Intended purpose
The SALSA MLPA Probemix P163 GJB-WFS1-POU3F4 is an in vitro diagnostic (IVD)
1 or research use only (RUO) semi-quantitative assay
2 for the detection of deletions or duplications in the
GJB2,
GJB3,
GJB6,
WFS1, and
POU3F4 genes, microdeletions upstream of
POU3F4, and the presence 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 and clinical diagnosis of hereditary hearing loss and Wolfram Syndrome type 1. This assay can also be used for molecular genetic testing of at-risk family members.
Copy number variations (CNVs) detected with P163 GJB-WFS1-POU3F4 should be confirmed with a different technique. In particular, CNVs detected by only a single probe always require confirmation by another method. Most defects in the
GJB2,
GJB3,
POU3F4 and
WFS1 genes are point mutations, the majority of 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
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 nonsyndromic; 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 nonsyndromic hearing loss are caused by mutations in the DFNB1 (Deafness, Nonsyndromic, autosomal recessive 1) locus on 13q12 (
https://www.ncbi.nlm.nih.gov/books/NBK1272/). This locus contains the
GJB2 and
GJB6 genes, encoding connexin 26 and 30 protein, respectively. In Caucasians, the
GJB2 c.35delG is the most frequent mutation, comprising 70% of mutated DFNB1 alleles with a carrier rate of 2-4% in the northern European population. The c.235delC mutation is the most common variant in the Japanese population (carrier rate of 1-2%). With a carrier rate of ~7.5%, the c.167delT mutation is the most frequently occurring mutation in the Ashkenazi Jewish population. The c.101T>C mutation is associated with mild hearing loss and has a minor allele frequency of 1-2%. The IVS1+1G>A and c.313del14 mutations have been found with relatively high frequency among various populations, mainly in eastern Europe, Russia, the Middle East and Asia. Six large deletions in
GJB2 and/or
GJB6 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 kilobases (kb) [del(GJB6-D13S1830)] and 232 kb [del(GJB6-D13S1854)] truncate
GJB6 and the upstream
CRYL1 gene.
GJB3 is located on 1p34, which is the DFNA2B (Deafness, Nonsyndromic, autosomal dominant 2B) locus. Mutations in
GJB3 can cause nonsyndromic hearing loss, as well as the skin disorder erythrokeratodermia variabilis(
https://www.ncbi.nlm.nih.gov/books/NBK1434/).
The
GJB2 and
GJB6 genes are also involved in DFNA3 (Deafness, Nonsyndromic, autosomal dominant 3). DFNA3 is caused by a heterozygous pathogenic mutation in one of the two genes. The relative prevalence of DFNA3 as a cause of autosomal dominant nonsyndromic hearing loss is not known, but it is extremely rare; only 14 pathogenic variants have been described worldwide. The majority of these pathogenic variants are described only in single families or simplex cases. Copy number changes in
GJB2 and
GJB6 related to DFNA3 have not been described (
https://www.ncbi.nlm.nih.gov/books/NBK1536/).
About half of all X-linked hearing loss cases are caused by pathogenic mutations in the
POU3F4 gene and upstream region of the gene. Several X-linked hearing loss patients and families possess deletions that do not affect the transcribed region of
POU3F4, but instead remove different portions of DNA upstream of
POU3F4 where putative regulatory elements reside.
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 HCNRs at 970, 920, 170 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 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. 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/).
Probemix content
The SALSA MLPA Probemix P163-E1 GJB-WFS1-POU3F4 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 seven probes targeting the conserved regions up to 1 Mb upstream of
POU3F4. This probemix contains nine probes for the
WFS1 gene; one probe for each exon and two probes for exon 8. Furthermore, this probemix contains six probes detecting the wild type sequence of the c.313del14, c.235delC, c.167delT, c.101T>C, c.35delG and IVS1+1G>A mutations in the
GJB2 gene and a reduced signal can point towards the presence of this mutation or a (partial) deletion of
GJB2. In addition, ten 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.