Research Paper

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Jenna Huynh
Physiology
November 26, 201
Advanced Methodologies in Diagnosis of Duchenes Muscular Dystrophy
Introduction
Duchennes Muscular Dystrophy (DMD) is an X-linked recessive degenerative
disorder of the muscle, which means that the gene is inherited from mothers to their
sons (Nathalie et al., 2011). This disease occurs mostly in boys, affecting 1 in 3500
newborn boys. Some symptoms of DMD include severe, progressive muscle wasting,
leading to early death. Death usually occurs from chronic respiratory insufficiency or
cardiac failure. Even though the predominant symptom of DMD is skeletal muscle
weakness, progressive cardiomyopathy is common and can be severe (Marek et al.,
2001).The most common sign observed from young affected boys is the Gowers sign,
which means they use their hands to push on their legs to get up (Ann 2011). Young
boys start walking at very late age and have difficulties arising from the floor. The main
cause of DMD is due to mutations in the dystrophin gene including point mutation, small
deletions,insertions, duplications but mainly deletion which results in disruption of the
open reading frame leading to dystrophin protein with little or no function (Dent et
al.,2005) . Although specific treatments for DMD have not yet reached the clinic, some

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interventions including corticosteroid, respiratory, cardiac, orthopedic, and rehabilitation

have led to improvements in function, quality of life, health, and longevity, with children
who are diagnosed today having the possibility of a life expectancy into their fourth
decade (Katherine et al., 2009). DMD is described to be the largest gene to date, which
is composed of 8 promoters and 79 exons spread over 2.2 million base pairs of genomic
DNA. Testing for dystrophin point mutations usually requires sequencing of the
enormous DMD gene, there are no particularly common point mutations or point
mutation hotspots, and a unique mutation is correlated with a specific family. Other
techniques have been used such as multiplex PCR, protein truncation test, but there are
drawbacks to each of these methods including high cost, time-consuming, labor
intensive, or psychological distress for children. In the journal article Improved
Molecular Diagnosis of Dystrophinopathies in an Unselected Clinical Cohort, the
authors report the development of SCAIP (single condition amplification/internal
primer) method that determines the sequence of the dystrophin gene in a rapid,
accurate, and economical fashion. The major step used in SCAIP method is to amplify a
large number of exons at a specific PCR temperature. Some advantages of this method
include sufficient sequencing specificity-all known coding regions and seven in eight
tissue-specific promoters are covered in the resulting complete double-stranded DNA,
less time-3 days, and at a reasonable cost. In addition, this method can detect the 2%

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of patients with exonic deletions not detected by the widely available multiplex PCR
technique (Kevin et al., 2003).
In this journal article, the frequency of mutations in a group of random
dystrophinopathy patients was collected using single condition amplification/internal
primer(SCAIP) sequencing analysis and multiplex amplifiable probe hybridization
(MAPH) analysis of duplications from peripheral blood samples. Other available
methods can only detect deletions of one exon or greater. By using these modern
methods, scientists want to discover the percentage of dystrophy mutations in noncoding regions.
Methods
Eighty four patients from sixty eight families were ascertained from a single clinic
at the University of Utah. Their genomic DNA was extracted from peripheral blood
leukocytes. The samples then underwent SCAIP sequencing analysis. Each DMD exon
and seven of the eight promoters were amplified at a uniform set of PCR temperatures
and the presence of exonic fragments were visually confirmed. Primer-site
polymorphisms were prevented by using alternate primer pairs to amplify the most
distant exons presumed to be deleted. If no deletion was identified, the second, internal
set of sequencing primers was performed to sequence each amplicon. Complete double
stranded sequencing coverage of all known coding regions and in seven of the eight

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tissue specific promoters for each non-deleted patient was obtained. An aliquot of
template DNA from each original sample was used as a control to re-amplify and resequence either the specific mutation or a combination of polymorphisms. Next step,
MAPH analysis was used to test the samples in which no mutations were detected in
SCAIP analysis. Aliquots of genomic DNA were analyzed for exon duplication, missense
or splice site mutation.
Results
Of the 68 index cases, 45 had DMD, 21 had BMD( Becker muscular dystrophy-a
milder variant of DMD, less common than DMD, and approximately 1 incidence in
18500 births), and 3 were manifesting carriers of DMA. Using SCAIP method, 45
probands were identified with deletions of one or more exons (66%); point mutation in
12(18%) including 9 premature stop codons(13% of total mutations) and 3 missense
mutations(4% of total); and frameshift mutations in 2 (3%). They also detected 14
subexonic mutations which include 9 nonsense mutations(64%), 3 missense
mutations(21%), and frameshift mutations(14%). Of the 45 DMD mutations, 3 (7%)
underwent undetected.
MAPH analysis detected 4 duplications(6%), 5(7%) of the evaluated patients had
no disease-causing mutation identified using either SCAIP or MAPH. Table I shows the

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distribution of mutation types among phenotypes. Table II and III catalogue mutations
and phenotypes.

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Discussion
Until recently, only the two-thirds of DMD gene mutations were identified due to
exonic or multi-exonic deletions or duplications. Many methodologies have been used to

test for dystrophinopathy patients. Reverse transcription-polymerase chain reaction(RTPCR) is used to sequence DMD cDNA from mRNA obtained from muscle biopsy, which
is accompanied by some risks of infection, bleeding, and complications associated with
anesthesia. Single-stranded conformational polymorphism screening, denaturing highperformance liquid chromatography (dHPLC) screening, and denaturing gel gradient
electrophoresis are also used to identify regions of the gene for follow-up sequencing
analysis. However, these methods are limited to detect single nucleotide changes. For

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these reasons, SCAIP analysis was developed, which allows direct sequencing of all
exons and flanking inronic sequences of the DMD gene without using any screening
steps. Another benefit of SCAIP analysis is that it can detect 7% exonic deletions that
are not detected using multiplex PCR tests.
Five patients (7%) were diagnosed with no DMD mutations . However, three of
these patients show symptoms of DMD, two had BMD. Immunoblot analysis showed
abnormal dystrophin expression in three ( one DMD and two BMD). The remaining two
showed clear X-linked family histories of disease but had not undergone muscle biopsy.
Based on SCAIP analysis, the authors state that no subexonic mutations or deletions
exist in the coding and flanking splice regions of the gene in five patients. MAPH
analysis is proved to be a sensitive technique which could detect some duplications not
detected by Southern blot analysis (White et al., 2002). Furthermore, polymorphisms
were not detected within the coding regions, which prove that exonic cryptic splice site
activation is the mechanism of molecular pathogenesis. The authors conclude that midintronic substitutions activating cryptic splice cites, with subsequent incorporation of
pseudoexons are more common than previously reported. More studies of muscle
biopsy tissues from the five patients are needed for confirmation. Several other
possibilities the authors came up with include mutations in under fined promoters that
suppress DMD transcription or mutations exist in a gene coding another protein

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responsible for the stability, localization, or function of the DMD gene or its message,
especially in the patients without an X-linked family histories.

Conclusion
The purpose of the experiment is to discover the percentage of non-coding region
mutations and the result indicates that 7% of dystrophinopathy patients do not have
coding region mutations. Development of SCAIP- a direct sequence analysis and
MAPH-a highly sensitive technique for duplications is beneficial for diagnosis, genetic
counseling, and therapy of the dystrophinopathies. 93% of patients are diagnosed from
genomic DNA samples using these two methods without having to use muscle biopsy in
most but not all patients. Potential DMD carriers can be detected by direct sequencing
analysis in female relatives, which show ascertainment of point mutations in probands.
Finally, probands gene mutation category is helpful for determining candidates for
mutation-specific therapeutic trials.

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References
Ann R (2011) Muscular Dystrophy. The Journal of the American Medical
Association. Vol 306, No. 22.
Dent K.M, Dunn D.M, Kerr L et al. (2005) Improved Molecular Diagnosis of
Dystrophinopathies in an Unselected Clinical Cohort. American Journal of Medical
Genetics 134A: 295-298.
Kevin M, Rober B.W, Jerry R.M et al. (2003) Rapid Direct Sequence Analysis of
the Dystrophin Gene. Am. J. hum. Genet. 72: 931-939.
Katharine B, Richard F et al. (2009) Diagnosis and Management of Duchenne
Muscular Dystrophy, part 1: diagnosis, and pharmacological and psychosocial
management. 1-17.

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Marek M, Michal O. (2001) Duchenne Muscular Dystrophy. Encyclopedia of Life

Sciences. Nature Publishing Group. 1-5.
Nathalie M, Mar T, Johanna T et al. (2011) Systemic Administration of PR0051 in
Dunchennes Muscular Dystrophy. The New England Journal of Medicine. 1513-1522.
White S, Kalf M, Liu Q, Villerius M, Engelsma D, Kriek M, Vollebregt E, Bakker B,
van Ommen GJ, Breuning MH, den Dunnen JT. 2002. Comprehensive detection of
genomic duplications and deletions in the DMD gene, by use of multiplex amplifiable
probe hybridization. Am J Hum Genet 71:365374.