Clinical Diagnosis

Calpainopathy (also known as limb-girdle muscular dystrophy 2A [LGMD2A]) is suspected in individuals with the following:

• Proximal muscle weakness (pelvic and/or shoulder girdle) with early onset (before age 12 years), adult onset, or late onset (after age 30 years)

• Atrophy of limb and trunk muscles; possible calf hypertrophy

• Scapular winging, scoliosis, Achilles tendon contracture, and other joint contractures (including hip, knee, elbow, finger, and spine)

• Waddling gait; tip-toe walking; difficulty in running, climbing stairs, lifting weights, and getting up from the floor or from a chair

• Sparing of facial, ocular, tongue, and neck muscles

• Absence of cardiomyopathy and intellectual disability

• Family history consistent with autosomal recessive inheritance

Testing

Serum creatine kinase (CK) concentration is elevated (5-80 times normal) from early infancy on, particularly during the active stage of the disease. Serum CK concentration decreases with disease progression.

Muscle CT scan or MRI

• Wasting of muscles predominantly from the posterior compartment of the limbs, evident on clinical examination, is observed by muscle CT scan [Fardeau et al 1996]. The difference in distribution of muscle involvement between calpainopathy and other limb-girdle muscular dystrophies (LGMDs) (i.e., sarcoglycanopathies and dysferlinopathies) makes CT scan analysis an important element in clinical diagnosis (see Differential Diagnosis).

• MRI scan confirms this selective involvement [Mercuri et al 2005].

Electromyogram (EMG) pattern is typically myopathic (showing small polyphasic potentials), although a normal EMG can also be observed in presymptomatic individuals. Myotonia and spontaneous discharges are not present.

Muscle biopsy

• Histopathology. A variety of morphologic changes and variable muscle involvement may be observed, irrespective of the age of the individual at the time of biopsy. Most individuals have the typical features of an active dystrophic process (increased fiber size variability, increased fibrosis, regenerating fibers, degenerating and necrotic fibers); others have mild and nonspecific myopathic features (increased central nuclei, fiber splitting, lobulated fibers, type 1 fiber predominance). Wide variability can be observed among individuals who are homozygous for the same missense mutation [Chae et al 2001, Fanin et al 2003].

• Calpain-3 immunohistochemical analysis. Absent immunohistochemical reaction was reported in muscle from patients in whom the absent protein had previously been detected by immunoblotting [Kolski et al 2008, Charlton et al 2009]. This method appears however less sensitive than immunoblotting in detecting partial protein reduction.

• Calpain-3 immunoblot analysis of muscle biopsy is the most useful diagnostic tool in calpainopathy. Approximately 80% of individuals with a CAPN3 mutation show variable levels of calpain-3 protein deficiency (approximately 58% of affected individuals have no detectable calpain-3 protein; 22% have partial reduction of the amount of protein) and 20% have a normal amount of protein (but with loss of protein function) [Groen et al 2007].
  
  Note: The results of calpain-3 immunoblot analysis need to be interpreted with caution, as the analysis is neither completely sensitive (i.e., it can yield false negative results) nor completely specific (i.e., it can yield false positive results). In particular, calpain-3 protein levels can be partially reduced in other muscular dystrophies such as dysferlinopathy and Udd muscular dystrophy (titinopathy) [Anderson et al 2000, Fanin et al 2001, Haravuori et al 2001]. Moreover, although calpain-3 protein is extremely stable in human muscle over time [Anderson et al 1998], the amount of protein can be partially reduced by the degradation that occurs when muscle tissue is handled or stored under conditions that promote rapid calpain-3 autolysis (especially partial thawing and exposure to moisture) [Anderson et al 1998, Fanin et al 2003].

• Assay of calpain-3 autolytic function in muscle (available only on a research basis) is better than conventional immunoblot analysis in identifying those affected individuals in whom CAPN3 mutations affect the autolytic function of calpain-3 protein rather than the quantity of calpain-3 protein [Fanin et al 2007].

Molecular Genetic Testing

Gene. CAPN3, which encodes proteolytic enzyme calpain-3, is the only gene known to be associated with calpainopathy.

Clinical testing

• Mutation scanning or sequencing of select exons

  • Although CAPN3 mutations are distributed throughout the length of the gene [Richard et al 1999], in some populations most mutant alleles are clustered in a limited numbers of exons [Anderson et al 1998, De Paula et al 2002, Zatz et al 2003, Fanin et al 2004, Piluso et al 2005, Leiden Muscular Dystrophy Pages].

    • Approximately 80% of mutations reported in Brazil are clustered in only exons 1, 2, 4, 5, 11, and 22 [Zatz et al 2003].

    • Approximately 87% of mutations reported in Italy are found in exons 1, 4, 5, 8, 10, 11, and 21 [Fanin et al 2004].

    • Approximately 80% of mutations reported in France are clustered in exons 1,4,7,10,11,13,19,22 [Krahn et al 2006a].

  • The probability of identifying calpainopathy or causative gene mutations varies depending on the calpain protein level detected on calpain immunoblot testing [Fanin et al 2003, Fanin et al 2004].

• Sequence analysis. Sequence analysis of the entire CAPN3 coding region (24 exons) is typically performed because of the ease of automation, increased turnaround time, and high sensitivity.

• Deletion/duplication analysis. Large genomic mutations [Richard et al 1999] including the frame-shift deletion of exons 2-8 [Todorova et al 2007] are an additional cause of calpainopathy.

Intronic nucleotide changes. The analysis of mutations at mRNA level (from muscle or blood) may be more efficient than exon-by-exon DNA screening and [Richard & Beckmann 1995] detect functional consequences of variants detected in non-coding regions and deep-intronic mutations [Blazquez et al 2008].

Table 1. Summary of Molecular Genetic Testing Used in Calpainopathy

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Gene Symbol	Test Method	Mutations Detected	Mutation Detection Frequency by Test Method 1	Test Availability
CAPN3	Mutation scanning	Sequence variants	90% 2	Clinical
	Sequence analysis	Sequence variants	~99%
	Deletion/duplication analysis 3	Partial- and whole-gene deletions/duplications	Unknown

Test Availability refers to availability in the GeneTests Laboratory Directory. GeneReviews designates a molecular genetic test as clinically available only if the test is listed in the GeneTests Laboratory Directory by either a US CLIA-licensed laboratory or a non-US clinical laboratory. GeneTests does not verify laboratory-submitted information or warrant any aspect of a laboratory's licensure or performance. Clinicians must communicate directly with the laboratories to verify information.

1. The ability of the test method used to detect a mutation that is present in the indicated gene

2. Detection frequency varies by exons scanned, ethnicity of the individual being tested, scanning method used, and laboratory Sequence analysis and mutation scanning of the entire gene can have high detection frequencies, although mutation scanning detection rates may vary considerably among testing laboratories as that method is highly dependent on details of the methodology employed.

3. Testing that identifies deletions/duplications not readily detectable by sequence analysis of genomic DNA; a variety of methods including quantitative PCR, long-range PCR, multiplex ligation-dependent probe amplification (MLPA), or targeted array GH (gene/segment-specific) may be used. A full array GH analysis that detects deletions/duplications across the genome may also include this gene/segment. See array GH.

Interpretation of test results. For issues to consider in interpretation of sequence analysis results, click here.

Testing Strategy

Testing strategy used in the diagnosis of calpainopathy in a proband differs depending on the availability of diagnostic muscle biopsy.

• Individuals for whom muscle biopsy is available. Diagnostic muscle biopsy allows screening by western blot of multiple proteins that are responsible for different forms of LGMD. After exclusion of other protein defects (i.e., sarcoglycans, dystrophin, dysferlin, caveolin), calpain-3 protein immunoblot remains the most useful strategy in the diagnosis of calpainopathy [Pollitt et al 2001].
  
  The subsequent steps, aimed at identifying gene mutations, vary according to the findings obtained from protein analysis.

  • In individuals with immunoblot-confirmed calpain-3 deficiency, the detection rate for one or two pathogenic CAPN3 mutations is approximately 84%. If two mutant alleles are identified, the genetic diagnosis is confirmed.

  • In individuals with immunoblot-confirmed calpain-3 protein deficiency and no identified CAPN3 mutations, genetic diagnosis of calpainopathy can neither be established nor excluded.

  • In individuals with normal amounts of calpain-3 protein on immunoblot analysis of muscle biopsy tissue, the diagnosis of calpainopathy appears unlikely but cannot be excluded because of possible functional enzyme defects. Such individuals have a residual risk of calpainopathy of approximately 9%. In these individuals, sequencing CAPN3 can be pursued but the yield will be low.

• Individuals for whom muscle biopsy is unavailable. Sequence analysis of CAPN3 is the optimal diagnostic step in these individuals because mutation scanning of CAPN3 is expected to identify a mutation in fewer than 40% of individuals with LGMD (of which LGMD2A is the most common form), in non-consanguineous populations [Kawai et al 1998, Chou et al 1999, Zatz et al 2000, Bushby & Beckmann 2003].

Carrier testing for at-risk relatives requires prior identification of the disease-causing mutations in the family.

Note: Carriers are heterozygotes and are not at risk of developing the disorder.

Prenatal diagnosis for at-risk pregnancies requires prior identification of the disease-causing mutations in the family.

Note: It is the policy of GeneReviews to include clinical uses of testing available from laboratories listed in the GeneTests Laboratory Directory; inclusion does not necessarily reflect the endorsement of such uses by the author(s), editor(s), or reviewer(s).

Genetically Related (Allelic) Disorders

No other phenotypes are associated with mutations in CAPN3.

Natural History

Calpainopathy is characterized by symmetric and progressive weakness of proximal (limb-girdle) muscles. The age at onset of muscle weakness ranges from two to 40 years. Early motor milestones are usually normal. Intra- and interfamilial clinical variability ranges from severe to mild [Richard et al 1999].

Three calpainopathy phenotypes have been identified based on the distribution of muscle weakness and age at onset:

• Pelvofemoral LGMD (Leyden-Möbius) phenotype, the most frequently observed calpainopathy phenotype Muscle weakness is first evident in the pelvic girdle and later in the shoulder girdle. Onset can be early (age <12 years), adult, or late (age >30 years). Individuals with early onset and rapid disease course usually have pelvofemoral LGMD.

• Scapulohumeral LGMD (Erb) phenotype. Muscle weakness is first evident in the shoulder girdle and later in the pelvic girdle. Early onset is infrequent; the disease course is variable, but usually milder than that in the pelvofemoral phenotype.

• HyperCKemia. Asymptomatic individuals have high serum CK concentrations only. HyperCKemia may be considered a presymptomatic stage of calpainopathy, as it is usually observed in children or young individuals.

The first clinical findings of calpainopathy are usually the tendency to walk on tiptoes, difficulty in running, and scapular winging. Waddling gait and slight hyperlordosis are frequently observed in the early stage of the disease. Symmetric weakness of proximal more than distal muscles is evident in the limbs, trunk, and periscapular area. Marked laxity of the abdominal muscles is frequently seen [Bushby 1999, Pollitt et al 2001]. The gluteus maximus, thigh adductors, and posterior compartment of the limbs are most affected [Fardeau et al 1996, Dincer et al 1997, Topaloglu et al 1997, Urtasun et al 1998]. Early Achilles tendon shortening and scoliosis may be present.

Some individuals report muscle pain and exercise intolerance [Penisson-Besnier et al 1998]. In some individuals, eosinophilic myositis was also found [Krahn et al 2006b]. Calf hypertrophy is rarely observed, and in a number of cases, significant atrophy is observed. Facial and neck muscles are usually spared. Macroglossia has not been described.

In the advanced stage of the disease, the inability to climb stairs, to rise up from a chair, or to get up from the floor is common. Joint contractures (in the hips, knees, elbows, and fingers) are common; rigid spine is occasionally observed on occasion [Pollitt et al 2001]. Respiratory insufficiency with reduced lung vital capacity may be present. Cardiomyopathy is uncommon. Intelligence is normal.

The disease is invariably progressive and loss of ambulation occurs approximately ten to 30 years after the onset of symptoms (between ages 10 and 48 years) [Richard et al 1999, Zatz et al 2003, Saenz et al 2005].

Genotype-Phenotype Correlations

Genotype-phenotype correlation in calpainopathy is complex and often complicated by the fact that most individuals are compound heterozygotes for CAPN3 mutations.

Null homozygous mutations are generally associated with a severe phenotype.

Although it has been suggested that homozygous missense mutations are usually associated with a milder phenotype than null mutations [Fardeau et al 1996, Anderson et al 1998, Richard et al 1999, Chae et al 2001], the phenotypic consequences of homozygous missense mutations are more difficult to predict [Richard et al 1997, Bushby 1999, De Paula et al 2002]. Wide clinical variability has been described among individuals homozygous for the same missense mutation, even among individuals within the same family [Fardeau et al 1996, Kawai et al 1998, Penisson-Besnier et al 1998, Richard et al 1999].

No direct correlations have been observed between the severity of the phenotype and the amount of calpain-3 protein detected by calpain-3 immunoblot analysis [Anderson et al 1998, Zatz et al 2003]. Affected individuals with either no detectable protein or normal amounts of protein have varying severity of the clinical phenotype [De Paula et al 2002, Fanin et al 2004]. Whereas null mutations are usually associated with a lack of detectable protein, missense mutations have variable and unpredictable consequences, which depend at least partially on the quantity of protein.

Penetrance

Nearly full penetrance is observed in adulthood. Some individuals remain asymptomatic until adulthood. Serum CK concentration is always increased.

Anticipation

Anticipation has not been described.

Nomenclature

Calpainopathy was formerly called LGMD2A because it was the first form of autosomal recessive LGMD to be mapped [Beckmann et al 1991].

Prevalence

Calpainopathy is considered the most common form of LGMD [Bushby & Beckmann 2003], representing approximately 30% of LGMD cases, depending on the geographic region [Chou et al 1999, Zatz et al 2000]. Estimates based on molecular data indicate that the frequency ranges from 10% of LGMD cases in a Caucasian population from the US [Chou et al 1999, Moore et al 2006] to 21% in the Netherlands [van der Kooi et al 2007], 25-28% in Italy [Guglieri et al 2008, Fanin et al 2009], 26% in Japan [Kawai et al 1998], 50% in Turkey [Dincer et al 1997], and 80% in the Basque country [Urtasun et al 1998] and Russia [Pogoda et al 2000]. In the latter countries and in inbred populations (La Reunion Island and the Old Order Amish community in North America), the disease can be ten times more common.

A founder effect for the 550delA mutation has been reported in Croatia [Milic & Canki-Klain 2005] and in Russia [Pogoda et al 2000].

A genetic epidemiologic study in northeastern Italy estimated that calpainopathy has a prevalence of approximately 1:100,000 inhabitants (corresponding to a carrier frequency of ~1:160) [Fanin et al 2005].

Evaluations Following Initial Diagnosis

To establish the extent of disease in an individual diagnosed with calpainopathy, a complete physical evaluation including the grading of muscle strength in single muscles and the analysis of several functional performances is recommended.

Treatment of Manifestations

Appropriate management, tailored to each individual, can improve quality of life and prolong survival. The general approach is based on the typical progression and complications of individuals with LGMD as described by McDonald et al [1995] and Bushby [1999].

• Physical therapy and stretching exercises can promote mobility and slow the disease progression, in particular by maintaining joint flexibility.

• Technical aids can also compensate for the loss of certain motor abilities; canes, walkers, orthotics, and wheelchairs enable individuals to regain independence.

• Surgical intervention may be required for correction of orthopedic complications including foot deformities, scoliosis, and Achilles tendon contractures.

• In the late stage of the disease, chronic respiratory insufficiency may occur and the use of respiratory aids may be indicated to prolong survival.

• Social and emotional support help to maximize a sense of social involvement and productivity and to reduce the sense of social isolation [Eggers & Zatz 1998].

Prevention of Secondary Manifestations

Physical therapy and stretching exercises can prevent contractures and muscle atrophy and thus help to slow disease progression.

Surveillance

Annual monitoring of muscle strength, joint range of motion, and respiratory function is recommended.

Monitoring for orthopedic complications, such as foot deformities, scoliosis, and Achilles tendon contractures, is recommended.

Agents/Circumstances to Avoid

Strenuous and eccentric muscle exercises should be discouraged as it exacerbates muscle necrosis and could precipitate the onset of weakness or accelerate muscle wasting.

Body weight should be controlled to avoid obesity and excessive weight loss (atrophy of muscles can be accelerated by loss of muscle proteins).

Physical trauma, bone fractures, and immobility can induce disuse atrophy and thus should be avoided.

Although no association of the disease with malignant hyperthermia is reported, the use of succinylcholine and halogenated anesthetic agents should be avoided when possible. (See Malignant Hyperthermia Susceptibility.)

While the specific mechanism whereby cholesterol-lowering agents (e.g., statins) may produce muscle lesions is unknown, such drugs should be avoided when possible.

Testing of Relatives at Risk

Relatives at risk (e.g., sibs of probands) should be clinically examined, especially if they may be affected. Molecular genetic testing can be offered to at-risk relatives when clinical findings suggest calpainopathy and the disease-causing mutations in the family are known.

See Genetic Counseling for issues related to testing of at-risk relatives for genetic counseling purposes.

Therapies Under Investigation

The safety and efficacy of AAV-mediated calpain-3 gene transfer in a mouse model of LGMD2A has been reported [Bartoli et al 2006].

Search ClinicalTrials.gov for access to information on clinical studies for a wide range of diseases and conditions.

Other

Genetics clinics, staffed by genetics professionals, provide information for individuals and families regarding the natural history, treatment, mode of inheritance, and genetic risks to other family members as well as information about available consumer-oriented resources. See the GeneTests Clinic Directory.

See Consumer Resources for disease-specific and/or umbrella support organizations for this disorder. These organizations have been established for individuals and families to provide information, support, and contact with other affected individuals.

Mode of Inheritance

Calpainopathy is inherited in an autosomal recessive manner.

Risk to Family Members

Parents of a proband

• The parents of an affected individual are obligate heterozygotes and therefore carry one mutant allele.

• Heterozygotes (carriers) are asymptomatic.

Sibs of a proband

• At conception, each sib of an affected individual has a 25% chance of being affected, a 50% chance of being an asymptomatic carrier, and a 25% chance of being unaffected and not a carrier.

• Once an at-risk sib is known to be unaffected, the risk of his/her being a carrier is 2/3.

• Heterozygotes (carriers) are asymptomatic.

Offspring of a proband

• The offspring of an affected individual are obligate heterozygotes (carriers) for a disease-causing mutation in CAPN3.

• In populations with a high rate of consanguinity, the reproductive partner of an affected individual may be a carrier, in which case the risk to the offspring of being affected is 50%.

Other family members of a proband. Each sib of the proband's parents is at a 50% risk of being a carrier.

Carrier Detection

Carrier testing for at-risk family members is available on a clinical basis once the mutations have been identified in the family.

Carrier testing for the reproductive partners of affected individuals and known carriers is available on a clinical basis and is generally done by sequence analysis of the exons in which mutations are most commonly found.

Related Genetic Counseling Issues

Family planning. The optimal time for determination of genetic risk, clarification of carrier status, and discussion of the availability of prenatal testing is before pregnancy. It is appropriate to offer genetic counseling (including discussion of potential risks to offspring and reproductive options) to young adults who are affected or at risk of being carriers.

DNA banking is the storage of DNA (typically extracted from white blood cells) for possible future use. Because it is likely that testing methodology and our understanding of genes, mutations, and diseases will improve in the future, consideration should be given to banking DNA of affected individuals. See for a list of laboratories offering DNA banking.

Prenatal Testing

Prenatal diagnosis for pregnancies at increased risk is possible by analysis of DNA extracted from fetal cells obtained by amniocentesis usually performed at approximately 15-18 weeks' gestation or chorionic villus sampling (CVS) at approximately ten to 12 weeks' gestation. Both disease-causing alleles of an affected family member must be identified before prenatal testing can be performed.

Note: Gestational age is expressed as menstrual weeks calculated either from the first day of the last normal menstrual period or by ultrasound measurements.

Preimplantation genetic diagnosis (PGD) may be available for families in which the disease-causing mutations have been identified. For laboratories offering PGD, see .

Note: It is the policy of GeneReviews to include clinical uses of testing available from laboratories listed in the GeneTests Laboratory Directory; inclusion does not necessarily reflect the endorsement of such uses by the author(s), editor(s), or reviewer(s).

Literature Cited

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Suggested Reading

1. Kang PB, Kunkel LM. The muscular dystrophies. In: Scriver CR, Beaudet AL, Sly WS, Valle D, Vogelstein B (eds) The Metabolic and Molecular Bases of Inherited Disease (OMMBID), McGraw-Hill, New York, Chap 216. Available at www.ommbid.com. Accessed 7-6-10.

Revision History

• 8 July 2010 (cd) Revision: deletion/duplication analysis available clinically

• 3 December 2007 (me) Comprehensive update posted to live Web site

• 15 December 2005 (ca) Revision: prenatal diagnosis available

• 10 May 2005 (me) Review posted to live Web site

• 29 November 2004 (ca) Original submission