Tetrahydrobiopterin deficiencies without hyperphenylalaninemia: diagnosis and genetics of dopa-responsive dystonia and sepiapterin reductase deficiency

DOPA responsive dystonia (DRD) and sepiapterin reductase (SR) deficiency are inherited disorders of tetrahydrobiopterin (BH4) metabolism characterized by the signs and symptoms related to monoamine neurotransmitter deficiency. In contrast to classical forms of BH4 deficiency DRD and SR deficiency present without hyperphenylalaninemia and thus cannot be detected by the neonatal screening for phenylketonuria (PKU). While DRD is mostly caused by autosomal dominant mutations in the GTP cyclohydrolase I gene (GCH1), SR deficiency is an autosomal recessive disease. The most important biochemical investigations for the diagnosis of these neurological diseases includes CSF investigations for neurotransmitter metabolites and pterins as well as neopterin and biopterin production in cytokine-stimulated fibroblasts. Discovery of SR deficiency opened new insights into alternative pathways of the cofactor BH4 via carbonyl, aldose, and dihydrofolate reductases. As a consequence of the low dihydrofolate reductase activity in the brain, dihydrobiopterin intermediate accumulates and inhibits tyrosine and tryptophan hydroxylases and uncouples nitric oxide synthase (nNOS), leading to neurotransmitter deficiency and possibly also to neuronal cell death.     

Detection of sepiapterin in CSF of patients with sepiapterin reductase deficiency

Sepiapterin reductase (SR) deficiency was recently described in patients with a severe biogenic amine deficiency presenting without hyperphenylalaninemia and it was suggested that the tetrahydrobiopterin (BH(4)) pathway may be different in different cells and tissues. We now developed a HPLC method for the measurement of yellow fluorescing sepiapterin for the rapid diagnosis of SR deficiency. Sepiapterin was elevated in CSF from two patients with SR deficiency (5.6 and 11.4 nmol/L) when compared with healthy controls (<0.5 nmol/L). Our data further support the hypothesis that sepiapterin is an intermediate in the salvage pathway of BH(4) and that it accumulates in the brain of patients with SR deficiency.     

Mutations in the BH4-metabolizing genes GTP cyclohydrolase I, 6-pyruvoyl-tetrahydropterin synthase, sepiapterin reductase, carbinolamine-4a-dehydratase, and dihydropteridine reductase

Tetrahydrobiopterin (BH(4)) deficiencies are a highly heterogeneous group of disorders with several hundred patients, and so far a total of 193 different mutant alleles or molecular lesions identified in the GTP cyclohydrolase I (GTPCH), 6-pyruvoyl-tetrahydropterin synthase (PTPS), sepiapterin reductase (SR), carbinolamine-4a-dehydratase (PCD), or dihydropteridine reductase (DHPR) genes. The spectrum of mutations causing a reduction in one of the three biosynthetic (GTPCH, PTPS, and SR) or the two regenerating enzymes (PCD and DHPR) is tabulated and reviewed. Furthermore, current genomic variations or SNPs are also compiled. Mutations in GCH1 are scattered over the entire gene, and only 5 out of 104 mutant alleles, present in a homozygous state, are reported to cause the autosomal recessive form of inheritable hyperphenylalaninemia (HPA) associated with monoamine neurotransmitter deficiency. Almost all other 99 different mutant alleles in GCH1 are observed together with a wild-type allele and cause Dopa-responsive dystonia (DRD, Segawa disease) in a dominant fashion with reduced penetrance. Compound heterozygous or homozygous mutations are spread over the entire genes for PTS with 44 mutant alleles, for PCBD with nine mutant alleles, and for QDPR with 29 mutant alleles. These mutations cause an autosomal recessive inherited form of HPA, mostly accompanied by a deficiency of the neurotransmitters dopamine and serotonin. Lack of sepiapterin reductase activity, an autosomal recessive variant of BH(4) deficiency presenting without HPA, was diagnosed in patients with seven different mutant alleles in the SPR gene in exons 2 or 3 or in intron 2. Details on all mutations presented here are constantly updated in the BIOMDB database (www.bh4.org).     

Whole-genome sequencing for optimized patient management

Whole-genome sequencing of patient DNA can facilitate diagnosis of a disease, but its potential for guiding treatment has been under-realized. We interrogated the complete genome sequences of a 14-year-old fraternal twin pair diagnosed with dopa (3,4-dihydroxyphenylalanine)-responsive dystonia (DRD; Mendelian Inheritance in Man #128230). DRD is a genetically heterogeneous and clinically complex movement disorder that is usually treated with l-dopa, a precursor of the neurotransmitter dopamine. Whole-genome sequencing identified compound heterozygous mutations in the SPR gene encoding sepiapterin reductase. Disruption of SPR causes a decrease in tetrahydrobiopterin, a cofactor required for the hydroxylase enzymes that synthesize the neurotransmitters dopamine and serotonin. Supplementation of l-dopa therapy with 5-hydroxytryptophan, a serotonin precursor, resulted in clinical improvements in both twins.     

Molecular and metabolic bases of tetrahydrobiopterin (BH4) deficiencies

Tetrahydrobiopterin (BH₄) deficiency is caused by genetic variants in the three genes involved in de novo cofactor biosynthesis, GTP cyclohydrolase I (GTPCH/GCH1), 6-pyruvoyl-tetrahydropterin synthase (PTPS/PTS), sepiapterin reductase (SR/SPR), and the two genes involved in cofactor recycling, carbinolamine-4α-dehydratase (PCD/PCBD1) and dihydropteridine reductase (DHPR/QDPR). Dysfunction in BH₄ metabolism leads to reduced cofactor levels and may result in systemic hyperphenylalaninemia and/or neurological sequelae due to secondary deficiency in monoamine neurotransmitters in the central nervous system. More than 1100 patients with BH₄ deficiency and 800 different allelic variants distributed throughout the individual genes are tabulated in database of pediatric neurotransmitter disorders PNDdb. Here we provide an update on the molecular-genetic analysis and structural considerations of these variants, including the clinical courses of the genotypes. From a total of 324 alleles, 11 are associated with the autosomal recessive form of GTPCH deficiency presenting with hyperphenylalaninemia (HPA) and neurotransmitter deficiency, 295 GCH1 variant alleles are detected in the dominant form of L-dopa-responsive dystonia (DRD or Segawa disease) while phenotypes of 18 alleles remained undefined. Autosomal recessive variants observed in the PTS (199 variants), PCBD1 (32 variants), and QDPR (141 variants) genes lead to HPA concomitant with central monoamine neurotransmitter deficiency, while SPR deficiency (104 variants) presents without hyperphenylalaninemia. The clinical impact of reported variants is essential for genetic counseling and important for development of precision medicine.     

Pitfalls in phenylalanine loading test in the diagnosis of dopa-responsive dystonia

Phenylalanine (Phe) loading test is a useful tool in the differential diagnosis of dopa-responsive dystonia due to autosomal dominant or recessive GTP cyclohydrolase I (GTPCH) deficiency or autosomal recessive sepiapterin reductase (SR) deficiency. In these patients hepatic phenylalanine hydroxylase system is compromised due to subnormal tetrahydrobiopterin (BH₄) levels and hydroxylation of phenylalanine (Phe) to tyrosine (Tyr) is reduced with elevated Phe/Tyr ratio 1–2 h after oral Phe administration (100 mg/kg bw) administration. In healthy persons there is only a modest increase in Tyr production and blood Phe normalizes after 4 h. We report on a challenge with Phe (100 mg/kg bw) in a patient with dopa-responsive dystonia while on therapy with BH₄ and l-dopa. During Phe challenge Phe concentration remained below the normal range while a transient mild hypertyrosinemia was observed, leading to an extremely low Phe/Tyr ratio. A repeated test, after BH₄ withdrawal, reversed the findings and resulted normal. These data suggest activation of hepatic phenylalanine hydroxylase by BH₄. Thus, the Phe loading test should not be performed during substitution with BH₄.     

甲基丙二酸尿症一家系MMACHC基因突变分析

目的 研究甲基丙二酸尿症(methymalonic aciduria,MMA)MMACHC基冈的突变.方法 应用聚合酶链反应及DNA直接测序技术对甲基丙二酸尿症一家系进行MMACHC基因突变位点检测,并与50名健康人的MMACHC基冈进行对照.结果 患者及其父亲的MMACHC基因中检测到一个新的整码突变,MMACHC基因第2外显子146_154缺失CCTTCCTGG,导致P.49_51位缺失丙氨酸苯丙氨酸亮氨酸(AFL),50名对照者的等位基因无此突变.结论 MMACHC基因的146_154缺失CCTTCCTGG也可能是该家系引起甲基丙二酸尿症的病因.     

Dopa-responsive dystonia: a syndrome of selective nigrostriatal dopaminergic deficiency.

Dopa-responsive dystonia (DRD) is no longer a rare oddity. For the clinician, DRD poses a diagnostic challenge as its clinical presentation can be quite diverse. Marked and sustained response to L-dopa is the most crucial and absolute hallmark in confirming a diagnosis. Absence of degenerative nigral cell loss underlies the remarkable L-dopa response. The broadening spectrum of the clinical presentations, progress in molecular genetics with evidence of incomplete penetrance and phenotypic variability, biochemistry, utility of nuclear imaging in differential diagnosis, and treatment are discussed. I propose the concept of DRD as a syndrome, defined as selective nigrostriatal dopamine deficiency caused by genetic defects in dopamine synthesis without degenerative cell loss. I further propose the term DRD-plus, defined as inherited metabolic disorders which have symptomatic features of DRD, and those features not seen in DRD as well.     

Autosomal recessive GTP cyclohydrolase I deficiency without hyperphenylalaninemia: evidence of a phenotypic continuum between dominant and recessive forms

We describe a unique presentation of autosomal recessive (AR) GTP cyclohydrolase I (GTPCH) deficiency, with severe CNS involvement but without hyperphenylalaninemia. A male infant presented with progressive spasticity, dystonia and oculogyric episodes. Blood phenylalanine levels were persistently normal: whereas an oral phenylalanine loading test revealed impaired phenylalanine clearance. CSF neopterin and tetrahydrobiopterin (BH(4)) were low, homovanillic acid marginally low and 5-hydroxyindoleacetic acid normal. Fibroblasts showed decreased GTPCH enzyme activity. A homozygous novel mutation of GCH1, p.V206A, was identified. On treatment (BH(4), L-Dopa/Carbidopa and 5-hydroxytryptophan), motor development improved. Mutational analysis provided neonatal diagnosis of a younger brother who, after 18 months on treatment, shows normal development. AR GTPCH I deficiency can present without hyperphenylalaninemia and with normal or subtle CSF neurotransmitter profiles. Testing for GTPCH deficiency should be considered for patients with unexplained neurological symptoms and extrapyramidal movement disorder.     

Unusually mild phenotype of AADC deficiency in 2 siblings

Aromatic L-amino acid decarboxylase deficiency is a rare neurotransmitter defect leading to serotonin, dopamine and norepinephrine deficiency. Affected individuals usually present in infancy with severe developmental delay, oculogyric crises and extrapyramidal movements. We present the clinical, molecular and biochemical features of a pair of siblings who presented with fatigability, hypersomnolence and dystonia and who showed excellent response to treatment. Analysis of CSF biogenic amines, plasma AADC levels and direct sequencing of the DDC gene was performed. CSF catecholamine metabolites were reduced, with elevation of 3-O-methyldopa. Plasma AADC activity was undetectable in both siblings, and decreased in their carrier parents. One missense mutation (853C>T) was found in exon 8, and a donor splice site mutation was found in the intron after exon 6 (IVS6+4A>T). Both siblings showed excellent response to MAO inhibitor and dopamine agonist treatment. This report expands the clinical spectrum of AADC deficiency and contributes to the knowledge of the genotype and phenotype correlation for the DDC gene. It is important to recognize the milder phenotypes of the disease as these patients might respond well to therapy.     

Homocystinuria and megaloblastic anemia responsive to vitamin B12 therapy. An inborn error of metabolism due to a defect in cobalamin metabolism

We describe an inborn error of vitamin B12 metabolism in an infant who had severe developmental delay, megaloblastic anemia, and homocystinuria. There was no evidence of methylmalonic aciduria or deficiency of folate or vitamin B12. Treatment with hydroxocobalamin, but not with cyanocobalamin and folic acid, resulted in rapid clinical and biochemical improvement. Cultured fibroblasts showed an absolute growth requirement for methionine, defective incorporation of radioactivity from [14C]5-methyltetrahydrofolate into protein, and normal incorporation of radioactivity from [14C]propionate, thus assigning the intracellular defect to methionine synthesis. The proportion of intracellular methylcobalamin in the fibroblasts was decreased, but that of 5'-deoxyadenosylcobalamin was normal. Methionine synthetase activity in cell extracts was normal, as was cobalamin incorporation into cultured cells. This defect differs from those described previously in being limited to methylcobalamin accumulation and defective use of 5-methyltetrahydrofolate by intact cells with normal activity of methylmalonyl CoA mutase.     

Diagnosis and treatment of methylmalonic aciduria: a case report

The methylmalonic aciduria is an organic acidemia, inherited as autosomic recessive trait, caused by a deficiency of the methylmalonyl-CoA mutase, or by defects in the biosynthesis of the cofactor adenosylcobalamin. Regarding the enzymatic defect, there are two forms: mut(o) with no detectable enzymatic activity and mut(-) with reduced activity. Its clinical presentation may vary from a severe neonatal form with acidosis and death, up to a progressive chronic form. Here we describe the case of a four year-old boy, with diagnosis of methylmalonyl-CoA mutase deficiency type mut(-) with an acute presentation. Molecular analysis of MUT gene identified two mutations c.607G>A (G203R) and c.2080C>T (R694W), later confirmed in the parents. The aim of this report is to highlight the importance of including the organic acid analysis in urine among the first line exams in acutely and severely ill children with undefined etiology. The definitive diagnosis is important because it may allow a specific treatment and a favorable evolution to prevent the secuelae.     

Genetic and biochemical approach to early prenatal diagnosis in a family with mut methylmalonic aciduria

Genetic and biochemical prenatal diagnosis was performed at 11 weeks of gestation in a family with a proband affected by mut methylmalonic aciduria (MMA) and homozygotes for the MUT gene c.643G>A (p.Gly215Ser) mutation. Both chorionic villus and amniotic fluid samples were used. The presence of high levels of methylmalonic acid and propionylcarnitine determined by gas chromatography/mass spectrometry and LC/MS/MS analysis, respectively, and the identification of the p.Gly215Ser at a homozygous level in foetal DNA allowed a certain, rapid and early diagnosis. To our knowledge, this is the first mut MMA prenatal diagnosis carried out by genetic and biochemical approach.     

Homozygous nonsense mutation in the MCEE gene and siRNA suppression of methylmalonyl-CoA epimerase expression: a novel cause of mild methylmalonic aciduria

Methylmalonyl-CoA epimerase (MCE) catalyzes the interconversion of D- and L-methylmalonyl-CoA in the pathway responsible for the degradation of branched chain amino acids, odd chain-length fatty acids, and other metabolites. Despite the occurrence of metabolic disorders in the enzymatic step occurring immediately upstream of MCE (propionyl-CoA carboxylase) and downstream of MCE (adenosylcobalamin-dependent methylmalonyl-CoA mutase), no disease-causing mutations have been described affecting MCE itself. A patient, formerly identified as belonging to the cblA complementation group of vitamin B12 disorders but lacking mutations in the affected gene, MMAA, was tested for mutations in the MCEE gene. The patient's fibroblasts had normal levels of adenosylcobalamin compared to controls, whereas other cblA cell lines typically had reduced levels of the cofactor. As well, this patient had a milder form of methylmalonic aciduria than usually observed in cblA patients. The patient was found to be homozygous for a c.139C>T (p.R47X) mutation in MCEE by sequence analysis that was confirmed by restriction digestion of PCR products. One sibling, also with mild methylmalonic aciduria, was homozygous for the mutation. Both parents and one other sibling were heterozygous. A nearby insertion polymorphism, c.41-160_161insT, heterozygous in both parents, showed the wild-type configuration on the mutant alleles. To assess the impact of isolated MCE deficiency in cultured cells, HeLa cells were transfected with a selectable vector containing MCEE-specific small interfering RNA (siRNA) to suppress gene expression. The reduced level of MCEE mRNA resulted in the reduction of [14C]-propionate incorporation into cellular macromolecules. However, siRNA only led to a small reduction in pathway activity, suggesting that previously postulated non-enzymatic conversion of D- to L-methylmalonyl-CoA may contribute to some flux through the pathway. We conclude that the patient's MCEE defect was responsible for the mild methylmalonic aciduria, confirming a partial requirement for the enzymatic activity in humans.     

Motor abnormalities during sleep in patients with childhood hereditary progressive dystonia, and their unaffected family members

The structure of sleep and number of body movements (BMS) and periodic leg movements during sleep (PMS), were studied in three unrelated girls suffering from L-DOPA responsive hereditary dystonia with marked diurnal fluctuation and in their 11 healthy, close relatives. All three girls had an increased number of BMS during rapid eye movement (REM) sleep. Five of the six parents and three siblings had abnormal PMS. One pair of parents had BMS similar to those of their affected daughter. The occurrence of BMS and PMS in the families studied may indicate a common mechanism for both. Because familial PMS is quite rare in its pure form, and this type of dystonia is also rarely encountered, the occurrence of BMS and PMS in members of these families may imply a causative relation between these two sleep-related motor phenomena.     

Dopa-responsive dystonia with a novel initiation codon mutation in the GCH1 gene misdiagnosed as cerebral palsy

Dopa-responsive dystonia (DRD) is a clinical syndrome characterized by childhood-onset dystonia and a dramatic response to relatively low doses of levodopa. However, patients with DRD can be misdiagnosed as cerebral palsy or spastic diplegia due to phenotypic variation. Here we report a young woman with DRD who were severely disabled and misdiagnosed as cerebral palsy for over 10 yr. A small dose of levodopa restored wheelchair-bound state to normality. However, thoracolumbar scoliosis has remained as a sequel due to late detection of DRD. Genetic analysis by using PCR-direct sequencing revealed a novel initiation codon mutation (c.1A>T; p.Met1Leu) in GTP cyclohydrolase 1 (GCH1) gene. Although it is known that DRD can be misdiagnosed as cerebral palsy, this case reinforces the importance of differential diagnosis of DRD from cerebral palsy.     

儿童运动及智能障碍者四氢生物蝶呤代谢病筛查及基因研究

目的探讨四氢生物蝶呤（tetrahydrobiopterin，BH4）代谢中各酶缺乏在儿童运动及智能发育障碍者中发生率及基因突变。方法对100例运动及智能障碍患者进行苯丙氨酸（phenylalanine，Phe）及BH4负荷试验、尿蝶呤谱分析、红细胞二氢蝶啶还原酶测定，并对部分患者进行多巴治疗性诊断；对诊断为多巴反应性肌张力障碍（dopa-responsive dystonia，DRD）及6-丙酮酰四氢蝶呤合成酶（6-pyruvoyl tetrahydropterin synthase，PTS）缺乏者做基因突变检测。结果100例中70例基础血Phe浓度正常，6例（6％）诊断为DRD；30例有高苯丙氨酸血症[Phe（1022±290）μmol／L]，8例（8％）诊断为VIS缺乏症，22例（22％）诊断为苯丙氨酸羟化酶缺乏症。发现2例DRD患者其三磷酸鸟苷环化酶基因（GTP cyclohydrolase 1 gene，GCHI）突变为IVS5＋3insT，8例FIS缺乏症患者存在PTS基因7种突变类型，其中259C→T，286G→A，155A→G最常见，占75％。结论一些肌张力障碍或智能障碍者是由于BH4代谢障碍所致，有必要做筛查诊断以明确诊断。     

Cor pulmonale as a complication of methylmalonic acidemia and homocystinuria (Cb1-C type)

We report an infant with a bronchiolitis-like illness and rapid deterioration who developed a cor pulmonale-like picture with a dilated right ventricle. Urinary organic acid assays established a probable diagnosis of Cbl-C-type methylmalonic aciduria, later confirmed by complementation studies. Despite medical intervention and cyanocobalamin treatment the patient died on his tenth hospital day. Postmortem examination showed the presence of thromboemboli in the pulmonary circulation. We hypothesize that acute cor pulmonale developed in this infant secondary to thromboembolism of his pulmonary circulation. A review of the literature shows that thromboembolism may be a part of this disease process.     

Defective lysosomal release of vitamin B12 (cb1F): a hereditary cobalamin metabolic disorder associated with sudden death

Here we report on a girl who presented with failure to thrive, developmental delay, minor facial anomalies, stomatitis, skin rashes, macrocytosis, mild homocystinemia(uria), and methylmalonic acidemia(uria). Fibroblast studies showed abnormal intracellular cobalamin (vitamin B12) metabolism. Reduced incorporation of 14C from [14C] propionate and [14C] methyltetrahydrofolate into TCA-precipitable macromolecules reflected decreased synthesis of adenosylcobalamin and methylcobalamin respectively. The diagnosis of cb1F mutation was established by demonstrating the accumulation of unmetabolized free cyanocobalamin in fibroblasts and by lack of genetic complementation with fibroblasts from the only other known cb1F patient. The defect is in the lysosomal release of endocytosed cobalamin. Administration of hydroxocobalamin resulted in clinical and biochemical improvement but sudden death occurred at age 5 months. The absence of brain pathological changes suggests that early treatment may prevent the neurological complications in cobalamin cofactor deficiency.