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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

Illustration of the homozygous regions and the IBD interval in the four probands. The regions of homozygosity, which range from 330 kb to 10.4 Mb, include a 330 kb IBD interval common to all probands (red line). This interval contains ZDHHC21 and CER1, and is 16 kb 3′ of FREM1’s last exon. Homozygosity mapping defined one IBD interval suggesting that mutation responsible for MOTA lies within the narrow 330 kb region.
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f4: Illustration of the homozygous regions and the IBD interval in the four probands. The regions of homozygosity, which range from 330 kb to 10.4 Mb, include a 330 kb IBD interval common to all probands (red line). This interval contains ZDHHC21 and CER1, and is 16 kb 3′ of FREM1’s last exon. Homozygosity mapping defined one IBD interval suggesting that mutation responsible for MOTA lies within the narrow 330 kb region.

Mentions: 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).


Evidence for additional FREM1 heterogeneity in Manitoba oculotrichoanal syndrome.

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

Illustration of the homozygous regions and the IBD interval in the four probands. The regions of homozygosity, which range from 330 kb to 10.4 Mb, include a 330 kb IBD interval common to all probands (red line). This interval contains ZDHHC21 and CER1, and is 16 kb 3′ of FREM1’s last exon. Homozygosity mapping defined one IBD interval suggesting that mutation responsible for MOTA lies within the narrow 330 kb region.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Illustration of the homozygous regions and the IBD interval in the four probands. The regions of homozygosity, which range from 330 kb to 10.4 Mb, include a 330 kb IBD interval common to all probands (red line). This interval contains ZDHHC21 and CER1, and is 16 kb 3′ of FREM1’s last exon. Homozygosity mapping defined one IBD interval suggesting that mutation responsible for MOTA lies within the narrow 330 kb region.
Mentions: 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).

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