Amelogenin Locus Analysis Essay

Paralogous gene pairs on the mammalian sex chromosomes can provide useful information on the tempo and mode of X-Y differentiation. Recently, Lahn and Page (1999) reported intriguing results on the chromosomal locations and synonymous sequence divergences (ks with multiple-hit correction) of 19 pairs of X-Y-paralogous cDNA sequences in humans or squirrel monkeys. It was found that there are four distinct groups (strata) of the gene pairs in terms of the ks value. Of these, seven belong to stratum 4, and the X paralogs are clustered in a single chromosomal region which is adjacent to the pseudoautosomal boundary in the short arm of chromosome X. In contrast, the seven Y paralogs are scattered on chromosome Y, presumably owing to frequent chromosomal rearrangements. Together with similar observations on strata 1, 2, and 3, it was hypothesized that X-Y-chromosomal differentiation was initiated one stratum at a time and that stratum 1 was the first to embark on the differentiation and stratum 4 was the most recent. Lahn and Page (1999) invoked chromosomal inversions as the most likely cause for formation of these strata by suppressing sex-chromosomal recombination.

Bengtsson and Goodfellow (1987) argued that X-Y differentiation had occurred only after X-Y recombination ceased and that the most likely mechanism of suppressing recombination was chromosomal inversions. Navarro et al. (1997) theoretically demonstrated that there should be a strong reduction in recombination in the proximal zone of an inverted region owing to the production of unbalanced gametes. Here we report that the junction between stratum 3 (ks = ∼30%) and stratum 4 (ks = ∼10%) in the human and the chimpanzee resides in the second intron of the amelogenin gene, arguing that this junction cannot be a direct breakpoint of any chromosomal inversion. We also make a similar comparison of amelogenin genes in other mammals and show that formation of stratum 4 is specific to mammalian orders.

Eutherian mammals generally possess a pair of amelogenin genes on the X and Y chromosomes (e.g., Lau et al. 1989 ; Nakahori, Takenaka, and Nakagome 1991 ; Salido, Yen, and Koprivnikar 1992 ) which are designated AMGX and AMGY, respectively. In humans, the AMGX locus is oriented from the centromere to the telomere. Among the seven loci examined for stratum 4, the AMGX locus is most proximal and nearest to stratum 3. We retrieved two human BAC clone sequences which contained AMGX (AC002366) and AMGY (AC013412) and also determined the genomic DNA sequences of the chimpanzee homologs (data will be presented elsewhere). Quite unexpectedly, it turns out that the upstream region from intron 2 exhibits 25% sequence differences per silent site (ps without multiple-hit corrections), the same level as the corrected ks value of 30% in stratum 3 (fig. 1 ). In sharp contrast, the region downstream of intron 2 exhibits ps = 10%, as in intron 3 (Huang et al. 1997 ) as well as the six other paralogous gene pairs belonging to stratum 4 (Lahn and Page 1999 ). The same pattern and degree of sequence differences were also found in the comparison between the chimpanzee AMGX and AMGY. These findings convincingly indicate that the junction between strata 3 and 4 arranged side by side on chromosome X occurs in the middle of the human and chimpanzee AMGX locus.

The first and second exons of the amelogenin gene almost exclusively encode the 5′ untranslated region so that the cDNA or amino acid sequences represent the feature of stratum 4. Accordingly, the phylogenetic analysis showed the sister relationship of X and Y paralogs within both cattle and pigs (Gibson et al. 1991, 1992 ; Hu et al. 1996 ; Chen et al. 1998 ; Girondot and Sire 1998 ; Toyosawa et al. 1998 ). The synonymous differences are about 6% in cattle, which is not significantly different from that in humans. However, since the promoter region and exon 1 in the cow AMGX and AMGY differ by ps = 30% (Chen et al. 1998 ), there is again an abrupt reduction in the extent of sequence differences, as in humans (fig. 1 ). To gain some insight into the position in the step reduction, we determined the genomic DNA sequences of the cattle paralogs. A preliminary result showed that the reduction occurred at almost the same position in intron 2 as in humans. It is also to be noted that the cDNA sequence differences (ps = 3%) between the pig AMGX and AMGY were significantly lower than those in humans and cattle. More extreme are the horse AMGX and AMGY genes, in which no nucleotide difference is found (Hasegawa et al.2000) . Since the genomic DNA sequences of the pig and horse AMGX and AMGY are unavailable at present, it is not clear to what extent they differ from each other in the upstream region of the gene. Yet, the clocklike accumulation of synonymous differences suggests that differentiation of AMGX and AMGY occurred relatively recently in the pig and has not yet occurred in the horse. Thus, the initiation timing of stratum 4 differentiation differs greatly among mammalian orders, being most ancient in humans (Primates) and most recent in horse (Perissodactyla). Rodents are exceptional in that they may not possess AMGY at all: it is either deleted or altered to an undetectable level (Snead et al. 1985 ; Lau et al. 1989 ; Bonass et al. 1994 ). In any case, these amelogenin sequences suggest that the downstream region of the gene, and probably stratum 4 as a whole, began to differentiate after the mammalian radiation. On the contrary, X-Y differentiation in stratum 3 must have occurred well before the mammalian radiation (Lahn and Page 1999 ). This is supported by the relatively large sequence differences between X and Y paralogs in stratum 3, such as those between ZFX and ZFY (Schneider-Gädicke et al. 1989 ).

As argued by Bengtsson and Goodfellow (1987) and by Lahn and Page (1999) , X-Y differentiation may result from suppression of X-Y recombination mediated through chromosomal inversions. Also, this process may be a common, irreversible evolutionary step in mammals (Jegalian and Page 1998 ). However, the deposit of stratum 4 in mammals cannot be easily explained as a direct consequence of a single chromosomal inversion for two reasons. First, if the junction between strata 3 and 4 is a breakpoint of a chromosomal inversion, the amelogenin gene on chromosome X or Y should have been disrupted, suggesting that any such inversion, if present, must have occurred somewhere else. Second, the initiation of stratum 4 differentiation is generally mammalian-order-specific, but the junctions between strata 3 and 4 are almost identical, at least between humans and cattle. This finding can be explained by a single inversion that might occur in the common ancestor of humans and cattle. However, since pigs and horses are more closely related to cattle than to humans (Goodman, Czelusniak, and Beeber 1985 ) and the inversion is likely to be shared by all four mammalian species, it is difficult to account for the observed large differences in the ks or ps value among mammalian AMGX and AMGY paralogs.

Nonetheless, it is still conceivable that independent chromosomal inversions, particularly on chromosome Y, were responsible for differential initiations of recombination suppression in mammals. Although such inversions must have occurred in regions other than the amelogenin locus itself, they could happen to suppress recombination around the locus. According to Navarro et al. (1997) , the proximal region of chromosomal inversions is expected to undergo a strong reduction in recombination. Recombination appears to be a complex event involving a number of enzymes and proteins, and the event is initiated by the specific binding of one of these proteins to the DNA molecule of the chromosome (Klein and Takahata 1990 ). If this interaction is highly specific, recombination would be initiated only in limited regions of a chromosome. While the molecular mechanism of recombination is still poorly understood, it is tempting to conclude that the upstream region of the amelogenin locus may contain a sequence or structural signal responsible for initiating or suppressing recombination, thereby repeatedly triggering subsequent X-Y differentiation in different mammals.

We thank two anonymous reviewers for their constructive criticism on an early version of this paper. This work was supported in part by Monkasho grant 12304046 to N.T.

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Stephen Palumbi, Reviewing Editor

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Keywords: recombination chromosomal inversion nucleotide differences BAC clone evolutionary strata

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Address for correspondence and reprints: Naoyuki Takahata, Department of Biosystems Science, Graduate University for Advanced Studies, Hayama, Kanagawa 240-0193, Japan. takahata@soken.ac.jp .

Fig. 1.—Percentages of sequence difference between the human AMGX and AMGY genes and their 5′ and 3′ flanking regions in the comparison between the two BAC clones (AC002366 and AC013412). Plotted are the nucleotide differences in nonoverlapping windows (100-bp window size). The stepwise regression (thick line) is determined by the least-squares method. The decline in the sequence differences is located in the second intron, and the reduced level extends beyond the downstream region of the gene. The exon (numbered from 1 through 7) and intron structure (in a horizontal line) is depicted at the bottom. Various insertion elements in chromosome X (above) and Y (below) are indicated by open triangles, and they are excluded from the analysis

Fig. 1.—Percentages of sequence difference between the human AMGX and AMGY genes and their 5′ and 3′ flanking regions in the comparison between the two BAC clones (AC002366 and AC013412). Plotted are the nucleotide differences in nonoverlapping windows (100-bp window size). The stepwise regression (thick line) is determined by the least-squares method. The decline in the sequence differences is located in the second intron, and the reduced level extends beyond the downstream region of the gene. The exon (numbered from 1 through 7) and intron structure (in a horizontal line) is depicted at the bottom. Various insertion elements in chromosome X (above) and Y (below) are indicated by open triangles, and they are excluded from the analysis

Gender Identification with Amelogenin

The ability to designate whether a sample originated from a male or a female source is useful in sexual assault cases, where distinguishing between the victim and the perpetrator’s evidence is important. Likewise, missing persons and mass disaster investigations can benefit from gender identification of the remains. Over the years a number of gender identification assays have been demonstrated using PCR methods (Sullivan et al. 1993, Eng et al. 1994, Reynolds & Varlaro 1996). By far the most popular method for sex-typing today is the amelogenin system as it can be performed in conjunction with STR analysis.

Amelogenin is a gene that codes for proteins found in tooth enamel. The British Forensic Science Service was the first to describe the particular PCR primer sets that are used so prevalently in forensic DNA laboratories today (Sullivan et al. 1993). These primers flank a 6bp deletion within intron 1 of the amelogenin gene on the X homologue (Figure 5.8). PCR amplification of this area with their primers results in 106bp and 112bp amplicons from the X and Y chromosomes, respectively. Primers, which yield a 212bp X-specific amplicon and a 218bp Y-specific product by bracketing the same 6bp deletion, were also described in the original amelogenin paper (Sullivan et al. 1993) and have been used in conjunction with the D1S80 VNTR system (Budowle et al. 1996).

An advantage with the above approach, i.e., using a single primer set to amplify both chromosomes, is that the X chromosome product itself plays a role as a positive control. This PCR-based assay is extremely sensitive. Mannucci and co-workers were able to detect as little as 20 pg (≈3 diploid copies) as well as sample mixtures where female DNA was in 100-fold excess of male DNA (Mannucci et al. 1994).

Other regions of the amelogenin gene have size differences between the X and Y homologues and may be exploited for sex-typing purposes. For example, Eng and co-workers (1994) used a single set of primers that generated a 977bp product for the X-chromosome and a 788bp fragment for the Y-chromosome. In this case, a 189bp deletion in the Y relative to the X-chromosome was used to differentiate the two chromosomes.

A careful study found that 19 regions of absolute homology, ranging in size from 22 bp to 80 bp, exist between the human amelogenin X and Y genes that can be used to design a variety of primer sets (Haas-Rochholz & Weiler 1997). Thus, by spanning various deletions of the X and/or Y chromosome, it is possible to generate PCR products from the X and Y homologues that differ in size and contain size ranges that can be integrated into future multiplex STR amplifications.

While amelogenin is an effective method for sex-typing biological samples in most cases, the results are not foolproof either due to primer binding sites that lead to null alleles or chromosomal deletions. Amelogenin Y allele dropouts have been observed due to loss of portions of the Y chromosome in some population groups. Amelogenin X allele dropouts have been seen primarily due to primer binding site mutations.

Amelogenin Y Allele Dropout

A rare deletion of the amelogenin gene on the Y chromosome can cause the Y chromosome amplicon to be absent (Santos et al. 1998). In such a case, a male sample would falsely appear as a female with only the amelogenin X allele being amplified. It appears that this deletion of the Y chromosome amelogenin region is more common in Indian populations (Thangaraj et al. 2002) than those of European or African origins. A study of almost 30,000 males in the Austrian National DNA database revealed that only six individuals lacked the amelogenin Y-amplicon (Steinlechner et al. 2002). These individuals were verified to be male with Y-STRs and amplification of the SRY region (see Chapter 13).

More recent studies have attempted to map the Y deletions in detail and to track the specific biogeographic ancestry of these interesting variants (Cadenas et al. 2007, Jobling et al. 2007). Through examining adjacent STR markers and sequence-specific tag sites, the extent of the Y chromosome deletion can be mapped (Takayama et al. 2009). When amplifying Y-STR loci, the locus DYS458 in Yfiler (see Chapter 13) is the most likely one to be lost with amelogenin Y deletions due to its close proximity to the AMEL Y (see Figure 13.6).

Amelogenin X Allele Dropout

Amelogenin X allele dropout has also been observed in males. In this case only the amelogenin Y-amplicon is present (Shewale et al. 2000, Alves et al. 2006, Maciejewska & Pawłowski 2009). In one study, this phenomenon was observed only three times out of almost 7000 males examined (Shewale et al. 2000). The authors of this study felt that the AMEL X null was most likely a result of a rare polymorphism in the primer binding sites for the amelogenin primers used in commercial STR kits. A different set of amelogenin primers targeting the same 6bp deletion on the X chromosome amplified both the X and Y alleles of amelogenin (Shewale et al. 2000). However, in some populations, this loss of the AMEL X allele is more common. In a study of 503 individuals from São Tomé Island (West Africa), 10 male individuals displayed only the Y allele from amelogenin amplification due to a primer binding site mutation in the AMEL X allele (Alves et al. 2006). A different mutation caused one male out of 5534 Polish males tested to display only the AMEL Y allele (Maciejewska & Pawłowski 2009).

A report of males examined in paternity testing labs using Applied Biosystems STR typing kits found that there were a higher number of African American males showing only the AMEL Y allele (i.e., the AMEL X allele dropped out). Still, this AMEL X null is fairly rare being seen only 48 times in 144,391 males tested or 0.03% of the time (AABB 2008).

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