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# A proposal for comprehensive newborn hearing screening to improve identification of deaf and hard-of-hearing children

## Abstract

Early intervention for newborns who are deaf or hard-of-hearing leads to improved language, communication, and social–emotional outcomes. Universal physiologic newborn hearing screening has been widely implemented across the United States with the goal of identifying newborns who are deaf or hard-of-hearing, thereby reducing time to diagnosis and intervention. The current physiologic newborn hearing screen is generally successful in accomplishing its goals but improvements could be made. In the past ten years, genetic testing has emerged as the most important etiological diagnostic test for evaluation of children with deafness and congenital cytomegalovirus has been recognized as a major cause of childhood deafness that may be treatable. A comprehensive newborn hearing screen that includes physiologic, genetic, and cytomegalovirus testing would have multiple benefits, including (1) identifying newborns with deafness missed by the current physiologic screen, (2) providing etiologic information, and (3) possibly decreasing the number of children lost to follow up. We present a framework for integrating limited genetic testing and cytomegalovirus screening into the current physiologic newborn hearing screening. We identify needed areas of research and include an overview of genome sequencing, which we believe will become available over the next decade as a complement to universal physiologic newborn hearing screening.

## INTRODUCTION

The impact of permanent deafness on a child’s development is profound. It affects not only language acquisition but also social development and quality of life.1 Early detection of congenital deafness with targeted intervention significantly reduces negative impacts in these areas.2,3,4 In 1994, the Joint Committee on Infant Hearing (JCIH) published a position statement that endorsed the goal of universal detection of infants with deafness and encouraged continuing research and development to improve techniques for detection of and intervention for deafness as early as possible.5,6 Today, the crucial role of newborn hearing screening (NBHS) is emphasized by the fact that 43 states and territories of the United States have passed laws mandating NBHS, with the remainder of states having implemented universal NBHS without legislation. Currently, the JCIH recommends universal NBHS by 1 month of age, diagnosis by 3 months of age, and early intervention by 6 months of age to allow optimal intervention for children with deafness, if warranted and if desired by the family.7

The most recent data show that 98.2% of newborns in the United States receive NBHS.8 Universal screening has led to a significant reduction in the average age at which newborns with congenital deafness are identified in this country.9 Although the current universal NBHS has been remarkably successful in improving the early diagnosis and intervention in newborns who are deaf or hard-of-hearing (DHH), as will be detailed below, knowledge gained from the universal NBHS, an improved understanding of the genetics of hearing loss, as well as an increased recognition of the contribution of congenital cytomegalovirus (cCMV) to childhood deafness have provided an opportunity to improve the current NBHS.

Over the past 20 years, our understanding of genetic deafness has greatly improved. Along with diagnostic audiologic evaluation, diagnostic genetic testing platforms now form a cornerstone for evaluation of DHH newborns and children.10,11,12 An etiological diagnosis is provided in 49% of DHH newborns who undergo genetic testing.13 Genetic testing can also identify mild deafness, later-onset childhood deafness, syndromic forms of deafness, risk factors for aminoglycoside-induced deafness, and auditory neuropathy that may not be detected by the current physiologic NBHS. A genetic test for deafness that could be translated into a universal genetic screening test would form a powerful complement to the current NBHS; however, to date no genetic screening has been incorporated as part of a NBHS program in the United States.

An additional important contributor to childhood deafness is congenital cytomegalovirus infection (cCMV), estimated to be a cause of ~10% of congenital deafness and 15–20% of childhood deafness.14,15 Because cCMV often presents as mild, fluctuating, and progressive deafness, detection with a physiologic screen can be challenging.16 The incorporation of cCMV screening in a NBHS program would provide etiological information, improve ascertainment, and further complement the physiologic NBHS.

Creating a comprehensive newborn hearing screen that includes physiologic, genetic, and cytomegalovirus screening would have multiple benefits including (1) identifying newborns at risk for deafness and who could benefit from early intervention but are missed by the current physiologic screen, (2) providing etiologic information as part of the screen, (3) possibly decreasing the number of children who are lost to follow up, and (4) potentially saving costs by reducing the frequency of later testing. In this paper, we aim to provide a conceptual framework for a comprehensive NBHS program that incorporates the current physiologic screening as well as molecular screening that includes genetic screening and cCMV screening (Fig. 1). A comprehensive NBHS of this nature would require three concurrent screening methods, with results that inform the final screening result. Its implementation will likely require incremental steps and goals, particularly with respect to the incorporation of a form of nucleotide-level genetic screening, as there is no precedent in the United States. Targeted cCMV screening for those newborns who fail the physiologic screen has been incorporated in several states and therefore there is precedent for this form of molecular screening.

We first provide an overview of the current physiologic NBHS, including examination of areas of needed improvement. We next examine the available evidence that genetic and cCMV testing could improve the NBHS. Finally, we address challenges and barriers to accomplishing the goal of an improved NBHS, including patient and family perspectives. We believe that a comprehensive NBHS would significantly improve detection of newborns with deafness, advance our understanding of deafness in children, improve time to diagnosis and time-to-intervention, provide more intervention options for families across the childhood age span, and lay the foundation for improved treatment and support for children who are DHH.

## THE CURRENT NBHS: STRENGTHS AND WEAKNESSES

The prevalence of permanent deafness identified by state-based Early Hearing Detection and Intervention (EHDI) programs and reported to the Centers for Disease Control and Prevention (CDC) is 1.7 per 1000 births.8 This prevalence makes deafness the most common sensory deficit in humans. Universal NBHS is widely implemented in the United States, with 98.2% of newborns undergoing screening. From this perspective, the physiologic NBHS has been tremendously successful in regard to universal implementation and in its primary goal of decreasing the average age of identification of children with deafness.9

The goal of universal NBHS is to identify all newborns with permanent deafness. Both detecting and establishing an etiologic diagnosis for deafness in children are challenging because of the large number of possible causes and the wide clinical variability in presentation (Table 1). Hearing loss is described as slight (16–25 decibel hearing level, dB HL), mild (26–40 dB HL), moderate (41–55 dB HL), moderately severe (56–70 dB HL), severe (71–90 dB HL), or profound (>90 dB HL); can be unilateral or bilateral; and is either asymmetric or symmetric (Fig. 2). Select frequencies can be affected that can give an audiogram a specific shape or profile associated with specific genetic forms of deafness (for example, downsloping if high frequencies are impacted more than low frequencies, or upsloping if the reverse is true; Fig. 2). Deafness can also be defined by the site of impairment in the auditory system. A conductive hearing loss (CHL, see a list of definitions in Box 1) implies that transmission of sound through the external ear canal or middle ear is impaired, as in the case of a middle ear effusion, stenosis of the ear canal, or fixation of the ossicular chain. A sensorineural hearing loss (SNHL), in comparison, reflects compromised transmission of the neural signal along the auditory pathway, be it in the cochlea, the auditory nerve, or more proximally in the brainstem and cortex. In the United States and other developed countries, most permanent congenital deafness is SNHL and the majority of SNHL (~70%) is due to a genetic cause (Table S1).14,17 Environmental causes of SNHL, such as infections, hypoxia, and trauma, are a significant but smaller contributor to congenital deafness compared with genetic causes.

The ideal NBHS, as is the case for any screening test, must be inexpensive, easy to learn and to administer by screeners, acceptable to the screened individuals, and able to identify the presence or absence of deafness with few false positives (high specificity) and few false negatives (high sensitivity). Nearly all current NBHS programs in the United States use a physiologic evaluation of auditory function in response to sound. Methods used include an otoacoustic emission (OAE) screening test, which measures responses from the outer hair cells of the cochlea; an automated auditory brainstem response (AABR) screening test, which records response to sound based on the neural transmission of a signal from the cochlea to the brainstem; or a combination of both. These screening tests are low cost and can be administered within minutes by a trained screener. As highly validated testing methods, they have been widely adopted. The NBHS results in a “pass” (screened negative) or a “refer/fail/did not pass” (screened positive) indicating possible deafness and the need for further evaluation.

For newborns who do not pass the NBHS, diagnostic/confirmatory audiological testing is required to determine the type, degree, and configuration of hearing loss. Further clinical evaluation focuses on determining the etiological cause of deafness and is performed in concert between the primary care provider and otolaryngologist. This evaluation varies based on type of deafness but by current guidelines should include clinical examination and genetic testing, followed by imaging, other laboratory tests, and referrals to specialists as indicated.11 Further referrals and follow ups in the health-care system are guided by findings determined by evaluation and frequently include referral to a geneticist and other relevant medical specialists, as determined by clinical findings. Typically, the finding of deafness is also reported to the state EHDI program. A treatment or habilitation plan is then developed that can range from preferential seating in school and speech therapy, to sign language, assistive devices, and/or surgical intervention such as cochlear implantation. Although it has been remarkably successful in improving identification of children with hearing loss, there are several ways in which the current physiologic NBHS could be improved, and these are detailed below.

### The current physiologic NBHS has a low positive predictive value

Any screening test must weigh false negatives versus false positives to best serve the population screened. “False positive” may refer to the actual screening device used but here we refer to the overall result given to parents—“pass” or “refer”—versus the subsequent diagnostic confirmatory physiologic testing, which serves as the “gold standard.” The positive predictive value (PPV) is the probability that subjects who screen positive truly have the disease. An ideal screening test would have a high PPV and a high sensitivity. While negative predictive value and specificity are also considered in design of screening tests, a confirmatory or diagnostic test should aim for a high negative predictive value and a high specificity as false negatives are reduced.

Data show that of the 3,866,820 newborns who underwent NBHS in 2015, 64,978 (1.7%) did not pass. Of these children, 39,468 (60.7%) went on to have confirmatory audiologic testing.8 Of these, only 6442 (16.3%) were found to have deafness on confirmatory diagnostic audiometric testing. Therefore, 83.7% of children who screened positive did not have deafness, equating to a PPV of 16.3% and a specificity of 98.6% for the NBHS. The true sensitivity of this screening program is not known, as there are no large studies that have performed diagnostic testing on all newborns to identify the number of false negative instances (i.e., newborns who passed screening but were found to have deafness). From a practical sense, this means that the vast majority of newborns who refer on the physiologic NBHS do not have permanent deafness.

While the false positive rate is a concern for the physiologic NBHS, other newborn screening tests have similar performance. For example, a study in California showed that of 755,673 newborns screened for a panel of inborn errors of metabolism using tandem mass spectrometry, 461 newborns screened positive (0.13%) (ref. 18). Of the 386 children who underwent confirmatory testing, 335 (86.7%) were found to be false positives and have no inborn errors of metabolism. For this panel, the overall PPV is 13.2%. Other newborn screenings for inborn errors of metabolism have similar false positives rates and positive predictive values.19,20

As a universal screening test performed on a vulnerable population, reducing the false positive rate for the NBHS is important. False positives have the immediate effect of not only unnecessarily worsening parental anxiety21 and increasing health-care costs but also may lead to a decrease in follow up (discussed in a separate section below). Increasing the PPV should be a goal to improve the current physiologic NBHS but is not an inherent benefit of adding genetic screening.

### Types of deafness that are not identified by the current NBHS

The effect of mild-to-moderate hearing loss on language development has been heavily debated, but new data and review of previous studies indicate that it is in fact a significant risk factor for communication difficulties.22 Data from the CDC show that the majority (56.1%) of bilateral deafness in newborns is mild to moderate,8 however an even larger percentage of newborns may be affected, as very mild deafness can go undetected by NBHS (depending on the thresholds and testing methods that are used). Although implementing physiologic screening at lower hearing levels would detect more mild-to-moderate deafness, it would also increase false positive rates and burden confirmatory diagnostic audiology evaluation. The precise sound intensity used in the NBHS is difficult to define accurately because screening equipment manufacturers choose stimulus levels and characteristics for their own equipment that cannot be adjusted by the operator. The screening level of AABR is typically programmed into the screening equipment; this level is based on research indicating the optimal screening level for AABR. There are no calibration standards for transient stimuli or for determining the actual stimulus levels in ear canals of newborns, which may be greater than specified by the manufacturer because of the small size of a newborn ear canal, possibly leading to false negative results. Identification of infants with permanent and/or progressive mild deafness is integral to improving the current physiologic NBHS.

Children who develop deafness after the newborn period will not be detected by the current NBHS. This limitation is important as the number of children with significant hearing loss increases throughout childhood from a congenital prevalence of 1.7 per 1000 births8 to a best-estimated prevalence during childhood of 31 per 1000 (ref. 23). Studies of the prevalence of childhood deafness are primarily based on surveys and although further research in this area is needed, particularly in regard to prevalence in preschool-aged children; it is clear that the prevalence of deafness increases with age during childhood.23 Importantly, a significant number of children who develop prelingual deafness following the newborn period are not detected by the currently implemented NBHS. Dedhia and coauthors estimated that nearly 25% of all children with SNHL are not identified by the NBHS and that nearly two-thirds of these had severe-to-profound deafness.24 Young et al. found that 30% of all cochlear implant candidates passed their NBHS and these children had significantly delayed treatment.25 It is unclear based on current studies whether these children had congenital deafness or deafness that began soon after the newborn period and further study is needed to clarify this point. However, because these children are not identified by the current universal NBHS, the detection of their deafness currently occurs through a patchwork of school-age hearing screening programs that vary significantly by state. Establishing a universal NBHS that is able to identify children with deafness that occurs outside the newborn period would prevent a delay in diagnosis and treatment.

Auditory neuropathy spectrum disorder (ANSD) is characterized by absent or severely abnormal inner hair cell (IHC), synaptic, and/or spiral ganglion function as measured by ABR testing, with preservation of outer hair cell (OHC) function as measured by OAE testing.26 Therefore, a newborn with ANSD manifesting as severe-to-profound deafness may go undetected by OAE screening when OAE screening is performed alone. The presentation of ANSD is also highly variable, with some infants and children with ANSD having asymmetric or unilateral deafness. At older ages, ANSD is accompanied by poor speech discrimination and poor word understanding, especially in the presence of noise. The prevalence of ANSD is reported to be 2.7% of DHH newborns identified by NBHS programs based on data from the CDC.8 Other reported data indicate the prevalence of ANSD to be 1.2%, 5.1%, or 8.4% depending on the population.27,28,29 Forty percent (40%) of ANSD is estimated to have a genetic basis, with the remainder due to acquired causes like hypoxia, prematurity, and jaundice, underscoring the increased rate of ANSD in neonatal intensive care units. The list of causative genes for ANSD includes DIAPH3, OTOF, PJVK, and mitochondrial DNA (mtDNA) variants (m.1095T>C) for nonsyndromic ANSD, and AIFM1, DDDP, MPZ, OPA1, PMP22, and TMEM126A for syndromic ANSD, although based on prevalence data there are likely other genes involved. The gene most frequently implicated in nonsyndromic ANSD is OTOF, which is estimated to be responsible for 0.5–3.5% of prelingual deafness across multiethnic cohorts.30,31,32 NBHS that relies only on OAE will not identify these babies, but fortunately the majority of neonatal intensive care units (where the rate of ANSD is highest) perform screening with AABR, as recommended by JCIH, and half of all NBHS is now performed with AABR. The actual number of newborns missed by the current NBHS due to ANSD is not known but ensuring these babies are screened is key to improving screening outcomes.

The current NBHS does not screen for a relatively common risk factor that can cause deafness in the newborn period. Several mitochondrial DNA variants lead to exquisite sensitivity to aminoglycoside-induced deafness. Aminoglycosides, in particular gentamicin, are commonly used in the neonatal period due to low cost and effectiveness against Gram-negative bacteria.33 Newborns who carry certain genetic variants in the mitochondrial gene MT-RNR1 can experience significant deafness with a single dose of these commonly used antibiotics. In addition, animal studies show that there is a synergistic effect of these genetic variants, aminoglycosides, and noise, which further predisposes these newborns to deafness. Prevalence of these mitochondrial variants was found to be 0.2% in one study of 703 children from a neonatal intensive care unit in the United States and 0.19% in a study of 58,397 Chinese children.34,35 Identifying these newborns with screening could have immediate treatment implications. In addition, diagnosis could prevent aminoglycoside-induced later-onset deafness in vulnerable individuals and lead to targeted cost-effective evaluation in maternal relatives after positive screening results from one individual.

### Limited etiological information is included in the current NBHS

The current NBHS provides a simple result: “pass” or “refer.” Subsequent audiologic and diagnostic evaluation provides information on degree of deafness and etiology. An extremely important goal of diagnostic testing for deafness in children is to identify etiologies that have further diagnostic and treatment implications. Among these are syndromic forms of deafness, the most common of which are described in Table S2 and include Usher syndrome (deafness–blindness), Pendred syndrome (deafness including inner ear malformations and thyroid goiter), branchiootorenal syndrome (BOR, branchial cleft anomalies, deafness, and renal abnormalities), Jervell and Lange-Nielsen syndrome (deafness and cardiac arrhythmias), and Alport syndrome (deafness, renal disease, and eye abnormalities). These syndromic forms of deafness are referred to as nonsyndromic hearing loss (NSHL) mimics because they present at birth as nonsyndromic deafness (no other associated abnormalities). Recent data show that these syndromes are more common than previously reported. In a cohort of 2460 individuals of all ages with deafness, the diagnoses of NSHL mimics totaled 25% of all diagnoses and most commonly included Usher syndrome (10%), followed by Pendred syndrome (5%), deafness–infertility syndrome (4%), and branchiootorenal syndrome (2%) (R.J.S. unpublished data). Further data are required on the clinical and ethical repercussions of early diagnosis of syndromic forms of hearing loss. Early genetic diagnosis of syndromic forms of deafness would significantly reduce other testing and provide opportunities for early intervention.

Identification of a genetic etiology has not only the potential to inform prognostic monitoring of deafness and/or associated syndromic features, but may also refine estimates of recurrence risk for family members. The information may influence family reproductive and financial planning.

### There are a large number of newborns lost to follow up in the current NBHS

A significant number of newborns are lost to diagnostic follow up in the current NBHS protocol. The most recent data from 2015 showed that 27.9% of newborns who referred on NBHS were lost to follow up prior to diagnostic audiology.8 Of these newborns, 54.7% were due to unknown reasons; 14.7% were cases in which there was an inability to contact the newborn’s guardian; and in the remaining 30.6% of cases, the parents or family were contacted but were not responsive to follow-up requests.8 There are several reasons that children are lost to follow up including social, economic, and geographic factors.36 Data show that altering testing methodologies, including a screen–rescreen policy as well as increasing time from birth can improve positive predictive value, but further research is needed in this area37,38,39 It is not unreasonable to assume that by providing more etiologic information to parents during the screening process and improving the positive predictive value of screening, fewer children would be lost to follow up. Providing an etiologic diagnosis sooner may motivate parents to seek early intervention services but further study in this area is needed.

## Incorporating genetic screening into the NBHS

Our understanding of the molecular physiology of hearing and deafness has improved dramatically in the past two decades, in part through the study of genetic causes of deafness. The first gene to be implicated in nonsyndromic human deafness, GJB2, was discovered in 1997.40 Since that time, over 130 genes and nearly 8000 genetic variations have been identified that are associated with nonsyndromic deafness (Table S1). These variations differ significantly based on ethnic population examined. In addition, there are more than 600 clinical syndromes that include deafness; in several, deafness is the first presenting clinical feature (NSHL mimics) (Table S2).

The extreme genetic heterogeneity of deafness has made single-gene targeted genetic testing inefficient compared with its use in disorders where a single primary underlying genetic cause predominates (e.g., testing the CFTR gene to diagnose cystic fibrosis). In addition, with respect to deafness, sequential single-gene sequencing tests are costly, time-consuming, and relatively low in their diagnostic yield.41 The first clinical diagnostic genetic test for deafness, evaluation for variants in GJB2, began in 1999. Initial tests were based on variant detection assays, but these assays quickly gave way to single-gene Sanger sequencing, which has now been replaced by comprehensive genetic testing using massively parallel sequencing techniques. Multiple genes are sequenced in parallel to provide a complete analysis of the genetic landscape, which is essential for a heterogeneous condition like deafness. Massively parallel sequencing was first demonstrated for genetic testing for deafness in 2010 and was shown to have an analytical sensitivity and specificity of >99% for single-nucleotide variant detection making it suitable for clinical genetic testing.42 Massively parallel sequencing is now the standard of care for the genetic evaluation of children with deafness as recommended by the American College of Medical Genetics and Genomics and the International Pediatric Otolaryngology Working Group.11,12

Recent data show that when a comprehensive genetic testing platform is used to evaluate persons with deafness, including all ages and types of deafness with no exclusionary criteria, a diagnosis is provided in ~40% of cases.10,30 Data from 2460 individuals with deafness run on the OtoSCOPE platform from the University of Iowa Molecular Otolaryngology and Renal Research Labs (MORL) show a diagnostic rate of 39.9% (R.J.S., unpublished data), which is consistent with a review of other published studies.10 This diagnostic rate varies based on gene content, variant interpretation standards, and patient population studied. In comparison, data from the Laboratory of Molecular Medicine (LMM) at Partners HealthCare show a diagnostic rate of 25.2%, an inconclusive rate of 57.8%, and a negative rate of 17.0% in a cohort of 1156 patients presenting for multigene panel testing (S.A. and J.S., unpublished data).

The number of individuals who are DHH that are provided a diagnosis from genetic testing varies based on clinical history such as the type of deafness, age of patient, family history, and ethnicity.30 For the large cohort of clinical samples from the University of Iowa, the diagnostic rate for those with congenital deafness was 53.6% (R.J.S., unpublished data). Published data show that in those affected by congenital deafness, if there is also a family history consistent with dominant or recessive inheritance, the diagnostic rates are 67% and 55%, respectively. It is likely that these percentages will increase as additional genetic causes of deafness are identified. Data from the University of Iowa show that diagnostic rates decrease with age of onset of deafness, and are 39.6% at 3–6 years and 27.3% at 12–18 years, paralleling the increase in nongenetic causes of deafness with increasing age (rates based on age at testing as precise data on age of onset are not available).

In comparison, other commonly used diagnostic tests for evaluation of children with deafness have lower diagnostic rates: computed tomography imaging, 30% (ref. 43); magnetic resonance imaging, 26% (ref. 44); ophthalmologic evaluation, 8%; renal ultrasound, 4%; and electrocardiogram, 1% (ref. 45). Genetic testing therefore has the highest diagnostic rate of any test used to identify the etiological cause of childhood deafness and is now recommended as the first-line test in evaluation of children with documented bilateral SNHL.10

There are several commercially available comprehensive genetic testing platforms (Genetic Testing Registry). The number of genes included, cost, and turnaround time vary, but in general most platforms rely on targeted genomic enrichment and massively parallel sequencing technologies, which allow sequencing of many genes simultaneously.46 Average turnaround time per test is 2–3 months and listed prices range from ~$1500 to$5000 per test. More importantly, however, is the downstream analysis, which can result in differences at the level of the identification and/or interpretation of genetic variants.

The inclusion of copy-number variant (CNV) analysis in comprehensive genetic testing is crucial, as has been illustrated in a study of 686 patients in which at least one CNV was identified in 104 persons (15.2%) and implicated in the genetic diagnosis 18.7% of the time.47 CNVs were most commonly identified in STRC (73% of CNVs identified) followed by OTOA (13% of CNVs identified). More recent, unpublished data confirm that CNVs in STRC are one of the most commonly detected pathogenic variants causing deafness and the second most common gene implicated in deafness behind GJB2 (Table 2, 3). If only mild-to-moderate deafness is considered, STRC deletion is the most common cause of deafness.

Importantly, comprehensive genetic testing for deafness also includes testing for NSHL mimics. These diagnoses comprise up to 25% of all genetic diagnoses in children undergoing genetic testing for deafness (R.J.S., unpublished data). Identification of these syndromic forms of deafness in newborns directly affects follow-up testing and care and reduces time to diagnosis. In addition, comprehensive genetic testing evaluates for mitochondrial variants that predispose to aminoglycoside-induced deafness and therefore can aid in prevention of this medication-related side effect.

Comprehensive genetic testing has several strengths that make it particularly valuable for the evaluation of newborns with deafness: (1) it provides an etiological diagnosis, (2) it tests for all severities of deafness (mild–profound), (3) it tests for syndromic forms of deafness, and (4) it can assess for risk of aminoglycoside-induced deafness. Therefore, incorporating some form of genetic testing as an additional screening component in the NBHS would (1) identify additional newborns and young children with deafness and at risk for deafness, (2) improve time to diagnosis and intervention, (3) identify comorbidities of congenital deafness that have important implications for medical management of children who are DHH, and (4) possibly reduce those lost to follow up.

Currently available comprehensive genetic tests used in the diagnostic evaluation of deafness are not suitable for the NBHS because of (1) high cost, (2) long turnaround time (weeks to months), (3) complexity of bioinformatics (including the identification of CNVs), and (4) challenges in variant interpretation (Table 4). Notwithstanding these challenges, genetic screening would add opportunities to improve detection of deafness thus justifying strategies to integrate its use into the NBHS. Three strategies—limited genetic evaluation for screening, comprehensive genetic sequencing, and genome evaluation—are discussed below.

### Limited genetic screening for NBHS

Implementation of limited genetic screening requires the selection of specific genes and/or variants, a challenging task given the extreme genetic heterogeneity of deafness. For example, in a cohort of 1119 patients with deafness, the gene most frequently implicated in deafness was GJB2.23 It accounted for 21.6% of diagnoses. In aggregate, however, ten genes accounted for 72.3% of all diagnoses (Table 2). At the variant level, in comparison, the ten most frequently encountered causative variants accounted for only 30.4% of diagnoses. The most commonly identified single causative variant in this cohort was a deletion involving STRC, which accounted for 11.8% of causative variants in individuals who were identified to have genetic hearing loss and can only be detected by CNV analysis. This type of genetic change cannot be identified using a nucleotide variant detection assay and would require a secondary screening assay.48

Data from 2460 individuals with deafness from a clinical genetic testing lab show the diminishing returns associated with sequencing an ever-higher number of genes (R.J.S., unpublished data, Figure S1). In this group of 2460 individuals, 79 different genes were identified as causative, with 34 genes accounting for only one or two diagnoses each. Therefore, there appear to be several genes that could be selected for targeted genetic screening, but there are diminishing returns as more genes are added (Figure S1). A limited panel of genes may provide an avenue for developing genetic NBHS. However, as described below, the logistics of this type of screening would prove challenging.

Several groups have studied the value of limited panels of genes or variants to augment NBHS in large cohorts. In a recent study of 5173 Chinese newborns, screening of four genetic variants resulted in identification of 46 newborns who passed the physiologic NBHS but had a genetic cause of deafness identified and therefore were not detected by physiologic NBHS.49 This study is limited in that long-term follow up to evaluate the degree of deafness in these children was not provided and so the exact type of deafness caused by these genetic variants is not known. In addition, one of the variants evaluated, GJB2 p.V37I, while very common particularly in Asian populations, has variable penetrance and causes mild-to-moderate hearing loss, making definitive interpretation of screening results difficult. A larger study of 58,397 Chinese newborns who were screened for 20 common genetic variants identified a genetic carrier result in 5.28% and the identification of five newborns with a positive genetic diagnosis of deafness who passed their NBHS in both ears.35 Another recent study that included concurrent genetic and physiologic screening in 1716 Chinese newborns found that 47% of those with a definitive GJB2 genetic diagnosis passed their physiologic screening.50 In contrast, three similar studies of 14,913, 10,043, and 2500 newborns that used a combination of sequencing approaches to detect common variants found no newborns who passed their physiologic NBHS but had a positive genetic diagnosis of deafness.51,52,53 The largest study to date of 142,417 Chinese newborns identified a carrier rate of 3.01% for the four most common genetic causes of deafness but did not include audiologic data to determine how genetic screening could augment physiologic screening.54 This variability in detection rates might reflect differences in sample sizes, variants screened, and screening methodologies; however, the data do indicate that even limited genetic screening can identify newborns who otherwise would have been missed by physiologic screening alone.

Limited genetic screens have inherent ethnic bias, which is an important drawback in a racially and ethnically diverse population like that of the United States. Table S3 shows the top 20 most commonly encountered pathogenic variants in GJB2, illustrating the significant differences among populations with respect to the most commonly encountered pathogenic variants. Note that in these six populations, there are 15 different variants that are rarely shared between populations. Of the top five variants, only one is shared among the East Asian, Latino, European, and African populations. These data underscore the challenge in implementing limited genetic variant screening in an ethnically diverse population. Screening for deafness genes and not variants could circumvent some of this ethnic bias because many genetic variants are ethnically specific. However, the candidate genes or variants for screening would need to be thoughtfully selected to provide the best screening for a given population.

A limited genetic variant screening tool would ideally use only a single methodology. Polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP) analysis, single-nucleotide polymorphism (SNP) microarray, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) have all been used for genetic screening, each with its own advantages and disadvantages. New methods of screening for deafness variants continue to be developed. A recent study using a custom PCR-based, multiplex ligation assay to screen for 115 variants cost $25 per sample.55 Another recently developed method using real-time PCR and melting curve analysis to detect 12 variants costs less than$10 (ref. 56). A third method that relies on multiplex PCR followed by a “reverse dot blot assay” can screen for 20 variants at a reported cost of $3 per sample.57 Finally, a fluidic microarray-based screening for nine common variants for deafness in patients of European ancestry has been validated and costs$30 per sample.58 All methods would need to be compared with gold standard genetic diagnostics prior to implementation as part of a genetic NBHS. In addition, the variants used for screening would need to be carefully selected based on goals of the screening and the population to be screened.

### Disclosure

The authors declare no conflicts of interest.

## Author information

Authors

### Corresponding authors

Correspondence to Cynthia C. Morton PhD or Richard J. Smith MD.

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Shearer, A.E., Shen, J., Amr, S. et al. A proposal for comprehensive newborn hearing screening to improve identification of deaf and hard-of-hearing children. Genet Med 21, 2614–2630 (2019). https://doi.org/10.1038/s41436-019-0563-5

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### Keywords

• newborn hearing screening
• deafness
• genetics
• genomics
• cytomegalovirus

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Human Genetics (2021)

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• , Rebecca K. Siegert
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• , Alex Chapin
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• , Karen B. Avraham
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• , Andrew J. Griffith
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• , Sami S. Amr
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