Copy number variants analysis in a cohort of isolated and syndromic developmental delay/intellectual disability reveals novel genomic disorders, position effects and candidate disease genes.

BACKGROUND: Array-comparative genomic hybridization (array-CGH) is a widely used technique to detect copy number variants (CNVs) associated with developmental delay/intellectual disability (DD/ID). AIMS: Identification of genomic disorders in DD/ID. MATERIALS AND METHODS: We performed a comprehensive array-CGH investigation of 1,015 consecutive cases with DD/ID and combined literature mining, genetic evidence, evolutionary constraint scores, and functional information in order to assess the pathogenicity of the CNVs. RESULTS: We identified non-benign CNVs in 29% of patients. Amongst the pathogenic variants (11%), detected with a yield consistent with the literature, we found rare genomic disorders and CNVs spanning known disease genes. We further identified and discussed 51 cases with likely pathogenic CNVs spanning novel candidate genes, including genes encoding synaptic components and/or proteins involved in corticogenesis. Additionally, we identified two deletions spanning potential Topological Associated Domain (TAD) boundaries probably affecting the regulatory landscape. DISCUSSION AND CONCLUSION: We show how phenotypic and genetic analyses of array-CGH data allow unraveling complex cases, identifying rare disease genes, and revealing unexpected position effects.

A novel synonymous mutation in the MPZ gene causing an aberrant splicing pattern and Charcot-Marie-Tooth disease type 1b.

Charcot-Marie-Tooth disease (CMT) is an inherited peripheral neuropathy with a heterogeneous genetic background. Here, we describe two CMT1B families with a mild sensory-motor neuropathy and a novel synonymous variant (c.309G > T, p.G103G) in exon 3 of the MPZ gene. Next generation sequencing analysis on a 94 CMT gene panel showed no mutations in other disease genes. In vitro splicing assay and mRNA expression analysis indicated that the c.309T variant enhances a cryptic donor splice site at position c.304 resulting in the markedly increased expression of the r.304_448del alternative transcript in patients' cells. This transcript is predicted to encode a truncated P0 protein (p.V102Cfs11*) lacking the transmembrane domain, thus suggesting a possible haploinsufficiency mechanism for this mutation. This is the third reported synonymous MPZ variant associated with CMT1 and affecting splicing. These data confirm the functional impact of synonymous variants on MPZ splicing and their possible role as disease-causing mutations rather than silent polymorphisms.

Directed evolution of an enantioselective lipase.

BACKGROUND: The biocatalytic production of enantiopure compounds is of steadily increasing importance to the chemical and biotechnological industry. In most cases, however, it is impossible to identify an enzyme that possesses the desired enantioselectivity. Therefore, there is a strong need to create by molecular biological methods novel enzymes which display high enantioselectivity. RESULTS: A bacterial lipase from Pseudomonas aeruginosa (PAL) was evolved to catalyze with high enantioselectivity the hydrolysis of the chiral model substrate 2-methyldecanoic acid p-nitrophenyl ester. Successive rounds of random mutagenesis by ep-PCR and saturation mutagenesis resulted in an increase in enantioselectivity from E=1.1 for the wild-type enzyme to E=25.8 for the best variant which carried five amino acid substitutions. The recently solved three-dimensional structure of PAL allowed us to analyze the structural consequences of these substitutions. CONCLUSIONS: A highly enantioselective lipase was created by increasing the flexibility of distinct loops of the enzyme. Our results demonstrate that enantioselective enzymes can be created by directed evolution, thereby opening up a large area of novel applications in biotechnology.

Combined immunosuppressive therapy with tacrolimus and mycophenolate mofetil for small bowel transplantation in pigs.

In a swine model of orthotopic small bowel transplantation, we assessed the efficacy of combined therapy with a low dose of tacrolimus plus mycophenolate mofetil, compared with high-dose tacrolimus monotherapy. The bowel was replaced in 25 piglets: group 1 (n = 5), no immunosuppression; group 2 (n = 10), tacrolimus, 0.3 mg/kg daily i.m. for 7 days, followed by b.i.d. oral doses to maintain blood levels of 15-25 ng/ml; and group 3 (n = 10), tacrolimus, 0.1 mg/kg i.m., in a single dose on day 0 and thereafter oral doses to maintain blood levels of 5-15 ng/ml, plus oral mycophenolate mofetil (10 mg/kg twice daily). Follow-up time was limited to 60 days. Median survival time as 11, 27, and > 60 days in groups 1, 2, and 3, respectively (P = 0.001). Survival rates were 0%, 40%, and 80% at 30 days and 0%, 0%, and 70% at 60 days in groups 1, 2, and 3, respectively (P = 0.03), group 1 vs. group 2; P = 0.003, group 1 vs. group 3; P = 0.02, group 2 vs. group 3). One animal in group 1 (20%) and two animals each in groups 2 and 3 (20%) died of technical complications. Rejection was the cause of death of 80% of animals of group 1 and of no animals in either group 2 or 3. None of the immunosuppressed animals developed clinical or histopathological evidence of graft-versus-host disease. Sixty percent of animals in group 2 (n = 6) and 10% in group 3 (n = 1) died from infections; two other animals in group 2 died of emaciation. The seven animals of group 3 that were alive at 60 days had immunosuppression stopped at that time. All died of rejection within 1 month. In conclusion, double-drug therapy with tacrolimus and mycophenolate mofetil consistently allowed extended survival after small bowel transplantation in swine, preventing or controlling acute cellular rejection without a high incidence of lethal complications related to overimmunosuppression.

Insulin-like growth factor-I ameliorates delayed kidney graft function and the acute nephrotoxic effects of cyclosporine.

BACKGROUND: Delayed graft function (DGF) is a relatively common complication after cadaveric renal transplantation. The adverse effect of DGF on long-term graft survival has lead to intensive efforts to reduce ischemic graft injury. In this study we examined the effects of a new protective treatment based on insulin growth factor (IGF)-I. We evaluated the impact of the treatment on renal recovery and on the nephrotoxicity that is a common side effect of mainstream immunosuppressants. Because therapy with IGF-I or the analog des(1-3)IGF-I is effective in treating experimental ischemic renal failure, these peptides may be useful as perspective clinical treatments. METHODS: We have addressed three areas relating to the potential use of IGF-I and its analog des(1-3)IGF-I. First, because of the immunogenic properties of IGF-I, we assessed the effect of des(1-3)IGF-I on the rejection of skin allografts in Lewis rats. Next we determined whether treatment with des(1-3)IGF-I influences the early function of transplanted kidneys in a model of DGF induced by a combination of warm and cold ischemia. Finally we tested whether IGF-I protects against acute cyclosporine nephrotoxicity. RESULTS: Des(1-3)IGF-I did not accelerate the rejection of the skin grafts (P=0.57). The administration of this peptide in a model of syngenic renal transplant improved the early function of the graft. Postoperative values of creatinine and blood urea nitrogen were significantly better (P<0.05) in treated animals. IGF-I also ameliorated the nephrotoxicity of cyclosporine, with better values of creatinine and blood urea nitrogen (P<0.05). CONCLUSIONS: In evaluating this study it should be recognized that the animal models studied, although widely used, differ from the human condition. However, IGF-I and des(1-3)IGF-I exhibit properties that strongly suggest their value in preventing clinical DGF, and they deserve further studies.

Chromosome abnormalities in lymphocytes and fibroblasts of subjects with multiple endocrine neoplasia type 1.

A consistent degree of chromosome instability was found in cultured lymphocytes and in fibroblasts derived from skin biopsies of three patients with multiple endocrine neoplasia type 1 (MEN1). In both tissues, there was a significant increase in chromosomal breakage. Dicentrics and tricentrics, rings, translocations, deletions, acentric fragments, and presumptive double minutes were the most frequent abnormalities in lymphocytes. The fibroblasts of two patients had large clones consisting of trisomy 7 and trisomy 18 cells, respectively.

Radioimmunoassisted follow-up and surgery vs traditional examinations and surgery after radical excision of colorectal cancer.

The authors report their experience in the radioimmunodetection of recurrent colorectal cancer in comparison with traditional examinations. 485 patients were studied after radical surgery for colorectal cancer: 168 (Group 1) were studied with a radioimmunoassisted follow-up plan including: Immunoscintigraphy (IS), serum markers assays, Radioimmunoguided Endoscopy (RIGE), Intraoperative Radioimmuno-localization (IORIL) in case of reoperation; 317 (Group 2) underwent a protocol with traditional examinations. In 24 patients of Group 1 IS was performed even at the time of their admission for primary cancer. 7 patients underwent RIGE for primary cancer and 16 in the follow-up. IORIL was performed in 12 patients with primary cancer and in 16 in the course of reoperation. The radioimmunodiagnostic methods were performed after a single administration of the radiolabeled MAb (111In F(ab')2 a-CEA and 111In B72.3). The radioimmunoassisted follow-up plan detected a greater number of recurrences than traditional examinations (27% vs 13%). The rates of radical reoperation in Group 1 and 2 were 61% and 37% respectively. 33% of the patients of Group 2 were alive 24 months after radical reoperation vs 62.5% of the patients of Group 1. Immunoscintigraphy demonstrated a good sensitivity and specificity, mainly in the detection of pelvic recurrences (sens. 92%, spec. 84%). The radioimmunoassisted follow-up plan was well accepted by the patients. RIGE led to the detection of 3 periluminal recurrences of rectal cancer that traditional investigations failed to demonstrate and in 5 cases influenced the patients management. IORIL detected minimal tumor foci (2 mm.) where pre and intraoperative study were negative, while the histopathologic examination gave evidence of tumor. Considering that the costs of the radioimmunodiagnostic methods and of traditional examinations are very similar, we can conclude that the radioimmunoassisted follow-up plan has a favorable cost/benefit rate and a remarkable impact on the treatment of patients with colorectal cancer.

Successful bronchial revascularization in experimental single lung transplantation.

It is the purpose of this paper to report our experience with bronchial artery revascularization in an experimental model of single lung transplantation in swine. Thirty-three large white pigs weighing 20-40 kg underwent left lung allotransplantation. In 24 animals, bronchial artery revascularization was attempted by anastomizing the aortic patch containing the bronchial artery orifice with the recipient descending aorta. Eight survivors were put to death on postoperative days 11-15; five animals were put to death or died on postoperative days 2-9; the other animals died intra-operatively or within a few hours. Preservation of left bronchial vascularization was achieved in all cases attempted, as documented by post-mortem injection of dye (methylene blue) or contrast medium. Five of the 8 animals surviving for 11-15 days showed diffuse graft hepatization, associated with diffuse vascular thrombosis. Whether this was caused by damage to the endothelium due to poor graft preservation or by rejection was unclear. In animals surviving for 11-15 days without gross lung pathology, the anastomosis and bronchial mucosa were completely normal; in contrast, bronchial ischaemic changes were found in nonrevascularized animals and in survivors with graft hepatization. Our experience confirms that re-anastomosis of the bronchial arteries can prevent bronchial healing problems in single lung transplantation. The pig is an ideal model for these experiments since the bronchial arteries have a constant common aortic origin, allowing easy identification and preservation of left bronchial vascularization.

How to study boron biodistribution in liver metastases from colorectal cancer.

The purpose of this study was to evaluate boron distribution for a safe and effective BNCT (Boron Neutron Capture Therapy) of liver metastases. Samples both from healthy and tumour liver parenchyma were analysed, after i.v. boron administration, by: alpha particles counting under neutron irradiation; morphological analysis by standard haematoxylin-eosin staining; neutron autoradiography. Our method was unaffected by the cytological heterogeneity inside tumour nodules; it demonstrated selective boron distribution in tumour tissue and predicted estimated mean therapeutic doses in tumour and safety doses in healthy tissue. The time interval for efficient BNCT was 2 to 4 hours after i.v. boron administration.

Catheter seeding of hepatocellular carcinoma following placement of a total implantable access port system.

A 61-year-old cirrhotic patient underwent hepatic resection for hepatocellular carcinoma and placement of a total implantable access port system in the hepatic artery for chemotherapy infusion. A year later, he developed a parietal metastasis at the port site as a consequence of tumor seeding along the arterial catheter. The metastasis was excised but the patient died because of disseminated disease two years after the first operation. Tumor seeding along the catheter should be included in the group of potential complications after placement of total implantable access port systems for intrahepatic chemotherapy The possible causes of this rare but life-threatening complication are discussed.

A rare case of pancreatic lipoma.

During CT scanning, a pancreatic lipoma was diagnosed in a 70 year-old woman presenting with abdominal pain, elevated amylase and abnormal liver function tests. The patient underwent surgical excision of the lipoma located in the head of the pancreas. The postoperative course was uneventful. This is the third case of pancreatic lipoma described in the literature. It is a very rare neoplasm that should be included in the category of "non-ductal" tumors of the pancreas. The role of different diagnostic tools for the differential diagnosis of ductal pancreatic adenocarcinoma is discussed.

[Simple ulcer of the colon. Considerations on a case]. / L'ucera simplex del colon. Considerazioni su di un caso.

A case of "simple ulcer" of the colon is reported. The lesion is uncommon and not well known. As far as pathogenesis is concerned, the Authors emphasize the role of peptic, neurogenic, circulatory, bacterial, toxic and mechanical factors. The following clinical picture was observed: sudden and acute abdominat pain due to colonic perforation with pneumoperitoneum. As already described by literature preoperative correct diagnosis was not possible; this is usually due to the fact that the ulcer tends to perforate before other symptoms arise. During surgery suture of the ulcer, temporary colostomy and Mikulicz's drainage were performed; after 4 months colostomy was closed.

[Possible causal factors in postoperative immunodepression]. / Studio di alcuni possibili fattori causali dell"immunodepressione postoperatoria.

The relationships between postoperative immunodepression and duration of general anesthesia, volume of blood transfusions, cortico-steroid administrations and amount of surgical trauma have ben studied, in cancer patients undergoing radical operations. Postoperative depression of cellular immunity, assayed by the lymphocyte blastogenic response to phytohemagglutinin, was significantly correlated with the amount of surgical trauma and did not correlate with blood transfusions and corticosteroid administrations, nor with the duration of anesthesia.

[Experimental study of colonic anastomoses. Comparison of different suture methods in the rat]. / Studio sperimentale delle anastomosi coliche. Confronto tra differenti metodi di sutura (nel ratto).

A comparative experimental study was conducted on three different suture techniques for end-to-end colonic anastomoses. The development of stenosis, perivisceral adhesions and resistance to endoluminal pressure were assessed. The results show that the one layer all-coat interrupted stitch anastomosis displays less resistance to endoluminal pressure during the first seven days but is followed by fewer complications.

[Experimental use of intraoperative pulse wave Doppler in the study of vascular microanastomosis]. / Impiego sperimentale del Doppler pulsato intraoperatorio nello studio delle microanastomosi vascolari.

Personal experience with pulse wave Doppler (PWD) at 20 MHz with real time signal spectral analysis in FFT (Fast Fourier Transform), for the valuation of the vascular microanastomosis patency degree is reported. Fifty-nine Sprague-Dawley rats have been studied after abdominal aorta dissection: in 19 (group A) an organic stenosis has been simulated varying from 5% to 95% by a 8/0 silk suture; in 40 rats (group B and C) we the abdominal aorta was stitched with interrupted suture after transversal section. In the group C (20 rats) a longitudinal arteriotomy on abdominal aorta within 10-15 min from anastomosis was performed to value under microscopic control the suture line. The PWD technique is able to recognize stenoses more than 10% of intraluminal diameter, showing a higher sensibility than traditional techniques.

[Choledochocele: a rare cause of chronic pancreatitis of the head of the pancreas]. / Coledococele: rara causa di cefalo-pancreatite cronica.

The authors critically review the literature in order to ascertain the current state of knowledge regarding the anatomopathological, clinical and therapeutic characteristics of choledochocele, or cystic dilation of the terminal portion of the biliary tract. In particular, the Authors examine the etiopathogenetic aspects to explain how the presence of a choledochocele may lead to the onset of chronic pancreatitis. A clinical case which was recently brought to their attention is examined in detail. It concerns a 27-year-old patient in whom the presence of a large choledochocele had led to the onset of severe cephalopancreatitis which resisted all forms of treatment. In the case in question, ERCP played a decisive role in the diagnosis of the choledochocele, whereas ultrasonography, CT and selective arteriography were useful above all in relation to pancreatitis. In the case described ultraradical surgery, namely duodenocephalopancreatectomy, allowed the severe pancreatopathy and its etiological cause (choledochocele) to be efficaciously treated, confirming that this type of surgery currently plays an important role in the treatment of chronic pancreatitis.

[Hemodynamic changes in liver and multi-organ transplantation]. / Modificazioni emodinamiche nel trapianto epatico e nel trapianto multiviscerale.

Multivisceral transplantation is a surgical technique developed as treatment for abdominal metastatic and/or multifocal malignancies. At present its clinical employment is reduced by our fragmentary knowledge of the intraoperative and postoperative outcome. The aim of this study is to compare intraoperative hemodynamic and respiratory changes during multivisceral transplantation (MTV, n = 12) and liver transplantation (OLTX, n = 14). The observations have been carried out a 4 phases: basal (I), visceral (II), reperfusion (III), final (IV). Phase I does not show differences between MTV and OLTX. In phase II MTV presents a lower temperature (T) and pulmonary arterial pressure (PAP) (p > 0.05). Phase III is marked by increasing T differences (p < 0.05), lower cardiac frequency (CF), pH and base excess (BE) (p < 0.05). PAP and cardiac output (CO) show a higher value in MTV (p < 0.05). Phase IV reports the vital signs close to normality in both groups, except pH in MTV (p > 0.05).

[Treatment of the nephrotoxicity of immunosuppressive drugs with insulin-like growth factor-I]. / Trattamento della nefrotossicità da farmaci immunosoppressori con insulin-like growth factor-I.

BACKGROUND: Delayed graft function is a common and severe complication after cadaveric kidney transplantation. Besides a more complicated postoperative course, DGF can worsen the overall graft survival. In particular, DGF enhances the nephrotoxicity of mainstream immunosuppressants cyclosporine and FK506. This study evaluates a new therapeutical approach to the treatment of DGF related nephrotoxicity, based on the administration of IGF-I. METHODS: Sixty inbred Lewis rats underwent a bilateral clamping of the renal pedicles (20') as standard damage. The animals were stratified in six groups, according to the postoperative treatment. Group 1 served as control and received only the standard ischemic injury. Cyclosporine and FK506 were added in groups 3 and 5. Groups 2, 4 and 6 had the same treatment of groups 1, 3, 5 respectively, plus the administration of IGF-I. Blood samples were drawn daily to evaluate creatinine and BUN for 7 days. RESULTS: The rats treated with IGF-I had significantly better values compared to the respective controls (2-way ANOVA, p < 0.05). CONCLUSIONS: In conclusion, IGF-I improves the nephrotoxicity of mainstream immunosuppressants in this model. Its use is potentially beneficial for transplantation.

[Duodenal schwannoma. Clinical considerations]. / Schwannoma duodenale. Considerazioni cliniche.

The recent observation of a case of schwannoma in the second part of the duodenum lead the authors to a literature survey in order to state the present knowledge of this subject. Schwannoma is an ectodermal neoplasm arising from the nerve sheath that envelop axons. In the gastrointestinal tract these tumours can concern cells of the myentericus and submucosus plexus of the intestinal wall. A duodenal location is extremely rare. Gastroduodenal endoscopy, abdominal ultrasonography and CT scan were found as important investigatory tools, but only histological examination of the excised tissue suggests the definitive diagnosis. Therapy consists in the radical excision of the tumour; this is rarely obtained by endoscopic way because of the submucosal neoplastic growth. Then a surgical laparotomic approach is more often required and it allows to perform local excisions through a duodenotomy, partial duodenectomy or ampullectomy and more complex operations, like a cephalic duodenopancreatectomy, in front of extended papillar involvement. The latter was the suitable treatment for radical removing of the juxtapapillar schwannoma reported by the authors; the uneventful postoperative course, the patient's excellent general conditions and nutriture at 12 months confirm the validity of the therapeutic policy.

[Postoperative infections. A prospective analysis of 1396 cases]. / Le infezioni postoperatorie. Analisi prospettica di 1396 casi.

Postoperative infections are the most frequent complications in surgery and are the commonest cause of the lengthening of hospital stay. The purpose of this study is to prospectively evaluate the incidence and predisposing factors of postoperative infections in 1396 surgical patients admitted to our Institute from 1984 to 1988. Patients undergoing minor surgical procedures (wound less than 2 cm) were excluded from the study. Patients were evaluated daily during hospital stay for onset of infections and results recorded on data sheet. Hemocultures in septic patients and samples of exudate at site of infection were taken whenever possible for aerobic and anaerobic cultures. 368 patients (26.36%) had at least one postoperative septic complication; (79 of them [5.65%] had two or more infections). The following infections were recorded: wound infections: 148 (10.60%); respiratory tract infections: 144 (10.31%); urinary tract infections 125 (8.95%); miscellaneous infections 11 (0.78%); thrombophlebitis 23 (1.64%); FUO 10 (0.71%). The most important predisposing factor for wound infection was endogenous contamination (wound infections: 18/499 [3.60%] in clean, 42/594 [7.67%] in potentially contaminated, 57/217 [26.26%] in contaminated and 31/86 [36.04%] in dirty operations). The duration of the anaesthesia was found to correlate with an increased incidence of respiratory tract infections (4.49% anaesthesia less than 60 min; 7.21% anaesthesia greater than 60 less than 120 min; 15.31% greater than 120 min anaesthesia). Urinary infections were more frequent when the patients where catheterized at least once in the postoperative period (24.86% vs 3.2%).