Hearing impairment in Estonia: an algorithm to investigate genetic causes in pediatric patients.

PURPOSE: The present study was initiated to establish the etiological causes of early onset hearing loss (HL) among Estonian children between 2000-2009. METHODS: The study group consisted of 233 probands who were first tested with an arrayed primer extension assay, which covers 199 mutations in 7 genes (GJB2, GJB6, GJB3, SLC26A4, SLC26A5 genes, and two mitochondrial genes - 12S rRNA, tRNASer(UCN)). From probands whose etiology of HL remained unknown, DNA analysis of congenital cytomegalovirus (CMV) infection and G-banded karyotype and/or chromosomal microarray analysis (CMA) were performed. RESULTS: In 110 (47%) cases, the etiology of HL was genetic and in 5 (2%) congenital CMV infection was diagnosed. We found mutations with clinical significance in GJB2 (100 children, 43%) and in 2 mitochondrial genes (2 patients, 1%). A single mutation in SLC26A4 gene was detected in 5 probands (2.2%) and was considered diagnostic. In 4 probands a heterozygous IVS2-2A>G change in the SLC26A5 gene was found. We did not find any instances of homozygosity for this splice variant in the probands. CMA identified in 4 probands chromosomal regions with the loss of one allele. In 2 of them we were able to conclude that the found abnormalities are definitely pathogenic (12q13.3-q14.2 and 17q22-23.2 microdeletion), but the pathogenity of 2 other findings (3p26.2 and 1p33 microdeletion) remained unknown. CONCLUSION: This practical diagnostic algorithm confirmed the etiology of early onset HL for 115 Estonian patients (49%). This algorithm may be generalized to other populations for clinical application.

A Diagnostic Algorithm for Mitochondrial Disorders in Estonian Children.

Mitochondrial disorders are a heterogeneous group of disorders affecting energy production of the body. Different consensus diagnostic criteria for mitochondrial disorders in childhood are available - Wolfson, Nijmegen and modified Walker criteria. Due to the extreme complexity of mitochondrial disorders in children, we decided to develop a diagnostic algorithm, applicable in clinical practice in Estonia, in order to identify patients with mitochondrial disorders among pediatric neonatology and neurology patients. Additionally, it was aimed to evaluate the live-birth prevalence of mitochondrial disorders in childhood. During the study period (2003-2009), a total of 22 children were referred to a muscle biopsy in suspicion of mitochondrial disorder based on the preliminary biochemical, metabolic and instrumental investigations. Enzymatic and/or molecular analysis confirmed mitochondrial disease in 5 of them - an SCO2 gene (synthesis of cytochrome c oxidase, subunit 2) defect, 2 cases of pyruvate dehydrogenase complex deficiency and 2 cases of combined complex I and IV deficiency. The live-birth prevalence for mitochondrial defects observed in our cohort was 1/20,764 live births. Our epidemiological data correlate well with previously published epidemiology data on mitochondrial diseases in childhood from Sweden and Australia, but are lower than in Finland.

Prevalence of Long-Chain 3-Hydroxyacyl-CoA Dehydrogenase Deficiency in Estonia.

The aim of our study was to evaluate the prevalence of long chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) in the general Estonian population and among patients with symptoms suggestive of fatty acid oxidation (FAO) defects. We collected DNA from a cohort of 1,040 anonymous newborn blood spot samples. We screened these samples for the presence of the common c.1528G>C mutation in the HADHA gene. Based on the clinical suspicion of FAO defects, we screened suspected individuals since 2004 for the common c.1528G>C mutation in the HADHA gene and since 2008 in addition by tandem mass spectrometric analysis of plasma acylcarnitines. Our results showed that the carrier frequency of the c.1528G>C mutation in the Estonian population is high - 1:173. During the screening of symptomatic patients, we identified five LCHADD patients in four families. Three patients were retrospectively identified by molecular screening of the HADHA gene. One patient was homozygous for the c.1528G>C mutation in the HADHA gene, and two siblings were compound heterozygotes with HADHA genotype c.[1528G>C]+[1690-2A>G]. Among patients tested using acylcarnitine profiling, we identified two cases with an abnormal acylcarnitine profile typical to LCHADD. Molecular analysis showed homozygosity for c.1528G>C mutation. Based on a carrier frequency of 1:173 (95% Confidence Interval 1:76-1:454) and taking into account that the c.1528G>C mutation makes up 87.5% of disease alleles in Estonian LCHADD patients, the estimated prevalence of LCHADD in Estonia would be 1: 91,700.

Whole Xp Deletion in a Girl with Mental Retardation, Epilepsy, and Biochemical Features of OTC Deficiency.

Background: Females with a total or partial deletion of the short arm of the X chromosome have variable features of Turner syndrome, but mental retardation (MR) rarely occurs. The haploinsufficiency of deleted genes that escape X-inactivation may explain the occurrence of MR and autism. Ornithine transcarbamylase (OTC) deficiency is the most common urea cycle disorder and is inherited in an X-linked semi-dominant trait, and the OTC gene maps to Xp21. Methods: We report on a girl with MR, epilepsy and biochemical changes characteristic of OTC deficiency but no identifiable point mutation in the OTC gene. Standard G-banding cytogenetic analysis, whole genome karyotyping, and X-inactivation studies were performed to determine the genetic etiology of the OTC deficiency in the patient. Results: Cytogenetic analysis and molecular karyotyping using SNP array revealed a deletion of the whole short arm of the X chromosome (Xp22.33-p11.1). Inactivation studies also revealed a completely skewed X-inactivation. Conclusion: Our patient presented with MR, epilepsy, and some evidence of reduced OTC activity, but performed genetic studies gave no explanation for this phenotype. We hope that this case report contributes to the understanding of the underlying genetic factors of the manifestation of X-linked disorders in female patients.

Long-term complications in Estonian galactosemia patients with a less strict lactose-free diet and metabolic control.

The main aim of our study was to retrospectively evaluate long-term complications and measure urinary galactose and galactitol excretion in classical galactosemia patients in Estonia who have been treated with a less restricted lactose-free diet and metabolic control. Our study group consisted of five classical galactosemia patients aged 7-14 years and diagnosed since 1996 in Estonia. Their diet eliminates lactose present in dairy foods, but we did not restrict the consumption of mature cheeses, fruits and vegetables. All patients had normal growth, except for one patient who was overweight at the last evaluation. In three patients mental and speech development was normal. One patient, number 1, who was diagnosed latest (at 6 weeks of age), had moderate mental retardation, verbal dyspraxia, extrapyramidal signs and bilateral cataracts. In both patients with developmental problems, a brain MRI showed bilateral subcortical changes in the cerebral white matter. Of four females, only patient 4 (p.Q188R homozygote) has premature ovarian insufficiency. Urinary galactose and galactitol content were retrospectively measured using high-performance liquid chromatography and refractive-index detection from urinary samples that were preserved during the years 1996-2009. Galactose ranged from 60 to 600 mmol/mol creatinine (normal=4-6), and galactitol ranged from 70 to 1200 mmol/mol creatinine (normal=2-4), which was 10-100 and 17-300 times higher than the respective reference ranges for galactose and galactitol. We conclude that a less strict lactose-free diet and metabolic control performed in Estonian classical galactosemia patients does not change long-term outcome compared to previously published studies.

Lipid vesicles in capillary electrophoretic techniques: characterization of structural properties and associated membrane-molecule interactions.

This paper reviews the use of lipid vesicles as model membranes in capillary electrophoresis (CE). The history and utility of CE in the characterization of microparticles is summarized, focusing on the application of colloidal electromigration theories to lipid vesicles. For instance, CE experiments have been used to characterize the size, surface properties, enclosed volumes, and electrophoretic mobilities of lipid vesicles and of lipoprotein particles. Several techniques involving small molecules or macromolecules separated in the presence of lipid vesicles are discussed. Interactions between the analytes and the lipid vesicles - acting as a pseudostationary phase or coated stationary phase in electrokinetic chromatography (EKC) - can be used to obtain additional information on the characteristics of the vesicles and analytes, and to study the biophysical properties of membrane-molecule interactions in lipid vesicles and lipoproteins. Different methods of determining binding constants by EKC are reviewed, along with the relevant binding constant calculations and a discussion of the application and limitations of these techniques as they apply to lipid vesicle systems.

Study of lipid and apolipoprotein binding interactions using vesicle affinity capillary electrophoresis.

Vesicle affinity capillary electrophoresis (VCE), a newly developed technique, was designed to assess the effect of physicochemical properties of apolipoprotein (apo) on the binding to lipoproteins, under physiological conditions (phosphate-saline buffer system at pH 7.4 and 37 degrees C), using vesicle as a model. The technique results in similar lipid binding properties of apo CIII (CIII) and its peptides compared to other techniques. It also offers a fast and more sensitive tool in determining the lipid affinity of apos in a unique system simulating the dynamic binding properties of apo in vivo. A noncompetitive binding model is used to determine the multiple binding properties of CIII and its peptides to vesicle. The VCE binding constants are dependent on temperature, physicochemical properties of the protein (hydrophobicity and charge), and nature of the vesicle. The vesicles used in the VCE experiments described here have been fully characterized and found to be stable under different temperatures (4 and 37 degrees C) and voltage conditions. Migration behavior of CIII and related peptides is reported in terms of relative mobility in order to correct for variability in viscosity at different vesicle concentrations. The VCE method provides very precise data on the migration time from 0.1 to 3.3% RSD at the highest concentration of vesicle. The model and current data have been used to determine VCE binding constants and protein-to-lipid binding ratios. The model predicts that higher lipid affinity (K(B)), protein-lipid binding ratio (n), and lower protein concentration result in a shift of the binding isotherm toward a lower concentration range of vesicle. A higher vesicle mobility, reflecting the size and charge of the vesicle, results in a larger separation window between the migration time of the free protein and the complex. The value of VCE for structure-function studies and drug design for peptides and proteins that are strongly bound to lipids has been illustrated.