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1.  Nitisinone Arrests but Does Not Reverse Ochronosis in Alkaptonuric Mice 
JIMD Reports  2015;24:45-50.
Alkaptonuria (AKU) is an ultrarare autosomal recessive disorder resulting from a deficiency of homogentisate 1,2 dioxygenase (HGD), an enzyme involved in the catabolism of phenylalanine and tyrosine. Loss of HGD function prevents metabolism of homogentisic acid (HGA), leading to increased levels of plasma HGA and urinary excretion. Excess HGA becomes deposited in collagenous tissues and subsequently undergoes polymerisation, principally in the cartilages of loaded joints, in a process known as ochronosis. This results in an early-onset, devastating osteoarthropathy for which there is currently no effective treatment. We recently described the natural history of ochronosis in a murine model of AKU, demonstrating that deposition of ochronotic pigment begins very early in life and accumulates with age. Using this model, we were able to show that lifetime treatment with nitisinone, a potential therapy for AKU, was able to completely prevent deposition of ochronotic pigment. However, although nitisinone has been shown to inhibit ochronotic deposition, whether it can also facilitate removal of existing pigment has not yet been examined. We describe here that midlife administration of nitisinone to AKU mice arrests further deposition of ochronotic pigment in the tibiofemoral joint, but does not result in the clearance of existing pigment. We also demonstrate the dose-dependent response of plasma HGA to nitisinone, highlighting its efficacy for personalised medicine, where dosage can be tailored to the individual AKU patient.
Electronic supplementary material
The online version of this chapter (doi:10.1007/8904_2015_437) contains supplementary material, which is available to authorized users.
PMCID: PMC4582025  PMID: 25940034
2.  Age-Related Deviation of Gait from Normality in Alkaptonuria 
JIMD Reports  2015;24:39-44.
Alkaptonuria is a rare metabolic disease leading to systemic changes including early and severe arthropathy which affects mobility. For unknown reasons, the onset of degenerative changes is delayed to around 30 years of age when both objective and subjective symptoms develop. In order to complement description of the structural changes in alkaptonuria with measures of movement function, clinical gait analysis was added to the list of assessments in 2013. The aim of this study was to describe the deviation of gait from normality as a function of age in patients with alkaptonuria. Three-dimensional movement of reflective markers attached to joints were captured during walking in 39 patients and 10 controls. Subsequent to processing the data to emphasise the shape of marker trajectories, the mean Movement Deviation Profile was generated for all participants. This single number measure gives the deviation of a patient’s gait from a distributed definition of gait normality. Results showed that gait deviation roughly follows a sigmoid profile with minimal increase of gait deviations in a younger patient group and an abrupt large increase around the second half of the 4th decade of life. Larger variations of gait deviations were found in the older group than in the younger group suggesting a complex interaction of multiple factors which determine gait function after symptoms manifest. Continued gait analysis of adults with AKU, extended to younger adults and children with AKU, is expected to complete understanding of both the natural history of alkaptonuria and how interventions can affect movement function.
PMCID: PMC4582030  PMID: 25786642
3.  Relationship Between Serum Concentrations of Nitisinone and Its Effect on Homogentisic Acid and Tyrosine in Patients with Alkaptonuria 
JIMD Reports  2015;24:21-27.
Background: Alkaptonuria (AKU) is a serious genetic disease due to a defect in tyrosine metabolism, leading to increased serum levels of homogentisic acid (HGA). Nitisinone decreases HGA in AKU, but the concentration–response relationship has not been previously reported.
Objectives: To determine the relationship between serum concentrations of nitisinone and the effect on both HGA and tyrosine; secondly to determine steady-state pharmacokinetics of nitisinone in AKU patients.
Method: Thirty-two patients with AKU received either 1, 2, 4, or 8 mg nitisinone daily. Urine and serum HGA and serum tyrosine and nitisinone were measured during 24 h at baseline (before first dose) and after 4 weeks of treatment.
Results: Nitisinone pharmacokinetics (area under the curve [AUC] and maximum concentrations [Cmax]) were dose proportional. The median oral clearance determined in all patients, irrespective of dose, was 3.18 mL/h·kg (range 1.6–6.7).
Nitisinone decreased urinary excretion of HGA in a concentration-dependent manner, with a maximum effect seen at average nitisinone concentrations of 3 μmol/L. The association between nitisinone and tyrosine concentrations was less pronounced. Serum levels of HGA at Week 4 were below the limit of quantitation in 65% of samples, which prevented determination of the relationship with nitisinone concentrations.
Conclusion: Nitisinone exhibits dose-proportional pharmacokinetics in the studied dosage interval. Urinary excretion of HGA decreases in a concentration-dependent manner, while the increase in tyrosine is less clearly related to nitisinone concentrations.
Electronic supplementary material
The online version of this chapter (doi:10.1007/8904_2015_412) contains supplementary material, which is available to authorized users.
PMCID: PMC4582028  PMID: 25772318
4.  Analysis of HGD Gene Mutations in Patients with Alkaptonuria from the United Kingdom: Identification of Novel Mutations 
JIMD Reports  2015;24:3-11.
Alkaptonuria (AKU) is a rare autosomal recessive disorder with incidence ranging from 1:100,000 to 1:250,000. The disorder is caused by a deficiency of the enzyme homogentisate 1,2-dioxygenase (HGD), which results from defects in the HGD gene. This enzyme converts homogentisic acid to maleylacetoacetate and has a major role in the catabolism of phenylalanine and tyrosine. To elucidate the mutation spectrum of the HGD gene in patients with alkaptonuria from 42 patients attending the National Alkaptonuria Centre, 14 exons of the HGD gene and the intron–exon boundaries were analysed by PCR-based sequencing. A total of 34 sequence variants was observed, confirming the genetic heterogeneity of AKU. Of these mutations, 26 were missense substitutions and four splice site mutations. There were two deletions and one duplication giving rise to frame shifts and one substitution abolishing the translation termination codon (no stop). Nine of the mutations were previously unreported novel variants. Using computational approaches based on the 3D structure, these novel mutations are predicted to affect the activity of the protein complex through destabilisation of the individual protomer structure or through disruption of protomer–protomer interactions.
Electronic supplementary material: The online version of this chapter (doi:10.1007/8904_2014_380) contains supplementary material, which is available to authorized users.
PMCID: PMC4582018  PMID: 25681086
Alkaptonuria; Homogentisic acid; Novel mutation; Rare genetic disorder; Sequencing
5.  Receptors and effects of gut hormones in three osteoblastic cell lines 
BMC Physiology  2011;11:12.
In recent years the interest on the relationship of gut hormones to bone processes has increased and represents one of the most interesting aspects in skeletal research. The proportion of bone mass to soft tissue is a relationship that seems to be controlled by delicate and subtle regulations that imply "cross-talks" between the nutrient intake and tissues like fat. Thus, recognition of the mechanisms that integrate a gastrointestinal-fat-bone axis and its application to several aspects of human health is vital for improving treatments related to bone diseases. This work analysed the effects of gut hormones in cell cultures of three osteoblastic cell lines which represent different stages in osteoblastic development. Also, this is the first time that there is a report on the direct effects of glucagon-like peptide 2, and obestatin on osteoblast-like cells.
mRNA expression levels of five gut hormone receptors (glucose-dependent insulinotropic peptide [GIP], glucagon-like peptide 1 [GLP-1], glucagon-like peptide 2 [GLP-2], ghrelin [GHR] and obestatin [OB]) were analysed in three osteoblastic cell lines (Saos-2, TE-85 and MG-63) showing different stages of osteoblast development using reverse transcription and real time polymerase chain reaction. The responses to the gut peptides were studied using assays for cell viability, and biochemical bone markers: alkaline phosphatase (ALP), procollagen type 1 amino-terminal propeptides (P1NP), and osteocalcin production.
The gut hormone receptor mRNA displayed the highest levels for GIP in Saos-2 and the lowest levels in MG-63, whereas GHR and GPR39 (the putative obestatin receptor) expression was higher in TE-85 and MG-63 and lower in Saos-2. GLP-1 and GLP-2 were expressed only in MG-63 and TE-85. Treatment of gut hormones to cell lines showed differential responses: higher levels in cell viability in Saos-2 after GIP, in TE-85 and MG-63 after GLP-1, GLP-2, ghrelin and obestatin. ALP showed higher levels in Saos-2 after GIP, GHR and OB and in TE-85 after GHR. P1NP showed higher levels after GIP and OB in Saos-2. Decreased levels of P1NP were observed in TE-85 and MG-63 after GLP-1, GLP-2 and OB. MG-63 showed opposite responses in osteocalcin levels after GLP-2.
These results suggest that osteoblast activity modulation varies according to different development stage under different nutrition related-peptides.
PMCID: PMC3162581  PMID: 21801348

Results 1-5 (5)