442
tion by PCR were carried out. The cycles consisted of 10 s at 941C, 20 s at S20C, 1 s at 600C, and 10 s at 740C, with a ramping of 2 s/ 0C between the annealing temperature and the step at 600C, to improve the stability of the mismatched primer. Five to ten microliters of the amplified DNA were digested with MspI for 2 h, and the digested DNA was analyzed by electrophoresis on 3% agarose gel. As shown in figure 1, the size difference (3 bp) between the products of mutant and normal alleles was well resolved and, together with the presence of heteroduplexes, allowed the identification of the dupLl54 mutant allele. The T-to-C transition at codon 260, resulting in the L260P mutant allele, was identified by mismatched PCR. As shown in figure 2, MspI digestion of amplified DNA from subjects heterozygous for the mutation showed the presence of two bands -one of 161 bp and one of 142 bp-corresponding, respectively, to the products of normal and mutant alleles. Thus, two mutations of the Sp a gene that are responsible for HE in populations from western Africa can be identified rapidly by using the methods described above. Screening large populations from various ethnic groups in Africa for both these mutations will allow better estimation of their actual frequency. These findings will be of interest with regard to a possible role of dupL154 in malaria resistance (Lecomte et al. 1991).
Acknowledgment Part of this work was funded by the Association Francaise contre les Myopathies.
LAURENT BOULANGER, MARIE-CHRISTINE LECOMPTE, DIDIER DHERMY, AND MICHEL GARBARZ INSERM U160 Hopital Beaujon Clichy, France References Jinks DC, Minter M, Tarver DA, Vanderford M, Hejtmanick JF, McCabe RB ( 1989) Molecular genetic diagnosis of sickle cell disease using dried blood specimens in blotters used for newborn screening. Hum Genet 81:363-366 Kumar R. Dunn LL (1989) Designed diagnosis restriction fragment length polymorphism for the detection of point mutation in ras oncogenes. Oncol Res 4:235-241 Lecomte MC, Barrault C, Deguercy A, Boivin P. Schrevel J. Dhermy D (1991) Inhibition of P. falciparum growth in
Letters to the Editor elliptocytes bearing spectrin al-domains mutations. Nouv Rev Fr Hematol 33:133 Lecomte MC, Dhermy D, Gautero H, Bournier 0, Galand C, Boivin P (1988) L'elliptocytose hereditaire en Afrique de I'Ouest: frequence et repartition des variants de la spectrine. C R Acad Sci [III] 306:43-46 Palek J, Lambert S (1990) Genetics of the red cell membrane skeleton. Semin Hematol 27:290-332 Sahr KE, Garbarz M, Dhermy D, Lecomte MC, Boivin P, Agre P, Laughinghouse K, et al (1990) Use of the polymerase chain reaction for the detection and characterization of mutations causing hereditary elliptocytosis. In: Palek J, Cohen C (eds) Cellular and molecular biology of normal and abnormal erythroid membranes. Alan R Liss, New York, pp 201-210 Sahr KE, Tobe T, Scarpa A, Laughinghouse K, Marchesi SL, Agre P, Linnenbach AJ, et al (1989) Sequence and exon-intron organization of the DNA encoding the al domain of human spectrin: application to the study of mutations causing hereditary elliptocytosis. J Clin Invest 84:1243-1252 Saiki RK, Gelfand DH, Stoffel S, Scharf S, Higuchi R. Horn GT, Mullis KB, et al (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487-489 O 1992 by The American Society of Human Genetics. All rights reserved. 0002-9297/92/5102-0027$02.00
Am. J. Hum. Genet. 51:442-444, 1992
Van der Woude Syndrome and Nonsyndromic Cleft Lip and Palate To the Editor:
Nonsyndromic cleft lip with or without cleft palate (CLP) is a common craniofacial birth defect occurring in approximately 1/1,000 live births (Fraser 1970; Bonaiti et al. 1982). Recent case reports (Hecht 1988, 1990; De Paepe 1989; Temple et al. 1989) and complex segregation analyses (Marazita et al. 1984, 1986; Chung et al. 1986; Hecht et al. 1991 b) have suggested that CLP in some multiplex families may be caused by a dominant/codominant gene. Association studies have suggested transforming growth factor alpha on chromosome 2pl3 as a causal gene (Ardinger et al. 1989; Chenevix-Trench et al. 1991; Vintiner et al. 1991); however, linkage studies have not confirmed this in multiplex CLP families (Hecht et al. 1991a; Vintiner et al. 1991).
Letters to the Editor Van der Woude syndrome (VWS) is an autosomal dominant condition in which CLP, cleft palate (CP), and lip pits occur together or singly (Schinzel and Klausler 1989). VWS has been mapped to chromosome 1 q32 and is linked to renin and to an anonymous DNA marker D1S53 (Murray et al. 1990). Renin and DlS53 flank the VWS gene at distances of 4 cM and 10 cM, respectively (Murray et al. 1990). We tested linkage to renin and to DlS53 in 12 multiplex CLP families, to determine whether clefting in these families represents variable expression of the VWS gene or expression of one or more as yet undetermined clefting genes. None of the family members had clinical evidence of VWS, i.e., lip pits. The CLP families have been described elsewhere (Hecht et al. 1 99la). A short tandem PCR repeat marker from renin, HUMRENA4 (Edwards et al. 1991) and from DlS53, L673 (Nishimura et al. 1992), were used in the present study. HUMRENA4 and L673 were amplified and run on sequencing gels, by following previously published methods. The sequencing gels were silver stained using the Gelcode system. Linkage was tested using MLINK and LINKMAP; part of the LINKAGE package used previously described linkage parameters (Lathrop et al. 1984; Hecht et al. 1991a). Of 12 families analyzed, 5 were informative for renin and eight for D1S53. When the conventional LOD score ( - 2.0 or less) for exclusion (Ott 1985) was used, linkage to renin at a distance of 2 cM and to DlS53 at a distance of 4 cM was excluded. The results of the multipoint analysis strongly suggest that, in the informative families, the CLP gene does not lie between these two markers. Although the LOD score does not stay below the required - 2.0 to be excluded from the entire interval (Ott 1985), it does not exceed - 1.73 in the interval between these two markers. Although a few families gave small positive LOD scores, a test for homogeneity was not significant. Recently, we have tested a four-generation family in which isolated nonsyndromic CP was segregating. Seven of 24 individuals in this family have CP. Linkage to renin was excluded at less than 1 cM, and to D1S53 at 10 cM. The LOD score in the multipoint analysis did not exceed - 1.33 in the interval between the markers. In summary, the gene that is causing clefting and that is segregating in these CLP and CP families does not demonstrate linkage to the chromosomal interval containing the VWS gene. JACQUELINE T. HECHT,* YAPING WANG*, SUSAN H. BLANTONJ'$ AND STEPHEN P. DAIGERt
443 University of Texas Medical School at Houston and tGraduate School of Biomedical Sciences, Houston; and tDepartment of Pediatrics, University of Virginia, Charlottesville References Ardinger HH, Buetow KH, Bell GI, Bardach J, VanDemark DR, Murray JC (1989) Association of genetic variation of the transforming growth factor alpha gene with cleft lip and palate. Am J Hum Genet 45:348-353 Bonaiti C, Briard ML, FeingoldJ, Pavy B, PsaumeJ, MigneTufferand G, Kaplan J (1982) An epidemiological and genetic study of facial clefting in France. I. Epidemiology and frequency in relatives. J Med Genet 19:8-15 Chenevix-Trench G, Jones K, Green A, 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 Chung CS, Bixler D, Watanabe T, Koguchi H, FoghAndersen P (1986) Segregation analysis of cleft lip with or without cleft palate: a comparison of Danish andJapanese data. Am J Hum Genet 39:603-611 De Paepe A (1989) Dominantly inherited cleft lip and palate. Med Genet 26:794 Edwards A, Civitello A, Hammond HA, Caskey CT (1991) DNA typing and genetic mapping with trimeric and tetrameric tandem repeats. Am J Hum Genet 49:746-756 Fraser FC (1970) The genetics of cleft lip and palate. Am J Hum Genet 22:336-352 Hecht JT (1988) Familial component of epilepsy in cleft lip and palate. PhD thesis, University of Texas, Houston (1990) Dominant CLP families. J Med Genet 27:597 Hecht JT, Wang Y, Blanton SH, Michels VV, Daiger SP (1991a) Cleft lip and palate: no evidence of linkage to transforming growth factor alpha. Am J Hum Genet 49: 682-686 Hecht JT, Yang P, Michels VV, Buetow KH (1991 b) Complex segregation analysis of nonsyndromic cleft lip and palate. Am J Hum Genet 49:674-681 Lathrop GM, Lalouel JM, Julier C, Ott J (1984) Strategies for multilocus linkage analysis in humans. Proc Natl Acad Sci USA 81:3443-3446 Marazita ML, Spence MA, Melnick M (1984) Genetic analysis of cleft lip with or without cleft palate in Danish kindreds. Am J Med Genet 19:9-18 (1986) Major gene determination of liability to cleft lip with or without cleft palate: a multiracial view. J Craniofac Genet Dev Biol 2:89-97 Murray JC, Nishimura DY, Buetow KH, Ardinger HH, Spence MA, Sparkes RS, Falk RE, et al (1990) Linkage of an autosomal dominant clefting syndrome (Van der Woude) to loci on chromosome 1 q. Am J Hum Genet 46: 486-491
Letters to the Editor
444 Nishimura DY, Leysens NJ, Murray JC (1992) A dinucleotide repeat for the DlS53 locus. Nucleic Acids Res 20: 1167 Ott J (1985) Analysis of human genetic linkage. Johns Hopkins University Press, Baltimore Schinzel A, Klausler M (1989) The Van der Woude syndrome (dominantly inherited lip pits and clefts). J Med Genet 23:291-294 Temple K, Calvert M, Plint D, Thompson E, Pembrey M (1989) Dominantly inherited cleft lip and palate in two families. J Med Genet 26:386-389 Vintiner J, Holder SE, Malcolm S, Winter RM (1991) Nonsyndromic cleft lip and palate: association and linkage studies with the transforming growth factor alpha gene. Am J Hum Genet 49 [Suppl]: A190 i 1992 by The American Society of Human Genetics. Alt rights reserved. 0002-9297/92/5102-0028$02.00
Am. J. Hum. Genet. 51:444-446, 1992
Human Tritanopia Associated with a Third Amino Acid Substitution in the Blue-sensitive Visual Pigment To the Editor: Tritanopia is an autosomal dominant color-vision disorder characterized by insensitivity to the blue region of the spectrum (Boynton 1979). Recently, we used
PCR and denaturing gradient gel electrophoresis (DGGE) to screen for mutations in the coding region and intron-exon boundaries of the gene encoding the blue-sensitive visual pigment in nine unrelated tritanopes and available members of their families (Weitz et al. 1992). In four of the nine probands we found two examples each of two point mutations leading, respectively, to the amino acid substitutions G79R and S214P. The mutationo segregated with tritanopia in the relevant pedigrees, and no example of either of these mutations were detected in 43 and 84 control subjects, respectively. In five of the nine probands we failed to detect any sequence variants that were candidates for causal mutations, although in one proband (C2; for numbering of subjects, see Weitz et al. [1992]) we detected, within the splice-donor consensus sequence of intron 3, a single-base-pair deletion that did not segregate with tritanopia. We now report three independent examples of a third point mutation, leading to the amino acid substitution P264S. This mutation was not detected in our
original experiments in which exon 4 (including its flanking sequences and "GC-clamp") was amplified as a single product of 423 bp, but it is clearly revealed by DGGE when the exon 4 sequence is analyzed as two smaller overlapping products of 253 bp (exon 4A) and 257 bp (exon 4B). Figure 1 shows the DGGE analysis of exon 4A PCR products from relevant subjects in our original study (Weitz et al. 1992). Tritanopes from family C (lanes 3 and 7), proband D1 (lane 8), and a tritanope studied by Wooten and Wald (1973) (lane 9) are shown to be heterozygous for exon 4A sequences, with one allele having a mobility indistinguishable from the control (lane 1) and with the other having a lower mobility (apparently the same in all four tritanopes); an unresolved pair of heteroduplexes is evident as a band of yet lower mobility. Unaffected subjects from family C (lanes 2 and 4-6) show a single allele indistinguishable from that of the control. The exon 4 PCR products corresponding to the wild-type and variant alleles from proband C2 were subcloned and sequenced as described by Weitz et al. (1992; see fig. 2 legend). The variant allele differed from the wild-type allele only in the substitution of C'199 by T (fig. 2) (for numbering, see Nathans et al. 1986), leading to the amino acid substitution P264S in-the predicted sixth membrane-spanning segment of the blue-sensitive visual pigment (fig. 3). Exon 4 PCR products from the subjects described in figure 1 and from 64 control subjects of northern European ancestry were "slot blotted" and probed, as
12 3 4 5 6 7 8 9 ....X E w 11 w ~~T-
!i,r-1
DGGE profile of exon 4A PCR products from subFigure I jects described by Weitz et al. (1992). Lane 1, Wild-type control. Lanes 2, 4, 5, and 6, Unaffected subjects C1, C3, C4, and C5, respectively. Lanes 3 and 7, Affected subjects C2 and C6, respectively. Lane 8, Affected subject DI. Lane 9, Tritanope studied by Wooten and Wald (1973). PCR and DGGE were performed as described for exon 4 (Weitz et al. 1992), except that the following primer was substituted for the second member of the primer pair in PCR amplification of genomic DNA: 5'-ACGGTTGTTGACCATGTACAT-3'.
tion by PCR were carried out. The cycles consisted of 10 s at 941C, 20 s at S20C, 1 s at 600C, and 10 s at 740C, with a ramping of 2 s/ 0C between the annealing temperature and the step at 600C, to improve the stability of the mismatched primer. Five to ten microliters of the amplified DNA were digested with MspI for 2 h, and the digested DNA was analyzed by electrophoresis on 3% agarose gel. As shown in figure 1, the size difference (3 bp) between the products of mutant and normal alleles was well resolved and, together with the presence of heteroduplexes, allowed the identification of the dupLl54 mutant allele. The T-to-C transition at codon 260, resulting in the L260P mutant allele, was identified by mismatched PCR. As shown in figure 2, MspI digestion of amplified DNA from subjects heterozygous for the mutation showed the presence of two bands -one of 161 bp and one of 142 bp-corresponding, respectively, to the products of normal and mutant alleles. Thus, two mutations of the Sp a gene that are responsible for HE in populations from western Africa can be identified rapidly by using the methods described above. Screening large populations from various ethnic groups in Africa for both these mutations will allow better estimation of their actual frequency. These findings will be of interest with regard to a possible role of dupL154 in malaria resistance (Lecomte et al. 1991).
Acknowledgment Part of this work was funded by the Association Francaise contre les Myopathies.
LAURENT BOULANGER, MARIE-CHRISTINE LECOMPTE, DIDIER DHERMY, AND MICHEL GARBARZ INSERM U160 Hopital Beaujon Clichy, France References Jinks DC, Minter M, Tarver DA, Vanderford M, Hejtmanick JF, McCabe RB ( 1989) Molecular genetic diagnosis of sickle cell disease using dried blood specimens in blotters used for newborn screening. Hum Genet 81:363-366 Kumar R. Dunn LL (1989) Designed diagnosis restriction fragment length polymorphism for the detection of point mutation in ras oncogenes. Oncol Res 4:235-241 Lecomte MC, Barrault C, Deguercy A, Boivin P. Schrevel J. Dhermy D (1991) Inhibition of P. falciparum growth in
Letters to the Editor elliptocytes bearing spectrin al-domains mutations. Nouv Rev Fr Hematol 33:133 Lecomte MC, Dhermy D, Gautero H, Bournier 0, Galand C, Boivin P (1988) L'elliptocytose hereditaire en Afrique de I'Ouest: frequence et repartition des variants de la spectrine. C R Acad Sci [III] 306:43-46 Palek J, Lambert S (1990) Genetics of the red cell membrane skeleton. Semin Hematol 27:290-332 Sahr KE, Garbarz M, Dhermy D, Lecomte MC, Boivin P, Agre P, Laughinghouse K, et al (1990) Use of the polymerase chain reaction for the detection and characterization of mutations causing hereditary elliptocytosis. In: Palek J, Cohen C (eds) Cellular and molecular biology of normal and abnormal erythroid membranes. Alan R Liss, New York, pp 201-210 Sahr KE, Tobe T, Scarpa A, Laughinghouse K, Marchesi SL, Agre P, Linnenbach AJ, et al (1989) Sequence and exon-intron organization of the DNA encoding the al domain of human spectrin: application to the study of mutations causing hereditary elliptocytosis. J Clin Invest 84:1243-1252 Saiki RK, Gelfand DH, Stoffel S, Scharf S, Higuchi R. Horn GT, Mullis KB, et al (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487-489 O 1992 by The American Society of Human Genetics. All rights reserved. 0002-9297/92/5102-0027$02.00
Am. J. Hum. Genet. 51:442-444, 1992
Van der Woude Syndrome and Nonsyndromic Cleft Lip and Palate To the Editor:
Nonsyndromic cleft lip with or without cleft palate (CLP) is a common craniofacial birth defect occurring in approximately 1/1,000 live births (Fraser 1970; Bonaiti et al. 1982). Recent case reports (Hecht 1988, 1990; De Paepe 1989; Temple et al. 1989) and complex segregation analyses (Marazita et al. 1984, 1986; Chung et al. 1986; Hecht et al. 1991 b) have suggested that CLP in some multiplex families may be caused by a dominant/codominant gene. Association studies have suggested transforming growth factor alpha on chromosome 2pl3 as a causal gene (Ardinger et al. 1989; Chenevix-Trench et al. 1991; Vintiner et al. 1991); however, linkage studies have not confirmed this in multiplex CLP families (Hecht et al. 1991a; Vintiner et al. 1991).
Letters to the Editor Van der Woude syndrome (VWS) is an autosomal dominant condition in which CLP, cleft palate (CP), and lip pits occur together or singly (Schinzel and Klausler 1989). VWS has been mapped to chromosome 1 q32 and is linked to renin and to an anonymous DNA marker D1S53 (Murray et al. 1990). Renin and DlS53 flank the VWS gene at distances of 4 cM and 10 cM, respectively (Murray et al. 1990). We tested linkage to renin and to DlS53 in 12 multiplex CLP families, to determine whether clefting in these families represents variable expression of the VWS gene or expression of one or more as yet undetermined clefting genes. None of the family members had clinical evidence of VWS, i.e., lip pits. The CLP families have been described elsewhere (Hecht et al. 1 99la). A short tandem PCR repeat marker from renin, HUMRENA4 (Edwards et al. 1991) and from DlS53, L673 (Nishimura et al. 1992), were used in the present study. HUMRENA4 and L673 were amplified and run on sequencing gels, by following previously published methods. The sequencing gels were silver stained using the Gelcode system. Linkage was tested using MLINK and LINKMAP; part of the LINKAGE package used previously described linkage parameters (Lathrop et al. 1984; Hecht et al. 1991a). Of 12 families analyzed, 5 were informative for renin and eight for D1S53. When the conventional LOD score ( - 2.0 or less) for exclusion (Ott 1985) was used, linkage to renin at a distance of 2 cM and to DlS53 at a distance of 4 cM was excluded. The results of the multipoint analysis strongly suggest that, in the informative families, the CLP gene does not lie between these two markers. Although the LOD score does not stay below the required - 2.0 to be excluded from the entire interval (Ott 1985), it does not exceed - 1.73 in the interval between these two markers. Although a few families gave small positive LOD scores, a test for homogeneity was not significant. Recently, we have tested a four-generation family in which isolated nonsyndromic CP was segregating. Seven of 24 individuals in this family have CP. Linkage to renin was excluded at less than 1 cM, and to D1S53 at 10 cM. The LOD score in the multipoint analysis did not exceed - 1.33 in the interval between the markers. In summary, the gene that is causing clefting and that is segregating in these CLP and CP families does not demonstrate linkage to the chromosomal interval containing the VWS gene. JACQUELINE T. HECHT,* YAPING WANG*, SUSAN H. BLANTONJ'$ AND STEPHEN P. DAIGERt
443 University of Texas Medical School at Houston and tGraduate School of Biomedical Sciences, Houston; and tDepartment of Pediatrics, University of Virginia, Charlottesville References Ardinger HH, Buetow KH, Bell GI, Bardach J, VanDemark DR, Murray JC (1989) Association of genetic variation of the transforming growth factor alpha gene with cleft lip and palate. Am J Hum Genet 45:348-353 Bonaiti C, Briard ML, FeingoldJ, Pavy B, PsaumeJ, MigneTufferand G, Kaplan J (1982) An epidemiological and genetic study of facial clefting in France. I. Epidemiology and frequency in relatives. J Med Genet 19:8-15 Chenevix-Trench G, Jones K, Green A, 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 Chung CS, Bixler D, Watanabe T, Koguchi H, FoghAndersen P (1986) Segregation analysis of cleft lip with or without cleft palate: a comparison of Danish andJapanese data. Am J Hum Genet 39:603-611 De Paepe A (1989) Dominantly inherited cleft lip and palate. Med Genet 26:794 Edwards A, Civitello A, Hammond HA, Caskey CT (1991) DNA typing and genetic mapping with trimeric and tetrameric tandem repeats. Am J Hum Genet 49:746-756 Fraser FC (1970) The genetics of cleft lip and palate. Am J Hum Genet 22:336-352 Hecht JT (1988) Familial component of epilepsy in cleft lip and palate. PhD thesis, University of Texas, Houston (1990) Dominant CLP families. J Med Genet 27:597 Hecht JT, Wang Y, Blanton SH, Michels VV, Daiger SP (1991a) Cleft lip and palate: no evidence of linkage to transforming growth factor alpha. Am J Hum Genet 49: 682-686 Hecht JT, Yang P, Michels VV, Buetow KH (1991 b) Complex segregation analysis of nonsyndromic cleft lip and palate. Am J Hum Genet 49:674-681 Lathrop GM, Lalouel JM, Julier C, Ott J (1984) Strategies for multilocus linkage analysis in humans. Proc Natl Acad Sci USA 81:3443-3446 Marazita ML, Spence MA, Melnick M (1984) Genetic analysis of cleft lip with or without cleft palate in Danish kindreds. Am J Med Genet 19:9-18 (1986) Major gene determination of liability to cleft lip with or without cleft palate: a multiracial view. J Craniofac Genet Dev Biol 2:89-97 Murray JC, Nishimura DY, Buetow KH, Ardinger HH, Spence MA, Sparkes RS, Falk RE, et al (1990) Linkage of an autosomal dominant clefting syndrome (Van der Woude) to loci on chromosome 1 q. Am J Hum Genet 46: 486-491
Letters to the Editor
444 Nishimura DY, Leysens NJ, Murray JC (1992) A dinucleotide repeat for the DlS53 locus. Nucleic Acids Res 20: 1167 Ott J (1985) Analysis of human genetic linkage. Johns Hopkins University Press, Baltimore Schinzel A, Klausler M (1989) The Van der Woude syndrome (dominantly inherited lip pits and clefts). J Med Genet 23:291-294 Temple K, Calvert M, Plint D, Thompson E, Pembrey M (1989) Dominantly inherited cleft lip and palate in two families. J Med Genet 26:386-389 Vintiner J, Holder SE, Malcolm S, Winter RM (1991) Nonsyndromic cleft lip and palate: association and linkage studies with the transforming growth factor alpha gene. Am J Hum Genet 49 [Suppl]: A190 i 1992 by The American Society of Human Genetics. Alt rights reserved. 0002-9297/92/5102-0028$02.00
Am. J. Hum. Genet. 51:444-446, 1992
Human Tritanopia Associated with a Third Amino Acid Substitution in the Blue-sensitive Visual Pigment To the Editor: Tritanopia is an autosomal dominant color-vision disorder characterized by insensitivity to the blue region of the spectrum (Boynton 1979). Recently, we used
PCR and denaturing gradient gel electrophoresis (DGGE) to screen for mutations in the coding region and intron-exon boundaries of the gene encoding the blue-sensitive visual pigment in nine unrelated tritanopes and available members of their families (Weitz et al. 1992). In four of the nine probands we found two examples each of two point mutations leading, respectively, to the amino acid substitutions G79R and S214P. The mutationo segregated with tritanopia in the relevant pedigrees, and no example of either of these mutations were detected in 43 and 84 control subjects, respectively. In five of the nine probands we failed to detect any sequence variants that were candidates for causal mutations, although in one proband (C2; for numbering of subjects, see Weitz et al. [1992]) we detected, within the splice-donor consensus sequence of intron 3, a single-base-pair deletion that did not segregate with tritanopia. We now report three independent examples of a third point mutation, leading to the amino acid substitution P264S. This mutation was not detected in our
original experiments in which exon 4 (including its flanking sequences and "GC-clamp") was amplified as a single product of 423 bp, but it is clearly revealed by DGGE when the exon 4 sequence is analyzed as two smaller overlapping products of 253 bp (exon 4A) and 257 bp (exon 4B). Figure 1 shows the DGGE analysis of exon 4A PCR products from relevant subjects in our original study (Weitz et al. 1992). Tritanopes from family C (lanes 3 and 7), proband D1 (lane 8), and a tritanope studied by Wooten and Wald (1973) (lane 9) are shown to be heterozygous for exon 4A sequences, with one allele having a mobility indistinguishable from the control (lane 1) and with the other having a lower mobility (apparently the same in all four tritanopes); an unresolved pair of heteroduplexes is evident as a band of yet lower mobility. Unaffected subjects from family C (lanes 2 and 4-6) show a single allele indistinguishable from that of the control. The exon 4 PCR products corresponding to the wild-type and variant alleles from proband C2 were subcloned and sequenced as described by Weitz et al. (1992; see fig. 2 legend). The variant allele differed from the wild-type allele only in the substitution of C'199 by T (fig. 2) (for numbering, see Nathans et al. 1986), leading to the amino acid substitution P264S in-the predicted sixth membrane-spanning segment of the blue-sensitive visual pigment (fig. 3). Exon 4 PCR products from the subjects described in figure 1 and from 64 control subjects of northern European ancestry were "slot blotted" and probed, as
12 3 4 5 6 7 8 9 ....X E w 11 w ~~T-
!i,r-1
DGGE profile of exon 4A PCR products from subFigure I jects described by Weitz et al. (1992). Lane 1, Wild-type control. Lanes 2, 4, 5, and 6, Unaffected subjects C1, C3, C4, and C5, respectively. Lanes 3 and 7, Affected subjects C2 and C6, respectively. Lane 8, Affected subject DI. Lane 9, Tritanope studied by Wooten and Wald (1973). PCR and DGGE were performed as described for exon 4 (Weitz et al. 1992), except that the following primer was substituted for the second member of the primer pair in PCR amplification of genomic DNA: 5'-ACGGTTGTTGACCATGTACAT-3'.
