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Published in final edited form as: Hum Mutat. 2018 Nov 18;40(1):53–72. doi: 10.1002/humu.23666

Global genetic insight contributed by consanguineous Pakistani families segregating hearing loss

Elodie M Richard 1,#, Regie LP Santos-Cortez 2,$,#, Rabia Faridi 3,4,#, Atteeq U Rehman 3,%, Kwanghyuk Lee 2, Mohsin Shahzad 1,5, Anushree Acharya 2, Asma A Khan 4, Ayesha Imtiaz 3, Imen Chakchouk 2, Christina Takla 3, Izoduwa Abbe 2, Maria Rafeeq 4, Khurram Liaqat 6, Taimur Chaudhry 3, Michael J Bamshad 7; University of Washington Center for Mendelian Genomics7, Isabelle Schrauwen 2, Shaheen N Khan 4, Robert J Morell 8, Saba Zafar 9, Muhammad Ansar 6, Zubair M Ahmed 1, Wasim Ahmad 6, Sheik Riazuddin 5,10, Thomas B Friedman 3,#, Suzanne M Leal 2,#, Saima Riazuddin 1,5,#
PMCID: PMC6296877  NIHMSID: NIHMS992637  PMID: 30303587

Abstract

Consanguineous Pakistani pedigrees segregating deafness have contributed decisively to the discovery of 31 of the 68 genes associated with nonsyndromic autosomal recessive hearing loss (HL) worldwide. In this study, we utilized genome-wide genotyping, Sanger and exome sequencing to identify 163 DNA variants in 41 previously reported HL genes segregating in 321 Pakistani families. Of these, 71 (43.6%) variants identified in 29 genes are novel. As expected from genetic studies of disorders segregating in consanguineous families, the majority of affected individuals (94.4%) are homozygous for HL-associated variants, with the other variants being compound heterozygotes. The five most common HL genes in the Pakistani population are SLC26A4, MYO7A, GJB2, CIB2 and HGF, respectively. Our study provides a profile of the genetic etiology of HL in Pakistani families, which will allow for the development of more efficient genetic diagnostic tools, aid in accurate genetic counseling and guide application of future gene-based therapies. These findings are also valuable in interpreting pathogenicity of variants that are potentially associated with HL in individuals of all ancestries. The Pakistani population, and its infrastructure for studying human genetics, will continue to be valuable to gene discovery for HL and other inherited disorders.

Keywords: Deafness, genetic spectrum, Pakistan, allelic heterogeneity, autosomal recessive hearing loss, DFNB, pathogenic variant

Introduction

Normal hearing ability requires many specialized cell types. In the inner ear there are intricately-structured mechanosensory hair cells overlaid by an acellular tectorial membrane, the stria vascularis, which generates the potassium-rich endolymph, and several supporting cell types, which in concert are necessary for transduction of sound (Burns, Kelly, Hoa, Morell, & Kelley, 2015). A failure of normal development, loss of any one of several essential physiological or biochemical functions or degeneration of one or more of these cell types in the inner ear can cause hearing loss (HL).

Today, ~466 million people have disabling HL (WHO, March 2018), including 1 in 500 newborns (Morton & Nance, 2006), 34 million children and one-half of all individuals over the age of 75 (Yamasoba et al., 2013). HL can be one feature of a more complex clinical syndrome (Gettelfinger & Dahl, 2018). Isolated hearing loss, referred to as nonsyndromic autosomal recessive hearing loss (NSARHL), is also genetically heterogenous. As of June 2018, 105 NSARHL loci have been mapped and 68 identified (http://hereditaryhearingloss.org). Close to half of these genes (31 out of 68) were identified through studies of large consanguineous families from Pakistan. Subsequently, additional mutations in these same genes were recognized in diverse populations across the world.

The genetic heterogeneity of hearing disorders can present a challenge for identification of causative molecular lesions and expeditious molecular diagnosis which is critical for prognosis and informed genetic counseling (Askew et al., 2015; Shibata et al., 2016). The availability of next generation sequencing (NGS), particularly exome sequencing, has improved the success for the identification of possible pathogenic variants for inherited disorders, including HL (Gao & Dai, 2014; Sloan-Heggen & Smith, 2016; Vona, Nanda, Hofrichter, Shehata-Dieler, & Haaf, 2015). Using a combination of experimental strategies from classical homozygosity mapping (Friedman et al., 1995) to exome sequencing, we identified 163 variants (71 of which are novel) in 41 reported hearing-associated genes co-segregating with HL in 321 Pakistani families. Our present and previous genetic profiling provides an improved understanding of the causes of inherited HL in the Pakistani population while revealing genes with high population-specific prevalence rates.

Material and Methods

Human subjects

This study followed the tenets of the Declaration of Helsinki. Approval for this study was obtained from institutional review boards at the University of Maryland, School of Medicine, Baltimore, Maryland, USA (HP-00059851), the Baylor College of Medicine and Affiliated Hospitals (H-17566), the Combined Neurosciences Blue Panel at the National Institutes of Health (OH-93-N-016), the National Centre of Excellence in Molecular Biology at the University of the Punjab, Quaid-i-Azam University, and Liaquat University of Medical and Health Sciences. Written informed consent was obtained from all participating individuals in this study.

Screening and identification of HL variants

Three new cohorts of Pakistani families segregating HL were examined by NGS. Cohorts 1, 2 and 3 have 261, 40, and 20 families, respectively, for a total of 321 families. For cohort 1, DNA samples from two affected individuals were first screened for variants in HGF (Schultz et al., 2009), CIB2 [c.272T>C; p.(Phe91Ser)] (Riazuddin et al., 2012), and GJB2 (Santos, Wajid, Pham, et al., 2005), using Sanger sequencing. HL-associated variants were found in 94 of these families. DNA samples from the family members of these 94 families were also Sanger-sequenced to test co-segregation of variants with HL. For the remaining 167 families that were negative for variants of GJB2, CIB2 or HGF, DNA samples for 129 families were genotyped using microsatellite or single nucleotide polymorphism (SNP) markers. Genotype data were checked for errors using PedCheck (O’Connell and Weeks, 1998) and MERLIN (Abecasis, et al. 2002). Analysis was performed using HomozygosityMapper (Seelow, Schuelke, Hildebrandt, & Nurnberg, 2009) and Allegro for linkage analysis (Gubjartsson, et al., 2005). Out of 129 families with genotype data, 46 families were mapped to a region containing a known HL gene and the putatively pathogenic variant was identified and co-segregation with HL confirmed using Sanger sequencing.

The remaining 121 families from cohort 1 included those who were not mapped to known HL genes and were negative when followed-up with Sanger sequencing. Forty additional families (cohort 2) did not have genome-scan genotype data. A DNA sample from an affected individual from each of these families (total 161 DNA samples) were exome sequenced either at the University of Maryland School of Medicine (UMSOM), University of Washington Center for Mendelian Genomics (UWCMG), or at the National Institute of Deafness and Other Communication Disorders (NIDCD) Genomic and Computational Biology Core.

Aside from the first two cohorts of 301 families, an additional 20 families (cohort 3) were screened using a gene panel that includes all known nonsyndromic and selected syndromic HL genes (n=101) (Zein et al., 2014) and the segregation of pathogenic variants identified from this panel was verified by Sanger sequencing the DNA of all informative family members.

Variants were considered further if (a) they have allele frequency <1% and (b) Combined Annotation Dependent Depletion (CADD; (Kircher et al., 2014)) scaled score was ≥20 and/or (c) at least two of the following algorithms predict the mutation to be deleterious: PROVEAN (Y. Choi & Chan, 2015), SIFT (Kumar, Henikoff, & Ng, 2009), MutationTaster (Schwarz, Rodelsperger, Schuelke, & Seelow, 2010), MutationAssessor (Reva, Antipin, & Sander, 2007), FATHMM (Shihab et al., 2013; Shihab et al., 2015) and PolyPhen2 (Adzhubei et al., 2010). Splice site and splice region variants were evaluated using splice site-altering scoring algorithms such as dbscSNV (Jian, Boerwinkle, & Liu, 2014) and Human Splice Finder (Desmet et al., 2009). Further details of bioinformatics analyses are described in Supp. Tables S1 and S2. All variants were tested for co-segregation with HL in the corresponding family via Sanger sequencing. All novel variants have been submitted to ClinVar public databasis (https://www.ncbi.nlm.nih.gov/clinvar/).

Results

Genetic spectrum

For 41 of the 68 reported NSARHL genes, we identified 163 variants co-segregating with severe-to-profound HL in 321 families (Fig. 1, Table 1). All of these consanguineous families segregate NSARHL associated genes either with homozygous (94.4%; Fig. 2, Table 1) or compound heterozygous (5.6%) variants (Fig. 3, Supp. Table S3). While 92 (56.4%) of the variants have been previously reported to cause HL in humans, we also identified 71 (43.6 %) novel variants associated with HL (Fig. 1). Almost 80% (127 of 163) of the identified variants are present only once in our cohort (Table 1). However, 57.1% of all causative variants identified in this study are found in five genes: SLC26A4, GJB2, MYO7A, HGF, and TMC1 (Fig. 4A).

Figure 1. Overview of genetic studies of 321 Pakistani families segregating recessive hearing loss.

Figure 1.

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Each box has the number of families over the number of variants found in our study. Of 163 variants, 11 of these variants were identified in multiple families as either homozygous or compound heterozygous. In the bottom two rows, only the number of variants is indicated. MS: missense, NS: nonsense, FS: frameshift, SS: splice site.

Table 1.

Novel and reported variants in this study of genes known to be associated with autosomal recessive hearing loss.

Family Gene Variant Protein change Reference Allele frequency
Pakistan 1000G ExAC
PKDF1551 ADGRV1 c.1055C>T p.(Pro352Leu) This study - - 2.5E-05
DEM4304, DEM4384, DEM4558, DEM4150, DEM4576, DEM4592, DEM4593, PKDF970b, PKDF1673, PKSR20 BSND c.35T>C p.(Ile12Thr) Riazuddin et al., 2009 - - 2E-05
DEM4545 CABP2 c.637+1G>T Splice error Schrauwen et al., 2012 - 0.0008 0.001
PKDF1767 CDH23† c.2866G>A p.(Glu956Lys) Miyagawa, Naito, Nishio, Kamatani, & Usami, 2013 - - -
PKDF055 CDH23† c.2968G>A p.(Asp990Asn) Bork et al., 2001 - - -
DEM4556 CDH23† c.3481C>T p.(Arg1161*) Roux et al., 2006 - - -
DEM4504 CDH23† c.3880C>T p.(Gln1294*) Bork et al., 2001 - - -
DEM4324 CDH23† c.4892C>T p.(Ala1631Val) Shearer et al., 2013 0/188 0.0010 0.0009
DEM4108 CDH23† c.5149T>C p.(Cys1717Arg) Sloan-Heggen et al., 2015 0/188 - 8.E-06
PKDF055 CDH23† c.6049+1G>A Splice error Bork et al., 2001 - - -
DEM4363 CDH23† c.6050–9G>A Splice error von Brederlow et al., 2002 - - -
LUAB14 CDH23† c.6083A>C p.(Asp2028Ala) This study 0/100 - -
DEM4112, PKDF1781 CDH23† c.6133G>A p.(Asp2045Asn) Bork et al., 2001 - - 8E-06
DEM4544 CDH23† c.6202A>C p.(Thr2068Pro) This study 0/202 - 8E-06
DEM4710 CDH23† c.6604G>A p.(Asp2202Asn) Bork et al., 2001 - - 8E-06
HL007 CDH23† c.7814A>G p.(Asn2605Ser) Naz et al., 2017 0/125 - -
DEM4491 CDH23† c.7987_7989delTTC p.(Phe2664del) Schultz et al., 2011 0/188 - -
DEM4305 CDH23† c.8351_8352insCGAT p.(Leu2785Aspfs*43) This study 0/284 - -
DEM4042, DEM4053, DEM4630, DEM4722, DEM4749, DEM4785, DEM4789, DEM4791, DEM4793, DEM4868, DEM4870, PKDF1093, PKDF1285, PKDF520, PKDF781 CIB2† c.272T>C p.(Phe91Ser) Riazuddin et al., 2012 - - 5E-05
PKZA13 CIB2† c.297C>G p.(Cys99Trp) Riazuddin et al., 2012 - - -
DEM4312 CLCNKA c.1985G>T p.(Gly662Val) This study 0/198 - -
DEM4101, DEM4210, PKDF958, PKDF1660 CLDN14† c.254T>A p.(Val85Asp) Lee, Ansar, et al., 2012 - - 8E-06
DEM4005 EDNRB c.553G>A p.(Val185Met) Puffenberger et al., 1994 - - 0.0001
DEM4134 EPS8 c.205–8A>G Splice error This study - - -
PKDF1550 ESPN† c.2496_2496delC p.(Tyr832*) This study - - 8.4E-06
DEM4704 ESRRB† c.536G>A p.(Arg179His) Miyagawa et al., 2013 - - 2E-05
PKDF969, PKDF1442, PKDF1573 GIPC3† c.662C>T p.(Thr221Ile) Rehman et al., 2011 - - 4E-05
DEM4551B, DEM4582 GJB2 c.−23+1G>A Splice error Denoyelle et al., 1997 - - -
DEM4347, DEM4648, DEM4659, DEM4352A, DEM4871, PKSR8a, PKDF1621 GJB2 c.35delG p.(Gly12Valfs*2) Zelante et al., 1997 - 0.002 0.006
DEM4307, DEM4339, DEM4346, DEM4351, DEM4409, DEM4550, DEM4573, DEM4582, DEM4583, DEM4656, DEM4717, DEM4438A, DEM4866, DEM4981, PKDF021, PKDF936, PKDF939, PKDF1368, PKDF1553, PKDF1655, PKDF1760 GJB2 c.71G>A p.(Trp24*) Kelsell et al., 1997; Santos, Wajid, Pham, et al., 2005 - 0.0004 0.0006
PKDF1666 GJB2 c.223C>T p.(Arg75Trp) Richard et al., 1998 - - -
DEM4528, DEM4537 GJB2 c.224G>A p.(Arg75Gln) Uyguner et al., 2002 - - -
DEM4015A, DEM4143, DEM4255, DEM4262, DEM4479, DEM4660, DEM4670, DEM4696, DEM4737, DEM4769, DEM4770, DEM4972, PKDF936, PKDF1588, PKDF1633, PKDF1665, PKDF1675, PKDF1790 GJB2 c.231G>A p.(Trp77*) Santos, Wajid, Pham, et al., 2005 - - 0.0001
DEM4614 GJB2 c.355G>T p.(Glu119*) This study 0/454 - 7E-05
PKDF1694, PKDF1764 GJB2 c.457G>A p.(Val153Ile) Marlin et al., 2001 - - -
DEM4431, DEM4653 GPSM2 c.138C>A p.(Phe46Leu) This study 0/328 - 8E-06
PKDF1679 GRXCR1† c.229C>T p.(Gln77*) Schraders et al., 2010 - - -
DEM4153 GRXCR1† c.655G>A p.(Glu219Lys) This study 0/114 - -
DEM4494, DEM4521, DEM4522, DEM4562, DEM4564, DEM4586, DEM4622, DEM4628, DEM4642, DEM4665, DEM4701, DEM4865, DEM4861, DEM4880, DEM4925, DEM4943, PKDF970a, PKDF1017, PKDF1331, PKDF1579, PKDF1604, PKDF1637, PKDF1668, PKDF1754, PKDF1787, PKDF1791 HGF† c.482 +1986_88delTGA Schultz et al., 2009 - - -
PKDF1775 HGF† c.482 +1991_2000delGATGATGAAA Schultz et al., 2009 - - -
PKDF1651 ILDR1 c.3G>A p.(Met1Ile) Borck et al., 2011 - - -
DEM4175 ILDR1 c.900delG p.(Thr301Profs*20) This study 0/188 - -
PKDF1455 ILDR1 c.1005_1005delG p.(Glu335Aspfs*30)) This study - - -
DEM4353 ILDR1 c.1029_1030delTT p.(Trp344Glyfs*17) This study 0/282 - -
PKDF1428 ILDR1 c.1384C>T p.(Arg462*) This study - - 6E-05
PKDF1311, PKDF1549 LHFPL5† c.250delC p.(Leu84*) Shabbir et al., 2006 - - -
PKDF1178 LHFPL5† c.452 G>T p.(Gly151Val) This study - - 8.00E-06
PKDF1662 MARVELD2† c.1138C>T p.(Gln380*) Sloan-Heggen et al., 2015 - - -
DEM4204 MARVELD2† c.1223_1224insA p.(Glu408Argfs*4) This study 0/280 - -
DEM4393, DEM4533, PKDF1639, PKDF1641 MARVELD2† c.1295+2T>C Splice error Riazuddin et al., 2006 - - 4E-05
PKDF1111, PKDF1489, PKDF1606, PKDF1610, PKDF1727 MSRB3† c.265T>G p.(Cys89Gly) Ahmed et al., 2011 - - 2E-05
PKDF1789 MSRB3† c.412–1G>A Splice error This study - - 8.2E-06
PKDF019A MYO7A c.20G>T p.(Gly7Val) This study 0/170 - 1E-05
PKDF173 MYO7A c.93C>A p.(Cys31*) Weston et al., 1996 - 0.017 5E-05
DEM4493 MYO7A c.247C>A p.(Arg83Ser) This study 0/112 - -
PKDF312 MYO7A c.397dupC p.(His133Profs*7) Riazuddin et al., 2008 - - 0.0006
PKDF1465, PKDF1490 MYO7A c.496delG p.(Glu166Argfs*5) Riazuddin et al., 2008 - - -
DEM4141, DEM4299, DEM4365 MYO7A c.722G>A p.(Arg241His) Cremers et al., 2007 - 0.0002 2E-05
PKDF370 MYO7A c.977T>A p.(Leu326Gln) Riazuddin et al., 2008 - - -
PKDF537, PKDF1786 MYO7A c.1183C>T p.(Arg395Cys) Shahzad et al., 2013 0/176 - -
DEM4190, DEM4652 MYO7A c.1258A>T p.(Lys420*) Le Quesne Stabej et al., 2012 - - 4E-05
DEM4331 MYO7A c.1591C>T p.(Gln531*) Riazuddin et al., 2008 - - 8E-06
PKDF019A MYO7A c.1849T>C p.(Ser617Pro) Carss et al., 2017 0/160 - 5E-05
PKDF721 MYO7A c.2339delG p.(Gly780Valfs*10) This study 0/154 - -
PKDF312 MYO7A c.3136dupC p.(Leu1046Profs*9) Riazuddin et al., 2008 - - -
DEM4561 MYO7A c.3364C>A p.(Leu1122Ile) This study - - -
DEM4281, DEM4692, PKDF628 MYO7A c.3502C>T p.(Arg1168Trp) Le Guedard-Mereuze et al., 2010 - 0.0002 0.0001
PKDF1484 MYO7A c.3508G>A p.(Glu1170Lys) Riazuddin et al., 2008 - - -
DEM4052A MYO7A c.3590T>C p.(Leu1197Pro) This study 0/282 - -
PKDF173 MYO7A c.3728C>G p.(Pro1243Arg) This study 0/164 - -
DEM4652 MYO7A c.4505A>G p.(Asp1502Gly) This study 0/454 - 2E-05
DEM4182, DEM4190, DEM4281, DEM4513, DEM4692, DEM4694 MYO7A c.4838delA p.(Asp1613Valfs*32) Riazuddin et al., 2008 - - -
DEM4433 MYO7A c.5326+3A>G Splice error This study 0/118 - -
DEM4561 MYO7A c.5345G>C p.(Gly1782Ala) This study - - 4E-05
DEM4206 MYO7A c.5434G>A p.(Glu1812Lys) Roux et al., 2006 - - -
DEM4327 MYO7A c.5522C>G p.(Thr1841Arg) This study 0/178 - -
DEM4725 MYO7A c.5660C>T p.(Pro1887Leu) Ben-Salem et al., 2014; Bharadwaj, Kasztejna, Huq, Berson, & Dryja, 2000 - - 2E-05
PKDF974 MYO7A c.5856+5G>C Splice error This study 0/148 - -
PKDF1382 MYO15A c.2456C>A p.(Ser819*) Naz et al., 2017 - - -
PKDF1657 MYO15A c.3843C>T p.(Arg1169*) This study - - 8E-06
PKDF1693 MYO15A c.4528C>T p.(Gln1510*) Rehman, et al., 2016 - - 8E-06
PKDF1693 MYO15A c.4570C>T p.(Gln1524*) This study - - -
PKDF1780 MYO15A c.3505C>T p.(Gln1669*) This study - - -
PKDF016 MYO15A c.8224+3A>G Splice error Rehman et al., 2016 - - -
DEM4725 OTOA c.1148C>T p.(Ala383Val) EVS variant - - 8E-04
PKDF889 OTOA c.1352G>A p.(Gly451Asp) Lee et al., 2013 - - 8E-06
DEM4362, DEM4366 OTOF c.1550T>C p.(Leu517Pro) This study 0/188 - -
PKDF1723 OTOF c.1904T>A p.(Val635Asp) This study - - -
PKDF1535 OTOF c.2122C>T p.(Arg708*) Choi et al., 2009 - - -
DEM4078 OTOF c.2508C>A p.(Tyr836*) Choi et al., 2009 - - 0.0006
DEM4512 OTOF c.2896G>A p.(Glu966Lys) Choi et al., 2009 - - 8E-06
DEM4548 OTOF c.2965_2967delTTC p.(Phe989del) Naz et al., 2017 0/188 - -
DEM4756 OTOF c.3376dupA p.(Ile1126Asnfs*5 This study - - -
PKDF1336 OTOF c.3514C>T p.(Arg1172Trp) This study - 0.0002 2.7E-05
PKDF1450 OTOF c.4799+1G>A Splice error This study - - 8.24e-06
PKDF1066a OTOF c.5714G>T p.(Gly1905Val) This study 0/168 -
DEM4372B OTOG c.7235delG p.(Arg2412Hisfs*77) This study 1/326 - 0.0009
PKDF627 PCDH15† deletion of exons 14 and 15 This study 0/100 - -
PKDF1440 PCDH15† c.400C>G p.(Arg134Gly) Ahmed et al., 2003 - - -
PKDF1499 PCDH15† c.788C>A p.(Pro263Gln) This study - - -
DEM4711 PCDH15† c.1737C>A p.(Tyr584*) Jaijo et al., 2006 - - -
DEM4040 PJVK c.158C>G p.(Ser53*) This study 0/284 - -
DEM4529 PJVK c.162_172del p.(Pro55Glufs*23) This study 0/454 - -
DEM4752 PJVK c.406C>T p.(Arg136*) This study - - -
PKDF880 PJVK c.908_910delACA p.(Asn303del) This study 0/168 - -
PKDF908 POU3F4 c.478C>T p.(Gln160*) This study 0/71 - -
PKDF1595 POU4F3 c.374C>T p.(Pro125Leu) This study - - -
DEM4437 PTPRQ c.5158_5159delAT p.(Ile1720Glnfs*7) This study 0/180 - -
DEM4123 PTPRQ c.6739–1G>A Splice error This study 0/172 - -
DEM4465 SLC26A4 c.42delC p.(Glu15Serfs*51) This study 0/454 - -
DEM4149B, HL005 SLC26A4 c.71G>T p.(Arg24Leu) Anwar et al., 2009 - - 0.0003
DEM4549 SLC26A4 c.154A>T p.(Lys52*) This study 0/454 - -
DEM4511 SLC26A4 c.317C>T p.(Ala106Val) This study 0/454 - -
DEM4014A SLC26A4 c.413T>A p.(Val138Asp) Chen, Wang, Sun, & Jiang, 2012
DEM4436 SLC26A4 c.416G>T p.(Gly139Val) Anwar et al., 2009 - - 4E-05
DEM4092, DEM4149B, DEM4152, DEM4220, DEM4241, DEM4271, DEM4290, DEM4292, DEM4293, DEM4295, DEM4296, DEM4429, DEM4482, DEM4483, DEM4523, DEM4575, DEM4726, DEM4766, DEM4192, DEM4291, DEM4481, DEM4982, PKDF1161, PKDF1664 SLC26A4 c.716T>A p.(Val239Asp) Park et al., 2003 - 0.0002 0.0003
DEM4186, DEM4454, DEM4455 SLC26A4 c.919–2A>G Splice error Coucke et al., 1999 - - 0.0003
DEM4336 SLC26A4 c.1001G>T p.(Gly334Val) Kahrizi et al., 2009 - - 8E-06
DEM4109 SLC26A4 c.1198delT p.(Cys400Valfs*32) Everett et al., 1997 - - 2.00E-05
PKDF384b SLC26A4 c.1226G>A p.(Arg409His) Van Hauwe et al., 1998 - - 0.0001
PKDF128, PKDF384b, PKDF910 SLC26A4 c.1226G>C p.(Arg409Pro) Park et al., 2003 - - -
DEM4283, PKDF384b SLC26A4 c.1229C>T p.(Thr410Met) Coucke et al., 1999 - 0.0002 0.0002
DEM4355 SLC26A4 c.1238A>G p.(Gln413Arg) Ji et al., 2009 - - 8E-06
DEM4200 SLC26A4 c.1264–3C>G Splice error This study 0/454 - -
DEM4004, DEM4094A, DEM4220, DEM4276, DEM4574, DEM4695, DEM4702, DEM4966 SLC26A4 c.1337A>G p.(Gln446Arg) Reardon, CF, Trembath, Jan, & Phelps, 2000 - -
DEM4286, DEM4287, DEM4294, DEM4392, DEM4425, DEM4553 SLC26A4 c.2106delG p.(Lys702Asnfs*19) Yazdanpanahi et al., 2012 - - 8E-06
PKDF1283 TECTA c.840_841insT p.(Val281Cysfs*11) This study 0/162 - -
HL04 TECTA c.1247_1248delGG p.(Gly416Aspfs*24) This study 0/100 - -
DEM4242A TECTA c.1774G>A p.(Val592Met) This study 0/132 - -
PKDF1452 TECTA c.2736C>A p.(Cys912*) This study - - -
PKDF1452 TECTA c.6162+5G>A Splice error This study
DEM4367, PKDF1584 TMC1† c.100C>T p.(Arg34*) Kurima et al., 2002 - - -
PKSR39a TMC1† c.596A>T p.(Asn199Ile) Imtiaz et al., 2016 0/148 - 8E-06
DEM4451 TMC1† c.945G>A p.(Trp315*) This study 0/454 - -
DEM4396 TMC1† c.1114G>A p.(Val372Met) Santos, Wajid, Khan, et al., 2005 - - 7E-05
PKSR13b TMC1† c.1143C>G p.(Tyr381*) This study 0/166 - -
DEM4147 TMC1† c.1209G>A p.(Trp403*) This study - - -
DEM4132 TMC1† c.1224+2T>C Splice error This study 0/112 - -
HL006 TMC1† c.1259G>A p.(Cys420Tyr) This study 0/125 - -
PKDF025 TMC1† c.1333C>T p.(Arg445Cys) Sirmaci et al., 2009 - - -
DEM4325 TMC1† c.1363T>C p.(Tyr454His) NSHI - - -
PKDF1449 TMC1† c.1543C>T p.(Cys515Arg) Kitajiri et al., 2007 - - -
DEM4350 TMC1† c.1728C>G p.(Asn576Lys) This study 0/454 - -
PKDF1617 TMC1† c. 1753_1754insA p.(Asn407Lysfs*2) This study - - -
HL006 TMC1† c.1788C>A p.(Ser596Arg) Imtiaz et al., 2016 0/125 - -
PKSR13b TMC1† c.1810C>T p.(Arg604*) Sirmaci et al., 2009 8E-06 - -
DEM4370A, DEM4724 TMC1† c.2035G>A p.(Glu679Lys) Santos, Wajid, Khan, et al., 2005 - - -
DEM4486 TMIE† c.241C>T p.(Arg81Cys) Santos et al., 2006 - - -
DEM4687 TMIE† c.250C>T p.(Arg84Trp) Naz et al., 2002 - - 2E-05
DEM4020, PKDF1053 TMPRSS3† c.271C>T p.(Arg91*) Bademci et al., 2016 - - 2E-05
DEM4003 TMPRSS3† c.323–6G>A Splice error Scott et al., 2001 - - 6E-05
PKSR31 TMPRSS3† c.581G>T p.(Cys194Phe) Ben-Yosef et al., 2001 - - -
DEM4106, DEM4124 TMPRSS3† c.767C>T p.(Ala256Val) Lee, Khan, et al., 2012 - - -
PKDF765 TMPRSS3† c.783–1G>T Splice error This study 0/130 -
DEM4046, DEM4202, DEM4372A, DEM4560, LUAB13, HL003, PKDF1057, PKDF1330, PKDF1634 TMPRSS3† c.1219T>C p.(Cys407Arg) Ben-Yosef et al., 2001 - - 5E-05
HL007, PKDF1756, PKDF1757, PKDF1758 TPRN† c.1056G>A p.(Trp352*) Rehman et al., 2010 - - -
DEM4492 TRIOBP† c.3460_3461delCT p.(Leu1154Alafs*29) This study 0/454 - -
PKDF1430 TRIOBP† c.3634_3646delCTGATCCCCCAAG p.(Leu1212Cysfs*22) This study - - -
DEM4707 USH1C c.238dup p.(Arg80Profs*69) Bitner-Glindzicz et al., 2000 - - 0.0003
DEM4335B USH1C c.463C>T p.(Arg155*) Ebermann et al., 2007 - - 8E-06
DEM4675 USH1G c.511G>T p.(Glu171*) This study - 0.0002 2E-05
DEM4268 USH1G c.812delC p.(Pro271Argfs*52) This study 0/454 - -
DEM4689 USH2A c.3661C>T p.(Gln1221*) This study 0/134 - 8E-06
DEM4171 WHRN c.2388_2389delCG p.(Asn796Lysfs*46) This study 0/284
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Novel mutations from this study are in bold font.

(†)

indicates genes associated with non-syndromic autosomal recessive hearing loss that were discovered through studies in Pakistani pedigrees.

Pak: Pakistani controls, 1000G: 1000 Genomes Project database, ExAc: Exome Aggregation Consortium database.

(-)

indicates that the variant is absent from the respective database.

Figure 2. Pakistani families co-segregating autosomal recessive HL due to multiple homozygous variants.

Figure 2.

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Families HL007 and DEM4372 are examples in which there is intra-familial locus heterogeneity of variants in genes known to cause HL. Circles indicate females, squares indicate males. Black-filled symbols represent affected individuals. Double lines indicate consanguineous marriages. In family HL007, homozygous variants in two different genes TPRN and CDH23 co-segregate with HL. Affected individuals are homozygous for either the TPRN (V:6, VI:1, VI:2, VI:13) or the CDH23 variant (V:11, V:12), except for affected individual VII:3 who is double homozygous. In family DEM4372, variants in two different genes TMPRSS3 and OTOG co-segregate with HL in different branches of this family. Novel mutations from this study are in bold font.

Figure 3. Consanguineous Pakistani families with compound heterozygous variants of reported HL genes.

Figure 3.

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Eight pedigrees segregating HL due to compound heterozygous variants in MYO7A (PKDF173, DEM4561, DEM4652, PKDF019), TMC1 (HL006, PKDF13B), MYO15A (PKDF1693) and TECTA (PKDF1452) genes. In family DEM4652, the normal hearing father III:2 is heterozygous for two different variants of MYO7A suggesting that the newly identified c.4505A>G variant might be non-pathogenic. Novel mutations from this study are in bold font.

Figure 4. Distribution of variants in the five most prevalent genes associated with nonsyndromic recessively inherited HL in Pakistani families.

Figure 4.

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(A) Percentage of identified alleles by gene. (B) Percentage of variants by type and occurrence of identified variants. The percentages are based on the total number of variants identified in this study.

Causal variants

Of the causative variants identified in this study 48.7% are missense, 23% are nonsense, 21.6% are small insertion or deletions and 6.7% are predicted to alter splice sites (Fig. 4B). In the five most common HL genes in this study, the distribution of mutation types appears to be gene-specific (Fig. 4B). Pathogenic alleles of HGF are all small deletion variants of an alternative 3’UTR (Schultz et al., 2009), while nonsense variants represent more than 70% of the HL alleles for GJB2 with two nonsense variants [c.71G>A, p.(Trp24*); c.231G>A, p.(Trp77*)] representing 38.9% and 33.3%, respectively, of the GJB2-associated HL mutations (Fig. 4).

A homozygous genomic deletion spanning exons 14 and 15 of PCDH15 was identified in family PKDF627, which causes a shift in the wild type translation reading frame and is predicted to result in a premature truncation of the encoded protein. This deletion has not been previously reported in humans. In mice that are homozygous for the Pcdh15av−5J (Ames Waltzer) allele, skipping of exon 14 due to a splice site variant results in disorganized stereocilia bundles, hearing loss, balance problems (Zheng et al., 2006) and delayed development of retinal sensory function (Haywood-Watson et al., 2006). CNV analyses of other families in our HL cohorts did not reveal any known or potentially HL causal indels. Thus, contrary to previous observations in other cohorts (Shearer et al., 2014), large indels probably rarely cause HL within the Pakistani population.

An unreported variant of ADGRV1 (also known as VLGR1, MASS1, KIAA0686 and GPR98 MIM# 602851), c.1055C>T (p.(Pro352Leu)), co-segregates with HL in family PKDF1551 (Fig. 5A). So far, mutations in ADGRV1 have been exclusively associated with Usher syndrome type 2 (USH2C, MIM# 605472) (Besnard et al., 2012; Reddy et al., 2014; Weston, Luijendijk, Humphrey, Moller, & Kimberling, 2004), characterized by congenital mild to severe sensorineural hearing loss, intact vestibular responses and progressive retinitis pigmentosa. In family PKDF1551, affected individuals exhibit mild to severe bilateral sensorineural hearing loss with no vision defects revealed by electroretinogram (Fig. 5B). Usher syndrome type 2 is a progressive disorder and the oldest affected individual in the family was only 17 years old at the time of clinical evaluation. However, USH2C, due to a large deletion in ADGRV1 was previously diagnosed in a 14-year old patient with vision defects (Hilgert et al., 2009).

Figure 5. A new variant of ADGRV1 associated with non-syndromic hearing loss in Pakistani family PKDF1551.

Figure 5.

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(A) Pedigree of family PKDF1551 segregating HL due to a missense mutation (c.1055C>T, p.(Pro352Leu)) in ADGRV1 (also referred to VLGR1, MIM# 602851, at the USH2C locus). The filled symbols represent affected individuals, and a double horizontal line connecting parents represents a consanguineous marriage. Half-filled symbol represents an individual with potential middle ear infection and/or carrying another variant responsible for his hearing impairment. Exome sequencing was performed on a DNA sample from individual VI:5. (B) Full-field electroretinograms of affected (VI:5, 17 years old) and non-affected individuals (VI:1, 22 years old) revealed no significant difference in a- and b-waves amplitudes among the two subjects, suggesting normal visual function. To show reproducibility, two responses (annotated 1 and 2, green and orange respectively) are superimposed per test and individual. The first crosshair indicates the a-wave while the second crosshair shows the b-wave. No differences in amplitude nor latency for waves a and b were detected in any of the performed tests.

Out of 321 families, a total of 19 families (5.9%) have evidence of intra-familial locus heterogeneity in that some deaf siblings or deaf relatives in other branches of the family are not segregating the same HL-causal variant (data not shown). For instance, two families (HL007 and DEM4372) display locus heterogeneity, within the same or different branches, segregating homozygous variants in two different known NSARHL genes (Fig. 2), which further highlights the unexpected complexity of the genetic diagnosis of HL in consanguineous families. In family HL007, most of the affected individuals are homozygous either for a reported c.1056G>A, p.(Trp352*) variant of TPRN, (Rehman et al., 2010), or another reported c.7814A>G, p.(Asn2605Ser) variant in CDH23 (Naz et al., 2017) a gene known to be involved in either NSARHL or Usher syndrome type 1D (MIM# 601067) (Bork et al., 2001). Affected individual VII:3 is homozygous for both of the likely pathogenic variants of TPRN and CDH23 (Fig. 2). Family DEM4372 is segregating variants for two different known NSARHL genes: OTOG and TMPRSS3 (Fig. 2). Affected individuals V:2 and V:3 are homozygous for a novel OTOG deletion [c.7235delG, p.(Arg2412Hisfs*77)]. Four affected individuals, IV:2, IV:6, IV:8, and IV:9, are homozygous for the recurrent c.1219T>C [p.(Cys407Arg)] variant (Ben-Yosef et al., 2001) in TMPRSS3. These two families (HL007 and DEM4372) exhibit intra-familial locus heterogeneity, which we previously demonstrated in several consanguineous Pakistani families with NSARHL (Rehman, et al., 2015).

Regardless of whether causal variants of one or more HL genes are segregating in a single family, the variants associated with HL are usually but not always homozygous. Affected individuals of family DEM4652 are compound heterozygous for three variants c.1258A>T [p.(Lys420*)], c.3502C>T [p.(Arg1168Trp)] and c.4505A>G [p.(Asp1502Gly)] variants in MYO7A (Fig. 3). While predicted damaging by various algorithms (Mutation Assessor, Mutation Taster, PolyPhen2, SIFT and PROVEAN), we cannot conclude that MYO7A c.4505A>G is pathogenic because normal hearing father III:2 is heterozygous for two different MYO7A variants: the known HL-causing variant c.1258A>T and the newly identified c.4505A>G variant. This family shows that even though multiple rare, predicted-as-damaging variants co-segregate with HL, it is likely that only a subset of these variants are causal, which further highlights the need to evaluate pathogenicity of variants based on multiple lines of evidence even when the variants occur in reported HL genes.

In order to obtain a more precise assessment of the genetic spectrum of HL in this population, we included in our counts not only the data from this specific study but also already published HL variants identified in our previously reported cohorts of Pakistani families (61 publications). Together, our studies have revealed 875 mutant alleles (368 unique variants) in 54 genes (Supp. table S4) and the 10 most prevalent genes are SLC26A4 (14.2%), MYO7A (10.5%), GJB2 (9.9%), CIB2 (8.6%), HGF (8%), TMC1 (6.4%), MYO15A (5.7%), CDH23 (4.6%), TMPRSS3 (4.3%) and OTOF (3.1%). Mutations in these ten genes represent 75.3% of all known causative variants identified in Pakistani families we have studied (Fig. 6, Supp. table S4).

Figure 6. Distribution of variants in the Pakistani population from 62 studies on hearing loss.

Figure 6.

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(A) Percentage of identified variants by gene. (B) Distribution of the 10 most prevalent genes in our collaborative studies. The percentages are based on the total number of alleles identified in our collaborative studies.

Discussion

Consanguineous families with recessively inherited HL have been a valuable resource for genetic mapping, gene and variant identification and genotype-phenotype correlations. Subsequently, functional studies and knockout animal models that follow gene identification aid in understanding the biology of inner ear development and function. In Pakistan, 45 to 60% of marriages are between relatives (Bittles, 2005; E. M. Scott, 1973), which has increased the coefficient of inbreeding in this population. According to a 2012 World Health Organization report, the prevalence of recessive disorders such as HL is higher in Pakistan (2.4%) compared to a world prevalence of 1.7%. In this study that includes a large cohort of 321 unpublished Pakistani families, we identified 163 unique variants in 41 genes responsible for autosomal recessive hearing loss. As expected, autosomal recessive inheritance due to homozygous variants is demonstrated in the majority of our HL cohort (94.4%), however 5.6% of the variants segregate in a compound heterozygous fashion even in consanguineous families (Fig. 3). Of the 163 identified variants, 71 (43.6%) are novel. In addition, 98.6% of these novel variants are found only once in our cohort. Given the rarity of these private variants, reporting them as damaging based on bioinformatics tools and co-segregation with HL in mostly consanguineous families aids in determining variant pathogenicity (Supp. Table S5) (Richards et al., 2015). The data provided here will also help variant curation in diagnostic laboratories where physician-referred genetic testing is usually performed on singletons.

Despite the heterogeneity of genetic causes of HL, in our study, variants in five known HL genes, SLC26A4 (58), GJB2 (54), MYO7A (39), HGF (27), and TMC1 (18), account for 57.1% (95% CI: 51.7–62.4%) of the identified alleles (Table 1). Variants in SLC26A4 alone account for 16.9% (95% CI: 13.2–21.4%) of variants in our 321 families. SLC26A4 encodes pendrin, a transmembrane anion exchanger for chloride, iodide, bicarbonate and other ions, essential for the development of inner ear and ionic homeostasis (Chattaraj et al., 2017; B. Y. Choi et al., 2011; Everett et al., 2001; Rehman, Friedman, & Griffith, 2017). Consistent with our previous findings (Anwar et al., 2009), c.716T>A [p.(Val239Asp)] is the prevalent variant responsible for SLC26A4-associated HL in Pakistani families. The c.2106delG [p.(Lys702Asnfs*19)] variant, which so far has only been reported in the Iranian population (Yazdanpanahi et al., 2015), is the third most common variant of SLC26A4 in our study, segregating with HL in six Pakistani families from Baluchistan, a province that shares a border with neighboring Iran. Besides these and other common alleles, we also found four novel variants [c.42delC, p.(Glu15Serfs*51); c.154A>T, p.(Lys52*); c.317C>T, p.(Ala106Val); c.1264–3C>G] of SLC26A4. Taken together, our results further confirm the high prevalence of SLC26A4 alleles in Pakistani families with HL (Anwar et al., 2009; Tsukada, Nishio, Hattori, & Usami, 2015).

GJB2 variants are the second most common (15.7%; 95% CI: 12.1–20.1%) cause of HL in our cohort of 321 families. The GJB2 variant spectrum is ancestry-specific and varies significantly around the world. Consistent with previous studies in the Pakistani population, two variants [c.71G>A, p.(Trp24*); c.231G>A, p.(Trp77*)] represent the majority of the GJB2-associated HL (Anjum et al., 2014; Bukhari, Mujtaba, & Naz, 2013; Salman et al., 2015; Santos, Wajid, Pham, et al., 2005; Shafique et al., 2014). We also identified a new nonsense allele [c.355G>T, p.(Glu119*)] segregating with HL in one family (DEM4614), which expands the list of more than 200 variants in the single protein-coding exon of GJB2 (Duman & Tekin, 2012).

Variants of MYO7A account for 11.4% (95% CI: 8.3–15.3%) of HL in our cohort of 321 families, slightly more frequent than in previous reports (Riazuddin et al., 2008; Shahzad et al., 2013). Contrary to SLC26A4 and GJB2, no predominant MYO7A variant in the Pakistani population was uncovered in our study. However, six MYO7A variants (c.496delG, c.722G>A, c.1183C>T, c.1258A>T c.3502C>T, c.4838delA) were present in more than one family (Table 1 and Supp. Table S3). We also identified eight families harboring causative compound heterozygous variants in MYO7A (Supp. Table S3).

Similar to MYO7A, no prevalent allele of TMC1 was revealed in our study and 14 out of 18 identified variants of this gene were found in single families. Seven new likely pathogenic variants (c.945G>A [p.(Trp315*)], c.1143C>G [p.(Tyr381*)], c.1209G>A [p.(Trp403*)], c.1224+2T>C, c.1259G>A [p.(Cys420Tyr)], c.1728C>G [p.(Asn576Lys)] and c. 1753_1754insA [p.( Asn407Lysfs*2)]) were identified in our cohort (Table 1 and Supp. Table S3). In contrast, the c.482 +1986_88delTGA allele of HGF accounts for HL segregating in 26 families (Table 1), which was already identified as a founder allele in 50 other Pakistani families in our collaborative studies (Naz et al., 2017; Rehman et al., 2015; Schultz et al., 2009). Similarly, the c.272T>C allele of CIB2 accounts for the HL phenotype segregating in 15 families (Table 1). Studies in mouse have highlighted the crucial role of CIB2 in the mechanotransduction process and the deleterious effect of this specific missense mutation on hearing (Giese et al., 2017; Michel et al., 2017). Variants of CIB2 have been found in hearing-impaired individuals of Turkish, Caribbean Hispanic, Iranian, Palestinian Arab, European and Dutch origin (Booth et al., 2017; Michel et al., 2017; Patel et al., 2015; Riazuddin et al., 2012; Seco et al., 2016; Shaikh et al., 2017). Together with previously published studies, the CIB2 c.272T>C allele has been found in 85 hearing-impaired Pakistani families (Rehman et al., 2015; Riazuddin et al., 2012; Seco et al., 2016; Shaikh et al., 2017) and is likely to be a founder allele (Riazuddin et al., 2012).

Likely pathogenic variants of other genes associated with hearing loss in our cohort of 321 families include CDH23 (n=16 variants), TMPRSS3 (n=16), OTOF (n=10) and BSND (n=10) (B. Y. Choi et al., 2009; Naz et al., 2017; Rehman et al., 2015; Riazuddin et al., 2012; Shafique et al., 2014). This study highlights the genetic heterogeneity of HL-associated variants as 127 out of 163 (77.1%) identified alleles occur only once in our cohort. The diversity and the growing numbers of identified rare and non-recurrent variants increase the complexity of a molecular genetic diagnosis of HL (Gao & Dai, 2014). These data also predict that the Pakistani population will continue to be valuable for discovering the molecular genetics not only of HL but also of a multitude of other understudied monogenic and complex disorders such as persistent stuttering (Raza, Amjad, Riazuddin, & Drayna, 2012; Raza, Riazuddin, & Drayna, 2010; Riaz et al., 2005).

To better evaluate the genetic spectrum of recessive nonsyndromic HL in the Pakistani population, we combined data from these 321 new families with data from 61 of our previously published collaborative studies in Pakistan since 1999 (Fig. 6, Supp. table S4). Out of 875 identified mutant alleles in 54 genes (368 unique variants), more than 75% (75.3%) of the pathogenic alleles were found in 10 genes: SLC26A4, MYO7A, GJB2, CIB2, HGF, TMC1, MYO15A, CDH23, TMPRSS3 and OTOF. MYO15A variants were recently reviewed (Rehman et al., 2016) and represent 5.7% of the total predicted causative mutations, while alleles in BSND a gene involved in either Bartter syndrome or sensorineural HL with mild renal dysfunction (MIM# 602522), occur in 1.8% of the identified variants. We emphasize that the families in these studies were recruited on the initial assumption that they were segregating non-syndromic HL although a syndromic deafness often becomes evident only after identifying the responsible variants and completing phenotypic analyses and functional studies of the gene in mutant and wild type animal models (Imtiaz et al., 2018).

When the Pakistani population is compared to other populations i.e. Iranian (Ghasemnejad, Shekari Khaniani, Zarei, Farbodnia, & Mansoori Derakhshan, 2017; Sloan-Heggen et al., 2015), Indian (Yan et al., 2015), Chinese (Jiang et al., 2015; Xin et al., 2013; Zhang et al., 2016), African-descent (Rudman et al., 2017), European ancestry (Yan et al., 2017) or multi-ethnic (Sloan-Heggen et al., 2016; Yan et al., 2016), the distribution of genes associated with nonsyndromic HL and the variants reveal differences and commonalities. Some of the most prevalent “HL genes” in all of these populations are SLC26A4, GJB2 and MYO7A.

In these three genes, hundreds of pathogenic variants associated with HL have been identified while only 7 to 50 variants have occurred in the other most prevalent genes in our cohort with the exception of CDH23 (Human Gene Mutation Databases). Increased mutational events might explain the higher incidence of HL-associated variants, independently of the ethnicity of the considered population.

In conclusion, this study expands our understanding and knowledge of the allelic spectrum of HL genes in the Pakistani population, highlighting the extreme locus and variant heterogeneity of this disorder, sometimes even among siblings (e.g. see Fig. 3 in (Rehman et al., 2015)). This implies that even though we identified the genetic etiology of HL in hundreds of families within coding regions of known NSARHL genes, due to intra-familial genetic heterogeneity, we expect to identify at least 1000 additional variants in perhaps hundreds more novel genes in our unsolved families. Given the complexity of inner ear structure and function of the mechano-chemical transduction of sound, it has been speculated that there will be as many as 1000 genes that are involved in HL in mammals (Steel, 2014), which is further supported by yet undiscovered genetic lesions in hundreds of individuals and families with HL in our cohorts. With the rapid evolution of sequencing technologies and bioinformatics tools (He et al., 2017; Ott, Wang, & Leal, 2015), and with whole-genome sequencing more affordable, we anticipate the identification of more rare variants within HL genes. Identifying the molecular lesions will be critical for a more complete understanding of how we hear and for accomplishing personalized therapies (Askew et al., 2015; Chien et al., 2016; Shibata et al., 2016).

Supplementary Material

Supp info

Acknowledgments

We would like to thank all the families for participating in the study. We thank AM Waryah, R. Yousaf and S. Yousaf for their technical help. We also would like to thank the University of Washington Center for Mendelian Genomics.

Contract grant sponsors: NIDCD/NIH extramural research grants (R56 DC011803 to SR, R01 DC011651 to SML, R01 DC003594 to SML, R01 DC012564 to ZMA); and in part from Intramural Research Program of the National Institute on Deafness and Other Communication Disorders at the NIH (DC000039–20 to T.B.F. and DC000086 to R.J.M.); and Higher Education Commission of Pakistan (HEC). Genotyping and exome sequencing performed at the UWCMG was funded by the National Human Genome Research Institute and the National Heart, Lung and Blood Institute grant HG006493 (to D.A.N., M.J.B. and S.M.L.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This work utilized computational resources of the NIH HPC Biowulf cluster.

Footnotes

Disclosure statement: All authors declare no conflicts of interest.

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