Suggestive Linkage between Markers on Chromosome 19q13.2 and Nonsyndromic Orofacial Cleft Malformation

GENOMICS

51, 177–181 (1998) GE985384

ARTICLE NO.

Suggestive Linkage between Markers on Chromosome 19q13.2 and Nonsyndromic Orofacial Cleft Malformation Marcella Martinelli,* Luca Scapoli,* Furio Pezzetti,* Francesco Carinci,† Paolo Carinci,‡ Ugo Baciliero,§ Ernesto Padula,§ and Mauro Tognon*,1 *Department of Morphology and Embryology, Section of Histology and Embryology, and †Department of Maxillo-Facial Surgery, School of Medicine, University of Ferrara, Via Fossato di Mortara 64/B, 44100 Ferrara, Italy; ‡Institute of Histology and General Embryology, School of Medicine, University of Bologna, Via Belmeloro 8, 40126 Bologna, Italy; and §Department of Maxillo-Facial Surgery, San Bortolo Hospital, Via Rodolfi, 36100 Vicenza, Italy Received March 17, 1998; accepted May 12, 1998

Nonsyndromic cleft lip with or without cleft palate (OFC) is a common birth defect that has genetic bases. The nature of the genetic contribution is still to be clarified; however, some chromosome regions and candidate genes have been proposed for this malformation. We examined linkage between BCL3, a protooncogene located in 19q13.2, and OFC in a sample composed of 40 multiplex pedigrees using both nonparametric and parametric methods. The affected pedigree member statistics and the transmission disequilibrium test supported a role for BCL3 in causing OFC, while no evidence of linkage or genetic heterogeneity was found with the lod score method. © 1998 Academic Press

INTRODUCTION

Nonsyndromic cleft lip with or without secondary clefting of the palate (OFC), with an incidence in the range of 1/700 –1/1000, is one of the most common malformations among live births (Fraser, 1970; Bonaiti-Pellie et al., 1982). Even though environmental influences on facial development have been described, a strong genetic component has been demonstrated (Murray, 1995; Wyszynski et al., 1996). The nature of the genetic contribution to the etiology of nonsyndromic OFC is still being studied. Although earlier investigations suggested a multifactorial threshold model (Fraser, 1970; Carter, 1976), more recently, complex segregation analysis of several populations provided mixed models with a major gene influence (Marazita et al., 1984, 1986, 1992; Chung et al., 1986; Temple et al., 1989; De Paepe, 1989; Hecht et al., 1991; Ray et al., 1993) or with oligogenic modes of inheritance (Mitchell and Risch, 1992; Farral and Holder, 1992). Recently, different groups have investigated the 1 To whom correspondence should be addressed. Telephone: 139532-291538. Fax 139-532-291533. E-mail: [email protected].

localization of a putative OFC major gene, sometimes providing conflicting results. Several studies have indicated that a gene on 6p plays a role in clefting. Indeed, chromosomal aberrations involving this region have been observed in patients affected by OFC malformation (Korman-Bortolotto et al., 1990; Donnai et al., 1992; Davies et al., 1995). Our studies (Carinci et al., 1995; Scapoli et al., 1997), together with that of Eiberg et al. (1987), showed positive linkage results between 6p and the disease, while other investigations obtained negative data (Vintiner et al., 1993; Blanton et al., 1996). Evidence of linkage and locus heterogeneity with 60% of a family linked to D6S259, using markers mapping on 6p23, was observed during our studies; moreover, we confirmed genetic heterogeneity using markers that map in 2p13, a region containing the TGFA as candidate OFC gene, and found linkage with the subset of families that were in linkage with 6p23 (Pezzetti et al., 1998). Additional investigations indicated, in addition to TGFA, the RARA mapping in 17q as a candidate gene of particular interest and a locus that maps in the 4q region (Murray, 1995). Recently, Stein et al. (1995) investigated the involvement of 23 genes, which also encode growth factors, collagen, and homeodomain-containing proteins, as a possible cause of OFC. This linkage study, using an autosomal dominant model, yielded evidence of locus heterogeneity and of linkage with BCL3, a proto-oncogene mapping in 19q13.2, in 17 multigeneration families of the 39 examined. These authors, assuming both heterogeneity and a reduced penetrance model, found a maximum multipoint lod score of 1.85 for ApoC2, a marker which is 2.5 cM telomeric to BCL3. The same data were analyzed with model-free linkage methods. The affected pedigree member (APM) linkage method provided a significance level (P 5 0.007) adopting the multilocus approach, while the transmission disequilibrium test (TDT) did not support any linkage (Amos et al. 1996a,b). However, analyzing 30 sporadic OFC cases and their parents yielded evidence of linkage

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disequilibrium between BCL3 alleles and OFC (Amos et al., 1996a). Using independent sample data composed of 30 U.S. and 11 Mexican multiplex families, Wyszynski et al. (1997) investigated the BCL3 locus. While evidence of linkage disequilibrium was found, no linkage or heterogeneity was obtained using the lod score method. Since no highly significant statistical data were obtained from previous studies as regards the BCL3 role in clefting, we performed linkage analyses on a group of 40 Italian families that we had previously used to investigate the 6p23 and 2p13 chromosome regions. Given the heterogeneity of the disease, we thought that it would be interesting to verify whether the BCL3 was a significant gene in our family sample. MATERIALS AND METHODS Families. Our pedigree collection, composed of 40 families, has already been described in a previous paper (Scapoli et al., 1998). Briefly, all families were collected from regions of northeastern Italy and included subjects that presented OFC as the only familial disease. All the patients were nonsyndromic, and no use of clefting drugs had been made in pregnancy. Of the 40 families, we studied two generations for 10 of them, 3 generations for 15, 4 generations for 12, and 5 generations for the remaining 3 families for a total of 384 individuals, 100 of whom had OFC. After informed consent was obtained, blood samples were drawn from 274 individuals, 82 of whom were affected. Two families of the 40 were not informative for lod score analysis. Markers and DNA typing. Three dinucleotide repeats were used to study the linkage between OFC and the BCL3 gene; the intragenic marker BCL3 and two flanking Ge´ne´thon markers, at loci D19S574 and D19S412, having an interlocus distance of less than 1 cM, were used (Gyapay et al., 1994). The centromeric D19D574 was only 200 kb far from BCL3 (Ashworth et al., 1995). The DNA was prepared from peripheral blood cells (Higuchi, 1989) and then used as a template for standard PCR. DNA samples were denatured at 94°C for 30 s, annealed at 57°C for 20 s, and extended at 72°C for 10 s. The annealing temperature was 63°C for D19S574. All amplifications were processed through 35 cycles. Small aliquots of PCR-amplified products were separated by polyacrylamide gel electrophoresis and visualized by silver staining. The estimations of marker allele frequencies were carried out from our pedigree data using the ILINK program of the LINKAGE package (Lathrop et al., 1984).

Linkage analyses. The linkage between markers and OFC was tested with three different methods: (i) the usual parametric lod score analysis, which requires the mode of inheritance and the marker allele frequency specifications; (ii) the model-free technique called affected pedigree member, which only requires marker allele frequency specifications; and (iii) the nonparametric transmission disequilibrium test, in which no assumptions about either the disease-gene model or the marker allele frequencies are required. Two-point and multipoint lod scores were calculated with the LINKAGE package using different models. A complex segregation analysis was performed on our family set; the results were consistent with a two-locus model having a major dominant locus and one modifier locus that might be either dominant or recessive (manuscript in preparation). Based on those analyses, for lod score calculations, we used a dominant mode of inheritance with a disease allele frequency of 0.0035 or a recessive model with a disease allele frequency of 0.187. The penetrance values, for both models, were set to 0.12 for males and 0.06 for females; moreover, other calculations were carried out with penetrances alternatively set at 0.9, 0.6, 0.3, and 0.001; the latter value was employed for the affected-only method (Terwillinger and Ott, 1994). The homogeneity test was performed using the HOMOG computer program (Ott, 1989). Power calculations using only the dominant model with parameter values obtained by the segregation analysis were performed. One thousand replicates of the sample were simulated using the SLINK program, for a marker having allele frequencies of D19S574, at recombination fraction u 5 0. The replicates were analyzed using the MSIM and HELODHET programs (Ott, 1989; Weeks et al., 1990). The power to detect linkage with our sample, i.e., lod score 3, was good; in fact, during the analysis, all the replicates exceeded this value as far as locus homogeneity was concerned. However, the power decreased significantly when heterogeneity was present; with a proportion of unlinked families of 50%, the percentage of replicates that gave lod scores . 3 was 53% and the percentage that gave lod scores . 2 was 76%. The APM is a model-free linkage test that compares marker similarity among all the affected members of the pedigrees to that expected on the basis of the allele frequency of the marker and the relationships among the affected individuals. We used the APM package version 2.0 to perform both single-locus and multilocus analyses (Weeks and Lange, 1988, 1992). The significance level (P value) for each APM statistic was determined empirically by carrying out 2000 simulated trials under the assumption of no linkage. All P values reported for the APM results were calculated using the weight function 1/square root of allele frequencies. The TDT compares the alleles transmitted from heterozygous parents to affected individuals to those alleles that are not transmitted and permits the detection of linkage disequilibrium (Spielman et al., 1993). Several statistics were proposed for TDT with multiallelic markers. In the present study, we used the likelihood ratio method proposed by Sham and Curtis (1995) as implemented in the ETDT

TABLE 1 Two-Point Lod Score for OFC versus Chromosome 19 Markers in 40 Families, Calculated under Autosomal Dominant (Dom) and Recessive (Rec) Models, with Reduced Penetrance Lod score at recombination fraction of Marker D19S412 Dom Rec BCL3 Dom Rec D19S574 Dom Rec

0.00

0.01

0.05

0.10

0.20

0.30

0.40

217.02 24.43

212.58 24.01

26.57 22.74

23.56 21.74

20.92 20.64

0.05 20.14

0.26 0.04

25.10 21.81

23.72 21.63

21.77 21.08

20.79 20.64

0.01 20.18

0.23 0.01

0.19 0.06

216.00 23.28

212.09 22.93

26.64 21.90

23.68 21.10

20.95 20.27

0.05 0.05

0.25 0.10

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LINKAGE BETWEEN 19q13.2 AND OFC

TABLE 2 Pairwise Affected Pedigree Member Analyses Marker

Weight. fun.

Statistic

P value

P empiric

D19S412

o)1 1/sqrt(p) 1/p 1 1/sqrt(p) 1/p 1 1/sqrt(p) 1/p

20.4712 0.1006 0.2262 0.1352 20.3541 0.7863 4.0593 3.7929 2.2413

0.68125 0.45992 0.41051 0.44622 0.63835 0.78414 0.00002 0.00008 0.01251

0.41250

BCL3

D19S574

0.63900

0.00050

program. In fact, this program calculates the log likelihood under the null hypothesis (L0) and under the alternative hypothesis that transmission probabilities may deviate from 50% in an allele-specific (L1) or genotype-specific (L2) manner. Twice the log likelihood ratio are x2 statistics having a degree of freedom equal to the number of alleles or equal to the number of genotypes observed in the genotype-wise analysis. The TDT was applied in two different ways: (i) All affected individuals were included to test the hypothesis of no linkage; in this way, only limited information about allelic association can be obtained. (ii) The TDT was performed using just one affected per pedigree to verify whether allelic association was present.

RESULTS

For all of the markers tested, the observed gene frequencies were in agreement with the value reported by the current version of the Genome Data Base; however, two additional rare alleles, of 139 and 133 bp, were found for BCL3. The calculated heterozygosity for markers, D19S574, BCL3, and D19S412 were of 0.88, 0.44, and 0.82, respectively. No evidence of linkage was detected in the analyses of the entire data set. In fact, lod scores , 0.5 were detected in pairwise (Table 1) and multipoint analyses, under both dominant and recessive modes of inheritance, by using different penetrance values. Moreover, the admixture test performed by the HOMOG computer program did not provide any significant level of heterogeneity. In a recent study, using the same 38 multigenerational families, we demonstrated linkage between OFC and the 6p23 chromosome region 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 (Pezzetti et al., 1998). Herein, the two groups of families selected a priori, i.e., the 6p23linked group and the 6p23-unlinked group, were analyzed separately for linkage to the BCL3 locus. The pairwise linkage analyses gave similar results for the two family groups, without increments of lod scores with respect to the entire data set. The number of useful pedigrees for the APM method was 24, while that of the affected individuals studied was 58. The single-locus analysis gave a statistically significant excess sharing of alleles identity by descent

at marker D19S574 (P 5 0.0005), while for BCL3 and D19S412 the statistics provided nonsignificant results (Table 2). The negative data obtained using the BCL3 and D19S412 markers was probably due to low BCL3 gene polymorphism and to the distance between D19S412 and the candidate disease locus. Indeed, no recombination was observed between the D19S574 and the BCL3 intragenic marker, while the distance between these two loci and D19S412 reported by the Ge´ne´thon map was 1 cM. Interestingly, supportive results for linkage were also obtained using the multilocus version of the APM test, which combines all the markers’ data (P 5 0.019) (Table 3). The numbers of heterozygous parents used for the TDT, when all affected individuals were used, were 54 for BCL3, 105 for D19S574, and 104 for D19S412. No significant transmission disequilibrium was found for the BCL3 and D19S412 marker alleles. In particular, the most common 135-bp allele at the BCL3 locus, previously found to be in linkage disequilibrium with OFC (Amos et al., 1996a; Wyszynski et al., 1997), was transmitted 24 times versus not transmitted 22 times (P 5 0.768). For D19S574, the TDT gave significant results using the genotype-wise model (P 5 0.015) but not with the allele-wise model (P 5 0.065). To verify whether any allelic association was present, a reanalysis of the data including only one affected and his/her parents for each pedigree was performed. Once again, significant linkage disequilibrium was obtained only for the D19S574 marker with the genotype-wise model (P 5 0.0254). The McNemar test performed for each allele gave significant results for the 188-bp allele, which was transmitted 14 times versus not transmitted 4 times (P 5 0.0184). Interestingly, the 135-bp allele at the BCL3 locus was in perfect equilibrium, being transmitted 15 times versus not transmitted 15 times. DISCUSSION

Although OFC is a common birth defect that has been extensively studied, the nature of the genetic contribution to the etiology of this malformation remains to be elucidated. Several complex segregation analyses performed on different populations favor the influence of an autosomal major gene, in some cases dominant, in others recessive. Recently, Clementi et al. (1995), by analyzing a large sample from two congenital malformation registers operative in northeastern Italy, found data in agreement with a two-locus model, TABLE 3 Multilocus Affected Pedigree Member Analyses Weight. fun.

Statistic

P value

1 1/sqrt(p) 1/p

1.7940 2.0691 0.8902

0.03641 0.01928 0.18667

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with a major dominant locus and at least one modifier. As reviewed by Murray (1995), several loci are claimed to contain a major clefting gene: 6p21–p25, 4q, 1q21, and some candidate genes, such as TGFA, RARA, BCL3, were investigated for their possible involvement in causing OFC. BCL3 is a transcription factor involved in the cell-lineage determination and in cellcycle regulation; its involvement was first proposed by Stein et al. (1995), who found suggestive linkage with the disease, under the hypothesis of genetic heterogeneity. Subsequently, evidence of linkage disequilibrium between BCL3 alleles both in sporadic and in familial OFC cases has been reported (Amos et al., 1996a; Wyszynski et al., 1997). In a previous report, we used 38 multiplex families to find evidence of linkage and genetic heterogeneity with markers on 6p23 (Scapoli et al., 1997) and heterogeneity with markers on 2p13 (Pezzetti et al., 1998); in this investigation we used the same family samples to verify whether linkage with BCL3 could be detected. We were unable to confirm the results of Stein et al. (1995) when using the same approach. Indeed, only lod scores ,0.5 were obtained, and no evidence of genetic heterogeneity was found by traditional parametric analyses. This result was replicated when either 6p23linked families or 6p23-unlinked families were analyzed separately. However, different results were obtained using the TDT and APM tests; indeed, support of linkage was obtained using both methods for D19S574, a highly polymorphic marker tightly linked to BCL3 gene. Moreover, significant linkage results were also obtained using the multilocus APM statistics. Although nonparametric and model-free methods are less powerful in their ability to detect linkage than parametric approaches under optimal conditions, nonparametric methods of analysis may be more useful when a probable disease gene model cannot be assumed, as in the case of OFC and other genetically complex disorders. There are empirical examples in which TDT has detected genetic effects that have a sound biological basis, whereas linkage analyses did not provide the same results. For example, two studies demonstrated that TDT detected linkage disequilibrium between insulin-dependent diabetes mellitus and the insulin-gene region, whereas linkage analyses, using the same data set, were negative (Spielman et al., 1993; Bennet et al., 1995). According to the standard proposed for mapping complex diseases (Lander and Kruglyak, 1995), the data previously reported by Stein et al. (1995) for the chromosome region 19q13.2 could provide ‘‘suggestive’’ linkage, i.e., statistical evidence expected to occur one time at random in a genome scan. Even though significant lod scores were not obtained in the two extension studies by Wyszynski et al. (1997) and our group, other suggestive linkage results were obtained using model-free linkage methods. Although suggestive linkage is, by definition, only indicative, so far three different groups have found suggestive link-

age for this locus, an encouraging sign that the locus is ‘‘real’’ and that it is relevant for different populations. Thus, BCL3 or a nearby gene seems to be implicated in some way in this congenital facial malformation. However, the difficulties in obtaining significant linkage indicate that the 19q13.2 gene is not a major clefting gene. In conclusion, it appears that BCL3 plays a role in the etiology of OFC; however, it is not known, at present, whether it acts as a modifier or as an additive gene for this malformation. ACKNOWLEDGMENTS This study was supported, in part, by grants from TELETHON (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.).

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