Identification and Characterization of Novel Founder Mutations in NDRG1: Refining the Genetic Landscape of Charcot-Marie-Tooth Disease Type 4D in Bulgaria.

Charcot-Marie-Tooth neuropathy type 4D (CMT4D) is a rare genetic disorder of the peripheral nervous system caused by biallelic mutations in the N-Myc Downstream Regulated 1 gene (NDRG1). Patients present with an early onset demyelinating peripheral neuropathy causing severe distal muscle weakness and sensory loss, leading to loss of ambulation and progressive sensorineural hearing loss. The disorder was initially described in the Roma community due to a common founder mutation, and only a handful of disease-causing variants have been described in this gene so far. Here, we present genetic and clinical findings from a large Bulgarian cohort of demyelinating CMT patients harboring recurrent and novel variants in the NDRG1 gene. Notably, two splice-site variants are exclusive to Bulgarian Muslims and reside in ancestral haplotypes, suggesting a founder effect. Functional characterization of these novel variants implicates a loss-of-function mechanism due to shorter gene products. Our findings contribute to a deeper understanding of the genetic and clinical heterogeneity of CMT4D and highlight novel founder mutations in the ethnic minority of Bulgarian Muslims.

Characterization of Clinical Phenotypes in Congenital Myasthenic Syndrome Associated with the c.1327delG Frameshift Mutation in CHRNE Encoding the Acetylcholine Receptor Epsilon Subunit.

Background: Congenital myasthenic syndromes (CMS) are a group of rare but often treatable inherited disorders of neuromuscular transmission characterized by fatigable skeletal muscle weakness. In this paper we present the largest phenotypic analysis to date of a cohort of patients carrying the pathogenic variant c.1327delG in the CHRNE gene, leading to CHRNE-CMS. Objective: This study aims to identify the phenotypic variability in CMS associated with c.1327delG mutation in the CHRNE gene. Methods: Disease specific symptoms were assessed using specific standardized tests for autoimmune myasthenia (Quantitative Myasthenia Gravis score) as well as patient-reported scales for symptom severity. Evaluated clinical manifestations included ocular symptoms (ophthalmoparesis and ptosis), bulbar weakness, axial muscle weakness, proximal and distal muscle weakness, and respiratory function. Patients were allocated into three groups according to clinical impression of disease severity: mild, moderate, and severe. Results: We studied 91 Bulgarian Roma patients, carrying the same causative homozygous CHRNE c.1327delG mutation. Bulbar weakness was present in patients throughout all levels of severity of CHRNE-CMS in this study. However, difficulties in eating and swallowing are more prominent characteristics in the moderate and severe clinical phenotypes. Diplopia and ptosis resulting from fatigue of the extraocular muscles were permanent features regardless of disease severity or age. Levels of axial, proximal and distal muscle weakness were variable between disease groups. The statistical analysis showed significant differences between the patients in the three groups, emphasizing a possible variation in symptom manifestation in the evaluated patient population despite the disease originating from the same genetic mutation. Impairment of respiratory function was more prominent in severely affected patients, which might result from loss of compensatory muscle function in those individuals. Conclusion: Results from our study indicate significant phenotypic heterogeneity leading to mild, moderate, or severe clinical manifestation in CHRNE-CMS, despite the genotypic homogeneity.

Seven Years of Selective Genetic Screening Program and Follow-Up of Asymptomatic Carriers With Hereditary Transthyretin Amyloidosis in Bulgaria.

Hereditary transthyretin amyloidosis (ATTRv amyloidosis) is a rare, autosomal-dominant (AD) multisystem disorder resulting from the extracellular deposition of amyloid fibrils formed by a destabilized mutant form of transthyretin (TTR), a transport protein predominantly produced by the liver. Aim: The aims of the current study are to demonstrate the Bulgarian experience with the screening programs among the high-risk patient population over the last 7 years, to present the results from the therapy with TTR stabilizer in our cohort, as well as to stress on the importance of a follow-up of asymptomatic carriers with TTR pathogenic variants by a multidisciplinary team of specialists. Materials and Methods: In 2014, a screening program among the high-risk patient population for ATTRv was initiated in Bulgaria. On one hand, it was conducted to identify new patients and families among people with "red flag" clinical features, while on the other hand, the program aimed to identify TTR mutation carriers among the families with already genetically proven diagnoses. Sanger sequencing methodology was used to make fast target testing for mutations in the TTR gene in the suspected individuals. All of the identified carriers underwent subsequent evaluation for neurological, cardiac, gastroenterological, and neuro-ophthalmological involvement. Those considered affected were provided with multidisciplinary treatment and a follow-up. Results: As a result of a 7-year selective screening program among the high-risk patient population and relatives of genetically verified affected individuals, 340 carriers of TTR mutations were identified in Bulgaria with the following gene defects: 78.53% with Glu89Gln, 10.29% with Val30Met, 8.24% with Ser77Phe, 2.06% with Gly47Glu, and 0.59% with Ser52Pro. All of these affected displayed a mixed phenotype with variable ages at onset and rate of progression, according to their mutation. From the 150 patients treated with TTR stabilizer, 84 remained stable, while in other 66 patients the treatment was terminated either because of polyneuropathy progression or due to death. A program for a regular follow-up of asymptomatic carriers in the last 3 years enabled us to detect the transition of 39/65 to symptomatic patients and to initiate treatment in a timely manner. Conclusion: Bulgarian ATTRv patients display a mixed phenotype with some clinical peculiarities for each mutation that should be considered when treating the affected and the follow-up of the asymptomatic carriers of a specific gene defect.

Limb girdle muscular dystrophy 2G in a religious minority of Bulgarian Muslims homozygous for the c.75G>A, p.Trp25X mutation.

Mutations in TCAP gene cause autosomal recessive limb-girdle muscular dystrophy type 2G (LGMD2G), congenital muscular dystrophy and autosomal dominant dilated and hypertrophic cardiomyopathy. We studied 18 affected individuals from 12 pedigrees, belonging to a Bulgarian Muslim minority from the South-West of Bulgaria, homozygous for the c.75G>A, p.Trp25X mutation in TCAP gene. The heterozygous carrier rate of p.Trp25X among 100 newborns in this region was found to be 2%. The clinical features in the Bulgarian TCAP group include disease onset in the first to the third decade of life, proximal muscle weakness in the lower limbs, followed or accompanied by difficulties in ankle dorsiflexion and involvement of the proximal muscles of the upper limbs 5-9 years after the disease onset. Asymmetry between left and right was present in more than 20% of the affected. Respiratory and cardiac functions were not affected. On the MRI the muscles of the posterior pelvic area, thigh and anterior leg were predominantly affected, while sartorius, gracilis and biceps femoris muscles remained relatively spared. In conclusion, LGMD2G appears to be a common form among Bulgarian Muslims. Homozygosity for c.75G>A, p.Trp25X is associated with a homogeneous clinical presentation, but the clinical course and severity of the disease show inter- and intra-familial variation.