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Multicolour FISH and quantitative PCR can detect submicroscopic deletions in holoprosencephaly patients with a normal karyotype
  1. C Bendavid1,
  2. B R Haddad3,
  3. A Griffin1,
  4. M Huizing1,
  5. C Dubourg2,
  6. I Gicquel2,
  7. L R Cavalli3,
  8. L Pasquier4,
  9. A L Shanske5,
  10. R Long1,
  11. M Ouspenskaia1,
  12. S Odent4,
  13. F Lacbawan1,
  14. V David2,
  15. M Muenke1
  1. 1Medical Genetics Branch, National Human Genome Research Institute, NIH, Bethesda, MD, USA
  2. 2CNRS UMR 6061 Génétique et Développement, Université de Rennes 1, Groupe Génétique Humaine, IFR140 GFAS, Faculté de Médecine, Rennes cédex, France
  3. 3Institute for Molecular and Human Genetics/Lombardi Comprehensive Cancer Center, and Departments of Oncology and Obstetrics and Gynecology, Georgetown University Medical Center, Washington DC, USA
  4. 4Unité de Génétique médicale, Hôpital Sud, Rennes, France
  5. 5Children’s Hospital Montefiore, Center for Craniofacial Disorders, Bronx, NY, USA
  1. Correspondence to:
 Dr M Muenke
 Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, 35 Convent Drive, MSC 3717, Building 35, Room 1B-203, Bethesda, MD 20892-3717, US; mmuenke{at}


Holoprosencephaly (HPE) is the most common structural malformation of the developing forebrain. At birth, nearly 50% of children with HPE have cytogenetic anomalies. Approximately 20% of infants with normal chromosomes have sequence mutations in one of the four main HPE genes (SHH, ZIC2, SIX3, and TGIF). The other non-syndromic forms of HPE may be due to environmental factors or mutations in other genes, or potentially due to submicroscopic deletions of HPE genes. We used two complementary assays to test for HPE associated submicroscopic deletions. Firstly, we developed a multicolour fluorescent in situ hybridisation (FISH) assay using probes for the four major HPE genes and for two candidate genes (DISP1 and FOXA2). We analysed lymphoblastoid cell lines (LCL) from 103 patients who had CNS findings of HPE, normal karyotypes, and no point mutations, and found seven microdeletions. We subsequently applied quantitative PCR to 424 HPE DNA samples, including the 103 samples studied by FISH: 339 with CNS findings of HPE, and 85 with normal CNS and characteristic HPE facial findings. Microdeletions for either SHH, ZIC2, SIX3, or TGIF were found in 16 of the 339 severe HPE cases (that is, with CNS findings; 4.7%). In contrast, no microdeletion was found in the 85 patients at the mildest end of the HPE spectrum. Based on our data, microdeletion testing should be considered as part of an evaluation of holoprosencephaly, especially in severe HPE cases.

  • BAC, bacterial artificial chromosome
  • CNS, central nervous system
  • Ct, threshold cycle number
  • FISH, fluorescent in situ hybridisation
  • HPE, holoprosencephaly
  • LCL, lymphoblastoid cell lines
  • qPCR, quantitative PCR
  • holoprosencephaly
  • microdeletion
  • FISH
  • quantitative PCR

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  • Published Online First 11 March 2006

  • Competing interests: there are no competing interests.