jurnal kelompok 2

Comprehensive genetic testing for hereditary hearingloss using massively parallel sequencingA. Eliot Shearera,b,1, Adam P. DeLucac,d,1, Michael S. Hildebranda,1, Kyle R. Taylorc,d, Jos Gurrola IIa, Steve Scherere,2,Todd E. Scheetzc,d,f,2, and Richard J. H. Smitha,b,g,2,3

aDepartment of Otolaryngology, Head and Neck Surgery, University of Iowa, Iowa City, IA 52242; bDepartment of Molecular Physiology and Biophysics,University of Iowa Carver College of Medicine, Iowa City, IA 52242; cDepartment of Biomedical Engineering, University of Iowa, Iowa City, IA 52242; dCenterfor Bioinformatics and Computational Biology, University of Iowa, Iowa City, IA 52242; eHuman Genome Sequencing Center, Baylor College of Medicine,Houston, TX 77030; fDepartment of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA 52242; and gInterdepartmental PhD Program inGenetics, University of Iowa, Iowa City, IA 52242

Edited* by Mary-Claire King, University of Washington, Seattle, WA, and approved October 14, 2010 (received for review August 31, 2010)

The extreme genetic heterogeneity of nonsyndromic hearing loss(NSHL) makes genetic diagnosis expensive and time consumingusing available methods. To assess the feasibility of target-enrichment and massively parallel sequencing technologies tointerrogate all exons of all genes implicated in NSHL, we testednine patients diagnosed with hearing loss. Solid-phase (Nimble-Gen) or solution-based (SureSelect) sequence capture, followed by454 or Illumina sequencing, respectively, were compared. Sequenc-ing reads weremapped using GSMAPPER, BFAST, and BOWTIE, andpathogenic variants were identied using a custom-variant callingand annotation pipeline (ASAP) that incorporates publicly availablein silico pathogenicity prediction tools (SIFT, BLOSUM, Polyphen2,and Align-GVGD). Samples included one negative control, threepositive controls (one biological replicate), and six unknowns (10samples total), in which we genotyped 605 single nucleotidepolymorphisms (SNPs) by Sanger sequencing tomeasure sensitivityand specicity for SureSelect-Illumina andNimbleGen-454methodsat saturating sequence coverage. Causative mutations were iden-tied in the positive controls but not in the negative control. In veof six idiopathic hearing loss patients we identied the pathogenicmutation. Massively parallel sequencing technologies providesensitivity, specicity, and reproducibility at levels sufcient toperform genetic diagnosis of hearing loss.

deafness | genomics | Usher syndrome | diagnostics | next-generationsequencing

Hereditary sensorineural hearing loss (SNHL) is the mostcommon sensory impairment in humans (1, 2). In developedcountries, two-thirds of prelingual-onset SNHL is estimated tohave a genetic etiology, of which 70% is nonsyndromic hearingloss (NSHL). Eighty percent of NSHL is autosomal recessivenonsyndromic hearing loss (ARNSHL), 20% is autosomal dom-inant (AD), and the remainder is composed of X-linked and mi-tochondrial forms (1, 3). To date, 134 deafness loci have beenidentied, and 32 recessive (DFNB), 23 dominant (DFNA) and 2X-linked (DFNX) genes have been cloned; 8 genes are associatedwith both ARNSHL and ADNSHL (4).Establishing a genetic diagnosis of NSHL is a critical compo-

nent of the clinical evaluation of deaf and hard-of-hearing personsand their families. If a genetic cause of hearing loss is determined,it is possible to provide families with prognostic information, re-currence risks, and improved habilitation options. For personsdiagnosed with Usher syndrome, preventative measures includingsunlight protection and vitamin therapy can be implemented tominimize the rate of progression of retinitis pigmentosa (5). Mostcurrent genetic testing strategies for NSHL rely on a gene-specicSanger sequencing approach. Because mutations in a single gene,GJB2 (DFNB1), account for up to 50% of ARNSHL in manyworld populations (6), this approach has changed the evaluation ofpatients with presumed ARNSHL. However, the mutation fre-quency in other genes in personswithNSHL inoutbred populationsis unknown,making sequential gene screening problematic (7). The

extreme heterogeneity of NSHL also makes serial sequencingapproaches unfavorable in terms of efciency and cost.The advent of new technologies that target and enrich specic

regions of the genome coupled with massively parallel sequencingoffers an alternative approach to genetic testing for deafness. Al-though genetic diagnoses can also be made by whole genome se-quencing (8) or targeted sequence capture of the entire exome (9,10), these approaches are expensive, and time-consuming dataanalysis is required. Therefore, our aim was to develop and testa streamlined, comprehensive genetic diagnostic platform that tar-gets only the0.014%of thegenomecurrently associatedwithNSHL.

ResultsWe elected to compare the two most widely used target enrichmentapproaches (NimbleGen solid-phase and SureSelect solution-based sequence capture) and two massively parallel sequencingtechnologies (454 GS FLX pyrosequencing and Illumina GAIIcyclic reversible termination sequencing) using genomic DNAfrom NSHL families. The optimized platform and diagnosticpipeline, which we refer to as OtoSCOPE (otologic sequencecapture of pathogenic exons), targets the exons of all 54 knowndeafness genes (Table S1). The subjects were nine individuals withpresumed NSHL (Table 1). Sanger sequencing-based genetictesting had been completed for GJB2 and SLC26A4 in all auto-somal recessive (AR) cases. Positive controls had been geneticallydiagnosed by Sanger sequencing prior to this study.We performed array and solution-based targeted capture of all

exons of the 54 genes known to cause NSHL, also including Ushersyndromegenes because in infants and children,Usher syndrome isnot readily distinguishable fromARNSHL (Materials andMethodsand Table S1). Some requested target regions were not covered bythe NimbleGen and SureSelect designs (9.3% and 8.3%, re-spectively; Table 2) due to repetitive regions. These percentagesare consistentwith other reports focusedon exon sequence capture(11, 12). However, the proportion of protein coding regions in-cluded in the SureSelect bait design was comparatively better thanthat covered by NimbleGen (97.7 and 93.6%, respectively; Table2), reecting different methods for dening repetitive regions andcomplementary oligonucleotide selection.

Author contributions: A.E.S., M.S.H., S.S., T.E.S., and R.J.H.S. designed research; A.E.S.,A.P.D., M.S.H., and K.R.T. performed research; A.P.D., K.R.T., J.G., S.S., and T.E.S. contrib-uted new reagents/analytic tools; A.E.S., A.P.D., M.S.H., K.R.T., J.G., T.E.S., and R.J.H.S.analyzed data; and A.E.S., M.S.H., and R.J.H.S. wrote the paper.

The authors declare no conict of interest.

*This Direct Submission article had a prearranged editor.

Freely available online through the PNAS open access option.1A.E.S., A.P.D., and M.S.H. contributed equally to this work.2S.S., T.E.S., and R.J.H.S. contributed equally to this work.3To whom correspondence should be addressed. E-mail: [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1012989107/-/DCSupplemental.

2110421109 | PNAS | December 7, 2010 | vol. 107 | no. 49 www.pnas.org/cgi/doi/10.1073/pnas.1012989107

Table

1.Samplesstudiedan

ddiagnosticsequen

cingresultsbyallele

Sample

Status

Hea

ringloss

inheritan

ceCan

didate

varian

tsGen

eNucleo

tide

chan

ge

Protein

chan

ge

Gen

omic

coordinates

(Hg19

)

Sequen

cingread

svarian

tread

s/total

coverage(%

)Status

1Po

sitive

control

ADNSH

L3

COCH

c.15

1C>T

p.P51

S*Chr14:

31,346

,846

463/90

2(51.3)

PosCtrlmutation

EYA4

c.39

0C>A

p.Tyr13

0XChr6:13

3,78

2,27

123

4/67

3(34.77

)RO-seg

regation

OTO

Fc.44

84G>C

p.R14

95P

Chr2:26

,689

,598

639/1,26

6(50.5)

RO-seg

regation

2Po

sitive

control

ARNSH

L1

GJB2

c.10

9G>A

p.V37

IChr13:

20,763

,612

1,07

5/1,07

8(99.7)

PosCtrlmutation

GJB2

c.10

9G>A

p.V37

IChr13:

20,763

,612

1,07

5/1,07

8(99.7)

PosCtrlmutation

3Rep

licateof2

ARNSH

L1

GJB2

c.10

9G>A

p.V37

IChr13:

20,763

,612

1,11

3/1,11

4(99.9)

PosCtrlmutation

GJB2

c.10

9G>A

p.V37

IChr13:

20,763

,612

1,11

3/1,11

4(99.9)

PosCtrlmutation

4Neg

ativecontrol

n/a

0

5IdiopathicSN

HL

ARNSH

L1

STRC

c.40

57C>T

p.Q13

53X

Chr15:

43,896

,918

44/47(93.6)

Cau

sative

mutation

STRC

10

0-kb

deletion

Chr15:

q15

Cau

sative

mutation

6IdiopathicSN

HL

ADNSH

L2

MYO6

c.86

2_86

5delACAA

p.D28

8DfsX17

Chr6:76

,554

,660

76,554

,663

109/30

6(35.6)

Cau

sative

mutation

MYO6

c.36

67G>A

p.D12

23N

Chr6:76

,624

,538

427/89

0(48.0)

RO-seg

regation

7IdiopathicSN

HL

ADNSH

L4

MYO7A

c.51

56A>G

p.Y17

19C

Chr11:

76,913

,457

439/90

8(48.3)

RO-seg

regation

PCDH15

c.10

39C>T

p.L34

7FChr10:

55,973

,755

215/45

9(46.8)

RO-seg

regation

USH

2Ac.67

13A>C

p.E22

38A

Chr1:21

6,16

6,45

437

2/75

2(49.47

)RO-seg

regation

OTO

Fc.37

51T>G

p.C12

51G

Chr2:26

,695

,500

706/72

0(98.1)

RO-seg

regation

8IdiopathicSN

HL

ADNSH

L2

KCNQ4

c.84

2T>C

p.L28

1S

Chr1:41

,285

,554

348/89

3(39.0)

Cau

sative

mutation

WFS1

c.23

27A>T

p.E77

6VChr4:6,30

3,84

916

1/32

1(50.1)

RO-seg

regation

9IdiopathicSN

HL

ADNSH

L2

MYH14

c.58

93G>T

p.E19

65X

Chr19:

50,812

,952

587/1,25

5(46.8)

Cau

sative

mutation

MYH14

c.41

93A>G

p.E13

98G

Chr19:

50,784

,876

413/1,33

4(31.0)

RO-controls

10IdiopathicSN

HL

ARNSH

L2

CDH23

c.10

96G>A

p.A36

6T

Chr10:

73,377

,112

2,23

3/4,52

9(49.3)

Cau

sative

mutation

CDH23

c.0.32

93A>G

p.N10

98S

Chr10:

73,472

,494

2,70

8/5,44

5(49.7)

Cau

sative

mutation

Can

didatevarian

tsfollo

winheritan

cepattern

(ifkn

own)an

darepredicteddeleteriousbyPo

lyphen

2,SIFT,B

LOSU

M62

,andalign-GVGD.C

andidatemutationswereruledoutorin

bysegregationan

alysisan

d/

orpresence

incontrols.C

ausative

mutationsarepredicteddam

aging,segregatewithhea

ringlossin

family,a

renotfoundin

controls,a

nd/orarekn

ownto

cause

hea

ringloss.N

SHL,nonsyndromichea

ringloss;

ADNSH

L,au

tosomal

dominan

tnonsyndromichea

ringloss;ARNSH

L,au

tosomal

recessivenonsyndromichea

ringloss;Po

sCtrl,positive

control;RO-seg

regation,mutationruledoutbysegregationan

alysis;RO-

controls,mutationruledoutbycontrolsan

alysis.

*Previouslyreported

(16).

Previouslyreported

(17).

Previouslyreported

(18).

Previouslyreported

(19).

Deletiondetermined

bysequen

cingread

san

alysis,conrm

edbyPC

Ran

darray-CGH,previouslyreported

(ref.20

andFig.1).

Shearer et al. PNAS | December 7, 2010 | vol. 107 | no. 49 | 21105

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We paired NimbleGen solid phase sequence capture with 454GS FLX pyrosequencing (NimbleGen-454 method) and Sure-Select solution-based sequence capture with Illumina GAII se-quencing (SureSelect-Illumina method) to determine the efcacyof these approaches for clinical diagnostics. Samples 1 and 2were sequenced using both the NimbleGen-454 and SureSelect-Illumina to provide direct comparison of these methods, whereassamples 310 were examined only using SureSelect-Illumina.The results of our study show that due to thedisparity in sequence

output between the two sequencing platforms (13), the overall se-quence depth of coverage was on average 13-fold higher for theSureSelect-Illuminamethod as compared with theNimbleGen-454method (903 and 71 depth of coverage, respectively (Materialsand Methods); Table 3). However the percent of on-target reads(i.e., capture efciency) was signicantly lower (P < 0.0001) for theSureSelect-Illuminamethod (average 19.6%) than theNimbleGen-454 method (average 64.2%), which may reect PCR-induced biasduring Illumina library preparation, small target size, and sequenceroversaturation (Tables 2 and 3).As a threshold for variant detection, we required 3 reads

(multidirectional) for 454 and 40 reads for Illumina, with theadded stipulation that the variant had to be present in at least30% of reads to be considered high quality. Similar thresholdshave been used in other studies (14) and reect the need for highsensitivity in diagnostic testing (false positives are preferred overfalse negatives) (15). At these thresholds, 96.0% and 95.3% ofprotein coding bases were covered by the NimbleGen-454 andSureSelect-Illumina platforms, respectively (Table 2).To validate the platforms as diagnostic tests we genotyped 605

highly heterozygous SNPs (average heterozygosity, 0.46; average55 SNPs per sample) in the targeted regions by Sanger se-quencing. Homozygous-alternative allele calls (n = 149; average14 per sample) and heterozygous allele calls (n= 199; average 18per sample) were used as true positive variants; homozygous-reference allele calls (n= 257; average 23 per sample) were usedas true negative variants (simulated nonvariants). Variants not

covered at variant-calling threshold for either platform wereconsidered false negatives.With both the NimbleGen-454 and SureSelect-Illumina meth-

ods, we identied the causative mutations in samples 1 and 2.Specicity was 98% for NimbleGen-454 and >99% forSureSelect-Illumina (Table 3). For the SureSelect-Illuminamethod, one false negative was found across all 10 samples(sensitivity 99.72%) reecting high coverage depth of targetedregions. For the NimbleGen-454 method, a mean depth of cov-erage of 71 was associated with an increased false-negative rate(average two per sample) and a decrease in sensitivity (93.98%).This difference may be attributable to a relatively lower qualitydepth-of-coverage threshold for the NimbleGen-454 method (3versus 40 for SureSelect-Illumina) as mean sequence coveragefor protein-coding sequence was similar.On the basis of data from samples 1 and 2, we analyzed the

remaining eight samples using the SureSelect-Illumina methodand a sequence analysis protocol we developed (Materials andMethods). In total, we detected causative mutations in the threepositive controls and in ve of six persons with idiopathic hearingloss (Table 1). Analysis of controls (samples 14) was completedin a blinded fashion with only the mode of inheritance known.Sample 1 was from a family segregating ADNSHL. Of three can-didate variants, the known hearing loss mutation in COCH(c.151C > T, p.P51S, rs28938175; DFNA9) was the only variantthat segregated with the phenotype in this family (16). In samples2 and 3 (biological replicates), the only candidate variant deter-mined in each sample was the known deafness mutation in GJB2(c.109G > A, p.V37I; DFNB1) (17). In sample 4, the negativecontrol, none of the nine nonsynonymous/splice site/indel var-iations identied was predicted to be pathogenic.Of the six persons with idiopathic hearing loss, two were from

families segregating ARNSHL. Sample 5 was from a person withprofound SNHL who was shown to carry a variant of unknownsignicance (VUS) in STRC (c.4057C > T, p.Q1353; rs2614824;DFNB16) in 44/47 Illumina reads. This variant was not observed

Table 2. Sequence capture performance results

NimbleGen-454 SureSelect-Illumina

All regions (%)Protein codingregions (%) All regions (%) Protein coding regions

Number of bases requested* 421,741 bp 187,017 bp 421,741 bp 187,017 bpBases covered by complementaryoligonucleotides (%)

386,880 bp (91.7) 266,947 bp (93.6) 382,627 bp (90.7) 182,749 bp (97.7)

Mean sequence coverage in bases (%) 406,756 bp (96.4) 180,489 bp (96.5) 401,252 bp (95.1) 182,727 bp (97.7)Mean sequence coverage at variantcalling threshold in bases (%)

403,873 bp (95.8) 179,468 bp (96.0) 385,101 bp (91.3) 178,230 bp (95.3)

Values given are a mean of all samples for each method.*The same bases were requested for targeting using either NimbleGen or SureSelect and default repetitive regions were avoided in both methods.Variant calling threshold: for Illumina-SureSelect, 40 coverage and >30% of reads; for 454, 3 reads (multidirectional) and >30% of reads.

Table 3. Sequencing results

Sequencingmethod

Capturemethod

Totalsequencing

reads

Uniquelymappingreads

% of uniquelymappingreads

Uniquely mappingreads on target

(99% 0*/0

Illumina sequencing was performed using one sample per ow cell channel; 454 sequencing was performed using one sample per quarter of a four-waygasket. Values given are the mean of all samples for a given method: Illumina, 8 samples; 454, 2 samples. Sens, sensitivity; spec, specicity; FN, false negatives;FP, false positives.*One FN was found across all ten samples.

21106 | www.pnas.org/cgi/doi/10.1073/pnas.1012989107 Shearer et al.

in 100 ethnically matched controls (200 chromosomes). A hemi-zygous deletion of 100 kb involving the STRCSTRC regionwas also detected (Fig. 1) and independently veried by PCR andarray-comparative genomic hybridization (CGH). Homozygosityfor this contiguous gene deletion is known to cause autosomalrecessive deafnessinfertility syndrome (20), making this persona compound heterozygote for a novel point mutation in STRC intrans with a large contiguous gene deletion that includes STRC(DFNB16). Sample 10, from a 4-y-old cochlear implant recipientwith no family history of hearing loss, was compound heterozygousfor known USH1Dmutations in CDH23 (c.1096G >A, p.A366T;c.3293A > G, p.N1098S) (19).The remaining four samples were from families segregating

ADNSHL. For these samples, we also used, when possible, thecomputerprogramAudioGene (21) as a phenotypiclter topredictprobable ADNSHL genotypes on the basis of audiometric criteriathrough audioproling. Sample 6 carried two candidate variants inMYO6 (DFNA22). The missense mutation was ruled out by seg-regation analysis; however, the 4-bp deletion (c.862_865delACAA,p.D288DfsX17) segregated with the phenotype and was not foundin 135 ethnically matched controls (270 chromosomes). Becausethe DFNA22 locus is not represented on AudioGene, phenotypicltering was not applied; however, the phenotype was consistentwith reported DFNA22 audioproles (22). Sample 8 carried twocandidate variantsone mutation was ruled out by segregationanalysis and the other is a known ADNSHL mutation [(DFNA2)c.842C > T, p.L281S] in KCNQ4 (18). This nding was consistentwith AudioGene analysis, which predicted DFNA2 as the mostlikely cause of hearing loss in this family. Sample 9 carried twocandidate variantsboth detected in MYH14that segregatedwith the phenotype in the extended family.Onemutationwas ruledout by analysis of controls, whereas the other, a stop mutation(c.5893G>T, p.E1965), was not found in 119 ethnically matchedcontrols (238 chromosomes) and was consistent with the predictedDFNA4 audioprole. Four candidate variants were detected insample 7, all of which were ruled out by segregation analysis.AudioGene analysis predictedKCNQ4 (DFNA2) as themost likelycause of hearing loss in this family; however, no variants inKCNQ4were identied in this analysis or by prior Sanger sequencing. Thisnding suggests that this familymay segregate a novel genetic cause

of ADNSHL, which is possible because causative genes have notbeen identied formore thanhalf of themappedADNSHL loci (4).

DiscussionIn this study, we identied NSHL mutations, three of which havenot been reported, in eight of nine persons tested. Our sequencingresults are consistent with other studies reporting utility of thesetechnologies for diagnosis of other genetic diseases (11, 15, 23)and suggest that massively parallel sequencing is suitable for ge-netic testing of NSHL. A key concern for any diagnostic test issensitivity, as it is critical that pathogenic mutations are not missed.This requirement could be viewedas a potential limitationof target-enrichmentmethodologies, as a signicant portion of targeted basesin repetitive regions cannot be captured (Table 2). However, thislimitation must be weighed against the decreased cost and time insimultaneously sequencing a large number of genes.Multiplexing samples offers the opportunity to increase

throughput, but to maintain read depth, capture efciency mustbe increased (24, 25). Scalability and cost also must be consideredfor large-scale sequencing projects. Our results show that whenadequate target coverage and depth of coverage are maintained,both the SureSelect-Illumina and NimbleGen-454 platforms offerrelatively high specicity and sensitivity. However, the SureSelect-Illumina method is superior in terms of scalability, cost, and in-creased sensitivity. Our results compare very favorably with recentreports of high-throughput diagnostic tests for NSHL that rely onprimer extension arrays (26) or targeted resequencing arrays (27),which are appealing due to low cost but are limited in terms ofcapacity (not all NSHL genes can be screened simultaneously)and therefore sensitivity.In summary, we have demonstrated that OtoSCOPE has the

potential to improve the efciency of genetic testing for NSHL andUsher syndrome. Our results show that targeted capture plusmassively parallel sequencing has a sensitivity and specicity com-parable toSanger sequencing.Comprehensive genetic screening fordeafness using platforms like OtoSCOPE would allow clinicians toimprove patient care by providing prognostic information and ge-netic counseling, and in cases like Usher syndrome, offer familiespreventative strategies to minimize the rate of progression of reti-nitis pigmentosa. Some of the novel habilitation options under

Fig. 1. Deletion analysis of sample 5 using massively parallel sequencing. Location on chromosome 15 is shown, with the highlighted region containing STRC;ve exons are indicated by blue bars on x axis. Gray line is average sequencing depth of coverage for nine samples (all samples excluding sample 5); thick blackline represents SD for these samples. Red line is depth of coverage for sample 5.


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development to slow progression of hearing loss are also gene andevenmutation specic (28), suggesting that comprehensive genetictesting will be an integral part of the care of deaf and hard-of-hearing patients in the future.

Materials and MethodsPatients. The subjects were nine individuals with presumed NSHL (Table 1)who provided informed consent for this study approved by the University ofIowa International Review Board. Detailed family histories, clinical evalua-tions, and audiograms were available for each patient.

Targeted Capture and DNA Sequencing. Genomic DNA (gDNA) was extractedfrom whole blood using standard procedures (29), quality assessed on anagarose gel and spectrophotometer, and quantied using the Qubit (Invi-trogen) system. Three or 5 g of gDNA were used for the SureSelect orNimbleGen capture methods, respectively.

Exons in all isoforms of the 54 known deafness genes were identied in theRefSeq and Ensembl databases using the University of California Santa Cruztable browser (http://genome.ucsc.edu/). An additional 50 bp of ankingintronic sequence were added to each exon and genomic intervals weremerged using Galaxy software (http://galaxy.psu.edu). In total, we targeted1,258 regions comprising 421,741 bp using both NimbleGen and SureSelectmethods. The same genomic coordinates were sent to Roche for comple-mentary oligonucleotide microarray design or uploaded to Agilents eArraywebsite for cRNA bait design.

DNA isolated using NimbleGen solid phase sequence capture was se-quenced using 454 GS FLX pyrosequencing (454 Life Sciences); DNA obtainedfrom SureSelect solution-based sequence capture was subjected to IlluminaGAII sequencing (Illumina). In both cases sequencing was performed ac-cording to manufacturers protocols.

Sequence Analysis. Sequencing depth of coverage was dened as the numberof sequencing reads, which had been ltered andmapped, per base. Averagedepth of coverage for each sequencing method was dened as the totaldepth of coverage per base, averaged over all requested bases. Depth ofcoverage per variant was dened as number of reads in which a variant wasseen divided by the total depth of coverage for that base.

To prioritize a VUS, we developed a ranking algorithm that included thevariant-calling threshold for each platform, location within the targetedregion, type of change (nonsynonymous, splice-site, or frameshift deletion)and observed population frequency if the VUS was a reported SNP (Table 4).Other investigators have ltered variants on the basis of absence from thedbSNP database (9, 10, 14), a criterion we did not include as several muta-tions known to cause hearing loss have been assigned RefSNP (RS) numbers.Instead, we ltered variants on the basis of quantitative data fromdbSNP130 such that any variant with an allele frequency >1% was consid-ered a benign polymorphism (excluding known ARNSHL-associated variantsof GJB2), whereas any variant with an allele frequency

19. Oshima A, et al. (2008) Mutation prole of the CDH23 gene in 56 probands with

Usher syndrome type I. Hum Mutat 29:E37E46.20. Zhang Y, et al. (2007) Sensorineural deafness and male infertility: A contiguous gene

deletion syndrome. J Med Genet 44:233240.21. Hildebrand MS, et al. (2008) Audioprole-directed screening identies novel

mutations in KCNQ4 causing hearing loss at the DFNA2 locus. Genet Med 10:797804.22. Hilgert N, et al. (2008) A splice-site mutation and overexpression of MYO6 cause

a similar phenotype in two families with autosomal dominant hearing loss. Eur J Hum

Genet 16:593602.23. Morgan JE, et al. (2010) Genetic diagnosis of familial breast cancer using clonal

sequencing. Hum Mutat 31:484491.24. Mamanova L, et al. (2010) Target-enrichment strategies for next-generation

sequencing. Nat Methods 7:111118.

25. Lee H, et al. (2009) Improving the efciency of genomic loci capture usingoligonucleotide arrays for high throughput resequencing. BMC Genomics 10:646.

26. Rodriguez-Paris J, et al. (2010) Genotyping with a 198 mutation arrayed primerextension array for hereditary hearing loss: Assessment of its diagnostic value formedical practice. PLoS ONE 5:e11804.

27. Kothiyal P, et al. (2010) High-throughput detection of mutations responsible forchildhood hearing loss using resequencing microarrays. BMC Biotechnol 10:10.

28. Hildebrand MS, et al. (2008) Advances in molecular and cellular therapies for hearingloss. Mol Ther 16:224236.

29. Grimberg J, et al. (1989) A simple and efcient non-organic procedure for theisolation of genomic DNA from blood. Nucleic Acids Res 17:8390.

30. Chan PA, et al. (2007) Interpreting missense variants: Comparing computationalmethods in human disease genes CDKN2A, MLH1, MSH2, MECP2, and tyrosinase(TYR). Hum Mutat 28:683693.


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Supporting InformationShearer et al. 10.1073/pnas.1012989107

Table

S1.

Ove

rview

ofgen

esincluded

onOtoSC

OPE

version1

Gen

eFu

llnam

eRolefunction

Dea

fnesslocus

Chromosomal

locus

Prim

ary

reference

sequen

ce

Prim

ary

sequen

ceexons

Prim

ary

sequen

cemRNABP

Autosomal

recessivegen

esCDH23

cadherin-related

23Hairbundle,ad

hesionprotein

DFN

B12

10q21q22

NM_022

124.5

6911

,134

CLD

N14

clau

din

14Ionhomeo

stasistightjunctions

DFN

B29

21q22

NM_012

130.2

31,23

3COL11A

2collagen

,typeXI,alpha2

Extracellularmatrixprotein

brillarcollagen

DFN

B53

/DFN

A13

6p21

.3NM_080

680.2

666,42

5

ESPN

espin

Hairbundle,cytoskeletal

form

ation

DFN

B36

1p36

.3NM_031

475.2

133,53

1ESRRB

estrogen

-related

receptorbeta

Tran

scriptionfactorsimilarto

estrogen

receptor

DFN

B35

14q24

.124

.3NM_004

452.3

123,02

9

GJB2

gap

junctionprotein,beta2

Ionhomeo

stasisgap

junction

DFN

B1/DFN

A3

13q12

NM_004

004.5

22,34

7GJB3

gap


Ionhomeo

stasisgap

junction

DFN

A2

1p34

NM_024

009.2

22,22

0GJB6

gap


Ionhomeo

stasisgap

junction

DFN

B1/DFN

A3

13q12

NM_006

783.4

52,11

0LH

FPL5

lipomaHMGIC

fusion

partner-like5

Hairbundle,ad

hesionprotein

DFN

B66

/67

6p21

.2p22

.3NM_182

548.3

42,14

7

MARVELD2

MARVEL

domaincontaining2

Ionhomeo

stasistightjunctions

DFN

B49

5q12

.3q14

.1NM_001

0386

03.1

72,28

2MYO3A

myo

sinIIIA

Hairbundle,motorprotein

DFN

B30

10p12

.1NM_017

433.4

355,79

8MYO6

myo

sinVI


DFN

B37

/DFN

A22

6q13

NM_004

999.3

358,66

2MYO7A

myo

sinVIIA

Hairbundle,motorproteins

DFN

B2/DFN

A11

11q13

.5NM_000

260.3

497,46

5MYO15A

myo

sinXVA


DFN

B3

17p11

.2NM_016

239.3

6511

,876

OTO

Aotoan

corin


DFN

B22

16p12

.2NM_144

672.3

283,62

4OTO

Fotoferlin

Exocytosisat

auditory

ribbonsynap

seDFN

B9

2p22p23

NM_194

248.2

477,17

1

PCDH15

protocadherin-related

15Hairbundle,ad

hesionprotein

DFN

B23

10p11

.2q21

NM_033

056.3

357,02

1PJVK

pejvakin

Signalingofhaircells

andneu

rons

DFN

B59

2q31

.1q31

.3NM_001

0427

02.3

71,53

4RDX

radixin


form

ation

DFN

B24

11q23

NM_002

906.3

144,49

8SLC26A4

solute

carrierfamily

26,

mem

ber

4Ionhomeo

stasis

DFN

B4

7q31

NM_000

441.1

214,93

0

SLC26A5

solute

carrierfamily

26,

mem

ber

5MolecularmotorofOHC

7q

22.1

NM_198

999.2

202,69

7

STRC

stereo

cilin


DFN

B16

15q21q22

NM_153

700.2

295,51

5TECTA

tectorinalpha


DFN

B21

/DFN

A8/DFN

A12

11q

NM_005

422.2

246,46

8TM

C1

tran

smem

branechan

nel-like1

Unkn

ownfunction

DFN

B7/DFN

B11

/DFN

A36

9q13q21

NM_138

691.2

243,20

1TM

IEtran

smem

braneinner

ear

Unkn

ownfunction

DFN

B6

3p14p21

NM_147

196.2

41,86

1TM

PRSS3

tran

smem

brane

protease,

serine3

Unkn

ownfunction

DFN

B8/DFN

B10

21q22

NM_024

022.2

132,46

3

TRIOBP

TRIO

andF-actin

bindingprotein


form

ation

DFN

B28

22q13

NM_001

0391

41.2

2410

,159

USH

1CUsher

syndrome1C

homolog

Hairbundle,scaffoldingprotein

DFN

B18

11p14p15

.1NM_153

676.3

273,24

6WHRN

whirlin


DFN

B31

9q32q34

NM_015

404.2

124,10

2Autosomal

dominan

tgen

esACTG

1actin,gam

ma1


form

ation

DFN

A20

/26

17q25

NM_001

614.2

61,91

9CCDC50

coiled-coildomain

containing50


form

ation

DFN

A44

3q28

-q29

NM_178

335.2

128,94

9

COCH

coag

ulationfactorChomolog,

cochlin


DFN

A9

14q12

-q13

NM_004

086.2

122,55

8

CRYM

crystallin,mu

Ionhomeo

stasis

16

p12

.2NM_001

888.2

91,30

3DFN

A5

dea

fness,au

tosomal

dominan

t5

Unkn

ownfunction

DFN

A5

7p15

NM_004

403.2

102,52

1

Shearer et al. www.pnas.org/cgi/content/short/1012989107 1 of 3

Table

S1.Cont.

Gen

eFu

llnam

eRolefunction

Dea

fnesslocus

Chromosomal

locus

Prim

ary

reference

sequen

ce

Prim

ary

sequen

ceexons

Prim

ary

sequen

cemRNABP

DIAPH

1diaphan

oushomolog1


form

ation

DFN

A1

5q31

NM_005

219.4

285,80

4

DSPP

den

tinsialophosphoprotein


DFN

A39

4q21

.3NM_014

208.3

54,33

1EY

A4

eyes

absenthomolog4

Tran

scriptionfactor

DFN

A10

6q22

-q23

NM_004

100.4

205,69

7GRHL2

grainyh

ead-like2

Tran

scriptionfactor

DFN

A28

8q22

NM_024

915.3

165,23

1KCNQ4

potassium

voltag

e-gated

chan

nel

Ionhomeo

stasis

DFN

A2

1p34

NM_004

700.2

142,33

5

MYH14

myo

sin,hea

vychain14

,nonmuscle

Unkn

ownfunction

DFN

A4

19q13

NM_024

729.3

426,80

7

MYH9

myo

sin,hea

vychain9,

nonmuscle


DFN

A17

22q

NM_002

473.4

417,50

5

MYO1A

myo

sinIA

Unkn

ownfunction

DFN

A48

12q13

-q14

NM_005

379.2

283,62

4PO

U4F3

POUclass4homeo

box3

Tran

scriptionfactor

DFN

A15

5q31

NM_002

700.2

21,18

2WFS1

Wolfram

syndrome1

(wolframin)

Ionhomeo

stasis

DFN

A6/DFN

A14

4p16

.3NM_006

005.3

83,64

0

X-linke

dan

dmitochondrial

gen

es;microRNAsinvo

lved

indea

fness

POU3F4

POUclass3

homeo

box4

Tran

scriptionfactor

DFN

3Xq21

.1NM_000

307.3

11,50

7

MT-RNR1

mitochondrially

encoded

12SRNA

RNAtran

slation

chrM

171

MT-TS1

mitochondrially

encoded

tRNA

serine1

RNAtran

slation

chrM

196

7

miR-96

microRNA96

Reg

ulationofgen

eexpression

inhaircells

DFN

A50

7q32

.21

78

miR-182

microRNA18

2Related

tomiRNA-96,

not

curren

tlyim

plicated

indea

fness(1)

7q

32.2

111

0

miR-183

microRNA18

3Related

tomiRNA-96,

notcurren

tlyim

plicated

indea

fness(1)

7q

32.2

111

0

Usher

syndromegen

esCLRN1

clarin

1Unkn

owntran

smem

braneprotein

3q

25.1

NM_174

878.2

42,35

9GPR98

Gprotein-coupledreceptor98

Signalingtran

smem

brane

protein

5q

14.3

NM_032

119.3

9019

,333

USH

1GUsher

syndrome1G


17

q25

.1NM_173

477.2

33,56

1USH

2AUsher

syndrome2A


1q

41NM_206

933.2

7218

,883

Total

541,12

424

9,13

4

1.Hild

ebrandMS,

etal.(201

0)miRNAmutationsarenotacommoncause

ofdea

fness.Am

JMed

Genet

A15

2A:64665

2.

Shearer et al. www.pnas.org/cgi/content/short/1012989107 2 of 3

Table S2. Complete variants list for Illumina-SureSelect data (expanded version of Table 4)

1 2 3 4 5 6 7 8 9 10 Mean (range)

Variations in targeted genes at callingthreshold*

780 745 836 1112 1109 796 824 732 925 898 876 (7321112)

Variations with

jurnal kelompok 2

Documents