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Multiple genetic mutations implicate spectrum of phenotypes in Bardet-Biedl syndrome
Journal Pre-proofs Short communication Multiple genetic mutations implicate spectrum of phenotypes in Bardet-Biedl syndrome Sanjiban Chakrabarty, Swheta B. Savantre, C. Ramachandra Bhat, Kapaettu Satyamoorthy PII: DOI: Reference:
S0378-1119(19)30823-6 https://doi.org/10.1016/j.gene.2019.144164 GENE 144164
To appear in:
Gene Gene
Received Date: Revised Date: Accepted Date:
4 May 2019 14 September 2019 8 October 2019
Please cite this article as: S. Chakrabarty, S.B. Savantre, C. Ramachandra Bhat, K. Satyamoorthy, Multiple genetic mutations implicate spectrum of phenotypes in Bardet-Biedl syndrome, Gene Gene (2019), doi: https://doi.org/ 10.1016/j.gene.2019.144164
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier B.V.
Multiple genetic mutations implicate spectrum of phenotypes in Bardet-Biedl syndrome Sanjiban Chakrabarty1, Swheta. B. Savantre2, C. Ramachandra Bhat2, Kapaettu Satyamoorthy1* 1Department
of Cell and Molecular Biology, Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal, Karnataka, India-576104 2Department
of Medicine, K.V.G. Medical College & Hospital, Dakshina Kannada, Sullia,
India-574327 *Corresponding author: Dr. Kapaettu Satyamoorthy Professor Manipal School of Life Sciences Manipal Academy of Higher Education, Manipal Karnataka, India-576104 Telephone: 0820-2922058 Fax: 0820-2571919 Email: [email protected] Short title: BBS10 mutation in Bardet Biedl Syndrome
Multiple genetic mutations implicate spectrum of phenotypes in Bardet-Biedl syndrome Sanjiban Chakrabarty1, Swheta. B. Savantre2, C. Ramachandra Bhat2, Kapaettu Satyamoorthy1* 1Department
of Cell and Molecular Biology, Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal, Karnataka, India-576104 2Department
of Medicine, K.V.G. Medical College & Hospital, Dakshina Kannada, Sullia,
India-574327 *Corresponding author: Dr. Kapaettu Satyamoorthy Professor Manipal School of Life Sciences Manipal Academy of Higher Education, Manipal Karnataka, India-576104 Telephone: 0820-2922058 Fax: 0820-2571919 Email: [email protected] Short title: BBS10 mutation in Bardet Biedl Syndrome 1
Abstract Bardet–Biedl syndrome (BBS) is a clinically and genetically heterogeneous ciliopathy with several clinical features including retinitis pigmentosa, obesity, kidney dysfunction, postaxial polydactyly, behavioral dysfunction and hypogonadism with wide spectrum of additional features. With multiple phenotypes and heterogeneous distribution, it is unlikely that BBS is caused by single gene defect. We have performed clinical and genetic diagnosis of two individuals from an Indian family with classical BBS symptoms. Whole exome sequencing identified homozygous missense mutation in BBS10 gene, hemizygous missense AR and homozygous missense PDE6B mutations in the proband and affected sibling with BBS. Identification of BBS10 mutation along with AR and PDE6B gene mutation will expand the genetic and phenotypic spectrum in individuals with BBS. Keywords: Bardet–Biedl syndrome, BBS, retinitis pigmentosa, PDE6B, hypogonadism, AR
2
1. Introduction BBS is grouped one among the ciliopathies with wide range of clinical features which includes retinitis pigmentosa, obesity, kidney dysfunction, postaxial polydactyly, behavioral dysfunction and hypogonadism first described by ophthalmologists Laurence and Moon in the year 1866 (Blacque and Leroux., 2006). Other features such as cardiac abnormalities, diabetes, hypertension, hearing defects and anosmia are also reported (Forsythe and Beales., 2013, Forsythe et al., 2018). Clinical diagnosis of BBS requires presence of either a) three primary and two secondary features or b) only four primary features (Beales et al., 1999). The prevalence of BBS is 1:140,000 to 1: 160,000 in north American and European population with 1:165,000 in consanguineous Arab population. Molecular analysis identified mutations with autosomal recessive mode of inheritance in 20 different genes (BBS1-BBS20) responsible for majority of patients with BBS phenotype. BBS genes are primarily involved in ciliary biogenesis and trafficking by forming chaperonin complex (Tobin and Beales., 2009, Alvarez-Satta et al., 2017). The role of BBS10 gene (12q21.2) in the BBS clinical 3
phenotype was first identified in a consanguineous pedigree of Lebanese origin (Stoetzel et al., 2006). However, more than 25% of the BBS patients do not harbor BBS gene mutation suggesting involvement of additional genes in the BBS phenotype. Here, we describe clinical and genetic analysis of two siblings from an Indian family with BBS phenotype. Exome sequencing identified homozygous missense mutation in BBS10 gene in the proband and affected sibling. In addition to BBS10 mutation, we identified AR and PDE6B gene mutations in the proband and affected sibling with BBS. Along with causal BBS10 mutation, AR and PDE6B mutation may contribute to the severity of the BBS phenotype in the proband and sibling. 2. Methods 2.1 Clinical report Proband is 23-year-old male born out of second-degree consanguineous marriage, with history of 3 first trimester abortions prior to the index case (Fig. 1A). Proband showed history of delayed developmental milestones, frequent volatile outbursts and emotional lability with severe progressive visual impairment in the past 5 years. He was a school dropout due to his learning difficulties and visual impairment. His younger male sibling was 19 yrs. age and a school dropout due to poor scholastic performance. Both the parents were asymptomatic and had normal fundus examination. No similar history was found in other family members. 2.2 Genetic analysis The study follows the principles outlined in the Helsinki Declaration and patients gave written informed consent for molecular study and publication. Clinical specimens were collected after obtaining written informed consent from the proband and parents. Genomic DNA was extracted from the peripheral blood of the proband and other family members using QIAamp DNA Mini Kit (Qiagen GmbH, Hilden, Germany). Purity of the DNA was determined using NanoDrop ND1000 Spectrophotometer (ThermoFisher Scientific, 4
Waltham, MA, USA). Purified DNA was quantified using Qubit® dsDNA HS (High Sensitivity) Assay Kit (ThermoFisher Scientific) and used for AmpliSeq exome library preparation. Whole exome sequencing was performed using Ion Proton PIv2 chip in Ion Proton next generation sequencing platform (ThermoFisher Scientific). Sequencing reads obtained from Ion Proton were aligned to human reference sequence (human genome build19) and variant calling was performed using Ion Torrent Variant Caller v5.0 (TVC) in Torrent Suite software v5.0 (ThermoFisher Scientific). Alignment reads and variants called, with respect to the reference human genome sequence, were viewed using Integrative Genomic Viewer software and to check for strand biases, homopolymer length and sequencing errors (Thorvaldsdottir et al., 2013). 3. Results 3.1 Clinical examination On physical evaluation, acanthoses nigricans and post-axial polydactyl in all 4 limbs with traumatic amputation of right-hand finger was noted in the proband. (Fig. 1B). His BMI was 29.3 kg/m2. However, we did not find any visible neurocutaneous markers in the proband. He had grade-1 right beating nystagmus. Visual acuity is impaired to finger count near face, fundoscopy showed pigmentary abnormalities in the peripheral retina (Fig. 1B). He had single testis with micropenis but well developed pubic and axillary hair. (Fig. 1B). Proband was anemic with hemoglobin 9.8 g/dL, while other hematological parameters were normal. His biochemical profile (RFT and FLP) was normal. His testicular ultra-sonogram revealed absence of left testis and hypoplastic left seminal vesicle and prostrate. His younger male sibling was found to have finger counting at 3 meters and on fundoscopy had macular degeneration with peripheral pigmented spicules. Based on the clinical analysis, our proband had 4 major and 2 secondary features as per modified diagnostic criteria for BBS (Beales et
5
al., 1999). Clinical specimens were collected after obtaining written informed consent from the proband and parents. 3.2 Mutation analysis Whole exome sequencing of the proband identified homozygous missense mutation in BBS10 gene (H395R). In addition, we have identified hemizygous missense mutation in AR (P392S) and PDE6B (R100C) (Fig. 2, Table 1). Sanger sequencing of BBS10, AR and PDE6B gene mutations were confirmed in the proband and affected young male sibling. Both parents of the proband and affected male sibling were carrier of the BBS10 and PDE6B mutations in heterozygote state (Fig. 2). Hemizygous AR mutation analysis by Sanger sequencing showed X-linked inheritance in both the proband and affected sibling. To evaluate the pathogenicity of identified mutations in BBS10, AR, and PDE6B gene, independent prediction algorithms including SIFT (http://sift.jcvi.org/), PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/), MutationTaster2 (http://www.mutationtaster.org/), M-CAP, CADD score and Grantham score were employed for analysis (Kumar et al., 2009, Adzhubei et al., 2013, Schwarz et al., 2014, Jagadeesh et al., 2016). We analyzed the BBS10 gene mutation (NM_024685.3; c.1184A>G) in dbSNP 150, 1000 Genomes Project (http://www.1000genomes.org/), ExAc and NHLBI GO Exome Sequencing Project (ESP) databases to confirm BBS10 gene mutation in proband as a pathogenic variant. BBS mutation in the proband and affected sibling c.1184A>G has an allele frequency of 0.000003980 (1/251242 alleles) in the GnomAD database. Protein sequence alignment showed that mutated amino acid residues in BBS10, AR, and PDE6B gene is evolutionary conserved across the species (Fig. 2D). Mutations were mapped to BBS10, AR and PDE6B gene protein domains (Supplementary figure 1). BBS10 mutation (H395R) was found in the Cpn60_TCP1 domain which is important for its interaction with other BBS proteins (Supplementary figure 1).
AR gene mutation was
identified in the exon 1 encompassing N-terminal domain critical for AR function 6
(Supplementary figure 1). PDE6B, β subunit of rod photoreceptor cyclic GMP phosphodiesterase (PDE) plays a central role in the visual photo transduction cascade. We have identified PDE6B mutation (R100C) in the N-terminal GAF domain in proband and affected sibling (Supplementary figure 1). 4. Discussion BBS10 gene is structurally similar to type II chaperonins and involved in protein folding (Alvarez-Satta et al., 2017). Mutation in BBS10 gene is reported in patients with wide range of clinical presentation including obesity, rod-cone dystrophy and polydactyly (Janssen et al., 2011). Additionally, we have identified hemizygous AR mutation (P392S) previously reported in androgen insensitivity syndrome and homozygous PDE6B (R100C) mutation (Gottlieb et al., 2012, Kalfa et al., 2013). AR mutation identified in the current study have implications to disease phenotypes or co-morbid conditions. Since the hemizygous mutation found in exon 1 of AR gene, which encompasses DNA binding domain, its role in partial androgen insensitivity syndrome (PAIS) or complete AIS (CAIS) are possible (Gottlieb et al., 2012). Hemizygous AR mutation (P392S) identified in proband and sibling in our study is previously reported in two Indian families with complete androgen insensitivity (CAIS) and partial androgen insensitivity (PAIS) suggesting it to be a causal variant in patients with androgen insensitivity (Akella. 2017). Both proband and the affected sibling had hemizygous AR (P392S) mutation with absence of left testis and hypoplastic left seminal vesicle and prostrate (Figure 2) suggesting the role of AR gene mutation in the genital manifestation. However, these remains to be experimentally established. Mutations in phosphodiestaerase 6B (PDE6B) are reported to be involved in various visual impairment disorders (Gopalakrishna et al., 2017). Mouse model with homozygous mutation in PDE6B has shown elevated cGMP levels with retinal degeneration phenotype (Bowes et al., 1990). Additionally, PDE6B recessive mutation have been reported in patients with retinitis pigmentosa (Cheng et 7
al., 2016). However, these observations from mouse studies (Bowes et al., 1990) remain to be functionally validated for the specific role of PDE6B in the retinal degeneration of individuals with BBS10 (H395R) mutation. Bardet-Biedl syndrome is complex genetic disorder affecting multiple organs with wide range of clinical phenotype. Recent studies have reported patients with BBS harbor mutations in additional genes which indicate possible oligogenic inheritance (Lindstrand A et al., 2014, Manara et al., 2019). In the present study, we have identified homozygous missense BBS10 gene mutation with hemizygous mutation in AR and homozygous missense in PDE6B genes in the proband and his affected sibling suggesting their possible role in BBS clinical phenotype and expands the phenotypic and genetic spectrum of BBS. Our report explains the possibility of bipartite independent phenotypes due to two or more distinct mutations and potential interactions between them contributing to the complex clinical phenotype such as BBS. 5. Conclusion Homozygous missense mutation in BBS10 gene along with hemizygous missense mutation in AR gene and homozygous missense mutation in PDE6B gene identified in the proband and affected sibling in an Indian family expanding the genetic spectrum of BBS. Contribution of AR and PDE6B mutation in BBS phenotype alone or in combination with other BBS genes can be studied to understand the functional role for improved diagnosis in BBS patients. Statement of contribution Sanjiban Chakrabarty and Kapaettu Satyamoorthy conceptualized and designed the study, drafted and reviewed the manuscript. Swheta B Savantre and C. Ramachandra Bhat have coordinated data collection and critically reviewed the manuscript for important intellectual content. All authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work. Acknowledgments 8
The authors thank the patient and his family members who participated in this study. The infrastructure funding from DST-FIST, TIFAC-CORE and Manipal Academy of Higher Education is gratefully acknowledged. Conflict of interest All the authors declare no conflict of interest. Reference Adzhubei I, Jordan DM, Sunyaev SR 2013. Predicting functional effect of human missense mutations using PolyPhen-2. Curr Protoc Hum Genet. Chapter 7, Unit7 20. Akella RR 2017. Mutational Analysis of Androgen Receptor Gene in Two Families with Androgen Insensitivity. Indian J Endocrinol Metab. 21, 520-523. Alvarez-Satta M, Castro-Sanchez S, and Valverde, D 2017. Bardet-Biedl Syndrome as a Chaperonopathy: Dissecting the Major Role of Chaperonin-Like BBS Proteins (BBS6BBS10-BBS12). Front Mol Biosci. 4, 55. Beales PL, Elcioglu N, Woolf AS, Parker D, Flinter, FA 1999. New criteria for improved diagnosis of Bardet-Biedl syndrome: results of a population survey. J Med Genet. 36, 437446. Blacque OE, Leroux MR 2006. Bardet-Biedl syndrome: an emerging pathomechanism of intracellular transport. Cell Mol Life Sci. 63, 2145-2161. Bowes C, LiT, Danciger M, et al. 1990. Retinal degeneration in the rd mouse is caused by a defect in the beta subunit of rod cGMP-phosphodiesterase. Nature. 347, 677–680. Cheng LL, Han RY, Yang FY, Yu XP, Xu JL, Min QJ, Tian J, Ge XL, Zheng SS, Lin YW, Zheng YH, Qu J, Gu F 2016. Novel mutations in PDE6B causing human retinitis pigmentosa. Int J Ophthalmol. 9,1094-1099. Forsythe E, Beales PL 2013. Bardet-Biedl syndrome. Eur J Hum Genet. 21, 8-13.
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Forsythe E, Kenny J, Bacchelli C, Beales PL 2018. Managing Bardet-Biedl Syndrome-Now and in the Future. Front Pediatr. 13;6:23. Gopalakrishna KN, Boyd K, Artemyev NO 2017. Mechanisms of mutant PDE6 proteins underlying retinal diseases. Cell Signal. 37, 74-80. Gottlieb B, Beitel LK, Nadarajah A, Paliouras M, Trifiro M 2012. The androgen receptor gene mutations database: 2012 update. Hum Mutat. 33, 887-894. Jagadeesh KA, Wenger AM, Berger MJ, Guturu H, Stenson PD, Cooper DN, Bernstein JA, Bejerano G 2016. M-CAP eliminates a majority of variants of uncertain significance in clinical exomes at high sensitivity. Nat Genet. 48, 1581-1586. Janssen S, Ramaswami G, Davis EE, et al. 2011. Mutation analysis in Bardet-Biedl syndrome by DNA pooling and massively parallel resequencing in 105 individuals. Hum Genet. 129, 79-90. Kalfa N, Philibert P, Werner R, Audran F, Bashamboo A, Lehors H, Haddad M, Guys JM, Reynaud R, Alessandrini P, et al. 2013. Minor hypospadias: the "tip of the iceberg" of the partial androgen insensitivity syndrome. PLoS One. 8, e61824. Kumar P, Henikoff S, Ng PC 2009. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat Protoc. 4, 1073-1081. Lindstrand A, Davis EE, Carvalho CM, et al., 2014. Recurrent CNVs and SNVs at the NPHP1 locus contribute pathogenic alleles to Bardet-Biedl syndrome. Am J Hum Genet. 94, 745-754. Manara E, Paolacci S, D'Esposito F, et al 2019. Mutation profile of BBS genes in patients with Bardet-Biedl syndrome: an Italian study. Ital J Pediatr. 45,72. Schwarz JM, Cooper DN, Schuelke M, Seelow D 2014. MutationTaster2: mutation prediction for the deep-sequencing age. Nat Methods. 11, 361-362.
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Stoetzel C, Laurier V, Davis EE, et al. 2006. BBS10 encodes a vertebrate-specific chaperonin-like protein and is a major BBS locus. Nat Genet. 38, 521-524. Thorvaldsdottir H, Robinson, JT, Mesirov JP 2013. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform. 14, 178192. Tobin JL, Beales PL 2009. The nonmotile ciliopathies. Genet Med. 11, 386-402.
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Table Table 1: Pathogenic variants identified by whole exome sequencing. Locus
Genotype
chr12:76740581
C/C
Gene Symbol BBS10
chrX:66766162
T/T
chr4:619713
T/T
Description
Phenotype
Coding
BBS10 gene
Bardet-Biedl syndrome 10
c.1184A>G
Amino Acid Change H395R
AR
Androgen receptor (dihydrotestoster one receptor)
Androgen insensitivity Abnormal male genitals, Reifenstein syndrome
c.1174C>T
P392S
0.02 (Damaging)
0.018 (Benign)
22.900
Disease causing
PDE6B
Phosphodiesteras e-6B, cGMP-specific, rod, beta
Night blindness, , Retinitis pigmentosa40
c.298C>T
R100C
0 (Damaging)
1 (Probably Damaging)
33.000
Disease causing
12
SIFT Score 0.01 (Damaging)
PolyPhen2 Score 0.999 (Probably Damaging)
CADD Score 14.610
Mutation Taster Disease causing
Figure legends Figure 1: Pedigree analysis and clinical features of the proband with BBS. (A) Family pedigree of the proband demonstrating the consanguinity between the proband’ s parents. Homozygous proband and his younger male sibling with BBS is indicated by the filled squares. Parents were heterozygous as indicated by the half-filled circles and half-filled squares. Arrow indicates the proband. (B) Post-axial polydactyl in upper limbs with traumatic amputation of right hand finger. Post-axial polydactyl in lower limbs. Fundoscopy showed pigmentary abnormalities in the peripheral retina. Proband has single testis with micropenis but well developed pubic and axillary hair. Figure 2: Whole exome sequencing of the proband identified homozygous BBS10 mutation with homozygous mutations in AR and PDE6B genes. Validation of BBS10, PDE6B and AR gene mutations by Sanger sequencing. (A) Homozygous missense mutation in BBS10 gene identified in proband and male sibling. Missense mutation in BBS10 gene was heterozygous in both the parents. (B) Homozygous missense mutation in AR gene identified in proband and male sibling. Missense mutation in AR gene was heterozygous in mother and homozygous wild type in father suggesting X-linked inheritance. (C) Homozygous missense mutation in PDE6B gene identified in proband and male sibling. PDE6B missense mutation was heterozygous in both the parents. (D) Protein sequence alignment showed evolutionary conservation of mutated residues in BBS10, PDE6B and AR genes. Supplementary figure 1: Mutation identified in the proband and affected siblings were mapped to BBS, AR and PDE6B protein domains. BBS10 mutation identified by whole exome sequencing was present in the Cpn60_TCP1 domain. Hemizygous AR mutation identified was present in N terminal androgen receptor domain and PDE6B mutation identified in regulatory GAF domain.
Highlights BBS10, AR and PDE6B mutation identified in two siblings from an Indian family. First report to show multiple gene mutation in BBS phenotype. Our study will enhance the knowledge in BBS diagnosis and therapeutics. 13
List of abbreviations
BBS- Bardet–Biedl syndrome
BMI- Body mass index
RFT- Renal Function Test
FLP- Fasting Lipid Profile
14
Conflict of interest
All the authors declare no conflict of interest
Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
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16
S0378-1119(19)30823-6 https://doi.org/10.1016/j.gene.2019.144164 GENE 144164
To appear in:
Gene Gene
Received Date: Revised Date: Accepted Date:
4 May 2019 14 September 2019 8 October 2019
Please cite this article as: S. Chakrabarty, S.B. Savantre, C. Ramachandra Bhat, K. Satyamoorthy, Multiple genetic mutations implicate spectrum of phenotypes in Bardet-Biedl syndrome, Gene Gene (2019), doi: https://doi.org/ 10.1016/j.gene.2019.144164
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier B.V.
Multiple genetic mutations implicate spectrum of phenotypes in Bardet-Biedl syndrome Sanjiban Chakrabarty1, Swheta. B. Savantre2, C. Ramachandra Bhat2, Kapaettu Satyamoorthy1* 1Department
of Cell and Molecular Biology, Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal, Karnataka, India-576104 2Department
of Medicine, K.V.G. Medical College & Hospital, Dakshina Kannada, Sullia,
India-574327 *Corresponding author: Dr. Kapaettu Satyamoorthy Professor Manipal School of Life Sciences Manipal Academy of Higher Education, Manipal Karnataka, India-576104 Telephone: 0820-2922058 Fax: 0820-2571919 Email: [email protected] Short title: BBS10 mutation in Bardet Biedl Syndrome
Multiple genetic mutations implicate spectrum of phenotypes in Bardet-Biedl syndrome Sanjiban Chakrabarty1, Swheta. B. Savantre2, C. Ramachandra Bhat2, Kapaettu Satyamoorthy1* 1Department
of Cell and Molecular Biology, Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal, Karnataka, India-576104 2Department
of Medicine, K.V.G. Medical College & Hospital, Dakshina Kannada, Sullia,
India-574327 *Corresponding author: Dr. Kapaettu Satyamoorthy Professor Manipal School of Life Sciences Manipal Academy of Higher Education, Manipal Karnataka, India-576104 Telephone: 0820-2922058 Fax: 0820-2571919 Email: [email protected] Short title: BBS10 mutation in Bardet Biedl Syndrome 1
Abstract Bardet–Biedl syndrome (BBS) is a clinically and genetically heterogeneous ciliopathy with several clinical features including retinitis pigmentosa, obesity, kidney dysfunction, postaxial polydactyly, behavioral dysfunction and hypogonadism with wide spectrum of additional features. With multiple phenotypes and heterogeneous distribution, it is unlikely that BBS is caused by single gene defect. We have performed clinical and genetic diagnosis of two individuals from an Indian family with classical BBS symptoms. Whole exome sequencing identified homozygous missense mutation in BBS10 gene, hemizygous missense AR and homozygous missense PDE6B mutations in the proband and affected sibling with BBS. Identification of BBS10 mutation along with AR and PDE6B gene mutation will expand the genetic and phenotypic spectrum in individuals with BBS. Keywords: Bardet–Biedl syndrome, BBS, retinitis pigmentosa, PDE6B, hypogonadism, AR
2
1. Introduction BBS is grouped one among the ciliopathies with wide range of clinical features which includes retinitis pigmentosa, obesity, kidney dysfunction, postaxial polydactyly, behavioral dysfunction and hypogonadism first described by ophthalmologists Laurence and Moon in the year 1866 (Blacque and Leroux., 2006). Other features such as cardiac abnormalities, diabetes, hypertension, hearing defects and anosmia are also reported (Forsythe and Beales., 2013, Forsythe et al., 2018). Clinical diagnosis of BBS requires presence of either a) three primary and two secondary features or b) only four primary features (Beales et al., 1999). The prevalence of BBS is 1:140,000 to 1: 160,000 in north American and European population with 1:165,000 in consanguineous Arab population. Molecular analysis identified mutations with autosomal recessive mode of inheritance in 20 different genes (BBS1-BBS20) responsible for majority of patients with BBS phenotype. BBS genes are primarily involved in ciliary biogenesis and trafficking by forming chaperonin complex (Tobin and Beales., 2009, Alvarez-Satta et al., 2017). The role of BBS10 gene (12q21.2) in the BBS clinical 3
phenotype was first identified in a consanguineous pedigree of Lebanese origin (Stoetzel et al., 2006). However, more than 25% of the BBS patients do not harbor BBS gene mutation suggesting involvement of additional genes in the BBS phenotype. Here, we describe clinical and genetic analysis of two siblings from an Indian family with BBS phenotype. Exome sequencing identified homozygous missense mutation in BBS10 gene in the proband and affected sibling. In addition to BBS10 mutation, we identified AR and PDE6B gene mutations in the proband and affected sibling with BBS. Along with causal BBS10 mutation, AR and PDE6B mutation may contribute to the severity of the BBS phenotype in the proband and sibling. 2. Methods 2.1 Clinical report Proband is 23-year-old male born out of second-degree consanguineous marriage, with history of 3 first trimester abortions prior to the index case (Fig. 1A). Proband showed history of delayed developmental milestones, frequent volatile outbursts and emotional lability with severe progressive visual impairment in the past 5 years. He was a school dropout due to his learning difficulties and visual impairment. His younger male sibling was 19 yrs. age and a school dropout due to poor scholastic performance. Both the parents were asymptomatic and had normal fundus examination. No similar history was found in other family members. 2.2 Genetic analysis The study follows the principles outlined in the Helsinki Declaration and patients gave written informed consent for molecular study and publication. Clinical specimens were collected after obtaining written informed consent from the proband and parents. Genomic DNA was extracted from the peripheral blood of the proband and other family members using QIAamp DNA Mini Kit (Qiagen GmbH, Hilden, Germany). Purity of the DNA was determined using NanoDrop ND1000 Spectrophotometer (ThermoFisher Scientific, 4
Waltham, MA, USA). Purified DNA was quantified using Qubit® dsDNA HS (High Sensitivity) Assay Kit (ThermoFisher Scientific) and used for AmpliSeq exome library preparation. Whole exome sequencing was performed using Ion Proton PIv2 chip in Ion Proton next generation sequencing platform (ThermoFisher Scientific). Sequencing reads obtained from Ion Proton were aligned to human reference sequence (human genome build19) and variant calling was performed using Ion Torrent Variant Caller v5.0 (TVC) in Torrent Suite software v5.0 (ThermoFisher Scientific). Alignment reads and variants called, with respect to the reference human genome sequence, were viewed using Integrative Genomic Viewer software and to check for strand biases, homopolymer length and sequencing errors (Thorvaldsdottir et al., 2013). 3. Results 3.1 Clinical examination On physical evaluation, acanthoses nigricans and post-axial polydactyl in all 4 limbs with traumatic amputation of right-hand finger was noted in the proband. (Fig. 1B). His BMI was 29.3 kg/m2. However, we did not find any visible neurocutaneous markers in the proband. He had grade-1 right beating nystagmus. Visual acuity is impaired to finger count near face, fundoscopy showed pigmentary abnormalities in the peripheral retina (Fig. 1B). He had single testis with micropenis but well developed pubic and axillary hair. (Fig. 1B). Proband was anemic with hemoglobin 9.8 g/dL, while other hematological parameters were normal. His biochemical profile (RFT and FLP) was normal. His testicular ultra-sonogram revealed absence of left testis and hypoplastic left seminal vesicle and prostrate. His younger male sibling was found to have finger counting at 3 meters and on fundoscopy had macular degeneration with peripheral pigmented spicules. Based on the clinical analysis, our proband had 4 major and 2 secondary features as per modified diagnostic criteria for BBS (Beales et
5
al., 1999). Clinical specimens were collected after obtaining written informed consent from the proband and parents. 3.2 Mutation analysis Whole exome sequencing of the proband identified homozygous missense mutation in BBS10 gene (H395R). In addition, we have identified hemizygous missense mutation in AR (P392S) and PDE6B (R100C) (Fig. 2, Table 1). Sanger sequencing of BBS10, AR and PDE6B gene mutations were confirmed in the proband and affected young male sibling. Both parents of the proband and affected male sibling were carrier of the BBS10 and PDE6B mutations in heterozygote state (Fig. 2). Hemizygous AR mutation analysis by Sanger sequencing showed X-linked inheritance in both the proband and affected sibling. To evaluate the pathogenicity of identified mutations in BBS10, AR, and PDE6B gene, independent prediction algorithms including SIFT (http://sift.jcvi.org/), PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/), MutationTaster2 (http://www.mutationtaster.org/), M-CAP, CADD score and Grantham score were employed for analysis (Kumar et al., 2009, Adzhubei et al., 2013, Schwarz et al., 2014, Jagadeesh et al., 2016). We analyzed the BBS10 gene mutation (NM_024685.3; c.1184A>G) in dbSNP 150, 1000 Genomes Project (http://www.1000genomes.org/), ExAc and NHLBI GO Exome Sequencing Project (ESP) databases to confirm BBS10 gene mutation in proband as a pathogenic variant. BBS mutation in the proband and affected sibling c.1184A>G has an allele frequency of 0.000003980 (1/251242 alleles) in the GnomAD database. Protein sequence alignment showed that mutated amino acid residues in BBS10, AR, and PDE6B gene is evolutionary conserved across the species (Fig. 2D). Mutations were mapped to BBS10, AR and PDE6B gene protein domains (Supplementary figure 1). BBS10 mutation (H395R) was found in the Cpn60_TCP1 domain which is important for its interaction with other BBS proteins (Supplementary figure 1).
AR gene mutation was
identified in the exon 1 encompassing N-terminal domain critical for AR function 6
(Supplementary figure 1). PDE6B, β subunit of rod photoreceptor cyclic GMP phosphodiesterase (PDE) plays a central role in the visual photo transduction cascade. We have identified PDE6B mutation (R100C) in the N-terminal GAF domain in proband and affected sibling (Supplementary figure 1). 4. Discussion BBS10 gene is structurally similar to type II chaperonins and involved in protein folding (Alvarez-Satta et al., 2017). Mutation in BBS10 gene is reported in patients with wide range of clinical presentation including obesity, rod-cone dystrophy and polydactyly (Janssen et al., 2011). Additionally, we have identified hemizygous AR mutation (P392S) previously reported in androgen insensitivity syndrome and homozygous PDE6B (R100C) mutation (Gottlieb et al., 2012, Kalfa et al., 2013). AR mutation identified in the current study have implications to disease phenotypes or co-morbid conditions. Since the hemizygous mutation found in exon 1 of AR gene, which encompasses DNA binding domain, its role in partial androgen insensitivity syndrome (PAIS) or complete AIS (CAIS) are possible (Gottlieb et al., 2012). Hemizygous AR mutation (P392S) identified in proband and sibling in our study is previously reported in two Indian families with complete androgen insensitivity (CAIS) and partial androgen insensitivity (PAIS) suggesting it to be a causal variant in patients with androgen insensitivity (Akella. 2017). Both proband and the affected sibling had hemizygous AR (P392S) mutation with absence of left testis and hypoplastic left seminal vesicle and prostrate (Figure 2) suggesting the role of AR gene mutation in the genital manifestation. However, these remains to be experimentally established. Mutations in phosphodiestaerase 6B (PDE6B) are reported to be involved in various visual impairment disorders (Gopalakrishna et al., 2017). Mouse model with homozygous mutation in PDE6B has shown elevated cGMP levels with retinal degeneration phenotype (Bowes et al., 1990). Additionally, PDE6B recessive mutation have been reported in patients with retinitis pigmentosa (Cheng et 7
al., 2016). However, these observations from mouse studies (Bowes et al., 1990) remain to be functionally validated for the specific role of PDE6B in the retinal degeneration of individuals with BBS10 (H395R) mutation. Bardet-Biedl syndrome is complex genetic disorder affecting multiple organs with wide range of clinical phenotype. Recent studies have reported patients with BBS harbor mutations in additional genes which indicate possible oligogenic inheritance (Lindstrand A et al., 2014, Manara et al., 2019). In the present study, we have identified homozygous missense BBS10 gene mutation with hemizygous mutation in AR and homozygous missense in PDE6B genes in the proband and his affected sibling suggesting their possible role in BBS clinical phenotype and expands the phenotypic and genetic spectrum of BBS. Our report explains the possibility of bipartite independent phenotypes due to two or more distinct mutations and potential interactions between them contributing to the complex clinical phenotype such as BBS. 5. Conclusion Homozygous missense mutation in BBS10 gene along with hemizygous missense mutation in AR gene and homozygous missense mutation in PDE6B gene identified in the proband and affected sibling in an Indian family expanding the genetic spectrum of BBS. Contribution of AR and PDE6B mutation in BBS phenotype alone or in combination with other BBS genes can be studied to understand the functional role for improved diagnosis in BBS patients. Statement of contribution Sanjiban Chakrabarty and Kapaettu Satyamoorthy conceptualized and designed the study, drafted and reviewed the manuscript. Swheta B Savantre and C. Ramachandra Bhat have coordinated data collection and critically reviewed the manuscript for important intellectual content. All authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work. Acknowledgments 8
The authors thank the patient and his family members who participated in this study. The infrastructure funding from DST-FIST, TIFAC-CORE and Manipal Academy of Higher Education is gratefully acknowledged. Conflict of interest All the authors declare no conflict of interest. Reference Adzhubei I, Jordan DM, Sunyaev SR 2013. Predicting functional effect of human missense mutations using PolyPhen-2. Curr Protoc Hum Genet. Chapter 7, Unit7 20. Akella RR 2017. Mutational Analysis of Androgen Receptor Gene in Two Families with Androgen Insensitivity. Indian J Endocrinol Metab. 21, 520-523. Alvarez-Satta M, Castro-Sanchez S, and Valverde, D 2017. Bardet-Biedl Syndrome as a Chaperonopathy: Dissecting the Major Role of Chaperonin-Like BBS Proteins (BBS6BBS10-BBS12). Front Mol Biosci. 4, 55. Beales PL, Elcioglu N, Woolf AS, Parker D, Flinter, FA 1999. New criteria for improved diagnosis of Bardet-Biedl syndrome: results of a population survey. J Med Genet. 36, 437446. Blacque OE, Leroux MR 2006. Bardet-Biedl syndrome: an emerging pathomechanism of intracellular transport. Cell Mol Life Sci. 63, 2145-2161. Bowes C, LiT, Danciger M, et al. 1990. Retinal degeneration in the rd mouse is caused by a defect in the beta subunit of rod cGMP-phosphodiesterase. Nature. 347, 677–680. Cheng LL, Han RY, Yang FY, Yu XP, Xu JL, Min QJ, Tian J, Ge XL, Zheng SS, Lin YW, Zheng YH, Qu J, Gu F 2016. Novel mutations in PDE6B causing human retinitis pigmentosa. Int J Ophthalmol. 9,1094-1099. Forsythe E, Beales PL 2013. Bardet-Biedl syndrome. Eur J Hum Genet. 21, 8-13.
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Forsythe E, Kenny J, Bacchelli C, Beales PL 2018. Managing Bardet-Biedl Syndrome-Now and in the Future. Front Pediatr. 13;6:23. Gopalakrishna KN, Boyd K, Artemyev NO 2017. Mechanisms of mutant PDE6 proteins underlying retinal diseases. Cell Signal. 37, 74-80. Gottlieb B, Beitel LK, Nadarajah A, Paliouras M, Trifiro M 2012. The androgen receptor gene mutations database: 2012 update. Hum Mutat. 33, 887-894. Jagadeesh KA, Wenger AM, Berger MJ, Guturu H, Stenson PD, Cooper DN, Bernstein JA, Bejerano G 2016. M-CAP eliminates a majority of variants of uncertain significance in clinical exomes at high sensitivity. Nat Genet. 48, 1581-1586. Janssen S, Ramaswami G, Davis EE, et al. 2011. Mutation analysis in Bardet-Biedl syndrome by DNA pooling and massively parallel resequencing in 105 individuals. Hum Genet. 129, 79-90. Kalfa N, Philibert P, Werner R, Audran F, Bashamboo A, Lehors H, Haddad M, Guys JM, Reynaud R, Alessandrini P, et al. 2013. Minor hypospadias: the "tip of the iceberg" of the partial androgen insensitivity syndrome. PLoS One. 8, e61824. Kumar P, Henikoff S, Ng PC 2009. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm. Nat Protoc. 4, 1073-1081. Lindstrand A, Davis EE, Carvalho CM, et al., 2014. Recurrent CNVs and SNVs at the NPHP1 locus contribute pathogenic alleles to Bardet-Biedl syndrome. Am J Hum Genet. 94, 745-754. Manara E, Paolacci S, D'Esposito F, et al 2019. Mutation profile of BBS genes in patients with Bardet-Biedl syndrome: an Italian study. Ital J Pediatr. 45,72. Schwarz JM, Cooper DN, Schuelke M, Seelow D 2014. MutationTaster2: mutation prediction for the deep-sequencing age. Nat Methods. 11, 361-362.
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Stoetzel C, Laurier V, Davis EE, et al. 2006. BBS10 encodes a vertebrate-specific chaperonin-like protein and is a major BBS locus. Nat Genet. 38, 521-524. Thorvaldsdottir H, Robinson, JT, Mesirov JP 2013. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform. 14, 178192. Tobin JL, Beales PL 2009. The nonmotile ciliopathies. Genet Med. 11, 386-402.
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Table Table 1: Pathogenic variants identified by whole exome sequencing. Locus
Genotype
chr12:76740581
C/C
Gene Symbol BBS10
chrX:66766162
T/T
chr4:619713
T/T
Description
Phenotype
Coding
BBS10 gene
Bardet-Biedl syndrome 10
c.1184A>G
Amino Acid Change H395R
AR
Androgen receptor (dihydrotestoster one receptor)
Androgen insensitivity Abnormal male genitals, Reifenstein syndrome
c.1174C>T
P392S
0.02 (Damaging)
0.018 (Benign)
22.900
Disease causing
PDE6B
Phosphodiesteras e-6B, cGMP-specific, rod, beta
Night blindness, , Retinitis pigmentosa40
c.298C>T
R100C
0 (Damaging)
1 (Probably Damaging)
33.000
Disease causing
12
SIFT Score 0.01 (Damaging)
PolyPhen2 Score 0.999 (Probably Damaging)
CADD Score 14.610
Mutation Taster Disease causing
Figure legends Figure 1: Pedigree analysis and clinical features of the proband with BBS. (A) Family pedigree of the proband demonstrating the consanguinity between the proband’ s parents. Homozygous proband and his younger male sibling with BBS is indicated by the filled squares. Parents were heterozygous as indicated by the half-filled circles and half-filled squares. Arrow indicates the proband. (B) Post-axial polydactyl in upper limbs with traumatic amputation of right hand finger. Post-axial polydactyl in lower limbs. Fundoscopy showed pigmentary abnormalities in the peripheral retina. Proband has single testis with micropenis but well developed pubic and axillary hair. Figure 2: Whole exome sequencing of the proband identified homozygous BBS10 mutation with homozygous mutations in AR and PDE6B genes. Validation of BBS10, PDE6B and AR gene mutations by Sanger sequencing. (A) Homozygous missense mutation in BBS10 gene identified in proband and male sibling. Missense mutation in BBS10 gene was heterozygous in both the parents. (B) Homozygous missense mutation in AR gene identified in proband and male sibling. Missense mutation in AR gene was heterozygous in mother and homozygous wild type in father suggesting X-linked inheritance. (C) Homozygous missense mutation in PDE6B gene identified in proband and male sibling. PDE6B missense mutation was heterozygous in both the parents. (D) Protein sequence alignment showed evolutionary conservation of mutated residues in BBS10, PDE6B and AR genes. Supplementary figure 1: Mutation identified in the proband and affected siblings were mapped to BBS, AR and PDE6B protein domains. BBS10 mutation identified by whole exome sequencing was present in the Cpn60_TCP1 domain. Hemizygous AR mutation identified was present in N terminal androgen receptor domain and PDE6B mutation identified in regulatory GAF domain.
Highlights BBS10, AR and PDE6B mutation identified in two siblings from an Indian family. First report to show multiple gene mutation in BBS phenotype. Our study will enhance the knowledge in BBS diagnosis and therapeutics. 13
List of abbreviations
BBS- Bardet–Biedl syndrome
BMI- Body mass index
RFT- Renal Function Test
FLP- Fasting Lipid Profile
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Conflict of interest
All the authors declare no conflict of interest
Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
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