Sophia Zachaki

Enteral L-Arginine Supplementation for Prevention of Necrotizing Enterocolitis in Very Low Birth Weight Neonates A Double-Blind Randomized Pilot Study of Efficacy and Safety

BACKGROUND Necrotizing enterocolitis (NEC) is the most common acquired gastrointestinal disease in premature infants and has high mortality and morbidity. Endothelial nitric oxide is an important regulator of vascular perfusion and is synthetized from the amino acid L-arginine. Hypoargininemia is frequently observed in preterm neonates and may predispose them to NEC. Our objective was to determine the effect of enteral L-arginine supplementation on the incidence and severity of NEC in very low birth weight (VLBW) neonates. MATERIALS AND METHODS We conducted a parallel blind randomized pilot study, comprising VLBW neonates with birth weight ≤1500 g and gestational age ≤34 weeks. VLBW neonates were randomly assigned to receive enteral L-arginine supplementation (1.5 mmol/kg/d bid) between the 3rd and 28th day of life or placebo. Diagnosis and classification of NEC were done according to modified Bells criteria. RESULTS Eighty-three neonates were randomized to the arginine (n = 40) or placebo (n = 43) group. No adverse effects were observed in neonates receiving L-arginine supplementation. The incidence of NEC stage III was significantly lower in the arginine-supplemented group (2.5% vs 18.6%, P = .030). CONCLUSIONS Enteral L-arginine supplementation of 1.5 mmol/kg/d bid can be safely administered in VLBW neonates from the 3rd to the 28th day of life. Enteral L-arginine supplementation appears to reduce the incidence of stage III NEC in VLBW infants. Larger studies are needed to further evaluate the effect of L-arginine supplementation in preventing NEC in VLBW infants.

Novel and known microsatellite markers within the β-globin cluster to support robust preimplantation genetic diagnosis of β-thalassemia and sickle cell syndromes.

Preimplantation genetic diagnosis (PGD) for β hemoglobinopathies has become the most common application among monogenic disorders. We present the identification of microsatellite markers [short tandem repeats (STRs)] closely linked to the β-globin gene for incorporation within PGD protocols, with the aim of increasing the number of transferable embryos. Nine candidate STRs were identified in-silico, of which three were selected based on rate-of-heterozygosity, polymerase chain reaction (PCR) efficiency and size. The multiplex reaction (β-globin gene and selected STRs, all within <0.4 Mb from the β gene) was optimized in single lymphocytes, and subsequently applied in 38 PGD cycles in couples at-risk for transmitting β hemoglobinopathies. In conclusion, incorporation of closely linked polymorphic microsatellite markers <0.4 Mb from the β-globin gene, facilitates robust assignment of β hemoglobinopathy genotypes, increasing the number of transferrable embryos otherwise rejected due to allele-drop-out (ADO), at the mutation-specific locus, compared to results based on disease-mutation genotyping alone (p <0.001).

The C609T inborn polymorphism in NAD(P)H:quinone oxidoreductase 1 is associated with susceptibility to multiple sclerosis and affects the risk of development of the primary progressive form of the disease

Oxidative stress plays a pivotal role in the pathogenesis of multiple sclerosis (MS). Inactivating polymorphisms of genes encoding detoxification enzymes, such as NAD(P)H:quinone oxidoreductase 1 (NQO1), could influence susceptibility to MS. To test this hypothesis we performed a case-control study in which we compared the distribution of NQO1 genotypes between 231 MS patients and 380 controls, using both PCR-RFLP and real-time PCR assays. Correlations with MS clinical subtype classification and gender were also evaluated. A significantly higher frequency of the homozygous (T/T) and heterozygous (C/T) NQO1 C(609)T variant genotypes was observed among MS patients compared to controls (P=0.01), with MS patients showing a 1.5-fold increased risk of carrying at least one variant T allele (P=0.009). Interestingly, patients belonging to the primary progressive subgroup exhibited a significantly higher incidence of the heterozygous C/T variant genotype, compared to the other forms of MS (P=0.019). There was no correlation of the NQO1 polymorphism with gender. These results provide the first evidence for a pathogenetic role for the NQO1 C(609)T polymorphism in MS susceptibility and suggest a possible role for the NQO1 genetic background in the development of primary progressive MS.

Combined GSTP1 and NQO1 germline polymorphisms in the susceptibility to Multiple Sclerosis

Germline polymorphisms of detoxification genes could influence susceptibility to Multiple Sclerosis (MS). Glutathione-S-transferases (GSTs) and NAD(P)H:quinone oxidoreductase 1 (NQO1) are detoxifying enzymes involved in biotransformation of metabolites preventing cells from oxidative damage. In order to evaluate the possible contribution of the A313G GSTP1 inactivating polymorphism, alone and in combination with the C609T NQO1 genetic variant in MS susceptibility, we performed a case-control study consisting of 254 MS patients and 370 healthy donors. Genotypes were investigated using a new Real-Time PCR and PCR-RFLP assays. The GSTP1 polymorphism was evaluated in relation to patients’ characteristics (clinical subtypes, age and gender) and the NQO1 gene status. GSTP1 genotype distribution was similar between cases and controls. Higher frequency of GSTP1 heterozygotes was observed in patients with relapsing remitting disease (RRMS) (p = 0.019), especially in those presenting a benign form (EDSS ≤2 after 10–15 years from the disease onset). Interestingly, genotype distribution analysis of combined GSTP1 and NQO1 polymorphisms revealed significantly higher frequency of GSTP1 heterozygous (A/G) and NQO1 variant genotypes (C/T and T/T) in patients as compared to the controls (p = 0.031). The increased incidence of combined GSTP1 and NQO1 variant genotypes in MS patients may suggest that defective function of detoxification enzymes might be an important determinant of susceptibility and clinical manifestation of the disease. Moreover, the results suggest a possible role for the GSTP1 heterozygous background in the development of RRMS.

Association of A(313)G glutathione S-transferase P1 germline polymorphism with susceptibility to de novo myelodysplastic syndrome.

Abstract Models for the pathogenesis of myelodysplastic syndrome (MDS) imply the role of individual genetic variations in genes involved in detoxification mechanisms. GSTP1 enzyme plays a key role in the biotransformation of a variety of carcinogens. The corresponding gene is subject to a single nucleotide polymorphism (A313G) leading to abolished enzyme activity. In order to evaluate whether the GSTP1 polymorphism influences MDS susceptibility, we conducted a case–control study comprising 310 de novo patients and 370 healthy controls using a real-time polymerase chain reaction (PCR) genotyping method. The GSTP1 gene status was also evaluated in relation to patients’ characteristics and chromosomal abnormalities. A significantly higher incidence of the GSTP1 variant genotypes was observed in patients with MDS compared to controls (p < 0.0001). The results revealed increased frequencies of heterozygotes in patients younger than 60 years old and of homozygotes G/G in older patients (p = 0.007). Our results provide evidence for a pathogenetic role of the GSTP1 polymorphism in MDS risk, probably in an age-dependent manner.

Association of C609T-Inborn Polymorphism of NAD(P)H: Quinone Oxidoreductase 1 with the Risk of Bronchopulmonary Dysplasia in Preterm Neonates

UNLABELLED Objectives In bronchopulmonary dysplasia (BPD), direct exposure to oxygen therapy can damage the pulmonary epithelium via oxidative stress. The NAD(P)H quinone oxidoreductase 1 (NQO1) enzyme detoxifies genotoxic products of oxidative stress. The corresponding gene is subject to an inactivating single-nucleotide polymorphism (C(609)T), which reduces detoxifying ability. The aim of this study was to investigate whether the C(609)T NQO1 inborn gene polymorphism is associated with an increased risk of BPD. Study Design Peripheral blood samples from 119 premature neonates ≤ 32 weeks of gestational age (42 BPD and 77 non-BPD) were used for DNA extraction. NQO1 genotyping was performed using the polymerase chain reaction-restriction fragment length polymorphism method. Results A significantly higher frequency of the NQO1 polymorphism was observed in BPD neonates compared with neonates without BPD. All neonates with ≤ 1,000 g birth weight who carried the mutant allele in heterozygous or homozygous state developed BPD. None of the BPD nonaffected group neonates with ≤ 1,000 g birth weight carried the NQO1 polymorphism. Conclusion The higher incidence of NQO1 mutants among BPD neonates as well as the presence of the mutant allele in all neonates with ≤ 1,000 g who developed BPD provided the first evidence for a possible pathogenetic role of the C(609)T polymorphism in BPD susceptibility due to the reduction or loss of NQO1 enzymatic activity.

The G516T CYP2B6 Germline Polymorphism Affects the Risk of Acute Myeloid Leukemia and Is Associated with Specific Chromosomal Abnormalities

The etiology of acute myeloid leukemia (AML) underlies the influence of genetic variants in candidate genes. The CYP2B6 enzyme detoxifies many genotoxic xenobiotics, protecting cells from oxidative damage. The CYP2B6 gene is subjected to a single-nucleotide polymorphism (G516T) with heterozygotes (GT) and homozygotes (TT) presenting decreased enzymatic activity. This case-control study aimed to investigate the association of CYP2B6 G516T polymorphism with the susceptibility of AML and its cytogenetic and clinical characteristics. Genotyping was performed on 619 AML patients and 430 healthy individuals using RCR-RFLP and a novel LightSNip assay. The major finding was a statistically higher frequency of the variant genotypes (GT and TT) in patients compared to the controls (GT:38.8% vs 29.8% and TT:9.3% vs 5.3% respectively) (p<0.001). More specifically, a significantly higher frequency of GT+TT genotypes in de novo AML patients (46.6%) and an immensely high frequency of TT in secondary AML (s-AML) (20.5%) were observed. The statistical analysis showed that the variant T allele was approximately 1.5-fold and 2.4-fold higher in de novo and s-AML respectively than controls. Concerning FAB subtypes, the T allele presented an almost 2-fold increased in AML-M2. Interestingly, a higher incidence of the TT genotype was observed in patients with abnormal karyotypes. In particular, positive correlations of the mutant allele were found in patients carrying specific chromosomal aberrations [-7/del(7q), -5/del(5q), +8, +21 or t(8;21)], complex or monosomal karyotypes. Finally, a strikingly higher frequency of TT genotype was also observed in patients stratified to the poor risk group. In conclusion, our results provide evidence for the involvement of the CYP2B6 polymorphism in AML susceptibility and suggest a possible role of the CYP2B6 genetic background on the development of specific chromosomal aberrations.

Polymorphisms and haplotypes of the CYP2B6 detoxification gene in the predisposition of Acute Myeloid Leukemia (AML) and induction of its cytogenetic abnormalities

CYP2B6 is a polymorphic detoxification gene which plays a vital role in the degradation of genotoxic compounds. In this study we hypothesized that inadequate detoxification due to CYP2B6 polymorphisms may contribute to AML. To evaluate the potential impact of CYP2B6 polymorphisms on AML development and induction of its specific chromosomal abnormalities we studied C777A and A785G polymorphisms for the first time in AML. Furthermore, we investigated the co-existence of the above polymorphisms with G516T polymorphism to determine the CYP2B6 high-risk haplotypes in AML susceptibility. Our study included 619 AML patients and 430 healthy donors. Concerning C777A CYP2B6 polymorphism, no significant difference was found between patients and controls. However, A785G CYP2B6 polymorphism showed a statistically higher frequency of the variant genotypes in patients (48.2%), mainly in secondary AML patients (49.1%) than in controls (26.1%). Moreover, an increased frequency of the variant genotypes was found in those with abnormal karyotypes, especially with -7/del(7q), -5/del(5q), +8, inv(16) and t(8;21). The combination of the three CYP2B6 polymorphisms (G516T, C777A & A785G) revealed seven haplotypes. Four out of six haplotypes with at least one mutant allele were significantly associated with an increased risk for AML. Interestingly, T516A777G785 haplotype, where the three mutant alleles co-existed, had ~3-fold increased risk to be found in patients than controls. The association between haplotypes and cytogenetic aberrations revealed a positive correlation between specific CYP2B6 haplotypes and AML cytogenetic abnormalities. Our data suggest that A785G CYP2B6 gene polymorphism and specific CYP2B6 haplotypes may contribute to AML and its specific chromosomal aberrations.

Enteral l-Arginine and Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is the most common acquired gastrointestinal disease in premature infants. The molecular basis of this multifactorial disease is poorly understood, and NEC diagnosis and treatment strategies still remain difficult and challenging. In current research, preventive approaches are of the utmost priority. Providing human milk to premature infants has been shown to be an effective intervention that decreases the incidence of NEC. Interestingly, recent reports reveal that l-arginine supplementation may be a promising prevention modality of NEC by promoting nitric oxide (NO) synthesis. Deficient or inadequate NO levels due to l-arginine deficiency or immaturity of NO synthase (NOS) activity in premature infants may lead to vasoconstriction and ischemia–reperfusion injury and may predispose the neonates to develop NEC. The role of l-arginine supplementation in NEC has been highlighted in numerous studies on experimental animal models and human neonates. These researches reveal that the prophylactic administration of l-arginine in low doses is safe with no adverse effects for the premature neonates and also that l-arginine supplementation appears to be effective in reducing the overall incidence of NEC in preterm infants and especially the incidence of Bell’s stage III NEC. The recent evidence on the l-arginine role in NEC pathophysiology opens up a new area in the field of NEC prevention and further supports the essential role of amino acids in nutrition and disease prevention.

Association of GSTP1 inactivating polymorphism with acute myeloid leukemia and its specific chromosomal abnormalities

Acute myeloid leukemia (AML) is a clinically and genetically heterogeneous disease and the most common acute leukemia in adults. Many genotoxic factors have been accused for AML development but onl...