Development of novel methods for investigating copy number

variation at the human salivary amylase locus

Sugandha Dhar and John AL Armour
School of Biology, The University of Nottingham
Background:
Copy number variations correspond to the changes (>1 kb) in number of copies of DNA
sequences in comparison to a reference chromosome. Located on chromosome 1p 21.1
amylase gene exhibits high copy number variation. In humans, there are two forms of
amylase -salivary (AMY1) and pancreatic (AMY2) which catalyse carbohydrate digestion but
are expressed in different tissues. Previous investigations reveal that in spite of a 96% AMY1-
AMY2 sequence similarity , proviral insertions were responsible for conferring AMY1 tissue
specificity. Current research suggests that AMY1 is a copy number variable locus exhibiting 2-
14 copies per diploid genome and has been positively correlated with high starch diet by
Perry et al. Previously, two Paralogue Ratio Tests (PRTs) have been constructed taking
advantage of the exclusive proviral AMY1 insertions . As proviral elements are scattered
throughout the genome therefore eliciting exclusive amplification from just test and reference
is theoretically questionable. The main focus of this study was to devise an alternative
method which would combine the strengths of PRTs and Real time PCR considering the
genomic complexities of AMY1 locus.


Aims:
1. Development of a novel ratio based copy number measurement assay
2. Characterization of sequence level variation by resolving the allelic architecture
Methods:
We have developed five assays to investigate AMY1 structural variation. Out of the five
assays, two comprise the paralogue ratio test (PRT), one is a microsatellite. They are run
on the AB13130 as triplex systems and used as copy number measuring tests. The other
two are novel methods run as duplex and are used as copy number validation Tests.



Chr 12A ForwardPrimer 5'-3' 12A ReversePrimer 3'-5'
AMY1(Test) 244 ATCTAGTCCTTTTCTATCAATG CTTGACAGATGTACTTATTTTCT
AMY1
(Ref) 249 ATCTAGTCCTTTTCTATCAATG CTTGACAGATGTACTTATTTTCT
5D 244 ATCTAGTCCTTTTCCATCAATG CTTGACAGATGTACTTATTTTAT
1G 244 ATCTAGTCCTTTTTCATCAATG CTTGACAGATGTACTTATTTTCT
1D 246 ATCTAGTCCTTTTCCATCAATG CTTGACAGATATACTTATTTTTT
6A 247 ATCTAGTCCTTTTCCATCAATG TTTGACAGATGTACTTATTTTCT
4B 243 ATCTAGTCCTTTTCCATCAATG TTTGACAGATGTACTTATTTTCT
4E 237 ATCTAGTCCTTTTCCATCAATG CTTGACAGATATACTTAATTTTT
5B 245 ATCTAGTCCTCTTCCATCAATG TTTGACAGATATACTTATTTTTT
Precise placement of
primers has a dual function
ensuring size differences
between Test and Reference
amplicons for easier
separation and creation of
mismatches to other
locations in the genome
Test copy number variable
Reference two copy
Results:
3. Non Paralogous Ratio Test (nPRT)
nPRT makes use of 2 pairs of 40 bp primers (tailed primers) to amplify 2 regions in the
genome (Test and reference) which have similar amplification properties.

A. Segregation derived haploid copy numbers
We have measured AMY1 copy number in 10 families of CEPH samples which are also part
of the Hap Map project. This was using the MS assay and 12A PRT. CEPH segregation data
was obtained from the CEPH genotype database V. 10 and linkage analysis on chromosome
1 was performed using the CHROMPIC option of CRIMAP





B. Selection of reference samples
Samples were selected from the segregation pattern deduced from the MS assay. The copy
number determined by the MS assay was examined for concordance with the rest of the
measurement systems.



AC
AB
CD
AD
BD
BC
8
7
11
6
4
9
1 2 3 2
2 2 2 1
2 3+2 2+2
3 2 1
1 2 1
1 2+2 2 2
Haplotype B
Haplotype B
Haplotype B Haplotype D
Haplotype D
Haplotype D
Haplotype A
Haplotype A
Haplotype A
Haplotype C
Haplotype C
Haplotype C
261
265
265
269
269
269
272
272
A B
269
272
272 276
C D
269
265
265
269
269
269
272
272
A
269
272
272 276
C D
269
265
265
269
269
269
272
272
A
269
272
272 276
C D
269
265
265
261
265
265
B
261
265
265
B
Sample Copies (MS) Ratio 12A PRT Ratio 1H PRT Ratio nPRTb Ratio nPRTc
1416_01
1416_11
1416_12
(Father)
(Grandfather)
(Grandmother)
8
8
8
7.5
7.7
7.9
8.8
9.2
9.4
9.0
10.7
8.4
5.9
6.6
5.2
C. Distribution of measured values


0
2
4
6
8
10
12
14
0
. 7
5

0
. 7
7

0
. 7
9

0
. 8
1

0
. 8
3

0
. 8
5

0
. 8
7

0
. 8
9

0
. 9
1

0
. 9
3

0
. 9
5

0
. 9
7

0
. 9
9

1
. 0
1

1
. 0
3

1
. 0
5

1
. 0
7

1
. 0
9

1
. 1
1

1
. 1
3

1
. 1
5

1
. 1
7

1
. 1
9

1
. 2
1

1
. 2
3

1
. 2
5

1
. 2
7

1
. 2
9

1
. 3
1

1
. 3
3

1
. 3
5

1
. 3
7

1
. 3
9

M
o
r e

F
r
e
q
u
e
n
c
y

Value relative to predicted
Figure 1: Structural arrangement of AMY1 locus illustrating 8Kb proviral insertions conferring
AMY1 tissue specificity. Sequence identity extends beyond 28Kb for the three AMY1 copies.
1. Paralogue ratio test (PRT)
Figure 2: PRT takes advantage of paralogous in the genome thereby only one pair of carefully
designed primers amplify different sized test and reference amplicons.
Figure 3: nPRT eliminates disadvantages of PRT by not relying of paralogous sequences and
solves the problems inherent in multiplex PCR. The products generated from the initial 5 cycles
are extended by the externally added synthetic primers, one of which is fluorescently labelled
which aids in capillary electrophoresis as seen in the trace.
Figure 5: The presence of variably sized
microsatellite marker within the AMY1 repeat
block ensured the development of the MS assay.
This microsatellite profiles suggest that this
marker is transmitted through generations as
seen in CEPH family 1416. A strong correlation
was observed between 12A and MS assay.
Table 1: Five fold determination of copy number as
measured by five independent systems. The DNA
samples used two independent sources of origin i.e.
HapMap and CEPH
Conclusions:
A. A novel ratio based assay, Non PRT was developed and successfully used in predicting
the AMY1 copy number.
B. Segregation analysis in families using the MS assay determined definitive copy numbers
which will be used as calibrator standards for future work.
C. The MS assay is a robust method to measure AMY1 CNV which substantiates previous
evidence.
D. As opposed to 2-14 copies, a diploid copy number of 2-16 was observed for HapMap
Japanese and a haploid copy number of 1-6 was observed for CEPH families.



Figure 6:Frequency distribution of the ratio of
ratios deduced from the MS assay representing the
deviation from the expected value for 182 ratios.
90% of the ratios lie within 85% to 115% of the
expected value highlighting the accuracy of the
assay.
Acknowledgements:
We would like to thank the University of Nottingham and the International office for funding Sugandhas PhD. Special vote of
thanks to Dr Jess tyson for her encouragement and also to the past and present members of C10 Lab.
References: 1. Redon et al. (2006) Nature, 444, 444-454 2. Zabel, et al. (1983) PNAS, 80(22), 6932-6936
3. Nishide et al. (1986) Gene, 41, 299-304 4.Perry et al. (2007) Nature Genetics, 39, 1256-1260
5. Ting et al. (1992) Genes & Development, 6, 1457-1465 6. Walker et al. (2009). Genomics, 93, 98-103.

MS CN 12A Ratio
7.22
8.30
11.75
6.31
4.33
9.05
Father
Mother
Child
Child
Child
Child
CEPH family 1416 CEPH collection.
2 1 3 + + 6 copies
Triplex system
Duplex system
1H Test (chr.1)
1H Reference (chr. 1)
12A Reference (chr. 12)
12A Test (chr. 1)
MS assay
(chr. 1)
Test 1 (chr. 1)
Reference 1
and 2 (chr. 11)
Test 2 (chr. 1)
1
2
4
5
3
6
2. Microsatellite Assay (MS assay)
This assay involves the amplification of microsatellite which vary in their length or number
and is spatially contained within the AMY1 ERV thus producing allelic ratios specific to AMY1
copy number.



D. Non conformity between previous published reports and the MS assay.

Our microsatellite measurement assay do not
conform with the copy number deduced by Perry et
al. This non conformity amounts to approximately
one to two integer copies more as measured by our
measurement systems.
Figure 4: Capillary trace of NA18999 predicting a copy number of six instead of five as
predicted by Perry et al.
MS assay
1+2+3+2=
2+2+2+1=
2+3+2+2+2=
3+2+1=
1+2+1=
1+2+2+2+2=
=
systems
Measurement
2 pairs of gene specific primers, but if used for too many
cycles amplification differences are exacerbated.

So used tailed primers

Quartet of tailed primers used at low concentration,
low annealing temperature for 5 cycles.

Addition of primers
matching the tails

Test Reference
Test chr. 1
Reference chr. 1
Test chr. 1
Reference
chr. 12
Microsatellite chr. 1 Test chr. 1
Reference
chr. 11
Reference
chr. 11
Test chr. 1