Stem cell and genetic therapies for the fetus

The prenatal diagnosis and management of congenital disease has made significant progress over the previous decade. Currently, fetal therapy (including open surgery and fetoscopic intervention) provides therapeutic options for a range of congenital anomalies; however, it is restricted to the treatment of fetal pathophysiology. Improvements in prenatal screening and the early diagnosis of genetic disease allow for preemptive treatment of anticipated postnatal disease by stem cell or genetic therapy. While currently awaiting clinical application, in utero stem cell therapy has made significant advances in overcoming the engraftment and immunologic barriers in both murine and pre-clinical large animal models. Likewise, proof in principle for fetal gene therapy has been demonstrated in rodent and large animal systems as a method to prevent the onset of inherited genetic disease; however, safety and ethical risks still need to be addressed prior to human application. In this review, we examine the current status and future direction of stem cell and genetic therapy for the fetus.     

Allogeneic hematopoietic stem‐cell engraftment and graft failure

Abstract: Two immunologically mediated reactions, the graft‐versus‐host (GvH) and host‐versus‐graft (HvG) responses, form primary and opposing barriers to successful transplantation of allogeneic hematopoietic stem‐cells (HSC). The HvG barrier is set by the strength of the allogeneic immune response, which is determined by antigenic stimulation provided by donor cells, owing to differences in histocompatibility antigens, and the capacity of host immune cells to generate a response. Risk of graft failure must be viewed as the interplay of multiple factors, including degree of human leukocyte antigen and minor histocompatibility antigen disparity, capacity of host immune response, and the capacity of donor hematopoietic and immunologic cells for overcoming residual host immunity.     

Fetal stem cell and gene therapy

Advances in our understanding of stem cells, gene editing, prenatal imaging and fetal interventions have opened up new opportunities for the treatment of congenital diseases either through in-utero stem cell transplantation or in-utero gene therapy. Improvements in ultrasound-guided access to the fetal vasculature have also enhanced the safety and efficacy of cell delivery. The fetal environment offers accessible stem cell niches, localized cell populations with large proliferative potential, and an immune system that is able to acquire donor-specific tolerance. In-utero therapy seeks to take advantage of these factors and has the potential to cure diseases prior to the onset of symptoms, a strategy that offers substantial social and economic benefits. In this article, we examine previous studies in animal models as well as clinical attempts at in-utero therapy. We also discuss the barriers to successful in-utero therapy and future strategies for overcoming these obstacles.     

Histochemical demonstration of ornithine transcarbamylase activity in fetal liver

A histochemical technique for demonstrating ornithine transcarbamylase (OTC) activity in human and rhesus monkey fetal liver is described. The proportion of OTC-positive cells increases in fetal monkey liver between 130 and 150 days of gestation. The histochemical assay can be used to verify the prenatal diagnosis of OTC deficiency in the human fetus.     

Stem cell transplantation for hemoglobinopathies

Hereditary anemias caused by beta-thalassemia and sickle cell disease are the most common genetic diseases worldwide. Supportive therapies such as chronic lifelong transfusions, iron chelation for thalassemia, and transfusions or hydroxyurea for sickle cell anemia have significantly ameliorated clinical manifestations of these diseases but cannot eliminate disease and treatment-related complications that result in end-organ damage. Allogeneic hematopoietic stem cell transplantation is the only cure for patients with hemoglobinopathies. Results of transplants have steadily improved over the last few decades due to effective control of transplant-related complications and development of new preparative regimens. Our understandings of mixed chimerism in patients with hemoglobinopathies provide a rationale for the use of less intensive conditioning regimens and gene therapy in these disorders. Although the role of stem cell transplantation for thalassemia major is well defined, few transplants have been carried out in sickle cell disease, and, in light of recent advances, the role of stem cell transplantation in this disease should be revised. This review summarizes the current status of stem cell transplantation for hemoglobinopathies.     

The late effects of fetal growth patterns

The immediate prenatal and postnatal consequences of reduced fetal growth have long been known. The longer term associations between reduced birth weight and adult disease risk are also now well established. Reduced fetal growth is usually detected late in gestation, and the assumption has been that this is the time when factors regulating fetal growth have their greatest effect. However, recent evidence suggests that both the growth trajectory of the fetus and its adaptive responses to the prenatal and postnatal environment may be determined in the period around the time of conception.     

Fetal surgery: for what purpose?]

Widespread use of prenatal ultrasonography allows accurate diagnosis of many fetal malformations. The natural history and pathophysiology of some fetal diseases will undoubtedly induce lethal issue. Prenatal ultrasounds allow to determine prognosis during the second trimester of pregnancy. Experimental model and clinical practice have shown that prenatal surgery could be proposed for some correctable malformations: congenital diaphragmatic hernia, cystic adenomatoid lung disease, sacrococcygeal teratoma, lower urinary tract obstruction. To purpose this fetal therapy, great attention has to be paid to maternal risk and her future fertility, keeping in mind the aim of such fetal surgery: a new hope for improved management of the fetus with life-threatening defect.     

Fanconi anemia and beta c deficiency-associated pulmonary alveolar proteinosis as two hereditary diseases of childhood which are potentially curable by stem cell gene therapy but require different therapeutic approaches.

Fanconi anemia (FANC) and pulmonary alveolar proteinosis associated with deficiency of the beta-chain common to the GM-CSF/IL3/IL5 receptors (beta c-PAP) are rare inherited disorders of childhood or adolescence. Hematopoietic stem cell gene therapy aiming at reintroducing the wildtype cDNA as a new concept for the treatment of hereditary diseases may be applicable to FANC and PAP, as both disorders can be successfully treated by allogeneic stem cell transplantation. However, there are important distinctions to be made between the two diseases: FANC seems to be a disorder with functional stem cell deficiency. Thus, introduction of the wildtype cDNA should provide an in vivo growth advantage to genetically corrected stem cells so that corrected cells and their progeny may expand in vivo and slowly repopulate the entire hematopoietic system. In beta c-PAP, the defect has no major impact on proliferation or differentiation of stem cells. Therefore, introduction of the wildtype gene will probably not provide any selective growth advantage and the percentage of corrected cells in the hematopoietic compartment depend on the percentage of stem cells initially transduced as the current technology only allows for transduction of stem cells with low efficiency. The introduction of a second selectable cDNA into the vector might be used to provide selective growth for modified cells and thus overcome a low gene transfer efficiency of stem cells. The correction of rare monogenetic diseases may serve as a model for gene therapy prior to attempts to treat more common and complex polygenetic diseases. The studies outlined here will be helpful envisioning new treatment strategies for other inherited monogenetic diseases such as mucopolysaccharidosis, Gauchers disease or adrenoleukodystrophy.     

Clinical features of polyhydramnios associated with fetal anomalies

The purpose of this study was to examine the clinical features of pregnancy complicated by polyhydramnios associated with fetal anomalies. Sixty-nine patients with a singleton pregnancy complicated by polyhydramnios were retrospectively analyzed. Based on prenatal ultrasonographic findings, 13 cases were considered to have idiopathic polyhydramnios and the remaining 56 cases were associated with fetal anomalies. Between these two groups, no significant difference was found in the gestational weeks when polyhydramnios developed. However, significant difference was noted in the maximum amniotic fluid index (AFI) values during the pregnancy period; 25.4 +/- 2.7 cm in the former, and 30.6 +/- 8.9 cm in the latter (P = 0.0004). In all of 13 cases with idiopathic polyhydramnios, AFI values remained less than 30 cm until delivery. Twenty-two patients (39%) with fetal anomalies required a prenatal treatment such as amnioreduction and tocolysis, whereas only one patient (7.7%) with idiopathic polyhydramnios needed tocolysis therapy (P = 0.03). There was a significant risk of premature delivery with fetal anomalies (35.6 +/- 3.9 weeks' gestation vs. 38.8 +/- 1.5 weeks' gestation, P = 0.004) because of refractory polyhydramnios, rupture of membranes, non-reassuring fetal status, and intrauterine fetal death, and although most infants with idiopathic polyhydramnios were appropriate-for-dates, many of the infants with congenital anomalies were small-for-dates. Significant risk of fetal anomalies should be considered in pregnant women with severe polyhydramnios (AFI > or = 30 cm), an increased trend of amniotic fluid during the pregnancy period, polyhydramnios requiring a prenatal treatment, or fetal growth restriction. On the other hand, based on our experience, a fetus without these conditions seems to have a low risk of congenital anomalies even if polyhydramnios is noted.     

Multiplex PCR in noninvasive prenatal diagnosis for FGFR3‐related disorders

Thanatophoric dysplasia and achondroplasia are allelic disorders caused by a constitutively active mutation in the FGFR3 gene. Because thanatophoric dysplasia is a lethal disorder and achondroplasia is non‐lethal, they need to be distinguished after ultrasound identification of fetal growth retardation with short limbs. Accordingly, we have developed a noninvasive prenatal test using cell‐free fetal DNA in the maternal circulation to distinguish thanatophoric dysplasia and achondroplasia. A multiplex PCR system encompassing five mutation hotspots in the FGFR3 gene allowed us to efficiently identify the responsible mutation in cell‐free DNA in all examined pregnancies with a suspected thanatophoric dysplasia or achondroplasia fetus. This system will be helpful in the differential diagnosis of thanatophoric dysplasia and achondroplasia in early gestation and in couples concerned about the recurrence of thanatophoric dysplasia due to germinal mosaicism.     

Reduced-intensity allogeneic hematopoietic cell transplantation: Graft versus tumor effects with decreased toxicity

The potentially curative role of allogeneic hematopoietic cell transplantation (HCT) in neoplastic and non-neoplastic diseases is offset by the substantial risks of morbidity and mortality from complications of the intensive myeloablative and immunosuppressive preparative regimen. These regimen-related toxicities have restricted allogeneic HCT to young, otherwise healthy individuals without comorbid diseases. Pediatric patients undergoing conventional allogeneic HCT have lower procedure-related mortality but are at risk for non-fatal late effects of the high-dose pretransplant chemoradiotherapy, such as growth retardation, sterility and other endocrine dysfunction. Evaluation of reduced-intensity preparative regimens is the major focus of current clinical research in allogeneic HCT. Reduced-intensity HCT (RI-HCT) relies on the use of immunosuppressive but non-myeloablative agents that allow engraftment of donor cells, which provide adoptive allogeneic cellular immunotherapy and graft versus tumor (GVT) effects, with decreased regimen-related toxicities. Although the experience with RI-HCT in pediatric patients is very limited at this time, results in adults indicate that attenuated-dose preparative regimens allow older patients and those with organ dysfunction to undergo successful allogeneic HCT with acceptable morbidity and mortality. In adults, the potency of the allogeneic GVT effect varies among neoplastic diseases, with better results observed in patients with indolent hematological malignancies or renal cell carcinoma. The effectiveness of RI-HCT as treatment for children with hemoglobinopathies, chronic granulomatous disease and cellular immunodeficiencies is encouraging, and the role of reduced-intensity preparative regimens for allogeneic HCT in pediatric malignancies is under investigation.     

Fetal germinal matrix and intraventricular hemorrhage

Subependymal germinal matrix with intraventricular hemorrhage (GMIVH) is a common complication associated with delivery in preterm neonates but has rarely been observed in the fetus. Clinical and neuroradiological findings of 5 patients who were diagnosed as having fetal GMIVH with prenatal ultrasonographic examinations (US) and MRI, and postnatal MRI were reviewed retrospectively. During a seemingly uneventful pregnancy, fetal GMIVH occurred at approximately 30-33 weeks of gestation, with the absence of any known factor predisposing to fetal hemorrhage. Routine obstetric US revealed an intraventricular lesion in the enlarged ventricles. Prenatal MRI clearly demonstrated parenchymal change such as intracerebral hematoma adjacent to the subependymal and intraventricular hematoma, and periventricular leukomalacia as well as GMIVH. Although patients without parenchymal destruction (hemosiderin deposit alone) had a favorable neurodevelopmental outcome, encephalomalacia and periventricular leukomalacia contributed to long-term neurodevelopmental deficits. Evaluating parenchymal damage with prenatal MRI can therefore help to predict neurodevelopmental prognosis of the fetus with GMIVH.     

COL1A2新突变致胎儿严重成骨不全症1例报告

目的 总结1例COL1A2新突变致胎儿严重成骨不全症（OI）的临床特征及基因突变的特点,为胎儿产前咨询提供依据。方法 对产检B超检查示OI可能的胎儿流产组织抽提DNA进行基因型分析,自行设计COL1A1和COL1A2所有外显子及剪接区域的引物。利用Sanger测序法对胎儿行COL1A1和COL1A2 基因外显子及剪接区域的测序分析并行父母验证。依据人类基因突变数据库（HGMD）专业版,对COL1A2突变所致疾病临床表型行文献复习。结果 胎儿的COL1A1和COL1A2基因均检测出变异位点。COL1A2基因检测到杂合突变（c.3142G>T, p. Glu1048Cys）在寡核苷酸多态性数据库、HGMD及Ⅰ型胶原蛋白突变数据库均未见报道,结合胎儿父母验证为新发突变,对比公共数据库及在线预测软件预测该突变类型为致病突变。在HGMD专业版中搜索COL1A2,共检索到387个COL1A2致病突变,与21种疾病及其亚型相关。92%的突变引起OI或其亚型,还可引起Ehlers-Danlos综合征或其亚型。结合COL1A2突变所致疾病临床表型行文献复习,本文报告胎儿符合Ⅱ型OI。结论 产前通过超声影像结合基因分型诊断胎儿为COL1A2基因新发突变（c.3142G>T, p. Glu1048Cys）所致Ⅱ型OI; COL1A2基因编码蛋白长链双螺旋的400～480氨基酸及MLBR 3区域中甘氨酸被天冬氨酸或谷氨酸替代,多导致严重表型的OI;本文为产前准确预测胎儿结局、指导临床决策提供依据。     

Hematological values of 163 normal fetuses between 18 and 30 weeks of gestation

Utilizing an easy and safe procedure for fetal blood sampling in utero we have studied 409 fetuses for prenatal diagnosis of rubella, toxoplasmosis, hemophilia, and hemoglobinopathies. Retrospectively we selected 163 fetuses confirmed as normal at birth and tested between 18 and 30 wk of gestation to establish normal hematological parameters and to follow the evolution of erythropoiesis, differential counts, hemoglobin synthesis, and hemostasis. Total white blood cell and platelet counts did not change during this period. The lymphocytes represented the main population and we observed a decrease of normoblasts during gestation. The results show a progressive increase of red blood cells and hemoglobin. This evolution is demonstrated by the ratio hemoglobin A to acetylated hemoglobin F. No significant modification of hemostasis was observed over a 12-wk intrauterine gestation. These results provide useful reference values for future investigations.     

Prenatal diagnosis of alpha- and beta-thalassaemias in Singapore--current status.

Prenatal diagnosis was performed in 31 pregnancies where the fetuses were at risk for either homozygous alpha(0) - or beta-thalassaemia. First-trimester prenatal diagnosis by DNA analysis using chorionic villi was carried out for 17 pregnancies at risk for homozygous alpha (0)-thalassaemia. The alpha-globin genes in fetal DNA were detected by gene mapping using restriction endonuclease mapping and hybridization with cloned alpha-globin probe. Homozygous alpha (0)-thalassaemia was detected in four fetuses and the results were subsequently confirmed by electrophoresis of the cord blood where only Hb Barts was detected. Prenatal diagnosis for beta-thalassaemia was carried out by globin chain biosynthesis using fetal blood at 18-20 weeks' gestation. Using carboxymethyl (CM) sepharose chromatography, homozygous beta-thalassaemia was predicted in six pregnancies, and one fetus carried Hb E-beta thalassaemia. The seven pregnancies were terminated and globin chain analysis using cord blood confirmed the prenatal diagnoses. The remaining seven fetuses were diagnosed as either normal or beta-thalassaemia carriers. Using DNA analysis and globin chain biosynthesis for prenatal diagnosis of homozygous alpha(0)- and beta-thalassaemia, a 100% correlation was achieved with fetuses predicted to possess the homozygous condition.     

微小RNA与胎儿异常发育的研究进展

微小RNA(microRNA,miRNA)是一类长度为22～28个核苷酸的非编码小分子RNA,它们通过与靶mRNA结合,导致靶mRNA降解或翻译抑制,从而在转录后水平调控蛋白表达。miRNA对胚胎细胞增殖、形态发生、分化和凋亡有重要的调节作用,参与胎儿疾病的发生和病理生理过程,在发育异常的胎儿中已发现各种miRNA表达异常。目前可通过检测妊娠母亲外周血miRNA水平来预测胎儿疾病,并在动物实验中成功干预miRNA异常,相信未来miRNA在胎儿疾病的诊断及治疗上将起更重要的作用。     

Prenatal revelation of Niemann–Pick disease type C in siblings

Objectives: To report two cases of prenatal Niemann–Pick disease type C in siblings, with different prenatal semiology and postnatal outcome.
Case reports: First fetus presented at 22 weeks'gestation with ascites, hepatosplenomegaly, then polyhydramnios. At birth, the infant developed severe cholestasis and died at day 5. His brother presented at 22 weeks'gestation an isolated hepatomegaly with cholestasis at birth showing favourable evolution. In first case, diagnosis of Niemann–Pick disease was confirmed by autopsy findings, biochemical tests on cultured skin fibroblasts and ascites fluid, then by molecular screening of NPC1 gene. Brother's molecular prenatal diagnosis was made at 14 weeks' gestation on cultured trophoblasts.
Conclusion: Prenatal screening of this disease is particularly indicated in case of fetal ascites with hypoferritinaemia. Tests on amniotic or ascites fluid cells allow to characterize the biochemical phenotype, leading to search for molecular abnormalities. Despite the same mutation identified in siblings, disease evolution is variable, which underlines complexity of genetic counselling.     

地中海贫血产前诊断操作规范建议

产前诊断是地中海贫血（简称地贫）防控最有效的二级预防手段, 其目的是预防重型地贫患儿出生。对有生育重型或中间型地贫患儿的高风险孕妇, 在妊娠10～
14周或17～26周通过介入性取材得到胎儿细胞,提取胎儿脱氧核糖核酸（DNA）后进行地贫基因突变检测,从而对胎儿出生后是否患病做出产前诊断。地贫产前诊断的临床及实验室工作均需要严格的技术要求和质量控制。地贫的植入前遗传诊断技术要求较高,β地贫基因检测结合HLA配型的植入前产前遗传学诊断技术对生育过重型或中间型β地贫患儿的家庭有较高预防和治疗价值;地贫的无创产前诊断技术尚处于研究阶段,目前仍不能取代介入性取材的产前诊断方法。     

Prenatal revelation of Niemann-Pick disease type C in siblings

OBJECTIVES: To report two cases of prenatal Niemann-Pick disease type C in siblings, with different prenatal semiology and postnatal outcome. CASE REPORTS: First fetus presented at 22 weeks'gestation with ascites, hepatosplenomegaly, then polyhydramnios. At birth, the infant developed severe cholestasis and died at day 5. His brother presented at 22 weeks'gestation an isolated hepatomegaly with cholestasis at birth showing favourable evolution. In first case, diagnosis of Niemann-Pick disease was confirmed by autopsy findings, biochemical tests on cultured skin fibroblasts and ascites fluid, then by molecular screening of NPC1 gene. Brother's molecular prenatal diagnosis was made at 14 weeks' gestation on cultured trophoblasts. CONCLUSION: Prenatal screening of this disease is particularly indicated in case of fetal ascites with hypoferritinaemia. Tests on amniotic or ascites fluid cells allow to characterize the biochemical phenotype, leading to search for molecular abnormalities. Despite the same mutation identified in siblings, disease evolution is variable, which underlines complexity of genetic counselling.     

单纯性完全型肺静脉异位引流产前8例超声心动图诊断

摘要 目的 探讨胎儿单纯性完全型肺静脉异位引流（TAPVC）的产前超声心动图特点,提高对本病的产前诊断准确率。方法 回顾性分析2011年5月至2014年2月经新生儿超声心动图、手术或尸解证实的8例单纯性TAPVC的胎儿期超声心动图检查结果,总结超声心动图特征。结果 8例单纯性TAPVC胎儿中,心下型2例,心内型1例,心上型5例,5例存在垂直静脉或共同肺静脉腔与垂直静脉连接处梗阻;8例胎儿中,引产1例（心下型）,余7例均于出生后行手术治疗,其中1例术后死亡（心下型）,6例均恢复良好。TAPVC胎儿超声心动图特征为：①正常的左心房形态消失,呈圆形或椭圆形,左心房光滑并多变小,在孕后期较为明显;②降主动脉与左心房间距离明显增大,多数在左心房后方可见一异常的腔隙（即共同肺静脉腔）,可显示左、右侧上升（心上型）或下降（心下型）的垂直静脉;③妊娠早期左、右心系统比值早期多正常,但妊娠中、晚期（孕26周后）可出现右心系统轻度扩大;④引流入冠状静脉窦时（心内型）,冠状静脉窦可有扩张;引流入上腔静脉时（心上型）,上腔静脉扩张;引流入肝内血管时（心下型）,肝内血管可有不同程度的扩张;⑤彩色多普勒可显示引流途径及是否合并垂直静脉梗阻。结论 在孕早期左、右心系统比值正常时,TAPVC易被漏诊及误诊,应注意多角度、多切面扫查,孕晚期超声心动图检查可减少漏诊。