Association of common genetic variants with breast cancer risk and clinicopathological characteristics in a Chinese population.

Genome-wide association studies (GWAS) have identified various genetic susceptibility loci for breast cancer based mainly on European-ancestry populations. Differing linkage disequilibrium patterns exist between European and Asian populations, and thus GWAS-identified single nucleotide polymorphisms (SNPs) in one population may not be of significance in another population. In order to explore the role of breast cancer susceptibility variants in a Chinese population of Southern Chinese descent, we analyzed 22 SNPs for 1,191 breast cancer cases and 1,534 female controls. Associations between the SNPs and clinicopathological features were also investigated. In addition, we evaluated the combined effects of associated SNPs by constructing risk models. Eight SNPs were associated with an elevated breast cancer risk. Rs2046210/6q25.1 increased breast cancer risk via an additive model [per-allele odds ratio (OR) = 1.43, 95 % confidence interval (CI) = 1.26-1.62], and was associated with estrogen receptor (ER)-positive (per-allele OR = 1.39, 95 % CI = 1.20-1.61) and ER-negative (per-allele OR = 1.55, 95 % CI = 1.28-1.89) disease. Rs2046210 was also associated with stage 1, stage 2, and stage 3 disease, with per-allele ORs of 1.38 (1.14-1.68), 1.48 (1.25-1.74), and 1.58 (1.28-1.94), respectively. Four SNPs mapped to 10q26.13/FGFR2 were associated with increased breast cancer risk via an additive model with per-allelic risks (95 % CI) of 1.26 (1.12-1.43) at rs1219648, 1.22 (1.07-1.38) at rs2981582, 1.21 (1.07-1.36) at rs2981579, and 1.18 (1.04-1.35) at rs11200014. Variants of rs7696175/TLR1, TLR6, rs13281615/8q24, and rs16886165/MAP3K1 were also associated with increased breast cancer risk, with per-allele ORs (95 % CI) of 1.16 (1.00-1.34), 1.15 (1.02-1.29), and 1.15 (1.01-1.29), respectively. Five SNPs associated with breast cancer risk predominantly among ER-positive tumors (rs2981582/FGFR2, rs4415084/MRPS30, rs1219648/FGFR2, rs2981579/FGFR2, and rs11200014/FGFR2). Among our Chinese population, the risk of developing breast cancer increased by 90 % for those with a combination of 6 or more risk alleles, compared to patients with ≤3 risk alleles.

Meteorological factors and aneurysmal subarachnoid haemorrhage in Hong Kong.

OBJECTIVE: To evaluate the influence of meteorological factors on the onset of aneurysmal subarachnoid haemorrhage in Hong Kong. DESIGN: Retrospective review of prospectively collected data. SETTING: University teaching hospital, Hong Kong. PATIENTS: A total of 135 consecutive patients with acute aneurysmal subarachnoid haemorrhage presenting to the hospital within 48 hours after ictus from October 2002 to October 2006. MAIN OUTCOME MEASURES: Occurrence of aneurysmal subarachnoid haemorrhage in relation to daily changes in atmospheric pressure, temperature, and humidity. RESULTS: The peak incidence of aneurysmal subarachnoid haemorrhage occurred in winter (December to February), especially January. The mean (+/-standard deviation) daily atmospheric pressure change was significantly higher on days with aneurysmal subarachnoid haemorrhage onset as opposed to days without (1.75+/-1.47 hPa vs 1.48+/-1.28 hPa; P=0.032). CONCLUSIONS: A seasonal variation and relationship to atmospheric pressure change in aneurysmal subarachnoid haemorrhage was noted in the current study carried out in Hong Kong. The mechanism linking atmospheric pressure change and aneurysmal rupture remained to be explored.

Genetic analysis of SCA2, 3 and 17 in idiopathic Parkinson's disease.

Recent reports of SCA2 and SCA3 patients who presented with levodopa responsive parkinsonism have generated considerable interest as they have implications for genetic testing. It is unclear whether ethnic race alone or founder effects within certain geographical region explain such an association. In this study, we conducted genetic analysis of SCA2, 3, 17 in an ethnic Chinese cohort with early onset and familial Parkinson's disease (PD) and healthy controls. A total of 191 subjects comprising of 91 PD and 100 healthy controls were examined. We identified one positive case of SCA2 in an early-onset sporadic PD patient who had CAG 36 repeats, yielding a prevalence of 2.2% in early-onset sporadic PD patients and less than 1.0% in our study PD population. The size of the repeats was lower than the expanded repeats (38-57) in SCA2 patients with ataxia in our population. All the children of the patient were physically normal even though some of them carried the repeat expansion of similar size. No cases and controls were positive for SCA3 and SCA17. We do not think routine screening of SCA2, SCA3 and SCA17 for all idiopathic PD patients is cost-effective in our ethnic Chinese population. However, SCA2 should be a differential diagnosis in young onset sporadic PD when genetic mutations of other known PD genes have been excluded.

SMN1 deletions among singaporean patients with spinal muscular atrophy.

INTRODUCTION: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder characterised by degeneration of spinal cord anterior horn cells, leading to muscular atrophy. It is the second most frequent autosomal recessive disease among Caucasian populations with a prevalence of between 1 in 6000 and 1 in 10,000 live births, and a carrier frequency of about 1 in 50. The International SMA Consortium classification defines several types of SMA depending on the age of onset and clinical severity. In the past, the diagnosis of SMA was confirmed by muscle biopsy and, sometimes, electromyography. In 1990, SMA was linked to the 5q13 region of chromosome 5. In 1995, it was found that >95% of patients with SMA have homozygous deletions of exons 7 and 8 of the survival motor neurone 1 (SMN1) gene, one of the candidate genes identified within 5q13. The purpose of our study was to determine the frequency of SMN1 deletions in patients with known SMA and the impact of this on the diagnosis of SMA. MATERIALS AND METHODS: Molecular analysis was performed on stored DNA and case notes were reviewed retrospectively. RESULTS: Twenty-two (91.7%) out of 24 patients with all types of SMA were homozygously deleted for exons 7 and/or 8 of SMN1. We also report our experience with prenatal diagnosis of SMA. CONCLUSIONS: Molecular studies can replace conventional investigations for SMA and have made the option of prenatal diagnosis possible for couples at risk.

The origin and distribution of cortical granules in human oocytes with reference to Golgi, nucleolar, and microfilament activity.

The origin and distribution of cortical granules were investigated in human preovulatory oocytes at various phases of maturation. Twenty-five oocytes obtained from unstimulated small antral follicles and from stimulated large antral and mature follicles were examined by transmission electron microscopy. Ovarian stimulation in women was accomplished by administering Clomid followed by hMG or hCG or both. Small antral follicle oocytes were dissected from ovarian biopsies, while the other oocytes were recovered by laparoscopy. Some oocytes were allowed to mature in Ham's F-10 or Whittingham's T-6 media before routine fixation in glutaraldehyde/osmium. Cortical granules originate from typical, hypertrophic Golgi complexes during early maturation and continue till its completion. Evidently there are two waves of cortical granule synthesis, the first more prolific than the second. The first occurred in small antral follicle oocytes, when there was a peak in Golgi activity, and the second was observed at the germinal vesicle stage, particularly at the onset of resumption of meiosis. Golgi complexes became progressively scarce as oocytes completed first maturation. Golgi membranes were also involved in the formation of lysosomes. A well-defined band of microfilaments was detected in small antral follicle oocytes which seemed to prevent the cortical granules, organized in a single layer, from migrating to the periphery. This band gradually became disorganized at the germinal vesicle stage as oocytes resumed meiosis, when cortical granules were apparently migrating to the surface. Metaphase I and mature oocytes had one to three discontinuous layers of cortical granules beneath the oolemma. The general organization of oocytes was also investigated and the roles of the nucleolus and endoplasmic reticulum in relation to Golgi activity and cell secretion were discussed.

Spinocerebellar ataxia type 3 presenting as an L-DOPA responsive dystonia phenotype in a Chinese family.

The clinical spectrum of spinocerebellar ataxia 3 (SCA 3) disease is wide and varied. We describe a Chinese patient with a mutation at the SCA 3 locus with clinical features of levodopa-responsive dystonia. The family history was suggestive of being autosomally dominant. Levodopa responsiveness though rare has been described in families with features of parkinsonism. Noteworthy is the relatively late onset of disease (>40 years) possibly explained by the low number of affected alleles at 59, the usual range being from 62 to 86, with the lowest recorded number at 56. This expands the wide and varied phenotypic manifestations of SCA 3, and highlights the observation that features suggestive of levodopa-responsive dystonia (DRD) such as focal dystonia, gait difficulty with diurnal fluctuation of symptoms, and a marked response to low doses of levodopa can be presenting features of SCA 3. SCA 3 should be considered a differential diagnosis in adult patients who present with DRD phenotype and with a positive family history.

The presymptomatic molecular diagnosis of familial adenomatous polyposis in Singapore.

Familial adenomatous polyposis (FAP) is an autosomal dominant disorder which predisposes to the development of colorectal cancer. The adenomatous polyposis coli (APC) gene, mutation of which is responsible for FAP, has been localised to chromosome 5q21. Linkage studies using DNA markers have proven useful for presymptomatic diagnosis of at-risk individuals. We have examined 8 FAP families from the Singapore Polyposis Registry by using 4 linked and 2 intragenic DNA markers. Presymptomatic diagnosis could be made in 84% (37 of 44) of at-risk individuals. Among these presymptomatically diagnosed cases, positive prediction was made in 32% (12 of 37) whereas negative prediction was possible in 68% (25 of 37). As the accuracy of genetic diagnosis is high and the test reliable in most cases, the major impact of these tests will be the reduction of unnecessary anxiety and a significant reduction in the frequency of screening for at-risk individuals who are not carrying the affected gene.

HLA and trophoblastic disease.

The susceptibility of trophoblastic diseases is not associated with HLA antigens. Compatibility studies between the patient and her husband, and the patient and the tumour suggest that it may not play an important role in the susceptibility or the progression of the tumour. This as well as other direct evidence indicate that the tumour cells probably do not express much HLA antigens on their cell surface. The expression of HLA antigens on normal and abnormal trophoblasts are discussed.

Molecular heterogeneity of beta-thalassaemia in Malaysia: a practical approach to diagnosis.

The beta-thalassaemia mutations in 20 Malaysian children with beta-thalassaemia major were characterised by using a multi-modal approach, consisting of a slot-blot hybridisation with selected allele-specific oligonucleotides (ASO), followed by reverse dot-blot assay (RDB), amplification refractory mutation system (ARMS) and genomic sequencing. This strategy yielded a 94.4% mutation detection rate. The 6 most common mutations were codons 41/42 (-TTCT), IVS II nt 654(C --> T), IVS I nt 5(G --> C), IVS I nt 1(G -->T), codon 35 (-C) and codon 19 (A --> G), which accounted for 83.3% of all mutations detected. A strategy of initial screening with the above 6 selected ASOs for slot-blot hybridisation followed by RDB assay for the less common Asian mutations would give a mutation identification of 91.7%. Another feasible approach would be to analyse alleles from a particular racial group, by a judicious selection of 4 ASOs common to that particular subpopulation and then supplement this with RDB assay. This could yield a 100% coverage for the Chinese subpopulation in Malaysia. With these strategies, a practical approach has been identified to overcome the pitfalls posed by the molecular heterogeneity of beta-thalassaemia to enable prenatal diagnosis and carrier screening to be carried out. Regional collaborative studies are to be encouraged as an indispensable tool in providing better health care services to our patients.

Trinucleotide repeat analysis of Huntington's disease gene in Singapore.

INTRODUCTION: Huntington's disease (HD) is an inherited neurodegenerative disorder characterised by chorea and progressive dementia. The mutation causing the disease has been identified as an unstable expansion of a trinucleotide (CAG)n. We have assessed the (CAG)n repeats in the patients and controls in our population. MATERIALS AND METHODS: Polymerase chain reactions (PCRs) for the repeat region were carried out for 116 individuals: 10 were asymptomatic at-risk members from 5 families; 53 symptomatic patients from various hospitals; and 53 normal unrelated Singaporeans. Estimation of the number of repeats was based on Metaphor gel electrophoresis, sizing using the GeneScan on ABI 310 Genetic Analyzer, and sequencing using the same equipment. RESULTS: Metaphor gel sizing generally gives an over-estimation, and GeneScan gives an under-estimation of repeat numbers compared with sequencing which is the gold standard. Of the 63 patients and family members tested, 25 had one expanded allele of 40 to 54 CAG repeats and the other allele in the normal range of 15 to 30 repeats. One patient had an allele in the intermediate range (38). CONCLUSION: The range of CAG repeats in the normal and HD alleles in our population is similar to those reported elsewhere. An accurate sizing can only be obtained with sequencing. For allele sizes in the intermediate range (37-40), sequencing should be carried out to confirm the carrier status of a patient.

Early prenatal diagnosis of beta-thalassaemia in Singapore.

beta-thalassaemia is one of the commonest autosomal recessive genetic diseases in the Singapore population. In the homozygous form, it results in a severe anaemia, requiring monthly transfusion for survival. Because of the less than satisfactory treatment available for the condition, prenatal diagnosis has always been an option for couples at-risk. The available method was globin chain analysis of foetal blood, obtained at 18 to 20 weeks of gestation. Affected pregnancies would then be diagnosed and require termination in the mid to late trimester. A relatively newer technique, chorionic villus sampling (CVS), allows foetal material to be obtained in the first trimester. However, analysis of the foetal tissue requires direct gene studies to be performed. The aim of this study was to evaluate the feasibility of this analysis in couples at-risk for beta-thalassaemia in Singapore. Sixteen couples who were at-risk for a child with beta-thalassaemia major were offered prenatal diagnosis. All of them opted for CVS as compared to foetal blood sampling. The mutations in the beta-globin gene in these couples at-risk were identified. Direct gene analysis was then performed on the foetal sample, using a variety of molecular techniques. These included reverse dot-blot hybridisation, allele-specific oligonucleotide hybridisation, restriction enzyme digests and direct analysis of amplified products. DNA profiling was done for each case to exclude definitively the possibility of maternal tissue contaminating the foetal sample. The results in all these cases were unequivocal. The procedure of CVS itself was uneventful in these 16 couples. Procedural-associated foetal loss was nil. Prenatal diagnosis in the first trimester allows early termination of an affected pregnancy with significantly less maternal morbidity and prenatal anxiety. It also results in greater patient acceptability of the procedure and plays a key role in the prevention of this devastating genetic disease.

DNA testing for fragile X syndrome in 255 males from special schools in Singapore.

INTRODUCTION: Fragile X syndrome, the most common cause of inherited mental retardation, results from unstable expansion of a trinucleotide (CGG)n repeat in the FMR1 gene. Phenotypic expression is variable making clinical diagnosis difficult, while diagnosis by Southern blotting is relatively expensive and labour intensive. The prevalence in Singapore has not been studied. MATERIALS AND METHODS: We developed a rapid screening test using a PCR analysis. We studied 255 males with unexplained cause for learning difficulties from 8 special schools. A clinical scoring system based on characteristic features described was devised. RESULTS: PCR analysis showed absence of the band for the normal allele in 11 samples, 6 of which were confirmed by Southern blotting to be positive for FMR1 expansion, giving a 2% false-positive rate with PCR. Sensitivity of the PCR test was evaluated by performing Southern blotting in all PCR-normal samples; all of which were confirmed to be normal. This PCR test was shown to be highly reproducible. Clinical criteria were not predictive. CONCLUSIONS: Six (2.4%) new cases of fragile X syndrome were detected. There is a need to incorporate fragile X testing in routine screening of patients with developmental delay and learning difficulties. The use of PCR could eliminate the need for Southern blotting in up to 95% of cases. PCR analysis provides a simple, reliable and rapid tool for screening.

Identification of integrated Epstein-Barr virus in nasopharyngeal carcinoma using pulse field gel electrophoresis.

Integrated Epstein-Barr virus (EBV) was found in 4 of 17 pathologically diagnosed, EBV-positive nasopharyngeal carcinoma (NPC) biopsy samples using pulse field gel electrophoresis (PFGE). The analyses were carried out after digestion of biopsy DNA with rare restriction enzymes such as PacI, which does not cleave EBV due to the absence of the recognition sequence, and ClaI. Employing this technique, EBV integration in IB4, through EcoI, and Namalwa through the terminal repeats, was confirmed. We also established that integration of EBV in AW Ramos was through the terminal repeats. The Raji cell line was also found to harbour integrated virus in addition to episomal DNA. However, the site of integration could not be confirmed, since as the Raji DNA appeared to be heavily methylated and could not be cleaved with CPG rare cutters. As in the Raji cell line, the DNA of EBV in nasopharyngeal-carcinoma (NPC) biopsies also appeared to be methylated.

Use of microimmobilization and microagglutination assays for attempted detection of HLA antigens and beta2 microglobulin on human sperm.

Microtechniques for detecting sperm agglutinating and immobilizing antibodies are described. These assays are proved to be useful in the study of anti-sperm antibodies in the sera of vaseectomized men and the serum of a rhesus monkey immunized with human sperm. However, using various antisera against beta2 microglobulin and HLA region antigens, including Ia antigens, in these assays, very little, if any, activity was found against sperm. Absorption and inhibition tests also could not show a significant amount of these antigens on sperm. Although no HLA region antigens could be detected in the seminal plasma by inhibition tests, a large amount of beta2 microglobulin was found. It is suggested that some beta2 microglobulin could be adsorbed onto sperm, since mouse sperm was shown to pick up beta2 microglobulin after incubation with human seminal plasma. The maximum amounts of HLA region antigens and beta2 microglobulin in the seminal plasma are estimated.

Use of capillary electrophoresis for thalassaemia screening.

This paper describes the use of high-performance capillary electrophoresis (HPCE) for screening of thalassaemias. Preparation of globin was performed by adding 1 mL of haemolysate to 20 volumes of cold acidified acetone. Separation of globins was accomplished in a 25 mM disodium phosphate buffer at pH 11.8 using an uncoated capillary column of 50 cm x 75 microns (i.d.). Distinct peaks of alpha, beta and gamma were resolved within 6 min. The coefficient of variations for within-day and between-day runs were 4.7% and 6.37%, respectively. Using this simple and rapid procedure up to 30 specimens could be analysed in a single day. This method appears to be reliable and can be used for mass screening of various haemoglobinopathies.

Beta-thalassemia mutations in Singapore--a strategy for prenatal diagnosis.

The strategy for early prenatal diagnosis of beta-thalassemia in Singapore by direct detection of the mutant beta-globin gene requires the spectrum of mutations producing the disorder in this population to be characterized. We analyzed 134 beta-thalassemia alleles from Singapore by specific oligonucleotide hybridization after DNA amplification, using a nonradioactive enhanced chemiluminescence detection system. The mutations were identified in 90% of the alleles using five oligonucleotide probes for the following mutations: codons 41/42 (deletion-TCTT), IVS II nt 654 (C-->T), codon 17 (A-->T), IVS I nt 5 (G-->C), and -28 TATA box (A-->G). Together with the strategy of direct sequencing, a total of 97% of the mutations were identified. In the Chinese subpopulation, 97% of the mutations were detected by the oligonucleotide probes. Using just four oligonucleotide probes would identify 96% of the mutations, and 76% of the mutations were accounted for by codon 41/42 (-TCTT) and IVS II nt 654 (C-->T) mutations. Thus in this subpopulation early prenatal diagnosis would be possible in virtually all the affected families.

Chromosome mediated gene transfer of six DNA markers linked to the cystic fibrosis locus on human chromosome seven.

The DNA probes met and pJ3.11 are derived from loci on chromosome seven that are closely linked to, and probably flanking, the gene mutation causing cystic fibrosis (CF). We have shown that mitotic chromosomes from the cell line MNNG-HOS, which contains an activated met oncogene, can induce morphological transformation of mouse NIH-3T3 cells. Southern analysis of isolated transfectant cell lines with cloned dispersed repetitive human DNA sequences as probes demonstrated that several lines of transformed NIH 3T3 cells had stabley incorporated large segments of chromosome seven DNA. Southern blot analysis also demonstrated the presence of met, pJ3.11 and several other single copy sequences that had been previously localised to chromosome 7 within the transgenomes. In this way a further four genetic markers were shown to be physically linked to met, and thus to CF. These probes may prove useful in confirming the order of loci around CF and in the prenatal diagnosis of this common autosomal recessive disease.