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High-density oligonucleotide array with sub-kilobase resolution reveals breakpoint information of submicroscopic deletions in nevoid basal cell carcinoma syndrome

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Abstract

Small submicroscopic genomic deletions and duplications constitute up to 15% of all mutations underlying human monogenic diseases. In this study, we used newly designed high-resolution oligonucleotide microarrays with a median distance between the probes of 776 bp (average probe interval 2,271 bp) to detect gene deletions in nevoid basal cell carcinoma syndrome (NBCCS) patients. NBCCS, also called Gorlin syndrome, is characterized by developmental defects and tumorigenesis such as medulloblastomas and basal cell carcinomas, caused by mutations of the human patched-1 (PTCH1) gene. Two out of three deletions could not be detected by a conventional chromosomal analysis. A submicroscopic deletion as small as 165 kb was detected affecting only PTCH1, whereas the other two deletions were much larger (5 and 11 Mb). We demonstrated not only the exact number of genes involved in the deletion but also rapidly determined the junction sequences after pinpointing the breakpoint regions in all individuals analyzed. This report of an array-based determination of junction sequences of long deletions circumvented a labor-intensive analysis such as Southern blotting or FISH. Alu-mediated recombination in one case and non-homologous end joining in the other two were probably implicated in the generation of deletions. This method will contribute to the understanding of molecular pathogenesis of gene deletions as well as rapid genetic testing.

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Acknowledgments

We thank Mayu Yamazaki-Inoue and Hiroko Meguro for their technical support, and Kayoko Saito for preparing the manuscript. This work was supported by the Naito Foundation (T.M.), Grants for Cancer Research from the Ministry of Health, Labour and Welfare (T.M.), a Grant-in-Aid for Scientific Research and the Budget for Nuclear Research from the Ministry of Education, Culture, Sports, Science and Technology (T.M.), the Core Research for Evolutional Science and Technology (CREST) from the Japan Science and Technology Agency (H.A.), Grants-in-Aid for Young Scientists, and a Grant-in-Aid for Scientific Research on Priority Areas ‘Applied Genomics’ from the Ministry of Education, Culture, Sports, Science and Technology of Japan (S.I.).

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Authors and Affiliations

  1. Department of Pediatrics, Chiba University Graduate School of Medicine, Chiba, 260-8670, Japan

    Katsunori Fujii, Hideki Uchikawa & Yoichi Kohno

  2. Division of Genome Science, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, 153-8904, Japan

    Shumpei Ishikawa & Hiroyuki Aburatani

  3. Department of Genetics, National Research Institute for Child Health and Development, 2-10-1 Okura, Setagaya-ku, Tokyo, 157-8535, Japan

    Hideki Uchikawa, Masao Yamada & Toshiyuki Miyashita

  4. Dependable and High Performance Computing, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, 153-8904, Japan

    Daisuke Komura

  5. Affymetrix, Inc., Santa Clara, CA, 95051, USA

    Michael H. Shapero, Fan Shen, Jing Hung & Keith W. Jones

  6. Department of Pediatric Neurology, Morinomiya Hospital, Osaka, 536-0025, Japan

    Hiroshi Arai

  7. Department of Pediatrics, Tokyo Dental College Ichikawa General Hospital, Chiba, 272-8513, Japan

    Yoko Tanaka

  8. Department of Pediatrics, Sapporo Ayumino-sono, Hokkaido, 003-0859, Japan

    Kimio Sasaki

  9. Japan Science and Technology Agency, Saitama, 332-0012, Japan

    Hiroyuki Aburatani

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  1. Katsunori Fujii
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  2. Shumpei Ishikawa
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  14. Hiroyuki Aburatani
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  15. Toshiyuki Miyashita
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Corresponding author

Correspondence to Toshiyuki Miyashita.

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439_2007_419_MOESM1_ESM.tif

Supplementary Fig. S1 Two-color FISH analyses using a BAC clone, RP11-163C13 (Invitrogen) (green), encompassing PTCH and an intrachromosomal control probe on 9p11-q11 (CEP9, Abbott) (red) confirmed a heterozygous 9q22.32 deletion in G5, G10 and G19 (A, B and C, respectively; arrows). Lymphoblastoid cells obtained from a healthy individual showed no evidence of deletion (D) (TIF 1.16 mb)

439_2007_419_MOESM2_ESM.tif

Supplementary Fig. S2 Junction-specific PCR. (A) Results of the agarose gel electrophoresis for junction-specific PCR analyses revealed amplification from patients but not from normal controls (Cont). Primers used are indicated at the bottom. Their sequences are listed in Table S2. Two PCR products from different primers gave identical junction sequence in each patient. (B) Junction-specific amplification was also observed in the mother of G19 (G19M). The junction sequence of G19M was revealed to be identical to that in G19. (C) Schematic representation of the position of primers used in this experiment (TIF 460 kb)

Supplementary Data, Table S1 (DOC 41 kb)

Supplementary Data, Table S2 (DOC 31 kb)

Supplementary Data, Table S3 (DOC 34.5 kb)

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Fujii, K., Ishikawa, S., Uchikawa, H. et al. High-density oligonucleotide array with sub-kilobase resolution reveals breakpoint information of submicroscopic deletions in nevoid basal cell carcinoma syndrome. Hum Genet 122, 459–466 (2007). https://doi.org/10.1007/s00439-007-0419-y

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