Exome sequencing data screening to identify undiagnosed Aromatic l-amino acid decarboxylase deficiency in neurodevelopmental disorders.

Aromatic l-amino acid decarboxylase (AADC) deficiency is a rare autosomal recessive neurometabolic disorder caused by biallelic pathogenic variants in the DDC gene and mainly characterized by developmental delay, hypotonia, and oculogyric crises. Early diagnosis is crucial for correct patient management; however, many patients remain misdiagnosed or undiagnosed due to the rarity and clinical heterogeneity of the disorder especially in the milder forms. Here, we applied exome sequencing approach by screening 2000 paediatric patients with neurodevelopmental disorders to identify possible new AADC variants and AADC deficiency patients. We identified five distinct DDC variants in two unrelated individuals. Patient #1 harboured two compound heterozygous DDC variants: c.436-12T > C and c.435 + 24A>C and presented with psychomotor delay, tonic spasms, and hyperreactivity. Patient #2 had three homozygous AADC variants: c.1385G > A; p.Arg462Gln, c.234C > T; p.Ala78 = , and c.201 + 37A > G and presented with developmental delay and myoclonic seizures. The variants were classified as benign class I variants and therefore non-causative according to the ACMG/AMP guidelines. Since the AADC protein is a structural and functional obligate homodimer, we evaluated the possible AADC polypeptide chain combinations in the two patients and determined the effects resulting from the amino acid substitution Arg462Gln. Our patients carrying DDC variants presented clinical manifestations not precisely overlapped to the classical symptoms exhibited by the most severe AADC deficiency cases. However, screening data derived from exome sequencing in patients featuring wide-range symptoms related to neurodevelopmental disorders may help to identify AADC deficiency patients, especially when applied to larger cohorts.

Growth hormone receptor blockade inhibits growth hormone-induced chemoresistance by restoring cytotoxic-induced apoptosis in breast cancer cells independently of estrogen receptor expression.

CONTEXT: GH and IGF-I play a role in breast cancer (BC) development. We previously demonstrated that GH protects the estrogen receptor (ER) positive BC-derived MCF7 cell line toward the cytotoxic effects of doxorubicin (D), independently of IGF-I. This issue may be important in ER negative BC cells that are more aggressive and more likely to develop chemoresistance. AIM OF THE STUDY: The aim of this study was to evaluate whether GH may impact chemoresistance phenotype of ER-negative BC-derived MDA-MB-231 cell line and investigate the possible mechanisms implicated in the protective action of GH toward the cytotoxic effects of D in both ER-positive and ER-negative BC-derived cell lines. RESULTS: GH protects ER-negative MDA-MB-231 cells from the cytotoxic effects of D and GH receptor antagonist pegvisomant reduces GH-induced DNA synthesis also in these cells. In both MDA-MB-231 and MCF7 cells, GH does not revert D-induced G2/M accumulation but significantly reduces basal and D-induced apoptosis, an effect blocked by pegvisomant. Glutathione S-transferase activity is not implicated in the protective effects of GH, whereas D-induced apoptosis depends on c-Jun N terminal kinase (JNK) activation. GH reduces both basal and D-stimulated JNK transcriptional activity and phosphorylation. CONCLUSIONS: In human BC cell lines, GH directly promotes resistance to apoptosis induced by chemotherapeutic drugs independently of ER expression by modulating JNK, further broadening the concept that GH excess may hamper cytotoxic BC treatment. These findings support the hypothesis that blocking GH receptor may be viewed as a potential new therapeutic approach to overcome chemoresistance, especially in ER-negative BC.

Heat shock protein 70-1 gene expression in pediatric heart surgery using blood cardioplegia.

BACKGROUND: In response to many stress stimuli, cardiomyocytes produce a common set of heat shock proteins (HSP). Up-regulation of HSP70-1 (the inducible isoform) is known to reduce the risk of myocardial cell damage during open-heart surgery and seems to be protective against ischemia. We assessed hsp70-1 gene expression during blood cardioplegic arrest in children undergoing surgical correction of congenital heart defects. METHODS: In tissue samples taken from the right atrium of 59 pediatric patients, we examined hsp70-1 gene expression using a real-time quantitative reverse transcription PCR, with 18S rRNA as internal standard. RESULTS: On average, hsp70-1 gene expression was higher than the baseline level by a factor of 1.44+/- 0.17 (mean+/-SEM). A significant relationship between hsp70-1 mRNA levels and aortic cross-clamp time was observed (R(2)=0.069, p=0.044). Conversely, no significant correlation was observed between hsp70-1 mRNA levels and temperature. CONCLUSIONS: These data suggest that blood cardioplegia can induce an increment in the expression of hsp70-1, confirming its protective role in ischemia/reperfusion injury.

QTc and autonomic neuropathy in diabetes: effects of acute hyperglycaemia and n-3 PUFA.

BACKGROUND AND AIMS: Autonomic function is also regulated by glycaemia and exerts a crucial role in the control of blood pressure and cardiac function. The disruption of this physiological mechanism impacts deeply on cardiovascular mortality in diabetes. We investigated the influence of autonomic dysfunction on QTc interval and on sympatho-vagal balance (LF/HF), in response to acute hyperglycaemia and to membrane electrical stabilization (n-3 PUFA). METHODS AND RESULTS: Twenty-four type 2 diabetic patients, without (N-: n=13) or with (N+: n=11) autonomic neuropathy and 13 healthy subjects (C) underwent BP and ECG monitoring during a 24-h period and during a 2-h hyperglycaemic clamp. Delta QTc during the night was blunted in diabetics (0.5+/-2.5 vs. C: 2.9+/-2.5%, p=0.001), and Delta LF/HF was decreased in N+ (-2.8+/-38.2 vs. C=34.8+/-28.1%, p=0.02). During hyperglycaemia, QTc increased in C; LF/HF significantly increased in C and N-. A 6-month treatment with n-3 PUFA partially restored Delta LF/HF and Delta QTc (2.1+/-1.40, p=0.04 vs. basal) only in N-. CONCLUSION: Hyperglycaemia increases QTc interval and sympathetic activity; electrical membrane stabilization improves autonomic function only in the absence of overt autonomic neuropathy. Strategies to prevent the disruption of autonomic function with newer approaches, other than just glucose control, should be implemented.

The Arg972 variant in insulin receptor substrate-1 is associated with an atherogenic profile in offspring of type 2 diabetic patients.

The insulin receptor substrate-1 (IRS-1) gene has been considered a candidate for insulin resistance, type 2 diabetes, and coronary artery disease. To investigate the relationship between the common Gly(972)Arg IRS-1 variant and the presence of cardiovascular risk factors, 153 glucose-tolerant, unrelated offspring of type 2 diabetic patients were studied. There were no differences between Arg(972) IRS-1 carriers and noncarriers in age, gender, body mass index, waist/hip ratio, body composition, fasting glucose and insulin levels, and glucose or insulin levels during the oral glucose tolerance test. Insulin sensitivity, assessed by hyperinsulinemic-euglycemic clamp, was significantly reduced in carriers of Arg(972) IRS-1 (P < 0.03). Carriers of Arg(972) IRS-1 displayed many features of the insulin resistance syndrome, including higher values for serum triglycerides (P < 0.01), total/high density lipoprotein cholesterol ratio (P < 0.01), free fatty acid levels (P < 0.04), systolic blood pressure (P < 0.04), microalbuminuria (P < 0.003), and intima-media thickness (P < 0.02). These results suggest that the Arg(972) IRS-1 variant could contribute to the risk for atherosclerotic cardiovascular diseases associated with type 2 diabetes by producing a cluster of insulin resistance-related metabolic abnormalities.

Analysis of the variation in the hsp70-1 and hsp90alpha mRNA expression in human myocardial tissue that has undergone surgical stress.

In the present work we reported a semiquantitative detection of messenger ribonucleic acids (mRNAs) encoding the human heat shock proteins Hsp70-1, the stress inducible member of the HSP70 family, and hsp90alpha, the inducible member of the HSP90 family. We investigated the change in the expression of these mRNAs in tissue samples taken from the right atrium of 48 pediatric patients, soon after the ischemic period during surgery to correct congenital heart diseases, in which a crystalloid cold cardioplegic solution was used. No significant variations were found for either hsp70-1 or hsp90alpha expressions. Moreover, we searched for an association between the hsp70-1 promoter region polymorphism and the expression of the hsp70-1 in a smaller group of these patients (n = 27). The -110AA genotype was on average significantly associated with a decrease in the hsp70-1 mRNA level (P < 0.05), whereas the other genotypes -110AC or -110CC did not seem to be associated with the hsp70-1 expression level. The lack of any observed increase in the hsp70-1 expression level may be due to the high basal level of the Hsp70 protein in the tissues examined.

Early autonomic dysfunction in glucose-tolerant but insulin-resistant offspring of type 2 diabetic patients.

In type 2 diabetes, both insulin resistance and hyperglycemia are considered responsible for autonomic dysfunction, but the specific role of these two abnormalities is not clear. To test the specific role of insulin resistance on autonomic dysfunction, we studied 69 glucose-tolerant offspring of type 2 diabetic patients, comparing the most insulin-resistant tertile (IR) with the most insulin-sensitive tertile (IS) and comparable control subjects, all undergoing the oral glucose tolerance test, impedentiometry, 24-hour blood pressure and ECG monitoring, and an intravenous glucose tolerance test (IVGTT) followed by a euglycemic hyperinsulinemic clamp, with continuous blood pressure and ECG measurements. Sympathovagal balance was evaluated as low- to high-frequency ratio (LF:HF) by spectral analysis on R-R intervals. The change of systolic and diastolic blood pressure was calculated as [(day-night/d)]x100. In IR, the changes of systolic and diastolic blood pressure were significantly lower versus IS (9.2+/-5.0% versus 12.4+/-3.6%, P<0.02; 13.2+/-6.5% versus 17.4+/-5.2%, P<0.02). During the night, LF:HF fall was reduced in IR (43.1+/-21.0 versus 61.4+/-16.9, P<0.02). Hyperinsulinemia (IVGTT) rapidly and significantly increased LF:HF in IR (4.9+/-3.3 versus basal: 2.3+/-1.4, P=0.03) but not in IS. In offspring of type 2 diabetic patients with normal glucose tolerance and normal blood pressure values, insulin resistance is associated with abnormal control of blood pressure and sympathetic activation. Insulin resistance may therefore be responsible for some early derangements of the autonomic nervous tone control and thus contributes to increase the incidence of arterial hypertension and/or diabetes.

Biochemical evaluation of vacuum-assisted venous drainage: a randomized, prospective study.

AIMS OF THE STUDY: In this prospective, randomized study, we investigate the potential advantages of vacuum-assisted venous drainage (VAVD), compared to gravitational drainage (GD), in patients undergoing first-time coronary artery bypass graft (CABG) surgery, concerning biochemical markers of organ and blood cell damage. MATERIALS AND METHODS: Seventy-two consecutive patients were randomized into two groups ['Vacuum' (VAVD) n=36; 'Not vacuum' (GD) n=36]. VAVD was achieved using a wall vacuum source and with a suction regulator connected to the vent port of the hardshell venous reservoir. In the VAVD group, we used 28-French venous cannulas, and 36-French in the GD group. In the VAVD group, we measured arterial perfusion flow (APF) and the venous reservoir volume (VRV) with and without vacuum application just after starting extracorporeal circulation (ECC). Six blood samples were drawn at different times before, during and after ECC. Routine blood tests were performed to evaluate hemolysis, and hepatic and renal function. RESULTS: The two groups were similar in terms of preoperative and operative characteristics. There were no significant differences in biochemical markers of organ function or hemolysis between the two groups. In the VAVD patients, platelet count was higher at 24 h after the end of the operation (VAVD 151.77+/-50.28 microl versus Not vacuum 124.93+/-41.60 microl, p=0.028). With the narrower venous cannulas (28-French), only VAVD achieved a satisfactory APF (VAVD 2.35+/-0.38 l/min/m2 versus GD 1.88+/-0.27 l/min/m2, p=0.002), with a larger VRV (VAVD 1091.67+/-421 ml versus GD 808.33+/-284.31 ml, p=0.025). CONCLUSION: Vacuum-assisted venous drainage is a technique comparable to gravitational drainage with regard to hemolysis and organ perfusion. It allows better perfusion flow and heart decompression with smaller venous cannulas. This study suggests reduced platelet consumption with VAVD.