Copper Toxicity Associated With an ATP7A-Related Complex Phenotype.

BACKGROUND: The ATP7A gene encodes a copper transporter whose mutations cause Menkes disease, occipital horn syndrome (OHS), and, less frequently, ATP7A-related distal hereditary motor neuropathy (dHMN). Here we describe a family with OHS caused by a novel mutation in the ATP7A gene, including a patient with a comorbid dHMN that worsened markedly after being treated with copper histidinate. METHODS: We studied in detail the clinical features of the patients and performed a genomic analysis by using TruSight One Expanded Sequencing Panel. Subsequently, we determined the ATP7A and ATP7B expression levels, mitochondrial membrane potential, and redox balance in cultured fibroblasts of Patient 1. RESULTS: We found a novel ATP7A late truncated mutation p.Lys1412AsnfsX15 in the two affected members of this family. The co-occurrence of OHS and dHMN in Patient 1 reveals the variable phenotypic expressivity of the variant. A severe clinical and neurophysiologic worsening was observed in the dHMN of Patient 1 when he was treated with copper replacement therapy, with a subsequent fast recovery after the copper histidinate was withdrawn. Functional studies revealed that the patient had low levels of both ATP7A and ATP7B, the other copper transporter, and high levels of superoxide ion in the mitochondria. CONCLUSIONS: Our findings broaden the clinical spectrum of ATP7A-related disorders and demonstrate that two clinical phenotypes can occur in the same patient. The copper-induced toxicity and low levels of both ATP7A and ATP7B in our patient suggest that copper accumulation in motor neurons is the pathogenic mechanism in ATP7A-related dHMN.

Measurement of serum tenascin-X in patients with congenital adrenal hyperplasia at risk for Ehlers-Danlos contiguous gene deletion syndrome CAH-X.

OBJECTIVE: Approximately 10% of patients with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency carry a mutation that disrupts CYP21A2 and the flanking TNXB gene resulting in CAH-X, a contiguous gene deletion syndrome. TNXB encodes tenascin-X (TNX), an extracellular matrix glycoprotein that plays an important role in collagen organization. TNXB impairment is associated with Ehlers-Danlos syndrome. Symptoms include joint hypermobility, hernias and cardiac defects. We measured serum TNX using an antibody targeting the amino-terminal of the TNX protein in 161 subjects, including extensively genotyped and phenotyped CAH patients, their relatives, and healthy controls. RESULTS: We evaluated the potential of serum TNX as a screening tool for CAH-X. CAH-X patients, especially haploinsufficient patients carrying the TNXA-TNXB chimeric gene CAH-X-CH-1 showed reduced TNX levels compared to controls (P < 0.05). TNX levels were similar in all subjects carrying a TNXB mutation. However, CAH patients who did not harbor a TNXB mutation also had reduced TNX compared to controls (P < 0.001). Thus, measuring serum TNX is not an effective screen for CAH-X amongst patients with CAH. TNXB genotyping is recommended for CAH patients who have symptoms of a connective tissue disorder. Epigenetic factors that influence TNX expression require further study.

Measurement of Serum Tenascin-X in Joint Hypermobility Syndrome Patients.

Joint hypermobility syndrome (JHS) (also termed hypermobility type Ehlers-Danlos syndrome, hEDS) is a heritable connective tissue disorder that is characterized by generalized joint hypermobility, chronic pain, fatigue, and minor skin changes. Initially, it was reported that there is a small subset of patients with JHS/hEDS who have haploinsufficiency of tenascin-X (TNX). However, the relationship between TNXB and JHS/hEDS has not been reported at all afterwards. EDS was reclassified into thirteen types in 2017, and the causative gene of JHS/hEDS remained to be identified. Therefore, in this study in order to determine whether JHS/hEDS can be diagnosed by the concentrations of serum form of TNX (sTNX), we measured the concentrations of sTNX in 17 JHS/hEDS patients. The sTNX concentrations in half of the JHS/hEDS patients were significantly lower than those in healthy individuals. No mutations, insertions or deletions were detected in the TNX exon sequence of the JHS/hEDS patients except for one in patient. That patient has a heterozygous mutation. A correlation between sTNX concentration and mutation of the TNXB genomic sequence was not found in the JHS/hEDS patients. These results indicate that the decrease in sTNX concentration could be used as a risk factor for JHS/hEDS.

Hemostatic abnormalities in patients with Ehlers-Danlos syndrome.

Essentials Ehlers-Danlos Syndrome (EDS) is a rare heterogeneous group of inherited collagen disorders. A cohort of EDS patients was investigated for bleeding tendency and hemostatic abnormalities. EDS is associated with an increased risk of bleeding. EDS patients have platelet function abnormalities, whose severity correlates with bleeding risk. SUMMARY: Background Ehlers-Danlos syndrome (EDS) includes a heterogeneous group of connective tissue disorders affecting skin, bones, vessels, and other organs. Patients with EDS have an increased risk of bleeding, but a comprehensive study of hemostasis in EDS patients is lacking. Objective To investigate the bleeding tendency of a cohort of patients with EDS by using the Bleeding Assessment Tool of the ISTH, the bleeding severity score (BSS). Methods The BSS was defined as abnormal when it was ≥ 4 in men and ≥ 6 in women. Patients with a bleeding tendency were compared with those without in terms of type and number of hemostatic abnormalities. Results Fifty-nine of 141 patients with EDS (41.7%) had an abnormal BSS. Prothrombin time and activated partial thromboplastin time were slightly prolonged in 10 patients (7.1%) because of mild coagulation factor deficiencies, which were not responsible for the bleeding diathesis. von Willebrand factor antigen, ristocetin cofactor, endogenous thrombin potential and platelet count were normal in all patients. At least one platelet function abnormality was found in 53 patients (90%) with an abnormal BSS and in 64 (78%) with a normal BSS (adjusted odds ratio [OR] 2.55, 95% confidence interval [CI] 0.87-7.48). The risk of bleeding progressively increased with the number of platelet function abnormalities, reaching an OR of 5.19 (95% CI 1.32-20.45) when more than three abnormalities were detected. Conclusions Our results show that nearly half of patients with EDS have an abnormal BSS, which, in 90% of cases, appear, at least in part, to be attributable to platelet function abnormalities. Abnormalities of primary hemostasis may contribute to the risk of bleeding in patients with EDS.

A novel truncating mutation in FLNA causes periventricular nodular heterotopia, Ehlers-Danlos-like collagenopathy and macrothrombocytopenia.

INTRODUCTION: Filamin A (FLNA) is located in Xq28, and encodes the actin binding protein, filamin A. A mutation in FLNA is the most common cause of periventricular nodular heterotopia (PVNH), but a clear phenotype-genotype correlation has not been established. Indeed, some patients with a FLNA mutation have recently been shown to additionally have Ehlers-Danlos-like collagenopathy or macrothrombocytopenia. In an attempt to establish a clearer correlation between clinical symptoms and genotype, we have investigated a phenotype that involves thrombocytopenia in a patient with a truncation of the FLNA gene. CASE REPORT: We present the case of a 4-year-old girl who, at birth, showed a ventral hernia. At 2 months of age, she was diagnosed with patent ductus arteriosus (PDA) and aortic valve regurgitation. At 11 months, she underwent ligation of the PDA. She was also diagnosed with diaphragmatic eventration by a preoperative test. At 19 months, motor developmental delay was noted, and brain MRI revealed bilateral PVNH with mega cisterna magna. Presently, there is no evidence of epilepsy, intellectual disability or motor developmental delay. She has chronic, mild thrombocytopenia, and a platelet count that transiently decreases after viral infection. Dilation of the ascending aorta is progressing gradually. Genetic testing revealed a de novo nonsense heterozygous mutation in FLNA (NM_001456.3: c.1621G > T; p.Glu541Ter). Immunofluorescence staining of a peripheral blood smear showed a lack of filamin A expression in 21.1% of her platelets. These filamin A-negative platelets were slightly larger than her normal platelets. CONCLUSION: Our data suggests immunofluorescence staining of peripheral blood smears is a convenient diagnostic approach to identify patients with a FLNA mutation, which will facilitate further investigation of the correlation between FLNA mutations and patient phenotype.

Recognizing the tenascin-X deficient type of Ehlers-Danlos syndrome: a cross-sectional study in 17 patients.

The tenascin-X (TNX) deficient type Ehlers-Danlos syndrome (EDS) is similar to the classical type of EDS. Because of the limited awareness among geneticists and the challenge of the molecular analysis of the TNXB gene, the TNX-deficient type EDS is probably to be under diagnosed. We therefore performed an observational, cross-sectional study. History and physical examination were performed. Results of serum TNX measurements were collected and mutation analysis was performed by a combination of next-generation sequencing (NGS), Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). Included were 17 patients of 11 families with autosomal recessive inheritance and childhood onset. All patients had hyperextensible skin without atrophic scarring. Hypermobility of the joints was observed in 16 of 17 patients. Deformities of the hands and feet were observed frequently. TNX serum level was tested and absent in 11 patients (seven families). Genetic testing was performed in all families; 12 different mutations were detected, most of which are suspected to lead to non-sense mRNA mediated decay. In short, patients with the TNX-deficient type EDS typically have generalized joint hypermobility, skin hyperextensibility and easy bruising. In contrast to the classical type, the inheritance pattern is autosomal recessive and atrophic scarring is absent. Molecular analysis of TNXB in a diagnostic setting is challenging.

Vascular type Ehlers-Danlos syndrome is associated with platelet dysfunction and low vitamin D serum concentration.

BACKGROUND: The vascular type represents a very rare, yet the clinically most fatal entity of Ehlers-Danlos syndrome (EDS). Patients are often admitted due to arterial bleedings and the friable tissue and the altered coagulation contribute to the challenge in treatment strategies. Until now there is little information about clotting characteristics that might influence hemostasis decisively and eventually worsen emergency situations. RESULTS: 22 vascular type EDS patients were studied for hemoglobin, platelet volume and count, Quick and activated partial thromboplastin time, fibrinogen, factor XIII, von Willebrand disease, vitamin D and platelet aggregation by modern standard laboratory methods. Results show a high prevalence of over 50 % for platelet aggregation disorders in vascular type EDS patients, especially for collagen and epinephrine induced tests, whereas the plasmatic cascade did not show any alterations. Additionally, more than half of the tested subjects showed low vitamin D serum levels, which might additionally affect vascular wall integrity. CONCLUSION: The presented data underline the importance of detailed laboratory screening methods in vascular type EDS patients in order to allow for targeted application of platelet-interacting substances that might be of decisive benefit in the emergency setting.

A method for quantification of serum tenascin-X by nano-LC/MS/MS.

BACKGROUND: Complete deficiency of an extracellular matrix tenascin-X (TNX) leads to a classical type of Ehlers-Danlos syndrome (EDS). TNX haploinsufficiency is a cause of hypermobility type of EDS. Human TNX is also present in a serum form (sTNX) with a molecular size of 140kDa. In this study, we established a method for quantification of sTNX using nano-liquid chromatography tandem mass spectrometry (LC/MS/MS) with selected/multiple reaction monitoring. METHODS: Twelve abundant protein-depleted sera were reduced, alkylated, and digested with Lys-C and trypsin. Subsequently, the digests were fractionated by strong cation exchange chromatography. Optimal and validated transitions of precursor and product ions of the peptides from sTNX were developed on a triple quadrupole mass spectrometer. RESULTS: Serum concentrations of sTNX of healthy individuals were quantified as an average of 144ng/ml. However, sTNX was not detected by this method in serum from a patient with a classical type of EDS in whom sTNX was not found by Western blot analysis. The limit of quantification (LOQ) of sTNX by nano-LC/MS/MS method was 2.8pg whereas the detection sensitivity of sTNX by Western blot analysis was 19pg. The nano-LC/MS/MS method is more sensitive than Western blot analysis. CONCLUSIONS: The quantification method will be useful for diagnosis and risk stratification of EDS caused by TNX deficiency and haploinsufficiency.

Comparative study of dermal components and plasma TGF-ß1 levels in Slc39a13/Zip13-KO mice.

Ehlers-Danlos syndrome (EDS) is a group of disorders caused by abnormalities that are identified in the extracellular matrix. Transforming growth factor-ß1 (TGF-ß1) plays a crucial role in formation of the extracellular matrix. It has been reported that the loss of function of zinc transporter ZRT/IRT-like protein 13 (ZIP13) causes the spondylocheiro dysplastic form of EDS (SCD-EDS: OMIM 612350), in which dysregulation of the TGF-ß1 signaling pathway is observed, although the relationship between the dermis abnormalities and peripheral TGF-ß1 level has been unclear. We investigated the characteristics of the dermis of the Zip13-knockout (KO) mouse, an animal model for SCD-EDS. Both the ratio of dermatan sulfate (DS) in glycosaminoglycan (GAG) components and the amount of collagen were decreased, and there were very few collagen fibrils with diameters of more than 150 nm in Zip13-KO mice dermis. We also found that the TGF-ß1 level was significantly higher in Zip13-KO mice serum. These results suggest that collagen synthesis and collagen fibril fusion might be impaired in Zip13-KO mice and that the possible decrease of decorin level by reduction of the DS ratio probably caused an increase of free TGF-ß1 in Zip13-KO mice. In conclusion, skin fragility due to defective ZIP13 protein may be attributable to impaired extracellular matrix synthesis accompanied by abnormal peripheral TGF-ß homeostasis.

[The hypoxic syndrome in children with cardiomyopathy].

The children with inherited cardiopathy including hypersensitive (n = 85) and dilatation (n=10) cardiopathy as well as cardiopathy under Ehlers-Danlos Syndrome (n = 70) combined with different inherited heart disease were examined to establish signs of hematic and tissue hypoxia. The most typical signs turned out periodic decrease of blood pCO2 with increasing of content of lactate and pyruvate in blood and saliva, multiple caries of teeth and high rate of systemic hypoplasia of enamel of both temporary and permanent teeth. The study established decrease in blood of level of macro-ergic compounds (ATP, ADF AMP) with increasing of excretion calcium and phosphates with urine. The increase of rate of mutations of hypoxanthine guanine phosphoribosyltransferase in lymphocytes with increasing of content of uric acid in blood and/or in urine was detected. The study revealed increasing of processes of peroxide oxidation, alterations of morphology of cells of skeletal muscles (RRF) and accumulation ofcalcium, lipids and alteration of structure of mitochondria.

Familial Ehlers-Danlos syndrome with lethal arterial events caused by a mutation in COL5A1.

Different forms of Ehlers-Danlos syndrome (EDS) exist, with specific phenotypes and associated genes. Vascular EDS, caused by heterozygous mutations in the COL3A1 gene, is characterized by fragile vasculature with a high risk of catastrophic vascular events at a young age. Classic EDS, caused by heterozygous mutations in the COL5A1 or COL5A2 genes, is characterized by fragile, hyperextensible skin and joint laxity. To date, vessel rupture in four unrelated classic EDS patients with a confirmed COL5A1 mutation has been reported. We describe familial occurrence of a phenotype resembling vascular EDS in a mother and her two sons, who all died at an early age from arterial ruptures. Diagnostic Sanger sequencing in the proband failed to detect aberrations in COL3A1, COL1A1, COL1A2, TGFBR1, TGFBR2, SMAD3, and ACTA2. Next, the proband's DNA was analyzed using a next-generation sequencing approach targeting 554 genes linked to vascular disease (VASCULOME project). A novel heterozygous mutation in COL5A1 was detected, resulting in an essential glycine substitution at the C-terminal end of the triple helix domain (NM_000093.4:c.4610G>T; p.Gly1537Val). This mutation was also present in DNA isolated from autopsy material of the index's brother. No material was available from the mother, but the mutation was excluded in her parents, siblings and in the father of her sons, suggesting that the COL5A1 mutation occurred in the mother's genome de novo. In conclusion, we report familial occurrence of lethal arterial events caused by a COL5A1 mutation.

Serum relaxin levels in benign hypermobility syndrome.

OBJECTIVE: In this study, we investigated the activity of serum relaxin in female patients with benign joint hypermobility syndrome (BJHS), locomotor system findings accompanying BJHS, and its relation to relaxin. METHODS: Into the study, female patients with BJHS and healthy women as the control group were included. The patients were diagnosed by using the Brighton 1998 criteria. Examination of the locomotor system for study groups were performed. Serum relaxin levels of both patient and control group were measured. RESULTS: There were 48 female patients with BJHS and 40 healthy women in the study. With respect to the control group, the level of serum relaxin was higher in the patients (47.1 ± 20.3, 34.4 ± 22.1; p> 0.05). Again compared with the control group, arthralgia (p= 0.00), myalgia (p= 0.01), shoulder impingement syndrome (p= 0.05), pes planus (p= 0.01), and hyperkyphosis (p= 0.000) were higher in the patients. The level of relaxin median was significantly higher in the patients with pesplanus and hyperkyphosis than those who did not have them (p= 0.05, p= 0.01, respectively). CONCLUSIONS: Although serum relaxin level is not considered a causative factor for BJHS, the significant increases found in those patients with hyperkyphosis and pes planus suggest the hypothesis that relaxin has a limited and indefinite role in patients with BJHS.

Evaluación de la hemostasia en niños con Síndrome de Ehlers-Danlos tipo III / Hemostasis evaluation in children with type III Ehlers-Danlos syndrome

Introducción: Aunque no se ha demostrado la existencia de alteraciones de la hemostasia que formen parte del cuadro clínico del síndrome de Ehlers-Danlos, se han reportado diversas alteraciones de la coagulación en casos aislados, como son eficiencia y alteraciones de la movilidad electroforética de la fibronectina, disfunción de la agregación plaquetaria con prolongación del tiempo de sangramiento, deficiencia de factores VIII, IX, XII y XIII y aumento de la sensibilidad a la aspirina, entre otras. Objetivo: Evaluar la existencia de alteraciones de la hemostasiaen niños con síndrome de Ehlers-Danlos tipo III. Métodos: Se realizó una investigación aplicada, observacional, descriptiva y transversal en 305 niños con síndrome de Ehlers-Danlos tipo III para evaluar, en aquellos con historia de manifestaciones hemorrágicas, la existencia de alteraciones de la hemostasia.Previa suspensión de drogas con acción antiagregante plaquetaria, a todos los pacientes se les realizaron estudios decoagulación y función y agregación plaquetaria. Resultados: En 181 pacientes se encontró historia de sangramiento espontáneo o traumático, predominantemente cutáneo-mucoso. Elcoagulograma fue normal en todos los casos y el extendido de sangre periférica mostró la presencia de macroplaquetas y escasa formación de grumos como alteración frecuente. Las pruebas de agregación y función plaquetaria evidenciaron la existencia de trastornos cualitativos con predominio de la disminución de la agregación con ADP, sola o combinada con epinefrina y colágeno, y con menor frecuencia trastornos de la disponibilidad de los fosfolípidos plaquetarios. La mayoría de estos pacientes habían utilizado antihistamínicos (ketotifeno) por diversas causas. Conclusiones: Se reporta la presencia de defectos cualitativos plaquetarios en niños con síndrome de Ehlers-Danlos tipo III destacándose el papel de la utilización de drogas antihistamínicas en la aparición de manifestaciones hemorrágicas en estos pacientes

Introduction: Although the existence of hemostasis disorders as part of type-III Ehlers-Danlos syndrome has not been confirmed, several coagulation alterations have been reported in isolated cases such as: deficiencies and modification in electrophoresis mobility of fibronectin, dysfunction of platelet aggregation with lengthening of bleeding time, deficiency of VIII, IX, XII and XIII factors and increase of aspirin sensitivity, among others. Objective: Evaluate the existence of hemostasis disorders in children with type III Ehlers-Danlos syndrome. Method: an applied, observational, descriptive and cross-sectional research was carried out in 305 children suffering from type-III Ehlers_Danlos syndrome to evaluate in those having history of hemorrhagic manifestations, the existence of alterations of the hemostasis. Previous suspension of drugs with platelet anti-aggregation action, coagulation and platelet aggregation function studies were carried out. Results: The study revealed that 181 patients presented history of spontaneous or traumatic bleeding mainly mucous-cutaneous. Coagulogram was normal in all cases and peripheral-blood smears showed the presence of macro-platelets and deficient formation of clots as the most frequent alteration. Aggregation and platelet function tests evidenced the presence of qualitative disorders, where a decrease of aggregation prevailed with the use of adenosine diphosphate (ADP), alone or combined with epinephrine and collagen, and with less frequency, disorders of of platelet phospholipids availability. The majority of these patients presented history of long-lasting use of antihistamines (ketotifen) due to diverse causes. Conclusions: The occurrence of these qualitative platelet defects in children with EDS-type III is reported, standing out the role of the use of antihistamine drugs on the onset of the hemorrhagic symptoms in these patients

Transforming growth factor-ß and inflammation in vascular (type IV) Ehlers-Danlos syndrome.

BACKGROUND: Vascular Ehlers-Danlos syndrome (VEDS) causes reduced life expectancy because of arterial dissections/rupture and hollow organ rupture. Although the causative gene, COL3A1, was identified >20 years ago, there has been limited progress in understanding the disease mechanisms or identifying treatments. METHODS AND RESULTS: We studied inflammatory and transforming growth factor-ß (TGF-ß) signaling biomarkers in plasma and from dermal fibroblasts from patients with VEDS. Analyses were done in terms of clinical disease severity, genotype-phenotype correlations, and body composition and fat deposition alterations. VEDS subjects had increased circulating TGF-ß1, TGF-ß2, monocyte chemotactic protein-1, C-reactive protein, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and leptin and decreased interleukin-8 versus controls. VEDS dermal fibroblasts secreted more TGF-ß2, whereas downstream canonical/noncanonical TGF-ß signaling was not different. Patients with COL3A1 exon skipping mutations had higher plasma intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, and VEDS probands had abnormally high plasma C-reactive protein versus affected patients identified through family members before any disease manifestations. Patients with VEDS had higher mean platelet volumes, suggesting increased platelet turnover because of ongoing vascular damage, as well as increased regional truncal adiposity. CONCLUSIONS: These findings suggest that VEDS is a systemic disease with a major inflammatory component. C-reactive protein is linked to disease state and may be a disease activity marker. No changes in downstream TGF-ß signaling and increased platelet turnover suggest that chronic vascular damage may partially explain increased plasma TGF-ß1. Finally, we found a novel role for dysregulated TGF-ß2, as well as adipocyte dysfunction, as demonstrated through reduced interleukin-8 and elevated leptin in VEDS.

Can elevated IGF-1 levels among patients with Ehlers-Danlos syndrome cause idiopathic intracranial hypertension?

We offer for consideration a possible association between hypermobility syndrome seen in Ehlers-Danlos syndrome and risk of potential development of idiopathic intracranial hypertension - mediated primarily through the effects of insulin-like growth factor-1.

Tenascin-X, collagen, and Ehlers-Danlos syndrome: tenascin-X gene defects can protect against adverse cardiovascular events.

Long thought to be two separate syndromes, Ehlers-Danlos syndrome hypermobility type (EDS-HT) and benign joint hypermobility syndrome (BJHS) appear on close examination to represent the same syndrome, with virtually identical clinical manifestations. While both EDS-HT and BJHS were long thought to lack the genetic loci of other connective tissue disorders, including all other types of EDS, researchers have discovered a genetic locus that accounts for manifestations of both EDS-HT and BJHS in a small population of patients. However, given the modest sample size of these studies and the strong correlation between serum levels of tenascin-X with clinical symptoms of both EDS-HT and BJHS, strong evidence exists for the origins of both types of hypermobility originating in haploinsufficiency or deficiency of the gene TNXB, responsible for tenascin-X. Tenascin-X regulates both the structure and stability of elastic fibers and organizes collagen fibrils in the extra-cellular matrix (ECM), impacting the rigidity or elasticity of virtually every cell in the body. While the impacts of tenascin-X insufficiency or deficiency on the skin and joints have received some attention, its potential cardiovascular impacts remain relatively unexplored. Here we set forth two novel hypotheses. First, TNXB haploinsufficiency or deficiency causes the range of clinical manifestations long identified with both EDS-HT and BJHS. And, second, that haploinsufficiency or deficiency of TNXB may provide some benefits against adverse cardiovascular events, including heart attack and stroke, by lowering levels of arterial stiffness associated with aging, as well as by enhancing accommodation of accrued atherosclerotic plaques. This two-fold hypothesis provides insights into the mechanisms underlying the syndromes previous identified with joint hypermobility, at the same time the hypothesis also sheds light on the role of the composition of the extracellular matrix and its impacts on endothelial sheer stress in adverse cardiovascular events.

Effects of disulfiram treatment in patients with Menkes disease and occipital horn syndrome.

The clinical and biochemical effects of disulfiram were evaluated in three boys with the disorders characterized by copper deficiency due to the defect of ATP7A. Two suffered from Menkes disease (MD) and one from occipital horn syndrome. Disulfiram was orally given, in addition to a parenteral administration of copper-histidine in the case of MD patients. Serum levels of copper and ceruloplasmin slightly increased in one MD patient, and he showed favorable emotional expression and behavior more often than before according to his caretakers. However, no obvious changes were observed in the other two patients. Serum ratios of noradrenaline to dopamine, and adrenaline to dopamine, which are thought to be the indicators of dopamine ß-hydroxylase activity, one of the copper requiring enzymes, were unaltered after disulfiram treatment. No adverse effects were recognized during the treatment period in all patients. Although the major improvement was not observed clinically or biochemically by disulfiram treatment so far, the trial will be continued to see the possible effects in these disorders with copper transport defect.

Determination of beta4-galactosyltransferase-7 activity using high-performance liquid chromatography-electrospray ionization tandem mass spectrometry.

OBJECTIVES: Patients with Ehlers-Danlos syndrome were described to contain reduced activities of beta4-galactosyltransferase-7 (beta4Gal-T7). Therefore, measurement of beta4Gal-T7 activity can help to characterize defects in proteoglycan biosynthesis in patients with connective tissue diseases. DESIGN AND METHODS: We developed a sensitive and specific method to assay beta4Gal-T7 which is based on the transfer of galactose from UDP-galactose to the synthetic peptide Bio-BIK-F-Xyl. RESULTS: Calibration curves exhibited consistent linearity in the range of 10-2000 microg/L Bio-BIK-F-Xyl-Gal, corresponding to a beta4Gal-T7 activity of 3.5-659 microU/L. The limit of detection and the lower limit of quantification were 3.70 microg/L (1.22 microU/L) and 4.50 microg/L Bio-BIK-F-Xyl-Gal (1.48 microU/L beta4Gal-T7 activity), respectively. Interassay imprecision (CV) was 8.1-13.1% in the range from 15.9 to 659 microU/L, and mean recovery was 85.3% (range 61.7-106.3%). CONCLUSIONS: This sensitive, robust and interference-free LC-MS/MS assay allows an accurate determination of beta4Gal-T7 activity in human body fluids.