Carrier detection in families affected by duchenne muscular dystrophy using multiplex ligation dependent probe amplification (2)

ABSTRACT
Duchenne muscular dystrophy (DMD) is a recessive disorder
associated with the chromosome X caused by mutations in the
dystrophin gene. It affects mainly boys. According to an analysis of
previous studies, two-thirds of cases the defective gene is passed on
to a son through the mother’s faulty X chromosome; the diagnosis of
female carriers (mother, aunt, sister) to detect mutations which would
enable medical staff to provide prenatal genetic counseling for them
is the most effective solution to reduce incidence. With many
advantages such as rapid, sensitive, cost effective, reliable so MLPA
is used as the first option and is a useful quantitative method for
detecting mutation for the analysis of both affected males and female
carriers.
In this study, we have succeeded in the application of the
MLPA method to identify female carriers. Using the MLPA method,
we detected 7 out of 10 female carriers in 5 affected DMD families.
Four of them show heterozygous deletion exons 45-52, 8-43, 3-47
and 48-53, in the DMD gene, respectively. The remaining three
female carriers have heterozygous duplication exons 11-20 and 51-60
in the DMD gene. Most of these mutations are located in the ‘hot
spot’ regions. In addition, we also detected one patient with
duplication exons 11-20 and 51-60 in the gene. MLPA assays are
performed according to manufacturer recommendations.

1


TÓM TẮT
Bệnh loạn dưỡng cơ Duchenne (Duchenne Muscular
Dystrophy- DMD) là bệnh di truyền lặn liên kết với nhiễm sắc thể X
gây ra bởi đột biến ở gen dystrophin.Theo một số nghiên cứu, khoảng

hai phần ba số trường hợp gen khiếm khuyết được truyền sang con
trai từ người mẹ có nhiễm sắc thể X bị lỗi; chẩn đoán người nữ dị
hợp tử (mẹ, cô, dì, chị gái) để phát hiện các đột biến sẽ cho phép
nhân viên y tế tư vấn di truyền trước khi sinh và là giải pháp hiệu quả
nhất để giảm tỷ lệ mắc căn bệnh này. Với nhiều ưu điểm như nhanh,
nhạy, chi phí hiệu quả, độ tin cậy cao nên MLPA được sử dụng như
là sự lựa chọn đầu tiên và là một phương pháp định lượng hữu ích
cho việc phát hiện đột biến trong phân tích của cả nam giới bị bệnh
và người nữ dị hợp tử.
Trong nghiên cứu này, chúng tôi đã thành công trong việc áp
dụng phương pháp MLPA để xác định người nữ dị hợp tử. Sử dụng
phương pháp MLPA, chúng tôi phát hiện được 7 phụ nữ có mang gen
dị hợp tử trong số 10 phụ nữ trong 5 gia đình bị ảnh hưởng bởi bệnh
DMD. Bốn người trong số họ có đột biến xóa đoạn dị hợp tử ở các
exon 45-52, 8-43, 3-47 và 48-53. Ba người nữ còn lại có đột biến dị
hợp tử lặp đoạn ở các exon 11-20 và 51-60 trong gen DMD. Hầu hết
những đột biến này được phát hiện tại các vùng “hot spot”. Ngoài ra,
chúng tôi cũng phát hiện một bệnh nhân có đột biến lặp đoạn ở các
exon 11-20 và 51-60 trong gen dystrophy. Kỹ thuật MLPA được thực
hiện theo khuyến nghị của nhà sản xuất.

2


PREFACE
Duchenne muscular dystrophy (DMD) is one of the most
common fatal genetic disorders affecting children around the world. The
causes of DMD are mutations in the dystrophin gene on chromosome X;
hence, it is diagnosed mostly in males. Although DMD is the most
common fatal genetic disorder to affect children, at the moment no

cures have been found. Researchers are still looking for treatments to
alter the course of the disease and improve the quality of life for
patients. Previous studies have shown that two thirds of patients
receive the mutation from their mothers and the other one third has
new mutation [14], [19], [47], [54]. Thus, detection of the
heterozygous status of mother as well as other female members of
family with suitable consequent antenatal screening of the fetus at
risk is highly appreciated active prevention (reduces new incidence
of the disease). In recent years, many studies show that Multiplex
Ligation- dependent Probe Amplification (MLPA) is a rapid and
accurate

technique,

which

allows

high-throughput

screening

mutations, especial deletions and duplications, in DMD and other
genetic diseases. This is a molecular biology method based on the
basic principle of PCR; nonetheless, it uses one pair of primer, two
reactions to detection mutation in all 79 exons. Thus, within 1 week,
MLPA can screen all mutations in the dystrophin gene. This is a
particular advantage of the MLPA compared with other methods. In
order to carry out the diagnosis, prognosis and genetic counseling for
female carriers, we carry out the study: "Carrier detection in

families affected by Duchenne muscular dystrophy using
Multiplex Ligation- dependent Probe Amplification”.

3


CHAPTER 1. INTRODUCTION
1.1. Duchenne muscular dystrophy
1.1.1. Characteristics of DMD
DMD is an X-linked recessive disease caused by mutations
in the DMD gene [56]. Therefore, it was found to be rather more
common in males than females. DMD is a progressive disease in
which the patient's muscle injury is due to a lack of dystrophin
protein. The dystrophin gene can be passed on from the carrier
woman to her child (complies with the rules of the genetic X- linked
inheritance). According to Mendelian inheritance, there is 50%
chance a mother who carries the DMD gene can pass the X
chromosome carrying DMD mutated gene to the sons and they will
develop disease and 50% chance that her daughters will carry the
gene. A carrier mother may or may not pass on the gene with the
mutation. Normally, the majority of female carriers usually have no
symptom. However, 2-20% of carriers have symptoms of muscle
weakness or clinical signs of disease.
1.1.2. Treatment and management of DMD
It is nearly 30 years since the discovery of the genetic defect
causing DMD, but the disease has yet to be cured. To date only one
treatment, the use of corticosteroids, has been shown to be effective
in DMD patients [5], [55]. At present, some areas in which research
is being focused include: gene therapy, read-through stop codon
strategies, stem cell therapy, vival vectors and utrophin [59].

However, these methods are only of partial support for patients and
they are not the complete cures. Therefore, diagnosis of women with
a high risk and genetic counseling for female carriers in patients’
4


families are still the best options to aid in the prevention of this
disease [32], [39].
1.2. The dystrophin gene
The dystrophin gene is the largest known human gene. It is
located on short arm of the X- chromosome at position Xp21.2
spaning approximately 2400 kb, consists of 79 exons and produces a
14.6 kb mRNA [6], [8], [17], [64]. It is also composed of at least 7
alternative promoters: brain (B) promoter, muscle (M) promoter,
cerebellum promoter, promoter Dp 260, Dp 140, Dp 116, Dp 71,
leading to a number of different isoforms (Figure 3) [35].
1.3. Protein dystrophin

The product of the dystrophin gene is dystrophin
protein. Dystrophin is a hydrophobic, rod-shaped protein that is
found typically in muscles and is used for muscle movement. It
is encoded by the DMD gene and it has a molecular weight of 427
kDa, and contains about 3685 amino acids [17], [23]. This protein is
located in the plasma membrane of muscle cells and is divided into
four domains [23]: the amino-terminal domain, the central- roddomain, the cystein- rich domain, he carboxy- terminal domain.
Dystrophin plays an important structural role as part of a large
complex in muscle fiber membranes. It provides a structural link
between the muscle cytoskeleton and extracellular matrix to maintain
muscle integrity [7], [42].
1.4. Mutations in the dystrophin gene

1.4.1 Deletion mutations

5


Deletion one or several exons is common mutations in
patients with DMD, accounting for 60-65% of DMD disease-causing
mutations [9]. Deletion mutations in DMD genes are the most
commonly found intragenic deletions and concentrated in two known
"hot spot" regions.
1.4.2. Point mutations
Point mutation, accounting for 25-30%, is the second largest
mutation in the dystrophin gene after deletion mutations [43]. Most point
mutations in the DMD gene created stop codon and caused seriously
disease. Point mutations are located along the entire gene and this is a
major obstacle in identifying these mutations.
1.4.3. Duplication mutations
Duplication mutations are the cause of DMD in most of the
remaining cases (approximately 5%-15%). Prior (2005) suggested that
80% of mutations occur in the 5 'end and 20% in the center. In addition,
a small percentage of DMD patients have small mutations scattered
along the length of the entire gene making them difficult to detect [39].
1.5. The methods to detection mutations in the dystrophin gene
1.5.1. PCR method

PCR (polymerase chain reaction) is based on the
synthesis of a target DNA segment under the catalysis of the
enzyme DNA-polymerase (taq polymerase), and occurs in
repeated cycles. Components in the PCR reaction include DNA
sample, deoxynucleotide triphosphate (dATP, dGTP, dCTP,

dTTP), MgCl2, primers, DNA polymerase and PCR buffer
6


solution [33]. PCR is a three-step process that is carried out in
repeated cycles. The number of cycles per PCR reaction
depends on the initial number of DNA template, usually, does
not exceed 40 cycles. In the case of DMD, PCR plays an especially
important role in the process of mutation detection. For example,
multiplex PCR is appreciated in the diagnosis of deletion and about
98% of deletions are easily detectable using multiplex PCR in
affected males [3].
1.5.2. Southern blot method
Southern blot is a method used in molecular biology for
detection of a specific DNA sequence in DNA samples. The principle
in this method combines agarose gel electrophoresis for size
separation of DNA with methods to transfer the size- separated DNA
to a filter membrane for probe hybridization. Prior (2005) used this
method to detect mutations in DMD patients as well as female
carriers [39].
1.5.3. Fluorescence in situ hybridization - FISH
FISH (fluorescence in situ hypridization) is a molecularcytogenetic technique that permits DNA sequences to be detected on
metaphase chromosomes, in interphase nuclei, in a tissue section, or
in blastomeres and gametes [40]. This method uses fluorescent
probes that bind to only those parts of the chromosome with which
they show a high degree of sequence complementarity. Many studies
indicate that FISH is an efficient, sensitive method that brings
confident results to detection, identification and to screen DMD
female carriers [27], [51], [57].

7


1.5.4. Sequencing method
Today, with advancement of technology, both of Sanger
dideoxy method and Maxam-Gilbert chemical cleavage method are
replaced by modern sequencing equipment (automatic sequencing).
The new technology is based on the same principles of Sanger's
method but four different fluorescent dye-labelled ddNTPs are used.
Thus each fluorescent label can be detected by its characteristic
spectrum. The products are separated by automated electrophoresis
and the bands detected by fluorescence spectroscopy. For DMD,
sequence analysis of the dystrophin gene is a rapid way to detect
small mutation that nearly entire of the 79 exons but this method is
very costly and time consuming.
1.5.4. Multiplex Ligation- dependent Probe Amplification (MLPA)
method
In the MLPA technique, genomic DNA is hybridized in
solution

to

probe

sets,

each

of

which

consists

of

two

oligonucleotides: one short synthetic oligonucleotide and one long
probe oligonucleotide. The short synthetic oligonucleotide contains a
target-specific sequence (20–30 nucleotides) at the 3’ end and a
common sequence (19 nucleotides) that is the primer binding sites, at
the 5’ end. The long MLPA probe contains the 25-43 nucleotides
target-specific sequence at the 5’ end, a 36 nucleotides sequence that
contains primer binding sites and is common to all probes, at the 3’
end and a suffer sequence (19–370 nucleotides)- a variable length
random fragment in between to generate the length differences. The
different lengths of the products allow separation on an automated
capillary sequencer, and the peak areas are quantified [46]. MLPA
8


assay has become a widely used technique in laboratories performing
genetic testing for the molecular diagnosis in general and DMD in
particular. Compared to other techniques, MLPA is rapid, sensitive,
reliable and very simple to perform. Therefore, MLPA is highly
recommended by scientists for detecting DMD and others genetic
deseases.
1.6. The aim of the study
DMD is an X- linked disease which has a 100% fatality rate.

Up to now, DMD still has no effective method of treatment. Several
previous studies show that two-third of patients receives DMD genes
from heterozygous mothers and one-third of patients are new (de
novo) mutations [19], [54]. Hence, diagnosis of carriers is the most
effective option to restrict the development of this disease. In
Vietnam, MLPA has been used commonly because MLPA is rapid,
sensitive and accurate. The long term objective of this research is to
apply MLPA to detect DMD mutated gene in carriers and from
which, we aim to further develop the project into early diagnosis
prenatal program. To achieve the goal, we propose to pursue the
following specifics aim: Apply MLPA to detect the carriers in
families affected by DMD.

CHAPTER 2. MATERIALS AND METHODS
9


2.1. Patient, female carrier and normal control
1 male patient; 10 female relatives from 5 different families
characterized by DMD patients with exons 45-50, 11-20, 51-60, 347, 8-43 and 48-50 dystrophin deletions and duplications; 2 healthy
female

and

1

healthy

male

without

family

history

of

dystrophinopathies were analyzed as control.
2.2. Reagents and equipment
2.2.1. Reagents
a. Reagents for DNA extraction from blood
Lysis buffer solution; K solution; proteinase K solution (20
mg/ml); SDS 10% solution; Phenol: chloroform: isoamyl (25:24:1),
Chloroform: isoamyl (24:1); ethanol 100% (cold); ethanol 70%
(cold); sodium acetate 3M (pH 5.2), TE (Tris- EDTA).
b. Reagents for MLPA: SALSA MLPA probe mix P034 (DMD
exons 1-10, 21-30, 41-50, 61-70) and P035 (DMD exons 11-20, 3140, 51-60, 71-79) DMD/Becker kit is purchased by MRC Holland,
Amsterdam, The Netherlands.
2.2.2. Equipment
Thermal cycler (Eppendorf branch); GenomeLabTM GeXP Genetic
Analysis System (Applied Biosystems); Genemarker software v.1.95
(Softgenetic, State College, PA, USA); Automatic pipettes (range:
0.5-1000µl with tips matched together); Centrifuge suitable for 1.5
ml eppendorf tubes; Micro centrifuge; Thermomixer R (Eppendorf
branch); Nano drop spectrophotometer; Vortex machine; Timer,
Freezer (2-8oC, -20oC); 0.5, 0.2 ml PCR tubes.
10

2.3. Methods
2.3.1. Sampling process
Blood samples were collected in EDTA vacutainer tube (3 ml
quantities) from female relative, patients and matched control. The
process ensures absolute sterility.
2.3.2. DNA extraction from blood
The DNA was extracted from peripheral blood by
phenol/chloroform method. Then, the DNA will be tested by
measurement of optical density at wavelength of 260nm and 280 nm.
DNA samples, which has to concentrations > 20ng/μl and purity
(A260/A280) from 1.8 ÷ 2.0, will be used to carry out the next steps
of this study.
2.3.3. Calculation of DNA concentration
Calculation

of

DNA

concentration

depends

on

Spectrophotometric method. A ratio of OD260nm/OD280nm in the
range of 1.8 – 2 indicates highest -quality DNA whereas lower values
indicate protein contamination. Higher values indicate RNA
contamination [45].
2.3.4. Multiplex Ligation- dependent Probe Amplification (MLPA)

method
The MLPA DMD test kit (SALSA P34/P035) was purchased
from MRC Holland, Amsterdam, the Netherlands. The procedure
was performed according to the protocol provided by the
manufacturer’s recommendation [6]. Before carry out of MLPA,
prepare DNA sample: 20 ng/ml (DNA working stock) and
11


thermocycler (Eppendorf branch). The process is described as
follows:
1. DNA denaturation
Add 5 μl of DNA working stock to each PCR tube. Denature sample
DNA for 5 minutes at 98°C and cool the samples to 25°C.
2. Hybridization reaction
- Prepare hybridisation master mix for hybridization reaction.
- Add 3 μl of the hybridisation master mix to each PCR sample
tube above. Continue incubate for 1 minute at 95 °C, then for 16 – 20
hours at 60 °C following the thermocycler program.
3. Ligation reaction
- Prepare ligase mix for ligation reaction.
- Continue the thermocycler program: pause at 54°C. When the
samples are at 54°C, add 32 µl of ligase mix and incubate 15 minutes
at 54oC for ligation;
- Heat to inactivate the ligase for 5 minutes at 98 oC and then
pause at 15 oC.
4. PCR reaction
- Label new tubes for the PCR reaction.
- Prepare PCR buffer mix (well mix)
- Add 30µl of PCR buffer mix to each new tube and well mix

by pipetting
- At room temperature, transfer 10 μl of each ligation product
to its corresponding PCR tube. Spin.
- Prepare PCR master mix (in icebox)

12


- Continue the thermocycler program: pause at 60 °C and place
the PCR tubes in the thermocycler
- While these tubes are in a thermocycler at 60 °C, add 10 μl
mastermix to each tube. Mix by pipetting gently (up and down) and
continue the thermocycler program immediately: 35 cycles: 30 seconds
95 °C; 30 seconds 60 °C; 60 seconds 72 °C. End with 20 minutes
incubation at 72 °C and then pause at 15 °C.
Table 2.1 PCR program for the MLPA reaction
Stage

Temperature

Time

98 °C

5 minutes

25 °C

pause

Hybridisation

95 °C

1 minute

reaction

60 °C

pause

54 °C

pause

54 °C

15 minutes

98 °C

5 minutes

15 °C

pause

60 °C

pause

DNA denaturation

Ligation reaction

PCR reaction
35 cycles

95 °C 30 seconds
60 °C 30 seconds

x 35 cycles

72 °C 60 seconds
72 °C

20 minutes

15 °C

pause

5. Capillary electrophoresis
PCR products were analyzed on the GenomeLabTM GeXP Genetic
Analysis System (Applied Biosystems) using Genemarker software
13


v.1.95 (Softgenetic, State College, PA, USA). The peaks achieved

after capillary sequencing could easily be differentiated and assigned
to particular exons on the basis of their different lengths representing
the variability of their stuffer sequences. The company protocol
recommends that the relative probe signals of each probe was
detemined by dividing each measured peak area (As) by the sum of
all 45 peaks area (ΣAs) of that sample. To obtain the relative peak
ratio, the relative peak area (As/ΣAs) is then divided by the relative
peak area of the corresponding probe obtained from a control DNA
sample. For analysis of the test samples, the relative peak areas of the
amplified probes were analyzed using Microsoft Excel and the peak
area of each fragment was compared to that of a control sample. As
the DMD gene is located on the X chromosome, deletion of one or
more exons will result in a complete lack of one or more of the
corresponding peaks in male patients and a 35-55% reduction of the
relative peak ratio of the expected value in female carriers.
Duplications will give an approximately two-fold greater relative
peak area in male patients and a 30-55% increase in female carriers.
Identified deletions and duplications were regarded as reliable when
they involved two or more adjacent exons [25].

CHAPTER 3. RESULTS AND DISCUSSION
3.1. DNA extraction

14


DNA was extracted from 14 samples: 10 female relatives and 1
patient from 5 different families characterized by DMD and 1 healthy
male and 2 healthy female as positive control. Then we determined the
quality,

concentration

and

purity

of

DNA

by

using

the

spectrophotometric method. All samples were obtained with high purity
(concentrations > 100 ng/μl and purity- A260/A280 from 1.8 to 2.0).
3.2. MLPA results
3.2.1. MLPA results of patient A’s family
a. MLPA result of patient A’s mother (D1)

Figure 11: MLPA results of DMD female control (C2) and
mother (D1) in using DMD Probe set P034 the dystrophin gene
b. MLPA result of the patient’s second aunt (D2)

15

Figure 12: MLPA results of DMD female control (C2) and second
aunt (D2) in using DMD Probe set P034 the dystrophin gene
c. MLPA result of the first aunt’s daughter (D3)

Figure 13: MLPA results of DMD female control (C2) and first
aunt’s daughter (D3) in using DMD Probe set P034 the
dystrophin gene
In the family of patient A, analysis of the MLPA results of
the mother, the second aunt and the first aunt’s daughter (figure: 11,
12, 13) show that the peak height of exons 45-52 (reflecting the
concentration in the PCR product) of the sample D1 (mother)
decreased by approximately a half compared with that of the female
control sample; the peak height of exons 45-52 of the sample D2
(second aunt), D3 (first aunt’s daughter) equals the height of
corresponding exons in the female control sample. According to Hwa
H. L. (2007), the female samples were considered pathological (female
carrier) when the following criteria was fulfilled: the peak height of the
mutation exons in the female decreased by approprimately a half (3565%) compared with the peak height of corresponding to exons in the
healthy female control [20]. In addition, according to Lai K.K. (2006),
16


the height of the peak signal from the mother reduced by 35-55%
compared with the peak signal of female control sample meaning that
the mother is heterozygous female [25]. Thus, based on the results of
the MLPA analysis (figure 11, 12, 13) it is confirm that the mother of
patient A is a female carrier and the aunt, first aunt’s daughter are
healthy females. It is proved that the mutation of the dystrophin gene
was transmitted to the patient from his mother.
3.2.2. MLPA results of patient B’s family

a. MLPA result of patient B

Figure 15: MLPA results of DMD male control (C1) and patient
B (D8) in using DMD Probe set P035 the dystrophin gene
b. MLPA result of patient's first aunt (D4)

17


Figure 16: MLPA results of DMD female control (C2) and first
aunt (D4) in using DMD Probe set P035 the dystrophin gene
c. MLPA result of patient's second aunt (D5)

Figure 17: MLPA results of DMD female control (C2) and second
aunt (D5) in using DMD Probe set P035 the dystrophin gene
d. MLPA result of first aunt’s daughter (D6)

18


Figure 18: MLPA results of DMD female control (C2) and first
aunt’s daughter (D6) in using DMD Probe set P035 the
dystrophin gene
e. MLPA result of daughter of second aunt (D7)

Figure 19: MLPA results of DMD female control (C2) and second
aunt’s daughter (D7) in using DMD Probe set P035 the
dystrophin gene
In this family, MLPA results (Figure 16,17,18) of the female
members (D4, D5, D6) showed that these members have duplication

mutations in exons 11-20 and 51-60 because the peak height of exons
11-20 and 51-60 increased by 30 to 50% compare with the height of
these corresponding exons in the female control samples. According
to K.K Lai (2006), the height of the peak signal from the mother
increased by 30-50% compared with the peak signal of female
control sample meaning that the mother has deletions mutation exon
corresponding to that peak signal [25]. This proves that D4, D5 and
D6 are female carirrers. In addition, as mentioned above, based on
19


pedigree we found that the second aunt is an enforced heterozygous
carrier so that it is confirmed that the female carries the heterozygous
genotype again. In particular, the daughter of the second aunt (D7)
has a mother carrier but MLPA results did not show the peak height
of this sample exon increasing or decreasing compared with the
corresponding peak of female control sample (Figure 19). Thus, it
was considered the daughter of the second aunt is normal and not a
carrier. From the family pedigree of patient B, we found that the
patient has a cousin affected (deceased), this proved to be genetic
factors in this family. So that the mother and patient's second aunt are
obligate carriers and their sons receive gene from mothers, this is a
perfect fit when genetic analysis of patients with mom and second
aunt. So, five members in the patient's family are female carriers,
particularly grandmother, she transmitted gene for all 3 of her
daughters. All daughters will be unaffected, but each has a 50%
chance of being a carrier like her mother. However, in family of
patient B all of the daughters of his grandmother (his mother and two
aunts) are female carriers (heterozygous gene). This suggests the
possibility of genetic of the disease of mother carriers has been

spreading quite high and quite fast in the community. This family
should be genetic counseling thorough and detailed, especially
provide the advices about prenatal diagnosis when the females want
pregnant to avoid giving birth to children continue to be affected or
carriers, in order to reduce the burden on families and society.
Fortunately, the daughter of the second aunt is healthy female,
although her mother is a carrier. This is of great significance,
psychological help relieve anxiety for her and her family. However,
in the family with so many people which are female carriers
20


(grandmother, mother and aunts), after marriage she should perform
prenatal diagnosis methods when she has pregnancy to help give
birth healthy babies.
3.2.3. MLPA result of patient E’s mother (D9)

Figure 20: MLPA results of DMD female control (C3) and
patient E’s mother (D9) in using DMD Probe set P035 the
dystrophin gene
3.2.4. MLPA result of patient F’mother (D10)

Figure 21: MLPA results of DMD female control (C3) and
patient F’s mother (D10) in using DMD Probe set P035 the
dystrophin gene

21


3.2.5. MLPA results of patient G’s mother (D11)

Figure 22: MLPA results of DMD female control (C3) and
patient G’s mother (D11) in DMD Probe set P034 the dystrophin
gene
The patients in these 3 families (E, F and G) have been found
with deletion mutations in the other study. MLPA results (Figure 20,
21, 22) of the mothers (D9, D10, D11) of patients E, F, G,
respectively, showed that these mothers have deletion mutations in
exons 11-20, 31-40 and 48-50 because the peak height of exons 1120, 31-40 and 48-50 decreased by approximately 35-50% comparison
with the peak height of these corresponding exons in the female
control samples. According to K.K Lai (2006), in female
heterozygotes, a 35-55% reduced relative peak area of the
amplification product of that probe is expected [25]. It is proves that
D9, D10 and D11 are female carriers and they transmit gene to their
sons.
In all three families, the mothers have a son with DMD so when
they intend to have more children, the prenatal diagnosis is extremely
important and meaningful to prevent birth of affected children.

22


Table 3.2 Results of multiplex ligation-dependent probe amplification
(MLPA)
Patient
A

Female relative

Mutation

Mother (D1)

Carrier deletion exons 45-52

Second aunt (D2)

None carrier

First aunt’s daughter (D3)

None carrier
Carrier duplication exons

First aunt (D4)

11-20 and exons 51-60

B

Carrier duplication exons

Second aunt (D5)

11-20 and exons 51-60

First aunt’s daughter (D6)
Second aunt’s daughter

Carrier duplication exons

11-20 and exons 51-60
None carrier

(D7)
E

Mother (D9)

Carrier deletion exons 8-43

F

Mother (D10)

Carrier deletion exons 3-47

G

Mother (D11)

Carrier deletion exon 48-50

Table 3.2 shows that 7 out of 10 female members have
heterozygous mutation of the DMD gene: four cases have
heterozygous deletion exons 45-52, 8-43, 3-47 and 48-50,
respectively; three cases have heterozygous duplication exons 11-20,
51-60. Deletions and duplications can happen almost anywhere in the
dystrophin gene. However, deletion mutations are the most
commonly found in two “hot spot” regions. In this study, most of
deletions were confined to these regions (exons 2-20 and exons 4453).

CONCLUSION AND SUGGESTION
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We have successfully applied MLPA technique to detect the
carriers in ten female members of DMD patient families. Seven out
of them were detected to have heterozygous deletion or duplication
(4 heterozygous deletions, 3 heterozygous duplications). Carrier
detection is important for genetic counseling in order to reduce the
incidence of this disease.
The study was only carried out on a small sample size of
Vietnamese DMD female carriers and their family. We emphasize
the need for applying the MLPA technique further for studying and
detecting female carriers in order to provide better diagnostic,
prognostic and prenatal services to the suffering patients and their
families.

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