Yeast as a tool to select inhibitors of the cullin deneddylating enzyme Csn5.

The CSN complex plays a key role in various cellular pathways: through a metalloprotease activity of its Csn5 deneddylating enzyme, it regulates the activity of Cullin-RING ligases (CRLs). Indeed, Csn5 has been found amplified in many tumors, but, due to its pleiotropic effects, it is difficult to dissect its function and the involvement in cancer progression. Moreover, while growing evidences point to the neddylation function as a good target for drug development; specific inhibitors have not yet been developed for the CSN. Here, we propose the yeast Saccharomyces cerevisiae as a model system to screen libraries of small molecules as inhibitors of cullins deneddylation, taking advantage of the unique feature of this organism to survive without a functional CSN5 gene and to accumulate a fully neddylated cullin substrate. By combining molecular modeling and simple genetic tools, we were able to identify two small molecular fragments as selective inhibitors of Csn5 deneddylation function.

Implementing PGD/PGD-A in IVF clinics: considerations for the best laboratory approach and management.

For an IVF clinic that wishes to implement preimplantation genetic diagnosis for monogenic diseases (PGD) and for aneuploidy testing (PGD-A), a global improvement is required through all the steps of an IVF treatment and patient care. At present, CCS (Comprehensive Chromosome Screening)-based trophectoderm (TE) biopsy has been demonstrated as a safe, accurate and reproducible approach to conduct PGD-A and possibly also PGD from the same biopsy. Key challenges in PGD/PGD-A implementation cover genetic and reproductive counselling, selection of the most efficient approach for blastocyst biopsy as well as of the best performing molecular technique to conduct CCS and monogenic disease analysis. Three different approaches for TE biopsy can be compared. However, among them, the application of TE biopsy approaches, entailing the zona opening when the expanded blastocyst stage is reached, represent the only biopsy methods suited with a totally undisturbed embryo culture strategy (time lapse-based incubation in a single media). Moreover, contemporary CCS technologies show a different spectrum of capabilities and limits that potentially impact the clinical outcomes, the management and the applicability of the PGD-A itself. In general, CCS approaches that avoid the use of whole genome amplification (WGA) can provide higher reliability of results with lower costs and turnaround time of analysis. The future perspectives are focused on the scrupulous and rigorous clinical validations of novel CCS methods based on targeted approaches that avoid the use of WGA, such as targeted next-generation sequencing technology, to further improve the throughput of analysis and the overall cost-effectiveness of PGD/PGD-A.

NT, NPY and PGP 9.5 presence in myomeytrium and in fibroid pseudocapsule and their possible impact on muscular physiology.

The uterine myoma pseudocapsule is a neurovascular bundle surrounding fibroid, containing neuropeptides, probably involved in uterine scar healing. We studied neurotensin (NT), neuropeptide tyrosine (NPY), and protein gene product 9.5 (PGP 9.5) nerve fibres in the pseudocapsule neurovascular bundle of intramural uterine fibroids on 67 no pregnant women by intracapsular myomectomy sparing the neurovascular bundle, sampling full thickness specimens of the pseudocapsule of uterine fibroids (PUF) and normal myometrium (NM) obtained from the fundus uteri (FU) and the uterine body (UB). The samples were sent for histological and immunofluorescent analyses and compared by morphometrical quantification. The Conventional Unit (C.U.) difference of NT, NPY, and PGP 9.5 nerve fibres was statistically analyzed. Our results showed that NT, NPY, and PGP 9.5 neurofibers are almost equally present in PUF as in NM of a no pregnant uterus. As all of these neuropeptides are present in the uterine muscle and can affect muscle contractility, uterine peristalsis and muscular healing. A myomectomy respecting the pseudocapsule neurofibers should facilitate smooth muscle scarring and promote restoration of normal uterine peristalsis with a possible positive influence on fertility.

Ultrasound evaluation of uterine healing after laparoscopic intracapsular myomectomy: an observational study.

STUDY QUESTION: Can uterine scar healing after laparoscopic intracapsular myomectomy (LIM) be adequately monitored by traditional two-dimensional (2D) ultrasound (US) and Doppler velocimetry? SUMMARY ANSWER: The myometrial area of the scar after LIM can be followed by 2D US and Doppler velocimetry. WHAT IS KNOWN ALREADY: Apart from post-surgical adhesions, the main concern linked to laparoscopic myomectomy is the quality of healing of the myometrial incision: it has been suggested that US could be useful for assessing uterine scars after myomectomy. However, no diagnostic method has yet been widely accepted to assess the healing process. STUDY DESIGN, SIZE, DURATION: A cohort prospective study (level of evidence II-2), run in University-affiliated hospitals: 149 women with symptomatic uterine fibroids (UFs) underwent LIM, between January 2007 and October 2011. During follow up 13 patients withdrew from the study. PARTICIPANTS/MATERIALS, SETTING, METHODS: After LIM, all patients were followed by traditional 2D US scanning and Doppler velocimetry on Days: 0, 1, 7, 30 and 45. Authors evaluated: number, size and location of UFs, scar diameter and Doppler velocimetry and resistance index (RI) of the uterine arteries, at their ascending branch. MAIN RESULTS AND THE ROLE OF CHANCE: The uterine examination showed a significant (P < 0.05) progressive reduction of uterine scar area from 78% of the previous UF location on the first day, to 19% on 30th day, and <4% on the 45th day. There was no correlation with the size of the fibroid or the relative reduction in the size of the scar, on both Days 1 and 45. There was a significant (P < 0.05) increase in the RI of the ipsilateral uterine arteries from 0.65 on the first post-operative day to 0.83 after 7 days followed by a decrease to 0.71 on the 30th and 0.61 on the 45th post-operative day. LIMITATIONS, REASONS FOR CAUTION: This is a cohort investigation on a limited number of patients and it does not surgically compare LIM and 'classic' myomectomy in the scar US follow up. WIDER IMPLICATIONS OF THE FINDINGS: LIM avoided intraoperative bleeding and excessive tissue damage, as post-operative US follow up showed, with just two intra-myometrial hematomas (1.5%). The 2D US and Doppler velocimetry, a non-invasive safe method to check the myometrium after LIM, can detect post-operative hematoma and disechogenic, heterogeneous or ill-defined scar area, all unfavorable signs for myometrial scarring. Moreover, Doppler transvaginal monitoring, evaluating the pulsatility index (PI) and RI of the uterine arteries at their ascending branch, could identify patients with altered PI and RI parameters, possible markers of impaired wound healing.

Prevention of lymphocele in female pelvic lymphadenectomy by a collagen patch coated with the human coagulation factors: a pilot study.

BACKGROUND AND OBJECTIVES: The prevention of lymphoceles was tested using collagen patch coated with the human coagulation factors (TachoSil) on 58 consecutive patients with endometrial cancer who had undergone hysterectomy and pelvic lymphadenectomy (PL). METHODS: Patients were randomized in two groups: standard technique plus TachoSil (30 patients, group 1) and standard technique only (28, group 2). All surgical parameters were collected and patients underwent ultrasound examination on postoperative days 7, 14, and 28. The main outcome measures were: the development of symptomatic or asymptomatic lymphoceles, the need for further surgical intervention, as adverse effect of surgery and the drainage volume and duration. RESULTS: Same number of lymph nodes in both groups was removed; group 1 showed a lower drainage volume. Lymphoceles developed in 7 patients in group 1 and 16 in group 2, but only 3 were symptomatic in group 1 and 9 symptomatic in group 2, with statistical difference. Percutaneous drainage proved necessary in five cases: only one was in group 1 and four in group 2. CONCLUSIONS: Intraoperative application of TachoSil reduced rate of postoperative lymphocysts after PL, and it seems to provide a useful additional treatment option for reducing drainage volume and preventing lymphocele development after PL.

Surgical management of neurovascular bundle in uterine fibroid pseudocapsule.

The uterine fibroid pseudocapsule is a fibro-neurovascular structure surrounding a leiomyoma, separating it from normal peripheral myometrium. The fibroid pseudocapsule is composed of a neurovascular network rich in neurofibers similar to the neurovascular bundle surrounding a prostate. The nerve-sparing radical prostatectomy has several intriguing parallels to myomectomy. It may serve either as a useful model in modern fibroid surgical removal, or it may accelerate our understanding of the role of the fibrovascular bundle and neurotransmitters in the healing and restoration of reproductive potential after intracapsular myomectomy. Surgical innovations, such as laparoscopic or robotic myomectomy applied to the intracapsular technique with magnification of the fibroid pseudocapsule surrounding a leiomyoma, originated from the radical prostatectomy method that highlighted a careful dissection of the neurovascular bundle to preserve sexual functioning after prostatectomy. Gentle uterine leiomyoma detachment from the pseudocapsule neurovascular bundle has allowed a reduction in uterine bleeding and uterine musculature trauma with sparing of the pseudocapsule neuropeptide fibers. This technique has had a favorable impact on functionality in reproduction and has improved fertility outcomes. Further research should determine the role of the myoma pseudocapsule neurovascular bundle in the formation, growth, and pathophysiological consequences of fibroids, including pain, infertility, and reproductive outcomes.

Molecular methods for a correct diagnosis of multiple HPV infections and clinical implications for vaccine.

INTRODUCTION: The human papillomavirus (HPV) family is characterized by minimal genotypic differences corresponding to different virus types. The aim of this study was to detect the HPV coinfections and the inner genotype in a series of 336 cervical-vaginal samples. METHODS: A total of 336 cervical-vaginal samples were taken from 2007 to 2009 using specific molecular techniques such as molecular sequencing and hybridizations. The genome amplification of the L1 open reading frame was analyzed by real-time polymerase chain reaction; direct sequencing was performed by SYBR green fluorescent molecule and degenerate primers MY09 and MY11. The HPV genotyping was accomplished via oligonucleotide probe hybridization. The phylogenetic correlations in coinfections were analyzed by sequence homology of the L1 genomic region. Identified genotypes were then compared. RESULTS: Human papillomavirus positivity was observed in 125 cases (37.2%), with 21 cases (16.8%) of HPV presence in coinfections. Coinfections involved HPV 16 genotype (8 cases) and HPV 18 (5 cases). The HPV 16 infection was mainly associated with genotypes with a lower-than-broad sequence homology, so the HPV 18 was linked to genotypes represented in the opposite phylogenetic tree. CONCLUSIONS: The combined and steady use of diagnostic procedures, such as real-time polymerase chain reaction, molecular hybridization, direct sequencing, and HPV genotyping test, allow accurate diagnosis of monoinfections and coinfections. This may faciliate the development of specific viral tests and prophylactic anti-HPV vaccines.

[Importance of the molecular diagnosis in the screening of alpha-thalassemia]. / Importanza della diagnosi molecolare nel riconoscimento delle alfa talassemie. Descrizione di un caso.

The term alpha thalassemia refers to inherited disorders of hemoglobin caused by reduced or absent synthesis of alpha globin chains. This paper highlights that in the presence of a alfa2-Tal (-α/αα), called the silent, the biochemical diagnosis turns out to be insufficient. In these cases, the molecular study of alpha-globin genes is necessary for identification. In reason of this we present the following case report. A woman of 29 years, pregnant at 12(a) weeks, arrived at our observation to undergo prenatal screening test for Down and Edwards syndromes (bitest). The medical history of the couple revealed that both had doubts haematological indices: Mr. T.G. had a biochemical framework related to alpha1-Tal (MCV 58.8fl, MCH 19.8pg, HbA2: 1.9, HbF:0.4, erythrocytes 6.58x10(6)/ul ed Hb 13g/dl), which was confirmed by molecular analysis (genotype alfa(0-20.5Kb)). Particular difficulties of interpretation presented the C.F. patient who had a biochemical phenotype border line (MCV 79.8fl, MCH 27.2pg, HbA2: 2.9, HbF: 0.6, erythrocytes 5.11x10(6)/ul, Hb 12.8 g/dl). Only molecular analysis has found with certainty that Mrs. C.F. appeared to be phenotypically alpha2-TAL (-α/αα) for the presence of the mutation "alfa2 init.Cd(T>C) NcoI". In the event, as in our case, there is a couple where one spouse is alpha2-TAL (-α/αα) and the other alpha1-TAL (--/αα), must have to inform the couple about the possibility of conceiving a child with hemoglobin H (HbH). Far from the authors refer to the idea of prenatal diagnosis for couples at risk to bear children with HbH, but it is worth highlighting the importance of a careful study of the blood parameters and an extensive and precise information about the clinical implications related to complications of alpha thalassemia.

Mayer-Rokitansky-Küster-Hauser syndrome with 22q11.21 microduplication: a case report.

BACKGROUND: Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome (Online Mendelian Inheritance in Man [OMIM] #277000) is a congenital condition characterized by the total or partial agenesis of vagina and uterus. Agenesis can be isolated (MRKH 1) or associated with other renal, vertebral or cardiac defects (MRKH 2). CASE PRESENTATION: In this paper, we report a case of a Caucasian patient showing the clinical signs associated with MRKH. Array-based comparative genomic hybridization (a-CGH) analysis revealed a microduplication of approximately 3.01 megabases (Mb) located on the long arm of chromosome 22 (22q11.21). Microduplications affecting the 22q11.21 region have been shown to be associated with MRKH syndrome and Müllerian aplasia. The phenotype of patients with 22q11.2 duplication (OMIM #608363) appears extremely variable, ranging from apparently normal to mild learning difficulties or with multiple defects, sharing features with DiGeorge/velocardiofacial (DGS/VCFS) syndrome. CONCLUSIONS: The altered gene expression together with other genetic, nongenetic, epigenetic or environmental factors can cause the extremely variable phenotype in patients carrying such duplication. Therefore, we can consider MRKH syndrome to be one of the clinical features of DGS/VCFS syndrome.

Cardiofaciocutaneous syndrome with rare structural variant in DOCK8 gene associated with neurodevelopmental disorders.

We describe a girl with clinical signs of cardiofaciocutaneous syndrome who simultaneously presents a mutation in the BRAF gene and a 9p24.3 microduplication. This genetic condition has never been described in the literature and could explain the phenotypic variability observed in the girl.

Long term efficacy and safety of rivaroxaban plus cilostazol in the treatment of critical ischemia of the lower limbs in a frail, elderly patient with non valvular atrial fibrillation.

BACKGROUND: Many patients with critical lower limb ischemia are not eligible for revascularization procedures. Still, given the emerging role of both platelet and coagulation activation in the formation of arterial thrombi, they may benefit from the novel anticoagulant and antiplatelet drugs. CASE PRESENTATION: We describe the case of a male with critical lower limb ischemia complicated by older age, frailty, polymorbidity and non valvular atrial fibrillation, who was deemed as non eligible for surgery. The patient was successfully treated with the combination of rivaroxaban and cilostazol, and the clinical benefit was maintained throughout 32 months, with no occurrence of major or minor hemorrhagic or thrombotic events. CONCLUSIONS: To our knowledge, this is the first report on the efficacy and safety of such a combination therapy in critical lower limb ischemia. In a clinical setting in which alternative pharmacological approaches are urgently needed, the association of rivaroxaban and cilostazol warrants further investigations.

Methylenetetrahydrofolate reductase gene C677T and A1298C polymorphisms and susceptibility to recurrent pregnancy loss.

Several studies have investigated the link between two different polymorphisms (C677T and A1298T) of the gene encoding methylenetetrahydrofolate reductase (MTHFR) and the risk of recurrent pregnancy loss (RPL); however, the results remain controversial. This study aimed to provide greater insight into this debated topic. In the current study, two groups of pregnant women (group A: RPL women; group B: non-RPL women), each of which were subdivided further into two subgroups based on their gestational age, were screened for C677T and A1298T variants of the MTHFR gene. The resulting data were analyzed using receiver operating characteristic (ROC) curve and Z test methods to compare the two groups. These ROC curve and Z test analyses indicated that there were no differences between the groups regarding C677T and A1298T expression. RPL is primarily caused by mutations in prothrombin or factor V Leiden genes. However, a low percentage of RPL cannot be attributed to these mutations. In the last five years, research has focused on the MTHFR gene, the two major variants of which (C677T and A1298T) have been associated with an increased risk of cardiovascular diseases (thrombotic events) in homozygous individuals. In addition, these mutations may be related to an increased rate of neural tube defects in fetuses. While a link between MTHFR mutation and RPL may be expected based on previous findings, the present study indicated the absence of an association between the polymorphisms of the MTHFR gene and RPL risk.

Five cases of supernumerary small ring chromosomes 1: heterogeneity and genotype-phenotype correlation.

Genetic counselling of patients with small supernumerary ring chromosomes (sSRCs) can be difficult, especially in prenatal testing, due to the complexity in establishing a karyotype-phenotype correlation. In fact, it has been estimated that about 10% of extra ring(1) chromosomes are associated with an unremarkable phenotype. We report on five new cases of extra ring chromosomes(1) manifesting different clinical outcome. One case was familial, segregating from a mother with mosaic karyotype, while the others were de novo. Ring chromosomes were characterised by FISH. In three subjects the involvement of the same euchromatic 1p region was demonstrated. Present observations corroborate previous results and provide some insight into the identification of the harmless ring(1) structures.

The influence of D-chiro-inositol and D-myo-inositol in pregnant women with glucose intolerance.

The aim of the present study was to demonstrate that the use of inositol and folic acid from the first trimester of pregnancy, counteracts the onset of gestational diabetes mellitus (GDM) in women at risk, preserving the infants from macrosomia, hypoglycemia and preterm delivery. The authors collected data from the pregnant women at the laboratory (Unit of Cytogenic and Molecular Genetics), from January 2014 to April 2016, all with first trimester fasting plasma glucose (FPG) >92 mg/dl. A total of 40 women were treated with 250 mg/day D-chiro-inositol, 1.75 g/day D-myo-inositol, 12.5 mg/day zinc, 10 mg/day methylsulfonylmethane, 400 µg/day 5-methyltetrahydrofolic acid. The other 43 women (control group) were treated with only 400 µg/day folic acid. The primary outcome measure was the incidence of maternal GDM. The secondary outcome measures were the incidence of fetal macrosomia, preterm delivery and neonatal hypoglycemia. At the 24th week of pregnancy, the incidence of maternal GDM was recorded in 18 women in the control group and in 5 women in the treated group [relative risk (RR)=3.35; 95% confidence interval (CI)=1.37-8.17; P=0.0028). A significant difference was observed between treated and control groups in terms of risk of macrosomia. A total of seven infants in the control group, and two in the treated group, weighed >4,000 g (RR=5,12; 95% CI=1.21-21.68; P=0.0099). No significant difference was identified between two groups, regarding the other two secondary outcomes, neonatal hypoglycemia (RR=4.650; 95% CI=0.57-38.11; P=0.1086) and preterm delivery (RR=1.74; 95% CI=0.83-3.66; P=0.1301). The current study demonstrated the potential benefit of supplementation with the association of D-chiro-inositol and D-myo-inositol in pregnant 'at risk' women, with first trimester FPG >92 mg/dl, in preventing the onset of maternal GDM and macrosomia in newborns.

Analysis of cystic fibrosis gene mutations in children with cystic fibrosis and in 964 infertile couples within the region of Basilicata, Italy: a research study.

INTRODUCTION: Cystic fibrosis is the most common autosomal recessive genetic disease in the Caucasian population. Extending knowledge about the molecular pathology on the one hand allows better delineation of the mutations in the CFTR gene and the other to dramatically increase the predictive power of molecular testing. METHODS: This study reports the results of a molecular screening of cystic fibrosis using DNA samples of patients enrolled from January 2009 to December 2013. Patients were referred to our laboratory for cystic fibrosis screening for infertile couples. In addition, we identified the gene mutations present in 76 patients affected by cystic fibrosis in the pediatric population of Basilicata. RESULTS: In the 964 infertile couples examined, 132 subjects (69 women and 63 men) resulted heterozygous for one of the CFTR mutations, with a recurrence of carriers of 6.85%. The recurrence of carriers in infertile couples is significantly higher from the hypothetical value of the general population (4%). CONCLUSIONS: This study shows that in the Basilicata region of Italy the CFTR phenotype is caused by a small number of mutations. Our aim is to develop a kit able to detect not less than 96% of CTFR gene mutations so that the relative risk for screened couples is superimposable with respect to the general population.

Efficacy and safety of 0.5% levobupivacaine versus 0.5% bupivacaine for peribulbar anesthesia.

BACKGROUND: This randomized double-blind study examined the use of a new anesthetic agent, levobupivacaine 0.5%, which is the S(-)-enantiomer of a racemic mixture of bupivacaine, for peribulbar anesthesia and compared it with racemic bupivacaine 0.5% alone or in combination with hyaluronidase 10 IU/mL. METHODS: A total of 160 patients undergoing ophthalmic surgery were randomized into four groups (n = 40 each) to receive inferotemporal peribulbar injection of levobupivacaine 0.5% (group L), racemic bupivacaine 0.5% (group B), levobupivacaine + hyaluronidase 10 IU/mL (group LH), or racemic bupivacaine + hyaluronidase 10 IU/mL (group BH) by two anesthetists and two ophthalmologists in a ratio of 25% each. Ocular akinesia and orbicularis oculi function were evaluated using a three-point scale; a value < 5 points was considered as requiring surgery, and movements were re-evaluated the day following surgery to confirm regression of the block. RESULTS: The time to onset (12 ± 2.6 minutes versus 13 ± 2.8 minutes) and duration of anesthesia (185 ± 33.2 minutes versus 188 ± 35.7 minutes) were similar between groups L and B. Complete akinesia (score 0) was obtained more frequently when hyaluronidase was used in addition to the anesthetic, with occurrences of 72.5% versus 57.5% in group LH versus L, respectively, and 67.5% versus 45% in group BH versus B. Moderate hypotension (<30% of baseline) was observed in four patients (10%) in group L, two (5.0%) in group B, one (2.5%) in group LH, and three (7.5%) in group BH. The time to onset was significantly different between groups L and BH, B and BH, and LH and BH, and the duration of anesthesia differed significantly between groups B and LH, B and BH, and L and LH. The akinesia score differed significantly between groups L and LH and between groups B and LH (P = 0.043 and P = 0.018, respectively), and the number of patients with a score of 0 differed significantly between groups B and LH and between groups B and BH (P = 0.004 and P = 0.017, respectively). CONCLUSION: Levobupivacaine is a long-lasting local anesthetic with limited cardiotoxicity and neurotoxicity, and may be considered the landmark for vitreoretinal surgery in elderly patients.

Incidence of ß-thalassemia carrier on 1495 couples in preconceptional period.

OBJECTIVE: This research, conducted on 1495 couples in preconceptional period, demonstrates how the study of globular resistance of erythrocytes (GRO) is not a first choice test and not useful as other more accurate tests to identify subjects with ß-thalassemia trait. Instead, the complete blood count (CBC) and the evaluation of HbA, HbA2 and HbF by high pressure liquid chromatography (HPLC) are essential. METHODS: Each couple arrived in our laboratory to screen for ß thalassemia. In case of patients with positive (240) or doubtful (112) results, we studied ß-globin gene. RESULTS: Of the 2990 subjects examined, we found 280 subjects with ß-thalassemia trait (9.36%). During biochemical tests, among 112 subjects with doubtful--normal GRO or altered GRO--results, 40 of them resulted positive for the molecular analysis, while 72 of them did not show mutations in ß-globin genes. The 2710 samples with non-carriers of ß-thalassemia trait presented as mean evaluation of HbA2 2.6%, while the 280 subjects with ß-thalassemia trait presented as mean evaluation of HbA2 4.8%. Molecular study showed that the ß thalassemia phenotype is caused by a small number of mutations, whose regional distribution is typical. CONCLUSIONS: In the presence of thalassemic parameters in the CBC, the accurate and precise quantification of hemoglobin HbA2 is essential for the diagnosis of ß-thalassemia trait. DNA mutation analysis provides the most effective way to detect primary gene mutations. The mutations identified in this work can be identified with a simple and inexpensive kit. This means, in economic terms, a significant savings for health spending.

Effect of multivitamins on plasma homocysteine in patients with the 5,10 methylenetetrahydrofolate reductase C677T homozygous state.

The role of hyperhomocysteinemia (HHcy) as a cardiovascular risk factor remains a matter of debate, while it correlates with folates, it demonstrates inverse correlation with plasma homocysteine (Hcy) levels and vitamin B12 levels and reduces plasma Hcy levels following supplementation with multivitamins. The purpose of this study was to demonstrate that administering multivitamins at specific doses for 90 days restores normal plasma Hcy levels in women who are homozygous for the thermolabile variant of 5,10 methylenetetrahydrofolate reductase (MTHFR C677T). We enrolled 106 healthy females aged between 30 and 42 years, who were non-smokers, non-vegetarian, normotensive and who had no history of food abuse in the previous months. Only females were enrolled in order to rule out any bias due to the variation in Hcy plasma concentrations between males and females. Patient blood sampling was performed in order to determine plasma Hcy, serum folic acid and vitamin B12 levels. Furthermore, molecular characterization of the C677T polymorphism present in the MTHFR gene, was also performed. The results of this study demonstrated that supplementation with specific multivitamins restores normal plasma Hcy levels, regardless of the MTHFR genotype. Furthermore, it is unnecessary to adminster high doses of folate to reduce plasma Hcy levels, and administering high doses of folate may cause pro-inflammatory and pro-proliferative effects.

Identification of patients with defects in the globin genes.

INTRODUCTION: hemoglobinopathies constitute a major health problem worldwide. These disorders are characterized by a clinical and hematological phenotypic heterogeneity. The increase of HbA2 is an invaluable hematological marker of the beta-thalassemia heterozygosis and of double heterozygosis for the alleles of delta and alpha globin genes or for the alleles of delta and beta globin genes which can cause the increase of HbA2 up to normal or borderline values. CASE REPORT: we report the case of a 30-year-old woman (first pregnant) who was admitted to our Unit at 12 weeks for a screening for thalassemia. The outcomes of the biochemical and haematological exams (MCV, MCH, HbA2, HbF) highlighted that the patient was a carrier of a beta-thalassemic trait. Molecular analysis of the beta globin genes highlighted a ß(0)39C>T heterozygous mutation. Biochemical and hematological parameters of the husband (MCV, MCH, HbA2, HbF) were normal except for the level of HbA2 (3,6%). The molecular analysis of the beta globin genes highlighted a IVS2 nt844 C>G heterozygous mutation. Furthermore, the heterozygous mutation δ(+)cod.27G>T was detected in his δ globin gene. For this reason, he was diagnosed a δ+ß Thal. CONCLUSIONS: the aim of this paper is to highlight that biochemical diagnosis could not exhaustive and a molecular diagnostic widening is required to detect the genetic deficiency causing the thalassemic trait.