The SALSA MLPA probemix P163 GJB-WFS1-POU3F4 is an in vitro diagnostic (IVD)1
or a research use only (RUO) assay for the detection of deletions or duplications in the human GJB2
genes, genomic microdeletions upstream of POU3F4
, and the presence of six specific mutations in the GJB2
gene in order to confirm a potential cause and clinical diagnosis for hereditary hearing loss. This assay can also be used for the detection of deletions or duplications in the human WFS1
gene in order to confirm a cause and clinical diagnosis for Wolfram Syndrome type 1. This assay is for use with human DNA extracted from peripheral blood. This product can also be used for molecular genetic testing of at-risk family members.
Deletions or duplications obtained with the P163 GJB-WFS1-POU3F4 probemix must be confirmed by another technique. In particular, deletions or duplications detected by only a single probe always require confirmation by another method. Most defects in GJB2
genes are point mutations, the majority of which will not be detected by MLPA. It is therefore recommended to use this SALSA MLPA probemix in combination with sequence analysis of the aforementioned genes. This assay is not intended to be used as a standalone assay for clinical decisions. The results of this test must be interpreted by a clinical molecular geneticist or equivalent.
Please note that this probemix is for In Vitro Diagnostic use (IVD) in the countries specified at the end of this product description. In all other countries, the product is for Research Use Only (RUO).
Hearing loss is a common congenital defect. The estimated incidence of permanent hearing loss at birth, defined as a sensorineural loss of ≥35 dB, is 1 in 500-1000 newborns, and approximately 50 to 60% of all hearing loss cases have been attributed to inherited or genetic factors. Inheritance of hearing loss can be autosomal recessive (75-85%), autosomal dominant (15-24%), X-linked (1-2%) or mitochondrial. More than 70% of hereditary hearing loss is non-syndromic; the remaining 30% is accompanied by additional clinical findings and is considered syndromic.
In many populations, up to 50% of all cases of autosomal recessive non-syndromic hearing loss are caused by mutations in the DFNB1 (Deafness, Neurosensory, autosomal recessive 1) locus on 13q12 (https://www.ncbi.nlm.nih.gov/books/NBK1536/
). This locus contains the genes GJB2
, encoding connexin 26 and 30 protein, respectively. In Caucasians, c.35delG is the most frequent mutation in the GJB2
gene comprising 70% of mutated DFNB1 alleles in some populations with a carrier rate of 1-3% in the general population. Six large deletions in GJB2
have been shown to contribute to DFNB1 hearing loss and account for 1-10% of all the mutated DFNB1 alleles. The most common deletions of 309 kb [del(GJB6-D13S1830)] and 232 kb [del(GJB6-D13S1854)] truncate GJB6
and the upstream CRYL1
gene mutations can cause non-syndromic hearing loss, as well as the skin disorder erythrokeratodermia variabilis (https://www.ncbi.nlm.nih.gov/books/NBK1434/
About half of all X-linked hearing loss cases are caused by pathogenic mutations in the POU3F4
gene and upstream of the gene. Several X-linked hearing loss patients and families contain deletions that do not affect the transcribed region of POU3F4
, but instead remove different portions of DNA upstream of POU3F4
where a putative regulatory element resides. POU3F4
is located on the X chromosome, in a 3-Mb gene desert region enriched for highly conserved non-coding regions (HCNRs). Multiple enhancers, important for POU3F4
expression, are defined within these HNCRs at 970 kb, 920 kb, 170 kb and 12 kb upstream of POU3F4
(Naranjo et al. 2010, Song et al. 2010, Vore et al. 2005).
Wolfram Syndrome is a progressive neurodegenerative disorder characterized by the onset of diabetes mellitus and optic atrophy before the age of 15 and is typically associated with sensorineural hearing loss, progressive neurologic abnormalities, and other endocrine abnormalities. The estimated prevalence of Wolfram syndrome type 1 is 1 in 500,000 people worldwide. Mutations in WFS1
cause more than 90% of Wolfram syndrome type 1 cases (https://www.ncbi.nlm.nih.gov/books/NBK4144/
The SALSA MLPA Probemix P163-E1 GJB-WFS1-POU3F4 probemix contains 51 MLPA probes with amplification products between 121 and 500 nucleotides (nt). This includes two probes for GJB2
and five probes for GJB6
, and several other probes are present for the 13q12 region. Four GJB3
probes are present, covering the two exons. Two probes are included covering exon 1 of the POU3F4
gene, as well as 7 probes targeting the conserved regions up to 1 Mb upstream of POU3F4
. This probemix contains probes for each exon of the WFS1
gene. Furthermore, this probemix contains six probes detecting the wild type sequence of the 313del14, 235delC, 167delT, 101T>C, c.35delG, IVS1+1G>A mutations in the GJB2
gene and a reduced signal can point towards the presence of this mutation or a deletion of GJB2
. In addition, ten reference probes are included that detect autosomal chromosomal locations. Complete probe sequences are available online (www.mlpa.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.mlpa.com