An oligonucleotide based array‐CGH system for detection of genome wide copy number changes including subtelomeric regions for genetic evaluation of mental retardation

Abstract

Developmental delay (DD) and mental retardation (MR) are important child heath issues with a one percent prevalence. Karyotyping with or without subtelomeric FISH (fluorescent in situ hybridization), unless the phenotype of the patient suggests a specific aberration for a specific FISH assay, is the most common procedure in cytogenetic evaluation of MR/DD. In addition, there are several platforms utilizing microarray based comparative genomic hybridization technology (array-CGH) for genetic testing. Array-CGH can detect deletions or duplications in very small segments of chromosomes and the use of this technology is expected to increase the diagnostic yield. The major limitation of the current BAC based array technologies is the low resolution ( approximately 1 Mb) of the chip and suboptimal coverage particularly in the subtelomeric regions. Our aim was to design a novel array-CGH chip with high-density of probes in the subtelomeric regions as well as to maintain sufficient density in other regions of the genome to provide comprehensive coverage for DD/MR. For this purpose, we used Human Genome CGH Microarray 44B chip (Agilent) as the template for the novel design. Using e-array 4.0 (Agilent), one third of the probes were randomly removed from the array and replaced by 14,000 subtelomeric probes. The average density of the probe coverage is 125 kb and 250-400 probes interrogate subtelomeric regions. To evaluate the array, we tested 15 samples (including subtelomeric aberrations and other microdeletion syndromes), which were previously analyzed by karyotyping and/or FISH. The concordance rate between array results and previous results is 100%. In addition we detected two novel aberrations that were not detected by karyotyping. These results demonstrate the utility of this format of array-CGH in detecting genome wide submicroscopic copy number changes as well as providing comprehensive coverage of all subteleomeric regions.

Molecular and immunohistochemical evidence for the origin of uterine leiomyosarcomas from associated leiomyoma and symplastic leiomyoma-like areas

“…The leiomyoma-like and uterine leiomyosarcoma areas from six cases were examined using highdensity oligonucleotide array-CGH (comparative genomic hybridization) technology 10 (Agilent 44K arrays) to compare genetic aberrations in these two areas.…”

A functional network module for Smith–Magenis syndrome

“…A value of $1.8-2.0 was considered good and used for downstream assays. Firststrand cDNA synthesis was carried out with 500 ng of total RNA using MMLV reverse transcriptase and 1 ml oligo(dT) [12][13][14][15][16][17][18] (500 mg/ml) primers according to manufacturer's instructions (Invitrogen). For quantitative real-time PCR, pre-designed Assays on Demand Gene Expression Products (Applied Biosystems, Foster City, CA) Taqman Ò MGB probes and primers for 10 genes (RAI1, ZIC1, LIPE, HMGCS1, INSIG1, SMN4, NR1D2, ZNF236, PIK3R1, and GAPDH) were used (Applied Biosystems Inc., Foster City, CA).…”

“…Recently identified 9q34 deletion (similarity in craniofacial profile) and Sotos syndrome cases (overgrowth phenotypes in RAI1 mutation cases) are also good candidates for SMS-like phenotypes (2). We then screened published reports of chromosomal rearrangements in cases with mental retardation/developmental delay/behavioral deficit for phenotypes that were similar to SMS (10)(11)(12)(13)(14)(15)(16)(17). These cases were ruled out previously for common chromosomal anomalies by high-resolution banding or karyotyping.…”

Oligonucleotide arrays for high-resolution analysis of copy number alteration in mental retardation/multiple congenital anomalies

“…67 Higher-density arrays are now available in both preconfigured and custom formats from Agilent for array CGH applications. A customized array created on the backbone of Agilent's Human Genome CGH microarray kit 44B was used to evaluate genome-wide copy number alterations, with special emphasis on subtelomeric regions, in patients with developmental delay and MR. 68 This approach accurately detected 15 previously well-characterized subtelomeric aberrations and microdeletion syndromes that ranged in size from 600 Kb to 154 Mb. 68 The utility of this array in clinical diagnosis was demonstrated by the successful detection of two novel, previously undetectable aberrations in patients with MR.…”

“…This included a 3-Mb deletion in 14q11 and a 3.5-Mb deletion in 17q24-q25.1. 68 High-resolution, oligonucleotide-based array platforms for CGH are also available commercially from NimbleGen Systems Inc. (Madison, WI) and Illumina Inc. (San Diego, CA). NimbleGen offers preconfigured genome-wide and custom tiling arrays containing long oligonucleotides (45-85 mers) for direct CGH.…”