Abstract
Non-syndromic cleft lip and palate (NS CLP) is a complex birth defect resulting from multiple genetic and environmental factors. We have previously reported the sequencing of the coding region of genes in the fibroblast growth factor (FGF) signaling pathway, in which missense and non-sense mutations contribute to approximately 5%–6% NS CLP cases. In this article we report the sequencing of conserved non-coding elements (CNEs) in and around 11 of the FGF and FGFR genes, which identified 55 novel variants. Seven of variants are highly conserved among ≥8 species and 31 variants alter transcription factor binding sites, 8 of which are important for craniofacial development. Additionally, 15 NS CLP patients had a combination of coding mutations and CNE variants, suggesting that an accumulation of variants in the FGF signaling pathway may contribute to clefting.
Keywords: cleft lip and palate, fibroblast growth factor, conserved non-coding elements, sequencing
INTRODUCTION
Comparing the human genome with distantly related species is a productive method to identify evolutionarily conserved non-coding elements (CNEs) [Venkatesh et al., 2006]. CNEs contain regulatory regions important for transcription, translation, recombination, replication and repair; however, a large portion of these CNEs contain sequence whose function is not understood [Emison et al., 2005; Ponting and Lunter, 2006]. Although conservation over evolutionary distance does suggest functionality, not all functional sequence will be conserved [Ponting and Lunter, 2006]. It has been suggested that 3–15 mammalian genomes are sufficient to detect transcription factor binding sites, although comparisons among vertebrates such as chicken or zebrafish are regularly utilized [Eddy, 2005; Emison et al., 2005]. The identification of functional elements through CNEs does have obstacles; nevertheless, cross-species comparison is the best approach available.
Studies using comparative genomics have shown that 1.5% of the human genome codes for exons whereas 3% is devoted to CNEs, suggesting CNEs are highly important locations to explore for mutations [Emison et al., 2005]. Interestingly, approximately 20–30% of patients with rare Mendelian diseases have no identifiable mutations in the coding region of the gene associated with the disease, suggesting non-coding regions may harbor the etiologic mutation in a significant fraction of cases [Emison et al., 2005]. Using systematic comparisons of orthologous sequences, a common non-coding variant was identified in the receptor tyrosine kinase, RET, gene in families with Hirschsprung disease (HSCR), a multifactorial, non-Mendelian disorder [Emison et al., 2005]. HSCR is one of the first complex diseases to be genetically dissected; the complex, multigenic nature of HSCR makes it comparable to non-syndromic cleft lip and palate (NS CLP) and suggests we should utilize similar approaches to identify genes and mutations in NS CLP patients.
NS CLP is a common, complex disease affecting ~1/700 births worldwide, with substantial variation based on geographic origin and socioeconomic status [Jugessur and Murray, 2005; Lidral and Moreno, 2005]. We previously reported on the sequencing of the coding regions of the fibroblast growth factors (FGFs) and receptor (FGFR) genes in NS CLP patients [Riley et al., 2007]. Thirty-seven point mutations were identified in the exons or at the intron–exon junctions of the FGF and FGFR genes, nine of the mutations were either missense or nonsense, accounting for about 5–6% of the cases examined. Comprehensive examination of the functional elements within each FGF or FGFR locus affords an opportunity to identify additional variation and/or genes playing a critical role in CLP and could have substantial clinical implications. This study explores the CNEs in FGF and FGFR genes for contributions to NS CLP.
METHODS
Samples
Ninety-one DNA samples from cases in the Philippines and another 93 from cases born in Iowa were utilized for the sequencing studies. The 184 cases are isolated, non-syndromic unilateral cleft lip (n = 1), bilateral cleft lip (n = 2), unilateral CLP (n = 60), bilateral CLP (n=102), and CLP with unknown laterality (n= 19). Whole blood samples were collected by venipuncture. Subjects were reviewed by JCM to exclude any with syndromic features and have been described in more detail [Vieira et al., 2005]. Clinical aspects of sample collection have been previously described [Murray et al., 1997; Schultz et al., 2004]. The University of Iowa IRB gave approval for sample collection (approval numbers 9701068, 199804081, and 200003065) in conjunction with local approval in the Philippines.
Conserved Non-Coding Elements Selection
CNEs (2–10) were selected for the following genes: FGF2, FGF3, FGF4, FGF7, FGF8, FGF9, FGF10, FGF18, FGFR1, FGFR2, FGFR3 (see the online Supplementary Table I at http://www.interscience.wiley.com/jpages/1552-4825/suppmat/index.html.). Regions in the introns of the gene and up to 140 kb outside the gene were chosen using VISTA genome browser and the UCSC genome browser (see the online Supplementary Figs. 1–11 at http://www.interscience.wiley.com/jpages/1552-4825/suppmat/index.html for Vista plots). Regions were first selected based on greater than 50% conservation between human and frog. Next, regions were selected that had greater than 80% conservation between human and mouse and were longer than 100 bp in length. Finally, 3′- and 5′-UTRs were selected (FGFR2 and FGFR3) that were not fully sequenced in the original coding region sequencing project. A total of 52 regions were sequenced.
Sequencing
Cycle sequencing was performed in a 10 µl reaction using 0.25 µl of ABI Big Dye Terminator sequencing reagent (version 1.1), 0.5 µl of 5 µM sequencing primer, 0.5 µl DMSO, 1 µl of 5× buffer, and 6.75 µl of ddH2O. Primers were designed from public sequence and are available on the Murray lab web site at http://genetics.uiowa.edu. Following a denaturation step at 96° for 30 sec, reactions were cycle sequenced at 96°C for 10 sec, 55°C for 5 sec, and 60°C for 4 min for 40 cycles. Cleanup with magnetic beads by standard protocols and injected on an Applied Biosystems 3730 capillary sequencer.
Sequence Analysis
Chromatograms were transferred to a Unix workstation (Sun Microsystems, Inc., Mountain View, CA), base called with PHRED (version 0.961028), assembled with PHRAP (version 0.960731), scanned by POLYPHRED (version 0.970312) and the results viewed with the CONSED program (version 4.0) [Nickerson et al., 1997].
RESULTS
Fifty-two CNEs were sequenced encompassing approximately 30,000 nucleotides of sequence in 184 NS CLP patients. Fifty-five novel variants that have not been previously reported in dbSNP were identified (listed in the online Supplementary Table II at http://www.interscience.wiley.com/jpages/1552-4825/suppmat/index.html) as well as 33 previously reported SNPs. Seven of the novel variants (found in five different CNEs) are conserved across eight species or more and are highlighted in bold in Table I. The five regions containing the seven highly conserved variants were chosen for additional sequencing in 184 controls from the Philippines (90) and a European ancestral population (94). Four of the seven variants were found in only one case and no controls, and the other three variants were found in multiple cases and controls.
TABLE I.
Novel Variants Conserved Across Six Species or More
| Gene | CNE | Variant | Population | Case frequency | Control frequency | UCSC genome browser positiona | Base pair conservation |
|---|---|---|---|---|---|---|---|
| FGF2 | 2 | G > C | Iowa | 6/167 | Chr 4:123,972,190 | M, R, Ra, D, A, E | |
| 3 | G > A | Iowa | 7/171 | 13/176 | Chr 4:123,981,273 | M, R, Ra, D, A, E | |
| *3 | G > T | Iowa | 1/171 | 0/176 | Chr 4:123,981,030 | M, R, Ra, D, A, E, O, Xt, T | |
| 4 | G > A | Iowa | 1/177 | Chr 4:123,995,377 | M, R, D, A, E, O | ||
| 5 | C > T | Iowa | 8/158 | 4/159 | Chr 4:124,025,315 | M, R, Ra, D, E, A | |
| FGF7 | 2 | G > T | Phil | 1/140 | Chr 15:47,472,855 | M, R, Ra, D, A, E, O | |
| 2 | A > T | Phil | 2/140 | Chr 15:47,473,045 | M, R, Ra, D, A, E, O | ||
| 3 | T > C | Phil | 19/142 | Chr 15:47,490,373 | M, R, D, A, O, Xt | ||
| 4 | G > T | Phil | 2/149 | Chr 15:47,502,707 | Ra, D, A, E, O C, Xt | ||
| FGF8 | 2 | A > G | Phil | 1/164 | Chr 10:103,523,734 | M, R, Ra, D, O, C, Xt | |
| 2 | G > A | Iowa | 1/164 | Chr 10:103,523,419 | M, R, Ra, O, C, Xt | ||
| FGF9 | 3 | T > C | Phil | 1/175 | Chr 13:21,174,985 | M, Ra, D, A, E, O, C | |
| FGF10 | *1 | G > A | Phil | 3/173 | 6/174 | Chr 5:44,427,863 | M, R, Ra, D, E, O, C, Xt |
| *1 | A > G | Iowa | 1/173 | 0/174 | Chr 5:44,427,841 | M, R, Ra, D, E, O, C, Xt | |
| *1 | C > T | Iowa | 4/173 | 3/174 | Chr 5:44,427,983 | M, R, D, E, O, C, Xt, T | |
| *3 | G > C | Iowa | 1/181 | 4/29 | Chr 5:44,340,627 | M, R, Ra, D, A, E, O, C, Xt | |
| FGFR2 | 1 | T > C | Iowa | 5/102 | Chr 10:123.458,864 | M, R, Ra, D, A, O | |
| 1 | G > A | Phil | 8/102 | Chr 10:123,458,555 | M, R, A, E, O, C | ||
| *5 | C > A | Iowa | 1/145 | 0/154 | Chr 10:123,345,664 | M, R, Ra, D, A, E, O, C | |
| *8 | G > A | Phil | 1/152 | 0/155 | Chr 10:123,274,030 | M, R, Ra, D, A, E, O, C | |
| 10 | A > G | Iowa | 2/151 | Chr 10:123,229,037 | M, R, Ra, D, A, E | ||
| FGFR3 | 2 | C > T | Iowa | 1/129 | Chr 4:1,772,043 | M, R, D, A, E, C |
Those variants highlighted in bold and indicated with an asterisk are conserved across eight species or more.
M, mouse; R, rat; Ra, rabbit; D, dog; A, armadillo; E, elephant; O, opossum; C, chicken; Xt, X. tropicalis; T, tetradon.
UCSC genome browser position from the human March 2006 assembly.
Of the 55 novel variants, only one (FGF2 CNE 5 C > T) falls into a transcription factor binding site (FOXO1A) identified by the UCSC genome browser in the HMR Conserved Transcription Factor Binding Sites track. This track contains the location and score of transcription factor binding sites conserved in the human/mouse/rat alignment. The C > T nucleotide change in the FOXOIA binding site was found in 8 unrelated Iowa NS CLP cases and 4 of 159 controls (all 4 are Caucasian).
Additional analyses using the Genomatix MatInspector program (http://www.genomatix.de) were performed to search for transcription factor binding sites disrupted or created by the novel variants. These analyses predict 14 of the variants create new transcription factor binding sites, 16 of the variants remove transcription factor binding sites, and 3 of the variants change a binding site to a new binding site. Of the predicted transcription factor binding sites affected by the novel variants, eight are implicated in craniofacial development or the FGF signaling pathway: CBFA1/2, ARNT, PAX3, SOX5, STAT, MSX1/2, DLX, and MAZR. These transcription factors are highlighted in bold in Table II.
TABLE II.
Predicted Transcription Factor Binding Sites
| Gene | CNE | Variant | UCSC genome browser positiona | UCSC HMR conserved transcription factor binding sites | Genomatix Matlnspector predictionb created binding sites | Genomatix Matlnspector predictionb removed binding sites |
|---|---|---|---|---|---|---|
| FGF2 | 2 | G > C | Chr 4:123,971,990 | Changes CBFA1 (runt-related trx factor) to a CBFA2 | Removes INSM1 (zinc finger protein insulinoma-associated trx repressor) | |
| 2 | G > C | Ch r4:123,972,190 | E4F (GLI-Krueppel regulator of adenovirus E4 promoter) | |||
| 3 | G > A | Chr 4:123,981,273 | RREB (Ras responsive element binding protein) | |||
| 4 | G > A | Chr 4:123,995,377 | MEL1 (MDSl/EVIl-like gene) DNA binding domain 2 | |||
| 4 | C > A | Chr 4:123,995,164 | ATBF1 (AT binding trx factor 1) | |||
| 5 | C > T | Chr 4:124,025,315 | FOXO1A | |||
| FGF3 | 1 | A > G | Chr 11:69,346,124 | TCF11/LCRF1/Nrf1 | ||
| 1 | T > C | Chr 11:69,345,933 | IK2 (Ikaros 2) | |||
| FGF7 | 2 | G > T | Chr 15:47,472,855 | CREB (cAMP responsive element site) | ||
| 2 | A > T | Chr 15:47,473,045 | Changes MTBF (muscle specific MT binding site) to Lentivirus LTR TATA box | |||
| FGF7 | 4 | G > T | Chr 15:47,502,707 | Changes CDP (trx repressor) and HNF1 (hepatic nuclear factor 1) to BCL6 (zinc finger trx repressor) | ||
| FGF8 | 2 | A > G | Chr 10:103,523,734 | ARNT (AhR nuclear translocator) | ||
| 2 | T > A | Chr 10:103,523,780 | E2F (involved in cell cycle regulation) | |||
| 2 | G > A | Chr 10:103,523,419 | PAX3 (paired domain protein) | |||
| FGF9 | 3 | T > C | Chr 13:21,174,985 | OCT1, SOX5, and GCMF (glial chorionspecific trx factor) | ||
| FGF10 | 1 | C > T | Chr 5:44,427,983 | STAT (signal transducer and activator of trx) | ||
| FGFR1 | 1 | C > A | Chr 8:38,448,960 | CKROX (collagen krox protein) | ||
| 5 | T > C | Chr 8:38,473,226 | E2F | |||
| 6 | A > G | Chr 8:38,449,599 | OCT1 | |||
| FGFR2 | 1 | G > A | Chr 10:123,458,555 | MSX1/2 binding sites | GTF2I repeat 4 (TFII-IR4) | |
| 3 | G > T | Chr 10:123,365,553 | SMARCA3 (SWI/SNF related) | |||
| 3 | C > T | Chr 10:123,365,264 | NRSE (neural restrictive silencer element) | |||
| 5 | C > A | Chr 10:123,345,664 | GKLF (Gut enriched Krueppel-like factor) and NKX5-1 | PSE/SNAP (proximal sequence element) | ||
| 8 | G > A | Chr 10:123,274,030 | DMTF1 (cyclin D-interacting myblike protein) | |||
| 10 | A > G | Chr 10:123,229,037 | S8 homeodomain proteins (DLX and MSX), NKX2.5 and BARX2 | |||
| FGFR3 | 3 | C > T | Chr 4:1,766,606 | NMP4 (nuclear matrix protein 4) | ||
| 3 | C > T | Chr 4:1,769,420 | HIC1 (hypermethylated in cancer) | |||
| 3 | C > G | Chr 4:1,769,576 | WHN (winged helix protein) | |||
| 6 | G > A | Chr 4:1,759,904 | DMTF1 and ZAS domain trx factor (NFKB/HIVEP1) | |||
| 10 | A > G | Chr 4:1,732,481 | Meisb and Hoxa9 | |||
| 10 | C > T | Chr 4:1,732,679 | WT1 (Wilms tumor suppressor) and MAZR (myc associated zinc finger protein) |
Transcription factor binding sites highlighted in bold are implicated in craniofacial development. Only CNEs listed have identified transcription factor binding sites.
UCSC genome browser position from the human March 2006 assembly.
To further characterize possible functions of the novel variants, each variant was examined for presence in a human miRNA target site using the Memorial Sloan Kettering Human miRNA Targets—Search and View (http://cbio.mskcc.org/cgi-bin/mirnaviewer/mirnaviewer.pl). This online program creates a track on the UCSC genome browser. None of the identified variants were located in predicted miRNA target sites.
DISCUSSION
Mutation searches in human disease should include both coding regions of genes and neighboring non-coding elements [Emison et al., 2005]. We have thoroughly searched both the exons and non-coding conserved elements of 12 FGF and FGFR genes for mutations in NS CLP patients. Although many novel mutations in the exons have been identified in patients with NS CLP, these are likely rare mutations that explain, or partially explain, the clefting phenotype in those individuals. The results from CNE sequencing also identified many novel mutations, 31 of which are located in transcription factor binding sites (Table II). The remaining 25 novel variants should not be excluded from playing a role in etiology as these variants may be located in elements whose functional consequences that have not yet been characterized.
Of the 31 variants that alter transcription factor binding sites, 8 are located in binding sites important for craniofacial development. The change of CBFA1 to a CBFA2 site in the FGF2 CNE2 may be important as mutations in CBFA1 (OMIM 600211) cause cleidocranial dysplasia (defects in the development of the skull and collar bone) and can be associated with cleft palate. A change such as this may affect the regulation of the FGF2 gene in the craniofacial region. ARNT (AhR nuclear translocator) has been significantly associated with non-syndromic clefting [Kayano et al., 2004]. Mutations in PAX3 cause craniofacial-deafness-hand syndrome and it has been demonstrated that Pax3 is a downstream target of Fgf signaling, suggesting a potential feedback loop [Asher et al., 1996; Firnberg and Neubuser, 2002; Monsoro-Burq et al., 2005]. Knockout mice for Sox5 have cleft palate and it has been shown that SOX proteins regulate the expression of Fgf genes [Smits et al., 2001; Luster and Rizzino, 2003; Murakami et al., 2004]. The signal transducer and activator of transcription (STAT) proteins are downstream regulators of the FGF signaling pathway. Mutations in both MSX1 and MSX2 have been identified in NS CLP patients and are critical regulators of craniofacial development [van den Boogaard et al., 2000; Jezewski et al., 2003; Vieira et al., 2005]. The DLX genes are involved in craniofacial development and knockout mice for Dlxl, Dlx2, Dlxl/Dlx2, Dlx5, and Dlx5/Dlx6 all have cleft palate in a proportion of the embryos [Qiu et al., 1997; Depew et al., 1999; Robledo et al., 2002]. The MAZR protein pairs with BACH2 to bind DNA and activate transcription; this pair is known to bind upstream of the FGF4 gene and may regulate other FGF and FGFR genes [Kobayashi etal.,2000].
The seven variants that were highly conserved between eight species or more were also sequenced in control individuals and four of the seven variants which were found in only one case and were not found in the controls sequenced (Table I). The other three variants were found in both cases and controls. It is interesting to note that four of the highly conserved novel variants were identified in the FGF10 gene and association studies for FGF10 had borderline significant results with a P-value of 0.02 [Riley et al., 2007].
Kryukov et al. [2005] suggest that most individual variants in conserved non-coding regions are only slightly deleterious and do not have a large effect on fitness. However, they also propose that cumulatively, these CNE variants may have a significant impact on the fitness of the individual [Kryukov et al., 2005]. Of the highly conserved (≥8 species) CNE variants and the CNE variants located in transcription factor binding sites implicated in craniofacial development, we find only two individuals with multiple CNE variants (Iowa-5 and Iowa-10, Table III). The Iowa-10 individual has two CNE variants in transcription factor binding sites (MAZR and FOXOIA) and the Iowa-5 individual has three CNE variants in transcription factor binding sites (PAX3, BARX2, and FOXOIA). Expanding the data to include all CNE variants identifies many individuals with multiple accumulated variants; however, it is difficult to determine if this accumulation has an affect on the fitness of the individual without functional testing. Further studies investigating the accumulation of CNE variants in individuals with NS CLP will help to determine the component of contributions of this class of variant to this common, complex disease.
TABLE III.
Individuals With Multiple FGF/FGFR CNE Variants and Coding Variants
| Individual | Phenotype | Variant | Variant | Variant | Variant |
|---|---|---|---|---|---|
| Phil-1 | BCLP | FGF8 CNE2 A > G (ARNT) | FGF3 G30R | ||
| Phil-2 | BCLP | FGF9 CNE3 T > C (SOX5) | FGF18 P138P | ||
| Phil-3 | BCLP | FGFR2 CNE1 G > A (MSX1/2) | FGF18 P138P | ||
| Phil-4 | UCLP | FGFR2 CNE1 G > A (MSX1/2) | FGFR1 S107L | ||
| Phil-5 | BCLP | FGFR2 CNE8 G > A (DMTF1) | FGFR3 V329I | ||
| Iowa-1 | UCLP | FGF2 CNE2 G > C (CBFA1/2) | FGFR1 D200D | ||
| Iowa-2 | BCLP | FGF2 CNE2 G > C (CBFA1/2) | FGFR1 R609X | FGF4 A98A | |
| Iowa-3 | ?CLP | FGF2 CNE2 G > C (CBFA1/2) | NUDT6 K172N | ||
| Iowa-4 | ?CLP | FGF2 CNE2 G > C (CBFA1/2) | FGF3 A23A | ||
| Iowa-5 | BCLP | FGF8 CNE2 G > A (PAX3) | FGFR2 CNE10 A> G (BARX2) | FGF2 CNE5 C > T (FOXOIA) | FGFR1 L754L |
| Iowa-6 | UCLP | FGF10 CNE1 C > T (STAT) | FGF3 A23A | ||
| Iowa-7 | BCLP | FGF10 CNE1 C > T (STAT) | FGF18 P138P | FGF3 A23A | |
| Iowa-8 | ?CLP | FGF10 CNE1 C > T (STAT) | FGF3 A23A | ||
| Iowa-9 | ?CLP | FGFR2 CNE5 C > A (PSE, GKLF, NKX) | FGF3 A23A | ||
| Iowa-10 | BCLP | FGFR3 CNE10 C > T (MAZR) | FGF2 CNE 5 C > T (FOXOIA) | FGF3 A23A |
Overlapping variants include: highly conserved (bold), located in transcription factor binding sites involved in craniofacial development (gray), coding variants (black).
BCLP, bilateral cleft lip and palate; UCLP, unilateral cleft lip and palate; ? CLP, unknown laterality CLP.
Next, it has been suggested that coding and non-coding mutations work synergistically [Emison et al., 2005]. Table III lists 5 Filipino and 10 Iowa individuals that have both CNE variants and coding variants. While this is consistent with the hypothesis that coding and non-coding variants may work synergistically, functional testing is needed to accurately predict their effects.
Currently, it is not possible to discriminate between those mutations that contribute to disease and those that are normal polymorphisms without testing for function. We can make generalizations based on frequency in case versus control populations, however, this criterion may not be appropriate for a significant fraction of mutations because of penetrance and gene–gene interaction issues that must be accounted for in complex diseases such as CLP. In the future it will be necessary to design an assay to screen these CNE variants for functionality. Most recently, Fisher et al. [2006] developed an efficient method to test CNE function using a transposon-based transgenic assay in which the CNE can be screened for control of GFP expression in vivo in the zebrafish [Fisher et al., 2006]. An extension of this method would be to compare the wild-type CNE expression to the mutant CNE expression to determine if the identified variants alter gene expression.
Supplementary Material
This article contains supplementary material, which may be viewed at the American Journal of Medical Genetics website at http://www.interscience.wiley.com/jpages/1552-4825/suppmat/index.html.
ACKNOWLEDGMENTS
We would like to thank all of the families who participated and the many Operation Smile and HOPE volunteers who make this work possible. Thanks also to Brenna Trump and Lisa Raffensperger who helped perform some of the sequencing. And a special thanks to our lab administrators Susie McConnell, Melanie DeVore, and Dan Benton. This research was supported by NIH grants ES1087, R37-DE08559, P50-DE016215, and 5T32GM008629; and by support from the Center for Inherited Disease Research CIDR NIH contract N01-HG-65403.
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