General information
The SALSA MLPA Probemix P180 Limb Malformations-2 is a research use only (RUO) assay for the detection of deletions or duplications in the SALL1, SALL4, and TBX5 genes, which are associated with limb malformations.

The SALL1 gene product is a C2H2 zinc finger DNA binding protein which is associated with pericentromeric heterochromatin, and is a strong translational repressor. Mutations in SALL1 cause Townes-Brocks syndrome (TBS), characterised by radial limb malformation, anal abnormalities, abnormal shaped pinnae, and renal anomalies. All cases of TBS described so far are caused by point mutations or small deletions/insertions leading to a truncated protein product.

The SALL4 gene product is a C2H2 zinc finger DNA binding protein, which is thought to function as a transcription factor. Mutations in SALL4 cause a variety of phenotypes, including Okihiro syndrome (Duane-radial ray syndrome), characterised by an inability to abduct the eye and radial dysplasia, acro-renal-ocular syndrome, Holt-Oram Syndrome (see below), and malformations previously attributed to thalidomide embryopathy. Most mutations are point mutations or small deletions/insertions leading to a truncated protein product, however recently six large deletions have been associated with Okihiro syndrome.

The TBX5 gene product is a transcription factor of the T-box family which is particularly important for heart and upper limb development. Point mutations and deletions in TBX5 have been shown to result in Holt-Oram syndrome (HOS), characterised by radial ray and cardiac (ASD, VSD, Mitral valve prolapse) defects. As the last three exons of TBX5 are separated by large introns, two probes have been included for these exons.

More information is available at https://www.ncbi.nlm.nih.gov/books/NBK1373/, https://www.ncbi.nlm.nih.gov/books/NBK1111/ and https://www.ncbi.nlm.nih.gov/books/NBK1445/.

Probemix content
The SALSA MLPA Probemix P180-B4 Limb Malformations-2 contains 29 MLPA probes with amplification products between 128 and 382 nucleotides (nt). This includes 13 probes for the TBX5 gene, three probes for the SALL1 gene and four probes for the SALL4 gene. In addition, nine reference probes are included that detect autosomal chromosomal locations. Complete probe sequences and the identity of the genes detected by the reference probes are available online (www.mrcholland.com).

This probemix contains nine quality control fragments generating amplification products between 64 and 105 nt: four DNA Quantity fragments (Q-fragments), two DNA Denaturation fragments (D-fragments), one Benchmark fragment, and one chromosome X and one chromosome Y-specific fragment. More information on how to interpret observations on these control fragments can be found in the MLPA General Protocol and online at www.mrcholland.com.