Socio-economic inequalities in the effect of public policies and the COVID-19 pandemic on exclusive breastfeeding in Chile.

OBJECTIVE: To assess the impact of the addition of 12 maternity leave (ML) weeks (2011), a pay for performance (P4P) exclusive breastfeeding (EBF) promotion strategy (2015), and the COVID-19 pandemic in EBF inequalities in Chile. STUDY DESIGN: Interrupted time-series analyses (ITSAs). METHODS: Aggregated national EBF data by municipality and month were collected from 2009 to 2020. We assess the impact of the three events in EBF inequalities using two procedures: 1. ITSA stratified by municipal SES quintiles (Q1-Q5); 2. Calculating the EBF slope index of inequality (SII). RESULTS: The EBF prevalence was higher in lower SES municipalities before and after the three time-events. No impact in EBF inequalities was observed after the extended ML. The P4P strategy increased EBF at six months in all SES quintiles (effect size between 4% and 5%), but in a higher level in poorer municipalities (SII: -0.36% and -1.05%). During COVID-19, wealthier municipalities showed a slightly higher EBF at six months prevalence (SII: 1.44%). CONCLUSION: The null impact of the extended ML in EBF inequalities could be explained by a low access to ML among affiliated to the public health system (20%). The P4P strategy includes multiple interventions that seemed effective in increasing EBF across all SES quintiles, but further in lower quintiles. The restrictions in healthcare access in poorer municipalities could explain EBF inequalities during COVID-19.

An update of the WCRF/AICR systematic literature review and meta-analysis on dietary and anthropometric factors and esophageal cancer risk.

BACKGROUND: In the 2007 World Cancer Research Fund/American Institute for Cancer Research Second Expert Report, the expert panel judged that there was strong evidence that alcoholic drinks and body fatness increased esophageal cancer risk, whereas fruits and vegetables probably decreased its risk. The judgments were mainly based on case-control studies. As part of the Continuous Update Project, we updated the scientific evidence accumulated from cohort studies in this topic. METHODS: We updated the Continuous Update Project database up to 10 January 2017 by searching in PubMed and conducted dose-response meta-analyses to estimate summary relative risks (RRs) and 95% confidence intervals (CIs) using random effects model. RESULTS: A total of 57 cohort studies were included in 13 meta-analyses. Esophageal adenocarcinoma risk was inversely related to vegetable intake (RR per 100 g/day: 0.89, 95% CI: 0.80-0.99, n = 3) and directly associated with body mass index (RR per 5 kg/m2: 1.47, 95% CI: 1.34-1.61, n = 9). For esophageal squamous cell carcinoma, inverse associations were observed with fruit intake (RR for 100 g/day increment: 0.84, 95% CI: 0.75-0.94, n = 3) and body mass index (RR for 5 kg/m2 increment: 0.64, 95% CI: 0.56-0.73, n = 8), and direct associations with intakes of processed meats (RR for 50 g/day increment: 1.59, 95% CI: 1.11-2.28, n = 3), processed and red meats (RR for 100 g/day increment: 1.37, 95% CI: 1.04-1.82, n = 3) and alcohol (RR for 10 g/day increment: 1.25, 95% CI: 1.12-1.41, n = 6). CONCLUSIONS: Evidence from cohort studies suggested a protective role of vegetables and body weight control in esophageal adenocarcinomas development. For squamous cell carcinomas, higher intakes of red and processed meats and alcohol may increase the risk, whereas fruits intake may play a protective role.

Fruits, vegetables and lung cancer risk: a systematic review and meta-analysis.

BACKGROUND: Lung cancer is the most common cause of cancer death. Fruits and vegetables containing carotenoids and other antioxidants have been hypothesized to decrease lung cancer risk. As part of the World Cancer Research Fund International Continuous Update Project, we conducted a systematic review and meta-analysis of prospective studies. METHODS: We searched PubMed and several databases up to December 2014 for prospective studies. We conducted meta-analyses comparing the highest and lowest intakes and dose-response meta-analyses to estimate summary relative risks (RRs) and 95% confidence intervals (CIs), and examine possible non-linear associations. We combined results from the Pooling Project with the studies we identified to increase the statistical power of our analysis. RESULTS: When comparing the highest with the lowest intakes, the summary RR estimates were 0.86 [95% CI 0.78-0.94; n (studies) = 18] for fruits and vegetables, 0.92 (95% CI 0.87-0.97; n = 25) for vegetables and 0.82 (95% CI 0.76-0.89; n = 29) for fruits. The association with fruit and vegetable intake was marginally significant in current smokers and inverse but not significant in former or never smokers. Significant inverse dose-response associations were observed for each 100 g/day increase: for fruits and vegetables [RR: 0.96; 95% CI 0.94-0.98, I(2) = 64%, n = 14, N (cases) = 9609], vegetables (RR: 0.94; 95% CI 0.89-0.98, I(2) = 48%, n = 20, N = 12 563) and fruits (RR: 0.92; 95% CI 0.89-0.95, I(2) = 57%, n = 23, N = 14 506). Our results were consistent among the different types of fruits and vegetables. The strength of the association differed across locations. There was evidence of a non-linear relationship (P < 0.01) between fruit and vegetable intake and lung cancer risk showing that no further benefit is obtained when increasing consumption above ∼400 g per day. CONCLUSIONS: Eliminating tobacco smoking is the best strategy to prevent lung cancer. Although residual confounding by smoking cannot be ruled out, the current evidence from prospective studies is consistent with a protective role of fruit and vegetables in lung cancer aetiology.

Anthropometric factors and endometrial cancer risk: a systematic review and dose-response meta-analysis of prospective studies.

BACKGROUND: Greater body mass index (BMI) has been convincingly related to increased endometrial cancer risk, however, whether adiposity earlier in life or abdominal fatness is an independent risk factor and whether weight gain or greater height increases the risk is not clear. METHODS: As part of the Continuous Update Project of the World Cancer Research Fund International, we conducted a systematic review and meta-analysis of prospective studies of the association between anthropometric measures and endometrial cancer risk and searched PubMed and several other databases up to February 2015. Summary relative risks (RRs) were calculated using a random-effects model. RESULTS: Thirty prospective studies of BMI and endometrial cancer risk with 22 320 cases among 6 445 402 participants were included. The summary RR for a 5-unit increment was 1.54 [95% confidence interval (CI) 1.47-1.61, I(2) = 81%]. Although the test for non-linearity was significant, Pnon-linearity < 0.0001, and the curve was steeper within the overweight and obese BMI ranges, there was evidence of increased risk even within the high normal BMI range. The summary RR was 1.45 (95% CI 1.28-1.64, I(2) = 76%) per 5 BMI units for BMI in young adulthood, 1.18 (95% CI 1.14-1.23, I(2) = 67%) per 5 kg increase of weight, and 1.16 (95% CI 1.12-1.20, I(2) = 51%) per 5 kg of weight gained between young adulthood and study baseline, 1.27 (95% CI 1.17-1.39, I(2) = 71%) per 10 cm increase in waist circumference, 1.21 (95% CI 1.13-1.29, I(2) = 0%) per 0.1-unit increment in waist-to-hip ratio and 1.30 (95% CI 1.19-1.41, I(2) = 0%) per 10-cm increase in hips circumference. The summary RR was 1.15 (95% CI 1.09-1.22, I(2) = 61%) for a 10-cm increase in height. CONCLUSIONS: All measures of adiposity were associated with increased risk of endometrial cancer, and in addition increasing height was associated with increased risk.

Characterization and review of MTHFD1 deficiency: four new patients, cellular delineation and response to folic and folinic acid treatment.

In the folate cycle MTHFD1, encoded by MTHFD1, is a trifunctional enzyme containing 5,10-methylenetetrahydrofolate dehydrogenase, 5,10-methenyltetrahydrofolate cyclohydrolase and 10-formyltetrahydrofolate synthetase activity. To date, only one patient with MTHFD1 deficiency, presenting with hyperhomocysteinemia, megaloblastic anaemia, hemolytic uremic syndrome (HUS) and severe combined immunodeficiency, has been identified (Watkins et al J Med Genet 48:590-2, 2011). We now describe four additional patients from two different families. The second patient presented with hyperhomocysteinemia, megaloblastic anaemia, HUS, microangiopathy and retinopathy; all except the retinopathy resolved after treatment with hydroxocobalamin, betaine and folinic acid. The third patient developed megaloblastic anaemia, infection, autoimmune disease and moderate liver fibrosis but not hyperhomocysteinemia, and was successfully treated with a regime that included and was eventually reduced to folic acid. The other two, elder siblings of the third patient, died at 9 weeks of age with megaloblastic anaemia, infection and severe acidosis and had MTFHD1 deficiency diagnosed retrospectively. We identified a missense mutation (c.806C > T, p.Thr296Ile) and a splice site mutation (c.1674G > A) leading to exon skipping in the second patient, while the other three harboured a missense mutation (c.146C > T, p.Ser49Phe) and a premature stop mutation (c.673G > T, p.Glu225*), all of which were novel. Patient fibroblast studies revealed severely reduced methionine formation from [(14)C]-formate, which did not increase in cobalamin supplemented culture medium but was responsive to folic and folinic acid. These additional cases increase the clinical spectrum of this intriguing defect, provide in vitro evidence of disturbed methionine synthesis and substantiate the effectiveness of folic or folinic acid treatment.

Body mass index and survival in women with breast cancer-systematic literature review and meta-analysis of 82 follow-up studies.

BACKGROUND: Positive association between obesity and survival after breast cancer was demonstrated in previous meta-analyses of published data, but only the results for the comparison of obese versus non-obese was summarised. METHODS: We systematically searched in MEDLINE and EMBASE for follow-up studies of breast cancer survivors with body mass index (BMI) before and after diagnosis, and total and cause-specific mortality until June 2013, as part of the World Cancer Research Fund Continuous Update Project. Random-effects meta-analyses were conducted to explore the magnitude and the shape of the associations. RESULTS: Eighty-two studies, including 213 075 breast cancer survivors with 41 477 deaths (23 182 from breast cancer) were identified. For BMI before diagnosis, compared with normal weight women, the summary relative risks (RRs) of total mortality were 1.41 [95% confidence interval (CI) 1.29-1.53] for obese (BMI >30.0), 1.07 (95 CI 1.02-1.12) for overweight (BMI 25.0-<30.0) and 1.10 (95% CI 0.92-1.31) for underweight (BMI <18.5) women. For obese women, the summary RRs were 1.75 (95% CI 1.26-2.41) for pre-menopausal and 1.34 (95% CI 1.18-1.53) for post-menopausal breast cancer. For each 5 kg/m(2) increment of BMI before, <12 months after, and ≥12 months after diagnosis, increased risks of 17%, 11%, and 8% for total mortality, and 18%, 14%, and 29% for breast cancer mortality were observed, respectively. CONCLUSIONS: Obesity is associated with poorer overall and breast cancer survival in pre- and post-menopausal breast cancer, regardless of when BMI is ascertained. Being overweight is also related to a higher risk of mortality. Randomised clinical trials are needed to test interventions for weight loss and maintenance on survival in women with breast cancer.

Update on transcobalamin deficiency: clinical presentation, treatment and outcome.

Transcobalamin (TC) transports cobalamin from blood into cells. TC deficiency is a rare autosomal recessive disorder usually presenting in early infancy with failure to thrive, weakness, diarrhoea, pallor, anemia, and pancytopenia or agammaglobulinemia. It can sometimes resemble neonatal leukemia or severe combined immunodeficiency disease. Diagnosis of TC deficiency is suspected based on megaloblastic anemia, elevation of total plasma homocysteine, and blood or urine methylmalonic acid. It is confirmed by studying the synthesis of TC in cultured fibroblasts, or by molecular analysis of the TCN2 gene. TC deficiency is treatable with supplemental cobalamin, but the optimal type, route and frequency of cobalamin administration and long term patient outcomes are unknown. Here we present a series of 30 patients with TC deficiency, including an update on multiple previously published patients, in order to evaluate the different treatment strategies and provide information about long term outcome. Based on the data presented, current practice appears to favour treatment of individuals with TC deficiency by intramuscular injections of hydroxy- or cyanocobalamin. In most cases presented, at least weekly injections (1 mg IM) were necessary to ensure optimal treatment. Most centres adjusted the treatment regimen based on monitoring CBC, total plasma homocysteine, plasma and urine methylmalonic acid, as well as, clinical status. Finally, continuing IM treatment into adulthood appears to be beneficial.

Human methylenetetrahydrofolate reductase: isolation of cDNA, mapping and mutation identification.

Methylenetetrahydrofolate reductase (MTHFR) catalyses the reduction of methylenetetrahydrofolate to methyltetrahydrofolate, a cofactor for homocysteine methylation to methionine. MTHFR deficiency, an autosomal recessive disorder, results in homocysteinemia. Using degenerate oligonucleotides based on porcine peptide sequence data, we isolated a 90-bp cDNA by PCR from pig liver RNA. This cDNA was used to isolate a human cDNA, the predicted amino acid sequence of which shows strong homology to porcine MTHFR and to bacterial metF genes. The human gene has been localized to chromosome 1p36.3. Two mutations were identified in MTHFR-deficient patients: a missense mutation (Arg to Gln), in a residue conserved in bacterial enzymes, and a nonsense mutation (Arg to Ter).

Dietary fiber and breast cancer risk: a systematic review and meta-analysis of prospective studies.

BACKGROUND: Evidence from case-control studies suggest that dietary fiber may be inversely related to breast cancer risk, but it is unclear if this is supported by prospective data. We conducted a systematic review and meta-analysis of the evidence from prospective studies. METHODS: PubMed was searched for prospective studies of fiber intake and breast cancer risk until 31st August 2011. Random effects models were used to estimate summary relative risks (RRs). RESULTS: Sixteen prospective studies were included. The summary RR for the highest versus the lowest intake was 0.93 [95% confidence interval (CI) 0.89-0.98, I(2) = 0%] for dietary fiber, 0.95 (95% CI 0.86-1.06, I(2) = 4%) for fruit fiber, 0.99 (95% CI 0.92-1.07, I(2) = 1%) for vegetable fiber, 0.96 (95% CI 0.90-1.02, I(2) = 5%) for cereal fiber, 0.91 (95% CI 0.84-0.99, I(2) = 7%) for soluble fiber and 0.95 (95% CI 0.89-1.02, I(2) = 0%) for insoluble fiber. The summary RR per 10 g/day of dietary fiber was 0.95 (95% CI 0.91-0.98, I(2) = 0%, P(heterogeneity) = 0.82). In stratified analyses, the inverse association was only observed among studies with a large range (≥13 g/day) or high level of intake (≥25 g/day). CONCLUSION: In this meta-analysis of prospective studies, there was an inverse association between dietary fiber intake and breast cancer risk.

Dietary fructose, carbohydrates, glycemic indices and pancreatic cancer risk: a systematic review and meta-analysis of cohort studies.

BACKGROUND: Dietary carbohydrates, glycemic load and glycemic index have been hypothesized to influence pancreatic cancer risk, but epidemiological studies have been inconsistent. We conducted a systematic review and meta-analysis of prospective studies to clarify these results. METHODS: PubMed and several other databases were searched for prospective studies of intake of carbohydrates, glycemic index and glycemic load and pancreatic cancer up to September 2011. Summary relative risks were estimated using a random effects model. RESULTS: Ten cohort studies (13 publications) were included in the meta-analysis. The summary relative risk (RR) per 10 glycemic index units was 1.02 [95% confidence interval (CI): 0.93-1.12, I(2) = 0%], per 50 glycemic load units was 1.03 (95% CI: 0.93-1.14, I(2) = 10%), per 100 g/day of total carbohydrates was 0.97 (95% CI: 0.81-1.16, I(2) = 35%), and per 25 g/day of sucrose intake was 1.05 (95% CI: 0.85-1.23, I(2) = 53%). A positive association was observed with fructose intake, summary RR = 1.22 (95% CI: 1.08-1.37, I(2) = 0%) per 25 g/day. CONCLUSIONS: This meta-analysis does not support an association between diets high in glycemic index, glycemic load, total carbohydrates or sucrose and pancreatic cancer risk. The finding of an increased risk with fructose intake warrants further investigation in studies with better adjustment for confounding and in non-American populations.

Body mass index, abdominal fatness and pancreatic cancer risk: a systematic review and non-linear dose-response meta-analysis of prospective studies.

BACKGROUND: Questions remain about the shape of the dose-response relationship between body mass index (BMI) and pancreatic cancer risk, possible confounding by smoking, and differences by gender or geographic location. Whether abdominal obesity increases risk is unclear. METHODS: We conducted a systematic review and meta-analysis of prospective studies of the association between BMI, abdominal fatness and pancreatic cancer risk and searched PubMed and several other databases up to January 2011. Summary relative risks (RRs) were calculated using a random-effects model. RESULTS: Twenty-three prospective studies of BMI and pancreatic cancer risk with 9504 cases were included. The summary RR for a 5-unit increment was 1.10 [95% confidence interval (CI) 1.07-1.14, I(2) = 19%] and results were similar when stratified by gender and geographic location. There was evidence of a non-linear association, P(non-linearity) = 0.005; however, among nonsmokers, there was increased risk even within the 'normal' BMI range. The summary RR for a 10-cm increase in waist circumference was 1.11 (95% CI 1.05-1.18, I(2) = 0%) and for a 0.1-unit increment in waist-to-hip ratio was 1.19 (95% CI 1.09-1.31, I(2) = 11%). CONCLUSIONS: Both general and abdominal fatness increases pancreatic cancer risk. Among nonsmokers, risk increases even among persons within the normal BMI range.

Fruits, vegetables and breast cancer risk: a systematic review and meta-analysis of prospective studies.

Evidence for an association between fruit and vegetable intake and breast cancer risk is inconclusive. To clarify the association, we conducted a systematic review and meta-analysis of the evidence from prospective studies. We searched PubMed for prospective studies of fruit and vegetable intake and breast cancer risk until April 30, 2011. We included fifteen prospective studies that reported relative risk estimates and 95 % confidence intervals (CIs) of breast cancer associated with fruit and vegetable intake. Random effects models were used to estimate summary relative risks. The summary relative risk (RR) for the highest versus the lowest intake was 0.89 (95 % CI: 0.80-0.99, I (2) = 0 %) for fruits and vegetables combined, 0.92 (95 % CI: 0.86-0.98, I (2) = 9 %) for fruits, and 0.99 (95 % CI: 0.92-1.06, I (2) = 20 %) for vegetables. In dose-response analyses, the summary RR per 200 g/day was 0.96 (95 % CI: 0.93-1.00, I (2) = 2 %) for fruits and vegetables combined, 0.94 (95 % CI: 0.89-1.00, I (2) = 39 %) for fruits, and 1.00 (95 % CI: 0.95-1.06, I (2) = 17 %) for vegetables. In this meta-analysis of prospective studies, high intake of fruits, and fruits and vegetables combined, but not vegetables, is associated with a weak reduction in risk of breast cancer.

Defect in vitamin B12 release from lysosomes: newly described inborn error of vitamin B12 metabolism.

Cultured diploid fibroblasts from a patient with a previously undescribed inborn error of cobalamin metabolism accumulate unmetabolized, nonprotein-bound vitamin B12 in lysosomes. These cells are able to endocytose the transcobalamin II-B12 complex and to release B12 from transcobalamin II. The freed vitamin B12 is not released from lysosomes into the cytoplasm of the cell. This suggests that there is a specific lysosomal transport mechanism for vitamin B12 in the human.

Transcobalamin (TC) deficiency--potential cause of bone marrow failure in childhood.

It is unusual for inborn errors of metabolism to be considered in the investigative work-up of pancytopenia. We report a family in which the proband presented with failure to thrive at 2 months of age and subsequent bone marrow failure. A previous sibling had died at 7 months of age with suspected leukaemia. Haematological findings in the proband were significant for pancytopenia, and bone marrow aspiration showed dysplastic changes in all cell lineages. Urinary organic acid analysis revealed elevated methylmalonic acid. The synthesis of transcobalamin II (transcobalamin, TC) by cultured fibroblasts was markedly reduced, confirming the diagnosis of TC deficiency. The proband and his younger asymptomatic sister (also found to have TC deficiency) were homozygous for R399X (c.1195C>T), a novel mutation resulting in the loss of the C- terminal 29 amino acids of TC, a highly conserved region. Response to parenteral vitamin B(12) in the proband was dramatic. At 6 years 3 months of age, physical examination is normal and developmental level is age appropriate. His sister is clinically asymptomatic and is also developing normally. Propionylcarnitine concentrations were not elevated in the newborn screening cards from the proband and sister, but that was for specimens retrieved from storage after 7 years and 5 years, respectively. Inherited and acquired cobalamin disorders should both be considered in the differential diagnosis of bone marrow failure syndromes in young children. Early detection of the metabolic causes of bone marrow failure can ensure prompt recovery in some cases involving the vitamin B(12) pathway.

Genetic heterogeneity among patients with methylcobalamin deficiency. Definition of two complementation groups, cblE and cblG.

A number of patients with megaloblastic anemia and homocystinuria associated with low levels of methylcobalamin synthesis in cultured cells have been recognized. Methionine biosynthesis by intact cells, as determined by incorporation of label from 5-[14C]methyl-tetrahydrofolate into acid-precipitable material, was deficient in cultured skin fibroblasts that were derived from all of these patients. In one group of patients, activity of the methylcobalamin-dependent enzyme, methionine synthase, in cell extracts was within the normal range when the enzyme was assayed under standard conditions. In a second group of patients, methionine synthase activity was decreased under the same assay conditions. Genetic complementation analysis demonstrated the existence of two complementation classes that corresponded to these two groups of patients. The designation cblE has previously been proposed for normal methionine synthase activity. We propose the designation cblG for the mutation in those patients with methylcobalamin deficiency and decreased synthase activity. The results of these studies suggest that the products of at least two loci are required for cobalamin-dependent methionine biosynthesis in mammalian cells.

Altered vitamin B12 metabolism in fibroblasts from a patient with megaloblastic anemia and homocystinuria due to a new defect in methionine biosynthesis.

Cultured fibroblasts from a recently described patient with homocystinuria and megaloblastic anemia of infancy without methylmalonic aciduria were previously shown to have normal cobalamin uptake and a specific decrease in the proportion of intracellular methylcobalamin. As in control cells but unlike in those from patients with combined homocystinuria and methylmalonic aciduria (cobalamin C and cobalamin D), accumulated 57Co-labeled cobalamin was bound in appropriate amounts and proportion to intracellular binders which are known to be the two vitamin B12-dependent enzymes, methionine synthetase and methylmalonyl-CoA mutase. Despite the association of a normal quantity of intracellular cobalamin with methionine synthetase, the proportion of intracellular cobalamin which was methyl-B12 was below normal and in the range observed in cobalamin C and D cells. This methyl-B12 was decreased by exposure of fibroblasts in culture to nitrous oxide as was observed with control cells. Exposure of control fibroblasts during culture, but not of fibroblasts from this patient, to nitrous oxide significantly reduced the holoenzyme activity of methionine synthetase assayed in cell extracts. In addition, although methionine synthetase activity in cell extracts of control and cells from the patient were similar in the presence of standard assay concentrations of thiols, at low thiol concentrations, methionine synthetase activity in extracts of cells from the patient was much lower than in control extracts. Mixing of control patient extracts corrected this decreased activity in excess of that explained by addition of the individual activities added. The defect of this patient appears to be in a reducing system required for methionine synthesis.

Folate distribution in cultured human cells. Studies on 5,10-CH2-H4PteGlu reductase deficiency.

We have studied the distribution of folate coenzyme forms in cultured human fibroblasts from control lines and from lines derived from nine patients representing all of the published reports of 5,10-CH(2)-H(4)PteGlu reductase deficiency. Based on mobility on DEAE-Sephadex and differential microbiological assay the major folate fractions in extracts of human fibroblasts were 5-CH(3)-H(4)PteGlu, 10-CHO-H(4)PteGlu, and 5-CHO-H(4)PteGlu with smaller fractions, which included 5-CH(3)-H(2)PteGlu, 10-CHO-PteGlu, and H(4)PteGlu. Evidence that the 5-CHO-H(4)PteGlu may have been derived from 5,10-CH=H(4)PteGlu during extraction is presented. In most of the mutant fibroblasts the absolute concentration of 5-CH(3)-H(4)PteGlu was lower than in control cells but the proportion of intracellular folate which was 5-CH(3)-H(4)PteGlu was strikingly lower in mutant cells when determined by chromatography or differential microbiological assay. In both control and mutant cells most of the 5-CH(3)-H(4)-PteGlu was polyglutamate. The proportion of intracellular folate which was polyglutamate was similar in control and mutant cells. A direct relationship was observed between the proportion of cellular folate which was 5-CH(3)-H(4)PteGlu, and both the clinical severity of this disorder and the residual enzyme activity indicating that the distribution of different folates may be an important control of intracellular folate metabolism. These studies indicate that 5,10-CH(2)-H(4)PteGlu reductase is the only significant intracellular pathway for the generation of 5-CH(3)-H(4)PteGlu, that the activity of this enzyme regulates the level of this folate in control and mutant cells under conditions of culture used here, that the majority of intracellular folate is in the polyglutamate form, and that the relative distribution of folates may control folate metabolism by interaction in the various folate reactions.

Genetic characterization of a MUT locus mutation discriminating heterogeneity in mut0 and mut- methylmalonic aciduria by interallelic complementation.

Genetic complementation of fibroblasts from patients with methylmalonic aciduria (MMA) defines a unique class of allelic mutations arising from mutations at the locus encoding the methylmalonyl coenzyme A (CoA) mutase apoenzyme. Various phenotypes of MMA have been delineated including complete absence of enzyme activity (mut0) and abnormal enzyme activity with an elevated Km for adenosylcobalamin (mut-). We describe genetic studies on a cell line (WG1130) from a patient with mut0 MMA which exhibited an unusual complementation phenotype, complementing with three of nine mut0 cell lines and four of five mut- cell lines. This suggests that interallelic complementation occurs between mutant alleles in WG1130 and subsets of alleles associated with both mut0 and mut- phenotypes. The methylmalonyl CoA mutase cDNA was cloned from WG1130 and found to contain a G354----A (Arg93----His) mutation. Gene transfer of this mutant clone into primary fibroblasts from patients with MMA confirms that this mutation expresses a mut0 phenotype when transferred into a mut0 cell line with low levels of mRNA but can contribute to apoenzyme function when transferred into mut cell lines which show correction with WG1130 by somatic cell complementation. These results point to further heterogeneity within both mut0 and mut- and may enable identification of mutations affecting discrete components of apoenzyme function.