to that locally used or
desired.
Reactions of ARMS are reasonably
easily multiplexed and can also be used
to determine haplotypes.
ARMS is able to detect 1 mutant allele
from 40 normal alleles.

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As ARMS is a oligonucleotide
based test, care must be taken to
avoid polymorphism that might
affect the primer binding.

Cystic fibrosis, Thalassemia, Fragile X syndrome

TYPES OF ARMS
DOUBLE ARMS
MS PCR
MULTIPLEX ARMS

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Cystic fibrosis, Thalassemia, Fragile X syndrome

Fragile x syndrome and polymerase chain

reaction detection

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Cystic fibrosis, Thalassemia, Fragile X syndrome

What is fragile X syndrome?

Its a genetic condition that causes a range of developmental
problems including learning disabilities and cognitive impairment.
Males are more severely affected by this disorder than females.
Affected individuals :
1. Delayed development of speech & language
2. Males have mild to moderate intellectual disability,
3. ~1/3rd of affected females are intellectually disabled.
4. Children may also have anxiety and hyperactive behavior such
as fidgeting or impulsive actions.
5. ~1/3rd of individuals have features of autism spectrum
disorders that affect communication and social interaction.

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Cystic fibrosis, Thalassemia, Fragile X syndrome

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Cystic fibrosis, Thalassemia, Fragile X syndrome

Causes
Fragile X syndrome occurs as a result of a mutation of the fragile X
mental retardation 1 (FMR1) gene on the X chromosome, most
commonly an increase in the number of CGG trinucleotide repeats in
the 5' untranslated region of FMR1.
FXS can also occur as a result of point mutations affecting FMR1.
In unaffected individuals, the FMR1 gene contains 544 repeats of the
CGG codon, most commonly 29 or 30 repeats. Between 45 & 54
repeats considered a "grey zone", with a premutation allele generally
considered to be between 55 & 200 repeats in length.
Individuals with fragile X syndrome have a full mutation of
the FMR1 allele, with over 200 repeats of the CGG codon.
In these individuals with a repeat expansion greater than 200, there
is methylation of the CGG repeat expansion and FMR1 promoter,
leading to the silencing of the FMR1 gene and a lack of its product.
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Cystic fibrosis, Thalassemia, Fragile X syndrome

Signs and symptoms

Large, protruding ears.
Long face (vertical maxillary
excess)
High-arched palate.
Hyperextensible finger joints.
Double-jointed thumbs.
Flat feet.
Soft skin.
Post pubescent macroorchidism Large testis in men after puberty.
Low muscle tone.

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Cystic fibrosis, Thalassemia, Fragile X syndrome

Diagnosis
Lab Tests for Fragile X, The FMR1 DNA Test can be administered
with two different lab procedures.
PCR analysis can determine the actual number of CGG repeats (a
pattern of DNA) that are present in the Fragile X gene.
It is quite accurate in determining premutation and normal gene
repeat numbers.
However, PCR is less expensive and quicker than Southern blot, and
recent advances in technology have increased its ability to identify
Fragile X full mutations.
PCR may thus be the only test used in the near future.

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Cystic fibrosis, Thalassemia, Fragile X syndrome

Role of Polymerase chain reaction

One of the frequently used tests is based on PCR amplification of the
FMR1 CGG-repeat region.
Moreover, because it is relatively easy to collect and transport
bloodspot samples on filter paper, they are often used for molecular
screening purposes.
Methylation-sensitive PCR is another commonly used method for
verifying the aforementioned methylation status.
PCR analysis can determine the actual number of CGG repeats (a
pattern of DNA) that are present in the Fragile X gene.

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Cystic fibrosis, Thalassemia, Fragile X syndrome

Results
Molecular testing of 1399 males suspected of having fragile X syndrome.

Screening PCR
Venous blood
Weak
Failed

Methylation (+) Methylation ()

SBH MB-PCR
SBH MB-PCR
Strong 219
0
0
219
0
10*
10
10
0
0

Bloodspot
Strong
Weak # 16*
Failed # 14*

1140
2
12

2
12

14
2

14
2

Total

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24

235

76

27

1399

60

Thank You