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The common restriction fragment length polymorphisms (RFLPs) associated with the FIX gene: 5' BamH I, Dde I, BamH I (2), Taq I and 3' Hha I were absent or of low incidence in Southern Chinese and are therefore not useful for linkage analysis. No deletion was detected amongst seven consecutive unrelated haemophilia B patients, but one had an insertion of a 15 kb Pvu II fragment containing exon d. Using an alternate strategy of polymerase chain reaction (PCR) amplification and direct sequencing, the molecular defect in the other six patients was defined. The four novel mutations characterized were: nucleotide (nt) 6410 G----C (Gly12----Ala); nt 31261 delta T (stop codon 31 bp downstream); nt 31260 C----G (Thr380----Ser) and nt 31122 C----A (Ala34----Asp). Two patients had the same mutation at nt 6365, G----A (Arg-4----Gln), identical to one previously described in other ethnic groups, suggesting that this is a hotspot for mutation. Each of the mutations was found to affect an enzyme recognition site and could thus be identified by direct visualization of abnormal restriction fragments in amplified genomic DNA. This allows rapid and accurate DNA diagnosis of haemophilia B in an ethnic group which otherwise shows little or no polymorphism for the common RFLP sites.

A reverse dot blot method based on membrane-bound allele-specific oligonucleotides as hybridization targets for amplified alpha-gene fragments has been developed for the rapid detection of four non-deletion alpha thalassaemia defects found in the Chinese. Since these non-deletion defects account for 22 8% of haemoglobin H disease, a sensitive, specific and rapid screening method should be of value.

Background: hRad9 is a cell cycle checkpoint gene that is up-regulated in breast cancer. We have previously shown that the mRNA up-regulation correlated with tumor size and local recurrence. Immunohistochemical studies were made to better define the role of hRad9 in breast carcinogenesis. Methods: Localisation of hRad9 protein were performed on paired tumor and normal breast tissues. Immunoblotting with and without dephosphorylation was used to define the protein isolated from breast cancer cells. Results: Increased hRad9 protein was observed in breast cancer cells nucleus compared to non-tumor epithelium. This nuclear protein existed in hyperphosphorylated forms which may be those of the hRad9-hRad1-hHus1 complex. Conclusion: Finding of hyperphosphorylated forms of hRad9 in the nucleus of cancer cells is in keeping with its function in ameliorating DNA instability, whereby it inadvertently assists tumor growth. © 2008 Chan et al; licensee BioMed Central Ltd., published_or_final_version

The pattern of malignant lymphomas in the Hong Kong Chinese population is characterized by a low incidence of Hodgkin's disease and follicular lymphomas. The authors studied the immunoglobulin (Ig), T-cell receptor (TCR), and bcl-2 gene rearrangement in 62 cases of malignant lymphoma in this population by Southern blot hybridization. Two cases of Hodgkin's disease showed no rearrangement of the Ig and TCR genes. All 42 cases of B-cell lymphoma had Ig heavy chain (JH) rearrangement with or without additional rearrangement of the light chains (C kappa and C lambda). One case of diffuse B-cell lymphoma had additional T-cell receptor beta-chain (C beta) rearrangement. Sixteen of 18 cases of T-cell lymphoma had C beta rearrangement, and one case of T-lymphoblastic lymphoma had additional JH rearrangement. Two of eight (25%) cases of follicular lymphoma but only one of the 34 (2.9%) cases of diffuse B-cell lymphoma had bcl-2 rearrangement that was detected by pFL-1 probe. None of the 62 cases showed bcl-2 rearrangement using the pFL-2 probe. In conclusion, the Ig and TCR gene rearrangement pattern of the lymphomas found in Hong Kong correlates well with the T-cell and B-cell lineage, which is similar to reports in the white population. However, the incidence of bcl-2 gene rearrangement in follicular B-cell lymphoma is lower than that reported in the US but comparable with that in Japan.

This descriptive study was undertaken to investigate patients' perceptions of the seven content areas commonly included in cardiac teaching in terms of how important and how realistic they were for being learned following the experience of an acute myocardial infarction (MI). Lazarus's stress-coping theory was used as a framework for the study. A sample of 30 MI subjects were interviewed before discharge and during early convalescence. Before discharge, subjects rated six out of the seven content areas as "important" for learning. Although the information was considered important, it was not rated as equally realistic to learn. During early convalescence, mean scores under 'important to learn' were slightly lower, but all seven content areas were rated as significantly more realistic to be learned (P less than 0.05). Little variation in patients' perceptions of the importance of teaching content was noted over time. However, there was major variation between their perceptions of how important and how realistic it was to learn the material before discharge. Both dimensions, how important and how realistic, are therefore crucial to consider in cardiac teaching.

The use of nursing conceptual frameworks and nursing diagnoses is generally acknowledged to hold immense promise for the further development of nursing's scientific base. The nursing profession to date has failed to link the two, thus diminishing their impact. A large community hospital in Canada adopted King's Conceptual Framework for Nursing Practice and advocated the use of NANDA's nursing diagnostic statements as the manner by which patient needs or problems would be identified. Nurses, however, required assistance in identifying more clearly the connection between the theoretical base and nursing diagnoses. During stage one of this project a tool was designed that categorized NANDA's nursing diagnoses according to the concepts and systems of King's conceptual framework. The difficulties encountered in categorizing the nursing diagnoses are discussed and the categorization system is outlined. The utility of the tool was tested during stage two. A total of 25 nurses were asked to describe how they arrived at a particular nursing diagnosis before and after using the categorization tool. Criteria were used to indicate whether a connection had been made between the formulation of the nursing diagnosis and King's conceptual framework throughout the nurse's thought processes. The results indicated that nurses could make a connection between NANDA's nursing diagnoses and King's framework with the assistance of the categorization tool.

Objective: Low-density lipoprotein receptor-related protein 5 (LRP5) is important for osteoblast differentiation and mutations of the gene are associated with both low and high bone mass syndromes. Our study aimed to evaluate the importance of LRP5 in the determination of peak bone mass acquisition in Chinese females in the general population. Methods: A total of 286 young southern Chinese females (aged 22–44 years) with low bone mineral density (BMD) (defined by a BMD Z score ≤–1.28 at either the hip or spine) or high BMD (Z score ≧+1) were studied. The LRP5 gene was sequenced for single nucleotide polymorphisms (SNPs) and 4 SNPs were tagged from 8 genotyped SNPs for this study. Results: Single locus allele association tests revealed significant associations of rs682429 and rs686921 with BMD variation (p < 0.05). Omnibus test (likelihood ratio test) revealed overall significant association between LRP5 gene locus and total hip BMD, with rs682429 being most predictive. rs682429 is located in 5′UTR, 2 bases adjacent to a consensus recognition site for the Elk-1 binding element. Conclusion: Common variations of the LRP5 promoter are associated with BMD in young women. These significant associations appear to be driven by rs682429. Functional studies are necessary to elucidate the role of this SNP on the effect of Elk-1 binding element transcriptional activity of LRP5 gene.

amniotic cell DNA showed the fetuses to be rhesus-Dpositive. All fetuses who were rhesus-D-negative on plasma PCR (n=13) were all correctly negative, confirmed by genotyping amniotic-cell or chrionic villus DNA (patient 3798). Knowledge of the fetal RHD genotype is important in the management of rhesus alloimmunisation during pregnancy. Previously, genotyping and phenotyping procedures have been invasive, with inherent risks of iatrogenic fetal loss of 0·3–1·0%. We show that fetal RHD genotyping can be done from maternal plasma. Fetal cells can persist in the maternal circulation for years and can, therefore, interfere with prenatal typing of later pregnancies, but this effect is unlikely for fetal DNA in maternal plasma. In our study, six of 13 women who were correctly determined to carry a rhesus-Dnegative child, had previously had a rhesus-D-positive child. For plasma samples that are negative on RHD PCR, an control PCR should be used to confirm the presence of fetal DNA. To prevent rhesus D immunisation, immunoprophylaxis should be given to all pregnant rhesusD-negative women at about 28 weeks’ gestation. This plasma PCR makes it possible to restrict prophylaxis to only those women who carry a rhesus-D-positive child. This approach will save anti-D immunoglobulin, which is available in limited amounts, and prevent unnecessary exposure to human immunoglobulin in pregnant women. From all but one patient, blood was taken in the second trimester. Lo and colleagues showed that fetal DNA can be detected in the maternal plasma in the first trimester, which makes the plasma PCR promising in prenatal tests early in pregnancy.

Blood counts, hemoglobin (Hb) high performance liquid chromatography (HPLC), and DNA analyses were performed on 260 children, aged 5 months to 16 years, at Siem Reap to assess the prevalence of thalassemia and other hemoglobinopathies in regional Cambodia. Hemoglobinopathies were present in 134 children (51.5%) with 20 abnormal genotypes identified. alpha-Thalassemia (thal) (35.4%) was the most prevalent disorder and the -alpha3.7 gene deletion was the most common alpha-globin gene abnormality. The - -SEA deletion and nondeletional forms of alpha-thal, Hb Constant Spring [Hb CS, alpha142, Term-->Gln, TAA-->CAA (alpha2)], Hb Pakse [alpha142, Term-->Tyr, TAA-->TAT (alpha2)] and triplicated alpha genes, were also present but at low frequencies. Hb E [beta26(B8)Glu-->Lys, GAG-->AAG] (28.8%) was the most common beta-globin gene abnormality, whilst beta-thal was only detected in two children (0.8% of cases). Although hemoglobinopathies were common, the majority of abnormalities detected (heterozygous -alpha3.7 and Hb E) were not clinically significant. On the basis of these findings, and with the majority of abnormalities being mild, it seems improbable that thalassemia represents a major health burden in this region of Cambodia.

To report our experience in preimplantation genetic diagnosis (PGD) for the exclusion of homozygous alpha degrees thalassemia.PGD was performed on nine couples with alpha degrees thalassemia genotype undergoing assisted reproduction. Oocytes were aspirated after ovarian stimulation and fertilized by intracytoplasmic sperm injection. One or two blastomeres were biopsied from the six- to eight-cell embryo. Single cell multiplex PCR of the normal and alpha degrees thalassemia alleles was performed for first round, followed by semi-nested PCR of the respective alleles using 5'-end labelled fluorescent primers. Only those embryos with a blastomere diagnosed as having at least one normal allele were selected for transfer.One hundred and twenty-six blastomeres from 82 embryos were analyzed. The rates of allele dropout was 10.2% and PCR failure 12.7%. Fifty-eight embryos (70.7%) had at least one normal allele, of which 31 were transferred to 13 prepared cycles and one triplet pregnancy achieved. The triplets showed no ultrasound features of homozygous alpha degrees thalassemia at 18 weeks and were delivered in healthy condition by caesarean section at 34 weeks. Their genotypes were confirmed by cord blood analysis.PGD for alpha degrees thalassemia is possible by single cell PCR. The transfer and successful implantation of unaffected embryos ensure birth of disease-free babies.

Summary Bone mineral density (BMD), a risk factor for osteoporosis, is believed to be under genetic control. The effect of environmental factors and gender on the heritability of BMD and bone size is ill-defined. In this study, heritability estimates (h 2 ) were determined in 3,320 southern Chinese subjects from 1,019 families using the variance components model. The h 2 for age, weight and height-adjusted BMD was 0.63‐0.71 for females, and 0.74‐0.79 for males; and for bone size, 0.44‐0.64 for females and 0.32‐0.86 for males. Adjustment for lifestyle factors including calcium and phytoestrogen intake, exercise, smoking and alcohol consumption altered the h 2 differently in males and females. The proportion of variance in BMD and bone size explained by all covariates varied between skeletal sites, but was consistently greater in females than males. A significant gender difference was observed in the genetic variance of BMD and bone size at the hip but not the spine. In conclusion, a gender difference was observed in the degree of heritability of BMD and bone size at specific skeletal sites. Environmental influences contributed variably at different sites in the two sexes.

A retrospective study was performed to compare the detection rate of chromosomal abnormalities by different approaches of full karyotyping, rapid aneuploidy diagnosis (RAD) or both when invasive prenatal testing is performed for diagnosis of thalassaemia. The karyotype results of 1120 prenatal samples obtained from thalassaemia couples from January 1985 to December 2002 in a referral centre for prenatal diagnosis were studied. The detection rate of chromosomal abnormalities by four different approaches were compared: (i) karyotyping for all samples; (ii) RAD (21,18,13,X,Y) for all samples; (iii) RAD for all samples + karyotyping for cases with ultrasound abnormalities; and (iv) RAD (21,18,13) for all + RAD (X,Y) for cases with ultrasound abnormalities consistent with Turner syndrome + karyotyping for cases with ultrasound abnormalities. Normal karyotypes were found in 1103 samples (98.5%). There were 17 cases (1.5%) of chromosomal abnormalities: four cases (0.36%) were clinically significant, eight cases (0.7%) were of borderline clinical significance and five cases (0.44%) were not confirmed by subsequent prenatal or postnatal tests. The incidences of autosomal (7/1120 = 0.63%) and sex chromosomal (5/1120 = 0.45%) abnormalities were not higher than those (0.41 and 0.22%, respectively) from newborn surveys (Hook and Hamerton, 1977) (P = 0.398 and 0.216, respectively). Approach 1 would detect all 17 chromosomal abnormalities. Approach 2 would detect three of four clinically significant chromosomal abnormalities but not detect six of eight chromosomal abnormalities of borderline clinical significance and three of five chromosomal abnormalities not confirmed by subsequent prenatal or postnatal tests. Approach 3, in addition, would be able to detect all four clinically significant chromosomal abnormalities. Approach 4 would detect all four clinically significant chromosomal abnormalities but would not detect seven of eight chromosomal abnormalities of borderline clinical significance and four of five chromosomal abnormalities not confirmed by subsequent prenatal or postnatal tests. RAD (21,18,13) for all + RAD (X,Y) for cases with ultrasound abnormalities consistent with Turner syndrome + karyotyping for cases with ultrasound abnormalities seemed to be the best approach for the detection of chromosomal abnormalities when invasive prenatal testing is performed for diagnosis of thalassaemia.

SummaryThe molecular basis of haemophilia B is heterogeneous and many mutations of the Factor IX (FIX) gene have been characterised. Using the allele-specific arrayed primer extension (ASAPEX) technology, we have designed a FIX array to simultaneously analyse 69 mutations found in British, Thai and Chinese patients. This technology overcomes the problem of multiple reverse dot-blot analysis and has a 100% accuracy in the detection of both affected subjects and carriers in families with known mutations. In seven unknown mutations from Thailand, the array could detect the specific mutation in five and in the remainders the normal primer at specific spots failed to extend due to a mutation a few nucleotides upstream, thus allowing their identification. Hence this FIX array can detect 53% of the 2891 mutation entries in the FIX database. Each of the microarray slide can be used for three different test samples and would be useful for carrier testing for common mutations and prenatal diagnosis. It is simpler and more cost effective than genome sequencing and would be particularly useful in laboratories with limited technical capabilities.

Tight junction (TJ) constitutes the barrier by controlling the passage of ions and molecules via paracellular pathway and the movement of proteins and lipids between apical and basolateral domains of the plasma membrane. Claudins, occludin, and junctional adhesion molecules are the major three transmembrane proteins at TJ. This study focuses a newly identified mammalian TJ gene, claudin-19, in kidneys. Mouse claudin-19 composes of 224 amino acids and shares 98.2% and 95% amino acid homology with rat and human, respectively; the most evolutionary-related claudins are claudin-1 and -7, which share approximately 75% DNA sequence homology with claudin-19. Claudin-19 is abundantly expressed in the mouse and rat kidneys among the organs examined by Northern blots, and to a much less extent, also found in brain by RT-PCR. Claudin-19 and zonula occludens-1 (ZO-1) are localized at junctional regions of Madin-Darby canine kidney (MDCK) cells by immunofluorescent microscopy. In addition, ZO-1 is found in the claudin-19-associated protein complexes in MDCK cells by co-immunoprecipitation. Using aquaporin-1 and aquaporin-2 antibodies as markers for different renal segment, strong expression of claudin-19 was observed in distal tubules of the cortex as well as in the collecting ducts of the medulla. To less extent, claudin-19 is also present in the proximal tubules (cortex) and in the loop of Henle (medulla). Furthermore, intense claudin-19 immunoreactivity is found co-localized with the ZO-1 in kidneys from postnatal day 15, day 45, and adult rats and mice. Similar localizations of claudin-19 and ZO-1 are also observed in human kidneys. Since these renal segments are mainly for controlling the paracellular cation transport, it is suggested that claudin-19 may participate in these processes. In human polycystic kidneys, decreased expression and dyslocalization of claudin-19 are noticed, suggesting a possible correlation between claudin-19 and renal disorders. Taken together, claudin-19 is a claudin isoform that is highly and specifically expressed in renal tubules with a putative role in TJ homeostasis in renal physiology.

Osteoporosis is a complex disease with a strong genetic component, but the genes involved are poorly defined. To determine whether estrogen receptor beta (ESR2) gene is an osteoporosis risk gene, we examined its association with bone mineral density (BMD) and fracture risk. Using a gene-based approach, a set of 12 polymorphisms of ESR2 was studied in 752 case–control pairs of southern Chinese in ethnicity. Among all polymorphisms, the most significant relation with BMD and fracture risk was observed with T-1213C. Subjects with low BMD had a higher frequency of the variant C allele of T-1213C (cases 11.4%, control 8.4%, P = 0.02). The C allele was associated with 4% reduction in BMD at both the spine and hip in women, and 11% reduction in spine BMD and 9% reduction in hip BMD in men. Similar results were seen with SNP haplotype analysis. Subjects with the C allele of T-1213C were associated with higher risks of osteoporosis and BMD T scores ≤ −2.5 (odds ratios: 2.2 at spine and 3.5 at femoral neck for women; 3.5 at lumbar spine for men). Postmenopausal women carrying this C allele were associated with 2.22-fold increased risk of osteoporotic fractures (95% confidence interval 1.26–4.25) even after adjusting for BMD. In conclusion, ESR2 is involved in BMD determination in both sexes. The T-1213C polymorphism influences the risk of fracture in postmenopausal women independent of BMD.

Bone mineral density (BMD), an important risk factor for osteoporosis, is a complex trait likely affected by multiple genes. The linkage and/or association of 13 polymorphic loci of seven candidate genes (estrogen receptor alpha [ERalpha] and beta [ERbeta], calcium-sensing receptor, vitamin D receptor, collagen type 1alpha1, low-density lipoprotein [LDL] receptor-related protein 5 [LRPS], and transforming growth factor beta1) were evaluated in 177 southern Chinese pedigrees of 674 subjects, with each pedigree identified through a proband having a BMD Z score of -1.28 or less at the hip or spine. A suggestive linkage was detected between the IVS1-351A/G polymorphism of ERalpha and spine BMD, and between the 1082G/A, 1730G/A, and D14S1026 polymorphisms of ERbeta and BMD at both spine and hip. The quantitative transmission disequilibrium test (QTDT) detected total family association between 1730G/A of ERbeta and BMD at spine and hip; between D14S1026 of ERbeta and hip BMD; and between the 266A/G and 2220C/T polymorphisms of LRP5 and hip BMD. Similar total family associations were detected when only the females were analyzed. In addition, the IVS1-397T/C polymorphism of ERalpha was associated with spine BMD, and the 266A/G and 2220C/T polymorphisms of LRP5 were associated with femoral neck BMD in the females. A within-family association was detected with the IVS1-397T/C polymorphism of ERalpha, and the 266A/G and 2220C/T polymorphisms of LRP5 in the females. The effect of each polymorphism on BMD variance ranged from 1% to 4%. In conclusion, ERalpha, ERbeta and LRP5 are important candidate genes determining BMD variation, especially in females.

Background. Members of the genus Helicobacter have been associated with colitis development in a number of immunodeficient animal models. While it is known that these organisms can initiate colitis development, the location and spatial distribution of these bacteria within the intestinal tract is currently unknown. In this study, we developed and optimized fluorescence in situ hybridization (FISH) probes specifically for Helicobacter species. Materials and Methods. Based on 16S-RNA gene alignments, two probes specific for the entire family Helicobacteraceae and two probes specific for Helicobacter ganmani and Helicobacter hepaticus were designed. Evaluation of these probes was determined using ATCC reference strains and cecum samples from ten IL-10 knockout mice. The presence of Helicobacter species was determined using FISH and verified using PCR-DGGE and microscopic examination of silver stained sections. Results. Analysis of the ATCC reference strains revealed that the probes HEL274/HEL717 were specific for the family Helicobacteraceae, while HEP642 was specific for H. hepaticus and GAN1237 for H. ganmani. Using these probes, a pattern of spatial localization of the two different Helicobacter species was observed in the cecum tissues of IL-10 knockout mice. This consistently showed that H. ganmani was localized to the lower regions and H. hepaticus to the mid-upper regions of the crypts. Conclusion. We have developed FISH probes specific for the family Helicobacteraceae as well as two individual Helicobacter species. This study will allow the future use of the FISH to better understand host-pathogen interactions in vitro.

In Caucasians, maturity-onset diabetes of the young (MODY) is mostly caused by mutations in the hepatocyte nuclear factor (HNF)-1alpha (MODY3) and glucokinase (MODY2) genes. Most Japanese MODY patients, however, are not linked to known MODY genes. In this study, we examined the genetic and clinical characteristics of Chinese subjects with MODY. The study included 146 unrelated families fulfilling the minimum criteria for MODY: two consecutive generations of type II diabetes with at least one member diagnosed under the age of 25. We screened for mutations in the HNF-4alpha (MODY1), MODY2 and MODY3 genes by direct sequencing. Antibody to glutamic acid decarboxylase (GAD-Ab) was measured in subjects with MODY of unknown cause (MODYX). Insulin resistance index and other clinical data were compared in sex-, age- and duration-matched MODY3 and MODYX patients. In all, 13 families had MODY3 mutations and two had MODY2 mutations. No MODY1 mutation was found. Four of the 12 different MODY3 mutations were newly identified novel mutations (Q243E, A311D, P379R and P488fsdelC). In subjects with MODYX, 3% were GAD-Ab positive and 60% were overweight. Compared to MODY3 patients, MODYX patients had higher body mass index (P

Objectives To determine the cost effectiveness of a universal prenatal screening program for α- and β-thalassaemia. Methods We retrospectively reviewed our program from 1998 to 2002, and calculated the direct and indirect costs of various components. Results 18 936 women were screened at our prenatal clinic and 153 couples were subsequently referred to our Prenatal Diagnostic Centre for counselling and further investigations. In addition, there were 238 tertiary referrals and 157 self-referrals. After investigations, 84 fetuses were at risk of β-thalassaemia major/β-E thalassaemia, 19 of them were affected and 18 were aborted. The total expenditure on our program (HK$10.0 million) would be less than the postnatal service costs (HK$40.4 million) for 18β-thalassaemia major fetuses if they were born. Of 361 women at risk of carrying a homozygous α0-thalassaemia fetus, 311 (86.2%) opted for the indirect approach (using serial ultrasound examinations to exclude Hb Bart's disease), and 76 (24.5%) subsequently underwent an invasive test for a definitive diagnosis. The sensitivity and false positive rate of this indirect approach was 100.0% and 2.9% respectively. Conclusion It is cost effective to run a universal prenatal screening program in an area where both β-thalassaemia and α-thalassaemia are prevalent. The indirect approach can effectively avoid an invasive test in unaffected pregnancies. Copyright © 2004 John Wiley & Sons, Ltd.

A real time quantitative PCR (QPCR) method using TaqMan technology was used to assess the copy number of the two survival motor neuron genes (SMN1 and SMN2) on chromosome 5q13. This allows the accurate determination of carriers for spinal muscular atrophy (SMA), with one copy of SMN1. Analysis of 569 normal southern Chinese individuals revealed a carrier incidence of 1.6%, similar to that found in the western society. Study of 42 obligatory carriers showed a (2 + 0) genotype in two (4.8 %). In 27 SMA patients with homozygous deletion of the SMN1 gene, the number of SMN2 gene correlated with disease phenotype, with 68% of type II and III patients carrying three or more SMN2 genes, whilst the incidence of three or more SMN2 genes in the normal population was 1.57%.

Hb Lepore-Hollandia, identified in a Thai patient, was found to be due to a new cross-over between IVS1-nt 42 and nt 56 of the delta- and beta-globin genes. This differs from a previous case which crossed-over between codon 22 and IVS1-nt 16. Two independent homologous unequal cross-over events account for these two Lepore-Hollandia genes.

The alpha and beta thalassaemias are the commonest genetic disorders worldwide. The homozygous state is associated with high morbidity and mortality, thus screening of at-risk pregnancies and prenatal testing are strongly advocated. A thalassaemia (thal) array has been designed using allele-specific arrayed primer extension (AS-APEX) for the simultaneous analysis of 15 non-deletion alpha-gene defects and 23 beta-gene mutations commonly found in southeast Asian countries, where thalassaemias are highly prevalent. This overcomes the problem of using multiple reverse dot blot analysis. The array showed 100% sensitivity and specificity in the detection of 120 beta-thal mutants and 35 non-deletion alpha-thal mutants. It is robust enough to be produced in a single place and shipped to other laboratories for use. The production cost of the array is low, each slide can be used for three different test samples and is therefore amenable to large scale antenatal screening in southeast Asian countries.

Context.—Bioelectronic sensors, which combine microchip and biological components, are an emerging technology in clinical diagnostic testing. An electronic detection platform using DNA biochip technology (eSensor) is under development for molecular diagnostic applications. Owing to the novelty of these devices, demonstrations of their successful use in practical diagnostic applications are limited. Objective.—To assess the performance of the eSensor bioelectronic method in the validation of 6 Epstein-Barr virus–transformed blood lymphocyte cell lines with clinically important mutations for use as sources of genetic material for positive controls in clinical molecular genetic testing. Two cell lines carry mutations in the CFTR gene (cystic fibrosis), and 4 carry mutations in the HFE gene (hereditary hemochromatosis). Design.—Samples from each cell line were sent for genotype determination to 6 different molecular genetic testing facilities, including the laboratory developing the DNA biochips. In addition to the bioelectronic method, at least 3 different molecular diagnostic methods were used in the analysis of each cell line. Detailed data were collected from the DNA biochip output, and the genetic results were compared with those obtained using the more established methods. Results.—We report the successful use of 2 applications of the bioelectronic platform, one for detection of CFTR mutations and the other for detection of HFE mutations. In all cases, the results obtained with the DNA biochip were in concordance with those reported for the other methods. Electronic signal output from the DNA biochips clearly differentiated between mutated and wild-type alleles. This is the first report of the use of the cystic fibrosis detection platform. Conclusions.—Bioelectronic sensors for the detection of disease-causing mutations performed well when used in a “real-life” situation, in this case, a validation study of positive control blood lymphocyte cell lines with mutations of public health importance. This study illustrates the practical potential of emerging bioelectronic DNA detection technologies for use in current molecular diagnostic applications.

In this study, 24 family caregivers of terminally ill patients participated in in-depth interviews regarding their experiences of giving care. The data were analyzed using grounded theory qualitative method. Commitment emerged as the precondition of the caregiving process. The caregivers did not perceive the work of caring as a burden. Rather, they felt that despite any personal hardships, what they were doing was important to their loved ones and therefore meaningful to them as caregivers. The components of commitment can be described as relational commitment, the act of showing love, and determination. The process of caregiving includes four phases: 1) holding onto hope for a miracle, 2) taking care, 3) preparing for death, and 4) adjusting to another phase of life. A patient-caregiver relationship, Confucian concepts of yi (appropriateness or rightness), and filial duty are reflected in the process of caregiving. Consequences of the process include finding meaning in life and peace of heart and mind. The emotional aspect of the caregiving experience can be described as an intense emotional experience filled with feelings of hope and hopelessness, guilt, fear, and regret. As a result of the caregiving experience, most participants found they have had a change of worldviews and treasure their lives. Findings show a significant need for interventions at each phase of the caregiving process designed to provide effective and culturally sensitive support and affirmation to family members as they care for their loved ones with terminal illnesses.

Iron overload was found to be the major cause of disability in Chinese HbH disease patients although they were not on regular blood transfusion. The transferrin receptor 2 (TFR2) and hereditary hemochromatosis (HFE) genes were examined to see if inheritance of these gene defects may be a possible cause of iron overload in 45 HbH patients. A novel intronic (IVS6 (+6) T→A) mutation of the TFR2 gene was identified in one patient, and six others were found to carry a known missense mutation (exon 5, I238M) that was also present in one normal control subject. One HbH patient and one normal control carried the H63D mutation of the HFE gene. Since only eight out of 45 iron-overloaded HbH patients carry a defect in the TFR2 or HFE gene in the heterozygote state and their iron loading status was comparable to the matched controls without such defects, it would appear that the accumulation of excess iron in HbH disease is more likely a result of increase dietary absorption secondary to ineffective erythropoiesis.

Background We used human papillomaviruses (HPV) as a model system to evaluate the utility of a nucleic acid, hybridization-based bioelectronic DNA detection platform (eSensor™) in identifying multiple pathogens. Methods Two chips were spotted with capture probes consisting of DNA oligonucleotide sequences specific for HPV types. Electrically conductive signal probes were synthesized to be complementary to a distinct region of the amplified HPV target DNA. A portion of the HPV L1 region that was amplified by using consensus primers served as target DNA. The amplified target was mixed with a cocktail of signal probes and added to a cartridge containing a DNA chip to allow for hybridization with complementary capture probes. Results Two bioelectric chips were designed and successfully detected 86% of the HPV types contained in clinical samples. Conclusions This model system demonstrates the potential of the eSensor platform for rapid and integrated detection of multiple pathogens.

Introduction The hemoglobin molecule is a tetramer consisting of 2 pairs of globin chains, each of which contains a heme group. During fetal development, the major hemoglobin is Hb F (α2γ2). In a normal adult, the major hemoglobin is Hb A (α2β2). The α-globin gene cluster is located on

The purpose of this study is to provide an in-depth description of the nurse-facilitated empowering experiences of family caregivers of terminally ill patients in Hong Kong. Data were collected through in-depth interviews with 24 family caregivers of patients with terminal cancer. Grounded theory strategies served as the basis for data collection and analysis. Data were analysed in terms of the strategies, consequences, and interactions involved in the empowering experience. Themes included establishing an engaged relationship; providing information, knowledge and skills; affirming self-worth; and giving reassurance that the patient is under good care. The consequences of empowerment included a trusting relationship, having confidence to carry out caregiving tasks, acceptance of patient’S death, sustained self-worth and feeling peace at heart. The focus of empowering experiences was related to the quality of life of the dying patients. Family caregivers require information, education, encouragement and support, and nurses are in an ideal position to meet this need. Fundamental in the empowering process is the formation of a trusting relationship and as a result the caregivers feeling they are not being abandoned or left alone in the caring process. The results of this study can help nurses interact more effectively with family caregivers, giving them direction for effective nursing practice when caring for patients with a terminal illness.

Maturity-onset diabetes of the young is an autosomal dominant form of diabetes characterised by an early age of onset (usually25 years). We investigated the prevalence and trans-activating activity of hepatocyte nuclear factor (HNF) -1 alpha mutations in southern Chinese families with MODY.We screened for mutations in the HNF-1 alpha gene in 50 unrelated southern Chinese families, which fulfilled the minimum criteria for MODY. Functional properties of the mutant proteins were investigated using site-directed mutagenesis and luciferase reporter assay.Five of the 50 (10%) families were found to have mutations in the coding region, including a new nonsense mutation Q176X and four reported mutations (frameshift mutation P379fsdelCT, nonsense mutation R171X, missense mutations G20R and P112L). These mutations had decreased trans-activating activity on the human insulin gene promoter. We also detected a new intronic sequence variation IVS7nt-6 G--A, which co-segregated with diabetes. The intronic variation creates a potential splice acceptor site and might alter the splicing of the HNF-1 alpha mRNA.Mutations in the HNF-1 alpha gene seem to be an important cause of MODY in southern Chinese. The mutations could affect normal islet function by altering the expression of target genes.

To evaluate the clinical utility of magnetic resonance imaging (MRI) in screening for iron overload in non-transfusion dependent Haemoglobin (Hb) H disease.Thirty-six non-transfusion dependent HbH patients were evaluated with axial spin echo T1 and gradient echo T2 MRI of the abdomen and heart. The ratios of signal intensities (SIR) of the liver, spleen, pancreas and heart to paraspinous muscles were calculated. SIR1 was taken as indicative of iron overload. Qualitative grading (0-4 scale) of iron overload was also performed. The relationship between T1 and T2 SIR and serum ferritin, and that between qualitative grading and serum ferritin were examined using standard statistical methods. Comparisons were also made between qualitative grading and quantitative T1 and T2 SIR data in diagnosing iron overload. Six patients underwent liver biopsies.T2 SIR was more sensitive in detecting iron overload than T1 SIR. Thirty-three livers, 13 spleens, six pancreas and one heart were diagnosed as having iron overload with T2 SIR, including three patients with normal serum ferritin. A positive diagnosis by T2 SIR was more closely related to that of qualitative grading than T1 SIR. Serum ferritin was negatively correlated with hepatic SIR (T1 and T2), and with T2 SIR of the spleen and pancreas, even after adjustment for age. Liver haemosiderosis was confirmed in all six patients who underwent liver biopsies. Liver iron concentration of only one and a half times the normal was found in one patient with positive MR findings.MR is a non-invasive, effective method for early detection of iron overload particularly in the liver and spleen. Qualitative grading and quantitative T2 SIR data are equivalent in diagnosing iron overload. Routine screening of non-transfusion dependent HbH patients will identify high risk patients in whom early therapeutic intervention may prevent further complications and morbidity.

Essential thrombocythemia (ET) was diagnosed clinically in three patients. Karyotypic analysis and reverse transcription polymerase chain reaction for the bcr-abl chimeric transcript showed that two were Philadelphia chromosome (Ph) positive, bcr-abl positive, whereas the third was Ph negative, bcr-abl positive. The first patient received an allogeneic bone marrow transplantation but relapsed as localized blastic transformation, thus behaving similarly to chronic myeloid leukemia (CML). However, the other patients showed clinical courses more in keeping with ET. Essential thrombocythemia with BCR rearrangements may resemble CML but there are clinical differences. These may be due to genetic changes in addition to the BCR rearrangement.

Translocation (8;21)(q22;q22) involves fusion of the AML1 gene with the ETO gene, generating an AML1/ETO fusion transcript that can be detected by the polymerase chain reaction (PCR). Persistence of the AML1/ETO transcript has been demonstrated by PCR in patients with t(8;21) in long-term remission, but the rearranged AML1 gene could not be detected by Southern analysis, showing that the t(8;21) clone existed as minimal residual disease (MRD). In one patient with t(8;21), AML1/ETO could be detected serially in the peripheral blood. However, rearrangement of the AML1 gene was also found to persist. Furthermore, the hybridization intensities of the rearrangement bands showed that some of the mature myeloid cells also possessed the AML1 rearrangement. Thus, the presence of AML1/ETO in this case appeared to be due to persistence of the mutated clone as mature myeloid cells instead of MRD, implying that the t(8;21) had occurred in a preleukemic myeloid progenitor cell capable of differentiation.

41 unrelated southern Chinese haemophilia A patients were studied. The 5' promoter region, all 26 exons, their immediate 5' and 3' flanking splice junctions and the 3' untranslated region of the FVIII gene were amplified (including 16 different segments of exon 14) using GC-clamped primers. The GC-clamped PCR products were screened by denaturing gradient gel electrophoresis (DGGE) and fragments showing an abnormal migration pattern were sequenced. 17 mutations were identified, of which four were splicing defects, involving the first 1-6 nucleotide (nt) in the intervening sequences (IVS), six missense mutations, three nonsense mutations and four frameshift mutations. 14 other patients carried the type 1 inversion, affecting the distal copy of the FSA gene at the telomere of the X chromosome and the same gene in intron 22 of the FVIII gene. All the mothers studied (12/14) were carriers of the inversion. Two of these patients with inversion also have a co-existing missense mutation. In most cases the clinical severity of the disease corresponds to the genotype.

Summary . Two multi-allelic microsatellite polymorphisms within the factor VIII gene were studied in 138 Chinese subjects. The allele sizes detected were higher than those found in Caucasian populations, whereas the heterozygosity rates were lower, being 0.5370 for intron 13 and 0.4444 for intron 22 repeats respectively. Their usefulness in diagnosis was compared to other intragenic and extragenic RFLPs, using previous data on the same 31 unrelated haemophilia A families. These intragenic microsatellite repeat polymorphisms were only informative for 18/31 families (58%); however, with the combined use of all existing RFLPs, the cumulative informativeness would be 100%.

Summary. A patient with mild FIX deficiency was found to have partial duplication of the 3' region of the gene, giving, in addition to the a normal gene, another piece of DNA containing exons 5', 6', 7' and 8' and the intervening sequences. Cloning and sequencing of the junction region revealed that crossover occurred at nt 31927 in the 3° untranslated region of one chromosome/chromatid and nt 10640 in intron 4 of the other. No homology or topoisomerase specific sequences were observed in the crossover region. PCR and sequencing of illegitimate FIX transcripts from the patient's lymphocytes showed at least three different species of mRNAs. Translation of two of these 'novel'mRNAs should result in truncated proteins. Possibilities for the splicing of the mature mRNA are offered to explain the translation of a normal-size FIX protein, which was the only product demonstrated on Western blot analysis.

OBJECTIVE: To study the use of slot blot hybridization as a method of prenatal quantication of the number of X-chromosomes in chorionic villi samples. METHOD: DNA of chorionic villi from fetuses with karyotypes of 46, XY; 45,X0; 47,XXX; 69,XXX and 48,XXXX were extracted, slot blotted and hybridized to the following radioactive probes β 0.9 , FVIII, pY 3.4 and pBLUR. DNA of chorionic villi from five other fetuses with unknown karytypes were similarly blotted and hybridized. Autoradiography with pre-exposed films was carried out and the density of each of the hybidized bands was scanned by a laser densitomer. RESULTS: It was found that with increasing amounts of DNA in the samples, the intensity of the bands hybridized with FVIII and β 0.9 probes increased proportionately. However, the intensity of the bands obtained with the probes pY 3.4 and pBLUR (probes containing multiple repeat sequences) varied little with increasing DNA concentrations. The ratios of radioactivity obtained for the two probes FVIII/β 0.9 , were well correlated with the number of X-chromosomes in the samples. Calculations of the FVIII/β 0.9 ratio for the five samples with the unknown karyotypes gave a correct prediction of the number of X-chromosomes in each case. CONCLUSION: Slot blot hybridization can be used as a method of prenatal quantitation of the number of X-chromosomes in chorionic villi samples. The method could be useful for rapid prenatal diagnosis of other numerical chromosomal abnormalities.

Abstract 1013 More than 3% of Chinese in Hong Kong are heterozygous carriers of β-thalassemia. Homozygotes or compound heterozygotes for β-thalassemia are usually severely ill and require monthly transfusions. Increased production of fetal hemoglobin (HbF) can modulate the disease severity by compensating for the shortfall of HbA caused by the β-thalassemia mutations. HbF level in adults varies and is regulated as a multigenic trait. Three major HbF quantitative trait loci (QTL) have been identified: the C/T SNP also known as the Xmn I site at the Gγ-globin gene promoter, the BCL11A polymorphism on chromosome 2p16, and the HBS1L-MYB intergenic polymorphism (HMIP) on chromosome 6q23. The functional motif for each of these 3 QTLs responsible for their effects upon HbF is not known. We undertook a genome-wide association study (GWAS), using Illumina Human 610-Quad BeadChip array, on 619 Chinese β-thalassemia heterozygotes from Hong Kong. In this population, the variance in HbF due to HMIP is 13.5%, significantly higher than that due to BCL11A polymorphism (6.4%). We used 1,000 Genomes Project data, SNP imputation, comparisons of association results across populations, predicted binding of transcription factors, and phylogenetic conservation to identify the functional variant in HMIP. Based on these lines of evidence, a hitherto unreported association between HbF expression and a 3-bp deletion on chromosome 6q23 was found. In 335 Chinese β-thalassemia heterozygotes, the 3-bp deletion polymorphism is in complete linkage disequilibrium with rs9399137, the SNP found in multiple GWAS to be most significantly associated with HbF (P=1.4E-24 in the Chinese cohort GWAS). Flanking this deletion are conserved binding sites for TAL1/SCL1, E47, GATA, and RUNX1/AML1, which are essential erythropoiesis-related transcription factors. The 3-bp deletion changes the normal DNA binding configuration of these transcription factors and spatial configuration for DNA-protein binding and/or protein-protein interactions. Furthermore, this 3-bp deletion polymorphism resides within a likely erythroid distal regulatory region manifested by DNase I hypersensitivity and GATA-1 binding (Wahlberg et al, Blood 114:1254, 2009). We hypothesized that a 61-bp fragment of DNA that encompasses the site of the 3-bp deletion polymorphism might have enhancer-like activity. When ligated to the Gγ-globin gene 1.4 kb proximal promoter linked to a luciferase reporter gene, the 61-bp fragment of DNA enhances the Gγ-globin gene promoter activity by more than 3-fold after transient transfection into K562 cells. A 58-bp fragment of DNA that includes the 3-bp deletion has 60% more enhancer-like activity than the 61-bp fragment without the deletion. These findings suggest that this 3-bp deletion polymorphism is most likely the functional motif accounting for HMIP modulation of HbF. Further studies are needed to identify target genes for this enhancer-like activity mediated by the DNA sequences encompassing the 3-bp deletion polymorphism in HMIP. These studies also suggest that this experimental approach could be used to identify functional motifs in other genotype-phenotype association studies. Disclosures: No relevant conflicts of interest to declare.

Polymerase chain reaction (PCR) was used to amplify the DNA fragments of the complementarity determining region 3 of the immunoglobulin (Ig) gene heavy chain from the leukemic cell specimens of patients with acute and chronic lymphoid leukemias of B-cell lineage. Two different pairs of primers were tested. Fourteen of the 17 (82%) cases of acute lymphoblastic leukemia (ALL), and all 15 cases (100%) of B-cell chronic lymphocytic leukemia, who had rearrangement of the Ig gene heavy chain by Southern analysis, were positive by PCR with either one or both pairs of primers. This technique was able to detect leukemic cells at the level of 0.1%. Applying it to study the remission marrow specimens following induction chemotherapy was more useful than morphology alone in predicting early relapse of the leukemia.

The pattern of immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements was determined in 87 patients with acute and chronic leukaemias and myelodysplastic syndromes by Southern blot hybridisation. All 31 cases of common, B cell and null cell acute lymphoblastic leukaemia, and B cell chronic lymphocytic leukaemia showed Ig heavy chain (JH) rearrangement, and TCR (beta-chain) rearrangement was seen in all 5 cases of T cell acute lymphoblastic leukaemia. Inappropriate JH and TCR (beta) rearrangements were present in some cases of T-ALL (60%) and common acute lymphoblastic leukaemia (18%), respectively. For the 19 patients with acute leukaemias following chronic myeloid leukaemia, blastic transformation, all 4 with lymphoid transformation and 3 of the 15 with myeloid transformation had JH rearrangement, and 3 CD10-positive lymphoid transformation and 2 myeloid transformation had their TCR (beta) genes rearranged. In conclusion, the pattern of Ig and TCR gene rearrangements correlated well with the cell lineage. However, cross-lineage rearrangements were more commonly seen in patients with acute leukaemias following chronic myeloid leukaemia blastic transformation, as compared to the de novo cases.

Methods and strategies are described for the prenatal diagnosis of three common congenital disorders--alpha and beta thalassemias and hemophilia A. It is important to study the frequency of restriction fragment length polymorphisms and types of molecular defects in specific ethnic groups, since they are known to differ significantly. The studies reported here were carried out in southern Chinese who form the majority of the Hong Kong population. The information obtained is also useful for prenatal diagnosis in southern Chinese migrant populations in Canada, USA, Australia, UK and southeast Asia.

The theory of 'homing' mechanism has been proposed to explain the association of malignant lymphoma involving multiple sites of mucosa-associated lymphoid tissue (MALT). DNA rearrangement in the peripheral blood and bone marrow specimens of 20 patients with MALT lymphoma were studied utilizing JH and C beta gene probes, aiming to detect circulating lymphoma cells and unrecognized bone marrow involvement. Our search for malignant cells in the peripheral blood was unrewarding and the 'homing' theory remained unproven. However, the study of DNA rearrangement showed to be useful in determining the malignant nature of abnormal lymphoid aggregates found in the bone marrow of these patients.