Sara Morais

Alteration of ornithine metabolism leads to dominant and recessive hereditary spastic paraplegia

Hereditary spastic paraplegias are heterogeneous neurological disorders characterized by a pyramidal syndrome with symptoms predominantly affecting the lower limbs. Some limited pyramidal involvement also occurs in patients with an autosomal recessive neurocutaneous syndrome due to ALDH18A1 mutations. ALDH18A1 encodes delta-1-pyrroline-5-carboxylate synthase (P5CS), an enzyme that catalyses the first and common step of proline and ornithine biosynthesis from glutamate. Through exome sequencing and candidate gene screening, we report two families with autosomal recessive transmission of ALDH18A1 mutations, and predominant complex hereditary spastic paraplegia with marked cognitive impairment, without any cutaneous abnormality. More interestingly, we also identified monoallelic ALDH18A1 mutations segregating in three independent families with autosomal dominant pure or complex hereditary spastic paraplegia, as well as in two sporadic patients. Low levels of plasma ornithine, citrulline, arginine and proline in four individuals from two families suggested P5CS deficiency. Glutamine loading tests in two fibroblast cultures from two related affected subjects confirmed a metabolic block at the level of P5CS in vivo. Besides expanding the clinical spectrum of ALDH18A1-related pathology, we describe mutations segregating in an autosomal dominant pattern. The latter are associated with a potential trait biomarker; we therefore suggest including amino acid chromatography in the clinico-genetic work-up of hereditary spastic paraplegia, particularly in dominant cases, as the associated phenotype is not distinct from other causative genes.

The gel test: some problems and solutions

SUMMARY. The gel centrifugation test (GT) is a method of transfusion serology, based on the fact that, after centrifugation, unagglutinated red blood cells (RBC) pass easily through a gel, while agglutinated RBC do not. The introduction of the GT to our blood bank transfusion routine [strictly following the manufacturers instructions (DiaMed ID Micro Typing System)] resulted in problems with the interpretation of the results. These were overcome after the introduction of modifications, which included; (1) the systematic use of 1% RBC suspensions; (2) the use of 50 μ1 of 1% RBC suspensions and 25 μ1 of serum in all tests; (3) the control of all negative indirect antiglobulin tests (IAT) and direct antiglobulin tests (DAT) by the addition of 50 μ1 of a 1% IgG coated RBC suspension followed by centrifugation; and (4) the systematic use of saline‐suspended RBC for ABO typing in patients with positive DAT.

Genomic mechanisms underlying PARK2 large deletions identified in a cohort of patients with PD

Objectives: To identify the genomic mechanisms that result in PARK2 large gene deletions. Methods: We conducted mutation screening using PCR amplification of PARK2-coding regions and exon-intron boundaries, followed by sequencing to evaluate a large series of 244 unrelated Portuguese patients with symptoms of Parkinson disease. For the detection of large gene rearrangements, we performed multiplex ligation-dependent probe amplification, followed by long-range PCR and sequencing to map deletion breakpoints. Results: We identified biallelic pathogenic parkin mutations in 40 of the 244 patients. There were 18 different mutations, some of them novel. This study included mapping of 17 deletion breakpoints showing that nonhomologous end joining is the most common mechanism responsible for these gene rearrangements. None of these deletion breakpoints were previously described, and only one was present in 2 unrelated families, indicating that most of the deletions result from independent events. Conclusions: The c.155delA mutation is highly prevalent in the Portuguese population (62.5% of the cases). Large deletions were present in 42.5% of the patients. We present the largest study on the molecular mechanisms that mediate PARK2 deletions in a homogeneous population.

Massive sequencing of 70 genes reveals a myriad of missing genes or mechanisms to be uncovered in hereditary spastic paraplegias

Hereditary spastic paraplegias (HSP) are neurodegenerative disorders characterized by lower limb spasticity and weakness that can be complicated by other neurological or non-neurological signs. Despite a high genetic heterogeneity (>60 causative genes), 40–70% of the families remain without a molecular diagnosis. Analysis of one of the pioneer cohorts of 193 HSP families generated in the early 1990s in Portugal highlighted that SPAST and SPG11 are the most frequent diagnoses. We have now explored 98 unsolved families from this series using custom next generation sequencing panels analyzing up to 70 candidate HSP genes. We identified the likely disease-causing variant in 20 of the 98 families with KIF5A being the most frequently mutated gene. We also found 52 variants of unknown significance (VUS) in 38% of the cases. These new diagnoses resulted in 42% of solved cases in the full Portuguese cohort (81/193). Segregation of the variants was not always compatible with the presumed inheritance, indicating that the analysis of all HSP genes regardless of the inheritance mode can help to explain some cases. Our results show that there is still a large set of unknown genes responsible for HSP and most likely novel mechanisms or inheritance modes leading to the disease to be uncovered, but this will require international collaborative efforts, particularly for the analysis of VUS.

Circulating endothelial cells in patients with venous thromboembolism and myeloproliferative neoplasms.

Background Circulating endothelial cells (CEC) may be a biomarker of vascular injury and pro-thrombotic tendency, while circulating endothelial progenitor cells (CEP) may be an indicator for angiogenesis and vascular remodelling. However, there is not a universally accepted standardized protocol to identify and quantify these cells and its clinical relevancy remains to be established. Objectives To quantify CEC and CEP in patients with venous thromboembolism (VTE) and with myeloproliferative neoplasms (MPN), to characterize the CEC for the expression of activation (CD54, CD62E) and procoagulant (CD142) markers and to investigate whether they correlate with other clinical and laboratory data. Patients and Methods Sixteen patients with VTE, 17 patients with MPN and 20 healthy individuals were studied. The CEC and CEP were quantified and characterized in the blood using flow cytometry, and the demographic, clinical and laboratory data were obtained from hospital records. Results We found the CEC counts were higher in both patient groups as compared to controls, whereas increased numbers of CEP were found only in patients with MPN. In addition, all disease groups had higher numbers of CD62E+ CEC as compared to controls, whereas only patients with VTE had increased numbers of CD142+ and CD54+ CEC. Moreover, the numbers of total and CD62+ CEC correlated positively with the white blood cells (WBC) counts in both groups of patients, while the numbers of CEP correlated positively with the WBC counts only in patients with MPN. In addition, in patients with VTE a positive correlation was found between the numbers of CD54+ CEC and the antithrombin levels, as well as between the CD142+ CEC counts and the number of thrombotic events. Conclusions Our study suggests that CEC counts may reveal endothelial injury in patients with VTE and MPN and that CEC may express different activation-related phenotypes depending on the disease status.

Soluble endothelial cell molecules and circulating endothelial cells in patients with venous thromboembolism

&NA; To evaluate the plasma levels of soluble endothelial cell molecules in patients with venous thromboembolism (VTE) out of the acute phase as compared with healthy individuals. We also investigated the possible associations of the soluble endothelial cell molecules among them, as well as with other clinical and laboratory data, including the numbers of circulating endothelial cells (CEC), circulating endothelial progenitor cells (CEP), and CEC expressing activation-related [cluster of differentiation (CD)54 and CD62E] and procoagulant (CD142) markers. In total, 15 patients with VTE and 20 normal individuals were studied. The CEC and CEP were quantified and characterized by flow cytometry. The soluble molecules studied included P-selectin, E-selectin, intercellular cell adhesion molecule 1, vascular cell adhesion molecule 1 and tissue factor (ELISA), and von Willebrand factor antigen (immunoturbidimetry). VTE patients had significantly higher levels of vascular cell adhesion molecule 1 and von Willebrand factor antigen and lower levels of soluble E-selectin than controls. They also showed significantly higher numbers of CEC, as of activated/procoagulant CEC and lower numbers of CEP, compared with controls. We did not find any correlation between the levels of soluble molecules and the numbers of endothelial cell in circulation, but there was with several clinical and laboratory data in VTE patients. Our results would suggest that in VTE patients, the endothelium remains activated and in some hypercoagulable state. The levels of soluble endothelial cell molecules did not seem to be directly related to the numbers of CEC and CEP neither reflected the number of activated CEC, which may be because of the different function that surface and soluble molecules may have.

Biomechanics, energetics and coordination during extreme swimming intensity: effect of performance level

ABSTRACT The present study aimed to examine how high- and low-speed swimmers organise biomechanical, energetic and coordinative factors throughout extreme intensity swim. Sixteen swimmers (eight high- and eight low-speed) performed, in free condition, 100-m front crawl at maximal intensity and 25, 50 and 75-m bouts (at same pace as the previous 100-m), and 100-m maximal front crawl on the measuring active drag system (MAD-system). A 3D dual-media optoelectronic system was used to assess speed, stroke frequency, stroke length, propelling efficiency and index of coordination (IdC), with power assessed by MAD-system and energy cost by quantifying oxygen consumption plus blood lactate. Both groups presented a similar profile in speed, power output, stroke frequency, stroke length, propelling efficiency and energy cost along the effort, while a distinct coordination profile was observed (F(3, 42) = 3.59, P = 0.04). Speed, power, stroke frequency and propelling efficiency (not significant, only a tendency) were higher in high-speed swimmers, while stroke length and energy cost were similar between groups. Performing at extreme intensity led better level swimmers to achieve superior speed due to higher power and propelling efficiency, with consequent ability to swim at higher stroke frequencies. This imposes specific constraints, resulting in a distinct IdC magnitude and profile between groups.

Large-Scale Functional RNAi Screen in C. elegans Identifies TGF-β and Notch Signaling Pathways as Modifiers of CACNA1A

Variants in CACNA1A that encodes the pore-forming α1-subunit of human voltage-gated Cav2.1 (P/Q-type) Ca2+ channels cause several autosomal-dominant neurologic disorders, including familial hemiplegic migraine type 1, episodic ataxia type 2, and spinocerebellar ataxia type 6. To identify modifiers of incoordination in movement disorders, we performed a large-scale functional RNAi screen, using the Caenorhabditis elegans strain CB55, which carries a truncating mutation in the unc-2 gene, the worm ortholog for the human CACNA1A. The screen was carried out by the feeding method in 96-well liquid culture format, using the ORFeome v1.1 feeding library, and time-lapse imaging of worms in liquid culture was used to assess changes in thrashing behavior. We looked for genes that, when silenced, either ameliorated the slow and uncoordinated phenotype of unc-2, or interacted to produce a more severe phenotype. Of the 350 putative hits from the primary screen, 37 genes consistently showed reproducible results. At least 75% of these are specifically expressed in the C. elegans neurons. Functional network analysis and gene ontology revealed overrepresentation of genes involved in development, growth, locomotion, signal transduction, and vesicle-mediated transport. We have expanded the functional network of genes involved in neurodegeneration leading to cerebellar ataxia related to unc-2/CACNA1A, further confirming the involvement of the transforming growth factor β pathway and adding a novel signaling cascade, the Notch pathway.

Functional shoulder ratios with high velocities of shoulder internal rotation are most sensitive to determine shoulder rotation torque imbalance: a cross-sectional study with elite handball players and controls

Abstract The aim of the present study was to determine which approach to calculating shoulder ratios is the most sensitive for determining shoulder torque imbalance in handball players. Twenty-six participants (handball athletes, n = 13; healthy controls, n = 13) performed isokinetic concentric and eccentric shoulder internal rotation (IR) and external rotation (ER) assessment at 60, 180 and 300°/s. We used eight approaches to calculating shoulder ratios: four concentric (i.e. concentric ER torque divided by concentric IR torque), and four functional (i.e. eccentric ER torque divided by concentric IR torque) at the velocities of 60, 180 and 300°/s for both IR and ER, and combining 60°/s of ER and 300°/s of IR. A three factorial ANOVA (factors: shoulder ratios, upper limb sides, and groups) along with Tukey’s post-hoc analysis, and effect sizes were calculated. The findings suggested the functional shoulder ratio combining 60°/s of ER and 300°/s of IR is the most sensitive to detect differences between upper limbs for handball players, and between players and controls for the dominant side. The functional shoulder ratio combining 60°/s of ER with 300°/s of IR seems to present advantages over the other approaches for identifying upper limb asymmetries and differences in shoulder torque balance related to throwing.

Rare Neurodegenerative Diseases: Clinical and Genetic Update

More than 600 human disorders afflict the nervous system. Of these, neurodegenerative diseases are usually characterised by onset in late adulthood, progressive clinical course, and neuronal loss with regional specificity in the central nervous system. They include Alzheimers disease and other less frequent dementias, brain cancer, degenerative nerve diseases, encephalitis, epilepsy, genetic brain disorders, head and brain malformations, hydrocephalus, stroke, Parkinsons disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS or Lou Gehrigs Disease), Huntingtons disease, and Prion diseases, among others. Neurodegeneration usually affects, but is not limited to, the cerebral cortex, intracranial white matter, basal ganglia, thalamus, hypothalamus, brain stem, and cerebellum. Although the majority of neurodegenerative diseases are sporadic, Mendelian inheritance is well documented. Intriguingly, the clinical presentations and neuropathological findings in inherited neurodegenerative forms are often indistinguishable from those of sporadic cases, suggesting that converging genomic signatures and pathophysiologic mechanisms underlie both hereditary and sporadic neurodegenerative diseases. Unfortunately, effective therapies for these diseases are scarce to non-existent. In this chapter, we highlight the clinical and genetic features associated with the rare inherited forms of neurodegenerative diseases, including ataxias, multiple system atrophy, spastic paraplegias, Parkinsons disease, dementias, motor neuron diseases, and rare metabolic disorders.