Maternal vitamin B12 status in early pregnancy and its association with birth outcomes in Canadian mother-newborn Dyads.

Vitamin B12 (B12) is a co-enzyme essential for fetal growth and development. Lower maternal B12 status has been associated with preterm birth (<37 gestational weeks) and low birth weight (<2500 g), which are linked to morbidity and mortality across the lifespan. In Canada, 17-25 % of women in early pregnancy had a serum total B12 concentration <148 pmol/l and maternal total B12 concentration decreased throughout pregnancy. This study aimed to determine the association between maternal B12 status and birth outcomes in Canadian mother-newborn dyads. A secondary analysis of 709 mother-newborn dyads in British Columbia (BC), Canada, was conducted. Bio-banked first- (n 656) and second-trimester (n 709) maternal serum samples of apparently healthy South Asian (50 %) and European (50 %) women from the BC Prenatal Genetic Screening Program were quantified for B12 biomarkers (total B12, holotranscobalamin (holoTC), methylmalonic acid (MMA) and total homocysteine (tHcy)). Obstetric history and birth outcome data were obtained from the BC Perinatal Data Registry. All associations were determined using multiple linear regression. Maternal serum total B12, holoTC, MMA and tHcy had a mean weekly decrease of 3·64 pmol/l, 1·04 pmol/l, 1·44 nmol/l and 0·104 µmol/l, respectively (P < 0·001). Despite a total B12 concentration <148 pmol/l among 20-25 % of the women, maternal B12 biomarker concentrations were not associated with birth weight z-score, head circumference z-score and gestational age at birth (P > 0·05). Additional research in women at high risk of adverse birth outcomes and the association between maternal B12 status and functional, for example, cognitive, outcomes is needed.

Serum Betaine and Dimethylglycine Are Higher in South Asian Compared with European Pregnant Women in Canada, with Betaine and Total Homocysteine Inversely Associated in Early and Midpregnancy, Independent of Ethnicity.

BACKGROUND: As a methyl donor required in the folate-vitamin B-12 independent remethylation of total homocysteine (tHcy) to methionine, betaine is critical for fetal development. Pregnant South Asian women living in Canada had a higher reported prevalence of low vitamin B-12 status compared with Europeans; betaine concentrations in this population are unknown. OBJECTIVES: We aimed to compare serum betaine concentrations between South Asian and European pregnant women, and to determine the relation between betaine and tHcy concentrations in early pregnancy. METHODS: A retrospective cohort study was conducted using biobanked serum samples of 723 apparently healthy pregnant women of South Asian (50%) and European ethnicity residing in British Columbia, Canada. Betaine, dimethylglycine (DMG), tHcy, and related metabolites were quantified in samples collected in the first (8-13 weeks of gestation) and second (14-20 weeks of gestation) trimesters. The relation between betaine and tHcy concentrations was assessed using a generalized regression model adjusted for weeks of gestation, ethnicity, prepregnancy BMI, maternal age, neonatal sex, parity, total vitamin B-12, folate, pyridoxal 5'-phosphate, and methionine concentrations. RESULTS: Median serum concentrations of betaine and its metabolite DMG were higher in South Asian women in the first (19.8 [IQR: 16.3-25.0] and 1.55 [IQR: 1.30-1.96] $\mu {\rm mol/L} $, respectively) and second trimesters (16.1 [IQR: 12.9-19.8] and 1.42 [IQR: 1.14-1.81] $\mu {\rm mol/L} $, respectively) compared with European women (17.6 [IQR: 13.7-22.6] and 1.38 [IQR: 1.12-1.77] $\mu {\rm mol/L} $, respectively) and (12.9 [IQR: 10.6-16.7] and 1.19 [IQR: 0.97-1.52] $\mu {\rm mol/L} $, respectively; all P values < 0.0001). Betaine was inversely associated with tHcy concentration (ß = -0.0208; 95% CI: -0.0341, -0.00742; P = 0.002). Additionally, total vitamin B-12 was associated with tHcy concentration (ß = -0.0312; 95% CI: -0.0401, -0.0224), after adjusting for confounding factors. CONCLUSIONS: Pregnant South Asian women residing in Canada had higher betaine and DMG concentrations, compared with women of European ethnicity, while betaine and total vitamin B-12 predicted tHcy independent of ethnicity. Our results emphasize the role of betaine, as methyl donor, in the remethylation of tHcy in a folate-replete population.

Reference intervals for serum total vitamin B12 and holotranscobalamin concentrations and their change points with methylmalonic acid concentration to assess vitamin B12 status during early and mid-pregnancy.

Background Maternal vitamin B12 (B-12) adequacy is important for maternal health and optimal fetal growth. However, pregnancy-specific cut-offs for B-12 biomarkers are lacking. Methods Reference intervals for serum total B-12, holotranscobalamin (holoTC) and methylmalonic acid (MMA) concentrations were calculated following CLSI EP28-A3c guidelines in 723 pregnant women of European (50%) and South Asian (50%) ethnicity, residing in British Columbia, Canada, at median (range) 11.4 (8.3-13.9) and 16.1 (14.9-20.9) weeks of gestation. Change point analyses described relationships between log serum MMA concentration with serum total B-12 and holoTC concentrations, assuming linear-linear relationships. Results The central 95% reference interval limits indicated that serum total B-12 <89.9 and <84.0 pmol/L, holoTC <29.5 and <26.0 pmol/L and MMA >371 and >374 nmol/L, in the first and second trimesters, respectively, may indicate B-12 deficiency in pregnant women. The lower limits of total B-12 and holoTC and the upper limits of MMA significantly differed by ethnicity in both trimesters. According to the change point analysis, total B-12 <186 and <180 pmol/L and holoTC <62.2 and <67.5 pmol/L in the first and second trimesters, respectively, suggested an increased probability of impaired intracellular B-12 status, with no difference between ethnicities. Conclusions We present novel reference limits and change points for B-12 biomarkers, which may be employed to identify possible B-12 deficiency in women during early and mid-pregnancy. Future research is needed to validate these cut-offs and determine the predictors and functional outcomes associated with impaired B-12 status in ethnically diverse populations.

A mitochondrial DNA D loop insertion detected almost exclusively in non-replicating tissues with maternal inheritance across three generations.

Muscle biopsy identified a possibly pathogenic, mitochondrial DNA D-loop insertion, in each of 5 family members from two generations, that was otherwise undetectable in most other tissues. The tissue specific regulation of heteroplasmy is reflected in an age related increase in muscle heteroplasmy level, across the pedigree. This latter finding is in keeping with previous reports (e.g. T408A, C16327) but differs in having a very high muscle heteroplasmy level, and appears maternally transmitted.

Pregnant women of South Asian ethnicity in Canada have substantially lower vitamin B12 status compared with pregnant women of European ethnicity.

Maternal vitamin B12 (B12) status has been inversely associated with adverse pregnancy outcomes and positively with fetal growth and infant development. South Asians, Canada's largest ethnic minority, are prone to B12 deficiency. Yet, data are lacking on B12 status in South Asian pregnant women in North America. We sought to determine B12 status, using multiple biomarkers, in 1st and 2nd trimester pregnant women of South Asian and, for comparison, European ethnicity living in Vancouver, Canada. In this retrospective cohort study, total B12, holotranscobalamin (holoTC), methylmalonic acid (MMA), and total homocysteine concentrations were quantified in two routinely collected (mean gestational week: 11·5 (range 8·3-13·9) and 16·5 (range 14·9-20·9)), banked serum samples of 748 healthy pregnant South Asian (n 371) and European (n 377) women. South Asian pregnant women had significantly lower B12 status than European pregnant women at both time points, as indicated by lower serum total B12 and holoTC concentrations, and higher MMA concentrations (all P≤0·001). The largest difference, which was substantial (Cohen's d≥0·5), was observed in mean serum total B12 concentrations (1st trimester: 189 (95 % CI 180, 199) v. 246 (95 % CI 236, 257) pmol/l; 2nd trimester: 176 (95 % CI 168, 185) v. 226 (95 % CI 216, 236) pmol/l). Further, South Asian ethnicity was a significant negative predictor of B12 status during pregnancy. South Asian women living in Vancouver have substantially lower B12 status during early pregnancy. Future research identifying predictors and health consequences of this observed difference is needed to allow for targeted interventions.

Reference interval of methylmalonic acid concentrations in dried blood spots of healthy, term newborns to facilitate neonatal screening of vitamin B12 deficiency.

OBJECTIVES: Unrecognized vitamin B12 (B-12) deficiency in early infancy can lead to poor development of the child. Methylmalonic acid (MMA) is the most specific functional biomarker of B-12 status. To facilitate timely diagnosis and treatment of neonatal B-12 deficiency, the objective of this study was to calculate a reference interval of MMA in dried blood spots (DBSs) of healthy, term newborns. DESIGN AND METHODS: MMA was quantified in 160 newborn DBSs, routinely collected for newborn screening, using LC-MS/MS. The reference interval of DBS MMA was calculated according to current guidelines (CLSI EP28-A3c). The effect of storage at room temperature on DBS MMA concentrations was determined. RESULTS: The mean DBS MMA concentration of 160 healthy, term newborns was 16.8pmol (95% CI: 15.9-17.6pmol)/8-mm spot. The reference interval (2.5th to 97.5th percentile) for MMA was 9.89 to 29.3pmol/8-mm spot (0.450 to 1.33µmol/L whole blood). DBS MMA concentrations correlated positively (p<0.005) with storage time at room temperature. DBS MMA concentrations of children with methylmalonic acidemia (n=4) were above the upper limit of the reference interval. CONCLUSIONS: This is the first study to present a reference interval for DBS MMA of healthy, term newborns utilizing a new highly sensitive MMA method. Analysis of DBS MMA collected during newborn screening may have the potential to identify newborns with acquired B-12 deficiency. Newborn DBS MMA concentrations increase with storage at room temperature, therefore, sample storage has to be monitored for evaluation of DBS MMA data.

Mitochondrial carbonic anhydrase VA deficiency resulting from CA5A alterations presents with hyperammonemia in early childhood.

Four children in three unrelated families (one consanguineous) presented with lethargy, hyperlactatemia, and hyperammonemia of unexplained origin during the neonatal period and early childhood. We identified and validated three different CA5A alterations, including a homozygous missense mutation (c.697T>C) in two siblings, a homozygous splice site mutation (c.555G>A) leading to skipping of exon 4, and a homozygous 4 kb deletion of exon 6. The deleterious nature of the homozygous mutation c.697T>C (p.Ser233Pro) was demonstrated by reduced enzymatic activity and increased temperature sensitivity. Carbonic anhydrase VA (CA-VA) was absent in liver in the child with the homozygous exon 6 deletion. The metabolite profiles in the affected individuals fit CA-VA deficiency, showing evidence of impaired provision of bicarbonate to the four enzymes that participate in key pathways in intermediary metabolism: carbamoylphosphate synthetase 1 (urea cycle), pyruvate carboxylase (anaplerosis, gluconeogenesis), propionyl-CoA carboxylase, and 3-methylcrotonyl-CoA carboxylase (branched chain amino acids catabolism). In the three children who were administered carglumic acid, hyperammonemia resolved. CA-VA deficiency should therefore be added to urea cycle defects, organic acidurias, and pyruvate carboxylase deficiency as a treatable condition in the differential diagnosis of hyperammonemia in the neonate and young child.

Carnitine palmitoyltransferase I and sudden unexpected infant death in British Columbia First Nations.

OBJECTIVE: Infant mortality in British Columbia (BC) First Nations remains elevated relative to other residents. The p.P479L (c.1436C>T) variant of carnitine palmitoyltransferase 1 (CPT1A) is frequent in some aboriginal populations and may be associated with increased infant deaths. This work was initiated to determine the performance of acylcarnitine profiling for detecting this variant, to determine its frequency in BC, and to determine if it is associated with sudden infant deaths in this population. METHODS: Newborn screening cards from all BC First Nations infants in 2004 and all sudden unexpected deaths in BC First Nations infants (1999-2009) were genotyped for the CPT1A p.P479L variant and linked to archival acylcarnitine data. RESULTS: The CPT1A p.P479L variant is frequent in BC First Nations but is not evenly distributed, with higher rates in coastal regions (up to 25% homozygosity) with historically increased infant mortality. There is also an overrepresentation of p.P479L homozygotes in unexpected infant deaths from these regions, with an odds ratio of 3.92 (95% confidence interval: 1.69-9.00). Acylcarnitine profiling will identify p.P479L homozygotes with a 94% sensitivity and specificity. CONCLUSIONS: The CPT1A p.P479L variant is common to some coastal BC First Nations, and homozygosity for this variant is associated with unexpected death in infancy. The high frequency of this variant in a wide range of coastal aboriginal communities, however, suggests a selective advantage, raising the possibility that this variant may have differing impacts on health depending on the environmental or developmental context.

Levator palpebrae biopsy and diagnosis of progressive external ophthalmoplegia.

BACKGROUND: Progressive external ophthalmoplegia (PEO) is a mitochondrial myopathy of ocular muscles. Diagnostic investigation usually involves limb skeletal muscle biopsy and molecular genetic studies, although diagnostic yield tends to be low. The purpose of this study was to evaluate the diagnostic yield obtained by analysis of levator palpebrae (LP) muscle tissue. METHODS: This is a clinicopathologic study of 8 patients with a diagnosis of PEO, who had LP muscle biopsies as part of oculoplastic procedures. Six of these patients also had limb muscle biopsies. Histopathology, electron microscopy and genetic studies were performed. RESULTS: Diagnostic histopathologic findings were present in 4/6 quadriceps biopsies, and 7/8 LP biopsies. Genetic testing on DNA extracted from LP muscle revealed abnormalities in 4 patients. CONCLUSION: In patients whose LP. muscle demonstrate both genetic defects and histopathological abnormalities, the diagnosis of PEO can be confirmed without limb muscle biopsy. Patients having LP resection during oculoplastics procedures for treatment of ptosis may therefore be able to avoid a separate procedure for limb muscle biopsy. Further study is required to determine the specificity of these findings.

Infantile cardioencephalopathy due to a COX15 gene defect: report and review.

We describe respiratory chain complex IV deficiency (cytochrome c oxidase deficiency) in a female infant with a neonatal rapidly progressive fatal course characterized by microcephaly, encephalopathy, persistent lactic acidosis, and hypertrophic cardiomyopathy. Postmortem cardiac muscle study showed marked complex IV deficiency. In contrast, complex IV activity was only slightly decreased in the skeletal muscle. Subsequent molecular investigations showed compound heterozygosity for two known pathogenic mutations in the COX15 gene. We compare the findings in our patient to those of the three previously reported cases.

The paradox of the carnitine palmitoyltransferase type Ia P479L variant in Canadian Aboriginal populations.

Investigation of seven patients from three families suspected of a fatty acid oxidation defect showed mean CPT-I enzyme activity of 5.9+/-4.9 percent of normal controls. The families, two Inuit, one First Nation, live in areas of Canada geographically very distant from each other. The CPT1 and CPT2 genes were fully sequenced in 5 of the patients. All were homozygous for the same P479L mutation in a highly conserved region of the CPT1 gene. Two patients from the first family were also homozygous for the CPT2 F352C polymorphism in the CPT2 gene. Genotyping the patients and their family members confirmed that all seven patients were homozygous for the P479L variant allele in the CPT1 gene, as were 27 of 32 family members. Three of the seven patients and two cousins had hypoketotic hypoglycemia attributable to CPT-Ia deficiency, but adults homozygous for the variant denied hypoglycemia. We screened 422 consecutive newborns from the region of one of the Inuit families for this variant; 294 were homozygous, 103 heterozygous, and only 25 homozygous normal; thus the frequency of this variant allele is 0.81. There was an infant death in one family and at least 10 more deaths in those infants (7 homozygous, 3 heterozygous) consecutively tested for the mutation at birth. Thus there is an astonishingly high frequency of CPT1 P479L variant and, judging from the enzyme analysis in the seven patients, also CPT-I deficiency in the areas of Canada inhabited by these families. Despite the deficiency of CPT-Ia which is the major rate-limiting enzyme for long chain fatty acid oxidation, clinical effects, with few exceptions, were slight or absent. One clue to explaining this paradox is that, judging from the fatty acid oxidation studies in whole blood and fibroblasts, the low residual activity of CPT-Ia is sufficient to allow a reasonable flux through the mitochondrial oxidation system. It is likely that the P479L variant is of ancient origin and presumably its preservation must have conveyed some advantage.

Delays in diagnosing cystic fibrosis: can we find ways to diagnose it earlier?

OBJECTIVE: To describe the clinical presentation and delays in diagnosis of patients with cystic fibrosis (CF) with the goal of raising physicians' awareness of CF and establishing baseline data for comparison with outcomes of patients who undergo newborn screening for CF. DESIGN: Retrospective review of hospital medical records and CF clinic charts of newly diagnosed CF patients younger than 18 years who had attended the CF clinic at the BC Children's Hospital in Vancouver between January 1, 1993, and January 1, 2005. Age at diagnosis of CF was ascertained for 24 adult patients diagnosed during the same period from the CF clinic at St Paul's Hospital in Vancouver, BC. SETTING: Cystic fibrosis clinic at the BC Children's Hospital. PARTICIPANTS: All newly diagnosed CF patients from mainland BC and northern Vancouver Island (N = 122). MAIN OUTCOME MEASURES: Mean age at diagnosis; mean delay in diagnosis; weight and height or length at diagnosis; vitamin E status; mean head circumference; types of symptoms before diagnosis; Pseudomonas aeruginosa status; and number of days spent in tertiary care hospitals before diagnosis. RESULTS: Excluding the adult patients and patients with meconium ileus, mean age at diagnosis of CF was 3.6 years, and mean delay in diagnosis after first symptoms was 2.1 years. Weight at diagnosis was < or = 5th percentile in 37% of cases, and height or length was < or = 5th percentile in 26% of cases. Excluding those with meconium ileus and those taking vitamin E supplementation, 70% of the children were vitamin E deficient at diagnosis. These children had a mean head circumference substantially smaller than that of children who had adequate levels of vitamin E. About 95% of children had gastrointestinal (GI) or malnutrition symptoms before diagnosis; 15% had GI symptoms only. About 81% of patients had respiratory symptoms, but only 4% had respiratory symptoms as the only evidence of CF before diagnosis. Around 9% were colonized with P aeruginosa at diagnosis. Before being diagnosed, 79% of patients had required tertiary care hospitalization for a group total of 320 hospital days. CONCLUSION: Considerable delays in diagnosis of children with CF occur when the disease is identified solely on clinical presentation. Morbidity is often severe enough to require hospital admission before CF is diagnosed. Symptoms that occurred before diagnosis were often GI or malnutritional in nature rather than respiratory, but all such symptoms were associated with diagnostic delays.

Screening for Fabry disease in patients with chronic kidney disease: limitations of plasma alpha-galactosidase assay as a screening test.

BACKGROUND AND OBJECTIVES: Fabry disease is a progressive X-linked disorder of glycosphingolipid metabolism that typically presents in childhood and progresses to heart failure and renal failure in adulthood. This study sought to determine the prevalence of Fabry disease in a multiethnic male chronic kidney disease population, involving dialysis-dependent, non-dialysis-dependent, and transplant patients. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: A total of 499 patients were screened with assay of alpha-galactosidase activity using fluorometric enzyme assay on plasma prepared from fresh heparinized blood, followed by leukocyte alpha-galactosidase activity in the subset of patients with plasma alpha-galactosidase activity below the second percentile (corresponding to a value <3.0 nmol/h per ml plasma). RESULTS: This study did not identify any new cases of Fabry disease; however, repeat testing of some of the study patients identified three limitations of the plasma enzyme assay that is commonly used as a high throughput screening method for Fabry disease: (1) False-negative results can occur; (2) these false-negative results are not prevented by use of inhibitors of alpha-galactosidase B activity; and (3) considerable intraindividual variation in plasma alpha-galactosidase levels reduces the discriminatory power of the screening test. CONCLUSION: Clinicians need to be aware that screening using plasma will fail to detect some patients with Fabry disease.

A hemizygous SCO2 mutation in an early onset rapidly progressive, fatal cardiomyopathy.

Mutations in SCO2, a metallochaperone involved in mitochondrial copper delivery, are associated with early onset, fatal hypertrophic cardiomyopathy. All reported patients carry at least one copy of the common 1541G>A (E140K) mutation. Whereas patients with one copy of the E140K allele, in combination with a more deleterious mutation, follow a severe clinical course, patients homozygous for the E140K mutation have a delayed onset of disease and a more prolonged survival. Here, we have investigated a patient who appeared homozygous for the common 1541G>A mutation based on DNA sequencing and restriction enzyme analysis of a PCR product, yet presented with early onset, severe cardiomyopathy. Restriction enzyme analysis of parental DNA revealed that the mother was heterozygous for 1541G>A, while the father was homozygous wild-type. The patient showed biparental inheritance for microsatellite markers spanning the length of chromosome 22, making isodisomy unlikely. Sequencing of several single nucleotide polymorphisms within the 5'-UTR, intron and single exon of the SCO2 gene was uninformative; however, a 16 bp deletion within the intron was present in the patient and the mother, but not the father. Restriction enzyme analysis confirmed that the mother was heterozygous and that the patient was hemizygous for the deletion. Southern blot, Northern blot, and FISH analyses were consistent with the de novo deletion of one allele of SCO2 in the patient. This is the first report of hemizygosity in a SCO2 patient. The patient phenotype underscores the strikingly similar clinical course in all patients with one copy of the E140K allele. Examination of both patient and parental genotypes by thorough molecular analyses can reveal information with important implications for genetic counseling.

Neonatal hyperphenylalaninemia, perinatal hemochromatosis, and renal tubulopathy: a unique patient or a novel metabolic disorder?

A neonate presented with hyperphenylalaninemia (HPA), with a persistently elevated phenylalanine/tyrosine ratio. The HPA was responsive to tetrahydrobiopterin (BH4). His clinical course was dominated by liver failure, associated with perinatal hemochromatosis. He also developed renal tubulopathy. HPA has not previously been reported in association with any of these features. We investigated the etiology of his condition, and discuss the possibility that this represents a novel single-gene disorder.

6-pyruvoyl-tetrahydropterin synthase deficiency with mild hyperphenylalaninemia.

Severe 6-pyruvoyl-tetrahydrobiopterin synthase deficiency is a tetrahydrobiopterin deficiency disorder that presents in infancy with developmental delay, seizures, and abnormal movements associated with hyperphenylalaninemia usually detectable by neonatal phenylketonuria screening programs. We describe an 8-year-old girl with delay, seizures, and dystonia with mild hyperphenylalaninemia detected in late childhood. The diagnosis of 6-pyruvoyl-tetrahydrobiopterin synthase deficiency was made by analysis of pterins in urine, pterins and neurotransmitters in cerebrospinal fluid, and enzyme assay. The patient improved clinically taking oral tetrahydrobiopterin, levodopa/carbidopa, and 5-hydroxytryptophan. This treatable condition may not always be detected by routine population screening for hyperphenylalaninemia.

A genomic approach to mutation analysis of holocarboxylase synthetase gene in three Chinese patients with late-onset holocarboxylase synthetase deficiency.

OBJECTIVE: Multiple carboxylase deficiency (MCD, MIM:253270) is a common organic aciduria and caused by deficiency of either biotinidase or holocarboxylase synthetase (HLCS; EC 6.3.4.10). Patients commonly present during early infancy with acute metabolic derangements and severe metabolic acidosis. Recently, a late onset form of HLCS deficiency was also described. The different phenotypes (early and late presenting) may be related to a spectrum of mutations in HLCS gene. Applications of mutation analysis in HLCS had been limited previously by the requirement of cDNA from living tissue for study. We described here a genomic approach for molecular diagnosis of HLCS deficiency which we have used to detect mutations in Chinese patients who had the late-onset form of HLCS deficiency. In addition, a fibroblast cell line with MCD from Coriell Cell repositories was also studied. DESIGN AND METHODS: Three Chinese patients with late onset HLCS deficiency were studied. The genomic sequence of HLCS was retrieved and newly designed primers were used to cover all coding sequences of the gene. PCR products were analyzed by direct sequencing. Population allelic frequencies of mutations detected were determined by genotyping of control samples by restriction fragment length polymorphism. RESULTS: We found a recurrent mutation, R508W, in the three unrelated Chinese patients. Two were homozygous for this mutation. The other patient was a compound heterozygote of R508W and a novel mutation, D634N. The results suggest that R508W may be an important and relatively prevalent disease-causing mutation in Chinese MCD patients. A fibroblast cell-line from an African patient revealed an additional novel mutation, R565X and a known mutation, V550M. CONCLUSION: R508W is a recurrent mutation in Chinese MCD patients which is associated with the late onset phenotype. This new genomic approach for mutation analysis of HLCS gene provides new opportunities in studies of MCD.