After mutation identification, segregation analysis of the family was performed. We investigated 26 patients from Latvia and 34 patients from Lithuania with clinical symptoms of LGMD. The aim of this study was to investigate LGMD type 2 mutation spectrum and allelic frequency in Latvian and Lithuanian populations. Limitations of this method are related to insufficient exon coverage, leading to a potential lack of a representative examination of all exons. The advantage of next generation sequencing lies in the capability of analyzing whole exomes at a reasonable cost without invasive muscle biopsies. Furthermore, several commercially available kits include loci for the diagnosis of neuromuscular disorders. Hence, the advancement of LGMD diagnostics was achieved through next generation sequencing of the genome or exome, or through targeted exome capture applications. Directly sequencing the coding regions of the gene is the most common method, but remains often insufficient, due to the size and number of genes involved. Confirming the diagnosis of LGMD, in addition to subtyping, is usually done using molecular techniques. Although common mutations have been identified for some causative genes (for example, calpain 3 ( CAPN3) c.550delA or fukutin related protein ( FKRP) c.826C > A), a lot of additional mutations were found in different populations. Diagnosing of specific types of LGMD is a challenging process due to the many genes involved, including unknown genes within the identified chromosomal loci. These genes encode proteins involved in many different aspects of muscle cell biology. The rarer autosomal dominant (LGMD type 1 LGMD-1) form accounts for only 10 % of all cases and has eight causative genes and chromosomal loci currently identified. The autosomal recessive form of LGMD (LGMD type 2 LGMD-2) is more common, with 23 causal genes and chromosomal loci identified to date. LGMD is a rare disorder, and the different forms of LGMD range in prevalence from 1 in 14,500 to 1 in 123,000, depending on the population. Limb-girdle muscular dystrophies (LGMD) are a heterogeneous group of diseases that predominantly affect pelvic and shoulder girdle muscle groups. The allele frequencies of CAPN3 gene mutation c.2288A > G and DYSF gene mutation c.4872delG are 0.003. The allele frequency of CAPN3 gene mutation c.550delA in Latvia is 0.0016 and in Lithuania - 0.0029.

Genetic diagnosis was possible in 12 of 60 patients (20 %). Additionally, three mutations within FKRP gene were found: homozygous c.826C > A, and two compound – c.826C > A/c.404_405insT and c.826C > A/c.204_206delCTC mutations, and one mutation within CLCN1 gene – c.2680C > T p.Arg894Ter. ResultsĪnalysis revealed a homozygous CAPN3 c.550delA mutation in eight patients and three heterozygous variants in controls: dysferlin ( DYSF) c.5028delG, CAPN3 c.2288A > G, and FKRP c.135C > T.

We investigated 26 patients from Latvia and 34 patients from Lithuania with clinical symptoms of limb-girdle muscular dystrophies, along with 565 healthy unrelated controls from general and ethnic populations using our developed test kit based on the Illumina VeraCode GoldenGate genotyping platform, Ion AmpliSeq Inherited Disease Panel and direct sequencing of mutations in calpain 3 ( CAPN3), anoctamin 5 ( ANO5) and fukutin related protein ( FKRP) genes. Currently, there are 31 genes implicated in the different forms of limb-girdle muscular dystrophies, which exhibit similar phenotypes and clinical overlap therefore, advanced molecular techniques are required to achieve differential diagnosis. Limb-girdle muscular dystrophies are characterized by predominant involvement of the shoulder and pelvic girdle and trunk muscle groups.

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