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Targeted next generation sequencing identified a novel mutation in MYO7A causing Usher syndrome type 1 in an Iranian consanguineous pedigree
International Journal of Pediatric Otorhinolaryngology 104 (2018) 10–13
Contents lists available at ScienceDirect
International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl
Targeted next generation sequencing identified a novel mutation in MYO7A causing Usher syndrome type 1 in an Iranian consanguineous pedigree
MARK
Daniz Kooshavara,1, Masoumeh Razipoura,1, Morteza Movasatb, Mohammad Keramatipoura,∗ a b
Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran Eye Research Center, Farabi Eye Hospital, Tehran University of Medical Sciences, Tehran, Iran
A R T I C L E I N F O
A B S T R A C T
Keywords: Usher syndrome MYO7A Mutation Targeted next generation sequencing
Background: Usher syndrome (USH) is characterized by congenital hearing loss and retinitis pigmentosa (RP) with a later onset. It is an autosomal recessive trait with clinical and genetic heterogeneity which makes the molecular diagnosis much difficult. In this study, we introduce a pedigree with two affected members with USH type 1 and represent a cost and time effective approach for genetic diagnosis of USH as a genetically heterogeneous disorder. Methods: Target region capture in the genes of interest, followed by next generation sequencing (NGS) was used to determine the causative mutations in one of the probands. Then segregation analysis in the pedigree was conducted using PCR-Sanger sequencing. Results: Targeted NGS detected a novel homozygous nonsense variant c.4513G > T (p.Glu1505Ter) in MYO7A. The variant is segregating in the pedigree with an autosomal recessive pattern. Conclusion: In this study, a novel stop gained variant c.4513G > T (p.Glu1505Ter) in MYO7A was found in an Iranian pedigree with two affected members with USH type 1. Bioinformatic as well as pedigree segregation analyses were in line with pathogenic nature of this variant. Targeted NGS panel was showed to be an efficient method for mutation detection in hereditary disorders with locus heterogeneity.
1. Introduction Usher syndrome (USH) is a genetically and clinically heterogeneous disorder with an autosomal recessive inheritance pattern that is characterized by congenital, bilateral deafness and a later onset of vision impairment, caused by retinitis pigmentosa (RP) [1–3]. USH accounts for about 50% of all deafblindness cases [1,4] and its prevalence ranges from 1/6000 to 1/10,000 [2]. According to the severity of the sensorineural hearing loss (SNHL), the age of onset of RP, and the presence or absence of vestibular dysfunction, USH is divided into three clinical subtypes as USH type 1, USH type 2 and USH type 3. USH type 1 is the most severe form of USH and is defined by congenital bilateral profound sensorineural hearing loss, severe vestibular dysfunction, and prepubertal onset of RP. USH type 2 patients suffer from mild to severe congenital hearing impairment and RP with prepubertal onset. Their vestibular function is normal. USH type 3 is characterized by mild and progressive hearing impairment and variability in age of onset of RP and vestibular function [1,2]. To date, 14 genes have been identified to be associated with Usher syndrome. The USH type 1 genes include MYO7A encoding the motor ∗
1
protein myosin VIIa, USH1C encoding harmonin, USH1G encoding SANS, CDH23 and PCDH15 encoding cadherin 23 and protocadherin 15 respectively, and CIB2 which encodes calcium and integrin binding protein 2. The USH type 2 genes are USH2A encoding usherin and ADGRV1 (GPR98) encoding adhesion G protein-coupled receptor VI. Another gene associated with USH type 2 is WHRN (DFNB31) encoding whirlin. The USH type 3 gene is CLRN1 encoding Clarin1 [2,5]. Furthermore, HARS encoding histidyl tRNA synthetase and ABHD12 encoding Lysophosphatidylserine (LPS) lipase have been identified in atypical USH [6,7]. In addition, two modifier genes have been recently identified, PDZD7 and CEP250 encoding PDZ domain containing protein 7 and centrosome associated protein 250, respectively [8,9]. The USH proteins are organized in a mutual “interactome” in retinal photoreceptors and inner ear hair cells. Dysfunction or absence of any of these molecules can cause the clinical symptoms of USH [10,11]. Because there are several genes that can cause USH with a large number of coding exons, identification of causative mutations is costly and time-consuming by traditional methods like direct Sanger sequencing. NGS has provided a high-throughput and cost-effective approach for detection of mutations in genetic diseases with high locus
Corresponding author. Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Poursina Ave., Keshavarz Blvd., Tehran 1417613151, Iran. E-mail address: [email protected] (M. Keramatipour). These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ijporl.2017.10.022 Received 3 September 2017; Received in revised form 13 October 2017; Accepted 13 October 2017 Available online 18 October 2017 0165-5876/ © 2017 Elsevier B.V. All rights reserved.
International Journal of Pediatric Otorhinolaryngology 104 (2018) 10–13
D. Kooshavar et al.
2.4. Validation of the detected variant
heterogeneity such as USH. In this study, a targeted NGS method was used for genetic investigation of an Iranian pedigree with two affected members suspected of USH type 1. Here we represent the detailed data about the identification of a novel variant in this pedigree.
Polymerase Chain Reaction (PCR) followed by Sanger sequencing was used to validate the detected variant and analyze the segregation in the pedigree. The sequences of designed primers are as follows; Forward primer: TGGCACGTAGCGAGTTTGT and Reverse primer: CCCCACCACTGTTATGCGAG. PCR reaction was performed in 30 μl total volume containing 15 μl the Taq DNA Polymerase 2× Master Mix (Ampliqon A/S, Odense, Denmark), 10.5 μl DH2O, 1.5 μl of each 5 pM primer and 1.5 μl of 50 ng/μl DNA. The PCR condition was based on the touchdown PCR protocol for efficient amplification of fragments. Initial denaturation was performed at 95 °C for 5 min, followed by 37 cycles of 95 °C for 30 s, annealing step for 30 s, 72 °C for 40 s, and a final extension of 72 °C for 7 min. The annealing temperature was decreased 1 °C in every cycle from 72 to a touchdown temperature at 60 °C; this final annealing temperature was maintained for the remaining 25 cycles. In addition, bioinformatic investigation was conducted by multiple in-silico predictive tools including Mutation Taster (http://www. mutationtaster.org) and CADD (Combined Annotation Dependent Depletion, http://cadd.gs.washington.edu/) to determine the pathogenicity of the novel variant which was not previously reported in any of the population/disease databases such as ExAC, 1000G, dbSNP, ClinVar, and HGMD®. Furthermore, the phylogenetic comparison was performed to show the conservation of the residue using the consurf server (http://consurf.tau.ac.il/).
2. Materials and methods 2.1. Subjects A pedigree with two affected members was investigated. A 16 years old male with profound hearing loss, bilateral rod-cone degeneration, and clinical diagnosis of Usher syndrome type 1 who was a result of a consanguineous marriage was referred to Farabi Eye Hospital, Tehran, Iran for genetic counseling/analysis. His hearing loss was documented at about the age of 1 year. According to the parents' reports, he had a delay in walking and language development. He started to walk at the age of 2 years. This history supports vestibular dysfunction in the proband. His ophthalmologic problems were initiated with night blindness and amblyopia at the age of 3–4 years. Eye examination including electroretinography (ERG) at the age of 14 reported bilateral rod-cone degeneration. The proband had a one-year-old cousin, also result of a consanguineous marriage, with profound hearing loss. Fig. 1 demonstrates the pedigree of the family. Written informed consent was taken from the parents of the patients and peripheral blood samples were collected from the patients and their family.
3. Results
2.2. DNA extraction
Targeted NGS of 13 Usher syndrome-related genes revealed a novel homozygous stop gained variant in the proband V:3. The variant was a G-to-T transition at the first base of codon 1505 in exon 34 of the MYO7A that causes the substitution of Glu (GAG) by a stop codon (TAG). As demonstrated in Fig. 1, segregation of the variant was investigated in the pedigree. The variant was well-cosegregated with the disease in the pedigree. Both affected pedigree members were TT homozygotes, their parents and some other pedigree members were G/T heterozygote carriers, and some other pedigree members were normal GG homozygotes. Fig. 2 demonstrates sequence chromatograms of the variant in two affected members and their parents. According to our survey, the variant c.4513G > T (p.Glu1505Ter) in MYO7A, has not been previously reported in any of the population/
Genomic DNA was extracted from whole peripheral blood using the GeneAll®Exgene™ kit (GeneAll Biotechnology Co., LTD, Seoul, Korea), according to the manufacturer's instructions. 2.3. Targeted NGS Target region capturing of 13 Usher syndrome-related genes (CDH23, DFNB31, GPR98, MYO7A, PCDH15, USH1C, USH1G, USH2A, CLRN1, HARS, PDZD7, CIB2, and ABHD12) was performed with Nimblegen chip. NGS was performed on an Illumina HiSeq NGS System (Illumina Inc., San Diego, CA, USA) provided by Beijing Genomics Institute (BGI).
Fig. 1. The pedigree of the Usher syndrome family. Genotype of the variant c.4513G > T in studied members is shown in the pedigree. Arrows indicate the probands.
11
International Journal of Pediatric Otorhinolaryngology 104 (2018) 10–13
D. Kooshavar et al.
Fig. 2. Sequence chromatograms of the variant c.4513G > T in A: 1. The proband (V:3) 2. His father 3. His mother. B: 1. The proband (V:8) 2. His father 3. His mother.
disease databases such as ExAC, 1000G, dbSNP, ClinVar, and HGMD®. In addition, this variant has a frequency of zero in the largest available local database of genomic variations in Iranian population (Pishgam Biotech Company, Tehran, Iran), which consists of whole exome sequencing (WES) data of more than 1000 individuals. The bioinformatic investigation was performed using online tools including Mutation Taster and CADD to predict the possible effect of the variant on the function of the protein. The variant was predicted with high confidence to be “disease causing” by Mutation Taster and with CADD PHRED Score of 51. Furthermore, the phylogenetic comparison was performed to show the conservation of the Glu1505- Myosin VIIa residue using the consurf server (Table 1). We found that Glu1505Myosin VIIa is considered a relatively highly conserved residue.
Table 1 Conservation status of position of the residue Glu1505- Myosin VIIa. Protein
Position
Amino acid
Conservation scorea (Scaleb)
MSA datac
Residue variety
Myosin VIIa
1505
Glu
−0.798 (8)
127/ 131
N/E/D
a
The normalized conservation scores. Scale representing the conservation scores (9 – conserved, 1 – variable). c The number of aligned sequences having an amino acid (non-gapped) from the overall number of sequences at each position. b
1. As a result, the novel homozygous variant c.4513G > T (p.Glu1505Ter) in MYO7A was detected which was segregated in an autosomal recessive pattern in the pedigree. Regarding the genetically intact nature of the Iranian population, identification of novel variants was not unexpected. MYO7A is located on the long arm of chromosome 11 (11q13.5). It
4. Discussion In this study, we attempted to target a group of genes responsible for USH to detect causative mutations in an Iranian patient with USH type 12
International Journal of Pediatric Otorhinolaryngology 104 (2018) 10–13
D. Kooshavar et al.
Fig. 3. Schematic representation of the Myosin VIIa and the position of the detected stop-gained variant.
genetic evaluation of disorders with genetic heterogeneity and speed up the clinical diagnosis and decision-making in a cost-effective manner.
has 49 exons (48 coding exons) containing 7462 base pairs encoding a 2215-amino acid protein, named Myosin VIIa [12]. Myosin VIIa consists of a motor head domain, a neck region composed of five calmodulin-binding IQ motifs, and a long tail composed of a short coiled-coil domain followed by two FERM domains, two MyTH4 domains and an Src homology 3 (SH3) domain [Fig. 3]. The first 17 coding exons of MYO7A code the head of Myosin VIIa, the next 3 exons code the neck and the last 28 exons code the tail of the protein [3,5,12]. Myosin VIIa is required for normal hearing and vision. MYO7A is the most common mutated gene underlying USH type 1 which is characterized by congenital profound hearing loss and progressive retinal degeneration. It causes 29% to about 50% of USH type 1 cases in different populations [4]. The variant has been expected to result in a truncated protein and activate nonsense mediated decay (NMD) pathway. NMD is a translation-coupled mechanism which targets mRNAs containing premature termination codons (PTCs) [13,14]. Several studies reported nonsense mutations in terminal exons of MYO7A that cause USH [15–25]. According to the Ensembl genome browser (https://www.ensembl.org/ index.html), more than 20 stop gained variants were reported downstream of residue Glu1505-Myosin VIIa that cause USH. These reports are in favor of the pathogenic nature of the novel variant c.4513G > T (p.Glu1505Ter), detected in our study. In addition, the variant c.4513G > T (p.Glu1505Ter), creates a stop codon in exon 34 of MYO7A which eliminates a large part of the protein tail, including a part of FERM 1 domain and also three other domains, SH3, MyTH4 2 and FERM 2. As the Myosin VIIa tail play a crucial role in determining the functional specificity of each myosin in binding to cargo molecules, regulatory factors, and components of the transduction pathways [12], elimination of the tail appears to be damaging to protein function. This effect supports the pathogenic nature of the variant, independent of NMD effect. Because of the financial constraints, in vitro or in vivo functional studies were not conducted to show the possible damaging effect of the novel variant on the gene or gene product. However, based on American College of Medical Genetics and Genomics (ACMG) guideline [26], there is enough evidence for pathogenicity of the novel variant c.4513G > T (p.Glu1505Ter) including a very strong evidence (being a null variant), a moderate evidence (absent from controls in population databases and also absent in Iranian local database) and two supporting evidence (cosegregation with the disease in both affected members in the pedigree and computational data). Therefore, according to ACMG guidelines, this novel variant can be classified as a pathogenic variant. Regarding that, clinical diagnosis of the disease is confirmed and prenatal diagnosis (PND) and pre-implantation genetic testing (PGD) can be recommended in the future pregnancies of the pedigree. In the present study, we have elucidated the genetic cause of USH in an Iranian pedigree that could extend the mutation spectrum of MYO7A in Iranian population. Furthermore, our study represents the advantages of targeted NGS to identify the causative variants in genetically heterogeneous diseases. In fact, targeted NGS can accelerate the
Acknowledgments We are grateful to all the individuals who participated in our study. The authors declare no conflict of interest. References [1] C. Bonnet, A. El-Amraoui, Usher syndrome (sensorineural deafness and retinitis pigmentosa): pathogenesis, molecular diagnosis and therapeutic approaches, Curr. Opin. Neurol. 25 (1) (2012) 42–49. [2] T. Koffler, K. Ushakov, K.B. Avraham, Genetics of hearing loss: Syndromic, Otolaryngol. Clin. N. Am. 48 (6) (2015) 1041–1061. [3] D. Yan, X.Z. Liu, Genetics and pathological mechanisms of Usher syndrome, J. Hum. Genet. 55 (6) (2010) 327–335. [4] J.M. Millán, et al., An update on the genetics of usher syndrome, J. Ophthalmol. 2011 (2011). [5] S. Dad, et al., Usher syndrome in Denmark: mutation spectrum and some clinical observations, Mol. Genet. Genomic Med. 4 (5) (2016) 527–539. [6] E.G. Puffenberger, et al., Genetic mapping and exome sequencing identify variants associated with five novel diseases, PLoS One 7 (1) (2012) e28936. [7] T. Eisenberger, et al., Targeted next-generation sequencing identifies a homozygous nonsense mutation in ABHD12, the gene underlying PHARC, in a family clinically diagnosed with Usher syndrome type 3, Orphanet J. Rare Dis. 7 (1) (2012) 59. [8] I. Ebermann, et al., PDZD7 is a modifier of retinal disease and a contributor to digenic Usher syndrome, J. Clin. Investig. 120 (6) (2010) 1812–1823. [9] S. Khateb, et al., A homozygous nonsense CEP250 mutation combined with a heterozygous nonsense C2orf71 mutation is associated with atypical Usher syndrome, J. Med. Genet. 51 (7) (2014) 460–469. [10] H. Kremer, et al., Usher syndrome: molecular links of pathogenesis, proteins and pathways, Hum. Mol. Genet. 15 (suppl 2) (2006) R262–R270. [11] J. Reiners, et al., Molecular basis of human Usher syndrome: deciphering the meshes of the Usher protein network provides insights into the pathomechanisms of the Usher disease, Exp. Eye Res. 83 (1) (2006) 97–119. [12] C. Petit, Usher syndrome: from genetics to pathogenesis, Annu. Rev. Genom Hum. Genet. 2 (1) (2001) 271–297. [13] N. Hug, D. Longman, J.F. Cáceres, Mechanism and regulation of the nonsense-mediated decay pathway, Nucleic acids Res. 44 (4) (2016) 1483–1495. [14] S. Brogna, J. Wen, Nonsense-mediated mRNA decay (NMD) mechanisms, Nat. Struct. Mol. Biol. 16 (2) (2009) 107–113. [15] T. Jaijo, et al., Mutation profile of the MYO7A gene in Spanish patients with Usher syndrome type I, Hum. Mutat. 27 (3) (2006) 290–291. [16] D. Baux, et al., UMD-USHbases: a comprehensive set of databases to record and analyse pathogenic mutations and unclassified variants in seven Usher syndrome causing genes, Hum. Mutat. 29 (8) (2008). [17] S. Gerber, et al., USH1A: chronicle of a slow death, Am. J. Hum. Genet. 78 (2) (2006) 357. [18] H. Vastinsalo, et al., Two Finnish USH1B patients with three novel mutations in myosin VIIA, Mol. Vis. 12 (2006) 1093–1097. [19] A.-F. Roux, et al., Four-year follow-up of diagnostic service in USH1 patients, Investig. Ophthalmol. Vis. Sci. 52 (7) (2011) 4063–4071. [20] A. Adato, et al., Mutation profile of all 49 exons of the human myosin VIIA gene, and haplotype analysis, in Usher 1B families from diverse origins, Am. J. Hum. Genet. 61 (4) (1997) 813–821. [21] A.R. Janecke, et al., Twelve novel myosin VIIA mutations in 34 patients with Usher syndrome type I: confirmation of genetic heterogeneity, Hum. Mutat. 13 (2) (1999) 133. [22] F.P. Cremers, et al., Development of a genotyping microarray for Usher syndrome, J. Med. Genet. 44 (2) (2007) 153–160. [23] S.G. Jacobson, et al., Usher syndromes due to MYO7A, PCDH15, USH2A or GPR98 mutations share retinal disease mechanism, Hum. Mol. Genet. 17 (15) (2008) 2405–2415. [24] S.G. Jacobson, et al., Retinal disease course in Usher syndrome 1B due to MYO7A mutations, Investig. Ophthalmol. Vis. Sci. 52 (11) (2011) 7924–7936. [25] S.G. Jacobson, et al., Disease boundaries in the retina of patients with Usher syndrome caused by MYO7A gene mutations, Investig. Ophthalmol. Vis. Sci. 50 (4) (2009) 1886–1894. [26] S. Richards, et al., Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the american College of medical genetics and genomics and the association for molecular pathology, Genet. Med. 17 (5) (2015) 405–423.
13
Contents lists available at ScienceDirect
International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl
Targeted next generation sequencing identified a novel mutation in MYO7A causing Usher syndrome type 1 in an Iranian consanguineous pedigree
MARK
Daniz Kooshavara,1, Masoumeh Razipoura,1, Morteza Movasatb, Mohammad Keramatipoura,∗ a b
Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran Eye Research Center, Farabi Eye Hospital, Tehran University of Medical Sciences, Tehran, Iran
A R T I C L E I N F O
A B S T R A C T
Keywords: Usher syndrome MYO7A Mutation Targeted next generation sequencing
Background: Usher syndrome (USH) is characterized by congenital hearing loss and retinitis pigmentosa (RP) with a later onset. It is an autosomal recessive trait with clinical and genetic heterogeneity which makes the molecular diagnosis much difficult. In this study, we introduce a pedigree with two affected members with USH type 1 and represent a cost and time effective approach for genetic diagnosis of USH as a genetically heterogeneous disorder. Methods: Target region capture in the genes of interest, followed by next generation sequencing (NGS) was used to determine the causative mutations in one of the probands. Then segregation analysis in the pedigree was conducted using PCR-Sanger sequencing. Results: Targeted NGS detected a novel homozygous nonsense variant c.4513G > T (p.Glu1505Ter) in MYO7A. The variant is segregating in the pedigree with an autosomal recessive pattern. Conclusion: In this study, a novel stop gained variant c.4513G > T (p.Glu1505Ter) in MYO7A was found in an Iranian pedigree with two affected members with USH type 1. Bioinformatic as well as pedigree segregation analyses were in line with pathogenic nature of this variant. Targeted NGS panel was showed to be an efficient method for mutation detection in hereditary disorders with locus heterogeneity.
1. Introduction Usher syndrome (USH) is a genetically and clinically heterogeneous disorder with an autosomal recessive inheritance pattern that is characterized by congenital, bilateral deafness and a later onset of vision impairment, caused by retinitis pigmentosa (RP) [1–3]. USH accounts for about 50% of all deafblindness cases [1,4] and its prevalence ranges from 1/6000 to 1/10,000 [2]. According to the severity of the sensorineural hearing loss (SNHL), the age of onset of RP, and the presence or absence of vestibular dysfunction, USH is divided into three clinical subtypes as USH type 1, USH type 2 and USH type 3. USH type 1 is the most severe form of USH and is defined by congenital bilateral profound sensorineural hearing loss, severe vestibular dysfunction, and prepubertal onset of RP. USH type 2 patients suffer from mild to severe congenital hearing impairment and RP with prepubertal onset. Their vestibular function is normal. USH type 3 is characterized by mild and progressive hearing impairment and variability in age of onset of RP and vestibular function [1,2]. To date, 14 genes have been identified to be associated with Usher syndrome. The USH type 1 genes include MYO7A encoding the motor ∗
1
protein myosin VIIa, USH1C encoding harmonin, USH1G encoding SANS, CDH23 and PCDH15 encoding cadherin 23 and protocadherin 15 respectively, and CIB2 which encodes calcium and integrin binding protein 2. The USH type 2 genes are USH2A encoding usherin and ADGRV1 (GPR98) encoding adhesion G protein-coupled receptor VI. Another gene associated with USH type 2 is WHRN (DFNB31) encoding whirlin. The USH type 3 gene is CLRN1 encoding Clarin1 [2,5]. Furthermore, HARS encoding histidyl tRNA synthetase and ABHD12 encoding Lysophosphatidylserine (LPS) lipase have been identified in atypical USH [6,7]. In addition, two modifier genes have been recently identified, PDZD7 and CEP250 encoding PDZ domain containing protein 7 and centrosome associated protein 250, respectively [8,9]. The USH proteins are organized in a mutual “interactome” in retinal photoreceptors and inner ear hair cells. Dysfunction or absence of any of these molecules can cause the clinical symptoms of USH [10,11]. Because there are several genes that can cause USH with a large number of coding exons, identification of causative mutations is costly and time-consuming by traditional methods like direct Sanger sequencing. NGS has provided a high-throughput and cost-effective approach for detection of mutations in genetic diseases with high locus
Corresponding author. Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Poursina Ave., Keshavarz Blvd., Tehran 1417613151, Iran. E-mail address: [email protected] (M. Keramatipour). These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ijporl.2017.10.022 Received 3 September 2017; Received in revised form 13 October 2017; Accepted 13 October 2017 Available online 18 October 2017 0165-5876/ © 2017 Elsevier B.V. All rights reserved.
International Journal of Pediatric Otorhinolaryngology 104 (2018) 10–13
D. Kooshavar et al.
2.4. Validation of the detected variant
heterogeneity such as USH. In this study, a targeted NGS method was used for genetic investigation of an Iranian pedigree with two affected members suspected of USH type 1. Here we represent the detailed data about the identification of a novel variant in this pedigree.
Polymerase Chain Reaction (PCR) followed by Sanger sequencing was used to validate the detected variant and analyze the segregation in the pedigree. The sequences of designed primers are as follows; Forward primer: TGGCACGTAGCGAGTTTGT and Reverse primer: CCCCACCACTGTTATGCGAG. PCR reaction was performed in 30 μl total volume containing 15 μl the Taq DNA Polymerase 2× Master Mix (Ampliqon A/S, Odense, Denmark), 10.5 μl DH2O, 1.5 μl of each 5 pM primer and 1.5 μl of 50 ng/μl DNA. The PCR condition was based on the touchdown PCR protocol for efficient amplification of fragments. Initial denaturation was performed at 95 °C for 5 min, followed by 37 cycles of 95 °C for 30 s, annealing step for 30 s, 72 °C for 40 s, and a final extension of 72 °C for 7 min. The annealing temperature was decreased 1 °C in every cycle from 72 to a touchdown temperature at 60 °C; this final annealing temperature was maintained for the remaining 25 cycles. In addition, bioinformatic investigation was conducted by multiple in-silico predictive tools including Mutation Taster (http://www. mutationtaster.org) and CADD (Combined Annotation Dependent Depletion, http://cadd.gs.washington.edu/) to determine the pathogenicity of the novel variant which was not previously reported in any of the population/disease databases such as ExAC, 1000G, dbSNP, ClinVar, and HGMD®. Furthermore, the phylogenetic comparison was performed to show the conservation of the residue using the consurf server (http://consurf.tau.ac.il/).
2. Materials and methods 2.1. Subjects A pedigree with two affected members was investigated. A 16 years old male with profound hearing loss, bilateral rod-cone degeneration, and clinical diagnosis of Usher syndrome type 1 who was a result of a consanguineous marriage was referred to Farabi Eye Hospital, Tehran, Iran for genetic counseling/analysis. His hearing loss was documented at about the age of 1 year. According to the parents' reports, he had a delay in walking and language development. He started to walk at the age of 2 years. This history supports vestibular dysfunction in the proband. His ophthalmologic problems were initiated with night blindness and amblyopia at the age of 3–4 years. Eye examination including electroretinography (ERG) at the age of 14 reported bilateral rod-cone degeneration. The proband had a one-year-old cousin, also result of a consanguineous marriage, with profound hearing loss. Fig. 1 demonstrates the pedigree of the family. Written informed consent was taken from the parents of the patients and peripheral blood samples were collected from the patients and their family.
3. Results
2.2. DNA extraction
Targeted NGS of 13 Usher syndrome-related genes revealed a novel homozygous stop gained variant in the proband V:3. The variant was a G-to-T transition at the first base of codon 1505 in exon 34 of the MYO7A that causes the substitution of Glu (GAG) by a stop codon (TAG). As demonstrated in Fig. 1, segregation of the variant was investigated in the pedigree. The variant was well-cosegregated with the disease in the pedigree. Both affected pedigree members were TT homozygotes, their parents and some other pedigree members were G/T heterozygote carriers, and some other pedigree members were normal GG homozygotes. Fig. 2 demonstrates sequence chromatograms of the variant in two affected members and their parents. According to our survey, the variant c.4513G > T (p.Glu1505Ter) in MYO7A, has not been previously reported in any of the population/
Genomic DNA was extracted from whole peripheral blood using the GeneAll®Exgene™ kit (GeneAll Biotechnology Co., LTD, Seoul, Korea), according to the manufacturer's instructions. 2.3. Targeted NGS Target region capturing of 13 Usher syndrome-related genes (CDH23, DFNB31, GPR98, MYO7A, PCDH15, USH1C, USH1G, USH2A, CLRN1, HARS, PDZD7, CIB2, and ABHD12) was performed with Nimblegen chip. NGS was performed on an Illumina HiSeq NGS System (Illumina Inc., San Diego, CA, USA) provided by Beijing Genomics Institute (BGI).
Fig. 1. The pedigree of the Usher syndrome family. Genotype of the variant c.4513G > T in studied members is shown in the pedigree. Arrows indicate the probands.
11
International Journal of Pediatric Otorhinolaryngology 104 (2018) 10–13
D. Kooshavar et al.
Fig. 2. Sequence chromatograms of the variant c.4513G > T in A: 1. The proband (V:3) 2. His father 3. His mother. B: 1. The proband (V:8) 2. His father 3. His mother.
disease databases such as ExAC, 1000G, dbSNP, ClinVar, and HGMD®. In addition, this variant has a frequency of zero in the largest available local database of genomic variations in Iranian population (Pishgam Biotech Company, Tehran, Iran), which consists of whole exome sequencing (WES) data of more than 1000 individuals. The bioinformatic investigation was performed using online tools including Mutation Taster and CADD to predict the possible effect of the variant on the function of the protein. The variant was predicted with high confidence to be “disease causing” by Mutation Taster and with CADD PHRED Score of 51. Furthermore, the phylogenetic comparison was performed to show the conservation of the Glu1505- Myosin VIIa residue using the consurf server (Table 1). We found that Glu1505Myosin VIIa is considered a relatively highly conserved residue.
Table 1 Conservation status of position of the residue Glu1505- Myosin VIIa. Protein
Position
Amino acid
Conservation scorea (Scaleb)
MSA datac
Residue variety
Myosin VIIa
1505
Glu
−0.798 (8)
127/ 131
N/E/D
a
The normalized conservation scores. Scale representing the conservation scores (9 – conserved, 1 – variable). c The number of aligned sequences having an amino acid (non-gapped) from the overall number of sequences at each position. b
1. As a result, the novel homozygous variant c.4513G > T (p.Glu1505Ter) in MYO7A was detected which was segregated in an autosomal recessive pattern in the pedigree. Regarding the genetically intact nature of the Iranian population, identification of novel variants was not unexpected. MYO7A is located on the long arm of chromosome 11 (11q13.5). It
4. Discussion In this study, we attempted to target a group of genes responsible for USH to detect causative mutations in an Iranian patient with USH type 12
International Journal of Pediatric Otorhinolaryngology 104 (2018) 10–13
D. Kooshavar et al.
Fig. 3. Schematic representation of the Myosin VIIa and the position of the detected stop-gained variant.
genetic evaluation of disorders with genetic heterogeneity and speed up the clinical diagnosis and decision-making in a cost-effective manner.
has 49 exons (48 coding exons) containing 7462 base pairs encoding a 2215-amino acid protein, named Myosin VIIa [12]. Myosin VIIa consists of a motor head domain, a neck region composed of five calmodulin-binding IQ motifs, and a long tail composed of a short coiled-coil domain followed by two FERM domains, two MyTH4 domains and an Src homology 3 (SH3) domain [Fig. 3]. The first 17 coding exons of MYO7A code the head of Myosin VIIa, the next 3 exons code the neck and the last 28 exons code the tail of the protein [3,5,12]. Myosin VIIa is required for normal hearing and vision. MYO7A is the most common mutated gene underlying USH type 1 which is characterized by congenital profound hearing loss and progressive retinal degeneration. It causes 29% to about 50% of USH type 1 cases in different populations [4]. The variant has been expected to result in a truncated protein and activate nonsense mediated decay (NMD) pathway. NMD is a translation-coupled mechanism which targets mRNAs containing premature termination codons (PTCs) [13,14]. Several studies reported nonsense mutations in terminal exons of MYO7A that cause USH [15–25]. According to the Ensembl genome browser (https://www.ensembl.org/ index.html), more than 20 stop gained variants were reported downstream of residue Glu1505-Myosin VIIa that cause USH. These reports are in favor of the pathogenic nature of the novel variant c.4513G > T (p.Glu1505Ter), detected in our study. In addition, the variant c.4513G > T (p.Glu1505Ter), creates a stop codon in exon 34 of MYO7A which eliminates a large part of the protein tail, including a part of FERM 1 domain and also three other domains, SH3, MyTH4 2 and FERM 2. As the Myosin VIIa tail play a crucial role in determining the functional specificity of each myosin in binding to cargo molecules, regulatory factors, and components of the transduction pathways [12], elimination of the tail appears to be damaging to protein function. This effect supports the pathogenic nature of the variant, independent of NMD effect. Because of the financial constraints, in vitro or in vivo functional studies were not conducted to show the possible damaging effect of the novel variant on the gene or gene product. However, based on American College of Medical Genetics and Genomics (ACMG) guideline [26], there is enough evidence for pathogenicity of the novel variant c.4513G > T (p.Glu1505Ter) including a very strong evidence (being a null variant), a moderate evidence (absent from controls in population databases and also absent in Iranian local database) and two supporting evidence (cosegregation with the disease in both affected members in the pedigree and computational data). Therefore, according to ACMG guidelines, this novel variant can be classified as a pathogenic variant. Regarding that, clinical diagnosis of the disease is confirmed and prenatal diagnosis (PND) and pre-implantation genetic testing (PGD) can be recommended in the future pregnancies of the pedigree. In the present study, we have elucidated the genetic cause of USH in an Iranian pedigree that could extend the mutation spectrum of MYO7A in Iranian population. Furthermore, our study represents the advantages of targeted NGS to identify the causative variants in genetically heterogeneous diseases. In fact, targeted NGS can accelerate the
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