Case Report: Transthyretin Glu54Leu-a rare mutation with predominant cardiac phenotype.

We report two unrelated Bulgarian families with hereditary transthyretin (ATTR) amyloidosis due to a rare p.Glu74Leu (Glu54Leu) pathogenic variant found in seven individuals-three of them symptomatic. Only one family with the same variant and with a Swedish origin has been clinically described so far. Our patients are characterized by predominant cardiac involvement, very much similar to the Swedish patients. Although the initial complaint was bilateral carpal tunnel syndrome, advanced amyloid cardiomyopathy was found in two symptomatic carriers at diagnosis with heart failure manifestations. The neurological involvement was considered as mild, with mainly sensory signs and symptoms being present. We followed a non-biopsy algorithm to confirm the diagnosis. Tafamidis 61 mg has been initiated as the only approved disease modifying treatment for ATTR cardiomyopathy. Clinical stability in the absence of adverse events has been observed at follow up.

Navigating the ALS Genetic Labyrinth: The Role of MAPT Haplotypes.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by wide clinical and biological heterogeneity, with a large proportion of ALS patients also exhibiting frontotemporal dementia (FTD) spectrum symptoms. This project aimed to characterize risk subtypes of the H1 haplotype within the MAPT (microtubule-associated protein tau) gene, according to their possible effect as a risk factor and as a modifying factor in relation to the age of disease onset. One hundred patients from Bulgaria with sporadic ALS were genotyped for the variants rs1467967, rs242557, rs1800547, rs3785883, rs2471738, and rs7521. Haploview 4.2 and SHEsisPlus were used to reconstruct haplotype frequencies using genotyping data from the 1000 Genomes project as controls. Genotype-phenotype correlation was investigated in the context of age of disease onset and risk of disease development. While the individual variants of the subtypes do not influence the age of onset of the disease, a correlation was found between the specific haplotype GGAGCA (H1b) and the risk of developing sALS, with results showing that individuals harboring this haplotype have a nearly two-fold increased risk of developing sALS compared to other H1 subtypes. The results from this study suggest that fine transcriptional regulation at the MAPT locus can influence the risk of ALS.

Novel insights on GTPBP3-associated hypertrophic cardiomyopathy.

About 100 genes have been associated with cardiomyopathies with genotype-phenotype correlations often hard to establish. Genetic testing may help to confirm the genetic diagnosis and assess the risk of inheritance in the family. A 25-year old male with hypertrophic cardiomyopathy and fasciculoventricular accessory pathway was referred for genetic testing by his cardiologist. Targeted PRKAG2 screening and whole-exome sequencing were performed, followed by Sanger sequencing segregation analysis in the family. The PRKAG2 gene screening was negative. Whole-exome sequencing revealed the following four variants in the patient: c.181G>C (p.Ala61Pro) and c.1199C>T (p.Thr400Met) in the GTPBP3 gene, as well as c.752C>T (p.Thr251Ile) and c.1760C>T (p.Pro587Leu) in the POLG gene. Family segregation analysis showed that the patient's mother is a carrier of variant c.181G>C and the patient's paternal grandmother is a carrier of variant c.1199C>T in the GTPBP3 gene, which is in accordance with an autosomal recessive model of inheritance of the disease. Both variants in the POLG are found paternally inherited in the patient's healthy half-brother, thus are not considered disease-causing. GTPBP3 variants have been reported in patients with hypertrophic cardiomyopathy, associated with combined oxidative phosphorylation deficiency 23. These novel variants represent the probable cause of the observed clinical symptoms in the patient.

Novel variant c.92T > G (p.Val31Gly) in the PFN1 gene (ALS18) responsible for a specific phenotype in a large Bulgarian amyotrophic lateral sclerosis pedigree.

Objectives: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive deterioration of motor function, disability, and death. Variants in the PFN1 gene, encoding the Profilin-1 protein, are related to ALS18. Methods: We present a pedigree consisting of 3 generations and 4 affected individuals, 3 of which carry a novel heterozygous variant: c.92T > G (p.Val31Gly) in the PFN1 gene. This variant was discovered through means of whole exome sequencing (WES) and targeted analysis of ALS-related genes. Results: The mean age of onset in our pedigree was 59.75 (±10.11 SD) years with a significant difference between the first two generations (females) and the third (male) of 22.33 (±3.4 SD) years. For this ALS form, we observed a longer disease progression of 4 (±1.87 SD) years (three of four affected are still alive). Clinical manifestations displayed predominant impairment of the lower motor neuron (LMN) in one limb, with gradual involvement of other limbs. A novel heterozygous missense variant c.92T > G, p. Val31Gly (NM_005022.4) in exon 1 in the PFN1 gene was discovered through means of whole exome sequencing (WES). Segregation analysis in the family showed that the detected variant was inherited from the affected mother, and the affected aunt also turned out to be a variant carrier. Conclusions: ALS18 is a very rare form of the disease. We report here a relatively large pedigree with a novel variant, leading to late onset (after 50 years), initial involvement of the lower limbs and relatively slow progression.

Arginase deficiency in Bulgaria: first cases and potential endemic region for the disorder.

Arginase deficiency is an autosomal recessive urea cycle disorder caused by pathogenic variants in the ARG1 gene. The clinical features of the disease include spasticity, tremour, ataxia, hypotonia, microcephaly and seizures. Growth delay can also be observed in the affected individuals. Here we describe the results from molecular-genetic analysis of two patients with arginase deficiency. In the first case, we reported a novel homozygous missense variant c.775G>A p.(Gly259Ser) in a patient with Bulgarian ethnic origin. In the second case, a novel homozygous splice site variant c.329+1G>A was detected in a patient from a consanguineous family of Roma ethnic origin. A hundred samples of newborns of Roma origin were screened for variant c.329+1G>A and one individual was found to be a heterozygous carrier of variant c.329+1G> A. The results from this study indicated the necessity for screening of the Roma population with respect to the disease arginase deficiency in Bulgaria.

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.

Screening for hereditary transthyretin amyloidosis in Bulgaria.

Transthyretin amyloid (ATTR) amyloidosis is a rare disorder with an adult-onset defined by the accumulation of misfolded fibrils predominantly in peripheral nerves, the heart, and the digestive tract. The disease is characterized by two forms - hereditary (ATTRv) or acquired (ATTRwt). Various point mutations in the transthyretin gene induce the hereditary form of the disease. For finding new cases of ATTR amyloidosis and proper screening, the establishment of a multidisciplinary team and a Centre of Excellence (CoE) is essential. CoE provides regular education and training for better diagnosis and treatment. In the current review, we focus on the importance of having a multidisciplinary team and CoE, the screening strategy for ATTR amyloidosis in Bulgaria, and assessments performed when a patient is first suspected of having this rare disease.

Characterization of population genetic structure of hereditary transthyretin amyloidosis in Bulgaria.

The hereditary transthyretin amyloidosis (ATTRv amyloidosis) is an autosomal dominant genetic disease characterized by amyloid formation in different tissues due to pathogenic variants in the TTR gene. Great heterogeneity in the penetrance and manifestation of ATTRv amyloidosis is observed. In Bulgaria, the most common TTR pathogenic variant is Glu89Gln. Other TTR pathogenic variants are also found - Val30Met, Ser77Phe, Gly47Glu and Ser52Pro. There is a proven founder effect for the Glu89Gln variant, thus the aim of the present study is to investigate the founder effect for the other TTR pathogenic variants in Bulgaria. Haplotype analysis was performed by using microsatellite markers close to the TTR gene. DNA samples from ATTRv amyloidosis patients and their healthy relatives were analyzed. Theoretical haplotype reconstruction was done with Arlequin v.3.01 software. The age of the most recent common ancestor (hypothetical founder) for the studied variants was calculated with the DMLE 2.2 software. In addition, DBS screening among 100 Roma newborns was done for the Gly47Glu TTR variant via direct Sanger sequencing. The reconstructed haplotypes of the patients were compared to their healthy relatives and to a control group of 40 healthy individuals. The results showed a possible founder effect for each of the studied variants. The Val30Met haplotype was compared to published haplotype data for this variant and no similarity was found. The result from the DBS screening showed no pathogenic TTR variants in exon 2 of the gene, so we considered the presence of the Gly47Glu variant in our population a sporadic event. With this study, we succeeded to gain a more complete picture of the population genetics of ATTRv amyloidosis in Bulgaria and made another step towards a more detailed understanding of the disease epidemiology.

MYH7-related disorders in two Bulgarian families: Novel variants in the same region associated with different clinical manifestation and disease penetrance.

Pathogenic variants in MYH7 cause a wide range of cardiac and skeletal muscle diseases with childhood or adult onset. These include dilated and/or hypertrophic cardiomyopathy, left ventricular non-compaction cardiomyopathy, congenital myopathies with multi-minicores and myofiber type disproportion, myosin storage myopathy, Laing distal myopathy and others (scapulo-peroneal or limb-girdle muscle forms). Here we report the results from molecular genetic analyses (NGS and Sanger sequencing) of 4 patients in two families with variable neuromuscular phenotypes with or without cardiac involvement. Interestingly, variants in MYH7 gene appeared to be the cause in all the cases. A novel nonsense variant c.5746C>T, p.(Gln1916Ter) was found in the patient in Family 1 who deceased at the age of 2 years 4 months with the clinical diagnosis of dilated cardiomyopathy, whose father died before the age of 40 years, due to cardiac failure with clinical diagnosis of suspected limb-girdle muscular dystrophy. A splice acceptor variant c.5560-2A>C in MYH7 was detected in the second proband and her sister, with late onset distal myopathy without cardiac involvement. These different phenotypes (muscular involvement with severe cardiomyopathy and pure late onset neuromuscular phenotype without heart involvement) may result from novel MYH7 variants, which most probably impact the LMM (light meromyosin) domain's function of the mature protein.

Human cytomegalovirus DNA detection in a recurrent glioblastoma multiforme tumour, but not in whole blood: a case report and discussion about the HCMV latency and therapy perspectives.

In the current study, a 58-year-old male patient presented with recurrent glioblastoma multiforme (GBM). The patient underwent surgical resection, 4 months earlier, followed by radiotherapy and chemotherapy. During the second surgical intervention, tumour tissue and whole blood were sampled and analysed for human cytomegalovirus (HCMV) DNA, immediate early (IE) mRNA and pp65 mRNA. HCMV DNA was detected only in the recurrent tumour tissue but not in the whole blood. Neither IE mRNA nor pp65 mRNA was expressed. Our result suggests HCMV latency in the brain tumour with detectable level of viral DNA. More data are needed to understand the HCMV infection chronology in the brain tumours but our data could be important for further studies of HCMV antigens on the tumour surface and anti-GBM therapy.

Cardiac involvement, morbidity and mortality in hereditary transthyretin amyloidosis because of p.Glu89Gln mutation.

BACKGROUND: Hereditary transthyretin amyloidosis is a systemic infiltrative disease, caused by a mutation in the transthyretin gene. p.Glu89Gln is the most common mutation in the Balkan countries. METHODS: We evaluated the clinical manifestations, cardiac involvement, morbidity and mortality in 78 patients with p.Glu89Gln mutation, verified through a DNA analysis. Clinical assessment, electrocardiogram and echocardiography were performed at the time of diagnosis. The patients have been followed for 30 months. RESULTS: All included patients were Caucasian, 39 (50%) - men, with median age at diagnosis of 56 years (42-73), median age at onset -- 53 years (35-69), starting significantly earlier in men (4.36, P = 0.004). Cardiac and neurological involvement was found in 74 (95%) patients. Pathological ECG was present in 65 (84%) patients, infarct pattern in 43 (56%), low voltage in 24 (31%). Echocardiography revealed an infiltrative cardiomyopathy with restrictive filling in 31 (40%) and ejection fraction less than 50% in 20 (27%) patients. Twenty-two patients (28%) died: 14 (64%) because of advanced heart failure, 6 (27%) died suddenly, 2 (9%) from an ischemic stroke. The median age at death was 58.5 years (52-72). No statistically significant sex difference in survival was observed; a significant difference in survival was found for the New York Heart Association class, familial amyloidotic polyneuropathy stage, ejection fraction, filling pattern and tafamidis treatment. CONCLUSION: Cardiac involvement is common and has significant prognostic implications in the evaluated patients with p.Glu89Gln mutation. Heart failure and rhythm disturbances are the main causes of death. An earlier identification of the disease is crucial to improve prognosis.

BACH2 drives quiescence and maintenance of resting Treg cells to promote homeostasis and cancer immunosuppression.

Regulatory T (Treg) cell populations are composed of functionally quiescent resting Treg (rTreg) cells which differentiate into activated Treg (aTreg) cells upon antigen stimulation. How rTreg cells remain quiescent despite chronic exposure to cognate self- and foreign antigens is unclear. The transcription factor BACH2 is critical for early Treg lineage specification, but its function following lineage commitment is unresolved. Here, we show that BACH2 is repurposed following Treg lineage commitment and promotes the quiescence and long-term maintenance of rTreg cells. Bach2 is highly expressed in rTreg cells but is down-regulated in aTreg cells and during inflammation. In rTreg cells, BACH2 binds to enhancers of genes involved in aTreg differentiation and represses their TCR-driven induction by competing with AP-1 factors for DNA binding. This function promotes rTreg cell quiescence and long-term maintenance and is required for immune homeostasis and durable immunosuppression in cancer. Thus, BACH2 supports a "division of labor" between quiescent rTreg cells and their activated progeny in Treg maintenance and function, respectively.

Transthyretin amyloidosis: Testing strategies and model for center of excellence support.

Appropriate testing strategies and strict model for Center Of Excellence (CoE) support are essential for the correct diagnosis, follow-up strategy and treatment plan for transthyretin (ATTR) amyloidosis. CoE is defined as a programme within a healthcare institution established to provide an exceptionally high concentration of expertise and related resources centred on a particular area of medicine, delivering associated care in a comprehensive, interdisciplinary fashion to afford the best patient outcome. Ideally, CoEs provide regular education and training for healthcare professionals and share knowledge and learning with other CoEs and specialists to ensure the highest standards of care. CoEs and testing strategies are of significant value to those with rare diseases and their families, as there is naturally low awareness among healthcare professionals, a phenomenon that potentially delays diagnosis and treatment. In this review, we focus on the importance of performing the most appropriate testing strategies for ATTR amyloidosis and establishing a CoE for this rare disease. We highlight our experience in establishing a CoE in Sofia, Bulgaria and define the fundamental steps needed to successfully launch a programme.

Fecal calprotectin levels are elevated in transthyretin amyloidosis patients with gastrointestinal manifestations.

Transthyretin amyloid (ATTR) amyloidosis is a rare systemic disorder characterized by amyloid deposits formed by misfolded monomers of the transthyretin. Gastrointestinal (GI) manifestations are common in ATTR amyloidosis; however, their pathogenesis is not fully elucidated. In the present study, we aim to evaluate the diagnostic role of fecal calprotectin (FC) in ATTR amyloidosis patients with GI manifestations.We recruited 21 consecutive ATTR amyloidosis patients and 42 sex and age-matched healthy controls. The presentation of GI symptoms and the severity of peripheral neuropathy were evaluated. Colonoscopy and FC assessment were performed in all subjects.Mean levels of FC in ATTR amyloidosis patients (184 µg/g [30-430]) were significantly higher thаn those of controls (40 µg/g [30-70]), P < .001. Receiver operating characteristic curve analysis indicated a FC cut-off level of 71 µg/g, which differentiates ATTR amyloidosis with GI manifestations from healthy subjects with 91% sensitivity, 100% specificity, 100% positive predictive value, 95% negative predictive value and 97% overall accuracy. FC values were significantly associated with the presence of neutrophilic granulocytic infiltration in the colonic mucosa (P = .002), with the presence of amyloid deposits in rectal mucosa (P = .007) and the presence of diarrhea (P = .046).FC levels are elevated in patients with ATTR amyloidosis with GI manifestations, which suggests an inflammatory component in the pathogenesis of the disease. The presence of elevated FC concentrations could help gastroenterologists to include ATTR amyloidosis in their diagnostic work-up.

Gastrointestinal Manifestations in Hereditary Transthyretin Amyloidosis associated with Glu89Gln Mutation.

AIMS: In the current study we aimed to explore the prevalence of gastrointestinal (GI) manifestations in hereditary transthyretin amyloid (hATTR) amyloidosis associated with Glu89Gln mutation. METHODS: We recruited 78 patients with hATTR amyloidosis associated with Glu89Gln mutation. The diagnosis of hATTR was defined by a documented transthyretin mutation through DNA analysis. Symptoms were recorded as present or absent at the time of enrollment into the study. The gastrointestinal (GI) symptoms checklist included the following items: early satiety, nausea, vomiting, constipation, alternating diarrhea/ constipation, diarrhea, fecal incontinence and unintentional weight loss. RESULTS: Forty-two patients (53.8%) reported at least one GI symptom or sign. Diarrhea was the most frequently reported (30.8%), followed by unintentional weight loss (28.2%) and nausea (21.8%). Fecal incontinence (3.8%) was the least common one. No significant gender related difference in overall GI symptom prevalence was found (females 52.16%, males 55%, p = 0.834). Type of disease onset was not related to GI prevalence (earlyonset 50%, late-onset 55.6%, p=0.650). After dividing the patients into groups with a disease duration of <5 years, 5-10 years and >10 years, respectively, the prevalence of GI symptoms was found to be significantly higher in later stages (26.3% vs. 55.0% vs. 78.9%, p = 0.005; OR 2.450, 95% CI 1.084-5.538). Gastrointestinal manifestations had no impact on survival (p=0.193). CONCLUSIONS: Gastrointestinal manifestations are very common in hATTR patients with Glu89Gln mutation and increase with disease duration. They are not associated with gender and onset of the disease and have no impact on patient survival. These results highlight the importance of a thorough evaluation of the GI function in patients with ATTR amyloidosis and should stimulate further studies on the phenotypic differences related to genotype and geographic origin.

Transthyretin Amyloidosis with Gastrointestinal Manifestation: a Case Report.

Transthyretin amyloidosis (ATTR) is a rare, progressive, life-threatening, hereditary disorder caused by mutations in the transthyretin gene. Due to the phenotypic heterogeneity, ATTR is difficult to recognize and it is often diagnosed very late. In ATTR gastrointestinal (GI) disorders play an important role in the patients' morbidity and mortality. In some cases, GI symptoms are present even before the onset of the peripheral polyneuropathy. However, the complaints are various and it is really difficult to differentiate them from other GI disorders. We present a 61-year old male referred for diarrhea, unintentional weight loss and early satiety. He had hypotension after longstanding hypertension, numbness and tingling in the feet. We considered a broad differential diagnosis spectrum of chronic diarrhea syndrome and performed numerous laboratory, biochemical, imaging, endoscopic, histological and genetic tests. Transthyretin amyloidosis with a Glu89Gln mutation was diagnosed. Transthyretin amyloidosis is frequently misdiagnosed, representing a diagnostic challenge in GI practice. The presence of certain clinical combinations could help gastroenterologists to include ATTR in their diagnostic work-up.

Founder effect of the Glu89Gln TTR mutation in the Bulgarian population.

Hereditary transthyretin amyloidosis is an autosomal dominant genetic disorder caused by missense mutations in the TTR gene resulting in amyloid formation of the transthyretin protein. Depending on the system affection, the manifestations may be different and high heterogeneity in the penetrance is observed. An endemic region in Bulgaria exists where the TTR mutation Glu89Gln is found with high frequency. This is a rare mutation and was probably introduced in the population by a common ancestor. This phenomenon, called "founder effect" was proved in carrier families by haplotype analysis of microsatellite markers showing linkage disequilibrium. Allele frequencies were analyzed and haplotype reconstruction was done with Arlequin v.3.01 software. The common ancestry of the carriers was demonstrated using additional data for their genealogies and microsatellite data from a control group of non-affected individuals. The results show that the mutation Glu89Gln is linked to one haplotype, called "hypothetical founder haplotype" which was compared to published haplotype data from other European patients and no similarity was found. Further population genetics studies of carriers of the Glu89Gln mutation from other endemic regions are required in order to clarify the geographical distribution of the mutation.

Monoallelic expression of the TTR gene as a contributor to the age at onset and penetrance of TTR-related amyloidosis.

TTR-related amyloidosis (ATTR) is manifested in two allelic forms: familial amyloid polyneuropathy (TTR-FAP) and cardiomyopathy (TTR-FAC), both caused by mutations in the TTR gene. The most prevalent mutation in Bulgaria is p.Glu89Gln. Markedly different age at onset and disease penetrance is noticed in Bulgarian p.Glu89Gln cases even in a single family or between genetically identical twins. The present study aimed to evaluate the transcription profile of the TTR gene in order to better understand the difference in disease onset and penetrance. Six p.Glu89Gln positive families were selected from our registry, based on intrafamilial differences in disease onset and penetrance. Plasma and urine specimens were collected from 13 patients and subjected to transcription analysis. Both mutant and wild type transcripts were visualized in a mixed transcription profile, which is the traditional model of autosomal gene expression. The results from a relative quantification of the mutant versus wild type transcript showed presence of the mutant transcript between 0.14 and 1.14 times against the wild type. In addition, monoallelic expression signature was also detected. Based on our results we propose a model of natural selection, which includes age-related allele exclusion or suppression: predominant expression of a wild type (at an early age) and mutant (at the process of ageing) alleles. The intrafamilial differences in disease onset and penetrance need to be considered in genetic counselling and in follow-up of mutation carriers.

First case of Roma ethnic origin with Andermann syndrome: A novel frameshift mutation in exon 20 of SLC12A6 gene.

Andermann syndrome (AS) is caused by mutation of SLC12A6 gene. It comprises severe progressive sensory and motor neuropathy with early onset, varying degree of agenesis of corpus callosum (ACC) and mental retardation. AS occurs occasionally among population outside the northeastern Quebec-Saguenay-Lac- St-Jean and Charlevoix regions, inhabited by French Canadians. None of the described patients were of Roma ethnic origin. We present an 8-month-old infant of Roma ethnic origin with AS, caused by a novel frame shift mutation c.2604delT,p.(Asp868GlufsTer11) in exon 20 of SLC12A6 gene. Our case presented with several atypical findings: clinical presentation resembling "spinal muscular atrophy plus" syndrome; tongue fasciculations, which are not reported in the literature; early contractures of the wrists; normal motor action potentials and preserved sensory action potentials. Our patient is the first of Roma origin from nonconsanguineous parents, which suggests that this mutation might be widespread in the Roma population, although screening for this mutation in 140 alleles from Roma individuals originating from the same geographic region did not reveal further carriers, implying the mutation is rare. We recommend that Roma patients presenting with the clinical phenotype of AS should be tested for this mutation primarily.

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.