Mutation in Transcription Factor POU4F3 Associated with Inherited Progressive Hearing Loss in Humans

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Science  20 Mar 1998:
Vol. 279, Issue 5358, pp. 1950-1954
DOI: 10.1126/science.279.5358.1950


The molecular basis for autosomal dominant progressive nonsyndromic hearing loss in an Israeli Jewish family, Family H, has been determined. Linkage analysis placed this deafness locus,DFNA15, on chromosome 5q31. The human homolog of mousePou4f3, a member of the POU-domain family of transcription factors whose targeted inactivation causes profound deafness in mice, was physically mapped to the 25-centimorgan DFNA15-linked region. An 8–base pair deletion in the POU homeodomain of humanPOU4F3 was identified in Family H. A truncated protein presumably impairs high-affinity binding of this transcription factor in a dominant negative fashion, leading to progressive hearing loss.

The inner ear is a complex structure requiring a large repertoire of genes to orchestrate sound acquisition and vestibular function on many levels, including neuronal innervation, structural integrity, and mechanoelectrical transduction. Although as many as 100 genes may be involved (1), only a small number of genes have been identified that cause nonsyndromic hearing loss: human connexin 26 (GJB2) (2), human myosin VIIA (MYO7A) (3), human diaphanous (4), and mouse myosin VI (Myo6) (5). Another POU-domain transcription factor,POU3F4 (POU domain, class 3, transcription factor 4), causes human X-linked mixed deafness, DFN3 (6).

We are studying the genetic basis of progressive hearing loss in an Israeli Jewish family. The family traces its ancestry to Italy and to subsequent migrations through various North African and Middle Eastern countries, including Tunisia, Libya, and Egypt, with branches of the family now living in Israel, the United States, and Belgium (7). Five generations demonstrate autosomal dominant inheritance of progressive deafness (Fig.1A). The earliest record of a hearing-impaired family member is from an individual born in 1843 (by family report) in Libya. He had four children, only one of whom was affected. Hearing loss in Family H begins between ages 18 and 30, with a moderate to severe defect in hearing by age 50 (Fig. 1B). On the audiologic examinations, 12 family members were identified as affected with progressive sensorineural hearing loss (8). These 12 individuals and 11 unaffected relatives older than age 40 were available for linkage analysis.

Figure 1

Family H of Israel. Hearing loss in this family is progressive and is not associated with any other phenotype. (A) Individuals with hearing loss are indicated by solid symbols, unaffected individuals by open symbols. Individuals younger than age 40 were omitted from the pedigree. DFNA15was mapped to chromosome 5q31-33 by linkage to polymorphic markers with genotypes indicated for each relative. All affected individuals in the family share the DFNA15 deletion in POU4F3, with the exception of 505, who may represent a phenocopy. All unaffected individuals over age 40 are wild-type at the comparablePOU4F3 site. (B) Representative pure-tone audiograms from two individuals, 511 (unaffected) and 504 (affected). Lines are drawn to show p95 ISO values (8), indicating mean hearing levels for the age and sex of these individuals. (C) Human POU4F3 on chromosome 5q31 linked to deafness in Family H. Markers D5S1979 and D5S422 define the 25-cM linked region. YACs 959E5 and 929D3 contain POU4F3, D5S436, and D5S210.

Deafness in Family H was mapped to chromosome 5q31-q33 by linkage analysis. Previously known loci for inherited deafness were excluded by linkage (9). However, lod scores (logarithm of the odds ratio) for linkage of deafness in Family H to DFNA1 on 5q31 were positive at distant recombination fractions, indicating an independent locus for hearing loss distal to DFNA1. The new locus was named DFNA15 and was localized further to the 25-cM region bounded by D5S1979 and D5S422 (Fig. 1A) (10). Critical meiotic recombination events were detected in affected individuals 518 and 515 and unaffected individuals 511 and 502. Individual 505 is hearing impaired, but he did not inherit any portion of the DFNA15-linked haplotype otherwise segregating with deafness in the family (Fig. 1A). Analysis of the maximum-likelihood genetic model for deafness in Family H indicated a rare autosomal dominant allele with full penetrance by age 40 and 0.05 probability of deafness from other causes by age 40 (in other words, 5% phenocopies). Multipoint analysis of linkage included all markers shown in Fig. 1A and yielded negative lod scores for the interval D5S658 to D5S1979 and lod scores of 3.63 for the interval D5S1979 to D5S2017, 4.04 for intervals between D5S2017 and D5S410, and 3.49 for the interval D5S410 to D5S422 (11).

Because few large families have inherited hearing loss, and because of the inaccessibility of the human ear, the mouse has proved to be an invaluable model for gaining access to deafness genes and studying human hearing loss. More than 60 mouse mutations exist with defects in the inner ear (12). The structure and function of the inner ear is similar between the two organisms, and the genomic organization is very well conserved. The first genes responsible for autosomal recessive deafness were initially identified in the mouse, demonstrating the value of the mouse for identifying and studying human deafness genes (5, 13).

To identify candidate genes in the DFNA15-linked region, we noted that the POU-domain transcription factor POU4F3 (POU domain, class 4, transcription factor 3; GenBank accession numbers U10060 andU10061) might lie on 5q, on the basis of the localization of mousePou4f3 (also referred to as Brn3c andBrn-3.1; GenBank accession number S69352) and the homology of human chromosomes 5 and 18 and mouse chromosome 18 (14).POU4F3 was an excellent candidate for a gene causing human deafness, because targeted deletion of both alleles ofPou4f3 causes complete deafness in mice (15, 16). To map POU4F3 more precisely, we synthesized primers for the human POU4F3 cDNA sequence and used these to amplify pools of the CEPH3 yeast artificial chromosome (YAC) library (Research Genetics). POU4F3 primers amplified DNA from YACs 959E5 and 929D3 (1610 and 1720 kb, respectively), both of which were also amplified by markers D5S436 and D5S210. Because these markers were linked to deafness in Family H (Fig. 1A), POU4F3must lie within the DFNA15-linked region (Fig. 1C).

To screen for POU4F3 mutations in Family H, we designed primers to amplify and sequence the entire coding region; these primers revealed an 8–base pair (bp) deletion in exon 2 (Fig.2) (17). The predicted result of this deletion is a frame shift beginning at codon 295 and a premature translation stop at position 299. The deletion segregated perfectly with the DFNA15-linked haplotype (Fig. 1A); all deaf individuals with the linked haplotype carried the mutation, and all unaffected relatives older than age 40 in Family H carried the wild-type POU4F3 sequence. However, deaf individual 505 did not inherit either the DFNA15-linked haplotype or the POU4F3deletion, which suggests that he is a phenocopy of the deafness caused by this deletion of POU4F3 (8).

Figure 2

Sequence analysis of the DFNA15 mutation of POU4F3. (A) Wild-type POU4F3 sequenced from genomic DNA of unaffected individuals. The underlined 8-bp sequence is deleted in members of Family H with genetic hearing loss (Fig. 1A). (B) The mutant DFNA15 allele ofPOU4F3 sequenced from genomic DNA of deaf individuals from Family H. DFNA15 is defined by an 8-bp deletion ofPOU4F3, whose site is based on GenBank sequences U10060 andU10061. (C) PCR products of normal versus mutant alleles ofPOU4F3 resolved on an agarose gel. The normal product is 310 bp; the mutant product is 302 bp. PCR products from the DNA of nine representative individuals (see Fig. 1A) are illustrated. As a test for the presence of the 8-bp deletion, PCR products were electrophoresed through a 3% MetaPhor gel at 10 V/cm for 2.5 hours in 0.5× tris-acetate buffer and at 4°C. The gel was stained with ethidium bromide and bands were visualized on an ultraviolet light box. (D) SSCP analysis distinguishes individuals with hearing loss, 504 and 524, from unaffected family H members, 511 and 528. (E) POU4F3 is expressed in the human cochlea, as shown by RT-PCR analysis of RNA samples from human fetal cochlea, brain, and kidney (+, reactions with reverse transcriptase; –, reactions without reverse transcriptase; C, PCR control with no DNA template). Primers 48F and 804R would amplify a 756-bp product from cDNA and a 1070-bp product from contaminating genomic DNA. The PCR reaction was run on a 1% agarose gel with 100-bp markers (M) (Gibco-BRL).

To determine whether the 8-bp deletion occurs in the hearing population, we tested 114 unrelated individuals of various North African and Middle Eastern Jewish ancestries for the mutation (18). These individuals were selected to represent the contribution of various ethnic Jewish populations to Family H. Their chromosomes are identified by origin as follows: 60 Moroccan, 54 Libyan, 20 Tunisian, 7 Algerian, 7 Egyptian, 42 Iraqi, 31 Sephardic (originally from Spain, mainly from Italy and the Balkan countries), and 7 Ashkenazic (mainly East European). None of the 228 control chromosomes tested carried the 8-bp deletion in POU4F3 found in Family H.

POU4F3 is not expressed in human brain, heart, placenta, skeletal muscle, lung, liver, kidney, pancreas, or lymphoblast tissues evaluated by Northern (RNA) blot analysis (19). Nor were any POU4F3 expressed sequence tags (ESTs) identified in human libraries found in public databases. We examined POU4F3 RNA expression in human fetal tissues by means of reverse transcription polymerase chain reaction (RT-PCR) with PCR primers from exon 1 and 2 spanning the intron (20). Complementary DNA amplification was only detected in fetal cochlea. This expression pattern corresponds to that observed in the mouse; namely, Pou4f3 is highly expressed in inner ear hair cells, but only in very defined regions of the brain and not in non-neural tissues including liver, kidney, heart, and skeletal muscle (15,16, 21). Exclusive POU4F3 expression in the human cochlea is consistent with the underlying cause of nonsyndromic deafness in Family H.

POU4F3 is a member of the family of POU transcription factors, a group of proteins identified by a well-conserved bipartite domain (22). The POU domain consists of a 75– to 82–amino acid NH2-terminal region, the POU-specific domain, and a ∼60–amino acid COOH-terminal region, the POU homeodomain. A large number of these proteins have been identified in humans, mice,Drosophila, C. elegans, Xenopus, and zebrafish, and are involved in development. In particular, they are known to be involved in terminal differentiation of neuronal cell types. The POU-specific domain is unique to this family of transcription factors, whereas the POU homeodomain is related to other DNA-binding homeodomains involved in developmental regulation. Binding to a 9-bp recognition site, ATAATTAAT, the POU-specific domain contacts the 5′ portion of this site and the POU homeodomain contacts the 3′ portion (23). However, unlike most homeodomain proteins, the POU homeodomain does not direct high-affinity binding of the POU-domain protein to its DNA target sites on its own, but requires the POU-specific domain for efficient recognition of response elements (24).

Targeted deletion of the Pou4f3 gene leads to vestibular dysfunction and profound deafness in mice (15, 16). At postnatal day 14, around the time mice begin to hear, neither hair cells nor supporting cells are present. Furthermore, there is extensive neuronal degeneration in inner ear sensory ganglia. These ganglia house the cell bodies of sensory neurons within the cochlea and vestibular apparatus; the hair cells of each portion of the inner ear receive primary afferent endings from these neurons, which send axons through the auditory nerve to the central nervous system (25). As is evident from the Pou4f3 null mice, this transcription factor is required for the terminal differentiation and trophic support of sensorineural cells.

In Family H, the deletion removes the 3′ portion of this POU transcription factor crucial for high-affinity binding to DNA. Because the deletion occurs in the final coding exon, it is possible that the transcript is translated (26). If so, the POU4F3 mutant peptide would be truncated at 298 amino acids rather than the normal 338 amino acids. The mutant protein would terminate at the end of the first helix, thereby removing the second and third helices of the POU homeodomain. The third helix, referred to as the recognition helix, makes base contacts with the 3′ half of the DNA complex subsite in the major groove of the DNA (27). A truncated protein, therefore, may not be able to bind the “right half” of the POU recognition sequence, and thus the binding of this transcription factor would be compromised. The first amino acid lost as a result of the 8-bp deletion in Family H is an isoleucine; this residue is responsible for another member of the class IV POU-domain factors, Pou4f2, acting as a repressor (28). Its loss may prevent POU4F3 from repressing the transcription of target genes crucial for hair cell function. The truncated peptide may also have novel DNA binding properties or may act as a dominant negative mutation by pairing with other POU transcription factors (29). Alternatively, haploinsufficiency ofPOU4F3 may down-regulate genes responsible for hair cell survival, causing a progressive loss of hair cells that is clinically significant only after extended time. However, brainstem-evoked responses have been evaluated in Pou4f3 knockout mice up to 24 months of age. The hearing of +/– animals is indistinguishable from that of +/+ animals (30). These experiments suggest that haploinsufficiency of POU4F3 is not an explanation for the deafness phenotype in Family H.

Screening for mutations in the POU4F3 gene is facilitated by its small size, and so it will be straightforward to determine whether mutations in this gene are responsible for deafness in other families. To facilitate testing of POU4F3 mutations in genomic DNA, we sequenced the genomic region encompassing both POU4F3 exons, 5′ and 3′ sequences, and the intron (GenBank accession numbers AF044575 and AF043452). Nearly half the general population loses part or all of their ability to hear by age 80, with a substantial loss in understanding of speech by age 65 (1). Defining the molecular events leading to deafness may form the basis for new ways to help the hearing-impaired population.

  • * These authors contributed equally to this report.

  • To whom correspondence should be addressed. E-mail: karena{at}


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