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Comparative genome analysis can provide novel insights into gene evolution and function. The VHL gene is highly conserved in primates, rodents, flies and worms [116]. In the fly, Drosophila Melanogaster, an overall 50% similarity between fly and human was calculated, and 76% similarity in the elongin C binding site [117, 118]. Figure 30 illustrates the nucleotide sequence alignment of the VHL open reading frame for human, rat and mouse.

H.Sapiens ATGCCCCGGAGGGCGGAGAACTGGGACGAGGCCGAGGTAGGCGCGGAGGAGGCAGGCGTC R.Norvegicas ATGCCCCGGAAGGC---AGCTAGTC-CAGAGGAGGCAG--- M.Musculus ATGCCCCGGAAGGC---AGCCAGTC-CAGAGGAGGCGG--- ********** *** ** ** * * ******** * H.Sapiens GAAGAGTACGGCCCTGAAGAAGACGGCGGGGAGGAGTCGGGCGCCGAGGAGTCCGGCCCG R.Norvegicas --AAAG---GATGCCG---GGCTCT M.Musculus --CGGG---GGAGCCC---GGTCCT * * *** ** *

H.Sapiens GAAGAGTCCGGCCCGGAGGAACTGGGCGCCGAGGAGGAGATGGAGGCCGGGCGGCCGCGG

R.Norvegicas GAAGAG---ATAGAGGCTGGGCGGCCGCGG

M.Musculus GAGGAG---ATGGAGGCTGGGCGGCCGCGG

** *** ** ***** ************ H.Sapiens CCCGTGCTGCGCTCGGTGAACTCGCGCGAGCCCTCCCAGGTCATCTTCTGCAATCGCAGT R.Norvegicas CCGGTTTTACGCTCTGTGAACTCGCGCGAACCCTCTCAGGTCATCTTCTGCAACCGCAGC M.Musculus CCGGTGCTGCGCTCGGTGAACTCGCGCGAGCCCTCTCAGGTCATCTTCTGCAACCGCAGC ** ** * ***** ************** ***** ***************** ***** H.Sapiens CCGCGCGTCGTGCTGCCCGTATGGCTCAACTTCGACGGCGAGCCGCAGCCCTACCCAACG

M.Musculus CCGCGCGTCGTGCTGCCTTTGTGGCTCAACTTCGACGGCGAGCCTCAGCCCTACCCGATC ***************** * *********** ** ** ***** *********** * H.Sapiens CTGCCGCCTGGCACGGGCCGCCGCATCCACAGCTACCGAGGTCACCTTTGGCTCTTCAGA R.Norvegicas TTACCACCGGGCACCGGCCGCCGCATCCACAGCTACCGAGGTCACCTTTGGCTCTTCAGG M.Musculus TTACCACCGGGCACCGGCCGCCGCATCCACAGCTACCGAGGTCATCTTTGGCTCTTCAGG * ** ** ***** ***************************** ************** H.Sapiens GATGCAGGGACACACGATGGGCTTCTGGTTAACCAAACTGAATTATTTGTGCCATCTCTC R.Norvegicas GATGCGGGGACCCATGATGGACTTCTGGTTAACCAAACGGAACTGTTTGTGCCATCCCTC M.Musculus GATGCGGGGACCCATGATGGACTTCTGGTTAACCAAACGGAGCTGTTTGTGCCATCCCTC ***** ***** ** ***** ***************** ** * *********** *** H.Sapiens AATGTTGACGGACAGCCTATTTTTGCCAATATCACACTGCCAGTGTATACTCTGAAAGAG R.Norvegicas AATGTTGATGGACAGCCTATTTTTGCCAACATCACATTGCCAGTGTATACCCTGAAAGAG M.Musculus AATGTCGATGGACAGCCTATTTTTGCCAACATCACATTGCCAGTGTATACCCTGAAAGAG ***** ** ******************** ****** ************* ********* H.Sapiens CGATGCCTCCAGGTTGTCCGGAGCCTAGTCAAGCCTGAGAATTACAGGAGACTGGACATC R.Norvegicas CGGTGCCTTCAGGTTGTACGGAGCCTGGTCAAGCCTGAGAACTACAGGAGGCTGGACATC M.Musculus CGGTGCCTTCAGGTTGTGCGGAGCCTGGTCAAGCCTGAGAACTACAGGAGACTGGACATC ** ***** ******** ******** ************** ******** ********* H.Sapiens GTCAGGTCGCTCTACGAAGATCTGGAAGACCACCCAAATGTGCAGAAAGACCTGGAGCGG R.Norvegicas GTCAGGTCGCTCTATGAAGACTTGGAAGACCACCCAAATGTGCGGAAAGACATACAGCGG M.Musculus GTCAGGTCACTCTATGAGGATTTGGAGGACTACCCAAGTGTGCGGAAGGACATACAGCGA ******** ***** ** ** **** *** ****** ***** *** *** * **** H.Sapiens CTGACACAGGAGCGCATTGCACATCAACGGATGGGAGA---T---TGA R.Norvegicas CTGACCCAAGAGCACCTCGAGAATCAGGCCCTGGGAGAGGAGCCTGAAGGAGTCCACTGA M.Musculus CTGAGCCAAGAGCACCTTGAGAGTCAGCACCTGGAAGAGGAGCCT---TGA **** ** **** * * * *** *** *** * *** Figure 30. Nucleotide sequence alignment of predicted VHL open reading frames in human (H.Sapiens), rat

(R.Norvegicus) and mouse (M.Musculus). Evolutionary analysis would suggest that the N-terminal repetitive

sequence in pVHL30 (red) is of less functional importance than those regions present in both pVHL30 and pVHL19 due

to lack of conservation. Identity with human: Mouse: 81.8%; Rat: 80.7%.

As already discussed, the N-terminal sequence of pVHL30 contains eight copies of

a GxEEx acidic repeat motif in human and higher primates, but only three copies were present in the marmoset, and only one copy was present in rodent VHL genes [116, 119]. Due to lack of conservation, evolutionary analysis would suggest that the N- terminal repetitive sequence in pVHL30 is of less functional importance than those

correlation between the pVHL domains that demonstrate most evolutionary conservation and those that were most frequently mutated in tumours.

Comparative analysis like that illustrated in Figure 30 has demonstrated conservation of the VHL gene product across many millions of years during evolution. Studies undertaken by Woodward et al. have highlighted not only this conservation throughout primates, but also as far back as to C.Elegans, demonstrating a much older evolutionary lineage dating from the beginnings of metazoan evolution [119]. The identification of a C.Elegans homologue is important for several reasons. First, conservation of amino acid sequence across such an evolutionary distance suggests the presence of a protein of significant functional importance. Second, C.Elegans provides an excellent experimental model organism in which to study the basic processes that are altered in human disease, and as we will see in chapter 4, the system from which the proline hydroxylases, proteins essential for HIFα regulation by pVHL, were isolated.

H.Sapiens MPRRAENWDEAEVGAEEAGVEEYGPEEDGGEESGAEESGPEESGPEELGAEEEMEAGRPR

M.Musculus MPRKAAS---PEEAAGE---P---GPE---EEMAGPRR

R.Norvegicus MPRKAAS---PEEAERM---P---GSE---EIEAGRPR

D.Melanogaster ---MALQI---AQNNRDG C.Elegans ---MSDGS---MDDDGRLF : .*

H.Sapiens PVLRSVNSRE-PSQVIFCNRSPRVVLPVWLNFDGEPQPYPTLPPGTGRRIHSYRGHLWLF

M.Musculus PVLRSVNSRE-PSQVIFCNRSPRVVLPLWLNFDGEPQPYPILPPGTGRRIHSYRGHLWLF

R.Norvegicus PVLRSVNSRE-PSQVIFCNRSPRVVLPLWLNFDGEPQPYPTLPPGTGRRIHSYRGHLWLF

D.Melanogaster QQLVGADQGKVEVYVLFANTTYRTLDLYWVCERERENMYLTLKPFEEVRVNTFTTHSWLF

C.Elegans PDLGSSTHDNREIRVRFLNRCAYPVDVFWLNPSKQPTKYGTLAQKKYLDIKTFKDHPWVA * . : * * * : *: . * * :::: * *:

H.Sapiens RDAGTHDGLLVNQTELFVPS---LNVDGQPIFAN---ITLPVYTLKERCLQVVRS

M.Musculus RDAGTHDGLLVNQTELFVPS---LNVDGQPIFAN---ITLPVYTLKERCLQVVRS

R.Norvegicus RDAGTHDGLLVNQTELFVPS---LNVDGQPIFAN---ITLPVYTLKERCLQVVRS

D.Melanogaster RDYYTGERMHVRSQRIFQPIRVRVPKSQQSPDQLVDVRSEVLIHFPMRSLRENCLWLVAR C.Elegans RRSFDGCKVLVNEKEVFWPE---PAPRMNLIVRNHCVITMKVQSLREIAGRSFLR * : *.. .:* * . . * : : :*:* . .

H.Sapiens -LVKPENYRRLDIVRSLYEDLEDHPNVQKDLERLTQERIAHQRMGD--- M.Musculus -LVKPENYRRLDIVRSLYEDLEDYPSVRKDIQRLSQEHLESQHLEEEP---- R.Norvegicus -LVKPENYRRLDIVRSLYEDLEDHPNVRKDIQRLTQEHLENQALGEEPEGVH D.Melanogaster WLIRTSNAPRRIIHGYHIPSTLKQQLLSLLTCIESYSRVAGTRRRR--- C.Elegans -HNPTEVPNKIKGLPRELQFEVKHFLDRKQEYSEIVCRSIPPPGPQRPQQ-- .. : . :

Figure 31. Amino acid sequence alignment of pVHL in human (H.Sapiens), mouse (M.Musculus), rat (R.Norvegicus), fly (D.Melanogaster) and worm (C.Elegans). Alignment according to ClastalW

As already mentioned, doubt has been cast on the importance of a second translational start site due to the lack of mutations found between the first and second methionine codons of the VHL gene in both sporadic and VHL-associated tumours. This observation suggests that mutation in this region might not lead to VHL inactivation if translation could be initiated at the second methionine codon, producing a functional VHL protein. Furthermore, both rat and mouse contain only 19 of the 53 amino acids present in this region of the human VHL ORF.

Interestingly, the acidic motif Gly-X-Glu-Glu-X, which is repeated eight times in the N-terminal 53-amino acid region of the human sequence (fig.31), is repeated only once in the rodents and not at all in the fly or worm [119]. However, according to Woodward et

el., all primates with available sequences contained eight acidic repeats, with the

exception of the marmoset, which only had three. The marmoset reflects evolutionarily, the oldest of the primates in this study. Therefore it is likely that this motif arose in progressively higher species by a series of duplications. Ironically, when the VHL gene was identified, the only significant homology between the predicted VHL gene product and other known proteins was the G-X-E-E-X repeat, showing similarity to a pro-cyclic surface membrane protein of Trypanasoma brucei. However, as already mentioned, subsequent investigations have cast doubt on the significance of this acidic repeat domain. Nonetheless, one study reports a significant pathogenic VHL mutation in the first 53 amino acids [5]. Here, 102 Swedish renal cell carcinomas were analysed for VHL mutations. In 47 patients, 70 new mutations were found, and most of them represented novel variations of the VHL gene.

Fig 33. Distributions of the detected VHL mutations according to Ma et al. [5]. The X-axis indicates the

number of the residue affected by mutations. The Y-axis indicates the number of renal cell carcinomas carrying mutations at designated residues. In red is the novel hotspot region located between residues 1-53.

1st _{Met 2}nd _Met

1 54

Figure 32. Amino acids 1-54 of human VHL. Eight acidic pentameric repeat sequences are present (GxEEx).

Only one repeat is present in rat and mouse, while all are conserved in primates.

GxEEx GxEEx GxEEx GxEEx GxEEx GxEEx GxEEx GxEEx

Three novel hotspots were detected in the study, among them, an interesting mutation located in the N-terminal sequence of VHL. Five clear cell renal cell carcinomas and one chromophilic renal cell carcinoma harboured a 15-nucleotide in-frame deletion (codons 41-45) at a duplex tandem repeat sequence site. In 5 out of 6 patients the wild- type allele was lost in the tumour samples, suggesting a causal role for the mutations in renal cell carcinoma. However, five patients with this deletion reside in the same hospital district. Unfortunately, the authors were unable to determine if the deletion was an inherited polymorphism or a sporadic mutation, because no other normal tissue was available from these patients or their biological relatives.

This study indicates a potentially significant role for the N-terminal region. Furthermore, the fact that several groups have shown that both translational initiation sites are utilised in vivo, and that two protein products, despite the acidic repeat discrepancies, are conserved from rodents through to humans, and that pVHL19 exhibits

functional activity as well as an alternative cellular localisation pattern suggests distinct functions for both protein products, pVHL30 and pVHL19 [102, 107, 120]. It could also be

suggested that mutations within the pVHL30 N-terminal region may not be tolerated,

thereby reflecting a critical role for this region in VHL biology in higher species.

Concluding Remarks

The comparative analysis of genomes is an important strategy in the molecular study of gene function. Such studies enable the reconstruction and understanding of functions that have been acquired, those that have been lost, and those that have a common heritage. The identification of specific mutations associated with human disease phenotypes complements the definition of regions of conserved homology across different animal species, and together these approaches provide a powerful molecular genetic tool toward the understanding of structure-function relationships in genes and proteins. Before looking at the functional implications of the VHL protein product, it is important to understand the fundamentals of VHL gene alterations, a topic which strikes at the core of VHL disease and the subject of consideration in the following chapter.