Sonic hedgehog mutation analysis in patients with VACTERL association

The hedgehog (Hh) genes encode secreted signaling molecules that play critical roles in numerous processes during embryonic and adult development [Bale, 2002]. During development of the human embryo, the gene encoding-Sonic Hedgehog (SHH) is expressed in the notochord, the floorplate of the neural tube, the brain, the zone of polarizing activity in the developing limbs, and the gut [Odent et al., 1999]. VATER, which is an acronym for a non-random association of malformations originally described by Quan and Smith [1973], stands for: vertebral defects (V), anal atresia (A), tracheoesophageal fistula with esophageal atresia (TE), and radial and renal dysplasia (R). Cardiac and limb defects can also be present, expanding the acronym to VACTERL. This spectrum of malformations affects 1 in 5,000 live births. The etiology is considered to be multifactorial, with environmental influences [Khoury et al., 1983]. Based on the observation that Shh and Gli mutant mice exhibited a spectrum of anomalies similar to those observed in VACTERL patients, Kim et al. [2001] proposed that some or most of the VACTERL phenotypes in humans could be explained by aberrations in SHH signaling. In humans, mutations in SHH have been associated with holoprosencephaly [Roessler et al., 1996], while mutations in GLI3 have been associated with Pallister–Hall syndrome [Johnston et al., 2005]. Here, we describe the mutation analysis of SHH in a group of 10 patients who fulfilled three or more of the criteria for VACTERL association. All 10 cases were sporadic, and all of the patients had normal karyotypes and no family history of congenital defects or teratogenic drug exposure. The clinical characteristics of the studied patients are shown in Table I. Informed consent was obtained from all patients, and this work was approved by the ethics committee of the Instituto Nacional de Perinatolog ıa. Blood samples were drawn by venipuncture, and genomic DNA was extracted using the GFX Genomic Purification kit (GE HealthCare Biosciences). The complete coding sequence of the SHH gene, including the exon–intron boundaries, was amplified by PCR. The utilized primer sequences were as follows: exon 1 (forward): TCT CCA GGC TTG CTA CCA TTT, (reverse) GGA CCT TTC GGT GTG TAA G (annealing temperature 54.6 C); (amplified fragment of 664 bp); exon 2 (forward): AGG CTA ACG TGT CCG TCG GT, (reverse) GGA GTT TCC ATC CCC ACT AAA (54.6 C; 495 bp); and exon 3 (forward): CTC CCT CTC GGA ACT CAA TG, (intermediate) TCG GGC TCG GGG CCG CCT T, and (reverse) TGA GTC AGA CCA AGC ACG CGG AA (58.7 C; 900 bp). Direct automated sequencing was performed using the Big Dye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA) in an ABI Prism 310 Genetic Analyzer (Applied Biosystems). Our direct sequence analysis of the complete SHH ORF did not demonstrate any evidence of sequence changes in the 10 studied patients. At present the etiology of the VACTERL association is unknown. It has been suggested that 5% of children may have Fanconi anemia [Esmer et al., 2004]. Alter et al. [2007] suggested that there may be specific BRCA2 mutations linked with the VACTERL association. Furthermore, VACTERL has been associated with a complex IV respiratory chain deficiency [Damian et al., 1996; Thauvin-Robinet