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A Locus in 2p13–p14 (OFC2), in Addition to That Mapped in 6p23, Is Involved in Nonsyndromic Familial Orofacial Cleft Malformation
GENOMICS
50, 299–305 (1998) GE985273
ARTICLE NO.
A Locus in 2p13–p14 (OFC2), in Addition to That Mapped in 6p23, Is Involved in Nonsyndromic Familial Orofacial Cleft Malformation Furio Pezzetti,* Luca Scapoli,* Marcella Martinelli,* Francesco Carinci,† Maria Bodo,* Paolo Carinci,‡ and Mauro Tognon*,1 *Department of Morphology and Embryology, Section of Histology and General Embryology, and †School of Medicine, University of Ferrara, Via Fossato di Mortara 64/B, 44100 Ferrara, Italy; and ‡Institute of Histology and General Embryology, School of Medicine, University of Bologna, Via Belmeloro 8, 40126 Bologna, Italy Received December 8, 1997; accepted February 16, 1998
An allelic association between the transforming growth factor a gene (TGFA) situated in the chromosome 2p13 region and nonsyndromic cleft lip with or without cleft palate, also named orofacial cleft (OFC), was found in several population studies. However, no linkage between gene and malformation has shown up until now, probably due to the presence of genetic heterogeneity and the small sample size analyzed. Previously, we employed a collection of 38 OFC families to demonstrate linkage to the 6p23 chromosome region with the presence of genetic heterogeneity. In the present study we tested whether, in the same sample, linkage between OFC and markers on 2p13 could be determined. Evidence for genetic heterogeneity in our family set was apparent, by both pairwise and multipoint linkage analyses. Moreover, lod scores ú3 were found for marker D2S378 when families linked to the 6p23 markers were analyzed. Taken together these results indicate a role for the TGFA, or for another gene physically close to it, and suggest an interaction between two different genes, OFC1 and OFC2, mapped in 6p23 and 2p13, respectively, in the development of the cleft. q 1998 Academic Press
INTRODUCTION
Nonsyndromic cleft lip with or without cleft palate, also named orofacial cleft (CL{P Å OFC) derives from an embryopathy with a consequent failure of fusion of the nasal process and palatal shelves. This severe birth defect is one of the most common malformations, affecting approximately 1/1000 live births among Caucasians. There is a large body of evidence that genetic factors are important in its etiology. However, the nature of the genetic contribution to the etiology of non1
To whom correspondence should be addressed at the School of Medicine, University of Ferrara, Via Fossato di Mortara 64/B, 44100 Ferrara, Italy. Telephone: /39-532-291538. Fax: /39-532-291533. E-mail: [email protected].
syndromic OFC is not completely understood (Murray, 1995). Ardinger et al. (1989), using restriction fragment length polymorphism (RFLP) analysis, reported a significant association between the transforming growth factor a gene (TGFA) located on 2p13 and the occurrence of clefting; since then, this locus has been investigated by several groups. Indeed, some authors confirmed this association (Chenevix-Trench et al., 1991, 1992; Holder et al., 1992; Sassani et al., 1993; Jara et al., 1995), while others found association only with the severity of the cleft (Stoll et al., 1993; Field et al., 1994). Recently, Mitchell (1997), through a meta-analysis study, confirmed the association between TGFA and OFC; however, his data were not conclusive, because heterogeneity was also observed. Shaw et al. (1996) did not detect allelic association between TGFA and OFC in a study based on a large population; however, they did find a strong association between maternal smoking and a genetic variant of TGFA at risk for cleft development. In fact, the risk of this malformation was approx twice as much if the mother smoked during pregnancy, but was even greater for infants when a negative synergistic effect occurred between smoking and a rare TGFA allele previously found associated with clefting. From the results of this study, one may infer that this is an example of gene–environment interaction in the occurrence of facial clefting. It seems that population-based studies support a role for TGFA, or a nearby gene (OFC2), as the susceptible gene involved in some way in OFC malformation. However, three different studies did not find linkage with TGFA using the lod score method of analysis (Hecht et al., 1991; Vintiner et al., 1992; Field et al., 1994). Since genetic heterogeneity was detected for OFC (Carinci et al., 1995; Stein et al., 1995; Scapoli et al., 1997), failure to detect linkage with TGFA could be due to the small number of families analyzed (Farrall et al., 1993). On the other hand, Feng et al. (1994), in a family-based association study, found significant positive linkage disequilibrium with the C2 allele of TGFA, thus sup-
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porting a role for this gene in OFC malformation. In the murine model, a TGFA gene knockout experiment indicated that the gene-deficient animals did not develop OFC (Luettke et al., 1993). This result suggests that TGFA may act as a modifier gene rather than being a necessary and sufficient agent on its own for OFC disease. Biological relevance for TGFA playing a role in clefting is provided by its expression pattern in palate formation (Dixon et al., 1991). A transgenic mouse, in which TGFA was overexpressed, had epithelial hyperplasia in a number of tissues, thus demonstrating that TGFA is a potent epithelial mitogen (Sandgren et al., 1990). Other chromosome regions were investigated for a putative OFC locus/loci; it is worth remembering the studies on the 6p region containing the OFC1 gene. Indeed, different groups testing the linkage with the HLA locus provided either negative (Van Dyke et al., 1980; Watanabe et al., 1984), or positive (Mehra and Verma, 1991) results. Other reports indicated that a clefting locus could be located on chromosome 6p close to different possible loci: factor 13A (F13A), which maps in the region 6p25–p24 (Eiberg et al., 1987), and the HGP22 and AP2 genes, which are potentially involved in face formation and which map at 6p24 (Scambler et al., 1992; Davies et al., 1995). Moreover, Kurihara et al. (1994) observed craniofacial abnormalities in transgenic mice deficient in the EDN1 gene product. Different data were recently reported by two other groups (Hecht et al., 1993; Vintiner et al., 1993), who found no linkage between OFC and the 6p region (between the HLA and the F13A loci). On the other hand, in a recent investigation we found, by studying the microsatellite markers and by linkage analysis, that an OFC locus maps to chromosome 6p23 (Carinci et al., 1995; Scapoli et al., 1997).
These data suggested to us that we test whether, in our collection of 38 nonsyndromic OFC families, previously employed for a similar investigation on chromosome region 6p23 (Carinci et al., 1995; Scapoli et al., 1997), any linkage between OFC and markers on 2p13 could be determined. MATERIAL AND METHODS Family descriptions. We studied 38 families who were previously analyzed for linkage to the 6p23 chromosome region (Scapoli et al., 1997) and were characterized by the presence, in each pedigree, of at least two OFC-affected individuals. The affected members were relatives of first, second, third, or fourth degree. All the families enrolled were of Italian ancestry, from northeastern regions, without immigrants and included subjects presenting OFC as the unique familial disease. Indeed, the cleft lip, with or without cleft palate, was the only anomaly detected after a detailed clinical examination performed by the dysmorphologist of our research group (F.C.). Moreover, those families using clefting drugs, such as phenitoin, warfarin, and ethanol, were excluded from the study. The pedigrees were composed of 8 OFC families with two generations, 15 with three generations, 12 with four generations, and 3 with five generations, for a total of 378 individuals, 96 affected by OFC. DNA studies. Blood samples, after obtaining consent, were drawn from 268 individuals, 78 of whom were affected. DNA, prepared from peripheral blood cells (Higuchi, 1989), was analyzed for seven dinucleotide repeats at loci D2S145, D2S285, D2S134, D2S380, D2S337, D2S378, and D2S123, having heterozygosity of 0.58, 0.85, 0.77, 0.85, 0.88, 0.81, and 0.76, respectively (Gyapay et al., 1994). Moreover, the TaqI polymorphism at the TGFA locus (heterozygosity Å 0.29), due to a 4-base insertion in intron V of the gene, was typed by PCR methods as reported before (Basart et al., 1994). DNA samples were denatured at 947C for 30 s, annealed at 577C for 20 s, and extended at 727C for 10 s. For D2S134 the annealing temperature was at 587C and for D2S380 and TGFA TaqI 597C. All amplifications were processed through 35 cycles. Small aliquots of PCR-amplified products were resolved by polyacrylamide gel electrophoresis and visualized by silver staining. Linkage studies. The eight markers were analyzed for linkage to OFC by using the computer program LINKAGE, version 5.1 (Lathrop et al., 1984), which includes MLINK and LINKMAP programs.
TABLE 1 Two-Point Lod Score for OFC versus Chromosome 2 Markers in 38 Families, Calculated under Autosomal Dominant Model with Reduced Penetrance (RP) and Affected Only (AO) Methods Lod score at recombination fraction of Marker
Method
0.001
0.01
0.05
0.10
0.20
0.30
0.40
RP AO RP AO RP AO RP AO RP AO RP AO RP AO RP AO
02.02 02.00 03.68 03.65 010.28 010.24 07.58 07.10 010.67 010.39 04.20 04.17 04.37 04.21 05.33 05.17
01.23 01.21 02.93 02.91 08.14 08.16 05.51 05.09 07.82 07.61 03.48 03.43 03.70 03.52 03.95 03.83
0.09 0.14 01.40 01.41 04.61 04.62 02.00 01.66 03.26 03.08 01.93 01.83 01.99 01.81 01.62 01.54
0.71 0.78 00.59 00.60 02.66 02.65 00.32 00.05 00.97 00.82 00.78 00.67 00.68 00.52 00.44 00.37
0.96 1.02 0.09 0.07 00.77 00.75 0.90 1.07 0.81 0.90 0.33 0.42 0.53 0.63 0.46 0.51
0.71 0.75 0.26 0.25 00.05 00.03 1.03 1.13 1.10 1.15 0.58 0.64 0.75 0.81 0.57 0.60
0.34 0.36 0.21 0.20 0.12 0.13 0.66 0.70 0.74 0.76 0.41 0.44 0.50 0.53 0.34 0.36
D2S145 TGFA TaqI D2S285 D2S134 D2S380 D2S337 D2S378 D2S123
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A LOCUS IN 2p13–p14 IS INVOLVED IN OFC (OFC2) A complex segregation analysis was performed in our family set; the results were consistent with a two-locus model with a major dominant locus and one modifier locus (C. Scapoli et al., submitted). Based on those analyses, we used for lod score calculations a dominant mode of inheritance with disease allele frequency of 0.0035; the penetrance was 0.12 for males and 0.06 for females (reduced penetrance model; RP). A two-locus model with a major dominant locus and at least one modifier locus was also proposed by Clementi et al. (1995), using a large sample of 636 newborns with OFC registered in northeastern Italy. Moreover, a combination of different genes working together to produce OFC was proposed by other authors (Farrall and Holder, 1992; Mitchell and Risch, 1992). If this is the case, then many unaffected individuals may possess the disease-predisposing genotype at one of the loci, but lack the required second disease locus genotype necessary for the development of the disease. For this reason it is advisable to consider all unaffected individuals to actually have ‘‘unknown’’ phenotype when performing linkage analyses. In this way, one bases the linkage analyses solely on the marker status of the affected individuals in the pedigree and does not apply any disease locus genotypic information to the unaffected individuals. To perform the ‘‘affected only’’ analyses (AO), all marker data were retained in the pedigree file, whereas the maximum penetrance values were set to 0.001 (Terwillinger and Ott, 1994). The multipoint analyses were performed using the selected markers in the following order: D2S145–TGFA TaqI–D2S285–D2S134– D2S380–D2S337–D2S378–D2S123. The interlocus distances in centimorgans, which are in the order: 1–4–3–0.1–5–4–4, were from the Ge´ne´thon map (Gyapay et al., 1994). The distances are in agreement with our study (data not shown). To verify the homogeneity of the pedigrees, the results obtained from linkage analysis were further analyzed using the HOMOG computer program.
RESULTS
Thirty-eight multiplex OFC families were tested with each of the eight 2p13–p14 markers. Low positive lod scores were found for all markers at recombination fractions between 0.2 and 0.3, using the AO model or the RP model (Table 1). The maximum lod score obtained in the pairwise linkage analysis was 1.15 for D2S380 at u Å 0.30. The homogeneity test performed by the HOMOG computer program on the two-point data under the AO model provided evidence of heterogeneity for the markers D2S145, D2S337, and D2S378, P values 0.0452, 0.0294, and 0.0301 at u Å 0.00 with a Å 0.50, 0.45, and 0.45, respectively. Interestingly, by adopting the RP model, the homogeneity test showed evidence of heterogeneity for the same markers, P values 0.0477, 0.0287, and 0.0340 with a Å 0.50, 0.45, and 0.45 at u Å 0.001, respectively. In multipoint linkage analyses of OFC, the maximum lod score was obtained at 30 cM telomeric to D2S123; the values were 1.30 under the AO model and 1.06 under the RP model. The homogeneity test performed by the HOMOG computer program on the multipoint data under the AO model provided evidence of heterogeneity in all the intervals between markers D2S285 and D2S123. The maximum significance level for the hypothesis of heterogeneity (P value 0.0072) was obtained with 35% of families linked at D2S378. For the RP model, the homogeneity test showed a P
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TABLE 2 Posterior Probabilities of Linkage Calculated for Chromosome Regions 6p and 2p, by Two Models of Inheritance Reduced penetrance
Affected only
Family
6p
2p
6p
2p
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
0.93 0.90 0.80 0.81 0.86 0.63 0.02 0.01 0.32 0.01 0.02 0.73 0.59 0.79 0.64 0.87 0.60 0.64 0.61 0.64 0.80 0.08 0.75 0.69 0.52 0.47 0.75 0.69 0.64 0.80 0.74 0.55 0.96 0.60 0.74 0.80 0.56 0.01
0.79 0.66 0.15 0.48 0.03 0.35 0.03 0.03 0.07 0.08 0.01 0.53 0.32 0.53 0.33 0.79 0.34 0.34 0.50 0.33 0.51 0.03 0.49 0.53 0.36 0.37 0.52 0.50 0.37 0.53 0.51 0.37 0.00 0.52 0.08 0.63 0.34 0.01
0.90 0.86 0.86 0.75 0.85 0.60 0.02 0.01 0.39 0.01 0.02 0.75 0.60 0.75 0.60 0.92 0.60 0.60 0.60 0.60 0.79 0.06 0.75 0.74 0.60 0.60 0.75 0.75 0.60 0.75 0.74 0.60 0.96 0.60 0.74 0.85 0.54 0.01
0.81 0.68 0.16 0.49 0.03 0.35 0.03 0.03 0.07 0.07 0.01 0.51 0.35 0.52 0.35 0.80 0.35 0.35 0.51 0.35 0.52 0.03 0.49 0.52 0.35 0.35 0.52 0.51 0.35 0.52 0.51 0.35 0.00 0.51 0.08 0.66 0.35 0.01
Note. The values were calculated on the multipoint data at markers D6S259 and D2S278.
value 0.0093 with 35% of families linked at u Å 0.00 for D2S378. With both models, 14 of the 38 families analyzed showed ¢50% likelihood of being linked (Table 2); under the reduced penetrance model, 21 families had positive lod scores, whereas 17 were negative. By adopting the affected-only model, 16 families were positive, 11 negative, and 11 not informative. In a recent study using the same 38 multigenerational families, we demonstrated linkage between OFC and chromosome region 6p23 with genetic heterogeneity (Scapoli et al., 1997); these results, obtained by the HOMOG program, indicated that 30 of 38 families had a posterior probability of ¢50% in favor of linkage to D6S259 using both models of inheritance (Table 2).
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TABLE 3 Two-Point Lod Score for OFC versus Chromosome 2 Markers in 30 6p23-Linked Families, Calculated under the Autosomal Dominant Model with Reduced Penetrance (RP) and Affected Only (AO) Methods Lod score at recombination fraction of Marker
Method
0.001
0.01
0.05
0.10
0.20
0.30
0.40
RP AO RP AO RP AO RP AO RP AO RP AO RP AO RP AO
0.63 0.74 03.02 02.99 04.92 04.82 00.50 00.24 02.46 02.25 1.33 1.48 3.64 3.81 00.92 00.77
1.20 1.30 02.43 02.40 03.45 03.39 0.37 0.61 00.81 00.64 1.60 1.73 3.79 3.96 00.05 0.09
1.92 2.01 01.23 01.22 01.28 01.23 1.55 1.75 1.29 1.43 1.84 1.97 3.78 3.94 1.08 1.20
1.99 2.07 00.58 00.57 00.29 00.24 1.97 2.12 2.04 2.16 1.90 2.03 3.52 3.64 1.45 1.55
1.54 1.58 00.02 00.02 0.44 0.46 1.98 2.08 2.21 2.29 1.71 1.82 2.78 2.85 1.46 1.52
0.92 0.94 0.15 0.15 0.50 0.52 1.52 1.57 1.71 1.75 1.24 1.31 1.88 1.92 1.08 1.12
0.38 0.39 0.14 0.13 0.29 0.30 0.83 0.85 0.92 0.94 0.65 0.68 0.94 0.96 0.56 0.58
D2S145 TGFA TaqI D2S285 D2S134 D2S380 D2S337 D2S378 D2S123
Herein, the two groups of families selected a priori, i.e., the 6p23-linked group and the 6p23-unlinked group, were analyzed separately for linkage to the 2p13 chromosome region. The pairwise linkage analyses performed on the two family groups gave different results. In fact, the 6p23-linked families showed a positive lod score with a maximum of 3.96 at u Å 0.01 for marker D2S378 under the AO model and a lod score of 3.79 at u Å 0.01 under the RP model (Table 3). On the other hand, negative lod scores were obtained for the 6p23unlinked families using either one of the two inheritance models (Table 4). DISCUSSION
Early attempts to localize the OFC gene(s) in the human genome were performed by different groups. These studies provided numerous and sometimes discordant information (Murray, 1995). Significant results regarding the chromosome region 2p account for the association between the TGFA locus and the occurrence of clefting by using RFLP analysis (Ardinger et al., 1989). Later, other studies confirmed this association (Chenevix-Trench et al., 1991, 1992; Holder et al., 1992; Sassani et al., 1993; Mitchell, 1997). Moreover, Feng et al. (1994) using the transmission disequilibrium test, showing a significant linkage disequilibrium with the C2 allele of the TGFA locus; however, our group, using the same approach, did not confirm these data (Scapoli et al., 1998), and the exclusion of TGFA as a candidate locus was obtained by linkage studies in a series of multiplex OFC families (Hecht et al., 1991; Vintiner et al., 1992). In addition, many molecular genetic investigations supported major gene/oligogenic models in the develop-
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ment of the cleft, by evidencing loci in different chromosome regions. Important studies indicated different loci on 6p23 (OFC1) (Eiberg et al., 1987; Carinci et al., 1995; Davies et al., 1995; Scapoli et al., 1997), 4q25–q31 (Beiraghi et al., 1994; Healey et al., 1994), 19q13 (BCL3 gene) (OFC3) (Stein et al., 1995), and 17q21 (RARA) (Chenevix-Trench et al., 1992; Shaw et al., 1993). Since the etiology of OFC seems quite complex, very likely several loci may contribute to this malformation. Farral and Holder (1992) predicted that four to seven different genes could be involved in developing the cleft malformation. In a recent investigation, a complex segregation analysis was performed in a sample of 636 newborns with OFC registered in northeastern Italy; a two-locus model with a major dominant locus and at least one modifier locus was proposed by the authors (Clementi et al., 1995). In linkage analyses, diseases are usually assumed to result from the action of a single gene, possibly modified by random environmental effects. Other models, such as oligogenic or multifactorial models, may be more realistic for OFC. Fortunately, numerous studies have shown that there is little danger of missing a true linkage if one analyzes the data by assuming the wrong overall genetic mechanism (e.g., assuming a single major locus when the disease is, in fact, oligogenic) (Greenberg, 1990; Vieland et al., 1992; Goldin and Weeks, 1993). Moreover, it is known that in an analysis for linkage with the lod score method assuming an incorrect mode of inheritance, the probability of type I error (getting a high lod score value by chance alone) is not inflated. Traditional linkage analyses of OFC disease identified some putative loci involved in this malformation. Beiraghi et al. (1994) reported suggestive evidence for
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TABLE 4 Two-Point Lod Score for OFC versus Chromosome 2 Markers in Eight 6p23-Unlinked Families, Calculated under Autosomal Dominant Model with Reduced Penetrance (RP) and Affected Only (AO) Methods Lod score at recombination fraction of Marker
Method
0.001
0.01
0.05
0.10
0.20
0.30
0.40
RP AO RP AO RP AO RP AO RP AO RP AO RP AO RP AO
02.65 02.74 00.66 00.66 05.36 05.42 07.08 06.86 08.21 08.14 05.53 05.65 08.01 08.02 04.41 04.40
02.43 02.51 00.50 00.51 04.69 04.77 05.88 05.70 07.01 06.97 05.08 05.16 07.49 07.48 03.90 03.92
01.83 01.87 00.17 00.19 03.33 03.39 03.55 03.41 04.55 04.51 03.77 03.80 05.77 05.75 02.70 02.74
01.28 01.29 00.01 00.03 02.37 02.41 02.29 02.17 03.01 02.98 02.68 02.70 04.20 04.16 01.89 01.92
00.58 00.56 0.11 0.09 01.21 01.21 01.08 01.01 01.40 01.39 01.38 01.40 02.25 02.22 01.00 01.01
00.21 00.19 0.11 0.10 00.55 00.55 00.49 00.44 00.61 00.60 00.66 00.67 01.13 01.11 00.51 00.52
00.04 00.03 0.07 0.07 00.17 00.17 00.17 00.15 00.18 00.18 00.24 00.24 00.44 00.43 00.22 00.22
D2S145 TGFA TaqI D2S285 D2S134 D2S380 D2S337 D2S378 D2S123
linkage using markers on 4q in a single large family with OFC. Genetic heterogeneity for OFC was reported by other authors in a sample of 39 families (Stein et al., 1995); in this study linkage was found with the BCL3 gene on chromosome 19, but for only 17 of 39 families. Moreover, in a recent study on OFC we found linkage and heterogeneity by analyzing markers on 6p23 (Scapoli et al., 1997). In this investigation, we tested linkage to the OFC2 region in the same sample previously analyzed, consisting of 38 families, with the same racial and ethnic origin from northeastern Italy. Evidence for genetic heterogeneity in our family set was apparent, whereas it was shown that markers in region 2p are tightly linked to OFC in 14 multigenerational families under both models. From these results we infer that a gene in the 2p region does play an etiologic role in OFC development and that the loci involved in this malformation can be detected by linkage study with a large number of families. It is of interest to note that the 6p23-linked families showed a positive lod score and reached the significance 3.96 at u Å 0.01 for the marker D2S378. This result suggests an interactive effect of the two disease loci mapping in 2p13 and 6p23; however, no attempts were made in this study to assay the nature of interaction of these genes. This hypothesis, i.e., two or more genes simultaneously involved in OFC, is supported by recent studies (Farrall and Holder, 1992; Clementi et al., 1995). Interestingly, Murray (1995) postulated that the strongest evidence implicates a primary role for OFC1 on 6p, whereas OFC2 on 2p13 is a modifier of clefting status. At present it is not clear why we obtained the highest probability of localization of this gene at D2S378,
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a marker that is not so close to TGFA. An explanation for this result could be that TGFA is not the OFC susceptibility gene in the 2p13 region, as otherwise suggested by association studies. Indeed, TGFA could be only a nearby gene. In this instance, it is not easy to explain the presence of linkage disequilibrium with a marker that maps several centimorgans away. Notwithstanding, the two-point analysis under a simple mode of inheritance seems to be quite robust for detecting linkage even when the true model is oligogenic or multifactorial. This approach is not that efficient in estimating recombination fractions (Risch and Giuffra, 1989). It should be pointed out that in a multipoint analysis, with a dense map of markers, the effects of parameter misspecifications have not been well studied. Our investigation indicates genetic heterogeneity and shows evidence of linkage to chromosome 2p in previously identified 6p23-linked families. Very likely, two distinct loci, OFC1 and OFC2, mapping in the two different chromosome regions 6p23 and 2p13–p14 are involved in this complex malformation. This is a very attractive hypothesis and will be the theoretical basis for further investigation. ACKNOWLEDGMENTS This study was supported, in part, by grants from Telethon (E.361) (P.C.), CNR, Target Project ‘‘Biotechnology’’ (M.T.) and bilateral projects (P.C. and M.T.), MURST (P.C. and M.T.), and Azienda Ospedaliera di Ferrara, Arcispedale Sant’Anna (M.T.).
REFERENCES Ardinger, H. H., Buetow, K. H., Bell, G. I., Bardach, J., Van Demark, D. R., and Murray, J. C. (1989). Association of genetic variation of
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the transforming growth factor-alpha gene with cleft lip and palate. Am. J. Hum. Genet. 45: 348–353. Basart, A. M., Quian, J. F., May, E., and Murray, J. C. (1994). A PCR method for detecting polymorphism in the TGFA gene. Hum. Mol. Genet. 3: 678. Beiraghi, S., Foroud, T., Dlouty, S., Bixler, D., Delohter-Blanchet, D., Conneally, P. M., and Hodes, M. E. (1994). Possible localization of a major gene for cleft lip and palate to 4q. Clin. Genet. 46: 255– 256. Carinci, F., Pezzetti, F., Scapoli, L., Padula, E., Baciliero, U., Curioni, C., and Tognon, M. (1995). Nonsyndromic cleft lip and palate: Evidence of linkage to microsatellite markers on 6p23. Am. J. Hum. Genet. 56: 337–339. Chenevix-Trench, G., Jones, K., Green, A. C., and Martin, N. (1991). Further evidence for an association between genetic variation in transforming growth factor alpha and cleft lip and palate. Am. J. Hum. Genet. 48: 1012–1013. Chenevix-Trench, G., Jones, K., Green, A. C., Duffy, D. L., and Martin, N. G. (1992). Cleft lip with or without cleft palate: Associations with transforming growth factor alpha and retinoic acid receptor loci. Am. J. Hum. Genet. 51: 1377–1385. Clementi, M., Tenconi, R., Collins, A., Calzolari, E., and Milan, M. (1995). Complex segregation analysis in a sample of consutive newborns with cleft lip with or without cleft palate in Italy. Hum. Hered. 45: 157–164. Davies, A. F., Stephens, R. J., Olavesen, M. G., Heather, L., Dixon, M. J., Magee, A., Flinter, F., and Ragoussis, J. (1995). Evidence of a locus for orofacial clefting on human chromosome 6p24 and STS content map of the region. Hum. Mol. Genet. 4: 121–128. Dixon, M. J., Garner, J., and Ferguson, M. W. J. (1991). Immunolocalisation of epidermal growth factor (EGF), EGF receptor and transforming growth factor alpha (TGFa) during murine palatogenesis in vivo and in vitro. Anat. Embryol. 184: 83–91. Eiberg, H., Bixler, D., Nielsen, L. S., Conneally, P. M., and Mohr, J. (1987). Suggestion of linkage of a major locus for nonsyndromic orofacial cleft with F13A and tentative assignment to chromosome 6. Clin. Genet. 32: 129–132. Farrall, M., and Holder, S. (1992). Familial recurrence—Pattern analysis of cleft lip with or without cleft palate. Am. J. Hum. Genet. 50: 270–277. Farrall, M., Buetow, K. H., and Murray, J. C. (1993). Resolving an apparent paradox concerning the role of TGFA in CL/P. Am. J. Hum. Genet. 52: 434–436. Feng, H., Sassani, R., Bartlett, S. P., Lee, A., Hecht, J. T., Malcom, S., Winter, R. M., Vintiner, G. M., Buetow, K. H., and Gasser, D. L. (1994). Evidence from family studies, for linkage disequilibrium between TGFA and a gene for nonsyndromic cleft lip with or without cleft palate. Am. J. Hum. Genet. 55: 932–936. Field, L. L., Ray, A. K., and Marazita, M. L. (1994). Transforming growth factor alpha: A modifying locus for nonsyndromic cleft lip with or without cleft palate? Eur. J. Hum. Genet. 2: 159–165. Goldin, L. R., and Weeks, D. E. (1993). Two-locus model of disease: Comparison of likelihood and nonparametric linkage methods. Am. J. Hum. Genet. 53: 908–915. Greenberg, D. A. (1990). Linkage analysis assuming a single-locus mode of inheritance for traits determined by two loci: Inferring mode of inheritance and estimating penetrance. Am. J. Hum. Genet. 55: 834–840. Gyapay, G., Morissette, J., Vignal, A., Dib, C., Fizames, C., Millasseau, P., Mare, S., Bernardi, G., Lathrop, M., and Weissenbach, J. (1994). The 1993–94 Ge´ne´thon human genetic linkage map. Nature Genet. 7: 246–249. Healey, S. C., Mitchell, L. E., and Chenevix-Trench, G. (1994). Evidence for an association between non-syndromic cleft lip with or without cleft palate and a gene located on the long arm of chromosome 4. Am. J. Hum. Genet. 55(Suppl): A47.
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Hecht, J. T., Wang, Y., Blanton, S. H., Michels, V. V., and Daiger, S. P. (1991). Cleft lip and palate: No evidence of linkage to transforming growth factor alpha. Am. J. Hum. Genet. 49: 682–686. Hecht, J. T., Wang, Y., Connor, B., Blanton, S. H., and Daiger, S. P. (1993). Nonsyndromic cleft lip and palate: No evidence of linkage to HLA or factor 13A. Am. J. Hum. Genet. 52: 1230–1233. Higuchi, R. (1989). Simple and rapid preparation of samples for PCR. In ‘‘PCR Technology’’ (H. A. Erlich, Ed.), pp. 31–38, Stockton Press, New York. Holder, S. E., Vintiner, G. M., Farren, B., Malcolm, S., and Winter, R. M. (1992). Confirmation of an association between RFLPs at the transforming growth factor-alpha locus and nonsyndromic cleft lip and palate. J. Med. Genet. 29: 390–392. Jara, L., Blanco, R., Chiffelle, I., Palomino, H., and Carreno, H. (1995). Associations between alleles of the transforming growth factor alpha locus and cleft lip and palate in the Chilean population. Am. J. Med. Genet. 57: 548–551. Kurihara, Y., Kurihara, H., Suzuki, H., Kodama, T., Maemura, K., Nagai, R., Oda, H., Kuwaki, T., Cao, W. H., Kamada, N., Jishage, K., Ouchi, Y., Azuma, S., Toyoda, Y., Ishikawa, T., Kumada, M., and Yazaki, Y. (1994). Elevated pressure and craniofacial abnormalities in mice deficient in endothelin-1. Nature 368: 703–710. Lathrop, G. M., Lalouel, J. M., Julier, C., and Ott, J. (1984). Strategies for multilocus linkage analysis in human. Proc. Natl. Acad. Sci. USA 81: 3443–3446. Luettke, N. C., Qiu, T. H., Peiffer, R. L., Oliver, P., Smithies, O., and Lee, D. C. (1993). TGF alpha deficiency results in a hair follicle and eye abnormalities in targeted and waved-1 mice. Cell 73: 263– 278. Mehra, S., and Verma, I. C. (1991). Ecogenetics of congenital craniofacial malformation. International Committee on the Human Genome. Am. J. Hum. Genet. 49(Suppl): A150. Mitchell, L. E. (1997). Transforming growth factor alpha locus and nonsyndromic cleft lip with or without cleft palate: A reappraisal. Genet. Epidemiol. 14: 231–240. Mitchell, L. E., and Risch, N. (1992). Mode of inheritance of nonsyndromic cleft lip with or without cleft palate: A reanalysis. Am. J. Hum. Genet. 51: 323–332. Murray, J. C. (1995). Face facts: Genes, environment, and cleft. Am. J. Hum. Genet. 57: 227–232. Risch, N., and Giuffra, L. (1989). Multipoint linkage analysis of genetically complex traits. Am. J. Hum. Genet. 47(Suppl.): A197. Sandgren, E. P., Luettke, N. C., Palmiter, R. D., Brinster, R. L., and Lee, D. C. (1990). Overexpression of TGFa in transgenic mice: Induction of epithelial hyperplasia, pancreatic metaplasia, and carcinoma of the breast. Cell 61: 1121–1135. Sassani, R., Bartlet, S. P., Feng, H., Goldner-Sauve, A., Haq, A. K., Buetow, K. H., and Gasser, D. L. (1993). Associations between alleles of the transforming growth factor-alpha locus and the occurrence of cleft lip. Am. J. Med. Genet. 45: 565–569. Scambler, P. J., Heather, L., Gorman, P., Lindsay, E., Yamamura, K. I., and Dixon, M. (1992). The human homolog of a sequence disrupted in a murine craniofacial dysmorphism maps to 6p: A candidate locus for nonsyndromic cleft lip/or palate. Am. J. Hum. Genet. 51(Suppl.): A50. Scapoli, L., Pezzetti, F., Carinci, F., Martinelli, M., Carinci, P., and Tognon, M. (1997). Genetic heterogeneity and evidence of linkage to 6p23 in nonsyndromic cleft lip with or without cleft palate. Genomics 43: 216–220. Scapoli, L., Pezzetti, F., Carinci, F., Martinelli, M., Carinci, P., and Tognon, M. (1998). Lack of linkage disequilibrium between transforming growth factor alpha Taq I polymorphism and cleft lip with or without cleft palate in families from northeastern Italy. Am. J. Med. Genet. 75: 203–206. Shaw, D., Ray, A., Marazita, M., and Field, L. (1993). Further evidence of a relationship between the retinoic acid receptor alpha
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A LOCUS IN 2p13–p14 IS INVOLVED IN OFC (OFC2) locus and nonsyndromic cleft lip with or without cleft palate (CL{P). Am. J. Hum. Genet. 53: 1156–1157. Shaw, G. M., Wasserman, C. R., Lammer, E. J., O’Malley, C. D., Murray, J. C., Basart, A. M., and Tolarova, M. M. (1996). Orofacial clefts, parental cigarette smoking, and transforming growth factoralpha gene variants. Am. J. Hum. Genet. 58: 551–561. Stein, J., Mulliken, J. B., Stal, S., Gasser, D. L., Malcolm, S., Winter, R., Blanton, S. H., Amos, C., Seemanova, E., and Hecht, J. T. (1995). Nonsyndromic cleft lip with or without cleft palate: Evidence of linkage to BCL3 in 17 multigenerational families. Am. J. Hum. Genet. 57: 257–272. Stoll, C., Quian, J. F., Feringold, J., Sauvage, P., and May, E. (1993). Genetic variation in transforming growth factor alpha: Possible association of Bam HI polymorphism with bilateral sporadic cleft lip and palate. Hum. Genet. 92: 81–82. Terwillinger, J. D., and Ott, J. (1994). ‘‘Handbook of Human Genetic Linkage,’’ pp.224–225, Johns Hopkins Univ. Press, Baltimore/London.
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Van Dyke, D. C., Goldman, A. S., Spielman, R. S., Zmijewski, C. M., and Oka, S. W. (1980). Segregation of HLA in sibs with cleft lip or cleft lip and palate: Evidence against linkage. Cleft Palate J. 17: 189–193. Vieland, V. J., Hodge, S. E., and Greenberg, D. A. (1992). Adequacy of single-locus approximations for linkage analyses of oligogenic traits. Genet. Epidemiol. 9: 45–59. Vintiner, G. M., Holder, S. E., Winter, R. M., and Malcolm, S. (1992). No evidence of linkage between the transforming growth factor alpha gene in families with apparently autosomal dominant inheritance of cleft lip and palate. J. Med. Genet. 29: 393–397. Vintiner, G. M., Lo, K. K., Holder, S. E., Minter, R. M., and Malcolm, S. (1993). Exclusion of candidate genes from a role in cleft lip with or without cleft palate: Linkage and association studies. J. Med. Genet. 30: 773–778. Watanabe, T., Ohishi, M., and Tashiro, H. (1984). Population and family studies of HLA in Japanese with cleft lip and palate. Cleft Palate J. 21: 293–300.
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ARTICLE NO.
A Locus in 2p13–p14 (OFC2), in Addition to That Mapped in 6p23, Is Involved in Nonsyndromic Familial Orofacial Cleft Malformation Furio Pezzetti,* Luca Scapoli,* Marcella Martinelli,* Francesco Carinci,† Maria Bodo,* Paolo Carinci,‡ and Mauro Tognon*,1 *Department of Morphology and Embryology, Section of Histology and General Embryology, and †School of Medicine, University of Ferrara, Via Fossato di Mortara 64/B, 44100 Ferrara, Italy; and ‡Institute of Histology and General Embryology, School of Medicine, University of Bologna, Via Belmeloro 8, 40126 Bologna, Italy Received December 8, 1997; accepted February 16, 1998
An allelic association between the transforming growth factor a gene (TGFA) situated in the chromosome 2p13 region and nonsyndromic cleft lip with or without cleft palate, also named orofacial cleft (OFC), was found in several population studies. However, no linkage between gene and malformation has shown up until now, probably due to the presence of genetic heterogeneity and the small sample size analyzed. Previously, we employed a collection of 38 OFC families to demonstrate linkage to the 6p23 chromosome region with the presence of genetic heterogeneity. In the present study we tested whether, in the same sample, linkage between OFC and markers on 2p13 could be determined. Evidence for genetic heterogeneity in our family set was apparent, by both pairwise and multipoint linkage analyses. Moreover, lod scores ú3 were found for marker D2S378 when families linked to the 6p23 markers were analyzed. Taken together these results indicate a role for the TGFA, or for another gene physically close to it, and suggest an interaction between two different genes, OFC1 and OFC2, mapped in 6p23 and 2p13, respectively, in the development of the cleft. q 1998 Academic Press
INTRODUCTION
Nonsyndromic cleft lip with or without cleft palate, also named orofacial cleft (CL{P Å OFC) derives from an embryopathy with a consequent failure of fusion of the nasal process and palatal shelves. This severe birth defect is one of the most common malformations, affecting approximately 1/1000 live births among Caucasians. There is a large body of evidence that genetic factors are important in its etiology. However, the nature of the genetic contribution to the etiology of non1
To whom correspondence should be addressed at the School of Medicine, University of Ferrara, Via Fossato di Mortara 64/B, 44100 Ferrara, Italy. Telephone: /39-532-291538. Fax: /39-532-291533. E-mail: [email protected].
syndromic OFC is not completely understood (Murray, 1995). Ardinger et al. (1989), using restriction fragment length polymorphism (RFLP) analysis, reported a significant association between the transforming growth factor a gene (TGFA) located on 2p13 and the occurrence of clefting; since then, this locus has been investigated by several groups. Indeed, some authors confirmed this association (Chenevix-Trench et al., 1991, 1992; Holder et al., 1992; Sassani et al., 1993; Jara et al., 1995), while others found association only with the severity of the cleft (Stoll et al., 1993; Field et al., 1994). Recently, Mitchell (1997), through a meta-analysis study, confirmed the association between TGFA and OFC; however, his data were not conclusive, because heterogeneity was also observed. Shaw et al. (1996) did not detect allelic association between TGFA and OFC in a study based on a large population; however, they did find a strong association between maternal smoking and a genetic variant of TGFA at risk for cleft development. In fact, the risk of this malformation was approx twice as much if the mother smoked during pregnancy, but was even greater for infants when a negative synergistic effect occurred between smoking and a rare TGFA allele previously found associated with clefting. From the results of this study, one may infer that this is an example of gene–environment interaction in the occurrence of facial clefting. It seems that population-based studies support a role for TGFA, or a nearby gene (OFC2), as the susceptible gene involved in some way in OFC malformation. However, three different studies did not find linkage with TGFA using the lod score method of analysis (Hecht et al., 1991; Vintiner et al., 1992; Field et al., 1994). Since genetic heterogeneity was detected for OFC (Carinci et al., 1995; Stein et al., 1995; Scapoli et al., 1997), failure to detect linkage with TGFA could be due to the small number of families analyzed (Farrall et al., 1993). On the other hand, Feng et al. (1994), in a family-based association study, found significant positive linkage disequilibrium with the C2 allele of TGFA, thus sup-
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porting a role for this gene in OFC malformation. In the murine model, a TGFA gene knockout experiment indicated that the gene-deficient animals did not develop OFC (Luettke et al., 1993). This result suggests that TGFA may act as a modifier gene rather than being a necessary and sufficient agent on its own for OFC disease. Biological relevance for TGFA playing a role in clefting is provided by its expression pattern in palate formation (Dixon et al., 1991). A transgenic mouse, in which TGFA was overexpressed, had epithelial hyperplasia in a number of tissues, thus demonstrating that TGFA is a potent epithelial mitogen (Sandgren et al., 1990). Other chromosome regions were investigated for a putative OFC locus/loci; it is worth remembering the studies on the 6p region containing the OFC1 gene. Indeed, different groups testing the linkage with the HLA locus provided either negative (Van Dyke et al., 1980; Watanabe et al., 1984), or positive (Mehra and Verma, 1991) results. Other reports indicated that a clefting locus could be located on chromosome 6p close to different possible loci: factor 13A (F13A), which maps in the region 6p25–p24 (Eiberg et al., 1987), and the HGP22 and AP2 genes, which are potentially involved in face formation and which map at 6p24 (Scambler et al., 1992; Davies et al., 1995). Moreover, Kurihara et al. (1994) observed craniofacial abnormalities in transgenic mice deficient in the EDN1 gene product. Different data were recently reported by two other groups (Hecht et al., 1993; Vintiner et al., 1993), who found no linkage between OFC and the 6p region (between the HLA and the F13A loci). On the other hand, in a recent investigation we found, by studying the microsatellite markers and by linkage analysis, that an OFC locus maps to chromosome 6p23 (Carinci et al., 1995; Scapoli et al., 1997).
These data suggested to us that we test whether, in our collection of 38 nonsyndromic OFC families, previously employed for a similar investigation on chromosome region 6p23 (Carinci et al., 1995; Scapoli et al., 1997), any linkage between OFC and markers on 2p13 could be determined. MATERIAL AND METHODS Family descriptions. We studied 38 families who were previously analyzed for linkage to the 6p23 chromosome region (Scapoli et al., 1997) and were characterized by the presence, in each pedigree, of at least two OFC-affected individuals. The affected members were relatives of first, second, third, or fourth degree. All the families enrolled were of Italian ancestry, from northeastern regions, without immigrants and included subjects presenting OFC as the unique familial disease. Indeed, the cleft lip, with or without cleft palate, was the only anomaly detected after a detailed clinical examination performed by the dysmorphologist of our research group (F.C.). Moreover, those families using clefting drugs, such as phenitoin, warfarin, and ethanol, were excluded from the study. The pedigrees were composed of 8 OFC families with two generations, 15 with three generations, 12 with four generations, and 3 with five generations, for a total of 378 individuals, 96 affected by OFC. DNA studies. Blood samples, after obtaining consent, were drawn from 268 individuals, 78 of whom were affected. DNA, prepared from peripheral blood cells (Higuchi, 1989), was analyzed for seven dinucleotide repeats at loci D2S145, D2S285, D2S134, D2S380, D2S337, D2S378, and D2S123, having heterozygosity of 0.58, 0.85, 0.77, 0.85, 0.88, 0.81, and 0.76, respectively (Gyapay et al., 1994). Moreover, the TaqI polymorphism at the TGFA locus (heterozygosity Å 0.29), due to a 4-base insertion in intron V of the gene, was typed by PCR methods as reported before (Basart et al., 1994). DNA samples were denatured at 947C for 30 s, annealed at 577C for 20 s, and extended at 727C for 10 s. For D2S134 the annealing temperature was at 587C and for D2S380 and TGFA TaqI 597C. All amplifications were processed through 35 cycles. Small aliquots of PCR-amplified products were resolved by polyacrylamide gel electrophoresis and visualized by silver staining. Linkage studies. The eight markers were analyzed for linkage to OFC by using the computer program LINKAGE, version 5.1 (Lathrop et al., 1984), which includes MLINK and LINKMAP programs.
TABLE 1 Two-Point Lod Score for OFC versus Chromosome 2 Markers in 38 Families, Calculated under Autosomal Dominant Model with Reduced Penetrance (RP) and Affected Only (AO) Methods Lod score at recombination fraction of Marker
Method
0.001
0.01
0.05
0.10
0.20
0.30
0.40
RP AO RP AO RP AO RP AO RP AO RP AO RP AO RP AO
02.02 02.00 03.68 03.65 010.28 010.24 07.58 07.10 010.67 010.39 04.20 04.17 04.37 04.21 05.33 05.17
01.23 01.21 02.93 02.91 08.14 08.16 05.51 05.09 07.82 07.61 03.48 03.43 03.70 03.52 03.95 03.83
0.09 0.14 01.40 01.41 04.61 04.62 02.00 01.66 03.26 03.08 01.93 01.83 01.99 01.81 01.62 01.54
0.71 0.78 00.59 00.60 02.66 02.65 00.32 00.05 00.97 00.82 00.78 00.67 00.68 00.52 00.44 00.37
0.96 1.02 0.09 0.07 00.77 00.75 0.90 1.07 0.81 0.90 0.33 0.42 0.53 0.63 0.46 0.51
0.71 0.75 0.26 0.25 00.05 00.03 1.03 1.13 1.10 1.15 0.58 0.64 0.75 0.81 0.57 0.60
0.34 0.36 0.21 0.20 0.12 0.13 0.66 0.70 0.74 0.76 0.41 0.44 0.50 0.53 0.34 0.36
D2S145 TGFA TaqI D2S285 D2S134 D2S380 D2S337 D2S378 D2S123
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A LOCUS IN 2p13–p14 IS INVOLVED IN OFC (OFC2) A complex segregation analysis was performed in our family set; the results were consistent with a two-locus model with a major dominant locus and one modifier locus (C. Scapoli et al., submitted). Based on those analyses, we used for lod score calculations a dominant mode of inheritance with disease allele frequency of 0.0035; the penetrance was 0.12 for males and 0.06 for females (reduced penetrance model; RP). A two-locus model with a major dominant locus and at least one modifier locus was also proposed by Clementi et al. (1995), using a large sample of 636 newborns with OFC registered in northeastern Italy. Moreover, a combination of different genes working together to produce OFC was proposed by other authors (Farrall and Holder, 1992; Mitchell and Risch, 1992). If this is the case, then many unaffected individuals may possess the disease-predisposing genotype at one of the loci, but lack the required second disease locus genotype necessary for the development of the disease. For this reason it is advisable to consider all unaffected individuals to actually have ‘‘unknown’’ phenotype when performing linkage analyses. In this way, one bases the linkage analyses solely on the marker status of the affected individuals in the pedigree and does not apply any disease locus genotypic information to the unaffected individuals. To perform the ‘‘affected only’’ analyses (AO), all marker data were retained in the pedigree file, whereas the maximum penetrance values were set to 0.001 (Terwillinger and Ott, 1994). The multipoint analyses were performed using the selected markers in the following order: D2S145–TGFA TaqI–D2S285–D2S134– D2S380–D2S337–D2S378–D2S123. The interlocus distances in centimorgans, which are in the order: 1–4–3–0.1–5–4–4, were from the Ge´ne´thon map (Gyapay et al., 1994). The distances are in agreement with our study (data not shown). To verify the homogeneity of the pedigrees, the results obtained from linkage analysis were further analyzed using the HOMOG computer program.
RESULTS
Thirty-eight multiplex OFC families were tested with each of the eight 2p13–p14 markers. Low positive lod scores were found for all markers at recombination fractions between 0.2 and 0.3, using the AO model or the RP model (Table 1). The maximum lod score obtained in the pairwise linkage analysis was 1.15 for D2S380 at u Å 0.30. The homogeneity test performed by the HOMOG computer program on the two-point data under the AO model provided evidence of heterogeneity for the markers D2S145, D2S337, and D2S378, P values 0.0452, 0.0294, and 0.0301 at u Å 0.00 with a Å 0.50, 0.45, and 0.45, respectively. Interestingly, by adopting the RP model, the homogeneity test showed evidence of heterogeneity for the same markers, P values 0.0477, 0.0287, and 0.0340 with a Å 0.50, 0.45, and 0.45 at u Å 0.001, respectively. In multipoint linkage analyses of OFC, the maximum lod score was obtained at 30 cM telomeric to D2S123; the values were 1.30 under the AO model and 1.06 under the RP model. The homogeneity test performed by the HOMOG computer program on the multipoint data under the AO model provided evidence of heterogeneity in all the intervals between markers D2S285 and D2S123. The maximum significance level for the hypothesis of heterogeneity (P value 0.0072) was obtained with 35% of families linked at D2S378. For the RP model, the homogeneity test showed a P
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TABLE 2 Posterior Probabilities of Linkage Calculated for Chromosome Regions 6p and 2p, by Two Models of Inheritance Reduced penetrance
Affected only
Family
6p
2p
6p
2p
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
0.93 0.90 0.80 0.81 0.86 0.63 0.02 0.01 0.32 0.01 0.02 0.73 0.59 0.79 0.64 0.87 0.60 0.64 0.61 0.64 0.80 0.08 0.75 0.69 0.52 0.47 0.75 0.69 0.64 0.80 0.74 0.55 0.96 0.60 0.74 0.80 0.56 0.01
0.79 0.66 0.15 0.48 0.03 0.35 0.03 0.03 0.07 0.08 0.01 0.53 0.32 0.53 0.33 0.79 0.34 0.34 0.50 0.33 0.51 0.03 0.49 0.53 0.36 0.37 0.52 0.50 0.37 0.53 0.51 0.37 0.00 0.52 0.08 0.63 0.34 0.01
0.90 0.86 0.86 0.75 0.85 0.60 0.02 0.01 0.39 0.01 0.02 0.75 0.60 0.75 0.60 0.92 0.60 0.60 0.60 0.60 0.79 0.06 0.75 0.74 0.60 0.60 0.75 0.75 0.60 0.75 0.74 0.60 0.96 0.60 0.74 0.85 0.54 0.01
0.81 0.68 0.16 0.49 0.03 0.35 0.03 0.03 0.07 0.07 0.01 0.51 0.35 0.52 0.35 0.80 0.35 0.35 0.51 0.35 0.52 0.03 0.49 0.52 0.35 0.35 0.52 0.51 0.35 0.52 0.51 0.35 0.00 0.51 0.08 0.66 0.35 0.01
Note. The values were calculated on the multipoint data at markers D6S259 and D2S278.
value 0.0093 with 35% of families linked at u Å 0.00 for D2S378. With both models, 14 of the 38 families analyzed showed ¢50% likelihood of being linked (Table 2); under the reduced penetrance model, 21 families had positive lod scores, whereas 17 were negative. By adopting the affected-only model, 16 families were positive, 11 negative, and 11 not informative. In a recent study using the same 38 multigenerational families, we demonstrated linkage between OFC and chromosome region 6p23 with genetic heterogeneity (Scapoli et al., 1997); these results, obtained by the HOMOG program, indicated that 30 of 38 families had a posterior probability of ¢50% in favor of linkage to D6S259 using both models of inheritance (Table 2).
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TABLE 3 Two-Point Lod Score for OFC versus Chromosome 2 Markers in 30 6p23-Linked Families, Calculated under the Autosomal Dominant Model with Reduced Penetrance (RP) and Affected Only (AO) Methods Lod score at recombination fraction of Marker
Method
0.001
0.01
0.05
0.10
0.20
0.30
0.40
RP AO RP AO RP AO RP AO RP AO RP AO RP AO RP AO
0.63 0.74 03.02 02.99 04.92 04.82 00.50 00.24 02.46 02.25 1.33 1.48 3.64 3.81 00.92 00.77
1.20 1.30 02.43 02.40 03.45 03.39 0.37 0.61 00.81 00.64 1.60 1.73 3.79 3.96 00.05 0.09
1.92 2.01 01.23 01.22 01.28 01.23 1.55 1.75 1.29 1.43 1.84 1.97 3.78 3.94 1.08 1.20
1.99 2.07 00.58 00.57 00.29 00.24 1.97 2.12 2.04 2.16 1.90 2.03 3.52 3.64 1.45 1.55
1.54 1.58 00.02 00.02 0.44 0.46 1.98 2.08 2.21 2.29 1.71 1.82 2.78 2.85 1.46 1.52
0.92 0.94 0.15 0.15 0.50 0.52 1.52 1.57 1.71 1.75 1.24 1.31 1.88 1.92 1.08 1.12
0.38 0.39 0.14 0.13 0.29 0.30 0.83 0.85 0.92 0.94 0.65 0.68 0.94 0.96 0.56 0.58
D2S145 TGFA TaqI D2S285 D2S134 D2S380 D2S337 D2S378 D2S123
Herein, the two groups of families selected a priori, i.e., the 6p23-linked group and the 6p23-unlinked group, were analyzed separately for linkage to the 2p13 chromosome region. The pairwise linkage analyses performed on the two family groups gave different results. In fact, the 6p23-linked families showed a positive lod score with a maximum of 3.96 at u Å 0.01 for marker D2S378 under the AO model and a lod score of 3.79 at u Å 0.01 under the RP model (Table 3). On the other hand, negative lod scores were obtained for the 6p23unlinked families using either one of the two inheritance models (Table 4). DISCUSSION
Early attempts to localize the OFC gene(s) in the human genome were performed by different groups. These studies provided numerous and sometimes discordant information (Murray, 1995). Significant results regarding the chromosome region 2p account for the association between the TGFA locus and the occurrence of clefting by using RFLP analysis (Ardinger et al., 1989). Later, other studies confirmed this association (Chenevix-Trench et al., 1991, 1992; Holder et al., 1992; Sassani et al., 1993; Mitchell, 1997). Moreover, Feng et al. (1994) using the transmission disequilibrium test, showing a significant linkage disequilibrium with the C2 allele of the TGFA locus; however, our group, using the same approach, did not confirm these data (Scapoli et al., 1998), and the exclusion of TGFA as a candidate locus was obtained by linkage studies in a series of multiplex OFC families (Hecht et al., 1991; Vintiner et al., 1992). In addition, many molecular genetic investigations supported major gene/oligogenic models in the develop-
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ment of the cleft, by evidencing loci in different chromosome regions. Important studies indicated different loci on 6p23 (OFC1) (Eiberg et al., 1987; Carinci et al., 1995; Davies et al., 1995; Scapoli et al., 1997), 4q25–q31 (Beiraghi et al., 1994; Healey et al., 1994), 19q13 (BCL3 gene) (OFC3) (Stein et al., 1995), and 17q21 (RARA) (Chenevix-Trench et al., 1992; Shaw et al., 1993). Since the etiology of OFC seems quite complex, very likely several loci may contribute to this malformation. Farral and Holder (1992) predicted that four to seven different genes could be involved in developing the cleft malformation. In a recent investigation, a complex segregation analysis was performed in a sample of 636 newborns with OFC registered in northeastern Italy; a two-locus model with a major dominant locus and at least one modifier locus was proposed by the authors (Clementi et al., 1995). In linkage analyses, diseases are usually assumed to result from the action of a single gene, possibly modified by random environmental effects. Other models, such as oligogenic or multifactorial models, may be more realistic for OFC. Fortunately, numerous studies have shown that there is little danger of missing a true linkage if one analyzes the data by assuming the wrong overall genetic mechanism (e.g., assuming a single major locus when the disease is, in fact, oligogenic) (Greenberg, 1990; Vieland et al., 1992; Goldin and Weeks, 1993). Moreover, it is known that in an analysis for linkage with the lod score method assuming an incorrect mode of inheritance, the probability of type I error (getting a high lod score value by chance alone) is not inflated. Traditional linkage analyses of OFC disease identified some putative loci involved in this malformation. Beiraghi et al. (1994) reported suggestive evidence for
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A LOCUS IN 2p13–p14 IS INVOLVED IN OFC (OFC2)
TABLE 4 Two-Point Lod Score for OFC versus Chromosome 2 Markers in Eight 6p23-Unlinked Families, Calculated under Autosomal Dominant Model with Reduced Penetrance (RP) and Affected Only (AO) Methods Lod score at recombination fraction of Marker
Method
0.001
0.01
0.05
0.10
0.20
0.30
0.40
RP AO RP AO RP AO RP AO RP AO RP AO RP AO RP AO
02.65 02.74 00.66 00.66 05.36 05.42 07.08 06.86 08.21 08.14 05.53 05.65 08.01 08.02 04.41 04.40
02.43 02.51 00.50 00.51 04.69 04.77 05.88 05.70 07.01 06.97 05.08 05.16 07.49 07.48 03.90 03.92
01.83 01.87 00.17 00.19 03.33 03.39 03.55 03.41 04.55 04.51 03.77 03.80 05.77 05.75 02.70 02.74
01.28 01.29 00.01 00.03 02.37 02.41 02.29 02.17 03.01 02.98 02.68 02.70 04.20 04.16 01.89 01.92
00.58 00.56 0.11 0.09 01.21 01.21 01.08 01.01 01.40 01.39 01.38 01.40 02.25 02.22 01.00 01.01
00.21 00.19 0.11 0.10 00.55 00.55 00.49 00.44 00.61 00.60 00.66 00.67 01.13 01.11 00.51 00.52
00.04 00.03 0.07 0.07 00.17 00.17 00.17 00.15 00.18 00.18 00.24 00.24 00.44 00.43 00.22 00.22
D2S145 TGFA TaqI D2S285 D2S134 D2S380 D2S337 D2S378 D2S123
linkage using markers on 4q in a single large family with OFC. Genetic heterogeneity for OFC was reported by other authors in a sample of 39 families (Stein et al., 1995); in this study linkage was found with the BCL3 gene on chromosome 19, but for only 17 of 39 families. Moreover, in a recent study on OFC we found linkage and heterogeneity by analyzing markers on 6p23 (Scapoli et al., 1997). In this investigation, we tested linkage to the OFC2 region in the same sample previously analyzed, consisting of 38 families, with the same racial and ethnic origin from northeastern Italy. Evidence for genetic heterogeneity in our family set was apparent, whereas it was shown that markers in region 2p are tightly linked to OFC in 14 multigenerational families under both models. From these results we infer that a gene in the 2p region does play an etiologic role in OFC development and that the loci involved in this malformation can be detected by linkage study with a large number of families. It is of interest to note that the 6p23-linked families showed a positive lod score and reached the significance 3.96 at u Å 0.01 for the marker D2S378. This result suggests an interactive effect of the two disease loci mapping in 2p13 and 6p23; however, no attempts were made in this study to assay the nature of interaction of these genes. This hypothesis, i.e., two or more genes simultaneously involved in OFC, is supported by recent studies (Farrall and Holder, 1992; Clementi et al., 1995). Interestingly, Murray (1995) postulated that the strongest evidence implicates a primary role for OFC1 on 6p, whereas OFC2 on 2p13 is a modifier of clefting status. At present it is not clear why we obtained the highest probability of localization of this gene at D2S378,
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a marker that is not so close to TGFA. An explanation for this result could be that TGFA is not the OFC susceptibility gene in the 2p13 region, as otherwise suggested by association studies. Indeed, TGFA could be only a nearby gene. In this instance, it is not easy to explain the presence of linkage disequilibrium with a marker that maps several centimorgans away. Notwithstanding, the two-point analysis under a simple mode of inheritance seems to be quite robust for detecting linkage even when the true model is oligogenic or multifactorial. This approach is not that efficient in estimating recombination fractions (Risch and Giuffra, 1989). It should be pointed out that in a multipoint analysis, with a dense map of markers, the effects of parameter misspecifications have not been well studied. Our investigation indicates genetic heterogeneity and shows evidence of linkage to chromosome 2p in previously identified 6p23-linked families. Very likely, two distinct loci, OFC1 and OFC2, mapping in the two different chromosome regions 6p23 and 2p13–p14 are involved in this complex malformation. This is a very attractive hypothesis and will be the theoretical basis for further investigation. ACKNOWLEDGMENTS This study was supported, in part, by grants from Telethon (E.361) (P.C.), CNR, Target Project ‘‘Biotechnology’’ (M.T.) and bilateral projects (P.C. and M.T.), MURST (P.C. and M.T.), and Azienda Ospedaliera di Ferrara, Arcispedale Sant’Anna (M.T.).
REFERENCES Ardinger, H. H., Buetow, K. H., Bell, G. I., Bardach, J., Van Demark, D. R., and Murray, J. C. (1989). Association of genetic variation of
gnmas
304
PEZZETTI ET AL.
the transforming growth factor-alpha gene with cleft lip and palate. Am. J. Hum. Genet. 45: 348–353. Basart, A. M., Quian, J. F., May, E., and Murray, J. C. (1994). A PCR method for detecting polymorphism in the TGFA gene. Hum. Mol. Genet. 3: 678. Beiraghi, S., Foroud, T., Dlouty, S., Bixler, D., Delohter-Blanchet, D., Conneally, P. M., and Hodes, M. E. (1994). Possible localization of a major gene for cleft lip and palate to 4q. Clin. Genet. 46: 255– 256. Carinci, F., Pezzetti, F., Scapoli, L., Padula, E., Baciliero, U., Curioni, C., and Tognon, M. (1995). Nonsyndromic cleft lip and palate: Evidence of linkage to microsatellite markers on 6p23. Am. J. Hum. Genet. 56: 337–339. Chenevix-Trench, G., Jones, K., Green, A. C., and Martin, N. (1991). Further evidence for an association between genetic variation in transforming growth factor alpha and cleft lip and palate. Am. J. Hum. Genet. 48: 1012–1013. Chenevix-Trench, G., Jones, K., Green, A. C., Duffy, D. L., and Martin, N. G. (1992). Cleft lip with or without cleft palate: Associations with transforming growth factor alpha and retinoic acid receptor loci. Am. J. Hum. Genet. 51: 1377–1385. Clementi, M., Tenconi, R., Collins, A., Calzolari, E., and Milan, M. (1995). Complex segregation analysis in a sample of consutive newborns with cleft lip with or without cleft palate in Italy. Hum. Hered. 45: 157–164. Davies, A. F., Stephens, R. J., Olavesen, M. G., Heather, L., Dixon, M. J., Magee, A., Flinter, F., and Ragoussis, J. (1995). Evidence of a locus for orofacial clefting on human chromosome 6p24 and STS content map of the region. Hum. Mol. Genet. 4: 121–128. Dixon, M. J., Garner, J., and Ferguson, M. W. J. (1991). Immunolocalisation of epidermal growth factor (EGF), EGF receptor and transforming growth factor alpha (TGFa) during murine palatogenesis in vivo and in vitro. Anat. Embryol. 184: 83–91. Eiberg, H., Bixler, D., Nielsen, L. S., Conneally, P. M., and Mohr, J. (1987). Suggestion of linkage of a major locus for nonsyndromic orofacial cleft with F13A and tentative assignment to chromosome 6. Clin. Genet. 32: 129–132. Farrall, M., and Holder, S. (1992). Familial recurrence—Pattern analysis of cleft lip with or without cleft palate. Am. J. Hum. Genet. 50: 270–277. Farrall, M., Buetow, K. H., and Murray, J. C. (1993). Resolving an apparent paradox concerning the role of TGFA in CL/P. Am. J. Hum. Genet. 52: 434–436. Feng, H., Sassani, R., Bartlett, S. P., Lee, A., Hecht, J. T., Malcom, S., Winter, R. M., Vintiner, G. M., Buetow, K. H., and Gasser, D. L. (1994). Evidence from family studies, for linkage disequilibrium between TGFA and a gene for nonsyndromic cleft lip with or without cleft palate. Am. J. Hum. Genet. 55: 932–936. Field, L. L., Ray, A. K., and Marazita, M. L. (1994). Transforming growth factor alpha: A modifying locus for nonsyndromic cleft lip with or without cleft palate? Eur. J. Hum. Genet. 2: 159–165. Goldin, L. R., and Weeks, D. E. (1993). Two-locus model of disease: Comparison of likelihood and nonparametric linkage methods. Am. J. Hum. Genet. 53: 908–915. Greenberg, D. A. (1990). Linkage analysis assuming a single-locus mode of inheritance for traits determined by two loci: Inferring mode of inheritance and estimating penetrance. Am. J. Hum. Genet. 55: 834–840. Gyapay, G., Morissette, J., Vignal, A., Dib, C., Fizames, C., Millasseau, P., Mare, S., Bernardi, G., Lathrop, M., and Weissenbach, J. (1994). The 1993–94 Ge´ne´thon human genetic linkage map. Nature Genet. 7: 246–249. Healey, S. C., Mitchell, L. E., and Chenevix-Trench, G. (1994). Evidence for an association between non-syndromic cleft lip with or without cleft palate and a gene located on the long arm of chromosome 4. Am. J. Hum. Genet. 55(Suppl): A47.
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Hecht, J. T., Wang, Y., Blanton, S. H., Michels, V. V., and Daiger, S. P. (1991). Cleft lip and palate: No evidence of linkage to transforming growth factor alpha. Am. J. Hum. Genet. 49: 682–686. Hecht, J. T., Wang, Y., Connor, B., Blanton, S. H., and Daiger, S. P. (1993). Nonsyndromic cleft lip and palate: No evidence of linkage to HLA or factor 13A. Am. J. Hum. Genet. 52: 1230–1233. Higuchi, R. (1989). Simple and rapid preparation of samples for PCR. In ‘‘PCR Technology’’ (H. A. Erlich, Ed.), pp. 31–38, Stockton Press, New York. Holder, S. E., Vintiner, G. M., Farren, B., Malcolm, S., and Winter, R. M. (1992). Confirmation of an association between RFLPs at the transforming growth factor-alpha locus and nonsyndromic cleft lip and palate. J. Med. Genet. 29: 390–392. Jara, L., Blanco, R., Chiffelle, I., Palomino, H., and Carreno, H. (1995). Associations between alleles of the transforming growth factor alpha locus and cleft lip and palate in the Chilean population. Am. J. Med. Genet. 57: 548–551. Kurihara, Y., Kurihara, H., Suzuki, H., Kodama, T., Maemura, K., Nagai, R., Oda, H., Kuwaki, T., Cao, W. H., Kamada, N., Jishage, K., Ouchi, Y., Azuma, S., Toyoda, Y., Ishikawa, T., Kumada, M., and Yazaki, Y. (1994). Elevated pressure and craniofacial abnormalities in mice deficient in endothelin-1. Nature 368: 703–710. Lathrop, G. M., Lalouel, J. M., Julier, C., and Ott, J. (1984). Strategies for multilocus linkage analysis in human. Proc. Natl. Acad. Sci. USA 81: 3443–3446. Luettke, N. C., Qiu, T. H., Peiffer, R. L., Oliver, P., Smithies, O., and Lee, D. C. (1993). TGF alpha deficiency results in a hair follicle and eye abnormalities in targeted and waved-1 mice. Cell 73: 263– 278. Mehra, S., and Verma, I. C. (1991). Ecogenetics of congenital craniofacial malformation. International Committee on the Human Genome. Am. J. Hum. Genet. 49(Suppl): A150. Mitchell, L. E. (1997). Transforming growth factor alpha locus and nonsyndromic cleft lip with or without cleft palate: A reappraisal. Genet. Epidemiol. 14: 231–240. Mitchell, L. E., and Risch, N. (1992). Mode of inheritance of nonsyndromic cleft lip with or without cleft palate: A reanalysis. Am. J. Hum. Genet. 51: 323–332. Murray, J. C. (1995). Face facts: Genes, environment, and cleft. Am. J. Hum. Genet. 57: 227–232. Risch, N., and Giuffra, L. (1989). Multipoint linkage analysis of genetically complex traits. Am. J. Hum. Genet. 47(Suppl.): A197. Sandgren, E. P., Luettke, N. C., Palmiter, R. D., Brinster, R. L., and Lee, D. C. (1990). Overexpression of TGFa in transgenic mice: Induction of epithelial hyperplasia, pancreatic metaplasia, and carcinoma of the breast. Cell 61: 1121–1135. Sassani, R., Bartlet, S. P., Feng, H., Goldner-Sauve, A., Haq, A. K., Buetow, K. H., and Gasser, D. L. (1993). Associations between alleles of the transforming growth factor-alpha locus and the occurrence of cleft lip. Am. J. Med. Genet. 45: 565–569. Scambler, P. J., Heather, L., Gorman, P., Lindsay, E., Yamamura, K. I., and Dixon, M. (1992). The human homolog of a sequence disrupted in a murine craniofacial dysmorphism maps to 6p: A candidate locus for nonsyndromic cleft lip/or palate. Am. J. Hum. Genet. 51(Suppl.): A50. Scapoli, L., Pezzetti, F., Carinci, F., Martinelli, M., Carinci, P., and Tognon, M. (1997). Genetic heterogeneity and evidence of linkage to 6p23 in nonsyndromic cleft lip with or without cleft palate. Genomics 43: 216–220. Scapoli, L., Pezzetti, F., Carinci, F., Martinelli, M., Carinci, P., and Tognon, M. (1998). Lack of linkage disequilibrium between transforming growth factor alpha Taq I polymorphism and cleft lip with or without cleft palate in families from northeastern Italy. Am. J. Med. Genet. 75: 203–206. Shaw, D., Ray, A., Marazita, M., and Field, L. (1993). Further evidence of a relationship between the retinoic acid receptor alpha
gnmas
A LOCUS IN 2p13–p14 IS INVOLVED IN OFC (OFC2) locus and nonsyndromic cleft lip with or without cleft palate (CL{P). Am. J. Hum. Genet. 53: 1156–1157. Shaw, G. M., Wasserman, C. R., Lammer, E. J., O’Malley, C. D., Murray, J. C., Basart, A. M., and Tolarova, M. M. (1996). Orofacial clefts, parental cigarette smoking, and transforming growth factoralpha gene variants. Am. J. Hum. Genet. 58: 551–561. Stein, J., Mulliken, J. B., Stal, S., Gasser, D. L., Malcolm, S., Winter, R., Blanton, S. H., Amos, C., Seemanova, E., and Hecht, J. T. (1995). Nonsyndromic cleft lip with or without cleft palate: Evidence of linkage to BCL3 in 17 multigenerational families. Am. J. Hum. Genet. 57: 257–272. Stoll, C., Quian, J. F., Feringold, J., Sauvage, P., and May, E. (1993). Genetic variation in transforming growth factor alpha: Possible association of Bam HI polymorphism with bilateral sporadic cleft lip and palate. Hum. Genet. 92: 81–82. Terwillinger, J. D., and Ott, J. (1994). ‘‘Handbook of Human Genetic Linkage,’’ pp.224–225, Johns Hopkins Univ. Press, Baltimore/London.
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305
Van Dyke, D. C., Goldman, A. S., Spielman, R. S., Zmijewski, C. M., and Oka, S. W. (1980). Segregation of HLA in sibs with cleft lip or cleft lip and palate: Evidence against linkage. Cleft Palate J. 17: 189–193. Vieland, V. J., Hodge, S. E., and Greenberg, D. A. (1992). Adequacy of single-locus approximations for linkage analyses of oligogenic traits. Genet. Epidemiol. 9: 45–59. Vintiner, G. M., Holder, S. E., Winter, R. M., and Malcolm, S. (1992). No evidence of linkage between the transforming growth factor alpha gene in families with apparently autosomal dominant inheritance of cleft lip and palate. J. Med. Genet. 29: 393–397. Vintiner, G. M., Lo, K. K., Holder, S. E., Minter, R. M., and Malcolm, S. (1993). Exclusion of candidate genes from a role in cleft lip with or without cleft palate: Linkage and association studies. J. Med. Genet. 30: 773–778. Watanabe, T., Ohishi, M., and Tashiro, H. (1984). Population and family studies of HLA in Japanese with cleft lip and palate. Cleft Palate J. 21: 293–300.
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