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Evidence for additional FREM1 heterogeneity in Manitoba oculotrichoanal syndrome.

Mateo RK, Johnson R, Lehmann OJ - Mol. Vis. (2012)

Bottom Line: Subsequent sequencing of both genes in the IBD region, followed by FREM1, did not reveal any mutations.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genetics, University of Alberta, Edmonton, Alberta, Canada. robertin@ualberta.ca

ABSTRACT

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 log(2) 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.

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Related in: MedlinePlus

Montage illustrating representative genotype and copy number data across the IBD interval and FREM1. A: Genotype status (upper panels) and copy number data (lower panels) are provided for the first 55 SNPs in the IBD region (Chr9: 14,377,817–14,484,388). The BAF plots demonstrate homozygosity in the probands (BAF=1 or 0) and heterozygosity in the unaffected parents (BAF=0.5). The LRR plots also suggest no CNVs are present (LRR ~0). B: The lack of CNVs in FREM1 (14,727,151–14,900,234) is evident from LRR plots. CnvPartition did not detect any CNVs in this region as all 96 SNPs in this region were assigned a normal CNV value of 2.
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f3: Montage illustrating representative genotype and copy number data across the IBD interval and FREM1. A: Genotype status (upper panels) and copy number data (lower panels) are provided for the first 55 SNPs in the IBD region (Chr9: 14,377,817–14,484,388). The BAF plots demonstrate homozygosity in the probands (BAF=1 or 0) and heterozygosity in the unaffected parents (BAF=0.5). The LRR plots also suggest no CNVs are present (LRR ~0). B: The lack of CNVs in FREM1 (14,727,151–14,900,234) is evident from LRR plots. CnvPartition did not detect any CNVs in this region as all 96 SNPs in this region were assigned a normal CNV value of 2.

Mentions: 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).


Evidence for additional FREM1 heterogeneity in Manitoba oculotrichoanal syndrome.

Mateo RK, Johnson R, Lehmann OJ - Mol. Vis. (2012)

Montage illustrating representative genotype and copy number data across the IBD interval and FREM1. A: Genotype status (upper panels) and copy number data (lower panels) are provided for the first 55 SNPs in the IBD region (Chr9: 14,377,817–14,484,388). The BAF plots demonstrate homozygosity in the probands (BAF=1 or 0) and heterozygosity in the unaffected parents (BAF=0.5). The LRR plots also suggest no CNVs are present (LRR ~0). B: The lack of CNVs in FREM1 (14,727,151–14,900,234) is evident from LRR plots. CnvPartition did not detect any CNVs in this region as all 96 SNPs in this region were assigned a normal CNV value of 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3369896&req=5

f3: Montage illustrating representative genotype and copy number data across the IBD interval and FREM1. A: Genotype status (upper panels) and copy number data (lower panels) are provided for the first 55 SNPs in the IBD region (Chr9: 14,377,817–14,484,388). The BAF plots demonstrate homozygosity in the probands (BAF=1 or 0) and heterozygosity in the unaffected parents (BAF=0.5). The LRR plots also suggest no CNVs are present (LRR ~0). B: The lack of CNVs in FREM1 (14,727,151–14,900,234) is evident from LRR plots. CnvPartition did not detect any CNVs in this region as all 96 SNPs in this region were assigned a normal CNV value of 2.
Mentions: 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).

Bottom Line: Subsequent sequencing of both genes in the IBD region, followed by FREM1, did not reveal any mutations.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Genetics, University of Alberta, Edmonton, Alberta, Canada. robertin@ualberta.ca

ABSTRACT

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 log(2) 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.

Show MeSH
Related in: MedlinePlus