Targeted exome sequencing identified a novel USH2A mutation in a Chinese Usher syndrome family: a case report

DOI: https://doi.org/10.21203/rs.3.rs-16667/v2

Abstract

Background: Usher syndrome is a king of phenotypic and genetic heterogeneous disease. Our purpose was to identify the gene mutation in a Chinese family with Usher syndrome type 2 and describe the clinical features.

Case presentation: A 23-year-old man complained of 10-year nyctalopia and a 3-year decline in visual acuity of both eyes accompanied by congenital dysaudia. To clarify the diagnosis, the clinical symptoms were observed and analysed in combination with comprehensive ophthalmologic examinations as well as genetic analysis (Targeted exome sequencing, TES). Typical clinical presentation of Usher syndrome on fundus was found including a wax yellow-like disc, bone-spicule formations and retinal vessel stenosis. Optical coherence tomography (OCT) and optical coherence tomography angiography (OCTA) showed the loss of ellipsoid zone and the reduction in paracaval vessel density of both eyes. Genetic analysis identified a novel homozygote of c.8483_8486del (p.Ser2828*) in USH2A. The mutation of premature translation termination causes the deletion of 19 fibronectin type 3 domains(FN3), transmembrane region (TM) and PDZ-binding motif domain, which plays an important role in protein binding. Combining the clinical manifestation and genetic result, the patient was diagnosed with Usher syndrome type 2.

Conclusions: We found a novel mutation of c.8483_8486del in USH2A gene through TES techniques. The result broaden the spectrum of mutations in Usher syndrome type 2 and suggest that the combination of clinical information and TES molecular diagnosis could help USH patients obtain a better diagnosis.

1 Background

Usher syndrome (USH) is an autosomal recessive disease that is characterized by retinal pigmentosa (RP), sensorineural hearing impairment and vestibule dysfunction. The prevalence is approximately 3.2 to 6.2 per 100,000 [1-4]. It is clinical and genetic heterogeneity. To date, 19 genes and loci are related to USH and atypical USH (RetNet [https://sph.uth.edu/retnet]; October 2019). Among them, 16 genes were identified as causative genes. USH could be divided into three types according to the age of onset, the severity of visual and hearing impairment and vestibule dysfunction. However, due to the point that genetic manifestation is currently not well understood, the rate of missed diagnosis (4%) is high in Asia, especially in China[5].

In patients with USH1, deafness occurs early, and their abnormal visual function is easily ignored. In patients with USH2 and USH3, visual function and hearing abnormalities are gradually progressive. Accurate clinical and molecular diagnoses are the basis of prognosis, treatment selection and eugenics. TES provides us with a new opportunity of revealing the genetic defects in usher syndrome patients[6]. Here, we screened 381 inherited retinal disease (IRD) related genes in a USH2 family and identified a novel mutation of c.8483_8486del (p.Ser2828*) in the USH2A gene.

Case presentation

A 23-year-old man visited our clinic and suffered from deafness from childhood with occasional dizziness, nyctalopia for 10 years and visual acuity declined for nearly 3 years of both eyes. Previously, he was diagnosed with "sensorineural deafness" by an otolarynologist. The patient and his family members gave informed consent for the study, which was approved by the Ethics Committee of Tianjin Medical University Eye Hospital (Tianjin, China). Then, peripheral venous blood samples were collected for NGS or Sanger sequencing. For clinical diagnosis, we performed a comprehensive ocular examination that contained best-corrected visual acuity (BCVA), slit-lamp examination, visual field tests, OCT, OCTA, ultra-wide field fundus photography, and fundus autofluorescence (FAF).

2 Methods

DNA Library Preparation

Genomic DNA was extracted from peripheral blood leukocytes of the patient and his family members using the DNA Extraction Kit (TIANGEN, Beijing, China) following the manufacturer’s instructions. The DNA was quantified with a Nanodrop 2000 (Thermal Fisher Scientific, DE). A minimum of 3 µg of DNA was used for the indexed Illumina libraries according to the manufacturer’s protocol (My Genostics, Inc., Beijing, China). DNA fragments with sizes ranging from 350 bp to 450 bp and those including the adaptor sequences were selected for the DNA libraries.

Targeted Gene Capture and Sequencing

Next, 381 known genes associated with IRD, including USH (Table 1), were selected by a gene capture strategy using the GenCapCustom Enrichment Kit (My Genostics Inc., Beijing, China) following the manufacturer’s protocol. The biotinylated capture probes were designed to tile all of the exons with non-repeated regions. Sequencing was performed on an Illumina HiSeq 2000 sequencer (Illumina, San Diego, CA, USA) for paired-end reads of 150 bp.

Bioinformatics Analysis

Following sequencing, raw image files were processed using Bcl2Fastq software (Bcl2Fastq, Illumina, Inc.) for base calling and raw data generation. Low-quality variations (score≥20) were filtered out. The clean reads were then aligned to the reference human genome using Short Oligonucleotide Analysis Package (SOAP) aligner software (SOAP2.21; soap.genomics.org.cn/soapsnp.html) (hg19). After removing polymerase chain reaction (PCR) duplicates using the Picard program, single nucleotide polymorphisms (SNPs) were determined using the SOAP SNP program, and the deletions and insertions (InDels) were detected using Genome Analysis Toolkit software 3.7. Subsequently, we annotated the identified SNPs and InDels with the Exome-assistant program (http://122.228.158.106/exomeassistant)and viewed the short read alignment using MagicViewer to confirm the candidate SNPs and InDels. Non-synonymous variants were evaluated for pathogenicity using Sorting Intolerant From Tolerant [SIFT; (http://sift.jcvi.org/)] and PolyPhen (http://genetics.bwh.harvard.edu/pph2/). Protein Analysis Through Evolutionary Relationships (PANTHER; www.pantherdb.org) and Pathogenic Mutation Prediction (Pmut; http://mmb.pcb.ub.es/PMut/) were also used.

Expanded Validation and Protein Function Prediction

Genomic DNA of the proband was subjected to TES. Filtered candidate variants identified by anIllumina HiSeq 2000sequencer were confirmed by Sanger sequencing. The coding exons containing the detected mutations were amplified using Ex Tag DNA polymerase (Takara, Dalian). The purified PCR samples were sequenced using the ABI PRISM 3730 genetic analyser (Applied Biosystems; Thermo Fisher Scientific, Inc.), and then sequence traces were analysed with the Mutation Surveyor (Softgenetics, PA). The mutation in the family members was confirmed by the same procedure. Multiple sequence alignments were performed using ClustalW2 with the default setting (http://www.ebi.ac.uk/Tools/clustalw2/).Protein structures were determined by SMART (http://smart.emblheidelberg.de). The 3D structure of the protein variation caused by gene mutation was analysed using Protein Data Bank(PDB) and the homology modelling software Swiss-Model. We collected all genomic DNA samples upon informed consent.

3 Results

Clinical Findings

A 23-year-old man presented a 10-year history of deafness and poor night vision. His best corrected visual acuity (BCVA) was 0.6/1.0 (R/L). His parents had a consanguineous marriage, and his grandparents had passed away, but they were healthy according to their past medical history and eye conditions. In addition, his parents and his sister were unaffected (Fig. 1). To better clarify the proband's condition, ophthalmologic investigations were performed. Slit-lamp examination showed that the anterior segment of the eyes was normal. The fundus was typical of RP: the appearance of the wax yellow-like disc, the large amount of osteoblast-like pigmentation, and tapering of the retinal vessels, of which the arteries were obvious (Fig. 2a). An abnormal parafoveal ring of increased autofluorescence of ultra-wide-angle images was observed, and there was a ring-like hypoautofluorescence region around the macular and optic disc on FAF imaging (Fig. 2b). We also found decreased retinal thickness and absence of the ellipsoid zone in macular (Fig. 2c). Macular OCTA revealed an enlarging foveal avascular area (FAZ) in the superficial capillary plexus and deep capillary plexus, while macular vascular flow density was also decreased (Fig. 3). Examination with Octopus perimeter device put up a tubular visual field in both eyes (Fig. 4).

Genetic and Molecular Analysis

DNA extracted from the peripheral blood was subjected to TES. Genetic tests showed that the patient had a novel mutation (c.8483_8486del) in the USH2A gene. Moreover, DNA samples extracted from proband’s sister and parents were used for Sanger sequencing. The results confirmed there was a pedigree genetic co-segregation in this family (Fig. 5a). A model structure for USH2A was generated from homology modelling (Fig. 5b). The mutation resulted in premature translation termination, and the stop-gain variant was predicted to remove 2375 amino acids from the encoded proteins, which would result in truncation of the α-βhydrolase domain (Fig. 5c). This may change the overall function of the folded state of the protein (Fig. 5d).

4 Discussion

We reported the case of a 23-year-old patient who presented a series of typical clinical features with a novel homozygous mutation, p.Ser2828* (rs1199684717) in USH2A, a gene responsible for USH2 (OMIM:276901). The frequency of the mutation is 0.000004 in the Genome Aggregation Database and it’s found at heterozygous state in one European non Finnish individual. Mutations in USH2A are associated with USH2, it is responsible for almost 50% of USH cases[7].

USH2A codes two alternatively spliced isoforms of usherin. Short ~170 kDa isoform a, consisting of 21 exons, is regarded as an extracellular protein. Full-length ~580 kDa isoform b is a complex transmembrane protein composed of three regions: a large extracellular region consisting of an N-terminal signal peptide, laminin G-like domain (LamGL), laminin domain N-terminal (LamNT), laminin-type EGF-like modules (EGF-Lam), fibronectin type III (FN3) repeats, laminin G domains (LamG); a transmembrane region(TM); and a cytoplasmic C-terminal domain containing a PDZ-binding motif[8, 9]. Usherin is distributed in the periciliary membrane complex and synapse in photoreceptors. All USH1 and USH2 proteins are organized as protein networks by the scaffold proteins harmonin (USH1C), whirlin (USH2D) and SANS (USH1G). Usherin(USH2A) and VLGR1b(USH2C) are part of the links that are intracellularly attached to the scaffold proteins. On the other hand, during the differentiation of the hair bundle, both USH1 and USH2 proteins contribute to the formation of side links located at the tip and the base of the stereocilia, respectively. They exist in multiprotein complexes that work together as molecular networks to anchor them to the stereocilia actin filaments[10-14].

The homozygous mutation (p.Ser2828*) in USH2A made the premature termination translation, as a result, 19 FN3, TM and PDZ-binding motif domains were deleted. FN3 plays a key role in cell adhesion, cell morphology, thrombosis, cell migration, and embryonic differentiation and pathophysiologic processes such as angiogenesis and vascular remodeling[15]. TM exists at the base of the differentiating stereocilia, and it makes up the mechanosensitive hair bundles receptive to sound. PDZ-binding motif domains provide the anchoring of interstereocilia lateral links to the F-acin core of stereocilia[16]. In this regard, we suppose that the absence these domains corresponding to the incompleteness of usherin, which might probably have in turn affected the process of stereocilia differentiation and maturation, resulting in a milder stereocilia dysmorphic phenotype. Several positions are found associated exclusively with pathogenic of FN3 in usherin[17], which support our hypothesis. However, the pathway needs to be confirmed by molecular experiments in the future.

Whole-genome sequencing (WGS), whole-exome sequencing (WES) and TES is three major methodologies for molecular diagnosis of IRD. WGS is useful for detecting copy number variations and structural variations[18]. WES is especially useful for identifying novel IRD related genes. TES is an accurate, rapid and cost-effective approach for screening of multiple genes[19], but still have some major limitations, such as detecting variants in low-depth regions and copy number variations[18, 20]. Because of the higher cost, both of the WGS and WES is less widely used than TES. TES is suitable for molecular diagnosis of USH. As the great diversity of various types of pathogenic genes and the frequent occurrence of new mutations, array-based diagnosis often could not accurately reflect the pathogenicity. USH pathogenic genes have many subtypes and numerous exons. At present, more than 400 coding exons have been commented.[21] Therefore, a higher diagnosis rate can be obtained by using sequence-based diagnosis method.

Conclusion

Here, we report a novel homozygous mutation, c.8483_8486del, in the USH2A gene through TES techniques. The mutation truncated the translation of the USH2A gene, and 19 FN3, TM and PDZ-binding motif domain were lost, which influenced the function of stereocilia. We broadened the spectrum of mutations in the disease and provided a new locus for gene therapy of the disease. The combination of TES molecular diagnosis and clinical information can help USH patients obtain more accurate diagnoses.

Declarations

Ethics approval and consent to participate

This study adhered to the tenets of the Declaration of Helsinki and was approved by institution review board of Tianjin Medical University Eye Hospital(Tianjin, China), Number´╝Ü2019KY-02. The

written informed consent to participate was obtained from the patients.

Consent to publication

Written informed consent for publication of their clinical images and genetic results in this manuscript was obtained from the patients according to Ethic Committee Regulations. 

Availability of data and materials

All data are fully available without restriction.

Competing interests

The authors declare that they have no competing interests.

Fundings

This study was supported by Natural Science Foundation of Tianjin City (Award number: 17JCYBJC27200; Recipient: Zhiqing Li) and Tianjin Clinical Key Subject Construction Project (Award number: TJLCZDXKQ022; Recipient: Dongjun Xing) in China.

Author Contributions

XL and ZL conceived the idea and take responsibility for the integrity of the data. DX and HZ collected the samples, performed data analyses and wrote the manuscript. Both of authors also contributed equally to this work. RY, LW and LH performed the experiments. All authors have read and approved the final manuscript.

Acknowledgements

We thank all the family members for their participation in this study.

Abbreviations

USH: Usher syndrome; OCT: Optical coherence tomography; OCTA: Optical coherence tomography angiography; IRD: inherited retinal disease; FN3: Fibronectin type 3 domains; TES: Targeted exome sequencing; RP: retinal pigmentosa; BCVA: best-corrected visual acuity; FAF: fundus autofluorescence; SOAP: Short Oligonucleotide Analysis Package; SNPs: single nucleotide polymorphisms; PCR: polymerase chain reaction; PDB: Protein Data Bank; FAZ: foveal avascular area; LamGL: laminin G-like domain; LamNT: laminin domain N-terminal; EGF-Lam: laminin-type EGF-like modules; LamG: laminin G domains 

References

  1. Blanco-Kelly F, Jaijo T, Aller E, et al. Clinical aspects of Usher syndrome and the USH2A gene in a cohort of 433 patients. JAMA Ophthalmol. 2015;133(2):157-64.
  2. Cohen M, Bitner-Glindzicz M, Luxon L. The changing face of Usher syndrome: clinical implications. Int J Audiol. 2007;46(2):82-93.
  3. Hartel BP, Löfgren M, Huygen PL, et al. A combination of two truncating mutations in USH2A causes more severe and progressive hearing impairment in Usher syndrome type IIa. Hear Res. 2016;339:60-8.
  4. Hope CI, Bundey S, Proops D, Fielder AR. Usher syndrome in the city of Birmingham--prevalence and clinical classification. Br J Ophthalmol. 1997;81(1):46-53.
  5. Pehere NK, Khanna RC, Marlapati R, Sannapaneni K. Prevalence of ophthalmic disorders among hearing-impaired school children in Guntur district of Andhra Pradesh. Indian J Ophthalmol. 2019. 67(4): 530-535.
  6. Huang, X.F., et al., 2013. Targeted exome sequencing identified novel USH2A mutations in Usher syndrome families. PLoS ONE 8 (5), e63832.
  7. Jouret G, Poirsier C, Spodenkiewicz M, et al. Genetics of Usher Syndrome: New Insights From a Meta-analysis. Otol Neurotol. 2019;40(1):121-129.
  8. Méndez-Vidal C, González-Del Pozo M, Vela-Boza A, et al. Whole-exome sequencing identifies novel compound heterozygous mutations in USH2A in Spanish patients with autosomal recessive retinitis pigmentosa. Mol Vis. 2013;19:2187-95.
  9. Pierrache LH, Hartel BP, van Wijk E, et al. Visual Prognosis in USH2A-Associated Retinitis Pigmentosa Is Worse for Patients with Usher Syndrome Type IIa Than for Those with Nonsyndromic Retinitis Pigmentosa. Ophthalmology. 2016;123(5):1151-60.
  10. Bonnet C, El-Amraoui A. Usher syndrome (sensorineural deafness and retinitis pigmentosa): pathogenesis, molecular diagnosis and therapeutic approaches. Curr Opin Neurol. 2012;25(1):42-9.
  11. Cosgrove D, Zallocchi M. Usher protein functions in hair cells and photoreceptors. Int J Biochem Cell Biol. 2014;46:80-9.
  12. Sorusch, N., et al., 2014. Usher syndrome protein network functions in the retina and their relation to other retinal ciliopathies. Adv. Exp. Med. Biol. 801 527-533.
  13. Mathur, P., Yang, J., 2015. Usher syndrome: Hearing loss, retinal degeneration and associated abnormalities. Biochim. Biophys. Acta 1852 (3), 406-420.
  14. Kremer, H., et al., 2006. Usher syndrome: molecular links of pathogenesis, proteins and pathways. Hum. Mol. Genet. 15 Spec No 2 R262-270.
  15. Maurer LM, Ma W, Mosher DF. Dynamic structure of plasma fibronectin. Crit Rev Biochem Mol Biol. 2015;51(4):213-27.
  16. Adato A, Lefèvre G, Delprat B, Michel V, Michalski N, Chardenoux S, et al. Usherin, the defective protein in Usher syndrome type IIA, is likely to be a component of interstereocilia ankle links in the inner ear sensory cells. Hum Mol Genet. 2005;14(24):3921-32. doi:10.1093/hmg/ddi416.
  17. Baux D, Blanchet C, Hamel C, et al. Enrichment of LOVD-USHbases with 152 USH2A genotypes defines an extensive mutational spectrum and highlights missense hotspots. Hum Mutat. 2014. 35(10): 1179-86.
  18. Huang XF, Mao JY, Huang ZQ, et al. Genome-Wide Detection of Copy Number Variations in Unsolved Inherited Retinal Disease. Invest Ophthalmol Vis Sci. 2017. 58(1): 424-429.
  19. Huang XF, Wu J, Lv JN, Zhang X, Jin ZB. Identification of false-negative mutations missed by next-generation sequencing in retinitis pigmentosa patients: a complementary approach to clinical genetic diagnostic testing. Genet Med. 2015. 17(4): 307-11.
  20. Huang XF, Huang F, Wu KC, et al. Genotype-phenotype correlation and mutation spectrum in a large cohort of patients with inherited retinal dystrophy revealed by next-generation sequencing. Genet Med. 2015. 17(4): 271-8.
  21. Jiang L, Liang X, Li Y, et al. Comprehensive molecular diagnosis of 67 Chinese Usher syndrome probands: high rate of ethnicity specific mutations in Chinese USH patients. Orphanet J Rare Dis. 2015;10:110.

Table

Table 1 List of 381 genes chosen for targeted gene capture

ABCA4 

ADAMTSL4 

ALMS1 

ARMS2 

BBS12 

BLOC1S6 

C5AR2 

CAPN5

CEP290 

CFI 

CLRN1 

COL11A1 

CRX 

CYP4V2 

DTNBP1 

ERCC8

FSCN2 

GNB3 

GUCA1B 

HK1 

HTRA1 

IMPG2 

KIAA1549 

LRIT3

MAPKAPK3 

MKS1 

NDP 

NRL 

OPN1MW2 

PCDH15 

PDE6H 

PEX16

PEX7 

PNPLA6 

PRPF31 

RABGGTA 

RD3 

RIMS1 

RPGR 

SEMA4A

SLC25A15 

SPATA7 

TENM3 

TMEM231 

TRPM1 

TUBGCP4 

USH2A 

YAP1

ABCB6 

ADGRA3 

ANO5 

ASRGL1 

BBS2 

BMP4 

C5orf42 

CC2D2A

CEP41 

CHM 

CLUAP1 

COL11A2 

CSPP1 

DHCR7 

EFEMP1 

EXOSC2

FXN 

GNPTG 

GUCY2D 

HMCN1 

IDH3B 

INPP5E 

KIF11 

LRP5

MC1R 

MLPH 

NEK2 

NYX 

OPN1SW 

PCYT1A 

PDZD7 

PEX19

PGK1 

POC1B 

PRPF4 

RABGGTB 

RDH11 

RLBP1 

RPGRIP1 

SGCD

SLC26A4 

SPP2 

TIMP3 

TMEM237 

TSPAN12 

TUBGCP6 

VCAN 

ZNF408

ABCC6 

ADGRV1 

AP3B1 

ATF6 

BBS4 

C10orf11 

C8orf37 

CCDC28B

CEP78 

CIB2 

CNGA1 

COL18A1 

CST3 

DHDDS 

ELOVL4 

EYA1

FZD4 

GPR143 

GUSB 

HMX1 

IDUA 

INVS 

KIF7 

LRPAP1

MCOLN1 

MPDZ 

NMNAT1 

OAT 

OR2W3 

PDCD2 

PEX1 

PEX2

PGR 

POMGNT1 

PRPF6 

RAX 

RDH12 

ROM1 

RPGRIP1L 

SHH

SLC38A8 

STRA6 

TINF2 

TMEM67 

TTC21B 

TULP1 

VHL 

ZNF423

ABHD12 

ADIPOR1 

APOE 

ATOH7 

BBS5 

C1QTNF5 

C9 

CDH23

CERKL 

CLDN19 

CNGA3 

COL2A1 

CTC1 

DHX38 

ERCC2 

EYS

GDF3 

GPR179 

HARS 

HPS1 

IFT140 

IQCB1 

KIZ 

LYST

MERTK 

MTHFR 

NPHP1 

OCA2 

OTX2 

PDE6A 

PEX10 

PEX26

PHYH 

PPT1 

PRPF8 

RAX2 

RDH5 

RP1 

RS1 

SHOX

SLC39A5 

TBK1 

TLR3 

TMEM98 

TTC8 

TYR 

VPS13B 

ZNF513

ACBD5 

AGBL5 

ARL13B 

ATXN7 

BBS7 

C2 

CA4 

CDH3

CFB 

CLN3 

CNGB1 

COL4A1 

CTNNA1 

DMD 

ERCC3 

FAM161A

GDF6 

GRK1 

HEXA 

HPS3 

IFT172 

ITGA2B 

KLHL7 

LZTFL1

MFRP 

MTTP 

NPHP3 

OFD1 

P3H2 

PDE6B 

PEX11B 

PEX3

PITPNM3 

PRCD 

PRPH2 

RB1 

RGR 

RP1L1 

SAG 

SIX5

SLC45A2 

TCTN1 

TLR4 

TOPORS 

TTLL5 

TYRP1 

VSX2 

ZNF644

ACO2 

AHI1 

ARL2BP 

BBIP1 

BBS9 

C21orf2 

CABP4 

CDHR1

CFH 

CLN5 

CNGB3 

COL9A1 

CTSD 

DNAJC5 

ERCC4 

FBLN5

GMPPB 

GRM6 

HEXB 

HPS4 

IFT27 

ITGB3 

LAMA1 

MAK

MFSD8 

MVK 

NPHP4 

OPA3 

PANK2 

PDE6C 

PEX12 

PEX5

PLA2G5 

PRDM13 

PRSS56 

RBP3 

RGS9 

RP2 

SALL2 

SIX6

SLC7A14 

TCTN2 

TMEM126A 

TPP1 

TTPA 

UNC119 

WDPCP 

ADAM9

AIPL1 

ARL3 

BBS1 

BEST1 

C2orf71 

CACNA1F 

CEP164 

CFHR1

CLN6 

CNNM4 

COL9A2 

CTSF 

DRAM2 

ERCC5 

FBN2 

GNAT1

GRN 

HFE 

HPS5 

IMPDH1 

ITM2B 

LCA5 

MAN2B1 

MITF

MYO5A 

NR2E1 

OPN1LW 

PAX2 

PDE6D 

PEX13 

PEX5L 

PLEKHA1

PROM1 

RAB27A 

RBP4 

RGS9BP 

RP9 

SCO2 

SLC24A1 

SNRNP200

TCTN3 

TMEM138 

TRIM32 

TTR 

USH1C 

WDR19 

ADAMTS18 

ALDH1A3

ARL6 

BBS10 

BLOC1S3 

C3 

CACNA2D4 

CEP250 

CFHR3 

CLN8

CNOT9 

CRB1 

CX3CR1 

DTHD1 

ERCC6 

FLVCR1 

GNAT2 

GUCA1A

HGSNAT 

HPS6 

IMPG1 

KCNV2 

LRAT 

MANBA 

MKKS 

MYO7A

NR2E3 

OPN1MW 

PAX6 

PDE6G 

PEX14 

PEX6 

PLK4 

PRPF3

RAB28 

RCBTB1 

RHO 

RPE65 

SDCCAG8 

SLC24A5 

SOD1 

TEAD1

TMEM216 

TRNT1 

TUB 

USH1G 

WHRN 

-