Amplify™ Sponsored Testing Program

Hearing loss (HL) or deafness is the most common sensory deficit in humans, affecting an estimated 5% of the world's population. HL may be caused by environmental or genetic factors (Azaiez et al. 2018. PubMed ID: 30245029). Hereditary hearing loss and deafness (HHLD) is classified by type (conductive, sensorineural, or mixed), age at onset (congenital, pre-lingual, post-lingual, or age-related), audiological characteristics (severity and affected frequencies), associated clinical features (syndromic or nonsyndromic), and mode of inheritance (autosomal dominant, autosomal recessive, X-linked, or mitochondrial).

Molecular genetic testing is available for many types of HHLD and plays a prominent role in diagnosis and genetic counseling (Hilgert et al. 2009. PubMed ID: 18804553; Shearer and Smith. 2012. PubMed ID: 23042251; Shearer et al. 2017. PubMed ID: 20301607; Smith et al. 2005. PubMed ID: 15752533).

This comprehensive panel consists of genes that have been associated with all types of HHLD. Importantly, both syndromic and nonsyndromic HL genes are included in this panel due to the variable or mild features associated with some syndromic HL genes.

In developed countries, HL is primarily caused by genetic factors and is nonsyndromic (Sloan-Heggen et al. 2016. PubMed ID: 26969326). The majority of HHLD is monogenic, with the exception of age-related HL, which is typically a complex genetic disorder (Azaiez et al. 2018. PubMed ID: 30245029). Congenital or pre-lingual HL is 80% genetic as compared to 20% acquired or environmental. Of pre-lingual genetic HL, 20% is syndromic and 80% nonsyndromic. Of pre-lingual genetic nonsyndromic hearing loss (NSHL), 80% is recessive, 19% is dominant, and the remainder is X-linked or mitochondrial (Shearer et al. 2017. PubMed ID: 20301607). To our knowledge, although de novo variants causing hearing loss have been documented, they are not a common cause of disease for the genes in this panel.

The gene most commonly associated with NSHL is GJB2 (also known as connexin 26), which accounts for 50% of congenital severe-to-profound autosomal recessive NSHL cases (Smith et al. 2016. PubMed ID: 20301449). The most prevalent pathogenic GJB2 variant is c.35delG, having a global population frequency of 0.62%, and a frequency of 0.96% in European populations (gnomAD population database).

The most common types of syndromic HL are Usher, Waardenburg, and Pendred syndromes (Koffler et al. 2015. PubMed ID: 26443487). Other less common syndromic causes of HL are also included in this panel (Perrault, Treacher Collins, Stickler, Jervell and Lange-Nielsen, branchio-oto-renal (BOR), Wolfram, Heimler, and Alport syndromes).

Copy number variant (CNV) detection is an important component of clinical genetic testing for HHLD, with CNVs contributing 18.7% of all positive genetic diagnoses in a large cohort of NSHL patients. The majority of CNVs detected in this study were in two genes: STRC (73%) and OTOA (13%). The carrier frequency of STRC CNVs was estimated at 1.1-1.6% in individuals without HL, indicating that STRC CNVs may be an equal or potentially larger contributor to autosomal recessive NSHL than GJB2 in some populations (Shearer et al. 2014. PubMed ID: 24963352). STRC CNVs often include the nearby CATSPER2 gene. Although CATSPER2 is not known to directly cause hearing loss, it is included in this panel because together with STRC, these two genes are associated with deafness-infertility syndrome. This test is able to detect CNVs in STRC, CATSPER2, and OTOA.

This test includes probes for CNV detection that cover all known GJB2 cis-regulatory element deletions associated with hearing loss, previously associated with digenic GJB2/GJB6 hearing loss and spanning from GJB6 to CRYL1. This includes the 309 kb GJB6-D13S1830 and 232 kb GJB6-D13S1854 deletions reported to be most common (del Castillo et al. 2003. PubMed ID: 14571368; del Castillo. 2005. PubMed ID: 15994881). The 95 kb region included in all published GJB2 regulatory element deletions has been especially targeted with CNV probes in this test (Tayoun et al. 2016. PubMed ID: 26444186). Additionally, a targeted test for the GJB6-D13S1830 and GJB6-D13S1854 deletions is available separately.

This panel also includes syndromic causes of HL affecting the external structure of the ear such as microtia (HOXA2) and aural atresia (TSHZ1). Genes that cause hearing loss as an early occurring feature (FITM2) and genes with new or rarely reported associations with HL are also included (AP1B1, ASIC5, ATOH1, CLDN9, DE1C, DMXL2, GRAP, IFNLR1, MAFB, MAP1B, MPZL2, REST, TBX1, TMEM126A, TMEM43, TRRAP, SLC44A4, NOG, PLS1, REEP6).

Recent additions to this panel are ACOX1 (February 2022) and CLRN2 (July 2022). A single recurrent ACOX1 de novo variant (p.Asn237Ser) has been reported in patients with autosomal dominant Mitchell syndrome, which is characterized by progressive ataxia with features of episodic demyelination, sensorimotor polyneuropathy, and in some cases, earlyonset hearing loss (Chung et al. 2020. PubMed ID: 32169171). A homozygous CLRN2 missense variant that also creates a cryptic splice acceptor site has been reported as pathogenic in three patients with hearing loss from a single extended consanguineous family (Vona et al. 2021. PubMed ID: 33496845).

In a study of 1,119 individuals with any type of hearing loss, 82% of positive diagnoses were due to 15 genes (GJB2, STRC, SLC26A4, TECTA, MYO15A, MYO7A, USH2A, CDH23, ADGRV1, TMC1, PCDH15, OTOF, TMPRSS3, LOXHD1, and OTOA) (SloanHeggen et al. 2016. PubMed ID: 26969326).

This test is performed using Next-Gen sequencing with additional Sanger sequencing as necessary. This panel provides 99.6% coverage of all coding exons of the genes plus 10 bases of flanking noncoding DNA in all available transcripts along with other non-coding regions in which pathogenic variants have been identified at PreventionGenetics or reported elsewhere. We define coverage as ?20X NGS reads or Sanger sequencing.

This program is available to patients in the U.S. (requirements below must be met for the patient to be eligible):

• Patient is less than 18 years of age
• Medical history consistent with auditory neuropathy diagnosis:
  • Absent or highly abnormal ABR
  • Presence of OAEs and/or cochlear microphonic (CM)

No-cost genetic counseling with a third party service (provided by Genome Medical), is available to patients through this sponsored testing program. One post-test genetic counseling appointment is available to individuals with identified variants classified as uncertain, likely pathogenic, or pathogenic.

SPECIMEN REQUIREMENTS

Whole Blood

Collect 3 ml - 5 ml of whole blood in EDTA (purple top tube) or ACD (yellow top tube), minimum 1 ml for small infants.

Saliva

Oragene™ or GeneFiX™ Saliva Collection kit used according to manufacturer instructions. DNA from saliva specimens is invariably contaminated with microbial and food DNA, which can impact specimen quality and may result in delayed testing and/or the need for a second specimen.

OCD-100 Buccal Swab

OCD-100 Buccal Swab used according to manufacturer instructions.

Buccal

Specimens may be shipped at room temperature.

Specimen collection kits: Buccal specimen collection kits, which contain the TRF and the shipping label, may be requested through the kit order form or via the online order form.

SHIPPING AND HANDLING INSTRUCTIONS

Label all specimen containers with the patient's name, date of birth, and/or ID number. At least two identifiers should be listed on specimen containers. Specimen deliveries are accepted Monday-Saturday for all specimen types. Holiday schedules will be posted on our website at least one week prior to major holidays.