Topical nerve growth factor prevents neurodegenerative and vascular stages of diabetic retinopathy.

Specific and effective preventive treatment for diabetic retinopathy (DR) is presently unavailable, mostly because the early stages of the complication have been, until recently, poorly understood. The recent demonstration that the vascular phase of DR is preceded and possibly caused by the neurodegeneration of retinal ganglion cells suggests that DR could, at least theoretically, be prevented through an early neuroprotective approach. The aims of our study were to clarify the natural history of diabetes-driven retinal neurodegeneration and to verify the possibility to prevent DR using topical nerve growth factor (NGF). The results of the study show that retinal neurodegeneration, characterized by the loss of retinal ganglion cells represents a relatively early phenomenon of diabetes (between 5 and 16 weeks of age), which tends to be self-limiting in the long run. Neurodegeneration is followed by the development of DR-related vascular dysfunctions, as confirmed by the development of acellular capillaries and the loss of retinal pericytes. Both retinal neurodegeneration and subsequent vascular dysfunction can be successfully prevented by topical NGF administration. These findings suggest that: 1) The first stage of DR consists in a self-limiting retinal neurodegeneration 2) The demonstrated effectiveness of topical NGF in the prevention of DR could be rapidly translated into clinical practice.

Rare variants in the TREX1 gene and susceptibility to autoimmune diseases.

TREX1 (DNase III) is an exonuclease involved in response to oxidative stress and apoptosis. Heterozygous mutations in TREX1 were previously observed in patients with systemic lupus erythematosus (SLE) and Sjögren's syndrome (SS). We performed a mutational analysis of the TREX1 gene on three autoimmune diseases: SLE (210 patients) and SS (58 patients), to confirm a TREX1 involvement in the Italian population, and systemic sclerosis (SSc, 150 patients) because it shares similarities with SLE (presence of antinuclear antibodies and connective tissue damage). We observed 7 variations; two of these are novel nonsynonymous variants (p.Glu198Lys and p.Met232Val). They were detected in one SS and in one SSc patient, respectively, and in none of the 200 healthy controls typed in this study and of the 1712 published controls. In silico analysis predicts a possibly damaging role on protein function for both variants. The other 5 variations are synonymous and only one of them is novel (p.Pro48Pro). This study contributes to the demonstration that TREX1 is involved in autoimmune diseases and proposes that the spectrum of involved autoimmune diseases can be broader and includes SSc. We do not confirm a role of TREX1 variants in SLE.

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.

ATXN-2 CAG repeat expansions are interrupted in ALS patients.

It has recently been suggested that short expansions of CAG repeat in the gene ATXN-2 causing SCA2 (spinocerebellar ataxia type 2) are associated with an increased risk of amyotrophic lateral sclerosis (ALS) in the populations of the USA and northern Europe. In this study, we investigated the role of ATXN-2 in Italian patients clinically diagnosed with ALS and characterized the molecular structure of ATXN-2 expansions. We assessed the size of the CAG repeat in ATXN-2 exon 1 in 232 Italian ALS patients and 395 matched controls. ATXN-2 expanded alleles containing > 30 repeats have been observed in seven sporadic ALS patients (3.0%), while being absent in the controls (p = 0.00089). Four out of the seven patients had an ATXN-2 allele in the intermediate-fully pathological range: one with 32 repeats, 2 with 33 repeats and 1 with 37 repeats, accounting for 1.7% of the ALS cohort. Sequencing of expanded (> 32) alleles showed that they were all interrupted with at least one CAA triplet. ATXN-2 alleles with the same length and structure have been reported in SCA2 patients with parkinsonism or in familial and sporadic Parkinson. Conversely, the phenotype of the present patients was typically ALS with no signs or symptoms of ataxia or parkinsonism. In conclusion, the findings of ATXN-2 expansions in pure ALS cases suggest that ALS may be a third phenotype (alongside ataxia/parkinsonism and pure Parkinson) associated with ATXN-2 interrupted alleles.

A novel HESX1 splice mutation causes isolated GH deficiency by interfering with mRNA processing.

OBJECTIVE: Mutations in HESX1 represent a rare cause of GH deficiency (GHD) associated with a broad spectrum of other anomalies. We searched for causative mutations in a cohort of 244 Italian patients affected by combined and isolated GHD (IGHD). METHODS: The HESX1 gene-coding region and exon-intron boundaries were screened by denaturing HPLC scanning. RESULTS: A novel mutation adjacent to the invariant donor splice site of intron 2 (c.357+3G>A) was identified at the heterozygous state in an IGHD patient. The in vitro and in vivo mRNA analysis of the wild-type HESX1 allele revealed the presence of the whole cDNA and two isoforms lacking exon 2 and exons 2-3 respectively. The mutant HESX1 allele yielded only two splicing products, the whole cDNA and the cDNA missing exons 2-3, whereas the mRNA lacking exon 2 was absent. An in vitro assay demonstrated that the exon 2-deleted mRNA, predicting a prematurely truncated protein, is subjected to nonsense-mediated mRNA decay (NMD). CONCLUSIONS: The c.357+3G>A mutation prevents the generation of one of the alternative isoforms normally produced by the wild-type allele, predicting a truncated HESX1 protein. The mutation is likely to cause IGHD in the heterozygous patient by interfering with the downregulation of HESX1 expression mediated by alternative splicing and NMD. Our results open new insight into the mechanism of HESX1 regulation suggesting that the coupling of alternative splicing and NMD might play a fundamental role in directing the HESX1 expression, and that the alteration of this process might lead to severe consequences.

A novel peripherin gene (PRPH) mutation identified in one sporadic amyotrophic lateral sclerosis patient.

Motor neurons in amyotrophic lateral sclerosis (ALS) are characterized by the presence of inclusion bodies composed of intermediate filament (IF) proteins. Peripherin protein is as components of these inclusions and rare mutations in peripherin gene (PRPH) were identified in sporadic ALS cases. The aim of this study was to further define the spectrum of PRPH mutations in a cohort of 122 Italian ALS patients. We screened the coding sequence, the exon/intron boundaries, and the 5'-3' un-translated regions (UTRs) in 122 ALS patients. Eighteen sequence variations were detected. Seven variants were not identified in a panel of at least 245 matched controls, including 2 missense variations, namely p.R133P and p.D141Y, each identified in one heterozygous patient. p.R133P was newly identified whereas p.D141Y was previously described in one homozygous sporadic ALS patient. These 2 variants were predicted to have a deleterious effect on protein structure or function. This work contributes to determine the role of PRPH gene variants in ALS. Further studies are necessary to define the mechanisms through which the mutant peripherin could cause ALS phenotype.

Growth hormone (GH)-releasing hormone increases the expression of the dominant-negative GH isoform in cases of isolated GH deficiency due to GH splice-site mutations.

An autosomal dominant form of isolated GH deficiency (IGHD II) can result from heterozygous splice site mutations that weaken recognition of exon 3 leading to aberrant splicing of GH-1 transcripts and production of a dominant-negative 17.5-kDa GH isoform. Previous studies suggested that the extent of missplicing varies with different mutations and the level of GH expression and/or secretion. To study this, wt-hGH and/or different hGH-splice site mutants (GH-IVS+2, GH-IVS+6, GH-ISE+28) were transfected in rat pituitary cells expressing human GHRH receptor (GC-GHRHR). Upon GHRH stimulation, GC-GHRHR cells coexpressing wt-hGH and each of the mutants displayed reduced hGH secretion and intracellular GH content when compared with cells expressing only wt-hGH, confirming the dominant-negative effect of 17.5-kDa isoform on the secretion of 22-kDa GH. Furthermore, increased amount of 17.5-kDa isoform produced after GHRH stimulation in cells expressing GH-splice site mutants reduced production of endogenous rat GH, which was not observed after GHRH-induced increase in wt-hGH. In conclusion, our results support the hypothesis that after GHRH stimulation, the severity of IGHD II depends on the position of splice site mutation leading to the production of increasing amounts of 17.5-kDa protein, which reduces the storage and secretion of wt-GH in the most severely affected cases. Due to the absence of GH and IGF-I-negative feedback in IGHD II, a chronic up-regulation of GHRH would lead to an increased stimulatory drive to somatotrophs to produce more 17.5-kDa GH from the severest mutant alleles, thereby accelerating autodestruction of somatotrophs in a vicious cycle.

Growth hormone (GH) deficiency type II: a novel GH-1 gene mutation (GH-R178H) affecting secretion and action.

CONTEXT AND OBJECTIVE: Main features of the autosomal dominant form of GH deficiency (IGHD II) include markedly reduced secretion of GH combined with low concentrations of IGF-I leading to short stature. DESIGN, SETTING, AND PATIENTS: A female patient presented with short stature (height -6.0 sd score) and a delayed bone age of 2 yr at the chronological age of 5 yr. Later, at the age of 9 yr, GHD was confirmed by standard GH provocation test, which revealed subnormal concentrations of GH and a very low IGF-I. Genetic analysis of the GH-1 gene revealed the presence of a heterozygous R178H mutation. INTERVENTIONS AND RESULTS: AtT-20 cells coexpressing both wt-GH and GH-R178H showed a reduced GH secretion after forskolin stimulation compared with the cells expressing only wt-GH, supporting the diagnosis of IGHD II. Because reduced GH concentrations found in the circulation of our untreated patient could not totally explain her severe short stature, functional characterization of the GH-R178H performed by studies of GH receptor binding and activation of the Janus kinase-2/signal transducer and activator of transcription-5 pathway revealed a reduced binding affinity of GH-R178H for GH receptor and signaling compared with the wt-GH. CONCLUSION: This is the first report of a patient suffering from short stature caused by a GH-1 gene alteration affecting not only GH secretion (IGHD II) but also GH binding and signaling, highlighting the necessity of functional analysis of any GH variant, even in the alleged situation of IGHD II.

A recurrent signal peptide mutation in the growth hormone releasing hormone receptor with defective translocation to the cell surface and isolated growth hormone deficiency.

CONTEXT: Mutations in the GHRH receptor (GHRHR) have been detected in the familial type-IB isolated GH deficiency (IGHD-IB) inherited as an autosomal recessive disorder and characterized by a low but detectable serum GH level and good response to substitutive GH therapy. OBJECTIVE: The aim of our study was the identification of mutations in sporadic patients with a IGHD-IB phenotype. SUBJECTS AND METHODS: The GHRHR gene was systematically screened by DHPLC in 134 IGHD patients with no family history of the disorder or declared parental consanguinity. RESULTS: We identified a novel variation, Val10Gly, within the signal peptide at the heterozygous state in three patients and in one of 1084 controls (P = 0.004), suggesting that it might contribute to IGHD. The functional analysis showed that the signal peptide is not cleaved from the mutant GHRHR, which in turn is not translocated to the cellular surface, demonstrating that 10Gly drastically affects the receptor correct processing. Because 10Gly was also present in normal-stature relatives of the patients as well as in a control, it is likely that it exerts its effects in the context of other genetic and environmental susceptibility factors. CONCLUSION: At difference from previous papers reporting GHRHR mutations in familial cases with a clear recessive mode of inheritance, our study was conducted on a large sample of sporadic patients and allowed to discover a novel mechanism of the disease caused by a recurrent dominant mutation in the GHRHR signal peptide associated with incomplete penetrance.

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.

A variation in a Pit-1 site in the growth hormone gene (GH1) promoter induces a differential transcriptional activity.

The proximal promoter of the human growth hormone gene (GH1) is highly polymorphic. We tested if promoter haplotypes differing at possibly functional sites, namely -278T/G (in the NF1 binding site), -75A/G (in the proximal Pit-1 binding site) and -57G/T (in the VDR binding site), induced a different luciferase activity when transfected in a rat pituitary cell line. The presence of a G instead of an A at position -75 induced a more than two-fold reduced activity (p<0.0001). In accordance with this findings the electrophoretic mobility shift assay demonstrated a reduced affinity of the -75G for the pituitary transcription factor Pit-1. Despite the strong effect of this polymorphism in vitro, the -75G variation was not associated to an impairment of the GH secretion in vivo.

The natural history of Cri du Chat Syndrome. A report from the Italian Register.

The aim of this report is to provide an update on the natural history of the Cri du Chat Syndrome by means of the Italian Register (I.R.). Two hundred twenty patients were diagnosed by standard cytogenetic methods and 112 of these were also characterised by molecular-cytogenetic investigation (FISH). FISH analysis showed interstitial deletions, short terminal deletions and other rare rearrangements not previously correctly diagnosed by standard cytogenetics. The diagnosis was made in the first month of life in 42% and within first year in 82% of cases. The remaining 18% were diagnosed at an age ranging from 13 months to 47 years. At the last follow-up, patient age ranged from 8 months to 61 years. Mortality, already low, has decreased over time as it is lower between 1984-2002 compared to 1965-1983. Mortality was higher in patients with unbalanced translocations resulting in 5p deletions. Our data confirm that the cat-like cry and peculiar timbre of voice are the most typical signs of the syndrome, not only at birth but also later and these are the only signs which might suggest the diagnosis in patients with small deletions and mild clinical picture. A cytogenetic and clinical variability must be underlined. Cardiac, cerebral, renal and gastrointestinal malformations were more frequent in the patients with unbalanced translocations resulting in 5p deletions. Sucking and feeding difficulties and respiratory infections are frequent in the first months or years of life. Intubation difficulties linked to larynx anomalies must be considered. Psychomotor development is delayed in all patients but there is a variability related to deletion size and type as well as other genetic and environmental factors. However, the results showed an improvement in the acquisition of the development skills and progress in social introduction which should encourage caregivers and parents to work together in carrying out the rehabilitative and educational interventions.

A novel deletion in the GH1 gene including the IVS3 branch site responsible for autosomal dominant isolated growth hormone deficiency.

CONTEXT: The majority of mutations responsible for isolated GH type II deficiency (IGHD II) lead to dominant negative deleteriously increased levels of the GH1 exon 3 skipped transcripts. OBJECTIVE: The aim of this study was the characterization of the molecular defect causing a familial case of IGHD II. PATIENTS: A 2-yr-old child and her mother with severe growth failure at diagnosis (-5.8 and -6.9 sd score, respectively) and IGHD were investigated for the presence of GH1 mutations. RESULTS: We identified a novel 22-bp deletion in IVS3 (IVS3 del+56-77) removing the putative branch point sequence (BPS). Analysis of patients' lymphocyte mRNA showed an excess exon 3 skipping. The mutated allele transfected into rat pituitary cells produced four differently spliced products: the exon 3 skipped mRNA as the main product and lower amounts of the full-length cDNA and of two novel mRNA aberrant isoforms, one with the first 86 bases of exon 4 deleted and the other lacking the entire exon 4. A mutagenized construct lacking exclusively the 7 bp of the BPS only generated the exon 4 skipped and the full-length isoforms. The presence of the full-length transcript in the absence of the canonical BPS points to an alternative BPS in IVS3. CONCLUSION: The IVS3 del+56-77 mutation, causing IGHD II in this family, has two separate effects on mRNA processing: 1) exon 3 skipping, analogous to most described cases of IGHD II, an effect likely caused by the reduction in size of the IVS3, and 2) partial or total exon 4 skipping, as a result of the removal of the BPS.