No abstract

The study of Lesch-Nyhan-diseased (LND) human brain is crucial for understanding how mutant hypoxanthine-phosphoribosyltransferase (HPRT) might lead to neuronal dysfunction. Since LND is a rare, inherited disorder caused by a deficiency of the enzyme HPRT, human neural stem cells (hNSCs) that carry this mutation are a precious source for delineating the consequences of HPRT deficiency and for developing new treatments. In our study we have examined the effect of HPRT deficiency on the differentiation of neurons in hNSCs isolated from human LND fetal brain. We have examined the expression of a number of transcription factors essential for neuronal differentiation and marker genes involved in dopamine (DA) biosynthetic pathway. LND hNSCs demonstrate aberrant expression of several transcription factors and DA markers. HPRT-deficient dopaminergic neurons also demonstrate a striking deficit in neurite outgrowth. These results represent direct experimental evidence for aberrant neurogenesis in LND hNSCs and suggest developmental roles for other housekeeping genes in neurodevelopmental disease. Moreover, exposure of the LND hNSCs to retinoic acid medium elicited the generation of dopaminergic neurons. The lack of precise understanding of the neurological dysfunction in LND has precluded development of useful therapies. These results evidence aberrant neurogenesis in LND hNSCs and suggest a role for HPRT gene in neurodevelopment. These cells combine the peculiarity of a neurodevelopmental model and a human, neural origin to provide an important tool to investigate the pathophysiology of HPRT deficiency and more broadly demonstrate the utility of human neural stem cells for studying the disease and identifying potential therapeutics.

Regulation of adipocyte differentiation is an important process in the control of adipose tissue development. So far, adipogenesis has been investigated through the use of various experimental models. In this work, we used human mesenchymal stem cells (hMSCs) obtained from amniotic fluid (AF) as an alternative model more representative of what naturally happens in vivo. In our opinion, these hMSCs are still not influenced by differentiation stimuli and could act in a way more correspondent to the physiological process of adipogenesis, representing also an ethically acceptable alternative to totipotent human embryonic stem cells (ES). Adipocyte differentiation was monitorated following the expressions of key genes. We measured the expression levels of PPARgamma2, PPARgamma-C1alpha, UCP-1, adipsin, and leptin genes using quantitative real-time PCR. We tested our experimental model with two different media. Understanding in vivo adipogenesis mechanisms will shed light on the pathophysiology of many diseases.

Through this study, it was determined that the deletion mechanisms in the AVPR2 region do not follow the rules of non-allelic homologous recombination. Two of the 13 deletions can be attributed to the fork stalling and template switching (FoSTeS) mechanism, whereas the remaining 11 deletions could be caused either by non-homologous end joining or by the FoSTeS mechanism. Although no recurrence was found, several groupings of deletion breakpoints were identified.

ATP7A is a copper-transporting P-type adenosine triphosphatase whose loss of function leads to the Menkes disease, an X-linked copper metabolism multi-organ disorder (1 in 100.000 births). Here we document our experience with the ATP7A linked diseases in Italy. We analyzed the exonic structure of the ATP7A gene in 25 unrelated Italian families and studied the variants of unknown significance. We identified 22 different DNA alterations, 13 of which first reported in this study. The classical Menkes phenotype was present in 21 of the 25 families and was linked with highly damaging mutations (7 nonsense; 4 frame-shift; 2 small in-frame deletions, 2 splice site alterations, 2 gross deletions, and 1 gross duplication). Of the 4 cases with milder variants of the Menkes disease two had a missense mutation, one a leaky splice site alteration and one a nonsense mutation in exon 22. We determined in silico that all the mutations leading to the classical Menkes disease leave no residual activity of ATP7A including the apparently less severe in-frame deletions. Whereas milder forms of the disease are characterized by mutations that allow a limited residual activity of ATP7A, including the nonsense mutation observed.