A CASE FOR ETIOLOGIC FOCUS IN AUDIOLOGY – GENETIC TESTING
Thesis
Presented in Partial Fulfillment of the Requirements for The Masters in Speech and Hearing Science in the Graduate School of The Ohio State University
By
Shelley Bloom, B.S.
*****
The Ohio State University 2009
Capstone Committee: Approved by
Christina Roup, PhD, Advisor
Gail Whitelaw, PhD
Christy Goodman, AuD ______Advisor
Speech & Hearing Sciences Graduate Program
ABSTRACT
Genetic testing can determine the etiology for hearing losses of genetic origin.
The genes for many common forms of hearing loss and some uncommon forms have been discovered and mapped. When the cause of the hearing loss is known, the treatment can be targeted and enables more accurate, complete information regarding the prognosis.
Therefore, it would be beneficial for audiologists to include a referral for genetic testing as a routine part of an audiologic evaluation. However, genetic counseling is crucial for enabling patient understanding of the genetic information and comprehending the benefits and risks of genetic testing. This fits into the goal of autonomy for the patient or caregivers by providing enough information to make an informed decision about treatment for the hearing loss.
ii DEDICATION
To my family and friends, who helped support me every step of the way.
iii VITA
April 12, 1979……………………………Born – TACOMA, WASHINGTON
June, 2003………………………………..BACHELORS OF SCIENCE WITH DISTICTION, UNIVERSITY OF WASHINGTON – Minors in Japanese and Music
2007-2008………………………………..INFANT HEARING PROGRAM GRADUATE INTERN The Ohio Department of Health
iv FIELDS OF STUDY
Major Field: Speech & Hearing Sciences with specialization in molecular genetics
v TABLE OF CONTENTS
Page Abstract………………………………………………………………………………….... ii
Dedication………………………………………………………………………………... iii
Vita………………………………………………………………………………………..iv
List of Abbreviations……………………………………………………………………...vii
Introduction……………………………………………………………………………….. 1
Genetic Testing for Hearing Loss………………………………………………………… 3
GBJ2 Gene – Connexin 26………………...………………………………………………6
SCL26A4 Gene: Enlarged Vestibular Aqueduct and Pendred Syndrome...……………..12
Mitochondrial DNA Hearing Loss……………………………………………………….20
Mitochondrial Multisystemic Syndromes……………………………………………...... 22
Mitochondrial DNA Mutations and Non-Syndromic Hearing Loss……………………..26
Age Related Hearing Loss – Presbyacusis….…………………………………………...29
Usher Syndrome………………………………………………………………………….32
Neurofibromatosis Type 2………..……………………………………………………...37
Other Genetic Hearing Losses…………………………………………………………...41
Concerns and Objections About Genetic Testing…..…………………………………...43
Conclusion……………………………………………………………………………….49
References…………………………………………………………………………….….51
vi LIST OF ABBREVIATIONS
ABI Auditory Brainstem Implant ABR Auditory Brainstem Response AN Auditory Nerve ARHL Age Related Hearing Loss ASL American Sign Language ATP Adenosine Triphosphate Bp Base Pair - CHO3 Bicarbonate CI Cochlear Implant CMV Cytomegalovirus CR Calorie Restriction CT Computed Tomography DPOAE Distortion Product Otoacoustic Emissions ENT Ear, Nose and Throat ERG Electroretinogram EVA Enlarged Vestibular Aqueduct GFP Green Fluorescent Protein GJB2 Gap Junction β 2 IAM Internal Acoustic Meatus INC Inner Hair Cells MELAS Mitochondrial Encephalopathy, Lactic Acidosis, and Strokelike Episodes MERRF Myoclonic Epilepsy and Ragged Red Fibers MIDD Maternally Inherited Diabetes and Deafness mL Milliliter MRI Magnetic Resonance Imaging mtDNA Mitochondrial Deoxyribonucleic Acid mV Millavolt NF2 Neurofibromatosis Type 2 ns-EVA Non-syndromic Enlarged Vestibular Aqueduct OHC Outer Hair Cells OTOF Otoferlin PEO Progressive External Ophthalmoplegia PS Pendred Syndrome PTA Pure Tone Average ROS Reactive Oxygen Species RP Retinitis Pigmentosa
vii SNHL Sensorineural Hearing Loss USH1B, C, D, F Usher Syndrome Type 1B, 1C, 1D, 1F USH2A Usher Syndrome Type 2A USH3A Usher Syndrome Type 3A WT Wild Type
viii INTRODUCTION
Hearing loss is one of the most common birth defects. The incidence of severe to profound hearing loss at birth in the United States is 0.8 per 1000, while moderate or unilateral hearing losses have an incidence of 1.1 per 1000 births. Genetic etiologies account for about half of the cases of pre-lingual hearing loss, which is defined as hearing loss present at birth or early childhood (Eisen & Ryugo, 2007). The remaining cases are attributed to environmental causes including pre- and post-natal infection, such as congenital rubella, cytomegalovirus (CMV), meningitis, along with perinatal complications and other factors, such as prematurity, anoxia, low birth weight, hyperbilirubinemia, ototoxic drugs, noise exposure and head injury (Nance & Dodson,
2007).
Additionally, genetics can also cause adult-onset hearing loss. These post-lingual onset hearing losses have purely genetic causes, or may be brought about by a mixture of genetic susceptibility and environmental factors, such as noise exposure and medication.
Even age-related hearing loss (ARHL) or Presbyacusis has a genetic component, resulting in detrimental anatomical and physiological changes to the auditory system
(Seidman et al., 2004; Dai et al., 2004).
Currently, audiologic testing is limited to determining the presence or absence of a hearing loss. Once a hearing loss is identified, the type, degree and configuration are
1 determined. All of this testing does not necessarily give definitive answers of why the patient has hearing loss. This results in limited information availability for the medical professional and patient in terms of prognosis and treatment for aural rehabilitation and medical intervention.
Audiology should now move from primarily diagnosing and categorizing hearing loss to the possibility of including the cause of a hearing loss by including referrals for genetic testing as part of an audiologic evaluation. Genetic testing, in conjunction with audiologic testing and medical evaluation can determine the etiology in hearing impairment that has a genetic cause. When the exact gene is ascertained, more accurate, comprehensive information can be given regarding prognosis and treatment.
Consequently, patients and families can make more informed decisions about the management of the hearing loss.
2 GENETIC TESTING FOR HEARING LOSS
Etiology of Hearing Loss
Genetic testing can provide a definitive reason for hearing loss with a genetic cause. Fifty to sixty percent of congenital hearing loss has a genetic cause and genetic factors could also play a part in adult-onset hearing loss (Arnos, 1997). Genetic testing also can enable specific information about the prognosis and clinical course of the hearing loss (Arnos, 2003). Genetic testing is non-invasive (Schrijver, 2004; Liu et al.,
2008) and can be obtained with buccal smears (Torkos et al., 2006). Once a genetic diagnosis is reached, future diagnostic invasive, costly or painful procedures may be reduced or eliminated (Kimberling & Lindenmuth, 2007) such as tests for diagnosing prenatal infections, electrocardiograms, thyroid testing, magnetic resonance imaging
(MRI), computed tomography (CT) scans of inner ear structures and electroretinograms
(ERG) (Arnos, 2003; Cohen, Bitner-Glindzicz, & Luxon, 2007).
Treatment Planning
Knowledge of the etiology of the hearing loss allows for timely, accurate treatment planning. For syndromic hearing losses, such as Usher Syndrome and
Neurofibromatosis 2, genetic testing also yields relevant medical information and any accompanying conditions to the hearing loss. This awareness of syndromic hearing loss enables appropriate medical referrals, care and support services (Arnos, Cunningham,
3 Israel, & Marazita, 1992). It is important to monitor known medical complications that may arise in the present and in the future, such as ocular or renal deficiencies (Bitner-
Glidzicz, 2002) along with the hearing loss. The genetic diagnosis assists the decision on whether the hearing loss would be better treated with medical management, hearing aids or both.
When the etiology of the hearing loss is known, planning for education and communication needs can be anticipated and made early in life. Patients and family members can evaluate the educational facilities and support programs available in the area of residence. Additionally, social and employment opportunities vary across the country and may affect where they choose to live. An option may be to relocate for medical or educational possibilities (Arnos, Cunningham, et al., 1992).
Once the genetic cause of hearing loss is established, the inheritance pattern, such as dominant, recessive, or mitochondrial can be determined, thereby addressing concerns regarding whether hearing loss or other associated conditions will be passed on to siblings, children and grandchildren (Arnos, Israel, Devlin, & Wilson, 1992). The reoccurrence risk is an estimate derived from empirical data of the likelihood that the progeny inherit the hearing loss. This estimate can be different from the actual reoccurrence risks when the genetic cause is known. Empirically, normal hearing parents with one deaf or hard-of-hearing child have a 5 to 17% chance of having another child who is deaf or hard-of-hearing. If one parent is deaf or hard-of-hearing, the chance is 7 to
20% that the child will be depending if the other parent is deaf. However, actual reoccurrence risks range from less than 1% to more than 50% or even 100% (Brunger,
4 Matthews, Smith, & Robin, 2001). The etiology allows for more precise estimate of recurrence risk counseling (Brunger et al., 2001).
5 GJB2 GENE - CONNEXIN 26
Hereditary non-syndromic sensorineural hearing loss (SNHL) is caused by mutations at least 50 different places or loci on the chromosomes (Gopalarao et al.,
2008). Despite the demonstrated locus heterogenetity of non-syndromic hearing loss, up to 50% of cases of genetic deafness can be attributed to mutations at a single locus, which contains the gene gap junction β 2 (GJB2), termed as DFNB1 (Petersen & Willems,
2006; Nance & Dodson, 2007). GJB2 gene mutations are the leading cause of pre-lingual, non-syndromic, recessive hearing loss in many populations (Petersen & Willems, 2006).
Audiologic Features
GJB2 mutations are associated with a broad range of phenotypes, although all expressed GJB2 mutations produce SNHL that is bilateral and symmetrical (Apps,
Rankin, & Kurmis, 2007). Individuals with GJB2 mutations have varying degrees of severity of the hearing impairment ranging from mild to profound and different audiometric configurations including downward sloping, flat, and u-shaped. Gopalarao et al. (2008) determined through a meta-analysis of seven studies that 19% of subjects who were homozygous for the GJB2 mutation had progressive hearing loss.
Pathophysiology
The GJB2 gene has been mapped to chromosome 13q11-q12. The GJB2 gene encodes the gap junction protein connexin 26 (Tang et al., 2006). The number 26 refers to the connexin protein’s molecular size (Nance & Dodson, 2007). Six connexin proteins
6 form hemichannels called connexons, which associate with adjacent connexons in adjacent cells to form a gap junction (Tang et al., 2006). The gap junctions assemble to form homologous or heterologus channels between adjacent cochlear cells that allow ions and small molecules to pass through (Nance & Dodson, 2007). Connexins and gap junctions are expressed in connective and epithelial tissue in the inner ear.
Gap junctions are essential for transduction in the cochlea and inner ear that changes sound waves from the mechanical form to an electrochemical signal. The large resting potential difference of 150mV between the endolymph and perilymph powers the transduction process. During transduction, the potassium ions flow into the perilymph by way of a concentration gradient and depolarize the hair cells. After transduction, the potassium ions are recycled back into the endolymph via gap junctions through the stria vascularis and spiral ligament. Gap junctions also maintain potassium homeostasis by keeping potassium away from the hair cells during transduction (Snoeckx et al., 2005).
Therefore, connexins and gap junctions are critical for maintaining the endocochlear potential and are essential for normal auditory function (Apps et al., 2007). GJB2 mutations disrupt the recycling of potassium ions to the endolymph. Potassium homeostasis is disrupted, which leads to the progressive intoxication of the Organ of
Corti and the hair cells. The consequences of GJB2 mutations are inner ear cellular dysfunction and cell death, resulting in hearing impairment (Apps et al., 2007).
Genotype/Phenotype Correlation
More than 100 mutations, polymorphisms and unclassified variants have been documented for GJB2 (Tang et al., 2006). The general conclusion is that biallelic GJB2 mutations, which consist of homozygous or compound heterozygous mutations, increase
7 the severity of the hearing loss (Liu et al., 2005). Furthermore, biallelic truncating mutations are associated with significantly more severe degree of hearing loss than biallelic non-truncating mutations (Snoeckx et al., 2005). Truncating mutations involve nonsense mutations and mutations caused by deletions or insertions that introduced a shift in the reading frame, resulting in a loss of connexin 26 expression. Non-truncating mutations consist of missense mutations, such as amino acid substitutions and inframe deletions. The abnormal resulting protein may produce nonfunctional channels or partially functioning channels with altered gating or trafficking of ions (Apps et al.,
2007).
In the GJB2 gene sequence, the 35delG truncating mutation results from a deletion of a guanine from a series of 6 consecutive guanines, extending from position
30-35, which leads to a frameshift mutation and a premature stop codon at nucleotide 38
(Apps et al., 2007; Petersen & Willems, 2006). Snoeckx et al. (2005) associated homozygous 35delG with severe to profound degree of SNHL in 70-95% of affected children. 35delG homozygotes were also found to have significantly more hearing impairment than 35delG/non-35delG compound heterozygotes (Cryns et al., 2004).
Ethnic Prevalence of the GJB2 Mutation
Specific combinations of GJB2 mutations exist in different populations. Two of the most common GJB2 mutations, 35delG and 235delC have been associated with the
Caucasian and Asian ethnicities, respectively. The high prevalence of these mutations in localized populations is due to a founder effect (Oguchi et al., 2005). The founder effect describes the high frequency of a particular disease allele in a specific population due to that population being derived from a small number of colonizing founders, one or more
8 of whom carried the mutation. Additionally, the carrier rate for 35delG GJB2 mutations is as much as 4% in families with severe and profound prelingual hearing loss in New
Zealand, France, and the United Kingdom (Apps et al., 2007).
The most prevalent GJB2 mutation in the Japanese and Chinese population is
235delC. 235delC is a truncating mutation, which is comparable to the 35delG mutation found in the northern European population. The phenotype is a severe to profound hearing loss for individuals who are homozygous for 235delC and those who are
235delC/non-235delC compound heterozygotes (Oguchi et al., 2005). In contrast, individuals who are homozygous or compound heterozygous for the second most frequent GJB2 mutation, the missense mutation V37I, showed a significantly milder hearing loss (Oguchi et al., 2005). The results of the Oguchi et al. (2005) study support the general rule that the phenotype observed with truncating GJB2 mutations are more severe than with missense mutations.
Nevertheless, GJB2 mutations may not be a common etiology for non-syndromic, autosomal recessive sensorineural hearing impairment for all populations. Samanich et al.
(2007) performed GJB2 sequencing and analysis for 93 Hispanic and 94 African
Americans, most with bilateral severe or profound hearing loss. Homozygous GJB2 mutations were found to be rare among African American and Caribbean Hispanic individuals compared to 15-40% found in North American populations. Therefore, the carrier rate for GJB2 mutations in African American and Caribbean Hispanic populations is a lot lower than the 1 in 50 carrier rate for North American populations. Samanich et al. (2007) concluded that pathologic GJB2 mutations are not a significant cause of hearing loss in these respective minority populations in America.
9 The high frequency of GJB2 mutations in many countries combined with the discovery that GJB2 mutations cause 50% of non-syndromic autosomal recessive hearing loss makes testing for GJB2 mutations one of the first steps that can be taken to determine the genetic etiology of a hearing loss. Additionally, audiograms of individuals with GBJ2 hearing loss are nonspecific and have been documented to cause mild through profound degrees of SNHL with a variety of configurations (Liu et al., 2005). Therefore,
GJB2 mutation testing is advised regardless of the degree or configuration of hearing loss
(Cryns et al., 2004).
Diagnosis and Treatment
It would be beneficial to know if the etiology of the hearing loss was due to a
GJB2 mutation. This information has implications for audiologic rehabilitation and for decisions regarding medical and educational management (Pandya & Arnos, 2006). For example, if GJB2 is the cause of the hearing loss, it is known that the mutation is localized in the cochlea and does not affect any other systems, such as cognitive abilities.
Since the damage from GJB2 is restricted to the cochlea, children perform well with amplification, such as hearing aids and cochlear implants (Gregoret, 2005).
Connell et al. (2007) studied speech acquisition and language development in children with cochlear implants and GJB2 hearing loss. They found that the primary variables for speech perception and language development were the duration of cochlear implant use and the age of implantation among children with cochlear implants.
However, the investigators concluded that cochlear implanted children with DFNB1 achieved greater verbal and expressive language scores at a faster rate than comparable cochlear implanted children without GJB2 hearing loss. These results suggest that
10 etiology of the hearing loss can be beneficial in predicting early cochlear implant outcomes (Connell et al., 2007).
11 SCL26A4 GENE: ENLARGED VESTIBULAR AQUEDUCT AND
PENDRED SYNDROME
Enlarged Vestibular Aqueduct - EVA
Enlarged vestibular aqueduct (EVA) is the most frequent inner ear radiological malformation connected to SNHL in children (Madden et al., 2007; Yoon et al., 2008), accounting for 5-10% of inherited hearing impairment (Berrettini et al., 2005). The criteria of the EVA is a diameter >1.5 mm measured at the midway point between the endolympathic sac and the vestibule (Berrettini et al., 2005; Yang et al, 2007; Suzuki et al., 2007). Bilateral EVA is more prevalent than unilateral EVA, accounting for between
55-94% of cases (Berrettini et al., 2005). This structural abnormality is associated with both syndromic and non-syndromic SNHL. Hearing loss caused by EVA is a distinguishing feature of both DFNB4 and Pendred syndome.
Non-Syndromic Enlarged Vestibular Aqueduct – DFNB4
Non-syndromic Enlarged Vestibular Aqueduct (ns-EVA), also known as DFNB4, is characterized by an abnormally large vestibular aqueduct and fluctuating, progressive
SNHL. The recessively inherited hearing loss is known to increase in severity accompanying activities involving minor head trauma and large sudden shifts in barometric pressure. The onset of the symptoms most frequently occurs in childhood and may start as soon as birth and as late as adolescence. There is wide variability of audiologic features encompassing hearing impairment and vestibular abnormalities.
12 Hearing loss in a study by Berrettini et al. (2005) varied from moderate to profound in sloping or u-shaped configuration. Vestibular symptoms are reported in ns-EVA and range widely from occasional unsteadiness to severe episodic vertigo. The diagnosis of
EVA is made by MRI and confirmed with a CT scan.
Pendred Syndrome
Pendred syndrome (PS) is an autosomal recessive disorder that consists of a combination of profound prelingual SNHL from inner ear malformations, such as EVA and Mondini deformity, defective iodide organification and goiter. Pendred syndrome is the most common form of syndromic hearing loss, accounting up to 10% of hereditary hearing loss (Yang et al., 2007). Enlarged vestibular aqueduct is the most common inner ear malformation in PS syndrome, which is found to be present in 80% of patients
(Azaiez et al., 2007). The consequences of PS results in reduced iodine transport across the membrane, thus trapping the iodide, resulting in high concentrations of thyroglobulin in the thyroid. Perchlorate tests measure the ability of iodine to bind to thyroglobulin.
Perchlorate testing and radiologic imaging are used to clinically diagnose PS.
Pathophysiology
Enlarged vestibular aqueduct is often connected to the malfunction of the
SLC26A4 gene, mapped to 7q23-q31. The SCL26A4 gene encodes a chloride-iodide transporter and mediator of chloride and formate called pendrin. Pendrin, a transmembrane protein, is expressed in the fetal inner ear, along with the thyroid and kidney. Defective pendrin can cause inner ear malformations, such as EVA, Mondini dysplasia and PS (Iwasaki et al., 2006; Azaiez et al., 2007).
13 The role of the transmembrane protein pendrin in the inner ear is to transport chloride, iodide, and bicarbonate ions in order to assist with pH control in the endolymph.
The SCL26A4 gene is expressed in areas where endolymph production occurs, such as the stria vascularis, external spiral sulcus, in Hensen’s cells, the spiral eminence and in the utricle. The organ of Corti structures like inner hair cells (IHCs) and outer hair cells
(OHCs), supporting cells and spiral ganglion also express the gene (Maciaszczyk &
Lewinski, 2008).
- Pendrin assists with bicarbonate (HCO3 ) secretion into the endolymph. When the pendrin is ineffective, bicarbonate production is decreased resulting in endolymph acidification. The imbalance in endolymph pH represses operation of the KCNJ10 potassium channels and the TRP5 and TRP6 calcium channels, which are crucial to maintain endocochlear resting potential. The increased concentration of potassium and decreased levels of calcium leads to a decreased endocochlear (intracochlear) potential.
The disturbances in inner ear fluid homeostasis result in structural abnormalities, such as vestibular aqueduct and endolymph sac enlargement (Maciaszczyk & Lewinski, 2008).
The role of the SLC26A4 gene and pendrin in the inner ear was further investigated using mice. SCL26A4-knockout mice, which are mice engineered with two mutated, defective SCL26A4 genes, had profound hearing loss with vestibular abnormalities, such as an unsteady gait, tilting of the head, circling, and abnormal reaching. The inner ear in these mice developed normally until embryonic day 15, when there was subsequent development of progressive endolymphatic system dilation leading to the complete degeneration of sensory cells and malformation of the otoconial membranes and otoconia (Kopp, Pesce, & Solis-S, 2008; Madeo et al., 2006).
14 Genotype/Phenotype Correlation
Albert et al. (2006) studied SLC26A4 mutations in participants with non- syndromic EVA. The onset of the hearing loss was prelingual or within the first decade of life. Biallelic mutations were associated with more severe HL, earlier age of discovery, fluctuating HL, and earlier walking age. A combination of other yet to be identified genetic factors, modifier genes and environmental factors are theorized to contribute to the heterozygous SLC26A4 mutation, resulting in EVA (Berrettini et al., 2005).
In a study from Suzuki et al. (2007), clinical findings were retrospectively analyzed from a 39-person study population with EVA and bialleleic SLC26A4 mutations. The hearing thresholds ranged from mild to profound, with an average pure tone average (PTA) of 74.6 dB HL. Progressive hearing loss and fluctuation of hearing were documented in 22 of 25 (88%) and 24 of 26 (92.3%) of the test group, respectively.
Twenty-four subjects complained of vertigo of the 34 participants for whom data was available, which comprises 70.6% of the study population.
Additionally, Madden et al. (2007) found that children with abnormal mutations in the SCL26A4 gene result in larger vestibular aqueduct diameter. Furthermore, subjects with bilateral EVA who are either homozygous or heterozygous for mutations in the
SCL26A4 gene had significantly more hearing loss, 64dB average PTA vs. 32dB average
PTA, compared to subjects with bilateral EVA without the mutation (Madden et al.,
2007).
Differential Diagnosis
Scott et al. (2000) performed a functional analysis of pendrin produced by PS alleles by simulating iodide and chloride uptake in Xenopus oocytes that were injected
15 with PS alleles. The iodide and chloride uptake of the 3 common PS alleles (L236, T416P and E384G) was not significantly different from the water control. In contrast, there was increasing amounts of anion transport by the SCL26A4 gene mutations that were found in subjects with non-syndromic hearing loss (V480D, V653A and I490L/G497S).
However, these DFNB4 mutations showed significantly less iodide and chloride uptake than the wild-type (wt) pendrin protein. These results suggested that mutations found in
PS cause complete loss of pendrin protein function while the DFNB4 mutation has enough residual pendrin function in the thyroid to prevent goiter.
Pendred Syndrome may be difficult to differentially diagnose from ns-EVA because goiter has various levels of penetrance, which may not be expressed phenotypically. Also, goiter does not develop until adolescence, so it is difficult to clinically differentially diagnose PS from ns-EVA before the age of 10 (Pryor et al.,
2005). Pryor et al. (2005) opted to use medical history, family history, thyroid ultrasonography, serologic testing and formal endocrinological evaluation to further analyze confounding perchlorate discharge tests inorder to assist with differential diagnosis. The perchlorate discharge values were significantly different between the patients with 2 mutant alleles and those with 1 or 0 mutant alleles (Pryor et al., 2005).
Individuals with 2 SLC26A4 mutations had measurable iodine organizational defect, while the other group’s results were normal and thus indicated that the iodine organizational defect was absent. Bilateral EVA was observed in participants with 2, 1 or
0 mutant alleles. Unilateral EVA was only seen in patients with 1 or 0 mutant alleles. All the patients with two mutant SLC26A4 alleles had PS and the subjects with ns-EVA had one or no mutant alleles. Pryor et al. (2005) concluded that molecular testing and
16 perchlorate discharge results show evidence that PS and ns-EVA are distinct clinical and genetic entities.
Conversely, Azaiez et al. (2007) and Suzuki et al. (2007) concluded that there was not a simple Mendelian genotype-phenotype correlation between SCL26A4 with ns-EVA and PS. Instead they made the argument that ns-EVA and Pendred syndrome were on the ends of a continuum of diseases caused by SCL26A4 mutations. Of the participants in
Azaiez et al. (2007) study with inner ear abnormalities, 65% had zero SCL26A4 mutations. Even though it was more likely that those with PS would have biallelic
SCL26A4 mutations, some subjects who have been clinically diagnosed with PS had one or zero mutations. Recently, Yang et al. (2007) reported that a mutation in the SCL26A4 promoter contains a key transcriptional regulatory element that binds FOXI1, which regulates transcription of the SCL26A4 gene. Mutations in the promoter region, in the
FOX11 gene and in double heterozygous combination mutations in both SCL26A4 and
FOX11 genes can also cause PS or EVA (Yang et al., 2007; Azaiez et al., 2007; Hilgert,
Smith, & Van Camp, 2009).
Ethnic Prevalence of SLC26A4 Mutations
Albert et al. (2006) attempted to establish the prevalence of the SLC26A4 mutation in a Caucasian population with non-syndromic hearing loss and inner ear radiologic abnormalities. One hundred and nine children from 100 families were recruited with bilateral non-syndromic SNHL associated with EVA as confirmed by CT scan. None of the patients had the GJB2 mutation. Mutations in SLC26A4 were found in
40% of the families and the prevalence of bilateral mutations was 24% (Albert, et al.,
2006). Albert et al. (2006) estimated that of up to 4% of non-syndromic hearing
17 impairment is due to mutations in the SLC26A4 gene, making SLC26A4 mutations the second most frequent gene for non-syndromic deafness in the Caucasian population.
Tsukamoto et al. (2003) recruited 10 Japanese families in which some members have been clinically diagnosed with PS and 32 Japanese families in which some individuals had EVA without goiter. SCL26A4 mutations were present in 90% of PS families and in 78.1% of families with ns-EVA. The H723R allele was found in 53% percent of the Japanese study population. Also, in a study using a Japanese population with bialleleic SLC26A4 mutations, Suzuki et al. (2007) found that the most common allele was H723R, which was present in 74.9% of the study population. The most frequent alleles found in the Caucasian population, L236P, T416P and IVS8+1G>A, were rarely found in the Japanese test subjects, that again emphasizes the importance of ethnicity in testing for genetic mutations and for making a genetic diagnosis (Tsukamoto et al., 2003).
Diagnosis and Treatment
The diagnosis of EVA is notable since it affects the counseling and medical intervention by audiologists and medical professionals. Patients with EVA are counseled to avoid even mild head trauma due to the association between head trauma and sudden decreases in hearing (Madeo, et al., 2006). For children with EVA who experience a sudden hearing loss, aggressive intervention with corticosteroids can preserve residual hearing with a response rate of 85% (Lin, Lin, Kao, & Wu, 2005).
The primary focus for patients with PS or ns-EVA should be aural rehabilitation through amplification with hearing aids or CIs (Madeo et al., 2006). Aggressive medical intervention and aural rehabilitation preserves residual hearing for speech and language
18 development (Lin et al., 2005), since children with more residual hearing tend to be more successful with amplification. Cochlear implantation is a viable option for EVA because the hearing loss is cochlear in origin and the retrocochlear structures do not appear to be affected (Berrettini et al., 2005).
Miyamoto, Bichey, Wynne, and Kirk (2002) conducted a retrospective study that compared PTA, speech detection thresholds and various, age appropriate open-set speech perception tasks between 23 CI patients with EVA and a control group of 46 CI users.
Both groups were matched by patient age, age of implantation, and whether the onset of the hearing loss was pre- or post-lingual. The results indicted that there were not any significant differences between the two groups in PTAs, speech detection thresholds, and open-set speech perception testing. The investigators concluded that CIs provide benefit to 14 adults and 9 children with EVA in the study and EVA alone is not a contraindication to CI implantation (Miyamoto et al. 2002.)
Goiter and hypothyroidism development in PS demonstrates intrafamilial and regional variability. Nutritional iodine intake is a significant modifier of the goiter phenotype (Kopp et al., 2008). Dysfunctional iodide transport can be treated by dietary alterations, such as adequate intake of vegetables and dairy products (Iwasaki et al.,
2006). Increased iodine intake results in very mild or absent enlargement of the thyroid
(Kopp et al., 2008). Using molecular genetic testing could diagnose PS before the goiter develops, which enables monitoring and earlier treatment of the symptoms.
19 MITOCHONDRIAL DNA HEARING LOSS
Mitocondrial Genetics
The hearing loss phenotype is not just the result of mutations in chromosomal, nuclear DNA. Inherited and acquired hearing impairment are also attributed to mitochondrial DNA (mtDNA) mutations (Hema Bindu & Reddy, 2008). Up to 20% of post-lingual hearing loss may be caused by mtDNA mutations (Kokotas, Petersen, &
Willems, 2007). The mtDNA is a 16,569 base-pair (bp), circular, double-stranded molecule. Each nucleated somatic cell contains 2-10 mtDNA molecules among hundreds of mitochondria, accounting for 0.5% of the total DNA in the cell (Kokotas et al., 2007).
Mitochondria DNA has 37 genes composed of 13 mRNAs, 2 rRNAs and 13 tRNAs, which enable the hundreds of mitocondria in each cell to fulfill a variety of metabolic functions (Hema Bindu & Reddy, 2008). Mitochondria produce energy in the form of cellular adenosine triphosphate (ATP) and is the primary source of ATP, creating more than 5 times the amount produced by glycolysis per glucose molecule (Kokotas et al.,
2007).
Mitochondria also play a central role calcium signaling, reactive oxygen species
(ROS) production and apoptosis (Kokotas et al., 2007). Reactive oxygen species are otherwise known as free radicals. Reactive oxygen species contain an unpaired number of electrons making them chemically reactive and extremely toxic to cellular and subcellular structures. Reactive oxygen species production results in mutations and deletions in
20 mtDNA genome by decreasing the production and the function of the endogenous enzymes that protect the cell against ROS damage (Seidman et al., 2004). Mitochondrial
DNA mutation accumulation is problematic because the mutation rate is 10 times greater than in nuclear DNA and a mtDNA repair system is virtually absent (Kokotas et al.,
2007). When mtDNA damages accrue, the cell becomes bioenergetically inefficient. The metabolic structures, such as hair cells and the stria vascularis become compromised by the deficiencies in intercellular ATP production (Forli et al., 2007).
Due to the widespread location of mitochondria, mitochondrial pathology results in heterogeneous disorders often with multi-system involvement. Mitochondrial function is genetically controlled by alterations in primary mtDNA or by mutations in nuclear genes affecting mitochondria performance. Phenotypic expression of mtDNA defects is impacted by environmental factors (Forli et al., 2007). Therefore, phenotypic expression for hearing loss is extremely variable and thus the same mtDNA genetic cause results in different phenotypes (Forli, et al., 2007). Mitochondrial DNA is inherited maternally by the microcondria inside the oocyte prior to fertilization. Males can be affected by mtDNA disorders, but do not continue to transmit the mutated mtDNA to their progeny.
Pathogenic mutations are usually heteroplasmic, or defined as a cell that has more than one mtDNA genotype. These heteroplasmic mtDNA mutations cells cause mitochondrial diseases, such as Mitochondrial encephalopathy, lactic acidosis, and strokelike episodes
(MELAS) and maternally inherited diabetes and deafness (MIDD). Only a couple of mtDNA diseases comprise of a pathogenic single mtDNA genotype or homoplasmic, one of which is non-syndromic hearing loss caused by the A1555G mutation (Kokotas et al.,
2007).
21 MITOCHONDRIAL MULTISYSTEMIC SYNDROMES
Heteroplasmic mtDNA mutations are implicated in a variety of multisystemic syndromes. Mitochondrial encephalopathy, lactic acidosis, and strokelike episodes
(MELAS) is one of several rare neuromuscular syndromes connected to mtDNA mutations. The diagnostic criteria for MELAS is stroke-like episodes prior to 40 years old, encephalopathy comprising of dementia, seizures or both, mitrocondria myopathy consisting of lactic acidosis, ragged-red muscle fibers, or both, and two of the following symptoms: normal motor development, recurrent vomiting, and recurrent headaches.
Additionally, hearing loss is present in at least 50% of MELAS cases (Sinnathuray, Raut,
Awa, Magee, & Toner, 2003).
Mutations in mtDNA can also lead to maternally inherited diabetes and deafness
(MIDD). Maternally inherited diabetes and deafness is a syndrome whose symptoms include diabetes mellitus and SNHL. In patients with MIDD, the hearing loss develops in early adulthood and usually precedes the diagnosis of diabetes (Murphy, Turnbull,
Walker, & Hattersley, 2008). The onset of diabetes mellitus is non-insulin dependent and then progresses to become insulin dependent with age (Kokotas et al., 2007).
Audiologic Features
The mtDNA pathology in systematic neuromuscular syndromes and MIDD result in degeneration of cochlear structures, such as the OHCs, the stria vascularis and the spiral ganglion. However, severely affected people with mtDNA pathology have signs of
22 retrocochlear pathologic changes. The possible explanations are that the mutation affects the cochlea before the retrocochlear structures and the retrocochlear effects are a result of an advanced disease process. Another interpretation is that the retrocochlear symptoms were secondary to a diseased cochlea.
The hearing loss in MELAS and MIDD has been classified as a gradual onset bilateral SNHL that affects the high frequencies first and then proceeds to the lower frequencies (Forli et al., 2007). The age of the onset of the hearing loss is reported to be between the teens and forties. Additionally, sudden onset hearing loss has been associated with the stroke-like episodes of MELAS (Forli et al., 2007). The hearing losses have also been documented as slowly or rapidly progressive with a 1.5 dB to 7.9 dB decrease per year (Sinnathuray et al., 2003). Variability in hearing loss severity and configuration were reported (Forli et al., 2007), with Chinnery et al. (2000) finding that the severity of hearing loss in MELAS patients was correlated with percentage of mutated mtDNA in the muscles. The hearing losses in MELAS and MIDD have similar features because of a similar genetic mutation.
A3243G Mutation
The MELAS and MIDD clinical features arise from the A3243G point mutation in the tRNALEU(UUR) gene, MTTL1 (Sinnathuray et al., 2003). The A3243G mutation is responsible for 80% of the cases of MELAS and 60% of MIDD (Sinnathuray et al.,
2003). The A3243G mutation also causes many additional mtDNA syndromes, such as myoclonic epilepsy and ragged red fibers (MERRF), progressive external ophthalmoplegia (PEO), Kearns-Sayre syndrome, Leigh syndrome, progressive non- diabetic kidney disease and cardiac diseases (Nagata et al., 2001). These diseases are
23 characterized by defective oxidative phosphorylation caused by a combination of mutations in mitochondrial and nuclear genes (Kokotas et al., 2007).
The prevalence is 16.3/100,000 in the general adult population for A3243G mutation, and 0.314-1.74% of that population was treated for SNHL in ear, nose and throat (ENT) clinics (Sinnathuray et al., 2003). Whittaker et al. (2007) found that 21 of
29 patients with mitochondrial diseases accompanied with hearing loss and diabetes tested positive for the A3243G mutation. The A3243G mutation expresses incomplete penetrance (Peterson et al., 2007), essentially meaning that not all of the people who inherit the mutation will develop hearing loss caused by the mutation. The threshold theory has been used to explain the phenotypic variability of mtDNA diseases.
Heteroplasmic mtDNA defects are only expressed when the mutation percentage level in mitochondra exceeds a critical threshold level in in vitro studies to where the damaging effects of the mutation are no longer offset by the normal, wild type mtDNA. The level of mutated mtDNA varies between and within individuals with mtDNA disease and thus the threshold of expression differs (Chinnery et al., 2000).
Diagnosis and Treatment
Hearing loss is often underestimated and undiagnosed because of the attention paid to the other symptoms in mitochondrial disorders, even though it could be the first symptom. Sixty-seven percent of patients with mtDNA disorders also have SNHL; therefore, mitochondrial diseases should be considered in cases of progressive SNHL especially those associated with multi-system involvement (Seidman et al. 2004).
Since mtDNA syndromic hearing loss is inherited maternally, a thorough case history especially noting any deafness and diabetes from the patient’s mother is
24 important. Mitochondrial DNA deletions preferentially accumulate in post-mitotic structures such as the inner ear, retina, skeletal muscle and brain (Seidman et al., 2004), so any diseases or symptoms affecting those parts of the body need to be noted and documented. Nagata et al. (2001) recommends genetic testing for A3243G mutations in patients with diabetes and/or cardiac disorders along with a family history of post-lingual, maternally transmitted hearing loss. Additionally, Whittaker et al. (2007) advised that if the results of the screening for the A3243G mutation are negative, that a referral to a neuromuscular specialist be made in order to investigate for other mtDNA mutations.
Cochlear implantation can be a good option since the hearing loss is post-lingual and cochlear, with the retrocochlear sites, such as nerve fibers, appearing to be intact
(Sinnathuray et al., 2003). Sinnathuray et al. (2003) reported that the cochlear implantees, who all have a variety of mtDNA mutations, have performed well, including most having good open set speech recognition and 58% of the study population are able to use the phone.
25 MITOCHONDRIAL DNA MUTATIONS AND NON-SYNDROMIC HEARING
LOSS
Audiologic Features
The A1555G mutation causes non-syndromic hearing loss and aminoglycoside induced hearing loss. The A1555G mutation, along with connexin 26 mutation, is the most common genetic cause of hearing loss (Usami, Abe, Shinkawa, & Kimberling,
1998). Audiological features commonly include bilateral, symmetrical SNHL, usually progressive with increased hearing loss in the high frequencies, and tinnitus (Usami et al.,
1998). The absence of distortion product otoacoustic emissions (DPOAEs) suggests a cochlear site of lesion for the hearing loss (Noguchi et al., 2004). The wide range of
A1555G clinical phenotypes encompasses profound congenital hearing loss and normal hearing acuity (Hobble et al., 2008). The configuration and degree of the hearing loss varies greatly within and across families (Usami et al., 1998; Kokotas et al., 2007).
Vestibular abnormalities (Usami et al., 1998; Noguchi et al., 2004) and inner ear malformations (Usami et al., 1998) are not characteristics of an A1555G mutation. The penetrance of mtDNA non-syndromic hearing loss is generally low, with about 3% of
SNHL caused by A1555G mutations within the Japanese population (Usami et al., 1998).
Aminoglycoside Ototoxicity
The A1555G mutation can increase susceptibility to aminoglycoside antibiotic ototoxicity. Aminoglycosides are used to in patients with chronic infections, such as
26 tuberculosis and cystic fibrosis, to combat aerobic gram negative bacterial infections.
Neomycin and kanamycin cause cochlear damage, streptomycin and gentamycin cause vestibular damage, and tobramycin affect both equally (Hema Bindu & Reddy, 2008).
Ototoxicity is usually considered irreversible. The hearing loss from even a small dosage of aminoglycoside injections can happen within 3 months of being injected and is unrelated to the patient’s age at the time of injection (Usami et al., 1998).
Aminoglycoside antibiotic ototoxicity risk factors include therapy that lasts more than 7 days, prior exposure to aminoglycosides, elevated serum levels, high daily dose, use in neonates, noise exposure, and a background of predisposing mutation, such as the
A1555G mutation (Kokotas et al., 2007).
Genetics of the A1555G Mutation
The A1555G mutation affects the MTRNR1 gene, which encodes the ribosomal
RNA small subunit, 12S rRNA (Kokotas, et al., 2007). Hobble et al. (2008) demonstrated that pathogenic mutation A1555G significantly reduces the accuracy of translation by the ribosomes, causing a ribosomal site that is increasingly susceptible to mutations from aminoglycosides. The translation of the RNA is disrupted enough to lead to changes in the resulting protein, thus leading to changes of phenotypic expression, which results in hearing loss (Hobble et al., 2008).
A1555G mutations are prevalent in populations all over the world and vary by ethnicity. The consequence of having such common mutation is the existence of high-risk
A1555G populations all over the world. Therefore, an A1555G mutation molecular screen could be beneficial just because of the sheer number of people in the world who have the mutation (Usami et al., 1998). The identification of the A1555G mutation can
27 also assist with counseling regarding the use and side effects of aminoglycoside antibiotic treatment. Hearing aids and cochlear implants have been shown to provide benefit to those with the mutation (Usami et al, 1998).
28 AGE RELATED HEARING LOSS – PRESBYACUSIS
Audiological Features
Presbyacusis is age-related hearing loss that involves a reduction in hearing sensitivity and changes in auditory processing (Boettcher, 2002). Presbyacusis is a very common problem affecting older adults, encompassing 83% of the elderly in the
Framingham cohort (Gates, Cooper, Kannel, & Miller, 1990) and ~45% overall prevalence in the Epidemiology of Hearing Loss Study (Cruickshanks et al., 1998).
Presbyacusis is typically characterized as a bilateral, high frequency sloping gradual hearing loss with difficulty understanding speech in noisy situations (Gates & Mills,
2005). The trouble with understanding speech in noise stems from the inability to discriminate the lower intensity, high frequency voiceless consonants due to the sloping configuration of the high frequency hearing loss (Gates & Mills, 2005) along with auditory and temporal processing declines (Pichora-Fuller & Souza, 2003).
Genotype/Phenotype Correlation
Presbycusis is associated with an increase in DNA damage and reduction in mitochondrial function (Yamasoba et al., 2007). Generally, the mtDNA 4977-bp deletion, called the common aging deletion, is found more frequently in aged tissues and in older subjects temporal bones (Yamasoba et al., 2007). Research by Dai et al. (2004) using human temporal bone results revealed that the mtDNA 4977 deletion in the temporal
29 bone was common in the presbyacusis group (50%), less common in the age-matched group (21%) and rare in the young and middle-aged group.
Presence of mtDNA 4977-bp deletion had a close correlation with the condition of the inner blood supply but no linear correlation with age or PTA threshold shift at high frequencies in the presbyacusis group (Dai et al., 2004). In the temporal bones with the mtDNA 4977 deletion, the lumen of the vasa nervorum of the internal acoustic meatus
(IAM) showed a more severe narrowing. Dai et al. (2004) concluded that there is an anatomic reason for correlation between the mtDNA 4977 and presbyacusis. The blood vessels are small and narrow, which makes it easier to cause hypoxia of the inner ear leading to ischemia. The resulting lower oxygen metabolism reduces the level of oxidative phosphorylation, which increases free radicals causing damage to mtDNA (Dai et al., 2004). Therefore, oxidative phosphorylation decreases and mtDNA mutation rate increases with age.
There is a significant reduction of blood supply and the ongoing need for energy generation through oxidative phosphorylation in the aging cochlea. Aging in the central nervous system is directly correlated with oxidative stress (Seidman et al., 2004). mtDNA damage is more frequent in age-related hearing loss and mtDNA lesions are frequently associated with congential and acquired deafness. Degeneration of hair cells, spiral ganglion cells and stria vascularis are pathological cochlear anatomical changes documented in those with age related hearing loss (ARHL) (Yamasoba et al., 2007).
Diagnosis and Treatment
Mitochondrial DNA disorders appear to follow the principles of the multifactoral model. Inherited nuclear and mtDNA susceptibility genes in combination with
30 environmental factors such as diet and other toxic factors might further contribute to the progressive breakdown of mitochondrial function with the aging process, resulting in late-onset disease (Yamasoba et al., 2007). The multifactorial model of disease holds implications for treatment of diseases by implementing a holistic approach, which would include lifestyle changes along with medication. This holistic approach also may apply to audiologic rehabilitation. Auditory rehabilitation may be more effective if the patients make lifestyle changes in addition to using amplification.
Calorie restriction (CR) has been shown to protect mtDNA against age-related decline by reversing much of the mtDNA damage, by reversing many changes in mtDNA gene expression, and by increasing energy metabolism (Yamasoba et al., 2007). Calorie restriction has been reported to slow ARHL in rats, and improve auditory brainstem response (ABR) thresholds, in essence by reducing the quantity of mtDNA deletions.
Effects of CR are variable and depend on the genetic background.
Additionally, ingesting dietary supplements could be helpful. Alpha-lipoic acid and acetyl-l-carnitine diet supplements reverse structural decay and reduced the common aging deletion in the stria vascularis and auditory nerve in guinea pigs and mice
(Yamasoba et al., 2007). Also, the dietary supplement Lecithin may preserve cochlear mitochondrial function and protect age related hearing loss in rats (Seidman et al., 2004).
Currently, the effect of the antioxidant resveratrol, a component of the red wine grape, is being investigated in diminishing age-related decreases in auditory capabilities (Seidman et al., 2004).
31 USHER SYNDROME
Usher syndrome is a recessively inherited syndrome, whose hallmarks are hearing loss and retina pigmentosa (RP), which results in progressive blindness from retina degeneration (Cohen et al., 2007). Night blindness is the first symptom of RP, which progresses to the narrowing of the visual field or “tunnel vision”, and then to complete blindness (Reiners, Nagel-Wolfrum, Jürgens, Märker, & Wolfrum, 2006). Vestibular dysfunction may also be involved. Usher syndrome is also associated with reduced odor sensitivity, sperm motility, mental deficiency, cerebral atrophy, and ataxia (Kremer, van
Wijk, Märker, Wolfrum, & Roepman, 2006; Reiners et al., 2006). This heterogeneous disease is mapped to 11 different chromosomal loci thus far (Liu et al., 2008).
The prevalence of Usher syndrome is 3-6.2 per 100,000, consists of 50% of the deaf-blind population (Kenner, Gallo, & Bryant, 2005; Kremer et al., 2006), 10% of the population in schools for the deaf and 3-6% of children with congenital deafness (Liu et al., 2008). There are three clinical types of Usher syndrome, classified by severity and progression of hearing loss along with the presence or absence of vestibular dysfunction.
Usher Syndrome Type I
Usher syndrome type 1 (USH1A-H) is the most severe form of Usher consisting of congenital severe to profound HL with absent vestibular responses. Patients with
USH1 tend to have a delay in walking age of more than 18 months old due to areflexia.
The pre-pubescent onset of retina pigmentosa is diagnosed by end of first decade
32 (Kremer et al., 2006). The hearing loss along with the onset and progression of retina pigmentosa shows considerable intrafamilial and interfamilial variation (Kenner et al,
2005), again due to the heterogeneity of the disease. Usher syndrome type 1 generally comprises 30-40% of Usher syndrome.
Usher syndrome type 1B (USH1B) is the most common subtype in the United
States and United Kingdom population, comprising between 1/2 and 1/3 of patients with
Usher syndrome type 1. The gene responsible is MYO7A, mapped to 11q13.5 (Kremer et al., 2006). The recessive form of MYO7A encodes an unconventional myosin VIIA, which is an actin-based motor protein that was found to be responsible for maintaining the mechanoelectric transduction sensitivity (Eisen & Ryugo, 2007). Mutant forms of
MYO7A account for both USH1B and non-syndromic hearing loss DFNB2. Riazuddin et al. (2008) performed research by tagging both the USH1B and DFNB2 aberrant forms of
MYO7A with the green fluorescent protein (GFP) in transfected mouse IHCs. The
USH1B myosin VIIA protein did not localize to the IHC stereocilia in mice. Conversely, the myosin VIIA protein with the DFNB2 mutation localized correctly in the mouse hair cells, which led to the conclusion that the DFNB2 has a less severe phenotype due to the protein preserving some degree of normal function (Riazuddin et al., 2008).
Mutations in the CDH23 gene, located at 10q22.1, can either produce Usher syndrome type 1D (USH1D) or non-syndromic hearing loss DFNB12. Usher syndrome type 1D is second most common form of USH1 accounting for 10-33% of the USH1 phenotype (Kremer et al., 2006). The CDH23 gene encodes the cadherin protein, which functions as a scaffold protein. Usher syndrome type 1D results from nonsense, frameshift, splice site, and missense mutations in families with USH1D. CDH23
33 mutations are also responsible for DFNB12, which results from missense mutations
(Cohen et al., 2007).
The PDCH15 gene, mapped to 10q21.1 (Kremer, 2006) produces the protein
Protocadherin 15 at the Usher syndrome type 1F (USH1F) locus and is the most common cause of Usher type 1 for Askenazi Jews. Usher syndrome type 1F comprises 11% of the
USH1 phenotype for the United States and United Kingdom population. Mutations in the
PDCH15 gene are also responsible for the non-syndromic hearing loss DFNB23.
Truncating alleles of the PCDH15 gene result in USH1F, while missense mutations are associated with non-syndromic deafness (Ahmed et al., 2008).
Usher Syndrome Type 2
Usher syndrome type 2 (USH2A-C) produces prelingual, relatively stable, moderate to severe high frequency SNHL, normal vestibular function, and retina pigmentosa diagnosed during adolescence (Kremer et al., 2006; Sadeghi et al., 2004).
Usher syndrome type 2 is the most prevalent form of Usher syndrome, responsible for half of all cases (Cohen et al., 2007). Greater than 85% of Type 2 is Usher Syndrome
Type 2A (USH2A), which is mapped to 1q41, which produces the usherin protein. The most common USH2A mutation is 2299delG mutation. The USH2A phenotype has worse hearing loss, more progressive hearing loss, with an earlier age of onset compared to the non-USH2A phenotype (Sadeghi, et al., 2004).
Usher Syndrome Type 3
Usher Syndrome Type 3 (USH3) involves non-linear progressive hearing loss and retina pigmentosa of variable age of onset, along with variable vestibular findings.
Sadeghi, Cohn, Kimberling, Tranebjærg, and Möller (2005) found that by age 4-9, 35%
34 of the USH3 study subjects had a moderate sloping to a severe-to-profound hearing loss in the high frequencies. The rapid progression of hearing loss before the age of 20 distinguishes USH2 and USH3. Approximately half of the study participants became profoundly deaf by age 40 (Sadeghi et al., 2005).
The Usher syndrome type 3A (USH3A) gene encodes the clarin-1 protein and is mapped to 3q21-q25 (Cohen et al., 2007; Kremer et al., 2006). Usher syndrome type 3 is common among Finnish population and Askenazi Jews, accounting for 40% of Usher syndrome cases for both populations. Researchers had documented a predominance of the nonsense mutation 528T→G in USH3A subjects of Swedish and Finnish ancestry and the
144T→G missense mutation found in the Askenazi Jewish population (Cohen et al.,
2007). The predominance of USH3A in these ethnic populations is explained the founder effect, which can be attributed to geographic and genetic isolation (Liu et al, 2008).
Diagnosis and Treatment
Genetic assessment of Usher syndrome is a good idea because it is difficult to assess vestibular function in a young children and the electroretinogram (ERG) is invasive, involving general anesthetic (Liu et al., 2008). The early diagnosis of Usher syndrome allows prompt early intervention services (Kenner et al., 2005), such as aural rehabilitation. Rehabilitative options can be chosen with the anticipation of vision and hearing impairments. The identification is important for educational decisions because it will become difficult to exclusively use a visual based communication system, such as
American Sign Language (ASL), when RP develops. Braille may be an option (Liu et al.,
2008). Those with USH1 reported to do well with cochlear implants with good open set speech recognition with visual cues if implanted prior to three years of age (Liu et al.,
35 2008). The prognosis of Usher syndrome makes it important to develop aural communication skills before onset of visual impairment (Liu et al, 2008).
36 NEUROFIBROMATOSIS TYPE 2
Neurofibromatosis Type 2 (NF2) results from inactivation of tumor suppressing gene, featuring predisposition to multiple peripheral nervous system benign tumors including vestibular schwannomas, meninginomas and ependymomas along with ocular abnormalities, and skin tumors (Welling, Packer, & Chang, 2007; McClatchey, 2007).
Bilateral vestibular schwannomas are a hallmark of NF2 (Evans et al., 2005). Vestibular schwannomas comprise 1/3 to 2/3 of the initial NF2 symptoms, which are more likely presented in adults than in children (Baser et al., 2004). Neurofibromatosis Type 2 patients are diagnosed between the second and fourth decade, but 18% of patients present with symptoms prior to age 15 (Neff &Welling, 2005). The incidence of NF2 is 1 in 35 to
40,000 people (Evans, 1999) and the prevalence is 1 in 100,000 (Ruggieri et al., 2005).
Audiologic Features
Up to 41% of NF2 patients can initially present with unilateral SNHL with tinnitus, dizziness, and imbalance. The hearing loss is progressive, but can be of sudden onset. If bilateral vestibular schwannomas exist, the audiometric result is symmetrical or asymmetrical bilateral SNHL with tinnitus, dizziness, and headaches (Evans et al., 2005;
Neff & Welling, 2005).
Genetics of NF2
Located on chromosome 22q12, the NF2 gene comprises of 17 exons and codes a protein with 595 amino acids (Ruttledge & Rouleau, 2005). The mode of transmission is
37 autosomal dominant with high degree of penetrance since almost all cases express symptoms by late 50s (Evans, 1999). There is a 50% chance of transmitting the disease to the second generation and beyond. If the NF2 mutation is inherited, there is a 95% chance that the offspring will develop bilateral vestibular schwannomas (Neff & Welling,
2005). Half of all NF2 patients have new mutations without any apparent family history of NF2 (Hanemann, 2008). Between 25-30% of these individuals may have a less than
50% transmission rate due to somatic mosaicism, in which only a portion of the patient’s cells acquire mutations after conception and replicate, leading to a mixture of normal and mutated cells (Hanemann, 2008; Evans et al., 2005).
Genotype/Phenotype Correlation
Nonsense and frameshift mutations, which result in a truncated protein product
(Evans et al., 2005), are associated with severe phenotypes. Truncated proteins might be caused by the dominant negative effect where the mutant protein dimerizes with the normal copy of the protein, therefore inhibiting the function of the tumor suppressor
(Evans, 1999). Neurofibromatosis Type 2 truncating mutations have a relatively higher mortality rate (Evans et al., 2005) compared to other classifications of mutations. Milder phenotypes of NF2 are caused by somatic mosaicism, missense mutations, which result in a complete protein product, and inframe deletions, which result in no protein product.
However, if the offspring does inherit NF2 from a parent whose NF2 is caused by somatic mosaic mutations, than the offspring’s phenotype will be more severe than in the parent since all of the cells will contain the mutated DNA (Evans et al., 2005).
People with nonsense and frameshift mutations had significantly younger age of onset (≤20 years of age) and there tended to be significantly more peripheral, spinal
38 tumors, and meningomas (Baser et al., 2004). Severity, age of onset of symptoms, progression, number of tumors, disability level are relatively persevered within families
(Ruttledge & Rouleau, 2005). Zhao et al. (2002) concluded that there was a significant intrafamilal correlation between age of first symptoms, age at onset of hearing loss, and number of intracranial meningiomas.
Diagnosis and Treatment
The first step of assessing an at-risk patient for developing NF2 is an audiologic evaluation. A referral for an MRI should be requested if there is an asymmetric hearing loss, a family history of NF2, or any other NF2 stigmata. Acoustic reflexes or ABR testing may be helpful, but have decreased sensitivity in detecting small tumors and should not replace a MRI as the primary screening tool (Neff & Welling, 2005).
Aural rehabilitation should include hearing aids for moderate degrees of hearing loss in the early course of NF2. As the hearing loss progresses to severe and profound thresholds, CIs may be used provided that the auditory nerve (AN) is preserved. Good open-set speech recognition scores and speech in noise scores, ranging from 83% to
100%, have been obtained with CIs following vestibular schwannoma removal (Neff &
Welling, 2005).
Vestibular schwannoma removal surgery involving tumors larger than 2.0 cm results in severe to profound hearing loss, since the surgery most likely destroys the AN
(Neff & Welling, 2005). If both ANs were sectioned during vestibular schwannoma removal surgery or if the ANs were preserved but the promontory did not respond the electrical stimulation, an auditory brainstem implant (ABI) is a good alternative, in conjunction with lip reading and signing (Neff & Welling, 2005). Open-set speech
39 recognition using auditory only modality with the ABI is very poor, 3% to 7%, compared to the performance obtained with a CI, although the lip reading cues and sound awareness are helpful. Furthermore, 85-96% of patients with ABIs achieved sound awareness, but did not have any speech understanding (Neff & Welling, 2005).
Genetics may provide useful information in the clinical management of NF2
(Baser et al., 2004) such as presymptomatic clinical and radiological testing and genetic screening testing of at-risk relatives. The sensitivity of genetic testing for relatives is near
100% because the mutation can be compared to the known mutation of the affected family member (Neff & Welling, 2005). If it can be determined that a relative does not carry a NF2 mutation then frequent MRIs can be avoided.
Neff and Welling (2005) are convinced that the early detection and treatment of
NF2 affords a better opportunity to preserve the hearing by removing the tumors when they are still small, even though early surgical intervention is a greatly debated topic.
Another reason to act early would be to enable CI use in select patients after the vestibular schwannoma removal, which may result in improved speech understanding outcomes. With increased tumor size, it becomes more difficult to maintain an intact AN during surgery (Neff & Welling, 2005).
40 OTHER GENETIC HEARING LOSSES
Otoferlin (OTOF) Gene – DFNB9
Hearing loss caused by the recessively inherited otoferlin (OTOF) gene is labeled as DFNB9. Mutations are hypothesized to result in a cochlear hearing loss that affects the only the IHCs without structural inner ear abnormalities. The phenotype is a non- syndromic, profound SNHL with non-syndromic or environmentally caused auditory neuropathy (Rodriguez-Balesteros et al., 2003). Biallelic mutations result in profound
SNHL, while heterozygotes have variable degrees of SNHL with pre- or post-lingual onset (Rodriguez-Balesteros et al., 2008).
The OTOF gene encodes otoferlin, a calcium-binding, membrane-anchored protein. Otoferlin is expressed in the cochlea, vestibule and brain. Defects in Otoferlin result in impaired neurotransmission due to defects in fusion of the synaptic vesicles in
IHCs (Rodriguez-Balesteros et al. 2003; Rodriguez-Balesteros et al. 2008). The loss of function mutation prevents transcription of protein that enables function of otoferlin.
There is a good outcome with cochlear implantation for those with biallelic mutations of the otoferlin gene.
COCH gene – DFNA9
The COCH gene is located at 14q12-13 (Bischoff et al., 2005). Mutations in the
COCH gene result in hearing loss and vestibular dysfunction. The hearing loss that results from a mutated COCH gene is bilateral high frequency SNHL that progresses to a
41 profound degree. The autosomal dominantly inherited adult onset hearing loss begins in the 2nd-4th decade with complete penetrance (Robertson et al., 2006; Usami et al., 2003), meaning that all who inherit the mutation will eventually develop the disease. Vestibular abnormalities such as recurrent vertigo attacks (Usami et al., 2003) are variable between patients (Robertson et al., 2006). Hearing loss due to the COCH gene mutation is documented on four continents, but the prevalence is unknown due to lack of systematic genetic testing of adults (Robertson et al., 2006).
Genetic testing can provide a differential diagnosis between DFNA9 and
Meniere’s disease. Recurrent episodes of dizziness, with tinnitus, aural fullness, nausea and vomiting have been reported in some individuals with COCH mutations. The similarity between the vestibular symptoms documented in DFNA9 and Meniere’s disease patients led some investigators to suggest COCH gene mutations for Meniere’s disease (Usami et al., 2003). However, individuals with documented Meniere’s disease did not have mutations in the COCH gene (Sanchez et al., 2004; Usami et al., 2003).
42 CONCERNS AND OBJECTIONS ABOUT GENETIC TESTING
Genetic Counseling
The genetic evaluation process consists of many steps that go beyond the specific action of performing the genetic testing, with genetic counseling being one of the most important stages. First, pre-test counseling is crucial in providing the patient with a clear understanding of the risks and limitations of genetic testing, along with the potential benefits before consenting to have a genetic evaluation (Brunger et al, 2000; Arnos,
2008). The counseling is administered by a genetic counselor (Arnos, 2003). The risks of genetic testing include psychological, financial, and ethical concerns that need to be addressed and seriously considered before progressing further (Arnos, 2003; Arnos,
2008).
Genetic counseling needs to address the psychological ramifications that come from learning of the genetic results including frustration and depression. Patients and their families may find out information not related to the original reason for testing, for which they may be unprepared for and wish not to know. For example, parents of Ushers syndrome patients may go through a grieving process regarding the hearing loss, which manifests earlier than the RP. Then they go through a second grieving process when confronted with the prospect that their child may end up deaf and blind (Kimberling &
Lindenmuth, 2007). Genetic counseling needs to address issues of blame and
43 responsibility (Withrow, Burton, Arnos, Kalfoglou, & Pandya, 2008) and provide medical, psychosocial, and educational referrals when needed (Arnos, 1997).
Conversely, learning about a genetic cause of hearing loss may alleviate guilt and anxiety from hearing parents of deaf children, who may have previously attributed the hearing loss to their own ignorance, neglect or misfortune during and after the pregnancy
(Brunger et al., 2001; Withrow et al., 2008). By providing parents with a specific etiology for deafness, misconceptions or inaccurate beliefs about the cause might be replaced by providing more precise information (Brunger et al., 2001). Determining the etiology of the hearing loss can ultimately help with moving through the grieving process cumulating in the acceptance of the hearing loss diagnosis. Once the hearing loss is accepted, more effort and energy can go into discussing communication and medical requirements for themselves or their families (Arnos, 2003).
One problem with genetic testing is that patients or guardians are receiving poor or nonexistent genetic counseling. Brunger et al. (2001) found that over 90% of parents did not correctly understand genetic mechanisms or reoccurrence risks including incorrectly estimating the chance that they or their children will have another child with hearing loss. This indicates that participants either did not have genetic counseling or did not comprehend the information. Misunderstanding of test results leads to drawing inaccurate conclusions. Genetic testing is specific, but not sensitive. Just because the genetic test for a specific gene came back with a negative result does not eliminate the possibility that the hearing loss has another genetic cause (Brunger et al., 2001). Genetic pre- and post-test counseling is important to induce feedback from patients, which helps to lead to the correct interpretation of the results from parents (Brunger et al., 2001).
44 Genetic testing without supportive counseling can leave the patients and families confused and guilty (Madeo et al., 2006). This is projected as a problem as genetic testing increases in popularity and is performed more frequently (Brunger et al., 2001; Arnos,
2008). There are concerns from patients and family that genetic counseling is going to be completed by professionals who are not qualified to interpret and explain test results, such as a physician who is unfamiliar with current discoveries and accepted protocols for genetic testing (Withrow et al., 2008; Brunger et al., 2001). A referral to the appropriate provider, such as a geneticist or genetic counselor is recommended (Withrow et al.,
2008).
Privacy and Confidentiality of Genetic Testing Results
The privacy and confidentiality concerns of genetic testing process may deter some to pursue genetic testing. The access and use of the genetic testing results by third parties, such as insurance companies and employers holds particular concern with patients and families. Negative financial effects manifest themselves as employment and insurance discrimination. Health insurers may raise premiums or deny coverage due to the results of the genetic evaluation. Employers may refuse employment to those they find medically susceptible to serious medical conditions. However, the federal government and many states realized the potential negative implications and have passed laws that restrict access to the genetic test result information for insurance companies and banned genetic screening for employment decisions (Arnos, 2003; Arnos, 2008).
Genetic testing can have a negative impact on social and family dynamics.
Genetic testing provides information about the patient’s family in addition to information about the patient (Brunger et al., 2001). Details that are gathered during a family history
45 interview can be threatening and stigmatizing to family members (Arnos, 2003), including private information that is unrelated to the original reason for the genetic evaluation. The patient’s right to privacy and respect for the patient’s decisions are of up- most importance. However, other family members may be contacted to provide more information in order to have the genetic testing correctly interpreted (Arnos, 2003).
Practical complications to delivering information can occur when family members lose contract by moving or by changing names (Harris, Winship, & Spriggs, 2005). There may also be a duty to correspond with other relatives if the genetic testing results have repercussions for the health and well-being of those family members (Arnos, 2003).
Genetic Testing and The Deaf Community
Ethical concerns are also paramount for the Deaf community concerning genetic testing for hearing loss. The Deaf community has profound hearing loss and use sign language as the primary mode of communication. They consider themselves part of a community with its own culture and beliefs (Arnos, 2003). The Deaf community dismisses the medical viewpoint that deafness is a pathology by viewing deafness as merely a sociocultural difference that should be understood and preserved. This negative attitude towards the medical community stems from the common belief that the medical profession’s goal is to “fix” or cure their hearing loss (Brunger et al., 2001). The prevailing opinion within the Deaf community is that genetics is a threat to the Deaf culture by reducing the size of the Deaf population, which is based on the awareness of the eugenics movement of the past (Stern et al., 2002). A majority of Deaf participants in a study by Middleton, Hewison and Mueller (1998) believed that genetic testing has the potential to do more harm than good. The Deaf community is worried about the
46 consequences of future genetic discoveries and treatments (Brunger et al., 2001) as a way for the medical community to eradicate hearing loss.
Nevertheless, members of the Deaf community may see the potential of genetics to guarantee or evade the arrival of a Deaf child by the conscious act of choosing a partner (Arnos, 2003) and may be interested in knowing the chances of having a deaf or hearing child. A Deaf child may be a welcome event. They are more likely to not have concerns about having either a deaf or hearing child than a person outside the Deaf culture (Arnos, 1997; Stern et al., 2002). Additionally, many Deaf couples who seek out genetic testing are curious and motivated to learn about their own cause of deafness
(Arnos, 1997; Withrow et al., 2008). The culture and communication differences influence the motivation to undergo or to forgo genetic testing (Arnos, 1997; Kaimal et al., 2007).
No Genetic Diagnosis
Another concern about genetic testing is that genetic testing may not provide a diagnosis. Hearing loss expresses locus heterogeneity. Locus Heterogeneity is defined as different genotypes that produce the same phenotype, such that mutations in different locations in the genome all cause hearing loss. Just for the GBJ2 hearing loss, there are estimated over 220 different mutations and not all of these mutations are tested (Hilgert et al., 2009). Additionally, not all disorders are inherited in a simple Mendelian fashion.
Modifier genes produce variations in phenotype (Hilgert et al., 2009) along with multifactorial inheritance, which is the interaction between genetic and environmental factors (Arnos, 2008). The negative results can be useful because they indicate that the cause the hearing loss is not any of the etiologies that are tested for in a genetic
47 assessment. This result does rule out common syndromic forms of hearing loss, which by definition involve medical dysfunctions and defects in systems in the body (Pandya &
Arnos, 2006).
No Cure for Hearing Loss
Another objection to genetic testing for hearing loss is that there is not any cure for hearing loss (Kaimal et al., 2007). Therefore, the purpose of confirming a genetic etiology is questioned, especially by patients and families with a family history of hearing loss. Other detractors do not see how genetic testing would change the treatment
(Withrow et al., 2008). However, the results of genetic testing can unveil any associated medical conditions, some of which may be life threatening. A study by Kaimal et al.
(2007) found that in the span of one year, one Deaf participant reversed her opinion regarding the usefulness of genetic testing for hearing loss when she found out that her son’s hearing loss is part of Jervell Lange-Nielsen syndrome. This syndrome is characterized by profound SNHL and prolongation of the QT interval on the electrocardiogram, representing abnormal ventricular repolarisation. The cardiac condition is life-threatening if untreated and genetic testing can be utilized to make informed and critical decisions.
48 CONCLUSION
Etiologic diagnosis is just the beginning; genetic factors are now facilitating development of future treatments and preventative strategies (Brunger et al., 2001). One option is stem cell treatment. Stem cells are pluripotent, undifferentiated cells that have the potential to replace the multiple types of damaged cells that comprise tissue (Van
Eyken, Van Camp, & Van Laer, 2007). The theory is that stem cells are transplanted into the inner ear where they integrate and respond to signals in the new environment, thereby differentiating into hair cells (Cotanche, 2008). Gene therapy is another treatment option.
Gene therapy is the exogenous manipulation of genes for the purpose of restoring the function of the auditory system. A non-functioning gene is replaced with a working gene.
Furthermore, the area of epigenetic manipulation, which is described as a change in phenotype not caused by a change or mutation in genotype holds more treatment possibilities; however, little is known in regards to hearing loss. Mechanisms, such as
DNA methylations and histone modifications interact to either promote or repress DNA transcription. Abnormal DNA expression results in phenotypical changes (Provenzano &
Domann, 2007).
Future treatments will most likely require gene specific information, so general and specific knowledge of the genetic mutation need to be obtained (Cremers et al.,
2007). There will be a need to understand normal cellular and molecular processes in inner ear (Van Eyken et al., 2007). Additionally, there will also be a need to identify
49 genetic and molecular pathogenesis of disease to develop diagnostic criteria, management strategies, animal models and clinical trials (McClatchey, 2007).
Genetic testing enables the idea of autonomy (Kimberling & Lindenmuth, 2007), which is defined as the freedom to dictate the progress and status of one’s life by determining the etiology of the hearing loss. When the cause of the hearing loss is known, treatment can be specifically targeted and the patient and family are enabled to make the best decisions for treatment. Audiologists should have an obligation to inform the patient and the family about a possible genetic etiology and that tests and counseling are available. Failure to do so negatively affects self-determination by withholding pertinent information about the hearing loss. Ultimately, genetic testing empowers patients and their families to take control of their health care.
50
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