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Background: Hereditary Tyrosinemia type 1 (HT1) is a rare metabolic disorder caused by a defect in the enzyme Fumarylacetoacetate Hydrolase. Due to this defect, toxic products accumulate which, in turn, cause liver and kidney dysfunction. Treatment with 2-(2-nitro-4-trifluoromethylbenoyl)-1,3-cyclohexanedione (NTBC) and diet has diminished these problems, but recent data indicate that HT1 patients have neurocognitive problems. However, the neuropsychological profile of these patients is unknown. Therefore, this study aimed to investigate this neuropsychological profile by comparing HT1 patients with healthy controls., Methods: Neurocognitive testing was performed in a heterogeneous group of 19 NTBC and dietary treated HT1 patients (five female, fourteen male; mean age 12.9 ± 4.8 years; range 7.9-23.6 years) and 19 age and gender matched controls (five female, fourteen male; mean age 13.2 ± 4.6 years; range 8.1-24.8 years). IQ scores were estimated and all participants performed the Amsterdam Neuropsychological Tasks, measuring executive functions (inhibition, cognitive flexibility and working memory) and social cognition (face recognition and identification of facial emotions)., Results: HT1 patients showed poorer estimated IQ, executive functioning (working memory and cognitive flexibility), and social cognition compared to healthy controls. Lower IQ scores in HT1 patients were mostly unrelated to scores on executive function- and social cognition tasks and therefore did not account for group differences in these domains. Further analyses within the HT1 patient group (comparing different groups of patients based on the age at diagnosis and the clinical symptoms at diagnosis) did not reveal any significant results. The duration of NTBC treatment was negatively correlated with IQ., Conclusions: Despite the heterogeneity of the patient group, these data clearly show that IQ, executive functioning and social cognition are affected in HT1 patients, and that IQ screening is not sufficient for cognitive monitoring of these patients. Further research should focus on the underlying pathophysiological mechanisms of these impairments to consequently try to improve treatment strategies.

In a 28-year-old male with a mild mitochondrial myopathy manifesting as exercise intolerance and early signs of cardiomyopathy without muscle weakness or ophthalmoplegia, we identified two novel mutations in the SLC25A4 gene: c.707G>C in exon 3 (p.(R236P)) and c.116_137del in exon 2 (p.(Q39Lfs*14)). Serum lactate levels at rest were elevated (12.7 mM). Both the patient's father and brother were heterozygous carriers of the c.707G>C mutation and were asymptomatic. The second mutation causes a 22 bp deletion leading to a frame shift likely giving rise to a premature stop codon and nonsense-mediated decay (NMD). The segregation of the mutations could not be tested directly as the mother had died before. However, indirect evidence from NMD experiments showed that the two mutations were situated on two different alleles in the patient. This case is unique compared to other previously reported patients with either progressive external ophthalmoplegia (PEO) or clear hypertrophic cardiomyopathy with exercise intolerance and/or muscle weakness carrying recessive mutations leading to a complete absence of the SLC25A4 protein. Most likely in our patient, although severely reduced, SLC25A4 is still partially present and functional.

Despite their good appetite, many females with Rett syndrome (RTT) meet the criteria for moderate to severe malnutrition. Although feeding difficulties may play a part in this, other constitutional factors such as altered metabolic processes are suspected. Irregular breathing is a common clinical feature, leading to chronic respiratory alkalosis or acidosis. We assumed that these changes in intracellular pH cause disturbances in the metabolic equilibrium, with important nutritional consequences. The study population consisted of a group of thirteen well-defined RTT girls with extended clinical, molecular and neurophysiological assessments. Despite normal levels of total dietary energy and protein intakes, malnutrition was confirmed based on significantly low fat-free mass index (FFMI) values. Biochemical screening of multiple metabolic pathways showed significantly elevated plasma creatine concentrations and increased urinary creatine/creatinine ratio in five RTT girls. Four girls, 10 years and older, were forceful breathers, one 13-year-old girl had an undetermined cardiorespiratory phenotype. An isolated increase of the urinary creatine/creatinine ratio was seen in two girls, a 9-year old forceful and a 4-year old feeble breather. Given that the young girls are feeble breathers and the older girls are forceful breathers, it is impossible to determine whether the elevated creatine concentrations are due to increasing age or cardiorespiratory phenotype. Furthermore, MeCP2 deficiency may cause epigenetic aberrations affecting the expression of the creatine-transporter gene, which is located at Xq28. Further studies are required to confirm these findings and to provide greater insight into the pathogenesis of the abnormal creatine metabolism in RTT.

Two young girls without a notable medical history except for asthma presented with an acute toxic encephalopathy with very low serine concentrations both in plasma and cerebrospinal fluid (CSF) comparable to patients with 3-phosphoglycerate dehydrogenase (3-PGDH) deficiency. Clinical symptoms and enzyme measurement (in one patient) excluded 3-PGDH deficiency. Deficiencies in other serine biosynthesis enzymes were highly unlikely on clinical grounds. On basis of the fasting state, ketone bodies and lactate in plasma, urine and CSF, we speculate that reduced serine levels were due to its use as gluconeogenic substrate, conversion to pyruvate by brain serine racemase or decreased L-serine production because of a lack of glucose. These are the first strikingly similar cases of patients with a clear secondary serine deficiency associated with a toxic encephalopathy.

Background: The hypothalamus regulates basic homeostasis such as appetite, circadian rhythm, autonomic and pituitary functions. Dysregulation in these functions results in the hypothalamic syndrome, a rare disorder of various origins. Since serotonin (5-HT) modulates most of the above-mentioned homeostasis, a defect in the serotonergic system can possibly participate in this syndrome., Methods: We describe a girl suffering from hypothalamic syndrome with a decreased concentration of 5-hydroxytryptophan (5-HTP) and a normal level of tryptophan in the cerebrospinal fluid (CSF) suggesting a functional defect in tryptophan hydroxylase (TPH). TPH is a rate-limiting enzyme in the synthesis of the neurotransmitter 5-HT., Results: Therapeutic intervention with 5-HTP, carbidopa and a specific serotonin reuptake inhibitor significantly improved her clinical symptoms and caused biochemical normalisation of neurotransmitters., Conclusion: The girl described had the typical symptoms of a hypothalamic disorder and a defective serotonergic metabolism, a relationship which has not been reported before. Therapeutic interventions to restore 5-HT metabolism resulted in clinical improvement. We suggest that investigation of 5-HT metabolism in CSF of patients with this rare disorder is included in the aetiological work-up.

Background: Urine microscopic analysis is hampered by its lack in standardisation and semi-quantitative reports, resulting in limited reliability. Automation of urinalysis could overcome these problems., Methods: We compared the performance of the iQ200 with traditional microscopy and strip analysis in routine urinalysis. A total of 1482 routine samples, positive in dipstick testing, were evaluated for erythrocytes, leukocytes, casts, dysmorphic erythrocytes and bacteria using the iQ200 and traditional microscopy. The results of 320 of these samples were linked to underlying urological pathology as well as results from bacterial culturing., Results: Analytically, the iQ200 surpasses traditional microscopy. The identification of casts and dysmorphic erythrocytes in routine samples improves when using the iQ200, although the sub-classification of casts required well-trained technicians. The auto-classification of particles was least reliable for yeast and bacterial cocci. The quantitative reports, and therefore the use of precise cut-off points allowed earlier and improved detection of urinary tract pathology., Conclusions: The performance of the iQ200 is equal to traditional microscopy, but it strongly improves the reliability of urinalysis by standardisation, quantitative reports and improved workflow. From a clinical point of view, renewed attention and improvement of routine urinalysis aids in the efficient detection of renal and urinary tract pathology.

The accuracy of detection and identification of low-concentration M proteins using capillary zone electrophoresis/immunosubtraction (CZE/IS) plots was systematically studied. To this end, 49 CZE plots (protein spectra; session I) and CZE/IS plots (spectra with corresponding immunosubtraction plots; session II) were judged by participants from 12 Dutch laboratories. Plots were based on mixed sera with a low concentration (0.4, 1 or 4 g/L) of one M protein, as well as normal sera, sera with an oligoclonal or polyclonal increase in immunoglobulins, and sera containing free monoclonal light chains. At 0.4 g/L, nine out of 12 (9/12) different M proteins were hard to detect. At 1 g/L, 6/12 M proteins were detected by > 85% of the participants. At 4 g/L, 10/12 M proteins were detected by all participants. Normal spectra were always judged correctly. At 1 g/L and 4 g/L, detected M proteins were identified correctly by 92% of the participants, versus only 63% at 0.4 g/L. The detection accuracy was only slightly (non-significantly) improved by the application of IS plots. Our data indicate that CZE/IS is applicable for most M proteins at concentrations > or = 4 g/L.

Thrombin generation has been studied in the plasma of severely factor XI deficient patients under conditions in which contact activation did not play a role. In platelet-rich as well as platelet-poor plasma, thrombin generation was dependent upon the presence of factor XI at tissue factor concentrations of between 1 and 20 pg/ml i.e. approximately 0.01 to 0.20% of the concentration normally present in the thromboplastin time determination. The requirement for factor XI is low; significant thrombin generation was seen at 1% factor XI; at 10%, thrombin formation was nearly normalised. A suspension of normal platelets in severely factor XI deficient plasma did not increase thrombin generation. This implies that there is no significant factor XI activity carried by normal platelets, although the presence of factor XI and factor XI inhibitors in platelets cannot be ruled out.

In von Willebrand disease (vWD) type 1 and mild haemophilia A patients we studied the effect of an infusion of DDAVP (0.3 microg/kg body weight) on thrombin generation in platelet-rich plasma (PRP) and platelet-poor plasma (PPP). Baseline thrombin generation in PRP was diminished both in the haemophilia A and vWD patients. It was normal in vWD plasma when sufficient procoagulant phospholipids were present, either via adding phospholipid vesicles to PPP or via scrambling of the platelet membrane with ionomycin in PRP. In haemophilia A plasma, thrombin generation did not normalize by providing procoagulant phospholipids. Treatment with DDAVP temporarily restored thrombin generation in PRP to normal in both diseases. To investigate the individual roles of von Willebrand factor (vWF) and factor VIII, we also studied the effect of factor VIII infusion on thrombin generation in a severe haemophilia patient. It appears that at a fixed normal vWF concentration, <25% factor VIII is sufficient for normal thrombin generation in PRP. At a sufficient factor VIII concentration, however, thrombin generation is still lower than normal in vWD patients; approximately 40% of vWF is required for half-normal thrombin generation in PRP. It thus appears that vWF is also a clotting factor, in the sense that it is required for normal thrombin generation. This underlines the importance of the interaction between coagulation and the platelets in normal haemostasis. Thrombin generation in PRP appears to be a suitable test to reflect the combined function.

alpha(2A)-Adrenergic receptor-mediated Ca(2+) signaling and integrin alpha(IIb)beta(3) exposure were investigated in human platelets under conditions where indirect, thromboxane- or ADP-mediated effects were absent. The alpha(2)-adrenergic receptor agonists, UK14304 and epinephrine (EPI), were unable to raise cytosolic levels of inositol 1,4,5-trisphosphate (InsP(3)) or Ca(2+) but potentiated the [Ca(2+)](i) rises evoked by other agonists that act through stimulation of phospholipase C (thrombin or platelet-activating factor) or stimulation of Ca(2+)-induced Ca(2+) release (CICR) in the absence of InsP(3) generation (thimerosal or thapsigargin). In addition, alpha(2)-adrenergic stimulation resulted in a 20% lowering in the cytosolic cAMP level. In platelets treated with G(salpha)-stimulating prostaglandin E(1), EPI increased the Ca(2+) signal evoked by either phospholipase C- or CICR-stimulating agonists mainly through modulation of the cAMP level. The stimulating effects of UK14304 and EPI on platelet Ca(2+) responses, and also on integrin alpha(IIb)beta(3) exposure and platelet aggregation, were abolished by pharmacological stimulation of cAMP-dependent protein kinase, and these effects were mimicked by inhibition of this activity. In permeabilized platelets, UK14304 and EPI potentiated InsP(3)-induced, CICR-mediated mobilization of Ca(2+) from internal stores in a similar way as did inhibition of cAMP-dependent protein kinase. In summary, a G(ialpha)-mediated decrease in cAMP level appears to play a major role in the platelet-activating effects of alpha(2A)-adrenergic receptor stimulation. Thus, in platelets, unlike other cell types, occupation of the G(ialpha)-coupled alpha(2A)-adrenergic receptors does not result in phospholipase C activation but rather in modulation of the Ca(2+) response by relieving cAMP-mediated suppression of InsP(3)-dependent CICR.

Infusion of the GPIIb/IIIa-inhibitor MK383 inhibits thrombin generation in platelet rich plasma by interfering with the production of platelet procoagulant phospholipid exposure. The effect is similar to that of 0.2 U/ml of heparin. Heparin infusion, well known to inhibit thrombin generation by fostering antithrombin activity, inhibits the formation of platelet-derived procoagulant microparticles, probably by decreasing the formation of free thrombin, which, under our circumstances, is the main platelet activator.