Leveraging Unique Chromosomal Microarray Probes to Accurately Detect Copy Number at the Highly Homologous 15q15.3 Deafness-Infertility Syndrome Locus.

BACKGROUND: Biallelic deletions at 15q15.3, including STRC and CATSPER2, cause autosomal recessive deafness-infertility syndrome (DIS), while biallelic deletions of STRC alone cause nonsyndromic hearing loss. These deletions are among the leading genetic causes of mild-moderate hearing loss, but their detection using chromosomal microarray (CMA) is impeded by a tandem duplication containing highly homologous pseudogenes. We sought to assess copy number variant (CNV) detection in this region by a commonly-employed CMA platform. METHODS: Twenty-two specimens with known 15q15.3 CNVs, determined by droplet digital PCR (ddPCR), were analyzed by CMA. To investigate the impact of pseudogene homology on CMA performance, a probe-level analysis of homology was performed, and log2 ratios of unique and pseudogene-homologous probes compared. RESULTS: Assessment of 15q15.3 CNVs by CMA compared to ddPCR revealed 40.9% concordance, with frequent mis-assignment of zygosity by the CMA automated calling software. Probe-level analysis of pseudogene homology suggested that probes with high homology contributed to this discordance, with significant differences in log2 ratios between unique and pseudogene-homologous CMA probes. Two clusters containing several unique probes could reliably detect CNVs involving STRC and CATSPER2, despite the noise of surrounding probes, discriminating between homozygous vs heterozygous losses and complex rearrangements. CNV detection by these probe clusters showed 100% concordance with ddPCR. CONCLUSIONS: Manual analysis of clusters containing unique CMA probes without significant pseudogene homology improves CNV detection and zygosity assignment in the highly homologous DIS region. Incorporation of this method into CMA analysis and reporting processes can improve DIS diagnosis and carrier detection.

The Burden and Benefits of Knowledge: Ethical Considerations Surrounding Population-Based Newborn Genome Screening for Hearing.

Recent advances in genomic sequencing technologies have expanded practitioners' utilization of genetic information in a timely and efficient manner for an accurate diagnosis. With an ever-increasing resource of genomic data from progress in the interpretation of genome sequences, clinicians face decisions about how and when genomic information should be presented to families, and at what potential expense. Presently, there is limited knowledge or experience in establishing the value of implementing genome sequencing into newborn screening. Herein we provide insight into the complexities and the burden and benefits of knowledge resulting from genome sequencing of newborns.

Genome-wide association meta-analysis identifies 48 risk variants and highlights the role of the stria vascularis in hearing loss.

Hearing loss is one of the top contributors to years lived with disability and is a risk factor for dementia. Molecular evidence on the cellular origins of hearing loss in humans is growing. Here, we performed a genome-wide association meta-analysis of clinically diagnosed and self-reported hearing impairment on 723,266 individuals and identified 48 significant loci, 10 of which are novel. A large proportion of associations comprised missense variants, half of which lie within known familial hearing loss loci. We used single-cell RNA-sequencing data from mouse cochlea and brain and mapped common-variant genomic results to spindle, root, and basal cells from the stria vascularis, a structure in the cochlea necessary for normal hearing. Our findings indicate the importance of the stria vascularis in the mechanism of hearing impairment, providing future paths for developing targets for therapeutic intervention in hearing loss.

Reporting of Diagnostic Cytogenetic Results.

This appendix, developed by the staff at the Center for Advanced Molecular Diagnostics in the Department of Pathology at the Brigham and Women's Hospital, includes a comprehensive list of current "macros" or standardized statements used to facilitate reporting of cytogenetic results. These are provided as a useful reference for other laboratories. The statements are organized under the general categories of constitutional or acquired abnormalities and subdivided into analysis type (GTG-banding, FISH, or chromosomal microarray). Multi-specimen usage macros are included that can be applied to two or more specimen types.

PSIP1/LEDGF: a new gene likely involved in sensorineural progressive hearing loss.

Hereditary Hearing Loss (HHL) is an extremely heterogeneous disorder. Approximately 30 out of 80 known HHL genes are associated with autosomal dominant forms. Here, we identified PSIP1/LEDGF (isoform p75) as a novel strong candidate gene involved in dominant HHL. Using exome sequencing we found a frameshift deletion (c.1554_1555del leading to p.E518Dfs*2) in an Italian pedigree affected by sensorineural mild-to-moderate HHL but also showing a variable eye phenotype (i.e. uveitis, optic neuropathy). This deletion led to a premature stop codon (p.T519X) with truncation of the last 12 amino acids. PSIP1 was recently described as a transcriptional co-activator regulated by miR-135b in vestibular hair cells of the mouse inner ear as well as a possible protector against photoreceptor degeneration. Here, we demonstrate that it is ubiquitously expressed in the mouse inner ear. The PSIP1 mutation is associated with a peculiar audiometric slope toward the high frequencies. These findings indicate that PSIP1 likely plays an important role in HHL.

Cochlin in normal middle ear and abnormal middle ear deposits in DFNA9 and Coch (G88E/G88E) mice.

DFNA9 sensorineural hearing loss and vestibular disorder, caused by mutations in COCH, has a unique identifying histopathology including prominent acellular deposits in cochlear and vestibular labyrinths. A recent study has shown presence of deposits also in middle ear structures of DFNA9-affected individuals (McCall et al., J Assoc Res Otolaryngol 12:141-149, 2004). To investigate the possible role of cochlin in the middle ear and in relation to aggregate formation, we evaluated middle ear histopathology in our Coch knock-in (Coch (G88E/G88E) ) mouse model, which harbors one of the DFNA9-causative mutations. Our findings reveal accumulation of acellular deposits in the incudomalleal and incudostapedial joints in Coch (G88E/G88E) mice, similar to those found in human DFNA9-affected temporal bones. Aggregates are absent in negative control Coch (+/+) and Coch (-/-) mice. Thickening of the tympanic membrane (TM) found in humans with DFNA9 was not appreciably detected in Coch (G88E/G88E) mice at the evaluated age. We investigated cochlin localization first in the Coch (+/+)mouse and in normal human middle ears, and found prominent and specific cochlin staining in the incudomalleal joint, incudostapedial joint, and the pars tensa of the TM, which are the three sites where abnormal deposits are detected in DFNA9-affected middle ears. Cochlin immunostaining of Coch (G88E/G88E) and DFNA9-affected middle ears showed mutant cochlin localization within areas of aggregates. Cochlin staining was heterogeneous throughout DFNA9 middle ear deposits, which appear as unorganized and overlapping mixtures of both eosinophilic and basophilic substances. Immunostaining for type II collagen colocalized with cochlin in pars tensa of the tympanic membrane. In contrast, immunostaining for type II collagen did not overlap with cochlin in interossicular joints, where type II collagen was localized in the region of the chondrocytes, but not in the thin layer of the articular surface of the ossicles nor in the eosinophilic deposits with specific cochlin staining.

Reporting of diagnostic cytogenetic results.

This appendix, developed by the staff at the Clinical Cytogenetics Laboratory at the Brigham and Women's Hospital, includes a comprehensive list of current "macros" or standardized statements used to facilitate reporting of cytogenetic results. These are provided as a reference for other laboratories. The statements are organized under the general categories of constitutional or acquired abnormalities and subdivided into analysis type (GTG-banding or FISH). Multi-specimen usage macros are included that can be applied to two or more specimen types.

Hearing and vestibular deficits in the Coch(-/-) null mouse model: comparison to the Coch(G88E/G88E) mouse and to DFNA9 hearing and balance disorder.

Two mouse models, the Coch(G88E/G88E) or "knock-in" and the Coch(-/-) or "knock-out" (Coch null), have been developed to study the human late-onset, progressive, sensorineural hearing loss and vestibular dysfunction known as DFNA9. This disorder results from missense and in-frame deletion mutations in COCH (coagulation factor C homology), encoding cochlin, the most abundantly detected protein in the inner ear. We have performed hearing and vestibular analyses by auditory brainstem response (ABR) and vestibular evoked potential (VsEP) testing of the Coch(-/-) and Coch(G88E/G88E) mouse models. Both Coch(-/-) and Coch(G88E/G88E) mice show substantially elevated ABRs at 21 months of age, but only at the highest frequency tested for the former and all frequencies for the latter. At 21 months, 9 of 11 Coch(-/-) mice and 4 of 8 Coch(G88E/G88E) mice have absent ABRs. Interestingly Coch(-/+) mice do not show hearing deficits, in contrast to Coch(G88E/+), which demonstrate elevated ABR thresholds similar to homozyotes. These results corroborate the DFNA9 autosomal dominant mode of inheritance, in addition to the observation that haploinsufficiency of Coch does not result in impaired hearing. Vestibular evoked potential (VsEP) thresholds were analyzed using a two factor ANOVA (Age X Genotype). Elevated VsEP thresholds are detected in Coch(-/-) mice at 13 and 21 months, the two ages tested, and as early as seven months in the Coch(G88E/G88E) mice. These results indicate that in both mouse models, vestibular function is compromised before cochlear function. Analysis and comparison of hearing and vestibular function in these two DFNA9 mouse models, where deficits occur at such an advanced age, provide insight into the pathology of DFNA9 and age-related hearing loss and vestibular dysfunction as well as an opportunity to investigate potential interventional therapies.

Reporting of diagnostic cytogenetic results.

This appendix, developed by the staff at the Clinical Cytogenetics Laboratory at the Brigham and Women's Hospital, includes a comprehensive list of current "macros" or standardized statements used to facilitate reporting of cytogenetic results. These are provided as a reference for other laboratories. The statements are organized under the general categories of constitutional or acquired abnormalities and subdivided into analysis type (GTG-banding or FISH). Multi-specimen usage macros are included that can be applied to two or more specimen types.

A targeted Coch missense mutation: a knock-in mouse model for DFNA9 late-onset hearing loss and vestibular dysfunction.

Mutations in COCH (coagulation factor C homology) are etiologic for the late-onset, progressive, sensorineural hearing loss and vestibular dysfunction known as DFNA9. We introduced the G88E mutation by gene targeting into the mouse and have created a Coch(G88E/G88E) mouse model for the study of DFNA9 pathogenesis and cochlin function. Vestibular-evoked potential (VsEP) thresholds of Coch(G88E/G88E) mice were elevated at all ages tested compared with wild-type littermates. At the oldest ages, two out of eight Coch(G88E/G88E) mice had no measurable VsEP. Auditory brainstem response (ABR) thresholds of Coch(G88E/G88E) mice were substantially elevated at 21 months but not at younger ages tested. At 21 months, four of eight Coch(G88E/G88E) mice had absent ABRs at all frequencies tested and two of three Coch(G88E)(/+) mice had absent ABRs at three of four frequencies tested. Distortion product otoacoustic emission amplitudes of Coch(G88E/G88E) mice were substantially lower than Coch(+/+) mice and absent in the same Coch(G88E/G88E) mice with absent ABRs. These results suggest that vestibular function is affected beginning as early as 11 months when cochlear function appears to be normal, and dysfunction increases with age. Hearing loss declines substantially at 21 months of age and progresses to profound hearing loss at some to all frequencies tested. This is the only mouse model developed to date where hearing loss begins at such an advanced age, providing an opportunity to study both progressive age-related hearing loss and possible interventional therapies.

Expression studies of osteoglycin/mimecan (OGN) in the cochlea and auditory phenotype of Ogn-deficient mice.

Genes involved in the hearing process have been identified through both positional cloning efforts following genetic linkage studies of families with heritable deafness and by candidate gene approaches based on known functional properties or inner ear expression. The latter method of gene discovery may employ a tissue- or organ-specific approach. Through characterization of a human fetal cochlear cDNA library, we have identified transcripts that are preferentially and/or highly expressed in the cochlea. High expression in the cochlea may be suggestive of a fundamental role for a transcript in the auditory system. Herein we report the identification and characterization of a transcript from the cochlear cDNA library with abundant cochlear expression and unknown function that was subsequently determined to represent osteoglycin (OGN). Ogn-deficient mice, when analyzed by auditory brainstem response and distortion product otoacoustic emissions, have normal hearing thresholds.

Ion channel gene expression in the inner ear.

The ion channel genome is still being defined despite numerous publications on the subject. The ion channel transcriptome is even more difficult to assess. Using high-throughput computational tools, we surveyed all available inner ear cDNA libraries to identify genes coding for ion channels. We mapped over 100,000 expressed sequence tags (ESTs) derived from human cochlea, mouse organ of Corti, mouse and zebrafish inner ear, and rat vestibular end organs to Homo sapiens, Mus musculus, Danio rerio, and Rattus norvegicus genomes. A survey of EST data alone reveals that at least a third of the ion channel genome is expressed in the inner ear, with highest expression occurring in hair cell-enriched mouse organ of Corti and rat vestibule. Our data and comparisons with other experimental techniques that measure gene expression show that every method has its limitations and does not per se provide a complete coverage of the inner ear ion channelome. In addition, the data show that most genes produce alternative transcripts with the same spectrum across multiple organisms, no ion channel gene variants are unique to the inner ear, and many splice variants have yet to be annotated. Our high-throughput approach offers a qualitative computational and experimental analysis of ion channel genes in inner ear cDNA collections. A lack of data and incomplete gene annotations prevent both rigorous statistical analyses and comparisons of entire ion channelomes derived from different tissues and organisms.

Characterization of an abundant COL9A1 transcript in the cochlea with a novel 3' UTR: Expression studies and detection of miRNA target sequence.

EST N66408 represents one of several large unique clusters expressed in the Morton human fetal cochlear cDNA library. N66408 is 575 bp in size and initial BLAST analysis of this sequence showed no homology to any known genes or expressed sequence tags (ESTs) from other organs or tissues. Sequence of the original cochlear clone from which N66408 was derived revealed that the corresponding cDNA was about 700 bp in size, including 125 bp at its 5' end with homology to the 3' end of COL9A1 in addition to 575 bp of novel sequence. RT-PCR analysis using primers specific to COL9A1 isoforms 1 and 2 detected expression of both isoforms in human fetal cochlea. Tissue in situ hybridization using the novel 3' UTR sequence as probe showed abundant expression in spiral limbus and spiral ligament, and a moderate level of expression in the organ of Corti. dbEST analysis of ESTs specific to the 3' UTR of COL9A1 showed 19 ESTs derived from various tissues; three polyadenylation sites were identified and the majority of these ESTs were derived from overlapping polyadenylation signals at the second site (position 749-758). Comparison of the 3' UTR of human COL9A1 with its orthologs as well as with dbEST uncovered a highly conserved region around the overlapping polyadenylation signals at position 749-758 in mammals. A search of the microRNA database revealed a highly conserved target sequence for miR-9 immediately preceding the overlapping polyadenylation signals in the novel 3' UTR of COL9A1, suggesting its role in posttranscriptional regulation of COL9A1.

Isolation from cochlea of a novel human intronless gene with predominant fetal expression.

We have cloned a novel human gene, designated PFET1 (predominantly fetal expressed T1 domain) (HUGO-approved symbol KCTD12 or C13orf2), by subtractive hybridization and differential screening of human fetal cochlear cDNA clones. Also, we have identified the mouse homolog, designated Pfet1. PFET1/Pfet1 encode a single transcript of approximately 6 kb in human, and three transcripts of approximately 4, 4.5, and 6 kb in mouse with a 70% GC-rich open reading frame (ORF) consisting of 978 bp in human and 984 bp in mouse. Both genes have unusually long 3' untranslated (3' UTR) regions (4996 bp in human PFET1, 3700 bp in mouse Pfet1) containing 12 and 5 putative polyadenylation consensus sequences, respectively. Pfetin, the protein encoded by PFET1/Pfet1, is predicted to have 325 amino acids in human and 327 amino acids in mouse and to contain a voltage-gated potassium (K+) channel tetramerization (T1) domain. Otherwise, to date these genes have no significant homology to any known gene. PFET1 maps to the long arm of human chromosome 13, in band q21 as shown by FISH analysis and STS mapping. Pfet1 maps to mouse chromosome 14 near the markers D14Mit8, D14Mit93, and D14Mit145.1. The human 6 kb transcript is present in a variety of fetal organs, with highest expression levels in the cochlea and brain and, in stark contrast, is detected only at extremely low levels in adult organs, such as brain and lung. Immunohistochemistry with a polyclonal antibody raised against a synthetic peptide to PFET1 sequence (pfetin) reveals immunostaining in a variety of cell types in human, monkey, mouse, and guinea pig cochleas and the vestibular system, including type I vestibular hair cells.

Reporting of diagnostic cytogenetic results.

This appendix, developed by the staff at the Clinical Cytogenetics Laboratory at the Brigham and Women's Hospital, provides a comprehensive list of the facilities' current "macros" or standardized statements, used to facilitate reporting of cytogenetic results. These are provided as a reference for other laboratories. The statements are organized under the general categories of constitutional or acquired abnormalities and subdivided into analysis type (GTG-banding or FISH). Multi-specimen usage macros are included that can be applied to two or more specimen types.