Tsang Ming Ko

Molecular genetic study of Pompe disease in Chinese patients in Taiwan

Pompe disease is caused by mutations in the acid α‐glucosidase (GAA) gene. Multiple kinds of mutations in the GAA gene have been reported worldwide. In order to elucidate the molecular basis of the disease in Taiwanese patients of Chinese origin, we have recruited 11 unrelated families who had at least one member with Pompe disease for study. We used 16 pairs of oligonucleotide primers to amplify all the coding regions from exon 2 to 20 in the family members. The coding regions were sequenced on both the sense and antisense strands. We identified 7 different mutations in 17 alleles but failed to identify the defects in the other 5 alleles. The most common defect was D645E (Asp645Glu), accounting for 36% (8/22 alleles) of mutations, followed by G615R (Gly615Arg) (3 alleles); 1411del4 (Glu471‐shift) (2 alleles); and one allele each of R600H (Arg600His); ΔN675 (ΔAsn675); 2380delC (Arg794‐shift) and 2815delGT (Val939‐shift). The molecular defects of Pompe disease are highly heterogeneous in Chinese. Characterization of the molecular defects of the disease is useful for a genotype‐phenotype correlation and for genetic counseling and prenatal diagnosis. Hum Mutat 13:380–384, 1999.

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

SummaryAlpha-thalassemia of Southeast Asian deletion (-- SEA/) is very common in Southeast Asia. Homozygosity of this genotype is the major cause of Hb Barts 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 φζ 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.

Thirteen X-chromosomal short tandem repeat loci multiplex data from Taiwanese

Study results of variations in the X chromosome are useful tools in researching the genetic diversity of human populations and individual identification. We developed a 13 X chromosomal short tandem repeat (STR) multiplex system (DXS6807, DXS8378, DSX9902, DXS7132, DXS9898, DXS6809, DXS6789, DXS7424, DXS101, GATA172D05, HPRTB, DXS8377, DXS7423) amplified in one single polymerase chain reaction. DNA samples of 113 male and 108 female Taiwanese Han subjects were successfully analyzed using this 13 X-STR multiplex system. The distributions of allele frequencies were examined for independence. DXS8377, DXS101, DXS6789, and DXS6809 were found to be the most polymorphic markers in this study. High values of discrimination power and mean exclusion chance without significant evidence of association between these markers were obtained. In conclusion, this 13 X chromosomal STR multiplex system offers considerable forensic efficiency and may be useful in forensic identification casework.

Molecular characterization and PCR diagnosis of Thailand deletion of α‐globin gene cluster

Thailand deletion of α‐Thalassemia (thal) 1 involves the ζ2‐, γζ1‐, α2‐, α1‐, and υ1‐globin genes. In Southeast Asians and Taiwanese, this mutation is the second most common long‐segment deletion of two α‐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 pCR®2.1 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 ζ2‐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. Am. J. Hematol. 57:124–130, 1998.

Resolving the ancestry of Austronesian-speaking populations

There are two very different interpretations of the prehistory of Island Southeast Asia (ISEA), with genetic evidence invoked in support of both. The “out-of-Taiwan” model proposes a major Late Holocene expansion of Neolithic Austronesian speakers from Taiwan. An alternative, proposing that Late Glacial/postglacial sea-level rises triggered largely autochthonous dispersals, accounts for some otherwise enigmatic genetic patterns, but fails to explain the Austronesian language dispersal. Combining mitochondrial DNA (mtDNA), Y-chromosome and genome-wide data, we performed the most comprehensive analysis of the region to date, obtaining highly consistent results across all three systems and allowing us to reconcile the models. We infer a primarily common ancestry for Taiwan/ISEA populations established before the Neolithic, but also detected clear signals of two minor Late Holocene migrations, probably representing Neolithic input from both Mainland Southeast Asia and South China, via Taiwan. This latter may therefore have mediated the Austronesian language dispersal, implying small-scale migration and language shift rather than large-scale expansion.

Recurrent Down's syndrome due to maternal ovarian trisomy 21 mosaicism.

A family with three children who had Downs syndrome and one healthy child is reported. Cytogenetic studies of the peripheral blood revealed trisomy 21 in the affected children, and normal karyotypes in both the parents and the healthy child. However, a biopsy of the mothers right ovary showed a mosaic trisomy 21 cell line (8/20 cells). By DNA polymorphism analysis, segregation of trisomy oogonia appeared to be the cause of recurrent trisomy 21.[/p]

Alpha-thalassemia in the four major aboriginal groups in Taiwan

A total of 1309 unrelated blood samples from four major Taiwan aboriginal groups, including 522 of the Ami, 246 of the Bunun, 227 of the Atayal, and 214 of the Paiwan groups, were collected. Subjects with a mean corpuscular volume below 85 fl and Hb A2 values below 3.5% were further studied with Southern hybridization to determine the status of α-globin genes. In the Ami, 43 (4.1%) chromosomes had α-thalassemia 1 and 43 (4.1%) had α-thalassemia 2. Of the 43 α-thalassemia 1 chromosomes, 33 were of the Thailand, one of the Philippine, and nine of the Southeast Asian deletion. Of the 43 α-thalassemia 2 chromosomes, 42 were of the type I rightward deletion and one was of leftward deletion. In the Bunun group, one chromosome (0.2%) was of the Thailand deletion and two (0.4%) were of type I rightward deletion. In the Atayal group, only one chromosome (0.2%) was of the Philippine deletion. In the Paiwan group, four chromosomes (0.9%) were of the Southeast Asian deletion and three (0.7%) were of the Thailand deletion. Among the four groups, the Ami had the highest prevalence of α-thalassemia, which was also higher than that of the Chinese living in Taiwan.

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.

Norplant® subdermal contraceptive system: Experience in Taiwan

From November 1988 to December 1994, a total of 567 female volunteers were enrolled in Norplant implant studies at the National Taiwan University Hospital. After a median follow-up of 29 months, only 3 of the 529 available cases became pregnant (a cumulative rate of 1.2 pregnancies per 100 users over 5 years). Chromosome analysis of 2 of the 3 abortuses revealed 46,XX/46,XX,inv(3) and 46,XX. Menstrual problems were the most common adverse effects and were also the main reason for discontinuation (65%, 108/166). The continuation rate was 90%, 78%, 70%, 61%, and 42% at the end of 1, 2, 3, 4, and 5 years after insertion, respectively. In the 21 patients who wished to become pregnant, fertility recurred soon after removal of the Norplant implants. The data suggested that the Norplant implants system is a highly effective, safe, and long-acting method of reversible contraception. It would be worthwhile to introduce this contraceptive system to Taiwans family planning program.

Cost‐effectiveness analysis of triple test in second‐trimester maternal serum screening for Down's syndrome: an experience from Taiwan with decreasing birth rate but increasing population of old pregnant women

OBJECTIVESnWe intended to assess the cost-effectiveness of adding unconjugated oestriol (uE3) in maternal serum screening for Downs syndrome in Taiwan, where there is a decreasing birth rate but an increasing trend of old women having pregnancies.nnnMETHODSnWe used logistic regressions to estimate the risk of Downs syndrome with maternal age and different combinations of biomarkers. Cost-effectiveness analysis was presented in terms of the average and incremental cost-effectiveness ratios. Sensitivity analyses with different parameters were performed.nnnRESULTSnGiven a cut-off point of 1:270 for the confirmation of Downs syndrome with amniocentesis, the average cost per case averted for maternal age above 35 years only, double test [alpha-fetoprotein (AFP) and human chorionic gonadotrophin (hCG)] and triple test (AFP, hCG and uE3) were estimated as