Ovarian Cyst Fluid of Serous Ovarian Tumors Contains Large Quantities of the Brain Amino Acid N-acetylaspartate

BACKGROUND: In humans, N-acetyl L-aspartate (NAA) has not been detected in other tissues than the brain. The physiological function of NAA is yet undefined. Recently, it has been suggested that NAA may function as a molecular water pump, responsible for the removal of large amounts of water from the human brain. Ovarian tumors typically present as large cystic masses with considerable fluid accumulation. METHODOLOGY AND PRINCIPAL FINDINGS: Using Gas Chromatography-Mass Spectrometry, we demonstrated that NAA was present in a high micromolar concentration in oCF of epithelial ovarian tumors (EOTs) of serous histology, sometimes in the same range as found in the extracellular space of the human brain. In contrast, oCF of EOTs with a mucinous, endometrioid and clear cell histological subtype contained a low micromolar concentration of NAA. Serous EOTs have a cellular differentiation pattern which resembles the lining of the fallopian tube and differs from the other histological subtypes. The NAA concentration in two samples of fluid accumulation in the fallopian tube (hydrosalpinx) was in the same ranges as NAA found in oCF of serous EOTs. The NAA concentration in oCF of patients with serous EOTs was mostly 10 to 50 fold higher than their normal serum NAA concentration, whereas in patients with other EOT subtypes, serum and cyst fluid NAA concentration was comparable. CONCLUSIONS AND SIGNIFICANCE: The high concentration of NAA in cyst fluid of serous EOTs and low serum concentrations of NAA in these patients, suggest a local production of NAA in serous EOTs. Our findings provide the first identification of NAA concentrations high enough to suggest local production outside the human brain. Our findings contribute to the ongoing research understanding the physiological function of NAA in the human body

Mултисистемные поражение у детей: синдром NARP – митохондриальная болезнь (клинический случай)

Institute of Mother and Child, Chisinau, Republic of Moldova, Translational Metabolic Laboratory, Radboudumc, Nijmegen,Netherland, Institute of Psyhiology and Sancreatology, of Academy of Science, Chisinau, Republic of Moldova, State University of Medicine and Pharmacy, ”Nicolae Testemițanu”, Chisinau, Republic of MoldovaIntroducere. Maladiile mitocondriale fac parte din grupul tulburărilor neurodegenerative cauzate de dereglările ale generării energiei mitocondriale celulare. Sindromul NARP (neuropatie, ataxie și retinită pigmentară) este determinat de mutații punctiforme în gena MT-ATP6 din cadrul ADN-ului mitocondrial și este caracterizat prin variabilitatea manifestărilor clinice. Transmiterea mutației se realizează pe cale maternă, iar incidența este 1: 12.000 nașterii. Gravitatea manifestărilor clinice este asociată cu gradul de heteroplasmie a mutației ce determină patologia în cauză. Materiale și metode. Se raportează cazul unei fetițe, născută la termen, care s-a dezvoltat normal până la vârsta de 3 luni, însă ulterior a dezvoltat hipotonie, deficit ponderal, retard psiho-motor sever, dificultăți de alimentare, convulsii rezistente la terapie anticonvulsivantă, atrofie parțială a nervului optic și hepatomegalie. Investigațiile metabolice de bază, RMN-ul cerebral și analiza molecular-genetică au fost utilizate pentru diagnosticul patologiei mitocondriale. Rezultate. Luând în considerarea afectarea multisistemică și prezența manifestărilor clinice preponderent neurologice, pacientul a fost suspectat pentru o eroare înnăscută de metabolism. Inițial, considerând prezența hipotoniei progresive marcate ca simptom clinic cheie, s-a exclus Atrofia musculară spinală. În același timp a fost inițiat work-up-ul metabolic, dezvăluindu-se schimbări relevante pentru o maladie mitocondrială în sânge: hiperlactatacidemie [lactat 3.7-7.8 mmol/L, x 3 ori la rând, val. ref. 0.7-2.1 mmol/L], hiperalaninemie Ala [1038, val. ref. < 450 μmol/L], raportul Ala/Lys [11.8, abnormal dacă >3] și în urină: hiperaminoacidurie parțială. RMN cerebral (3,0T) – focare patologice la nivelul nucleilor bazali bilaterali. În urma sumării manifestărilor clinice și paraclinice s-au obținut 8 puncte ca scor specific pentru maladie mitocondrială definită (Criteriile Nijmegen). Nu a fost efectuată biopsia musculară, ca test confirmativ de diagnostic. Copilul a decedat la 9 luni de viață, iar diagnosticul molecular genetic la nivelul ADN-ului mitocondrial a fost efectuat postmortem în RadboudUMC (Nijmegen, Olanda), determinându-se mutația punctiformă m.8993T>G (Leu156Arg), cunoscută ca fiind determinantă în dezvoltarea sindromului NARP. Nu a fost posibilă aprecierea heteroplasmiei. Discuții. Sindromul NARP se caracterizează printr-o varietate de simptome și semne clinice cu preponderent de afectare neurologică. Diagnosticul acestei patologii deseori reprezintă o provocare pentru clinicieni determinată heterogenitate clinică care se suprapune cu alte maladii genetice. Debutul și evoluția simptomelor clinice depinde de gradul de heteroplasmie a mutației. Algoritmul de diagnosticul include inițierea work-upului metabolic, efectuarea RMN cerebral, biopsia musculară și determinarea mutației la nivel molecular genetic. Managmentul terapeutic rămâne a fi simptomatic pentru îmbunătățirea calității vieții pacientului. Concluzii. Debutul precoce, polimorfismul manifestărilor clinice, cum ar fi afectarea sistemului nervos central, slăbiciunea musculară, retardul psihomotor și convulsii în cazul unui copil ar trebui să determine clinicianul să ia în considerare sindromul NARP cu efectuarea investigațiilor suplimentare, cum ar fi măsurarea acidului lactic în sânge, efectuarea electromiografiei, rezonanței magnetice nucleare și testarea genetică.Introduction. Mitochondrial diseases are part of the group of neurodegenerative disorders caused by disruptions of cellular mitochondrial energy generation. NARP syndrome (Neurogenic weakness, ataxia, and retinitis pigmentosa) is caused by point mutations in the MT-ATP6 gene in mitochondrial DNA and is characterized by variability in clinical manifestations. The mutation is transmitted maternally, and the incidence is 1: 12,000 births. The severity of clinical manifestations is associated with the degree of heteroplasmia of the disease-causing mutation. Material and methods. We report on a case of a girl, born at term, who developed normally until the age of 3 months, but later developed hypotonia, weight deficit, severe psychomotor retardation, eating difficulties, seizures resistant to anticonvulsant therapy, partial atrophy of the optic nerve and hepatomegaly. Basic metabolic investigations, brain MRI and molecular-genetic analysis were used to diagnose mitochondrial pathology. Results. Considering the multisystemic impairment and the presence of predominantly neurological clinical manifestations, the patient was suspected of an innate metabolic error. Initially, considering the presence of marked progressive hypotonia as a key clinical symptom, spinal muscular atrophy was excluded. At the same time, the metabolic work-up was initiated, revealing relevant changes for a mitochondrial disease in the blood: hyperlactatacidemia [lactate 3.7-7.8 mmol/L, x 3 times in a row, ref. val. 0.7-2.1mmol / l], hyperalaninemia Ala [1038, ref. val. <450 μmol/L], Ala/ Lys ratio [11.8, abnormal if >3] and in urine: partial hyperaminoaciduria. Brain MRI (3.0T) – pathological foci in the bilateral basal nuclei. Following the summation of clinical and paraclinical manifestations, 8 points were obtained as a specific score for defined mitochondrial disease (Nijmegen Criteria). No muscle biopsy was performed as a confirmatory diagnostic test. The child died at 9 months of age, and the genetic molecular diagnosis of mitochondrial DNA was performed postmortem in RadboudUMC (Nijmegen, Netherlands), determining the point mutation m.8993T> G (Leu156Arg), known to be decisive in development of NARP syndrome. It was not possible to assess level of heteroplasmy. Discussions. NARP syndrome is characterized by a variety of symptoms and clinical signs, with predominantly neurological impairment. Diagnosis of this pathology is often a challenge for clinicians due to clinical heterogeneity that overlaps with other genetic diseases. The onset and development of clinical symptoms depends on the degree of heteroplasmic mutation. The diagnostic algorithm includes performing of metabolic work-up, brain MRI, muscle biopsy and genetic analysis. Therapeutic treatment is symptomatic and supportive of improving the patient’s quality of life. Conclusions. Early onset in the presence of complete health, the polymorphism of clinical manifestations, such as a central nervous system lesion, muscle weakness, impaired psychomotor development, and seizures in a child should prompt the clinician to consider NARP syndrome and conduct further investigations such as measurement of blood lactate, performing electromyography, magnetic resonance imaging, and genetic analysis.Введение. Митохондриальные заболевания относятся к группе нейродегенеративных заболеваний, вызванных нарушением выработки клеточной митохондриальной энергии. синдром NARP (Невропатия, атаксия, пигментная дегенерация сетчатки) вызывается точечными мутациями в гене MT-ATP6 в митохондриальной ДНК и характеризуется вариабельностью клинических проявлений. Мутаций передаются от матери, частота встречаемости составляет 1:12 000 рождении. Выраженность клинических проявлений связана со степенью гетероплазмии болезнетворной мутации.Материалы и методы. Мы сообщаем о клиническом случае доношенной девочке, которая нормально развивалась до 3 месяцев, но позже у нее развилась гипотония, дефицит веса, тяжелая психомоторная отсталость, трудности с питанием, судороги, устойчивые к противосудорожной терапии, частичная атрофия оптических нервов и гепатомегалия. Для диагностики митохондриальной патологии использовались базовые метаболические исследования, МРТ головного мозга и молекулярно-генетический анализ. Результаты. Учитывая мультисистемное нарушение и наличие преимущественно неврологических клинических проявлений, у пациента заподозрили врожденную метаболическую ошибку. Первоначально, учитывая наличие выраженной прогрессирующей гипотонии как ключевой клинический симптом, спинальная мышечная атрофия была исключена. В то же время, было начато исследование метаболизма, выявившее соответствующие изменения для митохондриального заболевания в крови: гиперлактатацидемия [лактат 3.7-7.8 ммоль/л, x 3 раза подряд, референсные значения 0.7-2.1 ммоль / л], гипераланинемия Ala [1038, референсные значения 3] и в моче: частичная гипераминоацидурия. МРТ головного мозга (3.0Т) – патологические очаги в двухсторонних базальных ядрах. После суммирования клинических и параклинических проявлений, было получено 8 баллов в качестве специфической оценки для определенного митохондриального заболевания (Неймегенские критерии). Биопсия мышц в качестве подтверждающего диагностического теста не проводилась. Ребенок умер в возрасте 9 месяцев, и генетическая молекулярная диагностика митохондриальной ДНК была проведена посмертно в RadboudUMC (Неймеген, Нидерланды), определив точечную мутацию m.8993T>G (Leu156Arg), которая, как известно, является решающей в развитии синдрома NARP. Оценка уровня гетероплазмии не удалось. Обсуждения. Синдром НАРП характеризуется множеством симптомов и клинических признаков с преимущественно неврологическими нарушениями. Диагностика этой патологии часто представляет собой проблему для клиницистов из-за клинической неоднородности, которая частично совпадает с другими генетическими заболеваниями. Возникновение и развитие клинических симптомов зависит от степени гетероплазмы мутации. Диагностический алгоритм включает в себя начало метаболических исследований, выполнение МРТ головного мозга, биопсию мышц и определение мутации на генетическом молекулярном уровне. Терапевтическое лечение остается симптомом улучшения качества жизни пациента. Заключение: Ранний дебют на фоне полного здоровья, полиморфизм клинических проявлений: поражение центральной нервной системы, мышечная слабость, нарушение психомоторного развития, судороги у ребенка, должны побуждать клинициста к рассмотрению синдрома NARP и проводить дальнейшие исследования, такие как измерение лактата в крови, выполнение электромиографии, магнитно-резонансной томографии и генетического анализа

Creatine Transporter Defect Diagnosed by Proton NMR Spectroscopy in Males With Intellectual Disability

Creatine deficiency syndrome due to mutations in X-linked SLC6A8 gene results in nonspecific intellectual disability (ID). Diagnosis cannot be established on clinical grounds and is often based on the assessment of brain creatine levels by magnetic resonance spectroscopy (MRS). Considering high costs of MRS and necessity of sedation, this technique cannot be used as a first level-screening test. Likewise, gene test analysis is time consuming and not easily accessible to all laboratories. In this article feasibility of urine analysis (creatine/creatinine (Cr/Crn) ratio) performed by nuclear magnetic resonance (NMR) as a first level-screening test is explored. Before running a systematic selection of cases a preliminary study for further molecular analysis is shown. NMR urine spectra (n = 1,347) of male patients with an ID without a clinically recognizable syndrome were measured. On the basis of abnormal Cr/Crn ratio, three patients with the highest values were selected for molecular analysis. A confirmatory second urine test was positive in two patients and diagnosis was further confirmed by a decreased brain creatine level and by SLC6A8 gene analysis. A de novo mutation was identified in one. Another patient inherited a novel mutation from the mother who also has a mild ID. A repeat urine test was negative in the third patient and accordingly creatine level in the brain and SLC6A8 gene analysis both gave a normal result. We conclude that Cr/Crn ratio measured by NMR for male patients represents a rapid and useful first level screening test preceding molecular analysis. © 2011 Wiley-Liss, Inc

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 &gt; 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

Характеристика диагностики врожденных нарушений гликозилирований у 40 подозреваемые больных из Молдовы

Institute of Mother and Child, Chisinau, Republic of Moldova, Translational Metabolic Laboratory, RadboudUMC, Nijmegen, Netherlands, “N. Testemitanu” State University of Medicine and Pharmacy, Chisinau, Republic of Moldova, “Petru Poni” Institute of Macromolecular Chemistry, Romanian Academy, Iasi, Romania, “C.D. Nenitescu” Centre of Organic Chemistry, Romanian Academy, Bucharest, RomaniaIntroducere: Erorile în sinteza, asamblarea și / sau procesarea glicanilor provoacă o familie de patologii genetice grupate într-o unitate nosologică sub denumirea de Deraglări Congenitale ale Glicozilării (CDG), actualmente fiind descrise în jur de 150 de tipuri. În orice stare clinică inexplicabilă este necesar de suspectat CDG, în special în cazul afectărilor multisistemice cu implicare neurologică. Metoda obișnuită pentru diagnosticarea CDG este investigarea transferinei serice prin focalizare izoelectrică (IEF). Scopul: Diagnosticul CDG la pacienții suspecți cu simptome de afectare multisistemică, bazată pe screeningul transferinei serice prin focalizare izoelectrică. Materiale și metode: În studiul prezent au fost utilizate probe de ser recoltate de la 40 de pacienți cu vârste variate (2 luni - 15 ani) suspectați pentru CDG, care aveau hipotonie, convulsii, retard psihoneuromotor, caracteristici dismorfice cu implicare multisistemică. Pentru diagnosticul CDG, IEF al transferinei serice a fost efectuat în colaborare cu RadboudUMC, Nijmegen, Olanda. În unele cazuri, s-a utilizat tratamentul cu neuraminidază pentru a detecta polimorfismul genetic al transferinei care poate imita structura anormală a glicanului. În plus, s-a efectuat spectroscopia RMN a urinei pacienților cercetaţi pentru diagnosticarea erorilor înnăscute de metabolism, care pot imita un profil caracteristic pentru CDG. Rezultate: Ca urmare a screeningului selectiv, 37 de pacienți aveau un profil normal al transferinei, în timp ce 3 probe au fost identificate cu profil anormal, sugestiv pentru CDG I. Galactozemia, fructozemia, alcoolismul pot exprima același profil de IEF ca și pentru CDG I. Probele celor trei pacienți au fost analizate prin metode biochimice și molecular-genetice care au identificat că la un pacient paternul anormal IEF a transferinei a fost cauzat de galactozemie, în timp ce la un altul de fructozemie. În cazul celui de-al treilea pacient rezultatele sugerează prezența CDG I și necesită o analiză avansată a profilului glicomic prin spectroscopia de masă. Concluzie: Focalizarea izoelectrică a transferrinei este instrumentul principal pentru diagnosticul CDG pentru multe laboratoare de screening datorită eficienței crescute la preţ rezonabil, în comparație cu alte metode.Введение: Нарушения синтеза, сборки и/или процессинга гликанов являются причиной группы генетических патологий метаболизма, называемых врождёнными нарушениями гликозилирования (ВНГ), типов которых на данный момент описано около 150. ВНГ следует подозревать при любой необъяснённой клинической патологии, особенно с полиорганным поражением с вовлечением нервной системы. Обычный метод диагностики ВНГ – исследование трансферрина в сыворотке крови методом изоэлектрического фокусирования (ИЭФ). Цель: Представление результатов ИЭФ трансферрина сыворотки крови 40 пациентов с полиорганными поражениями, с подозрением на ВНГ. Материалы и методы: В представленном исследовании использовались сыворотки 40 педиатрических пациентов с подозрением на ВНГ, различного возраста (2 мес – 15 лет), с гипотонией, судорогами, задержкой психомоторного развития, признаками дизморфизма, нарушениями развития с полиорганной патологией. Для диагностики ВНГ ИЭФ трансферрина сыворотки производилось в сотрудничестве с RadboudUMC, Неймеген, Нидерланды. B некоторых случаях, использовалось лечение нейраминидазой с целью обнаружения генетического полиморфизма трансферрина. Результаты: В результате селективного скрининга обнаружено, что у 37 пациентов с подозрением на ВНГ был нормальный профиль трансферрина, а в 3 образцах был обнаружен аномальный профиль, говорящий о возможности ВНГ I. К сожалению, анализ трансферрина методом ИЭФ имеет некоторые ограничения, в связи с фактом, что при галактоземии, фруктоземии и алкоголизме может обнаруживаться такой же профиль, как и при ВНГ I. Таким образом, данные пациенты были проанализированы с использованием биохимических и молекулярно-генетических методов, обнаруживших, что у одного пациента аномальный профиль ИЭФ трансферрина вызван галактоземией, а у другого пациента – фруктоземией. Последний пациент успешно прощёл данный тест, что говорит о наличии ВНГ I и необходимости определения профиля гликомики. Заключение: ИЭФ трансферрина представляет собой основное средство диагностики ВНГ во многих лабораториях скрининга, в связи с экономической эффективностью по сравнению с другими методам

Clinical presentation and long-term follow-up of dopamine beta hydroxylase deficiency

Dopamine beta hydroxylase (DBH) deficiency is an extremely rare autosomal recessive disorder with severe orthostatic hypotension, that can be treated with L-threo-3,4-dihydroxyphenylserine (L-DOPS). We aimed to summarize clinical, biochemical, and genetic data of all world-wide reported patients with DBH-deficiency, and to present detailed new data on long-term follow-up of a relatively large Dutch cohort. We retrospectively describe 10 patients from a Dutch cohort and 15 additional patients from the literature. We identified 25 patients (15 females) from 20 families. Ten patients were diagnosed in the Netherlands. Duration of follow-up of Dutch patients ranged from 1 to 21 years (median 13 years). All patients had severe orthostatic hypotension. Severely decreased or absent (nor)epinephrine, and increased dopamine plasma concentrations were found in 24/25 patients. Impaired kidney function and anemia were present in all Dutch patients, hypomagnesaemia in 5 out of 10. Clinically, all patients responded very well to L-DOPS, with marked reduction of orthostatic complaints. However, orthostatic hypotension remained present, and kidney function, anemia, and hypomagnesaemia only partially improved. Plasma norepinephrine increased and became detectable, while epinephrine remained undetectable in most patients. We confirm the core clinical characteristics of DBH-deficiency and the pathognomonic profile of catecholamines in body fluids. Impaired renal function, anemia, and hypomagnesaemia can be part of the clinical presentation. The subjective response to L-DOPS treatment is excellent and sustained, although the neurotransmitter profile in plasma does not normalize completely. Furthermore, orthostatic hypotension as well as renal function, anemia, and hypomagnesaemia improve only partially