Molecular
Vision 2012; 18:1301-1311
<http://www.molvis.org/molvis/v18/a137> Received 17 April 2012 | Accepted 24 May 2012 | Published 30 May 2012
Molecular
Vision 2012; 18:1301-1311
<http://www.molvis.org/molvis/v18/a137>
Received 17 April 2012 | Accepted 24 May 2012 | Published 30 May
2012
Robertino Karlo Mateo,1 Royce Johnson,2 Ordan J. Lehmann1,2
1Department of Medical Genetics, University of Alberta, Edmonton, Canada; 2Department of Ophthalmology, University of Alberta, Edmonton, Canada
Correspondence to: Robertino Karlo Mateo, Department of Medical Genetics, University of Alberta, 8-29 Medical Sciences Building, Edmonton, AB, Canada, T6G 2H7; Phone: (780) 492-8550; FAX: (780) 492-6934; email: robertin@ualberta.ca
Purpose: Manitoba Oculotrichoanal (MOTA) syndrome is an autosomal recessive disorder present in First Nations families that is characterized by ocular (cryptophthalmos), facial, and genital anomalies. At the commencement of this study, its genetic basis was undefined.
Methods: Homozygosity analysis was employed to map the causative locus using DNA samples from four probands of Cree ancestry. After single nucleotide polymorphism (SNP) genotyping, data were analyzed and exported to PLINK to identify regions identical by descent (IBD) and common to the probands. Candidate genes within and adjacent to the IBD interval were sequenced to identify pathogenic variants, with analyses of potential deletions or duplications undertaken using the B-allele frequency and log2 ratio of SNP signal intensity.
Results: Although no shared IBD region >1 Mb was evident on preliminary analysis, adjusting the criteria to permit the detection of smaller homozygous IBD regions revealed one 330 Kb segment on chromosome 9p22.3 present in all 4 probands. This interval comprising 152 SNPs, lies 16 Kb downstream of FRAS1-related extracellular matrix protein 1 (FREM1), and no copy number variations were detected either in the IBD region or FREM1. Subsequent sequencing of both genes in the IBD region, followed by FREM1, did not reveal any mutations.
Conclusions: This study illustrates the utility of studying geographically isolated populations to identify genomic regions responsible for disease through analysis of small numbers of affected individuals. The location of the IBD region 16 kb from FREM1 suggests the phenotype in these patients is attributable to a variant outside of FREM1, potentially in a regulatory element, whose identification may prove tractable to next generation sequencing. In the context of recent identification of FREM1 coding mutations in a proportion of MOTA cases, characterization of such additional variants offers scope both to enhance understanding of FREM1’s role in cranio-facial biology and may facilitate genetic counselling in populations with high prevalences of MOTA to reduce the incidence of this disorder.
Manitoba Oculotrichoanal (MOTA) syndrome is a rare autosomal recessive disorder, first documented in the Island Lake region of Northern Manitoba [1]. Individuals of native Aboriginal descent (Canada’s First Nations peoples) exhibited ocular anomalies, most notably a fusion of the upper eyelid to the globe, known as subtotal cryptophthalmos or hidden eye. Associated phenotypes included facial anomalies with aberrant hair distribution extending below the brow, nasal dimpling, as well as ano-genital anomalies [2]. The existence of a similar disorder in the Inuit [3], who are ancestrally related to the First Nations, suggested a common genetic etiology. MOTA syndrome is phenotypically similar to Fraser Syndrome (FS), with common features including cryptophthalmos, nasal and genital anomalies [4]; however MOTA probands are less severely affected and to our knowledge do not exhibit cognitive impairment, syndactyly, renal, auricular, or limb defects.
Both disorders are autosomal recessively inherited [2]. Fraser syndrome cases are attributable to mutations in either FRAS1 (Fraser syndrome 1) or FREM2 (FRAS1-related extracellular matrix protein 2) [5-7], with these genes accounting for approximately 40% of cases. Other FRAS/FREM gene family members (FREM1 and FREM3) form multi-protein complexes in the extracellular matrix that interact with GRIP1, (glutamate receptor-interacting protein 1) which serves to anchor FRAS/FREM proteins [8,9], and in which mutations were recently detected in FS probands [10]. Linkage analysis of Fraser Syndrome to the vicinity of FREM1 (chromosome 9p22.3) was reported 5 years ago, however no disease causing mutations were identified [11]. More recently, homozygous FREM1 mutations were shown in a Middle Eastern sibship [12] to be associated with a bifid nose, anorectal, and renal anomaly phenotype, but which lacked cryptophthalmos, suggesting that FRAS/FREM variants may contribute to a diverse spectrum of related disorders [13].
The Fras/Frem, and Grip1 genes have been extensively studied in murine models, strains, collectively referred to as “bleb” mutants due to epidermal blistering during embryonic development [14-17]. These exhibit cryptophthalmos, syndactyly and renal defects that correspond with those phenotypes observed in FS patients. Fras/Frem genes, which are expressed in a tissue specific manner and encode proteins that are secreted into the extracellular matrix, regulate the bioavailability of growth factors during development [18] and so have key roles in tissue morphogenesis [19,20]. FRAS/FREM proteins contain chondroitin sulfate proteoglycan (CSPG) domains, and their tissue specific expression is thought to maintain epithelial-mesenchymal integrity during development via a mechanism similar to CSPG4 (or NG2), directly binding collagens V and VI as well as fibroblast growth factor (FGF) and epidermal growth factor (EGF) [21,22].
At the commencement of this study MOTA syndrome was molecularly undefined, with no FRAS/FREM family members known to underlie MOTA. We used homozygosity mapping, an approach that permits mapping of genes responsible for autosomal recessive disorders [23-26]. Single nucleotide polymorphisms (SNPs) were used to identify regions that are Identical By Descent (IBD) in multiple affected individuals and so determine the genomic interval responsible for disease [27,28]. This methodology takes advantage of the geographically isolated nature of the First Nations community studied and MOTA’s reported inheritance pattern, enabling the molecular basis to be elucidated using a very small number of patient samples.
Affected individuals were derived from three pedigrees of Cree ancestry living in a geographically isolated region in Northern Alberta (Figure 1). Since the area is only accessible during the winter by ice roads, this was anticipated to result in high levels of consanguinity in the approximately 1,000 inhabitants. Blood samples were collected from four probands (1.III-1, 2.V-2, 3.III-1, and 3.III-7) and the unaffected parent (mother) that accompanied each child for oculoplastic surgery at the regional ophthalmic center, followed by genomic DNA extraction. Ethical approval was provided by the University of Alberta Hospital Health Research Ethics Board, and informed consent was obtained from all participants.
Genotyping was performed using a 610-Quad SNP array (Illumina Inc., San Diego, CA) comprising approximately 28,000 Copy Number Variant (CNV) probes and 592,000 single nucleotide polymorphisms (SNPs), spaced at a mean distance of 1 SNP per 2.7 kb across the genome, and processed by deCODE genetics in Reykjavík, Iceland. Raw data were analyzed using GenomeStudio software (Illumina), non-Mendelian genotypes removed using the software’s Heritability Report algorithm, and then exported to PLINK v1.07 for homozygosity analysis [29]. Homozygous regions were then analyzed to define IBD intervals common among the probands. Initial homozygosity analysis performed using default PLINK parameters (homozygous region >1 Mb) did not identify an IBD interval common to all four probands. Subsequently, criteria were altered to permit detection of smaller homozygous segments (>300 kb) comprising at least 100 consecutive homozygous SNPs. In addition the percentage homozygosity of each genome was calculated using the total length of homozygous regions >300 kb divided by that of the autosomes (NCBI Build 36) [30].
Two values were calculated from the array data to determine if any copy number variants were present. The first (B-Allele Frequency [BAF]) is derived from the relative ratio of fluorescent intensities of the two alleles at each SNP (Cy5 [green] A allele; Cy3 [red] B allele) with a heterozygous SNP having a BAF of 0.5, while homozygous SNPs are either 1 or 0. The second criterion used, is the logarithm of the ratio of the observed to the expected intensities at each SNP (Log2R ratio [LRR]), with deviations from zero (log21) providing evidence of a CNV (deletion=-1, duplication=0.5, normal=0). Additional software (CnvPartition 3.1.6; Illumina) was used in parallel with LRR data to assign a CNV value for each SNP, and so detect any potential deletions or duplications.
The coding and splice junctions of three genes lying in or adjacent to the genomic region of interest (FREM1, cerberus 1 (CER1) [31], and zinc finger, DHHC-type containing 21 (ZDHHC21) [32]) were sequenced using published primers [12] or those designed with Primer3 (Appendix 1). Genomic DNA from a single affected individual (1.III-1) was used as template and sequence data generated (ABI Prism 3100, Applied Biosystems, Foster City, CA) was analyzed relative to the ENSEMBL reference sequence (Sequencher 4.6; GeneCodes, Madison, WI).
In an effort to identify potential regulatory elements within the IBD interval, non-coding genomic sequences conserved in vertebrates were defined using ECR Browser [33], with appropriate correction for the different genomic builds (SNP array, Build 36, ECR Brower, Build 37) using the UCSC LiftOver tool [30]. Criteria consisted of ECRs with a minimum length of 90 bp and greater than 70% conservation of the human sequence against chimpanzee, rhesus monkey, cow, dog, opossum, rat, mouse, chicken, frog, pufferfish, or zebrafish genomes. ECRs conserved between human and Xenopus (Table 1) were selected for further analysis and sequenced with primers designed by Primer 3 (Appendix 2).
The four MOTA cases displayed a spectrum of ocular anomalies with considerable variation in phenotypic severity. There was a greater proportion of bilateral (n=3) than unilateral involvement (Figure 2), and cases with partial upper eyelid involvement most frequently affected the medial segment. Additional features included fusion of the eyelid to the cornea, which ranged in severity from total fusion (Figure 2D) to focal synechiae (Figure 2E,F), as well as frequent corneal opacification and corneal vascularisation (Figure 2G,H). Aberrant facial development was evident from extension of hair distribution from the scalp to reach the eyebrow (Figure 2B,C) as well as nasal dimpling (Figure 2B,D).
Only a small number of SNPs (67 – 95 [~0.01%]; Appendix 3) were excluded due to non-Mendelian errors, indicating that the genotyping data were of high quality. High homozygosity levels were observed in the four affected individuals (range: 9.3% – 15.8%; Appendix 4), indicating very substantial degrees of consanguinity that contrast with the ~6% theoretically calculated for the offspring of a first cousin marriage [34,35]. Homozygosity mapping analysis identified only a single segment that is identical by descent in the four affected individuals. This 330kb interval on 9p22.3 (Chr.9: 14,377,817 - 14,711,766, flanking SNPs rs2382470 and rs1494359) lies approximately 16 kb 3′ to the last exon of FREM1 and the SNPs in this IBD interval display BAF values of 1 or 0 in the probands (demonstrating homozygosity) while the unaffected parents are heterozygous (BAF=~0.5) (Figure 3A; upper panels). Equally, the LRR values cluster around zero for the 152 SNPs in the IBD region, demonstrating the absence of any CNVs (Figure 3A; lower panels). In particular, the SNPs encompassing FREM1 (9: 14,727,151 - 14,900,234) have normal LRR values and additional automated CNV analysis (CnvPartition) demonstrated that no CNVs were detectable in either the IBD (data not shown) or FREM1 intervals (Figure 3B). Similarly, no CNVs or additional IBD regions were detected in the intervals encompassing FRAS1, FREM2, FREM3, or GRIP1 (data not shown).
The IBD region contains two genes CER1 (a TGF-β signaling antagonist) [31] and ZDHCC21 (a regulator of hair follicle development) [32] and as illustrated (Figure 4) its border is distinct from that of FREM1. Sequencing was performed initially for CER1 and ZDHCC21, with no coding or splice site mutations identified. Notwithstanding the homozygosity mapping data, the 38 exons of FREM1 were next sequenced and did not identify any causative variants. Ten homozygous variants were present: seven that result in synonymous amino acid substitutions, one non-synonymous SNP (A1212S) present in 28% of controls (dbSNP rs35870000), and a 5′UTR variant (Table 2). Notably a variant (c.5556A>G) that was recently described as contributing to MOTA [35], did not segregate in an autosomal recessive pattern (homozygous 1.III-1; heterozygous 3.III-1 and 3.III-7; homozygous wildtype 2.V-2). Seven non-coding regions within the IBD interval were found to be evolutionarily conserved with >70% identity between humans and Xenopus. Sequencing these seven regions identified a homozygous T>C base pair substitution that segregated with the disease phenotype (all probands: C/C, unaffected parents T/C; Appendix 5).
This study’s key finding is the identification of a 330 kb region on chromosome 9p22.3 that is associated with MOTA syndrome. This illustrates the value of studying consanguineous populations such as the First Nations with homozygosity analysis. These findings localize the causative variant to an interval adjacent to FREM1, which represents an excellent candidate on the basis of the recapitulation of the human phenotypes in Frem1 mutant mice [36,37], and related phenotypes induced by mutation of other FRAS/FREM gene family members. This study was predicated on the assumption that the level of homozygosity in a geographically isolated population, living on a remote reserve, would be increased. The range of autosomal homozygosity observed (9.3% - 15.8%), which in some cases exceeds that observed in other consanguineous populations or in the offspring of first cousin marriages [34], validates the approach used and contrasts with the far lower rates observed in a general population (1.9% - 4.6%) [38]. These data, derived from a very small number of affected individuals, illustrate the applicability of homozygosity mapping in the First Nations and suggest that it may permit other causes of this population’s disproportionately large disease burden to be identified.
While this manuscript was in preparation, two papers were published that substantially advanced understanding of FREM1’s role in these disorders [13,35]. The first, reported several FREM1 mutations in MOTA cases of either First Nations or European ancestry [35] including: an inframe deletion of exons 8–23, one nonsense, two missense, and a synonymous alteration (c.5556A>G). Notably, neither of the two variants identified in First Nations patients (deletion of exons 8 – 23 or c.5556A>G [G1853G]) is the cause in our cases, in view of the absence of CNVs in the 330 kb region (Figure 3) and the fact that c.5556A>G’s does not segregate with disease (data not shown). Since a second causative allele was not identified in some individuals of Oji-Cree ancestry reported in Slavotinek et al. [35], the possibility therefore exists that a still to be identified allele is common to both the Oji-Cree and First Nations populations. The second publication describes heterozygous FREM1 deletions and 3 missense variants that associate with metopic craniosynostosis as well as documenting the contribution of FREM1 in patterning the murine cranial skeleton [13]. Accordingly our study demonstrates additional genetic heterogeneity among the First Nations, who would have been anticipated to have a single cause for the phenotype.
The most parsimonious explanation for our findings is that a sequence variant within the 330 kb IBD interval, which is located 16 kb 3′ of FREM1’s last known exon, causes MOTA. This is most likely to represent a regulatory element; however the possibility that an additional exon remains to be defined, cannot be excluded. Support for the concept of a regulatory variant is provided by the Frem1bfd murine strain, which lacks a coding Frem1 mutation and is believed to have a variant in a control region that causes cryptophthalmos-like phenotypes [37]. Frem1’s role during development suggests that its temporal-spatial expression is tightly controlled, in keeping with the regulatory elements and tissue specific enhancers defined for a range of other developmental regulatory genes [39]. There are several examples of such mutated sequences in both ocular and systemic diseases, with regulatory mutations 3′ to PAX6 causing aniridia and demonstrated to be functionally relevant by murine transgenesis rescue experiments [40,41]. In an attempt to define such elements, seven regions conserved across vertebrates were sequenced, identifying a homozygous T>C base pair substitution (ECR-7) that segregated with the phenotype. Bioinformatics analysis for regulatory elements using the VISTA Enhancer Browser online database [42] yielded no tissue specific enhancers for this region (data not shown). The most likely explanation is that this variant is in linkage disequilibrium with the true mutation, and it should be noted that sequence conservation is not necessarily a criteria of all regulatory elements [43]. Future research directions to support the relevancy of the identified 330 kb IBD region could include assaying FREM1 expression from mRNA isolated from skin fibroblasts of MOTA probands, relative to a housekeeping gene and control samples. In parallel, next generation sequencing of the 330 kb IBD interval is increasingly feasible. It is interesting to note that the transcription factor delta-Np63 has been shown to control expression of members of the Fras/Frem gene family and displays enhancer activity in the murine nose, eyelids, genitals, and digits [44,45], the tissue domains affected in FS, BNAR, and MOTA.
In summary, this study extends FREM1 heterogeneity in MOTA syndrome of First Nations ancestry. Homozygosity mapping defined one 330 kb IBD region on chromosome 9p22.3 comprising 152 SNPs in 4 probands. Sequencing the genes in or adjacent to this interval (FREM1, CER1, and ZDHHC21) revealed no disease-causing mutations. Accordingly, we infer that a variant within this region is responsible for MOTA syndrome, and suggest that future studies are indicated to define the causative mutation and by facilitating genetic counselling, reduce the high prevalence of MOTA syndrome in these isolated populations.
Appendix 1. Primers used to amplify CER1 and ZDHHC21.
Appendix 2. Primers used to amplify ECRs.
Appendix 3. Erroneous SNPs identified.
Appendix 4. Percentage genome homozygosity.
Appendix 5. A homozygous point mutation within ECR-7 segregates with MOTA.
This work was supported by the Women and Children’s Health Research Institute and the Canadian Institutes of Health Research. We thank Azam Khorshidi and Dr. Sameer Pant for assistance with bioinformatic analysis. Data presented at Women and Children Health Research Institute Research Day (November 24, 2010), and the Department of Medical Genetics Research Day (March 9, 2011).