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.

Fragile X premutation alleles in SCA, ET, and parkinsonism in an Asian cohort.

Among 367 subjects, the authors analyzed 167 patients with essential tremor, sporadic progressive cerebellar ataxia, multiple-system atrophy, and atypical parkinsonism and 200 healthy control subjects for FMR1 premutation alleles. None of the subjects carried alleles within the premutation range. These findings suggest that in the absence of other supportive clinical or imaging features, the cost-effectiveness of routine fragile X tremor/ataxia syndrome screening in this Asian cohort with movement disorders was low.

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 simultaneous presence of alpha- and beta-thalassaemia alleles: a pitfall of thalassaemia screening.

OBJECTIVE: To compare the efficacy of routine haematological tests and molecular analysis in the diagnosis of double heterozygous alpha- and beta-thalassaemia. METHODS: Screening was carried out in extended family members from 125 families registered in the National Thalassaemia Registry, known to have both alpha- and beta-thalassaemia carriers. RESULTS: Eighty-three individuals from 59 families were identified to be double heterozygous for alpha- and beta-thalassaemia only upon molecular analyses. Among 40 married individuals, 1 was at 25% risk for having beta-thalassaemia major children and 6 for having Bart's hydrops pregnancies. CONCLUSION: Molecular analysis must be used for the accurate diagnosis of double heterozygous alpha- and beta-thalassaemia for proper risk ascertainment, especially in regions with a high prevalence of both types of thalassaemia.

Prevalence and ethnic differences of autosomal-dominant cerebellar ataxia in Singapore.

We report the prevalence and ethnic differences of autosomal-dominant cerebellar ataxia (ADCA) in Singapore. Amongst 204 patients with ataxia who underwent genetic testing for dentatorubral-pallidoluysian atrophy (DRPLA) and for spinocerebellar ataxias (SCA) 1, 2, 3, 6, 7, 8, 10 and 12, 58 (28.4%) patients from 36 families tested positive. SCA 3 was identified in 31 (53.4%) patients from 15 families, SCA 2 in 17 (29.3%) patients from 12 families and SCA 1 in four (6.9%) patients from four families. Other SCA subtypes were rare. SCA 2 was the only subtype identified amongst ethnic Malay and ethnic Indian families. The estimated prevalence of ADCA in Singaporean families was at least 1 : 27,000. Based on the history and ancestry of Singaporeans, our study supported a founder effect for specific SCA subtypes and the association of ethnicity-specific SCA subtypes. Our findings suggest that SCA 2 is relatively common amongst the Malay race and that priority testing for SCA 3 and SCA 2 for ethnic Chinese, and SCA 2 for ethnic Malay, may be cost effective and relevant for the region.

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.

An anemic patient with phenotypical beta-thalassemic trait has elevated level of structurally normal beta-globin mRNA in reticulocytes.

Of the numerous beta-thalassemic mutations linked or unlinked to the beta-globin gene, all invariably cause a decrease in or an absence of structurally normal beta-globin mRNA when assayed. Here we report an anemic patient with an elevated alpha-/beta globin synthesis ratio of 2.0 in his reticulocytes. The patient's blood film showed marked red cell anisopoikilocytosis, microcytosis, and hypochromia, consistent with a typical beta-thalassemic trait phenotype. Acid-eluted erythrocytes contained numerous Heinz bodies. Molecular analysis of the patient's reticulocyte mRNA indicated that, compared to normal controls, there was a 3-fold elevation of beta-globin mRNA when assayed by RT-PCR and a 1.5-fold elevation of beta-globin mRNA when assayed by RNA slot blotting. The level of alpha-globin mRNA was normal when compared to that of normal adult controls. Extensive structural analysis of the beta-globin mRNA and gene by sequencing of RT-PCR and PCR products, respectively, did not detect any mutations. Tryptic mapping of purified beta-globin chains also did not show any abnormal tryptic fragments. These data indicated that a relative insufficiency of structurally normal beta-globin mRNA was not a cause of this beta-thalassemic phenotype. Therefore, the lesion that caused this particular thalassemic phenotype is not linked to the beta-globin allele.

Spinocerebellar ataxia in Singapore: predictive features of a positive DNA test?

Significant differences in frequency of the different spinocerebellar ataxia (SCA) subtypes have been described to occur in different populations. A 'blunderbuss' diagnostic DNA testing approach would entail unnecessary healthcare cost. In this study, we determine the prevalence of SCA subtypes and predictive features of a positive DNA test in consecutive clinically diagnosed SCA cases in Singapore. Twenty-one consecutive patients from 14 families were evaluated over a 3-year period. Thirteen patients (61.9%) from 6 families had a positive DNA test. Eleven of these (all ethnic Chinese) had SCA 3 (abnormal CAG size ranged from 61 to 71), and 2 ethnic Malays had SCA 2 (abnormal CAG size of 39). Clinical features which were highly predictive of a positive DNA SCA test in our population included presence of a positive family history, chorea and dystonia, muscle and tongue fasciculations, gaze-evoked nystagmus, and hypertonia. Our study draws attention to the observation that knowledge of relatively specific features of the most common SCA subtype in a local population can greatly enhance the practical accuracy of the choice of which SCA DNA test to order.

Novel germline BRCA1 mutations detected in women in singapore who developed breast carcinoma before the age of 36 years.

BACKGROUND: In recent years, although BRCA1 has been extensively investigated, the contribution of inherited BRCA1 mutations to breast carcinoma in Asian populations is largely unknown. The authors undertook this study to determine the prevalence and spectrum of germline BRCA1 mutations among women in Singapore with early onset breast carcinoma. METHODS: Forty-three of 72 eligible patients whose breast carcinoma was diagnosed before the age of 36 years were studied, independent of family history. DNA samples from 50 unrelated individuals randomly selected from the National Thalassemia Registry served as controls. Mutational screening was performed by single-strand conformation polymorphism analysis and protein truncation test, and alterations were confirmed by sequencing. First-degree relatives of patients with definite BRCA1 mutations were offered screening. RESULTS: A total of 6 novel alterations in BRCA1 were identified, including 2 frameshift mutations in exon 11 (2846insA and 2885delA), 3 rare sequence variants, and 1 polymorphism. Three women (7%) carried deleterious mutations, and the mutation was present in at least 1 unaffected first-degree relative of the proband. The same mutation (2846insA) was identified in 2 of the 7 unrelated subjects of Malay ethnicity. One mutation and three rare variants were identified in four women with no family history of breast or ovarian carcinoma whereas all women with affected first-degree relatives did not harbor BRCA1 mutations. No mutation was identified in the controls. CONCLUSIONS: The spectrum of germline BRCA1 mutations among the patients in this study was distinct from that in Caucasian populations although a similar prevalence was observed. Larger studies are necessary to clarify the significance of the mutation 2846insA in the Malay community and the penetrance of specific mutations in the Singapore population.

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.

Loss of c-met protooncogene in primary and metastatic sites of breast cancer.

BACKGROUND: It has been proposed that clones of tumor cells acquire higher metastatic potential as a result of specific genetic alterations. This study was designed to determine the role of the c-met protooncogene in systemic spread by comparing the loss of the c-met protooncogene between primary and metastatic breast carcinomas. METHODS: Only patients who had not received chemotherapy or radiotherapy in the preceding 6 months were included in this study. Histologically proven malignant tissue was obtained from the primary tumor, involved nodes, and distant metastatic and recurrent tumors of patients with breast carcinomas. Allelic loss of the c-met protooncogene in tumor tissue was determined by Southern blotting using a polymerase chain reaction-generated 347-bp human met-H probe. Restriction digestion was performed using Taq I and Msp I, with the patient's lymphocyte DNA as controls. RESULTS: Of 52 patients, lymphocyte DNA from 36 patients was heterozygous for the c-met protooncogene (69% informative). Forty-six tumors from these 36 patients were analyzed. Four of 30 primary tumors (13%) showed allelic loss of c-met. Of the nine nodal metastases examined, three (33%) showed allelic loss of the c-met protooncogene. Of seven distant metastatic breast tumors or recurrent disease, two (29%) showed allelic loss (both in patients with skin metastasis in the chest wall). CONCLUSIONS: Allelic loss of the c-met protooncogene was detected in both primary (13%) and metastatic sites (31%) of breast cancer. Although a higher proportion of allelic loss of c-met was noted in nodal and distant/recurrent disease, the difference when compared with the primary tumor was not statistically significant. These findings indicate a limited role of the c-met protooncogene in breast cancer metastases.

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.

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.

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.

Rapid antenatal diagnosis of beta-thalassemia in Chinese caused by the common 4-bp deletion in codons 41/42 using high-resolution agarose gel electrophoresis and heteroduplex detection.

The 4-bp deletion in codons 41/42 (-TTCT) in the beta-globin gene is a common mutation that causes beta-thalassemia in Chinese. A simple method, which involved PCR amplification of the relevant region, was used for the antenatal diagnosis of a fetus at risk for this mutation. The fetal PCR product showed a single fragment of normal size on MetaPhor gel. The homozygous normal status was further confirmed by the generation of heteroduplexes only after addition of homozygous mutant DNA.