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Epidemiology of Cleft Palate in Europe: Implications for Genetic Research ELISA CALZOLARI, M.D. FABRIZIO BIANCHI, B.SC., PH.D. MICHELE RUBINI, PH.D. ANNUKKA RITVANEN, M.D. AMANDA J. NEVILLE, B.SC. EUROCAT WORKING GROUP Objective: To describe the epidemiology of cleft palate (CP) in Europe. Design and Setting: A descriptive epidemiological study on 3852 cases of CP, identified (1980 through 1996) from more than 6 million births from the EUROCAT network of 30 registers in 16 European countries. Results: Significant differences in prevalence in Europe between registries and within countries were observed. A total of 2112 (54.8%) CP cases occurred as isolated, 694 (18.0%) were associated with other defects such as multiple congenital anomalies, and 1046 (27.2%) were in recognized conditions. The study confirmed the tendency toward female prevalence (sex ratio [SR] 5 0.83), particularly among isolated cases (SR 5 0.78) even if SR inversion is reported in some registries. A specific association with neural tube defects (NTDs) in some registers is reported. Conclusion: The differences identified in Europe (prevalence, sex, associated anomalies) can be only partially explained by methodological reasons because a common methodology was shared among all registries for case ascertainment and collection, and CP is an easy detectable condition with few induced abortions. The complex model of inheritance and the frequently conflicting results in different populations on the role of genes that constitute risk factors suggest the presence of real biological differences. The association of CP/NTD in an area with a high prevalence of NTDs can identify a group of conditions that can be considered etiologically homogeneous. The epidemiological evaluation can guide genetic research to specify the role of etiological factors in each different population KEY WORDS: cleft palate, genetics, epidemiology, etiology, neural tube defects Cleft palate (CP) is a common congenital anomaly affecting about 1 in 2000 births, with significant variation depending on geographical location, racial and ethnic background, and socioeconomic status (Schuttle and Murray, 1999). The frequent occurrence, as well as psychological, surgical, and dental involvement, emphasize the importance of understanding the causes. Most CP cases occur as an isolated congenital malformation, but CPs are often part of chromosomal and monogenic syndromes or associated with other congenital malformation (multiple congenital anomalies, [MCA]; Shaw et al., 1995; Shuttle and Murray, 1999; Prescott et al., 2001, Wilkie and MorrissKay, 2001; Beaty et al., 2002). Nonetheless, CP also occurs as part of many single gene syndromes, and some of these same genes may also play roles in nonsyndromic orofacial clefts (Sozen et al., 2001). The role of genetic factors in determining CP is documented by recurrence risk (Fraser, 1970) and monozygotic twin concordance (Nordstrom et al., 1996), but thus far there is no evidence of any single gene acting as a major factor in the etiology of this orofacial malformation. In isolated CP, a major genetic component with a relatively small number of interacting causative loci has been suggested, and the final phenotype is the result of gene products that interact in many ways with Dr. Calzolari is Professor of Genetic Medicine, Ms. Neville is a Scientific Secretary, and Dr. Rubini is a Senior Researcher, Genetic Medicine Section, Department of Experimental Medicine and Diagnostics, University of Ferrara, Ferrara, Italy. Dr. Bianchi is a Senior Researcher, Unit of Epidemiology, Istituto di Fisiologia Clinica, Consiglio Nazionale delle Ricerche, Pisa, Italy. Dr. Ritvanen is a Senior Researcher, The Finnish Register of Congenital Malformations, The National Research and Development Centre for Welfare and Health, Helsinki, Finland. The EUROCAT Working group: Helen Dolk, Professor of Epidemiology, EUROCAT Central Registry Leader, University of Ulster, Co Antrim, Northern Ireland, United Kingdom. Data from this paper were presented orally at the World Health Organization Meeting on Craniofacial Anomalies, Bauru, Brazil, December 4–6 2001. Submitted June 2002; Accepted March 2003. Address correspondence to: Professor E. Calzolari, Professor of Genetic Medicine, Genetic Medicine Section, Department of Experimental Medicine and Diagnostics, University of Ferrara, Via L. Borsari 46, 44110 Ferrara, Italy. E-mail [email protected]. 244 Calzolari et al., EPIDEMIOLOGY OF CLEFT PALATE IN EUROPE one another and the environment (multifactorial-complex disease). The objectives of this study were to establish the epidemiology of CP in Europe and determine differences in CP prevalence that may be attributed to etiological factors (genetics, environmental, or both) having a heterogeneous distribution across European countries. PATIENTS AND METHODS Data Collection Data from 30 centers in the 16 European countries of the European Concerted Action on Congenital Anomalies and Twins (EUROCAT) network of regional registers of congenital anomalies in Europe were considered for the study period 1980 through 1996 (Table 1). Recorded cases included live births, stillbirths, and induced abortions following prenatal diagnosis. Cases of CP born in the study period from mothers resident in the study area were considered and classified as the following: (1) isolated CP in which no other anomaly or anomalies are present, but the presence of minor anomalies such as low set ears or clinodactyly did not change the eligibility of the case to be considered isolated; and (2) associated CP in which two or more unrelated anomalies were present. This group was further classified into CP in recognized conditions, which comprise CP in chromosomal, monogenic, and environmental syndromes, or part of malformative sequences; or CP in MCA of unknown etiology, including all cases in which at least one major anomaly occurred in addition to CP. All cases were classified according to the EUROCAT definitions (EUROCAT, 1997) and coded according to the British Pediatric Association Classification of diseases (1977). Multiple sources of ascertainment were used, including birth certificates, pediatric records, maternity records, hospital discharge summaries, and cytology and pathology reports. Reliance on different sources varied according to which information sources were available in each area. Completeness of data was ensured by verifying medical records. Data Validation Validation of the database was carried out by a medical geneticist (EC) checking each individual record. The EUROCAT Registry leaders were involved in the data validation process, in both specification and classification of some complex cases and the interpretation and general discussion of the results. Registers and methods have also been described by Dolk et al. (1991) and in a EUROCAT (2002) report. Statistical Methods Prevalence rate was defined as the number of affected cases in live births, stillbirths, and induced abortions divided by the total number of births. For geographical comparisons, differences among centers 245 were analyzed using the chi-square test for heterogeneity (Armitage, 1955). Prevalence rate ratios (PRRs) were calculated to compare prevalence rates observed in a group of registers, compared with other groups of registers. The 95% confidence intervals (CIs) were calculated using the Cornfield (1956) method. Sex ratios (SRs) were calculated by dividing the male prevalence by the female prevalence. RESULTS Of the 3852 cases of CP derived from 6,181,499 births (prevalence 6.2 3 10,000; 95% CI 5 6.0 to 6.4), 2112 (54.8%) cases were isolated, 694 (18.0%) were associated with other defects such as MCAs, and 1046 (27.2%) were in recognized conditions (Table 1). There was a significant difference in prevalence rate of CP among European centers not only for total cases (chi-square 5 254.5; df 5 29; p , .001) but also for isolated CP (chi-square 5 220.9; df 5 29; p , .001), MCA (chi-square 5 81.7; df 5 29; p , .001), and recognized conditions (chi-square 5 234.2; df 5 29; p , .001). Relevant differences between registries were observed even within the same country (Table 1), and a significant north-south decreasing trend was not visible. High CP frequency was observed in Finland (PRR 5 2.60; 95% CI 5 2.34 to 2.89) when compared with other registers, essentially due to an excess of isolated CP (PRR 5 2.95; 95% CI 5 2.58 to 3.38) and recognized conditions (PRR 5 2.97; 95% CI 5 2.45 to 3.60) but not MCA (PRR 5 1.09; 95% CI 5 0.76 to 1.57). The study confirmed a female excess (SR 5 0.83) with a higher female excess among isolated CP (SR 5 0.78) than MCA cases (SR 5 0.87) and recognized conditions (SR 5 0.91). A male excess and SR inversion were reported in some registers. As reported in the literature, CP cases occur frequently associated with other defects (1740 of 3852 cases; 45.2%). Three registers in the United Kingdom (Belfast, Glasgow, and Liverpool) and the registers of Odense (Denmark) and Northern Netherlands accounted for 38.8% of MCA cases and showed a prevalence ratio of 1.7 per 10,000, compared with 1.0 in the other EUROCAT registers (PRR 1.8; 95% CI 5 1.5 to 2.1; Table 2). In these five registers, a relatively high rate of NTDassociated CP cases was observed, with a prevalence rate of 0. 31/10,000, compared with the 0.09/10,000 of the other EUROCAT registers (PRR 5 3.5; 95% CI 5 2.3 to 5.7; Table 2). These registers have an NTD prevalence rate of 21.2 3 10,000, compared with 9.2/10,000 of the other EUROCAT registers (PRR 5 2.3; 95% CI 5 2.2 to 2.4; EUROCAT, 1995, 1997, 2002). The registry of Dublin, with a prevalence rate of CP in MCA of 0.8/10,000 (Table 1) and an NTD prevalence rate of 24.0/10,000, presents the specific association NTD/CP (5 of 30 cases). In the same group of registers in the CP/NTD cases, females were more frequent (SR 5 0.43; 95% CI 5 0.19 to 0.68), compared with the other EUROCAT registers for the same CP/ 95% CI 1.1–4.2 1.4–2.3 1.2–2.0 2.7–4.3 0.9–2.0 0.3–1.5 3.8–5.5 1.6–2.8 0.6–1.7 0.0–1.8 1.1–2.3 0.1–0.9 1.3–2.0 1.5–2.5 1.7–3.1 0.0–0.4 1.8–2.6 0.7–1.7 0.0–0.8 1.0–1.6 1.2–2.0 0.4–1.0 0.0–0.8 0.5–1.4 0.5–2.9 0.8–3.3 0.2–1.7 1.4–3.1 0.0–1.0 0.4–2.3 1.6–1.8 Rate 2.6 1.8 1.6 3.5 1.5 0.9 4.6 2.2 1.2 0.7 1.7 0.5 1.6 2.0 2.4 0.2 2.2 1.2 0.4 1.3 1.6 0.7 0.4 1.0 1.7 2.0 1.0 2.2 0.4 1.4 1.7 No. 11 65 66 75 27 8 118 50 18 2 30 7 96 56 48 5 97 19 3 93 58 16 3 17 8 10 6 25 1 8 1,046 95% CI 0.0–0.0 0.5–1.1 1.1–1.9 1.3–2.5 1.2–2.5 1.0–2.8 0.8–1.7 1.2–2.3 0.3–1.2 0.0–1.1 0.9–2.0 0.0–0.6 0.9–1.5 0.8–1.6 1.1–2.2 0.7–1.6 0.6–1.1 0.7–1.7 0.4–1.8 0.6–1.0 0.8–1.5 0.4–1.1 0.2–1.6 0.1–0.7 0.3–2.3 0.2–2.2 0.0–1.0 0.2–1.2 0.0–1.7 0.7–2.8 1.0–1.2 Rate 0.0 0.8 1.5 1.9 1.8 1.9 1.2 1.8 0.8 0.4 1.4 0.3 1.2 1.2 1.6 1.2 0.9 1.2 1.1 0.8 1.2 0.8 0.9 0.4 1.3 1.2 0.5 0.7 0.7 1.7 1.1 No. 0 30 62 40 34 17 31 41 12 1 25 4 70 33 32 27 38 19 9 58 44 18 7 7 6 6 3 8 2 10 694 95% CI 0.8–3.6 4.0–5.4 2.0–3.0 2.7–4.3 2.7–4.4 3.2–6.0 8.1–10.5 2.2–3.5 2.7–4.6 0.7–4.6 2.4–4.0 1.9–3.6 1.9–2.6 3.4–5.0 3.8–5.7 1.9–3.2 2.8–3.8 2.5–4.4 2.6–5.2 2.5–3.2 2.7–3.8 2.5–3.9 1.3–3.5 1.8–3.3 1.1–4.0 1.2–4.0 0.9–3.2 1.4–3.1 0.0–2.8 2.8–6.2 3.3–3.6 Rate 2.2 4.7 2.5 3.5 3.5 4.6 9.3 2.8 3.6 2.6 3.2 2.7 2.3 4.2 4.8 2.6 3.3 3.5 3.9 2.8 3.2 3.2 2.4 2.6 2.6 2.6 2.1 2.2 1.4 4.5 3.4 No. 9 169 100 75 65 41 237 65 56 7 57 38 132 117 95 60 146 55 33 204 120 74 19 45 12 13 13 25 4 26 2,112 95% CI 2.7–6.9 6.5–8.3 4.9–6.4 7.7–10.2 5.6–8.0 5.6–9.2 13.7–16.7 5.8–7.9 4.4–6.7 1.4–6.1 5.1–7.5 2.5–4.5 4.5–5.7 6.4–8.4 7.5–10.1 3.1–4.7 5.6–7.1 4.7–7.0 3.8–6.9 4.4–5.5 5.2–6.8 3.8–5.6 2.4–5.1 3.0–4.9 3.4–7.7 3.7–8.0 2.0–4.9 3.9–6.5 0.6–4.3 5.4–9.9 6.0–6.4 Rate 4.8 7.4 5.6 8.9 6.8 7.4 15.2 6.8 5.5 3.7 6.3 3.5 5.1 7.4 8.8 3.9 6.4 5.9 5.3 5.0 6.0 4.7 3.7 4.0 5.6 5.8 3.5 5.2 2.5 7.6 6.2 20 264 228 190 126 66 386 156 86 10 112 49 298 206 175 92 281 93 45 355 222 108 29 69 26 29 22 58 7 44 3,852 Recognized Conditions No. MCA 41,549 357,457 405,352 212,677 184,530 89,349 253,847 228,599 155,215 26,680 177,885 140,061 585,049 276,574 199,055 233,877 442,197 158,754 84,715 716,939 371,499 230,455 77,957 174,082 46,558 49,587 63,054 111,750 28,506 57,640 6,181,449 * EUROCAT 5 European Concerted Action on Congenital Anomalies and Twins. Period 1981–1994 1980–1996 1980–1994 1980–1996 1980–1988 1980–1996 1993–1996 1981–1996 1991–1996 1980–1989 1980–1996 1980–1990 1981–1996 1985–1996 1982–1996 1987–1996 1988–1996 1985–1996 1983–1996 1981–1996 1981–1996 1980–1996 1980–1990 1993–1996 1990–1995 1990–1996 1992–1996 1990–1996 1993–1996 1986–1996 Centers Galway (Ireland) Dublin (Ireland) Belfast (UK) Glasgow (UK) Liverpool (UK) Odense (Denmark) Finland Northern Netherlands Southwest Netherlands Luxemburg HainautNamur (Belgium) West Flanders (Belgium) Paris (France) Bouches-du-Rhône (France) Strasbourg (France) Saxony-Anhalt (Germany) Switzerland Styrian (Austria) Zagreb (Croatia) Northeast Italy Emilia-Romagna (Italy) Tuscany (Italy) Umbria (Italy) Campania (Italy) Southern Portugal Asturias (Spain) Barcelona (Spain) Basque Country (Spain) El Valles (Spain) Malta EUROCAT mean rate Isolated Total Births All Cleft Palate TABLE 1 EUROCAT Registers: Prevalence Rates of Cleft Palate (Live Births, Stillbirths, and Interrupted Pregnancies) per 10,000 Births 246 Cleft Palate–Craniofacial Journal, May 2004, Vol. 41 No. 3 0.12–0.14 0.13 80 11.1–11.6 11.3 694 6,181,449 1.1 1.0–1.2 7011 8.9–9.4 9.2 4635 0.9–1.1 500 5,060,942 * Includes the registers of Odense, Denmark; Northern Netherlands; and Liverpool, Glasgow, and Belfast, United Kingdom. Rates are per 10,000 births. NTD data are from EUROCAT (1995, 1997, 2002). CP 5 cleft palate; MCA 5 multiple congenital anomalies; NTD 5 neural tube defects; EUROCAT 5 European Concerted Action on Congenital Anomalies and Twins; CI 5 confidence interval; PRR 5 prevalence rate ratio. 45 0.09 0.08–0.10 2.3–5.7 3.5 0.28–0.35 0.31 35 2.2–2.4 2.3 1.0 NTD 20.4–22.1 21.2 2376 1.5–2.1 1.8 1.5–2.0 1.7 194 1,120,507 Odense, Northern Netherland, Glasgow, Liverpool, Belfast Other EUROCAT Registers Total CP/NTD PRR 95% CI PRR No. CP in MCA 95% CI Rate No. Total Births Groups of Registers TABLE 2 Prevalence of CP in MCA, NTD, and NTD-Associated CP in European Area* Rate 95% CI PRR 95% CI No. Rate 95% CI 95% CI Calzolari et al., EPIDEMIOLOGY OF CLEFT PALATE IN EUROPE 247 NTD association (SR 5 1.06, 95% CI 5 0.72 to 1.38; Fisher p 5 0.10). In Finland, the register with the highest CP prevalence rate, the CP/NTD association had a low prevalence rate of 0.12/ 10,000, which is similar to the other EUROCAT register rate of 0.13/10,000 (Table 2). DISCUSSION Efforts have been made to record the global frequency of CP, and many different figures are available. In the present study, covering more than 6 million births, prevalence of CP varied significantly in Europe, not only between registries but also within countries, with a European mean value of 6.2 3 10,000. The highest prevalence of isolated CP was confirmed in Finland during a longer study (ICBDMS, 2000). Our study showed a tendency toward female prevalence (Schuttle and Murray, 1999,) although a male excess was reported in some registers. No generally accepted explanation for these sex differences is reported. The recent discovery of the X-linked CP and ankyloglossia gene TBX22 (Braybrook et al., 2001) may suggest a candidate gene that might be relevant for palate morphogenesis and possibly play a role in the imbalanced sex prevalence of nonsyndromic CP cases. An association of CP and NTD in registers of Belfast, Liverpool, Glasgow, Dublin, northern Netherlands, and Odense was found with epidemiologic characteristics (spatial and sex distribution) similar to those of isolated NTD. Our data suggest that common factors may be involved in the etiology of NTD and other anomalies, this factor varying geographically (Calzolari et al., 1997). Dolk et al. (1991) reported that the association between NTD and a number of anomaly groups was at least two times more prevalent in the United Kingdom and Ireland than in continental Europe and Malta. Three possible non–mutually exclusive explanations of these data might be offered. Ascertainment Procedure This factor could contribute, but in a quite marginal manner because common methodology was shared among all registries for case ascertainment and collection; multiple sources of information were used by most registries; and CP is an easily diagnosed and reported malformation with a reduced impact from cases of induced abortion. Birth cut-off dates are not a critical factor, producing only small differences between registries with different cut-off dates for registration following diagnosis. Genetics The growth of the detailed structures of the head and face is largely determined genetically (Wilkie and Morriss-Kay, 2001), and these processes are known to be dependent on a spectrum of signaling molecules, transcription factors, and growth factors interacting with environmental factors. 248 Cleft Palate–Craniofacial Journal, May 2004, Vol. 41 No. 3 Many genes have been indicated to play a role in CP etiology (Murray, 1995; Schuttle and Murray, 1999; Beatty et al., 2002), each one possibly contributing to the genetic susceptibility in a complex network of gene-gene and gene-environment interactions. CP also occurs as part of many single gene syndromes, and some of these same genes may also play roles in nonsyndromic CP (Sozen et al., 2001). Association studies have looked at various CP candidate genes, chosen for their known biology or by positional localization within genomic regions implied in linkage studies. As in many other association studies, nonreplication in subsequent, independent studies has been reported (Spritz, 2001; Murray, 2002). No simple explanation for these conflicting data is possible. Chance associations, variations in study design, or differences in the frequencies of allelic variants that truly predispose to CP among different populations can be hypothesized. In the latter case, the same candidate gene or DNA variants may be associated with different relative risks in different populations, and the nonreplication might result from real biological differences. Studies on many other multifactorial-complex diseases (Reich and Lander, 2001) have pointed to the need to know not only the loci at which a mutation occurs but also the relation between the genes involved and the allele frequencies within and between populations. Geographical variations in gene frequencies of mutations or polymorphisms that are risk factors for CP could explain the observed differences in the prevalence of this condition. According to the model for complex diseases by Greenspan (2001), if several factors contribute to the phenotype (CP has a complex heterogeneous etiology), the same output can be produced in different ways (Fig. 1a and 1b) with the clinical phenotype being highly influenced by the type of mutation, the effect of other genes, and environmental factors. Complex pathways through this network of gene-gene and gene-environment interaction may provide alternative routes through which CP may be developed or avoided. Recently considerable attention has been paid to the possible role of folic acid in orofacial clefts. The manner in which folic acid is involved is less clear than that demonstrated for NTDs. Alteration of the metabolism of folate could be a shared susceptibility factor (Fig. 1c) and can contribute to explain the association between CP and NTDs in an area with high prevalence of NTDs. Homozygosity for the 677T variant in methylene-tetrahydrofolate reductase in CP patients (Mills et al., 1999) can be one of the possible link factors between alteration of folic acid metabolism and the occurrence of oral clefts or NTDs in offspring as a specific association, part of a common network. Folate could be linked to the etiology of NTDs and other anomalies such as omphalocele (Calzolari et al., 1997). Alternatively, the presence of a defect may predispose to subsequent malformation of specific structures or may be an essential component of sequence or field defects (neural crest). Environment Genes involved in the development of the face are influenced by environmental teratogens (e.g., smoke, alcohol; Shaw FIGURE 1 Proposed gene network interaction in determining cleft palate (CP). A. Normal interaction between genes prevents CP from developing. B. Mutations (yellow dots) in genes along a molecular network can lead to cleft palate. C. Shared mutations can determine CP associated with neural tube defects (NTDs). Modified from Greenspan (2001). et al., 1996) and maternal nutrition (Murray, 2002). The precise mechanisms by which these environmental teratogens lead to clefts are unknown, even if it could be related to the genetic background linked to functional variation in gene expression. CONCLUSIONS Gene/environment research should seek to establish the risk of CP associated with different populations and ethnic groups and be able to differentiate exposure and the genetic predisposition. Identification of those at risk could then lead to selective counseling. Integration of new genetic information (biological sample collection and genetic analysis) into epidemiological studies in the same population can help clarify links between etiological factors and different prevalence in different populations so providing the direction for genetic research. Acknowledgments. The EUROCAT project is funded under the Rare Diseases Program of the European Commission. Funding was also received from the Emilia-Romagna Region and the Tuscany Region and for which we are grateful. The authors thank all the physicians, nurses, midwives, and Registry clerks for their assistance in collecting the data. The EUROCAT Registries that participated in the study are: Styria, Austria, Martin Haeusler; West Flanders, Belgium, Vera Nelen; Hainaut, Belgium, Yves Gillerot; Zagreb, Croatia, Ingeborg Calzolari et al., EPIDEMIOLOGY OF CLEFT PALATE IN EUROPE Barisic; Odense, Denmark, Ester Garne; Finland, Annukka Ritvanen, Jorma Rautio; Bouche du Rhone, France, Nicole Philipe, Segolene Ayme; Paris, France, Catherine De Vigan, Janine Goujard; Strasbourg, France, Claude Stoll; Saxony-Anhalt, Germany, Volker Steinbicker; Dublin, Ireland, Bob McDonnell, Zachary Johnson; Galway, Ireland, David Lillis; Campania, Italy, Gioacchino Scarano; Emilia Romagna, Italy, Elisa Calzolari, Amanda Neville, Gianni Astolfi; North East Italy, Romano Tenconi; Tuscany, Italy, Fabrizio Bianchi, Sonia Catalano, Anna Pierini; Umbria, Italy, Anna Calabro; Luxembourg, D. HansenKoenig, M. 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