Prevalence of DDC genotypes in patients with aromatic L-amino acid decarboxylase (AADC) deficiency and in silico prediction of structural protein changes.

Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare autosomal recessive genetic disorder affecting the biosynthesis of dopamine, a precursor of both norepinephrine and epinephrine, and serotonin. Diagnosis is based on the analysis of CSF or plasma metabolites, AADC activity in plasma and genetic testing for variants in the DDC gene. The exact prevalence of AADC deficiency, the number of patients, and the variant and genotype prevalence are not known. Here, we present the DDC variant (n = 143) and genotype (n = 151) prevalence of 348 patients with AADC deficiency, 121 of whom were previously not reported. In addition, we report 26 new DDC variants, classify them according to the ACMG/AMP/ACGS recommendations for pathogenicity and score them based on the predicted structural effect. The splice variant c.714+4A>T, with a founder effect in Taiwan and China, was the most common variant (allele frequency = 32.4%), and c.[714+4A>T];[714+4A>T] was the most common genotype (genotype frequency = 21.3%). Approximately 90% of genotypes had variants classified as pathogenic or likely pathogenic, while 7% had one VUS allele and 3% had two VUS alleles. Only one benign variant was reported. Homozygous and compound heterozygous genotypes were interpreted in terms of AADC protein and categorized as: i) devoid of full-length AADC, ii) bearing one type of AADC homodimeric variant or iii) producing an AADC protein population composed of two homodimeric and one heterodimeric variant. Based on structural features, a score was attributed for all homodimers, and a tentative prediction was advanced for the heterodimer. Almost all AADC protein variants were pathogenic or likely pathogenic.

The novel P330L pathogenic variant of aromatic amino acid decarboxylase maps on the catalytic flexible loop underlying its crucial role.

Aromatic amino acid decarboxylase (AADC) deficiency is a rare monogenic disease, often fatal in the first decade, causing severe intellectual disability, movement disorders and autonomic dysfunction. It is due to mutations in the gene coding for the AADC enzyme responsible for the synthesis of dopamine and serotonin. Using whole exome sequencing, we have identified a novel homozygous c.989C > T (p.Pro330Leu) variant of AADC causing AADC deficiency. Pro330 is part of an essential structural and functional element: the flexible catalytic loop suggested to cover the active site as a lid and properly position the catalytic residues. Our investigations provide evidence that Pro330 concurs in the achievement of an optimal catalytic competence. Through a combination of bioinformatic approaches, dynamic light scattering measurements, limited proteolysis experiments, spectroscopic and in solution analyses, we demonstrate that the substitution of Pro330 with Leu, although not determining gross conformational changes, results in an enzymatic species that is highly affected in catalysis with a decarboxylase catalytic efficiency decreased by 674- and 194-fold for the two aromatic substrates. This defect does not lead to active site structural disassembling, nor to the inability to bind the pyridoxal 5'-phosphate (PLP) cofactor. The molecular basis for the pathogenic effect of this variant is rather due to a mispositioning of the catalytically competent external aldimine intermediate, as corroborated by spectroscopic analyses and pH dependence of the kinetic parameters. Altogether, we determined the structural basis for the severity of the manifestation of AADC deficiency in this patient and discussed the rationale for a precision therapy.

Age and Gender-Related Changes in Biogenic Amine Metabolites in Cerebrospinal Fluid in Children.

Metabolites of cerebrospinal biogenic amines (dopamine and serotonin)are an important tool in clinical research and diagnosis of children with neurotransmitter disorders. In this article we focused on finding relationships between the concentration of biogenic amine metabolites, age, and gender. We analyzed 148 samples from children with drug resistant seizures of unknown etiology and children with mild stable encephalopathy aged 0-18 years. A normal profile of biogenic amineswas found in 107 children and those children were enrolled to the study group. The CSF samples were analyzed by HPLC with an electrochemical detector. The concentrations of the dopamine and serotonin metabolites homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA), respectively, were high at birth, gradually decreasing afterward until the 18 years of age. Nevertheless, the HVA/5-HIAA ratio did not vary with age, except in the children below 1 year of age. In the youngest group we observed a strong relationship between the HVA/5-HIAA ratio and age (r = 0.69, p < 0.001). There were no statistical differences in the level of both dopamine and serotonin metabolites between boys and girls, although a tread toward lower HVA and 5-HIAA in the boys was noticeable. Significant inter-gender differences in the level of HVA and 5-HIAA were noted only in the age-group of 1-4 years, with 5-HIAA being higher in the girls than boys (p = 0.004). In conclusion, the study revealed that the concentration of biogenic amine metabolites is age and sex dependent.

Inherited disorders of brain neurotransmitters: pathogenesis and diagnostic approach.

Neurotransmitters (NTs) play a central role in the efficient communication between neurons necessary for normal functioning of the nervous system. NTs can be divided into two groups: small molecule NTs and larger neuropeptide NTs. Inherited disorders of NTs result from a primary disturbance of NTs metabolism or transport. This group of disorders requires sophisticated diagnostic procedures. In this review we discuss disturbances in the metabolism of tetrahydrobiopterin, biogenic amines, γ-aminobutyric acid, foliate, pyridoxine-dependent enzymes, and also the glycine-dependent encephalopathy. We point to pathologic alterations of proteins involved in synaptic neurotransmission that may cause neurological and psychiatric symptoms. We postulate that synaptic receptors and transporter proteins for neurotransmitters should be investigated in unresolved cases. Patients with inherited neurotransmitters disorders present various clinical presentations such as mental retardation, refractory seizures, pyramidal and extrapyramidal syndromes, impaired locomotor patterns, and progressive encephalopathy. Every patient with suspected inherited neurotransmitter disorder should undergo a structured interview and a careful examination including neurological, biochemical, and imaging.

Sepiapterin reductase deficiency: a treatable mimic of cerebral palsy.

OBJECTIVE: Sepiapterin reductase deficiency (SRD) is an under-recognized levodopa-responsive disorder. We describe clinical, biochemical, and molecular findings in a cohort of patients with this treatable condition. We aim to improve awareness of the phenotype and available diagnostic and therapeutic strategies to reduce delayed diagnosis or misdiagnosis, optimize management, and improve understanding of pathophysiologic mechanisms. METHODS: Forty-three individuals with SRD were identified from 23 international medical centers. The phenotype and treatment response were assessed by chart review using a detailed standardized instrument and by literature review for cases for which records were unavailable. RESULTS: In most cases, motor and language delays, axial hypotonia, dystonia, weakness, oculogyric crises, and diurnal fluctuation of symptoms with sleep benefit become evident in infancy or childhood. Average age of onset is 7 months, with delay to diagnosis of 9.1 years. Misdiagnoses of cerebral palsy (CP) are common. Most patients benefit dramatically from levodopa/carbidopa, often with further improvement with the addition of 5-hydroxytryptophan. Cerebrospinal fluid findings are distinctive. Diagnosis is confirmed by mutation analysis and/or enzyme activity measurement in cultured fibroblasts. INTERPRETATION: Common, clinical findings of SRD, aside from oculogyric crises and diurnal fluctuation, are nonspecific and mimic CP with hypotonia or dystonia. Patients usually improve dramatically with treatment. Consequently, we recommend consideration of SRD not only in patients with levodopa-responsive motor disorders, but also in patients with developmental delays with axial hypotonia, and patients with unexplained or atypical presumed CP. Biochemical investigation of cerebrospinal fluid is the preferred method of initial investigation. Early diagnosis and treatment are recommended to prevent ongoing brain dysfunction.

Dietary Intake and Circulating Amino Acid Concentrations in Relation with Bone Metabolism Markers in Children Following Vegetarian and Omnivorous Diets.

Scientific studies reported that most vegetarians meet the total protein requirements; however, little is known about their amino acid intakes. We aimed to assess dietary intake and serum amino acid levels in relation to bone metabolism markers in prepubertal children on vegetarian and traditional diets. Data from 51 vegetarian and 25 omnivorous children aged 4-9 years were analyzed. Dietary intake of macro- and micronutrients were assessed using the nutritional program Dieta 5®. Serum amino acid analysis was performed using high-pressure liquid chromatography technique, 25-hydroxyvitamin D and parathormone-electrochemiluminescent immunoassay, and bone metabolism markers, albumin, and prealbumin levels using enzyme-linked immunosorbent assay. Vegetarian children had a significantly lower intake of protein and amino acids with median differences of about 30-50% compared to omnivores. Concentrations of four amino acids (valine, lysine, leucine, isoleucine) in serum varied significantly by diet groups and were lower by 10-15% in vegetarians than meat-eaters. Vegetarian children also had lower (p < 0.001) serum albumin levels compared to omnivores. Among bone markers, they had higher (p < 0.05) levels of C-terminal telopeptide of collagen type I (CTX-I) than omnivores. Correlation patterns between amino acids and bone metabolism markers differed in the vegetarian and omnivore groups. Out of bone markers, especially osteoprotegerin was positively correlated with several amino acids, such as tryptophan, alanine, aspartate, glutamine, and serine, and ornithine in vegetarians. Vegetarian children consumed apparently sufficient but lower protein and amino acids compared to omnivores. In circulation these differences were less marked than in the diet. Significantly lower amino acid intake and serum levels of valine, lysine, leucine, and isoleucine as well as the observed correlations between serum amino acids and biochemical bone marker levels indicated the relations between diet, protein quality, and bone metabolism.

Clinical improvement of the aggressive neurobehavioral phenotype in a patient with a deletion of PITX3 and the absence of L-DOPA in the cerebrospinal fluid.

The development of midbrain dopamine (DA) neurons is regulated by several transcription factors, including Nurr1, Wnt1, Lmx1a/1b, En1, En2, Foxa1, Foxa2, and Pitx3. PITX3 is an upstream co-activator of the TH (tyrosine hydroxylase) promoter. Pitx3(-/-) mice have a selective loss of dopaminergic neurons in the substantia nigra and ventral tegmental area, leading to the significantly reduced DA levels in the nigrostriatal pathway and in the dorsal striatum and manifest anomalous striatum-dependent cognitive impairment and neurobehavioral activity. Treatment with L-DOPA, dopamine, or dopamine receptor agonists in these mice reversed several of their sensorimotor impairments. Heterozygous missense mutations in PITX3 have been reported in patients with autosomal dominant congenital cataract and anterior segment (ocular) mesenchymal dysgenesis (ASMD) whereas homozygous missense mutations have been found in patients with microphthalmia and neurological impairment. Using a clinical oligonucleotide array comparative genomic hybridization (aCGH), we have identified an ∼317 kb hemizygous deletion in 10q24.32, involving PITX3 in a 17-year-old male with a Smith-Magenis syndrome-like phenotype, including mild intellectual impairment, sleep disturbance, hyperactivity, and aggressive and self-destructive behavior. Interestingly, no eye anomalies were found in our patient. Analysis of neurotransmitters in his cerebrospinal fluid revealed an absence of L-DOPA and significantly decreased levels of catecholamine metabolites. Importantly, L-DOPA treatment of our patient has led to mild mitigation of his aggressive behavior and mild improvement of his attention span, extended time periods of concentration, and better sleep.

Molecular Background and Disease Prevalence of Biotinidase Deficiency in a Polish Population-Data Based on the National Newborn Screening Programme.

Biotinidase deficiency (BD) is a rare autosomal recessive metabolic disease. Previously the disease was identified only by clinical signs and symptoms, and since recently, it has been included in newborn screening programs (NBS) worldwide, though not commonly. In Europe, BD prevalence varies highly among different countries, e.g., from 1:7 116 in Turkey to 1:75 842 in Switzerland. This paper aimed to present the molecular spectrum of BD (profound and partial forms) in Polish patients diagnosed within the national NBS of 1,071,463 newborns. The initial suspicion of BD was based on an abnormal biotinidase activity result determined in a dry blood spot (DBS) by colorimetric and by fluorimetric methods while biochemical verification was determined by serum biotinidase activity (as quantitative analysis). The final diagnosis of BD was established by serum enzyme activity and the BTD gene direct sequencing. The obtained results allowed for the estimation of disease prevalence (1:66,966 births, while 1:178,577 for profound and 1:107,146 for partial forms), and gave novel data on the molecular etiology of BD.

Urgent metabolic service improves survival in long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency detected by symptomatic identification and pilot newborn screening.

UNLABELLED: Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) is a fatty acid oxidation disorder with especially high mortality and uncertain long-term outcome. The aim of the study was to analyze the influence of diagnostic approach on survival in 59 affected children. Referral to a metabolic center was replaced over time by urine/blood testing in centralized metabolic laboratory (selective screening) and by pilot tandem mass spectrometry newborn screening (NBS). Molecular analysis revealed the prevalent mutation in the HADHA gene in all 58 examined cases. Twenty patients died. The number of detections and number of deaths were respectively 9 and 4 (44%) in the patients recognized by differential diagnosis, 28 and 9 (32%) - by selective screening, and 11 and 1 (9%) - by NBS. In 80% of cases the death occurred before or within 3 weeks from the identification. Urgent and active metabolic service remarkably influenced the surviving. The current age of 39 survivors is 0.5 to 23 yrs (mean 7.2 yrs). The disease frequency estimated on the patients number was 1: 115 450, whereas in the pilot NBS - 1: 109 750 (658 492 neonates tested). Interestingly, the phenylalanine level in asymptomatic neonates frequently exceeded the cut-off values. CONCLUSIONS: 1) Urgent metabolic intervention decreases mortality of LCHAD-deficient patients, but the prognosis is still uncertain. 2) Emergent metabolic reporting and service are crucial also for the survival of neonates detected by NBS. 3) The nationwide selective screening appeared efficient in LCHADD detection in the country. 4) Transient mild hyperphenylalaninaemia may occur in LCHAD-deficient newborns.

Proteins Structure Models in the Evaluation of Novel Variant (C.472_477del) in the MOCS2 Gene.

(1) Background: Molybdenum cofactor deficiency type B (MOCODB, #252160) is a rare autosomal recessive metabolic disorder characterized by intractable seizures of neonatal-onset, muscular spasticity, accompanying with hypouricemia, elevated urinary sulfite levels and craniofacial dysmorphism. Thirty-five patients were reported to date. (2) Methods: Our paper aimed to delineate the disease genotype by presenting another patient, in whom a novel, in-frame variant within the MOCS2 gene was identified. (3) Results: Exome sequencing led to the identification of a novel variant in the MOCS2 gene-c.472_477del of unknown significance (VUS). (4) Conclusions: To prove the clinical significance of the mentioned variant, analysis of the possible mutation consequences on molecular level with the use of the available crystal structure of the human molybdopterin synthase complex was of great importance. Moreover, a potential pathomechanism resulting from a molecular defect was presented, giving original insight into the current knowledge on this rare disease, including treatment options.

Doppler velocimetry of the materno-fetal circulation in preterm delivered pregnancies complicated with hypertension.

BACKGROUND: Hypertension is one of the most frequent complications of pregnancy. Due to high risk of morbidity and mortality in both mothers and children, it is necessary to continuously monitor the pregnancy, principally with biophysical methods. Particularly, doppler velocimetry of the materno-fetal circulation proves useful. THE AIM of the study was to assess the usefulness of doppler test in monitoring the condition of the foetus in preterm delivered pregnancy complicated with hypertension. MATERIAL AND METHODS: The retrospective analysis comprised the data of 116 women who delivered prematurely at the Clinics of the Department of Gynaecology and Obstetrics at the Collegium Medicum of the Jagiellonian University in the years 2006-2007, resulting in creation of Group I involving 38 pregnant women with preeclampsia, and Group II of 36 women whose pregnancy was complicated with gestational hypertension. Control group was formed of 42 women with correct arterial blood pressure. When describing the groups, the differences in the birth weight and Apgar score were indicated. RESULTS: A significant statistical difference was found in the area of pulsation rate in the umbilical artery and cerebro-placental ratio (CPR). In the case of preterm delivery complicated with arterial pressure disorders, the foetus is characterised with worse organic perfusion and slower somatic growth than if no concomitant hypertension is present. Hypertension forms an additional risk factor in the course of preterm delivery, and doppler velocimetry is a good method for monitoring the condition of the foetus, as it allows for detection of irregularities and for implementation of relevant treatment to improve the newborn's condition at birth.

[Feto-placental blood flow--practical remarks]. / Zaburzenia przeplywu lozyskowego--praktyczne zastosowanie kliniczne.

Pregnancy is a physiological state which still needs close observation from the very beginning till the delivery to decrease the potential risk of complications for the mother and her baby. The finding of pathology is possible thanks to advanced diagnostic technology--cardiotocograph and ultrasonography, especially Doppler technique. Monitoring of the fetus is based not only on the routine obstetric examination, but also on the observation and analysis of the fetal heart rate, biometry measurements with expected fetus weight, as well as assessment of blood flow in the fetal, the umbilical and uterine vessels. Assuming that pathology in fetal heart rate can predict intrauterine discomfort, early examination and appropriate reaction to the situation is essential. Early identification of intrauterine growth restriction enables intensive treatment which improves the outcome and reduces perinatal mortality. Information concerning biophysical monitoring of a fetus and basic rules of test result interpretations of are presented in this article.

Clinical, biochemical, neuropathological and molecular findings of the first Polish case of adenylosuccinase deficiency.

Adenylosuccinase (ADSL) deficiency is an autosomal recessive disorder affecting mainly the nervous system. The disease causes psychomotor retardation, frequently with autistic features and epilepsy. ADSL deficiency may be diagnosed by detection of two abnormal metabolites in body fluids--succinyladenosine (S-Ado) and succinylaminoimidazole carboxamide riboside (SAICAr). It is assumed that the former metabolite is neurotoxic. We present clinical, biochemical and neuropathological findings of a child affected by a severe form of ADSL deficiency. She had progressive neurological symptoms that started immediately after birth and died at 2.5 months of age. Macroscopically the brain showed signs of moderate atrophy. Histological examination of all grey matter structures showed widespread damage of neurons accompanied by microspongiosis of neuropile. Cerebral white matter showed lack of myelination in the centrum semiovale and diffuse spongiosis of neuropile. Myelination appropriate for the age was visible in posterior limb of internal capsule, in striatum, thalamus and in brain stem structures but diffuse destruction of myelin sheets was seen with severe marked astroglial reaction with signs of destruction of the cells and their processes. Ultrastructural examination showed enormous destruction of all cellular elements, but astonishingly mitochondria were relatively spared. The neuropathological changes can be considered as the neurotoxic result of metabolic disturbances connected with adenylosuccinase deficiency.

Clinico-pathological correlation in case of BRAT1 mutation.

The clinical picture of BRCA1-associated protein required for ATM activation-1 (BRAT1) comprises retractable early-onset epileptic encephalopathy, progressive microcephaly, and early demise. Both, inter- and intrafamilial variations of features of BRAT1-associated disease have been described. Here, the familial case of a brother and sister with homozygous pathogenic variants in BRAT1 is presented with special emphasis on differences in seizure type/onset and central nervous system lesions. The neuropathology is extensively discussed and hypotheses put forward that may shed light on etiology of brain symptomatology within the context of BRAT1 mutations.

Serum amino acid profile in patients with Parkinson's disease.

Amino acids play numerous roles in the central nervous system, serving as neurotransmitters, neuromodulators and regulators of energy metabolism. The free amino acid profile in serum of Parkinson's disease (PD) patients may be influenced by neurodegeneration, mitochondrial dysfunction, malabsorption in the gastroenteric tract and received treatment. The aim of our study was the evaluation of the profile of amino acid concentrations against disease progression. We assessed the amino acid profile in the serum of 73 patients divided into groups with early PD, late PD with dyskinesia and late PD without dyskinesia. Serum amino acid analysis was performed by high-pressure liquid chromatography with fluorescence detection. We observed some significant differences amongst the groups with respect to concentrations of alanine, arginine, phenylalanine and threonine, although no significant differences were observed between patients with advanced PD with and without dyskinesia. We conclude that this specific amino acid profile could serve as biochemical marker of PD progression.

Adenylosuccinate lyase deficiency: the first identified polish patient.

Adenylosuccinate lyase (ADSL) deficiency is a rare disease of de novo purine synthesis. The main symptoms are psychomotor retardation, epilepsy, autistic features, occasionally associated with muscular hypotonia. Diagnosis is made by detection of abnormal purine metabolites (succinyladenosine - S-Ado and succinylaminoimidazole carboxamide riboside - SAICAr) in body fluids. The severity of the clinical features correlates with low S-Ado/SAICAr ratio. We report clinical, biochemical and brain MRI findings of a female infant with severe early epilepsy and hypotonia, who died at the age of 10 weeks.

Phenotypic features of children with neurodevelopmental diseases in relation to biogenic amines.

Disruption of monoamines metabolism leads to diverse manifestations, including developmental, movement and respiratory dysfunctions. We aimed to correlate clinical phenotypes of 55 children with neurodevelopmental disorders with dopamine (HVA) and serotonin (5-HIIA) metabolites in CSF. Decreased level of at least one metabolite was documented in 49.1% patients. Both metabolites were significantly lower in progressive disorder and extrapyramidal syndrome (p<0.05). HVA was significantly lower in hypokinetic and regulatory disorders (p<0.05). In univariate analysis, only progressive course, extrapyramidal syndrome and dystonia were significantly associated with decreased 5-HIAA. In multivariate regression only progressive course remained significant (p=0.005). Progressive disease, extrapyramidal syndrome, dystonia, tremor and rigidity were positively associated with low HVA. In multivariate analysis only: progressive course and rigidity remained significant. Progressive/rigid phenotype carries a high risk of monoamines deficiency, strongly implying need for their analysis. Psychomotor delay with epilepsy and hypotonia is rarely linked to low monoamines level. Irrespective of final diagnosis, different clinical presentations may be associated with impaired monoamines turnover.

The analysis of genetic aberrations in children with inherited neurometabolic and neurodevelopmental disorders.

Inherited encephalopathies include a broad spectrum of heterogeneous disorders. To provide a correct diagnosis, an integrated approach including genetic testing is warranted. We report seven patients with difficult to diagnose inborn paediatric encephalopathies. The diagnosis could not be attained only by means of clinical and laboratory investigations and MRI. Additional genetic testing was required. Cytogenetics, PCR based tests, and array-based comparative genome hybridization were performed. In 4 patients with impaired language abilities we found the presence of microduplication in the region 16q23.1 affecting two dose-sensitive genes: WWOX (OMIM 605131) and MAF (OMIM 177075) (1 case), an interstitial deletion of the 17p11.2 region (2 patients further diagnosed as Smith-Magenis syndrome), and deletion encompassing first three exons of Myocyte Enhancer Factor gene 2MEF2C (1 case). The two other cases represented progressing dystonia. Characteristic GAG deletion in DYT1 consistently with the diagnosis of torsion dystonia was confirmed in 1 case. Last enrolled patient presented with clinical picture consistent with Krabbe disease confirmed by finding of two pathogenic variants of GALC gene and the absence of mutations in PSAP. The integrated diagnostic approach including genetic testing in selected examples of complicated hereditary diseases of the brain is largely discussed in this paper.

Behavioral and intellectual functioning in patients with tyrosinemia type I.

INTRODUCTION: In tyrosinemia type I (TT1) increased level of tyrosine and phenylalanine (both precursors of neurotransmitters), may potentially influence patients' cognitive development. AIM OF THE STUDY: Was to evaluate if the children during the treatment with phenylalanine- and tyrosine-restricted diet and nitisinone present with cognitive, emotional or behavioral problems and to find out whether plasma tyrosine and phenylalanine levels may have impact on this. MATERIAL AND METHODS: Cognitive development and behavior, together with plasma tyrosine and phenylalanine levels, were analyzed in eight patients during their first five years of nitisinone treatment. Psychological examination has been done using standard diagnostic methods: the Wechsler Intelligence Scale for Children (WISC-R) and Child Behavior Checklist CBCL/4-18 (parents version). RESULTS: The results showed that in the patients with TT1, attention deficit is not rare, and may be connected with the variation of the plasma tyrosine level. Moreover the reverse correlation between attention deficit and results from verbal scale may suggest decreased ability to verbal reasoning, comprehension, verbal expression and school difficulties. CONCLUSIONS: What is significant for the presence of attention disorders and the related difficulties in using the intellectual potential is not the level of tyrosine (high vs. low), but its changes (stability vs. instability). Therapeutic trials to stabilize the tyrosine level could alleviate the difficulties in focusing attention. Following a diet is necessary for keeping the normal level of tyrosine.