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A novel compound heterozygous BEST1 gene mutation in two siblings causing autosomal recessive bestrophinopathy



To describe the clinical features, imaging characteristics, and genetic test results associated with a novel compound heterozygous mutation of the BEST1 gene in two siblings with autosomal recessive bestrophinopathy.


Two siblings underwent a complete ophthalmic examination, including dilated fundus examination, fundus photography, fundus autofluorescence imaging, spectral-domain optical coherence tomography, fluorescein angiography, electroretinography, and electrooculography. A clinical diagnosis of autosomal recessive bestrophinopathy was established based on ocular examination and multimodal retinal imaging. Subsequently, clinical exome sequencing consisting of a panel of 6670 genes was carried out to confirm the diagnosis and assess genetic alterations in the protein-coding region of the genome of the patients. The identified mutations were tested in the two affected siblings and one of their parents.


Two siblings (a 17-year-old female and a 15-year-old male) presented with reduced visual acuity and bilaterally symmetrical subretinal deposits of hyperautofluorescent materials in the posterior pole, which showed staining in the late phase of fluorescein angiogram. Spectral-domain optical coherence tomography demonstrated hyperreflective subretinal deposits and subretinal fluid accumulation. Both patients shared two mutations in the protein-coding region of the BEST1 gene, c.103G > A, p.(Glu35Lys) and c.313C > A, p.(Arg105Ser) (a novel disease-causing mutation). Sanger sequencing confirmed that the unaffected mother of the proband was carrying p.(Glu35Lys) variant in a heterozygous state.


We have identified and described the phenotype of a novel disease-causing mutation NM_004183.4:c.313C > A, p.(Arg105Ser) in a heterozygous state along with a previously reported mutation NM_004183.4:c.103G > A, p.(Glu35Lys) of the BEST1 gene in two related patients with autosomal recessive bestrophinopathy.

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The BEST1 (alternatively VMD2, RP50, BMD) gene located on chromosome GRCh38 11q12.3 encodes a transmembrane pentameric protein consisting of 585 amino acids with a highly conserved N-terminal region followed by four transmembrane domains (amino acids 1–390) and a carboxy-terminal region (amino acids 391–585) [1]. Structural models of the BEST1 propose the N- and C-termini as being cytosolic with four transmembrane domains (domains 1, 2, 5, and 6) and two cytoplasmic domains (domains 3 and 4) [2, 3]. The protein is predominantly expressed in the basolateral plasma membrane of the retinal pigment epithelium (RPE) and functions as a calcium-activated chloride channel (CaCC) which regulates the flow of chloride and other monovalent anions across cellular membranes in response to intracellular calcium levels [4,5,6,7,8]. Mutation of the BEST1 gene has been associated with lipofuscin accumulating within and beneath the RPE and degeneration of the RPE and the overlying photoreceptors [9]. A wide range of ocular phenotypes resulting from mutations in the BEST1 gene have been described and are collectively termed bestrophinopathies. [10, 11] Autosomal recessive bestrophinopathy (ARB) may result from a total absence (null phenotype) of functional BEST1 protein in the RPE, [12, 13] improper localization to the cell membrane with intact anion channel activity, [14] or lack of the anion channel activity [15].

Schatz et al., in 2006, first described a variant of Best macular dystrophy in two members of a Swedish family presenting with reduced vision, multifocal retinal deposits, and intraretinal cystic changes, harboring biallelic mutations in the BEST1 gene. [16] In 2008, Burgess et al. coined the term autosomal recessive bestrophinopathy (ARB) and identified it as the third distinct phenotype resulting from mutations in the BEST1 gene. [12] Other described phenotypes associated with pathogenic variants of the BEST1 gene include Best vitelliform macular dystrophy (BVMD) [17, 18], adult vitelliform macular dystrophy (AVMD), autosomal dominant vitreoretinochoroidopathy (ADVIRC), [19, 20] autosomal dominant microcornea, rod-cone dystrophy, early-onset cataract, and posterior staphyloma (MRCS) [11], rod-cone dystrophy and retinitis pigmentosa [11, 21]. In contrast to other phenotypes of bestrophinopathies that result from dominant mutations, ARB is associated with recessive biallelic mutations in the BEST1 gene [12, 22, 23].

Patients with ARB typically present in the first two decades of life but may remain asymptomatic as late as the fifth decade [12, 15, 24]. The clinical features of ARB include a gradual and progressive visual loss, hyperopia, predominantly peri-macular sub-retinal yellowish deposits of lipofuscin, seen as hyperautofluorescent areas, accumulation of subretinal and/or intraretinal fluid, absence of light peak in electrooculography, normal or reduced electroretinogram, and sometimes associated with shallow anterior chambers and reduced axial length predisposing the affected patients to angle-closure glaucoma. [12, 25, 26] Full-field electroretinography is typically normal early on in the disease and shows abnormal results from late childhood or adolescence, indicating generalized rod and cone dysfunction. In addition, pattern electroretinography evidence of macular dysfunction is also seen. [12] This article presents the results of clinical evaluation, multimodal imaging, electrophysiological tests, and genetic investigations of two siblings with ARB.


Clinical investigation

Clinical investigations in patients included a detailed history and physical examination, slit-lamp biomicroscopy, indirect ophthalmoscopy, fundus photography, fundus autofluorescence imaging (FAF), optical coherence tomography (OCT), fluorescein angiography (FA), full-field electroretinography (ERG), and electrooculography (EOG). The ERG and EOG were performed per the guidelines of the International Society for Clinical Electrophysiology of Vision (

Genetic analysis

Whole exome sequencing

DNA isolation, exome library preparation, and sequencing

DNA was isolated from the patient’s whole blood sample using QIAamp DNA Blood Mini Kit (QIAGEN, CA, US) and subjected to targeted gene capture using MedGenome Clinical Exome (Ver. 4) which captures a panel of 6670 protein-coding genes. The libraries thus generated were sequenced to mean coverage of > 80-100X on the Illumina HiSeq 4000 sequencing platform (Illumina, CA, US). 100% of the protein-coding region of the BEST1 gene was covered.

Variant calling and annotation

The Genome Analysis Toolkit (GATK) best practices framework was followed to identify the variants in the sample using Sentieon (v201808.07). The sequencing reads were aligned to the human reference genome (GRCh38.p13) using the Sentieon aligner. Sentieon’s version of GATK (IndelRealigner) was used to perform local realignment in regions containing potential indels. Sentieon’s version of GATK Toolkit – BaseRecalibrator was used to recalibrate the quality scores of all the reads [27]. Sentieon DNASeq (v201808.07) HaplotypeCaller was used to identify variants. Gene annotation of the variants was performed using the VEP program against the Ensembl release 99 human gene model [28, 29]. In addition to SNVs and small Indels, copy number variants (CNVs) were detected from targeted sequence data using the ExomeDepth (v1.1.10) method [30]. Clinically relevant mutations were annotated using published variants in literature and a set of disease databases—ClinVar [31], OMIM [32] (updated on 11th May 2020), GWAS [33], HGMD (v2020.2) [34], and SwissVar [35].

Variant filtering and analysis

To identify candidate variants, we selected the variations if their minor allele frequencies are less than 0.05 in 1000 Genome Project [36], gnomAD [37], dbSNP [38], Exome Variant Server [39], 1000 Japanese Genome [40], and internal Indian population database. The identified variations were classified into pathogenic, likely pathogenic, VUS, likely benign, and benign groups according to the variant interpretation guidelines of the American College of Medical Genetics and Genomics (ACMG) [41]. Furthermore, all nucleotide variants present in BEST1 were reviewed. The genes and corresponding variants that qualified these filtering criteria were investigated to determine their significance and relevance in Bestrophinopathy. 8.65 Gb of raw sequencing data was generated, of which > 88% of raw reads passed the on-target alignment. > 90% of the targeted base qualified the Phred score Q30.

Sanger sequencing

Sanger sequencing was performed in the proband and the mother to validate the variants identified by whole exome sequencing and identify the mutation in the parent. Sanger sequencing was performed with these primers:


Bioinformatics analysis

The potential functional impact of all the candidate variants was investigated using three programs, including PolyPhen2 (, in the public domain), Mutation Taster (, in the public domain), and SIFT (, in the public domain).


Clinical findings

Patient A (proband), a 17-year-old female, reported blurred distance vision in both eyes for three years. Her best-corrected visual acuity was 6/9 in both eyes, which did not change during the follow-up of 1 year. Slit-lamp examination of the anterior segment of both patients was unremarkable. The axial length measured by optical biometry was 21.80 mm and 21.64 mm in the right and the left eye, respectively. Dilated fundus examination revealed bilateral and symmetrical, multifocal subretinal yellowish deposits in the posterior pole and upper nasal region, with peripapillary sparing (Fig. 1A, B). On FAF, the yellowish deposits appeared as hyper-autofluorescent spots surrounding an area of hypo-autofluorescence (Fig. 1C, D). The deposits showed staining in the late phase of the fluorescein angiogram (Fig. 1E, F). On OCT, subretinal fluid and intraretinal hyporeflective spaces (schisis) located predominantly in the outer nuclear layer (ONL) were seen along with elongated photoreceptors and hyperreflective deposits in the subretinal space bilaterally (Fig. 1G, H). The electroretinogram (ERG) was normal, and an absent light peak was noted on EOG.

Fig. 1
figure 1

Clinical features of patient A (proband). A 17-year-old otherwise healthy female presented with blurred distance vision in both eyes, which she first noticed when she was 14 years old. Colour fundus photographs (A, B) show bilateral and symmetrical, multifocal subretinal yellowish deposits in the posterior pole and upper nasal region, with notable peripapillary sparing. The yellowish deposits are hyperautofluorescent on blue light fundus autofluorescence (C, D) and circumscribe areas of hypoautofluorescence. On fluorescein angiography (E, F), the yellowish deposits show diffuse staining in the late phase. Horizontal spectral-domain optical coherence tomography images through the right and left fovea (G, H) show center-involving subretinal fluid and thickening of the ellipsoid zone, with elongation of the photoreceptor outer segments and deposits in the subretinal space. Additionally, intraretinal hyporreflective areas predominantly located in the outer nuclear layer can be seen

Patient B, a 15-year-old male, reported a unilateral decrease in visual acuity and inward deviation of the left eye since the age of four. On ocular examination, he was found to have left esotropia of 15 prism diopters with prescribed correction and 25 prism diopters without the correction for distance. His best-corrected visual acuity was 6/6 in his right eye and 5/60 in his left eye, with an accommodative-convergence over accommodation (AC/A) ratio of 2:1. Slit-lamp examination of the anterior segment was unremarkable. The axial length measured by optical biometry was 21.61 mm and 21.54 mm in the right and the left eye, respectively. Dilated fundus examination revealed two circumscribed areas of bilaterally symmetrical, multifocal subretinal yellowish deposits, one in the posterior pole and the other in the upper nasal region, with peripapillary sparing. (Fig. 2A, B). The yellowish lesions were hyper-autofluorescent surrounding an area of hypo-autofluorescence on FAF (Fig. 2C, D) and showed staining in the late phase of fluorescein angiogram (Fig. 2E, F). On OCT, subretinal fluid (seen as subfoveal hyporeflective space), elongated photoreceptors along with hyperreflective deposits in the subretinal area were observed bilaterally (Fig. 2G, H). The electroretinogram (ERG) was normal, and an absent light peak was noted on EOG.

Fig. 2
figure 2

Clinical features of patient B. A 15-year-old otherwise healthy male presented with a unilateral decrease in visual acuity and inward deviation of the left eye, which was first noticed by his parents when he was four. On colour fundus photographs of the right and the left eye (A, B), two separate areas of bilateral and symmetrical, multifocal subretinal yellowish deposits are seen in the posterior pole and upper nasal region, with notable peripapillary sparing. On blue light fundus autofluorescence (C, D), the yellowish deposits are hyperautofluorescent, and circumscribed areas show hypoautofluorescence. In the late phase of fluorescein angiography (E, F), diffuse staining, seen as diffuse hyperfluorescence of the yellowish deposits is observed. Horizontal spectral-domain optical coherence tomography scans through the fovea (G, H) show center-involving subretinal fluid and thickening of the ellipsoid zone, with elongation of the photoreceptor outer segments and deposits in the subretinal area

The patients were born of a non-consanguineous marriage from parents of North Indian descent. The parents and unaffected siblings were examined; however, no ocular or systemic abnormalities were observed. (Fig. 3). Both affected siblings were treated with topical carbonic anhydrase inhibitors and followed up for one year. The subfoveal fluid did not improve after one year of treatment. The clinical, imaging, and electrophysiological findings of the affected patients are summarized in Table 1.

Fig. 3
figure 3

Pedigree of the family with two affected members. Patient A, a 17-year-old female (black arrow), and Patient B, a 15-year-old male

Table 1 Clinical profile of the patients

Genetic findings

A heterozygous missense mutation, NM_004183.4(BEST1):c.103G > A, was found in exon 2 of the BEST1 gene in both patients (chr11: g.61951909G > A; c.103G > A) and was further validated by Sanger sequencing (Fig. 4A). It resulted in the amino acid substitution of Glutamic acid (negatively charged) for Lysine (positively charged) at codon 35, NM_004183.4:p.(Glu35Lys) (Table 2). The variant is classified as likely pathogenic in the ClinVar database [31] and lies in the RFP-TM, chloride channel domain of the bestrophin protein. The NM_004183.4:p.(Glu35Lys) variant has not been reported in the 1000 genomes [36] and gnomAD databases [37] (accessed 30th January 2022). The in-silico predictions according to PolyPhen-2 is to be probably damaging; and deleterious according to SIFT and MutationTaster2. The reference codon is evolutionarily conserved in mammals.

Fig. 4
figure 4

A Sequence chromatogram and alignment to the reference sequence showing the variant in exon 2 of the BEST1 gene (chr11:g.61951909G > A; c.103G > A; p.Glu35Lys) detected in heterozygous condition in the proband and the unaffected mother. B The variant in exon 4 of the BEST1 gene (chr11:g.61955783C > A; c.313C > A), was detected in the proband but not in the unaffected mother. The reference sequence NM_004183 of BEST1 was used

Table 2 Whole Exome Sequencing results of the BEST1 gene of patients A and B

Another heterozygous missense mutation, NM_004183.4(BEST1):c.313C > A, was found in exon 4 of the BEST1 gene in both patients (chr11:g.61955783C > A; c.313C > A), and was further validated by Sanger sequencing (Fig. 4B). It resulted in the amino acid substitution of Arginine (positively charged) for Serine (uncharged) at codon 105, NM_004183.4:p.(Arg105Ser) (Table 2). The variant lies in the RFP-TM, chloride channel domain of the bestrophin protein. The NM_004183.4:p.(Arg105Ser) variant has not been reported in the 1000 genomes databases [36] and has a minor allele frequency of 0.0007% in the gnomAD database (accessed 30th January 2022) [37]. The in-silico predictions of the variant according to PolyPhen-2 is to be probably damaging, and deleterious according to SIFT and MutationTaster2. The reference codon is evolutionarily conserved in mammals. This mutation has not been previously reported in patients with ARB or VMD. The results of next-gen sequencing are summarized in Table 2. The IGV depicting the distribution of alternate allele and wild type allele for both variations is shown in Supplementary figure S 1A and S 1B.

We analyzed the mother for both variants through Sanger sequencing to clarify whether the two mutations were located on separate BEST1 alleles. The NM_004183.4:p.(Glu35Lys) was detected in a heterozygous condition along with the wild type in the unaffected mother of the proband (Fig. 4A, B).


This report analyzed the clinical and imaging characteristics along with the genetic test results of two siblings with ARB. The diagnosis of ARB was established based on clinical observation and multimodal retinal imaging and further confirmed by whole exome sequencing and Sanger sequencing. Patient A demonstrated good central acuity, as seen in other patients with ARB in the first and second decade of life [42]. We noted that the sibling (patient B) had poor visual acuity in one eye due to amblyopia resulting from uncorrected esotropia. In addition, both the patients had short axial lengths in both eye without any abnormal iridocorneal anatomic features or shallow anterior chamber depth. Reduced axial length predisposes patients to angle-closure glaucoma, potentially leading to a further visual decline [42].

The whole exome sequencing revealed a likely compound heterozygous mutations in the BEST1 gene shared by both siblings that likely led to ARB. The variants were validated by Sanger sequencing. One of the alleles carried a missense mutation in exon 2 NM_004183.4(BEST1):c.103G > A, which resulted in the amino acid substitution from negatively-charged Glutamic acid to positively-charged Lysine at the 35th amino acid residue, NM_004183.4:p.(Glu35Lys). This variant was detected in the unaffected mother in a heterozygous state along with the wild type using Sanger sequencing. The variant has been submitted to ClinVar (accession number- RCV000356527) and has previously been reported by Tian et al. and Habibi et al., albeit in a homozygous state [43, 44]. To our knowledge, ours is the first study to report this variant in a compound heterozygous state.

Another variation was observed to be a transversion in exon 4 NM_004183.4(BEST1):c.313C > A, which resulted in the amino acid substitution from positively-charged Arginine to uncharged Serine at 105th amino acid residue, NM_004183.4:p.(Arg105Ser) (Table 2). To our knowledge, this mutation has not been reported previously in patients with either ARB or VMD. This variant is predicted to be pathogenic. The NM_004183.4(BEST1):c.313C > A (p.Arg105Ser) has been submitted to ClinVar previously (accession number- RCV002025371.1) [31]. Two other disease-causing mutations affecting the same codon, p.Arg105Gly in patients with BMD, and p.Arg105Cys in a 69-year-old patient with age-related macular degeneration, have been reported [45, 46]. Interestingly, the p.Arg105Gly mutation resulted in additional extramacular multifocal deposits similar to ARB in three patients with BVMD [45].

Notably, the mutations discovered in this study are localized to the N-terminal region (Fig. 5A-C). The mutation NM_004183.4:p.(Glu35Lys) localizes to the first transmembrane domain, while the NM_004183.4:p.(Arg105Ser) mutation alters an amino acid in the cytoplasmic region distal to the second transmembrane domain (Fig. 5B). The amino acids at these positions are conserved among mammals (Fig. 5D). Among the roughly 335 mutations reported in BEST1 thus far, only about 40 compound heterozygous and homozygous mutations are associated with ARB. [26, 34]

Fig. 5
figure 5

A Schematic representation of BEST1 (NM_004183.4) transcript. Grey boxes represent exons, and lines connecting them represent introns. Green boxes represent the translation start site, while red boxes represent the translation termination site. B Topological representation of BEST1 (Milenkovic et al.) representing mutation sites denoted by red circles. C Generated structural model of BEST1 wild type, E35K, and R105S. D Multiple sequence alignment of BEST1 from different species

Although the detailed pathophysiology that leads to the disease is poorly understood, most of the characterized BEST1 mutations alter the electrophysiological properties of the calcium-activated chloride channel (CaCC), which is thought to be determined by the N-terminus portion of BEST1, affecting the flow to chloride across the RPE [1, 5]. Crystallographic studies of the wild type and mutated proteins suggest that BEST1 variants alter the cytoplasmic pore structure, which affects the permeability of anions or anion-cation selectivity, leading to lipofuscin accumulation and degeneration of the RPE (Fig. 5C) [47].

What this study adds

This study expands the genetic spectrum of the BEST1 variants associated with an ARB phenotype by reporting a novel variant p.(Arg105Ser), found in compound heterozygosity with another clinically significant variant in two affected siblings. Furthermore, the reported variant p.(Arg105Ser) variant appears to contribute to the ARB phenotype as the other variant alone did not cause any disease in the carrier (unaffected mother).


Although it does not affect the diagnosis, the genetic testing of the unaffected father and other unaffected siblings would have been ideal but could not be carried out.

Availability of data and materials

All data generated or analyzed during this study are included in this published article. Accession number for the variants: SRR20990872.



Anterior Chamber


Autosomal recessive bestrophinopathy


Best-corrected visual acuity


Best macular dystrophy/Best vitelliform macular dystrophy






Fluorescein angiography


Fundus autofluorescence


Genome-wide association studies


Human Gene Mutation Database


Next-gen sequencing


Optical coherence tomography


Online Mendelian Inheritance in Man


Retinal pigment epithelium


Sorting Intolerant From Tolerant


Subretinal fluid


Vitelliform macular dystrophy


Variant of uncertain significance


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MedGenome Labs Limited, Bengaluru, India for the assistance in the genetic workup of the study participants. Dr. Shaik Mohammed Zakir and Dr. Abdul Waris, Institute of Ophthalmology, Jawaharlal Nehru Medical College, Aligarh, India for helping to acquire fundus images and patient care.



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Authors and Affiliations



OIH contributed to the patient’s care, design and implementation of the research, IRB approval, clinical workup, and follow-up of the subjects, investigations, genetic testing of siblings and the parent, analysis of the results, draft preparation, acquiring images, preparing Figs. 1– 5, editing, submission, and revision of the manuscript. AC contributed towards the genetic analysis of the mother and proband, preparing Fig. 4, supplementary Fig. 1 AB, submission of genetic data, and revision of the manuscript; FN analyzed genetic test results and prepared the topological BEST1 protein model in Fig. 5; OA edited the genetic section of the manuscript, analyzed genetic test results and prepared Fig. 5; JPM reviewed, edited, and revised the manuscript and wrote the captions of Figs. 1 and 2, and provided inputs as a consultant for clinical insight into the disease; TA was involved in the genetic testing of the unaffected mother and revision of the manuscript. The author(s) read and approved the final manuscript.

Corresponding author

Correspondence to Obaid Imtiyazul Haque.

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The study was approved by the Jawaharlal Nehru Medical College Institutional Ethics Committee. It was performed per institutional ethics guidelines per the tenets of the Declaration of Helsinki. Written informed consent for the study was obtained from the parents. The assent of the minors was obtained prior to the study.

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The authors declare that they have no competing interests.

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Haque, O.I., Chandrasekaran, A., Nabi, F. et al. A novel compound heterozygous BEST1 gene mutation in two siblings causing autosomal recessive bestrophinopathy. BMC Ophthalmol 22, 493 (2022).

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