Autosomal Recessive Limb Girdle Muscular Dystrophy In A Complex Consanguineous Family: The First Cases Series In Indonesia

Article Info History: Received: 06 Sept 2017 Accepted: 22 Dec 2017 Available: 31 Dec 2017 Abstract Background: Limb g irdle muscular dystrophy (LGMD) is a neuromuscular abnormality with clin ical heterogeneity and various severity, where over 30 subtypes have been identified. Meanwhile, molecular diagnosis of LGMD is not commonly carried out in Indonesia. We present a large pedigree of familial LGMD, with over 14 years of fo llow-up. Case Presentation: A 12-year old female patient came with muscle weakness. She had toe walking since age of 6, followed by calf hypertrophy for over three years. Family h istory revealed complex consanguinity . Her younger sister and her parents’ cousin had similar condition, with the latter was already bedridden. Physical examination results were waddling gait, lordotic spine, and absent deep tendon reflexes. Muscle biopsy showed sign of dystrophic process. Immunoperoxidase staining of some proteins resulted normal. Single nucleotide polymorphis m (SNP) array in two siblings revealed homozygosity on chromosome 15 containing CAPN3 gene of LGMD2A subtype. Recently, the patient is wheelchair bound and undergoes rehabilitation. Her sister is still able to walk with abnormal gait, while her parents’ cousin had passed away in age 55. From the multiple consanguinity, it could be concluded as autosomal recessive type LGMD. Conclusion: A large family with LGMD from Indonesia was presented with more than 14 years of care. Clinical diagnosis was made based on physical and additional examination, however molecular analysis for establishing definitive diagnosis is still limited. Further studies such as targeted or whole exome sequencing is warranted to elucidate the cause of disease. Long-term evaluation and supportive care, in addition to proper counseling may increase quality of life.


INTRODUCTION
Limb girdle muscular dystrophy (LGMD) is a neuromuscular disorder with clinical heterogeneity and slow muscle deterioration progressivity.The overall prevalence of LGMD is estimated between 1 in 14,500 to 45,000 indiv iduals, with more than 30 subtypes have been known related to various severity and phenotypic spectrums.LGM D1 is a fo rm of autosomal do minant, with known subtypes including LGMD1A, LGM D1B, and LGM D1C.Meanwhile, LGMD 2 consists of various subtypes from LGM D2A to LGM D2Q, and recently found subtypes with rare prevalence from LGMD2R to LGM D2W.The most prevalent subtype among all is LGM D2A, wh ich compro mise 26.5-30% of all LGMD cases and is inherited in autosomal recessive manner. 1,2The disorder is due to deficiency of calpain-3 protein that involves in sarcomere remodeling process in the muscle. 3Lack of calpain-3 may cause a significant Index by : decrease in post-fusion incorporation of neonatal myosin heavy chain (nMHC), a developmental myogenic markers in muscle fiber regeneration. 4The mo lecular d iagnosis scope of limb gird le muscular dystrophy is not commonly performed in Indonesia.Thus, the etiological diagnosis of many neurodegenerative disorders remains overlooked by the clinicians.Nevertheless, clinical and supportive care of the affected family member are equally important.We present a large pedigree of familial limb gird le muscular dystrophy, with more than 14 years history of follow-up.This case, to the best of our knowledge, is the first reported cases of familial LGMD in Indonesia.

CAS E PRES ENTATION
A 12-years old female patient with muscle weakness was referred to the Center for Bio med ical Research (CEBIOR) in 2002 for a genetic testing and genetic counseling regarding her condition.Since she was 6 years old, she had toe walking and was unable to squat.The condition was follo wed by pseudo hypertrophy of the calves for over three years.Her walking gait deteriorated, despite of undergoing Achilles tendon lengthening surgery to imp rove feet posture and gait.She experienced fatigable weakness, had mild arm weakness and lordosis.There were no cardiovascular or respiratory abnormalities.
Family history revealed complex consanguinity, in which her parents are of second cousins' marriage.Her younger sister had similar condition starting from age 7, while her parents' cousin had started toe walking since age 20 and was already bedridden at 49 years old.All three family members had elevated creatinine kinase (CK) level, where index patient and her sister showed significantly higher level (4,715 U/L and 13,545 U/ L [normal female value 145 U/L], respectively).Meanwhile, CK level of the uncle increased, but not as high as other family members upon checkup (278 U/ L; normal male value 171 U/L).Multiple consanguinity of the family is shown in Figure 1.Her younger sister occasionally walked on her toes, and received routine physiotherapy.There was no difficu lty on going up on a staircase.Meanwhile, her uncle had started toe walking at 20 years old.By the time of examination, he was already bedridden.
Upon the latest follow up, the patient is now 26 years old.She has already graduated from university, and is now wheelchair bound.Her younger sister is still able to walk with abnormal gait, while her parents' cousin has passed away in age of 59 years old.The patient received short term steroid treatment, and until recently, she still undergoes physiotherapy.

DISCUSS ION
The most plausible subtype of LGMD2A in this patient is first demonstrated by the multiple consanguinity in the family, wh ich exp lains the autosomal recessive inheritance.LGM D2A onset ranges from 2 to 40 years old, with the average of 8-15 years old. 2 In this case, the age of onset were 6 years old (index case), 9 years old (proband sibling), and 20 years old (proband uncle).Loss of ambulation typically occurred fro m 5 to 39 years.The long range of onset and slow progressivity distinguished this case to other autosomal recessive LGMD subtypes.On LGMD2B cases, there is similarly slow progressivity (10-20 years) but with different muscle involvement.Meanwhile in LGM D2C, there is a rapid progressivity similar to Duchenne muscular dystrophy. 1 LGMD2A is caused by missense, splice site, frameshift, or nonsense mutation on calpain-3 gene (CAPN3) wh ich located in 15q 15-21.A missense mutation in the exon 22 of calpain-3 gene was shown in three cases of Brazilian and Amish consanguineous families. 5This region was particularly found homozygous in the two siblings, suggestive to the same cause of disorder.Phenotype manifestations are mainly in proximal muscles involvement, with specific biopsy results commonly found.There is no facial, card iac, and respiratory involvement on the LGMD2A subtype. 6n this case, the uncle has lower level of CK serum (278 U/ L) co mpared to other affected members, in the age of 45 years old.In the LGM D2A patients, CK serum is typically raised to 3-20 times higher than normal level.Decreasing CK seru m level may be caused by the progressivity of disease and the increasing activity of muscle cell degeneration. 4

CONCLUS ION
A large family with LGM D fro m Indonesia was presented with over 14 years of follow-up, wh ich included physical examination, laboratory workups, rehabilitation and genetic counseling.Clin ical diagnosis was made based on physical and additional examination, however molecular analysis for establishing definit ive diagnosis is still limited.This case emphasizes the importance of genetic testing and counseling in Indonesia, especially in patients with neurodegenerative disorders, where diagnostic workup remains as a challenge.To establish LGMD2A diagnosis, or to elucidate other possible cause of disease and protein involvement, fu rther studies such as targeted sequencing on CAPN3 gene or whole exo me sequencing are warranted.Our longterm evaluation and supportive care, in addition to proper genetic counseling may enhance the family quality of life.Proper diagnosis may shed light to future prevention on next generation, and may increase the efficacy of genetic counseling.

Figure 1 .
Figure 1.Pedigree of family member with limb girdle muscular dystrophy.V:3 is index case with suspected LGM D2A.V:5 is her younger sister with starting similar condition.IV:22 is her uncle (parents' cousin) who was bedridden.

Table 1 .
Differences between subtypes of LGM D2 are shown on Table1.Various subtypes of autosomal recessive LGM D. The clinical features, type of muscle weakness and age onset, along with probable involvement of protein showed LGM D2A as the most possible cause of disorder.