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Prenatal molecular diagnosis and carrier detection of Duchenne muscular dystrophy in Korea
Journal of Genetic Medicine 2020;17:27-33
Published online June 30, 2020;  https://doi.org/10.5734/JGM.2020.17.1.27
© 2020 Korean Society of Medical Genetics and Genomics.

Min Ji Kang1,2, Moon-Woo Seong3, Sung Im Cho3, Joong Shin Park1, Jong Kwan Jun1,4,*, and Sung Sup Park3

1Department of Obstetrics and Gynecology, Seoul National University College of Medicine, Seoul, Korea
2Department of Obstetrics and Gynecology, Gimcheon Jeil Hospital, Gimcheon, Korea
3Department of Laboratory Medicine, Seoul National University College of Medicine, Seoul, Korea
4Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University College of Medicine, Seoul, Korea
Jong Kwan Jun, M.D., Ph.D. https://orcid.org/0000-0002-0242-1736
Department of Obstetrics & Gynecology, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul 03080, Korea.
Tel: +82-2-2072-3744, Fax: +82-2-762-3599, E-mail: jhs0927@snu.ac.kr
Received June 2, 2020; Revised June 9, 2020; Accepted June 10, 2020.
cc This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Purpose: Duchenne muscular dystrophy (DMD) is the most common lethal muscular dystrophy and is caused by the genetic variants of DMD gene. Because DMD is X-linked recessive and shows familial aggregates, prenatal diagnosis is an important role in the management of DMD family. We present our experience of prenatal molecular diagnosis and carrier detection based on multiplex polymerase chain reaction (PCR), multiplex ligation-dependent probe amplification (MLPA), and linkage analysis.
Materials and Methods: During study period, 34 cases of prenatal diagnosis and 21 cases of carrier detection were performed at the Seoul National University Hospital. Multiplex PCR and MLPA was used to detect the exon deletions or duplications. When the DMD pathogenic variant in the affected males is unknown and no DMD pathogenic variant is detected in atrisk females, linkage analysis was used.
Results: The prenatal molecular diagnosis was offered to 34 fetuses. Twenty-five fetuses were male and 6 fetuses (24.0%) were affected. Remaining cases had no pathogenic mutation. We had 24 (80.0%) cases of known proband results; exon deletion mutation in 19 (79.2%) cases and duplication in 5 (20.8%) cases. Linkage analysis was performed in 4 cases in which 2 cases (50.0%) were found to be affected. In the carrier testing, among 21 cases including 15 cases of mother and 6 cases of female relative, 9 (42.9%) cases showed positive results and 12 (57.1%) cases showed negative results.
Conclusion: Prenatal molecular diagnosis and carrier detection of DMD are effective and feasible. They are useful in genetic counseling for DMD families.
Keywords : Prenatal diagnosis, Muscular dystrophy, Duchenne, Multiplex polymerase chain reaction, Multiplex ligationdependent probe amplification, Linkage analysis.
Introduction

Duchenne muscular dystrophy (DMD) is one of neuromuscular diseases characterized by progressive muscular weakness and degeneration of skeletal muscle [1]. The onset of DMD symptoms is observed ages between 2 and 3. Usually, affected boys have difficulty in motor development, approximately half of affected fail to walk until 18 months of age. It is the most common X-linked recessive fatal disease occurring in 1 in 3,500 newborns, and results from pathogenic variants in the dystrophin gene encoding the dystrophin protein [2,3]. The dystrophin gene is found in the short arm of the X chromosome (X-p21.2) [4]. Its 11.3 kb long coding sequence embraces 79 exons and 7 unique promoters and introns which comprise 99.5% of the DMD gene [5].

Prenatal diagnosis for DMD was first introduced in 1984 [6]. Prenatal tests for DMD are performed for a male fetus whereas no testing is mandate for female fetuses [7]. Traditional genetic diagnosis for DMD detects large mutations at first and smaller mutations subsequently by applying two or more analytical methods. Conservative prenatal diagnosis detects any known familial mutation in male fetal DNA by chorionic villus sampling or amniocentesis [8]. The same approach can be applied to detect the mutation of the proband and female carriers [7]. If the familial mutation is not observed, haplotyping is another method for prenatal diagnosis [9].

Since the locus of the DMD gene identified in 1986 by independent experimental methods [10,11], clinicians have heavily concentrated on the molecular diagnosis of DMD. Definite treatment is not available. Therapies under investigation; pregnancy management for female carriers; genetic counseling for risk to family members and family planning; and prenatal testing and preimplantation genetic diagnosis (PGD) are all dependent on the precise molecular diagnosis of the disorders.

Many diagnostic testing procedures are employed for DMD mutation tests including multiplex polymerase chain reaction (PCR), multiplex ligation-dependent probe amplification (MLPA), array comparative genome hybridization (CGH), PCR-based Sanger sequencing, and others [12].

Some laboratories still use Multiplex PCR to identify common dystrophin deletions due to its convenience and cost-efficiency to perform [12]. Since the introduction of MLPA by Schouten in 2002, there has been a substantial increase in detection rate of dystrophin mutations as the majority of mutations developing DMD are intragenic deletions or duplications representing 65% and 5% of total cases, respectively [5].

The aim of this study is to show our clinical experience in prenatal diagnosis and carrier detection of DMD using molecular biologic techniques.

Materials and Methods

1. Study subjects

The prenatal diagnosis data of 34 at-risk pregnancies in DMD families referred to the department of obstetrics and gynecology in Seoul National University Hospital from September 1997 to January 2010 were retrospectively reviewed. Peripheral blood was collected from 24 male probands clinically diagnosed with DMD and 21 corresponding female relatives for carrier tests. This study was approved by the ethics committee of Seoul National University Hospital. Written informed consent was obtained from the patients or their guardians.

2. Specimens

For the pregnant women, chorionic villi biopsy was performed at 11 to 14 week gestation, 10-20 mL of amniotic fluid was collected via amniocentesis at 14 to 18 week gestation to obtain genomic DNA from fetus. Chorionic villi and amniocyte culture was done for prenatal genetic diagnosis. A total of 2 mL of peripheral blood was collected from patients to obtain genomic DNA for proband and carrier tests.

3. Mutation analysis

Genomic DNA was extracted from chorionic villi, amniocytes, and the patients’ peripheral blood using Gentra PureGene DNA Isolation Kits (Gentra Systems Inc., Minneapolis, MN, USA). Multiplex PCR was used to detect exon deletion in 26 sites. Exon deletion or duplication of dystrophin was screened using MLPA. MLPA was performed using the SALSA P034 DMD mix 1 Kit and P035 DMD mix 2 Kit (MRC Holland, Amsterdam, The Netherlands). PCR products were analyzed on an ABI 3130 analyzer using Genemarker v1.51 (Softgenetics, State College, PA, USA). Peak heights were normalized, and a deletion or duplication was suspected when a normalized peak ratio was under 0.75 or above 1.30. Linkage analysis was done using 14 different short tandem markers spanning the DMD gene, including DXS1049, DXS1242, and etc. PCR-restriction fragment length polymorphism, and detection of ZF gene in X chromosome/ZF gene in Y chromosome were used to identify sex of fetus.

Results

The prenatal molecular diagnosis was offered to 34 fetuses (Table 1). To detect the exon deletion or duplication, multiplex PCR was used (Fig. 1) and after 2005, MLPA was introduced for screening (Fig. 2). Twenty-five fetuses were male and 6 fetuses (24.0%) were affected. We had 24 (80.0%) cases of known proband results; exon deletion mutation in 19 (79.2%) cases and duplication in 5 (20.8%) cases (Fig. 3). Remaining cases had no pathogenic mutation. However, the prenatal tests could not be performed if no patients survived in their households. When the fetus was a male (Case no. 1, 8, 10, and 17), a possible test only showed that there was no known deletion/duplication of the DMD gene exon. In this context, DMD/BMD due to point mutation cannot be excluded. Linkage analysis was performed in 4 cases which resulted in 2 affected cases (50.0%) (Fig. 4). In the carrier testing, among 21 cases including 15 cases of mother and 6 cases of female relative, 9 (42.9%) cases showed positive results and 12 (57.1%) cases showed negative results (Fig. 5). Of 12 cases which showed negative results in the carrier testing, 7 (58.3%) cases were male cases and all of these cases were negative in prenatal diagnosis.

Discussion

This study showed current status of prenatal molecular diagnosis and carrier detection of DMD in Korean.

As a result of the study, affected male fetus accounted for 6 of 25 (24.0%) male fetuses, which is somewhat lower compared to 34 of 91 (37.4%) male fetuses in the previous study [13]. This may be due to the variance in patient group and race, the percentage of patients without proband results, and the diagnostic method used for prenatal diagnosis.

Among 30 fetuses analyzed by Multiplex PCR and MLPA, the percentage of patients without proband results was 6 of 30 (20.0%) fetuses. In particular, there was not much genetic information about probands in the initial cases during study periods, because genetic counselling for DMD in the early stages of the study was relatively uncommon compared to the present, and there might be possibilities of early death of proband prior to molecular diagnosis. For another reason, it is considered that muscle biopsies were common in the early 2000s accounting for more than two thirds among various diagnostic tools for DMD.

When analyzed chronologically over 10 years, the rate of muscle biopsies in total diagnosed cases has gradually fallen from 96.8% in 1990s to 19.3% in 2010s as molecular diagnosis techniques improved [14]. Because of the improvement of molecular diagnosis techniques, most DMD patients are diagnosed genetically without muscle biopsies recently.

As MLPA was first described by Schouten et al. [15] in 2002, multiplex PCR was used for detection of deletions in dystrophin gene in this study without MLPA. Multiplex PCR is a method to test the presence or absence of exon deletion of the DMD gene dystrophin; Xp21.2 and the test confirmed the exon deletion of 26 sites. About two thirds of DMD patients are caused by exon deletion and multiplex PCR can diagnose more than 99% of exon deletion patients. Therefore, some laboratories still use this method because of its convenience and cost-efficiency to perform [12].

Multiplex PCR is used to identify deletions at a hemizygous locus but it is impossible to completely describe deletions because of its limitation to cover the whole gene and incapacity to detect duplications or female carriers. These are the critical drawbacks of Multiplex PCR [16]. It also has difficulties to differentiate whether the deletion detected is in-frame or out-of-frame, and to apply exon skipping therapy consequently. Additionally, it is impossible to determine boundaries of the deletion [12]. In present study, no duplication was found in the mutation of the proband when multiplex PCR was used but the duplication was found after MLPA introduction. In 24 total cases in which proband results exist, 19 (79.2%) cases were exon deletion mutation and 5 (20.8%) cases were duplication mutation. This rate is similar to the results of previous studies reporting mutation spectrum in the dystrophin gene of 507 Korean boys [14].

When the DMD gene variant observed in an affected male, carrier diagnosis could be applicable to the mother and female relatives at risk, who are heterozygotes for the pathogenic gene variant. In this study, carrier test was performed in 21 cases except 3 cases among 24 cases in which DMD gene variants of affected males were identified. Carrier test results provide information in deciding whether to perform a prenatal diagnosis. Also these results are clinically important as abnormal cardiac monitoring is essential for asymptomatic heterozygous females and a cardiac evaluation should be considered prior to conception or during pregnancy [12].

In the present cohort, the results revealed that 9 of 21 (42.9%) mothers were carriers. Of the 21 cases with both proband and carrier results, 12 of 21 (57.1%) were carrier negative. The de novo mutation of the DMD gene and germline mosaicism can be considered for this discrepancy. If the mother was not a carrier, her son with DMD mostly had a de novo mutation. However, germline mosaicism in DMD should not be ignored as described in theonferring a recurrence risk for the next son [17,18].

Although 7 male fetuses of carrier negative patients were all negative in the prenatal diagnosis and the 7 male fetuses who were born healthily in this study, the possibility of germline mosaicism cannot be completely excluded. Wang et al. reported 3 cases of germline mosaicism in 50 mothers with no pathogenic mutations [19]. Furthermore, Bermúdez-López et al. [20] verified that germinal mosaicism deletions have been reported in at least 11.6% of families of DMD patients, suggesting that mosaicism should be considered in the genetic counseling. In our center, prenatal diagnosis was suggested to at-risk pregnant women in DMD families regardless of their carrier status.

Because recent prenatal diagnosis is offered based upon the presence of an affected boy, it is insufficient to detect fetuses with de novo mutations. De novo mutations represents approximately one third of DMD patients. This issue can be resolved by offering prenatal diagnostic test for the dystrophin gene to every pregnant woman. Advancing in noninvasive prenatal diagnosis will possibly improve the current conditions despite there are still many challenges to overcome [9,21]. Moreover, cost-effectiveness should be considered.

Point mutations, microdeletions, microinsertions, or splicing mutations are known as the cause for 30% of all DMD gene mutations [5]. These mutations can be detected by dystrophin gene sequencing performed at the genomic or complementary DNA level. In the present study, multiplex PCR and MLPA are employed for the detection of large and small mutations. However, our study does not use sequencing to detect small mutations because of high costs [22]. Limitation of this study is that some variants such as deep intronic mutations or complex rearrangements may not be identified through traditional DNA-based mutation screening techniques [7]. In the future, studies using Array CGH, PCR-based Sanger sequencing in prenatal diagnosis that can detect point mutations are expected.

PGD is another option for carrier women. With PGD, either unaffected male embryos or females can be selected and implanted into the uterus. Additionally, germline mosaicism is an issue and in order to find it developing new diagnostic methods such as measuring creatinine phosphokinase level in amniotic fluid and fetal blood or establishing a direct diagnostic tool through fetal muscle biopsy are essential. Chorionic villus sampling or amniocentesis used for sample collection in this study are invasive methods carrying associated risks. In the future, research and development of noninvasive prenatal diagnostic tests will be required providing a basis for prenatal diagnosis of DMD such as the discovery of cell-free fetal DNA in maternal plasma [21].

In conclusion, this study showed prenatal diagnosis and carrier detection using multiplex PCR, MLPA, and linkage analysis were effective measures to detect DMD.

Acknowledgements

This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI08C0537).

Figures
Fig. 1. Multiplex polymerase chain reaction. DMD, Duchenne muscular dystrophy.
Fig. 2. Multiplex ligation-dependent probe amplification.
Fig. 3. Cases analyzed by multiplex polymerase chain reaction and multiplex ligation-dependent probe amplification.
Fig. 4. Linkage analysis.
Fig. 5. Cases with or without carrier test.
TABLES

Prenatal diagnosis of 34 fetuses in DMD high-risk families

No. Date Sample Tests Proband Fetus


Results Relation Carrier status Sex Results
1 1997. 9 AF Multiplex PCR None M Normal
2 2000. 9 AF Multiplex PCR None Aunt F
3 2002. 1 AF Multiplex PCR 50 del Mother M DMD
4 2004. 1 AF Multiplex PCR 49-52 del Mother M Normal
5 2004. 3 CVS Multiplex PCR 45-52 del Aunt + M DMD
6 2004. 7 CVS Multiplex PCR None F
7 2004. 9 AF Multiplex PCR 48 del Mother M Normal
8 2005. 1 AF Multiplex PCR None Sister M Normal
9 2005. 1 AF Multiplex PCR 47-52 del Mother M Normal
10 2005. 1 AF Multiplex PCR None Aunt M Normal
11 2005. 3 CVS Multiplex PCR 45-52 del Aunt + M Normal
12 2005. 5 AF Multiplex PCR 6-8 del Mother F
13 2005. 8 AF Multiplex PCR 46-47 del Mother + F
14 2006. 2 AF MLPA+Multiplex PCR 45-52dup Aunt F
15 2006. 5 AF MLPA+Multiplex PCR 45-54 del Mother M Normal
16 2006. 9 AF MLPA+Multiplex PCR 46-61 dup Sister M Normal
17 2007. 1 AF MLPA+Multiplex PCR None Sister M Normal
18 2007. 2 AF MLPA+Multiplex PCR 2 dup Mother M Normal
19 2007. 9 AF MLPA+Multiplex PCR 54 dup Mother M Normal
20 2007. 9 AF MLPA+Multiplex PCR 46-48 del Mother F
21 2007. 11 AF MLPA+Multiplex PCR 45-50 del Mother + M DMD
22 2007. 12 AF MLPA+Multiplex PCR 48-52 del Mother + F
23 2008. 3 AF MLPA+Multiplex PCR 12-17 del Mother M Normal
24 2008. 3 AF MLPA+Multiplex PCR 8-26 del Mother F
25 2008. 5 AF MLPA+Multiplex PCR 41-43 del Mother F
26 2008. 6 AF MLPA+Multiplex PCR 46-61 dup Sister M Normal
27 2008. 7 AF MLPA+Multiplex PCR 44 del Mother + M Normal
28 2008. 9 AF MLPA+Multiplex PCR 3-44 del Mother + M DMD
29 2009. 1 AF MLPA+Multiplex PCR 3-17 del Aunt + M Normal
30 2009. 9 CVS MLPA+Multiplex PCR 48-52 del Mother + M Normal
31 2007. 7 AF Linkage analysis Mother DMD
32 2007. 9 AF Linkage analysis Aunt DMD
33 2007. 11 AF Linkage analysis Mother Normal
34 2008. 12 AF Linkage analysis Sister Normal

DMD, Duchenne muscular dystrophy; AF, amniotic fluid; PCR, polymerase chain reaction; M, male; F, female; CVS, chorionic villi sampling; MLPA, multiplex ligation-dependent probe amplification.


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