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Characterisation of breakpoints in disease-associated balanced chromosome rearrangements (DBCRs), which disrupt or inactivate specific genes, has facilitated the molecular elucidation of a wide variety of genetic disorders. However, conventional methods for mapping chromosome breakpoints, such as in situ hybridisation with fluorescent dye-labelled bacterial artificial chromosome clones (BAC-FISH), are laborious, time consuming and often with insufficient resolution to unequivocally identify the disrupted gene. By combining DNA array hybridisation with chromosome sorting, the efficiency of breakpoint mapping has dramatically improved. However, this can only be applied when the physical properties of the derivative chromosomes allow them to be flow sorted. To characterise the breakpoints in all types of balanced chromosome rearrangements more efficiently and more accurately, we performed massively parallel sequencing using Illumina 1G analyser and ABI SOLiD systems to generate short sequencing reads from both ends of DNA fragments. We applied this method to four different DBCRs, including two reciprocal translocations and two inversions. By identifying read pairs spanning the breakpoints, we were able to map the breakpoints to a region of a few hundred base pairs that could be confirmed by subsequent PCR amplification and Sanger sequencing of the junction fragments. Our results show the feasibility of paired-end sequencing of systematic breakpoint mapping and gene finding in patients with disease-associated chromosome rearrangements. European Journal of Human Genetics (2010) 18, 539-543; doi:10.1038/ejhg.2009.211; published online 2 December 2009

We report on a 2-year-old girl with situs ambiguus comprising right-sided stomach and spleen, left-sided liver and complex cardiac defect. Psychomotor development of this patient was normal, and no other major abnormalities were present. Chromosome analysis revealed a de novo balanced chromosome translocation t(X;1)(q26;p13.1). Molecular cytogenetic investigations identified a breakpoint spanning BAC clone on the X-chromosome containing the ZIC3 gene. Mutations in ZIC3 are associated with situs ambiguus and cardiac defects predominantly in males. This is the first report of a live born girl with an X-autosome translocation involving the ZIC3 region.

We have identified a girl with characteristic features of Rett syndrome (RTT) who carries a de novo balanced translocation involving chromosomes 1 and 7. Both breakpoints were mapped by fluorescence in situ hybridization with selected genomic clones from the regions of interest. Southern blot hybridisations, utilizing probes derived from breakpoint spanning BACs, detected several aberrant fragments specific for the patient. Sequence analysis of the cloned junction fragment indicated that on chromosome 1 the predominantly brain-expressed Netrin G1 (NTNG1) gene is disrupted, whereas on chromosome 7 there was no indication for a truncated gene. The chromosome 1 breakpoint lies within the 3' part of NTNG1 and affects alternatively spliced transcripts, suggesting that the phenotype in this patient is the result of disturbed NTNG1 expression. In silico translation of the NTNG1 splice variants predicted protein isoforms with different C-termini: one membrane bound through a glycosylphosphatidylinositol anchor and the other soluble. The membrane-bound protein isoform would be affected by the breakpoint, whereas the soluble form would remain intact. Our results suggest that the central nervous system is sensitive to NTNG1 expression levels and that NTNG1 is a novel candidate disease gene for RTT.

Cryptic subtelomeric chromosome rearrangements play an important role in the aetiology of mental retardation, congenital anomalies, miscarriages and neoplasia. To facilitate a comprehensive molecular-cytogenetic analysis of these extremely gene-rich and mutation-prone chromosome regions, novel multicolour fluorescence in situ hybridisation (FISH) techniques are being developed. As yet, subtelomeric FISH methods have either had limited multiplicities, making it necessary to perform many hybridisations per patient, or a limited scope of analysable chromosome mutation types, thus not detecting some aberration types such as pericentric inversions or very small aberrations. COBRA (COmbined Binary RAtio) labelling is a generic multicolour FISH technique that combines ratio and combinatorial labelling to attain especially high multiplicities with few fluorochromes. The Subtelomere COBRA FISH method ("S-COBRA FISH") described here detects efficiently all 41 BAC and PAC FISH probes necessary for a complete subtelomere screening in only two hybridisations. It was applied to the analysis of 10 cases with known and partially known aberrations and successfully detected balanced and unbalanced translocations, deletions and an unbalanced pericentric inversion in a mosaic situation. The ability of S-COBRA FISH to efficiently detect all types of balanced and unbalanced subtelomeric chromosome aberrations makes it the most comprehensive diagnostic procedure for human subtelomeric chromosome regions described to date.

Approximately 15-20% of all clinically recognised pregnancies abort, most commonly between 8-12 gestational weeks. While the majority of early pregnancy losses is attributed to cytogenetic abnormalities, the aetiology of approximately 40% of early abortions remains unclear. To determine additional factors causing spontaneous abortions we retrospectively searched for uniparental disomies (UPD) in 77 cytogenetically normal diploid spontaneous abortions. In all cases an unbalanced chromosome anomaly was ruled out by cytogenetic investigation of chorionic/amniotic membranes and/or chorionic villi. For UPD screening microsatellite analyses were performed on DNA of abortion specimens and parental blood using highly polymorphic markers showing UPD in two cases. The distribution of markers analysed indicated maternal heterodisomy for chromosome 9 (UPhD(9)mat) in case 1 and paternal isodisomy for chromosome 21 (UPiD(21)pat) in case 2. The originating mechanism suggested was monosomy complementation in UPiD(21)pat and trisomy rescue in UPhD(9)mat. In the case of UPhD(9)mat purulent chorioamnionitis was noted and a distinctly growth retarded embryo of 3 cm crown-rump length showing no gross external malformations. Histological analysis in the case of UPiD(21)pat suggested a primary anlage defect. Our results indicate that less than 3% of genetically unexplained pregnancy wastage is associated with total chromosome UPD. UPD may contribute to anlage defects of human conception. Chromosome aneuploidy correction can occur in very early cleavage stages. More research, however, ought to be performed into placental mosaicism to further clarify timing and mechanisms involved in foetal UPD.