Chapter 4.1 A 400 kb duplication, 2.4 Mb triplication and 130 kb duplication of 9q34.3 in a patient with severe mental retardation

The presence of a duplication as well as a triplication in one chromosome is a rare rearrangement and not easy to distinguish with routine chromosomal analysis. Recent developments in array technologies, however, not only allow screening of the whole genome at a higher resolution, but also make it possible to characterize complex chromosomal rearrangements in more detail. Here we report a molecular cytogenetic analysis of a 16-year old female with severe mental retardation and an abnormality on the end of the long arm of chromosome 9. Subtelomeric multiplex ligation-dependent probe amplification (MLPA) analysis revealed that the extra material originated from the telomeric end of chromosome 9q. Fine mapping using a high-resolution single nucleotide polymorphism (SNP) array detected a duplication of ~400 kb upstream of a ~2.4 Mb triplication followed by a duplication of ~130 kb of chromosome 9q34.3. This study underscores the value of combining conventional karyotyping with novel array technologies to unravel complex chromosomal alterations in order to study their phenotypic impact. 1. Methods of detection 1.1 Cytogenetic analysis Conventional cytogenetic analysis on GTG-banded chromosomes from cultured lymphocytes of the patient was performed according to standard techniques. All metaphases studied demonstrated a 46,XX, add(9)(q34) karyotype (Fig. 4.1.1a). 1.2 MLPA Two specifically designed sets of probes for testing subtelomeric chromosomal imbalances, SALSA P036B and P070 Human Telomere Test Kit (MRC Holland, Amsterdam, The Netherlands), were used for subtelomere screening of the patient and the parents. MLPA experiments were performed as described by MRC Holland (http://www.mlpa.com/pages/indexpag.html) with slight modifications. Amplification products were identified and quantified by capillary electrophoresis on an ABI 3130 genetic analyzer (Applied Biosystems, Nieuwerkerk aan de IJssel, The Netherlands). Peak analysis was performed with the GeneMarker Software V1.51 (SoftGenetics, USA). 1.3 SNParray The Affymetrix GeneChip Human Mapping 238K StyI array was used. This SNP array contains ~238.000 25-mer oligonucleotides with a ~12 kb resolution. A sample of 250 ng DNA was processed according to the instruction provided in the Affymetrix GeneChip Human Mapping 500K Manual (http://www.affymetrix.com). SNP copy number was assessed in the patient using DNA-Chip Analyzer (dChip) software (version release 02-16-06) [8]. Regions of copy number gain and loss were detected using the hidden Markov model output of dChip. 1.4 Chromosomal anomaly To further characterize the extent of the aberration found by karyotyping, subtelomeric MLPA was performed. A duplication of both 9q probes, located in the MRPL41 gene and EHMT1 gene, was observed (Fig. 4.1.1b). Fine mapping of the duplication was performed with a 238K SNP array. This array illustrated a terminal duplication of ~2.93 Mb (Fig. 4.1.1c). Due to exceptional high intensity values from both, the MLPA and the SNP array analyses, a 9q34.3