
==== Front
Mol Genet Genomic Med
Mol Genet Genomic Med
10.1002/(ISSN)2324-9269
MGG3
Molecular Genetics & Genomic Medicine
2324-9269
John Wiley and Sons Inc. Hoboken

10.1002/mgg3.2478
MGG32478
MGG3-2024-02-0150.R1
Original Article
Original Article
Identification of rare missense variants in the BSN gene co‐segregating with chronic otitis media in a consanguineous Pakistani family
Yousaf et al.
Yousaf Ayesha 1
Yousaf Sairah 2
Shabbir Asra S. 3 4
Yousaf Rafia 5
Riazuddin Saima 2 6
Shaikh Rehan S. 1 7
Santos‐Cortez Regie Lyn P. 8
Ahmed Zubair M. https://orcid.org/0000-0003-2914-4502
2 6 9 zmahmed@som.umaryland.edu

1 Institute of Molecular Biology and Biotechnology Bahauddin Zakariya University Multan Pakistan
2 Department of Otorhinolaryngology—Head and Neck Surgery School of Medicine University of Maryland Baltimore Maryland USA
3 School of Pharmacy University of Management and Technology Lahore Pakistan
4 Department of Pharmacy Government College University Faisalabad Pakistan
5 Nishter Hospital Multan Pakistan
6 Department of Biochemistry and Molecular Biology School of Medicine University of Maryland Baltimore Maryland USA
7 Centre of Applied Molecular Biology University of the Punjab Lahore Punjab Pakistan
8 Department of Otolaryngology—Head and Neck Surgery, School of Medicine University of Colorado Anschutz Medical Campus Aurora Colorado USA
9 Department of Ophthalmology and Visual Sciences School of Medicine University of Maryland Baltimore Maryland USA
* Correspondence
Zubair M. Ahmed, Department of Otorhinolaryngology—Head and Neck Surgery, School of Medicine, University of Maryland, Baltimore, MD, USA.
Email: zmahmed@som.umaryland.edu

20 9 2024
9 2024
12 9 10.1002/mgg3.v12.9 e247807 5 2024
05 2 2024
21 5 2024
© 2024 The Author(s). Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.

Abstract

Background

Otitis media (OM) is the most frequent and complex middle ear condition with multifactorial etiology including genetic predisposition. OM depicts a variable clinical spectrum, leading to speech, developmental delay, and hearing loss. Here, we report the clinical and genetic findings of chronic suppurative otitis media (CSOM) segregating in a six‐generation consanguineous Pakistani family PKOM08.

Methods

Clinical evaluations, including audio and tympanometry, were conducted to assess OM manifestation and their impact on hearing function. Exome sequencing was performed to identify potential genetic variants underlying CSOM in the study participants.

Results

Clinical evaluation of participating individuals revealed varying degrees of disease severity, with mild to moderate hearing loss. All the affected individuals had CSOM with no other apparent comorbidity. Whole exome followed by Sanger sequencing revealed two rare heterozygous variants [c.1867C>T, p.(Pro623Ser) and c.11015G>A, p.(Arg3672Gln)] of BSN gene in most of the affected individuals of family PKOM08. BSN encodes a scaffold bassoon protein involved in synaptic vesicle trafficking. The identified variants replaced evolutionary conserved amino acid residues in the encoded protein and are predicted to impact the ionic interactions in the secondary structure.

Conclusion

A deep intronic variant of BSN has been previously implicated in the etiology of childhood ear infections. Our study further supports a link between BSN‐impaired function and ear infection and CSOM in children.

Large consanguineous family segregating chronic suppurative otitis media (CSOM) associated with biallelic variants of BSN. Our study further supports a link between BSN‐impaired function and ear infection and CSOM in children.

chronic suppurative otitis media
BSN
ear infection
missense variants
otitis media
National Institute on Deafness and Other Communication Disorders 10.13039/100000055 R01DC016295 source-schema-version-number2.0
cover-dateSeptember 2024
details-of-publishers-convertorConverter:WILEY_ML3GV2_TO_JATSPMC version:6.4.8 mode:remove_FC converted:20.09.2024
Yousaf, A. , Yousaf, S. , Shabbir, A. S. , Yousaf, R. , Riazuddin, S. , Shaikh, R. S. , Santos‐Cortez, R. L. P. , & Ahmed, Z. M. (2024). Identification of rare missense variants in the BSN gene co‐segregating with chronic otitis media in a consanguineous Pakistani family. Molecular Genetics & Genomic Medicine, 12 , e2478. 10.1002/mgg3.2478
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pmc1 INTRODUCTION

Otitis media (OM) is the most frequent and complex middle ear condition, primarily affecting infants and preschoolers worldwide (Chonmaitree et al., 2016). Based on clinical aberrations, OM can be categorized as either acute or chronic. Acute otitis media (AOM) is characterized by inflammation, bulging of the tympanic membrane (TM), erythema, fullness, otalgia, and fever (Mittal et al., 2015). The estimated global incidence of AOM is 10.85% (Monasta et al., 2012). The progression from AOM to chronic suppurative otitis media (CSOM) might commence with a tympanic membrane perforation and is associated with persistent otorrhea over 2–3 weeks. The approximate worldwide prevalence of CSOM is 4.76% (Monasta et al., 2012), impairing the development, conduct, hearing, and learning achievements of children (Lilian, 2018).

The complex pathophysiology/etiology of OM includes upper respiratory tract infections (URTIs), immunological deterioration, anatomical elements, environmental factors, genetic predisposition, and otopathogens (Thornton et al., 2011; Zhang et al., 2014). Accurate diagnosis of OM encompasses otoscopy, pneumatic otoscopy, audiometry, or tympanometry for the prevention of patients suffering from life‐threatening issues (Schilder et al., 2016). Treatment strategies comprise antibiotics, anti‐inflammatory drugs, and the surgical insertion of ventilation tubes (Qureishi et al., 2014; Ramakrishnan et al., 2007; To, K, et al., 2013), but 4.5%–8.5% result in treatment failure. Thus, alternative management approaches are desired (Mustafa et al., 2017).

Molecular genetic analysis established the association of numerous candidate susceptible genes in humans as well as in mice models with otitis media including FBXO11 (Rye et al., 2011), BPIFA1, CAPN14, GALNT14 (Rye et al., 2012), FNDC1 (Van Ingen et al., 2016), TGF1 (Ilia et al., 2014), TBX1, ABO, BSN, AUTS2, CDHR3, EFEMP1 (Tian et al., 2017), FUT2 (Santos‐Cortez et al., 2018), PLG (Bootpetch et al., 2020; Eriksson et al., 2006), and SPINK5 (Frank et al., 2020) for OM.

In Pakistan, the prevalence of AOM at tertiary health care among febrile children was noted to be 22.9% (Raza et al., 2022), and the prevalence of otitis media with effusion is estimated to range from 6.9% to 11.5% (Najeeb et al., 2008; Tallat et al., 2013), whereas others place it as high as 27% (Riaz et al., 2022). Chronic/persistent infection is also the leading cause of ear disease (Mansoor et al., 2009). The OM‐related hearing loss is >40/10,000, and the mortality rate is 50–79·9/10 × 106 population (Shakoor et al., 2016).

Here, we report the ascertainment of a large consanguineous Pakistani family (PKOM08) with multiple affected individuals manifesting OM. Exome sequencing (ES) identified candidate variants in BSN (OMIM: 604020) that segregated with the OM phenotype in the family PKOM08.

2 MATERIALS AND METHODS

2.1 Ethical compliance

This study was approved by the Institutional Review Board (IRB) Committees of the Institute of Molecular Biology and Biotechnology, Bahauddin Zakariya University, Multan, Pakistan and the University of Maryland School of Medicine, Baltimore, MD, USA. All procedures complied with the Declaration of Helsinki.

2.2 Participants and clinical evaluation

Probands with a history of OM were identified and ascertained from Nishtar Hospital, Multan, Pakistan. Written informed consent/assent from the participants were obtained before enrollment into the study. Multiple participants were interviewed to determine the family structure, OM disorder history, consanguinity, age, and other demographic information. Otological examinations, including otoscopy, pure tone audiometry (PTA), and tympanometry, were performed at the health facilities in Multan, Pakistan. Venous blood samples were collected for genomic DNA extraction.

2.3 Exome sequencing and segregation analysis

Genomic DNA sample of proband (V:1) underwent ES at the genomics core facility of the University of Maryland Baltimore. Exome enriched libraries were generated using the Agilent Sure‐Select Human Expanded All Exon V5 kit and sequenced on Illumina HiSeq4000 instrument with an average coverage of 100X, as previously reported (Riazuddin et al., 2017; Richard et al., 2019). Sequence reads were processed and aligned to the human genome (hg19/GRCh37) with the Burrows‐Wheeler Aligner (Li & Durbin, 2010), and variants were identified through UnifiedGenotyper GATK module. Multi‐layer filtration and variant prioritization criteria were used to narrow down candidate OM‐associated variants for segregation analysis through Sanger sequencing (Riazuddin et al., 2017).

2.4 Three‐dimensional protein modeling and bioinformatic analysis

To evaluate the impact of amino acid alteration on protein folding and secondary structure, various in silico tools were employed. For instance, Phyre2 was implemented to generate 3D protein structures (Kelley et al., 2015), and CHIMERA (https://www.cgl.ucsf.edu/chimera) and HOPE tool were used to visually evaluate the protein structures and amino acids bonding patterns (Venselaar et al., 2010). We also used SIFT (https://sift.bii.a‐star.edu.sg/), Polyphen2 (http://genetics.bwh.harvard.edu/pph2/), Mutation Taster (http://www.mutationtaster.org/), and Mutation Assessor (http://mutationassessor.org/r3/) in silico algorithms to determine the impact of identified variants on the encoded BSN.

Conservation of amino acid residues was analyzed by importing analogous nonhuman proteins from Ensembl (https://asia.ensembl.org/index.html) and aligned using Clustal Omega tool (https://www.ebi.ac.uk/Tools/msa/clustalo/). The genetic tolerance of identified variations was predicted through online resource, MetaDome (https://stuart.radboudumc.nl/metadome/). Two‐point Logrithim of the odds (LOD) score for the BSN variants was calculated with Superlink by EasyLinkage 5.02 v GUI. An autosomal recessive inheritance and a disease allele frequency of 0.001 were used for linkage analysis.

3 RESULTS

3.1 Ascertainment and clinical phenotyping

After approval from institutional review boards of participating institutes, we ascertained a multi‐generational family (PKOM08) segregating OM from the Southern Punjab province of Pakistan (Figure 1a). In total, there are 12 affected individuals, including four females and eight males, that had a history of CSOM. Although the specific age of disease onset for the affected participants was unknown, however, according to the family history interviews, all of the participating affected individuals had a history of otorrhea extending back at least 2–3 years. Next, to further characterize the disease phenotype and comorbidities, two of the participating affected individuals (IV:2 and V:1) underwent a detailed medical examination (Table 1). Otoscopic examination in these individuals revealed uni‐ or bi‐lateral CSOM (Table 1). No other common comorbidities, including vertigo, fever, URTIs, and mastoiditis, were observed in these two affected individuals (Table 1). TM in the right and left ears of patients were assessed for perforation and retraction pockets. TM perforation was observed in both ears of affected individual IV:2, while individual V:1 only had perforation in the right ear (Table 1). Tympanometry and pure tone audiometry (PTA) profiles of assessed patients revealed varying degrees of hearing loss in these affected individuals (Table 1).

FIGURE 1 BSN variants co‐segregate in family PKOM08. (a) Pedigree of family PKOM08. The filled symbols represent affected individuals, and a double horizontal line connecting parents represents a consanguineous marriage. Genotypes for the identified BSN variants are given for the participating individuals. (b) Representative Sanger sequencing chromatograms from one normal (VI:1) and two affected individuals (V:5, V:6). Affected individual V:5 has wild‐type allele at c.1867 position, while he is homozygous for the c.11015G>A variant. In contrast, affected individual V:6 is compound heterozygous for both variants. (c) Tolerance landscape visualization of BSN via MetaDome with relative positions of the two missense variants (red) identified in the PKOM08 family. The p.(Pro623Ser) variant was found to be present in a slightly intolerant region, whereas the p.(Arg3672Gln) is located in the tolerated region of the protein.

TABLE 1 Clinical evaluation of affected individuals of family PKOM08.

Individual ID	IV:2	V:1	
Ethnicity	Matilay	
Gender	Male	Female	
Phenotype	Affected	Affected	
Age a	72 years	30 years	
Fever	No	No	
Disturbed sleep	No	No	
Vomiting	No	No	
Vertigo	No	No	
Balance	No	No	
Tinnitus	No	No	
URTIs	No	No	
Mastoiditis	No	No	
Adenoid	No	No	
Ear	Right	Left	Right	Left	
Type of OM	CSOM	CSOM	CSOM	CSOM	
TM color	White	White	White	White	
TM position	Neutral	Neutral	Neutral	Neutral	
TM mobility	Normal	Normal	Normal	Normal	
TM translucency	Translucent	Translucent	Semi‐opaque	Semi‐opaque	
TM perforation	Yes	Yes	Yes	No	
Air fluid levels	No	No	No	No	
Retraction pockets	No	No	No	No	
Cholesteatoma	No	No	No	No	
Tympanosclerosis	No	No	Yes	No	
Hearing loss	Moderate to profound	Moderate	Moderate	Moderate to mild	
a At the time of enrollment into the current study.

Abbreviations: CSOM, chronic suppurative otitis media; TM, tympanic membrane; URTI, upper respiratory tract infections.

3.2 Genetic findings

To decipher the underlying genetic deficits contributing to CSOM phenotype in family PKOM08, we performed ES on the proband. After quality control, we used an in silico panel to filter variants in the known genes associated with OM in humans or animal models. Next, we selected variants of genes that had (a) a minor allele frequency of ≤0.001, (b) had CADD score of ≥10, (c) predicted damage by at least one in silico algorithm, or (d) had been associated with OM in prior studies.

Table 2 lists the four variants of OM genes that passed these criteria. All these variants were found in the heterozygous state in the affected individuals. However, two of these variants were present within the same gene, BSN, thus the proband could be compound heterozygous (Table 2). Sanger sequencing of all the participating individuals of family PKOM08 revealed that four (IV:2, V:1, V:2, and V:6) of eight affected individuals were compound heterozygous for the c.1867C>T [p.(Pro623Ser)] and c.11015G>A [p.(Arg3672Gln)] variants (Figure 1a,b). Additional two affected individuals (V:5 and V:8) inherited c.11015G>A variant in homozygous fashion, while the remaining two affected (V:9 and VI:2) were heterozygous for this variant (Figure 1a,b). Parametric linkage analysis was performed using the BSN variants genotype, which resulted in a two‐point LOD score of 1.25 at θ = 0. The p.(Pro623Ser) and p.(Arg3672Gln) variants found in PKOM08 family are located in the relatively intolerant regions of encoded protein when assessed by the MetaDome intolerance plot (Figure 1c).

TABLE 2 Genetic analysis of PKOM08 proband for known otitis media candidate gene variants.

Gene	Proband genotype	Transcript ID	cDNA change	Amino acid change	gnomAD	CADD	SIFT	Polyphen2	Mutation taster	Mutation assessor	References	
BSN	Het	NM_003458	c.1867C>T	p.(Pro623Ser)	0.000152	10.46	T	B	N	N	Pickrell et al., (2016)	
BSN	Het	NM_003458	c.11015G>A	p.(Arg3672Gln)	0.000116	12.67	T	P	D	L	Tian et al. (2017)	
FBXO11	Het	NM_001190274	c.160C>A	p.(Gln54Lys)	0	2.70	D	‐	D	N	Rye et al. (2011)	
CHD7	Het	NM_001316690	c.2341G>A	p.(Ala781Thr)	0.000333	10.64	T	B	D	L	Tian et al., (2012)	
Abbreviations: B, benign; CADD, combined annotation‐dependent depletion; D, damaging; Het, heterozygous; gnomAD, Genome Aggregation Database; L, likely pathogenic; N, Neutral; P, probably damaging; T, tolerated.

The BSN gene consists of 12 exons and encodes a 420 kDa scaffold bassoon protein (Figure 2a, b), involved in synaptic vesicle trafficking and establishment of the cytomatrix at the active zone of photoreceptors (Dick et al., 2003) and inner hair cells (Khimich et al., 2005). The p.(Pro623Ser) and p.(Arg3672Gln) variants found in the PKOM08 family are located in evolutionarily conserved regions (Figure 2c). To decipher how these variations might disrupt BSN secondary structure, three‐dimensional (3D) protein modeling was performed using Phyre2 in silico tool. In the wild‐type protein, p.Pro623 is predicted to induce a distinct backbone conformation due to its rigid structure. Replacement of p.Pro623 with p.Ser623 alters the protein's conformation and disrupts hydrogen bonding with p.Gln120 and p.Ser392 (Figure 2d) and thus might impact protein folding. The p.Arg3672 position of BSN was not modeled through the Phyre2 server; therefore, we used the HOPE prediction tool to analyze the impact of p.Arg3672Gln substitution. The positively charged p.Arg3672 is significantly larger than the neutral p.Gln3672, and thus, replacement was predicted to disrupt interactions with other molecules or residues.

FIGURE 2 BSN gene and protein structures along with the in silico analysis of impacts of identified variants. (a) Schematic representation of BSN gene structure, the position of identified rare variants is indicated. (b) Schematic representation of BSN protein marks mutated amino acids. (c) Alignment of amino acid sequences among different species exhibited the conservation of mutated amino acid residues at positions 623 and 3672. (d) The three‐dimensional arrangement of BSN protein. Helix and coil regions are presented with purple and green colors, respectively. Wild‐type and mutant amino acid residues in a protein are highlighted in yellow. Proline is the only amino acid where the protein side chain binds to the backbone twice, generating a nitrogen‐containing ring with five members. Substituting proline with serine at position 623 is predicted to affect the protein conformation due to its smaller size and less hydrophobic interaction.

As indicated in Figure 1a, two of the evaluated affected individuals (V:9, VI:2) were only carriers of one allele, indicating interfamilial genetic heterogeneity. We also found rare variants of two other known OM‐associated genes, FBXO11 and CHD7, present in a heterozygous state in the affected individual of family PKOM08 (Table 2). Although various in silico algorithms predicted low effect size for these variants individually (Table 2), clustering of them in various zygosity combinations among the affected individuals might increase the disease risk burden and contribute to the OM phenotype and genetic heterogeneity observed in family PKOM08.

4 DISCUSSION

There are few studies on the incidence and genetic analysis of individuals with CSOM from Pakistan. A recent study reported a rare A2ML1 frameshift variant c.3676_3677delGC (p.(Ala1226Glnfs*34)) co‐segregated with OM in a Pakistani family (Larson et al., 2019). Additionally, two other variants in FUT2 (c.311C>T (p.Ala104Val)) and PLG (c.1414G>A, (p.Ala104Val)) were found to confer susceptibility to familial OM (Bootpetch et al., 2020; Santos‐Cortez et al., 2018). In this study, we examined a large family with multiple affected individuals showing variable expression of otorrhea, perforated tympanic membrane, and hearing loss. We described two missense variants of BSN conferring risk for CSOM susceptibility in PKOM08 ascertained from the Southern Punjab province of Pakistan. Prior studies in children reported a significant (p = 1.56 × 10−8) association of a deep intronic variant (rs67035515) of BSN with ear infection (Tian et al., 2017).

BSN encodes a scaffold bassoon protein involved in synaptic vesicle trafficking and establishing the cytomatrix at the active zone of photoreceptors (Dick et al., 2003) and inner hair cells (Khimich et al., 2005). Studies in Bsn‐mutant mice demonstrated an essential role of bassoons in anchoring synaptic ribbons in inner hair cells and auditory coding (Jing et al., 2013). Reduced bassoon expression or complete absence of full‐length bassoon disrupts the anchoring of ribbons to the active zones, diminished readily releasable vesicle pool, and impaired synchronous auditory signaling and sound‐evoked stimulation of spiral ganglion neurons (Khimich et al., 2005), so crucial for normal hearing.

In silico protein analysis predicted the slightly intolerated impact of the variant p.Pro623Ser that affects the protein conformation. Other variant p.Arg3672Gln, located in the C‐terminus of bassoon, the terminus attached to the piccolo‐bassoon transport vesicles membrane and Golgi membranes along the trafficking route (Gundelfinger et al., 2016). The damages of the C‐terminus might disturb the docking, fusing, and trafficking of synaptic vesicles (Ye et al., 2023), possibly explaining the implication of BSN in our observed phenotype. The identified variants might interact in a novel way that confers CSOM susceptibility in patients. However, further studies would be required in appropriate animal models to establish their pathological effect on otorrhea and hearing loss.

The “disease causing” variants [c.4444T>A (p.Ser1481Thr), c.4445C>T (p.Pro1482Leu)] in BSN are also considered a candidate gene for Landau–Kleffner syndrome, which is a rare childhood epileptic encephalopathy (Conroy et al., 2014). Furthermore, rare heterogeneous variants of BSN are reported to be implicated in neurodegenerative diseases, progressive supranuclear palsy‐like syndrome, and Parkinson's (Yabe et al., 2018; Yemni et al., 2019). Similarly, variants in BSN are reported to cause epilepsy in humans, a common neurological disorder featured by unprovoked seizures (Ye et al., 2023). A single nucleotide polymorphism in BSN is co‐segregating with schizophrenia in a multiplex family of Taiwan (Chen et al., 2021). Furthermore, recently, a rare loss of function BSN variant has been reported to be associated with obesity in an autosomal‐dominant manner (Zhu et al., 2023). The affected individuals of PKOM08 did not have obesity and above‐mentioned neurological phenotypes, thus suggesting the pleiotropic effects of BSN gene and implicated two compound heterozygous missense variants [p.(Pro623Ser) and p.(Arg 3672Gln)] of BSN in OM etiology. Currently, the sample size of CSOM cases is insufficient for a meaningful evaluation of genotype–phenotype variations. However, this study suggests BSN a potential candidate gene for CSOM genetic susceptibility.

AUTHOR CONTRIBUTIONS

A.Y.: Subject enrollement, Investigation, formal analysis, writing—original draft preparation. S.Y., A.S.S., R.Y.: Clinical evaluation, formal analysis, writing—review and editing. S.R., R.S.S.: Supervision, methogoloy, writing—review and editing. R.L.P.S.C.: Methodology, formal analysis, writing—review and editing. Z.M.A: Conceptualization, investigation, formal analysis, supervision, writing—review and editing.

FUNDING INFORMATION

This study was partially supported by the National Institute of Deafness and Other Communication Disorders (NIDCD), National Institutes of Health, Bethesda, MD, USA (R01DC016295 to ZMA).

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.

ETHICS STATEMENT

The study was conducted in accordance with the Declaration of Helsinki, and approved by the In‐stitutional Review Board of UMB (1 May 2023) for studies involving humans.

ACKNOWLEDGMENTS

We are thankful to all the participants of the study and healthcare professionals for their cooperation in clinical investigations. We are thankful to Ms. S. Rehman and Dr. F. Kabir for their technical assistance.

DATA AVAILABILITY STATEMENT

Data are available in the manuscript in figures and tables. Exome‐sequencing data will be available through dbGAP.
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