Priscilla M.K. Poon

Quantitative Analysis of Fetal DNA in Maternal Plasma and Serum: Implications for Noninvasive Prenatal Diagnosis

We have developed a real-time quantitative PCR assay to measure the concentration of fetal DNA in maternal plasma and serum. Our results show that fetal DNA is present in high concentrations in maternal plasma, reaching a mean of 25.4 genome equivalents/ml (range 3.3-69. 4) in early pregnancy and 292.2 genome equivalents/ml (range 76. 9-769) in late pregnancy. These concentrations correspond to 3.4% (range 0.39%-11.9%) and 6.2% (range 2.33%-11.4%) of the total plasma DNA in early and late pregnancy, respectively. Sequential follow-up study of women who conceived by in vitro fertilization shows that fetal DNA can be detected in maternal serum as early as the 7th wk of gestation and that it then increases in concentration as pregnancy progresses. These data suggest that fetal DNA can be readily detected in maternal plasma and serum and may be a valuable source of material for noninvasive prenatal diagnosis.

MECP2 mutation in male patients with non‐specific X‐linked mental retardation

In contrast to the preponderance of affected males in families with X‐linked mental retardation, Rett syndrome (RTT) is a neurological disorder occurring almost exclusively in females. The near complete absence of affected males in RTT families has been explained by the lethal effect of an X‐linked gene mutation in hemizygous affected males. We report here on a novel mutation (A140V) in the MECP2 gene detected in one female with mild mental retardation. In a family study, the A140V mutation was found to segregate in the affected daughter and in four adult sons with severe mental retardation. These results indicate that MECP2 mutations are not necessarily lethal in males and that they can be causative of non‐specific X‐linked mental retardation.

The Apolipoprotein E ε4 Allele Is Unlikely to Be a Major Risk Factor of Age-Related Macular Degeneration in Chinese

Apolipoprotein E (ApoE) is a major transporter of lipids and cholesterol in the nervous system. Age-related macular degeneration (ARMD), characterized by drusen containing lipids, was reported to show a lower frequency of the ApoE ε4 allele than control subjects. We sought to examine the association of this polymorphism with ARMD in Hong Kong Chinese. Among 98 ARMD subjects, the frequency of ε4 carriers showed a trend toward a decrease compared to controls, but it was not significant (11.2 vs. 15.0%, p < 0.52). The association of ε4 with an apparent reduced risk of ARMD was reported previously in the exudative form of the disease, however among 39 exudative ARMD patients there was also no significant difference in ε4 frequency (12.8%, p < 0.93). The lack of a statistically significant effect of ε4 may be due to the lower frequency of ε4 in Chinese than Europeans. Thus we cannot exclude a possible effect of this allele on ARMD risk, but we can conclude that this allele is likely not a major factor influencing ARMD risk in the Chinese.

Prenatal diagnosis of glycogen storage disease type 1b using denaturing high performance liquid chromatography

Glycogen storage disease type 1b (GSD1b) is an autosomal recessive inborn error of metabolism caused by deficiency of glucose‐6‐phosphate translocase (G6PT1). Current laboratory diagnosis for GSD1b is established by a functional enzyme assay of glucose‐6‐phosphatase in both fresh and detergent‐treated liver homogenates. This procedure requires liver biopsy and is impractical for routine prenatal diagnosis owing to the high morbidity of fetal liver biopsy. Recently, the gene for GSD1b has been cloned and the prevalent mutations in different ethnic groups have been determined. In this study, prenatal molecular diagnosis was performed for a Chinese family in which a previous child was born homozygous for the G149E mutation. We detected genomic sequence variants by heteroduplex formation, followed by denaturing high performance liquid chromatography (DHPLC). With this method, post‐PCR analysis was shortened to 7 min. In the case we analysed, PCR products amplified from the fetal DNA yielded a single peak in the chromatogram, indicating a homozygous state in the fetus. When wild‐type PCR products were mixed with fetal PCR products, two peaks were observed, indicating that the fetus was homozygous for the parental (G149E) mutation. Sequencing results confirmed this diagnosis. As a result, the pregnancy was terminated and the diagnosis was confirmed on DNA analysis of the aborted fetus. We show here that DNA mutation analysis can be used in the prenatal diagnosis of GSD1b and that DHPLC promises to be a robust technique for this and other prenatal molecular diagnoses. Copyright

Frequency of the fragile X syndrome in Chinese mentally retarded populations is similar to that in Caucasians

Fragile X syndrome is recognized as the most common inherited cause of mental retardation in western countries. The prevalence of the fragile X syndrome in Asian populations is uncertain. We report a multi-institutional collaborative study of molecular screening for the fragile X syndrome from 1,127 Chinese mentally retarded (MR) individuals. We found that 2.8% of the Chinese MR population screened by DNA analysis had the fragile X full mutation. Our screening indicated that the fragile X syndrome prevalence was very close to that of Caucasian subjects. In addition, we found that 62.5% of fragile X chromosomes had a single haplotype for DXS548-FRAXAC1 (21-18 repeats) which was present in only 9.7% of controls. This unique distribution of microsatellite markers flanking the FMR1 CGG repeats suggests that the fragile X syndrome in Chinese populations, as in the Caucasian, may also be derived from founder chromosomes.

The Asian project for collaborative derivation of reference intervals: (1) strategy and major results of standardized analytes

Abstract Background: A multicenter study conducted in Southeast Asia to derive reference intervals (RIs) for 72 commonly measured analytes (general chemistry, inflammatory markers, hormones, etc.) featured centralized measurement to clearly detect regionality in test results. The results of 31 standardized analytes are reported, with the remaining analytes presented in the next report. Method: The study included 63 clinical laboratories from South Korea, China, Vietnam, Malaysia, Indonesia, and seven areas in Japan. A total of 3541 healthy individuals aged 20–65 years (Japan 2082, others 1459) were recruited mostly from hospital workers using a well-defined common protocol. All serum specimens were transported to Tokyo at –80°C and collectively measured using reagents from four manufacturers. Three-level nested ANOVA was used to quantitate variation (SD) of test results due to region, sex, and age. A ratio of SD for a given factor over residual SD (representing net between-individual variations) (SDR) exceeding 0.3 was considered significant. Traceability of RIs was ensured by recalibration using value-assigned reference materials. RIs were derived parametrically. Results: SDRs for sex and age were significant for 19 and 16 analytes, respectively. Regional difference was significant for 11 analytes, including high density lipoprotein (HDL)-cholesterol and inflammatory markers. However, when the data were limited to those from Japan, regionality was not observed in any of the analytes. Accordingly, RIs were derived with or without partition by sex and region. Conclusions: RIs applicable to a wide area in Asia were established for the majority of analytes with traceability to reference measuring systems, whereas regional partitioning was required for RIs of the other analytes.

A survey of FRAXE allele sizes in three populations

FRAXE is a fragile site located at Xq27-8, which contains polymorphic triplet GCC repeats associated with a CpG island. Similar to FRAXA, expansion of the GCC repeats results in an abnormal methylation of the CpG island and is associated with a mild mental retardation syndrome (FRAXE-MR). We surveyed the GCC repeat alleles of FRAXE from 3 populations. A total of 665 X chromosomes including 416 from a New York Euro-American sample (259 normal and 157 with FRAXA mutations), 157 from a Chinese sample (144 normal and 13 FRAXA), and 92 from a Finnish sample (56 normal and 36 FRAXA) were analyzed by polymerase chain reaction. Twenty-seven alleles, ranging from 4 to 39 GCC repeats, were observed. The modal repeat number was 16 in the New York and Finnish samples and accounted for 24% of all the chromosomes tested (162/665). The modal repeat number in the Chinese sample was 18. A founder effect for FRAXA was suggested among the Finnish FRAXA samples in that 75% had the FRAXE 16 repeat allele versus only 30% of controls. Sequencing of the FRAXE region showed no imperfections within the GCC repeat region, such as those commonly seen in FRAXA. The smaller size and limited range of repeats and the lack of imperfections suggests the molecular mechanisms underlying FRAXE triplet mutations may be different from those underlying FRAXA.

Detection of an intense resonance at 2.4 ppm in 1H mr spectra of patients with severe late-delayed, radiation induced brain injuries

Proton MRS and MRI were used to monitor the progression of severe cerebral radiation injuries in 10 patients over a period of 18 months. An unknown resonance (Px) in the 2.37–2.40 ppm region was consistently detected in the affected temporal lobes of four patients. The detection of Px was only confined to spectra with lactate (Lac) and in patients with the highest severity grading of radiation injury. The incidence of Px in Lac‐positive spectra was 42.8% (15/35) and in lesions with highest injury grading was 46.8% (15/32). Lesions with Px had significantly higher Lac/creatine (Cr) ratios and more extensive mass effect changes when compared to lesions without Px. The probable identity of Px was examined in the context of anaerobic glycolysis producing pyruvate (2.37 ppm) and the model of metabolic changes in brain abscess formation implicating succinate (2.40 ppm). Magn Reson Med 45:994–1000, 2001.

FRAXAC1 and DXS548 polymorphisms in the Chinese population

The fragile X syndrome is the most common inherited form of mental retardation. Haplotype studies using FRAXAC1 and DXS548 polymorphic markers flanking the fragile site have demonstrated linkage disequilibrium at the FMR1 locus. We investigated the association of the FRAXAC1, DXS548 and CGG alleles between normal subjects and mentally retarded (MR) patients of unspecified cause who do have fragile X syndrome. We have evaluated the FRAXAC1 site in 390 normal subjects and 321 MR patients and the DXS548 site in 146 normal and 319 MR subjects. Both FRAXAC1 and DXS548 alleles were determined by application of the polymerase chain reaction. When compared with Caucasians, the normal Chinese population has a different FRAXAC1 allele distribution. There are more AC18 repeat alleles and fewer AC19 repeat alleles. The DXS548 allele distributions were similar between Chinese and Caucasians. The same distribution pattern of FRAXAC1 alleles was found in both normal subjects and MR patients, but there were significant differences in the distribution patterns of DXS548 alleles. The FMR1 CGG-DXS548 and FRAXAC1-DXS548 haplotype distribution between normal subjects and MR patients also differed significantly. Our results suggest a possible association between DXS548 alleles and non-FRAXA mental retardation.

Gas chromatographic-mass fragmentographic determination of serum 1α,25 dihydroxyvitamin D3

We extracted 1α,25-dihydroxyvitamin D 3 [1α,25(OH) 2 D 3 ] from 10 mL serum using Sep-Pak C18 and Sep-Pak Silica mini-columns and normal-phase high performance liquid chromatography (HPLC) separation for analysis by gas chromatography-mass fragmentography (GC-MS). A GC-MS method was optimised using manual tuning for ion mass calibration and selective ion monitoring (SIM) for quantitation. Serum 1α,25(OH) 2 D 3 was identified by superimposition of the m/z 452 and 501 ion peaks and by overlapping the m/z 452 ion peak with that of its authentic standard. It was quantitated from the relative peak areas of its m/z 452 ion and the m/z 363 ion of vitamin D 2 , the internal standard. Twenty picograms of 1α,25(OH) 2 D 3 gave a peak with a signal-to-noise ratio of 26:1. Between-batch coefficient of variation (CV) for 1α,25(OH) 2 D 3 standard was 2 D 3 .