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  • 1.
    Böttiger, A. K.
    et al.
    Örebro University, School of Health and Medical Sciences.
    Nilsson, Torbjörn K.
    Örebro University, School of Health and Medical Sciences.
    Pyrosequencing assay for genotyping of the Transcobalamin II 776C>G polymorphism2007In: Scandinavian Journal of Clinical and Laboratory Investigation, ISSN 0036-5513, E-ISSN 1502-7686, Vol. 67, no 2, p. 247-251Article in journal (Refereed)
    Abstract [en]

    The 776C>G polymorphism of the Transcobalamin II gene is located in a GC-rich region and TaqMan real-time polymerase chain reaction (PCR) does not yield satisfactory genotyping results. We therefore hypothesized that a method based on DNA sequencing would be needed for this single nucleotide polymorphism (SNP) analysis; Pyrosequencing technology was tested for this purpose. A Pyrosequencing protocol was developed, optimized and applied to a sample of 389 Swedish senior citizens. The three genotypes CC, CG, and GG consistently yielded typical programs that were readily distinguishable from each other. The prevalence of the mutated allele in the studied Swedish population was q=0.445. It is concluded that the TC II 776C>G polymorphism can be reliably genotyped by Pyrosequencing technology.

  • 2.
    Böttiger, Anna
    Örebro University, School of Health and Medical Sciences.
    Genetic variation in the folate receptor-alpha and methylenetetrahydrofolate reductase genes as determinants of plasma homocysteine concentrations2008Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Elevated total plasma homocysteine (tHcy) is a risk factor for cardiovascular disease and neurocognitive disease such as dementia. The B vitamins folate and B12 are the main de terminants of tHcy. tHcy concentration can also be affected by mutations in genes coding for receptors, enzymes and transporters important in the metabolism of Hcy. This thesis focuses on mutations in the genes for folate receptor-alpha and methylenetetrahydrofolate reductase (MTHFR) and the effect they have on tHcy concentrations.

    Six novel mutations in the gene for folate receptor-alpha were described in Paper I. Taken together they exist in a population with a prevalence of approximately 1% and thus are not unusual. There may be an association of –69dupA and –18C>T to tHcy but for the 25-bp deletion, –856C>T, –921T>C and –1043G>A there is probably no association to tHcy. Mutation screening was continued and four additional mutations, 1314G>A, 1816delC, 1841G>A and 1928C>T, were described in Paper II. The prevalences for the heterozygotes were between 0.5% and 13% in an elderly population. There was no significant difference in prevalence between the elderly subjects and patients with dementia. The 1816(–)-allele and the 1841A-allele were in complete linkage and the haplotype 1816(–)-1841A may possibly have a tHcy raising effect. The 1314G>A and 1928C>T mutations had no association to tHcy.

    The genotype prevalences and haplotype frequencies of the MTHFR 677C>T, 1298A>C and 1793G>A polymorphisms were determined in a population sample of Swedish children and adolescents (Paper III). The MTHFR 677T-allele was associated with increased tHcy concentrations in both children and adolescents. A small elevating effect of the 1298C-allele and a small lowering effect of the 1793A-allele could be shown. In an epidemiological sample of adults from the Canary Islands, Spain, data for serum folate and vitamin B12 were used for a broader study of the nutrigenetic impact on tHcy (Paper IV). The 677T-allele had a significant tHcy increasing effect in men but not in women. The 1298C-allele had a minor elevating effect on tHcy in men with the 677CT genotype. It was not possible to document any effect of the 1793A-allele on tHcy due to its low prevalence. A slightly superior explanatory power for the genetic impact was obtained using the MTHFR haplotypes in the analysis compared to the MTHFR 677C>T genotype-based approach in both the Swedish children and adolescents and in the Spanish adults. Therefore MTHFR haplotypes should be considered when analysing the impact of the MTHFR 677C>T, 1298A>C and 1793G>A polymorphisms on tHcy.

    Notwithstanding the large geographical distance between our study populations the haplotype composition is quite similar. The MTHFR 677T-allele is slightly more prevalent in Spain compared to Sweden but it has only an effect on tHcy in the Spanish men. Age, gender and factors linked to the ethnicity of the studied subjects, seem to be able to override the nutrigenetic impact of tHcy-raising genotypes or haplotypes in particular settings, such as in the Spanish women in our study. Gene-nutrient interactions on plasma tHcy levels thus may or may not exist in a certain population. The transferability of nutrigenetic findings may therefore be limited, and must be re-evaluated for each particular setting of age-gender-ethnicity.

    List of papers
    1. Novel mutations in the 5'-UTR of the FOLR1 gene
    Open this publication in new window or tab >>Novel mutations in the 5'-UTR of the FOLR1 gene
    2006 (English)In: Clinical Chemistry and Laboratory Medicine, ISSN 1434-6621, E-ISSN 1437-4331, Vol. 44, no 2, p. 161-167Article in journal (Refereed) Published
    Abstract [en]

    We have previously reported two novel mutations in the 5'-untranslated region (UTR) of the gene for folate receptor-alpha (FOLR1). In our search for additional mutations, 92 patient samples with elevated levels of homocysteine were screened by single-strand conformation polymorphism (SSCP) between nt -425 and -782, and -712 and -1110. Between nt -425 and -782 we did not find any mutations. Between nt -712 and -1110 there were three novel mutations. One subject had two mutations very close to each other, c.-856C>T and c.-921T>C. Two subjects had a c.-1043G>A mutation. To get an idea of the prevalence of FOLR1 mutations in an unselected population, we also screened 692 healthy school children for mutations. In this cohort, between nt -188 and +272 we discovered one novel mutation, a single nucleotide substitution, c.-18C>T, in addition to five children with the 25-bp deletion mutation previously described by us. Thus, so far we have discovered six novel mutations in the 5'-UTR region of the gene for folate receptor-alpha. We genotyped all 17 subjects with a FOLR1 mutation for the methylenetetrahydrofolate reductase (MTHFR) 677C>T polymorphism, and developed new single-nucleotide polymorphism (SNP) genotyping protocols for MTHFR 1298A>C and 1793G>A utilising Pyrosequencing technology. None of the 17 subjects had the 677TT genotype, which ruled out this as a cause of elevated homocysteine levels, which was observed in some of the subjects. Further studies of mutations in the 5'-UTR of FOLR1, and in particular of their interplay with folate intake status, are warranted.

    National Category
    Medical and Health Sciences
    Research subject
    Medicine
    Identifiers
    urn:nbn:se:oru:diva-3035 (URN)10.1515/CCLM.2006.029 (DOI)16475900 (PubMedID)
    Available from: 2008-11-15 Created: 2008-11-15 Last updated: 2020-01-29Bibliographically approved
    2. Mutations in exons 2 and 3 of the FOLR1 gene in demented and non-demented elderly subjects
    Open this publication in new window or tab >>Mutations in exons 2 and 3 of the FOLR1 gene in demented and non-demented elderly subjects
    2007 (English)In: International Journal of Molecular Medicine, ISSN 1107-3756, E-ISSN 1791-244X, Vol. 20, no 5, p. 653-662Article in journal (Refereed) Published
    Abstract [en]

    We have previously reported six novel mutations in the 5'-UTR of the gene for folate receptor-alpha (FOLR1). In our search for additional mutations we screened patients, referred for investigation of suspected dementia (DGM subgroup) by SSCP and DNA sequencing from the end of exon 1 to the first bases of intron 3. We found 4 sequence variations, FOLR1 g.1314G>A, g.1816delC, g.1841G>A, and g.1928C>T. Pyrosequencing genotyping assays were developed for all of them, and 389 active seniors (AS subgroup) and the 202 DGM patients were genotyped for these mutations. The frequency q of the mutated allele was, among the AS subjects, 0.068, 0.0026, 0.0026, and 0.024 respectively, and among the DGM subjects, 0.067, 0.0076, 0.0078, and 0.023. The g.1816delC and g.1841G>A mutations thus were more frequent in the DGM than in the AS subgroup, but the difference did not reach statistical significance. The mutated alleles, FOLR1 1816(-) and 1841A, always occurred together in the same subjects, suggestive of a rare double-mutant haplotype. The two common polymorphisms, FOLR1 g. 1314G>A and g.1928C>T seemed not to raise tHcy plasma levels, whereas the double-mutated g.1816(-)-g.1841A haplotype may possibly have a slight tHcy-raising effect. Thus, so far 8 novel rare FOLR1 mutations with a combined prevalence of approximately 1.3% in Whites as well as two common polymorphisms with 5% and 13%, respectively, have been demonstrated. Only a few of the rare mutations may potentially be associated with raised plasma tHcy concentrations. No association with dementia was found.

    Place, publisher, year, edition, pages
    Athens, Greece: D.A. Spandidos, 2007
    National Category
    Medical and Health Sciences Geriatrics
    Research subject
    Medicine
    Identifiers
    urn:nbn:se:oru:diva-3036 (URN)17912458 (PubMedID)
    Available from: 2008-11-15 Created: 2008-11-15 Last updated: 2017-12-14Bibliographically approved
    3. Association of total plasma homocysteine with methylenetetrahydrofolate reductase genotypes 677C>T, 1298A>C, and 1793G>A and the corresponding haplotypes in Swedish children and adolescents
    Open this publication in new window or tab >>Association of total plasma homocysteine with methylenetetrahydrofolate reductase genotypes 677C>T, 1298A>C, and 1793G>A and the corresponding haplotypes in Swedish children and adolescents
    Show others...
    2007 (English)In: International Journal of Molecular Medicine, ISSN 1107-3756, E-ISSN 1791-244X, Vol. 19, no 4, p. 659-665Article in journal (Refereed) Published
    Abstract [en]

    We studied 692 Swedish children and adolescents (aged 9-10 or 15-16 years, respectively), in order to evaluate the effect of the methylenetetrahydrofolate reductase (MTHFR) 677C>T, 1298A>C, and 1793G>A polymorphisms on total plasma homocysteine concentrations (tHcy). Genotyping was performed with Pyrosequencing technology. The MTHFR 677C>T polymorphism was associated with increased tHcy concentrations in both the children and the adolescents (P<0.001 for both age groups) in both genders. The effect of MTHFR 1298A>C was studied separately in subjects with the 677CC and 677CT genotypes, and the 1298C allele was found to be associated with higher tHcy levels both when children were stratified according to 677C>T genotypes, and when using haplotype analyses and diplotype reconstructions. The 1793A allele was in complete linkage disequilibrium with the 1298C allele. It was still possible to show that the 1793A allele was associated with lower tHcy levels, statistically significant in the adolescents. In conclusion, a haplotype-based approach was slightly superior in explaining the genetic interaction on tHcy plasma levels in children and adolescents than a simple genotype based approach (R2 adj 0.44 vs. 0.40). The major genetic impact on tHcy concentrations is attributable to the MTHFR 677C>T polymorphism. The common 1298A>C polymorphism had a minor elevating effect on tHcy, whereas the 1793G>A polymorphism had a lowering effect on tHcy.

    Place, publisher, year, edition, pages
    Athens, Greece: D.A. Spandidos, 2007
    Keywords
    Molecular medicine
    National Category
    Medical and Health Sciences Medical Genetics
    Research subject
    Medicine
    Identifiers
    urn:nbn:se:oru:diva-3037 (URN)17334642 (PubMedID)
    Available from: 2008-11-15 Created: 2008-11-15 Last updated: 2020-01-29Bibliographically approved
    4. Plasma homocysteine and MTHFR genotypes and haplotypes: gene-nutrient interactions in the Canary Islands Nutrition Study (ENCA)
    Open this publication in new window or tab >>Plasma homocysteine and MTHFR genotypes and haplotypes: gene-nutrient interactions in the Canary Islands Nutrition Study (ENCA)
    (English)Manuscript (preprint) (Other (popular science, discussion, etc.))
    Keywords
    molecular medicine, medical genetics
    National Category
    Medical and Health Sciences Medical Genetics
    Research subject
    Medicine
    Identifiers
    urn:nbn:se:oru:diva-3038 (URN)
    Available from: 2008-11-15 Created: 2008-11-15 Last updated: 2018-01-13Bibliographically approved
  • 3.
    Böttiger, Anna K.
    et al.
    Örebro University, School of Health and Medical Sciences.
    Hagnelius, Nils-Olof
    Örebro University, School of Health and Medical Sciences.
    Nilsson, Torbjörn K.
    Örebro University, School of Health and Medical Sciences.
    Mutations in exons 2 and 3 of the FOLR1 gene in demented and non-demented elderly subjects2007In: International Journal of Molecular Medicine, ISSN 1107-3756, E-ISSN 1791-244X, Vol. 20, no 5, p. 653-662Article in journal (Refereed)
    Abstract [en]

    We have previously reported six novel mutations in the 5'-UTR of the gene for folate receptor-alpha (FOLR1). In our search for additional mutations we screened patients, referred for investigation of suspected dementia (DGM subgroup) by SSCP and DNA sequencing from the end of exon 1 to the first bases of intron 3. We found 4 sequence variations, FOLR1 g.1314G>A, g.1816delC, g.1841G>A, and g.1928C>T. Pyrosequencing genotyping assays were developed for all of them, and 389 active seniors (AS subgroup) and the 202 DGM patients were genotyped for these mutations. The frequency q of the mutated allele was, among the AS subjects, 0.068, 0.0026, 0.0026, and 0.024 respectively, and among the DGM subjects, 0.067, 0.0076, 0.0078, and 0.023. The g.1816delC and g.1841G>A mutations thus were more frequent in the DGM than in the AS subgroup, but the difference did not reach statistical significance. The mutated alleles, FOLR1 1816(-) and 1841A, always occurred together in the same subjects, suggestive of a rare double-mutant haplotype. The two common polymorphisms, FOLR1 g. 1314G>A and g.1928C>T seemed not to raise tHcy plasma levels, whereas the double-mutated g.1816(-)-g.1841A haplotype may possibly have a slight tHcy-raising effect. Thus, so far 8 novel rare FOLR1 mutations with a combined prevalence of approximately 1.3% in Whites as well as two common polymorphisms with 5% and 13%, respectively, have been demonstrated. Only a few of the rare mutations may potentially be associated with raised plasma tHcy concentrations. No association with dementia was found.

  • 4.
    Böttiger, Anna K.
    et al.
    Orebro Univ Hosp, Dept Clin Chem, SE-70185 Orebro, Sweden.
    Hurtig-Wennlöf, Anita
    Sjöström, Michael
    Yngve, Agneta
    Karolinska Inst, Dept Biosci & Nutr, SE-14157 Huddinge, Sweden.
    Nilsson, Torbjorn K.
    Association of total plasma homocysteine with methylenetetrahydrofolate reductase genotypes 677C > T, 1298A > C, and 1793G > A and the corresponding haplotypes in Swedish children and adolescents2007In: International Journal of Molecular Medicine, ISSN 1107-3756, E-ISSN 1791-244X, Vol. 19, no 4, p. 659-665Article in journal (Refereed)
    Abstract [en]

    We studied 692 Swedish children and adolescents (aged 9-10 or 15-16 years, respectively), in order to evaluate the effect of the methylenetetrahydrofolate reductase (MTHFR) 677C > T, 1298A > C, and 1793G > A polymorphisms on total plasma homocysteine concentrations (tHcy). Genotyping was performed with Pyrosequencing (TM) technology. The MTHFR 677C > T polymorphism was associated with increased tHcy concentrations in both the children and the adolescents (P < 0.001 for both age groups) in both genders. The effect of MTHFR 1298A > C was studied separately in subjects with the 677CC and 677CT genotypes, and the 1298C allele was found to be associated with higher tHcy levels both when children were stratified according to 677C > T genotypes, and when using haplotype analyses and diplotype reconstructions. The 1793A allele was in complete linkage disequilibrium with the 1298C allele. It was still possible to show that the 1793A allele was associated with lower tHcy levels, statistically significant in the adolescents. In conclusion, a haplotype-based approach was slightly superior in explaining the genetic interaction on tHcy plasma levels in children and adolescents than a simple genotype based approach (R-2 adj 0.44 vs. 0.40). The major genetic impact on tHcy concentrations is attributable to the MTHFR 677C > T polymorphism. The common 1298A > C polymorphism had a minor elevating effect on tHcy, whereas the 1793G > A polymorphism had a lowering effect on tHcy.

  • 5.
    Böttiger, Anna K.
    et al.
    Örebro University, School of Health and Medical Sciences.
    Hurtig-Wennlöf, Anita
    Örebro University, School of Health and Medical Sciences.
    Sjöström, Michael
    Yngve, Agneta
    Nilsson, Torbjörn K.
    Örebro University, School of Health and Medical Sciences.
    Association of total plasma homocysteine with methylenetetrahydrofolate reductase genotypes 677C>T, 1298A>C, and 1793G>A and the corresponding haplotypes in Swedish children and adolescents2007In: International Journal of Molecular Medicine, ISSN 1107-3756, E-ISSN 1791-244X, Vol. 19, no 4, p. 659-665Article in journal (Refereed)
    Abstract [en]

    We studied 692 Swedish children and adolescents (aged 9-10 or 15-16 years, respectively), in order to evaluate the effect of the methylenetetrahydrofolate reductase (MTHFR) 677C>T, 1298A>C, and 1793G>A polymorphisms on total plasma homocysteine concentrations (tHcy). Genotyping was performed with Pyrosequencing technology. The MTHFR 677C>T polymorphism was associated with increased tHcy concentrations in both the children and the adolescents (P<0.001 for both age groups) in both genders. The effect of MTHFR 1298A>C was studied separately in subjects with the 677CC and 677CT genotypes, and the 1298C allele was found to be associated with higher tHcy levels both when children were stratified according to 677C>T genotypes, and when using haplotype analyses and diplotype reconstructions. The 1793A allele was in complete linkage disequilibrium with the 1298C allele. It was still possible to show that the 1793A allele was associated with lower tHcy levels, statistically significant in the adolescents. In conclusion, a haplotype-based approach was slightly superior in explaining the genetic interaction on tHcy plasma levels in children and adolescents than a simple genotype based approach (R2 adj 0.44 vs. 0.40). The major genetic impact on tHcy concentrations is attributable to the MTHFR 677C>T polymorphism. The common 1298A>C polymorphism had a minor elevating effect on tHcy, whereas the 1793G>A polymorphism had a lowering effect on tHcy.

  • 6.
    Böttiger, Anna K.
    et al.
    Örebro University, School of Health and Medical Sciences.
    Nilsson, Torbjörn K.
    Henriquez, Patricia
    Serra-Majem, Lluis
    Plasma homocysteine and MTHFR genotypes and haplotypes: gene-nutrient interactions in the Canary Islands Nutrition Study (ENCA)Manuscript (preprint) (Other (popular science, discussion, etc.))
  • 7.
    Farkas, Sanja A.
    et al.
    Department of Laboratory Medicine, Örebro University Hospital, Region Örebro County, Örebro, Sweden.
    Böttiger, Anna K.
    Department of Laboratory Medicine, Örebro University Hospital, Region Örebro County, Örebro, Sweden.
    Isaksson, Helena S.
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden. Department of Laboratory Medicine, Örebro University Hospital, Region Örebro County, Örebro, Sweden.
    Finnell, Richard H.
    Department of Nutritional sciences, Dell pediatric Research Institute, University of Texas, Austin, USA.
    Ren, Aiguo
    Institute of Reproductive and Child health, Ministry of health Key Laboratory of Reproductive health, Peking University health science center, Beijing, P.R. China.
    Nilsson, Torbjorn K.
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden. Department of Laboratory Medicine, Örebro University Hospital, Region Örebro County, Örebro, Sweden.
    Epigenetic alterations in folate transport genes in placental tissue from fetuses with neural tube defects and in leukocytes from subjects with hyperhomocysteinemia2013In: Epigenetics, ISSN 1559-2294, E-ISSN 1559-2308, Vol. 8, no 3, p. 303-316Article in journal (Refereed)
    Abstract [en]

    The objectives of this study were to identify tissue-specific differentially methylated regions (T-DMR's) in the folate transport genes in placental tissue compared with leukocytes, and from placental tissues obtained from normal infants or with neural tube defects (NTDs). Using pyrosequencing, we developed methylation assays for the CpG islands (CGIs) and the CGI shore regions of the folate receptor a (FOLR1), proton-coupled folate transporter (PCFT) and reduced folate carrier 1 (RFC1) genes. The T-DMRs differed in location for each gene and the difference in methylation ranged between 2 and 54%. A higher T-DMR methylated fraction was associated with a lower mRNA level of the FOLR1 and RFC1 genes. Methylation fractions differed according to RFC1 80G > A genotype in the NTD cases and in leukocytes from subjects with high total plasma homocysteine (tHcy). There were no differences in methylated fraction of folate transporter genes between NTD cases and controls. We suggest that T-DMRs participate in the regulation of expression of the FOLR1 and RFC1 genes, that the RFC1 80G > A polymorphism exerts a gene-nutrition interaction on DNA methylation in the RFC1 gene, and that this interaction appears to be most prominent in NTD-affected births and in subjects with high tHcy concentrations.

  • 8.
    Nilsson, Torbjörn K.
    et al.
    Örebro University, School of Health and Medical Sciences.
    Löf-Öhlin, Z. M.
    Böttiger, A. K.
    Genotyping of the reduced folate carrier-1 c.80G>A polymorphism by pyrosequencing technology: importance of PCR and pre-PCR optimization2008In: Scandinavian Journal of Clinical and Laboratory Investigation, ISSN 0036-5513, E-ISSN 1502-7686, Vol. 68, no 2, p. 166-170Article in journal (Refereed)
    Abstract [en]

    When developing a genotyping assay by Pyrosequencing technology for the RFC1 (SLC19A1) c.80G>A polymorphism (rs1051266), unequal peak heights in the pyrograms were observed, probably due to unequal amplification of the mutated and wild-type alleles. This rarely occurring problem could potentially render assignment of heterozygous genotypes uncertain. When the PCR conditions were studied, it was found that substitution of the dGTP nucleotide in the master mix by dGTP and dITP in proportion 1:1 largely overcame this problem. Heat denaturation of the DNA at 95°C before PCR also counteracted the problem. A combination of these two modifications of the standard pyrosequencing PCR protocol gave the best results. We conclude that, with these modifications, the RFC1 c.80G>A SNP can be reliably assayed by pyrosequencing.

  • 9.
    Nilsson, Torbjörn K.
    et al.
    Örebro University, School of Health and Medical Sciences.
    Yngve, Agneta
    Böttiger, Anna K.
    Hurtig-Wennlöf, Anita
    Örebro University, School of Health and Medical Sciences.
    Sjöström, Michael
    High folate intake is related to better academic achievement in Swedish adolescents2011In: Pediatrics, ISSN 0031-4005, E-ISSN 1098-4275, Vol. 128, no 2, p. e358-e365Article in journal (Refereed)
    Abstract [en]

    Background: Adolescents are vulnerable to increased plasma total homocysteine (tHcy) and to insufficient folate status. Folate status and Hcy metabolism are linked to cognitive functions, but academic achievement by adolescents has not been studied in this respect.

    Objective: To assess a possible link between academic achievement in adolescents and tHcy and its determinants, dietary folate intake, MTHFR 677 TT homozygosity, and socioeconomic status (SES).

    Subjects and Methods: A study of 386 Swedish adolescents aged 15 years in whom plasma tHcy and MTHFR 677C →T genotype were assayed. The sum of school grades in 10 core subjects obtained in the final semester of compulsory 9 years of schooling was used as outcome measure of academic achievement. Lifestyle and SES data were obtained from questionnaires.

    Results: Academic achievement was strongly correlated to tertiles of tHcy (negatively; P = .023) and to tertiles of folate intake (positively; P < .001). Other significant predictors were gender, smoking, and SES (proxied by school, mother's education, and father's income). When these were controlled for, tertiles of folate intake (P < .002) but not tertiles of tHcy (P = .523) or MTHFR genotype remained significantly related to academic achievement.

    Conclusion: Folate intake had a positive association with academic achievement in the 15-year-olds, which was not attenuated by SES or MTHFR 677 TT homozygosity. These results provide new information that points to the importance of keeping a closer watch on folate status in childhood and adolescence. They may also have direct implications for school meal provisions, school teaching programs, and information to parents.

  • 10.
    Stenberg, Reidun
    et al.
    Örebro University, School of Medical Sciences. Örebro University Hospital. University Health Care Research Center.
    Böttiger, Anna K.
    Örebro University, School of Medical Sciences. Örebro University Hospital. Department of Laboratory Medicine.
    Nilsson, Torbjörn K.
    Department of Medical Biosciences/Clinical Chemistry, Umeå University, Umeå, Sweden.
    High levels of vitamin B12 are fairly common in children with cerebral palsy2020In: Acta Paediatrica, ISSN 0803-5253, E-ISSN 1651-2227Article in journal (Refereed)
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