Muhammad Waqar Arshad ( Department of Bioinformatics and Biotechnology, International Islamic University, Islamabad, Pakistan, Department of Biology )
Yujin Lee ( Sungshin Women's University, Seoul, Republic of Korea. )
Muhammad Arshad Malik ( Department of Bioinformatics and Biotechnology, International Islamic University, Islamabad, Pakistan, Department of Biology )
Jahangir Khan ( Department of Bioinformatics and Biotechnology, International Islamic University, Islamabad, Pakistan, Department of Biology )
Aslam Khan ( Department of Bioinformatics and Biotechnology, International Islamic University, Islamabad, Pakistan, Department of Biology )
Abdul Kareem ( Department of Bioinformatics and Biotechnology, International Islamic University, Islamabad, Pakistan, Department of Biology )
Changsoo Kang ( Sungshin Women's University, Seoul, Republic of Korea )
Muhammad Imran Shabbir ( Department of Bioinformatics and Biotechnology, International Islamic University, Islamabad, Pakistan, Department of Biology )
Objective: To identify the underlying genetic anomalies in two consanguineous Pakistani families with autosomal recessive achromatopsia.
Methods: The exploratory study was conducted under the patronage of International Islamic University, Islamabad, Pakistan, and Sungshin Women University, Seoul, South Korea, after two families coded PKCN-02 and PKCN-07 belonging to different ethnic groups were recruited from different areas of Khyber Pakhtunkhawa province of Pakistan in July 2016. The families were originally diagnosed with nystagmus upon medical examination. Exome sequencing was performed to identify the possible
causative gene which was found to be cyclic nucleotide-gated channel alpha-3. Sanger sequencing was performed to confirm the mutations. After genetic analysis, clinical analysis was re-evaluated for colour vision using Ishihara 26 plates. Pathogenic potential of these mutations was evaluated using algorithmic mutation prediction tools. In-silico analysis was performed to predict effect of these mutations on protein structure of the gene in question.
Results: Exome sequencing revealed a reported missense mutation c .1306C>T (p.R436W) in family PKCN-02 and a novel missense mutation c.1540G>A (p.D514N) in family PKCN-07. After mutational analysis, clinical re-evaluation revealed that both families were segregating autosomal recessive achromatopsia. Further, the topological model of the cyclic nucleotide-gated channel alpha-3 polypeptide describes these missense mutations primarily affecting the C-linker and cyclic guanosine monophosphate-binding sites, respectively. Protein structure modelling of cyclic nucleotide-gated channel alpha-3 protein revealed abnormal structure produced by p.R436W and p.D514N.
Conclusions: Exome sequencing approach was used to first identify the genetic alteration in families with nystagmus. Two mutations in cyclic nucleotide-gated channel alpha-3gene were uncovered, including one novel mutation. Clinical re-evaluation uncovered that both families had achromatopsia.
KeyWords: Exome sequencing, Genotype-phenotype correlation, Novel mutation, Achromatopsia, Genetic diagnosis. (JPMA 69: 183; 2019)
Achromatopsia (ACHM; OMIM 216900) is an autosomal recessive retinal dystrophy affecting cone cells, characterised by photophobia, decreased visual acuity, nystagmus and colour blindness.1 ACHM occurs in complete and incomplete forms based on inability to distinguish colours. Individuals with complete ACHM have total colour blindness, nystagmus, amblyopia (greatly reduced visual acuity; 6/60) and severe photophobia. The fundus of the eye show completely normal appearance. Individuals with incomplete ACHM have reduced or fractional colour discrimination ability, reduced visual acuity with or without nystagmus or photophobia.2-4 In general, the symptoms in both the cases are almost the same, with incomplete ACHM being less severe than the other.5 With a prevalence of 1 in 30,000 people, it is a rare genetic disorder, but consanguinity elevates the chances of its occurrence.6 The onset is recorded at birth or early infant stages. Onset of symptoms is dependent upon dysfunction of the cone cells solely arising by the genetic mutations.7 ACHM is genetically heterogeneous. To date, molecular genetic analyses have identified numerous mutations in six genes that are associated with total ACHM (ACHM2 to ACHM7). A type of achromatopsia previously identified as ACHM1 was later found to be the same as ACHM3 caused by cyclic nucleotide-gated channel beta-3 (CNGB3) gene (MIM 605080) 8Among other genes are included cyclic nucleotide-gated channel alpha-3 (CNGA3; MIM 600053) causing ACHM29, Guanine nucleotide-binding protein G subunit -2 (GNAT2; MIM 139340) causing ACHM4,10 phosphodiesterase 6C
(PDE6C; MIM 613093) causing ACHM5,11 cone inhibitory phosphodiesterase 6H (PDE6H; MIM 601190) causing ACHM612 and activating transcription factor 6 (ATF6; MIM 605537) causing ACHM7.13 The current study was planned to report two Pakistani families that were initially evaluated as having nystagmus phenotype. However, after performing genetic analysis, using exome sequencing, the phenotype was reevaluated as ACHM2. In-silico analysis further described the effect of mutations on the polypeptide protein structure.
Materials and Methods
The exploratory study was conducted under the patronage of International Islamic University, Islamabad, Pakistan, and Sungshin Women University, Seoul, South Korea, after two families, coded PKCN-02 and PKCN-07 and belonging to different ethnic groups, were recruited from different areas of Khyber Pakhtunkhwa province of Pakistan in July 2016. The approval was obtained from the institutional review boards of both the universities. Informed written consent was obtained from the adult subjects and the parents of minor subjects. Medical histories from both families were taken and initial diagnosis was established that both families were affected with nystagmus. Peripheral blood samples were taken from each participating individual of both the families and genomic deoxyribonucleic acid (DNA) extraction was performed
using standard procedures.14 Whole exomes of nine individuals 5[55%] from PKCN-02 and 4[45%] from PKCN-07)) were captured by using the SureSelct kit (Agilent Technologies, Santa Clara, CA, USA), then sequenced as 100-bp paired reads on an Illumina HiSeq2000 machine (Illumina, San Diego, CA, USA). Raw data in FASTQ file format were aligned to hg19 (National center for biotechnology information (NCBI) build GRCh37) using SeqMan next generation 12(NGen12). ArrayStar v.12 was used to call the variant alleles based on dbSNP142. To screen potential candidate mutations, filtration criteria were applied.1 For an autosomal recessive disease, affected individuals must have both alleles in mutated state (homozygous mutant) whereas normal parents of such affected individuals should carry one mutated and one normal allele (heterozygous carrier) of the gene under test. Achromatopsia inherits as autosomal recessive pattern in both PKCN-02 and PKCN-07 families; that is, affected members (PKCN-02: IV-3, -4, -5 and -6, PKCN-07: III-4 and -5) are homozygous for the mutation; unaffected parents (PKCN-02: III-3, PKCN-07: II-4, -5) carry one copy of the mutation.2 Polymorphisms with a minor allele frequency higher than 0.05 in the 1000 Human Genome database (http://www.1000genomes.org/) were excluded from candidate mutations.3 Homozygous variants in our in-house exome data obtained from 17 normal unrelated Pakistani individuals were excluded.4 Synonymous and intronic variants at non-splicing junctions were also excluded.
Normal gene and protein sequence of CNGA3 was retrieved from ensemblegenome browser (http://www.ensembl.org/Homo_sapiens/Info/Index). The pathogenicity of identified variants was evaluated byin-silico prediction tools such as scale-invariant feature transform (SIFT ),27 Polymorphism phenotyping v2 (PolyPhen-2),28 Mutation taster and Protein Variation Effect Analyzer (PROVEAN)29 and SuSPect30 was used to find the scores to rank the amino acid variants in CNGA3 polypeptide from 0 (Neutral) to 100 (Disease-associated). Further SuSpect-P was used to find the disease-specific
pathogenicity scores for these variants. Together, all of these assessments provided useful information about the pathogenicity of the variants. The topological model of the CNGA3 polypeptide was drawn with the help of prediction using SMART.31 The Protein structures prediction of the wild-type and mutant polypeptides was done using Phyre215 and visualized with Discovery Studio Visualizer 2016 (Dassault Systèmes BIOVIA, San Diego, California, USA). After genetic analysis, due to identification of alterations in CNGA3 gene, the affected members of both families were revisited and detailed available clinical examination was performed in order to confirm the disease status. Both examined patients of PKCN-02 had nystagmus and decreased visual acuity measuring 6/60 Snellen in both eyes. Complete achromatopsia was noted on Ishihara plate testing. Fundus examination in individual IV:3 revealed loss of the foveal reflex and mild pigmentary mottling at the macula whereas individual IV:4 revealed no significant pathology with normal retinal vessels and fundus pigmentation, healthy macular reflexes and normal optic disc appearances with no pallor in both eyes. Colour vision testing and visual acuity were performed for affected individuals from both families. Family PKCN-07 could not be visited for funduscopy testing. Electroretinography (ERG) was not performed due to unavailability of the facility in the province.
Affected subjects from families PKCN-02 and PKCN-07 (Figure 1A-1B)
were confirmed by the ophthalmological examinations in the nearby eye hospital. Examined patients had nystagmus and decreased visual acuity measuring 6/60 Snellen in both eyes. Complete achromatopsia was noted on Ishihara plate testing. Fundus examination in both patients IV:3 and IV:4 from family PKCN-02 revealed no significant pathology with normal retinal vessels and fundus pigmentation. It had healthy macular reflexes and normal optic disc appearances without any pallor noted in either eye (Figure 1C) (Table 1).
Whole exomes of nine individuals in PKCN-02 and PKCN- 07 were sequenced. A total number of bases in the reads was 9.9 Gb, and mean depth of the target region was 123. Among the nine individuals, 409,135 variants qualified for quality control criteria (minimum Q call >20, minimum depth >10). Of these, 113,323 were missense, silent, insertion and deleterious variants. Applying filtration criteria revealed two homozygous missense mutations in CNGA3 gene. In family PKCN-02, a missense mutation c.1306C>T (p.R436W) was identified and all the affected individuals IV:3, IV:4, IV:5, and IV:6 were found homozygous for the mutation. However, their healthy parents and one sister was observed to be heterozygous for c.1306C>T variant (Figure 1A-2A). This mutation has been identified in previous studies (rs104893621).
16 In the other family PKCN- 07, a missense mutation c.1540G>A (p.D514N) was identified in affected individuals III:4, III:5, III:8, and III:9. Among healthy subject of the family, individuals II:4, II:5, II: 6, II:11 and II:12 were found heterozygous, but individual II:10 was homozygous for the wild type allele (Figure 1C; Figure 2A). To the best of our knowledge, this is the first report of D514N mutation. These two mutations were predicted to be deleterious/disease causing based on six types of predictive software (Table 2).
These two pathogenic variants were tested to exclude them as rare polymorphism in the normal population by performing Sanger sequencing in 200 healthy controls. Both of these potential mutations were absent in the 200 controls. The first substitution from C-T substitution at c.1306 is predicted to be a missense mutation that changes amino acid arginine to tryptophan at amino acid (a.a.) position 436. Second substitution mutation from G -A at c. 1540 also produces a missense at a.a. position 514 from aspartic acid to asparagine. ClustalW multiple a.a. sequence alignment of CNGA3 orthologs showed that both p.Arg436 and p.Asp514 were highly conserved among species (Figure 2B). However, among human cyclic nucleotidegated (CNG) channel subunits, CNGA3 homologs show varied conservation of p.Arg436 (not conserved in CNGA2) and p.Asp514 (conserved in CNGA but not in CNGB) (Figure 2C). Both of the mutations originated from the largest exon (E8) of the CNGA3 gene (Figure 2D). Topological model of the CNGA3 protein included the six transmembrane helices (1-6) that consist of a pore region responsible for transport of ions. Here, the helix-6 was connected to cyclic guanosine monophosphate (cGMP)-binding domain via C-linker 16,17 The variant R436W is located in the C-linker whereas D514N affected the cGMP-binding domain (Figure 2E, 3A). Model structure of R436W described the change in the structure of C-linker at residue 436 from arginine to tryptophan (Figure 3C, 3D). Structural modelling of novel variant D514N revealed the absence of a bond between the mutated asparagine at residue 514 and isoleucine at residue 515 (Figure3D, 3E).
The study describes the genetic basis of ACHM segregating in an autosomal recessive pattern in two families (PKCN- 02 and PKCN-07) of Pakistani origin (Figure 1A, 1B). All affected families had nystagmus, reduced visual acuity, severe photophobia and total loss of colour perception. Whole- exome sequencing revealed two genetic alterations in CNGA3 gene, including a reported variantc.1306 C>T (p.R436W) in family PKCN-02 and a novel variant c.1540G>A (p.D514N) in family PKCN-07 (Figure 2A). In-silico analysis through evolutionary conservation status of altered amino acids (Figure 2B, 2C), algorithmic mutation effect prediction ( Table 2) and structural modelling (Figure 3)
describe the observed variants to be deleterious/pathogenic which establish the genotypephenotype correlation in both families. Conventional techniques can be employed for identifying the causative variants in human pedigrees. These may include linkage analysis to find the co-segregation of causative locus/gene with the ACHM and then using Sanger sequencing for molecular diagnosis. However, these methodologies have several limitations being time and labour intensive and are also not suitable for largescale analysis. On the other hand, next-generation sequencing (NGS) has been shown to identify variants more systemically with less time and labour. NGS also provides extremely large-scale analysis which has greatly increased the pace of gene discovery.18 It is increasingly being used to discover causatie genes for Mendelian disorders and for screening for mutations in loci for genetically heterogeneous diseases as well as for molecular genetic diagnosis.19,20 It is also less costly than whole genome sequencing (WGS) making it more advantageous over other approaches.21 There are some studies with identifying causative mutations of ACHM that used exome sequencing.22,23 Both of the observed variants p.R436W and p.D514N were found to be evolutionary conserved among CNGA3
orthologs (Figure 2B). In mammals, six CNG channels are present in the form of two subfamilies, the A or subunits (CNGA1-4) and the B or subunits (CNGB1 and CNGB3). Through heterologous expression studies it was observed that -subunits are mainly responsible for the ionconducting activity of the channel, whereas modulator functions are performed by -subunits.17 On analysis of
human CNG channels subunits, the variant p.R436W was found to be conserved among all except CNGA2 which is found in olfactory neurons.24 However, the variant p.D514N was found conserved in all four CNGA subunits but not in CNGB subunits (Figure 2C). Further studies can uncover the importance of these evolutionary conservational differences. The CNGA3 gene located on chromosome 2q11 contains eight exons with 141 pathogenic variants reported in Human Gene Mutation Database (HGMD®) Professional 2017.1 database. Among 123 mutations were identified
missense/nonsensence, 2 splice site mutation, 5 small insertions, 1 small indel, 1 gross insertions. All the information shows that all affected persons were suffering from Albinisam, Oculocutaneous albinism (OCA) and ACHM. Both of the observed mutations originate from coding part of the largest exon 8 of the CNGA3 gene (Figure 2D). In CNGA3, p.R436W mutation is located in a conserved region (Figure 2B) which is located in C-linker site that connects the transmembrane helix-6 to the cGMP-binding site (Figure 2E, 3A).This variant is predicted to be deleterious by different mutation prediction tools (Table 2). Structural model of R436W describe the change in the structure of C-linker at residue 436 from a polar positive basic amino acid arginine to a non-polar aromatic amino acid Tryptophan (Figure 3C, 3D). This variant is previously reported and is also predicted to disrupt the channel function.16 The c.1540G>A variant in exon 8 is a missense mutation leading to change a polar negative amino acid aspartic acid to a polar amino acid with uncharged amide side chain aspartame (p.D514N). This mutation is located in a
conserved region (Figure 2B) of cGMP-binding site (Figure 2E, 3A) of CNGA3 protein. This variant is predicted to be deleterious by different algorithmic mutation prediction tools (Table 2). In-silico analysis for protein structural modelling of novel variant D514N revealed the absence of a bond between the mutated asparagine at residue 514 and isoleucine at residue 515 (Figure 3D, 3E). The cGMP domain is the binding site for cyclic guanosine mono-phosphate, and is therefore involved in the cyclic nucleotide mediated activation of the protein. It is hypothesised that this may lead to the loss-of-function of CNGA3 due to problem in interaction between cGMP at its binding site. Further analysis involving ligandreceptor interaction for cGMP and cGMP-binding site will reveal the important insights into D514N genetic alteration. At D514, a compound heterozygous mutation has been reported to be a heterozygote of two different missense mutations inclu ding c.1228C>T (p.R410W ) and c.1541A>T(p.D514V).9,16,25,26 In p.D514V, a polar negative amino acid aspartic acid has been substituted to a nonpolar aliphatic amino acid valine which disrupted the normal function of cGMP-binding site of CNGA3 polypeptide produced from one allele of the CNGA3 gene.26 This further strengthens the importance of aspartic acid at 514 position of CNGA3 protein. In both pedigrees PKCN-02 and PKCN-07, identification of causative mutations helped in reaching to the correct diagnosis of ACHM. Both of these families were initially identified as having nystagmus phenotype. After genetic analysis through exome sequencing, causative mutations were identified in CNGA3 gene. Clinical re-evaluation uncovered that both families had decreased visual acuity, photophobia and total colour blindness along with nystagmus. All of these are indicators of complete achromatopsia-2 (ACHM2) known to be caused by CNGA3 gene mutations. In-silico analysis with algorithmic mutation prediction tools and structural modelling for wild type and mutant sequences further describe the
effect of mutations on the polypeptide protein structure.
The study further strengthens the perception that exome sequencing is a superior diagnostic approach in conjunction with routine testing, especially for ACHM and other such diseases where these could easily be misdiagnosed when relying only on clinical examinations.
Conflict of Interest: None.
Source of Funding: None.
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