Ana Spreiz

Whole Genome Amplification from Microdissected Chromosomes

Over the last years various whole genome amplification (WGA) methods have been established for genetic investigations from a limited number of cells or small quantities of DNA but not for molecular analysis of isolated chromosomes, which is important for the direct investigation of haplotypes or molecular rearrangements of derivative chromosomes in clinical cytogenetics and oncology. Here, the results of a pilot study in which the GenomePlex Single Cell Kit® linker adapter PCR approach (Sigma-Aldrich, Vienna, Austria) was modified for WGA of glass needle based microdissected chromosomes are presented. Compared with two other WGA strategies (Improved-Primer Extension Preamplification PCR and Multiple Displacement Amplification) the GenomePlex Single Cell Kit® shows a higher rate of successfully amplified markers, a lower WGA drop out rate and faster feasibility.

Novel Mutation in Potassium Channel related Gene KCTD7 and Progressive Myoclonic Epilepsy

Progressive myoclonic epilepsy (PME) is a heterogeneous group of epilepsies characterized by myoclonus, seizures and progressive neurological symptoms.

Phenotypic variability of a deletion and duplication 6q16.1 → q21 due to a paternal balanced ins(7;6)(p15;q16.1q21)†‡

Constitutional insertional translocations are rare findings in clinical cytogenetics. Here, we report on the unbalanced segregation of a balanced paternal insertional translocation ins(7;6)(p15;q16.1q21) to three children. Investigations by conventional karyotyping, FISH with locus‐specific probes, microsatellite marker analysis, and SNP‐array based copy number analysis revealed a direct orientation of the inserted segment, a size of 11.3 Mb, and breakpoints between rs4370337 and rs12660854 and rs12110990 and rs4946730 on 6q16.1 and 6q21, respectively, as well as within BAC clone RP11–182J2 on 7p15. A 17‐year‐old daughter inherited the der(6) chromosome and was affected by severe mental retardation, obesity, and minor anomalies. Two further children inherited the der(7) chromosome. A daughter shows an almost unremarkable phenotype and only minor features in neuropsychological testing at 19 years of age. Her 14‐year‐old half‐brother demonstrates a mild delay in cognitive development most likely jointly caused by the chromosomal rearrangement and asphyxia during delivery. The patient with the deletion confirms the previously reported phenotype of severe mental retardation and obesity in patients with del(6)(q16.2), while both patients with partial trisomy for the same segment of chromosome 6 are further examples for a generally less severe phenotype associated with duplications than with deletions, and even for the recent insight that chromosomal aneusomies of several megabases may go without major clinical consequences.

Disruption of EXOC6B in a patient with developmental delay, epilepsy, and a de novo balanced t(2;8) translocation.

Most balanced chromosomal aberrations are not associated with a clinical phenotype, however, in some patients they may disrupt gene structure. With the development of various next-generation sequencing techniques, fast and specific analyses of the breakpoint regions of chromosomal rearrangements are possible. Here, we report on a 19-year-old woman with a de novo balanced translocation t(2;8)(p13.2;q22.1) and a severe clinical phenotype including intellectual disability, epilepsy, behavioral features resembling autism, and minor dysmorphic features. By next-generation sequencing, we defined the breakpoints and found disruption of the exocyst complex component 6B (EXOC6B) gene in intron 1 on chromosome 2p13.2 involving two Alu elements with a homology of 81%. No gene was found at the respective breakpoint on chromosome 8. Expression analysis of the EXOC6B in blood lymphocytes and buccal smear revealed reduced expression in the patient in comparison with the control. Our findings in combination with one recently published case and one other patient listed in DECIPHER v5.1 indicate EXOC6B as a gene relevant for intellectual development and electrophysiological stability.

The mitochondrial solute carrier SLC25A5 at Xq24 is a novel candidate gene for non-syndromic intellectual disability

Loss-of-function mutations in several different neuronal pathways have been related to intellectual disability (ID). Such mutations often are found on the X chromosome in males since they result in functional null alleles. So far, microdeletions at Xq24 reported in males always have been associated with a syndromic form of ID due to the loss of UBE2A. Here, we report on overlapping microdeletions at Xq24 that do not include UBE2A or affect its expression, in patients with non-syndromic ID plus some additional features from three unrelated families. The smallest region of overlap, confirmed by junction sequencing, harbors two members of the mitochondrial solute carrier family 25, SLC25A5 and SLC25A43. However, identification of an intragenic microdeletion including SLC25A43 but not SLC25A5 in a healthy boy excluded a role for SLC25A43 in cognition. Therefore, our findings point to SLC25A5 as a novel gene for non-syndromic ID. This highly conserved gene is expressed ubiquitously with high levels in cortex and hippocampus, and a presumed role in mitochondrial exchange of ADP/ATP. Our data indicate that SLC25A5 is involved in memory formation or establishment, which could add mitochondrial processes to the wide array of pathways that regulate normal cognitive functions.

Parental origin of apparently balanced de novo complex chromosomal rearrangements investigated by microdissection, whole genome amplification, and microsatellite-mediated haplotype analysis

Grossmann V, Höckner M, Karmous‐Benailly H, Liang D, Puttinger R, Quadrelli R, Röthlisberger B, Huber A, Wu L, Spreiz A, Fauth C, Erdel M, Zschocke J, Utermann G, Kotzot D. Parental origin of apparently balanced de novo complex chromosomal rearrangements investigated by microdissection, whole genome amplification, and microsatellite‐mediated haplotype analysis.

Chromosomal microaberrations in patients with epilepsy, intellectual disability, and congenital anomalies

Epilepsy is a common finding in patients with chromosomal macro‐ and micro‐rearrangements but only few aberrations show a constant pattern of seizures. DNA array‐based studies have reported causative copy number variations (CNVs) in 5–30% of patients with epilepsy with or without co‐morbidities. The interpretation of many of the detected CNVs remains challenging. In order to identify CNVs carrying epilepsy‐related genes we investigated 43 children with various patterns of epileptic seizures, intellectual disability (ID), and minor dysmorphism, using the Illumina® Infinium Human1M‐DuoV1 array. In three patients we found likely causative de novo CNVs, i.e. deletions in 1q41q42.12 (3.4 Mb) and 19p13.2 (834 kb), and a mosaic two‐segment duplication in 17p13.2 (218 kb) and 17p13.1 (422 kb). In six additional patients there were aberrations (a deletion in one and duplications in five patients) with uncertain clinical consequences. In total, the finding of causative chromosomal micro‐rearrangements in 3 out of 43 patients (7%) and potentially causative CNVs in 6 additional patients (14%) with epilepsy and ID but without major malformations confirms the power of DNA arrays for the detection of new disease‐related genetic regions.

Parental Origin of de novo Cytogenetically Balanced Reciprocal Non-Robertsonian Translocations

De novo cytogenetically balanced reciprocal non-Robertsonian translocations are rare findings in clinical cytogenetics and might be associated with an abnormal phenotype. Knowledge of the parental origin and mechanisms of formation is still limited. By microdissection of the derivative chromosomes and their normal homologs from metaphases followed by microsatellite-mediated marker analysis we identified 7 cases of paternal and 3 cases of maternal origin in a cohort of 10 patients with de novo cytogenetically balanced reciprocal non-Robertsonian translocations. Neither in the maternal nor in the paternal group of our study parental age seems to be increased. Together with the data from the literature our results confirm that the majority of de novo cytogenetically balanced reciprocal translocations are of paternal origin, but the preponderance does not appear to be as distinct as previously thought and the paternal age does not seem to be necessarily a major contributing factor.

Microdeletion 19p13.2 in an almost 5-year-old boy.

Deletions of the short arm of chromosome 19 are rarely found by conventional cytogenetic techniques. This region has a high gene density and this is likely the reason why deletions in this region are associated with a severe phenotype. Since the implementation of modern high‐resolution SNP‐ and CGH‐array techniques more cases have been reported. Here, we present an almost 5‐year‐old boy with intellectual disability, minor dysmorphisms, febrile seizures, and a de novo deletion of 834.2 kb on 19p13.2 encompassing 32 genes. The deletion was found by the Illumina® Infinium HD Human1M‐Duo v1 BeadChip SNP‐array and confirmed by the NimbleGen Human CGH 2.1M Whole Genome Tiling v2.0D oligonucleotide array. PCR amplification of the junction fragment and subsequent sequencing defined the breakpoints and indicated that formation was mediated by non‐allelic homologous recombination (NAHR). The phenotype of our patient shows that microrearrangements even at gene‐dense chromosomes may result in mild clinical consequences.

Parental origin and mechanism of formation of a 46,X,der(X)(pter--<q21.1::p11.4--<pter)/45,X karyotype in a woman with mild Turner syndrome

OBJECTIVE To describe the parental origin and the mechanism of formation of a 46,X,der(X)(pter-->q21.1::p11.4-->pter)[23]/45,X[8] karyotype in a patient with mild Turner syndrome. DESIGN Case report. SETTING A university hospital. PATIENT(S) A 23-year-old woman with normal height, gonadal dysgenesis, and mild Turner stigmata. INTERVENTION(S) Genotype-phenotype correlation, array-based copy number analysis, fluorescence in situ hybridization with locus-specific probes, and microsatellite marker-mediated haplotype analysis subsequent to whole genome amplification of microdissected chromosomes. MAIN OUTCOME MEASURES Genotype-phenotype correlation, mechanism of formation, and parental origin. RESULT(S) Formation in paternal meiosis by refolding in itself and unequal recombination between Xp and Xq were found as the most likely mechanism of formation. CONCLUSION(S) Formation of der(X) chromosomes in females can be more complex than previously thought. The nearly normal height of this patient could be explained by a combination of trisomy of the Xp-located SHOX gene and mosaicism with a 45,X cell line.