The cell cycle checkpoint gene Rad9 is a novel oncogene activated by 11q13 amplification and DNA methylation in breast cancer.

Human Rad9 (hRad9), a structural homologue of yeast Schizosaccharomyces pombe rad9, is involved in cell cycle checkpoints and apoptosis. hRad9 can serve as a corepressor of androgen receptor in prostate cancer cells, but little is known about its role in the development of breast or other cancers. In the present study, semiquantitative reverse transcription-PCR showed that Rad9 mRNA levels were up-regulated in 52.1% (25 of 48) of breast tumors, and this up-regulation correlated with tumor size (P = 0.037) and local recurrence (P = 0.033). Overexpression of Rad9 mRNA was partly due to an increase in Rad9 gene number as measured by quantitative PCR. In other breast tumors with Rad9 mRNA overexpression but without increase in gene number, there was differential methylation of two putative Sp1/3 binding sites within the first and second introns of the Rad9 gene, which was similarly found in MCF-7 breast cancer cell line with increased Rad9 mRNA. Silencing Rad9 expression by RNA interference in MCF-7 cell line inhibited its proliferation in vitro. Promoter assays indicated that the Sp1/3 site in intron 2 may act as a silencer. In vivo binding of Sp3 to intron 2 was shown by chromatin immunoprecipitation assays. Treatment of MCF-7 cell line with 5'-aza-2'-deoxycytidine reduced Rad9 mRNA expression and also increased binding of Sp3 to the demethylated intron 2 region. Collectively, these findings suggest that Rad9 is a novel oncogene candidate activated by 11q13 amplification and DNA hypermethylation in breast cancer and may play a role in tumor proliferation and local invasion.

Detection of known haemophilia B mutations and carrier testing by microarray.

The 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 (AS-APEX) 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.

A new cross-over region for hemogloboin-Lepore-Hollandia.

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.

Carrier incidence for spinal muscular atrophy in southern Chinese.

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%.

EGFR array: uses in the detection of plasma EGFR mutations in non-small cell lung cancer patients.

INTRODUCTION: We aim to develop a simple and sensitive array-based method for the detection of epidermal growth factor receptor (EGFR) gene mutations in the plasma of non-small-cell lung cancer patients and determine its use in the follow-up of those on tyrosine-kinase inhibitor (TKI) therapy. METHOD: DNA from 100 µl of plasma was amplified in the presence of peptide nucleic acid clamp to provide single-stranded template for the allele-specific arrayed primer extension reaction, incorporating cyanine-5-deoxycytidine triphosphate in the newly synthesized strands. The fluorescent product was visualized by laser at 670 nm. RESULTS: Eleven different types of EGFR TKI drug-sensitive mutants (SM) were identified in plasma-DNA from 46 of 51 patients. Five patients carried only wild-type sequence. Plasma-DNA finding was concordant in 36 of 37 cases with tumor-sequencing data. This method could detect as little as 62.5 copies of mutant L858R; 125 copies of E709K + G719A or 625 copies of del 746-750 in the presence of 100,000 copies of wild-type EGFR. In 21 patients on longitudinal follow-up for up to 18 months, SM was found in all initial plasma samples, except for three samples collected after recent chemotherapy. Nine of 16 patients (56%) who responded to TKI had undetectable plasma EGFR mutant. SM was present concurrently with drug-resistant mutant in 44% of patients with disease progression while on TKI, the remaining 56% might have other mechanisms of resistance. CONCLUSION: The EGFR array provides a sensitive, inexpensive, and robust method for monitoring non-small-cell lung cancer patients' response to TKI, and obviates the need of repeated lung biopsy.

A comprehensive HBV array for the detection of HBV mutants and genotype.

OBJECTIVE: To develop a comprehensive hepatitis B virus (HBV) array providing simultaneous analysis of 8 genotypes, 47 mutations of reverse-transcriptase polymerase gene and 18 mutations of S gene. METHOD: Oligonucleotides corresponding to various HBV-normal and -mutant sequences were spotted onto pre-treated glass slides. Single-stranded templates of the HBV gene fragment were prepared from serum-DNA of HBV-infected patients by 2-staged PCR and subjected to allele-specific arrayed-primer extension with Cy5-dCTP. Fluorescein-labelled products were scanned at 670nm. RESULTS: Comparative analysis of 100 unrelated samples using the array and a commercial kit, revealed 44 with additional mutations from the array, these were confirmed by sequencing. Analysis of 381 samples from 45 patients during 1-3 years of anti-viral therapy showed improved sensitivity with detection of drug-resistant mutations months before clinical relapse. The lower detection limit was 28 copies/mL. CONCLUSION: The array is better than many existing methods as it provides both mutations and genotype data in a single analysis.

A complex MLL rearrangement identified five years after initial MDS diagnosis results in out-of-frame fusions without progression to acute leukemia.

Chromosomal rearrangements of the MLL gene are uncommon in myelodysplastic syndromes (MDSs), and few studies of their molecular structures and oncogenic mechanisms exist. Here, we present a case of de novo MDS with a normal karyotype at initial diagnosis and a mild clinical course. Five years after the initial diagnosis, investigators identified a complex rearrangement of the MLL gene without progression to acute leukemia. The 5' part of the MLL gene is fused out of frame with the LOC100131626 gene, and the 3' part of the MLL gene out of frame with the TCF12 gene. Rapid amplification of complementary DNA 3' ends yielded two main fusion transcripts, which is in concordance with the two described isoforms of the LOC100131626 gene. For both isoform-fusion transcripts, the open reading frame terminates shortly after the breakpoint that is predicted to form two de facto truncated MLL proteins and disrupts the open reading frame of the LOC100131626, TCF12, and UBE4A genes. Neither dimerization nor a transcriptional activation domain, each of which is causally linked to MLL protein-mediated transformation, is present. This and other unusual MLL rearrangements probably represent a subclass of MLL gene abnormalities that have intrinsically no ability or only a weak ability to transform hematopoeitic cells and are identified only in the context of other hematopoetic malignancies.

Use of the oral chelator deferiprone in the treatment of iron overload in patients with Hb H disease.

Seventeen non-transfusion-dependent Chinese haemoglobin H (Hb H) disease patients (age 29-76 years) with serum ferritin >900 microg/l were treated with deferiprone for up to 18 months. One patient withdrew and data from 16 patients were analysed. Sixteen other Hb H patients with ferritin <900 microg/l, matched for age and genotype, acted as controls. Treatment was well tolerated except for mild arthralgia. Serum ferritin fell with treatment, reaching significance at 6 and 18 months (from 1492.3 +/- 901.4 to 519.4 +/- 405.4 microg/l at 18 months, P = 0.0008). Nine of 16 patients had levels below 397 microg/l before 18 months. Serum ferritin remained stable 6 months after stopping treatment. In contrast, there was no change in ferritin levels in the control group. Magnetic resonance imaging was used for measurement of liver iron content. Spin echo T(1)-signal intensity ratio (T(1)-SIR) and gradient echo T(2)-signal intensity ratio (T(2)-SIR) increased with treatment. T(2)-SIR rose from 0.17 +/- 0.08 pretreatment to 0.58 +/- 0.50 at 2 years (P = 0.0055). Improvement occurred in 12 of 16 patients, reaching normal in three patients. Using echocardiography, peak early diastolic : late diastolic blood flow (E/A) remained unchanged with treatment, but isovolumic relaxation time (IVRT) was prolonged at 2 years indicating mild impairment of diastolic function. All systolic function parameters were normal. A longer treatment period is desirable to demonstrate improvement in cardiac function.

A genetic locus for adolescent idiopathic scoliosis linked to chromosome 19p13.3.

Adolescent idiopathic scoliosis (AIS) is one of the most common orthopedic disorders, affecting up to 4% of schoolchildren worldwide. We studied seven unrelated multiplex families of southern Chinese descent with AIS, consisting of 25 affected members. A genomewide scan with >400 fluorescent microsatellite markers was performed. Multipoint linkage analysis by GENEHUNTER revealed significant linkage of the abnormal phenotype to the distal short arm of chromosome 19, with both a maximum multipoint LOD score and a nonparametric LOD score of 4.93. Two-point linkage analysis by MLINK gave a LOD score of 3.63 (recombination fraction theta[m=f]=0.00) at D19S216. Further high-density mapping and informative recombinations defined the AIS critical region in the vicinity of D19S216, flanked by D19S894 and D19S1034, spanning 5.2 cM on the sex-averaged genetic map on chromosome 19p13.3.