Congenital adrenal hyperplasia. Molecular characterization.

OBJECTIVE: To study the molecular defects of congenital adrenal hyperplasia (CAH). STUDY DESIGN: Twenty Chinese patients, including 8 with salt-wasting (SW) type CAH, 11 with simple virilizing (SV) type CAH and 1 with nonclassical (NC) type CAH, were recruited. Two rounds of the polymerase chain reaction (PCR) were used to study the 21-hydroxylase gene (CYP21). The primary PCR amplified CYP21-specific DNA fragments, and the secondary PCR used products from the primary PCR for analysis of amplification-created restriction sites (ACRS) and direct DNA sequencing. In all patients, ACRS analysis was done at 12 possible mutation sites, and then direct DNA sequencing was performed to confirm or define the molecular defects. RESULTS: Ten different mutations, including nine point mutations and gross gene deletion or conversion, were found in this study. Of the nine point mutations, eight could be easily detected by ACRS analysis. The three most common mutations were codon (CD)172 t-->a (I172N), IVS-II 656 c/a-->g, and gross gene deletion or conversion, accounting for 27.5% (11/40 alleles), 25% (10/40) and 20% (8/40) of all identified mutations, respectively. All SW patients were compound heterozygotes of IVS-II 656, gross gene deletion or conversion, or other severe defects, including CDs236 (t-->a) (I236N)+ 237 (t-->a) (V237E)+ 239 (t-->a) (M239K), CD306 (+t), CD318 (c-->t) (Q318X) and CD356 (c-->t) (R356W) mutations. All SV patients had one allele with a CD172 (I172N) mutation. One allele of an NC patient had a CD183 (c-->g) (D183E) mutation, and the other allele was not defined. In the whole series, four alleles (10%) had more than one mutation. CONCLUSION: We found 10 different mutations in this study. The correlation between genotypes and phenotypes was compatible with the reported data. Two rounds of PCR and ACRS analysis may provide important information for genetic counseling, prenatal diagnosis and management of families at risk for CAH.

Molecular characterization and PCR diagnosis of Thailand deletion of alpha-globin gene cluster.

Thailand deletion of alpha-Thalassemia (thal) 1 involves the zeta2-, phi zeta1-, alpha2-, alpha1-, and theta1-globin genes. In Southeast Asians and Taiwanese, this mutation is the second most common long-segment deletion of two alpha-globin genes, after the Southeast Asian deletion. To define the Thailand deletion breakpoints, we used polymerase chain reaction (PCR) to amplify the normal-sequence DNA fragments across the breakpoints. The amplified products were sequenced directly or after cloning into pGem-3Z or pCR2.1 vectors. Comparison of the normal and mutant sequences revealed that the 5' breakpoint lies between nucleotides 1,269 and 1,290 upstream of the initiator codon adenine of the zeta2-globin gene, and the 3' breakpoint lies between nucleotides 29,387 and 29,408 downstream of it. A total of 30,677 nucleotides were deleted. Both breakpoints mentioned above lie within the Alu repetitive sequences and an extensive sequence homology is present around the two breakpoints. These findings suggest that homologous recombination is the mechanism by which the deletion occurs. Based on our data, we used three oligonucleotide primers to amplify the regions across the deletion and its corresponding normal sequence. The feasibility of PCR diagnosis was confirmed in 20 carriers with this deletion.

Prevalence and molecular characterization of beta-thalassemia in Filipinos.

Beta-thalassemia (thal) is a common single-gene disease worldwide. However, the prevalence of beta-thal and the spectrum of beta-globin gene mutations in Filipinos remain unclear. This study sought to answer these two questions. A total of 2954 apparently healthy Filipinos in Taiwan were recruited for a prevalence study. A complete blood count was done in every subject. Those with microcytosis were studied with hemoglobin (Hb) high-performance liquid chromatography to determine the levels of Hb A2 and Hb F. Twenty-seven subjects had elevated levels of Hb A2 (>4.0%). These 27 suspected beta-thal carriers and another 16 beta-thal major patients who were being treated in the Philippines were studied to determine the spectrum of beta-globin gene mutations. Gap-PCR was used to detect the Filipino deletion of beta-thal, and direct sequencing was used to detect point or small mutations in the beta-globin gene. All of the 27 suspected beta-thal carriers had one mutation in the beta-globin gene, resulting in an overall prevalence of 0.9%. The spectrum of beta-thal mutations was similar in the carrier and patient groups. Analysis of the pooled identified seven different mutations in the study population. The Filipino deletion was the most common mutation, accounting for 45.8% (27/59) of the alleles, followed by codon 67 (-TG) (16 alleles), and Hb E (11 alleles). These three mutations accounted for 92% of the Filipino beta-thal alleles. Elucidation of the beta-thal mutations in Filipinos is useful for the genetic counseling and prenatal diagnosis of this disease.

Complete androgen insensitivity syndrome. Molecular characterization in two Chinese women.

OBJECTIVE: To study the molecular basis of complete androgen insensitivity syndrome (AIS). STUDY DESIGN: The coding region of the human androgen receptor (hAR) gene in two women with AIS was amplified with polymerase chain reaction using 12 pairs of oligonucleotide primers and then sequenced with a dye terminator method. RESULTS: Both patients had mutation in exon E of the androgen-binding domain. In one patient, codon 732 GAC (aspartic acid) was changed to ACC (asparagine), and her CAG polyglutamine tract had 27 repeats. In the other patient, codon 765 GCC (alanine) was changed to ACC (threonine), and her CAG polyglutamine tract in exon A had 19 repeats. CONCLUSION: Except for CAG polyglutamine polymorphism, these two missense mutations were the only differences detected in the coding region of the hAR gene. Both mutations involved the CpG sequence, which has been regarded as a mutation hotspot. To the best of our knowledge, these two mutations have not been observed before in Chinese women. Elucidation of the molecular defects of AIS patients would be very helpful for genetic counseling and prenatal diagnosis.

Misdiagnosis of homozygous alpha-thalassaemia 1 may occur if polymerase chain reaction alone is used in prenatal diagnosis.

The polymerase chain reaction (PCR) is a quite sensitive diagnostic tool but its specificity may be hampered because of contamination of foreign DNA. In order to determine the diagnostic accuracy of PCR in diseases due to gross gene deletion, a total of 180 fetuses at risk of homozygous South-East Asian deletion (SEA) of alpha-globin genes were included for study. Both PCR and Southern hybridization (SH) were performed. By PCR, three of 43 affected fetuses were misdiagnosed as heterozygotes; four of 50 normal fetuses were misdiagnosed as heterozygotes; and four of 87 heterozygotes were misdiagnosed, two as normal and two as affected. Misdiagnosis in affected and normal fetuses was most likely due to maternal DNA contamination, while misdiagnosis in heterozygotes was probably due to a failed PCR. In the experiments with PCR in which we added DNA from a carrier woman to an affected or a normal fetus, a level of 1/64 and 1/16 contamination resulted in the appearance of normal and SEA breakpoint sequences, respectively. In the SH experiments using artificially contaminated DNA, a level of 1/4 contamination showed the normal and SEA bands, respectively, while a contamination level lower than 1/8 and 1/16 respectively did not reveal contamination bands. The high sensitivity of PCR makes it easier to amplify contaminated DNA. For accurate prenatal diagnosis, PCR should be performed very carefully and SH seems to be a useful back-up.

Molecular characterization of beta-thalassemia in Taiwan and the identification of two new mutations.

Polymerase chain reaction-based techniques were used to study the molecular defects of 480 unrelated beta-thalassemia heterozygotes in Taiwan. Analysis of artificially created restriction sites and gap-polymerase chain reaction were performed to detect four common mutations, i.e. IVS-II-654 (C-->T), codons 41/42 (-TCTT), codon 17 (A-->T), -28 (A-->G), and a deletional form of delta beta-thalassemia in the Chinese population. In cases with negative or ambiguous results with the aforementioned methods, direct DNA cycle sequencing using either S35-dATP or a fluorescent dye terminator, was carried out to determine the defects. A total of 14 different mutations have been found in this series. The IVS-II-654 mutation was the most common (39.6%), followed by the codons 41/42 mutation (37.9%). The four common genotypes accounted for 92.3% of defects. Two new mutations were detected: codon 31 (-C) and codons 40/41 (+T). Both defects resulted in a frameshift and a premature terminator, the former at codon 60, the latter at codon 43. Although we have studied our cases extensively, the molecular defects in seven alleles are still unknown.

Ultrasonographic scanning of placental thickness and the prenatal diagnosis of homozygous alpha-thalassaemia 1 in the second trimester.

In order to evaluate the association between placental thickness (PT) and fetal homozygous alpha-thalassaemia 1 before the appearance of classic ultrasound findings of haemoglobin (Hb) Bart's hydrops fetalis, a total of 473 pregnancies were collected. The control group included 422 normal pregnancies with a gestational age from 14 to 23 weeks and the study group included 51 affected fetuses in the same gestational period. Fetal biparietal diameter (BPD) and PT were measured by high-resolution ultrasound. PT was evaluated against BPD. In the control group, the PT generally increased in parallel with the advancement of gestational age. All PT measurements in the study group were above the mean PT of their respective gestational week in the control group. Forty-six (90 per cent) of the pregnancies in the study group had PT larger than the mean plus two standard deviations of the control group. This study suggests that ultrasound measurement of PT may be a useful aid in the prenatal diagnosis of Hb Bart's hydrops fetalis before its classic findings become apparent in the late second trimester or third trimester.

Carrier detection and prenatal diagnosis of alpha-thalassemia of Southeast Asian deletion by polymerase chain reaction.

Alpha-thalassemia of Southeast Asian deletion (-- SEA/) is very common in Southeast Asia. Homozygosity of this genotype is the major cause of Hb Bart's hydrops fetalis in Taiwan. With polymerase chain reaction using three oligonucleotide primers bridging the common deletion breakpoint, a DNA fragment of 194 basepairs (bp) was amplified in chromosomes with the -- SEA determinant and a DNA fragment of 287 bp was amplified in chromosomes without this deletion. In our pilot study including 8 normal subjects, 20 obligate carriers, and 11 homozygotes of the deletion, all the genotypes were determined and then confirmed by Southern blotting and DNA hybridization with phi zeta globin gene probe. For prenatal diagnosis, 55 at-risk pregnancies were collected. Chorionic villus sampling was done in 51 cases and early amniocentesis was done in 4 cases. Fourteen cases (25.5%) were diagnosed as normal, 25 (45.5%) as heterozygotes, and 16 (29%) as homozygotes of -- SEA. All of the diagnoses were also confirmed as aforementioned. With polymerase chain reaction, the determination of the -- SEA deletion is straightforward and is much quicker and easier than with conventional Southern blotting and DNA hybridization. In areas with a high prevalence of -- SEA deletion, this method provides a rapid tool for carrier detection and prenatal diagnosis.

Molecular characterization of severe alpha-thalassemias causing hydrops fetalis in Taiwan.

alpha-Thalassemia hydrops fetalis is a common disorder in Taiwan. The condition causes perinatal death and many maternal obstetrical complications. In order to determine the molecular defects of this condition in Chinese, 87 unrelated families with this disorder were collected in the past 4 years. The molecular defects were studied by Southern blotting and DNA hybridization with phi zeta 1-globin gene and LO (a 0.4 kb BamHI/EcoRI fragment in the 5' flanking region of the zeta 2-globin gene) probes. Eighty-one (93.1%) fetuses had homozygous Southeast Asian deletion (- -SEA/- -SEA). Five (5.7%) fetuses were compound heterozygotes for the Southeast Asian deletion and Thailand deletion (- -SEA/- -THAI). The remaining fetus was a compound heterozygote for the Southeast Asian deletion and an uncharacterized nondeletional defect (- -SEA/(alpha alpha)Th). The molecular defects of alpha-thalassemia hydrops fetalis in Chinese are heterogeneous. This fact has important implications for genetic counseling and prenatal diagnosis.

Prenatal diagnosis of Chinese homozygous alpha-thalassaemia 1 and haemoglobin H disease by analysis of alpha- and phi zeta-globin genes in chorionic villi and amniocytes.

Eighty-eight high-risk pregnancies, 81 for homozygous alpha-thalassaemia 1 and 7 for haemoglobin (Hb) H disease, were collected in this study. Chorionic villus sampling (CVS) was done in 63 cases and amniocentesis in 25 cases to obtain fetal cells. Southern blotting and DNA hybridization with alpha- and phi zeta-globin gene probes were used to determine the alpha-globin gene status. In two non-informative families with non-deletional mutations, DNA analysis failed to rule out the affected condition, and fetal blood sampling (FBS) and Hb electrophoresis were used for the final diagnosis. In the 81 fetuses at risk for homozygous alpha-thalassaemia 1, 17 (13 by CVS and 4 by amniocentesis) were affected, 30 were alpha-thalassaemia 1 heterozygotes, 19 were normal, and the remaining 15 were either normal or heterozygous. In the seven fetuses at risk for Hb H disease, one was normal, three were alpha-thalassaemia 1 heterozygotes, two were alpha-thalassaemia 2 heterozygotes, and one was affected with Hb H disease and developed hydrops fetalis. DNA analysis on fetal cells enabled us to diagnose prenatally severe alpha-thalassaemias, to prevent the birth of infants with Hb H disease, and to minimize maternal obstetrical complications from harbouring a fetus with Hb Bart's hydrops fetalis.

Incidence study of heterozygous beta-thalassemia in northern Taiwan.

Determined with automated cell counters, mean corpuscular volume (MCV) of erythrocytes was screened in 4,100 consecutive gravidas who delivered at the National Taiwan University Hospital during the period from July 1986 through August 1988. In total, 248 (6%) women had MCV less than 80 fl and were recruited for further study, including Hb A2 quantitation using microcolumn chromatography, cord blood Hb electrophoresis on cellulose acetate membrane, and alpha-globin gene mapping in some cases. In the first control group of 51 cases of obligate carriers of heterozygous beta-thalassemia, the mean MCV +/- standard deviation (SD) was 67 +/- 4.5fl (range 59 to 76fl) and the mean Hb A2 +/- SD was 6.53 +/- 1.31% (range 4.3 to 8.0%). In the second control group of 40 cases of obligate carriers of heterozygous alpha-thalassemia 1, the mean MCV +/- SD was 70 +/- 3.7fl (range 63 to 78fl) and the mean Hb A2 +/- SD was 2.28 +/- 0.54% (range 1.5 to 3.8). Forty-five microcytic women (1.1%) had Hb A2 over 4%, a cut-off level for heterozygous beta-thalassemia in our laboratory. In this study group, the mean MCV +/- SD was 68 +/- 5.4 and the mean Hb A2 +/- SD was 6.03 +/- 1.41. None of the newborns delivered by these women had Hb Bart's in cord blood. alpha-Globin gene mapping using DNA hybridization was done in 14 women, and the results were all compatible with the exclusion of deletional alpha-thalassemia. It is concluded that approximately 1% of people in northern Taiwan are beta-thalassemia heterozygotes.

DNA polymorphism haplotype analysis of beta-thalassemia genes in Taiwan.

Twenty unrelated families of homozygous beta-thalassemia were collected for restriction polymorphism haplotype study of the beta-thalassemia (beta T) genes. DNA was extracted from WBC of the family members. With Southern blotting and DNA hybridization, restriction fragment length polymorphisms (RFLPs) were studied at 7 sites within the beta-globin gene cluster, including 5' epsilon-HincII, G gamma-HindIII, A gamma-HindIII, psi beta-HincII,3' psi beta-HincII, beta-AvaII,3' beta-BamHI. From RFLPs of the family members, restriction polymorphism haplotypes of the 40 beta T chromosomes and the 40 normal beta-globin gene (beta A)-bearing chromosomes were constructed. In beta T chromosomes. 26 (65%) belonged to haplotype I (+----++), 12 (30%) to haplotype II (+-----+), 1 (2.5%) to haplotype III (-++-+-+) and 1 (2.5%) to haplotype IV (++---++) which has not been reported in Chinese before. In beta A chromosomes, 12 different haplotypes were found. Seven (17.5%) belonged to haplotype I, 13 (32.5%) to haplotype II, and 6 (15%) to haplotype V (+----+-). The implications of RFLP and haplotyping of beta T genes in terms of molecular defects and prenatal diagnosis are discussed.

New 75Se-labeled radiopharmaceuticals: 2. Selenium derivatives of aralkylamines.

Two 75Se-labeled aralkylamines, 2-(3,4-methylenedioxyphenylseleno)ethylamine hydrochloride (75Se-6) and 1-methylseleno-1-phenylethylmethylamine hydrochloride (75Se-9), were prepared with high specific activity applying two different chemical means starting from [75Se]selenious acid. Tissue distribution studies in rats show high uptake in the lungs with lung/blood ratios of 27/1 and 2.3/1 at 10 min for compound 75Se-9 and 75Se-6, respectively. High adrenal uptake and adrenal-to-blood ratio (15/1 at 2 h) of compound 75Se-9 were observed. This study indicates that aralkylamines can accommodate a Se group and still show high uptake by the organs that contain appreciable amount of dopaminergic receptors.

New selenium-75 labeled radiopharmaceuticals: selenonium analogues of dopamine.

Selenium-75 labeled selenonium analogues of dopamine, [2-(3,4-dimethoxyphenyl)ethyl]dimethylselenonium iodide (4) and its dihydroxy analogue (7), were prepared by reducing [75Se]selenious acid with sodium borohydride at pH 6.0 and reacting the NaSeH produced with 1-(3,4-dimethoxyphenyl)-2-(p-toluenesulfonyloxy)ethane. Tissue distribution studies in rats given the 75Se-labeled selenonium agents intravenously demonstrated high initial heart uptake (2.38% dose/g at 5 min). Prolonged adrenal retention (t1/2 = 10 h) and high adrenal to blood ratio of compound 4 (21/1 at 4 h after injection) were observed. The high uptake and adrenal to blood ratio suggest the potential use of compound 4 as a radiopharmaceutical for the adrenal gland.