Custom oligonucleotide array-based CGH: a reliable diagnostic tool for detection of exonic copy-number changes in multiple targeted genes.

The frequency of disease-related large rearrangements (referred to as copy-number mutations, CNMs) varies among genes, and search for these mutations has an important place in diagnostic strategies. In recent years, CGH method using custom-designed high-density oligonucleotide-based arrays allowed the development of a powerful tool for detection of alterations at the level of exons and made it possible to provide flexibility through the possibility of modeling chips. The aim of our study was to test custom-designed oligonucleotide CGH array in a diagnostic laboratory setting that analyses several genes involved in various genetic diseases, and to compare it with conventional strategies. To this end, we designed a 12-plex CGH array (135k; 135 000 probes/subarray) (Roche Nimblegen) with exonic and intronic oligonucleotide probes covering 26 genes routinely analyzed in the laboratory. We tested control samples with known CNMs and patients for whom genetic causes underlying their disorders were unknown. The contribution of this technique is undeniable. Indeed, it appeared reproducible, reliable and sensitive enough to detect heterozygous single-exon deletions or duplications, complex rearrangements and somatic mosaicism. In addition, it improves reliability of CNM detection and allows determination of boundaries precisely enough to direct targeted sequencing of breakpoints. All of these points, associated with the possibility of a simultaneous analysis of several genes and scalability 'homemade' make it a valuable tool as a new diagnostic approach of CNMs.

Detection of exonic copy-number changes using a highly efficient oligonucleotide-based comparative genomic hybridization-array method.

Genomic copy-number variations (CNVs) involving large DNA segments are known to cause many genetic disorders. Depending on the changes, they are predicted to lead either to decreased or an increased gene expression. However, the ability to detect smaller exonic copy-number changes has not been explored. Here we describe a new oligonucleotide-based comparative genomic hybridization (CGH)-array approach for high-throughput detection of exonic deletions or duplications and its application to deletion/duplication analyses of the genes encoding CFTR, six sarcoglycans (SGCA, SGCB, SGCG, SGCD, SGCE, and SGCZ), and DMD. In this work we show the successful development of an array format containing 158 exons that collectively span eight genes and its clinical application for the rapid screening of deletions and duplications in a diagnostic setting. We have analyzed a series of 35 DNA samples from patients affected with cystic fibrosis (CF), Duchenne and Becker muscular dystrophies (DMD/BMD), or sarcoglycanopathies, and have characterized exonic copy-number changes that have been validated with other methods. Interestingly, even heterozygous deletions and duplications of only one exon, as well as mosaic deletions, were detected by this CGH approach. Our results showed that the resolution is very high, as abnormalities of about 1.5-2 kb could be detected. Since this approach is completely scalable, this new molecular tool will allow the screening of combinations of genes involved in a particular group of clinically and genetically heterogeneous disorders such as mental retardation, muscular dystrophies and brain malformations.

Revised spectrum of mutations in sarcoglycanopathies.

To define the spectrum of mutations in alpha-, beta-, gamma-, and delta-sarcoglycan (SG) genes, we analyzed these genes in 69 probands with clinical and biological criteria compatible with the diagnosis of autosomal recessive limb-girdle muscular dystrophy. For 48 patients, muscle biopsies were available and multiplex western blot analysis of muscle proteins showed significant abnormalities of alpha- and gamma-SG. Our diagnostic strategy includes multiplex western blot, sequencing of SG genes, multiplex quantitative-fluorescent PCR and RT-PCR analyses. Mutations were detected in 57 patients and homozygous or compound heterozygous mutations were identified in 75% (36/48) of the patients with abnormal western blot, and in 52% (11/21) of the patients without muscle biopsy. Involvement of alpha-SG was demonstrated in 55.3% of cases (26/47), whereas gamma- and beta-SG were implicated in 25.5% (12/47) and in 17% (8/47) of cases, respectively. Interestingly, we identified 25 novel mutations, and a significant proportion of these mutations correspond to deletions (identified in 14 patients) of complete exon(s) of alpha- or gamma-SG genes, and partial duplications (identified in 5 patients) of exon 1 of beta-SG gene. This study highlights the high frequency of exonic deletions of alpha- and gamma-SG genes, as well as the presence of a hotspot of duplications affecting exon 1 of the beta-SG gene. In addition, protein analysis by multiplex western blot in combination with mutation screening and genotyping results allowed to propose a comprehensive and efficient diagnostic strategy and strongly suggested the implication of additional genes, yet to be identified, in sarcoglycanopathy-like disorders.