Download Triplet repeat primed PCR assay for the screening and diagnosis of

Triplet repeat primed PCR assay for the screening and diagnosis of Friedreich ataxia, and Spinocerebellar ataxia types
1, 2, 3, 6, 7, 8, & 17
Mohamed Jama , Samuel Egbert , and Elaine Lyon
1
ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah,
1
1
1,2
An enterprise of the University of
Utah and its Department of Pathology
INTRODUCTION:
Friedreich (FA) and Spinocerebellar ataxia (SCA) are a group of genetically inherited
neurodegenerative diseases caused by dynamic expansions of trinucleotide repeats in different
genes (Table 1). These diseases are characterized by progressive incoordination of gait, poor
coordination of hands, speech, and eye movements. They are either inherited recessively (FA) or
dominantly (SCA).
Friedreich ataxia is the most prevalent form of autosomal recessive cerebellar ataxia in
Caucasians associated with unstable expansion of GAA trinucleotide repeats in the first intron of
the FXN gene. SCA classification is based on different regions of the cerebellum affected. The
severity of the diseases correlates with the repeat number in the less expanded allele.
There are 28 subtypes of SCA characterized by autosomal dominant inheritance. SCA1, 2, 3,
6 and 7 are caused by CAG expansion and account for 50–65% of all cases of autosomal
dominant ataxia, with SCA3 as the most common subtype.
Current molecular methods for the diagnosis of trinucleotide expansions include PCR and/or
Southern blot. The major disadvantage of the Southern blots analysis is its difficulty to accurately
size alleles, while current PCR techniques have decreased sensitivity for detecting large
expansions. Although the combination of Southern and PCR methods enables reliable diagnosis
of trinucleotide repeats, the Southern blot analysis is expensive and time consuming.
Triplet primed PCR (TP PCR) has been described to screen for fragile X alleles reducing the
need for Southern blots. TP PCR uses a locus-specific and chimeric primer to amplify from
multiple priming sites within the trinucleotide repeat. TP PCR gives a characteristic ladder on a
fluorescence electropherogram enabling the rapid and inexpensive identification of expanded
repeats that may have been missed using current PCR methods.
The aim of this study was to develop a simple, robust, and rapid TP PCR-based test capable
of detecting large trinucleotide repeats in FA and SCA types 1, 2, 3, 6, 7, 8, & 17. Normal and
expanded alleles were evaluated for each of the diseases. Expanded alleles were detected by the
characteristic ladder while normal alleles showed no amplification beyond the highest repeat
number. This TP PCR methodology can be used to screen and detect alleles in FA and SCA types
1, 2, 3, 6, 7, 8, & 17.
One microliter of the generated PCR products was added to a mixture of 1.0 µL of LIZ 500
internal size standard (Celera diagnostics) and 9 µL of HiDi formamide (Invitrogen
Corporation, CA, USA). The mixture was heated at 95ºC for 2 minutes and resolved by
electrophoresis on an automated ABI Prism 3100 Genetic Analyzer using POP-6 (performance
optimized polymer), on a 50 cm array (Applied Biosystems).
Samples were electrokinetically injected at 15 kV for 10 seconds and electrophoresed at
15 kV for 5400 seconds at 60ºC under filter set D. Raw data were analyzed with GeneMarker
1.85 (SoftGenetics, State College, PA), programmed for automatic allele calling based on
fragment sizes. Allele calls on GeneMarker 1.85 software were calculated using mean and
2 standard deviation of each allele type from different runs and by setting a minimum and
maximum size range for each marker.
RESULTS:
DISCUSSION:
Figure 1. SCA 1 electropherogram results analysis of sample #4 showing homozygous
normal allele call of 29/29 with a flat baseline denoting lack of expanded allele in this
samples. Note the presences of CAT interruptions, they are important feature in diagnosis of
SCA-1
Gene/gene product
Locus
Mean age
at onset
(range)
Repeat
expansion
FA
SCA1
FA1/9q13-q21
ATXN1/ataxin-1
Frataxin
6p23
GAA
CAG
SCA2
ATXN1/ataxin-2
12q24
2–48
37 (<10 to
>70)
32 (<10 to
Figure 2. SCA 1 electropherogram results analysis of normal sample showing a heterozygous
allele call of 30/31.
SCA6
SCA7
SCA8
ATXN3/ataxin-3;
Machado Joseph disease
(MJD) protein 1
14q24.3-
36 (<10 to
q31
>60)
CACNA1A/voltagedependent
P/Q-type calcium channel
alpha-1A subunit
ATXN7/ataxin-7
19p13
52 (20 to
KLHL1AS
CAG
Lack of CAT interruptions within CAG repeats in an affected sample
TBP/TATA-box binding-
3p21.1-
35 (< to
p12
>60)
13q21
40 (1 to
CTA/CTG
6q27
33 (6 to >
•
This assay offers a quick TAT as all the listed assay can be perform concurrently.
•
This methodology is valuable in screening individuals suspected to be affected with genetically
inherited neurodegenerative diseases caused by dynamic expansions of trinucleotide repeats.
•
Electropherogram results are easy to interpret for normal and expanded alleles as shown in figure
1,2, 3 & 4.
•
SCA 1 assay can detect samples with or without CAT interruptions, an important feature in
determining individuals at risk of transmitting mutable alleles to their offspring, see figure 2.
This work was funded by ARUP Institute for Clinical and Experimental Pathology.
Figure 3. SCA 1 electropherogram results analysis of sample NA13537 showing heterozygous
affect allele call of 32/60
Reverse primer sequence
/5FAM/GCT GGG ATT ACA GGC GCG CGA
TAC GCA TCC CAG TTT GAG ACG GAA GAA GAA GAA
GAA GAA GAA
SCA-1
/5HEX/CTG AGC CAG ACG CCG GGA CAC AAG GCT
GAG
/5NED/GAGCCGGTGTATGGGCCCCTCACCATGTCG
/5PET/CCA GTG ACT ACT TTG ATT CGT GAA ACA
ATG
/5FAM/CAC GTG TCC TAT TCC CCT GTG ATC CGT
AAG
5PET/TGT TAC ATT GTA GGA GCG GAA AGA ATG
TCG
/5NED/ AAA TGC AGC AGC AGC AGC AGC
/5HEX/CCT TAT GGC ACT GGA CTG ACC CCA CAG
CCT
AGC CCT GCT GAG GTG CTG CTG CTG CTG CTG
SCA-8
SCA-17
This technique has been applied to FA, SCA1, 2, 3, 6, 7, 8 & 17.
CAG
Forward primer sequence with fluorescent dye label on 5΄
SCA-7
•
ACKNOWLEDGMENTS:
CAG
Primer
name
FA
SCA-6
Fluoresently repeat-primed PCR is a robust, sensitive, specific and reliable methodology to detect
normal and expanded triplet repeats.
>70)
Table 2
SCA-2
SCA-3
•
CAG
>60)
SCA17
CONCLUSION:
CAG
>60)
SCA3
Screening for expanded allels in FA and SCA types 1, 2, 3, 6, 7, 8, & 17 by TP PCR gave either
a single peak with a flat baseline denoting a homozygous genotype (Figure 1), two peaks showing the
sample to be heterozygous (Figure 2) or an expanded allele that the sample carries an expanded allele
(Figure 4) that will require further testing to determine the genotype.
This assay makes it very easy to characterize any sample thereby enabling a rapid, inexpensive
identification of normal, intermediate and expanded repeats hence reducing the need for further testing
for most samples when used to screen for expanded alleles in affected populations with this kind of
neurodegenerative diseases.
This SCA 1 assay can also determine the presence or absences of CAT repeats. Alleles with 21-35 CAG
repeats in SCA 1 without CAT interruptions are known to expand in future generations, uninterrupted
CAT is also a critical determinant of age of disease onset and pathogenesis of SCA 1.
CAT interruptions within CAG repeats
Table 1.
Name
Genotype
Expanded allele
Normal and affected allele
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Negative
CAT interruptions within CAG repeats
PCR amplification
PCR was performed using GeneAmp 9700 thermal reaction cycler (Applied Biosystems,
Foster City, CA) in a 20 µL PCR mixture containing, 1 µL of extracted DNA (15-50 ng/µl), 0.5
µM of each forward primer fluorescently labeled with FAM, HEX , NED or PET as shown on
table 2, 1 X FailSafe Premix K containing PCR buffer, dNTPs and MgCl2 (Epicentre, Madison,
WI, USA), and 1.0 unit of Platinum Taq DNA polymerase (Invitrogen Corporation, CA, USA).
Amplification was performed with an initial denaturation at 95°C for 5 minutes, followed by
35 cycles of denaturation at 94°C for 1 minute, annealing at 67°C for 1 minute and extension at
72°C for 3 minutes and a final 15 minute extension at 72°C.
Assay
FA
SCA-1
Please note that the above 10 samples were used to optimize the assays and affected alleles were noted in FA and
SCA-1 assays, see figure 3 & 4
Electropherograms showing typical results are shown below:
MATERIALS AND METHODS:
A total of 10 samples were analyzed for each assay. Samples with expanded allele for FRDA
and SCA 1 were purchased from Coriell Cell Repositories (http://ccr.coriell.org).
Sample Name
NA15847
NA13537
Sample 3.
Sample 4.
Sample 5.
Sample 6.
Sample 7.
Sample 8.
Sample 9.
Sample 10.
No Template.
REFERENCES:
1.Dorschner, M. O., D. Barden, et al. (2002). "Diagnosis of five spinocerebellar ataxia disorders by
multiplex amplification and capillary electrophoresis." J Mol Diagn 4(2): 108-113.
AGC CGC GGG CGG CGG CTG CTG CTG CTG CTG
TCC TGA TAG GTC CCC CTG CTG CTG CTG CTG
2. Bird TD: Ataxia overview. GeneClinics: Clinical Genetic Information Resource [database online].
University of Washington, Seattle, updated 07/25/00, www.geneclinics.org/profiles/ataxias.
CGG CCT GGC CAC CGC CTG CTG CTG CTG CTG
CTG CGG AGG CGG CGG CTG CTG CTG CTG CTG
3. Schelhaas HJ, Ippel PF, Beemer FA, Hageman G: Similarities and differences in the phenotype,
genotype, and pathogenesis of different spinocerebellar ataxias. Eur J Neurol 2000, 7:309-314.
GGT CCT TCA TGT TAG AAA ACC TGG CT
TGC CAC TGC CTG TTG CTG CTG CTG CTG CTG
Bold areas are type of fluorescent dye used and triplet repeat region where the primer can be specific or
chimeric
Figure 4. FA electropherogram results analysis of sample NA115847 showing an expanded
affect alleles that could not be genotyped by this assay.