A functional common polymorphism in the vitamin D-responsive element of the GH1 promoter contributes to isolated growth hormone deficiency.

CONTEXT: Causal mutations have been detected only in a minority of isolated GH deficiency (IGHD) patients. Idiopathic IGHD might be the result of the interaction between several low-penetrance genetic factors and the environment. OBJECTIVE: The aim of this study was to test the contribution to IGHD of genetic variations in the GH1 gene regulatory regions. DESIGN AND PATIENTS: A case-control association study was performed including 118 sporadic IGHD patients with a nonsevere phenotype (height -4/-1 sd score and partial GH deficiency) and two control groups, normal stature (n=200) and short-stature individuals with normal GH secretion (n=113). Seven single-nucleotide polymorphisms in the GH1 promoter, one in the IVS4 region, and two in the locus control region were analyzed. RESULTS: The -57T allele within the vitamin D-responsive element showed a positive significant association when comparing patients with normal (P=0.006) or short stature (P=0.0011) controls. The genotype -57TT showed an odds ratio of 2.93 (1.44-5.99) and 2.99 (1.42-6.31), respectively. The functional relevance of the -57 variation was demonstrated by the luciferase assay in the presence of vitamin D. The vitamin D-induced inhibition of luciferase activity was significantly (P=0.012) stronger for the promoter haplotype carrying the associated variation -57T [haplotype #1 (hp#1)] with respect to hp#2, bearing -57G. Replacement of the T with a G at -57 on hp#1 abolished the repression, demonstrating that the T at position -57 is necessary to determine the greater vitamin D-induced inhibitory effect of hp#1. EMSA experiments showed a different band-shift pattern of the T and G sequences. CONCLUSION: The common -57G-->T polymorphism contributes to IGHD susceptibility, indicating that it may have a multifactorial etiology.

Pervasive supply of therapeutic lysosomal enzymes in the CNS of normal and Krabbe-affected non-human primates by intracerebral lentiviral gene therapy.

Metachromatic leukodystrophy (MLD) and globoid cell leukodystrophy (GLD or Krabbe disease) are severe neurodegenerative lysosomal storage diseases (LSD) caused by arylsulfatase A (ARSA) and galactosylceramidase (GALC) deficiency, respectively. Our previous studies established lentiviral gene therapy (GT) as a rapid and effective intervention to provide pervasive supply of therapeutic lysosomal enzymes in CNS tissues of MLD and GLD mice. Here, we investigated whether this strategy is similarly effective in juvenile non-human primates (NHP). To provide proof of principle for tolerability and biological efficacy of the strategy, we established a comprehensive study in normal NHP delivering a clinically relevant lentiviral vector encoding for the human ARSA transgene. Then, we injected a lentiviral vector coding for the human GALC transgene in Krabbe-affected rhesus macaques, evaluating for the first time the therapeutic potential of lentiviral GT in this unique LSD model. We showed favorable safety profile and consistent pattern of LV transduction and enzyme biodistribution in the two models, supporting the robustness of the proposed GT platform. We documented moderate inflammation at the injection sites, mild immune response to vector particles in few treated animals, no indication of immune response against transgenic products, and no molecular evidence of insertional genotoxicity. Efficient gene transfer in neurons, astrocytes, and oligodendrocytes close to the injection sites resulted in robust production and extensive spreading of transgenic enzymes in the whole CNS and in CSF, leading to supraphysiological ARSA activity in normal NHP and close to physiological GALC activity in the Krabbe NHP, in which biological efficacy was associated with preliminary indication of therapeutic benefit. These results support the rationale for the clinical translation of intracerebral lentiviral GT to address CNS pathology in MLD, GLD, and other neurodegenerative LSD.

Functional SNPs within the intron 1 of the PROP1 gene contribute to combined growth hormone deficiency (CPHD).

CONTEXT: Mutations within the PROP1 gene represent one of the main causes of familial combined pituitary hormone deficiency (CPHD). However, most of the cases are sporadic with an unknown genetic cause. OBJECTIVE: The aim of this study was the search for low penetrance variations within and around a conserved regulatory element in the intron 1 of PROP1, contributing to a multifactorial form of the disease in sporadic patients. METHODS AND PATIENTS: A fragment of 570 bp encompassing the conserved region was sequenced in 107 CPHD patients and 294 controls, and an association study was performed with the four identified variants, namely c.109+435G>A (rs73346254), c.109+463C>T (rs4498267), c.109+768C>G (rs4431364), and c.109+915_917ins/delTAG (rs148607624). The functional role of the associated polymorphisms was evaluated by luciferase reporter gene expression analyses and EMSA. RESULTS: A statistically significant increased frequency was observed in the patients for rs73346254A (P = 5 × 10(-4)) and rs148607624delTAG (P = 0.01) alleles. Among all the possible allele combinations, only the haplotype bearing both risk alleles showed a significantly higher frequency in the patients vs. controls (P = 4.7 × 10(-4)) and conferred a carrier risk of 4.19 (P = 1.2 × 10(-4)). This haplotype determined a significant decrease of the luciferase activity in comparison with a basal promoter and the other allelic combinations in GH4C and MCF7 cells (P = 4.6 × 10(-6); P = 5.5 × 10(-4), respectively). The EMSA showed a differential affinity for nuclear proteins for the alternative alleles of the two associated variations. CONCLUSIONS: Variations with a functional significance conferring susceptibility to CPHD have been identified in the PROP1 gene, indicating a multifactorial origin of this disorder in sporadic cases.