Copy-Number Variation Contributes to the Mutational Load of Bardet-Biedl Syndrome

Abstract

Bardet-Biedl syndrome (BBS) is a defining ciliopathy, notable for extensive allelic and genetic heterogeneity, almost all of which has been identified through sequencing. Recent data have suggested that copy-number variants (CNVs) also contribute to BBS. We used a custom oligonucleotide array comparative genomic hybridization (aCGH) covering 20 genes that encode intraflagellar transport (IFT) components and 74 ciliopathy loci to screen 92 unrelated individuals with BBS, irrespective of their known mutational burden. We identified 17 individuals with exon-disruptive CNVs (18.5%), including 13 different deletions in eight BBS genes (BBS1, BBS2, ARL6/BBS3, BBS4, BBS5, BBS7, BBS9, and NPHP1) and a deletion and a duplication in other ciliopathy-associated genes (ALMS1 and NPHP4, respectively). By contrast, we found a single heterozygous exon-disruptive event in a BBS-associated gene (BBS9) in 229 control subjects. Superimposing these data with resequencing revealed CNVs to (1) be sufficient to cause disease, (2) Mendelize heterozygous deleterious alleles, and (3) contribute oligogenic alleles by combining point mutations and exonic CNVs in multiple genes. Finally, we report a deletion and a splice site mutation in IFT74, inherited under a recessive paradigm, defining a candidate BBS locus. Our data suggest that CNVs contribute pathogenic alleles to a substantial fraction of BBS-affected individuals and highlight how either deletions or point mutations in discrete splice isoforms can induce hypomorphic mutations in genes otherwise intolerant to deleterious variation. Our data also suggest that CNV analyses and resequencing studies unbiased for previous mutational burden is necessary to delineate the complexity of disease architecture.

Introduction

Bardet-Biedl syndrome (BBS [MIM: 209900]) is a rare (1:13,500–1:160,000) multisystemic developmental disorder characterized by retinal dystrophy, obesity, polydactyly, intellectual disability, renal dysfunction, and hypogonadism. During the past 15 years, causal variants in 21 different genes have been identified,^{1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22} and in vivo and in vitro studies have established the cellular basis of BBS as a defect of ciliary function.² Biochemical characterization of the cilium has shown that it is composed of at least four different functional complexes: the BBSome, the transition zone, and two intraflagellar transport complexes.²³ BBS proteins are thought to localize mainly to the BBSome and the transition zone,²⁴ with the exception of recent reports of pathogenic mutations in IFT27 (MIM: 6158700)¹ and IFT172 (MIM: 607386)²² encoding components of IFT complex B, that localize primarily to cilia. Furthermore, some BBS genes have also been linked to other ciliopathies, including Meckel-Gruber syndrome (MKS [MIM: 249000]), Joubert syndrome (JBTS [MIM: 213300]), Senior-Løken syndrome (SLS [MIM: 266900]), nephronophthisis (NPHP [MIM: 256100]), and Leber congenital amaurosis (LCA [MIM: 204000]), illustrating the genetic, biological, and clinical overlap within these genetic disorders.^{9, 25, 26, 27, 28} Alström syndrome (ALMS [MIM: 203800]), caused by mutations in ALMS1 (MIM: 606844), overlaps clinically with BBS, sharing common characteristics such as obesity, diabetes, and retinal dystrophy.^{29, 30}

Although BBS is viewed traditionally as an autosomal-recessive Mendelian trait, oligogenic inheritance has been documented for most BBS-associated genes. In rare cases, at least one deleterious (typically heterozygous) allele in a different BBS-associated gene modifies the penetrance of the disorder.^{2, 30, 31, 32, 33, 34} More commonly, trans modifier alleles contribute to variable expressivity, not only in BBS, but across the ciliopathy spectrum.^{35, 36} For example, a common c.685G>A (p.Ala229Thr) mutation in RPGRIP1L (MIM: 610937) is associated with retinitis pigmentosa in ciliopathies;³⁶ similarly, nephronophthisis-affected individuals with a homozygous NPHP1 (MIM: 607100) deletion are more likely to develop retinal degeneration if they also bear the AHI1 p.Arg830Trp variant in the heterozygous state.³⁷ Moreover, resequencing across different ciliopathy cohorts unbiased for known mutations has demonstrated an enrichment of deleterious heterozygous changes in TTC21B (MIM: 612014), a key component of the retrograde IFT machinery, in affected individuals versus control subjects.³⁵ These data have created a model in which, in addition to primary causal alleles, the mutational burden or “load” in the ciliary proteome, defined as the total number, mutational type, and locus distribution of deleterious alleles, has a role in defining disease presentation, especially the elaboration of endophenotypes.²³ Notably, trans modifying alleles can be deleterious or protective, as evidenced by the ability of homozygous loss-of-function Mkks/Bbs6 alleles to ameliorate retinal degeneration in a Cep290 mouse model,³⁸ as well as the postulated protective effect of CEP290 (MIM: 610142) haploinsufficiency in the context of de novo deletions in 16p11.2 (MIM: 611913) that are associated with complex neurocognitive traits.³⁹

In the past decade, copy-number variants (CNVs) have emerged as major contributors to the genetic burden of both rare and common disorders.⁴⁰ A number of CNVs have been associated with human genomic disorders, such as microdeletion and duplication syndromes,^{41, 42} as well as dose changes involving single loci^{43, 44} and recessive carrier states.⁴⁵ In BBS, CNVs have been reported episodically.^{46, 47} More recently, we have shown that the 290 kb recurrent deletion of NPHP1, usually underlying renal ciliopathies including NPHP^{48, 49} and SLS,⁵⁰ is both a likely driver (primary causal locus under a recessive paradigm) of BBS in one family and is also enriched in BBS-affected case subjects.¹¹ These observations prompted us to investigate systematically whether CNVs contribute to mutational load in BBS. Here, through high-resolution analysis of bona fide genes involved in BBS, other ciliopathies, and/or core ciliary components, we report exon-disruptive CNVs in 18.5% of unrelated BBS-affected individuals, most of which are driven by recombination at intronic repetitive elements. When combined with sequencing data from all assayed loci, our analyses indicate that (1) these events can represent recessive mutational drivers, with homozygous exonic or whole gene deletions; (2) they can Mendelize heterozygous deleterious variants; and (3) they can unmask genetic interactions with recessive mutations in other BBS genes. When combined with in vivo modeling of likely pathogenic SNVs, our observations improve the resolution of BBS-associated gene contribution to disease; support further a mutational burden model that includes distinct classes of variant alleles (SNV plus CNV); facilitate the identification of candidate loci through a mutational mechanism that involves the ablation of a specific splice isoform; and illuminate further the genetic complexity of this disorder.

Subjects and Methods

Research Participants

A total of 92 unrelated BBS-affected individuals and 229 non-BBS control subjects were analyzed by aCGH. Affected individuals were selected irrespective of previously identified pathogenic changes. The control samples included 137 individuals of northern European descent from our Age Related Macular Degeneration control cohort,^{51, 52} five HapMap samples of western European ancestry, and anonymized samples from 87 healthy internal lab control subjects of northern European descent. We used standard methods to isolate genomic DNA from peripheral blood. Informed consent was obtained from all participating individuals, with approval from the Institutional Review Boards of the Duke University Medical Center and the Baylor College of Medicine.

Whole-Genome Amplification

30 ng of genomic DNA from affected participants and gender-matched male (NA10851) and female (NA15510) control DNAs (obtained from Coriell Cell Repositories) were amplified by whole-genome amplification (GenomePlex Whole Genome Amplification Kit, Sigma). Amplified product was verified by gel electrophoresis and quantified by UV absorbance at 260 nm with a NanoDrop (ND-1000) spectrophotometer (NanoDrop Technologies).

Oligonucleotide aCGH Analyses

We used a 4×180k array format at an average coverage of one probe per 100 base pairs (bp) in coding sequences and one probe per 500 bp in intragenic non-coding sequences. The high-resolution targeted aCGH design was created in Agilent’s web portal eArray and slides were ordered from Agilent Technologies. In brief, 2 μg of each amplified BBS DNA sample and 1.2 μg of unamplified control DNA sample were then labeled and hybridized as described.⁵³

Junction Sequence Analyses

Long-range PCR was conducted with the Phusion High-Fidelity PCR Kit (NEB, E0553L) according to the manufacturer’s protocol, and PCR products that were unique to carriers and not observed in control subjects were Sanger sequenced.

Quantitative PCR Confirmation of CNVs

When junction fragments failed to amplify by multiple primer pairs, we used quantitative (q)PCR to confirm CNVs. TaqMan Copy Number Assays (Life Technologies) were selected from within the CNV regions. We conducted triplicate reactions according to an ABI protocol on an ABI7900HT Fast Real-Time PCR System for both case subjects and matched control subjects. A copy neutral reference assay was performed in parallel (RNase P) for normalization purposes. Data were analyzed and visualized with CopyCaller (v.1.0).

Resequencing of BBS Genes

We amplified all exons and splice junctions of BBS1 (MIM: 2099010), BBS2 (MIM: 6061510), ARL6/BBS3 (MIM: 608845), BBS4 (MIM: 600374), BBS5 (MIM: 603650), MKKS/BBS6 (MIM: 604896), BBS7 (MIM: 607590), TTC8/BBS8 (MIM: 608132), BBS9 (MIM: 607968), BBS10 (MIM: 610148), TRIM32/BBS11 (MIM: 602290), BBS12 (MIM: 610683), MKS1/BBS13 (MIM: 609883), CEP290/BBS14 (MIM: 609883), WDPCP/BBS15 (MIM: 613580), SDCCAG8/BBS16 (MIM: 613524), IFT27/BBS19, and NPHP1 in the 17 individuals harboring CNVs, and we conducted bidirectional Sanger sequencing on an ABI3730 according to standard protocols. Identified mutations were segregated by Sanger sequencing in all available family members. Variants (CNVs or SNVs) were named according to the following NCBI GenBank reference transcript accession numbers: BBS1, NM_024649.4; BBS2, NM_031885.3; ARL/BBS3, NM_032146.4; BBS4, NM_033028.4; BBS5, NM_152384.2; MKKS/BBS6, NM_018848.3; BBS7, NM_176824.2; TTC8/BBS8, NM_144596.3; BBS9, NM_198428.2; BBS10, NM_024685.3; ALMS1, NM_015120.4; CEP290, NM_025114.3; SDCCAG8, NM_006642.3; NPHP1, NM_000272.3; IFT74, NM_025103.2.

Transient Gene Suppression in Zebrafish Embryos

All zebrafish studies were approved by the Duke University Institutional Animal Care and Use Committee. Translation-blocking and splice-blocking morpholino (MO) antisense oligonucleotides were designed by and obtained from Gene Tools. To determine the optimal dose for in vivo complementation assays and genetic interaction studies, we injected increasing doses of MO into 1- to 4-cell-stage wild-type (WT) zebrafish embryos. To determine MO efficiency (splice-blocking MOs), we harvested injected embryos in Trizol (Invitrogen), extracted total RNA according to the manufacturer’s instructions, and generated oligo-dT-primed cDNA with the QuantiTect reverse transcription kit (QIAGEN) for use as a template for RT-PCR; subsequent Sanger sequencing of RT-PCR products identified the precise alteration of endogenous transcript.

In Vivo Complementation and Genetic Interaction Studies in Zebrafish

For rescue experiments, human IFT74 (MIM: 608040) transcript variant 1 (long; GenBank: NM_025103.2) and IFT74 transcript variant 2 (short; NM_001099224.1) messages were amplified from cDNA generated from a human fibroblast cell line; SDCCAG8 (NM_006642.3; clone T8331) was obtained commercially (Genecopoeia); and all ORFs were subcloned into the pCS2+ vector and transcribed in vitro via the mMessageMachine kit (Ambion). All other BBS gene ORF clones and mutagenesis procedures have been described.^{9, 11, 54} For genetic interaction studies, subeffective doses of MO (producing <30% affected embryos/batch when injected alone) were injected in single, pairwise, or triple combinations to assess additive or epistatic effects.

qPCR to Monitor Endogenous bbs Gene Expression

We harvested embryo batches (n = 20 embryos/batch) at the mid-somite stage in Trizol (Invitrogen), extracted total RNA, and generated oligo-dT-primed cDNA with the QuantiTect reverse transcription kit (QIAGEN) for use as a template for RT-PCR (30 ng template/reaction). We used SYBR Green PCR Master Mix (ThermoFisher) to monitor gene expression profiles in real-time on an ABI7900HT instrument, and cycle threshold (Ct) values were computed with SDS 2.3 software (Applied Biosystems) according to manufacturer’s instructions. All experiments were conducted in triplicate wells corresponding to each gene with at least one biological replicate. Mean Ct values for reference genes (acta1b [GenBank: NM_214784.2], actb1 [NM_131031.1], or actb2 [NM_181601.4]) were used to normalize mean Ct values for test genes (bbs2 [NM_152887.1], bbs4 [NM_001077466.2], mkks/bbs6 [NM_200165.1], bbs5 [NM_200299.1], and bbs10 [NM_001089463.1]) and Kruskal-Wallis tests were used to compare differences between experimental conditions. Primer sequences are available upon request.

CRISPR/CAS9 Genome Editing of ift74

We used the CRISPR Design tool to identify ift74 (GenBank: NM_001002385.1) guide (g)RNA target sequences, for which two oligonucleotides were synthesized and annealed. Annealed oligos were then ligated into the T7cas9sgRNA2 vector as described,⁵⁵ and plasmids prepped from individual clones were sequence confirmed. Template vector was linearized with BamHI and gRNA was transcribed using the MEGAshortscript T7 kit (Life Technologies). A total of 50 pg of ift74 gRNA and 200 pg of CAS9 protein (PNA Bio) was co-injected into individual cells of 1-cell-stage embryos. To determine targeting efficiency in F0 mutants, we harvested single embryos in Trizol (Invitrogen) at 2 dpf (three controls and eight F0 mutants), extracted total RNA according to manufacturer’s instructions, reverse transcribed cDNA using the QuantiTect reverse transcription kit (QIAGEN), and PCR amplified a region flanking the gRNA target site. PCR products were denatured, reannealed slowly, and separated on a 15% TBE 1.0 mm precast polyacrylamide gel (BioRad) as described.⁵⁶ The resulting PAGE gel was incubated in ethidium bromide and imaged on a Biorad ChemiDoc system. To estimate the percent mosaicism, RT-PCR products were gel purified (QIAGEN) and cloned into a TOPO-TA vector (ThermoFisher). Plasmid was prepped from multiple individual colonies per embryo (n = 8–15) and Sanger sequenced on an ABI3730 instrument according to standard protocols.

Phenotypic Analyses in Zebrafish

Embryo batches were assessed live at the 8- to 10-somite stage for early developmental defects in gastrulation (shortened body axis, widened and kinked notochord, and widened and thinned somites) and were categorized into class I (moderately affected) or class II (severely affected) according to previously established objective criteria.^{9, 11, 20, 21, 35, 36, 54} Live embryo images were acquired on a Nikon AZ100 microscope at 8× magnification facilitated by NIS Elements software and statistical comparisons were conducted with χ² tests. Renal structure was assessed in bbs or ciliopathy gene models as described.^{11, 57} In brief, larvae were fixed in Dent’s solution at 4 days post fertilization (dpf) and immunostained with anti-Na⁺/K⁺ ATPase antibody (a6F, Developmental Studies Hybridoma Bank) to demarcate renal tubules. Larvae were imaged on a Nikon AZ100 microscope at 4× magnification facilitated by NIS Elements software; renal structures (area of the proximal convoluted tubule, or tubule diameter adjacent to the proximal convoluted tubule) were measured with ImageJ software (NIH); statistical comparisons were conducted with a Student’s t test.

Results

Identification of Intragenic CNVs in BBS

To evaluate the contribution of CNVs to BBS, we investigated a cohort of 92 unrelated BBS-affected individuals (Table 1), without preselection based on known mutation status. To improve the accuracy of CNV detection and to achieve sub-kb resolution, we focused on a genetically and biologically plausible preselected set of genes. We therefore generated a custom high-resolution oligonucleotide aCGH targeting of a total of 94 genes. These included the 17 known BBS genes (at the time of the design of the array); 20 genes encoding components of the IFT complex; and 57 additional loci associated with ciliopathies (Table S1). With this defined gene set, we designed probes at a density of one probe per 100 bp of coding sequence and one probe per 500 bp in intragenic regions.

Table 1.

Clinical Phenotype Summary of BBS-Affected Case Subjects with CNVs

Individual	Primary Locus	Consanguinity	Sex	Age	RP	Ob	PD	HG	RD	ID	DD	Others
AR240-03	BBS1	−	F	21	✓	✓	✓	ND	−	✓	−	S, OM
AR246-03	BBS1	−	M	12	✓	✓	✓	✓	−	✓	✓	M, DC, MT, E
AR380-03	BBS1	−	F	9	✓	✓	✓	ND	−	✓	−	PS, OM, H
AR888-0311	BBS1	−	M	4	✓	✓	✓	ND	✓	✓	✓	OM, S, HL, Ns, Sz, SA
KK047-05	BBS2	✓	M	9	✓	✓	✓	✓	ND	✓	ND
KK11-04	ARL6/BBS3	✓	M	17	✓	✓	✓	✓	ND	ND	✓	Hs, UT
KK11-09	ARL6/BBS3	✓	F	7 months	ND	ND	✓	ND	ND	ND	ND
DM034-004	ARL6/BBS3	−	M	10	✓	✓	✓	ND	ND	−	−	As, DM1, FL
32/3	BBS4	−	M	8	✓	ND	ND	ND	ND	ND	ND
AR800-03	BBS4	−	M	5	✓	✓	✓	✓	ND	✓	ND	St, SS, F
AR400-03	BBS4	−	F	11	✓	✓	✓	ND	ND	✓	✓	A, M, As, OM, UH, ST
AR634-03	BBS7	−	F	17	✓	✓	−	ND	−	✓	✓	M, As, DM2
AR634-04	BBS7	−	M	15	✓	✓	−	✓	ND	✓	ND	M, AS, HL
44/311	BBS7	−	M		✓	✓	✓	✓	✓	ND	✓
AR883-04	BBS10	−	M	4	✓	✓	✓	ND	✓	✓	ND	SA, Sz, OM
AR672-04	IFT74	−	M	36	✓	✓	✓	✓	−	✓	−	MC
RC2-0311	NPHP1	✓	M	6	✓	−	✓	✓	✓	ND	✓	Ns, F
AR811-03	unknown	✓	M	4	✓	✓	ND	✓	ND	ND	✓	SA
AR704-0311	unknown	−	F		✓	✓	✓	ND	ND	✓	ND

Phenotypes are indicated as present (✓) or absent (−). Age at assessment is indicated in years, unless otherwise noted. ND indicates no data because subject was not evaluated. Phenotypes assessed: RP, retinitis pigmentosa; Ob, obesity; PD, polydactyly; HG, hypogonadism; ID, intellectual disability; RD, renal disease; DD, developmental delay. Others: MC, microcephaly; M, myopia; S, scoliosis; OM, otitis media; DM1, diabetes mellitus type 1; DM2, diabetes mellitus type 2; HL, hearing loss; DC, dental crowding; MT, missing teeth; E, eczema; PS, pulmonary stenosis; H, hypertension; Ns, nystagmus; Sz, seizures; St; strabismus; SS, short stature; A, asthma; UH, umbilical hernia; ST, sickle trait; As, astigmatism; FL, fatty liver; F, farsighted; SA, sleep apnea; Hs, hypospadias; UT, undescended testis.

In addition to the previously reported recurrent deletion at the NPHP1 locus (found in one homozygous and two heterozygous BBS-affected individuals) and a 17 kb heterozygous deletion in BBS1 identified in the same cohort,¹¹ we detected another 14 unique intragenic CNVs in 13 individuals with BBS: 13 deletions (ranging in size from 730 bp to 172 kb) and 1 complex intragenic duplication (Tables 1, 2, and 3; Figures 1, 2, and S1). These data, combined with our recent report,¹¹ total 16 different CNVs among 17 unrelated individuals from the same BBS cohort.

Table 2.

Mutational Findings of 17 Individuals Affected with BBS Harboring CNVs Encompassing BBS Genes

Sample ID	Primary Causal Locus	Allele 1	Allele 2	Additional Mutational Burden	Genotype Counts in 6,503 Controls (EVS All)	In Vivo Pathogenicity Score
Primary Causal Locus: Homozygous CNV
KK047-05	BBS2	del: exon8_15 (pat)a,b	del: exon8_15 (mat)b	none	NA	NA
DM034-004	ARL6/BBS3	del: exon4_9 (pat)b	del: exon4_9 (mat)b	none	NA	NA
KK11-04	ARL6/BBS3	del: exon8 (pat)b	del: exon8 (mat)b	SDCCAG8: c.1594G>A (p.Glu532Lys) het (mat)	0	hypomorph
32/3	BBS4	del: exon3_4 (U)b	del: exon3_4 (U)b	none	NA	NA
AR800-03	BBS4	del: exon5_6 (pat)b	del: exon5_6 (mat)b	MKKS/BBS6: c.724G>T (p.Ala242Ser) het73	AA = 0, AC = 58, CC = 6,445	dominant-negative54
				BBS2: c.367A > G (p.Ile123Val) hom (Pat, Mat)	CC = 240, CT = 2,060, TT = 4,198	hypomorph54
AR400-03	BBS4	del: exon5_6 (pat)a,b	del: exon5_6 (mat)b	BBS9 c.1993C>T (p.Leu665Phe) het (pat)a	TT = 0, TC = 42, CC = 6,461	null
				ALMS1 del exon16_17 het (pat)a,b	NA	NA
RC2-0311	NPHP1	del: whole gene (pat)b	del: whole gene (mat)b	BBS2: c.367A>G (p.Ile123Val) hom (pat, mat)	CC = 240, CT = 2,060, TT = 4,198	hypomorph54
Primary Causal Locus: Compound Heterozygous CNV + SNV
AR380-03	BBS1	del: exon9_12 (U)b	c.1169T>G (p.Met390Arg) (U); (rs113624356)	none	NA	NA
AR888-0311	BBS1	del: exon1_11 (pat)b	c.1645G>T (p.Glu549∗) (mat); (rs121917777)	NPHP1: c.14G>T (p.Arg5Leu) het (mat)	AA = 0, AC = 6, CC = 6,497	hypomorph11
				BBS4: c.439T>A (p.Tyr147Asn) het (pat)	0	null
				BBS5: c.584A > G (p.Asp195Gly) het (mat)	GG = 0, GA = 4, AA = 6,497	null
				BBS7: c.442A>C (p.Asn148His) het (mat)	0	null
AR240-03	BBS1	del: exon10_17 (mat)b	c.1169T>G (p.Met390Arg) (pat); (rs113624356)	BBS5: c.551A>G (p.Asn184Ser) het (mat)	GG = 0, GA = 61, AA = 6,440	null54
AR246-03	BBS1	del: exon14_17 (U)b	c.1169T>G (p.Met390Arg) (U); (rs113624356)	BBS10: c.2119_2120delGT (p.Val707∗) het (U)20	0	ND
AR634-03	BBS7	del: exon16_17 (pat)b	c.878A>C (p.Gln293Pro) (mat)	BBS4: c.137A>G (p.Lys46Arg) het (mat)	GG = 0, GA = 92, AA = 6,403	null54
				IFT74: c.1735G>A (p.Val579Met) het (U)	AA = 0, AG = 97, GG = 5,884	benign
AR672-04	IFT74	del: exon14_19 (mat)b	c.1685–1G>T (pat); (rs200699377)	none	NA	NA
Heterozygous CNV Contributes Additional Mutational Burden
AR883-04	BBS10	c.2119_2120delGT (p.Val707∗) (pat)	c.2118_2119delTG (p.Val707fs∗) (mat)	BBS5: del: exon8_12 het (mat)b	NA	NA
44/311	BBS7	c.87_88delCA (p.His29Glnfs∗12) (pat)	c.87_88delCA (p.His29Glnfs∗12) (mat)	NPHP1: del whole gene het (mat)b	NA	NA
				CEP290 c.6727G>C (p.Glu2243Gln); c.6401T>C (p.Ile2134Thr) het (mat)	0; GG = 0, GA = 64, AA = 5,768	hypomorph
AR704-0311	unknown	NA	NA	NPHP1: del whole gene het (mat)b	NA	NA
				BBS10: c.145C>T (p.Arg49Trp) het (pat)	0	null54
				TTC8/BBS8: c.1253A>G (p.Gln418Arg) het (mat)	GG = 0, GA = 27, AA = 6,476	hypomorph
AR811-03	unknown	NA	NA	NPHP4: het dup exon1_28; normal exon29; het dup exon 30 (U)b	NA	NA

Abbreviations are as follows: pat, paternal; mat, maternal; U, unknown inheritance; hom, homozygous; het, heterozygous; del, deletion; dup, duplication; EVS, Exome Variant Server; NA, not applicable; ND, no data.

^aNo DNA available from the father, deletion detected in the grandfather.

^bCNVs.

Table 3.

Breakpoint Features of 18 CNVs Detected in 17 BBS-Affected Case Subjects

Sample ID	Gene	hg19 CNV Coordinates	Array Result	Size (bp)	Rearrangement Type	Breakpoint Feature	% Identical Nucleotides	Mechanism
AR888-03	BBS1	chr11: 66,274,840–66,292,574	het del: exon1_11	17,734	simple non-recurrent	AluY - AluY	92%	Alu-Alu
AR380-03	BBS1	chr11: 66,288,383–66,293,649	het del: exon9_12	5,266	simple non-recurrent	1 bp microhomology (T)	no homology	NHEJ/ RBM
AR240-03	BBS1	chr11: 66,290,130–66,305,022a	het del: exon10_17	14,892a	NA	-	-	-
AR246-03	BBS1	chr11: 66,296,361–66,304,267	het del: exon14_17	7,906	simple non-recurrent	AluSp - AluSp	87%	Alu-Alu
KK047-05	BBS2	chr16: 56,529,926–56,538,197	hom del: exon8_15b	8,271	simple non-recurrent	no homology	no homology	NHEJ
DM034-004	ARL6/BBS3	chr3: 97,498,355–97,583,359	hom del: exon4_9b	85,004	simple non-recurrent	5 bp microhomology	no homology	NHEJ/ RBM
KK11-04	ARL6/BBS3	chr3: 97,508,904–97,513,012	hom del: exon8b	4,108	simple non-recurrent	(TG)n simple repeat 41 bp homology	100%	RBM/SSA
32/3	BBS4	chr15: 73,000,565–73,006,826	hom del: exon3_4b	6,261	simple non-recurrent	AluSc8 - AluSx	83%	Alu-Alu
AR800-03	BBS4	chr15: 73,006,570–73,014,289	hom del: exon5_6b	7,719	simple non-recurrent	1 bp microhomology	no homology	NHEJ/ RBM
AR400-03	BBS4	chr15: 73,006,584–73,012,788	hom del: exon5_6b	6,204	simple non-recurrent	AluSx - AluSg	82%	Alu-Alu
AR400-03	ALMS1	chr2: 73,617,393–73,789,869	het del: exon2_15	172,476	simple non-recurrent	5 bp insertion	no homology	NHEJ/ RBM
AR883-04	BBS5	chr2: 170,355,189–170,364,951	het del: exon8_12	9,762	simple non-recurrent	AluYc3 - AluSg	82%	Alu-Alu
AR634-03	BBS7	chr4: 122,749,644–122,750,374	het del: exon16_17	730	simple non-recurrent	5 bp insertion	no homology	NHEJ/RBM
RC2-03	NPHP1	chr2: 110,875,689–110,967,529a	hom del: whole geneb	91,840a	recurrent	NA	NA	NAHR
44/3	NPHP1	chr2: 110,875,689–110,967,529a	het del: whole gene	91,840a	recurrent	NA	NA	NAHR
AR704-03	NPHP1	chr2: 110,875,689–110,967,529a	het del: whole gene	91,840a	recurrent	NA	NA	NAHR
AR811-03	NPHP4	chr1: 5,910,699–6,038,368a	het dup: exon1_28	120,535a	NA	NA	NA	NA
AR811-03	NPHP4		normal: exon29		NA	NA	NA	NA
AR811-03	NPHP4	chr1: 6,051,187–6,158,763a	het dup: exon30	79,389a	NA	NA	NA	NA
AR672-04	IFT74	chr9: 27,040,563–27,060,990	het del: exon14_19	20,427	simple non-recurrent	AluSz - AluY	77%	Alu-Alu

Abbreviations are as follows: NHEJ, non-homologous end joining; RBM, replication-based mechanism; NA, not available; NAHR, nonallelic homologous recombination.

^aSize and coordinates estimated from the array analysis only.

^bHomozygous CNVs.

Figure 1.

Schematic of Non-recurrent Exon-Disruptive CNVs Identified in BBS-Affected Case Subjects

(A) Three heterozygous BBS1 deletions were identified in trans with deleterious point mutations.

(B–D) A single homozygous BBS2 deletion (B); two homozygous ARL6/BBS3 deletions (C); and three homozygous BBS4 deletions (D) were identified in BBS-affected case subjects.

(E) A heterozygous deletion was detected in BBS5; no other deleterious variant was detected in trans, suggesting that it is a second-site contributor in this individual.

(F) A heterozygous single-exon deletion in BBS7 was identified in trans with a pathogenic SNV.

(G) A heterozygous ALMS1 deletion was detected in a BBS pedigree harboring a homozygous BBS4 exon 5–6 deletion (see D).

(H) Identification of a complex duplication encompassing the NPHP4 locus.

Chromosomal locations are indicated in red (top of each panel); genes, horizontal black lines; exons, vertical black bars (if different transcript isoforms exist, all putative exons are shown); CNVs, horizontal bars (blue, homozygous deletion; green, heterozygous deletion; red, duplication); SNVs, black stars. Repeat elements are shown for Alu-mediated CNVs in the relevant panels.

Figure 2.

Pedigrees and Segregation Data of Primary Causal BBS Gene CNVs in Our Cohort

Segregation analysis results indicate that CNVs can either be sufficient to cause disease (D–I; blue coloring) or Mendelize heterozygous deleterious alleles (A–C, J; green coloring). Squares, males; circles, females; black symbols, individuals affected with BBS; double lines, consanguinity.

Breakpoint Analysis Shows Multiple Mechanisms of CNV Formation

To refine CNV endpoints, to map precisely the genomic content, and to examine rearrangement end products as a means of inferring possible underlying formation mechanisms, we characterized 13 of 15 non-recurrent breakpoint junctions by long-range PCR and Sanger sequencing (Table 3; Figure S1).¹¹ None of the 15 CNVs detected by high-resolution aCGH in the BBS genes (BBS1, BBS2, ARL6/BBS3, BBS4, BBS5, BBS7), ALMS1, IFT74, and NPHP4 in our cohort have been reported previously or are present in the Database of Genomic Variants (DGV), except for the heterozygous deletion in BBS1 (AR888-03) that we published recently¹¹ and a BBS5 deletion of similar size and position to individual 32/3 that has been found (in heterozygosity) in two database entries: one subject with reported developmental delay and one with obesity. We found three individuals with unique CNVs in BBS4; two of them involve exons 5–6 as determined by breakpoint mapping. Remarkably, individuals with deletions and differing breakpoints in BBS4 involving the same exons have been reported,^{13, 47} suggesting that this region might be susceptible to genomic instability (Tables 2 and 3; Figure 1).

In 6 of 15 (40%) non-recurrent CNVs (AR888-03, BBS1; AR246-03, BBS1; 32/3, BBS4; AR400-03, BBS4; AR883-04, BBS5; AR672-04, IFT74), the junctions were located within Alu elements with variable percentage of sequence identity (77%–92%), suggesting that the deletions were mediated by Alu-Alu recombination. Alu-Alu recombination forming an Alu hybrid is a prominent mechanism underlying the formation of pathogenic CNVs associated with distinct diseases.^{58, 59, 60, 61, 62} In two junctions, substrate pairs of Alu repeats from the same family (AR888-03, AluY-AluY; AR246-03, AluSp-AluSp) are involved whereas the remaining four have substrate pairs consisting of distinct Alu repeat families mediating CNV formation (32/3, AluSc8 - AluSx; AR400-03, AluSx - AluSg; AR883-04, AluYc3 - AluSg; AR672-04, AluSx - AluY). Notably, a particular AluSx is present at the proximal junction of non-recurrent BBS4 deletions present in two unrelated BBS-affected individuals (AR400-03 and 32/3). In addition, a simple dinucleotide microsatellite repeat, (TG)n, 4 kb apart and consisting of 41 nt, seems to be involved in the formation of the CNV in individual KK11-04 (ARL6/BBS3).

The formation of complex rearrangements such as triplications and inversions mediated by Alu-Alu recombination and the co-occurrence of point mutations and indels at or near the CNV breakpoint junctions implicate replication-based mechanisms (RBMs),^{63, 64} but simple rearrangements may be caused by double-strand breaks repaired by single-strand annealing (SSA) by the sequence similarity for annealing the broken DNA.⁶⁵ In two Alu-Alu-mediated CNVs, additional point mutations were observed: a T>C SNV 178 bp from the junction of BBS1 in AR888-03¹¹ and a deletion of nine nucleotides in a poly(A) region involved in the formation of the breakpoint junction of IFT74 in AR672-04 (Figure 3), potentially implicating an error-prone DNA polymerase and a RBM underlying formation of those variants. Because those CNVs are inherited, we could not confirm whether the SNVs and the rearrangement were formed concomitantly.

Figure 3.

IFT74 Is a Candidate BBS Locus

(A) Chromosomal location of human IFT74 on chromosome 9p21 is indicated with a vertical red bar.

(B) Schematic of two IFT74 transcript variants (long, GenBank: NM_025103.2; short, NM_001099224.1) with alternate 3′ exon usage. Horizontal line, gene locus; vertical black bars, exons; black star, paternally inherited point mutation; green bar, heterozygous deletion.

(C) aCGH plot indicates a heterozygous deletion of ∼20 kb encompassing exons 14–19 of the long transcript.

(D) Enlarged view of the CNV- and SNV-bearing region of IFT74 (corresponds to the dashed blue box in B) and location of AluSz and AluY repeat elements.

(E) BBS pedigree AR672 and segregation of the paternally inherited c.1685−1G>T splice variant and maternally inherited exon 14–19 deletion.

(F) Breakpoint characterization of the IFT74 deletion. The junction sequence and corresponding reference location is highlighted in blue and microhomology is shown in red.

(G) Chromatogram corresponding to the microhomology region shown in (F).

Six additional deletion CNVs presented with no homology, 1 to 5 bp homology, or 5 bp nucleotide insertions at the breakpoint junctions (Table 3; Figures 1 and S1), indicative of canonical non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ),⁶⁶ or RBM as underlying mechanisms. Of note, breakpoint junctions of CNVs spanning ALMS1 (AR400-03) and BBS7 (AR634-03) presented five base pair insertions (Table 3; Figure S1) that could have originated from within the deleted region as a templated insertion (indicating either RBM or MMEJ); nonetheless, the short length of such insertions makes it difficult to rule out random bp insertion that can also occur during NHEJ.

In two cases we were unable to obtain junction fragments, and therefore confirmed the aberrations with TaqMan Copy Number Assays; both CNVs, the 14.9 kb BBS1 deletion (individual AR240-03) and the complex duplication in NPHP4 (MIM: 607215; individual AR811-03), displayed the expected dosage alteration (Figure S1).

Enrichment of Ciliary Gene CNVs in BBS-Affected Case Subjects

Subsequent to breakpoint analysis and CNV confirmation, we asked whether there was an increased prevalence of CNVs within our target ciliary gene set in BBS-affected case subjects. A search in the DGV revealed a single heterozygous CNV in BBS5 (9969)⁶⁷ overlapping with those identified in our BBS cohort; the CNV was of similar size and genomic location as the deletion detected in AR883-04. However, mindful of likely resolution differences between CNVs in this database and our study, we performed aCGH with the same reagent, hardware, and analytic software on 229 healthy control individuals. We did not detect any of the discovered CNVs in these controls; analysis for any exon-disrupting CNV identified a sole heterozygous deletion of exons 5–7 of BBS9, predicted to result in an in-frame deletion of 133 amino acids. These data, together with our previously reported NPHP1 and BBS1 CNVs,¹¹ suggest an incidence of exon-disruptive CNVs at 13.6% (25/184) of BBS chromosomes compared to 0.2% (1/458) of control chromosomes, respectively, constituting a significant enrichment (p < 0.0001, Fisher’s exact test).

BBS-Associated Gene CNVs Contribute Recessive Alleles

Next, we investigated how the CNVs disrupting BBS-associated genes contribute to disease manifestation. For cases in which parental samples were available (13 of 17), segregation analysis showed all CNVs to be inherited (Table 2; Figure 2).¹¹ Among the six BBS-affected pedigrees harboring non-recurrent homozygous deletions, familial DNAs segregated according to Mendelian expectations, demonstrating anticipated carrier status for both parents in three pedigrees (AR800, DM034, KK11) and a single carrier parent in two pedigrees (KK47 and AR400; DNA was unavailable for the other parent; Figure 2). Five of six pedigrees had only a single affected individual. Nonetheless, the nature of the gene disruptive deletion, ranging from two to eight exons, suggested strongly that these CNVs are likely BBS drivers. For KK47, AR800, and AR400, the putative splicing of exons flanking the deletions is predicted to result in a frameshift and premature termination codon. In DM034, the six exons encoding the C-terminal end of ARL6/BBS3 are deleted, predicted to result in a 41 amino acid truncated protein bereft of the majority of the ADP-ribosylation factor domain. Affected individual 32/3 harbored a two-exon deletion that would not alter the reading frame if spliced, but would delete part of the first tetratricopeptide (TPR) domain in BBS4, known to be important for protein-protein interactions.⁶⁸ Finally, deletion of a single exon in Saudi pedigree KK11 is predicted to result in truncation of 23 amino acids from the C-terminal end of ARL6/BBS3; the presence of the same homozygous exon 8 deletion in affected sibling KK11-09 bolstered our confidence in the pathogenicity of this CNV as the primary driver of disease.

Unmasking a recessive allele on one chromosome by a deletion on the other is a recognized disease-causing mechanism.^{69, 70, 71, 72} For BBS, we reported previously that this phenomenon contributes to pathology in a BBS1 pedigree.¹¹ Consistent with this observation, segregation analysis of a previously reported⁷³ maternally inherited BBS7 c.878A>C (p.Gln293Pro) change in AR634 showed this variant to be in trans with the paternally inherited exon 16–17 deletion; both affected siblings carried the two BBS7 alleles (Figure 2; Table S2). To determine whether this mechanism could explain disease in any of the three pedigrees with heterozygous BBS1 deletions, we sequenced the coding exons and splice junctions in each of AR240, AR246, and AR380. In each case, we identified the c.1169T>G (p.Met390Arg) mutation, known to be one of the two most frequently mutated sites among BBS loci and a known hypomorphic allele,^{14, 54, 74, 75} in trans with the deletion and segregating according to Mendelian expectations with disease in each pedigree (Figure 2; Tables 2 and S2). Notably, Sanger sequencing of BBS1 exon 12 in AR380-03 showed c.1169T>G as an apparently homozygous mutation because the encompassing deletion masks the zygosity of the change. Sanger sequencing of BBS5 by a similar strategy in AR883, a pedigree carrying a heterozygous exon 8–12 deletion, yielded no additional functional variants in BBS5. Together, the presence of either a homozygous gene deletion (n = 7) or a heterozygous deletion in trans with a pathogenic SNV in the same gene (n = 5) assigned a primary genetic cause for a previously underappreciated proportion of our cohort (13%; Figure 2).¹¹

IFT74, a BBS Candidate Gene

Next, we asked whether CNV deletions could assist in the identification of BBS genetic drivers. Among the families studied, there was a single individual, AR672-04, who was bereft of known driver mutations, but harbored a heterozygous deletion CNV. Our array data, confirmed by breakpoint sequencing, showed a maternally inherited deletion that removed the coding exons 14–19 of IFT74 (Figure 3), a locus encoding a component of the IFT complex that to date has not been associated with BBS or any other ciliopathy. We sequenced all 20 coding exons and splice junctions of IFT74 in the index case subject and both parents. We identified a paternally inherited heterozygous c.1685−1G>T change (Figure 3). This change maps to the conserved intronic splice acceptor site of exon 20, is absent from 11,868 publicly available healthy control chromosomes (NHLBI Exome Variant Server [EVS]), and was found only once in ∼120,000 chromosomes in ExAC. Expanded resequencing of IFT74 in our 92 BBS-affected individuals yielded only a single additional heterozygous missense change with a minor allele frequency <1% (c.1735G>A [p.Val579Met]; present in 682/120,714 alleles in ExAC) in AR634-03 (Tables 2 and S2).

Data from Chlamydomonas and ciliated mammalian cells have shown that IFT74 and IFT81 (MIM: 605489) form a tetrameric complex required for the transport of tubulin within the cilium.^{76, 77} Ablation of IFT proteins and the concomitant loss of cilia are largely incompatible with life,⁷⁸ an expectation inconsistent with our discovery of deleterious mutations in BBS. To understand this apparent paradox, we investigated the pathogenicity of the putative protein product resulting from the 6-exon deletion in AR672. Database analyses showed that the IFT74 locus encodes two splice isoforms in both human and mouse that utilize different 3′ exons. Notably, the affected individual in AR672 is predicted to have ablated function only for the long isoform, by virtue of the position of both the CNV and the splice site mutation (Figure 3).

To test functionally, the protein resulting from the maternally inherited IFT74 deletion, we used in vivo complementation in zebrafish embryos.⁷⁹ Reciprocal BLAST identified a single zebrafish IFT74 ortholog (51% identity; 71% similarity to human), encoding one annotated transcript against which we designed two different splice-blocking MOs targeting either the splice donor site of ift74 exon 6 (sb1) or the splice acceptor site of ift74 exon 4 (sb2), that we used to inject 1- to 4-cell-stage WT embryos (Figure S2; Tables S3 and S4). Suppression of endogenous message conferred by either MO resulted in significant gastrulation defects (shortened body axes, broader and thinner somites, and broad and kinked notochords) in embryo batches scored at the mid-somitic stage (n = 50–86 embryos/injection, masked scoring, repeated at least twice, p < 0.0001 versus uninjected control), consistent with that reported for other BBS and IFT gene morphants.^{9, 35, 54, 80} The phenotype was specific for each of sb1 and sb2, since both could be rescued with the long WT human IFT74 mRNA isoform (p < 0.0001; Figure 4; Table S4) as well as the long isoform harboring a common missense change (c.165A>G [p.Ile55Met]; rs10812505; Table S4). In contrast, the short IFT74 isoform rescued either of the two ift74 MOs only partially (Figure 4; Table S4), consistent with the contention that the AR672 proband is hypomorphic for IFT74 function.

Figure 4.

In Vivo Knockdown or Genome Editing of ift74 Results in Gastrulation Defects and Renal Phenotypes in Zebrafish

(A) Representative live images of ift74 morphant zebrafish embryos at the mid-somitic stage (top, lateral; bottom, dorsal) display gastrulation defects typical of IFT and other ciliary gene suppression models.

(B and C) In vivo complementation studies indicate that the short IFT74 transcript is a hypomorphic allele; n = 39–86 embryos/injection batch; masked scoring, repeated at least twice; statistical significance was determined using a χ² test to compare injected batches versus controls; ^∗p < 0.0001; NS, not significant; WT, wild-type.

(D) Fixed 4 day post fertilization larvae were immunostained with anti-Na⁺/K⁺ ATPase antibody to mark renal tubules, representative images are shown for each of the ift74 morphant and mutant models; green dashed boxes indicate region of inset; blue lines indicates the location of the renal tubule diameter measured on lateral images.

(E) Quantification of renal phenotypes in larvae injected with 50 pg guide RNA alone; 50 pg guide RNA/200 pg CAS9 protein; 9 ng ift74 sb1 or 9 ng ift74 sb2 MO demonstrate an increased diameter of the proximal convoluted tubule in comparison to controls; n = 23–54 per condition, repeated; statistical significance was determined by a Student’s t test to compare injected batches versus controls. NS, not significant; ^∗∗∗∗p < 0.0001. Error bars represent standard error of the mean (SEM).

Cognizant that some MOs can cause non-specific phenotypes, particularly during gastrulation, we used CRISPR/CAS9 genome editing to target the ift74 locus in zebrafish. We injected CAS9 protein and guide RNA targeting ift74 exon 3 into 1-cell-stage embryos and harvested embryos at 2 dpf to determine: (1) a high percentage of embryos that expressed mRNA harboring targeting events in ift74 (8/8 F0 mutants with detectable heteroduplexes in PCR products amplified from the guide RNA target); and (2) an average mosaicism estimated at 43% (11%–75% mosaicism determined from 8–15 clones/embryo; n = 8 F0 mutants; Figure S3). The degree of mosaicism is a likely underestimate, since small deletions will often lead to unstable mRNA transcripts that are eliminated by nonsense-mediated decay. Injection of ift74 guide RNA and CAS9 protein produced gastrulation defects in midsomitic embryos, phenocopying both ift74 morphant models; injection of the same dose of guide RNA alone yielded no appreciable phenotypes (n = 39–50 embryos/injection batch, repeated twice; Figure 4).

Next, we sought to investigate the effects of ift74 depletion on a specific organ system relevant to the BBS clinical spectrum. For this purpose, we evaluated morphants and mutants at 4 dpf for renal abnormalities. Recent reports have shown that IFT complex B zebrafish models, including ift46 and ift54 mutants, display dilated pronephric tubules.^{81, 82} To determine whether this defect was present in ift74 morphants and mutants, we demarcated renal tubule structures by immunostaining with an anti-Na⁺/K⁺-ATPase antibody, imaged lateral views of larvae, and measured the diameter of a defined tubule region posterior to the proximal convolution (n = 23–54 larvae/batch, repeated; Figure 4). For each of the three ift74 models (sb1, sb2, and gRNA/CAS9), we observed a significant increase in tubule diameter in comparison to controls (p < 0.0001), possibly a consequence of altered fluid flow in the kidney as reported for other ift zebrafish models.⁸³

Extensive Oligogenic Load among Individuals Harboring BBS CNVs

Oligogenic inheritance is a hallmark feature of BBS.²⁴ However, exploration of even that mechanism has been based almost exclusively on sequencing data. Given our current observations on the CNV burden in BBS, and the fact that all affected individuals screened were not preselected for or against prior known pathogenic BBS alleles, we asked how exon-disruptive CNVs and pathogenic SNVs might coalesce in our cohort. First, we expanded our mutational screening efforts to the entire coding exons and splice junctions of BBS1-16, IFT27/BBS19, and NPHP1/BBS20 in all 17 individuals harboring CNVs. We confirmed variants in BBS2, MKKS/BBS6, BBS7, BBS10, and NPHP1/BBS20 that were identified in previous candidate gene^{11, 20} or targeted resequencing⁷³ studies. Further, we detected ten additional rare alleles that were predicted to disrupt amino acid sequence and were present at <1% minor allele frequency in publicly available databases (13,006 chromosomes; EVS; Tables 2 and S2). With the exceptions of a homozygous 2 bp deletion in BBS10 (c.2119_2120delGT [p.Val707^∗]) that segregated with disease in BBS pedigree AR883 and two heterozygous variants shown previously to be functional null alleles (BBS5 c.551A>G [p.Asn184Ser] in AR240 and BBS4 c.137A>G [p.Lys46Arg] in AR634),⁵⁴ all other variants were heterozygous missense changes of unknown pathogenic potential. We therefore employed in vivo complementation in zebrafish embryos by established, highly sensitive, and specific assays for BBS4, BBS5, BBS7, TTC8/BBS8, BBS9, CEP290/BBS14, and SDCCAG8/BBS16.^{9, 17, 54} Upon masked scoring of embryos rescued with either WT or mutant mRNA (in triplicate), we found that 7 of 7 were pathogenic (Tables 2, S2, S3, S4).

Next, we asked (1) whether CNVs contribute oligogenic alleles to BBS and (2) the extent to which pathogenic BBS gene variation (CNV or SNV) is present in addition to primary recessive loci. Supported by both genetic and in vivo functional data, we have shown previously that the common NPHP1 deletion can contribute oligogenic alleles to BBS.¹¹ We found additional such examples (Figures 5 and S4). For instance, in AR883, the proband harbors both a homozygous BBS10 c.2119_2120delGT mutation (the recessive driver) and a heterozygous BBS5 exon 8–12 deletion (Figure 5). In other families, we found a range of combinations of deleterious SNVs and CNVs across two or more BBS loci. Overall, of the 17 BBS-affected individuals bearing CNVs, 11 had additional pathogenic mutations in one or more BBS genes in addition to their driver locus. Some of these second sites were SNVs and some were CNVs, with one family harboring as many as four BBS-associated pathogenic alleles (Figures 5, 6, and S4). The enrichment of oligogenic CNVs in the BBS cohort remained significant after removing CNVs considered to be primary causal alleles under a recessive paradigm (Table 2). We observed a total of five heterozygous oligogenic CNVs in affected individuals (AR883-04/BBS5; AR811-03/NPHP4; AR400-03/ALMS1; and the previously reported NPHP1 deletion in 44/3 and AR704-03;¹¹ Table 2; Figure 6) and one heterozygous deletion in control subjects (5/184 for BBS chromosomes versus 1/458 for control chromosomes; p = 0.0085, Fisher’s exact test).

Figure 5.

Segregation and In Vivo Analysis of Oligogenic BBS Loci Demonstrate Epistatic Effects

AR883-03 harbors a heterozygous BBS5 CNV that is a likely second-site contributor to disease caused by mutation at the primary causal locus BBS10.

(A) Pedigree and segregation of each BBS gene variant (CNV or SNV; separate pedigrees for each gene) with the primary locus harboring causal variants shown on the far left. Gene name color indicates heterozygous deletion CNV (magenta) or point mutation (gray).

(B) Bar chart indicates in vivo assessment of bbs gene interaction by the comparison of either single-gene or pairwise injection of sub-effective doses of MOs and phenotypic scoring of zebrafish embryo batches at the mid-somitic stage. Objective scoring criteria correspond to images shown in Figure 4A and demonstrate an epistatic effect in the double-MO batch. See Table S5 for embryo counts and additional examples.

(C) A second phenotypic readout, area of proximal convoluted tubule measured at 4 days post fertilization, demonstrates epistatic effects of bbs gene interaction. Fixed larvae were immunostained with anti-Na⁺/K⁺ ATPase antibody; green dashed box indicates region of inset; blue dashed line indicates the region of the proximal convoluted tubule measured on lateral images.

(D) Quantification of renal phenotypes in single or double MO-injected larvae demonstrate a progressive reduction in the size of the proximal convoluted tubule; n = 34–46 per condition, repeated; statistical significance was determined by a Student’s t test to compare injected batches versus controls. NS, not significant; ^∗∗p < 0.01. Error bars represent standard error of the mean (SEM).

Figure 6.

Genetic Architecture of BBS Gene Variation in 17 BBS-Affected Case Subjects Harboring CNVs

Each slice of the pie chart represents one BBS-affected case subject and the primary BBS locus harboring causal variants is indicated with gene names and colors (homozygous CNV, blue; compound heterozygous CNV + SNV, green; homozygous or compound heterozygous SNV, gray). Circles overlapping each slice represent the additional mutational burden in each BBS-affected case subject (CNV, magenta; SNV, gray). 5/17 individuals (29%) harbor no additional BBS gene variants outside their primary causal driver; 12/17 individuals (71%) have 1–4 additional heterozygous deleterious variants in BBS1-16, IFT27/BBS19, or NPHP1. Asterisk (^∗) indicates duplication CNV.

In Vivo Analysis of Oligogenic BBS Loci

Genetic interaction has been assessed extensively among the BBS-associated genes and in concert with other ciliopathy loci.^{9, 11, 20, 21, 35, 54, 84, 85} To investigate whether the pairwise BBS-associated gene combinations seen here result in additive or epistatic effects, we co-injected sub-effective doses of MO targeting the orthologous zebrafish genes, and we compared the fraction of affected embryos scored at the mid-somitic stage to that of embryos injected with single-gene MO concentrations alone. To approximate the BBS mutational burden in affected individuals, we tested four pairwise gene suppression combinations; we also generated an additional model in which we suppressed three BBS-associated genes simultaneously (Figures 5 and S4; Tables 2 and S5). In three instances, and consistent with our previous observations of nphp1 interactions with bbs1, bbs2, bbs7, and bbs10,¹¹ we observed an additive effect. However, in one pairwise (bbs5 and bbs10) and one triple (bbs2, bbs4, and bbs6) gene suppression model, we saw an epistatic effect in which the combined gene suppression model exhibits increased percentages of phenotypic severity, especially class II (severe) in comparison to the addition of any of the single suppression models alone (Figures 5 and S4). We corroborated these epistatic interactions in 4 dpf zebrafish larvae, building on our previous study of bbs gene interaction with a BBS candidate locus, nphp1.¹¹ In contrast to ift models, which display dilated pronephric tubules, we have shown previously that suppression of bbs results in atrophy and diminished convolution of the proximal tubule.¹¹ Immunostaining of renal structures and measurement of lateral images of the proximal convoluted tubule from the bbs5/bbs10 or bbs2/bbs4/bbs6 genetic interaction models showed a reduction in the size of the proximal convoluted tubule in the combined model in comparison to any of the single MO-injected batches alone (Figures 5 and S4). These exacerbated phenotypic effects are not likely to be underscored by altered bbs expression in the presence of excess MO dosing; qPCR studies from representative single, double, or triple MO injected batches showed no significant differences in bbs expression in comparison to controls (Figure S5).

Finally, we used this approach to test the oligogenic contribution of the heterozygous ALMS1 CNV detected in BBS pedigree AR400. The proband harbors a recessive BBS4 locus (homozygous exon 5–6 deletion) in addition to a heterozygous BBS9 c.1993C>T (p.Leu665Phe) probably functional null allele and a heterozygous ALMS1 exon 2–15 deletion. To model the combined effects of these loci, we first developed a zebrafish model of alms1 suppression (Figure S2). We targeted the sole ALMS1 ortholog in zebrafish with an sb-MO and coinjected subeffective doses into embryo batches either alone or in combination with bbs4 and/or bbs9 MOs. In midsomite-stage embryos, we found an additive effect, suggesting that ALMS1 is neither redundant with the other two BBS-associated proteins nor does it exacerbate significantly the phenotypic severity (Table S5). Together, these data substantiate further the complexity of the genetic architecture of BBS and lend further support to the contribution of mutational burden, in addition to primary recessive loci.

Discussion

Genetic and functional studies of the ciliopathies have informed the complexity of biological systems with regard to phenotypic variability and have afforded us the opportunity to begin to understand how the distribution of mutations beyond the primary causal locus can influence penetrance and expressivity.²³ However, essentially all that work was driven by mutational data derived by sequencing; some examples notwithstanding, the contribution of CNVs to both causality and overall mutational burden has been under-recognized. Here, using a high-density aCGH with sub-kb resolution to test an unselected cohort of BBS individuals, we have found that 18% of these individuals harbor at least one exon-disruptive CNV. These lesions, which range in size from ∼700 bp to >100 kb, can contribute recessive alleles; can Mendelize pathogenic SNVs on the other chromosome; and probably can interact with both CNVs and SNVs in other BBS loci, a posit substantiated by genetic interaction of the discovered BBS-associated gene combinations in vivo. Of note, analyses of either control databases or intramural studies of control samples on the same platform as our discovery cohort showed a stark absence of such CNVs from the general population, indicating that this CNV enrichment is unlikely to represent random background variation. Even so, the study of larger cohorts, both for BBS and other ciliopathies, will be required to measure the precise contribution of CNVs to both recessive and oligogenic paradigms.

We also note that the observed genetic interactions did not always lead to the same magnitude of effect, as measured by the severity of the combinatorial suppression of two or more bbs genes in zebrafish embryos evaluated at two different stages of development. Acknowledging that the interaction experiments in zebrafish embryos are a coarse approximation of the genetic architecture of these specific BBS allelic contributions to trait manifestation in case subjects, we speculate that these patterns, once understood more fully, have the potential to inform the effect of trans alleles on phenotypic expression (an exercise that the current cohort is underpowered to accomplish). Moreover, we recognize that not all genetic interactions will necessarily be deleterious. In that context, the analysis of individuals with clinically mild ciliopathies, or individuals protected from a specific organ disorder within the ciliopathy spectrum, might be informative at documenting protective CNV/SNV combinations.

Finally, our studies highlight the continued utility of CNV analysis to identify driver loci. In addition to previous studies that identified recessive CNVs in NPHP1 in BBS and ARMC4 (MIM: 615408) in primary ciliary dyskinesia (PCD [MIM: 615451]^{11, 86}), examination of the non-deleted chromosome identified IFT74 as a likely BBS locus. We discovered a single nuclear family; sequencing of the remainder of our cohort identified a sole rare SNV at this locus, functional testing of which showed it to be benign (Table S4). Therefore, we remain cautious until additional affected individuals are identified. Nonetheless, several lines of evidence support the candidacy of this locus. First, this is not the first IFT component to be mutated in this disorder; mutations in IFT27 and IFT172 were shown recently in BBS sibships.^{1, 22} Second, not only are the mutations likely damaging and potentially clinically severe for their functional impact (deletion/splice), but both mutant alleles affect specifically the long isoform of this gene, rendering the index case a functional hypomorph for this locus, offering an attractive hypothesis to explain the milder phenotype in this family compared to the severe, often prenatal or perinatal, lethal phenotypes caused by recessive null alleles in IFT genes in humans or model organisms.^{78, 80, 87, 88} Thus, it will be important to determine the differential functions of the two IFT74 splice isoforms, including their spatiotemporal distribution (and relative ratios across different tissues); as well as their interacting partners and cargo, since these might inform pathomechanism. Of note, the renal pathology of our zebrafish ift74 model might predict a dilatation defect in humans, which is different from the dysplastic phenotypes seen in other BBS models; clinical assessment of additional BBS-affected families with IFT mutations will be required to test this hypothesis.

In conclusion, our studies have highlighted a substantial role for CNVs in BBS. In addition to improving our appreciation of the complexity of the genetic architecture of this and other ciliopathies, our observations also highlight two critical points for clinical molecular diagnosis. First, our data highlight the need to consider all types of genetic and genomic lesions that can impact gene and protein function. Computational algorithms that rely on depth of coverage^{89, 90} could not have detected most of the deletions that we discovered due to their small size, and the same is true for some of the commonly used techniques in molecular cytogenetic laboratories.⁹¹ It is possible that the eventual transition to whole-genome diagnostic sequencing will improve the accuracy of detection of such structural variants. Second, our data also emphasize why it remains important to continue the study of the exomes and genomes of affected individuals beyond the discovery of a primary disease driver. Even though most current human genetic studies lack the resolution to interpret the effect of alleles beyond the recessive locus, the accrual of bona fide deleterious lesions in biological systems will ultimately inform the management of individuals who have the same recessive alleles but divergent clinical presentations.

Published: August 4, 2016

Web Resources

• Coriell Cell Repositories, https://catalog.coriell.org/

• CRISPR design tool, http://crispr.mit.edu

• Database of Genomic Variants (DGV), http://dgv.tcag.ca/dgv/app/home

• ExAC Browser, http://exac.broadinstitute.org/

• GenBank, http://www.ncbi.nlm.nih.gov/genbank/

• MutationTaster, http://www.mutationtaster.org/

• NHLBI Exome Sequencing Project (ESP) Exome Variant Server, http://evs.gs.washington.edu/EVS/

• OMIM, http://www.omim.org/

• PolyPhen-2, http://genetics.bwh.harvard.edu/pph2/

• SIFT, http://sift.bii.a-star.edu.sg/