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Preimplantation diagnosis for homeobox gene HLXB9 mutation causing Currarino syndrome

Currarino syndrome (CS) is a severe autosomal dominant disorder caused by mutation of HLXB9 and involving partial sacral agenesis, presacral mass, and anorectal malformations [MIM, 176450]. This homeobox gene is located on chromosome 7q36, between microsatellite DNA markers D7S559-D7S2423 [Ross et al., 1998]. The abnormalities observed in CS are caused by the disturbances in early embryonic development of the human tail bud, leading to the formation or positioning defects of neural tube, notochord, somites, and hindgut. More than two dozens different HLXB9 intragenic mutations and microdeletions have been detected in patients with CS, including frameshift and nonsense mutations in intron 1 and missense mutations in the homeodomain, resulting in a nonconservative substitution of a highly conserved amino acid [MIM, 143994; Belloni et al., 2000; Hagan et al., 2000; Kochling et al., 2001]. Although a carrier screening and prenatal diagnosis of CS is currently available, the decision about termination of pregnancy will be controversial, as approximately half of the carriers of the mutation are asymptomatic [Kochling et al., 2001]. Therefore, the absence of genotype–phenotype correlation and an extremely high variability of phenotype in the carriers, from the above severe triad to minor sacral abnormalities undetectable without radiographs to even a completely asymptomatic carrier status, make preimplantation genetic diagnosis (PGD) an important alternative for the at-risk couples wishing to avoid CS in their offspring. The couple presented for PGD with a previous child diagnosed to have CS. The child was born with imperforate anus, an anterior meningocoele, and a typical sickleshaped hemisacrum revealed by X-ray of the sacrum region. The father was also born with an anal stricture, requiring anal dilatation, and also had a sacral defect, involving a central anomaly from S2 downward. One of his two sisters was born with imperforate anus and anterior meningocoele and also had rectovaginal fistula, and a vesico-ureteric reflux resulting in the need for renal transplantation; a sacral radiograph showed the same central defect with absence of the distal one-third of the sacrum. The other sister was clinically asymptomatic, but sacral radiograph revealed no coccyx, and an MRI scan disclosed an anterior meningocoele. His mother had undeveloped coccyx and urinary tract bilateral ureteropelvic junction obstruction. Of her three siblings, one of the two brothers had an anal stricture, while the asymptomatic sister was identified as a carrier of the mutation because of the finding of imperforate anus in her grandson. DNA analysis in this family demonstrated the presence of homeobox HLXB9 mutation due to frameshift insertion of a cytosine into a stretch of six cytosines at positions 125–130 in exon 1 of the gene, leading to the introduction of premature termination codon [Ross et al., 1998]. The primer sequences for mutation testing and their positions are presented in Fig. 1 and listed in Table I (see the online Table I at http://www. interscience.wiley.com/jpages/0148-7299/suppmat/index.html). PGD cycle was performed using a standard in vitro fertilization (IVF) protocol coupled with micromanipulation procedures, described elsewhere [Verlinsky and Kuliev, 2000]. Single blastomeres were removed from the eight-cell embryos and tested by multiplex nested PCR analysis, involving specific mutation testing simultaneously with linked marker analysis [Rechitsky et al., 1999; Verlinsky and Kuliev, 2000]. PCR product was identified by fragment length analysis using capillary elecrophoresis (Fig. 1). Prior to initiating PGD cycles a single sperm testing was performed to establish paternal haplotypes. To be able to identify a possible allele drop out (ADO) in the mutation analysis four closely linked microsatellite dinucleotide DNA markers, D7S559, D7S550, D7S637, and D7S594, were tested in the same reaction with the HLXB9 gene [Ross et al., 1998]. The mutant allele was linked to 119, 155, 124, and 162 bp, and the normal allele to 123, 159, 116, and 168 bp repeats of D7S559,D7S550, D7S637, andD7S594markers, respectively. Testing for these markers in the affected child and the mother showed that the parents shared the same size of two of four markers linked to normal allele, making these markers of limited value for preselection of mutation-free embryos for transfer. As per informed consent, approved by our Institutional Review board, based on both mutation and STR analysis, unaffected embryos were preselected for transfer back to patient, while those predicted mutant were exposed to confirmatory analysis using genomic DNA from these embryos to evaluate the accuracy of single cell based PGD. A total of 17 embryos were available for testing in a single PGD cycle. Single blastomeres were removed from these embryos, of which three failed to amplify either HLXB9 alleles or polymorphic markers, suggesting the lack of a nucleus in these blastomeres, and two showed amplification of polymorphic markers only with no signal detected for the HLXB9 gene. Of a total of 12 embryos with results for the mutation and linked marker analysis, 11 were with conclusive results, of which 5 were predicted to contain the mutant allele in agreement with the presence of repeat markers (one of the embryos, #5, is shown in Fig. 1). Another embryo contained only paternal alleles, which may be explained by the absence of the maternal chromosome 7. The remaining six embryos were predicted to be free of mutation, but only in three of them ADO of mutant allele may have been excluded based on the linked marker analysis (two of these embryos are shown in Fig. 1). Two of these embryos were transferred, yielding a singleton pregnancy and birth of a HLXB9 mutation-free child, following confirmation of diagnosis by amniocentesis. In the other three embryos predicted to contain the normal allele, ADO of the mutant gene could not be excluded, because of the parents’ sharing the same size of polymorphic markers linked to the normal allele. This article contains supplementary material, which may be viewed at the American Journal of Medical Genetics website at http://www.interscience.wiley.com/jpages/0148-7299/suppmat/ index.html.