Dr. Derakhshandeh, PhD

Mutation ScreeningMutation Screening

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Quantitative PCR and Dosage

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Quantitative PCR and Dosage

used in a number of diagnostic applications eg. SMA trisomy 21 X-linked

Agammaglobulinemia

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SMA

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CLASSIFICATIONSMA TYPE I

(Werdnig-Hoffmann)

SMA TYPE II (Classic)

SMA TYPE III (Kugelberg-Welander)

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SMA TYPE I

Severe form of SMA

Onset : first 6 months

Death : < 2 year

Never raising the head or sitting

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SMA TYPE I

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SMA TYPE II

Less severe

Clinical appearing : < 18 months

Able to sit un aid

Death : about 9 years

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SMA TYPE III

Mildest form of SMA

Onset : > 18 months

Walking without aid

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GENETIC MAP

Three candidate genes named SMN (Survival Motor Neuron), NAIP (Neuronal Apoptosis Inhibitory Protein) and P44 were identified in this locus

Up to 95% of SMA patients (SMNI-III) are homozygously deleted for two exons (7&8) of both telomeric copy of SMN gene (SMNt)

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Deletions

Up to 5% of SMA patients have frameshift mutations, gene conversions and point mutation

Exons 5 and 6 of NAIPt gene are deleted in approximately 50% of type I SMA and 18% of types II and III SMA

P44t is lacked or intrrupted in 73%

of SMA type I patients and 7% in types II and III

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The summery of normal alleles (N) , mutant alleles (M) and deletion types (D) of SMN

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MOLECULAR DIAGNOSIS & PND

PCR-SSCP or PCR-RFLP of SMN gene enables confirmation of a suspected clinical diagnosis of SMA or prenatal diagnosis

These two techniques based on nucleotide differences of both exon 7 and exon 8 of telomeric and centromeric copy of SMN

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Deletion Analysis of SMN gene

• Exones 7 and 8 of SMN gene were amplified and cut by Dra I and Dde I , respectively. (only centromeric copy is cutted)

• Absence of SMNt exone(s) 7 (and 8) confirm diagnosis of SMA

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188 bp

164 bp

188 bp

123 bp

65 bp

Exon 7, DraI

Exon 8, DdeI

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SMN Deletion AnalysisDerakhshande et al.

(Farhud Genetic Lab/ NRCGEB)

• SMNt Exon 7 is deleted in affected child

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NAIP Gene Deletion Analysis

• Exones 5 & 6 of NAIP gene were amplified with exon 13 which was the internal control

• Absence of exon 5 and exon 6 ( which only exist within the telomeric functional copy of NAIP) was detected in ~50% of type I SMA and 18% of types II and III SMA

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NAIP Deletion analysisDerakhshande et al.

(Farhud Genetic Lab/ NRCGEB)

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• The objective of this study was to genetically characterize the childhood onset spinal muscular

atrophy in Iran.

SMN NAIP

Deletion of exon 7 & 8

Deletion of exon 5 & 6

SMA type I (n=70) 70(100%) 61(87%)

SMA type II (n=3) 2(66%) 1(33%)

SMA type III (n=2) 1(50%) 0(0%)

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• Various deletion haplotypes were constructed by using genotypes of SMN and NAIP genes.

• Haplotype A, which has the deletions of all two involved genes, were deleted in approximately 83% of type I and II SMA but not in type III and was found predominantly in the severe group with an early onset at less than 6 month of age.

• we report Thirty four our experiences for prenatal diagnosis

Haplotypes (%)

A B C

SMA type I (n=70)

87 100 0

SMA type II (n=3)

33 66 33

SMA type III (n=2)

0 50 50

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• These studies suggested that the frequency of gene deletions of SMN1 and NAIP gene is a few higher than previous reports. It is may be due to high rate of consanguine marriage by Iranian Muslims (96 % in this families). Thus, the conformation of SMA related gene deletion will also be a useful tool for the pre and postnatal diagnostic. In addition to common PCR methods for SMN exon 7 and 8 and NAIP exons 4 and 5, we also conducted multiplex PCR of exon 5, 6 and 13 of the NAIP telomere in one reaction.

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Quantitative PCR and Dosage

• Products can be measured by image scanning of agarose gels or Polaroid pictures

• Method is not sensitive to template concentration (1.3g to 2.7pg DNA used) but can be affected by template quality so preparation is important.

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More complex methodSMA

This method allows quantitation of the copy number of SMNT and SMNC genes on chromosome 5q12

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The homologous gene quantitative polymerase chain reaction(HGQ-PCR)

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Detection of trisomy 21 by HGQ-PCRLanes 1–16 Serial dilutions for a normal individual and aDown’s syndrome patient used to evaluate

the optimal HGQPCR

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SequencingSequencing

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Top and bottom strand differences for a point

mutation

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Top and bottom strand differences for a heterozygous

base

                                                                             

(Click for full size image)

Figure 2. Top and bottom strand differences for a heterozygous base

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Sequencing

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Forensics

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ForensicsPCR analysis PCR

(polymerase chain reaction)RFLP analysisSTR analysis Short tandem

repeat (STR) technology is a forensic analysis

For example, the likelihood that any two individuals (except identical twins) will have the same 13-loci DNA profile can be as high as 1 in 1 billion or greater.

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ForensicsMitochondrial DNA Analysis

Mitochondrial DNA analysis (mtDNA) can be used to examine the DNA from samples that cannot be analyzed by RFLP or STR

Nuclear DNA must be extracted from samples for use in RFLP, PCR, and STR

mtDNA analysis uses DNA extracted from another cellular organelle called a mitochondrion

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ForensicsWhile older biological samples that

lack nucleated cellular material, such as hair, bones, and teeth, cannot be analyzed with STR and RFLP

they can be analyzed with mtDNAIn the investigation of cases that

have gone unsolved for many years, mtDNA is extremely valuable.

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ForensicsY-Chromosome Analysis

The Y chromosome is passed directly from father to son

so the analysis of genetic markers on the Y chromosome is especially useful for tracing relationships among males

this technique can be very valuable if the laboratory detects complex mixtures (multiple male contributors)

Reverse Dot Blot for Human Mutation

Detection

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Introduction

• Reverse dot blot (RDB)• or reverse allele specific

oligonucleotide (Reverse ASO)

• hybridization • important method for

genotyping common human mutations

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Commonly used in:

• a high mutation spectrum• high frequency disorders

such as:– cystic fibrosis– hemoglobin C (HbC)– hemoglobin E (HbE)– hemoglobin S (HbS)– ß-thalassemias

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Location of mutations in the -globin gene

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RDB procedure

• exons (or other regions of interest)

• amplified by the polymerase chain reaction (PCR)

• using labeled oligonucleotide primers

• 5' biotin label on PCR primers

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Amplicons• Amplification products• denatured • hybridized

– with mutation specific DNA probes

• covalently bound to solid membran

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Incubation• nucleic acids: incubated

with an enzyme conjugated to streptavidin.

• enzyme-conjugated, streptavidin-biotin-nucleic acid complex is then washed

• incubated with– a chromogenic – or luminogenic substrate,

which allows visualization of hybridized spots

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Materials and Methods

• Total genomic DNA• extracted from peripheral

blood leukocytes • Amniotic fluid cells (AF)• chorionic villi (CVS)

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A woman having amniocentesis

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Oligonucleotide probes

• A C6-amino-link phosphoramidite • amino moiety on the 5' end of the

product

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Oligonucleotides used for reverse dot blot (RDB)

53RDB

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Reverse dot (RDB) blot hybridization for detection of 10 common β-thalassaemia mutations

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-thalassemia Patients

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Screening for causal mutations

• genomic DNA from patient blood samples

• reverse dot blot (RDB) • amplification refractory

mutation system-polymerase chain reaction (ARMSPCR)

• DNA sequencing

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PCR from genomic DNA

720 bp

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Strips

N M

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3

4

5

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7

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1 2 3 4 5 6 7 8 9 10

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The Blots

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Comparison of different factors determining the efficiency of ARMS and reverse hybridization in beta thalassemia diagnosis

ARMS Reverse hybridization

Turnover time several days 6-8 hours

Equipment Expensive (large PCR machine, gel electrophoresis, photodocumentationsystem

Less expensive (small PCR machine, agarose gel, small shaking water bath)

Number of PCR reactions per sample

8-88 1

Documentation Requires documentation process after experiment

Self-documented

Technician time (number of patients: time in days)

1:1 10:1

Starting material Depending on the number of PCR reactions

0.5 μg genomic DNA for just one PCR reaction

Toxic materials Ethidium bromide (carcinogen)

None

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In the late stages of muscular dystrophy, fat and connective tissue often replace muscle fibers.

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DMD

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Orthopaedic management of patients with Duchenne's muscular dystrophy

67~95% of deletions can be detected in males using multiplex PCR

L A B C D E F G H L

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MAPH

•Detection of deletions/duplication mutations in Duchenne Muscular Dystrophy using: Multiplex Amplifiable Probe Hybridisation (MAPH)

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MAPH• Although ~95% of deletions can be detected

in males using multiplex PCR• other methods must be used to determine

duplications, as well as the carrier status of females

• The most commonly applied methods are quantitative multiplex PCR and quantitative Southern blotting

• The drawback of quantitative multiplex PCR is that often not all mutations are examined

• meaning that small and rare mutations are missed

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MAPH

• Using high-quality Southern blots it is possible to perform a quantitative analysis and detect duplications

• this technique is time consuming• it is difficult to exactly determine the

duplication • it can be difficult to detect duplications in

females and triplications will be missed

Armour et al (Nucl.Acids Res. 2000)

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• system for analysing all 79 exons of the DMD gene for deletions and duplications

• MAPH is based on a quantitative PCR of short DNA probes recovered after hybridisation to immobilized genomic DNA

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• 1 ug of denatured genomic DNA is spotted on a small nylon filter

• hybridized overnight in a solution containing one of the probe mixes

• Following stringent washing the next day the filter is placed in a PCR tube

• and a short PCR reaction is performed• This releases the specifically-bound

probes into the solution• An aliquot of this is transferred to a

second, quantitative PCR reaction