Liver mtDNA content increases during development: a comparison of methods and the importance of age- and tissue-specific controls for the diagnosis of mtDNA depletion.

BACKGROUND: The quantitative loss of mitochondrial DNA (mtDNA) known as mtDNA depletion, often gives rise to liver disease. The diagnosis of mtDNA depletion syndrome is frequently imprecise, both for technical reasons and because of the lack of established age-adjusted normal ranges. We aimed to refine quantitative methods for diagnosing the hepatic type of mtDNA depletion syndrome, firstly by establishing an age-matched reference range for mitochondrial to nuclear DNA ratio (henceforth "mtDNA content") and secondly by investigating mtDNA in fibroblasts. METHODS: By comparing realtime PCR with an established method for quantifying mtDNA content we established a reference range for young children using biopsy and post-mortem material from patients <15 years. In addition, we investigated the arrangement of mtDNA in nucleoids from fibroblasts using fluorescence microscopy. RESULTS: Both methods showed that the mtDNA content of liver increases rapidly over the perinatal period. In a patient whose liver mtDNA content fell, but remained within the reference range, early investigation and age-matched controls were essential, as we found a progressive increase in muscle mtDNA copy number, respiratory chain activity and muscle power with age. In three further patients, fluorescence microscopy of the fibroblasts proved diagnostic. In one case a movement disorder was an important pointer. CONCLUSIONS: These cases highlight the (i) need for comparing mtDNA copy number data generated from patients to DNA isolated from an age-matched normal range from the tissue of interest and (ii) the utility of mtDNA staining with PicoGreen as a method to detect aberrant nucleoid morphology in mtDNA depletion patient fibroblast lines when affected tissues are not available for measuring mtDNA copy number.

Sepiapterin reductase deficiency an autosomal recessive DOPA-responsive dystonia.

The diagnosis of a 14-year-old girl with a new homoallelic mutation in the sepiapterin reductase (SR) gene is reported. Initially she presented at the age of 2 with hypotonia and mild cognitive developmental delay, and was diagnosed as having mild methylmalonic aciduria, which was recently identified as methylmalonylCoA racemase deficiency, a new defect in valine-isoleucine metabolism. After a 12-year progression of her neurologic condition, which had made her wheelchair-bound at the age of 6, dystonia with diurnal variation had become apparent. At the age of 14 this finding led to rapid diagnosis of SR deficiency. The diagnostic approach with CSF neurotransmitter and pterins analysis and combined phenylalanine/BH(4) loading test, and finally measurement of sepiapterin in CSF is illustrative for the diagnosis of SR deficiency. As in all other patients with this new defect, very low levels of homovanillic acid and 5-hydroxyindoleacetic acid and high levels of biopterin and sepiapterin in the CSF are the diagnostic hallmark. The girl improved dramatically on treatment with L-DOPA and 5-hydroxytryptophan. The initial diagnosis of methylmalonic aciduria may afterwards be considered to have not significantly contributed to her clinical condition and only has led to a long delay of the clinically relevant diagnosis of SR deficiency. Although the clinical condition of this recently recognized autosomal recessive defect in pterin metabolism is complex and many symptoms can occur in variable severity and time of onset, dystonia with diurnal variation is a characteristic finding, as shown in nearly all patients described so far. The rapid and favourable response on treatment with L-DOPA warrants the classification of SR deficiency as another autosomal recessive type of DOPA-responsive dystonia (DRD). This classification is important to improve the awareness of clinicians that more than one metabolic defect can underlie the phenotype of a DOPA-responsive dystonic disorder and that dystonia should always trigger a rapid diagnosis of the underlying neurotransmitter synthesis defect, in view of the excellent treatability of a DRD.

Identification of novel mutations in classical galactosemia.

Classical galactosemia is an autosomal recessive disorder of galactose metabolism due to galactose-1-phosphate uridyltransferase (GALT) deficiency. Treatment through restriction of dietary galactose intake is lifesaving, but, in spite of this diet, most patients develop abnormalities. In this paper we report the mutational spectrum of classical galactosemia in a cohort of 123 Dutch patients, all with biochemically proven classical galactosemia. In the human GALT gene, which is located on chromosome 9p13, we identified 24 different mutations, including nine mutations that have not been reported previously. The novel mutations include five missense mutations (c.152G>A/p.R51Q, c.404C>T/p.S135W, c.687G>T/p.K229N, c.756G>T/p.Q252H, and c.1140A>C/p.X380C), a frame shift mutation (c.410dupT), a splice site mutation (c.821-2A>G), a possible branch point mutation (c.508-29delT), and a large deletion encompassing at least exons 1-11. Six of these novel mutations were found in patients of Dutch descent: p.R51Q, p.S135W, p.K229N, p.Q252H, p.X380C, and c.410dupT.

Efficacy and safety of statin therapy in children with familial hypercholesterolemia: a randomized controlled trial.

CONTEXT: Children with familial hypercholesterolemia have endothelial dysfunction and increased carotid intima-media thickness (IMT), which herald the premature atherosclerotic disease they develop later in life. Although intervention therapy in the causal pathway of this disorder has been available for more than a decade, the long-term efficacy and safety of cholesterol-lowering medication have not been evaluated in children. OBJECTIVE: To determine the 2-year efficacy and safety of pravastatin therapy in children with familial hypercholesterolemia. DESIGN: Randomized, double-blind, placebo-controlled trial that recruited children between December 7, 1997, and October 4, 1999, and followed them up for 2 years. SETTING AND PARTICIPANTS: Two hundred fourteen children with familial hypercholesterolemia, aged 8 to 18 years and recruited from an academic medical referral center in the Netherlands. INTERVENTION: After initiation of a fat-restricted diet and encouragement of regular physical activity, children were randomly assigned to receive treatment with pravastatin, 20 to 40 mg/d (n = 106), or a placebo tablet (n = 108). MAIN OUTCOME MEASURES: The primary efficacy outcome was the change from baseline in mean carotid IMT compared between the 2 groups over 2 years; the principal safety outcomes were growth, maturation, and hormone level measurements over 2 years as well as changes in muscle and liver enzyme levels. RESULTS: Compared with baseline, carotid IMT showed a trend toward regression with pravastatin (mean [SD], -0.010 [0.048] mm; P =.049), whereas a trend toward progression was observed in the placebo group (mean [SD], +0.005 [0.044] mm; P =.28). The mean (SD) change in IMT compared between the 2 groups (0.014 [0.046] mm) was significant (P =.02). Also, pravastatin significantly reduced mean low-density lipoprotein cholesterol levels compared with placebo (-24.1% vs +0.3%, respectively; P<.001). No differences were observed for growth, muscle or liver enzymes, endocrine function parameters, Tanner staging scores, onset of menses, or testicular volume between the 2 groups. CONCLUSION: Two years of pravastatin therapy induced a significant regression of carotid atherosclerosis in children with familial hypercholesterolemia, with no adverse effects on growth, sexual maturation, hormone levels, or liver or muscle tissue.

Living with classical galactosemia: health-related quality of life consequences.

OBJECTIVE: Classical galactosemia (McKusick 230400) is an autosomal recessive disorder of galactose metabolism caused by a deficiency of galactose-1-phosphate uridyltransferase (EC 2.7.712). Treatment, consisting of a severe restriction of dietary galactose, is life saving, but most patients develop abnormalities despite this diet. The aim of this study was to study the influence of galactosemia on the patients' health-related quality of life (HRQoL), on educational levels, and on the specific galactosemia-related concerns of these families. METHODS: Age-specific HRQoL questionnaires, a classical galactosemia-specific questionnaire designed by the authors, and a list of questions regarding educational attainment were handed out or sent to all 75 members of the Dutch Galactosemia Society and their families. RESULTS: Sixty-three (84%) patients with classical galactosemia from 58 families returned the questionnaire. Concerning HRQoL, significant differences between patients aged 1 to 5 and healthy children were found on the domains of abdominal complaints and communication. Patients aged 8 to 15 years differed from their healthy peers on the domain of cognitive function. Mothers of patients aged 6 to 15 reported a significantly lower HRQoL on the domains of motor and cognitive function. Patients 16 years and older had significant lower scores on the domains of cognitive and social function. The percentage of patients who attend special schools is significantly higher than in the general population, and the educational attainment is significantly lower in patients with classical galactosemia. CONCLUSIONS: This is the first study to describe the HRQoL of patients with classical galactosemia using well-developed and validated instruments in different age groups. The results of the present study indicate that having galactosemia negatively influences the HRQoL. Early and regular evaluation and support of possible cognitive problems should be a major part of the protocol for the follow-up of patients with classical galactosemia.

Arterial intima-media thickness in children heterozygous for familial hypercholesterolaemia.

Patients with familial hypercholesterolaemia have severe coronary-artery disease early in adult life. Whether lipid-lowering treatment should be started in childhood remains to be established. We therefore assessed 201 children heterozygous for familial hypercholesterolaemia and 80 unaffected siblings (both age ranges 8-18 years) with B-mode ultrasound to measure carotid wall intima-media thickness. Mean combined carotid intima-media thickness of heterozygotes was significantly greater than that of unaffected siblings (0.494 mm [SD 0.051] vs 0.472 [SD 0.049], p=0.002). A significant deviation in intima-media thickness was noted from age 12 years in children with familial hypercholesterolaemia. Findings on multivariate analysis showed LDL cholesterol, age, and sex to be strong and independent predictors of intima-media thickness. Since raised LDL cholesterol concentrations can be lowered efficiently, clinical studies are needed to investigate long-term safety and effectiveness of statin treatment in children with familial hypercholesterolaemia.

Steady-state pharmacokinetics of pravastatin in children with familial hypercholesterolaemia.

OBJECTIVE: To determine pharmacokinetic data for pravastatin in children, since current data are insufficient in this age group. SUBJECTS AND METHODS: A 2-week, multiple-dose, steady-state pharmacokinetic study was carried out with pravastatin 20mg daily in 24 children with familial hypercholesterolaemia (aged 8-16 years; 12 prepubertal, 12 pubertal). A plasma concentration-time curve was performed on day 14. Pharmacokinetic curves for each individual were constructed using nonparametric methods, yielding area under the plasma concentration-time curve (AUC), maximum plasma concentration (C(max)) and half-life (t((1/2))). Clearance values and volumes of distribution were calculated from these parameters. Cholesterol lowering was observed on day 14 and 6 weeks after the start of pravastatin. RESULTS: The C(max) in prepubertal (group A) children (52.1 +/- 27.0 mug/L [mean +/- SD]) differed, although not significantly (p = 0.09, unpaired two-tailed t-test), from the C(max) in adolescents (group B) [31.7 +/- 29.2 mug/L]. There was a moderate negative correlation between C(max) and age (Spearman correlation r = -0.42; p = 0.04). The AUC in prepubertal children (91.3 +/- 39.7 mug . h/L) did not differ significantly from the AUC in adolescents (69.3 +/- 57.0 mug . h/L). The t((1/2)) was the same for the two groups: 2.5 +/- 1.1h. Clearance values (CL/f) varied widely between the two groups (group A: 4.3 +/- 1.8 L/min; group B: 11.0 +/- 11.9 L/min; p = 0.08). A moderate positive correlation was found between clearance and age (Spearman correlation r = 0.36; p = 0.09). A large variation was found in the volumes of distribution within the two groups (group A: 31.2 mL/kg [SD 26.7], group B:37.0 mL/kg [SD 29.6]; p = 0.12). A very weak positive correlation was found between age and volume of distribution (Spearman correlation r = 0.11; p = 0.61). A 27% low-density lipoprotein-cholesterol reduction from baseline was achieved at day 14. CONCLUSIONS: Body surface area and gender did not influence the pharmacokinetics of pravastatin in children aged 8-16 years. On the basis of our findings there are no reasons to use pravastatin at a dosage according to bodyweight or to use different dosage regimens from those in adults. However, for prepubertal children half the advised starting dose for adults may be sufficient.

The natural course of infantile Pompe's disease: 20 original cases compared with 133 cases from the literature.

OBJECTIVE: Infantile Pompe's disease is a lethal cardiac and muscular disorder. Current developments toward enzyme replacement therapy are promising. The aim of our study is to delineate the natural course of the disease to verify endpoints of clinical studies. METHODS: A total of 20 infantile patients diagnosed by the collaborative Dutch centers and 133 cases reported in literature were included in the study. Information on clinical history, physical examination, and diagnostic parameters was collected. RESULTS: The course of Pompe's disease is essentially the same in the Dutch and the general patient population. Symptoms start at a median age of 1.6 months in both groups. The median age of death is 7.7 and 6 months, respectively. Five percent of the Dutch patients and 8% of all reported patients survive beyond 1 year of age. Only 2 patients from literature became older than 18 months. A progressive cardiac hypertrophy is characteristic for infantile Pompe's disease. The diastolic thickness of the left ventricular posterior wall and cardiac weight at autopsy increase significantly with age. Motor development is severely delayed and major developmental milestones are generally not achieved. For the Dutch patient group, growth deviates significantly from normal despite start of nasogastric tube feeding. Levels of aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, creatine kinase, or creatine kinase-myocardial band isoenzyme are typically elevated, although aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase increase significantly with age. The patients have fully deleterious mutations. Acid alpha-glucosidase activity is severely deficient. CONCLUSIONS: Survival, decrease of the diastolic thickness of the left ventricular posterior wall, and achievement of major motor milestones are valid endpoints for therapeutic studies of infantile Pompe's disease. Mutation analysis and measurement of the alpha-glucosidase activity should be part of the enrollment program.

The apolipoprotein epsilon4 allele confers additional risk in children with familial hypercholesterolemia.

Children with familial hypercholesterolemia (FH) exhibit substantial variance of LDL cholesterol. In previous studies, family members of children with FH were included, which may have influenced results. To avoid such bias, we studied phenotype in 450 unrelated children with FH and in 154 affected sib-pairs. In known families with classical FH, diagnosis was based on plasma LDL cholesterol above the age- and gender-specific 95th percentile. Girls had 0.47 +/- 0.15 mmol/L higher LDL cholesterol, compared with boys (p = 0.002). Also in girls, HDL cholesterol increased by 0.07 +/- 0.03 mmol/L per 5 y (pfor trend = 0.005); this age effect was not observed in boys. The distribution of apolipoprotein (apo) E genotypes was not significantly different between probands, their paired affected siblings, or a Dutch control population. Carriers with or without one epsilon4 allele had similar LDL and HDL cholesterol levels. Within the affected sib-pairs, the epsilon4 allele explained 72.4% of the variance of HDL cholesterol levels (-0.15 mmol/L, 95% confidence interval -0.24 to -0.05, p = 0.003). The effect of apoE4 on HDL cholesterol differed with an analysis based on probands or on affected sib-pairs. The affected sib-pair model used adjustment for shared environment, type of LDL receptor gene mutation, and a proportion of additional genetic factors and may, therefore, be more accurate in estimating effects of risk factors on complex traits. We conclude that the epsilon4 allele was associated with lower HDL cholesterol levels in an affected sib-pair analysis, which strongly suggests that apoE4 influences HDL cholesterol levels in FH children. Moreover, the strong association suggests that apoE4 carries an additional disadvantage for FH children.

Family history and cardiovascular risk in familial hypercholesterolemia: data in more than 1000 children.

BACKGROUND: Elevated LDL cholesterol (LDL-C) levels in childhood predict cardiovascular disease (CVD) later in life. Familial hypercholesterolemia (FH) represents the paradigm of this relation. METHODS AND RESULTS: The objectives of this study were to (1) establish the LDL-C level that provides the most accurate diagnosis of FH in children from families with known FH and (2) assess whether lipoprotein variation in these children is associated with premature CVD in relatives. Foremost, however, it was our objective to identify children with FH who are at high risk and in need of early intervention. A total of 1034 consecutive children from FH kindreds were investigated. First, LDL-C levels >3.50 mmol/L had a 0.98 post-test probability (95% CI, 0.96 to 0.99) of predicting the presence of an LDL receptor mutation. Second, children with FH in the highest LDL-C tertile (>6.23 mmol/L) had a 1.7-times higher incidence (95% CI, 1.24 to 2.36) of having a parent with FH suffering from premature CVD (P=0.001). In addition, such a parent was found 1.8 times more often (95% CI, 1.20 to 2.59) among children with FH who had HDL-C <1.00 mmol/L (P=0.004). Last, children with FH whose lipoprotein(a) was >300 mg/L had a 1.45-times higher incidence (95% CI, 0.99 to 2.13) of having a parent with FH suffering from premature CVD (P=0.05). CONCLUSIONS: In FH families, LDL-C levels allow accurate diagnosis of FH in childhood. Moreover, increased LDL-C and lipoprotein(a) and decreased HDL-C levels in children identify FH kindreds with the highest CVD risk.

Early statin therapy restores endothelial function in children with familial hypercholesterolemia.

OBJECTIVES: This study was designed to determine whether simvastatin improves endothelial function in children with familial hypercholesterolemia (FH). BACKGROUND: Endothelial function measured by flow-mediated dilation of the brachial artery (FMD) is used as a surrogate marker of cardiovascular disease (CVD). Adult studies have shown that statins reverse endothelial dysfunction and therefore reduce the risk for future CVD. METHODS: The study included 50 children with FH (9 to 18 years) and 19 healthy, non-FH controls. Children with FH were randomized to receive simvastatin or placebo for 28 weeks. The FMD was performed at baseline and at 28 weeks of treatment. RESULTS: At baseline, FMD was impaired in children with FH versus non-FH controls (p < 0.024). In the simvastatin FH group, FMD improved significantly, whereas the FMD remained unaltered in the placebo FH group throughout the study period (absolute increase 3.9% +/- 4.3% vs. 1.2% +/- 3.9%, p < 0.05). In the simvastatin FH group, FMD increased to a level similar to the non-FH controls (15.6% +/- 6.8% vs. 15.5% +/- 5.4%, p = 0.958). Upon treatment, the simvastatin FH group showed significant absolute reductions of total cholesterol (TC) (-2.16 +/- 1.04 mmol/l, 30.1%) and low-density lipoprotein cholesterol (LDL-C) (-2.13 +/- 0.99 mmol/l, 39.8%). The absolute change of FMD after 28 weeks of therapy was inversely correlated to changes of TC (r = -0.31, p < 0.05) and LDL-C (r = -0.31, p < 0.05). CONCLUSIONS: Our data show significant improvement of endothelial dysfunction towards normal levels after short-term simvastatin therapy in children with FH. These results emphasize the relevance of statin therapy in patients with FH at an early stage, when the atherosclerotic process is still reversible.

Efficacy and safety of statin therapy in children with familial hypercholesterolemia: a randomized, double-blind, placebo-controlled trial with simvastatin.

BACKGROUND: A multicenter, randomized, double-blind, placebo-controlled study was conducted to evaluate LDL cholesterol-lowering efficacy, overall safety, and tolerability and the influence on growth and pubertal development of simvastatin in a large cohort of boys and girls with heterozygous familial hypercholesterolemia (heFH). METHODS AND RESULTS: A total of 173 heFH children (98 boys and 75 girls) were included in this study. After a 4-week diet/placebo run-in period, children with heFH were randomized to either simvastatin or placebo in a ratio of 3:2. Simvastatin was started at 10 mg/d and titrated at 8-week intervals to 20 and then 40 mg/d. During a 24-week extension period, the patients continued to receive simvastatin (40 mg) or placebo according to their assignment. After 48 weeks of simvastatin therapy, there were significant reductions of LDL cholesterol (-41%), total cholesterol (-31%), apolipoprotein B (-34%), VLDL cholesterol (-21%), and triglyceride (-9%) levels. HDL cholesterol and apolipoprotein A-I levels were increased by 3.3% and 10.4%, respectively (not significant). No safety issues became evident. Except for small decreases in dehydroepiandrosterone sulfate compared with placebo, there were no significant changes from baseline in adrenal, gonadal, and pituitary hormones in either treatment group. CONCLUSIONS: Simvastatin significantly reduced LDL cholesterol, total cholesterol, triglyceride, VLDL cholesterol, and apolipoprotein B levels and was well tolerated in children with heFH. There was no evidence of any adverse effect of simvastatin on growth and pubertal development. Therefore, simvastatin at doses up to 40 mg is a well-tolerated and effective therapy for heFH children.

Defects in degradation of blood group A and B glycosphingolipids in Schindler and Fabry diseases.

Skin fibroblast cultures from patients with inherited lysosomal enzymopathies, alpha-N-acetylgalactosaminidase (alpha-NAGA) and alpha-galactosidase A deficiencies (Schindler and Fabry disease, respectively), and from normal controls were used to study in situ degradation of blood group A and B glycosphingolipids. Glycosphingolipids A-6-2 (GalNAc (alpha 1-->3)[Fuc alpha 1-->2]Gal(beta1-->4)GlcNAc(beta 1-->3)Gal(beta 1--> 4)Glc (beta 1-->1')Cer, IV(2)-alpha-fucosyl-IV(3)-alpha-N-acetylgalactosaminylneolactotetraosylceramide), B-6-2 (Gal(alpha 1-->3)[Fuc alpha 1--> 2] Gal (beta 1-->4)GlcNAc(beta 1-->3)Gal(beta 1-->4)Glc(beta 1-->1')Cer, IV(2)- alpha-fucosyl-IV(3)-alpha-galactosylneolactotetraosylceramide), and globoside (GalNAc(beta 1-->3)Gal(alpha 1-->4)Gal(beta 1-->4)Glc(beta 1-->1') Cer, globotetraosylceramide) were tritium labeled in their ceramide moiety and used as natural substrates. The degradation rate of glycolipid A-6-2 was very low in fibroblasts of all the alpha-NAGA-deficient patients (less than 7% of controls), despite very heterogeneous clinical pictures, ruling out different residual enzyme activities as an explanation for the clinical heterogeneity. Strongly elevated urinary excretion of blood group A glycolipids was detected in one patient with blood group A, secretor status (five times higher than upper limit of controls), in support of the notion that blood group A-active glycolipids may contribute as storage compounds in blood group A patients. When glycolipid B-6-2 was fed to alpha-galactosidase A-deficient cells, the degradation rate was surprisingly high (50% of controls), while that of globotriaosylceramide was reduced to less than 15% of control average, presumably reflecting differences in the lysosomal enzymology of polar glycolipids versus less-polar ones. Relatively high-degree degradation of substrates with alpha-D-Galactosyl moieties hints at a possible contribution of other enzymes.

Family history of cardiovascular events and endothelial dysfunction in children with familial hypercholesterolemia.

OBJECTIVES: in patients with familial hypercholesterolemia (FH), the propensity towards atherosclerosis may vary considerably. In the general population, a positive family history is associated with an increased risk for cardiovascular events. Since endothelial dysfunction is predictive for future cardiovascular events, we evaluated whether FH-children with a positive family history of premature cardiovascular disease have more pronounced endothelial dysfunction compared to children with a negative family history. STUDY DESIGN: 50 FH children, 10-18 years, participated in this study. Thirty-one children had a positive family history for cardiovascular events (fh(+)) and 19 children had no events in the family (fh(-)). Nineteen matched siblings participated as controls. Endothelial function was assessed by testing the flow mediated dilatation (FMD) of the brachial artery. RESULTS: baseline characteristics were comparable for fh(+), fh(-) and controls. Lipid levels were significantly higher in FH children. In FH, FMD was impaired compared to controls (11.7+/-4.4 vs. 15.6+/-6.8%, P<0.03). In addition, FMD was significantly lower in fh(+) compared to fh(-) (10.7+/-9.9 vs. 13.3+/-4.6%, P<0.05). CONCLUSION: In FH-children, endothelial function is impaired compared to matched controls. This impairment is most pronounced in FH children with a positive family history of premature cardiovascular disease.

Phenylketonuria and glycogen storage disease type III in sibs of one family.

Hyperphenylalaninemia result from a block in the conversion of phenylalanine into tyrosine due to a defect in either the enzyme phenylalanine hydroxylase (98% of subjects) or in the metabolism of the cofactor tetrahydrobiopterin. Phenylalanine hydroxylase deficiency is the most common form of inherited hyperphenylalaninemia disorders, with a prevalence between 1/4,000-1/40,000. Glycogen storage disease (GSD) type III is caused by debranching enzyme deficiency of glycogen degradation. The clinical features vary in relation to the localization of the enzyme defect. Two clinical entities exist: a combined hepatic myogenic form (GSD IIIa) and a purely hepatic form (GSD IIIb). The inheritance is autosomal recessive. We describe a Turkish family in which two girls were found to have phenylketonuria, while in two other sisters glycogen storage disease type III was diagnosed. The parents of these children are cousins and they have had 12 children.