Invasive molecular prenatal diagnosis of alpha and beta thalassemia among Hakka pregnant women.

This study is a retrospective analysis of the prenatal genetic diagnosis results of fetuses with high risk of major thalassemia to provide information for clinical genetic counseling and to better control the birth of major thalassemia child in Hakka population. Totally, 467 fetuses in at-risk pregnancies were collected from Meizhou people's hospital from January 2014 to December 2017. Genomic DNAs were extracted from peripheral blood of the couples and villus, amniotic fluid or cord blood of the fetuses. DNA-based diagnosis was performed using polymerase chain reaction (PCR) and flow-through hybridization technique. Follow-up visits were done half a year after the fetuses were born. Around 467 fetus at-risk pregnancies were performed prenatal diagnosis. We detected 88 CVS samples, 375 amniocentesis fluid samples and, 4 cord blood samples. The 356 fetuses in α-thalassemia families consisted of 69 (19.38%) with Bart's hydrops syndrome, 20 (5.62%) fetuses with Hb H disease, and 184 (51.68%) fetuses with heterozygote. And the 111 fetuses in ß-thalassemia families consisted of 31 (27.93%) thalassemia major, 51 (45.95%) fetuses with heterozygote. There are 13 fetuses with α+ß-thalassemia, including 2 cases with severe ß-thalassemia. DNA-based testing prenatal diagnosis of thalassemia was found to be highly reliable. Our findings provide key information for clinical genetic counseling of prenatal diagnosis for major thalassemia in Hakka pregnant women. Our work plays an important role in the prevention and control of thalassemia in Hakka population. We will also combine other techniques to further improve our molecular prenatal diagnostic capabilities, including the next-generation sequencing (NGS), Sanger sequencing and MLPA.

A comparison of intrauterine hemopoietic cell transplantation and lentiviral gene transfer for the correction of severe ß-thalassemia in a HbbTh3/+ murine model.

Major hemoglobinopathies place tremendous strain on global resources. Intrauterine hemopoietic cell transplantation (IUHCT) and gene transfer (IUGT) can potentially reduce perinatal morbidities with greater efficacy than postnatal therapy alone. We performed both procedures in the thalassemic HbbTh3/+ mouse. Intraperitoneal delivery of co-isogenic cells at embryonic days13-14 produced dose-dependent chimerism. High-dose adult bone marrow (BM) cells maintained 0.2-3.1% chimerism over ~24 weeks and treated heterozygotes (HET) demonstrated higher chimerism than wild-type (WT) pups (1.6% vs. 0.7%). Fetalliver (FL) cells produced higher chimerism than BM when transplanted at thesame doses, maintaining 1.8-2.4% chimerism over ~32 weeks. We boosted transplanted mice postnatally with BM cells after busulfan conditioning. Engraftment was maintained at >1% only in chimeras. IUHCT-treated nonchimeras and non-IUHCT mice showed microchimerism or no chimerism. Improved engraftment was observed with a higher initial chimerism, in HET mice and with the addition of fludarabine. Chimeric HET mice expressed 2.2-15.1% engraftment with eventual decline at 24 weeks (vs. <1% in nonchimeras) and demonstrated improved hematological indices and smaller spleens compared with untreated HETmice. Intravenous delivery of GLOBE lentiviral-vector expressing human ß-globin (HBB) resulted in a vector concentration of 0.001-0.6 copies/cell. Most hematological indices were higher in treated than untreated HET mice, including hemoglobin and mean corpuscular volume, but were still lower than in WT. Therefore, direct IUGT and IUHCT strategies can be used to achieve hematological improvement but require further dose optimization. IUHCT will be useful combined with postnatal transplantation to further enhance engraftment.

Pre-natal diagnosis of thalassaemia in Sri Lanka: A ten year review.

Thalassaemia is the commonest monogenic disease in Sri Lanka, affecting over 3500 children and half-a-million thalassaemia carriers. This is a review of 82 amniocenteses performed from 2006 to 2016, in the largest prenatal diagnoses study for thalassaemia carried out in Sri Lanka. Amniocenteses were performed between 11 and 12 weeks of ultrasonically confirmed gestation, on mothers with previous thalassaemia major children pregnant for the second time and nulliparous thalassaemia trait women married to trait partners. The Consultant Radiologist, using local analgesia, under ultrasound cover, performed these as an outpatient procedure, at the Teaching Hospital Kandy & Suwasevana Hospital Kandy. The amniotic fluid was analysed by the team of Senior Geneticists, at the Genetech Molecular Diagnostics and School of Gene Technology, Colombo, via the polymerase-chain-reaction based ARMS (Amplification Refractory Mutation Systems) assay. The genetic results indicated the presence of 21% thalassaemia major foetuses, 53% thalassaemia traits and 26% foetuses without thalassaemia mutations. The predominance of the IVS1-5(G-C) mutation in the Sri Lankan population is exemplified, with a low prevalence of HbE thalassaemia. Impact statement Thalassaemia is the commonest monogenic disease in Sri Lanka affecting over 3500 children and half-a-million thalassaemia carriers. Although pre-natal diagnosis by amniocentesis was practised universally for many years, this could not be performed in Sri Lanka as genetic diagnostic facilities were not available until 2005. Therefore, parents with a thalassaemia major child limited their families to one child, by choice or by termination. The results of this study point to a 21% probability of thalassaemia major in the next child, giving the parents a guarded optimism to conceive another child without thalassaemia disease. With siblings being the highest HLA compatibility for Bone Marrow Transplant, that is now being established in Sri Lanka as a permanent cure for thalassaemia, this will bring a ray of hope for these desperate parents to finally cure their previous sick child. Although, 95% of the Sri Lankan mutated genetic sites for thalassaemia are known, more research will be needed to identify the other rare sites. The publication of this paper, with its novelty for pre-natal diagnosis, would encourage clinicians to practice it in other centres and to extend it to families with other genetic disorders.

Non-invasive pre-implantation aneuploidy screening and diagnosis of beta thalassemia IVSII654 mutation using spent embryo culture medium.

BACKGROUND: Cell-free nuclear DNA has been isolated from spent embryo culture medium. Whether this small amount of DNA can be amplified at the whole genome level and the concordance rate of karyotypes and specific alleles between biopsied cells and media has not been evaluated. METHODS: Seven couples were recruited, 88 donated embryos and their corresponding media were collected for whole genome amplification (WGA). The efficiency of WGA, the concordance of chromosome status, and the HBB gene IVSII654 allele between biopsied cells and media were investigated. RESULTS: After WGA, the DNA detection rate was 90.90% with a mean concentration of 26.15 ng/µl. The full chromosome concordance rate between biopsied cells and medium was 64.52%, and it increased to 90.00% for diploid blastocyst samples. Analysis of the mutated IVSII654 locus and SNP linkage verified that the DNA present in the medium originated from embryonic cells. CONCLUSION: We confirmed that nuclear DNA is present in spent culture medium and that the majority of this DNA can be amplified for subsequent analysis. Our results showed that non-invasive embryo genetic testing at the chromosomal-level using medium can concordant to the biopsied cells, but it needs further optimized before use in clinical applications. KEY MESSAGES The aggressive biopsy step during PGD/PGS procedure would have a negative effect on the future development of the embryo. Cell-free nuclear DNA has been observed in spent embryo culture medium, which holds promise for the development of non-invasive PGD/PGS approaches. The presence of DNA in medium, its efficiency for WGA, and the concordance between chromosome status and the HBB gene IVSII654 allele as diagnosed from biopsied cells or medium were investigated. Non-invasive embryo genetic testing at the chromosomal-level and allele site using medium can concordant to the biopsied cells, but it needs further optimized before use in clinical applications.

[Preimplantation genetic diagnosis for carriers of thalassemia and chromosomal abnormality].

OBJECTIVE: To provide preimplantation genetic diagnosis(PGD) for two couples carrying thalassemia mutations and chromosomal abnormalities. METHODS: Couple 1 were both carriers of ß 41/42 thalassemia mutations, while the husband has carried a reciprocal translocation with a karyotype of 46,XY,inv(9)(p11;q13),t(11;22)(q25;q13). Couple 2 were both carriers of α (-SEA) thalassemia mutation. Their chromosome karyotypes were both normal, but had two spontaneous abortions. The couples had received 1 and 3 blastocysts respectively through in vitro fertilization(IVF) cycles. Following the biopsy, the cells underwent whole genome amplification, and the amplified DNA from each embryo was subjected to genetic testing and a 23-chromosome single nucleotide polymorphism(SNP) microarray assay. RESULTS: The embryo of couple 1 was diagnosed as carrier of ß 41/42 thalassemia with euploid chromosomes. The embryo was transferred and resulted in intrauterine pregnancy. Similarly, an embryo of couple 2 was verified as carrier of α (-SEA) thalassemia with euploid chromosomes. CONCLUSION: PGD for aneuploidy coupled with testing for single gene disorders via trophectoderm biopsy and whole genome amplification is feasible. The approach can attain diagnosis with minimal damage with sound clinical outcome.

Generation of Human Induced Pluripotent Stem Cells from Extraembryonic Tissues of Fetuses Affected by Monogenic Diseases.

The generation of human induced pluripotent stem cells (hiPSCs) derived from an autologous extraembryonic fetal source is an innovative personalized regenerative technology that can transform own-self cells into embryonic stem-like ones. These cells are regarded as a promising candidate for cell-based therapy, as well as an ideal target for disease modeling and drug discovery. Thus, hiPSCs enable researchers to undertake studies for treating diseases or for future applications of in utero therapy. We used a polycistronic lentiviral vector (hSTEMCCA-loxP) encoding OCT4, SOX2, KLF4, and cMYC genes and containing loxP sites, excisible by Cre recombinase, to reprogram patient-specific fetal cells derived from prenatal diagnosis for several genetic disorders, such as myotonic dystrophy type 1 (DM1), ß-thalassemia (ß-Thal), lymphedema-distichiasis syndrome (LDS), spinal muscular atrophy (SMA), cystic fibrosis (CF), as well as from wild-type (WT) fetal cells. Because cell types tested to create hiPSCs influence both the reprogramming process efficiency and the kinetics, we used chorionic villus (CV) and amniotic fluid (AF) cells, demonstrating how they represent an ideal cell resource for a more efficient generation of hiPSCs. The successful reprogramming of both CV and AF cells into hiPSCs was confirmed by specific morphological, molecular, and immunocytochemical markers and also by their teratogenic potential when inoculated in vivo. We further demonstrated the stability of reprogrammed cells over 10 and more passages and their capability to differentiate into the three embryonic germ layers, as well as into neural cells. These data suggest that hiPSCs-CV/AF can be considered a valid cellular model to accomplish pathogenesis studies and therapeutic applications.

ENU mutagenesis identifies the first mouse mutants reproducing human ß-thalassemia at the genomic level.

Forward genetic screens have been performed in many species to identify phenotypes in specific organ systems. We have undertaken a large-scale N-ethyl-N-nitrosourea (ENU) mutagenesis screen to identify dominant mutations that perturb erythropoiesis in mice. Mutant mice that displayed an erythrocyte mean cell volume (MCV) greater than three standard deviations from the population mean were identified. Two of these lines, RBC13 and RBC14, displayed a hypochromic, microcytic anemia, accompanied by a marked reticulocytosis, splenomegaly and diminished red cell survival. Timed pregnancies from heterozygous intercrosses revealed that a quarter of the embryos displayed severe anemia and did not survive beyond embryonic day (E) 18.5, consistent with homozygous ß-thalassemia. Genetic complementation studies with a ß-thalassemia mouse line reproduced the embryonic lethality in compound heterozygotes and a genomic custom capture array and massively parallel sequencing of the ß-globin locus identified the causative mutations. The RBC13 line displayed a nonsense mutation at codon 40 in exon 2 of the ß-major gene, invoking parallels with the common ß(0)39 thalassemia mutation seen in humans. The RBC14 line exhibited a mutation at the polyadenylation signal of the ß-major gene, exactly replicating a human ß-thalassemia mutation. The RBC13 and RBC14 lines are the first ß-thalassemia mouse models that reproduce human ß-thalassemia at the genomic level, and as such highlight the power of ENU mutagenesis screens in generating mouse models of human disease.

Generation of human ß-thalassemia induced pluripotent stem cells from amniotic fluid cells using a single excisable lentiviral stem cell cassette.

Induced pluripotent stem cells (iPSCs) derived from somatic cells of patients represent a powerful tool for biomedical research and may have a wide range of applications in cell and gene therapy. However, the safety issues and the low efficiency associated with generating human iPSCs have limited their usage in clinical settings. The cell type used to create iPSCs can significantly influence the reprogramming efficiency and kinetics. Here, we show that amniotic fluid cells from the prenatal diagnosis of a ß-thalassemia patient can be efficiently reprogrammed using a doxycycline (DOX)-inducible humanized version of the single lentiviral "stem cell cassette" vector flanked by loxP sites, which can be excised with Cre recombinase. We also demonstrated that the patient-derived iPSCs can be characterized based on the expression of pluripotency markers, and they can be differentiated into various somatic cell types in vitro and in vivo. Moreover, microarray analysis demonstrates a high correlation coefficient between human ß-thalassemia iPS cells and human embryonic stem (hES) cells but a low correlation coefficient between human ß-thalassemia amniotic fluid cells and human ß-thalassemia iPS cells. Our data suggest that amniotic fluid cells may be an ideal human somatic cell resource for rapid and efficient generation of patient-specific iPS cells.

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

Preimplantation genetic diagnosis (PGD) for ß 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 ß-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 (ß-globin gene and selected STRs, all within <0.4 Mb from the ß gene) was optimized in single lymphocytes, and subsequently applied in 38 PGD cycles in couples at-risk for transmitting ß hemoglobinopathies. In conclusion, incorporation of closely linked polymorphic microsatellite markers <0.4 Mb from the ß-globin gene, facilitates robust assignment of ß 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).

Peptide-nucleic acid-mediated enriched polymerase chain reaction as a key point for non-invasive prenatal diagnosis of beta-thalassemia.

The presence of fetal DNA in maternal plasma can be exploited to develop new procedures for non-invasive prenatal diagnosis. Tests to detect 7 frequent beta-globin gene mutations in people of Mediterranean origin were applied to the analysis of maternal plasma in couples where parents carried different mutations. A mutant enrichment amplification protocol was optimized by using peptide nucleic acids (PNAs) to clamp maternal wild-type alleles. By this approach, 41 prenatal diagnoses were performed by microelectronic microchip analysis, with total concordance of results obtained on fetal DNA extracted from chorionic villi. Among these, 27/28 were also confirmed by direct sequencing and 4 by pyrosequencing.

The codon 37 (TGG-->TAG) beta(0)-thalassemia mutation found in a Chinese family.

We have found an example of the nonsense beta-thalassemia (thal) mutation at codon 37 (TGG-->TAG; Trp-->Stop) in a Chinese family. The fetus, who inherited both parents' beta-thalassemic alleles, was a compound heterozygote for the codons 41/42 (-TCTT) and codon 37 (TGG-->TAG) mutations, and presented with the phenotype of severe beta-thal.

First detection of the splice donor site IVS-I-2 (T-->B) beta-thalassemia mutation in a Chinese patient.

We present the first description of a Chinese family with a rare b-thalassemia mutation commonly observed in black Americans. This mutation is a splice donor site mutation, and is associated with a phenotype of beta0-thalassemia.

Early prenatal diagnosis by celocentesis.

BACKGROUND: Celocentesis is the ultrasound-guided aspiration of fluid from the extra-amniotic cavity at 7-8 weeks of gestation. This paper reports on the clinical application of celocentesis for early prenatal diagnosis. METHODS: Celocentesis was successfully performed in nine pregnancies and 1-2 mL of fluid were obtained after one needle insertion. The indications were prenatal diagnosis of beta-thalassemia or sickle cell disease (n = 6), Marfan syndrome (n = 1) and paternity testing (n = 2). Molecular biological techniques were used to analyze the celomic fluid and this was successfully carried out in all cases. RESULTS: In two cases pregnancy termination was performed at the request of the mother because in one case the fetus was found to have sickle cell anemia and in the second case paternity testing demonstrated that the father was not the woman's husband. In both cases the results were confirmed using the placental samples collected after pregnancy termination. In six of the seven pregnancies with desirable results, amniocentesis was performed at 16 weeks and the results were concordant with those obtained from celocentesis. All pregnancies were uneventful and resulted in the delivery of healthy and appropriately grown babies. CONCLUSION: Celocentesis may be a viable alternative to the currently used tests of chorionic villus sampling and amniocentesis.

Unrelated umbilical cord blood transplantation for an infant with beta-thalassemia major.

BACKGROUND: beta-thalassemia major, one of the most prevalent hemoglobinopathies throughout the world, can be cured by allogeneic stem cell transplantation therapy. Many patients, however, lack a suitably matched related donor. Unrelated umbilical cord blood can be used as an alternative stem cell source for some of these patients. This report describes the successful transplantation of a 2-month-old infant with beta-thalassemia major using partially HLA-matched unrelated umbilical cord blood. METHODS: After cytoreduction with busulfan, cyclophosphamide, and antithymocyte globulin (ATG), the patient underwent transplantation at the age of 2 months with a 4/6 HLA matching umbilical cord blood unit from an unrelated donor. RESULTS: The patient engrafted promptly with 100% donor chimerism. His only major complication was an autoimmune hemolytic anemia that resolved 2 years after transplantation. He is currently surviving, event-free, 5 years after transplantation with normal growth and cognitive development and full donor chimerism without evidence of beta-thalassemia. CONCLUSIONS: Umbilical cord blood transplantation from related and unrelated donors should be considered for patients with beta-thalassemia major who lack traditional bone marrow donors. As most newborns undergo screening for hemoglobinopathies, those with disease could be transplanted early in life before experiencing the morbidity and mortality caused by transfusion therapy, alloimmunization, and iron overload, increasing the likelihood of successful transplantation therapy.

Postnatal cytokines and boosts improve chimerism and hematological parameters in beta-thalassemic mice transplanted in utero.

We have developed a murine model of in utero transplantation in nonanemic, beta-thalassemic mice to study chimerism, tolerance, and changes in hematological parameters in response to cytokines and postnatal boosts with donor cells. We have documented improved survival of homozygous fetuses by 40% as compared with controls. Low-level, mixed chimerism was improved by postnatal cytokine therapy and boosts and was associated with improvement in hemoglobin levels, reticulocyte counts, and iron stores. Cytotoxicity assays demonstrated higher responses to donor cells in control mice as compared with in utero transplanted animals (at 50:1 effector to target ratios, transplanted mice showed 8.66% target lysis and control mice showed 51.85% target lysis, P=0.0003), indicating tolerance. The combination of prenatal tolerance to allogeneic cells with postnatal boosts in primed hosts may become an effective, nontoxic strategy for the improvement of hemolytic anemia in beta-thalassemic patients.

Prenatal diagnosis of beta-thalassemia major by high-performance liquid chromatography analysis of hemoglobins in fetal blood samples.

In Thailand and adjacent countries, most of the beta-thalassemia genes are beta(0)-thalassemia mutations that prevent the production of Hb A. We propose the quantitation of the Hb A fraction in fetal blood in the mid-trimester of pregnancy by automated high performance liquid chromatography as a reasonable prenatal diagnostic method to be applied in areas with limited laboratory facilities. Forty pregnant women at risk of delivering a child with beta-thalassemia major were identified using an erythrocyte osmotic fragility test and quantitation of Hb A2. Cordocentesis was performed at the gestational age of 18-22 weeks and fetal blood was analyzed for hemoglobin fractions by automated high performance liquid chromatography. The beta-globin gene mutations were characterized by beta-globin gene sequencing. The 4 bp deletion at codons 41/42 (-TTCT) was the most frequent of the 40 beta-thalassemia mutations observed (20/40 = 50%), followed by the splice site mutation IVS-I-1 (G-->T) (7/40 = 17.5%), the nonsense mutation at codon 17 (A-->T) (7/40 = 17.5%), the nonsense mutation at codon 35 (C-->A) (3/40 = 7.5%), and the beta(+)-thalassemia promoter mutation at -28 (A-->G) (3/40 = 7.5%). High performance liquid chromatography revealed nine fetuses which had only Hb F and no Hb A. All were homozygotes or compound heterozygotes for beta(0)-thalassemia mutations. In the remaining 31 fetuses, a Hb A peak was present in the chromatograms. One fetus with 0.5% Hb A was a compound heterozygote for the -28 (A-->G) and codons 41/42 (-TTCT) mutations. In the remaining 30 fetuses, the Hb A values ranged between 0.8 and 7.4%. Twenty of these, with a Hb A concentration of 1.82 +/- 0.49% (range 0.8-2.8%), were beta-thalassemia heterozygotes. The remaining 10 fetuses had Hb A values of 4.89 +/- 1.47% (range 2.9-7.4%) and normal beta-globin genes. The absence of Hb A in homozygotes or compound heterozygotes for beta(0)-thalassemia mutations and the presence of measurable amounts of Hb A in heterozygotes and normal homozygotes, permits the diagnosis of fetuses expected to develop postnatal beta-thalassemia major.

[Molecular analysis and prenatal diagnosis of beta-thalassemia: about our experience in central Tunisia]. / Analyse moléculaire et diagnostic prénatal de la beta-thalassémie: à propos de notre expérience en Tunisie centrale.

Beta-thalassemia, by its high frequency and its heterogeneity, constitutes a real problem of health in Tunisia. Prenatal diagnosis by DNA analysis represents the only reality for couples at risk. The denaturant gradient (urea and formamide) on polyacrylamide gel electrophoresis has been performed in our laboratory, using psoralen as chemical clamps. This method is simple, reliable, safe, rapid, without radioactivity and has a reasonable cost (chemical clamps). Even if it needs an informatic modelization in other laboratories, this method seems to be adapted to our economic and work conditions and to the molecular heterogeneity of the Tunisian beta-thalassemia. We present the results of an epidemiological molecular study on 75 patients with beta-thalassemia and the results of ten prenatal diagnosis. The molecular lesions codon 39 (C-T) and IVS1 nt2 (T-G) are the most frequent in our study. This technical approach provides genetic counselling for at risk families by offering prenatal diagnosis (reducing as possible the cost and the delay of the result) after prealable family study and identification of the mutation(s).

Fetal nuchal translucency and prenatal diagnosis of beta-thalassaemia.

In Sardinia, fetal karyotyping for couples at risk for beta-thalassaemia is offered only to women >/=35 years and for specific risk of chromosomopathies. This policy is not easily accepted by the couples who incessantly request additional karyotyping. In order to select those at highest risk of chromosomal abnormalities among young women, fetal nuchal translucency (NT) thickness measurement was performed in 510 fetuses to assess the chromosomal risk before chorionic villus sampling. A risk >/=1/100 was judged positive and worthy of additional karyotyping. 126 cases interrupted the pregnancy after a result of homozygous beta-thalassaemia, hence 384 pregnancies were included in the study. 22 (5.7 per cent) fetuses were found NT positive. A total of three chromosomal abnormalities were detected. The NT test was positive in all three cases of chromosomopathies detected (100 per cent) and in 19 of 381 (4.98 per cent) normal karyotype fetuses. No features of major chromosomal abnormalities were reported among the newborns whose NT had resulted normal. These preliminary results have confirmed the efficacy of NT testing to assess the risk of trisomy 21 and other chromosomopathies and enhanced its utility in pregnancies already suited to sampling in the first-trimester for Mendelian disorders.