Genetic diagnosis of limb girdle muscular dystrophy type 2A, A Case Report

Transcription

1

2 Genetic diagnosis of limb girdle muscular dystrophy type 2A, A Case Report Roshanak Jazayeri, MD, PhD Assistant Professor of Medical Genetics Faculty of Medicine, Alborz University of Medical Sciences

3 Main Disorders of Motor Unit ANTERIOR HORN NERVE NM JUNCTION MUSCLE FIBER SPINAL MUSCULAR ATROPHIES (SMA) Types 1-4 (Werdnig-Hoffman) (Kugelberg-Welander) NEUROPATHIES H.S.M.N. (Charcot- Marie-Tooth, Déjerine-Sotas, others) H.M.N. H.S.N. MYASTHENIAS Myasthenia gravis Congenital myasthenic syndromes DYSTROPHIES Duchenne / Becker Limb girdle (LGMD) sarcoglycanopathies Emery-Dreifuss / FSH MYOTONIC Sd Steinert / Thomsen MISCELLANEOUS Mitochondrial Congenital Metabolic Dysimmune

4 Limb-girdle muscular dystrophies (LGMD) A group of rare progressive genetic disorders, characterized by atrophy and weakness of the voluntary muscles of the hip and shoulder areas (limb-girdle area), may spread to affect other muscles of the body. There are many different subtypes of LGMD, each one resulting from a mutation of a different disease gene (genetic heterogeneity). Most of these genes are involved in the production of certain muscle proteins. Autosomal dominant LGMD is known as LGMD1, 8 subtypes (LGMD1A-1H). Autosomal recessive LGMD is known as LGMD2, 17 subtypes(lgmd2a-2q).

5 Diagnosis clinical evaluation detailed patient history identification of characteristic symptoms (e.g., specific distribution of muscle weakness and atrophy) blood tests may reveal elevated levels of the creatine Kinase (CK) a variety of specialized tests including, muscle biopsy and IHC Molecular genetic testing, to identify a specific genetic mutation, is now the gold standard for diagnosis in LGMD and allows a specific diagnosis as well as specific testing for other family members.

6 Differential Diagnosis Dystrophinopathys (DMD, BMD) Facio scapulo humeral muscular dystrophy (FSHD) Emery-Dreifuss muscular dystrophy (EDMD) Spinal muscular atrophy (SMA) Myopathys metabolic myopathies such as Pompe disease; inflammatory myopathies such as dermatomyositis or polymyositis; distinct congenital myopathies such as nemaline myopathy.

7

8 Treatment No cure exists for any form of LGMD. Genetic counseling may be of benefit for affected individuals and their families. Other treatment is symptomatic and supportive.

9 Case presentation We report a young 24 years old boy with muscular disease, presented with gradual onset proximal muscle weakness in all four limbs and waddling gate since nine years. There was noticeable thinning of shoulders, arms and thighs, and he was on a wheelchair. Gower s sign was positive. His parents were first cousin and he has a similar affected sister. There was also positive family history in his cousins. Serum CK: elevated NCV & EMG: moderate myopathy Muscle biopsy was recommend (didn t do)

10 Approach History & Clinical Impression Pedigree Physical Exam EMG & NCV CPK Inheritance Pattern XL-R AD AR mit Weakness Proximxl Distal Limb Girdle Pseudo HT Ophthalmoplegia Macroglossia + Gower sign Hyporeflexia Neurogenic AH Myopathic Myasthenic SM neuropathic Muscle imaging Muscle biopsy Molecular study Elevated

11 Methods : Obtaining blood from the proband, DNA was extracted from lymphocytes. NGS with a panel of 79 genes related with hereditary muscular disease was performed. The result confirmed by Sanger sequencing. Results : One homozygous mutation c.946-2a>g on CAPN3 gene has been detected. This mutation has been previously reported for its pathogenicity

12

13

14 The human calpain-3 gene localizes to chromosome 15q and has 24 exons spanning some 53 kb of DNA. The gene is exceptional by having many rather small exons; 10 exons are only bp, while exons 12, 15 and 14 are even smaller, 12, 18 and 37 bp resp. Most introns vary in size between kb. Exceptional are introns 18 and 20, measuring below 100 bp, and intron 1, with a size of 24.3 kb covering about half of the gene.

15 CAPN3 gene Normal Function The CAPN3 gene provides instructions for making an enzyme called calpain-3, which is found within muscle cells in structures called sarcomeres. Sarcomeres are the basic unit of muscle contraction. They are made of proteins that generate the mechanical force needed for muscles to contract. The function of the calpain-3 enzyme is not well understood. Researchers suggest it may help cut (cleave) damaged proteins into shorter segments to facilitate their removal from the sarcomere. Studies have also shown that calpain-3 attaches (binds) to proteins involved in controlling the ability of muscle fibers to stretch (elasticity) and in cell signaling. However, its specific roles in these processes are unknown.

16 CAPN3 gene, disease related to genetic changes More than 300 mutations in the CAPN3 gene have been identified in people with limb-girdle muscular dystrophy type 2A. This form of limb-girdle muscular dystrophy is also called calpainopathy. Most CAPN3 gene mutations change one protein building block (amino acid) in the calpain-3 enzyme. These mutations result in a calpain-3 enzyme that is abnormally short or unstable. Disruption of the enzyme's ability to properly cleave proteins for removal from the sarcomere may allow these waste proteins to accumulate in muscle tissue and become toxic. Other mechanisms have also been suggested to account for the muscle damage that underlies limb-girdle muscular dystrophy in people with CAPN3 gene mutations.

17 CALPAIN 3; CAPN3 Allelic Variants (11 Selected Examples) : Number Phenotype Mutation.0001 MUSCULAR DYSTROPHY, LIMB-GIRDLE, AUTOSOMAL RECESSIVE 1 CAPN3, ARG769GLN.0002 MUSCULAR DYSTROPHY, LIMB-GIRDLE, AUTOSOMAL RECESSIVE MUSCULAR DYSTROPHY, LIMB-GIRDLE, AUTOSOMAL RECESSIVE MUSCULAR DYSTROPHY, LIMB-GIRDLE, AUTOSOMAL RECESSIVE MUSCULAR DYSTROPHY, LIMB-GIRDLE, AUTOSOMAL RECESSIVE MUSCULAR DYSTROPHY, LIMB-GIRDLE, AUTOSOMAL RECESSIVE 1 MYOSITIS, EOSINOPHILIC, INCLUDED CAPN3, ARG572GLN CAPN3, ARG110TER CAPN3, SER86PHE CAPN3, PRO319LEU CAPN3, 2362AG-TCATCT.0007 MUSCULAR DYSTROPHY, LIMB-GIRDLE, AUTOSOMAL RECESSIVE MUSCULAR DYSTROPHY, LIMB-GIRDLE, AUTOSOMAL RECESSIVE MUSCULAR DYSTROPHY, LIMB-GIRDLE, AUTOSOMAL RECESSIVE MUSCULAR DYSTROPHY, LIMB-GIRDLE, AUTOSOMAL RECESSIVE MUSCULAR DYSTROPHY, LIMB-GIRDLE, AUTOSOMAL DOMINANT 4 CAPN3, 1080G-C, TRP360CYS CAPN3, 1-BP INS, 1796A CAPN3, 1-BP DEL, 550A CAPN3, ARG490GLN CAPN3, 21-BP DEL, NT643

18 CAPN3 protein structure and the location of the identified mutations within each domain

19 LGMD2A (calpain-deficient LGMD; calpainopathy) the most common form, accounting for about 30 percent of cases affects children between the ages of 8-15, but may range from 2-40 years of age. progressive, symmetrical weakness of the proximal limb and girdle without heart involvement or intellectual disability. also known as primary calpainopathy, is caused by mutations in the CAPN3 gene encoding for calpain-3, resulting in total or partial loss of protein.

20 LGMD2A (calpain-deficient LGMD; calpainopathy) There are three subtypes of autosomal recessive disorders associated with mutations in the CAPN3 gene which differ by the distribution of muscle weakness and age at onset: Pelvifemoral limb-girdle muscular dystrophy (also known as Leyden-Mobius LGMD) is the most frequently observed subtype. In these cases, muscle weakness is first evident in the pelvic girdle and later in the shoulder girdle. Onset is usually before age 12 or after age 30; Scapulohumeral LGMD (also known as Erb LGMD) usually has milder symptoms with infrequent early onset. In most cases, muscle weakness is first evident in the shoulder girdle and later in the pelvic girdle; HyperCKemia is usually observed in children or young individuals. In most cases, those affected do not have symptoms, just high levels of creatine kinase in their blood.

21 DIAGNOSIS/TESTING The diagnosis of calpainopathy, which is suggested by clinical findings and elevated serum CK concentration, is established by identification of biallelic pathogenic variants in CAPN3 (encoding proteolytic enzyme calpain-3) or a dominantly acting heterozygous pathogenic variant for the CAPN3 21-bp deletion (c.643_663del21) by molecular genetic testing. If such testing is not available, muscle biopsy with protein immuno-analysis should be used for diagnostic confirmation

22 Genetic counseling Calpainopathy is typically inherited in an autosomal recessive manner. Less commonly, calpainopathy is inherited in an autosomal dominant manner. In the autosomal recessive form, 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. To date, all individuals diagnosed with autosomal dominant calpainopathy have inherited a pathogenic CAPN3 variant from a heterozygous parent. Testing for at-risk relatives and prenatal testing for pregnancies at increased risk are possible if the CAPN3 pathogenic variant(s) in the family are known.

23 Thank you for your attention