The prevalence and genotype of 21-hydroxylase deficiency in the Croatian Romani population.

Objective: Congenital adrenal hyperplasia (CAH) owing to 21-hydroxylase deficiency (21-OHD) is a rare autosomal recessive disorder caused by pathological variants in the CYP21A2 gene. After a high prevalence of classic 21-OHD CAH in the Romani population was reported in the Republic of North Macedonia, we decided to estimate the prevalence of 21-OHD in Croatia and, if high, assess the possible causes and estimate the frequency of particular CYP21A2 variants. Design: Cross-sectional study. Methods: Data from a Croatian 21-OHD genetic database was reviewed, and only Romani patients were included in the study. CYP21A2 genotyping was performed using allele-specific PCR, MLPA, and Sanger sequencing. Results: According to a survey conducted in 2017, Croatia had 22,500 Romani people and six of them had a salt-wasting (SW) form of 21-OHD. All were homozygous for the c.IVS2-13A/C-G pathological variant in intron 2 and descended from consanguineous families belonging to different Romani tribes. The calculated prevalence of 21-OHD in Croatian Romani is 1:3,750, while in the Croatian general population, it is 1:18,000. Three of the six Romani patients originated from two neighboring villages in North-western Croatia (Slavonia County), as well as the seventh patient who is of mixed Romani/Croatian descent and heterozygous for the c.IVS2-13A/C-G pathological variant (not included in the prevalence calculation). Conclusion: A high prevalence of SW 21-OHD in the Croatian Romani population caused by the homozygous cIVS2-13A/C-G pathological variant was found. In addition to isolation and consanguinity, other possible reasons could be the heterozygous advantage of the CYP21A2 gene pathological variant and the bottleneck effect as a result of the Romani Holocaust in World War II.

Biomarkers of aggression in dementia.

Dementia is a clinical syndrome defined by progressive global impairment of acquired cognitive abilities. It can be caused by a number of underlying conditions. The most common types of dementia are Alzheimer's disease (AD), frontotemporal dementia (FTD), vascular cognitive impairment (VCI) and dementia with Lewy bodies (DLB). Despite the fact that cognitive impairment is central to the dementia, noncognitive symptoms, most commonly described nowadays as neuropsychiatric symptoms (NPS) exist almost always at certain point of the illness. Aggression as one of the NPS represents danger both for patients and caregivers and the rate of aggression correlates with the loss of independence, cognitive decline and poor outcome. Therefore, biomarkers of aggression in dementia patients would be of a great importance. Studies have shown that different genetic factors, including monoamine signaling and processing, can be associated with various NPS including aggression. There have been significant and multiple neurotransmitter changes identified in the brains of patients with dementia and some of these changes have been involved in the etiology of NPS. Aggression specific changes have also been observed in neuropathological studies. The current consensus is that the best approach for development of such biomarkers may be incorporation of genetics (polymorphisms), neurobiology (neurotransmitters and neuropathology) and neuroimaging techniques.

Complex intrachromosomal rearrangement in 1q leading to 1q32.2 microdeletion: a potential role of SRGAP2 in the gyrification of cerebral cortex.

BACKGROUND: Van der Woude syndrome (MIM: 119300, VWS) is a dominantly inherited and the most common orofacial clefting syndrome; it accounts for ~2 % of all cleft lip and palate cases. Intellectual disability (ID) is characterized by significant limitations, both in intellectual functioning (cognitive deficit) and in adaptive behavior as expressed in conceptual, social and practical adaptive skills. Karyotyping has been the first standard test for the detection of genetic imbalance in patients with ID for more than 35 years. Advances in genetic diagnosis have laid chromosomal microarrays (CMA) as a new standard and first first-line test for diagnosis of patients with ID, as well as other conditions, such as autism spectrum disorders or multiple congenital anomalies. CASE PRESENTATION: The present case was initially studied due to unexplained cognitive deficit. Physical examination at the age of 18 years revealed cleft palate, lower lip pits and hypodontia, accompanied with other dysmorphic features and absence of speech. Brain MRI uncovered significantly reduced overall volume of gray matter and cortical gyrification. Banding cytogenetics revealed an indistinct intrachromosomal rearrangement in the long arm of one chromosome 1, and subsequent microarray analyses identified a 5.56 Mb deletion in 1q32.1-1q32.3, encompassing 52 genes; included were the entire IRF6 gene (whose mutations/deletions underlay VWS) and SRGAP2, a gene with an important role in neuronal migration during development of cerebral cortex. Besides, a duplication in 3q26.32 (1.9 Mb in size) comprising TBL1XR1 gene was identified. Multicolor banding for chromosome 1 and molecular cytogenetics applying a battery of locus-specific probes covering 1q32.1 to 1q44 characterized a four breakpoint-insertional-rearrangement-event, resulting in 1q32.1-1q32.3 deletion. CONCLUSIONS: Considering that the human-specific three-fold segmental duplication of SRGAP2 gene evolutionary corresponds to the beginning of neocortical expansion, we hypothesize that aberrations in SRGAP2 are strong candidates underlying specific brain abnormalities, namely reduced volume of grey matter and reduced gyrification.

Aberrant expression of shared master-key genes contributes to the immunopathogenesis in patients with juvenile spondyloarthritis.

Association of juvenile spondyloarthritis (jSpA) with the HLA-B27 genotype is well established, but there is little knowledge of other genetic factors with a role in the development of the disease. To date, only a few studies have tried to find those associated genes by obtaining expression profiles, but with inconsistent results due to various patient selection criteria and methodology. The aim of the present study was to identify and confirm gene signatures and novel biomarkers in highly homogeneous cohorts of untreated and treated patients diagnosed with jSpA and other forms of juvenile idiopathic arthritis (JIA) according to ILAR criteria. For the purposes of the research, total RNA was isolated from whole blood of 45 children with jSpA and known HLA genotype, 11 children with oligo- and polyarticular forms of JIA, as well as 12 age and sex matched control participants without diagnosis of inflammatory disease. DNA microarray gene expression was performed in 11 patients with jSpA and in four healthy controls, along with bioinformatical analysis of retrieved data. Carefully selected differentially expressed genes where analyzed by qRT-PCR in all participants of the study. Microarray results and bioinformatical analysis revealed 745 differentially expressed genes involved in various inflammatory processes, while qRT-PCR analysis of selected genes confirmed data universality and specificity of expression profiles in jSpA patients. The present study indicates that jSpA could be a polygenic disease with a possible malfunction in antigen recognition and activation of immunological response, migration of inflammatory cells and regulation of the immune system. Among genes involved in these processes TLR4, NLRP3, CXCR4 and PTPN12 showed almost consistent expression in study patients diagnosed with jSpA. Those genes and their products could therefore potentially be used as novel biomarkers, possibly predictive of disease prognosis and response to therapy, or even as a target for new therapeutic approaches.

Personalized medicine in neurodegenerative diseases: how far away?

Neurodegenerative diseases are characterized by progressive dysfunction of the nervous system as a result of neuronal loss in the brain and spinal cord. Despite extensive research efforts aimed at development of new disease-modifying therapeutics, there is still no effective treatment to halt neurodegenerative processes. Thus, modification of current therapeutic and diagnostic research strategies is a goal of increasing urgency. The biggest limitation in neurodegenerative disease research is the lack of appropriate biomarkers. Discovery of universal biomarkers capable of diagnosing patients with neurodegenerative diseases, monitoring their response to therapy, and predicting disease progression seems to be a tall order. Instead, a combination of different methodologies in the discovery of biomarkers specific for each described aspect of the disease seems to be a more viable approach. Although application of personalized medicine in diagnosis and treatment of neurodegenerative diseases may seem far off, some recent developments, such as utilizing specific biological therapies in multiple sclerosis, microRNA profiling as a source of novel biomarkers in Parkinson's disease, or combination of neuroimaging and proteomic analyses in diagnosis of Alzheimer's disease patients, already point to the way clinical neurology may integrate new achievements in everyday practice. Combination of genomic, proteomic, glycomic, and metabolomic approaches may yield novel insights into molecular mechanisms of disease pathophysiology, which could then be integrated and translated into clinical neurology. Based on the developments during the past decade, it is feasible to predict that a personalized approach to treating neurological disorders will become more widely applicable in the coming years.

Genetic changes observed in a case of adult pilocytic astrocytoma revealed by array CGH analysis.

BACKGROUND: A palette of copy number changes in a case of adult pilocytic astrocytoma analyzed by Array Comparative Genomic Hybridization (aCGH) is presented. Pilocytic astrocytomas are specific gliomas that are benign and biologically distinct and the molecular mechanisms responsible for their development remain unexplained. The aCGH was performed using SurePrint G3 Human CGH microarrays 4 × 180 K (Agilent Technologies). To ascertain whether some of the aberrations were of constitutive nature, we also analyzed the blood sample from the same patient. RESULTS: The result of aCGH analysis demonstrated differences in the tumor tissue when compared to normal control on the array and also to autologous DNA from patient's blood. The total number of aberrations found in our case was 41 including 37 deletions and 4 amplifications. Whole chromosomal gains and losses were not observed. Collectively, our results showed three deletions and one amplification at 1p, two deletions at 2q, two deletions at 4q, two deletion at 5q, two deletions at 7p and two deletions at 7q; there were also three deletions at 8q, one deletion at 9p, one deletion at 10p, three deletions and one amplification at 10q. Chromosome 11 showed two deletions at 11p, while there was one deletion at 12p and one at 12q. Four deletions at 14q; two deletions at 15q, one amplification at 17q and one deletion at 17q; one deletion at 18p, two deletions at 22q and finally one deletion at Xp and one deletion and one amplification at Xq. Among the signaling pathways, olfactory transduction, Fc gamma R-mediated phagocytosis and p53 signaling pathway showed significant enrichment ascertained by gene ontology (GO) analysis using the DAVID software. CONCLUSIONS: Our aCGH analysis is bringing subtle genomic alterations thus broadening genetic spectrum of adult pilocytic astrocytoma in order to offer new molecular biomarkers that will help in diagnostics and therapeutic decision-making.

Vagus nerve stimulation in Lafora body disease.

INTRODUCTION: Lafora body disease (LBD) is a rare autosomal recessive disorder characterized by progression to inexorable dementia and frequent occipital seizures, in addition to myoclonus and generalized tonic-clonic seizures (GTCSs). It belongs to the group of progressive myoclonus epilepsies (PMEs), rare inherited neurodegenerative diseases with great clinical and genetic differences, as well as poor prognosis. Since those patients have a pharmacoresistant disease, an adjunctive treatment option is vagus nerve stimulation (VNS). To date, there are four reported cases of the utility of VNS in PME - in Unverricht-Lundborg disease (ULD), myoclonic epilepsy with ragged-red fibers (MERRF), Gaucher's disease, and in one case that remained unclassified. CASE PRESENTATION: A 19-year-old male patient had progressive myoclonus, GTCSs that often progressed to status epilepticus (SE), progressive cerebellar and extrapyramidal symptomatology, and dementia, and his disease was pharmacoresistant. We confirmed the diagnosis of LBD by genetic testing. After VNS implantation, in the one-year follow-up period, there was a complete reduction of GTCS and SE, significant regression of myoclonus, and moderate regression of cerebellar symptomatology. CONCLUSION: To our knowledge, this is the first reported case of the utility of VNS in LBD. Vagus nerve stimulation therapy may be considered a treatment option for different clinical entities of PME. Further studies with a larger number of patients are needed.

Gene expression in visceral and subcutaneous adipose tissue in overweight women.

Excess weight and obesity are common health problems with multifactorial and polygenic causes. Abdominal or visceral obesity is associated with a higher risk of obesity related complications. The aim of the study was to evaluate differentially expressed genes in visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) of 10 overweight women undergoing elective laparoscopic cholecystectomy. Following expression profiling using microarrays, a set of 294 genes that exhibited differential expression between VAT and SAT was further analyzed to test the functional correlation of gene sets using the Gene Set Enrichment Analysis method. To confirm the functional pathways involved in differential expression between SAT and VAT, additional pathway analysis was done using the GeneGo MetaCore software and the Ingenuity Pathway Analysis. Ten differentially expressed genes were selected according to the microarray data, with seven exhibiting significant differential expression in the RT-PCR experiments. The data from this preliminary study suggest enrichment of inflammation and oxidative stress related pathways in VAT, while insulin homeostasis pathways as well as pathways pertaining to several growth factors are enriched in SAT.

Convergence of miRNA expression profiling, α-synuclein interacton and GWAS in Parkinson's disease.

miRNAs were recently implicated in the pathogenesis of numerous diseases, including neurological disorders such as Parkinson's disease (PD). miRNAs are abundant in the nervous system, essential for efficient brain function and play important roles in neuronal patterning and cell specification. To further investigate their involvement in the etiology of PD, we conducted miRNA expression profiling in peripheral blood mononuclear cells (PBMCs) of 19 patients and 13 controls using microarrays. We found 18 miRNAs differentially expressed, and pathway analysis of 662 predicted target genes of 11 of these miRNAs revealed an over-representation in pathways previously linked to PD as well as novel pathways. To narrow down the genes for further investigations, we undertook a parallel approach using chromatin immunoprecipitation-sequencing (ChIP-seq) analysis to uncover genome-wide interactions of α-synuclein, a molecule with a central role in both monogenic and idiopathic PD. Convergence of ChIP-seq and miRNomics data highlighted the glycosphingolipid biosynthesis and the ubiquitin proteasome system as key players in PD. We then tested the association of target genes belonging to these pathways with PD risk, and identified nine SNPs in USP37 consistently associated with PD susceptibility in three genome-wide association studies (GWAS) datasets (0.46≤OR≤0.63) and highly significant in the meta-dataset (3.36×10⁻4

Pilot study of the association between the HLA region and testicular carcinoma among Croatian patients.

OBJECTIVES: To analyze the distribution of HLA alleles and HLA microsatellite alleles in Croatian patients with testicular carcinoma, compare it with that of healthy controls and investigate whether the polymorphism within the HLA region could be associated with the development of testicular cancer. METHODS: Genomic DNA was isolated from the peripheral blood of 24 patients with testicular germ cell tumors (TGCT). Patients and controls were typed for HLA class I and class II polymorphism by the PCR-SSO method. Nine HLA microsatellites were analyzed by PCR and electrophoresis in an automated sequencer. RESULTS: No significant deviation in the distribution of frequencies at HLA class I alleles was observed between patients and controls. Among HLA class II alleles, a statistically significant increase in the frequency of the HLA-DPB1*1701 allele was found among patients. The frequency of the HLA-DRB1*07-DQA1*0201-DQB1*0202 haplotype was increased in patients in comparison to the controls. Analysis of HLA microsatellites showed an increased frequency of D6S291-3 allele (p(corr) = 0.0455, OR = 3.05) among patients. CONCLUSIONS: The observed association of the disease and the DPB1*1701 allele as well as with the D6S291-3 allele suggests that this part of the HLA region might be involved in the pathogenesis of TGCT. Our data provide a basis for further studies about the correlation between the HLA region and testicular cancer.