The story is about a young student of 24 years of age of North African ethnicity that noticeably has difficulty walking that is related to a genetic disease. He requires assistance to get around and is unable to climb upstairs on his own. He said that he had been suffering for at least 10 years from Duchenne muscular dystrophy (DMD), a recessive genetic disorder linked to the X chromosome affecting almost only males. I asked him if he wanted to be analyzed for the identification of the molecular defect related to his pathology since there may be cases of dystrophy not linked to the X chromosome, which are due to mutations in autosomal genes. Once he agreed, I looked into his previous medical history to be able to help him in his diagnostic odyssey.
Patient's phenotype and physical examination result
The original physician, Doctor Amel Boughammoura (Neurologist of Monastir Tunisia) described in her report that this 23-year-old patient was from a first-degree consanguineous marriage (between individuals who are closely related) and that his maternal aunt also has the same symptoms describing a progressive muscular dystrophy. Since his schooling, the patient has had difficulty running, climbing stairs, and getting up from the ground. This is gradually worsening, reducing the physical activities he can participate in. His motor deficit is rather proximal, affecting both girdles, with moderate atrophy of the hip and para-vertebral muscles. The muscle biopsy confirmed progressive muscular dystrophy. Actually, he presents a worsening of dandian walking disorders, sometimes needing help to get up from his chair.
Analysis by the Geneticist
Muscular dystrophy is a group of diseases that cause progressive weakness and loss of muscle mass. In muscular dystrophy, abnormal gene mutations interfere with the production of proteins needed to form healthy muscle. Symptoms of the most common variety begin in childhood, mostly in males. Other types don't surface until adulthood. There are many kinds of muscular dystrophy (MD). The main sign is progressive muscle weakness. Specific signs and symptoms begin at different ages and in different muscle groups, depending on the type of muscular dystrophy1:
- Duchenne type MD
- Becker MD
- Limb-girdle MD
- Myotonic MD
- Facio-scapulo-humeral (FSHD)
- Congenital muscular dystrophy
Need for Whole Exome Sequencing (WES) analysis
Faced with this great phenotypic variability, diagnosis was difficult. The original diagnosis was limited to revealing only a progressive muscular dystrophy without specifying the type of dystrophy, hence the interest of molecular exploration by WES. Does this patient have a dominant or recessive X-linked or autosomal form of dystrophy? What gene is involved?
WES can answer these questions by identifying the gene and the variant involved in the phenotype. WES helps pinpoint genetic diagnosis for patients2.
Diagnosis after WES
The WES test result came back positive. It revealed a functional variant: a deletion of one nucleotide (del A) on the c.550 position on the exon 4 of CAPN3 gene on chromosome 15. The CAPN3 gene encodes for calpain-3; this protein is a calcium-dependent intracellular protease. The variant creates a shift in the reading frame leading to the appearance of stop codon at the position of 220 (p.184ArgfsTer36) leading to aberrant mRNA.
According to the EVIDENCE software used by the company performing the WES, the variant found in my patient was identified as Muscular dystrophy, limb-girdle, autosomal recessive 1 (OMIM: 253600) or Muscular dystrophy, limb-girdle, autosomal dominant 4 (OMIM: 618129)3.
Family analysis result
Analysis of my patient's family tree showed first degree inbreeding between the parents who are phenotypically healthy. In addition, two maternal aunts, born from consanguineous parents, show the same signs as our patient (Figure 1). These findings support an autosomal recessive limb-girdle muscular dystrophy.
In this type of muscular dystrophy, hip and shoulder muscles are usually affected first. Patients might have difficulty lifting the front part of the foot and so might trip frequently. Onset usually begins in childhood or the teenage years. In our patient, it started at the age of 10. The autosomal recessive limb-girdle muscular dystrophy type 2A (LGMD2A) is caused by mutations in the calpain 3(CAPN3) gene, and is characterized by selective atrophy and weakness of proximal limb and girdle muscles4. Based on the dbSNP on the NCBI database (www.ncbi.nlm.nih.gov), this variant constitutes a very rare polymorphism (rs80338800) whose allele frequency is almost 0,000125.
This variant had never been described in Tunisian patients. However, other variants have been identified in Tunisia: a new homozygous mutation c.1536+1G>T in the donor splice site of intron 12 of the CAPN3 was found in patients with limb-girdle muscular dystrophy type 2A (LGMD2A), the most common form of LGMD5. Another novel missense mutation (c.T1681C/p.Y561H) of the 13th exon of the CAPN3 gene was also reported in a Tunisian patient suffering from LGMD2A6.
Outside Tunisia, the variant c.550delA was identified in a clinical, molecular, and biochemical findings from 238 LGMD2A patients, representing approximately 50 % (238 out of 484) of the suspected calpainopathy cases referred for the molecular study of the CAPN3 gene7. In this study, authors identified 105 different mutations in the CAPN3 gene of which 50 have not been described previously. The most frequent mutation was 2362AG-->TCATCT (22nd exon). Other recurrent mutations described were N50S, 550DelA, G222R, IVS6-1G--> A483D, IVS17+1G-->T, 2069-2070DelAC, R748Q and R748X, each of which was found in >5 chromosomes (>0,51 %).
Other genes were involved in the autosomal recessive LGDMD. The most described in the Tunisian population is the SGCG gene which encodes the gamma-sarcoglycan subunit8,9. The gamma-sarcoglycanopathy is prevalent in Tunisia where only one homozygous mutation (c.521-T deletion) has been identified10.
Is there treatment for the limb-girdle muscular dystrophy?
There's no cure for muscular dystrophy. But medications and therapy can help manage symptoms and slow the course of the disease. These include weight control to prevent obesity, physical therapy, stretching exercises to promote mobility and prevent contractures, and the use of mechanical aids to help with ambulation and movement mobility. Surgical intervention is suggested in the event of an orthopedic complication11.
Clinical trials of gene therapy in girdle myopathies are beginning to multiply as the first results arrive and approaches are diversified. The aim of gene therapy is to deliver a non-mutated version of a gene to cells in the body, particularly to the cells most affected by the disease12. Preclinical research conducted to date has provided early proof of concept for calpain 3 or CAPN-3 in LGMD2A and supports further advancement. CAPN-3 works using a modified viral vector called AAVrh74, which is designed to deliver the gene to skeletal muscle tissue, including the diaphragm. This vector is an engineered version of a type of virus called AAV (adeno-associated virus), which are commonly used in gene therapies because they are safe and easy to manipulate in a lab setting. This gene therapy is the sixth one Sarepta has developed for different types of LGMD12.
Life after diagnosis
These results have enlightened the patient and his family regarding his particular type of dystrophy and the molecular defect most probably involved. This variant could never have been identified without WES analysis because the number of genes involved in progressive muscular dystrophy is extremely large for direct sequencing. This result may also help in guiding the attending physician to tailor-made treatment for the patient. Identification of the c.550delA variant will allow all family members a possible molecular exploration and a precise and early diagnosis for genetic counseling, it will also allow the screening of heterozygote individuals and the prenatal diagnosis for the willing couples.
Final thoughts on rare genetic disease diagnosis
Whole genome sequencing from a single blood test picks up 31 % more cases of rare genetic disorders than standard tests, shortening the “diagnostic odyssey” that affected families’ experience, and providing genetic counseling and huge opportunities for future research.
Faced with the multitude of hereditary pathologies which show great clinical variability and a large number of genes involved, doctors have problems diagnosing these types of diseases. In these cases, WES analysis remains the only method to pinpoint the molecular defect in question. Unfortunately, this kind of analysis is expensive; it remains underused especially in poor or developing countries. In Tunisia, only a few laboratories or research institutes have the equipment for the NGS. For others, aware of the interest of WES in genetic explorations, orders for NGS systems are underway. The stakes are high and the challenge for the integration of WES into our institutions is great. The difficulties of molecular diagnosis for genetic diseases, especially monogenic, remain linked to the ignorance of the genes involved, the heaviness of NGS equipment and the high cost of WES analyzes. This is especially true in the face of the multitude of cases requiring this kind of analysis in countries where genetic diseases, especially recessive, are frequent due to the relatively high number of consanguineous marriages, our country is one of them.
I warmly thank the 3billion team which allowed us to benefit from some successful WES, including the one for the patient subject of this story.
- I am Amel HAJ KHELIL, Professor of Genetics at the Superior Institute of Biotechnology, University of Monastir, Tunisia and member of the research laboratory of “Human Genome and multifactorial diseases” at the Faculty of Pharmacy, University of Monastir, Tunisia.
- Muscular dystrophy. Mayo Clinic Family Health Book, 5th Edition. (https://order.store.mayoclinic.com/books/gnweb43?)
- Schon K R, Horvath R, Wei W, Calabrese C, Tucci A, Ibañez K et al. Use of whole genome sequencing to determine genetic basis of suspected mitochondrial disorders:cohort study. BMJ;375:e066288. (2021).
- Richards S, Aziz N, Bale S, Bick D, Das S et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med;17(5):405-24. (2015).
- Wang CH, Liang WC, Minami N, Nishino I, Jong YJ. Limb-girdle Muscular Dystrophy Type 2A with Mutation in CAPN3: The First Report in Taiwan. Pediatr Neonatol;56(1):62-5. (2015).
- Hadj Salem I, Kamoun F, Louhichi N, Rouis S,Mziou M et al. Mutations in LAMA2 and CAPN3 genes associated with genetic and phenotypic heterogeneities within a single consanguineous family involving both congenital and progressive muscular dystrophies. Biosci Rep;31(2):125-35. (2011).
- Rekik S,Sakka S,Ben Romdhan S,Farhat N,Baba Amer Y et al. Novel Missense CAPN3 Mutation Responsible for Adult-Onset Limb Girdle Muscular Dystrophy with Calves Hypertrophy. J Mol Neurosci;69(4):563-9. (2019).
- Sáenz A,Leturcq F,Cobo AM,Poza JJ,Ferrer X et al. LGMD2A: genotype-phenotype correlations based on a large mutational survey on the calpain 3 gene. Brain;128(Pt 4):732-42. (2005).
- Ben Othmane K, Ben Hamida M, Pericak-Vance MA, Ben Hamida C, Blel S et al. Linkage of Tunisian autosomal recessive Duchenne-like muscular dystrophy to the pericentromeric region of chromosome 13q. Nature Genet;2:315-7. (1992).
- Trabelsi M, Kavian N, Daoud F, Commere V, Deburgrave N et al. Revised spectrum of mutations in sarcoglycanopathies. Europ J Hum Genet;16:793-803. (2008).
- Kefi M, Amouri A, Driss A, Ben Hamida C, Hamida M et al. Phenotype and sarcoglycan expression in Tunisian LGMD 2C patients sharing the same del 521-T mutation. Neuromuscular Disorders;13(10):779-87. (2004).
- Orphanet. Dystrophie musculaire des ceintures.
- Muscular Dystrophy News. Sarepta Acquires Investigational Gene Therapy for LGMD2A.