Renata Gaillyová

The effect of the swim-up and hyaluronan-binding methods on the frequency of abnormal spermatozoa detected by FISH and SCSA in carriers of balanced chromosomal translocations

BACKGROUND The swim-up and hyaluronan (HA)-binding methods are used for the selection of good quality spermatozoa to improve pregnancy rates and embryo quality and to reduce the number of miscarriages after IVF. We evaluated whether the processing of sperm by these methods reduces the frequency of spermatozoa with abnormal karyotypes and altered chromatin quality in balanced translocation carriers. METHODS Semen samples of 12 carriers of balanced chromosomal translocations were analysed for the frequency of spermatozoa, which are chromosomally unbalanced due to the segregation of balanced translocations, aneuploidies for chromosomes 7, 8, 13, 18, 21, X or Y, diploid sperm or sperm with fragmented DNA and poorly condensed chromatin. Results obtained by fluorescence in situ hybridization (FISH) and sperm chromatin structure assay were compared between ejaculated (n = 12), swim-up (n = 12) and HA-binding processed (n = 6) semen samples of the translocation carriers and with the control group (n = 10). RESULTS The mean frequencies of unbalanced segregation products were 17.5 and 16.5% in neat and swim-up processed samples from Robertsonian translocation carriers, and 55.4, 54.5 and 50.9% in neat, swim-up and HA-bound sperm samples from reciprocal translocation carriers. Significant decreases in the frequency of sperm showing chromosome 18 and XY disomy and of diploidy, and in the rates of high-density staining sperm were observed in the motile swim-up fractions. There were significantly more sperm showing fragmented chromatin in the group of translocation carriers than in the control group, but no differences in the aneuploidy and diploidy rates were observed. CONCLUSIONS The swim-up method is suitable for selection of sperm with condensed chromatin and a lower frequency of some aneuploidies and of diploidy. The frequency of spermatozoa chromosomally unbalanced due to the segregation of reciprocal (but not Robertsonian) translocations is significantly lower in HA-bound sperm. However, the advantages of either method for selecting normal sperm are limited.

The homozygous KCNQ1 gene mutation associated with recessive Romano-Ward syndrome.

In a 7‐year‐old boy with normal hearing suffering from repeated syncope an extremely prolonged QTc interval (up to 700 ms) was found. The mother was completely asymptomatic and the father had an intermittently borderline QTc interval (maximum 470 ms) but no symptoms. In the proband a mutation analysis of KCNQ1 gene revealed a homozygous 1893insC mutation. The parents were heterozygous for this mutation. There was no consanguineous marriage in the family. The clinical relevance of these findings is that apparently normal individuals may have a latent reduction of repolarizing currents, a “reduced repolarization reserve,” because they are carriers of latent ion channel genes mutations.

Agammaglobulinaemia in a girl with a mosaic of ring 18 chromosome

A patient with a mosaic karyotype 45,XX,‐18/46,XX,dic r(18)/46, XX, r(18) with multiple phenotypic abnormalities and immunodeficiency was presented at the age of 14 years. Immunological investigation revealed markedly decreased IgG, IgA and in two of three evaluations also IgM levels. Although selective IgA deficiency is frequent in patients with a ring chromosome 18, this is the third patient described with decreased levels of other immunoglobulin isotypes. The association of chromosome 18 partial deletions and immunoglobulin abnormalities suggests the presence of an as yet unrecognised gene with a pivotal role for immunoglobulin production on chromosome 18.

Mutation Analysis Ion Channel Genes Ventricular Fibrillation Survivors with Coronary Artery Disease

Background: Observations from population‐based studies demonstrated a strong genetic component of sudden cardiac death. The aim of this study was to test the hypothesis that ion channel genes mutations are more common in ventricular fibrillation (VF) survivors with coronary artery disease (CAD) compared to controls.

A new homozygous mutation of the KCNQ1 gene associated with both Romano-Ward and incomplete Jervell Lange-Nielsen syndromes in two sisters

The mutations in the KCNQ1 gene (GenBank accession no. AF000571) encoding the subunit of the KCNQ1 channel can cause 2 different diseases: Romano-Ward syndrome (RWS), traditionally described as a combination of repeated syncope episodes and a prolonged QT interval, and the less frequent Jervell and Lange-Nielsen syndrome (JLNS), also associated (except for the abovementioned symptoms) with congenital bilateral deafness.1 The first one is usually associated with the heterozygous gene mutation, the latter one with the homozygous mutation. The disease prevalence is estimated at close to 1 in 2,500 live births, and JLNS has been reported to affect about 3 in 1 million individuals, which represents less than 1% of all long-QT syndrome (LQTS) patients.

Sperm meiotic segregation and aneuploidy in a 46,X,inv(Y),t(10;15) carrier: case report

OBJECTIVE To analyze the meiotic segregation and an interchromosomal effect in sperm of an inv(Y) (p11.1;q11.2),t(10;15) (q25.2;q12) carrier. DESIGN Case report. SETTING Research institute. PATIENT(S) Man with a karyotype 46,X,inv(Y),t(10;15), normal sperm parameters, and secondary infertility. INTERVENTION(S) Multicolor fluorescence in situ hybridization using probes for chromosomes 10, 15, 8, 18, 21, X, and Y. MAIN OUTCOME MEASURE(S) Frequencies of meiotic segregation products and aneuploidy of chromosomes 8, 18, 21, X, and Y. RESULT(S) The most frequent type of meiotic segregation was the alternate (40.82%), followed by the adjacent 1 (28.09%), adjacent 2 (16.33%), and 3:1 (9.91%) segregations. Neither deviation from the expected 1:1 ratio of the X- and Y-bearing spermatozoa nor any evidence of an interchromosomal effect on aneuploidy of chromosomes X, Y, 8, 18, and 21 and diploidy was observed in the carrier compared with control donors. The disomies of chromosomes 8 and 21 were equally frequent in X- and Y-bearing spermatozoa of the carrier. CONCLUSION(S) The fluorescence in situ hybridization analysis of meiotic segregation and aneuploidy helps to personalize the reproductive risk in carriers of balanced structural chromosomal aberrations. Complete information concerning the quality of spermatogenesis is necessary in all donors (both translocation carriers and controls) implicated in interchromosomal effect studies.

Clinical characteristics of 30 Czech families with long QT syndrome and KCNQ1 and KCNH2 gene mutations: importance of exercise testing

BACKGROUND Classic symptoms of long QT syndrome (LQTS) include prolongation of QT interval on electrocardiograph, syncope, and cardiac arrest due to a distinctive form of polymorphic ventricular tachycardia, known as Torsade de Pointes. We assessed occurrence of LQTS signs in individuals from 30 Czech families with mutations in KCNQ1 and KCNH2 genes. METHODS AND RESULTS One hundred five individuals from 30 Czech families with LQTS were genotyped for KCNQ1 and KCNH2. The occurrence of typical LQTS signs (pathologic prolongation of QT interval; syncope; cardiac arrest; Torsade de Pointes) was clinically assessed by exercise test with QT interval analysis. Family history of sudden cardiac death was taken. Statistical analysis was performed to determine correlation of clinical results and mutation status. KCNQ1 gene mutations were found in 23 families, and KCNH2 gene mutations in eight families. Only 46 (70%) of the 66 mutation carriers had at least two of the typical LQTS signs. The others were minimally or asymptomatic. From 39 noncarrier individuals, only 1 fulfilled the clinical criteria of LQTS diagnosis, another 4 had an intermediate probability of diagnosis. The exercise test had 92% sensitivity and 93% specificity for LQTS diagnosis. CONCLUSIONS Incidence of classical signs of LQTS was not high in Czech carriers of KCNQ1 and KCNH2 mutations. Therefore, proper diagnosis relies on detection of symptoms at presentation. The exercise test may be beneficial owing to its high sensitivity and specificity for LQTS diagnosis.

Clinical Characteristics and Mutational Analysis of the RyR2 Gene in Seven Czech Families with Catecholaminergic Polymorphic Ventricular Tachycardia

Background: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a rare hereditary arrhythmia. The onset of clinical symptoms usually occurs during childhood, and is typically related to exercise. The aim of our study was to describe the clinical characteristics of seven Czech families with CPVT and the results of mutational analysis of the RyR2 gene in these families.

Two Distinct De novo Pathogenic Sequence Variants in the SCN2A Gene in Children with Early Infantile Epileptic Encephalopathy/Ohtahara Syndrome

Early infantile epileptic encephalopathy/Ohtahara syndrome represents a group of genetically heterogeneous disorders affecting normal brain development and functioning. In this work, we present a case of two unrelated children diagnosed with early infantile epileptic encephalopathy and associated multiple congenital abnormalities. To obtain genetic diagnosis, these children were examined through multi-step diagnostic algorithm including G-banded karyotype analysis and whole-genomic screening using array-based comparative genomic hybridization (array-CGH) with negative results. Additionally, these children and their unaffected parents were enrolled for our pilot study of targeted nextgeneration sequencing technology (NGS) using commercial panel ClearSeq Inherited DiseaseXT (Agilent Technologies) and consequent validation by Sanger sequencing. Our analysis detected two distinct de novo pathogenic sequence variants in the SCN2A gene, resulting in p.Met1545Val and p.Ala263Val changes on the SCN2A protein level and explaining their pathological phenotypes. In conclusion, our findings indicate that NGS can become the optimal approach to the genetics diagnostics of disorders of central nervous system (CNS) which can lead to the discovery of new candidate genes.

WS11.1 Development of a comprehensive workflow for the analysis of CFTR gene using next generation sequencing technology

I. Valaskova1,2,3, J. Necasova1, R.M. Selingerova1, R. Spesna1, A. Holcikova4, L. Homola4, E. Pokojova5, R. Gaillyova1,2,3. 1University Hospital, Medical Genetics, Brno, Czech Republic; 2Masaryk University, Faculty of Medicine, Department of Biology, Brno, Czech Republic; 3Masaryk University, Faculty of Medicine, Department of Laboratory Methods, Brno, Czech Republic; 4University Hospital, Clinic of Pediatric Infectious Diseases, Brno, Czech Republic; 5University Hospital, Department of Pulmonary Diseases and Tuberculosis, Brno, Czech Republic