Blood, 1 May 2003, Vol. 101, No. 9, pp. 3749-3750
CORRESPONDENCE
To the editor:
The transcobalamin codon 259 polymorphism should be designated
776C>G, not 775G>C
We read with great interest the recent article by Miller et
al1 that examined the transcobalamin (TC) genetic
polymorphism encoding proline or arginine at codon 259 in the
TC gene (Pro259Arg) and its possible
influence on indices of vitamin B12 status in healthy older adults.
Based on serum levels of holoTC (the B12-TC complex) that were
significantly higher in individuals who were homozygous for the
wild-type gene encoding the proline form of TC compared with
heterozygotes and homozygotes for the arginine-encoding gene variant,
Miller et al concluded that the TC Pro259Arg polymorphism may influence the intracellular availability of vitamin B12 and thus
influence susceptibility to B12 deficiency.1 There are several ways of describing human genetic sequence variations, and
sometimes the inconsistent use of genetic terms may cause confusion.
In international guidelines for genetic nomenclature it is
preferable to give the nucleotide position of a polymorphism rather
than giving the codon number only.2 Miller et al mention both nucleotide position and codon number, but unfortunately the nucleotide substitution that is the basis for the TC phenotypic variability on codon 259 is assigned to the wrong nucleotide compared with the full-length complementary DNA sequence of the TC
gene (GenBank accession number M60396 for the proline-encoding sequence and NM000355 for the arginine-encoding sequence; Figure
1).
The polymorphism is a C-to-G substitution at position 776 in relation to the first nucleotide (+1) of the ATG-translation initiation codon,
not a G-to-C substitution at nucleotide 775. This mistake, however,
does not interfere with their genotyping method, and all results
reported in the investigation seem valid for the TC 776C>G
polymorphism. Nevertheless, we would like to make the following comment
regarding the method. The presence of a G nucleotide at position 776, which encodes arginine at codon 259, removes a recognition site for
MvaI. The polymerase chain reaction (PCR) product
remains uncleaved if the individual is homozygous for the 776G allele and partially cleaved if the individual is heterozygous. This method is
sensitive to insufficient cleavage due to excess of PCR product or poor
cleavage conditions in a specific tube and is for this reason not
suitable for routine diagnostic laboratory work. We suggest the use of
alternative methods not based on restriction enzyme digestion for
determining the TC 776C>G polymorphism. Recently, we
presented a method for TC genotyping based on the
minisequencing technique.3,4 This technique does not
depend on the presence of restriction enzyme recognition sites and also
is less sensitive to silent polymorphisms.
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| Figure 1.
The TC Pro259Arg polymorphism is a C-to-G
substitution at nucleotide 776.
(Top) Sequence analysis of the TC gene in one individual
homozygous for the wild-type 776CC genotype that encodes proline at
codon 259. (Bottom) Sequence analysis of the TC gene in one
individual homozygous for the 776GG genotype that encodes arginine at
codon 259.
|
|
Henrik Zetterberg, Mona Palmér, Cecilia Boreström, Lars Rymo, and Kaj Blennow
Correspondence: Henrik Zetterberg, Department of Clinical
Chemistry and Transfusion Medicine, Sahlgrenska University Hospital,
Göteborg University, S-413 45 Gothenburg, Sweden; e-mail:
henrik.zetterberg{at}clinchem.gu.se
References
1.
Miller JW, Ramos MI, Garrod MG, Flynn MA, Green R.
Transcobalamin II 775G>C polymorphism and indices of vitamin B12 status in healthy older adults.
Blood.
2002;100:718-720[Abstract/Free Full Text].
2.
den Dunnen JT, Antonarakis E.
Nomenclature for the description of human sequence variations.
Hum Genet.
2001;109:121-124[CrossRef][Medline]
[Order article via Infotrieve].
3.
McCaddon A, Blennow K, Hudson P, Regland B, Hill D.
Transcobalamin polymorphism and homocysteine.
Blood.
2001;98:3497-3499[Free Full Text].
4.
Zetterberg H, Regland B, Palmér M, et al.
The transcobalamin codon 259 polymorphism influences the risk of human spontaneous abortion.
Hum Reprod.
2002;17:3033-3036[Abstract/Free Full Text].
Response:
Transcobalamin 776C>G: significance and determination
We thank Dr Zetterberg and his colleagues for their comments
regarding our paper1 and for pointing out that the correct designation for the transcobalamin codon 259 polymorphism is 776C>G. On the issue of which isoform of the protein (proline or arginine) should be considered the wild type, we acknowledge that convention dictates that the more commonly occurring isoform (proline) should be
given this designation. It is noteworthy, however, that the arginine
isoform does have a relatively high prevalence (~ 20% homozygosity
among whites),1-5 despite the fact that it is associated with poorer vitamin B12 status compared with the proline
isoform.1-6 This observation implies that the mutation
does not afford any obvious selective advantage, at least as far as
standard methods of B12 status assessment are concerned. Therefore, the
relatively high prevalence of the arginine isoform remains unexplained.
Concerning methodology, the minisequencing technique for genotyping
transcobalamin does have the advantage of not requiring restriction
enzyme digestion. However, the minisequencing method may not be readily
available in all laboratories. In developing our method, which utilizes
the restriction enzyme MvaI, we validated it by direct
sequencing of the polymerase chain reaction (PCR) products
prior to digestion. We concluded that our method was robust and
accurate, and thus is a relatively inexpensive method for determining
transcobalamin genotype.
Joshua W. Miller and Ralph Green
Correspondence: Joshua W. Miller, UC Davis Medical Center,
Research 3, Room 3200A, 4645 Second Ave, Sacramento, CA 95817; e-mail:
jwmiller{at}ucdavis.edu
References
1.
Miller JW, Ramos MI, Garrod MG, Flynn MA, Green R.
Transcobalamin II 775G>C polymorphism and indices of vitamin B12 status in healthy older adults.
Blood.
2002;100:718-720[Abstract/Free Full Text].
2.
Namour F, Guy M, Aimone-Gastin I, de Nonancourt M, Mrabet N, Guéant J-L.
Isoelectric phenotype and relative concentration of transcobalamin II isoproteins related to the codon 259 Arg/Pro polymorphism.
Biochem Biophys Res Comm.
1998;251:769-774[CrossRef][Medline]
[Order article via Infotrieve].
3.
Namour F, Olivier J-L, Abdelmouttaleb I, et al.
Transcobalamin codon 259 polymorphism in HT-29 and Caco-2 cells and in Caucasians: relation to transcobalamin and homocysteine concentration in blood.
Blood.
2001;97:1092-1098[Abstract/Free Full Text].
4.
Afman LA, Van Der Put NMJ, Thomas CMG, Trijbels JMF, Blom HJ.
Reduced vitamin B12 binding by transcobalamin II increases risk of neural tube defects.
Q J Med.
2001;94:159-166.
5.
McCaddon A, Blennow K, Hudson P, Regland B, Hill D.
Transcobalamin polymorphism and homocysteine.
Blood.
2001;98:3497-3499[Free Full Text].
6.
Namour F, Guéant J-L.
Transcobalamin polymorphism, homocysteine, and aging [letter].
Blood.
2001;98:3499.
