Adam P. DeLuca

Comprehensive genetic testing for hereditary hearing loss using massively parallel sequencing

The extreme genetic heterogeneity of nonsyndromic hearing loss (NSHL) makes genetic diagnosis expensive and time consuming using available methods. To assess the feasibility of target-enrichment and massively parallel sequencing technologies to interrogate all exons of all genes implicated in NSHL, we tested nine patients diagnosed with hearing loss. Solid-phase (NimbleGen) or solution-based (SureSelect) sequence capture, followed by 454 or Illumina sequencing, respectively, were compared. Sequencing reads were mapped using GSMAPPER, BFAST, and BOWTIE, and pathogenic variants were identified using a custom-variant calling and annotation pipeline (ASAP) that incorporates publicly available in silico pathogenicity prediction tools (SIFT, BLOSUM, Polyphen2, and Align-GVGD). Samples included one negative control, three positive controls (one biological replicate), and six unknowns (10 samples total), in which we genotyped 605 single nucleotide polymorphisms (SNPs) by Sanger sequencing to measure sensitivity and specificity for SureSelect-Illumina and NimbleGen-454 methods at saturating sequence coverage. Causative mutations were identified in the positive controls but not in the negative control. In five of six idiopathic hearing loss patients we identified the pathogenic mutation. Massively parallel sequencing technologies provide sensitivity, specificity, and reproducibility at levels sufficient to perform genetic diagnosis of hearing loss.

Exome sequencing and analysis of induced pluripotent stem cells identify the cilia-related gene male germ cell-associated kinase (MAK) as a cause of retinitis pigmentosa

Retinitis pigmentosa (RP) is a genetically heterogeneous heritable disease characterized by apoptotic death of photoreceptor cells. We used exome sequencing to identify a homozygous Alu insertion in exon 9 of male germ cell-associated kinase (MAK) as the cause of disease in an isolated individual with RP. Screening of 1,798 unrelated RP patients identified 20 additional probands homozygous for this insertion (1.2%). All 21 affected probands are of Jewish ancestry. MAK encodes a kinase involved in the regulation of photoreceptor-connecting cilium length. Immunohistochemistry of human donor tissue revealed that MAK is expressed in the inner segments, cell bodies, and axons of rod and cone photoreceptors. Several isoforms of MAK that result from alternative splicing were identified. Induced pluripotent stem cells were derived from the skin of the proband and a patient with non-MAK–associated RP (RP control). In the RP control individual, we found that a transcript lacking exon 9 was predominant in undifferentiated cells, whereas a transcript bearing exon 9 and a previously unrecognized exon 12 predominated in cells that were differentiated into retinal precursors. However, in the proband with the Alu insertion, the developmental switch to the MAK transcript bearing exons 9 and 12 did not occur. In addition to showing the use of induced pluripotent stem cells to efficiently evaluate the pathogenicity of specific mutations in relatively inaccessible tissues like retina, this study reveals algorithmic and molecular obstacles to the discovery of pathogenic insertions and suggests specific changes in strategy that can be implemented to more fully harness the power of sequencing technologies.

Advancing genetic testing for deafness with genomic technology

Background Non-syndromic hearing loss (NSHL) is the most common sensory impairment in humans. Until recently its extreme genetic heterogeneity precluded comprehensive genetic testing. Using a platform that couples targeted genomic enrichment (TGE) and massively parallel sequencing (MPS) to sequence all exons of all genes implicated in NSHL, we tested 100 persons with presumed genetic NSHL and in so doing established sequencing requirements for maximum sensitivity and defined MPS quality score metrics that obviate Sanger validation of variants. Methods We examined DNA from 100 sequentially collected probands with presumed genetic NSHL without exclusions due to inheritance, previous genetic testing, or type of hearing loss. We performed TGE using post-capture multiplexing in variable pool sizes followed by Illumina sequencing. We developed a local Galaxy installation on a high performance computing cluster for bioinformatics analysis. Results To obtain maximum variant sensitivity with this platform 3.2–6.3 million total mapped sequencing reads per sample were required. Quality score analysis showed that Sanger validation was not required for 95% of variants. Our overall diagnostic rate was 42%, but this varied by clinical features from 0% for persons with asymmetric hearing loss to 56% for persons with bilateral autosomal recessive NSHL. Conclusions These findings will direct the use of TGE and MPS strategies for genetic diagnosis for NSHL. Our diagnostic rate highlights the need for further research on genetic deafness focused on novel gene identification and an improved understanding of the role of non-exonic mutations. The unsolved families we have identified provide a valuable resource to address these areas.

Non-exomic and synonymous variants in ABCA4 are an important cause of Stargardt disease

Mutations in ABCA4 cause Stargardt disease and other blinding autosomal recessive retinal disorders. However, sequencing of the complete coding sequence in patients with clinical features of Stargardt disease sometimes fails to detect one or both mutations. For example, among 208 individuals with clear clinical evidence of ABCA4 disease ascertained at a single institution, 28 had only one disease-causing allele identified in the exons and splice junctions of the primary retinal transcript of the gene. Haplotype analysis of these 28 probands revealed 3 haplotypes shared among ten families, suggesting that 18 of the 28 missing alleles were rare enough to be present only once in the cohort. We hypothesized that mutations near rare alternate splice junctions in ABCA4 might cause disease by increasing the probability of mis-splicing at these sites. Next-generation sequencing of RNA extracted from human donor eyes revealed more than a dozen alternate exons that are occasionally incorporated into the ABCA4 transcript in normal human retina. We sequenced the genomic DNA containing 15 of these minor exons in the 28 one-allele subjects and observed five instances of two different variations in the splice signals of exon 36.1 that were not present in normal individuals (P < 10−6). Analysis of RNA obtained from the keratinocytes of patients with these mutations revealed the predicted alternate transcript. This study illustrates the utility of RNA sequence analysis of human donor tissue and patient-derived cell lines to identify mutations that would be undetectable by exome sequencing.

Carcinoembryonic antigen-related cell adhesion molecule 16 interacts with α-tectorin and is mutated in autosomal dominant hearing loss (DFNA4)

We report on a secreted protein found in mammalian cochlear outer hair cells (OHC) that is a member of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family of adhesion proteins. Ceacam16 mRNA is expressed in OHC, and its protein product localizes to the tips of the tallest stereocilia and the tectorial membrane (TM). This specific localization suggests a role in maintaining the integrity of the TM as well as in the connection between the OHC stereocilia and TM, a linkage essential for mechanical amplification. In agreement with this role, CEACAM16 colocalizes and coimmunoprecipitates with the TM protein α-tectorin. In addition, we show that mutation of CEACAM16 leads to autosomal dominant nonsyndromic deafness (ADNSHL) at the autosomal dominant hearing loss (DFNA4) locus. In aggregate, these data identify CEACAM16 as an α-tectorin–interacting protein that concentrates at the point of attachment of the TM to the stereocilia and, when mutated, results in ADNSHL at the DFNA4 locus.

Exon-level expression profiling of ocular tissues.

The normal gene expression profiles of the tissues in the eye are a valuable resource for considering genes likely to be involved with disease processes. We profiled gene expression in ten ocular tissues from human donor eyes using Affymetrix Human Exon 1.0 ST arrays. Ten different tissues were obtained from six different individuals and RNA was pooled. The tissues included: retina, optic nerve head (ONH), optic nerve (ON), ciliary body (CB), trabecular meshwork (TM), sclera, lens, cornea, choroid/retinal pigment epithelium (RPE) and iris. Expression values were compared with publically available Expressed Sequence Tag (EST) and RNA-sequencing resources. Known tissue-specific genes were examined and they demonstrated correspondence of expression with the representative ocular tissues. The estimated gene and exon level abundances are available online at the Ocular Tissue Database.

DFNA8/12 caused by TECTA mutations is the most identified subtype of nonsyndromic autosomal dominant hearing loss.

The prevalence of DFNA8/DFNA12 (DFNA8/12), a type of autosomal dominant nonsyndromic hearing loss (ADNSHL), is unknown as comprehensive population‐based genetic screening has not been conducted. We therefore completed unbiased screening for TECTA mutations in a Spanish cohort of 372 probands from ADNSHL families. Three additional families (Spanish, Belgian, and English) known to be linked to DFNA8/12 were also included in the screening. In an additional cohort of 835 American ADNSHL families, we preselected 73 probands for TECTA screening based on audiometric data. In aggregate, we identified 23 TECTA mutations in this process. Remarkably, 20 of these mutations are novel, more than doubling the number of reported TECTA ADNSHL mutations from 13 to 33. Mutations lie in all domains of the α‐tectorin protein, including those for the first time identified in the entactin domain, as well as the vWFD1, vWFD2, and vWFD3 repeats, and the D1–D2 and TIL2 connectors. Although the majority are private mutations, four of them—p.Cys1036Tyr, p.Cys1837Gly, p.Thr1866Met, and p.Arg1890Cys—were observed in more than one unrelated family. For two of these mutations founder effects were also confirmed. Our data validate previously observed genotype–phenotype correlations in DFNA8/12 and introduce new correlations. Specifically, mutations in the N‐terminal region of α‐tectorin (entactin domain, vWFD1, and vWFD2) lead to mid‐frequency NSHL, a phenotype previously associated only with mutations in the ZP domain. Collectively, our results indicate that DFNA8/12 hearing loss is a frequent type of ADNSHL. Hum Mutat 32:1–10, 2011.

Mutation in the COCH gene is associated with superior semicircular canal dehiscence

To the Editor: The prevalence of significant hearing loss (≥ 25 db HL) is 15-20% in young adults and rises to approximately 50% in individuals 80 years of age or older [Morton 1991]. Autosomal dominant nonsyndromic hearing loss (ADSNHL) accounts for approximately 15% of inherited hearing loss. To date, 22 genes have been implicated as causative for ADNSHL, and a further 30 loci mapped to autosomal chromosomal regions [Van Camp and Smith 2007]. ADSNHL at the DFNA9 locus is unusual amongst ADNSHL forms of deafness in that it is also associated with vestibular dysfunction. This type of hearing loss is caused by mutations in the COCH gene (chromosome 14q12), which encodes cochlin, a protein that consists of a signal peptide, an LCCL module, and two von Willebrand factor A (vWFA) domains (Table I) [de Kok et al., 1999]. Cochlin is the most highly expressed protein in the human and mouse inner ear, but its precise function remains unclear [Dessens et al., 2004; Robertson et al., 2006]. A possible role in either structural integrity or antimicrobial activity is predicted by its protein structure [Liepinsh et al., 2001] and immunohistochemistry of a temporal bone from a person with DFNA9-related deafness showing abundant aggregation of homogeneous acellular eosinophilic deposits with loss of cellularity in the cochlea and the vestibular labyrinth [Robertson et al., 2006]. Table I DFNA9-causing mutations in the COCH gene Most DFNA9-causing mutations are located within the LCCL domain, which is predicted to be involved in protein folding or a host-defense function (Table I) [Trexler et al., 2000]. The p.P51S variant that affects the LCCL module is the most frequently observed mutation in DFNA9 families [Bischoff et al., 2005; de Kok et al., 1999; Fransen et al., 2001; Fransen et al., 1999; Lemaire et al., 2003]. The identification of a large number of DFNA9 families carrying this variant has facilitated a detailed longitudinal analysis of the audiometric and vestibular impairment in 74 mutation carriers, which has shown that vestibular failure precedes and progresses more rapidly than hearing impairment [Bischoff et al., 2005]. In this study (approved by the University of Iowa Institutional Review Board), we recruited American subjects with ADNSHL and obtained DNA samples and audiograms. Audiograms were formatted for audioprofile analysis, which was conducted using the AudioGene v2.0 system [Hildebrand et al., 2008]. This system predicts the likely underlying genetic cause of hearing loss based on a number of different phenotypic parameters, including auditory thresholds, and rate of decrease in hearing relative to age and frequency. AudioGene v2.0 was trained with audiometric data from families with known deafness-causing mutations in KCNQ4 (DFNA2), DFNA5 (DFNA5), WFS1 (DFNA6/14/38), TECTA (DFNA8/12), COCH (DFNA9) and COL11A2 (DFNA13). To validate AudioGene v2.0 as a clinical and research tool, we studied a cohort of 77 families segregating presumed ADSNHL represented by audiograms from 160 individuals. Individuals from seven families were identified by AudioGene v2.0 as having a DFNA9 profile, and in six families, mutation screening of the COCH gene was completed (genomic DNA was unavailable for screening in one family). While no mutations were identified in five families, one family was found to segregate the p.P51S mutation (positive predictive value, ∼16.7%) (Figs ​(Figs1,1, ​,2).2). To our knowledge, this family is the first outside Western Europe to be reported with this mutation. However by comparing highly heterozygous short tandem repeat polymorphisms (STRPs) tightly linked to the COCH gene, we could show that this family is related to five previously reported Belgian p.P51S families [Fransen et al., 2001] (data not shown), providing further evidence of a founder effect in the Benelux region of Western Europe. Figure 1 Pedigree of the five-generation American family with nonsyndromic autosomal dominant HFSNHL, vestibular dysfunction and SSCD. Genotypes for nuceotide c.151 in the COCH gene are shown for those individuals included in the genetic analysis. Individuals ... Figure 2 The causative mutation in the COCH gene in the American family. The mutation is a heterozygous C-to-T base change in exon 3 (c.151C→T) that results in substitution of a proline residue for a serine residue at position 51 (p.P51S). The clinical presentation of all p.P51S families is very similar (Table I) [de Kok et al., 1999; Fransen et al., 2001; Fransen et al., 1999; Lemaire et al., 2003]. The hearing loss initially preferentially affects high frequencies (down-sloping) and progresses to become profound across all frequencies at a rate of ∼1.8 dB annually [Bom et al., 1999]. In a few exceptional cases, however, hearing at low frequencies declines much more rapidly, at greater than 24 dB annually [Bom et al., 1999]. In the American family, the self-reported age-of-onset of hearing loss ranged from 20 to 60 years. The hearing loss was progressive, with a general trend of initial moderate-to-severe high frequency (> 2000 Hz) hearing loss (HFSNHL) that became profound (Fig.3). In younger persons, the audioprofile was down-sloping, preserving hearing in the low and mid frequencies; with advancing age, low and mid frequency loss developed and the audioprofile flattened. Individuals IV:5 and IV:6 represent phenocopies. Figure 3 Audiograms of representative affected family members at various ages. Affected individuals initially exhibit high frequency (> 2000 Hz) mild-to-moderate SNHL (HFSNHL) that progresses to become severe-to-profound before flattening out later in ... All hearing impaired persons in this family excluding IV:5 and IV:6 also had vestibular impairment characterized by intermittent unsteadiness, particularly in the dark, and sporadic periods of dizziness. In addition, one person was diagnosed with Meniere’s-like symptoms and another with autoimmune inner ear disease requiring treatment with a diuretic and a low-salt diet. This variety of complaints is similar to that reported in other p.P51S families [Bischoff et al., 2005]. In general, p.P51S mutation carriers over 40 years of age usually have prominent vestibular symptoms however these complaints only emerge from the medical history if a targeted vestibular history is obtained. Computed tomography (CT) scanning on a number of family members revealed intact ossicles, cochleae and semicircular canals with the exception of individual V:1. This person presented at age 31 in October, 2006, for an audiogram because of the family history of hearing loss. He has no complaints and by audiometry had 10 dB thresholds bilaterally through 2 kHz, a drop to 25 dB between 3-6 kHz, and normal hearing again at 8 kHz (Fig.4A). In February, 2008, he returned with a complaint of left aural fullness and persistent otalgia (6-9 months) and underwent temporal bone CT, which revealed bilateral superior semicircular canal dehiscence (SSCD) (Fig.4B). Figure 4 Audiometry and temporal bone CT of individual V:1. A Audiogram recorded at 31 years of age showing slightly elevated hearing thresholds at high frequencies. B Coronial high-resolution CT scan of the temporal bones showing bilateral superior semicircular ... SSCD is a rare disorder. Originally described by Minor and colleagues, it is characterized by the absence of bone overlying the superior semicircular canal, which creates a third labyrinthine window (with the oval and round windows) [Minor 2000; Minor 2005]. The consequence is the loss of acoustic energy and abnormal stimulation of the vestibular system; the clinical manifestations include Valsalva- and exercise-induced vertigo, sound-induced vertigo (Tullio phenomenon), and variable conductive hearing loss [Cox et al., 2003; Mikulec et al., 2004; Minor et al., 2003]. Diagnosis requires high resolution temporal bone CT [Belden et al., 2003; Hirvonen et al., 2003]. The genetics of SSCD have not been studied, and our finding of a COCH mutation in a single patient with SSCD is noteworthy since both DFNA9-related deafness and SSCD are rare. Although it seems unlikely that SSCD is directly related to the well-documented type of vestibular impairment that is part of the DFNA9 phenotype, SSCD may be present in other DFNA9 patients. We therefore recommend high resolution temporal bone CT in patients with DFNA9-related deafness and screening of COCH in sporadic or familial cases of SSCD. This diagnostic algorithm is important because SSCD and its associated symptoms can be partially or fully corrected with surgery [Carey et al., 2007; Limb et al., 2006; Mikulec et al., 2005; Wilkinson et al., 2008]. A remarkable finding in the process of identifying DFNA9/COCH as the underlying cause of deafness in this American family was not only that a dedicated computer program, AudioGene v2.0, hinted at this possibility, but that this occurred in the course of a screening procedure for DFNA2 families [Hildebrand et al., 2008]. It is reassuring to find that AudioGene v2.0 can distinguish between the audioprofiles of genetically different types of ADNSHL, especially when both DFNA2 and DFNA9 show remarkably similar high frequency hearing loss with steeply downsloping thresholds. Admittedly, in the case of p.P51S mutation carriers genetic screening should be preferentially directed to DFNA9 and DFNA11, on the basis of the vestibular impairment findings. However, it is realistic to acknowledge the possibility that vestibular symptoms can go unnoticed. Furthermore, with other DFNA9/COCH mutations vestibular failure can be less prominent. It is not invariably penetrant and/or occurs at a more advanced age [Kemperman et al., 2005; Pauw et al., 2007a].

cGMP production of patient-specific iPSCs and photoreceptor precursor cells to treat retinal degenerative blindness

Immunologically-matched, induced pluripotent stem cell (iPSC)-derived photoreceptor precursor cells have the potential to restore vision to patients with retinal degenerative diseases like retinitis pigmentosa. The purpose of this study was to develop clinically-compatible methods for manufacturing photoreceptor precursor cells from adult skin in a non-profit cGMP environment. Biopsies were obtained from 35 adult patients with inherited retinal degeneration and fibroblast lines were established under ISO class 5 cGMP conditions. Patient-specific iPSCs were then generated, clonally expanded and validated. Post-mitotic photoreceptor precursor cells were generated using a stepwise cGMP-compliant 3D differentiation protocol. The recapitulation of the enhanced S-cone phenotype in retinal organoids generated from a patient with NR2E3 mutations demonstrated the fidelity of these protocols. Transplantation into immune compromised animals revealed no evidence of abnormal proliferation or tumor formation. These studies will enable clinical trials to test the safety and efficiency of patient-specific photoreceptor cell replacement in humans.

Audioprofile-directed screening identifies novel mutations in KCNQ4 causing hearing loss at the DFNA2 locus.

Purpose: Gene identification in small families segregating autosomal dominant sensorineural hearing loss presents a significant challenge. To address this challenge, we have developed a machine learning-based software tool, AudioGene v2.0, to prioritize candidate genes for mutation screening based on audioprofiling.Methods: We analyzed audiometric data from a cohort of American families with high-frequency autosomal dominant sensorineural hearing loss. Those families predicted to have a DFNA2 audioprofile by AudioGene v2.0 were screened for mutations in the KCNQ4 gene.Results: Two novel missense mutations and a stop mutation were detected in three American families predicted to have DFNA2-related deafness for a positive predictive value of 6.3%. The false negative rate was 0%. The missense mutations were located in the channel pore region and the stop mutation was in transmembrane domain S5. The latter is the first DFNA2-causing stop mutation reported in KCNQ4.Conclusions: Our data suggest that the N-terminal end of the P-loop is crucial in maintaining the integrity of the KCNQ4 channel pore and AudioGene audioprofile analysis can effectively prioritize genes for mutation screening in small families segregating high-frequency autosomal dominant sensorineural hearing loss. AudioGene software will be made freely available to clinicians and researchers once it has been fully validated.