First and Second Level Haemoglobinopathies Diagnosis: Best Practices of the Italian Society of Thalassemia and Haemoglobinopathies (SITE)

The purpose of this best practice paper is to review the current recommendations for the identification and prenatal diagnosis of hemoglobinopathies. Methods: The management committee of SITE selected and gathered a multidisciplinary team in order to formulate recommendations based on the available scientific evidence integrated with the opinions of experts, with the purpose of supporting clinicians. Results: We provide recommendations for first level tests (complete blood count, hemoglobin separation and iron balance), second level tests (molecular diagnosis) and prenatal diagnosis. Five Italian experts in hemoglobinopathies were consulted regarding the orientation of prenatal diagnosis, and for each indication, the degree of agreement among the experts has been specified. Conclusions: Best practice recommendations are the final outcome of this translational research and allow transfer to daily clinical practice

Role of novel and rare nucleotide substitutions of the β-globin gene

The Laboratory for Molecular Prenatal Diagnosis of Hemoglobinopathies at the Villa Sofia-Cervello Hospital in Palermo, Italy, carries out an intensive screening program aimed at identifying the healthy carriers of thalassemia and, consequently, the couples at risk of bearing an affected fetus. The diagnostic process is basically divided into two phases: i) hematologic and hemoglobin data; ii) molecular analysis of globin genes and, when possible, a genetic study of the family. Since 2003, we have been performing DNA sequence analysis on those cases in which classical molecular methods failed to give a complete diagnostic response, particularly in phenotypes with borderline values of HbA2 with mild or absent microcytosis. During ten years of screening activities (from 2003 to 2012), twenty- seven unknown or rare nucleotide changes of the &beta;-globin gene have been identified; hematologic and hemoglobin data have been carefully evaluated and, wherever possible, we have conducted a family study to evaluate whether a phenotypic expression could be associated to these nucleotide changes. Because of the limited numbers of cases for each mutation, the significance of these nucleotide substitutions has still not been fully clarified, and this raises a number of questions that need to be answered when carrying out appropriate genetic counseling for couples presumed to be at risk.&nbsp;意大利巴勒莫Villa Sofia-Cervello医院血红蛋白病分子产前诊断实验室进行密集的筛选程序，旨在识别健康的地中海贫血携带者和有怀上地中海贫血胎儿风险的夫妇。 诊断过程基本上分为两个阶段：1）血液及血红蛋白数据；2）珠蛋白基因分子分析以及家族遗传研究（如有可能）。 自2003年以来，我们已对这类病例进行DNA序列分析：传统的分子方法无法给出完整的诊断响应，尤其是有轻微小红细胞症或缺乏小红细胞症的HbA2临界值表型。 通过十年筛选活动（2003年至2012年），已识别出&beta;珠蛋白基因的27个未知或罕见的核苷酸变化；仔细评估过血液和血红蛋白数据，在可能的情况下，我们已进行家系研究，以确定表型表达是否与这些核苷酸变化相关。由于只有有限的突变案例，所以至今尚未完全证实这些核苷酸置换的重要性，并对假定存在患病风险的夫妇开展适当的基因遗传咨询时，因此引起了大量需要解答的问题。</p

Very early prenatal diagnosis of Cockayne’s syndrome by coelocentesis

Cockayne’s syndrome (CS) is a rare autosomal recessive multisystem disease characterised by early severe progression of symptoms. This study reports the feasibility of earlier prenatal diagnosis of CS by coelocentesis at 8 weeks of gestation respect to amniocentesis or villocentesis. Three couples at risk for CS asked to perform prenatal diagnosis by coelocentesis. Coelomic fluid was aspired from coelomic cavity in four singleton pregnancy at 8 weeks of gestation and 40 foetal cells were recovered by micromanipulator. Maternal DNA contamination was evaluated by quantitative fluorescent PCR (QF-PCR) and target regions of foetal DNA containing parental mutations of ERCC6 gene were amplified and sequenced. In all these cases, molecular analysis was possible. One foetus resulted affected of CS and the diagnosis was confirmed on placental tissue after voluntary abortion. In three cases, foetuses resulted carrier of a parental mutation and the results were confirmed after the birth. This study suggests that reliable prenatal diagnosis of CS could be performed using foetal cells present in coelomatic fluid in earlier pregnancy. Coelocentesis could be applied in prenatal diagnosis of CSs as well as for other monogenic diseases, at very early stage of pregnancy, if parental mutations are already known.Impact Statement What is already know on this subject? Previous studies utilising coelocentesis for prenatal determination of foetal sex reported variable success ranging from 58% to 95%, because of low total DNA content and presence of maternal cell contamination. This procedure has never been reported for early prenatal diagnosis at 8 weeks of gestation for rare genetically transmitted diseases such as Cockayne’s syndrome. What do the results of this study add? This study demonstrates that coelomic fluid sampling combined with well-standardised laboratory procedures can be applied for prenatal diagnosis at eight weeks of gestation for any rare monogenic disease if molecular defects are known. What are the implications of these findings for clinical practice and/or further research? The findings of this study in at risk couples for monogenic diseases investigated by coelocentesis demonstrate that embryo-foetal cell selection from CF allows reliable and early prenatal diagnosis of diseases. This technique is attractive to parents because it provides prenatal diagnosis of genetic disease at least 4 weeks earlier than what can be achieved by the traditional procedures reducing anxiety of parents and provides the option for medical termination of affected cases at 8–10 weeks’ gestation, which is less traumatic and safer than second-trimester surgical termination. Further research concerns the possibility to obtain foetal karyotype at eight weeks of gestation and the possibility of intrauterine corrective therapy

Nonsense-mediated decay mechanism is a possible modifying factor of clinical outcome in nonsense cd39 beta thalassemia genotype

Nonsense-mediated mRNA decay (NMD) is a surveillance system to prevent the synthesis of non-functional proteins. In &beta;-thalassemia, NMD may have a role in clinical outcome. An example of premature translation stop codons appearing for the first time is the &beta;-globin cd39 mutation; when homozygous, this results in a severe phenotype. The aim of this study was to determine whether the homozygous nonsense cd39 may have a milder phenotype in comparison with IVS1,nt110/cd39 genotype. Genotypes have been identified from a cohort of 568 patients affected by &beta;-thalassemia. These genotypes were compared with those found in 577 affected fetuses detected among 2292 prenatal diagnoses. The nine most common genotypes, each with an incidence rate of 1.5% or over, and together accounting for 80% of genotype frequencies, underwent statistical analysis. Genotype prevalence was calculated within the overall group. Results are expressed as proportions with 95% confidence intervals; P&le;0.05 was considered statistically significant. A binomial distribution was assumed for each group; z-tests were used to compare genotype frequencies observed in the patient group with frequencies in the affected fetus group. In the absence of selecting factors, prevalence of these two genotypes was compared between a cohort of 568 &beta;-thalassemia patients (PTS) and 577 affected fetuses (FOET) detected during the same period. IVS1,nt110/cd39 was significantly more prevalent in FOET than PTS (P&lt;0.0001), while there was no significant difference in prevalence of cd39/cd39 in FOET compared with PTS (P=0.524). These results suggest a cd39 genotype NMD mechanism may be associated with improved clinical outcomes in thalassemia major. 无义介导的mRNA 降解（NMD）&nbsp;是一种预防非功能性蛋白质合成的监控系统。在&beta;地中海贫血中，NMD可能对临床结果有影响。第一次出现的过早终止密码子（PTC）为&beta;珠蛋白cd39突变；若为纯合子，则会导致严重的表型。本研究旨在确定与IVS1,nt110/cd39基因型相比，纯合子无义cd39能否有更轻度的表型。目前已确定568名&beta;地中海贫血患者的基因型，并与从2292个产前诊断中检测出的577名地中海贫血胎儿的基因型相比较。对9个最常见基因型进行统计分析，每个基因型的发生率均为1.5%或以上，共占基因型频率的80%。在整个组中计算基因型分布情况，其结果以95%置信区间表示；若P&le;0.05，则具有统计意义。各组均假定成一个二项分布；Z测试适用于比较患者组的基因型频率和地中海贫血胎儿的基因型频率。 若没有选择因子，则比较568名&beta;地中海贫血患者（PTS）和同一时期所检测到的577个地中海贫血胎儿（FOET）这两组基因型的发生率。IVS1,nt110/cd39在FOET中的发生率明显高于PTS（P&lt;0.0001），同时cd39/cd39 在FOET和PTS（P=0.524）中的发生率并没有明显不同。这些结果表明cd39基因型NMD机制可能与重型地中海贫血的临床结果相关。</p

Incidental Detection of a Chromosomal Aberration by Array-CGH in an Early Prenatal Diagnosis for Monogenic Disease on Coelomic Fluid

Background: Turner syndrome is a rare genetic condition in which a female is partly or completely missing an X chromosome. Signs and symptoms vary among those affected. In fetuses that survive at birth and without congenital malformations, the prognosis is usually positive, but it has high lethality in utero, especially in the first trimester of pregnancy. Methods: We report a case of monosomy X detected during a prenatal diagnosis for beta thalassemia on coelomic fluid (CF) at the VIII week of gestation. Beta globin gene analysis, whole genome amplification (WGA), quantitative fluorescent PCR and array comparative genomic hybridization (array-CGH) were performed on DNA extracted from CF. Results: A monoallelic pattern of all Short Tandem Repeats mapped on the X chromosome was found and array-CGH performed on WGA from a few fetal erythroblasts confirmed monosomy X. Conclusion: This report underlines the importance of an early prenatal diagnosis and the countless potentialities of array-CGH that could make definition of molecular karyotype possible from a few fetal cells, unlike conventional cytogenetic techniques that require a greater cellular content. This is the first report of a molecular karyotype obtained from two cells selected by micromanipulation of CF and defined at such an early gestational age

β-Thalassemia heterozygote state detrimentally affects health expectation

Background: Thalassemia minor (Tm) individuals, are generally considered healthy. However, the prognosis of Tm individuals has not been extensively studied. The aim of this study was to evaluate the prognosis of Tm versus controls without β-thalassemia carrier state. Methods: A total of 26,006 individuals seeking thalassemia screening at the AOOR Villa Sofia-V. Cervello, Palermo (Italy) were retrospectively studied. Logistic penalised regression model was used to estimate risk of potential complications and survival techniques were used to study mortality. Results: We identified a total of 4943 Tm and 21,063 controls. Tm was associated with significantly higher risks of hospitalisation for cirrhosis (OR 1·94, 95% CI 1·30 to 2·90, p = 0·001), kidney disorders (OR 2·11, 95% CI 1·27 to 3·51, p = 0·004), cholelithiatis (OR 1·39, 95% CI 1·08 to 1·79, p = 0·010), and mood disorders (OR 2·08, 95% CI 1·15 to 3·75, p = 0·015). No statistically difference in life expectancy between thalassemia minor and control group was found (HR 1·090, 95% CI 0·777 to 1·555, p < 0·590; log-rank test p =.426). Conclusion: This study shows that Tm affects the prognosis of Tm carriers regarding health expectation. Probably, iron overload and anaemia for several years may be at the basis of these effects