Intended purpose
The SALSA MLPA Probemix P018 SHOX is an in vitro diagnostic (IVD)¹ or research use only (RUO) semi-quantitative assay² for the detection of deletions or duplications in the human short stature homeobox (SHOX) gene and its regulatory regions on Xp22.33/Yp11.32 in genomic DNA isolated from human peripheral whole blood specimens or buccal swabs. P018 SHOX is intended to confirm a potential cause for disorders associated with short stature, including Leri-Weill dyschondrosteosis (LWD), Langer mesomelic dysplasia (LMD) or Idiopathic short stature (ISS).

Copy number variations (CNVs) detected with P018 SHOX should be confirmed with a different technique. In particular, CNVs detected by only a single probe always require confirmation by another method. In the majority of patients, defects in the SHOX gene region are deletions. However, point mutations can occur which will not be detected by MLPA. It is therefore recommended to use this assay in combination with sequence analysis.

Assay results are intended to be used in conjunction with other clinical and diagnostic findings, consistent with professional standards of practice, including confirmation by alternative methods, clinical genetic evaluation, and counselling, as appropriate. The results of this test should be interpreted by a clinical molecular geneticist or equivalent.

This device is not intended to be used for standalone diagnostic purposes, pre-implantation or prenatal testing, population screening, or for the detection of, or screening for, acquired or somatic genetic aberrations.

¹ Please note that this probemix is for in vitro diagnostic (IVD) use in the countries specified at the end of this product description. In all other countries, the product is for research use only (RUO).
² To be used in combination with a SALSA MLPA Reagent Kit and Coffalyser.Net analysis software.

Clinical background
SHOX is located in the pseudoautosomal region 1 (PAR1) on the short arm of the X and Y chromosomes. Located upstream and downstream of SHOX are highly conserved non-coding elements (CNEs), some of which have been shown to be important SHOX enhancer sequences. Mutations in SHOX or its regulatory regions cause a range of disorders associated with short stature, including LWD, LMD, and ISS, as SHOX is a known transcription factor highly expressed in tissues responsible for bone development (Benito-Sanz et al. 2012b).

LWD is a dominant skeletal disorder characterised by short stature, mesomelic shortening of the limbs, and the characteristic Madelung deformity. LMD is a more severe form of LWD and is a result of mutations in both SHOX alleles (Bertorelli et al. 2007, Campos-Barros et al. 2007, Shears et al. 2002, Zinn et al. 2002). ISS classifies individuals with a height below the third centile in whom no identifiable disorder is present. Heterozygous mutations of SHOX and/or its regulatory elements are detected in approximately 60% of LWD patients and approximately 5-15% of ISS cases. Homozygous or compound heterozygous mutations of SHOX and/or its enhancers are detected in 75% of LMD patients (Benito-Sanz et al. 2006, Benito-Sanz et al. 2012a, Chen et al. 2009, Huber et al. 2006).

In individuals with a SHOX related disorder, 70-80% of SHOX mutations are whole gene deletions, 2-6% are partial deletions, and 20-25% are point mutations (Binder 2011, Caliebe et al. 2012). Duplications have also been reported in LWD and ISS patients (Benito-Sanz et al. 2011b). The P018 SHOX probemix can detect most deletions and duplications and therefore complements sequence analysis of SHOX.

More information is available on http://www.ncbi.nlm.nih.gov/books/NBK1215/.

Probemix content
The SALSA MLPA Probemix P018-G2 SHOX contains 48 MLPA probes with amplification products between 124 and 504 nucleotides (nt). This includes 32 probes for the PAR1 region on chromosome Xp22 / Yp11, including at least one probe for each exon of SHOX transcript variant 1, and one probe for intron 6 only present in the SHOXb splice variant. Several probes are present for SHOX regulatory regions, located upstream and downstream of SHOX. Moreover, this probemix contains multiple flanking probes targeting the X chromosome outside the SHOX area: one probe detecting the area just before the SHOX upstream regulatory regions, five probes inside the PAR1 region but downstream of the SHOX area, and seven probes outside of PAR1. Flanking probes can be used to characterise larger deletions/duplications and to distinguish SHOX deletions from a Turner syndrome karyotype. 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 ten quality control fragments generating amplification products between 64 and 118 nt: four DNA Quantity fragments (Q-fragments), two DNA Denaturation fragments (D-fragments), one Benchmark fragment, and one chromosome X and two chromosome Y-specific fragments. 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.