The SALSA MLPA Probemix P461 STRC-CATSPER2-OTOA is an in vitro diagnostic (IVD) semi-quantitative manual assay to be used with genomic DNA isolated from human peripheral blood. The probemix is intended for the detection of deletions in the STRC gene, as well as gene conversions between STRC and its pseudogene STRCP1 to confirm a potential cause for and establish a clinical diagnosis of Autosomal recessive deafness 16 (DFNB16). Moreover, this assay can detect deletions of STRC and CATSPER2 genes, to confirm a potential cause for and establish a clinical diagnosis of STRC-related autosomal recessive hearing loss, including Deafness-infertility syndrome (DIS). Lastly, P461 STRC-CATSPER2-OTOA allows for the detection of deletions in the OTOA gene to confirm a potential cause for and establish a clinical diagnosis of Autosomal recessive deafness 22 (DFNB22). The probemix can also be used for molecular genetic testing of at-risk family members.

For the full intended purpose, see the product description.

Deafness-infertility syndrome (DIS; OMIM # 611102) is characterized by early-onset deafness in males and females and infertility exclusively in males. The hearing loss in DIS patients is non-progressive and the vestibular function is normal. Furthermore, the degree of hearing loss is moderate to severe. DIS is inherited in an autosomal recessive manner, as a contiguous gene deletion syndrome resulting from a homozygous deletion of the STRC-CATSPER2 genes.

The STRC gene has also been associated to autosomal recessive deafness 16 (DFNB16; OMIM # 603720), a nonsyndromic hearing loss, with prelingual onset. The auditory phenotype in DFNB16 is similar to the auditory phenotype in DIS, where the hearing loss is moderate to severe. DFNB16 is caused by homozygous or compound heterozygous mutations, small indels, multi-exon or complete STRC deletions, as well as gene conversions between the STRC gene and its pseudogene (STRCP1) which are located less than 100 kb from each other (Moteki et al. 2016; Vona et al. 2015).

CNVs are a common cause of nonsyndromic hearing loss, STRC CNVs are the most common followed by CNVs in OTOA and GJB6 (Shearer et al. 2014). Homozygous or compound heterozygous missense, splice site mutations or complete OTOA deletions cause autosomal recessive deafness 22 (DFNB22; OMIM # 607039). DFNB22 has a prelingual onset, and the degree of hearing loss has been reported as moderate to severe and severe to profound.

More information is available at:

• DIS: https://www.omim.org/entry/611102; https://www.ncbi.nlm.nih.gov/books/NBK598310/;
• Hereditary hearing loss and deafness: https://www.ncbi.nlm.nih.gov/books/NBK1434/;
• DFNB16: https://www.omim.org/entry/603720;
• DFNB22: https://www.omim.org/entry/607039.

Table 1 presents the results of five different samples with gene conversions, including the corresponding theoretical final ratios. To our knowledge, gene conversions can be considered benign unless a loss-of-function variant is introduced in STRC by gene-to-pseudogene conversion (Shearer et al. 2014; Vona et al. 2015). The combined ratio of the probe pairs is 2 for gene conversions without an additional deletion or duplication in STRC or STRCP1.

Table 2a and Table 2b present two examples of complex family-based genetic testing cases, highlighting how parental data can clarify initially ambiguous or unexpected MLPA findings in probands.

Please note both tables are intended solely as interpretation aid. They are not exhaustive and include only selected examples. MLPA users are responsible for the correct interpretation of their results, and no rights can be derived from the tables.

Please notify us of any mistakes and additional information to be included in the tables: info@mrcholland.com.

• Pattern 1: Observed pattern suggests a pseudogene-to-gene conversion affecting exons 19 to 28.
• Pattern 2: Observed pattern suggests a gene-to-pseudogene conversion affecting exons 19 to 28.
• Pattern 3: Observed pattern suggests a pseudogene-to-gene conversion affecting exon 28.
• Pattern 4: Observed pattern suggests a gene-to-pseudogene conversion affecting exon 28 and a pseudogene-to-gene conversion affecting exon 20.
• Pattern 5: Observed pattern suggests a pseudogene-to-gene conversion affecting exons 19 to 25.

• Pattern 6: The proband appears to have a non-contiguous heterozygous deletion involving CKMT1B, STRC exon 28, and CATSPER2, along with a deletion in STRCP1 excluding exon 28. While this initially seems inconsistent, analysis of the parental profiles reveals a clear inheritance pattern that explains the observed genotype.
• Pattern 7: The pattern in Parent 1 suggests a pseudogene-to-gene conversion affecting STRC exons 19 to 25, reflected by a duplication in this region and deletion for the corresponding probe pairs in STRCP1. The STRCP1 deletion excluding exon 28 present in Parent 1 is inherited by the proband. The STRC exon 19–25 duplication seen in Parent 1 is balanced out in the proband by a corresponding deletion inherited from Parent 2, resulting in a normal copy number in the proband for this region.
• Pattern 8: Parent 2 carries a contiguous heterozygous deletion spanning CKMT1B, STRC, and CATSPER2, which is inherited by the proband. The deletion includes STRC exons 19–25, which counterbalances the duplication from Parent 1, leading to a normal dosage in the proband for this segment.

• Pattern 9: The proband shows a duplication of STRC exons 19–25, a heterozygous deletion involving CATSPER2, CKMT1A, and STRCP1 exon 28, and a homozygous deletion of STRCP1 exons 19, 20, and 23. The duplication is not detected in either parent and is therefore likely de novo. However, it is also possible that the duplication is present on one allele in either parent but is being masked by a deletion on the other allele (see Allele 1 and 2 of Parent 1). Since MLPA measures the average copy number across both alleles, such a configuration could appear as a normal diploid signal, thereby masking the true allelic imbalance. Furthermore, such a masked configuration could itself be inherited from a grandparent, meaning the apparent de novo event in the proband may actually represent a familial rearrangement. Therefore, in rare cases, analysis of grandparental samples can provide additional clarity and improve the understanding of the proband’s genetic profile. In this case, we cannot definitively explain the origin of the duplication, but these represent plausible theoretical possibilities. The homozygous STRCP1 deletion results from inheritance of heterozygous deletions from both parents (see Allele 1 of Parent 1 and 2).
• Pattern 10: Parent 1 carries a heterozygous deletion involving CKMT1B, STRC exon 28, and CATSPER2, as well as a heterozygous deletion of STRCP1 exons 19, 20, and 23. These deletions contribute to the heterozygous losses observed in the proband. Parent 1 does not carry the STRC exon 19–25 duplication, indicating that the duplication in the proband is likely de novo or a masked imbalance (see point above, Pattern 9). The STRCP1 deletion from Parent 1, combined with a similar deletion from Parent 2, results in a homozygous deletion of STRCP1 exons 19, 20, and 23 in the proband.
• Pattern 11: Parent 2 carries a heterozygous deletion spanning CATSPER2, CKMT1A, and STRCP1, including exon 28. This deletion is inherited by the proband and overlaps partially with the deletion from Parent 1, resulting in a heterozygous deletion of this shared region. Parent 2 also carries a heterozygous deletion of STRCP1 exons 19, 20, and 23, which, together with the deletion from Parent 1, leads to a homozygous deletion of these exons in the proband.

SALSA MLPA Probemix P461 STRC-CATSPER2-OTOA 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 (B1) version of this product.

• Coriell NA03184: Heterozygous duplication affecting the probes for PPIP5K1, CKMT1B, STRC, CATSPER2, CKMT1A, STRCP1 and PDIA3 on chromosome 15q.
• Coriell NA08039: Heterozygous duplication affecting the probes for METTL9, OTOA and UQCRC2 on chromosome 16q.
• Coriell NA13031: Heterozygous deletion affecting the probes for METTL9 and OTOA on chromosome 16q.
• Coriell NA20317: Heterozygous duplication affecting the probes for STRC and CKMT1A on chromosome 15q. Heterozygous deletion affecting the probes for STRCP1 and CKMT1B on chromosome 15q.
• Coriell NA20511: Heterozygous deletion affecting the probes for CKMT1B, STRC and CATSPER2 on chromosome 15q.

Key related products

• P153 EYA1: Targets EYA1, involved in bronchi-oto-renal dysplasia, causing profound deafness in children.
• P163 GJB-WFS1-POU3F4: Targets GJB2, GJB3, GJB6, WFS1, and POU3F4, involved in prelingual deafness.
• P280 SLC26A4: Targets SLC26A4, involved in DFNB4 and Pendred syndrome.

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 P461 STRC-CATSPER2-OTOA

• Domínguez-Ruiz M et al. (2023). Novel Pathogenic Variants in the Gene Encoding Stereocilin (STRC) Causing Non-Syndromic Moderate Hearing Loss in Spanish and Argentinean Subjects. Biomedicines. 11:2943.
• Marková SP et al. (2018). STRC Gene Mutations, Mainly Large Deletions, are a Very Important Cause of Early-Onset Hereditary Hearing Loss in the Czech Population. Genet Test Mol Biomarkers. 22:127-34.
• Markova T et al. (2022). Audiological Evidence of Frequent Hereditary Mild, Moderate and Moderate-to-Severe Hearing Loss. J Pers Med. 12:1843.
• Morgan A et al. (2020). Lights and Shadows in the Genetics of Syndromic and Non-Syndromic Hearing Loss in the Italian Population. Genes (Basel). 11:1237.
• Nishio SY et al. (2022). Frequency of the STRC-CATSPER2 deletion in STRC-associated hearing loss patients. Sci Rep. 12:634.
• Plevova P et al. (2017). STRC Deletion is a Frequent Cause of Slight to Moderate Congenital Hearing Impairment in the Czech Republic. Otol Neurotol. 38:e393-400.
• Spedicati B et al. (2023). The Enigmatic Genetic Landscape of Hereditary Hearing Loss: A Multistep Diagnostic Strategy in the Italian Population. Biomedicines. 11:703.
• Van Heurck R et al. (2021). Benefits of Exome Sequencing in Children with Suspected Isolated Hearing Loss. Genes (Basel). 12:1277.

References

• Moteki H et al. (2016). Detection and Confirmation of Deafness-Causing Copy Number Variations in the STRC Gene by Massively Parallel Sequencing and Comparative Genomic Hybridization. Ann Otol Rhinol Laryngol. 125:918-23.
• Shearer AE et al. (2014). Copy number variants are a common cause of non-syndromic hearing loss. Genome Med. 6:37.
• Vona B et al. (2015). DFNB16 is a frequent cause of congenital hearing impairment: implementation of STRC mutation analysis in routine diagnostics. Clin Genet. 87:49-55.