Biallelic nonsense mutations in the otogelin-like gene (OTOGL) in a child affected by mild to moderate hearing impairment.

Hearing impairment is characterized by great genetic heterogeneity. We report the identification, by whole exome sequencing, of two different nonsense mutations (c.1558C>T; p.Gln520 and c.2773C>T; p.Arg925) in the otogelin-like gene (OTOGL), in a child affected by mild to moderate isolated deafness. Parental genotypes allowed us to conclude that these mutations are present in the compound heterozygous state in the patient. In addition, our clinical data establish that the tectorial membrane and/or the outer hair cells are defective in this form of deafness.

Mutations in a new gene encoding a protein of the hair bundle cause non-syndromic deafness at the DFNB16 locus.

Hearing impairment affects about 1 in 1,000 children at birth. Approximately 70 loci implicated in non-syndromic forms of deafness have been reported in humans and 24 causative genes have been identified (see also http://www.uia.ac.be/dnalab/hhh). We report a mouse transcript, isolated by a candidate deafness gene approach, that is expressed almost exclusively in the inner ear. Genomic analysis shows that the human ortholog STRC (so called owing to the name we have given its protein-stereocilin), which is located on chromosome 15q15, contains 29 exons encompassing approximately 19 kb. STRC is tandemly duplicated, with the coding sequence of the second copy interrupted by a stop codon in exon 20. We have identified two frameshift mutations and a large deletion in the copy containing 29 coding exons in two families affected by autosomal recessive non-syndromal sensorineural deafness linked to the DFNB16 locus. Stereocilin is made up of 1,809 amino acids, and contains a putative signal petide and several hydrophobic segments. Using immunohistolabeling, we demonstrate that, in the mouse inner ear, stereocilin is expressed only in the sensory hair cells and is associated with the stereocilia, the stiff microvilli forming the structure for mechanoreception of sound stimulation.

Identification of three novel mutations in the USH1C gene and detection of thirty-one polymorphisms used for haplotype analysis.

Usher syndrome (USH) is a clinically and genetically heterogeneous autosomal recessive disorder in which sensorineural hearing loss is associated with retinitis pigmentosa. Usher syndrome type 1, the most severe form, is characterized by profound congenital deafness, vestibular dysfunction, and prepubertal onset of retinitis pigmentosa. Six different USH1 genes have so far been mapped, of which two have already been identified. MYO7A, encoding the unconventional myosin VIIA, underlies USH1B. Recently, the USH1C gene was shown to encode harmonin, a PDZ domain-containing protein. A previous screening of 18 unrelated USH1 patients, without a detected MYO7A mutation, for the three USH1C mutations described to date had demonstrated the presence of the 238-239insC mutation in the heterozygous state in four of them. A complete USH1C mutation screening in these four carriers of the 238-239insC mutation resulted in the detection of the second mutation in all the individuals, and the identification of three novel mutations, namely two splice site mutations (IVS1+1G>T and IVS5+1G>A) and a nonsense mutation (R31X). Thirty-one polymorphisms were detected in the USH1C gene. We observed that the E519D substitution is non-pathogenic, which is of particular interest for molecular diagnosis. Our analysis indicated that all the carriers of the 238-239insC mutation share a common haplotype. A different common haplotype was found in the two IVS1+1G>T carriers. Future studies of additional carriers and non-carriers should document the here proposed founder effect of these two mutations.

Detection of mutations by fluorescence-assisted mismatch analysis (FAMA).

Fluorescence-assisted mismatch analysis (FAMA) methodology uses bifluorescent double-stranded DNA substrates to maximize the reliability and sensitivity of mutation-scanning procedures that rely on cleavage of mismatches using chemical. This unit presents a nested PCR procedure to fluorescently label each DNA strand, followed by chemical cleavage to detect the point mutations. Fluorescent end labeling with strand-specific fluorophores, electrophoretic separation of cleavage products in an automated Perkin-Elmer ABI 373 or 377 DNA sequencer and analysis using the Perkin-Elmer GENESCAN software expands sensitivity by highlighting weak signals through superimposition of strand-specific fluorescence electropherograms for different samples.

A defect in harmonin, a PDZ domain-containing protein expressed in the inner ear sensory hair cells, underlies Usher syndrome type 1C.

Usher syndrome type 1 (USH1) is an autosomal recessive sensory defect involving congenital profound sensorineural deafness, vestibular dysfunction and blindness (due to progressive retinitis pigmentosa)1. Six different USH1 loci have been reported. So far, only MYO7A (USH1B), encoding myosin VIIA, has been identified as a gene whose mutation causes the disease. Here, we report a gene underlying USH1C (MIM 276904), a USH1 subtype described in a population of Acadian descendants from Louisiana and in a Lebanese family. We identified this gene (USH1C), encoding a PDZ-domain-containing protein, harmonin, in a subtracted mouse cDNA library derived from inner ear sensory areas. In patients we found a splice-site mutation, a frameshift mutation and the expansion of an intronic variable number of tandem repeat (VNTR). We showed that, in the mouse inner ear, only the sensory hair cells express harmonin. The inner ear Ush1c transcripts predicted several harmonin isoforms, some containing an additional coiled-coil domain and a proline- and serine-rich region. As several of these transcripts were absent from the eye, we propose that USH1C also underlies the DFNB18 form of isolated deafness.

Fdp, a new fibrocyte-derived protein related to MIA/CD-RAP, has an in vitro effect on the early differentiation of the inner ear mesenchyme.

During the course of a study aimed at isolating transcripts specifically or preferentially expressed in the inner ear, we identified a novel gene, encoding a fibrocyte-derived protein, that we named Fdp. Fdp is predicted to be a secreted 128-amino acid protein, which is highly homologous to the melanoma-inhibiting activity/cartilage-derived retinoic acid-sensitive protein (MIA/CD-RAP), a cartilage-specific protein also expressed in several tumors. Fdp and MIA/CD-RAP thus define a new family of proteins. Fdp is expressed from embryonic day 10.5 in the mesenchyme surrounding the otic epithelium. During development, these cells progressively aggregate, condense, and differentiate into cartilaginous cells forming the otic capsule, which no longer expresses Fdp, and into fibrocytes surrounding the epithelia, which strongly express Fdp. In order to address the function of Fdp, we developed an in vitro antisense oligonucleotide approach using microdissected periotic mesenchyme micromass cultures, and showed that Fdp antisense oligonucleotide treatment results in a significant reduction in chondrogenesis. Our results demonstrate that Fdp plays a role in the initiation of periotic mesenchyme chondrogenesis. Accordingly, Fdp and its human ortholog FDP, which map to chromosome 2 and band 20p11, respectively, could be candidate genes for forms of deafness associated with malformations of the otic capsule.

Characterization of otoconin-95, the major protein of murine otoconia, provides insights into the formation of these inner ear biominerals.

During the course of a study aimed at identifying inner ear-specific transcripts, a 1,906-bp murine cDNA predicted to encode a secreted 469-aa protein with two domains of homology with the secreted phospholipases A2 was isolated. This transcript is specifically expressed in the inner ear from embryonic day 9.5. The encoded 95-kDa glycoprotein is the major protein of the utricular and saccular otoconia and thus was named otoconin-95. By immunohistofluorescence, otoconin-95 also was detected in the cupulae of the semicircular canals and in previously undescribed transient granular structures of the cochlea. Otoconin-95 was found to be synthesized by various nonsensory cell types, but not by the supporting cells of the sensory epithelia, which produce the otoconial precursor vesicles. In addition, multiple isoforms generated by differential splicing were observed in different combinations during development. Based on the present results, we propose a model for the formation of the otoconia.

Novel variants of human IFN-alpha detected in tumor cell lines and biopsy specimens.

Interferon-alpha constitutes a complex gene family with 14 genes clustered on the short arm of chromosome 9. More than 50 sequence variants have been described. However, an extensive genetic polymorphism has not been seen in the few population studies reported so far. As many of the sequence variants reported were derived from tumor cell lines, we have investigated whether IFN-alpha genes are unstable in tumor cells. Using fluorescence-assisted mismatch analysis (FAMA), combined with allele-specific primer extension, RFLP analysis, and direct sequencing, we detected in a panel of 14 tumor cell lines two new sequence variants of the IFNA1 and IFNA13 genes. Further two-point mutations were found in tumor samples from leukemias (n = 10) and renal cell carcinomas (n = 17) not seen in normal tissues. In the IFNA17 gene, three new sequence variants were detected, one in a tumor cell line and two in tumor biopsy specimens. Besides these individual point mutations, two new polymorphisms were found in each of the IFNA13 and IFNA17 genes. No new variants were found in the IFNA2 and IFNA10 genes. The results suggest that new sequence variants of the IFN-alpha genes occur relatively frequently in tumors or in tumor cell lines.

Exhaustive mutation scanning by fluorescence-assisted mismatch analysis discloses new genotype-phenotype correlations in angiodema.

A complete mutational scan of the gene coding for the serpin C1 inhibitor, comprising all eight exons and adjacent intron sequences and 550 bp preceding the transcription start site, was rapidly accomplished in 36 unrelated angioedema patients by using fluorescence-assisted mismatch analysis (FAMA). Mutations accounting for C1 inhibitor deficiency were identified in every one of 34 patients, with two failures turning out to be spurious cases. Two new substitution dimorphisms were also detected in introns. Changes affecting the C1 inhibitor protein, distributed throughout the seven coding exons, provide new insights into the molecular pathology of serpins. Six different splice-site and two promoter mutations were also found. Among the latter, a C-->T transition within one of two putative CAAT boxes of this TATA-less promoter, the sole idiomorphic nucleotide change in this kindred, was found homozygous in the proband, at variance with the dominant mode of transmission observed for structural mutations. FAMA, in the chemical probes configuration used in this study, is a rapid and robust mutation-scanning procedure, applicable to large DNA segments or transcripts and proved capable of 100% detection. Moreover, it provides accurate positional information--and hence recognition of multiple substitutions, precise relationship with those already known, and often immediate identification of the nucleotide change.

COOH-terminal substitutions in the serpin C1 inhibitor that cause loop overinsertion and subsequent multimerization.

The region COOH-terminal to the reactive center loop is highly conserved in the serine protease inhibitor (serpin) family. We have studied the structural consequences of three substitutions (Val451-->Met, Phe455-->Ser, and Pro476-->Ser) found in this region of C1 inhibitor in patients suffering from hereditary angioedema. Equivalent substitutions have been described in alpha 1-antitrypsin and antithrombin III. The mutant C1 inhibitor proteins were only partially secreted upon transient transfection into COS-7 cells and were found to be dysfunctional. Immunoprecipitation of conditioned media demonstrated that in the intact, uncleaved form they all bind to a monoclonal antibody which recognizes specifically the protease-complexed or reactive center-cleaved normal C1 inhibitor. A second indication for an intrinsic conformational change was the increased thermostability compared to the normal protein. Furthermore, gel filtration studies showed that the Val451-->Met and Pro476-->Ser mutant proteins, and to a lesser extent Phe455-->Ser, were prone to spontaneous multimerization. Finally, a reduced susceptibility to reactive center cleavage by trypsin was observed for all three mutants, and the cleaved Val451-->Met and Pro476-->Ser mutants failed to adopt the conformation recognized by a cleavage-specific monoclonal antibody. Investigation of plasmas of patients with the Val451-->Met or Pro476-->Ser substitutions showed that these dysfunctional proteins circulate at low levels and are recognized by the complex-specific antibody. These results strongly indicate a conformational change as a result of these carboxylterminal substitutions, such that anchoring of the reactive center loop at the COOH-terminal side is not achieved properly. We propose that this results in overinsertion of the loop into beta-sheet A, which subsequently leads to multimerization.

Crucial residues in the carboxy-terminal end of C1 inhibitor revealed by pathogenic mutants impaired in secretion or function.

The last exon of the C1-1NH gene was screened for point mutations in 36 unrelated hereditary angioedema patients. Mutations were found in eight patients, predicting changes in the short COOH-terminal region which anchors the reactive site loop on its COOH-terminal side. The effects of each of these mutations were examined in transiently transfected Cos-7 cells. Complete intracellular retention or degradation was observed with substitutions in the COOH-terminal strands 4B or 5B: Leu459-->Pro, Leu459-->Arg, and Pro467-->Arg were all blocked at early stages of intracellular transport, but differences in the immunofluorescence patterns indicated that a significant fraction of the Leu459-->Pro and of the Pro467-->Arg proteins reached a compartment distinct from the endoplasmic reticulum. In line with previous findings with alpha 1-antitrypsin, chain termination within strand 5B resulted in rapid degradation. Mutant Val451-->Met, in strand 1C, and mutant Pro476-->Ser, replacing the invariant proline near the COOH terminus, yielded reduced secretion, but these extracellular proteins were unable to bind the target protease C1s. Presence of low levels of both dysfunctional proteins in patient plasmas defies the conventional classification of C1 inhibitor deficiencies as type I or type II. These data point to a key role of certain residues in the conserved COOH-terminal region of serpins in determining the protein foldings compatible with transport and proper exposure of the reactive site loop.

Efficient detection of point mutations on color-coded strands of target DNA.

Presently available methods for screening large genetic regions for unknown point mutations are neither flawless nor particularly efficient. We describe an approach, especially well suited to identifying mutations present in the heterozygous state, that combines several improvements in a protocol called fluorescence-assisted mismatch analysis (FAMA). Appropriate gene regions of the wild-type and the putative mutant allele are simultaneously amplified from genomic DNA by using the polymerase chain reaction, and large DNA fragments, so far up to 800 bp, are end labeled with strand-specific fluorophores. Aliquots are denatured and reannealed to form heteroduplexes and subjected to conventional cytosine- and thymine-specific modifications. Cleavages occurring on opposite strands are detected by denaturing gel electrophoresis using an automated DNA sequencer. Since the DNA fragments derived from the mutant allele are also end labeled, the number of informative mispaired bases is doubled compared to conventional searches using wild-type probes. The sensitivity of detection is also increased, because differential fluorescent end labelling allows the identification and measurement of strand-specific background cleavages at matched cytosine or thymine residues. Automatic superimposition of tracings from different subjects allows mismatch detection at sites that, because of the nature of the bases involved and of the neighboring sequence, are known to be less susceptible to cleavage. The effects of the latter parameters have been studied quantitatively on a series of point mutations found in the human C1-inhibitor gene in patients affected by hereditary angioedema. Dilution experiments have demonstrated that most mutations are detected even when the mutant chromosome is diluted 10-fold or more compared with the normal one.

C1 inhibitor mutations which affect intracellular transport and secretion in type I hereditary angioedema.

A cluster of point mutations was found in the region of exon 8 of the C1 INH gene which codes for the 28 C-terminal amino acids. Seven of these mutations introduce amino acid changes and one results in a stop codon. Upon transient expression in monkey kidney Cos-7 cells all of these C1 inhibitor mutants showed an impaired intracellular transport and most of them failed to be secreted. Biochemical and immunofluorescence studies indicated that the defective proteins accumulate or are degraded mainly in the endoplasmic reticulum. The product of deletions of exons 4, which has an internal in frame deletion of 45 amino acids, also fails to be secreted.