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.

Age and Gender Trends in the Prevalence of Markers for Hepatitis E Virus Exposure in the Heterogeneous Bulgarian Population.

The prevalence of hepatitis E virus (HEV) in the Bulgarian population remains underestimated. The aim of the present study was to evaluate age and gender trends in HEV prevalence in the heterogeneous Bulgarian population. Stored serum samples from blood donors and different patient sub-populations-kidney recipients (KR), patients with Guillain-Barre syndrome (GBS), Lyme disease (LD), patients with liver involvement and a clinical diagnosis other than viral hepatitis A and E (non-AE), hemodialysis (HD) and HIV-positive patients (HIV)-were retrospectively investigated for markers of past and recent/ongoing HEV infection. The estimated overall seroprevalence of past infection was 10.6%, ranging from 5.9% to 24.5% for the sub-populations evaluated, while the seroprevalence of recent/ongoing HEV infection was 7.5%, ranging from 2.1% to 20.4%. The analysis of the individual sub-populations showed a different prevalence with respect to sex. In regard to age, the cohort effect was preserved, as a multimodal pattern was observed only for the GBS sub-population. Molecular analysis revealed HEV 3f and 3e. The type of the population is one of the main factors on which the anti-HEV prevalence depends, highlighting the need for the development of guidelines related to the detection and diagnosis of HEV infection with regard to specific patient populations.

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.

Varicella zoster virus infection in neurological patients in Bulgaria.

The clinical manifestations of neurological complications associated with varicella zoster virus (VZV) are non-specific and indistinguishable from those of other viral infections. Therefore, the definite diagnosis requires evidence of VZV infection in cerebrospinal fluid (CSF). The aim of this study was to determine the frequency of VZV DNA detection in CSF of patients with neurological diseases in order to obtain information concerning involvement of VZV infection in neuropathology in the country. This study is a retrospective survey of test results obtained from January 2015 to October 2019. During this period, 411 CSF specimens were tested for the presence of VZV DNA by nested PCR. Fisher's exact test was used to test for statistically significant difference in the frequency of VZV DNA positivity of CSF specimens from different groups. Of all 411 tested CSF samples, 11.2% were positive for VZV DNA. The highest VZV prevalence was detected in CFS from patients with meningitis-18.2%, followed by patients with cranial neuritis (15.4%), encephalitis (12.2%), Guillain-Barré syndrome (11.1%), myelitis (10%), and with other neurological syndromes (8.2%). The difference of VZV prevalence in CSF of patients according to the gender and age was not statistically significant. Our results indicated that VZV is a frequent causative agent of neurological diseases, suggesting an important role of VZV infection for neuropathology in the country. Therefore, efforts for wider application of VZV identification in CSF to facilitate faster onset of antiviral treatment and further strategies concerning varicella zoster virus vaccines in the country are needed.

Traditional practices and perceptions of epilepsy among people in Roma communities in Bulgaria.

PURPOSE: We attempted to identify cultural aspects of epilepsy among the Roma community in Bulgaria by elucidating cultural beliefs, traditional treatments, and potential markers of stigma. METHODS: We established representative discussion groups among five distinct Roma subgroups (Lom, Kalderas, Thracian Tinsmiths (Tinkers), Kyustendil Xoroxane and Kopanari) from different Bulgarian regions. Data about local beliefs and treatment strategies were gathered. RESULTS: Most people were familiar with convulsions but non-convulsive focal seizures were seen not as epileptic but mainly as a "mental problem". Beliefs about putative etiologies for epilepsy were not uniform as some considered environmental and external factors such as high environmental temperatures, electric shocks, loud music, and fever as causes of seizures while others listed bad experiences, stress, trauma, and fear as possible causes. Epilepsy was seen by some as a divine punishment or resulting from black magic. Most considered epilepsy shameful and an obstacle to children attending school. Despite local differences, there was a uniform belief that epilepsy is incurable by Western medicine and people usually resort to traditional healers. A variety of rituals performed by local healers to treat epilepsy were described. DISCUSSION: Misconceptions about epilepsy may contribute to stigmatization in this population; this may in turn contribute to a high treatment gap in this group. As a result, the majority of Roma children with epilepsy are likely to leave school early, are greatly limited in their choice of spouse (particularly girls), and marriages often occur between people with epilepsy or those with a family history of epilepsy.

Chromosomal microarray analysis of Bulgarian patients with epilepsy and intellectual disability.

High resolution chromosomal microarray analysis (CMA) has facilitated the identification of small chromosomal rearrangements throughout the genome, associated with various neurodevelopmental phenotypes, including ID/DD. Recently, it became evident that intellectual disability (ID)/developmental delay (DD) can occur with associated co-morbidities like epileptic seizures, autism and additional congenital anomalies. These observations require whole genome approach in order to detect the genetic causes of these complex disorders. In this study, we examined 92 patients of Bulgarian origin at age between 1 and 22 years with ID, generalized epilepsy, autistic signs and congenital anomalies. CMA was carried out using SurePrint G3 Human CGH Microarray Kit, 4 × 180 K and SurePrint G3 Unrestricted CGH ISCA v2, 4 × 180 K oligo platforms. Referral indications for selection of the patients were the presence of generalized refractory seizures disorders and co-morbid ID. Clearly pathogenic copy number variations (CNVs) were detected in eight patients (8.7%) from our cohort. Additionally, possibly pathogenic rearrangements of unclear clinical significance were detected in six individuals (6.5%), which make for an overall diagnostic yield of 15.2% among our cohort of patients. We report here the patients with clearly pathogenic CNVs, discuss the potential causality of the possibly pathogenic CNVs and make genotype - phenotype correlations. One novel possibly pathogenic heterozygous deletion in 15q22.31 region was detected in a case with ID/DD. Additionally, whole APBA2 gene duplication in 15q13.1 was found in three generations of a family with epilepsy, ID and psychiatric abnormalities. The results from this study allow us to define the genetic diagnosis in a subset of Bulgarian patients and improve the genetic counseling of the affected families. To our knowledge, this is the first aCGH evaluation of a Bulgarian cohort of children with epilepsy and ID so far.

Clinical Spectrum and Genetic Variability in Bulgarian Patients with Niemann-Pick Disease Type C.

BACKGROUND: Niemann-Pick disease type C (NP-C) is a rare autosomal-recessive lysosomal storage disorder caused by mutations in either the NPC1 (in 95% of cases) or the NPC2 gene. METHODS: In a prospective, observational cohort study, all Bulgarian patients diagnosed with NP-C to date (since 2010) underwent detailed neurological examination and neuro-ophthalmological, neuropsychological and psychiatric evaluations, as well as brain MRI, abdominal ultrasound and hearing tests. Plasma chitotriosidase was also measured, when possible. RESULTS: The Bulgarian national NP-C cohort comprised 11 patients who were diagnosed based on molecular genetic analysis (n = 9) and/or filipin staining of skin fibroblasts (n = 3). The mean age at onset was 14.4 (SD 8.3). Diagnoses were achieved 1-23 years after initial clinical presentation. All patients who underwent genetic mutation analysis were compound heterozygotes: a total of 12 NPC1 mutations were recorded, 5 of which were novel. Two patients had late-infantile onset, 4 had juvenile onset, and the remaining 5 had the adult-onset form of NP-C. Initial symptoms were neurological in 9 patients, visceral in one, and predominantly psychiatric in another. Vertical gaze palsy was present in all patients. Dysarthria, pyramidal involvement, cognitive impairment, and organomegaly with varied severity were observed in 10 of them. Ataxia was present in 9 and dystonia in 7. Four patients had epileptic seizures, and gelastic cataplexy was reported in 5. Brain MRI revealed hyperintense white matter lesions in 5 patients and cortical and/or cerebellar atrophy in 4. CONCLUSIONS: This Bulgarian NP-C cohort showed wide variability in terms of NPC1 mutations and predominant forms of neurological involvement. Diagnosing NP-C is challenging, and it was often delayed in this cohort due to the heterogeneity of patients' clinical signs and symptoms.

A novel GEFS+ locus on 12p13.33 in a large Roma family.

We report a clinical and genetic follow-up study of a large consanguineous family from an endogamous Roma/Gypsy sub-isolate, where previous analyses have been inconclusive. Detailed clinical information was collected through extensive field work, repeat interviews and electrophysiological and neuroimaging investigations on 18 affected subjects. The phenotype is compatible with GEFS+, with some unusual features, e.g. GTCS persisting into late adult life and high frequency of focal epilepsy. Updated genealogical information, a dense SNP genome scan and linkage analysis identified a novel GEFS+ locus on 12p13.33, where 13 affected individuals from two branches of the kindred shared an identical haplotype. This haplotype was not found in the 3rd branch or in the remaining 21 Roma epilepsy families in our collection. Genetic heterogeneity and evidence of bilineality were found despite the inbreeding and endogamous nature of the family and population of origin. These data add to the growing evidence of lack of founder effect and significant genetic heterogeneity in epilepsy in the Roma/Gypsy population. Sequencing of the coding regions of three genes linked to neurotransmitter transport and release, SLC6A12, SLC6A13 and ERC1, on 12p did not identify a causative mutation.

Focal epilepsy of probable temporal lobe origin in a Gypsy family showing linkage to a novel locus on 7p21.3.

We aimed to characterise the phenotype and perform genetic studies in a family of Roma/Gypsy ethnicity, affected by epilepsy. The mean age at onset of epilepsy was 9 years and seizures persisted into adulthood. Antecedent febrile convulsions were rare. Seizure semiology and EEG findings suggested mesial temporal lobe origin with no evidence of hippocampal sclerosis. Seizures frequently generalised. Family structure suggested autosomal-dominant inheritance with incomplete penetrance. Linkage analysis identified a single novel locus on 7p21.3, corresponding to the expected maximum in the family. Previously reported temporal lobe epilepsy (TLE) loci were definitely excluded. The minimal shared haplotype of 2.4cM (1.3Mb) was not observed in other affected families or controls from the same population. Three brain-expressed validated genes in the critical region represent potential candidates. We have identified an epilepsy syndrome with temporal lobe seizures commonly evolving to generalised convulsions. Linkage to 7p21.3 adds up to a total of five currently known FTLE loci.

Mosaicism of a missense SCN1A mutation and Dravet syndrome in a Roma/Gypsy family.

SCN1A mutations account for a large proportion of Dravet syndrome patients, and are reported in other cases of epilepsy, such as some families with genetic epilepsy with febrile seizures plus (GEFS+). While most Dravet syndrome cases are caused by de novo mutations, 5% inherit a mutation from a mildly affected or symptom-free parent. Parental mosaicism has been identified, with documented cases involving truncating mutations or gene rearrangements. We describe a Roma/Gypsy family, where a missense mutation in SCN1A, p.D194N, is transmitted from a mosaic GEFS+ father to a child with Dravet syndrome. Mosaicism may be more common than assumed and should be considered regardless of the nature of the mutation.