Identification of a submicroscopic deletion of SHH associated with the holoprosencephaly spectrum by array‐based CGH

To the Editor: Holoprosencephaly (HPE) results from failure of the cerebrum to form distinct left and right hemispheres and is associated with mental retardation, hypotelorism, nasal defects, single central maxillary incisor, cleft palate, congenital nasal pyriform aperture stenosis, and microcephaly. When these craniofacial findings occur in the absence of true HPE, they may be considered microforms of the HPE spectrum. HPE is etiologically heterogeneous and may be due to chromosomal abnormalities (1) ormutations in several genes (2–11), includingSonic Hedgehog (SHH). Array-based comparative genomic hybridization (aCGH) has been used to detect chromosomal alterations in association with cancers (12, 13), mental retardation (14–16), and fetal malformations (17, 18). In this study, we used aCGH to identify a deletion of SHH in a patient with the HPE spectrum and a normal karyotype. The proband was born to African-American non-consanguineous parents with a normal birth weight. At evaluation at 22 months, features characteristic of the HPE spectrum included single central maxillary incisor, absent upper midline labial frenulum, congenital nasal pyriform aperture stenosis, microcephaly (head circumference 40.7 cm), and global developmental delay. She also had bilateral ptosis and an increased partial thromboplastin time. A brain magnetic resonance imaging showed mild reduction in frontal lobe volume without frank HPE. The karyotype (46,XX, >500 band level), and the subtelomeric fluorescence in situ hybridization (FISH) study were normal. Mutation analyses of the SHH, SIX3, ZIC2, and TGIF genes were normal (GeneDx, Bethesda, MD). Informed consent was provided upon enrollment in the IRB-approved study. The aCGH and FISH analyses were performed as described (19, 20). aCGH was performed to assess for a submicroscopic rearrangement. In aCGH, the relative intensity ratio (Cy3:Cy5 ratio) of the labeled test and control samples indicates the relative copy number. For the patient, the Cy3:Cy5 ratio (when the patient sample was labeled with Cy3) for two bacterial artificial chromosome (BAC) clones, RP11-86L18 and GS1-121J16, were 0.62 and 0.68, respectively (Fig. 1a), consistent with a deletion. RP11-86L18 and GS1-121J16 map to genomic position 153.6–155.0 Mb on 7q36 (UCSCGenome Browser, http://genome.ucsc.edu, May 2004 freeze). This deletion was not present in the patient’s unaffected mother (Fig. 1b). The patient’s father was not available for study. The deleted region was refined by FISH (Table 1). RP11-86L18 showed a signal on only one of the chromosome 7 homologs, consistent with a deletion (Fig. 1c). Additional FISH analyses refined the deletion size to 3.33–4.55 Mb and confirm that the subtelomeric region is not deleted (Table 1, Figs 1d,e). These studies demonstrate that the genomic region containing the entire SHH gene was deleted. Using aCGH, we have detected a submicroscopic deletion of 7q36 that included SHH out of a panel of five patients with the HPE spectrum. Based on previous reports of SHH alterations in association with the HPE spectrum, many of the proband’s features are likely due to haploinsufficiency of SHH. Submicroscopic deletions of SHH have been reported relatively infrequently (21–23). Most patients with the HPE spectrum do not have a defined genetic cause of the condition (11, 24), and aCGH can be a useful method for assessing these patients and could be employed after using quantitative PCR to assess for alterations in known HPE genes. With aCGH, all of the known HPE loci (including those for which a specific HPE-associated gene has not been identified) can be interrogated at once, offering an advantage over FISH analysis. Because aCGH is a genome-wide analysis, Clin Genet 2006: 69: 367–369 # 2006 The Authors Printed in Singapore. All rights reserved Journal compilation# 2006 Blackwell Munksgaard