The Genomics of Hearing Loss Ian Krantz, M.D. The Children s Hospital of Philadelphia and the Perelman School of Medicine at the University of Pennsylvania
Objectives 1. Understand the clinical and genetic heterogeneity of hearing loss 2. Understand the complexity of genetic testing for hearing loss 3. Understand how new diagnostic tools in genetic testing will dramatically impact our understanding and clinical work-up for hearing loss
Anatomy of Hearing Conductive HL Sensorineural HL
How common is hearing loss? There are over 6 million individuals in the United States affected by some degree of hearing impairment. The incidence of hearing loss among children under the age of 3 years is approximately 1 in 500 The incidence of hearing loss among children under the age of 19 years is approximately 1 in 250 In the US, congenital SNHL is 3x more common than Down syndrome, 6x spina bifida, 50x PKU
Universal Infant Hearing Screening Benefit of Early Identification Yoshinaga-Itano, 1998
50% Environmental Hearing Loss 50% Genetic ototoxic drugs, acoustic trauma, bacterial or viral infection 30% Syndromic Alport Norrie Pendred Usher Waardenburg Branchio-Oto-Renal Jervell and Lange-Neilsen 70% Non-Syndromic 22% Dominant (DFNA 1-40) 77% Recessive (DFNB1-30) 1% X-linked (DFN1-8) <1% Mitochondrial
Why Study Hearing Loss? Common genetic disease (2:1000 births) Poster diagnosis for genetic heterogeneity : Over 100 loci identified Many genes identified, yet most causes of hearing loss remain unknown. Mutations are AD, AR, X-linked, mitochondrial, syndromic & non-syndromic Very few individual genes contribute to a significant proportion of the hearing loss population.
Bilateral Sensorineural Hearing Impairment (BLSNHI) SMAC/DIABLO RDX SIX1 PCDH15 ESRRB GJB2 TMC1 BSND DIAPH1 GJB6 TMPRSS3 LOXHD1 SMPX AUNX1 GJB3 GJB2 PTPRQ MYO6 OTOF TPRN Over 80 primary ACTG1 BLSNHI MSRB3 genes TJP2 KCNQ4 SERPINB6 SLC26A5 PJVK/pejvakin Over 250 associated BLSNHI genes COL11A2 CEACAM16 GRXCR1 CCDC50 MYO6 Majority WFS1 of pediatric BLSNHI is genetic WHRN AUNX1 MYO3A DSPP HGF GJB3 DFN4 TRIOBP COL11A2 TECTA GIPC3 MARVELD2 OTOA EYA4 COCH GPSM2 CLDN14 MYO7A LHFPL5 MYH14 POU4F3 CRYM GRHL2 MYH9 TMC1 TECTA TMIE USH1C POU3F4 STRC DFNA5 MYO1A CDH23 ESPN SLC26A4 LRTOMT MYO15A PRPS1 ILDR1 SLC17A8
Phenotypic Diversity Genes causing both syndromic and non-syndromic hearing loss: MYO7A - DFNA11, DFNB2, USH1B USH1C - DFNB18, USH1C CDH23 - DFNB12, USH1D PCDH15 - DFNB23, USH1F COL11A2 - DFNA13, Stickler SLC26A4/PDS - DFNB4, Pendred WFS1 - DFNA6/14, Wolfram rrna Ser(UCN) - NSHL, neurologic dysfunction
Phenotypic Diversity Genes causing both recessive and dominant non-syndromic hearing loss: MYO7A - DFNA11, DFNB2 GJB2/Cx26 - DFNB1, DFNA3 TECTA - DFNA8/12, DFNB21
Case Presentation
Chromosome abnormalities associated with hearing loss: Trisomy 21 Trisomy 18 Trisomy 13 Trisomy 8 mosaic Turner syndrome (monosomy X) Pallister- Killian syndrome (tetrasomy 12p) Cat-eye syndrome (isochromosome 22) 22q11 deletion 18q deletion
Case Presentation 9 year-old female with the following : Congenital sensorineural hearing loss bilateral, profound History of delayed walking/? Balance problems Eye exam revealed hypoplastic optic discs ERG showed retinal dysfunction Non-dysmorphic
Usher Syndrome Type I (70%) Congenital, profound SNHL Childhood onset of retinitis pigmentosa Clumsiness & developmental delay Absent vestibular function Type II (26%) Congenital SNHL, high frequency > low frequency, stable Later onset of retinitis pigmentosa Normal vestibular function Type III (4%) Normal hearing and vision at birth Deterioration of hearing and vision over the years
Case Presentation
Marshall/Stickler Syndrome Autosomal dominant COL2A1 (Stickler) (chromosome 12q13), COL11A1 (Stickler and Marshall)(chromosome 1p21) flat midface, cleft palate (Pierre-Robin sequence), high myopia with retinal detachment, hearing loss, arthropathy Marshall syndrome more severe manifestation or heterogeneous? hearing loss is sensorineural, mild to severe, and progressive
Case Presentation
LEOPARD Syndrome Lentigines, Electrocardiac defects, Ocular hypertelorism, Pulmonary stenosis, Abnormalities of genitalia, Retardation of growth, and sensorineural Deafness. Hearing loss in 25%- can range from mild to profound Dominant inheritance, significant overlap with Noonan Syndrome- 4 PTPN11 mutations known to cause LS 28/30 (90%) LS patients with PTPN11 mutations (Keren et al, 2004) Triangular facies, hypertelorism, ptosis, low-set ears, dark brown lentigines (increasing in number with age), valvular PS, QRS abnormalities in frontal planes (S1,S2,S3 pattern) on EKG (also some with heart blocks), growth retardation, hypospadias, cryptorchidism and mild MR in 20%
Case Presentation
Type I Waardenburg Syndrome Dominant: PAX3 gene (2q35) Type II Dominant: MITF gene (3p14) Klein-Waardenburg (Type III) Albinism, musculoskeletal abnormalities homozygosity for two PAX3, particular single PAX3, or contiguous gene deletions Waardenburg-Shah (Type IV) (Waardenburg with Hirschsprung) Recessive inheritance: endothelin B receptor (ENDRB 13q22) and endothelin 3 (END3 20q13) genes Dominant: SOX10 (22q12-13)
Case Presentation 5 year 6 month-old male with the following features: Bilateral mild to severe conductive hearing loss Branchial cysts Cochlear hypoplasia bilaterally External auditory canal atresia Pre-auricular pits and tags Inguinal hernia Family history of father with: Pre-auricular tag Hearing loss
Branchio-Oto-Renal (BOR) Syndrome Autosomal Dominant: EYA1 gene (8q13.3) or SIX5 gene (19q13.32) Branchial fistulae 49% Cupped ears 36% Ext. aud. canal stenosis 29% Pre-auricular pits 82% Renal abnormalities 67% Hearing loss 93% Mixed 52% Conductive 33% Sensorineural 29% Mild 27% Moderate 22% Severe 33% Profound 16%
Case Presentation
? * * Prolonged QT * Craniosynostosis Prolonged QT and Hearing Loss
Jervell and Lange-Neilsen Syndrome Prolongation of QT interval on EKG May develop arrhythmias leading to sudden death Profound sensorineural hearing loss Autosomal recessive inheritance Due to mutations in the KVLQT1 gene on chromosome 11p15 and KCNE1 gene on chromosome 21q22 potassium channel genes
GJB2 - Connexin 26 Connexin 26 is a beta-2 gap junction protein (GJB2) Coding region is contained in a single large exon which is separated from the 5 untranslated region by an intron of variable size Coding sequence is 681 base pairs which translates into a 226 amino acid protein In the cell membranes of epithelial cells, connexin 26 molecules are in groups of 6 around a central pore which forms a connexon. Connexons bond together to form intercellular channels. Groups of connexons make up gap junctions involved in potassium circulation (Smith et el, Lancet 2005)
Common mutations: GJB2 35delG (also reported as 30delG), found in ~ 2/3 of mutated alleles carrier frequency of 2.5%in N. European population 167delT, found specifically in the Ashkenazi Jewish population carrier frequency of 4% 235delC, found in Japanese population carrier rate of 1% Over 90 different variants have been reported
The CHOP Experience (1999-2005) Yaeger et al, Am J Med Genet 2006
The Genomic Era
Overview of SNP Array Workflows Bi-allelic SNPs (A or B) DNA is amplified and fragmented Fragmented DNA is hybridized to an array. Each SNP is represented ~20X on the array for technical replication. Genotype and Copy Number information are determined by color and intensity of fluorescence for each SNP on the array Illumina
Copy Number Variation Copy number variation (CNV) is normal within the genome. Thousands of sites have been identified, with highly variable numbers. CNV is population specific, suggesting heritability. Many detected CNV are in fact normal and not indicative of genetically significant change. Redon et al, 2006
Understanding the Data Log R ratio describes (R subject /R reference ) the normalized intensities of the B allele frequency is plotted below, and is derived from the theta and norm R plots.
Understanding the Data A duplication will be indicated by an increase in the LogR ratio and a spreading of the B allele frequencies.
Understanding the Data A deletion will be indicated by an decrease in the LogR ratio and a loss of the heterozgous B allele frequencies (LOH).
GWAS Approach: Is BLSNHL a Complex Trait?
GWAS in 600 BLSNHL probands with no known etiology 2 Loci with statistical significance (LOD scores >10-8 ) on 10q25 and 2p13 Neither replicated (due to presence of variant in AA populations) Some weak GWAS hits for age-related hearing loss (presbycusis) (Huyghe et al AJHG, 2008; Friedman et al, Hum Mol Genet, 2009) and otosclerosis (Schrauwen et al AJHG, 2009)
Homozygosity for the V37I GJB2 Mutation in Fifteen Probands with Mild to Moderate Sensorineural Hearing Impairment: Further Confirmation of Pathogenicity and Haplotype Analysis in Asian Populations. Gallant et al in press
The CNV Approach Candidate Gene List Find Causative Gene(s) Literature Search Screen cohort of probands Screen controls & parents
GJB2 Negative BLSNHL Array Cohort 659 Probands PennCNV (Research) CNV WorkShop (Clinical) 509 Illumina550k 150 Illumina610k 1,910 Controls Illumina610k 93 Illumina610k
PECONπ (PErl COpy Numbers of Potential Interest) Using PERL, devised a program to score CNV calls to help identify the most interesting ones. Scores based on: Literature references Gene involvement ( unique / not unique) Normal CNV (internal / external controls) Cohort redundancy (+/-) Basic information: size, #SNPs, state Summary statements report involvement of genes across whole cohort Features Input calls are algorithm independent Ability to create custom cohorts Outputs ranked and tabbed Excel files to view results and perform further analysis Direct links to UCSC browser Custom filtering
PECONπ
PECONPI Output
GJB6 deletions confirmed (rank 134-5/2057)
OTOF homozygous deletion (rank 6/2057)
USH2A (rank 15/2057)
PECONπ - Initial Findings 4.6 Mb deletion of 9q31.1-q31.2 (26 genes) 1.8 Mb deletion of 22q11.21-q11.23 (distal to DGCR) 1 Mb deletion of 2q11.2 2 Mb deletion within 17q23.2 Confirmed Known HL deletions within our patients Cx30/GJB6 Found new deletions of known HL genes OTOF CDH23 GPR98 USH2A MYO6 LOXHD1 OTOA STRC Found many new candidate genes using Scoring Algorithms
1 homozygous deletion 3 heterozygous deletions PECONPI Output STRC
Enter the Arrays: STRC Deletions
Stereocilin (STRC) Encodes protein that is expressed within stereocilia Located on chromosome 15q15.3 ~19 kb with 29 exons producing a transcript of ~5.5 kb Translates a protein with 1775 amino acids Part of tandem duplication on 15q with the second copy being a pseudogene Defects in STRC are a cause of Non-syndromic sensorineural deafness autosomal recessive type 16 (DFNB16) Deafness-infertility syndrome (DIS)
Stereocilin (STRC) Mouse Model (Verpy et al., Nature 2008) Absent horizontal top link connectors Results in disorganized hair bundle and failure to produce cochlear waveform distortions Strc +/+ Strc -/-
Literature Search 3 reported cases Verpy et al. 2001 2 consanguineous families with autosomal recessive deafness 1 large het deletion with frameshift 1 homozygous frameshift Zhang et al. 2007 3 consanguineous Iranian families Nonsyndromic deafness and male infertility caused by a homozygous contiguous gene deletion of STRC and CATSPER2 Knijnenburg et al. 2009 1 case with homozygous deletion of STRC with slowly progressive BLSNHL with a moderate hearing impairment for all frequencies
Sequencing Cohort 88 GJB2 negative probands (+3 sibs) 48 +3 sibs mild-moderate BLSNHL similar to the array deletion cases seen 40 moderately-severe to profound BLSNHL 96 Controls
Control Het del Hom del Control
Final Sequencing Results of Probands with Positive Findings *Heterozygous array deletions
STRC Mutations/Intragenic Deletions > 5% of sporadic GJB2 mutation negative BLSNHL cases 50% of GJB2 mutation negative familial cases Francey et al 2011
Bilateral Sensorineural Hearing Impairment (BLSNHI) SMAC/DIABLO RDX SIX1 PCDH15 ESRRB GJB2 TMC1 BSND DIAPH1 GJB6 TMPRSS3 LOXHD1 SMPX AUNX1 GJB3 GJB2 PTPRQ MYO6 OTOF TPRN Over 80 primary ACTG1 BLSNHI MSRB3 genes TJP2 KCNQ4 SERPINB6 SLC26A5 PJVK/pejvakin Over 250 associated BLSNHI genes COL11A2 CEACAM16 GRXCR1 CCDC50 MYO6 Majority WFS1 of pediatric BLSNHI is genetic WHRN AUNX1 MYO3A DSPP HGF GJB3 DFN4 TRIOBP COL11A2 TECTA GIPC3 MARVELD2 OTOA EYA4 COCH GPSM2 CLDN14 MYO7A LHFPL5 MYH14 POU4F3 CRYM GRHL2 MYH9 TMC1 TECTA TMIE USH1C POU3F4 STRC DFNA5 MYO1A CDH23 ESPN SLC26A4 LRTOMT MYO15A PRPS1 ILDR1 SLC17A8
Applying Genomic Sequencing in Pediatrics UO1HG006546
Library Construction for Whole Exome Sequencing
Next Generation Sequencing
Pre-sequencing Privacy Coordinator creates record in ehonest Broker Universal ID Creates linked, de-id ed records in REDCap and LIMS Phenotype capture Unified system across four target disorders Clinician/GC entry into REDCap ~2500 data elements with extensive branching logic Materials management Nautilus LIMS with protocol-based extension Distributed sample accrual/barcoding
Sequencing Whole exome Agilent capture Addition of custom titered mitochondrial baits Illumina HiSeq, BGI@CHOP Assembly: Novoalign Alignment: GATK Annotation: GATK/snpEff QC: FASTQC, BAMCHOP
Whole Exome Pipeline Raw Reads (fastq) NovoAlign Ref: Hg19- g1kv37 Picard AddOrReplace ReadGoups sorting and proper naming Picard Marking Duplicates QC: FastQC, GATK, Picard Mapped reads (bam) Sorted +RG (bam) Sorted mkdups (bam) GATK UnifiedGenotyper Variant Calling & dbsnp annotation GATK Recalibration Picard FixMateInformation Corrects any problems GATK Realignment Does a Local realignment around potential indels Recalibrated GATK reads (bam) Repaired GATK reads (bam) Realigned GATK reads (bam) SNPs (VCF) SnpEff Annotation (RefSeq) Database Ready VCF
Variant Management Varify: Contextual variant presentation and exploration platform Harvest Stack Query engine Hypermedia APIs HTML5 client Variant calls and annotations Patient- and cohort-based functionality Standard genetic models
Available Variant Annotations Snpeff: Functional class (silent, missense, nonsense, none) Codon change AA change Gene name Transcript ID Exon ID Region: Exon, Intron, UTR5,UT3, etc. dbsnp v135 SIFT, Polyphen2 HGMD HPO (human phenotype ontology) Allele frequency sources: 1000genomes EVS (NHLBI Exome Sequencing Project) CBMi s Exome Database Coming soon: Phylop PhastCons 75
Return of results Limitations of vendor-specific decision support Discussing EHR limitations for genetics w/ EMR WG Proposed formal evaluation trial with genetic counselors Proposed report delivery: CHOP Care Assistant Stand-alone system that delivers content into an EHR Vendor-agnostic Content type limited only by browser Associated rules engine customized clinical decision support
Exome Sequencing Workflow Sample Collection Phenotypes 8 Validation Samples SDOC BGI Exome Capture NGS Aligning QC Recalibration Variant Calling CBMi
First 8 BLSNHL Exomes 55,000 variants per sample Subset to analyze Hearing Loss Gene List MAF < 5.0% (1000 Genomes) Depth > 10 Added Tier and Inheritance annotations
Quality Filter (Phred Quality Score) HL Gene List Filter 4937 Rows 1325 Variants 209 Genes Quality Q 30 1273 Variants Q < 30 93 Variants
Variants with Q < 30 93 Variants 45 Genes Mostly introns Small number of UTR and synonymous variants Non-synonymous variants 4 variants in 4 genes Low coverage depth 4 x more ref calls than alt calls
Sample Summary Rows Variants Genes Average 593.5 284.9 122.8 Pseq 3-1 640 293 122 Pseq 7-1 512 256 115 Pseq 10-1 526 253 117 Pseq 14-1 628 297 135 Pseq 16-1 673 305 122 Pseq 17-1 568 284 124 Pseq 18-1 592 288 119 Pseq 28-1 609 303 128 Tier 1 Tier2 Tier3 Genes Genes Genes 56.3 57.5 9 54 59 9 55 51 9 52 57 8 60 65 10 51 61 10 61 55 8 53 56 10 64 56 8
Alignment Stats Sample Reads Aligned % Dups % Pseq 3-1 Pseq 7-1 Pseq 10-1 Pseq 14-1 Pseq 16-1 Pseq 17-1 Pseq 18-1 Pseq 28-1 75,071,144 97.87 6.30 73,701,870 97.97 6.89 74,031,188 98.06 10.78 70,116,278 98.06 6.82 83,874,152 97.94 6.21 75,463,750 98.06 8.45 79,201,490 98.04 8.66 83,429,110 98.04 7.50 CDL-444P CDL-559P CDL-160P 73,299,818 98.06 6.75 77,492,204 97.91 7.73 77,458,182 97.93 5.52 84
Average Depth of Coverage (revised) SureSelect V4 Regions Sample Selected Bases Mean SureSelectV4 Depth Pseq 3-1 86.06% 82.57 Pseq 7-1 86.92% 84.71 Pseq 10-1 88.05% 82.09 Pseq 14-1 87.13% 80.57 Pseq 16-1 85.03% 94.17 Pseq 17-1 87.44% 85.78 Pseq 18-1 86.77% 88.44 Pseq 28-1 86.16% 93.34 CDL-444P CDL-559P CDL-160P 87.38% 87.63 87.14% 91.69 87.37% 92.96 85
Depth of Coverage Cont. 100.00 80.00 60.00 40.00 5x 10x 20x 30x 80x 100x 20.00 0.00 IK01 IK02 IK03 IK04 IK05 IK06 IK07 IK08 IK09 IK10 IK011 IK01 IK02 IK03 IK04 IK05 IK06 IK07 IK08 IK09 IK10 IK011 The Exome Exons within the genes of interest
Missing HLS Genes No SureSelect V4 probe for: SLC52A3 DURS1 HTC2 MBS3 MCDR1 NAMSD OTSC1 OTSC4 OTSC7 WS2B 87
Results Details Cohort HLS HLS HLS HLS HLS HLS HLS HLS Sample Pseq 3-1 Pseq 7-1 Pseq 10-1 Pseq 14-1 Pseq 16-1 Pseq 17-1 Pseq 18-1 Pseq 28-1 Raw Variants 581309 519980 493552 506911 635232 501238 543571 599195 Krantz CDL-444P 496192 Krantz CDL-559P 566557 Krantz CDL-160P 524899 Variants 10x (SNV- Indel) 82530 (75054-7476) 75799 (69012-6787) 75922 (69017-6905) 73754 (67082-6672) 81589 (74093-7496) 76839 (69825-7014) 78736 (71421-7315) 83259 (75586-7673) 72652 (66098-6554) 90101 (81998-8103) 74435 (67889-6546) 1000g Rare (SNV- Indel) EVS Rare (<0.05) 1000g + EVS Rare (<0.05) 3960 (3816-144) 6192 1768 3409 (3269-140) 6161 1625 3497 (3345-152) 6241 1641 3371 (3249-122) 5997 1632 3256 (3139-117) 5915 1592 3161 (3039-122) 5927 1589 3059 (2929-130) 5823 1503 3411 (3279-132) 6030 1625 2941 (2826-115) 5772 1526 13073 (12515-558) 13000 4989 3001 (2881-120) 5779 1550 88
Mutation Type HL Gene List Filter MAF < 5% Depth > 10 Quality Q 30 Q < 30 Variant Effect ALL OTHER MUTATION TYPES Synonymous Introns 5 and 3 UTRs Ignore 432 310 370 9 298 188 Rows
Has the variant been reported? HL Gene List Filter MAF < 5% Depth > 10 Quality Q 30 Q < 30 Variant Effect ALL OTHER MUTATION TYPES Synonymous Introns 5 and 3 UTRs Ignore Previously Reported dbsnp 1000 Genomes Exome Variant Server No 312 120 Rows
Previously Reported Non-Synonymous 133 variants 73 genes 12 dbsnp Gene Tiers 1 58 variants in 37 genes 2 51 variants in 27 genes 3 9 variants in 5 genes 1000 Genomes 2 6 85 0 15 EVS 13 EVS = exome variant server
# of Variants Cohort Frequency ALMS1 DSPP SCARF2 SETBP1 # of Samples
Is the variant listed as a pathogenic mutation? HL Gene List Filter MAF < 5% Depth > 10 Quality Q 30 Q < 30 Variant Effect ALL OTHER MUTATION TYPES Synonymous Introns 5 and 3 UTRs Ignore Previously Reported Yes No Pathogenic OMIM HGMD dbsnp
Is the variant listed as a pathogenic mutation? Non-synonymous, splice site, frameshift variants Introns, UTRs, Synonymous 118 variants from 69 genes with dbsnp rs# 805 variants from 188 genes with dbsnp rs# OMIM 10 HGMD 23 OMIM 0 HGMD 2
Preliminary Results Pseq 10-1 GJB2, c.167delt -homozygous Pseq 14-1 GJB2, c.35delg heterozygous GJB2, p.m34t heterozygous Pseq 28-1 GJB2, c.35delg homozygous Pseq 17-1 SLC26A4, p.e384g heterozygous SLC26A4, p.r409h heterozygous
Some Issues with Exome Sequencing for Diagnostics number of variants identified what to report back what are we missing? sifting through the data incidental findings actionable variants (early/late onset) portability of data / integration with EHR
Summary Hearing loss is a highly specialized and complex sensory process that as a result is extremely genetically heterogeneous in etiology complicating our diagnostic ability Pediatric BLSNHL is primarily inherited in Mendelian patterns Genetic etiologic diagnosis greatly assists management and counseling New technologies are contributing to our ability to identify genetic causes with SNP arrays and exome/genome sequencing providing valuable diagnostic support as well as forwarding discovery into new causative genes and modifiers
Acknowledgements Lauren Francey Dinah Clark Alisha Wilkens Sarah Noon Maninder Kaur Aaron Dickinson Hanna Kadesch Emily Gallant Nancy Spinner Pete White Barbara Bernhardt Jeff Pennington Mike Italia Mahdi Samadi Matt Dulik Elizabeth DeChenne Josh Brunton Vijay Jayaraman Byron Ruth John Germiller E. Bryan Crenshaw Heidi Rehm Marly Kenna Funding NIH/NHGRI NIH/NIDCD CHOP IDF
Thank You!