http://www.raredr.com/publications/duchenne-muscular-dystrophy/2015/may-2015/dmd-early-diagnosis-testing
Duchenne Muscular Dystrophy: Early Diagnosis and Genetic Testing Can Improve Management


DUCHENNE MUSCULAR DYSTROPHY: A Genetic Disease

Patients with Duchenne muscular dystrophy (DMD) have a mutation in the dystrophin gene, which affects their ability to produce full-length, functional dystrophin, a structural protein that connects the cytoskeleton of a muscle fiber to proteins embedded in the cell membrane which in turn are connected to the surrounding extracellular matrix.
 
DMD is reported to occur in approximately 1 in 3500 live male births.1 Because inheritance of DMD is X-linked recessive, the disease almost exclusively affects boys, who have inherited the disease from their mothers (Figure 1) but up to 1/3 of cases may be due to spontaneous mutations. Girls can be carriers, with up to 10% of these patients exhibiting some symptoms, but with generally milder presentations.2 The reduction in disease severity in females is attributed to the presence of the second X chromosome carrying a normal dystrophin gene, which compensates for the deficiencies of the defective gene on the first X chromosome.


Figure 1. Inheritance pattern for Duchenne Muscular Dystrophy (DMD). DMD primarily affects male patients, who are likely to have inherited the disease from their mother.
*Approximately 10% of heterozygous females will show disease symptoms.

 

Genetic testing is the gold standard for diagnosing DMD, and it should be performed for all individuals with clinical signs and symptoms potentially indicative of DMD, as well as for those with a family history of the condition.2 Genetic testing can also make certain patients with DMD eligible for treatments that are currently under investigation.

The Genetic History of Duchenne Muscular Dystrophy
In 1868, French neurologist Guillaume-Benjamin Amand Duchenne described a progressive neuromuscular disorder in 13 children after visiting hospital wards throughout Paris in search of rare neuromuscular disorders.  Duchenne observed that the condition ran in families and mainly affected boys, leading him to surmise that the disease was inherited.4
However, beyond being identified as a unique disease entity, little progress was made in understanding Duchenne muscular dystrophy (DMD) until 1986, when the gene for DMD (known as the dystrophin gene) was discovered on the short arm of the X chromosome at position Xp21.4


 NATURAL HISTORY OF DMD

Although patients with DMD appear healthy at birth, they tend to reach such milestones as sitting and walking later than expected. They progress to show signs of muscle weakness and dete­rioration during early childhood, often becoming wheelchair-dependent by their early teens or sooner.3 By their late teens, many patients experience respiratory, cardiac, and orthopedic complications, necessitating ventilatory and other support.
 
To increase longevity and improve patients’ quality of life, appropriate supportive care with corticosteroid, respiratory, cardiac, orthopedic, and rehabilitative interventions is necessary to maintain muscle function and delay disease progression for as long as possible. Without such timely interventions, the mean age of death is approximately 19 years, but when such interven­tions are promptly initiated, patients have the greatest opportunity to maximize their life expectancy, potentially reaching their third or even fourth decade of life.2 Early initiation of supportive care requires recognition of the initial, sometimes subtle signs and symptoms of DMD, followed by a prompt diagnosis.

 EARLY SIGNS AND SYMPTOMS

The gold standard for diagnosing DMD is a genetic test but unfortunately that may not occur for several months or years after a child begins to show signs and symptoms that warrant testing. Signs and symptoms of DMD initially present in the muscles of the lower extremities, and eventually progress to affect all muscles in the body. Babies with DMD meet very early motor mile­stones but typically have a delay in their walking ability. The mean age for walking in patients with DMD is 18 months, compared with 12 to 15 months for most babies without DMD.5 However, signs and symptoms are often more noticeable after the age of 2 years,6 when lower-extremity weakness becomes more pronounced. Toddlers and preschoolers with DMD may have difficulty jumping, running, and/or climbing stairs, and may be prone to falling.7

The "MUSCLE" Mnemonic
Based on a study showing 1.5 years between first symptoms and diagnosis, van Ruiten and
colleagues6 developed the “MUSCLE” mnemonic to help primary care providers better
recognize Duchenne muscular dystrophy (DMD) and potentially make a more timely diagnosis:
M – Motor milestone delays
U – Unusual gait
S – Speech delay
C – CK testing, as soon as possible
L – Leads to
E – Early DMD diagnosis, which is confirmed by genetic testing, including:
S – Sequencing
 

These children may also have difficulty getting up from the floor or moving from a seated position to standing.7 To compensate for their proximal muscle weakness, children with DMD may use a Gowers’ maneuver when rising from the floor. The maneuver enables them to stand upright by using their hands to “walk” up their body, until reaching a standing position. This is due to weakness in the hips and thigh muscles. (Figure 2).

As lower extremity weakness increases, pa­tients often develop distinctly large calves; however, the size is not the result of muscle tissue development, but rather of pseudohypertrophy, in which accumulated fat and fibrotic tissue eventually replace the normal muscle tissue.5 As this process progresses, children may report calf pain.5
 
By the time children with DMD are school age, they may have begun to walk on their toes or the balls of their feet, demonstrating a slightly wad­dling gait, which further increases their propen­sity for falling.6,7 To maintain balance, they may shift their center of balance by sticking out their bellies and pulling back their shoulders. They may also demonstrate difficulty keeping up with their peers on the playground and in the classroom. Some children with DMD have been shown to ex­hibit impairments on multiple measures of cog­nition, including assessments of receptive lan­guage, expressive language, visuospatial ability, fine motor skills, attention, and memory skills.9

DELAYS IN DIAGNOSIS

Since newborn screening is currently not per­formed for DMD, and newborns do appear nor­mal, a diagnosis can be delayed by a few years. For example, although the average age at the time of first reported symptoms is 2.7 years (range, 8-72 months), van Ruiten and colleagues6 found that consultation for suspicious symptoms often does not occur until age 3.6 years, with a serum creatinine kinase (CK) test and DMD diagnosis not occurring until 4.2 years. Elevated CK levels indicate muscle damage, but a definitive DMD diagnosis requires genetic test­ing (see “Genetic Testing” section below). A flowchart showing the diagnostic workup to test for pos­sible DMD is shown in Figure 3.


Figure 3. Flow chart showing the diagnostic work-up of DMD. Adapted from Abbs et al. 2010.
BMD, Becker muscular dystrophy; DMD, Duchenne muscular dystrophy.

DYSTROPHIN GENE MUTATIONS

Three broad categories of dystrophin gene mutations have been described (Figure 4)10,11:
1. Deletions of 1 or more exons (~65% of cases)
2. Small mutations, including de­letions, insertions, missense, and nonsense (~25% of cases, with 10%-15% involving nonsense mutations)
3. Duplications (7%-10%)
 

Figure 4. There are 3 broad categories of mutations leading to truncated non-functional dystrophin, deletions, duplications
and small mutations. Nonsense mutations comprise 10% -15% of all the mutations.


Genetic testing assesses for these mutations and is able to detect 97.3% of DMD cases without the need for invasive muscle biopsies11; rarely (2.7% of DMD cases), a biopsy is still necessary to make a definitive diagnosis when genetic test results are inconclusive or when evaluation of levels of dystrophin in muscle is needed to distinguish DMD from milder phenotypes of the disease.2,5 In addition to being noninvasive, genetic testing is the only diagnostic method that enables determination of eligibility for certain clinical trials {i.e., for deletions and duplications or for nonsense mutations} and it can often help predict the course of the disease.
 
Researchers have developed a software tool to help predict the clinical course and severity of disease in the case of specific duplications and deletions, accessible at www.humgen.nl/scripts/ DMD_frame.php. The Center for Human and Clinical Genetics at Leiden University Medical Center in The Netherlands has an excellent website (www.dmd.nl) for clinicians who want to learn more about the many genetic mutations that can help diagnose patients with DMD.
 
Providing clinicians and caregivers with the projected course and severity of DMD can help better prepare them for the future and enable them to have supportive care measures in place that can be accessed as soon as necessary. It must be emphasized, of course, that such analytic projections cannot definitively determine a patient’s prognosis.

GENETIC TESTING: A 2-STEP PROCESS

The genetic testing can be a 2-step process (Figure 5). The initial test detects large changes (depletions and duplications) in the dystrophin gene. It can be performed by using multiplex polymerase chain reaction (PCR) to amplify the exons known to be affected in DMD, or by using quantitative methods such as multiplex ligation-dependent probe amplification (MLPA) and multiplex ampli­fiable probe hybridization (MAPH). Unlike multiplex PCR, the MLPA and MAPH methods cover all exons in the dystrophin gene; thus, they can also be used for carrier testing in female patients.2,9 This initial test can detect ~75% of mutations and is cost-effective.
 

Figure 5. Gene sequencing MUST be offered if MLPA test is negative, ~50% chance for nonsense mutation

If the first test does not identify a deletion or duplication, a second test that detects small mutations is required to confirm a DMD diagnosis. This test sequences the entire dystrophin gene, assessing for small insertions, small deletions, and missense and nonsense mutations, thereby confirming or excluding a diagnosis of DMD.2,10 ,11 Up to 25% of DMD cases, including patients with nonsense mutations, will be missed by the first test and identified only with the second.

WHEN TO RECOMMEND GENETIC TESTING

Healthcare providers should recommend genetic testing when 1 or more of the following factors is present:
Signs and symptoms of DMD—These include motor milestone delays, unusual gait, and speech delay, which make up the “M,” “U,” and “S” components of the “MUSCLE” mnemonic.6
Family history of DMD—This includes children suspected of having DMD; mothers and sisters of boys with confirmed DMD (to determine whether they are carriers); and brothers of patients with confirmed DMD, unless a boy is 10 years or older and has normal muscle function, as DMD is unlikely in such cases. Carrier testing is important for family planning decisions and for medical monitoring, because carriers are known to have an increased risk of cognitive dysfunction and cardiomyopathy compared with individuals without dystrophin gene mutations.2
 
Abnormal laboratory findings—In particular, elevated CK levels can point to a possible DMD diagnosis. Elevated serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels can also indicate occult muscle disease, and are not solely caused by hepatocellular injury.12
 
Muscle biopsy findings indicate DMD— When a muscle biopsy has yielded a DMD diagnosis, genetic testing can be used for confirmation and to determine which mutation is causing the disease, potentially opening the door to eligibility for certain personalized treatments.
 
An accredited laboratory should provide genetic testing for DMD. Healthcare providers in the United States can use www.genetests.org to locate laboratories. This website is sponsored by the University of Washington, but because listings are voluntary, it may not include all US laboratories providing such services.
 
In Europe, www.orpha.net maintains a listing of genetic-testing laboratories, which can be located by searching by disease name and narrowing the results by country and/or city. Healthcare providers can also use the website to locate expert centers that offer genetic counsel­ing for patients with DMD and for their families.

 CONCLUSIONS

Patients with DMD continue to experience delays in diagnosis, with diagnosis typically occurring up to 2 years after the onset of symptoms. The delay is attributable to the rarity of the condi­tion and to the subtlety of symptoms, until lower extremity weakness becomes more pronounced. Because newborn screening for DMD is not performed, and pediatricians are unlikely to encounter the disease in routine clinical practice, early diagnosis requires a high degree of suspicion when a male infant or toddler presents with de­layed walking and/or gait problems.
 
Findings of pseudohypertrophy, Gowers’ maneuver, speech delay, and elevated CK, AST, and ALT levels can provide additional support for a probable diagnosis of DMD; however, genetic testing is the gold standard for diagnosing the disease. It is also the only diagnostic modality that can determine a patient’s eligibility for treatment with new personalized medicine drugs as they become available. To ensure that genetic testing is used to its maximum potential, patients who are not found to have duplications or deletions on the first mutation test (eg, MLPA or MAPH test) should undergo gene sequencing to ensure that small point mutations, including nonsense mutations, are also detected. This is especially critical because patients with such mutations may, in time, benefit from a future drug that tar­gets the underlying cause of their type of DMD.

References

  1. Kalman L, Leonard J, Gerry N, et al. Quality assurance for Duchenne and Becker muscular dystrophy genetic testing: development of a genomic DNA reference material panel. J Mol Diagn. 2011;13(2):167-174.
  2. Bushby K, Finkel R, Birnkrant DJ, et al; DMD Care Considerations Working Group. Diagnosis and management of Duchenne muscular dystrophy, part 1: diagnosis, and pharmacological and psychosocial management [published online November 30, 2009]. Lancet Neurol. 2010;9(1):77-93.
  3. Parent A. Duchenne De Boulogne: a pioneer in neurology and medical photography. Can J Neurol Sci. 2005;32(3):369-377.
  4. Hinton VJ, Goldstein EM. Duchenne muscular dystro­phy. In: Mazzocco MM, Ross JL, eds. Neurogenetic Developmental Disorders: Variation of Manifestation in Childhood. Cambridge, MA: The MIT Press; 2007:105- 132.
  5. Learning about Duchenne muscular dystrophy. Natio nal Human Genome Research Institute of the National Institutes of Health website. http://www.genome. gov/19518854. Updated April 18, 2013. Accessed March 4, 2015.
  6. van Ruiten HJ, Straub V, Bushby K, Guglieri M. Improving recognition of Duchenne muscular dystro­phy: a retrospective case note review. Arch Dis Child. 2014;99(12):1074-1077.
  7. Duchenne muscular dystrophy (DMD): signs and symptoms. Muscular Dystrophy Association website. http://mda.org/disease/duchenne-muscular-dystro­phy/signs-and-symptoms. Accessed March 4, 2015.
  8. Punnoose AR, Burke AE. Muscular dystrophy. JAMA. 306(22):2526.
  9. Cyrulnik SE, Fee RJ, Batchelder A, Keifel J, Goldstein E, Hinton VJ. Cognitive and adaptive deficits in young children with Duchenne muscular dystrophy (DMD). J Int Neuropsychol Soc. 2008;14(5):853-861.
  10. Abbs S, Tuffery-Giraud S, Bakker E, Ferlini A, Sejersen T, Mueller R. Best practice guidelines on molecular di­agnostics in Duchenne/Becker muscular dystrophies. Neuromuscul Disord. 2010;20(6):422-427.
  11. Grimm T, Kress W, Meng G, Müller CR. Risk as­sessment and genetic counseling in families with Duchenne muscular dystrophy. Acta Myol. 2012;31(3):179-183.
  12. McMillan HJ, Gregas M, Darras BT, Kang PB. Se­rum transaminase levels in boys with Duchenne and Becker muscular dystrophy. Pediatrics. 2011;127(1):e132-e136.
 
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