Publications by authors named "Thomas B Friedman"

123 Publications

Semi-automated single-molecule microscopy screening of fast-dissociating specific antibodies directly from hybridoma cultures.

Cell Rep 2021 Feb;34(5):108708

Laboratory of Single-Molecule Cell Biology, Graduate School of Biostudies, Kyoto University, Kyoto 606-8501, Japan; Department of Pharmacology, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan. Electronic address:

Fast-dissociating, specific antibodies are single-molecule imaging probes that transiently interact with their targets and are used in biological applications including image reconstruction by integrating exchangeable single-molecule localization (IRIS), a multiplexable super-resolution microscopy technique. Here, we introduce a semi-automated screen based on single-molecule total internal reflection fluorescence (TIRF) microscopy of antibody-antigen binding, which allows for identification of fast-dissociating monoclonal antibodies directly from thousands of hybridoma cultures. We develop monoclonal antibodies against three epitope tags (FLAG-tag, S-tag, and V5-tag) and two F-actin crosslinking proteins (plastin and espin). Specific antibodies show fast dissociation with half-lives ranging from 0.98 to 2.2 s. Unexpectedly, fast-dissociating yet specific antibodies are not so rare. A combination of fluorescently labeled Fab probes synthesized from these antibodies and light-sheet microscopy, such as dual-view inverted selective plane illumination microscopy (diSPIM), reveal rapid turnover of espin within long-lived F-actin cores of inner-ear sensory hair cell stereocilia, demonstrating that fast-dissociating specific antibodies can identify novel biological phenomena.
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http://dx.doi.org/10.1016/j.celrep.2021.108708DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7904085PMC
February 2021

Actin at stereocilia tips is regulated by mechanotransduction and ADF/cofilin.

Curr Biol 2021 Mar 4;31(6):1141-1153.e7. Epub 2021 Jan 4.

Department of Biology, Indiana University-Purdue University Indianapolis, 723 West Michigan Street, Indianapolis, IN 46202, USA. Electronic address:

Stereocilia on auditory sensory cells are actin-based protrusions that mechanotransduce sound into an electrical signal. These stereocilia are arranged into a bundle with three rows of increasing length to form a staircase-like morphology that is required for hearing. Stereocilia in the shorter rows, but not the tallest row, are mechanotransducing because they have force-sensitive channels localized at their tips. The onset of mechanotransduction during mouse postnatal development refines stereocilia length and width. However, it is unclear how actin is differentially regulated between stereocilia in the tallest row of the bundle and the shorter, mechanotransducing rows. Here, we show actin turnover is increased at the tips of mechanotransducing stereocilia during bundle maturation. Correspondingly, from birth to postnatal day 6, these stereocilia had increasing amounts of available actin barbed ends, where monomers can be added or lost readily, as compared with the non-mechanotransducing stereocilia in the tallest row. The increase in available barbed ends depended on both mechanotransduction and MYO15 or EPS8, which are required for the normal specification and elongation of the tallest row of stereocilia. We also found that loss of the F-actin-severing proteins ADF and cofilin-1 decreased barbed end availability at stereocilia tips. These proteins enriched at mechanotransducing stereocilia tips, and their localization was perturbed by the loss of mechanotransduction, MYO15, or EPS8. Finally, stereocilia lengths and widths were dysregulated in Adf and Cfl1 mutants. Together, these data show that actin is remodeled, likely by a severing mechanism, in response to mechanotransduction.
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http://dx.doi.org/10.1016/j.cub.2020.12.006DOI Listing
March 2021

Myosin 18Aα targets the guanine nucleotide exchange factor β-Pix to the dendritic spines of cerebellar Purkinje neurons and promotes spine maturation.

FASEB J 2021 Jan;35(1):e21092

Molecular Cell Biology Laboratory, Cell and Developmental Biology Center, NHLBI, NIH, Bethesda, MD, USA.

Myosin 18Aα is a myosin 2-like protein containing unique N- and C-terminal protein interaction domains that co-assembles with myosin 2. One protein known to bind to myosin 18Aα is β-Pix, a guanine nucleotide exchange factor (GEF) for Rac1 and Cdc42 that has been shown to promote dendritic spine maturation by activating the assembly of actin and myosin filaments in spines. Here, we show that myosin 18A⍺ concentrates in the spines of cerebellar Purkinje neurons via co-assembly with myosin 2 and through an actin binding site in its N-terminal extension. miRNA-mediated knockdown of myosin 18A⍺ results in a significant defect in spine maturation that is rescued by an RNAi-immune version of myosin 18A⍺. Importantly, β-Pix co-localizes with myosin 18A⍺ in spines, and its spine localization is lost upon myosin 18A⍺ knockdown or when its myosin 18A⍺ binding site is deleted. Finally, we show that the spines of myosin 18A⍺ knockdown Purkinje neurons contain significantly less F-actin and myosin 2. Together, these data argue that mixed filaments of myosin 2 and myosin 18A⍺ form a complex with β-Pix in Purkinje neuron spines that promotes spine maturation by enhancing the assembly of actin and myosin filaments downstream of β-Pix's GEF activity.
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http://dx.doi.org/10.1096/fj.202001449RDOI Listing
January 2021

The ATPase mechanism of myosin 15, the molecular motor mutated in DFNB3 human deafness.

J Biol Chem 2021 Jan 9;296:100243. Epub 2021 Jan 9.

Department of Pharmacology and Therapeutics, and the Myology Institute, University of Florida College of Medicine, Gainesville, Florida, USA. Electronic address:

Cochlear hair cells each possess an exquisite bundle of actin-based stereocilia that detect sound. Unconventional myosin 15 (MYO15) traffics and delivers critical molecules required for stereocilia development and thus is essential for building the mechanosensory hair bundle. Mutations in the human MYO15A gene interfere with stereocilia trafficking and cause hereditary hearing loss, DFNB3, but the impact of these mutations is not known, as MYO15 itself is poorly characterized. To learn more, we performed a kinetic study of the ATPase motor domain to characterize its mechanochemical cycle. Using the baculovirus-Sf9 system, we purified a recombinant minimal motor domain (S1) by coexpressing the mouse MYO15 ATPase, essential and regulatory light chains that bind its IQ domains, and UNC45 and HSP90A chaperones required for correct folding of the ATPase. MYO15 purified with either UNC45A or UNC45B coexpression had similar ATPase activities (k = ∼ 6 s at 20 °C). Using stopped-flow and quenched-flow transient kinetic analyses, we measured the major rate constants describing the ATPase cycle, including ATP, ADP, and actin binding; hydrolysis; and phosphate release. Actin-attached ADP release was the slowest measured transition (∼12 s at 20 °C), although this did not rate-limit the ATPase cycle. The kinetic analysis shows the MYO15 motor domain has a moderate duty ratio (∼0.5) and weak thermodynamic coupling between ADP and actin binding. These findings are consistent with MYO15 being kinetically adapted for processive motility when oligomerized. Our kinetic characterization enables future studies into how deafness-causing mutations affect MYO15 and disrupt stereocilia trafficking necessary for hearing.
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http://dx.doi.org/10.1074/jbc.RA120.014903DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7948958PMC
January 2021

Vestibular phenotype-genotype correlation in a cohort of 90 patients with Usher syndrome.

Clin Genet 2021 Feb 3;99(2):226-235. Epub 2020 Nov 3.

Otolaryngology Branch, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland, USA.

Usher syndrome has been historically categorized into one of three classical types based on the patient phenotype. However, the vestibular phenotype does not infallibly predict which Usher genes are mutated. Conversely, the Usher syndrome genotype is not sufficient to reliably predict vestibular function. Here we present a characterization of the vestibular phenotype of 90 patients with clinical presentation of Usher syndrome (59 females), aged 10.9 to 75.5 years, with genetic variants in eight Usher syndromic genes and expand the description of atypical Usher syndrome. We identified unexpected horizontal semicircular canal reactivity in response to caloric and rotational stimuli in 12.5% (3 of 24) and 41.7% (10 of 24), respectively, of our USH1 cohort. These findings are not consistent with the classical phenotypic definition of vestibular areflexia in USH1. Similarly, 17% (6 of 35) of our cohort with USH2A mutations had saccular dysfunction as evidenced by absent cervical vestibular evoked myogenic potentials in contradiction to the classical assumption of normal vestibular function. The surprising lack of consistent genotypic to vestibular phenotypic findings as well as no clear vestibular phenotypic patterns among atypical USH cases, indicate that even rigorous vestibular phenotyping data will not reliably differentiate the three USH types.
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http://dx.doi.org/10.1111/cge.13868DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7821283PMC
February 2021

Mouse Models of Human Pathogenic Variants of Associated with Non-Syndromic Deafness DFNB86 and DFNA65 and Syndromes Involving Deafness.

Genes (Basel) 2020 Sep 24;11(10). Epub 2020 Sep 24.

Laboratory of Molecular Genetics, National Institute on Deafness and Other Communication Disorders, Porter Neuroscience Research Center, National Institutes of Health, Bethesda, MD 20892, USA.

Human pathogenic variants of are associated with clinically heterogeneous phenotypes, including recessive nonsyndromic deafness DFNB86, dominant nonsyndromic deafness DFNA65, seizure accompanied by deafness, a variety of isolated seizure phenotypes and DOORS syndrome, characterized by deafness, onychodystrophy, osteodystrophy, intellectual disability and seizures. Thirty-five pathogenic variants of human associated with deafness have been reported. However, functions of TBC1D24 in the inner ear and the pathophysiology of TBC1D24-related deafness are unknown. In this study, a novel splice-site variant of c.965 + 1G > A in compound heterozygosity with c.641G > A p.(Arg214His) was found to be segregating in a Pakistani family. Affected individuals exhibited, either a deafness-seizure syndrome or nonsyndromic deafness. In human temporal bones, TBC1D24 immunolocalized in hair cells and spiral ganglion neurons, whereas in mouse cochlea, expression was detected only in spiral ganglion neurons. We engineered mouse models of p.(Asp70Tyr) and p.(Ser178Leu) nonsyndromic deafness and syndromic forms of deafness p.(His336Glnfs*12) that have the same pathogenic variants that were reported for human . Unexpectedly, no auditory dysfunction was detected in mutant mice, although homozygosity for some of the variants caused seizures or lethality. We provide some insightful supporting data to explain the phenotypic differences resulting from equivalent pathogenic variants of mouse and human .
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http://dx.doi.org/10.3390/genes11101122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7598720PMC
September 2020

Myosins and Hearing.

Adv Exp Med Biol 2020 ;1239:317-330

Department of Physiology, University of Kentucky College of Medicine, Lexington, KY, USA.

Hearing loss is both genetically and clinically heterogeneous, and pathogenic variants of over a hundred different genes are associated with this common neurosensory disorder. A relatively large number of these "deafness genes" encode myosin super family members. The evidence that pathogenic variants of human MYO3A, MYO6, MYO7A, MYO15A, MYH14 and MYH9 are associated with deafness ranges from moderate to definitive. Additional evidence for the involvement of these six myosins for normal hearing also comes from animal models, usually mouse or zebra fish, where mutations of these genes cause hearing loss and from biochemical, physiological and cell biological studies of their roles in the inner ear. This chapter focuses on these six genes for which evidence of a causative role in deafness is substantial.
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http://dx.doi.org/10.1007/978-3-030-38062-5_13DOI Listing
August 2020

Atypical and ultra-rare Usher syndrome: a review.

Ophthalmic Genet 2020 10 6;41(5):401-412. Epub 2020 May 6.

Ophthalmic Genetics and Visual Function Branch, National Eye Institute, National Institutes of Health , Bethesda, MD, USA.

Usher syndrome has classically been described as a combination of hearing loss and rod-cone dystrophy; vestibular dysfunction is present in many patients. Three distinct clinical subtypes were documented in the late 1970s. Genotyping efforts have led to the identification of several genes associated with the disease. Recent literature has seen multiple publications referring to "atypical" Usher syndrome presentations. This manuscript reviews the molecular etiology of Usher syndrome, highlighting rare presentations and molecular causes. Reports of "atypical" disease are summarized noting the wide discrepancy in the spectrum of phenotypic deviations from the classical presentation. Guidelines for establishing a clear nomenclature system are suggested.
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http://dx.doi.org/10.1080/13816810.2020.1747090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8018527PMC
October 2020

Noncoding Microdeletion in Mouse Disrupts Neural Crest Migration into the Stria Vascularis, Reduces the Endocochlear Potential, and Suggests the Neuropathology for Human Nonsyndromic Deafness DFNB39.

J Neurosci 2020 04 9;40(15):2976-2992. Epub 2020 Mar 9.

Auditory Development and Restoration Program,

Hepatocyte growth factor (HGF) is a multifunctional protein that signals through the MET receptor. HGF stimulates cell proliferation, cell dispersion, neuronal survival, and wound healing. In the inner ear, levels of HGF must be fine-tuned for normal hearing. In mice, a deficiency of HGF expression limited to the auditory system, or an overexpression of HGF, causes neurosensory deafness. In humans, noncoding variants in are associated with nonsyndromic deafness However, the mechanism by which these noncoding variants causes deafness was unknown. Here, we reveal the cause of this deafness using a mouse model engineered with a noncoding intronic 10 bp deletion (del10) in Male and female mice homozygous for del10 exhibit moderate-to-profound hearing loss at 4 weeks of age as measured by tone burst auditory brainstem responses. The wild type (WT) 80 mV endocochlear potential was significantly reduced in homozygous del10 mice compared with WT littermates. In normal cochlea, endocochlear potentials are dependent on ion homeostasis mediated by the stria vascularis (SV). Previous studies showed that developmental incorporation of neural crest cells into the SV depends on signaling from HGF/MET. We show by immunohistochemistry that, in del10 homozygotes, neural crest cells fail to infiltrate the developing SV intermediate layer. Phenotyping and RNAseq analyses reveal no other significant abnormalities in other tissues. We conclude that, in the inner ear, the noncoding del10 mutation in leads to developmental defects of the SV and consequently dysfunctional ion homeostasis and a reduction in the EP, recapitulating human DFNB39 nonsyndromic deafness. Hereditary deafness is a common, clinically and genetically heterogeneous neurosensory disorder. Previously, we reported that human deafness DFNB39 is associated with noncoding variants in the 3'UTR of a short isoform of encoding hepatocyte growth factor. For normal hearing, HGF levels must be fine-tuned as an excess or deficiency of HGF cause deafness in mouse. Using a mutant mouse with a small 10 bp deletion recapitulating a human noncoding variant, we demonstrate that neural crest cells fail to migrate into the stria vascularis intermediate layer, resulting in a significantly reduced endocochlear potential, the driving force for sound transduction by inner ear hair cells. HGF-associated deafness is a neurocristopathy but, unlike many other neurocristopathies, it is not syndromic.
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http://dx.doi.org/10.1523/JNEUROSCI.2278-19.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7141880PMC
April 2020

Exosomes mediate sensory hair cell protection in the inner ear.

J Clin Invest 2020 05;130(5):2657-2672

National Institute on Deafness and Other Communication Disorders (NIDCD), NIH, Bethesda, Maryland, USA.

Hair cells, the mechanosensory receptors of the inner ear, are responsible for hearing and balance. Hair cell death and consequent hearing loss are common results of treatment with ototoxic drugs, including the widely used aminoglycoside antibiotics. Induction of heat shock proteins (HSPs) confers protection against aminoglycoside-induced hair cell death via paracrine signaling that requires extracellular heat shock 70-kDa protein (HSP70). We investigated the mechanisms underlying this non-cell-autonomous protective signaling in the inner ear. In response to heat stress, inner ear tissue releases exosomes that carry HSP70 in addition to canonical exosome markers and other proteins. Isolated exosomes from heat-shocked utricles were sufficient to improve survival of hair cells exposed to the aminoglycoside antibiotic neomycin, whereas inhibition or depletion of exosomes from the extracellular environment abolished the protective effect of heat shock. Hair cell-specific expression of the known HSP70 receptor TLR4 was required for the protective effect of exosomes, and exosomal HSP70 interacted with TLR4 on hair cells. Our results indicate that exosomes are a previously undescribed mechanism of intercellular communication in the inner ear that can mediate nonautonomous hair cell survival. Exosomes may hold potential as nanocarriers for delivery of therapeutics against hearing loss.
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http://dx.doi.org/10.1172/JCI128867DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7190999PMC
May 2020

Growth factor and receptor malfunctions associated with human genetic deafness.

Clin Genet 2020 01 23;97(1):138-155. Epub 2019 Oct 23.

Laboratory of Molecular Genetics, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland.

A variety of different signaling pathways are necessary for development and maintenance of the human auditory system. Normal hearing allows for the detection of soft sounds within the frequency range of 20 to 20 000 Hz, but more importantly to perceive the human voice frequency band of 250 to 6000 Hz. Loss of hearing is common, and is a clinically heterogeneous disorder that can be caused by environmental factors such as exposure to loud noise, infections and ototoxic drugs. In addition, variants of hundreds of genes have been reported to disrupt processes required for hearing. Noncoding regulatory variants and variants of additional genes necessary for hearing remain to be discovered as many individuals with inherited deafness are without a genetic diagnosis, despite the advent of whole exome sequencing. Here, we discuss in detail some of these deafness-causing variants of genes encoding a ligand or its receptor. Spotlighted in this review are three growth factor-receptor-pairs EDN3/EDNRB, HGF/MET and JAG/NOTCH, which individually are necessary for normal hearing. We also offer our perspective on unanswered questions, future challenges and potential opportunities for treatments emerging from molecular genetic and mechanistic studies of deafness due to these causes.
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http://dx.doi.org/10.1111/cge.13641DOI Listing
January 2020

TRIOBP-5 sculpts stereocilia rootlets and stiffens supporting cells enabling hearing.

JCI Insight 2019 06 20;4(12). Epub 2019 Jun 20.

Department of Otolaryngology-Head and Neck Surgery, Kyoto University Graduate School of Medicine, Kyoto, Japan.

TRIOBP remodels the cytoskeleton by forming unusually dense F-actin bundles and is implicated in human cancer, schizophrenia, and deafness. Mutations ablating human and mouse TRIOBP-4 and TRIOBP-5 isoforms are associated with profound deafness, as inner ear mechanosensory hair cells degenerate after stereocilia rootlets fail to develop. However, the mechanisms regulating formation of stereocilia rootlets by each TRIOBP isoform remain unknown. Using 3 new Triobp mouse models, we report that TRIOBP-5 is essential for thickening bundles of F-actin in rootlets, establishing their mature dimensions and for stiffening supporting cells of the auditory sensory epithelium. The coiled-coil domains of this isoform are required for reinforcement and maintenance of stereocilia rootlets. A loss of TRIOBP-5 in mouse results in dysmorphic rootlets that are abnormally thin in the cuticular plate but have increased widths and lengths within stereocilia cores, and causes progressive deafness recapitulating the human phenotype. Our study extends the current understanding of TRIOBP isoform-specific functions necessary for life-long hearing, with implications for insight into other TRIOBPopathies.
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http://dx.doi.org/10.1172/jci.insight.128561DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6629139PMC
June 2019

The phenotypic landscape of a Tbc1d24 mutant mouse includes convulsive seizures resembling human early infantile epileptic encephalopathy.

Hum Mol Genet 2019 05;28(9):1530-1547

Laboratory of Molecular Genetics, National Institute on Deafness and Other Communication Disorders, Porter Neuroscience Research Center, National Institutes of Health, Bethesda, MD, USA.

Epilepsy, deafness, onychodystrophy, osteodystrophy and intellectual disability are associated with a spectrum of mutations of human TBC1D24. The mechanisms underlying TBC1D24-associated disorders and the functions of TBC1D24 are not well understood. Using CRISPR-Cas9 genome editing, we engineered a mouse with a premature translation stop codon equivalent to human S324Tfs*3, a recessive mutation of TBC1D24 associated with early infantile epileptic encephalopathy (EIEE). Homozygous S324Tfs*3 mice have normal auditory and vestibular functions but show an abrupt onset of spontaneous seizures at postnatal day 15 recapitulating human EIEE. The S324Tfs*3 variant is located in an alternatively spliced micro-exon encoding six perfectly conserved amino acids incorporated postnatally into TBC1D24 protein due to a micro-exon utilization switch. During embryonic and early postnatal development, S324Tfs*3 homozygotes produce predominantly the shorter wild-type TBC1D24 protein isoform that omits the micro-exon. S324Tfs*3 homozygotes show an abrupt onset of seizures at P15 that correlates with a developmental switch to utilization of the micro-exon. A mouse deficient for alternative splice factor SRRM3 impairs incorporation of the Tbc1d24 micro-exon. Wild-type Tbc1d24 mRNA is abundantly expressed in the hippocampus using RNAscope in situ hybridization. Immunogold electron microscopy using a TBC1D24-specific antibody revealed that TBC1D24 is associated with clathrin-coated vesicles and synapses of hippocampal neurons, suggesting a crucial role of TBC1D24 in vesicle trafficking important for neuronal signal transmission. This is the first characterization of a mouse model of human TBC1D24-associated EIEE that can now be used to screen for antiepileptogenic drugs ameliorating TBCID24 seizure disorders.
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http://dx.doi.org/10.1093/hmg/ddy445DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6466106PMC
May 2019

Mutational and phenotypic spectra of KCNE1 deficiency in Jervell and Lange-Nielsen Syndrome and Romano-Ward Syndrome.

Hum Mutat 2019 02 12;40(2):162-176. Epub 2018 Dec 12.

Laboratory of Molecular Genetics, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland, USA.

KCNE1 encodes a regulatory subunit of the KCNQ1 potassium channel-complex. Both KCNE1 and KCNQ1 are necessary for normal hearing and cardiac ventricular repolarization. Recessive variants in these genes are associated with Jervell and Lange-Nielson syndrome (JLNS1 and JLNS2), a cardio-auditory syndrome characterized by congenital profound sensorineural deafness and a prolonged QT interval that can cause ventricular arrhythmias and sudden cardiac death. Some normal-hearing carriers of heterozygous missense variants of KCNE1 and KCNQ1 have prolonged QT intervals, a dominantly inherited phenotype designated Romano-Ward syndrome (RWS), which is also associated with arrhythmias and elevated risk of sudden death. Coassembly of certain mutant KCNE1 monomers with wild-type KCNQ1 subunits results in RWS by a dominant negative mechanism. This paper reviews variants of KCNE1 and their associated phenotypes, including biallelic truncating null variants of KCNE1 that have not been previously reported. We describe three homozygous nonsense mutations of KCNE1 segregating in families ascertained ostensibly for nonsyndromic deafness: c.50G>A (p.Trp17*), c.51G>A (p.Trp17*), and c.138C>A (p.Tyr46*). Some individuals carrying missense variants of KCNE1 have RWS. However, heterozygotes for loss-of-function variants of KCNE1 may have normal QT intervals while biallelic null alleles are associated with JLNS2, indicating a complex genotype-phenotype spectrum for KCNE1 variants.
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http://dx.doi.org/10.1002/humu.23689DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6328321PMC
February 2019

Global genetic insight contributed by consanguineous Pakistani families segregating hearing loss.

Hum Mutat 2019 01 18;40(1):53-72. Epub 2018 Nov 18.

Department of Otorhinolaryngology-Head and Neck Surgery, School of Medicine, University of Maryland, Baltimore, Maryland.

Consanguineous Pakistani pedigrees segregating deafness have contributed decisively to the discovery of 31 of the 68 genes associated with nonsyndromic autosomal recessive hearing loss (HL) worldwide. In this study, we utilized genome-wide genotyping, Sanger and exome sequencing to identify 163 DNA variants in 41 previously reported HL genes segregating in 321 Pakistani families. Of these, 70 (42.9%) variants identified in 29 genes are novel. As expected from genetic studies of disorders segregating in consanguineous families, the majority of affected individuals (94.4%) are homozygous for HL-associated variants, with the other variants being compound heterozygotes. The five most common HL genes in the Pakistani population are SLC26A4, MYO7A, GJB2, CIB2 and HGF, respectively. Our study provides a profile of the genetic etiology of HL in Pakistani families, which will allow for the development of more efficient genetic diagnostic tools, aid in accurate genetic counseling, and guide application of future gene-based therapies. These findings are also valuable in interpreting pathogenicity of variants that are potentially associated with HL in individuals of all ancestries. The Pakistani population, and its infrastructure for studying human genetics, will continue to be valuable to gene discovery for HL and other inherited disorders.
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http://dx.doi.org/10.1002/humu.23666DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6296877PMC
January 2019

Defects in the Alternative Splicing-Dependent Regulation of REST Cause Deafness.

Cell 2018 07 28;174(3):536-548.e21. Epub 2018 Jun 28.

Department of Anatomy and Cell Biology, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA; Inflammation Program, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA; Department of Otolaryngology-Head and Neck Surgery, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA; Department of Internal Medicine, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA. Electronic address:

The DNA-binding protein REST forms complexes with histone deacetylases (HDACs) to repress neuronal genes in non-neuronal cells. In differentiating neurons, REST is downregulated predominantly by transcriptional silencing. Here we report that post-transcriptional inactivation of REST by alternative splicing is required for hearing in humans and mice. We show that, in the mechanosensory hair cells of the mouse ear, regulated alternative splicing of a frameshift-causing exon into the Rest mRNA is essential for the derepression of many neuronal genes. Heterozygous deletion of this alternative exon of mouse Rest causes hair cell degeneration and deafness, and the HDAC inhibitor SAHA (Vorinostat) rescues the hearing of these mice. In humans, inhibition of the frameshifting splicing event by a novel REST variant is associated with dominantly inherited deafness. Our data reveal the necessity for alternative splicing-dependent regulation of REST in hair cells, and they identify a potential treatment for a group of hereditary deafness cases.
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http://dx.doi.org/10.1016/j.cell.2018.06.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6370011PMC
July 2018

Mutations in Diphosphoinositol-Pentakisphosphate Kinase PPIP5K2 are associated with hearing loss in human and mouse.

PLoS Genet 2018 03 28;14(3):e1007297. Epub 2018 Mar 28.

Laboratory of Molecular Genetics, Department of Otorhinolaryngology-Head & Neck Surgery, School of Medicine University of Maryland, Baltimore, MD, United States of America.

Autosomal recessive nonsyndromic hearing loss is a genetically heterogeneous disorder. Here, we report a severe-to-profound sensorineural hearing loss locus, DFNB100 on chromosome 5q13.2-q23.2. Exome enrichment followed by massive parallel sequencing revealed a c.2510G>A transition variant in PPIP5K2 that segregated with DFNB100-associated hearing loss in two large apparently unrelated Pakistani families. PPIP5Ks enzymes interconvert 5-IP7 and IP8, two key members of the inositol pyrophosphate (PP-IP) cell-signaling family. Their actions at the interface of cell signaling and bioenergetic homeostasis can impact many biological processes. The c.2510G>A transition variant is predicted to substitute a highly invariant arginine residue with histidine (p.Arg837His) in the phosphatase domain of PPIP5K2. Biochemical studies revealed that the p.Arg837His variant reduces the phosphatase activity of PPIP5K2 and elevates its kinase activity. We found that in mouse inner ear, PPIP5K2 is expressed in the cochlear and vestibular sensory hair cells, supporting cells and spiral ganglion neurons. Mice homozygous for a targeted deletion of the Ppip5k2 phosphatase domain exhibit degeneration of cochlear outer hair cells and elevated hearing thresholds. Our demonstration that PPIP5K2 has a role in hearing in humans indicates that PP-IP signaling is important to hair cell maintenance and function within inner ear.
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http://dx.doi.org/10.1371/journal.pgen.1007297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5891075PMC
March 2018

Inframe deletion of human is associated with deafness, vestibulopathy and vision impairment.

J Med Genet 2018 07 23;55(7):479-488. Epub 2018 Mar 23.

Department of Otorhinolaryngology Head and Neck Surgery, School of Medicine, University of Maryland, Baltimore, Maryland, USA.

Background: Usher syndrome (USH) is a neurosensory disorder characterised by deafness, variable vestibular areflexia and vision loss. The aim of the study was to identify the genetic defect in a Pakistani family (PKDF1051) segregating USH.

Methods: Genome-wide linkage analysis was performed by using an Illumina linkage array followed by Sanger and exome sequencing. Heterologous cells and mouse organ of Corti explant-based transfection assays were used for functional evaluations. Detailed clinical evaluations were performed to characterise the USH phenotype.

Results: Through homozygosity mapping, we genetically linked the USH phenotype segregating in family PKDF1051 to markers on chromosome 1p36.32-p36.22. The locus was designated . Using a combination of Sanger sequencing and exome sequencing, we identified a novel homozygous 18 base pair inframe deletion in Variants of , encoding the actin-bundling protein espin, have been previously associated with deafness and vestibular areflexia in humans with no apparent visual deficits. Our functional studies in heterologous cells and in mouse organ of Corti explant cultures revealed that the six deleted residues in affected individuals of family PKDF1051 are essential for the actin bundling function of espin demonstrated by ultracentrifugation actin binding and bundling assays. Funduscopic examination of the affected individuals of family PKDF1051 revealed irregular retinal contour, temporal flecks and disc pallor in both eyes. ERG revealed diminished rod photoreceptor function among affected individuals.

Conclusion: Our study uncovers an additional USH gene, assigns the USH1 phenotype to a variant of and provides a 12th molecular component to the USH proteome.
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http://dx.doi.org/10.1136/jmedgenet-2017-105221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6232856PMC
July 2018

Bipolar filaments of human nonmuscle myosin 2-A and 2-B have distinct motile and mechanical properties.

Elife 2018 02 8;7. Epub 2018 Feb 8.

Cell Biology and Physiology Center, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, United States.

Nonmusclemyosin 2 (NM-2) powers cell motility and tissue morphogenesis by assembling into bipolar filaments that interact with actin. Although the enzymatic properties of purified NM-2 motor fragments have been determined, the emergent properties of filament ensembles are unknown. Using single myosin filament in vitro motility assays, we report fundamental differences in filaments formed of different NM-2 motors. Filaments consisting of NM2-B moved processively along actin, while under identical conditions, NM2-A filaments did not. By more closely mimicking the physiological milieu, either by increasing solution viscosity or by co-polymerization with NM2-B, NM2-A containing filaments moved processively. Our data demonstrate that both the kinetic and mechanical properties of these two myosins, in addition to the stochiometry of NM-2 subunits, can tune filament mechanical output. We propose altering NM-2 filament composition is a general cellular strategy for tailoring force production of filaments to specific functions, such as maintaining tension or remodeling actin.
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http://dx.doi.org/10.7554/eLife.32871DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5829915PMC
February 2018

Modifier variant of METTL13 suppresses human GAB1-associated profound deafness.

J Clin Invest 2018 04 12;128(4):1509-1522. Epub 2018 Mar 12.

Laboratory of Molecular Genetics, Department of Otorhinolaryngology - Head and Neck Surgery, University of Maryland, Baltimore, Maryland, USA.

A modifier variant can abrogate the risk of a monogenic disorder. DFNM1 is a locus on chromosome 1 encoding a dominant suppressor of human DFNB26 recessive, profound deafness. Here, we report that DFNB26 is associated with a substitution (p.Gly116Glu) in the pleckstrin homology domain of GRB2-associated binding protein 1 (GAB1), an essential scaffold in the MET proto-oncogene, receptor tyrosine kinase/HGF (MET/HGF) pathway. A dominant substitution (p.Arg544Gln) of METTL13, encoding a predicted methyltransferase, is the DFNM1 suppressor of GAB1-associated deafness. In zebrafish, human METTL13 mRNA harboring the modifier allele rescued the GAB1-associated morphant phenotype. In mice, GAB1 and METTL13 colocalized in auditory sensory neurons, and METTL13 coimmunoprecipitated with GAB1 and SPRY2, indicating at least a tripartite complex. Expression of MET-signaling genes in human lymphoblastoid cells of individuals homozygous for p.Gly116Glu GAB1 revealed dysregulation of HGF, MET, SHP2, and SPRY2, all of which have reported variants associated with deafness. However, SPRY2 was not dysregulated in normal-hearing humans homozygous for both the GAB1 DFNB26 deafness variant and the dominant METTL13 deafness suppressor, indicating a plausible mechanism of suppression. Identification of METTL13-based modification of MET signaling offers a potential therapeutic strategy for a wide range of associated hearing disorders. Furthermore, MET signaling is essential for diverse functions in many tissues including the inner ear. Therefore, identification of the modifier of MET signaling is likely to have broad clinical implications.
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http://dx.doi.org/10.1172/JCI97350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5873844PMC
April 2018

CDC14A phosphatase is essential for hearing and male fertility in mouse and human.

Hum Mol Genet 2018 03;27(5):780-798

School of Biological Sciences, University of the Punjab, Lahore 54590, Pakistan.

The Cell Division-Cycle-14 gene encodes a dual-specificity phosphatase necessary in yeast for exit from mitosis. Numerous disparate roles of vertebrate Cell Division-Cycle-14 (CDC14A) have been proposed largely based on studies of cultured cancer cells in vitro. The in vivo functions of vertebrate CDC14A are largely unknown. We generated and analyzed mutations of zebrafish and mouse CDC14A, developed a computational structural model of human CDC14A protein and report four novel truncating and three missense alleles of CDC14A in human families segregating progressive, moderate-to-profound deafness. In five of these families segregating pathogenic variants of CDC14A, deaf males are infertile, while deaf females are fertile. Several recessive mutations of mouse Cdc14a, including a CRISPR/Cas9-edited phosphatase-dead p.C278S substitution, result in substantial perinatal lethality, but survivors recapitulate the human phenotype of deafness and male infertility. CDC14A protein localizes to inner ear hair cell kinocilia, basal bodies and sound-transducing stereocilia. Auditory hair cells of postnatal Cdc14a mutants develop normally, but subsequently degenerate causing deafness. Kinocilia of germ-line mutants of mouse and zebrafish have normal lengths, which does not recapitulate the published cdc14aa knockdown morphant phenotype of short kinocilia. In mutant male mice, degeneration of seminiferous tubules and spermiation defects result in low sperm count, and abnormal sperm motility and morphology. These findings for the first time define a new monogenic syndrome of deafness and male infertility revealing an absolute requirement in vivo of vertebrate CDC14A phosphatase activity for hearing and male fertility.
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http://dx.doi.org/10.1093/hmg/ddx440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6059191PMC
March 2018

A common -linked haplotype underlying non-syndromic hearing loss with enlargement of the vestibular aqueduct.

J Med Genet 2017 10 5;54(10):665-673. Epub 2017 Aug 5.

Otolaryngology Branch, National Institute on Deafness and Other Communication Disorders (NIDCD), Bethesda, Maryland, USA.

Background: Enlargement of the vestibular aqueduct (EVA) is the most common radiological abnormality in children with sensorineural hearing loss. Mutations in coding regions and splice sites of the gene are often detected in Caucasians with EVA. Approximately one-fourth of patients with EVA have two mutant alleles (M2), one-fourth have one mutant allele (M1) and one-half have no mutant alleles (M0). The M2 genotype is correlated with a more severe phenotype.

Methods: We performed genotype-haplotype analysis and massively parallel sequencing of the region in patients with M1 EVA and their families.

Results: We identified a shared novel haplotype, termed CEVA (Caucasian EVA), composed of 12 uncommon variants upstream of . The presence of the CEVA haplotype on seven of ten 'mutation-negative' chromosomes in a National Institutes of Health M1 EVA discovery cohort and six of six mutation-negative chromosomes in a Danish M1 EVA replication cohort is higher than the observed prevalence of 28 of 1006 Caucasian control chromosomes (p<0.0001 for each EVA cohort). The corresponding heterozygous carrier rate is 28/503 (5.6%). The prevalence of CEVA (11 of 126) is also increased among M0 EVA chromosomes (p=0.0042).

Conclusions: The CEVA haplotype causally contributes to most cases of Caucasian M1 EVA and, possibly, some cases of M0 EVA. The CEVA haplotype of defines the most common allele associated with hereditary hearing loss in Caucasians. The diagnostic yield and prognostic utility of sequence analysis of exons and splice sites will be markedly increased by addition of testing for the CEVA haplotype.
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http://dx.doi.org/10.1136/jmedgenet-2017-104721DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5880640PMC
October 2017

Defective Gpsm2/Gα signalling disrupts stereocilia development and growth cone actin dynamics in Chudley-McCullough syndrome.

Nat Commun 2017 04 7;8:14907. Epub 2017 Apr 7.

INSERM, Neurocentre Magendie, U1215, 146 rue Leo-Saignat, F-33077 Bordeaux, France.

Mutations in GPSM2 cause Chudley-McCullough syndrome (CMCS), an autosomal recessive neurological disorder characterized by early-onset sensorineural deafness and brain anomalies. Here, we show that mutation of the mouse orthologue of GPSM2 affects actin-rich stereocilia elongation in auditory and vestibular hair cells, causing deafness and balance defects. The G-protein subunit Gα, a well-documented partner of Gpsm2, participates in the elongation process, and its absence also causes hearing deficits. We show that Gpsm2 defines an ∼200 nm nanodomain at the tips of stereocilia and this localization requires the presence of Gα, myosin 15 and whirlin. Using single-molecule tracking, we report that loss of Gpsm2 leads to decreased outgrowth and a disruption of actin dynamics in neuronal growth cones. Our results elucidate the aetiology of CMCS and highlight a new molecular role for Gpsm2/Gα in the regulation of actin dynamics in epithelial and neuronal tissues.
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http://dx.doi.org/10.1038/ncomms14907DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5385604PMC
April 2017

Gene Therapy Restores Balance and Auditory Functions in a Mouse Model of Usher Syndrome.

Mol Ther 2017 03 21;25(3):780-791. Epub 2017 Feb 21.

Neurotology Program, National Institute on Deafness and Other Communication Disorders (NIDCD), NIH, Bethesda, MD 20892, USA; Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins School of Medicine, Baltimore, MD 21287, USA. Electronic address:

Dizziness and hearing loss are among the most common disabilities. Many forms of hereditary balance and hearing disorders are caused by abnormal development of stereocilia, mechanosensory organelles on the apical surface of hair cells in the inner ear. The deaf whirler mouse, a model of human Usher syndrome (manifested by hearing loss, dizziness, and blindness), has a recessive mutation in the whirlin gene, which renders hair cell stereocilia short and dysfunctional. In this study, wild-type whirlin cDNA was delivered to the inner ears of neonatal whirler mice using adeno-associated virus serotype 2/8 (AAV8-whirlin) by injection into the posterior semicircular canal. Unilateral whirlin gene therapy injection was able to restore balance function as well as improve hearing in whirler mice for at least 4 months. Our data indicate that gene therapy is likely to become a treatment option for hereditary disorders of balance and hearing.
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http://dx.doi.org/10.1016/j.ymthe.2017.01.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5363211PMC
March 2017

Harnessing molecular motors for nanoscale pulldown in live cells.

Mol Biol Cell 2017 Feb 8;28(3):463-475. Epub 2016 Dec 8.

Laboratory of Molecular Genetics, National Institutes of Health, Bethesda, MD 20814.

Protein-protein interactions (PPIs) regulate assembly of macromolecular complexes, yet remain challenging to study within the native cytoplasm where they normally exert their biological effect. Here we miniaturize the concept of affinity pulldown, a gold-standard in vitro PPI interrogation technique, to perform nanoscale pulldowns (NanoSPDs) within living cells. NanoSPD hijacks the normal process of intracellular trafficking by myosin motors to forcibly pull fluorescently tagged protein complexes along filopodial actin filaments. Using dual-color total internal reflection fluorescence microscopy, we demonstrate complex formation by showing that bait and prey molecules are simultaneously trafficked and actively concentrated into a nanoscopic volume at the tips of filopodia. The resulting molecular traffic jams at filopodial tips amplify fluorescence intensities and allow PPIs to be interrogated using standard epifluorescence microscopy. A rigorous quantification framework and software tool are provided to statistically evaluate NanoSPD data sets. We demonstrate the capabilities of NanoSPD for a range of nuclear and cytoplasmic PPIs implicated in human deafness, in addition to dissecting these interactions using domain mapping and mutagenesis experiments. The NanoSPD methodology is extensible for use with other fluorescent molecules, in addition to proteins, and the platform can be easily scaled for high-throughput applications.
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http://dx.doi.org/10.1091/mbc.E16-08-0583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5341729PMC
February 2017

Genetic causes of moderate to severe hearing loss point to modifiers.

Clin Genet 2017 Apr 6;91(4):589-598. Epub 2016 Oct 6.

Laboratory of Molecular Genetics, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, USA 20892.

The genetic underpinnings of recessively inherited moderate to severe sensorineural hearing loss are not well understood, despite its higher prevalence in comparison to profound deafness. We recruited 92 consanguineous families segregating stable or progressive, recessively inherited moderate or severe hearing loss. We utilized homozygosity mapping, Sanger sequencing, targeted capture of known deafness genes with massively parallel sequencing and whole exome sequencing to identify the molecular basis of hearing loss in these families. Variants of the known deafness genes were found in 69% of the participating families with the SLC26A4, GJB2, MYO15A, TMC1, TMPRSS3, OTOF, MYO7A and CLDN14 genes together accounting for hearing loss in 54% of the families. We identified 20 reported and 21 novel variants in 21 known deafness genes; 16 of the 20 reported variants, previously associated with stable, profound deafness were associated with moderate to severe or progressive hearing loss in our families. These data point to a prominent role for genetic background, environmental factors or both as modifiers of human hearing loss severity.
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http://dx.doi.org/10.1111/cge.12856DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5365349PMC
April 2017

Mutational Spectrum of MYO15A and the Molecular Mechanisms of DFNB3 Human Deafness.

Hum Mutat 2016 10 21;37(10):991-1003. Epub 2016 Aug 21.

Laboratory of Molecular Genetics, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland, 20892.

Deafness in humans is a common neurosensory disorder and is genetically heterogeneous. Across diverse ethnic groups, mutations of MYO15A at the DFNB3 locus appear to be the third or fourth most common cause of autosomal-recessive, nonsyndromic deafness. In 49 of the 67 exons of MYO15A, there are currently 192 recessive mutations identified, including 14 novel mutations reported here. These mutations are distributed uniformly across MYO15A with one enigmatic exception; the alternatively spliced giant exon 2, encoding 1,233 residues, has 17 truncating mutations but no convincing deafness-causing missense mutations. MYO15A encodes three distinct isoform classes, one of which is 395 kDa (3,530 residues), the largest member of the myosin superfamily of molecular motors. Studies of Myo15 mouse models that recapitulate DFNB3 revealed two different pathogenic mechanisms of hearing loss. In the inner ear, myosin 15 is necessary both for the development and the long-term maintenance of stereocilia, mechanosensory sound-transducing organelles that extend from the apical surface of hair cells. The goal of this Mutation Update is to provide a comprehensive review of mutations and functions of MYO15A.
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http://dx.doi.org/10.1002/humu.23042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5021573PMC
October 2016

Heritability of non-speech auditory processing skills.

Eur J Hum Genet 2016 08 17;24(8):1137-44. Epub 2016 Feb 17.

Medical Research Council, Institute of Hearing Research, Nottingham, UK.

Recent insight into the genetic bases for autism spectrum disorder, dyslexia, stuttering, and language disorders suggest that neurogenetic approaches may also reveal at least one etiology of auditory processing disorder (APD). A person with an APD typically has difficulty understanding speech in background noise despite having normal pure-tone hearing sensitivity. The estimated prevalence of APD may be as high as 10% in the pediatric population, yet the causes are unknown and have not been explored by molecular or genetic approaches. The aim of our study was to determine the heritability of frequency and temporal resolution for auditory signals and speech recognition in noise in 96 identical or fraternal twin pairs, aged 6-11 years. Measures of auditory processing (AP) of non-speech sounds included backward masking (temporal resolution), notched noise masking (spectral resolution), pure-tone frequency discrimination (temporal fine structure sensitivity), and nonsense syllable recognition in noise. We provide evidence of significant heritability, ranging from 0.32 to 0.74, for individual measures of these non-speech-based AP skills that are crucial for understanding spoken language. Identification of specific heritable AP traits such as these serve as a basis to pursue the genetic underpinnings of APD by identifying genetic variants associated with common AP disorders in children and adults.
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http://dx.doi.org/10.1038/ejhg.2015.277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4872837PMC
August 2016