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.

Clinical onset and course, response to treatment and outcome in 24 patients with the cblE or cblG remethylation defect complemented by genetic and in vitro enzyme study data.

BACKGROUND: The cobalamin E (cblE) (MTRR, methionine synthase reductase) and cobalamin G (cblG) (MTR, methionine synthase) defects are rare inborn errors of cobalamin metabolism leading to impairment of the remethylation of homocysteine to methionine. METHODS: Information on clinical and laboratory data at initial full assessment and during the course of the disease, treatment, outcome and quality of life was obtained in a survey-based, retrospective study from physicians caring for patients with the CblE or CblG defect. In addition, data on enzyme studies in cultured skin fibroblasts and mutations in the MTRR and MTR gene were analysed. RESULTS: In 11 cblE and 13 cblG patients, failure to thrive, feeding problems, delayed milestones, muscular hypotonia, cognitive impairment and macrocytic anaemia were the most frequent symptoms. Delay in diagnosis depended on age at first symptom and clinical pattern at presentation and correlated significantly with impaired communication abilities at follow-up. Eighteen/22 patients presented with brain atrophy or white matter disease. Biochemical response to treatment with variable combinations of betaine, cobalamin, folate was significant. The overall course was considered improving (n = 8) or stable (n = 15) in 96% of patients, however the average number of CNS symptoms per patient increased significantly over time and 16 of 23 patients were classified as developmentally delayed or severely handicapped. In vitro enzyme analysis data showed no correlation with outcome. Predominantly private mutations were detected and no genotype- phenotype correlations evident. CONCLUSIONS: The majority of patients with the cblE and cblG defect show limited clinical response to treatment and have neurocognitive impairment.

Propionic acidemia: neonatal versus selective metabolic screening.

BACKGROUND: Whereas propionic acidemia (PA) is a target disease of newborn screening (NBS) in many countries, it is not in others. Data on the benefit of NBS for PA are sparse. STUDY DESIGN: Twenty PA patients diagnosed through NBS were compared to 35 patients diagnosed by selective metabolic screening (SMS) prompted by clinical findings, family history, or routine laboratory test results. Clinical and biochemical data of patients from 16 metabolic centers in Germany, Austria, and Switzerland were evaluated retrospectively. Additionally, assessment of the intelligent quotient (IQ) was performed. In a second step, the number of PA patients who have died within the past 20 years was estimated based on information provided by the participating metabolic centers. RESULTS: Patients diagnosed through NBS had neither a milder clinical course regarding the number of metabolic crises nor a better neurological outcome. Among NBS patients, 63% were already symptomatic at the time of diagnosis, and <10% of all patients remained asymptomatic. Among all PA patients, 76% were found to be at least mildly mentally retarded, with an IQ <69. IQ was negatively correlated with the number of metabolic decompensations, but not simply with the patients' age. Physical development was also impaired in the majority of patients. Mortality rates tended to be lower in NBS patients compared with patients diagnosed by SMS. CONCLUSION: Early diagnosis of PA through NBS seems to be associated with a lower mortality rate. However, no significant benefit could be shown for surviving patients with regard to their clinical course, including the number of metabolic crises, physical and neurocognitive development, and long-term complications.

Mutation analysis in 54 propionic acidemia patients.

Deficiency of propionyl CoA carboxylase (PCC), a dodecamer of alpha and beta subunits, causes inherited propionic acidemia. We have studied, at the molecular level, PCC in 54 patients from 48 families comprised of 96 independent alleles. These patients of various ethnic backgrounds came from research centers and hospitals in Germany, Austria and Switzerland. The thorough clinical characterization of these patients was described in the accompanying paper (Grünert et al. 2012). In all 54 patients, many of whom originated from consanguineous families, the entire PCCB gene was examined by genomic DNA sequencing and in 39 individuals the PCCA gene was also studied. In three patients we found mutations in both PCC genes. In addition, in many patients RT-PCR analysis of lymphoblast RNA, lymphoblast enzyme assays, and expression of new mutations in E.coli were carried out. Eight new and eight previously detected mutations were identified in the PCCA gene while 15 new and 13 previously detected mutations were found in the PCCB gene. One missense mutation, p.V288I in the PCCB gene, when expressed in E.coli, yielded 134% of control activity and was consequently classified as a polymorphism in the coding region. Numerous new intronic polymorphisms in both PCC genes were identified. This study adds a considerable amount of new molecular data to the studies of this disease.

The molecular basis of human 3-methylcrotonyl-CoA carboxylase deficiency.

Isolated biotin-resistant 3-methylcrotonyl-CoA carboxylase (MCC) deficiency is an autosomal recessive disorder of leucine catabolism that appears to be the most frequent organic aciduria detected in tandem mass spectrometry-based neonatal screening programs. The phenotype is variable, ranging from neonatal onset with severe neurological involvement to asymptomatic adults. MCC is a heteromeric mitochondrial enzyme composed of biotin-containing alpha subunits and smaller beta subunits. Here, we report cloning of MCCA and MCCB cDNAs and the organization of their structural genes. We show that a series of 14 MCC-deficient probands defines two complementation groups, CG1 and 2, resulting from mutations in MCCB and MCCA, respectively. We identify five MCCA and nine MCCB mutant alleles and show that missense mutations in each result in loss of function.

Intermediate hyperhomocysteinaemia and compound heterozygosity for the common variant c.677C>T and a MTHFR gene mutation.

Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in the regulation of plasma homocysteine levels. MTHFR deficiency, an autosomal recessive disorder, results in homocystinuria and hypomethioninaemia and presents with highly variable symptoms affecting many organs but predominantly the central nervous system. The common polymorphism of the MTHFR gene, c.677C>T, a known risk factor for elevated plasma homocysteine levels, occurs frequently in the caucasian population. In this study we investigated three subjects with moderate hyperhomocysteinaemia (total plasma homocysteine 72 micromol/L in case 1 and 90 micromol/L in case 3, total non-protein-bound homocysteine 144-186 micromol/L in case 2) but different clinical presentation with no symptoms in case 1, muscle weakness at 17 years of age in case 2, and syncopes and cerebral convulsions at 18 years of age in case 3. Each subject was compound heterozygous for the c.677C>T polymorphism and a novel mutation of the MTHFR gene (case 1: c.883G>A [p.D291N]; case 2: c.1552_c.1553delGA [p.E514fsX536]; case 3: c.616C>T [p.P202S]). Moderately decreased fibroblast MTHFR activity was associated with severely reduced affinity for NADPH and increased sensitivity to inhibition by S-adenosylmethionine (AdoMet) in case 2, and with mild FAD responsiveness in case 3. In case 1, fibroblast MTHFR activity was normal but the sensitivity to inhibition by AdoMet was slightly reduced. This study indicates that the sequence alteration c.677C>T combined with severe MTHFR mutations in compound heterozygous state may lead to moderate biochemical and clinical abnormalities exceeding those attributed to the c.677TT genotype and might require in addition to folate substitution further therapy to normalize homocysteine levels.

Biotinidase deficiency associated with renal loss of biocytin and biotin.

Clinical and biochemical investigations in six patients with congenital biotinidase deficiency are presented. The time course of biotin depletion in relation to carboxylase activities and clinical onset of symptoms was studied after withdrawal of biotin supplementation. Renal biotin clearance studies were performed in patients and controls. Renal loss of biocytin and biotin itself are shown to be a major cause for the increased biotin requirement in patients with congenital biotinidase deficiency.

Rapid differential diagnosis of carboxylase deficiencies and evaluation for biotin-responsiveness in a single blood sample.

We have developed a method for rapid differential diagnosis of isolated or multiple deficiencies of the 3 mitochondrial biotin-dependent carboxylases: propionyl-CoA (PCC), 3-methylcrotonyl-CoA (MCC) and pyruvate carboxylase (PC), and for simultaneous evaluation of biotin-responsiveness using a single blood sample. Lymphocytes were isolated from heparinized blood and preincubated without and with 10(-5) mol/l biotin in medium before determination of PCC, MCC and PC activities. Plasma was used for estimation of biotin concentration and biotinidase activity. A definitive diagnosis could be made in 7 of 9 patients studied up to now: 4 patients suffered from biotin-nonresponsive isolated PCC-deficiency, and 3 patients from biotin-responsive multiple carboxylase deficiency caused by deficient biotinidase activity. In two patients, a carboxylase deficiency was excluded. These results were confirmed in studies using fibroblasts. In addition, a simple method for detection of deficiency in holocarboxylase synthesis is described.

Quantitative determination of biocytin in urine of patients with biotinidase deficiency using high-performance liquid chromatography (HPLC).

A specific method for the quantitative determination of biocytin from urine of biotinidase deficient patients is described using HPLC-separation and quantitative determination by an avidin binding method. Partial purification of biocytin from urine was achieved with an anion exchange resin and concentration of the eluate by lyophilization. The recovery of biocytin from urines was 95.3 +/- 5.9 (mean +/- SD). The precision of biocytin estimation in patients urines including the HPLC-sample preparation procedure varied between 5.9% and 10.5% (CV). Biocytin concentrations were measured in urine samples of 5 patients obtained during and/or before biotin therapy. Before treatment biocytin excretion ranged from 6.2-28.8 nmol/mmol creatinine. During therapy biocytin excretion increased to the 1.3 to 4-fold level in 3 out of 4 patients. However, there was no dose-related increase of biocytin excretion when pharmacological doses were administered. Apart from biocytin and biotin, patients excrete additional biotin derivatives. Some of these have been preliminary identified as bisnorbiotin and oxidation products of bisnorbiotin, biocytin and biotin.

Proliferation and differentiation of cultured human follicular keratinocytes are not influenced by biotin.

In humans and in animals, biotin deficiency causes pathological changes in the skin and its appendages. High doses of biotin may also have beneficial effects on skin, hair and fingernails in humans and animals with normal biotin status. Therefore, we investigated the effects of low and high concentrations of biotin on proliferation and differentiation of cultured outer root sheath cells from human hair follicles as an in vitro model for skin. The activities of biotin-dependent carboxylases were measured to evaluate the biotin status of the cells. In monolayer cultures of outer root sheath cells, proliferation and expression of the differentiation-specific keratins K1 and K10 were not influenced by extremely low concentrations of biotin (<2 x 10(-10) mol/l) or by pharmacological doses of biotin (10(-5) mol/l). Biotin deficiency of the cells was confirmed under the former condition by demonstrating decreased activities of the mitochondrial carboxylases. In organotypic cocultures of outer root sheath cells and dermal fibroblasts, in which stratified epithelia resembling epidermis were developed, the biotin concentration had no effect on the expression of all tested epidermal differentiation markers, including the suprabasal keratins K1 and K10, the hyperproliferation-associated keratin K16, involucrin and filaggrin.

Multiple carboxylase deficiency: inherited and acquired disorders of biotin metabolism.

Acquired biotin deficiency and the two known congenital disorders of biotin metabolism, biotinidase and holocarboxylase synthetase (HCS) deficiency, all lead to deficiency of the 4 biotin-dependent carboxylases, i.e. to multiple carboxylase deficiency (MCD). The underlying mechanism in HCS-deficiency, discovered in 1981, is decreased affinity of HCS for biotin impairing the formation of holocarboxylases at physiological biotin levels. In biotinidase deficiency, discovered in 1983, MCD results from progressive development of biotin-deficiency due to inability to liberate and recycle biotin which is lost in urine as biocytin. MCD leads to typical organic aciduria and severe life-threatening illness. Main symptoms and signs are feeding difficulties, neurologic abnormalities (hypotonia, impaired consciousness, seizures, ataxia) and cutaneous changes (rash, alopecia). However, the clinical presentation and age of onset are extremely variable, and organic aciduria may initially be absent in biotinidase deficiency. Therefore, the definitive diagnosis requires enzyme studies. MCD can be detected in lymphocytes obtained before treatment and biotinidase deficiency is confirmed or excluded by a colorimetric enzyme assay in plasma. Newborn screening for biotinidase deficiency has resulted in the detection of patients with partial deficiency (10-30% of mean normal activity) in addition to patients with profound deficiency (0-10%). Severe illness has been observed mainly in patients with O-activity or a Km-mutation, detection of which requires detailed investigation. HCS-deficiency has to be confirmed by enzyme assay in cultured cells. Both congenital disorders respond clinically and biochemically to oral biotin therapy. Whereas 10 mg/day or less is sufficient to treat profound biotinidase deficiency, the optimal biotin dose for patients with HCS-deficiency must be assessed individually. The prognosis of both disorders is good if biotin therapy is introduced early and continued throughout life. However, delayed commencement of therapy in biotinidase deficiency can result in irreversible neurological damage, and in HCS-deficiency a few patients have responded only partially even to massive biotin doses of up to 100 mg/day.

Detection of biocytin in urine of children with congenital biotinidase deficiency.

The possible excretion of biocytin or biotinyl-peptides was investigated in urines of children with congenital biotinidase deficiency. Treatment of urine samples of affected children with normal plasma (as source of biotinidase) liberated additional free biotin (L. plantarum assay), indicating the presence of biotinyl-compounds. Using thin-layer chromatography the occurrence of biocytin was demonstrated in all urine samples of the affected children. In contrast, no biocytin could be detected in urines of healthy subjects.

Biotin determination by three different methods: specificity and application to urine and plasma ultrafiltrates of patients with and without disorders in biotin metabolism.

A microbiological, an avidin-binding and a streptavidin-binding method for biotin determination were compared. All three methods detected biotin equally well but they exhibit different specificities for derivatives of biotin. The microbiological assay has the highest specificity and is the method of choice for biotin determination in biotinidase-deficient patients. The specificity of streptavidin-binding has not been investigated so far. Application of the three methods to urine samples of patients with and without biotin therapy indicated that only 50% of biotin equivalents measured with the avidin method correspond to authentic biotin as previously shown. The other 50% comprise mainly bisnorbiotin and biotin-d-sulfoxide. HPLC-separation of urine samples prior to assay confirmed this finding and revealed a bisnorbiotin oxidation product and an unknown compound as further biotin metabolites. The latter was measurable by all three methods and not detectable in plasma ultrafiltrate. This was the only metabolite which was able to restore deficient 3-methylcrotonyl-CoA carboxylase activity in biotin-deficient fibroblasts. The combination of the three methods together with HPLC-separation proved to be a valuable analytical tool for the identification of the main biotin metabolites in biological fluids.

Isolated biotin-resistant 3-methylcrotonyl-CoA carboxylase deficiency: long-term outcome in a case with neonatal onset.

UNLABELLED: A patient with early-onset 3-methylcrotonyl coenzyme A carboxylase (MCC) deficiency showing a severe clinical course is described. Abnormal eye and head movements suggestive of seizures were noticed soon after birth. Tonic convulsions at the age of 10 weeks led to admission. Urinary organic acid analysis using gas chromatography-mass spectrometry at 3 months of age revealed elevated concentrations of 3-hydroxyisovaleric acid (3HIVA) and 3-methylcrotonylglycine but normal levels of lactate, 3-hydroxypropionate and methylcitrate suggesting isolated MCC deficiency. This was confirmed by enzyme assays in lymphocytes and cultured skin fibroblasts: MCC activity was virtually undetectable whereas activities of propionyl-CoA and pyruvate carboxylases were within the normal range. A low protein (0.8-1.5 g/kg/day) diet supplemented with a leucine-free amino acid mixture resulted in a marked decrease of 3HIVA excretion. L-Carnitine and biotin administration had no effect on the clinical condition or metabolite excretion. Supplementation with glycine resulted in only a temporary fall of 3HIVA excretion and was therefore discontinued. L-Carnitine therapy was reintroduced later because of secondary carnitine deficiency. Compliance with treatment was poor until the age of 27 months resulting in a severe episode with seizures and coma. The general clinical condition of the patient was always good but his psychomotor development was delayed and seizures were not continuously under good control due to poor therapy compliance. The boy is now 10.5 years old and attending a school for children with learning handicaps. CONCLUSION: Isolated MCC deficiency of early-onset is a rare condition exhibiting a more severe clinical course than the later-onset form described in most other cases. The prognostic value of 3 HIVA measurements in CSF and serum should be evaluated in future cases.

Propionic acidaemia: clinical, biochemical and therapeutic aspects. Experience in 30 patients.

Comprehensive data on 30 patients with propionic acidaemia, diagnosed by selective screening for inborn errors of metabolism, are presented. The most valuable diagnostic metabolites found were methylcitric-, 3-hydroxypropionic-, and 2-methyl-3-oxovaleric acids. Hyperlysinaemia and hyperlysinuria are also characteristic findings in this disease. The metabolic pattern found in propionic acidaemia is discussed extensively as are enzymatic findings. Residual activity of propionyl-CoA carboxylase is neither a predictive marker for severity nor for outcome of the disease. Propionate fixation assays were less reliable for confirmation of propionic acidaemia and of no prognostic value. Clinical presentation of the disease is discussed in detail. Besides the well-known unspecific findings (poor appetite, feeding difficulties, vomiting, dehydration, weight loss, muscular hypotonia, dyspnoea, somnolence, apathy, convulsion, coma, severe metabolic acidosis, hyperammonaemia) various skin abnormalities have been detected in about 50% of all patients. In 27% "dermatitis acidemica" was found.

The gene coding for the alpha-chain of human propionyl-CoA carboxylase maps to chromosome band 13q32.

The human gene encoding the alpha-polypeptide of propionyl-CoA carboxylase (PCC) has hitherto been localized to the distal half of the long arm of chromosome 13, segment 13q22----q34. We studied the enzyme activities of mitochondrial carboxylases in cell cultures obtained from patients with different deletions of chromosome 13. By setting the PCC activity in normal diploid cell cultures (control group) at 100%, cell cultures with trisomy 13 showed 150% activity. In contrast, one of four patients with partial monosomy 13 had an enzyme activity of only 50%. Thus, by comparative deletion mapping, combined with studies of the gene-dosage effect, we have been able to assign the PCCA gene locus to chromosome band 13q32.

Low biotinidase activity in plasma of some preterm infants: possible source of false-positive screening results.

Screening for biotinidase deficiency has been added recently to some national screening programmes. To clarify the problem of false-positive screening tests in premature infants, we have studied biotinidase activities in the plasma of this population in more detail. In 64 newborns (premature and term babies) biotinidase activities correlated positively with gestational age from the 2nd to the 30th day of life. During the 1st-3rd day the activities were below the normal adult range in all 64 infants. In 56 infants the activities subsequently increased gradually and reached the normal adult range during the 4th-40th day of life. In contrast, the biotinidase activities in eight preterm infants dropped during the 3rd-7th day of life. Impaired liver function as a possible cause for this finding could be ruled out in these infants. The lowest activities in these infants were measured during the 4th-6th day of life, i.e. unfortunately at a time when samples for the screening are normally taken. According to our data, 4-8 out of 48 preterm or small-for-date infants with biotinidase activities ranging from 4.7%-26% of the mean adult value would have given false-positive screening tests. A positive screening test was also obtained in a newborn and in an older unrelated child with a partial biotinidase deficiency. In these children the biotinidase activity did not rise but remained slightly below or at the lower range for heterozygotes (at 31% and 38% of the mean adult value). Currently we do not know whether such individuals are heterozygotes, or whether they have a variant of biotinidase deficiency. However, these children have developed normally without biotin therapy.

Comparison of patients with complete and partial biotinidase deficiency: biochemical studies.

Seventeen partially biotinidase-deficient patients detected by neonatal screening or family studies were compared with four patients with classical biotinidase deficiency. Using a sensitive HPLC method for biotinidase in plasma (substrate: biocytin) the patients could be divided into two groups: one with residual biotinidase activity, and the second with undetectable biotinidase activity (0-activity). Biocytin excretion, characteristically elevated in 0-activity patients, decreased rapidly with increasing residual biotinidase activity and was almost normal when residual activity exceeded 2-3% of mean normal. In one patient with classical disease (0-activity) biotin deficiency, typical organic aciduria and multiple carboxylase deficiency were found as early as at the second week of life. In contrast, 13 infants with residual activities from 1.2% to 23% had no remarkable clinical or biochemical abnormalities. However, in three 5-, 14- and 15-year-old healthy siblings with residual biotinidase activities between 2.3% and 4.2%, biotin deficiency was proven by decreased activities of the mitochondrial carboxylases in lymphocytes (30-57% of mean normal) and, in the older siblings, also by subnormal plasma biotin concentrations. In biotinidase deficiency, biotin depletion presumably occurs earlier in the brain than in other tissues and may thus first affect the central nervous system. For this reason and because of discrete biochemical abnormalities found in a patient with residual biotinidase activity of 8%, we suggest that at least all patients with residual activities below 10% should be treated with biotin.

Intestinal absorption and renal excretion of biotin in patients with biotinidase deficiency.

We have investigated four patients from three unrelated families with typical clinical and biochemical features of "late-onset" multiple carboxylase deficiency. All patients suffered from biotinidase deficiency (plasma biotinidase activities 1.4%-3% of normal). Intestinal absorption of biotin, measured in three of the patients using a single load of 1.5 micrograms/kg, was found to be normal. Deficient activities of the mitochondrial biotin-dependent carboxylases in lymphocytes of one of these patients increased from 25% of mean basal control values to 33%-36% within 45 min and to 46%-47% within 2 h of the 1.5 micrograms/kg biotin load. After a high biotin load of 100 micrograms/kg, the values normalised within 45 min in all three patients studied. These results indicate normal cellular transport of biotin and normal holocarboxylase synthesis. After cessation of biotin supplementation, the plasma and urinary biotin in patients decreased to subnormal levels. In one patient, available for more detailed studies, both plasma and urinary biotin declined about twice as fast as in controls (apparent half-life 12-14 h in the patient and 26 h in controls). These results point to increased excretion of free biotin in our patient. Renal loss of biotin is one of the factors contributing to the high biotin requirement observed in patients with biotinidase deficiency.