for MLPA Analysis Using Luminex xMap®
MLPA has proven to be an important technique in the detection
of exon copy number changes associated with breast and colon cancers,
as well as trisomies found in Down Syndrome (3, 4, 5). Coupling
MLPA with Luminex microsphere technology has the potential to
also be as important in the detection of copy number changes associated
with Niemann-Pick type C (6, 7, 8), alpha-thalassemia (8) and
other genetic diseases. Accurate analysis of Luminex-microsphere
flow cytometry instrument generated data is critical to disease
SoftGenetics’ GeneMarker now includes a Luminex-MLPA module
that quickly and accurately analyses data from Luminex instruments
(Luminex 100 and Luminex 200). The software is compatible with
the Luminex instrument data format (*.csv).
GeneMarker® Luminex-MLPA analysis automatically
performs background subtraction, and flags suspect intensities
according to user specified thresholds. The software selects the
least variable sample, from the sample set to act as the reference
for determining copy number change within the patient sample set.
Analysis of Luminex MLPA data is easy to perform. In the menu
bar of GeneMarker click Tools and select ‘Luminex MLPA Analysis’
from the drop-down menu. Select and open the file to analyse.
The Luminex MLPA
Data window will activate (figure 1). The user may select their
own control probe or normal sample (right click on the sample
row or cell to activate the control edit menu) or bead count threshold.
Click OK to initiate analysis. GeneMarker will automatically perform
background subtraction, select control sample and calculate copy
Ratios are automatically calculated and displayed in graphical
and tabular forms. Default settings can easily be adjusted to
meet user specified requirements.
In addition to reading Luminex data, GeneMarker®
can perform fragment analysis and genotyping on four or five colour
data sets from any slab gel or capillary electrophoresis system.
This software automatically corrects for any common problems—instrument
spike, colour pull-up, peak pull-up, noisy data, saturated peaks
and stutter peaks—saving significant analysis time and cost,
efficiently analysing raw fragment data within seconds. GeneMarker
is robust software to analyse DNA fragment data labelled with
MegaBACE™ dyes (Amersham), Big Dye® (Applied
Biosystems Inc.) or Beckman dyes from a variety of platforms:
ABI DNA Analyser or Genetic Analyser, Amersham instruments or
Beckman instruments. GeneMarker is compatible with files from
all major capillary and slab gel electrophoresis systems including
ABI files (*.FSA,*AB1, *.ABI), SCF files, MegaBACE®
files (*.RSD, *.ESD), SpectruMedix files (*.SMD, *.SMR), Beckman
files and Li-Cor files (*.scf). The software features high efficiency
allele calling, adjustable parameters and a variety of reporting
Figure 1. Luminex MLPA Data analysis
window: The top field displays instrument and project information.
The bottom field lists samples and genes analysed in the project
(control sample edit menu is active), notice sample 14 has been
selected as the background (B) sample. The lower left corner contains
the Suspect bead Count dialog box (the default setting value is
After background subtraction, reference sample selection and
intensity ratio calculation, the data are presented in an intensity
ratio plot. Sample number 8, H1_M1, was selected as the reference,
designated by the # sign. Probes with a normal copy number are
indicated by green squares. Probes that may have a duplication
or deletion are indicated by red squares (figure 2).
Sample C1 (highlighted in the list of samples and indicated in
red above the intensity ratio plot) is from a female patient.
The DMD ratio is 1.699: one copy of the x-linked DMD gene in reference
sample H1_M1 and two copies of the DMD gene in sample C1. The
intensity of DMD is highlighted in blue in the intensity ratio
table, the peak corresponding to DMD in the comparison chart is
highlighted in light blue and the intensity ratio of DMD is indicated
by the yellow square (red if not selected) in the ratio plot.
The three red squares in the intensity ratio plot clearly indicate
a deletion of one copy of HBB exons 1, 2 and 3 (figure 2).
Samples in the MLPA analysis window that contain probes that did
not meet the minimum bead threshold (Suspect Count) will be identified
(Figure 3). Suspect probes will be labelled in red in the peak
comparison chart and the corresponding point in the intensity
ratio plot will be pink.
Figure 2. MLPA analysis window
displaying list of patient samples (left), peak height comparison
chart (top centre: red peaks are control sample; blue peaks are
patient sample), intensity ratio plot comparing patient sample
to control sample (bottom centre) and intensity ratio table (upper
Figure 3. MLPA analysis window
showing peak height comparison chart (labels are in red) and intensity
ratio plot (squares are labelled in pink for suspect probes that
contained too few beads).
GeneMarker for Luminex MLPA converts Luminex
bead intensities into easy-to-read chromatograms.
The intensity ratio table can be saved as a tab delimited *.txt
file. A patient report (Figure 4) can be printed or saved as *.MDI
or *.jpg formats.
Figure 4. Patient
report includes sample ID, analysis parameters, intensity ratio
report (copy number changes are shaded), intensity ratio plot
and peak height comparison chart by individual sample.
Various techniques including DGGE (Denaturing Gradient Gel Electrophoresis),
DHPLC (Denaturing High Performance Liquid Chromatography) and
SSCA (Single Strand Confirmation Analysis) effectively identify
SNPs and small insertions and deletions. MLPA is one of the only
accurate, time efficient techniques to detect genomic deletions
and insertions which are frequent causes of cancer and genetic
diseases. Luminex-MLPA can successfully and with high sensitivity
easily determine the relative copy number of exons within a gene.
GeneMarker provides a quick and easy-to-use tool for necessary
quantification and reporting of the data.
1. Fulton RJ, McDade RL, Smith PL, Kienker LJ,
Kettman JR Jr, 1997, Advanced Multiplexed analysis with the FlowMetrixTM
system. Clinical Chemistry, 43: 1749-1756
2. Sellner LN, Taylor GR, 2004, MLPA and MAPH: New techniques
for detection of gene deletions. Human Mutation, 23:413-419
3. JJP Gille, FBL Hogervorst, G Pals, JTh Wijnen, RJ van Schooten,CJ
Dommering, GA Meijer, ME Craanen, PM Nederlof, D de Jong, CJ McElgunn,
JP Schoutenand FH Menko, 2002, Genomic deletions
of MSH2 and MLH1 in colorectal cancer families detected by a novel
mutation detection approach. British Journal of Cancer, 82(892-897)
4. Frans B.L. Hogervorst et al., 2003, Large Genomic Deletions
and Duplications in the BRCA1 Gene Identified by a Novel Quantitative
Method. Cancer Research, 63 (1449-1453)
5. Jan P. Schouten, Cathal J. McElgunn, Raymond Waaijer, Danny
Zwijnenburg, Filip Diepvens and Gerard Pals, 2002, Relative quantification
of 40 nucleic acid sequences by multiplex ligationdependent
probe amplification. Nucleic Acids Research, Vol. 30, No.12 e57
6. Dawson DB, Lundquist P. FlexMAP MLPA identifies copy number
changes in the NPC1 gene for four patients with Niemann-Pick type
C. HGVS/HGNC Helsinki Meeting May 31, 2006.
7. Lundquist P, Flynn-Gilmer HC, Thorland E, Dawson DB, 2005,
Detection of chromosome 18q11-12 microdeletions in two Nieman-Pick
type C families using Luminex-based MLPA and FISH
(Abstract G43). J Mol Diagn Nov; 7(5):657-8.
8. Dawson DB, Marley VM, Bogenschutz JL, Lundquist PA, Bortolin
S, Janeczko R, Highsmith WE. Detection of gene dosage differences
in alpha-thalassemia and Niemann-Pick type C using tags/
anti-tags and FlexMap microspheres on the Luminex 100 platform.
Association for Medical Genetics Clinical Genetics Annual Meeting
We would like to thank Brian Dawson, Ph.D., Mayo Clinic, for
collaborating with us to develop the use of Luminex xMap technology
with MLPA analysis.
GeneMarker® for MLPA
Analysis Using Luminex xMap® Technology