EPHA4 Genetic Variant in a Patient with Epilepsy, Ophthalmological Anomalies, and Neurodevelopmental Delay.

We present the findings of a Whole Exome Sequencing in a 2-year-old boy, conceived via In Vitro Fertilization with donor sperm, who suffers from an undiagnosed neurological syndrome. The following heterozygous variant in the EPHA4 gene was identified and classified as likely pathogenic: c.1655_1656, p.(Ser552CysfsTer23). Subsequent segregation analysis showed that the variant was not inherited from the mother and the sperm donor is not accessible for genetic testing. The presented results can further expand upon the genetic variants considered when diagnosing complex neurological syndromes and shows the importance of access to biological samples from donor banks in genetically ambiguous cases.

Metabolic determinants of NAFLD in adults with type 1 diabetes.

AIM: To assess the main metabolic determinants of non-alcoholic fatty liver disease (NAFLD) in adult patients with type 1 diabetes (T1D). METHODS: 115 patients with T1D were divided into 4 groups according to NAFLD grade. NAFLD was diagnosed via transient elastography when CAP > 233 dB/m. Body composition was evaluated by Inbody720, Biospace. Serum lipids, liver enzymes, uric acid, creatinine, hsCRP and HbA1c were evaluated at fasting. RESULTS: The overall prevalence of NAFLD was 47% (n = 54). In the subgroup with BMI > 25 kg/m2 NAFLD prevalence was 66%; and positive family history of type 2 diabetes brought the risk up to 76%. 37% of the lean individuals also had NAFLD. HbA1c > 7% doubled the risk of NAFLD. Waist circumference > 82.5 cm was independently related to NAFLD, accounting for 24% of its variation in females. Accumulation of two and three metabolic syndrome (MetS) components, besides hyperglycemia, increased the risk of NAFLD by 14% (p < 0.0001) and 6% (p = 0.024), respectively. Lean NAFLD correlated with total insulin dose; NAFLD in overweight T1D patients correlated with triglycerides. CONCLUSIONS: NAFLD is highly prevalent in adults with T1D and obesity or other metabolic derangements and might be independently related to poor long-term glycemic control and waist circumference in females.

Novel Mutation in the COL11A1 Gene Causing Marshall-Stickler Syndrome in Three Generations of a Bulgarian Family.

Here we report the first familial case spread through at least three generations, genetically verified cases of Marshall-Stickler syndrome in Bulgaria. The proband, a 2-year-old girl, has craniofacial dysplasia, ocular hypertelorism, small saddle nose with a flat bridge and midface hypoplasia. The pedigree of the proband's family showed that her father has the same clinical manifestations of the disease. In addition, her father presented with a tall, thin stature and mild hearing loss, manifested with aging. The same dysmorphological symptoms were presented by the paternal grandfather. Both patients, the 2-year-old girl and her father, have been diagnosed to carry Marshall-Stickler syndrome. The COL2A1 gene tested negative in the family. Based on the higher percentage of mutations in the COL2A1 gene, we analyzed this gene as the first target in the family. The COL2A1 gene tested negative, and we sequenced the gene further. A novel splice site mutation c.3474+1G>A was found in intron 44. This variant is related to the clinical presentation in the patient and her father. The c.3474+1G>A mutation results in altered splicing affects at the donor splice site of intron 44, which most probably gives a nonfunctional protein. The variant affects the major triple-helical domain that represents a mutation hot-spot for the gene.

Relationship between cardiac autonomic neuropathy and cardio-metabolic risk profile in adults with type 1 diabetes.

AIM: The present study aims to determine the prevalence and association of cardiac autonomic neuropathy (CAN) with some traditional cardio-metabolic risk factors in adults with type 1 diabetes (T1D). MATERIAL AND METHODS: 235 adults with T1D, divided into three groups according to diabetes duration, were recruited in this cross-sectional study from May 2017 till December 2018. Anthropometric parameters and blood pressure were measured. Lipids, liver enzymes, uric acid, creatinine, HbA1c and high sensitive C-reactive protein (hsCRP) were measured at fasting. Albumin/creatinine ratio (ACR) was measured in a first spot urine sample. Body composition was evaluated using bio-impedance analysis, Inbody720 (Biospace, USA). Advanced glycation end products (AGEs) were assessed by autofluorescence method, AGE Reader (Diagnoptics, The Netherlands). CAN was assessed by ANX-3.0 monitoring technology (ANSAR Medical Technologies, Inc., Philadelphia, PA), applying standard clinical tests. 2005 IDF and 2009 JIS definitions were used to define Metabolic Syndrome (MetS). RESULTS: The prevalence of CAN was 23% and increased with diabetes duration. Sympathetic activity was independently related to age, albumin/creatinine ratio (ACR) and total body fat mass, and parasympathetic activity - to age and ACR. Elevated hsCRP, AGEs and body fat, diabetic retinopathy and nephropathy, as well as hypertension, dyslipidemia and metabolic syndrome were found to increase the risk of CAN in T1D. CONCLUSION: CAN appears to be a common complication of T1D, especially with longer duration, and is found to be related to diabetic microvascular disease and metabolic syndrome components.

Evaluation of anal carcinoma screening in male and female HIV patients at an interdisciplinary HIV therapy centre.

BACKGROUND: Incidence of anal carcinoma is increased in people living with HIV (PLWH). Due to the improved life expectancy in PLWH, identifying appropriate prevention strategies for non-AIDS-defining cancer types such as anal carcinoma has become a priority in managing PLWH today. OBJECTIVE: We aimed to evaluate anal cytology assessment as screening tool for anal dysplasia and/or carcinoma in PLWH, regardless of gender or sexual orientation. Additionally, we investigated the correlation between cancer risk factors and abnormal screening results in our patient cohort. METHODS: People living with HIV from the Interdisciplinary HIV Centre of the University Hospital rechts der Isar in Munich, Germany (IZAR), were screened for anal carcinoma by single cytobrush examination and anal Papanicolaou (PAP) smear assessment from 2013 to 2015. Patients with abnormal PAP smear result were offered a follow-up examination after 12 months. Differences between two groups were tested for statistical significance using Student's t-test and Mann-Whitney U-test, as appropriate. RESULTS: In total, 101 PLWH were included. 26.7% of subjects (n = 27) were PAP IIID, and 9.9% (n = 10) were PAP IVa. Seven female subjects had an abnormal finding at screening. Smoking was significantly associated with abnormal findings at screening (P = 0.005). In addition, our study found an association between sexually transmitted infections (STI) and anal dysplasia. Condylomata acuminata were increased in subjects with PAP IIID/PAP IVa (P = 0.045). Reactive syphilis serology was found to be significantly associated with abnormal screening results (P = 0.016), respectively. CONCLUSION: Our results demonstrate that smoking and two common STIs, condylomata acuminata and syphilis, are risk factors associated with advanced anal intraepithelial neoplasia (AIN) stages in our PLWH cohort. While further analysis is needed to determine diagnostic guidelines concerning AIN in PLWH, these results suggest that interdisciplinary lifestyle prevention strategies are required to reduce the risk factors for AIN in PLWH in an outpatient setting.

Molecular genetic diagnostics of tuberous sclerosis complex in Bulgaria: six novel mutations in the TSC1 and TSC2 genes.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by the development of hamartomas localized in various tissues which can occur in the skin, brain, kidney and other organs. TSC is caused by mutations in the TSC1 and TSC2 genes. Here we report the results from the first molecular testing of 16 Bulgarian patients and one Romanian patient in whom we found six novel mutations: four in the TSC22 gene, of which one is nonsense, two frame shift and one large deletion of 16 exons; and two in the TSC1 gene, one nonsense and other frame shift. In addition, we detected 10 previously reported mutations; some of which are described only once in the literature. Our data is similar to the previous studies with exception of the larger number of TSC1 mutations than that reported in the literature data. In total, 40% (4/10) of the mutation in the TSC2 gene are located in the GTPase-activating protein domain, while 50% (3/6) are in the TSC1 gene and clustered in exon 15. All the cases represent the typical clinical symptoms and meet the clinical criteria for TSC diagnosis. In 35% of our cases the family history was positive. Our results add novel findings in the genetic heterogeneity and pathogenesis of TSC. The genetic heterogeneity might correlate to the clinical variability among the TSC-affected families, which makes the genetic counselling a real challenge.

BARTH SYNDROME IN MALE AND FEMALE SIBLINGS CAUSED BY A NOVEL MUTATION IN THE TAZ GENE.

Barth syndrome (BTHS) is an X-linked recessive disease caused by mutations in tafazzin gene (TAZ) which lead to cardiolipin deficiency and mitochondrial dysfunction. Male patients have variable clinical findings, including cardiomyopathy, skeletal myopathy, prepubertal short stature, neutropenia and 3-methylglutaconic aciduria. Female carriers are usually asymptomatic. We report a novel TAZ gene mutation in male and female siblings with left ventricular noncompaction and hypotonia. Additionally, the brother presented an intermittent neutropenia and increased urinary levels of 3-methylglutaconic and 3-methylglutaric acid. The molecular genetic testing showed that both siblings carry the mutation: c.253insC, p.(Arg85Profs*54) in exon 3 of the TAZ gene. This article presents the first case of BTHS in a heterozygous female patient with normal karyotype.

Dissecting susceptibility from exogenous triggers: the model of alopecia areata and associated inflammatory skin diseases.

BACKGROUND: Alopecia areata (AA) is a T-cell-driven autoimmune disease of the hair follicle and frequently reported to be associated with inflammatory skin diseases (ISD) such as atopic eczema (AE) or psoriasis. Interestingly, AA on the one hand and both AE and psoriasis on the other hand are believed to be driven by mutually antagonistic T-cell subsets. OBJECTIVE: To characterize AA-specific T-cell profiles and inflammatory pattern by intra-individual comparison of AA and coexistent ISD. METHODS: 112 patients with AA were recruited and investigated for coexisting ISD. In-depth analyses were performed in patients with AA and AE (n = 2), AA and psoriasis (n = 1), AA and psoriasis and AE (n = 1) and AA and lichen planus (n = 1), using histology, immunohistochemistry and cytokine staining of T cells isolated from lesional skin. RESULTS: Of 112 AA patients investigated, 23 suffered from an ISD. The prevalence of AE, vitiligo, psoriasis and lichen planus was higher in the investigated AA cohort than in the normal population. The clinical as well as histological phenotype of AA the coexistent ISD were unequivocal. In line with this, T-cell infiltrates were found to be disease-characteristics with AA and lichen planus dominated by CD8+ and IFN-γ+ TNF-α+ producing T cells while psoriasis lesions in the same patients were dominated by IL-17+ and AE by IL-4+ T cells. CONCLUSION: AA patients have a higher incidence of various T-cell-driven inflammatory skin diseases than the normal population, a phenomenon which might relate to over-activation of skin-homing T cells and to specific immune triggers as the primary cause of inflammation. More importantly, we showed that by using AA as a model disease, our approach of intra-individual comparison of distinct inflammatory responses in the same patient is feasible and offers the unique possibility to gain insights into disease pathogenesis independent from genetic susceptibilities.

King's Parkinson's disease pain scale, the first scale for pain in PD: An international validation.

Pain is a key unmet need and a major aspect of non-motor symptoms of Parkinson's disease (PD). No specific validated scales exist to identify and grade the various types of pain in PD. We report an international, cross-sectional, open, multicenter, one-point-in-time evaluation with retest study of the first PD-specific pain scale, the King's PD Pain Scale. Its seven domains include 14 items, each item scored by severity (0-3) multiplied by frequency (0-4), resulting in a subscore of 0 to 12, with a total possible score range from 0 to 168. One hundred seventy-eight PD patients with otherwise unexplained pain (age [mean ± SD], 64.38 ± 11.38 y [range, 29-85]; 62.92% male; duration of disease, 5.40 ± 4.93 y) and 83 nonspousal non-PD controls, matched by age (64.25 ± 11.10 y) and sex (61.45% males) were studied. No missing data were noted, and floor effect was observed in all domains. The difference between mean and median King's PD Pain Scale total score was less than 10% of the maximum observed value. Skewness was marginally high (1.48 for patients). Factor analysis showed four factors in the King's PD Pain Scale, explaining 57% of the variance (Kaiser-Mayer-Olkin, 0.73; sphericity test). Cronbach's alpha was 0.78, item-total correlation mean value 0.40, and item homogeneity 0.22. Correlation coefficients of the King's PD Pain Scale domains and total score with other pain measures were high. Correlation with the Scale for Outcomes in PD-Motor, Non-Motor Symptoms Scale total score, and quality of life measures was high. The King's PD Pain Scale seems to be a reliable and valid scale for grade rating of various types of pain in PD.

k=0 magnetic structure and absence of ferroelectricity in SmFeO3.

SmFeO3 has attracted considerable attention very recently due to its reported multiferroic properties above room temperature. We have performed powder and single crystal neutron diffraction as well as complementary polarization dependent soft X-ray absorption spectroscopy measurements on floating-zone grown SmFeO3 single crystals in order to determine its magnetic structure. We found a k=0 G-type collinear antiferromagnetic structure that is not compatible with inverse Dzyaloshinskii-Moriya interaction driven ferroelectricity. While the structural data reveal a clear sign for magneto-elastic coupling at the Néel-temperature of ∼675 K, the dielectric measurements remain silent as far as ferroelectricity is concerned.

First Bulgarian case of citrin deficiency caused by one novel and one recurrent mutation in the SLC25A13 gene.

Citrin deficiency is an autosomal recessive disorder caused by mutations in the SLC25A13 gene and has three phenotypes: neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) in newborns, failure to thrive and dyslipidemia caused by citrin deficiency (FTTDCD) in older children, and recurrent hyperammonemia with neuropsychiatric symptoms in citrullinemia type II (CTLN2) in adults. NICCD presents in the first few weeks of life with cholestatic hepatitis syndrome, multiple aminoacidemia and hypergalactosemia. To date almost all reported patients were from East Asia and only few cases from Caucasian origin have been described. We report the first Bulgarian case of NICCD. Mutation screening of the SLC25A13 gene revealed the compound heterozygous mutations c.1081C>T (p.R361*) and c.74C>A (p. A25E) which confirmed the diagnosis of NICCD. The nonsense mutation c.1081C>T (p.R361*) is novel.

Characterizing motor and non-motor aspects of early-morning off periods in Parkinson's disease: an international multicenter study.

INTRODUCTION: The characteristic off periods that develop over time in subjects with Parkinson's disease (PD) on chronic levodopa therapy are usually considered to be motor complications but more recently the important contribution of non-motor off and non-motor fluctuations has also been acknowledged. Early-morning off (EMO) periods in PD patients are known to be a cause of significant disability, in addition to having a negative impact on quality of life. Yet EMOs are poorly defined, particularly in relation to non-motor symptoms (NMS). METHODS: This European, multicentre, observational study was undertaken to characterize the range and patterns of NMS that occur during EMO periods in a consecutive series of PD patients. RESULTS: The results demonstrate that EMO periods are common and occur in 59.7% of subjects across all disease stages in line with other reports. However, importantly, in 88.0% of those, EMOs were found to be associated with NMS. The predominant NMS associated with EMO were urinary urgency, anxiety, dribbling of saliva, pain, low mood, limb paresthesia and dizziness. The patterns of dopaminergic treatment being taken by patients in this study suggested that a prolonged-release or continuous drug delivery strategy can alleviate some NMS associated with EMO. CONCLUSIONS: In light of these findings it is suggested that greater awareness, recognition and appropriate treatment of EMO and NMS could improve the overall 24-h management of PD. An EMO-specific scale/questionnaire which captures both motor and NMS associated with EMO over the off time period is warranted.

A family with fragile X syndrome, Duchenne muscular dystrophy and ichthyosis transmitted by an asymptomatic carrier.

The human X chromosome carries regions prone to genomic instability: deletions in the Xp22.31 region, involving the steroid sulfatase gene (STS) cause X-linked ichthyosis; rearrangements in the Xp21.2 region are associated with Duchenne or Becker muscular dystrophies (DMD or BMD); and the Xq27.3 unstable region, containing the (CGG)n repeat expansion in the FMR1 gene is associated with fragile X syndrome. We report on a family with two affected boys, the elder diagnosed with fragile X syndrome, the younger with DMD, and both suffering from severe ichthyosis. The family was analyzed by polymerase chain reaction, multiplex ligation-dependent probe amplification and haplotype analysis. The mother proved to be an asymptomatic carrier of all three non-contiguous mutation events, involving the STS gene, the DMD gene and a FMR1 expansion. To the best of our knowledge, this is the first description of an asymptomatic carrier of three different X-linked disorders, involving severe genetic rearrangements on both long and short arms of the X chromosomes. The boy with fragile X syndrome has inherited a triple recombinant maternal X chromosome, this way inheriting the FMR1 expansion and ichthyosis, originating most probably from different maternal Xes and excluding the DMD gene deletion. The transmission of these extremely defective maternal chromosomes to the next generation involved several recombinations.

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.

[The experience in genetic laboratory Varna in amniocentesis and genetic analysis for the period 2006-2010].

The aim of this report is to present and discuss the results from diagnostic amniocenteses, performed in Varna. The test started as a part of a prophylaxis program for pregnant women with calculated high risk for chromosomal disorders after a screening test. Amniocentesis was performed in total of 283 pregnant women. Of all patients who underwent the screening test, amniocentesis was performed in 1.55% of women under 36 years of age and 5.0% of women over 36 years. In the selected group with calculated high risk for chromosomal disorder these percentages were 28.5% and 26% respectively. Fetal chromosomal disorder was found in 5% (in 7 out of 141) in women under 36 and 3.82% (in 7 out of 83) in women over 36 years. Genetic tests (DNA and cytogenetic analysis) of amniocytes revealed chromosomal disorders in 16 (5.65%) fetuses (8 with trisomy 21, 3 with trisomy 18, 1 with trisomy 13, 1 case with triploidy, 3 cases with structural chromosomal rearrangement). Three additional amniocenteses were performed, indicated by family history of monogenic disorder (thalassaemia, spinal muscular atrophy). The effect of the introduced method for prenatal diagnosis, its interaction with the screening tests and their future as genetic prophylaxis program are discussed.

Analysis of the genes encoding neuroligins NLGN3 and NLGN4 in Bulgarian patients with autism.

Many studies have supported a genetic aetiology for autism. Neuroligins are postsynaptically located cell-adhesion molecules. Mutations in two X-linked neuroligin genes, NLGN3 and NLGN4, have been implicated in pathogenesis of autism. In order to confirm these causative mutations in our autistic population and to determine their frequency we screened 20 individuals affected with autism. We identified one patient with a point mutation in NLGN4 gene that substituted a Met for Thr 787 - c.2360C > T, p.(Thr787Met) and three patients with identical polymorphisms in the same gene: c.933C > T, p.(Thr311Thr) in combination with c.[1777C > T+1779C > G, p.(Leu593Leu)]. All patients tested for NLGN3 mutations were negative. These results indicate that mutations in these genes are responsible for at most a small fraction of autism cases.