Variation of tafamidis plasma levels during the treatment of TTR amyloidosis patients with Glu89Gln mutation.

PURPOSE: The transthyretin kinetic stabilizer tafamidis, used as a first-line therapy of amyloidosis patients, binds selectively to the transthyretin protein structure and thus prevents its dissociation. The limited information regarding tafamidis application in Glu89Gln amyloidosis patients imposed our research team to determine and evaluate its individual mean plasma levels and their biological variation. METHODS: The present cohort study investigated Bulgarian amyloidosis patients, grouped by gender, age, and therapy duration. A total of sixty patients aged 40-75 years and therapy duration up to 9 years were included. A precise and accurate high-performance liquid chromatography method with ultraviolet detection was used for plasma concentration measurement. RESULTS: Mean plasma concentrations were 5.13 ± 2.64 µmol/L and showed low intra-individual (18.50%) and high inter-individual variability (51.43%). No significant difference was observed between tafamidis plasma levels and therapy duration with p = 0.5941 (p < 0.05 considered significant), but a significant positive correlation was found between plasma concentration, gender, and age with obtained results about p-value 0.0001 and 0.0235, respectively. CONCLUSION: The summary results of the study showed differences that may be based on some specific clinical features of the Glu89Gln mutation.

POGLUT1 biallelic mutations cause myopathy with reduced satellite cells, α-dystroglycan hypoglycosylation and a distinctive radiological pattern.

Protein O-glucosyltransferase 1 (POGLUT1) activity is critical for the Notch signaling pathway, being one of the main enzymes responsible for the glycosylation of the extracellular domain of Notch receptors. A biallelic mutation in the POGLUT1 gene has been reported in one family as the cause of an adult-onset limb-girdle muscular dystrophy (LGMD R21; OMIM# 617232). As the result of a collaborative international effort, we have identified the first cohort of 15 patients with LGMD R21, from nine unrelated families coming from different countries, providing a reliable phenotype-genotype and mechanistic insight. Patients carrying novel mutations in POGLUT1 all displayed a clinical picture of limb-girdle muscle weakness. However, the age at onset was broadened from adult to congenital and infantile onset. Moreover, we now report that the unique muscle imaging pattern of "inside-to-outside" fatty degeneration observed in the original cases is indeed a defining feature of POGLUT1 muscular dystrophy. Experiments on muscle biopsies from patients revealed a remarkable and consistent decrease in the level of the NOTCH1 intracellular domain, reduction of the pool of satellite cells (SC), and evidence of α-dystroglycan hypoglycosylation. In vitro biochemical and cell-based assays suggested a pathogenic role of the novel POGLUT1 mutations, leading to reduced enzymatic activity and/or protein stability. The association between the POGLUT1 variants and the muscular phenotype was established by in vivo experiments analyzing the indirect flight muscle development in transgenic Drosophila, showing that the human POGLUT1 mutations reduced its myogenic activity. In line with the well-known role of the Notch pathway in the homeostasis of SC and muscle regeneration, SC-derived myoblasts from patients' muscle samples showed decreased proliferation and facilitated differentiation. Together, these observations suggest that alterations in SC biology caused by reduced Notch1 signaling result in muscular dystrophy in LGMD R21 patients, likely with additional contribution from α-dystroglycan hypoglycosylation. This study settles the muscular clinical phenotype linked to POGLUT1 mutations and establishes the pathogenic mechanism underlying this muscle disorder. The description of a specific imaging pattern of fatty degeneration and muscle pathology with a decrease of α-dystroglycan glycosylation provides excellent tools which will help diagnose and follow up LGMD R21 patients.

Association between loss of dp140 and cognitive impairment in duchenne and becker dystrophies.

The presence of variable degrees of non progressive cognitive impairment is recognized as a clinical feature of patients with Duchenne and Becker muscular dystrophies (DMD and BMD), but its pathogenesis still remains a matter of debate. A number of findings have proved that rearrangements located in the second part of the dystrophin ( DMD ) gene seem to be preferentially associated with cognitive impairment. Dp140 is a distal dystrophin isoform, mainly expressed during fetal brain development, whose role for neuropsychological functioning was suggested. The aims of the current study were to explore the possible association between cognitive impairment and DNA mutations affecting the regulatory regions of Dp140, as well as to compare the neuropsychological functioning of patients affected with DMD and Intermediate muscular dystrophy (IMD) with those affected by Becker muscular dystrophy (BMD). Fiftythree patients genetically diagnosed with DMD, IMD and BMD, subdivided according to sites of mutations along the DMD gene, underwent a neuropsychological assessment, evaluating their general cognitive abilities, verbal memory, attention and executive functions. Twenty patients with mutations, terminating in exon 44 or starting at exon 45 were tested by polymerase chain reaction (PCR) amplification of microsatellites STR44, SK12, SK21 and P20 DXS269, in order to evaluate the integrity of the Dp140 promoter region. According to our statistical results, there was not a significant difference in terms of general intelligence between the allelic forms of the disease, a higher frequency of mental retardation was observed in DMD patients. The patients with BMD had better results on tests, measuring long-term verbal learning memory and executive functions. We found that patients lacking Dp140 performed more poorly on all neuropsychological tests compared to those with preserved Dp140. Overall, our findings suggest that the loss of Dp140 is associated with a higher risk of intellectual impairment among patients with dystrophinopathies and highlights the possible role of this distal isoform in normal cognitive development.

Real-world effectiveness of fingolimod in patients with multiple sclerosis in Bulgaria. / Eficacia en la vida real del fingolimod en pacientes con esclerosis múltiple en Bulgaria.

INTRODUCTION: Fingolimod, a sphingosine-1-phosphate receptor agonist used for the treatment of multiple sclerosis (MS). Our goal was to assess the impact of fingolimod on quality of life in patients with relapsing-remitting multiple sclerosis (RRMS) after 2 years of treatment in this real-world study. PATIENTS AND METHODS: This was a 2-year, prospective, observational study conducted in Bulgaria in RRMS patients treated with fingolimod. Quality of life was assessed using the Bulgarian-language version of the MSQoL-54 scale. The primary endpoint was the change from baseline in the MSQoL-54 score after 2 years of treatment. Secondary endpoints included the change from baseline in the MSQoL-54 score after one year of treatment, furthermore the assessment of depression level using the Hamilton D-17 score. RESULTS: A total of 87 eligible patients were included in the study with a mean age of 38.7 ± 8.45 years. The median Expanded Disability Status Scale (EDSS) score was 3.5 points. We found statistically significant improvement in 10 subscales at month 12 and in seven subscales at month 24. The mental health composite score increased from 64.0 ± 16.69 points to 67.5 ± 15.94 points at month 24 (p = 0.012). The physical health composite score increased from 61.7 ± 17.61 to 66.3 ± 16.70 (p = 0.001). Depression level measured by the HAM-D17 decreased significantly by month 12 and month 24. The EDSS score decreased or remained stable in more than half of the patients (61.6%). We detected better quality of life in patients with a lower EDSS score. CONCLUSIONS: Quality of life scores and the depression level are improved in RRMS patients treated with fingolimod over 2 years in real-life setting.

TITLE: Eficacia en la vida real del fingolimod en pacientes con esclerosis múltiple en Bulgaria.Introducción. El fingolimod es un agonista del receptor de esfingosina-1-fosfato utilizado para el tratamiento de la esclerosis múltiple (EM). Nuestro objetivo era evaluar los resultados del fingolimod en la calidad de vida de los pacientes con EM recurrente-remitente tras dos años de tratamiento en este estudio de la vida real. Pacientes y métodos. Se trata de un estudio observacional prospectivo de dos años de duración realizado en Bulgaria en pacientes con EM recurrente-remitente tratados con fingolimod. Se evaluó la calidad de vida mediante la versión en búlgaro de la escala Multiple Sclerosis Quality of Life-54 (MSQoL-54). El criterio de valoración principal fue el cambio respecto al valor inicial en la puntuación en la MSQoL-54 tras dos años de tratamiento. Los criterios de valoración secundarios fueron el cambio respecto al valor inicial en la puntuación en la MSQoL-54 tras un año de tratamiento, además de la evaluación del nivel de depresión mediante la puntuación de la escala de puntuación de la depresión de Hamilton (HAM-D17). Resultados. En el estudio se incluyó a 87 pacientes elegibles con una edad media de 38,7 ± 8,45 años. La mediana de la puntuación en la Expanded Disability Status Scale (EDSS) fue de 3,5 puntos. Se halló una mejora estadísticamente significativa en 10 subescalas en el mes 12 y en siete subescalas en el mes 24. La puntuación combinada de salud mental aumentó de 64 ± 16,69 puntos a 67,5 ± 15,94 puntos en el mes 24 (p = 0,012). La puntuación combinada de salud física aumentó de 61,7 ± 17,61 a 66,3 ± 16,7 (p = 0,001). El nivel de depresión medido por la HAM-D17 disminuyó considerablemente en el mes 12 y en el mes 24. La puntuación de la EDSS disminuyó o se mantuvo estable en más de la mitad de los pacientes (61,6%). Detectamos una mejor calidad de vida en los pacientes con una puntuación más baja en la EDSS. Conclusiones. Las puntuaciones de calidad de vida y el nivel de depresión mejoraron en los pacientes con EM recurrente-remitente tratados con fingolimod durante dos años en un entorno real.

Genetic heterogeneity and minor CYP1B1 involvement in the molecular basis of primary congenital glaucoma in Gypsies.

Primary congenital glaucoma (PCG) is a genetically heterogeneous disorder of autosomal recessive inheritance, with mutations in the cytochrome P450 1B1 (CYP1B1) gene detected in an average of approximately 50% of cases worldwide. The Roma/Gypsies are considered to be a rare example of a single founder CYP1B1 mutation, E387K (identified in the Slovak Roma), accounting for 100% of disease alleles. Contrary to this concept, unusual genetic heterogeneity was revealed in this study of 21 Gypsy PCG patients from Bulgaria and 715 controls from the general Gypsy population. In our small sample of affected subjects, we identified five different CYP1B1 mutations - four known (E229K, R368H, E387K and R390C) and one novel and potentially pathogenic (F445I), which together accounted for approximately 30% of disease alleles. E387K was rare in both the patient and the control group, indicating that its high frequency in the Slovak Roma is the product of local founder effect not representative of the overall molecular pattern of PCG in the Gypsy population. Data on other Mendelian disorders and on the population genetics of the Gypsies suggest that a true founder mutation is likely to exist and has remained undetected. Our analysis of another candidate gene, MYOC, and the GLC3B and GLC3C loci did not provide support for their involvement. The molecular basis of PCG in the Gypsies is thus unresolved, and diagnostic analyses should be extended beyond the E387K mutation.

Patterns of inter- and intra-group genetic diversity in the Vlax Roma as revealed by Y chromosome and mitochondrial DNA lineages.

Previous genetic studies, supported by linguistic and historical data, suggest that the European Roma, comprising a large number of socially divergent endogamous groups, may be a complex conglomerate of founder populations. The boundaries and characteristics of such founder populations and their relationship to the currently existing social stratification of the Roma have not been investigated. This study is an attempt to address the issues of common vs independent origins and the history of population fissioning in three Romani groups that are well defined and strictly endogamous relative to each other. According to linguistic classifications, these groups belong to the Vlax Roma, who account for a large proportion of the European Romani population. The analysis of mtDNA sequence variation has shown that a large proportion of maternal lineages are common to the three groups. The study of a set of Y chromosome markers of different mutability has revealed that over 70% of males belong to a single lineage that appears unique to the Roma and presents with closely related microsatellite haplotypes and MSY1 codes. The study unambiguously points to the common origins of the three Vlax groups and the recent nature of the population fissions, and provides preliminary evidence of limited genetic diversity in this young founder population.

Homogeneous phenotype of the gypsy limb-girdle MD with the gamma-sarcoglycan C283Y mutation.

OBJECTIVE: To characterize the clinical phenotype of LGMD2C in gypsies. BACKGROUND: Limb-girdle muscular dystrophy (LGMD) in gypsies of Western Europe is caused by a homozygous C283Y mutation on the same haplotype, suggesting a founder effect. METHODS: We performed clinical, laboratory, and muscle imaging studies of 40 patients. RESULTS: Mean age at onset was 5.3 years. One half of the patients had loss of ambulation by the age of 12; 13% still could walk after age 16. Calf hypertrophy, scapular winging, macroglossia, and lumbar hyperlordosis were common. Girdle, trunk, and proximal limb flexor muscles had earlier and more severe involvement. Cardiomyopathy was not observed. Five patients in the third decade of life required mechanical ventilation. Scoliosis was common in the nonambulatory stage. CONCLUSIONS: LGMD2C in gypsy patients with C283Y mutation presents a rather homogeneous phenotype, characterized by an initial Duchenne-like progressive course followed by a more prolonged survival rate possibly due to the absence of early respiratory impairment and cardiac failure.

Hereditary motor and sensory neuropathy--Lom (HMSNL): refined genetic mapping in Romani (Gypsy) families from several European countries.

Hereditary motor and sensory neuropathy type Lom, initially identified in Roma (Gypsy) families from Bulgaria, has been mapped to 8q24. Further refined mapping of the region has been undertaken on DNA from patients diagnosed across Europe. The refined map consists of 25 microsatellite markers over approximately 3 cM. In this collaborative study we have identified a number of historical recombinations resulting from the spread of the hereditary motor and sensory neuropathy type Lom gene through Europe with the migration and isolation of Gypsy groups. Recombination mapping and the minimal region of homozygosity reduced the original 3 cM hereditary motor and sensory neuropathy type Lom region to a critical interval of about 200 kb.

Distal myopathy with upper limb predominance caused by filamin C haploinsufficiency.

OBJECTIVE: In this study, we investigated the detailed clinical findings and underlying genetic defect in 3 presumably related Bulgarian families displaying dominantly transmitted adult onset distal myopathy with upper limb predominance. METHODS: We performed neurologic, electrophysiologic, radiologic, and histopathologic analyses of 13 patients and 13 at-risk but asymptomatic individuals from 3 generations. Genome-wide parametric linkage analysis was followed by bidirectional sequencing of the filamin C (FLNC) gene. We characterized the identified nonsense mutation at cDNA and protein level. RESULTS: Based on clinical findings, no known myopathy subtype was implicated in our distal myopathy patients. Light microscopic analysis of affected muscle tissue showed no specific hallmarks; however, the electron microscopy revealed changes compatible with myofibrillar myopathy. Linkage studies delineated a 9.76 Mb region on chromosome 7q22.1-q35 containing filamin C (FLNC), a gene previously associated with myofibrillar myopathy. Mutation analysis revealed a novel c.5160delC frameshift deletion in all patients of the 3 families. The mutation results in a premature stop codon (p.Phe1720LeufsX63) that triggers nonsense-mediated mRNA decay. FLNC transcript levels were reduced in muscle and lymphoblast cells from affected subjects and partial loss of FLNC in muscle tissue was confirmed by protein analysis. CONCLUSIONS: The FLNC mutation that we identified is distinct in terms of the associated phenotype, muscle morphology, and underlying molecular mechanism, thus extending the currently recognized clinical and genetic spectrum of filaminopathies. We conclude that filamin C is a dosage-sensitive gene and that FLNC haploinsufficiency can cause a specific type of myopathy in humans.

GNE protein expression and subcellular distribution are unaltered in HIBM.

Mutations in GNE encoding UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) cause hereditary inclusion body myopathy (HIBM). To define the role of GNE mutations in HIBM pathogenesis, GNE protein expression was analyzed. GNE protein is expressed at equal levels in HIBM patients and normal control subjects. Immunofluorescence detection of GNE did not reveal any mislocalization of GNE in skeletal muscle. We conclude that impaired GNE function, not lack of expression, may be the key pathogenic factor in HIBM. For diagnostic purposes, direct genetic analysis of the GNE gene in patients with IBM will remain the mainstay and is not aided by immunohistochemistry or immunoblotting using antibodies against the GNE protein.

Spastin gene mutations in Bulgarian patients with hereditary spastic paraplegia.

Hereditary spastic paraplegia (HSP) is an extremely heterogeneous group of neurodegenerative disorders affecting the longest axons in the central nervous system. The most common genetic form accounting for about 40% of the autosomal-dominant HSP (ADHSP) cases is spastin gene, SPG4. We performed mutation screening of the spastin gene on 36 unrelated HSP patients from three different ethnic groups (Bulgarian, Turks and Gypsies) and found four new mutations and one already reported. The phenotype-genotype correlations in Bulgarian SPG4 patients showed a great difference in the age at disease onset between patients with missense mutations and those harboring deletions and splice-site mutations. Our study is the first to present corroborative clinical data in favor of the general hypothesis that the clinical course of the disease is related to the type of the spastin mutation. The clinical and genealogical findings in Bulgarian SPG4 patients suggest that a positive family history for inheritance as an autosomal-dominant trait is a strong indication for spastin mutation screening.

C283Y gamma-sarcoglycan gene mutation in the Bulgarian Roma (Gypsy) population: prevalence study and carrier screening in a high-risk community.

Limb-girdle muscular dystrophy type 2C (LGMD2C) is caused by mutations in the gamma-sarcoglycan gene where a founder Gypsy mutation C283Y was detected. The Bulgarian Gypsy LGMD2C patients, as the Gypsy patients from other countries, were found to be homozygous for this mutation. Considering the large number of Gypsies in Bulgaria and the high percent of consanguinity and endogamy a raised carrier frequency of the C283Y mutation was expected especially in North-Eastern Bulgaria where most of the patients originate from. Here, we present the precise geographic distribution of the C283Y mutation in the general Roma population from the whole Bulgarian territory by determining the carrier frequency of the mutation in dry blood newborn samples. Our results show that the geographic distribution of this founder mutation and the disease are not geographically restricted only among Gypsies from North-Eastern Bulgaria. We stress upon the regions with detected high carrier and/or disease frequency and upon the results from the performed carrier screening on volunteers in one of these regions. The ongoing carrier-screening programs in isolated Gypsy groups would be of a great benefit for the genetic prophylaxis of the disease. Such regions should be with priority in the Bulgarian healthcare system for performing a carrier-screening program.

Ultrastructural changes in peripheral nerve in hereditary motor and sensory neuropathy-Lom.

Ultrastructural observations have been made on nerve biopsy specimens from five cases of hereditary motor and sensory neuropathy-Lom (HMSNL). A number of features that distinguish it from other hereditary demyelinating neuropathies were identified. Teased fibre studies were not feasible but examination of longitudinal sections by electron microscopy demonstrated demyelination/remyelination. Severe progressive axonal loss was a conspicuous feature. There was no indication of axonal atrophy. Hypertrophic onion bulb changes were present in the younger patients which later regressed, probably secondary to axonal loss. Myelin thickness was generally reduced in relation to axon diameter, indicating hypomyelination, and partial ensheathment of axons by Schwann cells was observed. The Schmidt-Lanterman incisures were atypical in extending for long lengths along the internode. Uncompacted myelin with a periodicity greater than that observed in other neuropathies in which it occurs was a feature, as was the accumulation of pleomorphic material in the adaxonal Schwann cell cytoplasm. An unusual finding was the presence of intra-axonal accumulations of irregularly arranged curvilinear profiles. These resemble those that have been described in experimental vitamin E deficiency. The amount of endoneurial collagen was markedly increased and some endoneurial blood vessels showed a non-specific basal laminal reduplication.

C283Y mutation and other C-terminal nucleotide changes in the gamma-sarcoglycan gene in the Bulgarian Gypsy population.

Sarcoglycanopathies, affecting the dystrophin-associated sarcoglycan (SG) complex, are a heterogeneous group of neuromuscular disorders. A subgroup of these disorders, limb-girdle muscular dystrophy type 2C (LGMD2C) is an autosomal recessive disorder, clinically manifested as an early onset, severe Duchenne-like muscular dystrophy. LGMD2C is caused by mutations in the gamma-SG gene, localized on 13q12. Recently, a number of mutations have been described in that gene, among which C283Y, a "private" Gypsy mutation (eight codons before the 3' end of the gene) is detected. In this article, we report on a single-strand conformation polymorphism (SSCP) method for fast C283Y mutation detection, using direct dry blood spot amplification. The method permits a large number of samples to be easily screened. To check heterozygote carriers of C283Y mutation among Gypsy population in Bulgaria, the SSCP analysis was applied on 400 Gypsy newborns from northeast Bulgaria. Our results show 2.25% of heterozygosity, which means that 1 in 50 Gypsies carries the mutation. Moreover, new SSCP migration patterns were detected that revealed two polymorphisms still unavailable in the literature. One of these changes was 984G-->A, leading to substitution of conserved serine at position 287 with asparagine and the second one is 1049C-->G at the 3' UTR (untranslated region). The present data could help the understanding the role of these sequences for the protein function.

A novel locus for autosomal recessive peripheral neuropathy in the EGR2 region on 10q23.

During our studies of Romany (Gypsy) families with hereditary motor and sensory neuropathy-Lom, we have identified a large kindred with two independently segregating autosomal recessive neuropathies. The novel disorder, named "hereditary motor and sensory neuropathy-Russe" (HMSNR), presented as a severe disabling form of Charcot-Marie-Tooth disease with prominent sensory loss, moderately reduced motor nerve conduction velocity, and a high threshold for electrical nerve stimulation. A genome scan in two branches of the large kindred detected linkage to the 10q22-q23 region containing the early growth response 2 gene (EGR2), a transcription factor with a key role in peripheral nerve myelination. The results of sequence analysis and the detection of an intragenic polymorphism allowed us to exclude EGR2 as the HMSNR gene. Further analysis done using linkage and recombination mapping refined the position of the HMSNR gene to a small interval on 10q23.2, flanked by markers D10S581 and D10S1742, telomeric to EGR2. In this interval, a conserved seven-marker haplotype is shared by all disease chromosomes, suggesting a single founder mutation. The homozygosity region is contained in bacterial-artificial-chromosome contig 1570 of the Sanger Centre physical map and has an estimated physical size of approximately 500 kb.

Peripheral nerve abnormalities in the congenital cataracts facial dysmorphism neuropathy (CCFDN) syndrome.

Observations have been made on the peripheral nerve changes in four patients, ranging in age from 4 to 32 years, with the congenital cataracts facial dysmorphism neuropathy syndrome. Myelinated fibre density was within normal limits. The salient abnormality was diffuse hypomyelination which, in the older patients, was associated with demyelination and then axonal degeneration. These findings could be correlated with the relative preservation of sensory action potential amplitude despite markedly reduced nerve conduction velocity. Unmyelinated axon density was preserved. The morphological observations suggest the operation of a developmental process affecting myelination with a later superimposed degenerative disorder.