Human heterotaxy syndrome, or situs ambigu(o)us, is characterised by a wide variety of cardiac and extracardiac congenital malformations that are primarily induced by disorders of the left-right axis determination during early embryonic development (Shiraishi et al. 2012). Several genes have now been implicated in heterotaxy and related isolated congenital heart malformations. Studies indicate that heterotaxy can be caused by single gene mutations and also demonstrate that there is probably extensive locus heterogeneity (Belmont et al. 2004). X-linked heterotaxy-1 (HTX1) is caused by mutations in the ZIC3 gene. Visceral heterotaxy-2 (HTX2) is caused by heterozygous mutations in the CFC1 gene, visceral heterotaxy-4 (HTX4) is caused by heterozygous mutations in the ACVR2B gene, and visceral heterotaxy-5 (HTX5) is caused by heterozygous mutations in the NODAL gene.
The ZIC3 gene comprises 3 exons, spanning 5.9 kb of genomic DNA on Xq26.3, 136 Mb from the p-telomere. The CFC1 gene comprises 6 exons, spanning 7.4 kb of genomic DNA on 2q21.1, 131 Mb from the p-telomere. The ACVR2B gene comprises 11 exons, spanning 38.8 kb of genomic DNA on 3p22.2, 38 Mb from the p-telomere. The NODAL gene comprises 3 exons, spanning 9.7 kb of genomic DNA on 10q22.1, 72 Mb from the p-telomere.
The P434-A1 probemix contains one probe for each exon of the ZIC3, ACVR2B and NODAL genes. Furthermore, the probemix contains four probes for three different exons of the CFC1 gene. Please note that CFC1 is almost completely identical to CFC1B, and that no probes specific for CFC1 could be designed. Therefore, these probes detect both genes. A heterozygous deletion of either gene will result in a reduced signal of approximately 75%. In addition, 8 reference probes are included in this probemix, detecting several different autosomal chromosomal locations.
This SALSA® MLPA® probemix is designed to detect deletions/duplications of one or more sequences in the aforementioned gene(s) in a DNA sample. Heterozygous deletions of recognition sequences should give a 35-50% reduced relative peak height of the amplification product of that probe. Deletions of a probe’s recognition sequence on the X-chromosome will lead to a complete absence of the corresponding probe amplification product in males, whereas female heterozygotes are recognisable by a 35 50% reduction in relative peak height. Note that a mutation or polymorphism in the sequence detected by a probe can also cause a reduction in relative peak height, even when not located exactly on the ligation site! In addition, some probe signals are more sensitive to sample purity and small changes in experimental conditions. Therefore, deletions and duplications detected by MLPA should always be confirmed by other methods. Not all deletions and duplications detected by MLPA will be pathogenic; users should always verify the latest scientific literature when interpreting their findings. We have no information on what percentage of defects in these genes is caused by deletions/duplications of complete exons. Finally, note that most defects in this gene are expected to be small (point) mutations which will not be detected by this SALSA® MLPA® test.