Hum Genet (1992) 90:413-416
human .. genetics 9 Springer-Verlag 1992
Identification of a nonsense mutation in the amelogenin gene (AMELX) in a family with X-linked amelogenesis imperfecta (AIH1) Michael J. Aldred 1, Peter J. M. Crawford 2, Enriqueta Roberts 1, Nicholas S.T. Thomas 1 IInstitute of Medical Genetics, Universityof Wales Collegeof Medicine, Heath Park, CardiffCF4 4XN, UK 2Department of Child Dental Health, Universityof Bristol Dental Schooland Hospital, Lower Maudlin Street, Bristol BS1 2LY, UK Received: 3 April 1992 / Revised: 7 July 1992
Abstract. A family with X-linked amelogenesis imperfecta (XAI) is described in which the disease is associated with a nonsense mutation in exon 5 of the amelogenin gene. This mutation involves a single base deletion (CCCC--~CCC) in the exon in an affected male, his sister and his mother. The effect of this deletion is to alter the reading frame and to introduce an inappropriate TGA stop codon (an opal mutation) into the exonic sequence of the amelogenin gene immediately 3' of the mutation. The clinical features in the examined members of this family indicate that, in some individuals, the most noticeable defect is of enamel hypoplasia. In others, the hypoplastic changes are subtle and might have been overlooked on cursory examination; the most noticeable change is of enamel colour, indicating a degree of hypomineralisation. We propose that the amelogenin gene is implicated in both the formation of enamel of normal thickness and in the normal mineralisation process.
Introduction Amelogenesis imperfecta refers to a group of inherited conditions affecting tooth enamel. Autosomal dominant, autosomal recessive and X-linked forms have been included in the different classification schemes proposed in recent years, with subdivision of these categories according to the perceived phenotype. These phenotypic features reflect either reduced enamel thickness (hypoplasia) or decreased mineralisation (hypomineralisation, which can be further subdivided into hypomaturation or hypocalcification according to the severity of the defect). X-linked amelogenesis imperfecta (XAI) provides one of the most striking morphological examples of lyonisation in humans. Affected (hemizygous) males and (heterozygous) females in the same family have different Correspondence to: M.J.Aldred, Division of Oral Biology and Pathology,Universityof Queensland, St. Lucia, Brisbane, Queensland 4072, Australia
clinical manifestations, with the teeth of heterozygous females exhibiting vertical markings of the enamel (Crawford and Aldred 1992). Of the proteins in developing enamel, the amelogenins are the best characterised (Fincham et al. 1983). The murine amelogenin gene has been cloned (Snead et al. 1983) and homologous sequences have been mapped (Lau et al. 1989) to the distal short arm of the human X chromosome (AMG, Xp22.1-p22.3) and to the pericentromeric region of the human Y chromosome (AMELY, Yqll). There is recent evidence that the AMELY gene is expressed (Fincham et al. 1991), albeit at low levels (Salido et al. 1992). Four families with XAI have been studied, and the disease in these families (AIH1) has been mapped by linkage analysis to the Xp22 region (Lagerstrom et al. 1990; Aldred et al. 1992), i.e. the region of the amelogenin gene. We have previously also found evidence that a second locus on the X chromosome (AIH3), situated in the Xq22-q28 region, is also associated with the expression of an XAI phenotype (Aldred et al. 1992). Nakahori et al. (1991) have identified human DNA sequences that are derived from the X and Y chromosomes and that demonstrate homology to the murine and bovine amelogenin cDNA sequences. A region of X-Y homology was sequenced and three open reading frames were recognised (Nakahori et al. 1991). The entire human amelogenin gene has now been cloned and seven exons identified (Salido et al. 1992), with exons 3, 5 and 6 corresponding to the open reading frames identified by Nakahori et al. (1991). Two exons (1 and 2) were found 5' of the region previously sequenced (Nakahori et al. 1991), with another exon (exon 4) recognised between the first and second open reading frames reported by Nakahori et al. 1991) and a further exon (exon 7) at the 3' end of the gene. A 5-kb interstitial deletion within the amelogenin gene has been identified in affected males and heterozygous females in one of the two XAI families described by Lagerstrom et al. (1991). We present here a previously undocumented pedigree with XAI (family XAI 5) in which the disease is associated
414
|
1.1
with a nonsense gene.
1.2
mutation in exon 5 of the amelogenin
Subjects
I1.1
11.2
1.3
11.4
Fig. 1. Abbreviated pedigree of family X A I 5 with X-linked amelogenesis imperfecta. Affected male represented by filled square: heterozygous females by vertical striped circles
Fig. 2a. Clinical photograph of individual I1.3 showing tipper permanent incisor teeth with a "square" morphology caused by a combination of reduced enamel thickness and attrition of the incisal edges. The teeth are opaque with some irregularity of the surface. b Clinical photograph of individual II.1 demonstrating vertical markings restllting from alterations both in contour and shade of the enamel
The family tree is shown in Fig. 1 and the clinical features illustrated in Fig. 2. All examined individuals were seen by two of the authors (MJA and PJMC). Individual II.3, the proband, a male aged 13 years, had upper permanent incisor teeth that were "square" in morphology (Fig. 2a) with a more tapering form than normal to the upper canines. These findings were felt to reflect reduced tooth size. The enamel was hard and slightly rough with a "ground glass" feel when a probe was passed over the surface. There was a suggestion of an alteration in the contour of the enamel on teeth 14, 24, 25, 47, 35, 37 (46 and 36 had previously been extracted). Abrasion and attrition were most evident on the upper incisor teeth. The teeth were "paint white" in colour, uniformly opaque but of slightly variable colour, most being A2 [standard shade guide (Vita, Lumin Vacuum porcelain shade guide; Vita Lumin Vaccum-Farbskala. Vita Zahnfabrik, H. Rauter. Bad Sfickingen, Germany)] in shade. This alteration in colouration was most noticeable on the canines (13, 23, 33, 43), tooth 21 and the lower premolars (34, 35, 44, 45). Further questioning of the family revealed that the older sister, individual II.l, aged 16 years, had teeth with vertical markings, described by the family as "tram-line teeth". On examination, the general impression was of teeth of normal outline and lucency with the overall colour assessed as A2. The enamel was glassy in appeareance but irregular in vertical contour, hard on probing and with no significant degree of tooth wear. All teeth had vertical markings resulting from alterations both in contour and shade of the enamel, although the changes in contour were more noticeable than those of shade (Fig. 2b). Tooth 11 (upper right permanent central incisor) had particularly marked brownish inclusions in an approximately vertical distribution. Teeth 31, 41 and 42 (lower right permanent central incisor and lower left permanent central and lateral incisors) were less affected than other teeth. Individual II,2, a female aged 15 years, had teeth of normal form and colour and was regarded as being unaffected. The tooth shade was recorded as AIIA2. Individual II.4, a boy 2 years old, had teeth of normal form and colour and was felt to be unaffected. The mother of the proband, individual I. 1 (aged 44 years) had teeth with vertical bands of opaque enamel alternating with enamel of normal colour and translucency. The affected enamel was paler than the normal enamel A I / A 2 , adjacent normal enamel A3. All the enamel was uniformly smooth and hard but with a very subtle undulation in the surface contour overlying the paler affected enamel. Teeth 21 and 22 (upper left central and lateral permanent incisors) were more severely affected than other standing teeth. The degree of attrition and abrasion was felt to be consistent with the patient's age and was not considered to be caused by any intrinsic defect in the enamel. Individual 1.2, the unrelated male spouse of 1.1 (aged 37 years), had teeth of normal form and marking and of shade A3.
Table 1. Conditions for PCR amplification of exons 3 . 5 and 6 of the amelogenin gene Exon
1 cycle
Primer pairs ~
Initial denaturation 1 cycle
30 cycles Annealing
Extension
Denaturation
Annealing
Extension
Denaturation
3
AM1 AM2
94~ 5 min
54~ 1 min
72~ 2 min
94~ 30 s
54~ 1 rain
72~ l0 rain
94~ 20 s
5
AM3 AM4
94~ 5 rain
54~ 1 rain
72~ 2 rain
94~ 30 s
54~ I min
72~ 10 min
94~ 30 s
3
AM5 AM6
94~ 5 rain
55~ 30 s
72~ 2 rain
94~ 30 s
55~ 1 rain
72~ 10 min
94~ 30 s
" From Lagerstrom et al. (1991)
415 Materials and methods Genomic DNA was extracted from peripheral blood leucocytes by standard methods. DNA from each of the three exons in the portion of the amelogenin gene showing X-Y homology (Nakahori et al. 1991) was amplified using the primers reported by Lagerstrom et al. (1991). Primers were used at a concentration of 1 p.M under the conditions shown in Table 1. The polymerase chain reaction (PCR) products were electrophoresed in 2% agarose gels containing ethidium bromide and viewed under ultraviolet light to detect any differences in product size. Aliquots of these PCR products were then directly sequenced. Following purification with GeneClean (Bio 101 Inc), the PCR products were annealed to either or both primers (AM1, AM4, AM5 and AM6) according to the size of the exons and labelled with [35S] ~-CTP (Amersham) using the Sequenase system (National Diagnostics). The labelled fragments were separated in 0.l-ram thick polyacrylamide gels (Sequagel, National Diagnostics) and the resulting bands visualised by autoradiography.
Results No obvious differences were detected in the size of the P C R product for any of the three exons amplified in the affected male and unaffected female in this family. Direct D N A sequencing showed no differences in exons 3 and 6 (Salido et al. 1992) between the affected male (individual II.3) and his unaffected sister (individual E). In exon 5, however, a single cytosine deletion was detected in the affected male (CCCC--~CCC) (Fig. 3) within the sequence of the exon. Heterozygous (manifesting) females showed three b a n d s of normal intensity in this region, with a fourth band of reduced strength, and duplication of subsequent bands (also of reduced intensity). This pattern was not observed in the father (I.2) or the clinically unaffected female II.2, indicating that the mutation segregated with the disease.
The effect of this deletion is an alteration of the reading frame of exon 5 and the introduction of an inappropriate T G A stop codon (an opal mutation) 3' of the mutation. The altered D N A sequence and the changes in the amino acid sequence are shown in Table 2.
Discussion Based on the recent data from Salido et al. (1992), the present mutation occurs in exon 5 (the second exon of Nakahori et al. 1991). In family X A I 5, therefore, translation of the amelogenin gene will be prematurely terminated. This should result in a truncated amelogenin gene product; we estimate that it would yield a protein product of only 74 amino acids. Over 200 amino acids encoded by the remainder of the amelogenin gene will be absent. Although we have not carried out linkage analysis in this family to establish linkage to the Xp22 region containing the A M G gene, we believe that the detection of a nonsense mutation within an exon of this gene in affected individuals provides sufficient evidence for its association in this family to be causal. Previously reported phenotypes of X A I have been described as being characterised predominantly either by hypoplasia (reduced thickness) or hypomineralisation (reduced mineralisation) of the enamel. We have observed that some X A I families described in the literature apparently demonstrate features of both reduced enamel thickness and reduced mineral content (Crawford and Aldred 1992), indicating that these two processes are probably operating together in affected individuals. The present family similarly displays concurrent features of hypoplasia and hypomineralisation, but they are expressed differently in different family members. Thus, hypoplasia was more
Fig. 3. Manual direct DNA sequence analysis of affected male (individual II.3) with X-linked amelogenesis imperfecta and his unaffected sister (individual II.2). PCR products were directly sequenced on the anti-sense strand. GGGG---,GGG (sense CCCC ~ CCC) transition identified in the affected male in PCR-amplified exon 5 of the amelogenin gene
Table 2. Amelogenin exon 5a DNA sequence and the corresponding amino acid sequence in normal individuals and in an affected male in family XAI 5 with X-linked amelogenesis imperfecta Wild type Mutant
GTG V GTG V
CTr L CTT L
a From Salido et al. (1992)
ACC T ACC T
CCT P CTT L
TTG L TGA STOP
AAG K AGT
TGG W GGT
TAC Y ACC
CAG Q AGA
AGC S GCA
ATA I TAA
AGG R GGC
CCA P CA
416 n o t i c e a b l e in i n d i v i d u a l II.1, similar to the h y p o p l a s t i c X A I p h e n o t y p e of Schulze (1956). I n d i v i d u a l 1.1, however, h a d c h a n g e s of e n a m e l c o l o u r , r a t h e r t h a n cont o u r , which is m o r e c o n s i s t e n t with the findings r e p o r t e d by W i t k o p (1957, 1967), and which he c h a r a c t e r i s e d as " X - l i n k e d recessive h y p o m a t u r a t i o n a m e l o g e n e s i s imp e r f e c t a " . This w o u l d i n d i c a t e that an a b n o r m a l i t y of e n a m e l q u a n t i t y is the p r e d o m i n a n t defect in i n d i v i d u a l II.1, w h e r e a s a d e f e c t of m i n e r a l i s a t i o n is the essential a b n o r m a l i t y in i n d i v i d u a l 1.1. T h e a f f e c t e d m a l e (indiv i d u a l II.3) has s o m e r e d u c t i o n in e n a m e l thickness, and m a r k e d changes in the lucency of the e n a m e l ; he, t h e r e fore, shows f e a t u r e s of b o t h p h e n o t y p e s . B e c a u s e of the c o n c u r r e n t p r e s e n t a t i o n of b o t h h y p o m i n e r a l i s a t i o n and h y p o p l a s i a in the p r e s e n t family ( a n d in o t h e r s r e p o r t e d in the l i t e r a t u r e ) , we p r o p o s e that the a m e l o g e n i n gene is i m p l i c a t e d in b o t h the n o r m a l m i n e r a l i s a t i o n p r o c e s s a n d in the f o r m a t i o n of e n a m e l of n o r m a l thickness. T h e i d e n t i f i c a t i o n and c h a r a c t e r i s a t i o n of f u r t h e r a m e l o g e n i n m u t a t i o n s in o t h e r A I H 1 families s h o u l d p r o v i d e evid e n c e for the relative i m p o r t a n c e of d i f f e r e n t r e g i o n s of the a m e l o g e n i n gene in the p r o c e s s of a m e l o g e n e s i s , tog e t h e r with a logical classification of these c o n d i t i o n s .
Acknowledgements. We wish to thank the family described in this paper for their support. This study was funded by grants from the Medical Research Council and Welsh Scheme for the Development of Health and Social Research. MJA is a Research Fellow support by a Medial Research Council Special Training Fellowship in Recombinant DNA Technology and a Medical Research Council Clinician Scientist Fellowship. The primers were kindly supplied by the Human Gene Mapping Project Resource Centre.
References
Aldred M J, Crawford PJM, Roberts E, Gillespie CM, Thomas NST, Fenton I, Sandkuijl LA, Harper PS (1992) Genetic
heterogeneity in X-linked amelogenesis imperfecta. Genomics (in press) Crawford PJM, Aldred MJ (1992) X-linked amelogenesis imperrecta. Presentation of two kindreds and a review of the literature. Oral Surg Oral Med Oral Pathol 73 : 449-455 Fincham AG, Belcourt AB, Termine JD, Butler WY, Cothran WC (1983) Amelogenins: sequence homologies in enamel-matrix proteins from three mammalian species. Biochem J 211: 149-154 Fincham AG, Bessem CC, Lau EC, Pavlova Z, Shuler C. Slavkin HC, Snead ML (1991) Human developing enamel proteins exhibit a sex-linked dimorphism. Calcif Tissue Int 48 : 288-990 Lagerstrom M, Dahl N, Iselius L, Backman B, Pettersson U (1990) Mapping of the gene for X-linked amelogenesis imperrecta by linkage analysis. Am J Hum Genet 46:120-125 Lagerstrom M, Dahl N, Nakahori Y, Nakagome Y, Backman B, Landegren U, Pettersson U (1991) A deletion in the amelogenin gene (AMG) causes X-linked amelogenesis imperfecta (AIHI). Genomics 10 : 971-975 Lau EC, Mohandas TK, Shapiro LJ, Slavkin HC, Snead ML (1989) Human and mouse amelogenin gene loci are on the sex chromosomes. Genomics 4:162-168 Nakahori Y, Takenaka O, Nakagome Y (1991) A human X-Y homologous region encodes "amelogenin". Genomics 9:264 269 Salido EC, Yen PH, Koprivnikar K, Yu L-C, Shapiro LJ (1992) The human enamel protein gene amelogenin is expressed from both the X and Y chromosomes. Am J Hum Genet 50:303316 Schulze C (1956) Erbbedingte Strukturanomalien menschlicher Zfihne. Urban and Schwarzenberg, Munich Berlin Snead ML, Zeichner-David M, Chandra T, Robson KJH, Woo SLC, Slavkin HC (1983) Construction and identification of mouse amelogenin cDNA clones. Proc Natl Acad Sci USA 80 : 7254-7258 Witkop CJ (1957) Hereditary defects in enamel and dentin. Acta Genet 7 : 236-239 Witkop CJ (1967) Partial expression of sex-linked recessive amelogenesis imperfecta in females compatible with the Lyon hypothesis. Oral Surg Oral Med Oral Pathol 22:174-182
human .. genetics 9 Springer-Verlag 1992
Identification of a nonsense mutation in the amelogenin gene (AMELX) in a family with X-linked amelogenesis imperfecta (AIH1) Michael J. Aldred 1, Peter J. M. Crawford 2, Enriqueta Roberts 1, Nicholas S.T. Thomas 1 IInstitute of Medical Genetics, Universityof Wales Collegeof Medicine, Heath Park, CardiffCF4 4XN, UK 2Department of Child Dental Health, Universityof Bristol Dental Schooland Hospital, Lower Maudlin Street, Bristol BS1 2LY, UK Received: 3 April 1992 / Revised: 7 July 1992
Abstract. A family with X-linked amelogenesis imperfecta (XAI) is described in which the disease is associated with a nonsense mutation in exon 5 of the amelogenin gene. This mutation involves a single base deletion (CCCC--~CCC) in the exon in an affected male, his sister and his mother. The effect of this deletion is to alter the reading frame and to introduce an inappropriate TGA stop codon (an opal mutation) into the exonic sequence of the amelogenin gene immediately 3' of the mutation. The clinical features in the examined members of this family indicate that, in some individuals, the most noticeable defect is of enamel hypoplasia. In others, the hypoplastic changes are subtle and might have been overlooked on cursory examination; the most noticeable change is of enamel colour, indicating a degree of hypomineralisation. We propose that the amelogenin gene is implicated in both the formation of enamel of normal thickness and in the normal mineralisation process.
Introduction Amelogenesis imperfecta refers to a group of inherited conditions affecting tooth enamel. Autosomal dominant, autosomal recessive and X-linked forms have been included in the different classification schemes proposed in recent years, with subdivision of these categories according to the perceived phenotype. These phenotypic features reflect either reduced enamel thickness (hypoplasia) or decreased mineralisation (hypomineralisation, which can be further subdivided into hypomaturation or hypocalcification according to the severity of the defect). X-linked amelogenesis imperfecta (XAI) provides one of the most striking morphological examples of lyonisation in humans. Affected (hemizygous) males and (heterozygous) females in the same family have different Correspondence to: M.J.Aldred, Division of Oral Biology and Pathology,Universityof Queensland, St. Lucia, Brisbane, Queensland 4072, Australia
clinical manifestations, with the teeth of heterozygous females exhibiting vertical markings of the enamel (Crawford and Aldred 1992). Of the proteins in developing enamel, the amelogenins are the best characterised (Fincham et al. 1983). The murine amelogenin gene has been cloned (Snead et al. 1983) and homologous sequences have been mapped (Lau et al. 1989) to the distal short arm of the human X chromosome (AMG, Xp22.1-p22.3) and to the pericentromeric region of the human Y chromosome (AMELY, Yqll). There is recent evidence that the AMELY gene is expressed (Fincham et al. 1991), albeit at low levels (Salido et al. 1992). Four families with XAI have been studied, and the disease in these families (AIH1) has been mapped by linkage analysis to the Xp22 region (Lagerstrom et al. 1990; Aldred et al. 1992), i.e. the region of the amelogenin gene. We have previously also found evidence that a second locus on the X chromosome (AIH3), situated in the Xq22-q28 region, is also associated with the expression of an XAI phenotype (Aldred et al. 1992). Nakahori et al. (1991) have identified human DNA sequences that are derived from the X and Y chromosomes and that demonstrate homology to the murine and bovine amelogenin cDNA sequences. A region of X-Y homology was sequenced and three open reading frames were recognised (Nakahori et al. 1991). The entire human amelogenin gene has now been cloned and seven exons identified (Salido et al. 1992), with exons 3, 5 and 6 corresponding to the open reading frames identified by Nakahori et al. (1991). Two exons (1 and 2) were found 5' of the region previously sequenced (Nakahori et al. 1991), with another exon (exon 4) recognised between the first and second open reading frames reported by Nakahori et al. 1991) and a further exon (exon 7) at the 3' end of the gene. A 5-kb interstitial deletion within the amelogenin gene has been identified in affected males and heterozygous females in one of the two XAI families described by Lagerstrom et al. (1991). We present here a previously undocumented pedigree with XAI (family XAI 5) in which the disease is associated
414
|
1.1
with a nonsense gene.
1.2
mutation in exon 5 of the amelogenin
Subjects
I1.1
11.2
1.3
11.4
Fig. 1. Abbreviated pedigree of family X A I 5 with X-linked amelogenesis imperfecta. Affected male represented by filled square: heterozygous females by vertical striped circles
Fig. 2a. Clinical photograph of individual I1.3 showing tipper permanent incisor teeth with a "square" morphology caused by a combination of reduced enamel thickness and attrition of the incisal edges. The teeth are opaque with some irregularity of the surface. b Clinical photograph of individual II.1 demonstrating vertical markings restllting from alterations both in contour and shade of the enamel
The family tree is shown in Fig. 1 and the clinical features illustrated in Fig. 2. All examined individuals were seen by two of the authors (MJA and PJMC). Individual II.3, the proband, a male aged 13 years, had upper permanent incisor teeth that were "square" in morphology (Fig. 2a) with a more tapering form than normal to the upper canines. These findings were felt to reflect reduced tooth size. The enamel was hard and slightly rough with a "ground glass" feel when a probe was passed over the surface. There was a suggestion of an alteration in the contour of the enamel on teeth 14, 24, 25, 47, 35, 37 (46 and 36 had previously been extracted). Abrasion and attrition were most evident on the upper incisor teeth. The teeth were "paint white" in colour, uniformly opaque but of slightly variable colour, most being A2 [standard shade guide (Vita, Lumin Vacuum porcelain shade guide; Vita Lumin Vaccum-Farbskala. Vita Zahnfabrik, H. Rauter. Bad Sfickingen, Germany)] in shade. This alteration in colouration was most noticeable on the canines (13, 23, 33, 43), tooth 21 and the lower premolars (34, 35, 44, 45). Further questioning of the family revealed that the older sister, individual II.l, aged 16 years, had teeth with vertical markings, described by the family as "tram-line teeth". On examination, the general impression was of teeth of normal outline and lucency with the overall colour assessed as A2. The enamel was glassy in appeareance but irregular in vertical contour, hard on probing and with no significant degree of tooth wear. All teeth had vertical markings resulting from alterations both in contour and shade of the enamel, although the changes in contour were more noticeable than those of shade (Fig. 2b). Tooth 11 (upper right permanent central incisor) had particularly marked brownish inclusions in an approximately vertical distribution. Teeth 31, 41 and 42 (lower right permanent central incisor and lower left permanent central and lateral incisors) were less affected than other teeth. Individual II,2, a female aged 15 years, had teeth of normal form and colour and was regarded as being unaffected. The tooth shade was recorded as AIIA2. Individual II.4, a boy 2 years old, had teeth of normal form and colour and was felt to be unaffected. The mother of the proband, individual I. 1 (aged 44 years) had teeth with vertical bands of opaque enamel alternating with enamel of normal colour and translucency. The affected enamel was paler than the normal enamel A I / A 2 , adjacent normal enamel A3. All the enamel was uniformly smooth and hard but with a very subtle undulation in the surface contour overlying the paler affected enamel. Teeth 21 and 22 (upper left central and lateral permanent incisors) were more severely affected than other standing teeth. The degree of attrition and abrasion was felt to be consistent with the patient's age and was not considered to be caused by any intrinsic defect in the enamel. Individual 1.2, the unrelated male spouse of 1.1 (aged 37 years), had teeth of normal form and marking and of shade A3.
Table 1. Conditions for PCR amplification of exons 3 . 5 and 6 of the amelogenin gene Exon
1 cycle
Primer pairs ~
Initial denaturation 1 cycle
30 cycles Annealing
Extension
Denaturation
Annealing
Extension
Denaturation
3
AM1 AM2
94~ 5 min
54~ 1 min
72~ 2 min
94~ 30 s
54~ 1 rain
72~ l0 rain
94~ 20 s
5
AM3 AM4
94~ 5 rain
54~ 1 rain
72~ 2 rain
94~ 30 s
54~ I min
72~ 10 min
94~ 30 s
3
AM5 AM6
94~ 5 rain
55~ 30 s
72~ 2 rain
94~ 30 s
55~ 1 rain
72~ 10 min
94~ 30 s
" From Lagerstrom et al. (1991)
415 Materials and methods Genomic DNA was extracted from peripheral blood leucocytes by standard methods. DNA from each of the three exons in the portion of the amelogenin gene showing X-Y homology (Nakahori et al. 1991) was amplified using the primers reported by Lagerstrom et al. (1991). Primers were used at a concentration of 1 p.M under the conditions shown in Table 1. The polymerase chain reaction (PCR) products were electrophoresed in 2% agarose gels containing ethidium bromide and viewed under ultraviolet light to detect any differences in product size. Aliquots of these PCR products were then directly sequenced. Following purification with GeneClean (Bio 101 Inc), the PCR products were annealed to either or both primers (AM1, AM4, AM5 and AM6) according to the size of the exons and labelled with [35S] ~-CTP (Amersham) using the Sequenase system (National Diagnostics). The labelled fragments were separated in 0.l-ram thick polyacrylamide gels (Sequagel, National Diagnostics) and the resulting bands visualised by autoradiography.
Results No obvious differences were detected in the size of the P C R product for any of the three exons amplified in the affected male and unaffected female in this family. Direct D N A sequencing showed no differences in exons 3 and 6 (Salido et al. 1992) between the affected male (individual II.3) and his unaffected sister (individual E). In exon 5, however, a single cytosine deletion was detected in the affected male (CCCC--~CCC) (Fig. 3) within the sequence of the exon. Heterozygous (manifesting) females showed three b a n d s of normal intensity in this region, with a fourth band of reduced strength, and duplication of subsequent bands (also of reduced intensity). This pattern was not observed in the father (I.2) or the clinically unaffected female II.2, indicating that the mutation segregated with the disease.
The effect of this deletion is an alteration of the reading frame of exon 5 and the introduction of an inappropriate T G A stop codon (an opal mutation) 3' of the mutation. The altered D N A sequence and the changes in the amino acid sequence are shown in Table 2.
Discussion Based on the recent data from Salido et al. (1992), the present mutation occurs in exon 5 (the second exon of Nakahori et al. 1991). In family X A I 5, therefore, translation of the amelogenin gene will be prematurely terminated. This should result in a truncated amelogenin gene product; we estimate that it would yield a protein product of only 74 amino acids. Over 200 amino acids encoded by the remainder of the amelogenin gene will be absent. Although we have not carried out linkage analysis in this family to establish linkage to the Xp22 region containing the A M G gene, we believe that the detection of a nonsense mutation within an exon of this gene in affected individuals provides sufficient evidence for its association in this family to be causal. Previously reported phenotypes of X A I have been described as being characterised predominantly either by hypoplasia (reduced thickness) or hypomineralisation (reduced mineralisation) of the enamel. We have observed that some X A I families described in the literature apparently demonstrate features of both reduced enamel thickness and reduced mineral content (Crawford and Aldred 1992), indicating that these two processes are probably operating together in affected individuals. The present family similarly displays concurrent features of hypoplasia and hypomineralisation, but they are expressed differently in different family members. Thus, hypoplasia was more
Fig. 3. Manual direct DNA sequence analysis of affected male (individual II.3) with X-linked amelogenesis imperfecta and his unaffected sister (individual II.2). PCR products were directly sequenced on the anti-sense strand. GGGG---,GGG (sense CCCC ~ CCC) transition identified in the affected male in PCR-amplified exon 5 of the amelogenin gene
Table 2. Amelogenin exon 5a DNA sequence and the corresponding amino acid sequence in normal individuals and in an affected male in family XAI 5 with X-linked amelogenesis imperfecta Wild type Mutant
GTG V GTG V
CTr L CTT L
a From Salido et al. (1992)
ACC T ACC T
CCT P CTT L
TTG L TGA STOP
AAG K AGT
TGG W GGT
TAC Y ACC
CAG Q AGA
AGC S GCA
ATA I TAA
AGG R GGC
CCA P CA
416 n o t i c e a b l e in i n d i v i d u a l II.1, similar to the h y p o p l a s t i c X A I p h e n o t y p e of Schulze (1956). I n d i v i d u a l 1.1, however, h a d c h a n g e s of e n a m e l c o l o u r , r a t h e r t h a n cont o u r , which is m o r e c o n s i s t e n t with the findings r e p o r t e d by W i t k o p (1957, 1967), and which he c h a r a c t e r i s e d as " X - l i n k e d recessive h y p o m a t u r a t i o n a m e l o g e n e s i s imp e r f e c t a " . This w o u l d i n d i c a t e that an a b n o r m a l i t y of e n a m e l q u a n t i t y is the p r e d o m i n a n t defect in i n d i v i d u a l II.1, w h e r e a s a d e f e c t of m i n e r a l i s a t i o n is the essential a b n o r m a l i t y in i n d i v i d u a l 1.1. T h e a f f e c t e d m a l e (indiv i d u a l II.3) has s o m e r e d u c t i o n in e n a m e l thickness, and m a r k e d changes in the lucency of the e n a m e l ; he, t h e r e fore, shows f e a t u r e s of b o t h p h e n o t y p e s . B e c a u s e of the c o n c u r r e n t p r e s e n t a t i o n of b o t h h y p o m i n e r a l i s a t i o n and h y p o p l a s i a in the p r e s e n t family ( a n d in o t h e r s r e p o r t e d in the l i t e r a t u r e ) , we p r o p o s e that the a m e l o g e n i n gene is i m p l i c a t e d in b o t h the n o r m a l m i n e r a l i s a t i o n p r o c e s s a n d in the f o r m a t i o n of e n a m e l of n o r m a l thickness. T h e i d e n t i f i c a t i o n and c h a r a c t e r i s a t i o n of f u r t h e r a m e l o g e n i n m u t a t i o n s in o t h e r A I H 1 families s h o u l d p r o v i d e evid e n c e for the relative i m p o r t a n c e of d i f f e r e n t r e g i o n s of the a m e l o g e n i n gene in the p r o c e s s of a m e l o g e n e s i s , tog e t h e r with a logical classification of these c o n d i t i o n s .
Acknowledgements. We wish to thank the family described in this paper for their support. This study was funded by grants from the Medical Research Council and Welsh Scheme for the Development of Health and Social Research. MJA is a Research Fellow support by a Medial Research Council Special Training Fellowship in Recombinant DNA Technology and a Medical Research Council Clinician Scientist Fellowship. The primers were kindly supplied by the Human Gene Mapping Project Resource Centre.
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