Detection and characterization of NF1 microdeletions by custom high resolution array CGH

Abstract

In 5% to 10% of cases, neurofibromatosis type 1 is caused by microdeletions scattered across the entire NF1 gene and various neighboring genes. The phenotype appears to be more severe in patients with NF1 microdeletions than in patients with NF1 single point mutations. We have developed a new method for detecting and characterizing NF1 microdeletions based on a custom high-resolution oligonucleotide array comparative genomic hybridization by using the custom 8x15K Agilent array format. The array comprised a total of 14,207 oligonucleotide probes spanning the whole of chromosome 17, including 12,314 probes spanning an approximately 8 Mb interval surrounding the NF1 locus. We validated this approach by testing NF1 microdeleted DNA samples previously characterized by means of microsatellites and real-time PCR methods. Our array comparative genomic hybridization provided enough information for subsequent long-range PCR and nucleotide sequencing of the microdeletion endpoints. Unlike previously described methods, our array comparative genomic hybridization was able to unambiguously differentiate between the three types of microdeletions (type I, type II, and atypical) and to characterize atypical microdeletions. Further comparative studies of patients with well-characterized genotypes and phenotypes and different microdeletions sizes and breakpoints will help determine whether haploinsufficiency of deleted genes and/or genes rearrangements influence clinical outcomes.

Figure 1

Multistep design of the high-resolution 15K NF1 array CGH. The array comprised 15,689 60-mer probes (with 1482 manufacturer control probes) spanning coding and non-coding genomic sequences of chromosome 17 with regions of various probe densities. DNA sequence information referred to the public UCSC database (Human Genome Browser, March 2006 assembly: hg 18, NCBI Build 36.1).

Figure 2

A: Array CGH profiles of four deletions of the eight characterized deletions: one type I (NF00437), one type II (NF00085), and two atypical (NF00028, NF00358) deletions were easily identified. B: Zoom on telomeric breakpoints: SUZ12 is entirely deleted in the type I deletion (NF01470) and partially deleted in type II (NF00085). C: Electrophoregram of part of the specific junction sequence generated by long-range PCR from atypical deletions NF00028 and NF00358.