Deletions involving long-range conserved nongenic sequences upstream and downstream of FOXL2 as a novel disease-causing mechanism in blepharophimosis syndrome

Abstract

The expression of a gene requires not only a normal coding sequence but also intact regulatory regions, which can be located at large distances from the target genes, as demonstrated for an increasing number of developmental genes. In previous mutation studies of the role of FOXL2 in blepharophimosis syndrome (BPES), we identified intragenic mutations in 70% of our patients. Three translocation breakpoints upstream of FOXL2 in patients with BPES suggested a position effect. Here, we identified novel microdeletions outside of FOXL2 in cases of sporadic and familial BPES. Specifically, four rearrangements, with an overlap of 126 kb, are located 230 kb upstream of FOXL2, telomeric to the reported translocation breakpoints. Moreover, the shortest region of deletion overlap (SRO) contains several conserved nongenic sequences (CNGs) harboring putative transcription-factor binding sites and representing potential long-range cis-regulatory elements. Interestingly, the human region orthologous to the 12-kb sequence deleted in the polled intersex syndrome in goat, which is an animal model for BPES, is contained in this SRO, providing evidence of human-goat conservation of FOXL2 expression and of the mutational mechanism. Surprisingly, in a fifth family with BPES, one rearrangement was found downstream of FOXL2. In addition, we report nine novel rearrangements encompassing FOXL2 that range from partial gene deletions to submicroscopic deletions. Overall, genomic rearrangements encompassing or outside of FOXL2 account for 16% of all molecular defects found in our families with BPES. In summary, this is the first report of extragenic deletions in BPES, providing further evidence of potential long-range cis-regulatory elements regulating FOXL2 expression. It contributes to the enlarging group of developmental diseases caused by defective distant regulation of gene expression. Finally, we demonstrate that CNGs are candidate regions for genomic rearrangements in developmental genes.

Figure 1

A, Physical map of a 1.8-Mb region flanking FOXL2 that shows the position of the BACs, PACs, cosmids, microsatellites, and SNPs that were used for identification and delineation of the deletions. The map is drawn to scale; the size equivalent to 50 kb is given at the bottom left corner. The location of FOXL2 is indicated by a vertical black line. The top line represents the localization of SNPs, and the second line contains the known and anonymous microsatellites used in this study. In the third line, the location of the genes in this region is shown, whereas the BACs, PACs, and cosmids used for FISH are shown below the line. The lower part of A is an overview of all deletions identified in this study, except one. Every line represents a patient or family in whom a deletion was found. The dashed red line shows the maximal extent of the deletion. The first 10 lines represent the deletions that encompass FOXL2. The deletion identified in BPES11 is not shown, since the extent of the deletion could not be determined because of a lack of material for FISH and microsatellite analysis. The second group represents the five extragenic deletions (four upstream and one downstream). Clinical details and molecular data on the patients with deletions are shown in table 2. In a box at the bottom right, a detailed view of the SRO of the four upstream extragenic deletions is shown. The position of FOXL2 is marked by a vertical dashed black line, and the SRO is shown by the dashed red line. The positions of the known three translocation breakpoints at 3q23 in BPES and of the orthologue of the PIS locus are indicated by vertical arrows. B and C, UCSC Genome Browser map of the SRO of the four upstream extragenic rearrangements in families BPES12, BPES13, BPES14, and BPES15. CNGs are highlighted at the bottom with red boxes. Their names, based on our arbitrary numbering, are given below the boxes. Details on all CNGs can be found in table 4. The CNGs shown in B are evolutionarily conserved up to chicken. C represents a more detailed view of CNG3–CNG7, and CNG3–CNG5 are conserved up to zebrafish and/or pufferfish. They all represent potential cis-regulatory elements of the FOXL2 region. D, UCSC Genome Browser map of the sequence contained in the downstream extragenic rearrangement in family BPES16. Five CNGs are indicated in this 3′ SRO, of which several are conserved up to zebrafish and/or pufferfish (CNG14, CNG18, and CNG22–CNG24).

Figure 2

Pedigrees with BPES and a rearrangement encompassing FOXL2 or located outside of the transcription unit. Intragenic FOXL2 mutations were excluded by sequencing of the ORF in all patients. MLPA was performed to assess changes in gene copy number, and haplotypes are based on microsatellite or SNP analysis. An exclamation mark (!) indicates hemizygosity of an allele. BPES9, Microsatellite analysis in two generations of this French family showed apparent linkage to FOXL2. However, no intragenic FOXL2 mutation was found in affected members. MLPA analysis revealed a total gene deletion of FOXL2. Long-range PCR revealed the breakpoints of an 8-kb deletion (fig. 3). The occurrence of secondary amenorrhea in II:1 suggests this family has type I BPES. In addition, III:2 has a heart malformation in association with BPES. BPES16, In this family, two affected half-sisters (II:1 and II:3) have BPES, whereas their father (I:2) is clinically unaffected, suggesting germline mosaicism. FISH and microsatellite analysis revealed an extragenic deletion of 188 kb located 3′ of FOXL2 in the half-sisters. Microsatellite analysis showed an apparent absence of this deletion in their father. However, quantitative studies revealed somatic mosaicism for this deletion in 5% of DNA from his leucocytes and 10% of DNA from his sperm cells, confirming germline mosaicism at the molecular level (table 3). BPES12, Upstream microdeletion in a 14-year-old male with sporadic BPES and additional features. MLPA showed normal FOXL2 and ATR gene copy numbers, whereas several microsatellites at a distance from FOXL2 were hemizygous, indicating a deletion outside of the transcription unit. The centromeric breakpoint of the deletion is located in a 69-kb interval between microsatellites EDB14 and DB17; at the telomeric side, the deletion extends beyond the borders of the physical map in figure 1A, but does not contain D3S1309. The deletion encompasses at least several known and unknown genes (LOC389152, BPESC1, MRPS22, COPB2, RBP2, ACTGP1, RBP1, NMNAT3, and CLSTN2) and extends beyond 1.9 Mb. The contribution of each gene to the psychomotor retardation and other associated features in this patient is unclear. BPES13, Upstream microdeletion of maternal origin in a 2-year-old female with sporadic BPES who had normal psychomotor development and microcephaly. The deletion spans 567 kb at the most; the centromeric breakpoint is located between microsatellites EDB14 and DB16, and the telomeric breakpoint is between markers DB19 and D3S2435. The deletion covers part of BPESC1, MRPS22, COPB2, RBP2, ACTGP1, RBP1, and NMNAT3. BPES14, Microdeletion of ∼200 kb 5′ of FOXL2 in two affected females, I:2 and II:1, of a two-generation family with type II BPES. FISH analysis with probes RP11-548O1, RP11-306L14, RP11-319G6, Pac204O7, and Cos11L10 showed normal hybridization signals on both chromosomes 3, whereas RP11-657M13, Cos12P5, and Pac208L5 were deleted. One normal and one lighter signal were seen with RP11-219D15 and PAC169C10, confirming that the deletion breakpoints are located in these probes. The centromeric breakpoint of the deletion is located between DB16 and D3S3586, and the telomeric breakpoint is between EDB2′ and DB5. BPES15, Extragenic microdeletion found in a five-generation Belgian family with BPES type II, previously linked to 3q23 (Messiaen et al. 1996). This 126-kb microdeletion is the smallest one upstream of FOXL2 and delineates the SRO of the different microdeletions located at this side of FOXL2 (detailed view in fig. 1A). FISH probes RP11-657M13 and PAC208L5 showed one lighter and one normal hybridization signal, thus indicating the borders of the deletion. Analysis with adjacent probes showed two normal signals. The hemizygous microsatellites indicate that the centromeric breakpoint is located between rs10935309 and rs955084 and the telomeric breakpoint is between rs6802174 and rs4894405 (fig. 1A).

Figure 3

A, Schematic diagram of FOXL2 gene and protein. The location of the three MLPA probes is indicated with respect to the nucleotide (top) and amino acid (bottom) numbering. B, Electropherogram of MLPA products. The amplification products of the three FOXL2 probes are highlighted by red stars. In the top panel, the first two peak heights are half of those of the control sample (bottom panel), revealing a partial FOXL2 deletion in patient BPES10. In the middle panel, the peak height of the three probes is ∼50% of that of the control sample (bottom panel), revealing a total gene deletion in family BPES9. C, Sequence electropherogram of the deletion junction fragment in family BPES9, defining the location of the deletion breakpoints. Long-range PCR revealed the junction fragment of an 8-kb deletion in this family. The junction is indicated by a bold rectangle. D, Nucleotide sequence of the breakpoint junctions (uppercase letters). The deleted DNA sequence is shown in lowercase letters.

Figure 4

Haplotypes of families BPES5 and BPES7, in which affected members carry a deletion encompassing FOXL2, which illustrates the stability of deletions in the different generations. In family BPES7, the deletion encompasses interval DB36–DB6 (412 kb) in II:1 and III:1, whereas, in family BPES5, the deletion involves the interval between RP11-80P20 and D3S3711 (3.2 Mb) in I:1 and II:1.