PRENAT CARDIO. 2013 MAR;3(1):15-21.
Lech Dudarewicz et. al.
Original paper
IS SUBTELOMERIC MLPA TEST (MULTIPLEX LIGATION-DEPENDENT PROBE
AMPLIFICATION) USEFUL IN PRENATAL DIAGNOSIS?
Authors:
Lech Dudarewicz1, Anna Krzymińska 2, Wanda Hawuła1, Magdalena Kozłowska1, Urszula Laskowska1,
Agnieszka Gach1, Maciej Borowiec3, Wojciech Młynarski3, Wojciech Ałaszewski1, Lucjusz Jakubowski1
1
2
Department of Genetics, Polish Mother’s Memorial Hospital Research Institute, Lodz, Poland
Department of Neuropeptides Research., Medical University of Lodz
PRENAT CARDIO 2013 MAR;3(1):15-21
DOI 10.12847/03133
Abstract
Objective of the study:
At the moment of study design, there was no data available on prevalence of subtelomeric imbalanced rearrangements
in fetuses with abnormal phenotype assessed by ultrasound and with normal classical karyotype, consequently this study
was initiated to fill in this gap.
Material & Method:
Amniotic fluid samples or chorionic villi from:
137 fetuses with abnormalities in two or more organ systems
96 fetuses with nuchal translucency above 3.5 mm (99th centile),
85 apparently healthy fetuses (control group) were studied by subtelomeric MLPA, using two kits (P036 and P070) in
all cases. Confirmation of a rearrangement was obtained by means of fluorescence in situ hybridization (FISH) studies.
Results:
In the group of fetuses with abnormalities in two or more organ systems, one subtelomeric deletion (de novo deletion
(del1p36).) was detected, yielding the detection rate of cryptic subtelomeric imbalances in these pregnancies of 0.84%.
In the control group and in the group of fetuses with NT measurement above 3.5 mm, no abnormalities were found.
Conclusion:
The low detection rate of subtelomeric rearrangements in the studied group, together with the low robustness of the method
(only one sequence per telomere is studied in one experiment) and necessity to confirm the pathological findings with
another method, imply low usefulness of the method in the prenatal setting. In the current era, there are genome-wide
methods, like CGH-arrays or SNP-array, which are better-suited for prenatal diagnosis, because of higher yields and lack
of necessity of confirmation of the pathological results.
Key words: subtelomeric deletions, subtelomeric rearrangements, submicroscopic chromosome aberrations, prenatal diagnosis,
congenital defects, fetal structural defects, abnormal fetal fenotype, fetal ultrasound, prenatal ultrasound, MLPA
INTRODUCTION
Prenatal diagnosis consists of various tests, aimed
at different variables, which predict fetal health status,
like presence or absence of structural defects or
genetic disorders. Prenatal diagnosis procedures can
be performed as screening tests or in a patient at risk
because of the anamnesis or because of the abnormal
result of the screening.
In the available bibliography there have been few studies
available on the frequency of subtelomeric imbalanced
rearrangements in fetuses with abnormal phenotype
assessed by ultrasound and with normal classical
karyotype, moreover the results of those studies were
contradictory1,2,3,4.
AIM OF STUDY
In most fetuses and even neonates with congenital
anomalies it is not possible to find out the ultimate cause
of the disease, which hampers genetic counselling.
Subtelomeric rearrangements are one of the known
causes of the abnormal phenotype in postnatal patients,
in this connection the aim of the present study was to
obtain data on their frequency in the group of fetuses with
Corresponding author: [email protected]
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Is subtelomeric MLPA test (Multiplex Ligation-Dependent Probe Amplification) useful in prenatal diagnosis?
the abnormal phenotype as assessed by ultrasound and
normal classical karyotype. The study was performed in
patients of the Department of Genetics, Polish Mother’s
Memorial Hospital. The study was financed from the
funds of the Polish Committee for Scientific Research,
later named Ministry of Science
and Informatization .
scan. The following cases were excluded from the
study group: Abnormalities detected by the routine
karyotyping, lack of complete data or non-informative
results. Also the fetuses with isolated abnormalities in
one organ system (e.g. urinary, cardiovascular, skeletal,
gastrointerstinal, abdominal wall,
etc.) were excluded. In most cases
the karyotype was assessed
The study outline and design
prenatally (from the amniotic cells
How to cite this article:
was approved by the Institutional
or from the trofoblast cells), but
Dudarewicz L, Krzymińska A,
Review Board (The Scientific
in some cases it was assessed
Hawuła W, Kozłowska M, Laskowska
Studies Ethical Committe at
or confirmed after birth. The
U, Gach A, Borowiec M, Młynarski W,
the Polish Mother’s Memorial
gestational age was calculated
Ałaszewski W, Jakubowski L.
Hospital. Prior to the study design
using early ultrasound dating, in
Is subtelomeric MLPA test (Multiplex
the method of choice to look for
almost all cases using first trimester
Ligation-Dependent Probe Amplification)
subtelomeric rearrangements
biometry. Before participation in
useful in prenatal diagnosis?.
(mainly deletions) was
the study all patients were provided
Prenat Cardio. 2013 Mar;3(1):15-21.
Fluorescent In Situ Hybridization
with extensive genetic counselling
(FISH) using appropriate
and with detailed information about
probes. This technique is costly
the study, including the information
and has low throughput. In fact, the idea of this study
on invasive prenatal testing. All patients gave explicit
originated because there appeared a new, cost-efficient
written consent to participate. Before sampling, all fetuses
technology, called MLPA (Multiple-Ligation-dependent
underwent the anomaly scan by an experienced operator
Probe Amplification), which enabled, among others, the
using GE Voluson 730 Pro ultrasound machine with
detection of subtelomeric imbalances in all chromosomes
multi-frequency convex probes. NT measurements were
in a single run with high throughput. The additional goal of
performed according to the FMF protocol, by an accredited
the present study was to assess any possible correlation
sonographer. Mainly the transabdominal route was used,
between the type of the observed phenotypic abnormalities
but in chosen cases also the transvaginal scanning was
and the kind of the subtelomeric rearrangement.
carried out. The fetuses who were qualified for the present
study were classified as multiple structural anomalies,
MATERIAL AND METHODS
malformation syndromes or early morphogenesis defects.
The study group consisted of the fetuses diagnosed
The separate group was made up of fetuses with the
because of the abnormalities observed during ultrasound
NT value increased over the 99th centile, without other
phenotypic abnormalities. The value of the 99th centile
was chosen because of the small percentage within the
Trimester
whole group of examined fetuses and because of the
relatively high risk of significant abnormalities 5. In the
Type of the anomaly
I
II/III
group of fetuses with the increased NT, the median was
5.1 mm and the range was between 3.5 and 11 mm.
Malformations in > 3 organ systems *
5
12
Malformations in 2 organ systems
7
37
Cardiovascular malformations combined with
dysmorphism
8
22
Abdominal wall defects combined with dysmorphism**
6
10
CNS defects combined with dysmorphism***
5
11
Craniofacial defects combined with extrafacial
dysmorphism
3
11
NT≥ 99 centile
96
-
Table I: Distribution of fetuses according to the gestational age and number
of anomalies
The gestational age at the moment of amniotic fluid
sampling was within the range of 15 and 31 weeks, and
at the moment of chorionic villi sampling was between
11 and 14 weeks.
The inclusion criteria were met by 137 fetuses, described
in table I. In 119 cases informative results of the MLPA
test were obtained.
Methods
DNA from the amniotic fluid or from the chorionic villi
was extracted using QIAamp DNA mini kit (Qiagen, USA).
MLPA reaction was performed in accordance with
manufacturer’s instructions. After 40 PCR amplification
cycles the product separation was performed using ABI
3100 machine (Applied Biosystems, USA). The principle
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PRENAT CARDIO. 2013 MAR;3(1):15-21.
source: www.mlpa.com
of the MLPA method is briefly explained in fig. 1.
MLPA reactions using subtelomeric rearrangements
kits P036 and P070 were performed according to
manufacturer’s protocol. Each of these kits contains
probes complementary to the subtelomeric regions of
every chromosome and was designed to detect deletions
or duplication of these regions. Most of these probes are
complementary to well characterized genes located in
subtelomeric regions. Chromosomes 13, 14, 15, 21 and
22 have more than 10 Mb of repetitive sequences, which
cover most of p their arms, therefore only sequences
from q arms were used in case of the above mentioned
chromosomes. For confirmation, samples from all
patients have been analyzed using both kits, so that for
the subtelomeric region of every chromosome, the results
of tests using two different, closely located sequences
were available (all probes used in P070 kit are different
from the probes in P036 kit).
Lech Dudarewicz et. al.
Data analysis
The data obtained from analysis of the products of MLPA
reaction was analyzed using GeneMarker (SoftGenetics.
USA) analysis software after data normalization within the
sample and between different samples. The normalization
of the data was performed according to MLPA kits
manufacturer’s instructions, conforming to the rule, that
only data obtained using the same DNA extraction method,
from the same experiment and the same reagents’ lots
may be compared. Dose ratio for every sample was
calculated by dividing peak heights of the sample after
normalization by the average peak height of the control
samples and were expressed as ‘MLPA ratio’.
Criteria of abnormality
The values of „MLPA ratio” were settled as close to 1,0
for wildtype peaks, below 0,75 for deletions and over 1.32
for duplications. The abnormal results were confirmed
by FISH technique (Fluorescent In Situ Hybridization)
using Chromoprobe Mulitiprobe-T system
(Cytocell).
RESULTS:
MLPA reactions using P036 and P070
kits for subtelomeric rearrangements were
performed on samples obtained between
11th and 31st weeks of gestational age in:
137 fetuses with normal classical
karyotype(GTG banding, resolution >400
bands) and abnormal penotype assessed
by ultrasound
96 fetuses with nuchat translucency
thickness abothe the 99th centile,
85 fetuses with normal phenotype
as assessed by ultrasound and normal
karyotype, karytotyped because of the
advanced maternal age.
All samples were tested using both P036
and P070 kits, so two different sequences
were analyzed for each subtelomeric region
of every chromosome. The abnormal
results were verified by FISH Chromoprobe
Mulitiprobe-T (Cytocell).
After performing the MLPA analysis,
copy number changes were detected in
29 fetuses from the study group and in
16 fetuses from the control group. In the
* - After exclusion of NT thickening
** - After exclusion of gastroschisis
*** - After exclusion of isolated dysrafic defects and their
associated findings
Fig. 1. MLPA – The principle
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Is subtelomeric MLPA test (Multiplex Ligation-Dependent Probe Amplification) useful in prenatal diagnosis?
Fig. 2 - Example of the normal result of subtelomeric MLPA analysis.
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PRENAT CARDIO. 2013 MAR;3(1):15-21.
Lech Dudarewicz et. al.
Fig. 3 - Result of a subtelomeric MLPA test showing 1p36 deletion.
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Is subtelomeric MLPA test (Multiplex Ligation-Dependent Probe Amplification) useful in prenatal diagnosis?
samples from 25 fetuses from the study group, in which
copy number changes were detected, they were not
confirmed by the other, alternative kit, therefore they were
considered an artefact. The three remaining samples were
not available for verification, either because of the fetal
death, or pregnancy termination. Finally in one sample
subtelomeric deletion (del1p36) which was detected by
MLPA was confirmed by FISH. It was a de novo deletion
(in both parents the result of the FISH study using the
appropriate probe was normal), and the fetal phenotype
(heart defect and craniofacial abnormality) correlated
with the clinical picture of patients with the same deletion
described in the literature, therefore this copy number
change was judged to be causative.
The results presented above reveled the frequency
of subtelomeric imbalanced rearrangements in fetuses
with multiple phenotypic abnormalities as 1 in 119, and
in fetuses with NT value above 99th centile as 0/96, using
subtelomeric MLPA test. An example of the normal result
of the MLPA subtelomeric analysis is shown in fig. 2. As
a comparison, the only abnormal, confirmed subtelomeric
deletion MLPA result is illustrated in fig. 3.
DISCUSSION
MLPA is a versatile method enabling quantitation of
several (up to fourty) different sequences in a single
experiment. It is used in many research and diagnostic
applications. Contrasting to many other PSR-based
methods, not the target sequence, but the probe,
complementary to the target sequence is amplified in
this type of analysis (the principle behind this method
is briefly explained in fig. 1). The number of amplified
sequences is proportional to the starting number of the
target sequences in the sample. The number of the
amplified sequences is calculated using a sequencer
or other device for capillary electrophoresis. There are
commercially available MLPA kits, designed for particular
types of analysis, like e.g. detection of aneuploidy, common
microdeletion syndromes, and many other, still more
numerous applications. MLPA has numerous advantages,
like: short turnaround time, possibility to confirm the
rearrangement by another kit, employing different, closely
linked probes, possibility to test multiple (up to 40)
sequences in a single run, low cost per sequence tested,
high amenability to automation and high throughput. There
are however drawbacks, which include the requirement to
use high quality source DNA and relatively large starting
number of source sequences in the sample (which is
a particular obstacle in prenatal diagnosis), frequent
occurrence of interpretation problems and difficulty in
detecting maternal contamination of the sample6. The
experience gained during the present study let us state,
that the disadvantages particularly troublesome in prenatal
diagnosis of subtelometic copy number changes involve
interpretation problems connected with low number of
sequences tested per telomere, actually in case of using
one kit there is only one sequence tested per telomere,
and when using the second, confirmatory kit, there
are just two segments tested per chromosome arm,
making this method prone to errors. Because of the high
percentage of false positive aneuploidy calls, another
confirmatory method is necessary (in our case FISH), which
increases the cost and lengthens turnaround time. In other
applications the robustness of the method is enhanced by
the fact of use of multiple sequences per target, reducing
the influence of a single false call on the final conclusion.
Basing on own experience we are not able to answer the
question about the origin of the numerous false positive
results, we are only able to guess, that a proportion of them
might have arisen from the existence of polymorphisms
or because of the maternal contamination. MLPA is very
sensitive not only to maternal contamination, but to minor
variations in experiment conditions as well. In the majority
of archived samples, collected in the years preceding
the current study, we were not able to achieve reliable
results, mainly because of DNA quality issues - the DNA
was too fragmented and degraded as a result of freezing
and thawing. The requirement to confirm the abnormal
result by an independent method may also be viewed as
a serious drawback, practically eliminating subtelomeric
MLPA from the clinical application in prenatal diagnosis.
In the presented study, MLPA was used to search for
copy number changes of the subtelomeric regions of all
chromosomes as a possible submicroscopic basis of the
phenotypic abnormalities in fetuses. Subtelomeric copy
number change was detected, confirmed and regarded as
causative in only one of 119 cases of multiple phenotypic
abnormalities in fetuses, in none of 96 fetuses with NT
above the 99th centile and in none of the 85 fetuses from
the control group.
In case of the 1p36 deletion, the relationship between the
copy number change and abnormal phenotype appears
proven, as it was a de novo mutation with anomalies from
the spectrum described in the literature7. The comparison
of our results with the results of other authors is limited
by the scarcity of reports present in bibliography, and
the small number of cases studied, nevertheless the
low percentage of causative subtelomeric copy number
changes falls within the scope described in literature 1,2,3,4,5.
It is worth mentioning, that several factors, e.g. previously
mentioned polymorphism or mutation located within the
sequence recognized by the probe, or even a minor
change in reaction conditions, may lead to change in
peaks area and therefore to the false result. The confidence
of making conclusions by using merely one or two probes
per telomere is limited and considerably lower than in
case of newer methods (microarrays or next generation
sequwncing) which utilize multiple sequences per given
chromosomal region.
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CONCLUSIONS
The results of the presented study have to be interpreted
with caution, because of the limited number of cases,
occurrence of unconfirmed results and possible selection
of cases connected with the fact of origin from the
single center.
Conflict of interest: The author declares no conflict of interest and
did not receive any remuneration
Subtelomeric MLPA analysis is more difficult to perform
and interpret even in comparison with other MLPA
applications because of the technical problems and low
reliability stemming from the small number of sequences
tested per chromosomal region. MLPA is alo impractical
for testing archived samples of the amniotic fluid.
The restraints mentioned above, combined with the
low yield of pathologies render subtelomeric MLPA not
practical in the scope of routine prenatal diagnosis.
The study group did not include sufficient number of
cases to perform any analysis of genotype-phenotype
correlation.
The study was supported by the research grant of the
National Center of Science no 407 0166 35
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