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Bardet-Biedl syndrome (BBS) is primarily an autosomal recessive disorder characterized by the five cardinal features retinitis pigmentosa, postaxial polydactyly, mental retardation, obesity and hypogenitalism. In addition, renal cysts and other anomalies of the kidney and urinary tract can be present. To date, mutations in 12 BBS genes as well as in MKS1 and CEP290 have been identified as causing BBS. The vast genetic heterogeneity of BBS renders molecular genetic diagnosis difficult in terms of both the time and cost required to screen all 204 coding exons. Here, we report the use of genome-wide homozygosity mapping as a tool to identify homozygous segments at known BBS loci in BBS individuals from inbred and outbred background. In a worldwide cohort of 45 families, we identified, via direct exon sequencing, causative homozygous mutations in 20 families. Eleven of these mutations were novel, thereby increasing the number of known BBS mutations by 5% (11/218). Thus, in the presence of extreme genetic locus heterogeneity, homozygosity mapping provides a valuable approach to the molecular genetic diagnosis of BBS and will facilitate the discovery of novel pathogenic mutations.
Bardet-Biedl syndrome (BBS, [OMIM #209900]) is a clinically heterogeneous primarily autosomal recessive disorder characterized by structural and functional defects in numerous organs. The five cardinal features are retinitis pigmentosa, postaxial polydactyly, mental retardation, obesity and hypogenitalism. In addition, renal cysts and other anomalies of the kidney and urinary tract can be present1–4. Secondary features include developmental delay, ataxia, diabetes mellitus, congenital heart defects, hearing loss, and anosmia4–7. Diagnosis of BBS is often achieved only once vision begins to deteriorate. Night blindness from rod-cone dystrophy usually manifests around eight years of age and loss of peripheral vision develops by the age of 15 years4. Clinical manifestations such as cystic kidneys, type II diabetes, hypertension, and hypercholesterolemia are frequent causes of morbidity and mortality in individuals suffering from BBS4.
In addition to the broad clinical heterogeneity of BBS, early linkage studies have also revealed a high level of gene locus heterogeneity. Thus far, a total of 12 genes have been identified that, when mutated, cause BBS (BBS1, BBS2, BBS3 [ARL6], BBS4, BBS5, BBS6 [MKKS], BBS7, BBS8 [TTC8], BBS9, BBS10, BBS11 [TRIM32], and BBS12)7–17. In addition, mutations in MKS1 and CEP290 have been implicated in BBS18. The proteins BBS1, 2, 4, 5, 7, 8, 9 and 10 are part of a protein complex, termed the “BBSome” and localize to primary cilia, and the basal body of primary cilia or the peri-centriolar region. This indicates a role of ciliogenesis and intraflagellar transport (IFT) in BBS pathogenesis7–13,19. BBS6, 10, and 12 are likely type II chaperonins, which may facilitate in the folding of proteins essential for ciliogenesis or IFT12,15,17. BBS9 encodes a parathyroid hormone-responsive protein14. Furthermore, BBS11 encodes an E3 ubiquitin ligase, suggesting a role for basal body protein dysregulation in the pathogenesis of BBS16.
Given the observed pleiotropy associated with BBS, resolving a correlation between phenotype and genotype poses a significant challenge. Moreover, the vast clinical and genetic heterogeneity of this syndrome makes molecular genetic diagnosis difficult with regards to the time, labor, and cost required to screen all 204 coding exons. Finally, only ~40% of known pathogenic alleles among BBS patient cohorts of Northern European descent are contributed by two common mutations (M390R in BBS1 and C91LfsX5 in BBS10), with the remainder composed of rare variants. In non-European families, in which the prevalence of BBS is typically higher, the contribution of M390R is modest, exacerbating the problem of molecular diagnosis (reviewed in20). Here, we report the use of prescreening by genome-wide homozygosity mapping, in a multiethnic cohort of 45 families from both inbred and outbred backgrounds, as a tool to identify homozygous segments at known BBS-loci. Using this strategy, we were able to identify, by subsequent exon sequencing, causative mutations of eight different BBS genes in 20 families. As eleven of these mutations are novel, we increase the number of known BBS mutations by 5% (11/218).
We performed a total genome search in a worldwide cohort of 45 families with 68 individuals clinically diagnosed with Bardet-Biedl syndrome. The diagnoses were ascertained on the basis of previously established criteria4. For all kindreds, blood samples, and pedigrees were ascertained following informed consent. The study was done in accordance with ethics approval from the Internal Review Boards of the University of Michigan, the Ethics Committee of the UCL Institute of Child Health and the Internal Review Board of the Johns Hopkins University.
For all affected individuals, genome-wide scans for homozygosity were performed and analyzed as described previously21. We used single nucleotide polymorphism (SNP) arrays (GeneChip) from Affymetrix™, Inc. All individuals were genotyped at 250 K resolution (Human Mapping 250 K StyI Array). Samples were processed, hybridized, and scanned using the manufacturer's standard methods at the University of Michigan Core Facility (www.michiganmicroarray.com). Using the program ALLEGRO22, non-parametric likelihood ratio Z-scores (ZLR scores) were calculated with the following parameters: a disease allele frequency of 0.001, a hypothetical standard pedigree structure assuming first cousin marriage for parents of affected individuals using marker allele frequencies for Northern Europeans from the Affymetrix data set. For single affected individuals a non-existent sibling was included to enable non-parametric ALLEGRO runs. ZLR scores were plotted over genetic distance across the entire human genome using GNUPLOT (http://www.gnuplot.info/). In this way, maxima of ZLR scores, “ZLR peaks”, are expected to reflect segments of homozygosity by descent, which may harbor the homozygous mutation of the recessive disease gene21. Using ALOHOMORA23, ZLR scores were calculated using one marker every 100,000 nucleotides of human genomic sequence, i.e. for minor allele frequencies of >0.2, >0.3, and >0.4. We have reported previously21 that ZLR peaks, which exceed the value of 2.0 under two out of the three conditions (minor allele frequencies of >0.2, >0.3, and >0.4), did in fact contain the known homozygous disease-causing mutation in one of these ZLR peaks in 93% of the cases and exhibited continuous segments of homozygosity upon haplotype inspection. We refer to peaks that fulfill these criteria as “consistent ZLR peaks” (“cZLR” peaks).
Individuals exhibiting a cZLR peak at a known BBS locus or showing homozygosity >500 kb at a known BBS locus upon inspection of their haplotype data underwent mutation analysis of the coding region of the respective BBS gene. However, no mutations were found in homozygous segments shorter than 2.1 Mb, the previously described detection limit for cZLR peaks (Hildebrandt et al. PloS Genet 5:e1000353, 2009). Following the previously established protocol exon PCR and direct sequencing was performed using exon flanking primers (Supplementary Table 2)24. For sequence analysis we used SEQUENCHER 3.8 (Gene Codes, Ann Arbor, Michigan); the reference sequences used are shown in Supplementary Table 1. All novel mutations were shown to be absent from at >90 ethnically matched healthy control individuals by direct sequencing.
We performed genome wide homozygosity mapping in an ethnically diverse cohort of 45 families with BBS (Table 1 and Supplementary Figure 1). 27 families were known to be consanguineous (group c; Table 1) and 18 were from outbred populations with no evidence for consanguinity (group o; Table 1). Total genome homozygosity mapping yielded segments of likely homozygosity by descent “cZLR peaks”21 in 32 of the 45 families. Of these 32 families total genome homozygosity mapping data are depicted in Supplementary Figure 1 for the 20 families, in whom we detected the causative homozygous BBS mutations. The number of cZLR peaks of homozygosity is compared for consanguineous (group c) and outbred (group o) families in Table 1 and was as follows: In the 27 families with known consanguinity (group c) all exhibited cZLR peaks of homozygosity and the median number of cZLR peaks of homozygosity was 13 (range 1 – 36), whereas in the 18 families from outbred populations and no known consanguinity (group o) only 5 (27%) exhibited cZLR peaks with a median number of cZLR peaks of homozygosity was 2 (range 1 – 4) (Table 1). This is consistent with the previous finding that individuals with homozygous mutations in recessive disease genes exhibit four or more cZLR peaks of homozygosity, if they are from inbred populations, whereas they exhibit four or less cZLR peaks of homozygosity if they are from outbred populations21. The 12 families that exhibited cZLR peaks but in whom we did not detect the causative mutations were not homozygous at any known BBS locus. These families are candidates for the identification of novel BBS-causing genes within those homozygous candidate regions.
For each family, homozygous segments that harbored a known BBS gene (BBS1–BBS12) (arrow heads in Supplementary Figure 1) were examined further using direct exon sequencing of all coding exons and splice sites of that gene. Using this strategy we detected both disease-causing alleles in 20 families. Eighteen of these families were knowingly consanguineous, two were not consanguineous. There were 17 homozygous mutations in BBS 1, 2, 4, 5, 7, 8, 10 and 12 in these 20 families (Table 2 and Figure 1). Three mutations occurred twice in two different families each; eleven of the 17 homozygous mutations were novel (Table 2 and Figure 1). These eleven novel mutations resulted in various predicted changes on the protein level (Table 2 and Figure 1): i) three presumed null mutations (p.M1K in BBS5, p.R238fsX296 in BBS7, and p.W41X in BBS8); ii) five missense mutations (p.I66M in BBS7, p.W320R in BBS7, p.E596K in BBS7, p.Y177C in BBS10, and p.L530P in BBS12) and; iii) three splice site mutations (IVS1−1 g>a and IVS2+1g>a in BBS1 and IVS3−3 c>g in BBS4). All 17 mutations were absent from >90 ethnically matched healthy control individuals. We thereby increased the number of known BBS mutations by 5% (11/218) using only homozygosity mapping information as a hierarchical tool for screening (http://www.biobase-international.com).
The novel homozygous missense BBS12 mutation (c.1589T>C, p.L530P) was found in two consanguineous Pakistani individuals (A2003-II1 and A2015-II1) (Table 2 and Figure 1). Haplotype analysis confirmed that this mutation represents a likely Pakistani founder effect, as it occurs on a shared 9-Mb haplotype that extends across rs7690104 to rs2049092 (data not shown). Two additional individuals from Pakistan, A2002-II1 and A2027-II1, harbored a novel homozygous missense mutation of the BBS5 start codon (p.M1K) (Table 2 and Figure 1). This mutation is likely to represent a null mutation, resulting either in failure of protein translation or in the translation of an illegitimate transcript. The parents of both individuals were known to be related. Since the alteration is embedded in a 0.89 Mb homozygous haplotype between markers rs10199676 and rs2355351 (data not shown), this mutation also likely represents a Pakistani founder effect (Supplementary Figure 1).
Disorders of striking genetic heterogeneity, such as BBS, represent a major diagnostic challenge as mutations at more than 12 loci have been shown to be necessary and sufficient for pathogenesis, rendering medical resequencing of >204 known exons laborious and expensive. Other viable alternatives, such as specific interrogation of known mutations by primer extension is practical, but can only capture less than 50% of the mutational load25–28 (our unpublished data). In the present study, we have demonstrated that whole genome homozygosity mapping followed by direct sequencing is an effective alternative means of identifying causative mutations in BBS. We have shown recently that homozygosity mapping can also be applied to individuals who reside within “outbred” populations but are “homozygous by descent“ from a remote ancestor at the locus of the disease-causing gene21. We here successfully apply this approach to patients from both, inbred and outbred populations, confirming that there can be broad utility of homozygosity mapping in the absence of ancestry information21. Specifically, in families A2875 and A2876 who derive from outbred populations (Table 2), we were able to detect the disease causing mutations (Supplementary Figure 1).
Alternatively, the “homozygosity peaks” could be generated by hemizygosity, i.e. a large heterozygous deletion. In a recessive disease, a second mutant allele would be expected to be present in the gene affected by the disease-relevant heterozygous deletion giving rise to a compound heterozygous state. However, especially in individuals with known consanguinity of the parents, compound heterozygosity is unlikely, because consanguinity poses an a priory risk for the affected individual for homozygous mutations but not for compound heterozygous mutations. In molecular diagnostics the presence of a large heterozygous deletion would have the same practical consequence, i.e. to perform exon sequencing to detect the second recessive mutation.
Notably, none of the families studied had a sufficient number of affected individuals to allow mapping of a disease locus by classic linkage analysis, further emphasizing the fact that homozygosity mapping can solve an otherwise experimentally intractable problem, especially as the cost for total genome SNP analysis has considerably dropped recently. In the families who do not yield cZLR peaks, which were 13 families in this study, mutation analysis for compound heterozygous mutations will still have to be performed for all exons of all known BBS genes.
In conclusion, we increased the number of BBS-associated mutations by 5% (11/218) employing homozygosity mapping. Our study will likely catalyze the identification of additional BBS loci, since 12 families exhibited homozygous regions outside a known BBS locus or were negative upon mutation analysis (data not shown). Alternatively, these families may have compound heterozygous mutations in known BBS genes, because there is a possibility of false positive “homozygosity peaks” at a rate of less than 35% in individuals from “outbred” background (Hildebrandt et al. PloS Genet 5:e1000353, 2009). This rate was been determined by examining parents of children with recessive mutations for “homozygosity peaks”. However, as parents of children with mutations homozygous by descent have themselves a some what increased likelihood that their own parents are related, this rate of 35% may be somewhat of an overestimate.”
Ultimately, comprehensive analysis of the entire BBS-associated gene spectrum will be necessary, because of the presence of modifier alleles (even in consanguineous families) that can modify the penetrance and expressivity of BBS endophenotypes18,29,30. Nonetheless, the rapid identification of the primary genetic lesion will have a clear and immediate benefit with regard to patient management and reproductive choices and is likely to prove useful in the study of other disorders of marked genetic heterogeneity such as retinitis pigmentosa, sensorineural deafness and others.
We thank the following physicians for their help in this study: Nursel Elcioglu, Andrea Nemeth, Helen Stewart, Denise Williams, and Kate Chandler. This work was supported by grants to FH from the National Institutes of Health (DK068306, DK064614, DK069274, HD045345), grant R01HD04260 from the National Institute of Child Health and Development (NK), R01DK072301 and R01DK075972 (NK) and NRSA fellowship F32 DK079541 (EED) from the National Institute of Diabetes, Digestive and Kidney disorders, the Macular Vision Research Foundation (NK), the Foundation for Fighting Blindness (NK), and by grants from the NEWLIFE and Wellcome Trust (PB). NK is a Brumley endowed Professor. FH is an investigator of the Howard Hughes Medical Institute, a Doris Duke Distinguished Clinical Scientist, and a Frederick G.L. Huetwell Professor.
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