Учебники / Genetic Hearing Loss Willems 2004

244	Coucke and Willems

be involved there in the removal of the K+ ions that accumulated in the hair cells after entering these cells at the apical side (16,24).

E.KCNQ4 Homologs and Their Involvement in Human Disease

Five di erent family members KCNQ1–5 have been identified so far, four of them being implicated in genetic disease in man (Table 2). The homology of these genes is high, certainly in the transmembrane domains and the pore region (16). The actual K+ channels are formed by four homomers or heteromers of KCNQ gene products that juxtaposition their pore loops to form the actual K+ channel. KCNQ4 forms tetramers only with itself or with KCNQ3.

1.KCNQ1

The KCNQ1 subunits associate with KCNE1 subunits to form a K+ channel expressed in the heart and the cochlea, that is the molecular correlate of the cardiac-delayed rectifier-like K+ current. This current is involved in repolarization of action potentials in the heart, with KCNQ1 and KCNE1 mutations leading to a prolonged QT interval in the long-QT syndrome, also called the Romano-Ward syndrome (27). Severe functional impairment of this K+ channel leads to Jervell and Lange-Nielsen syndrome, a combination of long-QT syndrome with congenital deafness (28). In the cochlea, K+ homeostasis is maintained not only by the KCNQ4 gene product and several connexins, but also by the KCNQ1/KCNE1-encoded K+ channel, which allows secretion of K+ions from the stria vascularis back into the endolymph.

2.KCNQ2 and KCNQ3

These two subunits form a heteromeric K+ channel involved in the M- current in neurons, which is important for neuronal excitability. Mutations

Table 2 KCNQ4 Genes Involved in Human Disease

Gene	Location	Disease	Inheritance	Ref.
KCNQ1	11p15	Romano-Ward syndrome	AD and AR	27
		Jervell and Lange-Nielsen	AR	28
		syndrome
KCNQ2	20q13	Epilepsy (BFNC)	AD	29
KCNQ3	8q24	Epilepsy (BFNC)	AD	30
KCNQ4	1p34	Hearing loss (DFNA2)	AD	16
				17

K+-Channel Gene KCNQ4

245

in either of these genes are responsible for benign familial neonatal convulsions (BFNC), a form of congenital epilepsy (29,30). HL has never been described in BFNC.

3.KCNQ5

This subunit is expressed in brain and muscle, and forms multimers with itself and KCNQ3. Mutations in this gene have not yet been identified.

F.KCNQ4 Mutations

KCNQ4 mutations have been identified in nine unrelated families (Table 1, Fig. 2 and 3). Although the pore region of the KCNQ4 gene only represents approximately 3% of the open reading frame, it harbors the disease-causing mutation in seven of these nine families, reflecting the importance of this domain in the function of this K+ channel. One of the pore region mutations

Figure 2 Schematic representation of the genomic structure of the KCNQ4 gene with exons indicated as boxes (above), and the KCNQ4 protein with six transmembrane domains, with a pore region between the fifth and sixth transmembrane regions, (below). The seven KCNQ4 mutations in the nine families (the W276S mutation is present in three unrelated families) are indicated.

246	Coucke and Willems

Figure 3 Alignment of the amino acid sequence of the pore region of the di erent members from the KCNQ gene family. The five missense mutations in the pore region of KCNQ4 typically involve amino acids strongly conserved between the di erent members of the KCNQ gene family. The W276S mutation is present in three unrelated families.

(W276S) was found in three di erent families (the Dutch 1, Japanese, and Dutch 4 families). As these three families are not related, and the W276S mutation is present on di erent genetic backgrounds in these families, the W276S mutation probably represents a mutational hot spot in the KCNQ4 gene (15). The pore region mutations are all missense mutations substituting amino acids that are strongly conserved between di erent members of the KCNQ gene family (Fig. 3). Functional studies of the K+ channel with such a heterozygous mutation in the pore region (G285S) expressed in Xenopus oocytes showed severe reduction of K+ currents to only 10% of normal, indicating a dominant-negative e ect of this mutation (17). As the K+ channel is formed by a tetramer of KCNQ4 subunits, any given mutation with a dominant-negative e ect can cause a severe reduction in K+-channel activity, which is compatible with the autosomal dominant inheritance pattern and complete penetrance of the hearing loss. In the eighth family (Dutch 2) a missense mutation in the sixth transmembrane domain has been identified. The nineth KCNQ4 mutation is an out-of-frame deletion of 13 bp causing a frameshift at amino acid 71, resulting in 63 novel amino acids with a premature stop codon at position 134. Most likely, the short mutant protein is degraded rapidly as it does not contain transmembrane regions.

K+-Channel Gene KCNQ4

247

Apart from these disease-associated mutations, 11 neutral polymorphisms and one polymorphic amino acid substitution have been identified in the KCNQ4 gene (10).

IV. HEARING LOSS CAUSED BY KCNQ4 MUTATIONS

Mutations in the KCNQ4 gene have been identified in nine di erent families with a nonsyndromic, sensorineural, and progressive HL that is inherited in an autosomal dominant mode with complete penetrance. The clinical features of these families are summarized in Table 3, and age-related typical audiograms made by De Leenheer et al. (31) are shown in Figure 4. The clinical features of eight of the nine families have been analyzed and compared by di erent authors (31–33).

A.The French Family

In this small family with autosomal dominant progressive HL in three patients belonging to three consecutive generations the first KCNQ4 mutation (G285S) was identified (16). The family is known with HL starting in the first decade progressing to severe losses between 90 and 120 dB in the 2000–4000 Hz interval. Although hearing impairment was always more

Table 3 Clinical Features of Hearing Loss Due to KCNQ4 Mutations

			Threshold (dB)
		250 Hz			8000 Hz
Family	Origin	20 years	60 years	20 years		60 years	Tinnitus	Ref.
1	French	NR	NR		NR	NR	+	16
2	USA 1	30–35	45–50		70–75	100–105	+	5
3	Belgian	15–20	20–25	>120		>120	+	7
4	Dutch 1	15–20	50–55		70–75	100–105		8
5	Dutch 2	10–15	45–50		55–60	100–105		9
6	USA 2	20–25	45–50		65–70	100–105	+	10
7	Dutch 3	15–20	40–45		70–75	95–100		11
8	Japanese	30–35	60–65		65–70	110–115		13
9	Dutch 4	25–30	60–65		70–75	110–115		14

NR, not reported.

Source: Data derived from Ref. 31.

248	Coucke and Willems

Figure 4 Typical age-related audiograms of eight of the nine families with a KCNQ4 mutation (no data were available on the small French family). The initial high-tone loss in younger individuals, and the progressive hearing loss with age in older patients, with loss of the middle and eventually low frequencies, are evident in all families. Note that at the age of 10 years significant HL already exists, indicating that the DFNA2 HL probably is prelingual, and not postlingual. The age-related audiograms of eight of the nine families are very similar, but di erent from that of the Belgian family. In the latter family the HL of the high tones is more pronounced, but low tones are conserved throughout life. The data have been published before by De Leenheer et al. (31).

K+-Channel Gene KCNQ4

249

severe in the high frequencies, the lower tones also showed significant losses (50–90 dB at 500 Hz) (16). Detailed audiological studies of this family have not been reported.

B.The U.S. 1 Family

This five-generation family was used together with the original Indonesian family to map DFNA2 to chromosome 1p by linkage analysis (5). At that time it contained 27 patients with nonsyndromic HL, which started before the age of 10. Tinnitus was reported by a fraction of the a ected family members (Table 3). Mutation analysis of the KCNQ4 gene identified a G285C mutation in the pore region of the gene (17).

C.The Belgian Family

This four-generation pedigree contains at least 27 family members a ected by a progressive hearing loss of the high frequencies (7), whereas the low tones remain relatively spared in comparison with the other families with a KCNQ4 mutation (31–33). This family is the only one of the nine with such conservation of the low tones. Below 1000 Hz the average annual threshold increase (ATI) was only between 0.2 and 0.5 dB, whereas the ATI was very high, above 1000 Hz (2–5 dB). This family was used, together with the Dutch 1 and 2 families, to narrow down the linkage interval of the DFNA2 gene (7). A deletion of 13 bp (FS71) was found in the KCNQ4 gene, leading to a premature stop codon resulting in truncation of the protein (18). This is the only definite loss-of-function mutation in the KCNQ4 gene, as all the other KCNQ4 mutations in the other families are missense mutations, which most likely have a dominant-negative e ect.

D.The Dutch Family 1

This large family contains at least 41 a ected patients in six generations. The HL is similar to that of the other families, but clearly di erent from that of the Belgian family (8,31–33). About one-third of the patients show vestibular hyperreactivity in response to rotatory stimulation tests. A missense mutation (W276S) was identified in the pore region of KCNQ4. The W276S mutation is also present in the Japanese and the Dutch 4 family.

E.The Dutch Family 2

This is an extended six-generation family with at least 24 a ected members. The audiological features are very similar to these of the other KCNQ4

250	Coucke and Willems

families, and have been described in detail (9,31–33). Coucke et al. (17) found a missense mutation (G321S) in the sixth transmembrane domain of the KCNQ4 gene.

F.The U.S. Family 2

This large U.S. family of Austrian ancestry consists of 51 patients in five generations with a HL that is very similar to that of the other families. The majority of patients have tinnitus. The family shows linkage to the DFNA2 locus, and a L281S missense mutation was identified in the pore region of the KCNQ4 gene (10).

G.The Dutch Family 3

In this five-generation family 20 family members are a ected by a typical KCNQ4-associated HL (11,31–33). The family was shown to be linked to the DFNA2 locus and a missense mutation (L274H) was found in the pore region of the KCNQ4 gene (12).

H.The Japanese Family

In this family the W276S mutation, which was also present in the Dutch 1 and Dutch 4 families, was found (13). The clinical picture is not di erent from that of the other families (13,31).

I.The Dutch 4 Family

This is a five-generation family with 33 a ected patients. The clinical picture of this family was studied extensively by De Leenheer et al. (14). The HL is typical for KCNQ4-associated pathology. Although the HL starts early, possibly congenitally, speech recognition only starts to deteriorate significantly in the third decade. Although none of the patients had vertigo, two of 11 patients who underwent vestibular testing showed motion sickness. These two patients, together with a third patient, had hyperactive vestibuloocular reflexes. Also in this family the W276S mutation is present (Van Camp et al., unpublished results).

V.GENOTYPE-PHENOTYPE CORRELATION

Nine di erent families with a KCNQ4 mutation have been described (Tables 1 and 3). In all cases the HL is autosomal dominant, nonsyndromic, and

K+-Channel Gene KCNQ4

251

sensorineural, a ecting both ears equally. Tinnitus has been reported in the Belgian, French, and both U.S. families (17,31–33). Vertigo has not been reported, but an increased vestibulo-ocular reflex activity has been found in a minority of the Dutch patients with the W276S mutation (8,31). Visualization of the middle and inner ear by CT scan or MRI has been performed in the French and Dutch 4 family, but could not reveal morphological anomalies. A detailed comparison of the audiological features of eight of the nine di erent DFNA2 families in which a KCNQ4 mutation has been found (excluding the French family, for which only limited information was available) has been made by several authors (31–33). These comparative studies indicated that the hearing loss of all the families but the Belgian family is very similar in age of onset, rate of progression, and a ected frequencies (Table 3, Fig. 4). Regression analysis of the HL of these families in function of time and frequency shows a linear progression of all frequencies with an average annual threshold deterioration of approximately 1 dB per year. The o set values are close to zero for the low frequencies, but increase progressively with higher frequencies. Although hardly any audiology data are available from very young a ected children, it is conceivable that there is high-tone loss already at birth in these families (11,31–33). In most patients with a KCNQ4 mutation a hearing aid becomes necessary between the age of 20 to 40 years, whereas HL is severe to profound by the age of 70. Although DFNA2 is classified with the postlingual types of HL, it is likely that prelingual HL occurs, as significant HL is already present at the age of 10 years. This probably remains undetected in most patients as only the high tones are a ected at that young age.

The Belgian family shows greater loss in the high frequencies than the other families, but the low tones are relatively spared (Fig. 4). The Belgian family, therefore, represents the purest form of inherited high-tone loss. In this family a loss-of-function mutation was identified that might have a less severe impact on the residual activity of the KCNQ4 channel activity than the missense mutations that are present in all the other families. At first sight this might explain the relative conservation of the low tones, but it is di cult to reconcile this with the more pronounced losses of the high tones in the Belgian family as compared to the other families.

VI. DISCUSSION

Progressive HL is a normal process of ageing, and hearing impairment to the level of limitation of communication is the most frequent sensory handicap present in half of the population by the age of 80. It is unclear why our auditory system does not seem to endure for a complete life span.

252	Coucke and Willems

Other sensory systems such as the eye also show progressive malfunctioning with age, but the proportion of elderly in whom the aging process leads to real functional impairment is considerably smaller than in the case of HL. It is possible that the paucity of auditory sensory cells (a mere 30– 40,000 hair cells) makes the auditory system more vulnerable than other sensory systems, such as the visual system and the olfactory system, which each contain millions of sensory cells. Therefore, it is not surprising that many of the inherited forms of progressive HL involve the cochlea, in particular the hair cells. The typical progressive HL of the elderly (presbycusis) is a gradual loss of high tones with conservation of the middle tones and the low tones. Presbycusis most likely is a multifactorial disorder caused by a complex interaction between multiple genetic factors and many environmental factors such as noise, ear infections or trauma, ototoxic medication such as aminoglycosides, etc. Many postlingual forms of monogenic HL due to mutation in a single gene o er paradigms to study presbycusis.

Among the Mendelian forms of postlingual, progressive HL of the high tones, HL due to mutations in the KCNQ4 gene is probably the most frequent type of postlingual HL, certainly in Western Europe. Nine extended and unrelated families with a KCNQ4 mutation have now been described, and it is remarkable that four of these families are of Dutch descent. The inheritance pattern in all families is autosomal dominant with full penetrance. The HL in these nine families is very similar, and always nonsyndromic, sensorineural, postlingual, and progressive, a ecting the high tones first, the middle tones later, and the low tones in a final stage. It is likely that the HL starts in the prelingual period in most patients, and proceeds to severe HL by the age of 70. In three of the nine families tinnitus is present. Vertigo has never been reported, but vestibular studies revealed abnormalities in about a third of the patients. This is not unexpected as the KCNQ4 gene is expressed in the type 1 hair cells of the vestibular organ. The KCNQ4 gene is also expressed in several auditory nuclei and the outer hair cells of the organ of Corti, but controversy still exists around the presence of KCNQ4 in the inner hair cells. Also, the function of the KCNQ4 gene is still a matter of debate. It encodes a tetrameric K+ channel that is involved in K+ homeostasis in the cochlea. In view of its localization at the basolateral side of cochlear hair cells, it is possible that the K+ channel formed by KCNQ4 is involved in the outflux of K+ ions that accumulate in hair cells where they lead to repolarization (16,23,24,34).

Influx of K+ ions from the K+-rich endolymph is triggered by mechanical stimulation when the vibrations of the basilar membrane induce shearing of the tectorial membrane (Fig. 1). This leads to bending of the stereocilia of the hair cells in the cochlea, resulting in the opening of un-

K+-Channel Gene KCNQ4

253

identified K+ channels involving the action of unconventional myosins such as myosin 6, 7A, and 15. The influx of potassium ions leads to depolarization of the hair cells, and subsequent Ca2+ influx triggering the release of the neurotransmitter glutamate, which stimulates the nerve endings of the acoustic nerve. The accumulated K+ ions in the outer hair cells leave these cells through K+ channels at the basolateral side, and are recirculated through the gap junctions between the supporting cells and cochlear fibrocytes to end up in the marginal cells of the stria vascularis. Here the K+ ions reenter the endolymph through K+ channels formed by the associated gene products of KCNQ1 and KCNE1. The K+ ions from the inner hair cells recirculate to the interdental cells of the spiral limbus, where they are taken up again by the endolymph (Fig. 1A). Mutations in many of the genes involved in cochlear K+ homeostasis lead to hereditary HL, including the K+ channels KCNQ4 (DFNA2), KCNQ1, and KCNE1 (Jervell and LangeNielsen syndrome), the gap junction proteins GJB2 (DFNA3 and DFNB1), GJB3 (DFNA2), GJB6 (DFNA3), and the tight junction protein claudin 14 (DFNB9) (for review, see Ref. 1).

Although treatment of genetic disease in general represents more ‘‘hype’’ than hope, it is not impossible that HL due to deficient KCNQ4 channel activity might be prevented or even treated by drugs that activate KCNQ4 channel activity. Components such as retigabine and BMS-204352, which are in clinical trials now to treat epilepsy and stroke, have been shown to activate KCNQ4 channels in vivo (35).

REFERENCES

1.Willems PJ. Genetic causes of hearing loss. N Engl J Med 2000; 342:1101–1109.

2.Resendes BL, Williamson RE, Morton CC. At the speed of sound: gene discovery in the auditory system. Am J Hum Genet 2001; 69:923–935. Review.

3.Steel KP, Kros CJ. A genetic approach to understanding auditory function. Nat Genet 2001; 27:143–149. Review.

4.Tekin M, Arnos KS, Pandya A. Advances in hereditary deafness. Lancet 2001; 358:1082–1090. Review.

5.Coucke P, Van Camp G, Djoyodiharjo B, Smith SD, Frants RR, Padberg GW, Darby JK, Huizing EH, Cremers CWRJ, Kimberling WJ, Oostra BA, Van de Heyning PH, Willems PJ. Linkage of autosomal dominant hearing loss to the short arm of chromosome 1 in two families. N Engl J Med 1994; 331:425–431.

6.Van Camp G, Coucke P, Speleman F, Van Roy N, Beyer EC, Oostra BA, Willems PJ. The gene for human gap junction protein connexin37 (GJA4) maps to chromosome 1p35.1, in the vicinity of D1S195. Genomics 1995; 30:402–403.

7.Van Camp G, Coucke PJ, Kunst H, Schatteman I, Van Velzen D, Marres H, van Ewijk M, Declau F, Van Hauwe P, Meyers J, Kenyon J, Smith SD, Smith