Improving Early Intervention via a Single Comprehensive Genetic Screening for Prelingual Children with Hearing Loss
X Lin1,2, D Qian2, C Haase2, B Wyatt2, M Rojas-Pena2, K Ewing2, P Chen1,2
1Departments of Otolaryngology and Cell Biology Emory University 2Otogenetics Corporation Atlanta, GA Newborn Hearing Screening and Genetics
Congential Hearing Loss is one of the most common conditions identified via Newborn Screening Above 2 to 3 out of every 1000 children in the US are born with a detectable level (mild to moderate) of hearing loss in one or both ears (CDC and NIDCD). Many times more are carriers of recessive deafness genes. A genetic etiology is suspected in two thirds of these patients. Genetic Contribution to Prelingual Deafness
GJB2 Newborn Hearing Screening
Joint Committee on Infant Hearing Loss Position Statement, 2007 No later than age 1 month, all infants screened No later than age 3 months, all infants not passing screen have a comprehensive evaluation No later than 6 months of age, all infants with confirmed hearing loss receive appropriate intervention Options for Babies with a Positive Screening Result
Follow up diagnostic test options for patients who have screened positive include [1,2]: Serological tests for the presence of rubella and CMV viruses High resolution computer tomography (CT) or magnetic resonance imaging (MRI) to check for gross abnormalities in the formation of the sensory organs and the cochleae Special tests, such as electrocardiogram (EKG) tests can be performed to check for Long QT interval, and kidney and urine tests can be carried out to check for branchio-oto-renal syndrome. Genetic tests for a single gene, such as connexin26 (Cx26), Cx30 and mitochondria DNA are offered in many medical centers. The percentage of deafness patients who are offered these limited tests are estimated at about 24% [3] to 50% [4].
1. Greinwald, J.H., Jr., and Hartnick, C.J. (2002). The evaluation of children with sensorineural hearing loss. Archives of otolaryngology--head & neck surgery 128, 84-87. 2. Preciado, D.A., Lawson, L., Madden, C., Myer, D., Ngo, C., Bradshaw, J.K., Choo, D.I., and Greinwald, J.H., Jr. (2005). Improved diagnostic effectiveness with a sequential diagnostic paradigm in idiopathic pediatric sensorineural hearing loss. Otol Neurotol 26, 610-615. 3. Putcha, G.V., Bejjani, B.A., Bleoo, S., Booker, J.K., Carey, J.C., Carson, N., Das, S., Dempsey, M.A., Gastier-Foster, J.M., Greinwald, J.H., Jr., et al. (2007). A multicenter study of the frequency and distribution of GJB2 and GJB6 mutations in a large North American cohort. Genet Med 9, 413-426. 4. Denoyelle, F., Weil, D., Maw, M.A., Wilcox, S.A., Lench, N.J., Allen-Powell, D.R., Osborn, A.H., Dahl, H.H., Middleton, A., Houseman, M.J., et al. (1997). Prelingual deafness: high prevalence of a 30delG mutation in the connexin 26 gene. Human molecular genetics 6, 2173-2177. Why Genetic Testing? Infant hearing screening has a high false-positive rate: after repeated tests, referral rate is more than 0.8%, whereas the true rate of congenital hearing impairment is estimated to be about 0.1-0.2%. Precise and timed identification and early pre-lingual intervention (progressive hearing loss, auditory neuronopathy-TIMM8a, nutri/medic) Prevention of deafness caused by antibiotic usage (MT-RNR1, MT- CO1) Pre-onset intervention of life-threatening or blindness for patients carrying gene variants in syndromic deafness, such as Jervell and Lange-Nielsen (hearing impairment and LQT), USH (deaf and progressive RP), Biotinidase deficiency (Biotin supplement), Refsum (dietary restriction of Phytanic acid intake), and male infertility (Strc). NF2 risk prediction, germ line haploid insufficiency of Merlin. Developing new therapies, mechanisms, & gene- or cell-based Both the Joint Committee on Infant Hearing (JCIH, 2007) and the American College of Medical Genetics (ACMG, 2002) recommend genetic testing. Monogenic & ~130 genes identified & 40 more loci-unknown genes Next Generation Sequencing (NGS) is effective for deafness genetic screen
Traditional one-gene=one-test cannot meet the efficacy or overcome the cost burden of using deafness genetic tests as a follow up for the newborn hearing screening tests. Thousands of mutations in more than 100 genes (~half a million total bps to cover) can cause hearing loss: Next Generation Sequencing is unlimited in the number of variants and genes tested
Accurate- Direct sequencing, not indirect chip-based methods Efficient- Can test all relevant genes in one test Flexible- Tailored to the need of the tests and the test does not have to be re-designed as new variants are discovered in these deafness genes. Otogenetics Deafness Panel Test was Developed with the Support of NIDCD
Allows the sequencing of ~130 deafness genes (including the entire mitochondria genome) in a single test. Minimally-invasive sample collection High throughput and secure report platform makes it possible for physicians and genetic counselors to translate the test results to the patients or the families of patients efficiently Otogenetics Deafness Panel Test Usage
Tests have been ordered for more than 2000 patients world-wide so far, and causal genetic variants were identified in >50% of the sporadic deafness patients. For confirmed congenital hearing loss cases, causal gene mutations were diagnosed in the majority of the cases Exome Sequencing has been used to identify novel mutations causing deafness CNV changes can be detected simultaneously Diagnosis and New Discovery Europe Usher syndrome in Denmark: mutation spectrum and some clinical observations. Dad S, Rendtorff ND, Tranebjærg L, Grønskov K, Karstensen HG, Brox V, Nilssen Ø, Roux AF, Rosenberg T, Jensen H, Møller LB. Mol Genet Genomic Med. 2016 Jun 28;4(5):527-539. Partial USH2A deletions contribute to Usher syndrome in Denmark. Dad S, Rendtorff ND, Kann E, Albrechtsen A, Mehrjouy MM, Bak M, Tommerup N, Tranebjærg L, Rosenberg T, Jensen H, Møller LB. Eur J Hum Genet. 2015 Dec;23(12):1646-51. doi: 10.1038/ejhg.2015.54. Erratum in: Eur J Hum Genet. 2015 Disruption of the ATE1 and SLC12A1 Genes by Balanced Translocation in a Boy with Non-Syndromic Hearing Loss. Vona B, Neuner C, El Hajj N, Schneider E, Farcas R, Beyer V, Zechner U, Keilmann A, Poot M, Bartsch O, Nanda I, Haaf T (2014) Molecular Syndromology 5, 3-10 Targeted next-generation sequencing of deafness genes inhearing-impaired individuals uncovers informative mutations. Vona B, Muller T, Nanda I, Neuner C, Hofrichter M, Schroder J, Bartsch O, Labig A, Keilmann A, Schraven S, Kraus F, Shehata-Dieler W, Haaf T. (2014) Genetics in Medicine 16, 945–953 Confirmation of GRHL2 as the gene for the DFNA28 locus. Vona B, Nanda I, Neuner C, Müller T, Haaf T (2013) American Journal of Medical Genetics Part A. 161A, 2060-5. Infantile mitochondrial hepatopathy is a cardinal feature of MEGDEL syndrome (3-methylglutaconic aciduria type IV with sensorineural deafness, encephalopathy, and Leigh-like syndrome) caused by novel mutations in SERAC1. Sarig O., Goldsher D., Nousbeck J., Fuchs-Telem D., Cohen-Katsenelson K., Iancu T.C., Manov I., Saada A., Sprecher E., Mandel H. (2013) American Journal of Medicine Genetics Part A 161(9), 2204–2215. Genetic heterogeneity and consanguinity lead to a “double hit”: Homozygous mutations of MYO7A and PDE6B in a patient with retinitis pigmentosa. Goldenberg-Cohen N., Banin E., Zalzstein Y., Cohen B., Rotenstreich Y., Rizel L., Basel-Vanagaite L., and Ben-Yosef T. (2013) Molecular Vision Biology and Genetics in Vision Research 19, 1565–1571. Exome Sequencing Identifies a Founder Frameshift Mutation in an Alternative Exon of USH1C as the Cause of Autosomal Recessive Retinitis Pigmentosa with Late-Onset Hearing Loss. Khateb S., Zelinger L., Ben-Yosef T., Merin S., Crystal-Shalit O., Gross M., Banin E., Sharon D. (2012) PLoS One. 7(12), e51566. Asia Integrating Cadaver Exome Sequencing Into a First-Year Medical Student Curriculum. Gerhard, GS, Paynton B, Popoff SN. (2016) . JAMA 315(6):555-556. doi:10.1001/jama.2015.19465 Discovery of MYH14 as an important and unique deafness gene causing prelingually severe autosomal dominant non-syndromic hearing loss. Kim BJ, Kim AR, Han JH, Lee C, Oh DY, Choi BY. J Gene Med. 2017 Feb 21. doi: 10.1002/jgm.2950. [Epub ahead of print] Phenotypic Heterogeneity in a DFNA20/26 family segregating a novel ACTG1 mutation.Yuan Y, Gao X, Huang B, Lu J, Wang G, Lin X, Qu Y, Dai P. BMC Genet. 2016 Feb 1;17:33. doi: 10.1186/s12863-016-0333-1. Functional characterization of a novel loss-of-function mutation of PRPS1 related to early-onset progressive nonsyndromic hearing loss in Koreans (DFNX1): Potential implications on future therapeutic intervention. Kim SY, Kim AR, Kim NK, Lee C, Han JH, Kim MY, Jeon EH, Park WY, Mittal R, Yan D, Liu XZ, Choi BY. J Gene Med. 2016 Nov;18(11-12):353-358. doi: 10.1002/jgm.2935. Targeted Exome Sequencing of Deafness Genes After Failure of Auditory Phenotype-Driven Candidate Gene Screening. Kim BJ, Kim AR, Park G, Park WY, Chang SO, Oh SH, Choi BY. Otol Neurotol. 2015 Jul;36(6):1096-102. doi: 10.1097/MAO.0000000000000747. Targeted gene capture and massively parallel sequencing identify TMC1 as the causative gene in a six-generation Chinese family with autosomal dominant hearing loss. Gao X, Huang SS, Yuan YY, Wang GJ, Xu JC, Ji YB, Han MY, Yu F, Kang DY, Lin X, Dai P. Am J Med Genet A. 2015 Oct;167A(10):2357-65. doi: 10.1002/ajmg.a.37206. Genetic testing for sporadic hearing loss using targeted massively parallel sequencing identifies 10 novel mutations. Gu X, Guo L, Ji H, Sun S, Chai R, Wang L, Li H. Clin Genet. 2015 Jun;87(6):588-93. doi: 10.1111/cge.12431. Combined examination of sequence and copy number variations in human deafness genes improves diagnosis for cases of genetic deafness. Ji H, Lu J, Wang J, Li H, Lin X. BMC Ear Nose Throat Disord. 2014 Sep 10;14:9. doi: 10.1186/1472-6815-14-9. Novel compound heterozygous mutations in MYO7A Associated with Usher syndrome 1 in a Chinese family. Gao X, Wang GJ, Yuan YY, Xin F, Han MY, Lu JQ, Zhao H, Yu F, Xu JC, Zhang MG, Dong J, Lin X, Dai P. PLoS One. 2014 Jul 31;9(7):e103415. doi: 10.1371/journal.pone.0103415. Erratum in: PLoS One. De novo mutation in ATP6V1B2 impairs lysosome acidification and causes dominant deafness-onychodystrophy syndrome. Yuan Y, Zhang J, Chang Q, Zeng J, Xin F, Wang J, Zhu Q, Wu J, Lu J, Guo W, Yan X, Jiang H, Zhou B, Li Q, Gao X, Yuan H, Yang S, Han D, Mao Z, Chen P, Lin X, Dai P. Cell Res. 2014 Nov;24(11):1370-3. doi: 10.1038/cr.2014.77. Application of Massively Parallel Sequencing to Genetic Diagnosis in Multiplex Families with Idiopathic Sensorineural Hearing Impairment. Wu C.C., Lin Y.H., Lu Y.C., Chen P.J., Yang W.S., et al. (2013) PLOS ONE 8(2), e57369. USA The future role of genetic screening to detect newborns at risk of childhood-onset hearing loss. Linden Phillips L., Bitner-Glindzicz M., Lench N., Steel K.P., Langford C., Dawson S.J., Davis A., and Simpson S. (2013) International Journal of Audiology 52(2), 124-133. (Case study) Patient Demographics
26-40 dB 41-55 & 56-70 dB 71-90 dB 90 dB Minimally-invasive sample collection
Only small amount of DNA required for testing all ~130 deafness genes, compatible with following collection methods: Dried blood spots (Blood in purple collection tubes) Already commonly used for Newborn Screens Oral Swab Test Overview – NGS Genetic Testing
Sample Collected and ship to Otogenetics
DNA extraction
Clarity NGS LIMS Not passed Get replacement QC from Patient Pass Target Gene Sequencing Library Preparation/QC
QC
Sequencing
QC
Data QC
Clinical Report Individually Focused Deafness Panel Data Quality
100%Target Coverage
•Average Coverage=490 •99.3％ target coverage>30x •95.3％ target coverage>100x Fidelity of the NGS Genetic Screen Am J Med Genet A. 2015, 167, 2357-2365 Identify novel mutations in patients prior to onset of deafness
Autosomal dominant postlingual onset 6-generation family
V45
TMC1 c.1714 G>A DFNA36 (DFNB7/11) Copy Number Variations (CNV) or duplications & deletions can be assessed in the same test qPCR validated)
Exon 20 deletion 48 Kb deletion
DFNB22 DFNB16 (potential male infertility)
Combined examination of sequence and copy number variations in human deafness genes improves diagnosis for cases of genetic deafness. Ji H, Lu J, Wang J, Li H, Lin X. BMC Ear Nose Throat Disord. 2014 Sep 10;14:9. doi: 10.1186/1472-6815-14-9 Effective deafness gene screen
In a research collaboration between Emory University Hospitals and Shanghai Xinhua Hospital, 189 newborns who failed 1st-round hearing screening and 481 normal hearing controls were tested using Oto-DA. Positive identification of the underlying genetic cause for hearing loss in 48.1 % of newborns who failed newborn hearing screening. Pathogenic variants were found in 58 genes and included missense, small insertions and deletions, nonsense and splice-site alterations mutations. In the larger study of 583 patients, mutations in 102 deafness genes were identified. Comparison of Deafness Gene Panel Test
A related work for sporadic patients published by Smith group, Univ. of Iowa (Hum Genet (2016) 135:441–450) Identification of the underlying genetic cause for hearing loss in 39 % (440 patients). Pathogenic variants were found in 49 genes and included missense (49 %), large copy number changes (18 %), small insertions and deletions (18 %), nonsense (8 %), splice-site alterations (6 %), and promoter variants (<1 %). Develop Low Cost NGS Deafness Gene Panel Tests
GJB2 SLC26A4 (Pendrin) GJB3 GJB6 Mt-Genome ACTG1 POU4F3 Oto-Deafness Gene Panel Test Report Oto-Deafness Gene Panel Test Report Conclusions The Otogenetics Deafness Gene Panel allows the sequencing of ~130 deafness genes in a single test. The high throughput and secure report platform makes it possible for physicians and genetic counselors to translate the test results efficiently to the patients or the families of patients The Effectiveness of the test supports its usage as a follow up evaluation option for newborns who failed initial hearing screen. This Genetic Screening Panel can improve: With an accurate diagnosis of underlying molecular pathology mechanism, better timed identification to facilitate early pre- lingual intervention is possible Prevention of deafness caused by antibiotic usages Pre-onset intervention for Usher patients Early identification of syndromic deafness for better management/intervention/treatment Candidates for Deafness gene testing
Newborns who fail the mandatory newborn hearing screen at the hospitals, patients may also be included Congenitally deaf patients and their relatives Patients with a need for ototoxic aminoglycoside antibiotics with reasons to suspect they may carry aminoglycoside-sensitive mutations in mitochondria genes Family members who want to determine whether they are carriers of hearing loss mutation(s) Moderate-severe hearing loss patients to determine the underlying genetic cause for management and treatment. Thank You!
This work was supported by the NIDCD. NIH/NIDCD R41, DC009713 NIH/NIDCD R21/R33, DC010476 ~130 Genes Sampled in Oto-Deafness Gene Panel + cCMV (to be included for newborn screen)
ACTB, ACTG1, ADGRV1/GPR98 (USH2), ATP6V1B1,ATP6V1B2, BCS1L, BSND CATSPER2, CCDC50, CDH23 (USH1D DFNB12), CEACAM16, CEMIP/KIAA1199/TMEM2L, CLDN14, CLRN1 (USH3), COCH, COL11A2 (STL3), COL9A2, COLA3, CRYM, DFNA5 (ICERE-1), DFNB31/WHRN (USH2), DFNB59, DIAPH1 (DFNA1), DSPP, ECE1, EDNRA, EDNRB (WS IV), ERCC2, ERCC3, ESPN, ESRRB, EYA4 FAS, FGF3, FGFR3, FOXI1 GATA3, GIPC3, GJA1, GJB1, GJB2 (DFNA3, GJB3 (DFNA2B), GJB4, GJB6 (DFNA3), GPSM2, GRHL2, GRXCR1, GSTP1, HAL, HGF, ILDR1, JAG1, KCNE1 (JLNS), KCNJ10, KCNQ1 (JLNS), KCNQ4 (DFNA2), LHFPL5, LHX3, LOXHD1, LRTOMT MARVELD2, MIR182, MIR183, MIR96 (MIR1974 and 1978, or MT-RNR1),, MITF (WSII), MSRB3, MTAP, MT-TD, MT-TH, MT-TI, MT-TK, MT-TL1, MT-TL2, MT-TM, MT-TQ, MT-TS1, MT-TS2, MYH14 (DFNA4), MYH9, MYO15A, MYO1A, MYO1C, MYO1F, MYO3A, MYO6, MYO7A (USH1B DFNB2 DFNA11), NDP, NR2F1,OTOA, OTOF, OTOR, P2RX2, PAX3 (WS I&III), PCDH15 (USH1), PDZD7, PMP22, POU3F4, POU4F3, PRPS1, PTPRQ, RDX SERPINB6, SIX1 (BOR), SIX5 (BOR), SLC17A8, SLC26A4 (Pendred DFNB4), SLC26A5, SL-C4A11, SMPX, SNAI2, SOX2, SPINK5, STRC TBL1X, TCF21, TECTA, TFCP2, TIMM8A (MTS), TJP2, TMC1, TMIE, TMPRSS3, TMPRSS5, TPRN, TRIOBP, USH1C (DFNB18), USH1G, USH2A, WFS1 (Wolfram DFNA6/14/38) CIB2 (USH1J, DFNB48), Merlin (NF2), Col2A1 (STL1), Col11A1 (STL2), EYA1 (Not Eya4), EDN3 (WS IV), Sox10 (WS IV), BTD (biotin deficiency), PHYH (90% Refsum), PEX7 (10% Refsum), Col4A3 Col4A4 Col4A5 (Alport), DNFA3 Data Analysis and Report Workflow
Mapping Exonic region HGMD, OMIM HG19: Indels/Variant calls & * BWA-MEM coverage (*.vcf) Targeted regions (ie BrCA) + GATK lite Coverage filter**
*Human Gene Mutation Database, Online Mendelian Inheritance in Man, 200 genomic knowledge sources. Tute Genomics platform **ACMG 10-20x minimum;10x filter; 10-20x visual Pathogenic/ clinical phenotype *** exam by genetic counselor, <10x sanger validation. Ethnicity/ frequency **** ***Frequency <1% (D) -2% (R) in the population; 1000 Human Genome carriers for certain genes, such as CF (4%) and some deafness genes are high; genetic counselor input for those special genes. Clinical Report by Otogenetics - Medical Geneticist (MD Signature) available ****Correlation of variants with the clinical indications for the proband conducted by medical geneticist (disease panel). Patient response to medications