ZNF687 mutations are frequently found in pagetic patients from South Italy: implication in the pathogenesis of Paget's disease of bone.

Paget's disease of bone (PDB) is a skeletal disorder whose molecular basis is not fully elucidated. However, 10% of patients show a familial PDB and 35% of them carry mutations in the SQSTM1 gene. We recently reported a founder mutation (p.Pro937Arg) in the ZNF687 gene, underlying PDB complicated by giant cell tumor (GCT/PDB) and rarely occurring in PDB patients without neoplastic degeneration. Since 80% of Italian GCT/PDB patients derive from Avellino, we hypothesized that ZNF687 mutation rate was higher in this region than elsewhere. Interestingly, our molecular analysis on 30 PDB patients showed that 33% hosted ZNF687 mutations, with the p.Pro937Arg identified in 8 familial cases. Two novel ZNF687 mutations (p.Pro665Leu and p.Gln784Glu) were detected in 2 sporadic patients. Only 2 subjects were positive for the p.Pro392Leu mutation in SQSTM1. ZNF687-mutated patients showed a severe PDB, with a remarkable number of affected sites. in vitro studies revealed that the ZNF687-mutant osteoclasts appeared as giant sized with up to 150 nuclei, never described in PDB. Finally, we also confirmed the causality of the p.Pro937Arg mutation in 4 additional GCT/PDB cases deriving from the same geographic area, indicating that PDB and GCT/PDB represent 2 sides of the same coin.

Autosomal-dominant myopia associated to a novel P4HA2 missense variant and defective collagen hydroxylation.

We recently described a complex multisystem syndrome in which mild-moderate myopia segregated as an independent trait. A plethora of genes has been related to sporadic and familial myopia. More recently, in Chinese patients severe myopia (MYP25, OMIM:617238) has been linked to mutations in P4HA2 gene. Seven family members complaining of reduced distance vision especially at dusk underwent complete ophthalmological examination. Whole-exome sequencing was performed to identify the gene responsible for myopia in the pedigree. Moderate myopia was diagnosed in the family which was associated to the novel missense variant c.1147A > G p.(Lys383Glu) in the prolyl 4-hydroxylase,alpha-polypeptide 2 (P4HA2) gene, which catalyzes the formation of 4-hydroxyproline residues in the collagen strands. In vitro studies demonstrated P4HA2 mRNA and protein reduced expression level as well as decreased collagen hydroxylation and deposition in mutated fibroblast primary cultures compared to healthy cell lines. This study suggests that P4HA2 mutations may lead to myopic axial elongation of eyeball as a consequence of quantitative and structural alterations of collagen. This is the first confirmatory study which associates a novel dominant missense variant in P4HA2 with myopia in Caucasian patients. Further studies in larger cohorts are advisable to fully clarify genotype-phenotype correlations.

Hormonal regulation and characterization of MHG30 gene, a desaturase-like gene of hamster harderian gland.

The harderian gland (HG) is an orbital gland of the vast majority of land vertebrates. In the Syrian hamster these glands display a marked sexual dimorphism. Here we present data on a male specific clone named MHG30. The MHG30 cDNA (1470 bp) has significant sequence homologies with human #15µ10#Δ6-desaturase enzymes. The expression of MHG30 has been found in male HG and in the liver of both sexes, no other tissue showing the presence of MHG30 mRNA. Castration brings the MHG30 levels below detectable level in about 7 days. In in vitro cultures of male hamster HG cells, androgens (A) determine an enhancement of MHG30 expression in a time-dependent manner. Conversely, a continuous decrement has been observed in control cells and in cells treated with A plus flutamide (F) or with A and cycloheximide (Cy). Incubation of cells in cultures supplemented with desamethason (Dex) or thyroid hormone (T3) also increases MHG30 expression while 17ß-estradiol prevents the stimulatory effect exerted by A, Dex and T3. Findings strongly suggest that the MHG30 gene could be involved in supporting the sexual dimorphism and its expression is likely triggered by a series of hormonal interactions.

Positive outcomes with negative pressure therapy over primarily closed large abdominal wall reconstruction reduces surgical site infection rates.

PURPOSE: There is a significant morbidity associated with abdominal wall reconstruction (AWR) with a need for overall improvement during the post-operative management. Scientific literature has proven the use of negative pressure therapy (NPT) in wound healing for orthopedic and cardiac surgery with limited data present on its role in AWR. The goal of this study was to examine whether primary wound events were different between patients who had primary closure with NPT versus patients who only had primary closure after AWR. METHODS: This retrospective study examined the rate of post-operative complications in all open-complex AWR that were done in a similar fashion between May 2008 and July 2011 at two large university teaching hospitals. Wound closure was stringent upon attending surgeon preference without randomization. RESULTS: There were a total of 61 patients who met inclusion criteria with an average age of 54 and 60 % were women. Thirty-two patients had primary closure and 29 patients had primary closure with NPT. The mean length of follow-up was 167 days for both groups. The type of wound closure had an effect on the rate of hernia recurrence and surgical site infections. The application of NPT leads to lower hernia recurrence rate of 25 versus 3% and the type of wound closure had a profound effect on the rate and type of SSI. CONCLUSIONS: The data presented in this study demonstrates a potential advantage for adjunctive NPT in patients undergoing AWR. There is an associated decreased incidence in the overall rate of SSI and hernia recurrence with the use of NPT in those patients undergoing AWR. These results show an advantage for adjunctive NPT.

Markers of insulin resistance and carotid atherosclerosis. A comparison of the homeostasis model assessment and triglyceride glucose index.

AIMS: The present investigation was designed to test the association between carotid atherosclerosis and two simple markers of insulin resistance, i.e. HOMA-Index and TyG-Index. MATERIALS AND METHODS: The study was performed in two different cohorts. In the first cohort, 330 individuals were enrolled. Blood pressure, lipids, glucose, waist and cigarette smoking were evaluated. HOMA-IR and TyG-Index were calculated as markers of prevalent hepatic and muscular insulin resistance respectively. Carotid atherosclerosis was assessed by Doppler ultrasonography. The association between cardiovascular risk factors, markers of insulin resistance and carotid atherosclerosis was assessed by multiple logistic regression analyses. In the second cohort, limited to the evaluation of TyG-Index, 1432 subjects were studied. RESULTS: In the first cohort, TyG-Index was significantly associated with carotid atherosclerosis in a model including age, sex, diabetes, cigarette smoking and LDL cholesterol, while HOMA-IR was not. When components of metabolic syndrome were added to the model as dichotomous variables (absent/present), TyG-Index retained its predictive power. The same result was obtained when the metabolic syndrome was added to the model (absence/presence). The association between TyG-Index and carotid atherosclerosis was confirmed in the second cohort. CONCLUSIONS: The present findings suggest that TyG-Index is better associated with carotid atherosclerosis than HOMA-IR.

Betamethasone therapy in ataxia telangiectasia: unraveling the rationale of this serendipitous observation on the basis of the pathogenesis.

Ataxia telangiectasia (A-T) is a rare autosomal recessive disorder characterized by progressive neurological dysfunction. To date, only supportive care aimed to halt the progressive neurodegeneration is available for the treatment. Recently, an improvement of neurological signs during short-term treatment with betamethasone has been reported. To date, the molecular and biochemical mechanisms by which the steroid produces such effects have not yet been elucidated. Therefore, a review of the literature was carried out to define the potential molecular and functional targets of the steroid effects in A-T. Glucocorticoids (GCs) are capable of diffusing into the CNS by crossing the blood-brain barrier (BBB) where they exert effects on the suppression of inflammation or as antioxidant. GCs have been shown to protect post-mitotic neurons from apoptosis. Eventually, GCs may also modulate synaptic plasticity. A better understanding of the mechanisms of action of GCs in the brain is needed, because in A-T during the initial phase of cell loss the neurological impairment may be rescued by interfering in the biochemical pathways. This would open a new window of intervention in this so far incurable disease.

Estrogen receptor polymorphism, estrogen content and idiopathic scoliosis in human: a possible genetic linkage.

Idiopathic scoliosis (IS) is a largely diffused disease in human population but its pathogenesis is still unknown. There is a relationship between scoliotic phenotype and the patient age, since in the early stage the pathology shows a ratio of 50% between male and female teenagers. During puberty the sex ratio is 8.4/1 (female/male), suggesting a sex-conditioned manifestation of the disease. Genetic inheritance of idiopathic scoliosis is still unclear although some authors claim for its X-linked dominant inheritance. There is large agreement in considering the IS as a sex-conditioned disease, in terms of steroid content and their receptor activity, although no evidence has been found yet. The blood content of 17beta-estradiol in teenagers with IS shows lower levels than teenagers of the same age without IS. Also testosterone and progesterone content are lower in IS girls with respect to the control girls. Furthermore, we extracted DNA from white blood cells of IS patients and their relatives until the third generation in order to examine estrogen receptor alpha polymorphisms, considering this tool a plausible molecular marker for IS prognosis. In this respect, we identified four polymorphisms in the exons encoding for the steroid binding domain and two other in the trans-activation domain. Our results show a clear relationship with clinical manifestation of IS.

ZPLD1 gene is disrupted in a patient with balanced translocation that exhibits cerebral cavernous malformations.

The past few years have seen rapid advances in our understanding of the genetics and molecular biology of cerebral cavernous malformations (CCM) with the identification of the CCM1, CCM2, and CCM3 genes. Recently, we have recruited a patient with an X/3 balanced translocation that exhibits CCM. By fluorescent in situ hybridization analysis, sequence analysis tools and database mining procedures, we refined the critical region to an interval of 200-kb and identified the interrupted ZPLD1 gene. We detected that the mRNA expression level of ZPLD1 gene is consistently decreased 2.5-fold versus control (P=0.0006) with allelic loss of gene expression suggesting that this protein may be part of the complex signaling pathway implicated in CCM formation.

Laparoscopic antireflux surgery: indications, preoperative evaluation, techniques, and outcomes.

BACKGROUND/AIMS: During the past decade, the development of mini-invasive surgery has determined a resurgence in popularity of the antireflux surgery. The purpose of this study is to examine indications, preoperative evaluation, surgical techniques, and outcomes after mini-invasive surgery. METHODOLOGY: From 1996 to 2000, 25 patients with gastroesophageal reflux disease associated to hiatal hernia underwent laparoscopic surgery. The indication for surgery was failure of long-term medical therapy. All patients had severe acid reflux on 24h-pH monitoring, endoscopic evidence of esophagitis, and defective lower esophageal sphincter. Nissen fundoplication was performed in 16 patients with normal esophageal body motility, and 270 degrees posterior fundoplication in 9 patients with low esophageal motility. RESULTS: Mortality and conversion rate were 0. Mean operative time was 130 minutes and mean postoperative hospital stay 5 days. Twenty-four (96%) patients were completely cured of reflux symptoms off all medications. Transient, mild postoperative dysphagia occurred in 3 patients (12%). There was a significant improvement of the results in postoperative esophageal manometry and 24h-pH monitoring. CONCLUSIONS: Despite the fact that few patients were treated by using laparoscopic approach, results are encouraging with less morbidity and great advantages for patients. Precise selection of patients and surgical techniques are essential.

Regulation of androgen receptor mRNA expression in primary culture of Harderian gland cells: cross-talk between steroid hormones.

The androgen receptor (AR) must be considered a transcription factor belonging to the steroid-thyroid hormones receptor superfamily. Previous results gained from the Harderian gland, a tubulo-alveolar gland located in the orbital cavity of the golden hamster, indicate that Harderian gland cells express mRNAs encoding for androgen, glucocorticoid, thyroid hormone (T(3)), and estrogen receptors, respectively. Since in other systems, these receptors have been related to the expression of the androgen receptor, we have studied the regulation of AR expression in primary cultures of the male hamster Harderian gland. Our in vitro experiments show that androgen, and thyroid hormones increase the expression of AR. Retinoic acids also show a positive effect on AR expression, while exposure to glucocorticoid or estrogen blocks AR expression. Since these steroids differently modulate AR expression, our results must be considered in the context of multi-hormonal control of gene expression that could act through cross-talk between members of the steroid-thyroid hormones.

Multiple pathogenic and benign genomic rearrangements occur at a 35 kb duplication involving the NEMO and LAGE2 genes.

The X-linked dominant and male-lethal disorder incontinentia pigmenti (IP) is caused by mutations in a gene called NEMO (IKK-gamma). We recently reported the structure of NEMO and demonstrated that most IP patients carry an identical deletion that arises due to misalignment between repeats. Affected male abortuses with the IP deletion had provided clues that a second, incomplete copy of NEMO was present in the genome. We have now identified clones containing this truncated copy (Delta NEMO) and incorporated them into a previously constructed physical contig in distal Xq28. Delta NEMO maps 22 kb distal to NEMO and only contains exons 3-10, confirming our proposed model. A sequence of 26 kb 3' of the NEMO coding sequence is also present in the same position relative to the Delta NEMO locus, bringing the total length of the duplication to 35.5 kb. The LAGE2 gene is also located within this duplicated region, and a similar but unique LAGE1 gene is located just distal to the duplicated loci. Mapping and sequence information indicated that the duplicated regions are in opposite orientation. Analysis of the great apes suggested that the NEMO/LAGE2 duplication occurred after divergence of the lineage leading to present day humans, chimpanzees and gorillas, approximately 10-15 million years ago. Intriguingly, despite this substantial evolutionary history, only 22 single nucleotide differences exist between the two copies over the entire 35.5 kb, making the duplications >99% identical. This high sequence identity and the inverted orientations of the two copies, along with duplications of smaller internal sections within each copy, predispose this region to various genomic alterations. We detected four rearrangements that involved NEMO, Delta NEMO or LAGE1 and LAGE2. The high sequence similarity between the two NEMO/LAGE2 copies may be due to frequent gene conversion, as we have detected evidence of sequence transfer between them. Together, these data describe an unusual and complex genomic region that is susceptible to various types of pathogenic and polymorphic rearrangements, including the recurrent lethal deletion associated with IP.

Survival of male patients with incontinentia pigmenti carrying a lethal mutation can be explained by somatic mosaicism or Klinefelter syndrome.

Incontinentia pigmenti (IP), or "Bloch-Sulzberger syndrome," is an X-linked dominant disorder characterized by abnormalities of skin, teeth, hair, and eyes; skewed X-inactivation; and recurrent miscarriages of male fetuses. IP results from mutations in the gene for NF-kappaB essential modulator (NEMO), with deletion of exons 4-10 of NEMO accounting for >80% of new mutations. Male fetuses inheriting this mutation and other "null" mutations of NEMO usually die in utero. Less deleterious mutations can result in survival of males subjects, but with ectodermal dysplasia and immunodeficiency. Male patients with skin, dental, and ocular abnormalities typical of those seen in female patients with IP (without immunodeficiency) are rare. We investigated four male patients with clinical hallmarks of IP. All four were found to carry the deletion normally associated with male lethality in utero. Survival in one patient is explained by a 47,XXY karyotype and skewed X inactivation. Three other patients possess a normal 46,XY karyotype. We demonstrate that these patients have both wild-type and deleted copies of the NEMO gene and are therefore mosaic for the common mutation. Therefore, the repeat-mediated rearrangement leading to the common deletion does not require meiotic division. Hypomorphic alleles, a 47,XXY karyotype, and somatic mosaicism therefore represent three mechanisms for survival of males carrying a NEMO mutation.

Sequence analysis and androgen regulation of MHG07 (Male harderian gland) mRNA in male hamster harderian gland.

The hamster Harderian gland (HG), a compound tubuloalveolar gland located in the orbital cavity, displays sex dimorphism. The present study focuses on the sequence analysis of a cDNA clone named MHG07 and on the regulation of its expression by steroid hormones. MHG07 mRNA (5.0 kb) is expressed in male HG only. The MHG07 cDNA (1.74 kb) shows an ORF of 94 amino acids and has no significant homologies with other polypeptides/genes. Castration leads to the disappearance of MHG07 mRNA after 4 days, whereas treatment with testosterone impairs the effect of castration. No MHG07 mRNA has been found in either rat or murine HGs. Androgen (A) administration to female hamsters induces the appearance of MHG07 mRNA. In primary culture of male hamster HG, androgens increase the MHG07 expression and this effect is blocked by both flutamide and cycloheximide. Dose-response experiments show that, at low A concentration (10(-12) M), the MHG07 was higher than that of the control (2-fold). This effect reaches its zenith at 10(-8) M (10-fold). This picture is paralleled by androgen receptor mRNA expression. It is argued that the expression of MHG07 is under androgenic control.

The expression of androgen receptor messenger RNA is regulated by tri-iodothyronine in lizard testis.

The network of hormonal and non-hormonal signals required for testicular activity during the reproductive cycle of the seasonal breeding lizard, Podarcis sicula, are not yet well understood. Androgens are significantly involved in meiosis and spermiogenesis, and such an effect is mediated through their receptor (AR). Estrogens also affect the testicular activity down-regulating the expression of AR mRNA. Since over the last few years, extensive works have reported, in mammals, a clear influence of tri-iodothyronine (T(3)), the biologically active thyroid hormone, on Sertoli cell activities, we carried out a study to shead light on the effect/s exerted by T(3) in lizard testis. A thyroid hormone receptor mRNA (TR mRNA) has been found in the testis indicating that T(3) might be involved in the regulation of gonadal activity. In in vivo experiments, injection of T(3) to male lizards, captured during the recrudescence period (March) and maintained under experimental photothermal conditions (24 degrees C and 15 h daylight), increased the expression of AR mRNA. The in vitro results confirmed the stimulatory effect of T(3) on AR mRNA levels. Thus, in testosterone (T) exposed cells, the highest values of AR mRNA were observed in T(3)-primed animals, indicating that T and T(3) increase AR gene transcription independently. The present data suggest that, in lizards, the combined action of androgens, estrogen and T(3) might regulate testicular activity, modulating AR mRNA levels.

[Renal arteriovenous fistulas after renal biopsy. Percutaneous embolization]. / Fistole artero-venose renali post-bioptiche. Embolizzazione percutanea transcatetere.

PURPOSE: We report our experience relative to transcatheter percutaneous embolization of post-biopsy renal intraparenchymal arteriovenous fistulas in patients with chronic renal insufficiency. MATERIAL AND METHODS: We observed 5 patients affected with post-bioptic fistulas for possible embolization. In three cases the symptoms were represented by intermittent macro-microhematuria; one patient had hypertension of nephrovascular origin and one patient was asymptomatic. In all cases we performed angiography and it was possible to catheterize the peripheral afferent branch of the fistula with a superselective technique using a hydrophilic guide of 0.035 F and a hydrophilic Cobra catheter of 4-5 F. The occlusion was obtained by the positioning of Granturco metal coils: in 1 case we adapted a coil of 3 mm diameter and 1 cm length; in 3 cases 2 coils of 3 mm and in 1 case 2 coils of 3 mm and 1 coil of 5 mm diameter and 1 cm length were necessary. The success of the procedure was always checked with an immediate angiogram and color Doppler US after 48 hrs. RESULTS: The diagnosis of arteriovenous fistulas was always confirmed by a preliminary angiography that demonstrated the normal anatomic disposition of the renal arteries except in one case in which the fistula was fed by a peripheral branch originating from an inferior polar artery. All the lesions were localized in the inferior pole, the site of biopsy, and ranged from 3 mm to 2.5 cm in diameter. We never had any difficulties in the positioning and placement of the coils. The arterial occlusion and exclusion of the fistula was accomplished in all cases. The induced parenchymal loss ranged from 10 to 30% of the renal volume. There was a complete disappearance of symptoms in 3 of the patients, with hematuria without any modification of the blood pressure values in the patient with hypertension. Considering the patient status renal function did not worsen after the embolization. Each patient was followed-up with color Doppler US every two months. CONCLUSIONS: The intrarenal arteriovenous fistula represents a relative frequent complication of renal needle biopsy in patients with arterial hypertension and nephroangiosclerosis as risk factors. Embolization is a valid alternative therapeutic option to surgical treatments. The use of small size catheters permits the successful embolization also of peripheral lesions, reducing the induced parenchymal ischemia. We believe that among the embolization material available metal coils represent a valid solution as they are easily positioned and permit definitive occlusion without any risks of systemic venous microembolization.

Trauma care fellowships: current status and future survival.

BACKGROUND AND METHODS: To determine the current status and future direction of trauma care fellowships, a phone survey was conducted with the 45 program directors reporting information to the American Association for the Surgery of Trauma and the Eastern Association for the Surgery of Trauma. RESULTS: Forty programs (89%) were operational, with 86 positions. The duration of the fellowship was 1 year for 16 (40%) and 2 or more years for 24 (60%). Accreditation Council for Graduate Medical Education accreditation (ACGME) (for surgical critical care) was held by 28 (70%). Mean salary was $39,600 at the first-year level. A funding shift from institutional to practice revenue sources is foreseen. Thirteen directors (32.5%) saw future recruitment potential as increasing and 11 (27.5%) saw it as decreasing. CONCLUSION: The essence, structure, and funding of trauma fellowships are changing. One-year exclusive trauma fellowships are being replaced by 1- to 2-year trauma or surgical critical care fellowships with Accreditation Council for Graduate Medical Education accreditation increasingly seen as essential. The challenge for fellowships in an era of budgetary constraints will be to provide adequate training in the full spectrum of tramatology within a reasonable time frame supported by a predictable funding mechanism.