The Cleft Palate–Craniofacial Journal 52(1) pp. 115–120 January 2015 Ó Copyright 2015 American Cleft Palate–Craniofacial Association
BRIEF COMMUNICATION Do Orofacial Clefts Represent Different Genetic Entities? Rudolf Reiter, M.D., Sibylle Brosch, M.D., Manuel Lu¨deke, Ph.D., Elena Fischbein, B.S., Antje Rinckleb, Stephan Haase, M.D., Anke Schwandt, Anja Pickhard, M.D., Christiane Maier, M.D., Josef Ho¨gel, Ph.D., Walther Vogel, M.D. Objective: To contribute to the understanding of potential genetic differences between different cleft types. Method: Analysis of family history concerning cleft type and search for cleft-type–specific associations in candidate genes performed in 98 individuals from 98 families. Results: In a given family, the cleft type of a second case was more often identical to the index case than expected by chance. Each type of cleft (cleft lip [CL], cleft lip and palate [CLP], cleft palate only [CP], and submucous cleft palate only [SMCP]) was associated with different genes. Conclusion: Family history indicates some specificity of cleft types. The observed phenotype-genotype associations were compatible with this interpretation in that significant associations occurred with disjoint sets of genes in each cleft type. These observations indicate that CL, CLP, CP, and SMCP might represent genetically different entities. KEY WORDS:
family history, genes, orofacial clefts, recurrence
Orofacial clefts (OFCs) can be classified anatomically as cleft lip (CL) of the primary palate; cleft lip and palate (CLP), often combined as CL6P (cleft lip with or without cleft palate), which belongs to the primary and secondary palate; and cleft palate (CP), including submucous cleft palate (SMCP), which belongs to the secondary palate. SMCP is anatomically a subgroup of CP with insufficient median fusion of the muscles of the soft palate hidden under the mucosa (Gosain et al., 1996; Bernheim et al., 2006). A tendency to repeat the same cleft type within a family has been observed in first-degree relatives with continuous clefts (Bernheim et al. 2006; Grosen et al., 2012) and was noticed recently in first- and second-degree relatives with SMCPs, too (Reiter et al., 2012). Whether CL6P and CP share the same genetic susceptibility is
controversial. Some authors assume that CL6P and CP have a different genetic etiology (Bernheim et al. 2006; Carinci et al., 2007), but there is also evidence that CL6P and CP have an etiologic overlap (Sivertson et al., 2008; Birnbaum et al., 2009). CP and SMCP are however mostly considered to have a common etiology and SMCP is assumed to represent a mild manifestation of CP (Friedman et al., 2011). A number of genes which are associated with OFCs appear to confer a potential susceptibility to specific types of clefts. Associations with different genes have been reported for non-syndromic CL6P or CP (Carinci et al., 2007; Jugessur et al., 2009; Dixon et al., 2011; Ludwig et al., 2012; Rattanasopha et al., 2012). Recently, two SMCP susceptibility genes (TGFB3, transforming growth factor b3 and MN1, Meningeoma 1 gene) have been reported (Reiter et al., 2012). One of these (TGFB3) is also associated with CL6P (Dixon et al., 2011) while the other (MN1) seems unique to SMCP and CP (Davidson et al., 2012; Reiter et al., 2012). The aim of the present study was to look for a relationship between different cleft types by considering family history and comparing the genetic associations in a small sample. The particular question was (1) if the cleft types indicate a distinct susceptibility for the same type of cleft to occur in a family and (2) if we can detect different associations for the different cleft types.
Dr. Reiter is Professor and Senior Physician and Dr. Brosch is Professor and Head, Section of Phoniatrics and Pedaudiology, University of Ulm, Ulm, Germany. Dr. Maier is Professor and Scientist (Group Leader), Dr. Ho¨gel is Professor and Scientist (Group Leader), and Dr. Vogel is Professor Emeritus, Institute of Human Genetics, University of Ulm, Ulm, Germany. Dr. Lu¨deke is Scientist and Mrs. Rinckleb, Mrs. Fischbein, and Mrs. Schwandt are PhD students, Institute of Human Genetics, University of Ulm, Ulm, Germany. Dr. Haase is Professor and Head, Department of Cranio-Maxillo-Facial Surgery, University of Ulm, Ulm, Germany. Dr. Pickhard is Senior Physician, Department of Otolaryngology Head and Neck Surgery, Technical University Munich, Munich, Germany. Submitted February 2013; Revised August 2013; Accepted October 2013. Address correspondence to: Dr. Rudolf Reiter, Section of Phoniatrics and Pedaudiology, Department of Otolaryngology, Head and Neck Surgery, University of Ulm, Frauensteige 12, 89070 Ulm, Germany. E-mail [email protected]. DOI: 10.1597/13-016
MATERIAL
AND
METHODS
We performed an association study with 23 single nucleotide polymorphisms (SNPs) in 12 candidate genes (Table 1) in a sample of 98 index patients from 98 families 115
rs7606793 rs7561997 rs6546610 rs3917192 rs12532 rs861019 rs6685182 rs2304000 rs6987534 rs1801132 rs1614972 rs3133158 rs1429591 rs195294 rs5752638 rs2189132 rs2360635 rs3754443 rs6819797 rs342311 rs6822796 rs1356292 rs7433760
TGFA
0.4 0.55 0.45 0.1 0.3 0.45 0.1 0.2 0.45 0.65 0.278 0.3 0.071 0.214 0.35 0.3 0.25 0.55 0.35 0.1 0.2 0.05 0.2
MAF Cases .22 .39 .96 .42 .94 .92 .02 (0.21, 0.05–0.89) .93 .91 .0028 (3.8, 1.49–9.7) .76 .89 .55 .28 .26 .79 .36 .16 .87 .35 .21 .11 .42
P Value (OR, 95% CI) 0.258 0.427 0.452 0.177 0.315 0.468 0.315 0.226 0.452 0.328 0.363 0.242 0.1 0.268 0.234 0.355 0.234 0.468 0.290 0.0726 0.323 0.218 0.371
MAF Cases .71 .60 .88 .81 .62 .56 .44 .66 .77 .99 .27 .32 .52 .14 .88 .57 .013 (0.57, 0.36–0.89) .13 .10 .003 (0.35, 0.17–0.72) .79 .57 .053 (1.5, 0.99–2.25)
P Value (OR, 95% CI)
CLP (n ¼ 60)
0.327 0.481 0.481 0.096 0.423 0.481 0.404 0.231 0.404 0.404 0.346 0.289 0.150 0.375 0.250 0.231 0.308 0.462 0.327 0.135 0.404 0.212 0.308
MAF Cases .42 .70 .61 .18 .049 (1.78, 1.0–3.18) .56 .45 .70 .64 0.27 .61 .97 .66 .78 .87 .15 .54 .34 .56 .40 .32 .78 .71
P Value (OR, 95% CI)
CP (n ¼ 26)
0.262 0.437 0.452 0.112 0.248 0.418 0.387 0.223 0.442 0.364 0.345 0.277 0.118 0.313 0.180 0.309 0.325 0.427 0.417 0.209 0.369 0.194 0.325
MAF Cases
.74 .68 .86 .053 (1.61, 0.99–2.63) .23 .59 .36 .56 .64 0.91 .35 .39 .80 .38 .075 (1.44, 0.96–2.17) .62 .53 .41 .22 .38 .38 .97 .26
P Value (OR, 95% CI)
SMCP (n ¼ 103)
Literature
þ
þ
þ þ þ
þ
Known Loci
* Reiter et al., 2012. † Dixon et al., 2011. ‡ CL ¼ cleft lip; CLP ¼ cleft lip and palate; CP ¼ cleft palate; SMCP ¼ submucous cleft palate; SNP ¼ single nucleotide polymorphism; MAF ¼ minor allele frequency; OR ¼ odds ratio; CI ¼ confidence interval (CI). TGFA ¼ transforming growth factor alpha; TGFB3 ¼ transforming growth factor beta 3; MSX1 ¼ MSH homeobox 1;IRF6 ¼ interferon regulatory factor 6; FGFR1 ¼ fibroblast growth factor receptor; MTHFR ¼ methylenetetrahydrofolate reductase; ADH1C ¼ alcohol dehydrogenase 1C; TBX22 ¼ T-box transcription factor gene 22; MN1 ¼ meningeoma 1 gene; ALX3 ¼ aristaless-like homeobox 3; PDGFC ¼ platelet-derived growth factor C; ETV5 ¼ ETS variant gene 5. § The observed associations appear in only one of the different cleft types. The ORs and 95% CI are presented for significant results (indicated in bold). P values were determined by 1 df v2 test.
ETV5
PDGFC
ALX3
MN1
TBX22
MTHFR ADH1C
FGFR1
TGFB3 MSX1 IRF6
0.274 0.453 0.444 0.169 0.292 0.439 0.351 0.208 0.437 0.328 0.312 0.286 0.126 0.353 0.240 0.328 0.350 0.394 0.367 0.181 0.335 0.195 0.283
SNP
Gene
CL (n ¼ 10)
Associations of CL, CLP, and CP With SNPs in Candidate Genes Compared With SMCP* and to Known Loci† From the Literature‡§
Controls (n ¼ 279) MAF Controls
TABLE 1
116 Cleft Palate–Craniofacial Journal, January 2015, Vol. 52 No. 1
Reiter et al., DIFFERENT GENETIC ENTITIES OF OFCS
TABLE 2 Type† CL CLP CP SMCP‡
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Characteristics of Patients Included in the Study* Total Number (Male/Female) 10 62 26 103
(5/5) (43 /19) (12/14) (56/45)
Mean Age at Examination 21.0 28.6 14.0 15.0
Age ,18 y
Age 18 y
Bilateral (Male/Female)
Unilateral (Male/Female)
5 22 12 75
5 40 14 28
2 (1/1) 12 (3/7) -
8 (4/4) 50 (40/12) -
(1.0–38.0) (1.0–91.0) (1.2–34.0) (3.2–68.0)
* Number of patients with different types of clefts and gender distribution for the entire sample. In the association study the age subgroups and bilateral versus unilateral were not analyzed separately to avoid numbers that were too small. † CL ¼ cleft lip; CLP ¼ cleft lip and palate; CP ¼ cleft palate only; SMCP ¼ submucous cleft palate only. ‡ From Reiter et al., 2012.
gender information. The patients with clefts consisted of 62 patients with CLP, 26 patients with CP, and 10 patients with CL. (For details see Table 2). Clinical data for orofacial clefts were collected as described before (Reiter et al., 2012). Family history (firstdegree and second-degree relatives; Table 3) was obtained
with nonsyndromic continuous OFCs of German origin. The family history for OFCs was recorded up to seconddegree relatives. Allele frequencies were compared with those of 279 healthy population control subjects from the same location as the case subjects. The controls were healthy young adults made completely anonymous for TABLE 3
Familial Cases and Affected Relative*† Second Case in Family: First (Second) Degree Relative
Index Case CL
CLP
CP
SMCP*
Total
Total Number
Sex
Age at Investigation (y)
Unilateral /Bilateral
CL
4
F F F F
42 65 45 67
U U U -
Mother (Grandfather)
15
10
9
38
F F F F M M F M M M F M M M M
71 34 32 37 45 26 27 29 31 12 16 8 9 70 5
B U B U U U U B B U U U U U -
M F F F M M M M M M
31 43 10 12 8 17 29 38 42 41
U U -
M F F M M M M M F
32 43 29 45 50 32 15 43 78
-
CLP
CP
SMCP
(Uncle) 1 (1) (Grandfather)
0 (1)
0
(Grandfather) 0 (1)
Brother 1
0 (0)
Father Father Father Father Father Mother Mother Mother Sister Sister Brother Brother (Grandfather) 0 (1) (Aunt) (Aunt)
12 (1)
Brother Brother Sister Sister
0 (2)
0
4
0
0 19
0 5
* Distribution of different cleft types occurring in a family (first- and second-degree relative) with sex and age at investigation. † CL ¼ cleft lip; CLP ¼ cleft lip and palate; CP ¼ cleft palate only; SMCP ¼ submucous cleft palate only; F ¼ female; M ¼ male; U ¼ unilateral; B ¼ bilateral. ‡ From Reiter et al., 2012.
Father Brother (Aunt) (Aunt) 2 (2) Mother Mother Father Father Father Sister Brother (Aunt) (Grandfather) 7 (2) 14
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TABLE 4
Concordance of Cleft Types Within Families*†f Second Case in the Family: n ¼ First-Degree Relative, (n) ¼ Second-Degree Relative
Index Case CL CLP CP SMCP‡ Total
Total Number
CL
CLP
CP
SMCP
4 15 10 9 38
1 (1) (1) 0 (2) 0 5
(1) 12 (1) 0 (0) 0 14
0 1 4 0 5
(1) 0 2 (2) 7 (2) 14
* Families with two affected members split by the different cleft types. Numbers represent first-degree relatives; second-degree relatives indicated in brackets. Note the preponderance of identical cleft types (bold), especially in CLP and SMCP and the comparably unspecific distribution of the few second-degree cases. Testing for identity of cleft types using Cohen’s kappa revealed the following in first-degree relatives: kappa ¼ 0.83 (95% confidence interval ¼ 0.68–1.0); when second-degree cases are included: kappa ¼ 0.59 (95% CI ¼ 0.40-0.79). Kappa runs from 0 (chance distribution of second cases among cleft types) to 1.0 (complete identity of second case with index case). † CL ¼ cleft lip; CLP ¼ cleft lip and palate; CP ¼ cleft palate only; SMCP ¼ submucous cleft palate only; CI ¼ confidence interval. † From Reiter et al., 2012.
by systematically interviewing the patients (or their relatives) after investigation, and about half of the affected relatives were also found to confirm the reported diagnosis. Any somatic abnormality, including lip pits or lower-lip whorls (Neiswanger et al., 2009), were recorded. Any suspicion of additional abnormalities prompted a genetic consultation by an experienced syndromologist and was an exclusion criterion. The genes for the association study were mostly known to be associated with OFCs (Dixon et al., 2011) (see Table 1). These genes do not completely coincide with the results from genome-wide association studies (Ludwig et al., 2012); therefore, we selected three additional genes: (1) TBX22, which is expressed in the growing palate during embryogenesis (Kim et al., 2009), and (2) MN1 (Meester-Smoor et al., 2005) and FGFR1 (Fujiwara et al., 2008), which cause OFCs when knocked out in mice. The 12 candidate genes and the genotyped SNPs are listed in Table 1. Genotyping was performed after DNA extraction from blood samples using predesigned allelic discrimination probes on a TaqMan 7900HT instrument (Applied Biosystems, Darmstadt, Germany) as described before (Reiter et al., 2012). All markers were checked for deviations from HardyWeinberg equilibrium. Allele frequencies were inferentially compared between cases and controls using the 1 df v2 test of association and genotype frequencies by means of the Cochran-Armitage test for trend. Allelic odds ratios are given. Each type of cleft (CL, CP, and CLP) was investigated separately. The occurrence of two cases with the same cleft type within a family (Table 4) was assessed by Cohen’s j, a statistical measure of agreement of paired events (Cohen, 1960). All analyses are considered to be explorative without adjustments for multiple testing. For simplicity, all tests with P values .05 were considered significant. Association testing was carried out using PLINK (version 1.07, http://pngu.mgh.harvard.edu/ purcell/plink) (Purcell et al., 2007). Written informed consent was obtained from all patients or their parents as approved by the institutional review board of the University of Ulm.
RESULTS Occurrence of Cleft Types in a Family Of the 98 patients, 38 had a first-degree (n ¼ 27) or second-degree (n ¼ 11) degree relative with a cleft (Table 3). Especially in first-degree relatives, both relatives were affected by the same cleft type much more frequently than expected by chance: Cohen’s j ¼ 0.83 (95% confidence interval [CI] ¼ 0.68–1.0) for first-degree relatives and Cohen’s j ¼ 0.59 (95% CI ¼ 0.40–0.79) for all cases (Table 4). Association of CL, CP, and CLP With the Different SNPs The results for the associations with the different SNPs are presented in Table 1, together with the previous results for SMCP (Reiter et al., 2012). Six of the 12 investigated genes revealed a significant (or close to significant) association: three in CLP, two in CL, and one in CP. In CL, MTHFR (P ¼ .002) and in CLP, PDGFC (P ¼ .003) were associated with P values coming close to the Bonferroni corrected significance threshold (0.05/23 ¼ 0.00217). In patients with isolated CP, the association with MSX1 (P ¼ .049) was significant. Although the observed associations each occurred only in one type of cleft and were not present in the SMCP data, there may be some overlap, as indicated by some minor allele frequencies in nonsignificant cleft types with smaller sample size. The clearest example is rs3917192 in TGFB3 and CP, for which the minor allele frequency (0.096) was almost identical to that observed in SMCP (0.112), but it is possible that the number of CP cases was too small to reach significance. DISCUSSION The family history of the patients with OFCs is very suggestive for at least some specificity of genetic factors involved. In the present study the same cleft type occurred
Reiter et al., DIFFERENT GENETIC ENTITIES OF OFCS
much more frequently in a given family than expected by chance. In a previous study on SMCP, a positive family history was observed in 13.6% of subjects, and most of the relatives also had SMCP (Reiter et al., 2012). As expected in a disease with complex inheritance, the occurrence of the same type of cleft was much more specific in first-degree relatives than in the entire sample. A tendency to identical cleft types was reported before for continuous clefts (Grosen et al., 2010). In consequence, one might expect that there are different genetic factors specific for CL, CLP, CP, or SMCP in addition to a set of common genes involved in the development of all four types of clefts. This assumption is further supported by the observation that CL6P can be subdivided into cleft lip (CL) and cleft lip and palate (CLP) that are associated with different genetic (etiologic) factors (Harville et al., 2005; Grosen et al., 2010; Brito et al., 2012), an observation that was confirmed by our study. Several genes have consistently been found to be associated with nonsyndromal OFCs (either with CP or CL6P) (Carnici et al., 2007; Jugessur et al., 2009; Dixon et al., 2011). In our small sample, in each type of cleft we observed associations with different genes. However, some genes have been found associated with CP and with CL6P in different studies. An association specific for isolated CP was found for ALX3 and PDGFC (Jugessur et al., 2009), whereas these genes were associated to CLP in our study. Recently, a specific role of PDGFRA (Platelet-Derived– Growth Factor Receptor A) was also found for CP (Rattanasopha et al., 2012), and in contrast, a SNP in the regulatory region of PDGFC was associated with CLP (Choi et al., 2009). In patients with isolated CP we observed an association with MSX1 (p¼0.049) which had been observed before (Alapat et al., 2003). However, associations of MSX1 with CL6P were also reported (Vieira et al., 2003; Ingersoll et al., 2010). Variants in the TGFB3 gene have been repeatedly associated with complete OFC (CL6P) (Ichekwawa et al., 2006), but other studies did not confirm these results (Salishourfar et al., 2011). In our study, TGFB3 was not associated to any of the complete OFCs but had been found associated in patients with a SMCP (Reiter et al., 2012). The other gene associated with SMCP (MN1, Reiter et al., 2012), did not show an association with any of the complete clefts in the present study. It was recently found deleted in a case with a Robin sequence and CP (Davidson et al., 2012). Not only the co-occurrence of the same cleft type within a family but also the preferential associations of genes with specific cleft types indicate that different cleft types may be of genetically different etiology. Our results indicate cleftspecific associations of IRF6, MTHFR, ALX3, and PDGFC with the primary palate (CL6P) and a potential role of MSX1, TGFB3, and MN1 with the secondary palate (CP and SMCP, respectively) in patients of German origin.
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Of course, these results of our pilot study need to be confirmed in larger samples, which are also needed to resolve the controversial findings reported in the literature. Acknowledgments. We thank all patients and their families for participations in the study. Margot Brugger and Irina Wiest are acknowledged for excellent technical assistance.
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BRIEF COMMUNICATION Do Orofacial Clefts Represent Different Genetic Entities? Rudolf Reiter, M.D., Sibylle Brosch, M.D., Manuel Lu¨deke, Ph.D., Elena Fischbein, B.S., Antje Rinckleb, Stephan Haase, M.D., Anke Schwandt, Anja Pickhard, M.D., Christiane Maier, M.D., Josef Ho¨gel, Ph.D., Walther Vogel, M.D. Objective: To contribute to the understanding of potential genetic differences between different cleft types. Method: Analysis of family history concerning cleft type and search for cleft-type–specific associations in candidate genes performed in 98 individuals from 98 families. Results: In a given family, the cleft type of a second case was more often identical to the index case than expected by chance. Each type of cleft (cleft lip [CL], cleft lip and palate [CLP], cleft palate only [CP], and submucous cleft palate only [SMCP]) was associated with different genes. Conclusion: Family history indicates some specificity of cleft types. The observed phenotype-genotype associations were compatible with this interpretation in that significant associations occurred with disjoint sets of genes in each cleft type. These observations indicate that CL, CLP, CP, and SMCP might represent genetically different entities. KEY WORDS:
family history, genes, orofacial clefts, recurrence
Orofacial clefts (OFCs) can be classified anatomically as cleft lip (CL) of the primary palate; cleft lip and palate (CLP), often combined as CL6P (cleft lip with or without cleft palate), which belongs to the primary and secondary palate; and cleft palate (CP), including submucous cleft palate (SMCP), which belongs to the secondary palate. SMCP is anatomically a subgroup of CP with insufficient median fusion of the muscles of the soft palate hidden under the mucosa (Gosain et al., 1996; Bernheim et al., 2006). A tendency to repeat the same cleft type within a family has been observed in first-degree relatives with continuous clefts (Bernheim et al. 2006; Grosen et al., 2012) and was noticed recently in first- and second-degree relatives with SMCPs, too (Reiter et al., 2012). Whether CL6P and CP share the same genetic susceptibility is
controversial. Some authors assume that CL6P and CP have a different genetic etiology (Bernheim et al. 2006; Carinci et al., 2007), but there is also evidence that CL6P and CP have an etiologic overlap (Sivertson et al., 2008; Birnbaum et al., 2009). CP and SMCP are however mostly considered to have a common etiology and SMCP is assumed to represent a mild manifestation of CP (Friedman et al., 2011). A number of genes which are associated with OFCs appear to confer a potential susceptibility to specific types of clefts. Associations with different genes have been reported for non-syndromic CL6P or CP (Carinci et al., 2007; Jugessur et al., 2009; Dixon et al., 2011; Ludwig et al., 2012; Rattanasopha et al., 2012). Recently, two SMCP susceptibility genes (TGFB3, transforming growth factor b3 and MN1, Meningeoma 1 gene) have been reported (Reiter et al., 2012). One of these (TGFB3) is also associated with CL6P (Dixon et al., 2011) while the other (MN1) seems unique to SMCP and CP (Davidson et al., 2012; Reiter et al., 2012). The aim of the present study was to look for a relationship between different cleft types by considering family history and comparing the genetic associations in a small sample. The particular question was (1) if the cleft types indicate a distinct susceptibility for the same type of cleft to occur in a family and (2) if we can detect different associations for the different cleft types.
Dr. Reiter is Professor and Senior Physician and Dr. Brosch is Professor and Head, Section of Phoniatrics and Pedaudiology, University of Ulm, Ulm, Germany. Dr. Maier is Professor and Scientist (Group Leader), Dr. Ho¨gel is Professor and Scientist (Group Leader), and Dr. Vogel is Professor Emeritus, Institute of Human Genetics, University of Ulm, Ulm, Germany. Dr. Lu¨deke is Scientist and Mrs. Rinckleb, Mrs. Fischbein, and Mrs. Schwandt are PhD students, Institute of Human Genetics, University of Ulm, Ulm, Germany. Dr. Haase is Professor and Head, Department of Cranio-Maxillo-Facial Surgery, University of Ulm, Ulm, Germany. Dr. Pickhard is Senior Physician, Department of Otolaryngology Head and Neck Surgery, Technical University Munich, Munich, Germany. Submitted February 2013; Revised August 2013; Accepted October 2013. Address correspondence to: Dr. Rudolf Reiter, Section of Phoniatrics and Pedaudiology, Department of Otolaryngology, Head and Neck Surgery, University of Ulm, Frauensteige 12, 89070 Ulm, Germany. E-mail [email protected]. DOI: 10.1597/13-016
MATERIAL
AND
METHODS
We performed an association study with 23 single nucleotide polymorphisms (SNPs) in 12 candidate genes (Table 1) in a sample of 98 index patients from 98 families 115
rs7606793 rs7561997 rs6546610 rs3917192 rs12532 rs861019 rs6685182 rs2304000 rs6987534 rs1801132 rs1614972 rs3133158 rs1429591 rs195294 rs5752638 rs2189132 rs2360635 rs3754443 rs6819797 rs342311 rs6822796 rs1356292 rs7433760
TGFA
0.4 0.55 0.45 0.1 0.3 0.45 0.1 0.2 0.45 0.65 0.278 0.3 0.071 0.214 0.35 0.3 0.25 0.55 0.35 0.1 0.2 0.05 0.2
MAF Cases .22 .39 .96 .42 .94 .92 .02 (0.21, 0.05–0.89) .93 .91 .0028 (3.8, 1.49–9.7) .76 .89 .55 .28 .26 .79 .36 .16 .87 .35 .21 .11 .42
P Value (OR, 95% CI) 0.258 0.427 0.452 0.177 0.315 0.468 0.315 0.226 0.452 0.328 0.363 0.242 0.1 0.268 0.234 0.355 0.234 0.468 0.290 0.0726 0.323 0.218 0.371
MAF Cases .71 .60 .88 .81 .62 .56 .44 .66 .77 .99 .27 .32 .52 .14 .88 .57 .013 (0.57, 0.36–0.89) .13 .10 .003 (0.35, 0.17–0.72) .79 .57 .053 (1.5, 0.99–2.25)
P Value (OR, 95% CI)
CLP (n ¼ 60)
0.327 0.481 0.481 0.096 0.423 0.481 0.404 0.231 0.404 0.404 0.346 0.289 0.150 0.375 0.250 0.231 0.308 0.462 0.327 0.135 0.404 0.212 0.308
MAF Cases .42 .70 .61 .18 .049 (1.78, 1.0–3.18) .56 .45 .70 .64 0.27 .61 .97 .66 .78 .87 .15 .54 .34 .56 .40 .32 .78 .71
P Value (OR, 95% CI)
CP (n ¼ 26)
0.262 0.437 0.452 0.112 0.248 0.418 0.387 0.223 0.442 0.364 0.345 0.277 0.118 0.313 0.180 0.309 0.325 0.427 0.417 0.209 0.369 0.194 0.325
MAF Cases
.74 .68 .86 .053 (1.61, 0.99–2.63) .23 .59 .36 .56 .64 0.91 .35 .39 .80 .38 .075 (1.44, 0.96–2.17) .62 .53 .41 .22 .38 .38 .97 .26
P Value (OR, 95% CI)
SMCP (n ¼ 103)
Literature
þ
þ
þ þ þ
þ
Known Loci
* Reiter et al., 2012. † Dixon et al., 2011. ‡ CL ¼ cleft lip; CLP ¼ cleft lip and palate; CP ¼ cleft palate; SMCP ¼ submucous cleft palate; SNP ¼ single nucleotide polymorphism; MAF ¼ minor allele frequency; OR ¼ odds ratio; CI ¼ confidence interval (CI). TGFA ¼ transforming growth factor alpha; TGFB3 ¼ transforming growth factor beta 3; MSX1 ¼ MSH homeobox 1;IRF6 ¼ interferon regulatory factor 6; FGFR1 ¼ fibroblast growth factor receptor; MTHFR ¼ methylenetetrahydrofolate reductase; ADH1C ¼ alcohol dehydrogenase 1C; TBX22 ¼ T-box transcription factor gene 22; MN1 ¼ meningeoma 1 gene; ALX3 ¼ aristaless-like homeobox 3; PDGFC ¼ platelet-derived growth factor C; ETV5 ¼ ETS variant gene 5. § The observed associations appear in only one of the different cleft types. The ORs and 95% CI are presented for significant results (indicated in bold). P values were determined by 1 df v2 test.
ETV5
PDGFC
ALX3
MN1
TBX22
MTHFR ADH1C
FGFR1
TGFB3 MSX1 IRF6
0.274 0.453 0.444 0.169 0.292 0.439 0.351 0.208 0.437 0.328 0.312 0.286 0.126 0.353 0.240 0.328 0.350 0.394 0.367 0.181 0.335 0.195 0.283
SNP
Gene
CL (n ¼ 10)
Associations of CL, CLP, and CP With SNPs in Candidate Genes Compared With SMCP* and to Known Loci† From the Literature‡§
Controls (n ¼ 279) MAF Controls
TABLE 1
116 Cleft Palate–Craniofacial Journal, January 2015, Vol. 52 No. 1
Reiter et al., DIFFERENT GENETIC ENTITIES OF OFCS
TABLE 2 Type† CL CLP CP SMCP‡
117
Characteristics of Patients Included in the Study* Total Number (Male/Female) 10 62 26 103
(5/5) (43 /19) (12/14) (56/45)
Mean Age at Examination 21.0 28.6 14.0 15.0
Age ,18 y
Age 18 y
Bilateral (Male/Female)
Unilateral (Male/Female)
5 22 12 75
5 40 14 28
2 (1/1) 12 (3/7) -
8 (4/4) 50 (40/12) -
(1.0–38.0) (1.0–91.0) (1.2–34.0) (3.2–68.0)
* Number of patients with different types of clefts and gender distribution for the entire sample. In the association study the age subgroups and bilateral versus unilateral were not analyzed separately to avoid numbers that were too small. † CL ¼ cleft lip; CLP ¼ cleft lip and palate; CP ¼ cleft palate only; SMCP ¼ submucous cleft palate only. ‡ From Reiter et al., 2012.
gender information. The patients with clefts consisted of 62 patients with CLP, 26 patients with CP, and 10 patients with CL. (For details see Table 2). Clinical data for orofacial clefts were collected as described before (Reiter et al., 2012). Family history (firstdegree and second-degree relatives; Table 3) was obtained
with nonsyndromic continuous OFCs of German origin. The family history for OFCs was recorded up to seconddegree relatives. Allele frequencies were compared with those of 279 healthy population control subjects from the same location as the case subjects. The controls were healthy young adults made completely anonymous for TABLE 3
Familial Cases and Affected Relative*† Second Case in Family: First (Second) Degree Relative
Index Case CL
CLP
CP
SMCP*
Total
Total Number
Sex
Age at Investigation (y)
Unilateral /Bilateral
CL
4
F F F F
42 65 45 67
U U U -
Mother (Grandfather)
15
10
9
38
F F F F M M F M M M F M M M M
71 34 32 37 45 26 27 29 31 12 16 8 9 70 5
B U B U U U U B B U U U U U -
M F F F M M M M M M
31 43 10 12 8 17 29 38 42 41
U U -
M F F M M M M M F
32 43 29 45 50 32 15 43 78
-
CLP
CP
SMCP
(Uncle) 1 (1) (Grandfather)
0 (1)
0
(Grandfather) 0 (1)
Brother 1
0 (0)
Father Father Father Father Father Mother Mother Mother Sister Sister Brother Brother (Grandfather) 0 (1) (Aunt) (Aunt)
12 (1)
Brother Brother Sister Sister
0 (2)
0
4
0
0 19
0 5
* Distribution of different cleft types occurring in a family (first- and second-degree relative) with sex and age at investigation. † CL ¼ cleft lip; CLP ¼ cleft lip and palate; CP ¼ cleft palate only; SMCP ¼ submucous cleft palate only; F ¼ female; M ¼ male; U ¼ unilateral; B ¼ bilateral. ‡ From Reiter et al., 2012.
Father Brother (Aunt) (Aunt) 2 (2) Mother Mother Father Father Father Sister Brother (Aunt) (Grandfather) 7 (2) 14
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TABLE 4
Concordance of Cleft Types Within Families*†f Second Case in the Family: n ¼ First-Degree Relative, (n) ¼ Second-Degree Relative
Index Case CL CLP CP SMCP‡ Total
Total Number
CL
CLP
CP
SMCP
4 15 10 9 38
1 (1) (1) 0 (2) 0 5
(1) 12 (1) 0 (0) 0 14
0 1 4 0 5
(1) 0 2 (2) 7 (2) 14
* Families with two affected members split by the different cleft types. Numbers represent first-degree relatives; second-degree relatives indicated in brackets. Note the preponderance of identical cleft types (bold), especially in CLP and SMCP and the comparably unspecific distribution of the few second-degree cases. Testing for identity of cleft types using Cohen’s kappa revealed the following in first-degree relatives: kappa ¼ 0.83 (95% confidence interval ¼ 0.68–1.0); when second-degree cases are included: kappa ¼ 0.59 (95% CI ¼ 0.40-0.79). Kappa runs from 0 (chance distribution of second cases among cleft types) to 1.0 (complete identity of second case with index case). † CL ¼ cleft lip; CLP ¼ cleft lip and palate; CP ¼ cleft palate only; SMCP ¼ submucous cleft palate only; CI ¼ confidence interval. † From Reiter et al., 2012.
by systematically interviewing the patients (or their relatives) after investigation, and about half of the affected relatives were also found to confirm the reported diagnosis. Any somatic abnormality, including lip pits or lower-lip whorls (Neiswanger et al., 2009), were recorded. Any suspicion of additional abnormalities prompted a genetic consultation by an experienced syndromologist and was an exclusion criterion. The genes for the association study were mostly known to be associated with OFCs (Dixon et al., 2011) (see Table 1). These genes do not completely coincide with the results from genome-wide association studies (Ludwig et al., 2012); therefore, we selected three additional genes: (1) TBX22, which is expressed in the growing palate during embryogenesis (Kim et al., 2009), and (2) MN1 (Meester-Smoor et al., 2005) and FGFR1 (Fujiwara et al., 2008), which cause OFCs when knocked out in mice. The 12 candidate genes and the genotyped SNPs are listed in Table 1. Genotyping was performed after DNA extraction from blood samples using predesigned allelic discrimination probes on a TaqMan 7900HT instrument (Applied Biosystems, Darmstadt, Germany) as described before (Reiter et al., 2012). All markers were checked for deviations from HardyWeinberg equilibrium. Allele frequencies were inferentially compared between cases and controls using the 1 df v2 test of association and genotype frequencies by means of the Cochran-Armitage test for trend. Allelic odds ratios are given. Each type of cleft (CL, CP, and CLP) was investigated separately. The occurrence of two cases with the same cleft type within a family (Table 4) was assessed by Cohen’s j, a statistical measure of agreement of paired events (Cohen, 1960). All analyses are considered to be explorative without adjustments for multiple testing. For simplicity, all tests with P values .05 were considered significant. Association testing was carried out using PLINK (version 1.07, http://pngu.mgh.harvard.edu/ purcell/plink) (Purcell et al., 2007). Written informed consent was obtained from all patients or their parents as approved by the institutional review board of the University of Ulm.
RESULTS Occurrence of Cleft Types in a Family Of the 98 patients, 38 had a first-degree (n ¼ 27) or second-degree (n ¼ 11) degree relative with a cleft (Table 3). Especially in first-degree relatives, both relatives were affected by the same cleft type much more frequently than expected by chance: Cohen’s j ¼ 0.83 (95% confidence interval [CI] ¼ 0.68–1.0) for first-degree relatives and Cohen’s j ¼ 0.59 (95% CI ¼ 0.40–0.79) for all cases (Table 4). Association of CL, CP, and CLP With the Different SNPs The results for the associations with the different SNPs are presented in Table 1, together with the previous results for SMCP (Reiter et al., 2012). Six of the 12 investigated genes revealed a significant (or close to significant) association: three in CLP, two in CL, and one in CP. In CL, MTHFR (P ¼ .002) and in CLP, PDGFC (P ¼ .003) were associated with P values coming close to the Bonferroni corrected significance threshold (0.05/23 ¼ 0.00217). In patients with isolated CP, the association with MSX1 (P ¼ .049) was significant. Although the observed associations each occurred only in one type of cleft and were not present in the SMCP data, there may be some overlap, as indicated by some minor allele frequencies in nonsignificant cleft types with smaller sample size. The clearest example is rs3917192 in TGFB3 and CP, for which the minor allele frequency (0.096) was almost identical to that observed in SMCP (0.112), but it is possible that the number of CP cases was too small to reach significance. DISCUSSION The family history of the patients with OFCs is very suggestive for at least some specificity of genetic factors involved. In the present study the same cleft type occurred
Reiter et al., DIFFERENT GENETIC ENTITIES OF OFCS
much more frequently in a given family than expected by chance. In a previous study on SMCP, a positive family history was observed in 13.6% of subjects, and most of the relatives also had SMCP (Reiter et al., 2012). As expected in a disease with complex inheritance, the occurrence of the same type of cleft was much more specific in first-degree relatives than in the entire sample. A tendency to identical cleft types was reported before for continuous clefts (Grosen et al., 2010). In consequence, one might expect that there are different genetic factors specific for CL, CLP, CP, or SMCP in addition to a set of common genes involved in the development of all four types of clefts. This assumption is further supported by the observation that CL6P can be subdivided into cleft lip (CL) and cleft lip and palate (CLP) that are associated with different genetic (etiologic) factors (Harville et al., 2005; Grosen et al., 2010; Brito et al., 2012), an observation that was confirmed by our study. Several genes have consistently been found to be associated with nonsyndromal OFCs (either with CP or CL6P) (Carnici et al., 2007; Jugessur et al., 2009; Dixon et al., 2011). In our small sample, in each type of cleft we observed associations with different genes. However, some genes have been found associated with CP and with CL6P in different studies. An association specific for isolated CP was found for ALX3 and PDGFC (Jugessur et al., 2009), whereas these genes were associated to CLP in our study. Recently, a specific role of PDGFRA (Platelet-Derived– Growth Factor Receptor A) was also found for CP (Rattanasopha et al., 2012), and in contrast, a SNP in the regulatory region of PDGFC was associated with CLP (Choi et al., 2009). In patients with isolated CP we observed an association with MSX1 (p¼0.049) which had been observed before (Alapat et al., 2003). However, associations of MSX1 with CL6P were also reported (Vieira et al., 2003; Ingersoll et al., 2010). Variants in the TGFB3 gene have been repeatedly associated with complete OFC (CL6P) (Ichekwawa et al., 2006), but other studies did not confirm these results (Salishourfar et al., 2011). In our study, TGFB3 was not associated to any of the complete OFCs but had been found associated in patients with a SMCP (Reiter et al., 2012). The other gene associated with SMCP (MN1, Reiter et al., 2012), did not show an association with any of the complete clefts in the present study. It was recently found deleted in a case with a Robin sequence and CP (Davidson et al., 2012). Not only the co-occurrence of the same cleft type within a family but also the preferential associations of genes with specific cleft types indicate that different cleft types may be of genetically different etiology. Our results indicate cleftspecific associations of IRF6, MTHFR, ALX3, and PDGFC with the primary palate (CL6P) and a potential role of MSX1, TGFB3, and MN1 with the secondary palate (CP and SMCP, respectively) in patients of German origin.
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Of course, these results of our pilot study need to be confirmed in larger samples, which are also needed to resolve the controversial findings reported in the literature. Acknowledgments. We thank all patients and their families for participations in the study. Margot Brugger and Irina Wiest are acknowledged for excellent technical assistance.
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