The SALSA MLPA Probemix P163 GJB-WFS1-POU3F4 is an in vitro diagnostic (IVD) or research use only (RUO) semi-quantitative manual assay for the detection of deletions in the GJB2, GJB6, WFS1, and POU3F4 genes, microdeletions upstream of POU3F4, and the wildtype sequence of six specific mutations in the GJB2 gene in genomic DNA isolated from human peripheral whole blood specimens. P163 GJB-WFS1-POU3F4 is intended to confirm a potential cause for hereditary hearing loss, and to confirm a potential cause for and clinical diagnosis of Wolfram syndrome type 1. This assay can also be used for molecular genetic testing of at-risk family members.

Certain probes targeting additional genes included in P163 GJB-WFS1-POU3F4 may only be used in a research setting. The following table summarises which probes are for IVD, and which are exclusively restricted to RUO use.

For the full intended purpose, see the product description.

Hearing loss is a common congenital defect and is defined as a sensorineural loss of 35 dB or more. The estimated incidence of permanent hearing loss at birth is one in 500 newborns. The origins of hearing loss may be genetic, environmental or multifactorial, with at least 50% of prelingual hearing loss in industrialised countries attributable to genetic abnormalities (GeneReviews). More than 70% of hereditary hearing loss is non-syndromic; the remaining 30% is accompanied by additional clinical findings and is therefore 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, Non-syndromic, autosomal recessive 1) locus on 13q12 (GeneReviews). This locus contains the GJB2 and GJB6 genes, encoding connexin 26 and 30 proteins, respectively. The majority of individuals (99%) with DFNB1 are either homozygous or compound heterozygous for GJB2 pathogenic SNV variants. The remaining 1% are compound heterozygous for one GJB2 pathogenic SNV variant and a deletion in the GJB2 gene or one of three large deletions that includes sequences upstream of GJB2, such as CRYL1 and a portion of GJB6. The vast majority of pathogenic variants in GJB2 are SNVs or indels. While the frequency of mutations in GJB2 vary significantly across populations, commonly occurring mutations include c.35delG, c.101T>C, c.167delT, c.235delC, c.-23+1G>A (IVS1+1G>A), and 313del14. Large deletions account for less than 1% of pathogenic variants in GJB2. Two large deletions upstream of the GJB2 gene, del(GJB6-D13S1830) and del(GJB6-D13S1854) in the GJB6 gene, are the most common GJB6 mutations in hereditary hearing loss patients, and most often co-occur with mutations in GJB2 (del Castillo et al. 2005; del Castillo et al. 2003).

It is estimated that roughly 1% of hereditary hearing loss is caused by genes on the X-chromosome and, of these, 50% are caused by mutations in the POU3F4 gene or its upstream regulatory elements, known as X-linked non-syndromic hearing impairment (DFNX2 or DFN3). The POU3F4 gene is located on the X chromosome in a 3 Mb gene desert region enriched for highly conserved non-coding regions. Mutations in this region are most commonly SNVs and indels, but large deletions have also been reported (Feng et al. 2024, Vore et al. 2005, Song et al. 2010).

Wolfram syndrome type 1 (WFS1) is a progressive neurodegenerative disorder characterised 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 (GeneReviews). WFS1 is caused by mutations of WFS1 gene, which encodes the wolframin protein. This is a very rare form of hereditary hearing loss and exists on a spectrum, with clinical severity often reflecting the underlying genetic variant. The majority (>95%) of pathogenic variants in the WFS1 gene are SNVs or indels, though large deletions in WFS1 have also been reported (Resmerita et al. 2020, Riza et al. 2022).

SALSA MLPA Probemix P163 GJB-WFS1-POU3F4 is CE-marked under the IVDR for in vitro diagnostic (IVD) use in Europe.

This assay is for research use only (RUO) in all other territories.

Inclusion of a positive sample is usually not required, but can be useful for the analysis of your experiments. MRC Holland has very limited access to positive samples and cannot supply such samples. We recommend using positive samples from your own collection. Alternatively, you can use positive samples from an online biorepository, such as the Coriell Institute.

The commercially available positive samples below can be used with the current (E1) version of this product.

• Coriell HG00478 (m): Heterozygous positive for the GJB2 c.235delC mutation.
• Coriell NA12606 (m): Large heterozygous duplication (42 Mb) affecting the probes for GJB2, GJB6, CRYL1 and flanking probes.
• Coriell NA22601 (m): Heterozygous deletion affecting the probes for WFS1.
• Coriell NA23835 (m): Heterozygous positive for the GJB2 35delG and the 101T>C mutations.

Related digitalMLPA probemixes

Target many more regions simultaneously using our digitalMLPA technology, which combines the trusted MLPA technology with the power of NGS.

• D028 Carrier Panel 1: Targets 9 regions associated with autosomal recessive or X-linked disorders that are frequently tested for in carrier analysis: SMN1, DMD, CYP21A2, HBA, HBB, CFTR, DFNB1, CLN3, and CTNS.

Other related products

• P153 EYA1: Targets EYA1, involved in branchio-oto-renal dysplasia (BOR) syndrome.
• P186 PAX3-MITF-SOX10: Targets PAX3, MITF and SOX10, involved in Waardenburg syndrome (WS).
• P191 X-linked Alport syndrome mix 1: Targets COL4A5, involved in X-linked Alport syndrome. Intended to be used together with P192.
• P192 X-linked Alport syndrome mix 2: Targets COL4A5, involved in X-linked Alport syndrome. Intended to be used together with P191.
• P280 SLC26A4: Targets SLC26A4, involved in Pendred syndrome and autosomal recessive non-syndromic defaness (DFNB4).
• P292 PCDH15: Targets PCDH15, involved in Usher syndrome.
• P361 USH2A mix 1: Targets USH2A, involved in Usher syndrome and retinitis pigmentosa (RP). Intended to be used together with P362.
• P362 USH2A mix 2: Targets USH2A, involved in Usher syndrome and retinitis pigmentosa (RP). Intended to be used together with P361.
• P381 COL11A1 mix 1: Targets COL11A1, involved in Marshall syndrome and type II Sticker syndrome. Intended to be used together with P382.
• P382 COL11A1 mix 2: Targets COL11A1, involved in Marshall syndrome and type II Sticker syndrome. Intended to be used together with P381.
• P461 STRC-CATSPER2-OTOA: Targets CATSPER2, STRC and OTOA, involved in deafness infertility syndrome (DIS), autosomal recessive deafness 16 (DFNB16) and autosomal recessive deafness 22 (DFNB22).

General MLPA resources

• MLPA technique overview
• Coffalyser.Net overview

MLPA education

• Getting started with MLPA
• Using Coffalyser.Net to analyse MLPA data
• Help centre with other learning resources

Selected publications using P163 GJB-WFS1-POU3F4

• Arunachalam RK et al. (2020). Mutation Analysis Using Multiplex Ligation-Dependent Probe Amplification in Consanguineous Families in South India with a Child with Profound Hearing Impairment. Lab Med. 51:56-65.
• Da Silva-Costa SM et al. (2009). Screening for the GJB2 c.-3170 G>A (IVS 1+1 G>A) mutation in Brazilian deaf individuals using multiplex ligation-dependent probe amplification. Genet Test Mol Biomarkers. 13:701-4.
• Da Silva-Costa SM et al. (2011). Searching for digenic inheritance in deaf Brazilian individuals using the multiplex ligation-dependent probe amplification technique. Genet Test Mol Biomarkers. 15:849-53.
• Küçük Kurtulgan H et al. (2019). The Analysis of GJB2, GJB3, and GJB6 Gene Mutations in Patients with Hereditary Non-Syndromic Hearing Loss Living in Sivas. J Int Adv Otol. 15:373-8.
• Özyilmaz B et al. (2019). First-Line Molecular Genetic Evaluation of Autosomal Recessive Non-Syndromic Hearing Loss. Turk Arch Otorhinolaryngol. 57:140-8.
• Petrova N et al. (2023). Hereditary etiology of non-syndromic sensorineural hearing loss in the Republic of North Ossetia-Alania. PeerJ. 11:e14514.
• Prochazkova D et al. (2016). A p.(Glu809Lys) Mutation in the WFS1 Gene Associated with Wolfram-like Syndrome: A Case Report. J Clin Res Pediatr Endocrinol. 8:482-3.
• Resmerita I et al. (2020). Genetics of Hearing Impairment in North-Eastern Romania-A Cost-Effective Improved Diagnosis and Literature Review. Genes (Basel). 11:1506.
• Sansovic I et al. (2009). GJB2 mutations in patients with nonsyndromic hearing loss from Croatia. Genet Test Mol Biomarkers. 13:693-9.
• Smeds H et al. (2022). X-linked Malformation Deafness: Neurodevelopmental Symptoms Are Common in Children With IP3 Malformation and Mutation in POU3F4. Ear Hear. 43:53-69.

References

• Naranjo S et al. (2010). Multiple enhancers located in a 1-Mb region upstream of POU3F4 promote expression during inner ear development and may be required for hearing. Hum Genet. 128:411-9.
• Song MH et al. (2010). Clinical evaluation of DFN3 patients with deletions in the POU3F4 locus and detection of carrier female using MLPA. Clin Genet. 78:524-32.
• Vore AP et al. (2005). Deletion of and novel missense mutation in POU3F4 in 2 families segregating X-linked nonsyndromic deafness. Arch Otolaryngol Head Neck Surg. 131:1057-63.