Prenatal diagnosis of homozygous alpha 0-thalassaemia by direct DNA analysis of chorionic villi in Singapore

First-trimester prenatal diagnosis by DNA analysis was carried out for seven pregnancies at risk for homozygous alpha 0-thalassaemia. Transabdominal placental biopsy was carried out at 10-12 weeks' gestation. The presence of alpha-globin genes in the fetal DNA was determined by restriction endonuclease mapping and hybridization with cloned alpha-globin probe. Homozygous alpha 0-thalassaemia was detected in two fetuses and the pregnancies were interrupted. Alpha 0-thalassaemia in both cases was confirmed by electrophoresis of the umbilical cord blood where only haemoglobin Bart's was detected. The remaining five fetuses were diagnosed as normal or as possessing alpha-thalassaemia-1 trait and the pregnancies are being carried to term. The use of DNA analysis in prenatal diagnosis of fetuses at risk for homozygous alpha 0-thalassaemia enables detection of the haemoglobinopathy at 10 weeks' gestation.     

Prenatal diagnosis of homozygous α°-thalassaemia by direct DNA analysis of chorionic villi in Singapore

Abstract First-trimester prenatal diagnosis by DNA analysis was carried out for seven pregnancies at risk for homozygous α°-thalassaemia. Transabdominal placental biopsy was carried out at 10–12 weeks'gestation. The presence of α-globin genes in the fetal DNA was determined by restriction endonuclease mapping and hybridization with cloned α-globin probe. Homozygous α°-thalassaemia was detected in two fetuses and the pregnancies were interrupted. α°-thalassaemiat in both cases was confirmed by electrophoresis of the umbilical cord blood where only haemoglobin Bart's was detected. The remaining five fetuses were diagnosed as normal or as possessing α°-thalassaemia-1 trait and the pregnancies are being carried to term. The use of DNA analysis in prenatal diagnosis of fetuses at risk for homozygous α°-thalassaemia enables detection of the haemoglobinopathy at 10 weeks'gestation.     

Prenatal diagnosis of alpha-thalassaemia by analysis of enzymatically amplified DNA sequences

A new method using enzymatically amplified DNA sequences for the prenatal diagnosis of alpha-thalassaemia was evaluated. DNA from a foetus at risk for alpha(0)-thalassaemia was analysed to detect the presence of alpha-globin genes. The procedure involved amplification of a 136-base-pair (bp) region of the alpha-globin gene complex between the psi alpha and alpha 2 region. Amplification was performed using a pair of oligonucleotide primers and a heat stable DNA polymerase which allowed repeated cycles of DNA synthesis at 72 degrees C. A 136 bp product was detected by gel electrophoresis indicating the foetus was not positive for Bart's hydrops foetalis. The result was confirmed using the gene mapping technique. Prenatal diagnosis of alpha-thalassaemia by DNA amplification offers two advantages over the gene mapping technique since radionucleotides are not used and results can be obtained in 3 days.     

Beta thalassemia: molecular pathogenesis and clinical variability

Clinically, homozygous beta-thalassaemia is characterised by a severe anaemia requiring regular transfusion therapy in most patients. However, there is a marked clinical variability ranging from this severe picture to the virtual absence of symptoms and haematological abnormalities. Biochemically, beta-globin synthesis in the erythroid precursors of the bone marrow is reduced or absent resulting in a relative excess of insoluble alpha-globin chains and dyserythropoiesis. The molecular genetics of this disorder is highly variable involving a multitude of different mutations of the beta-globin gene. These mutations can inactivate gene expression at all levels on its way from DNA to mature haemoglobin. The clinical picture is largely determined by the type of mutations inherited. Additionally the degree of alpha-globin chain excess can be influenced by the co-inheritance of alpha-thalassaemia or mutations resulting in the hereditary persistence of fetal globin synthesis (HPFH). This review discusses the relationship between the molecular defect and the clinical picture of patients with beta-thalassaemia.     

Prenatal diagnosis of hemoglobin H disease.

Hemoglobin H disease was diagnosed prior to the twenty-second week of gestation in a pregnancy at risk for homozygous alpha-thalassemia using the technique of DNA-DNA hybridization. Fetal DNA was obtained from amniotic fluid fibroblasts obtained during the thirteenth week of gestation and grown in culture. The fetal fibroblast DNA was hybridized to radioactive alpha-globin cDNA. The number of alpha-globin genes present in the fetus was determined by comparing results of hybridization studies on the fetal DNA to similar studies on subjects with well-defined alpha-thalassemia syndromes and with normal subjects. The diagnosis of hemoglobin H disease was confirmed at birth by studies of the cord blood. This study confirms the ability of DNA-DNA hybridization techniques to distinguish the three-gene defect of hemoglobin H disease from the lethal four-gene defect of homozygous alpha-thalassemia.     

Molecular characterization of /3-thalassaemia in Singaporean Chinese: Application to prenatal diagnosis

Abstract  Sixty-five j3-thalassaemia genes from 14 unrelated Chinese /3-thalassaemia major patients and 37 Chinese /3-carriers were analysed by allele-specific oligonucleotide (ASO) hybridization after DNA amplification by the polymerase chain reaction (PCR). Six mutations were studied and are represented by 49.2% of codon 41 -42, 30.8% of IVSII #654, 6.2% of 17)3, 3.1 % of IVSI #5 (G – C) and 1.5% of - 28 TATA box. The complete mutations responsible for /3-thalassaemia major in 13 of our 14 affected families were identified. For these families prenatal diagnosis at 10 weeks gestation using DNA amplification and ASO hybridization will replace the globin chain biosynthesis technique at 19 weeks gestation. Using ASO analysis, our results indicate that 5 oligo-probes (41 -42, ll-#654,170, IVSI-#5 and – 28) allow determination of /3-thalassaemia mutations in 59/65 (90.8%) of the Singaporean Chinese studied.     

PRENATAL DIAGNOSIS OF β-THALASSEMIA IN EGYPT: Implementing Accurate High-Tech Methods Did not Reflect Much on the Outcome

The clinical severity of thalassemia major makes it a priority genetic disease for prevention programs through prenatal diagnosis for carrier couples. Incorporation of automated DNA sequencing that enables the characterization of mutations not detected by other mutation specific detection procedures was a prime goal of this work. Automated DNA sequencing was offered on fetal tissues in 30 pregnancies during the year 2005. The pregnancies were at high risk for homozygosity or compound heterozygosity for β-thalassemia based on mutation analysis of both parents before prenatal diagnosis. Both parents have β-thalassemia trait. Fetal samples were collected by chorionic villus sampling (CVS) in the first trimester and by amniocentesis in the second trimester. The point mutations were characterized by PCR (ARMS). The absence of the expected fragment with all the mutant ARMS primers insinuated an uncharacterized DNA segment that was further subjected to direct automated fluorescent DNA sequencing in an attempt to know if the fetus was affected by parents’ mutations. If no mutation was detected using the PCR ARMS, the sample was further analyzed using direct automated fluorescent DNA sequencing. The mean gestation when carrying out the invasive procedure was 14 (10 –18) weeks. All mothers had a previous affected pregnancy, and 13 had two or more previous affected pregnancies. Pregnancies were: 8 carrier fetuses (trait) and 22 affected fetuses in which 2 were homozygous and 20 double heterozygous. Fourteen parents of affected fetuses preferred to continue pregnancy and the babies were born as diagnosed. The other 8 parents decided on termination and DNA of the abortuses proved to be as previously diagnosed by DNA sequencing. The use of PCR amplification and direct sequencing have permitted the accurate characterization of unidentified alleles and successfully solved 100% of the examined samples. However, it has resulted in minor changes of the outcome as the majority of couples preferred continuation of pregnancy.     

Prenatal diagnosis of beta-thalassemia in Egypt: implementing accurate high-tech methods did not reflect much on the outcome

The clinical severity of thalassemia major makes it a priority genetic disease for prevention programs through prenatal diagnosis for carrier couples. Incorporation of automated DNA sequencing that enables the characterization of mutations not detected by other mutation specific detection procedures was a prime goal of this work. Automated DNA sequencing was offered on fetal tissues in 30 pregnancies during the year 2005. The pregnancies were at high risk for homozygosity or compound heterozygosity for beta-thalassemia based on mutation analysis of both parents before prenatal diagnosis. Both parents have beta-thalassemia trait. Fetal samples were collected by chorionic villus sampling (CVS) in the first trimester and by amniocentesis in the second trimester. The point mutations were characterized by PCR (ARMS). The absence of the expected fragment with all the mutant ARMS primers insinuated an uncharacterized DNA segment that was further subjected to direct automated fluorescent DNA sequencing in an attempt to know if the fetus was affected by parents' mutations. If no mutation was detected using the PCR ARMS, the sample was further analyzed using direct automated fluorescent DNA sequencing. The mean gestation when carrying out the invasive procedure was 14 (10 -18) weeks. All mothers had a previous affected pregnancy, and 13 had two or more previous affected pregnancies. Pregnancies were: 8 carrier fetuses (trait) and 22 affected fetuses in which 2 were homozygous and 20 double heterozygous. Fourteen parents of affected fetuses preferred to continue pregnancy and the babies were born as diagnosed. The other 8 parents decided on termination and DNA of the abortuses proved to be as previously diagnosed by DNA sequencing. The use of PCR amplification and direct sequencing have permitted the accurate characterization of unidentified alleles and successfully solved 100% of the examined samples. However, it has resulted in minor changes of the outcome as the majority of couples preferred continuation of pregnancy.     

利用孕妇外周血浆小片段游离胎儿DNA检测Y染色体性别决定区基因

目的探索从孕妇外周血浆中提取小片段游离胎儿DNA（cffDNA）提高检测胎儿Y染色体性别决定区（SRY）基因准确率的方法,评估利用小片段cffDNA进行无创性产前诊断可行性。方法收集117例孕妇外周血,利用柱吸收方法提取其血浆cffDNA,经琼脂糖凝胶电泳分离富集小片段cffDNA,使用二重PCR反应（dulex-PCR）检测SRY基因和磷酸甘油醛脱氢酶（GAPDH）基因。结果来源于66例孕男胎孕妇血浆标本小片段cffDNA均检出SRY和GAPDH基因,来源于50例孕女胎孕妇血浆标本小片段cffDNA仅检出GAPDH基因。与绒毛/羊水标本检测结果相符。特异性和敏感性均为100%。结论利用琼脂糖凝胶电泳,切胶回收,可选择性富集孕妇外周血小片段cffDNA,相对提高胎儿DNA水平,结合二重PCR扩增SRY基因技术可用于无创性产前性连锁遗传疾病和单基因突变疾病产前诊断。     

Hydrops fetalis caused by severe alpha-thalassemia.

Alpha-thalassemia is a prevalent condition in South East Asia and spreading because of increasing immigration. Hemodynamic study of Hb Bart's hydrops fetalis, the most severe form of alpha-thalassemia, showed the fetuses were in a hyperdynamic circulatory state and were also more acidotic, hypoxic, and hypercarvic than normal fetuses. The most common molecular defects of Hb Bart's hydrops fetalis was the Southeast Asia type deletion. Prenatal diagnosis of Hb Bart's hydrops fetalis and HbH diseases has been achieved using chorionic villi sampling or fetal blood sampling.     

HPLC studies in hemoglobinopathies

An accurate diagnosis of β-thalassemia carriers, homozygous patients and identification of different structural hemoglobin variants is important for epidemiological studies as well as for management and prevention of the major hemoglobin disorders. There are many electrophoretic and chromatographic approaches for estimation of HbA₂ and Hb F but cation exchange HPLC (CE-HPLC)using automated dedicated machines like the Variant Hb testing system have become the method of choice for these investigations. CE-HPLC also helps in the presumptive identification of many abnormal hemoglobin variants and has been useful for both neonatal screening of sickle cell disease as well as second trimester prenatal diagnosis of thalassemia by fetal blood analysis. Other applications of HPLC in hemoglobinopathies include separation of globin chains, measuring the ratio of gamma globin chains (Gγ/Aγ) and the recently described denaturing HPLC for detecting mutations in both α and β globin genes.     

Thalassemias in Sardinia: molecular pathology, phenotype-genotype correlation, and prevention

This article reviews the molecular bases of alpha- and beta-thalassemias in Sardinia. In addition, it describes the characteristics and the effects of a genetic program designed to prevent homozygous beta-thalassemia. In the large majority of the cases (95.7%), beta-thalassemia is caused by the nonsense mutation at codon 39, followed by frameshifts at codon 6 (2.1%). Homozygous beta-thalassemia most commonly results in thalassemia major, but in a small proportion this genotype produces milder forms referred to as thalassemia intermedia. The reasons for the attenuated forms were determined only in a small proportion of the cases. The reduced clinical severity was due to either of two factors: (a) There could be the presence of cytosine-thymidine (C----T) substitution of position - 158 G gamma, which is able to increase the G gamma chain output (as in homozygotes for frameshift at codon 6 or compound heterozygote for frameshift at codon 6 and codon 39 nonsense mutation). (b) Reduced clinical severity could be the result of co-inheritance of alpha-thalassemia in the form of two alpha-globin gene deletions of functional loss of the alpha 2-globin gene (homozygote for codon 39 nonsense mutation). The most prominent clinical form of alpha-thalassemia is hemoglobin H disease, which may result from the compound heterozygous state for deletion alpha 0- and alpha(+)-thalassemia (in 83.1% of cases) or deletion and nondeletion alpha-thalassemia (in 16.9%). Deletion Hb disease shows a milder clinical picture as compared to the nondeletion form. The most common nondeletion alpha-thalassemia is the ATG----ACG substitution at the initiation codon of the alpha 2 gene. Double heterozygotes of alpha (-alpha/-alpha) and beta-thalassemia or delta- and beta-thalassemia are relatively common and may cause confusion in carrier identification. The preventive program aimed to control beta-thalassemia is based on voluntary carrier screening., counselling, and prenatal diagnosis. Prenatal diagnosis is carried out by dot blot analysis with allelic specific oligonucleotides on amplified trophoblast DNA. This procedure gave very reliable results; in fact, no misdiagnosis has occurred so far. The preventive program was highly effective, resulting in a decline of the incidence of thalassemia major from 1:250 to 1:1,000 live births.     

Molecular mechanism accounting for milder types of thalassemia major

We carried out alpha-globin gene analysis by restriction endonuclease mapping in 91 Sardinians with homozygous transfusion-dependent beta 0-thalassemia and correlated the clinical findings with the alpha-globin genotype. In patients (n = 6) with deletion of two alpha-globin structural genes, disease onset and transfusion dependence occur later than in those (n = 50) with a full complement of alpha-globin genes. There was no statistically significant difference in the group of patients (n = 35) with deletion of only one alpha-globin gene. Patients with deletion of two alpha-globin genes had significantly higher Hb A2 levels than those with a full complement of alpha-structural genes and those with deletion of a single alpha-globin gene. From this and other studies, it seems that the deletion of two alpha-globin structural genes may convert the common severe clinical picture associated with homozygous beta 0-thalassemia to milder forms, ranging from a later occurring but still transfusion-dependent type to a non-transfusion-dependent form.     

The biologic implications of a rare hemoglobin mutant that decreases oxygen affinity

Blood from seven newborns, a 13-y-old, and seven adult family members with a suspected hemoglobinopathy because of unexplained cyanosis was obtained for analysis to determine Hb oxygen affinity and to characterize and quantify the Hb variants. Their oxygen saturation was 76 to 84%. The P(50) was 30.3 +/- 2.9 for the newborns and 32.5 +/- 2.6 mm Hg for their related adults. In the same order, the plasma erythropoietin was 7.4 +/- 2.9 and 15.9 +/- 3.7 mU/mL, whereas 2,3-diphosphoglycerate was 16.1. +/- 2.9 and 15.9 +/- 3.7 micromol/g Hb. In four of the newborns with increased P(50), the mother had a normal P(50) (27 mm Hg), which indicated a greater maternal oxygen affinity than the fetus with no adverse effects on the fetus. Genetic analysis of alpha-globin genes demonstrated a heterozygous mutation on the alpha2 gene [alpha94(G1)Asp-->His] for each of the newborns and their related adults. The same mutation was found on the alpha1 gene in an adolescent and her father. The mRNA measurements showed that the alpha2- to alpha1-globin mRNA mean ratio was 2.5, alpha2 mutant globin mRNA/total alpha2-globin mRNA was 45.0%, whereas the alpha1 mutant globin mRNA/total alpha1-globin mRNA was 37.8%. The level of alpha2 mutant globin/total alpha-globin was 27.3 +/- 1%, and alpha1 mutant globin/total alpha-globin was 23.8 +/- 1%. The percentage of synthesized alpha2 and alpha1 mutant globins was 27.5 +/- 2 and 26.1 +/- 1, respectively. The ratio of the alpha2/alpha1 mutant globins was 1.1, which corresponded to a ratio at the mRNA level of alpha2/alpha1 of 2.5 +/- 0.5, which suggested that there is a less efficient translation of the alpha2 mRNA than alpha1 mRNA. The reversal of the physiologic fetomaternal oxygen affinity had no effects on fetal development.     

Fetal echogenic lung lesions: Prenatal ultrasound diagnosis and outcome

The differential diagnosis of echogenic areas in the fetal chest include congenital diaphragmatic hernia (CDH), cystic adenomatoid malformation (CAM), sequestrated lung and tracheal or bronchial atresia. The purpose of this study was to evaluate the accuracy of prenatal diagnosis and document outcome in fetuses with echogenic chest lesions. Seventeen fetuses with echogenic chest masses were seen in our unit between 17 and 36 weeks' gestation over a 5-year period. We reviewed these cases retrospectively for prenatal diagnosis, postnatal diagnosis and outcome. Prenatal diagnosis was correct in 13 fetuses, with CDH in 8, sequestrated lung in 4 and tracheal atresia in 1. Four fetuses had incorrect or uncertain prenatal diagnoses. In three fetuses CDH and CAM could not be differentiated. After delivery two of these had CDH and one had sequestrated lung. One fetus with bilateral lesions had prenatal diagnosis of bilateral CAM. Post-mortem examination revealed tracheal atresia as part of Fraser syndrome. All five babies with sequestrated lung are well and none required surgery. Ten fetuses had CDH, two pregnancies were terminated, one died in utero, five died as neonates and two babies survived following surgery. The study reveals that in a minority of fetuses CDH and CAM could not be differentiated prenatally. We agree with recent reports of fetal sequestrated lung describing sonographic improvement in utero. A large lesion on initial scan does not necessarily predict a poor neonatal outcome in this condition. This, together with the poor outcome in fetuses with echogenic CDH and tracheal atresia, has important implications for prenatal counselling.     

An examination of different fetal specific antibodies and magnetic activated cell sorting for the enrichment of fetal erythroblasts from maternal blood

ABSTRACT  The aim of the present study was to compare the rates of fetal cells obtained after separation from maternal blood by magnetic activated cell sorting (MACS) using different fetal specific antibodies, and to evaluate the potential role of this method in the prenatal diagnosis of fetal trisomies. Peripheral blood samples were obtained from 42 women carrying chromosomally normal fetuses and from 4 women with aneuploid fetuses (2 cases of 47,XX,+18 and 2 of 47,XY,+21) at 9–20 weeks of gestation. After fetal cells were enriched by MACS with three different monoclonal antibodies (GPA, CD71, CD14), fluorescence in situ hybridization (FISH) with chromosome X, and Y-specific probes was performed to detect the rates of fetal cells in the samples sorted. FISH with chromosome 13-, 18-, and 21-specific probes was carried out to compare proportions of cells with three-signal nuclei in chromosomally normal and abnormal groups. In male infants, X-and Y-positive cells were detected in 80%, 73.3%, and 66.6% of samples after the separation by antibodies CD14, GPA, and CD71, respectively. The percentage of nuclei with three signals was increased in pregnancies with trisomy, ranging between 2% and 5.18%. Pregnancies with normal fetuses showed 0 to 3.7% of nuclei with three signals. The data demonstrate that fetal cell detection varies depending on the antibodies used for cell sorting. This study provides further evidence on the feasibility of screening for fetal chromosomal abnormalities by enriching maternal blood for fetal cells and using FISH.     

Preserved umbilical cord facilitates antenatal diagnosis of spinal muscular atrophy

Spinal Muscular atrophy (SMA) Type I is a fatal autosomal recessive disease caused by homozygous deletion of telometric region of exon 7/8 of the SMN gene. Prenatal diagnosis is feasible and desirable by most families. We report on prenatal diagnosis of SMAI in a family where dried umbilical cord stump from the deceased affected baby was used to confirm the diagnosis. Prenatal diagnosis was provided in the subsequent pregnancy. We emphasize the need for storing DNA from individuals affected with suspected single gene disorders.     

Update on the prenatal diagnosis and treatment of congenital adrenal hyperplasia due to 11beta-hydroxylase deficiency

11beta-Hydroxylase deficiency is a common form of congenital adrenal hyperplasia causing virilization of the female fetus and hypertension. DNA analysis of the gene (CYP11B1) encoding 11beta-hydroxylase has been reported previously to be effective in the prenatal diagnosis of one affected female fetus. In that case, prenatal treatment with dexamethasone resulted in normal female genitalia. We now report five new pregnancies that underwent prenatal diagnosis for 11beta-hydroxylase deficiency. In the first family, the proband is homozygous for a T318M mutation and all fetuses from four subsequent pregnancies are carriers. In a second family, the mother is homozygous for a A331V mutation and was started on dexamethasone, but identification of a homozygous normal fetus led to the discontinuation of treatment. In another family, the fetus was a male homozygous for R384Q and treatment was discontinued. Lastly, a novel G444D mutation in exon 8 was identified and proven to reduce 11beta-hydroxylase activity.     

Alpha-thalassemia in premature newborns

In this study we have carried out alpha-globin gene mapping, hemoglobin (Hb) Bart's quantitation serum bilirubin, and red blood cell indices determination in a group of Sardinian appropriate for gestational age premature infants (from 32 to 35 wk gestation) in order to define the incidence in this population of the different alpha-thalassemia syndromes, their expression rate, and the correlation between the alpha-globin genotype and phenotype at this developmental stage. The gene frequencies of deletion (-alpha) and nondeletion (alpha alpha th) alpha-thalassemia were 0.29 and 0.04, respectively, and thus not different from those found in full-term newborns from the same population. The majority of premature newborns with a single alpha-globin gene deletion [(-alpha/alpha alpha) genotype] were hematologically silent. Those who manifested increased Hb Bart's (1.2 to 3.4%) had slightly reduced Hb levels (17.4 +/- 2.6 g/dl), mean corpuscular volume (102.6 +/- 6.3 fl), and mean corpuscular Hb (34.8 +/- 2.0 pg) values. Those infants with the deletion of two alpha-globin structural genes (-alpha/-alpha) showed without exception moderate amount of Hb Bart's in the 3.5-8.1% range and an obvious decrease of Hb levels (16.1 +/- 1.6 g/dl) mean corpuscular Hb (30.6 +/- 3.5 pg), and mean corpuscular volume (88.5 +/- 11.5 fl) values. The only infant with the deletion of 3 alpha-globin structural genes had 25% Hb Bart's associated with a moderate microcytic anemia at birth and developed the clinical picture of Hb H disease. Carriers of nondeletion alpha-thalassemia (alpha alpha/alpha alpha th) showed variable amount of Hb Bart's always associated with thalassemia-like red cell indices.(ABSTRACT TRUNCATED AT 250 WORDS)     

Wiskott-Aldrich综合征高危儿七例产前诊断

目的 探讨Wiskott-Aldrich综合征(WAS)高危孕妇羊水脱落细胞基因分析及脐静脉穿刺脐血WAS蛋白(WASP)检测在WAS高危儿产前诊断中的意义.方法 2008年至2010年我院经WASP流式检测和基因分析明确诊断的7例WAS患儿.详细记录先证者病史,进行家族相关亲属基因检测,建立7个WAS家系图谱.2008年至2011年对7个家系中携带异常基因的7个高危孕妇于孕18 ～ 20周经羊膜腔穿刺抽取羊水.其中部分羊水提取细胞DNA,经PCR扩增WAS基因.PCR产物进行双向序列重复测定.其中1例高危孕妇孕28周采集脐带血,采用流式细胞术检测WASP.培养羊水中胎儿脱落细胞,采用原位制片及G带染色技术进行染色体核型分析.产后采集高危儿外周血进行基因分析和WASP检测验证产前诊断结果.结果 7例WAS高危儿羊膜腔穿刺均成功,羊水细胞培养成功率100％.WAS基因和染色体核型分析结果显示1例正常男性胎儿,4例正常女性胎儿,2例为女性异常基因携带者.1例脐带血流式细胞术检测WASP显示正常表达.7例高危儿均顺利出生,产后WASP和基因分析结果均正常,与产前结果相同.结论 羊水脱落细胞核型、基因分析与脐带血WASP检测可为WAS高危孕妇提供可靠的产前诊断服务.