The mutational oncoprint of recurrent cytogenetic abnormalities in adult patients with de novo acute myeloid leukemia.

Recurrent chromosomal abnormalities and gene mutations detected at the time of diagnosis of acute myeloid leukemia (AML) are associated with particular disease features, treatment response and survival of AML patients, and are used to denote specific disease entities in the World Health Organization classification of myeloid neoplasms and acute leukemia. However, large studies that integrate cytogenetic and comprehensive mutational information are scarce. We created a comprehensive oncoprint of mutations associated with recurrent cytogenetic findings by combining the information on mutational patterns of 80 cancer- and leukemia-associated genes with cytogenetic findings in 1603 adult patients with de novo AML. We show unique differences in the mutational profiles among major cytogenetic subsets, identify novel associations between recurrent cytogenetic abnormalities and both specific gene mutations and gene functional groups, and reveal differences in cytogenetic and mutational features between patients younger than 60 years and those aged 60 years or older. The identified associations between cytogenetic and molecular genetic data may help guide mutation testing in AML, and result in more focused application of targeted therapy in patients with de novo AML.

Mutations in the CCND1 and CCND2 genes are frequent events in adult patients with t(8;21)(q22;q22) acute myeloid leukemia.

Core-binding factor acute myeloid leukemia (CBF-AML) is defined by the presence of either t(8;21)(q22;q22)/RUNX1-RUNX1T1 or inv(16)(p13.1q22)/t(16;16)(p13.1;q22)/CBFB-MYH11. The resulting fusion genes require a 'second hit' to initiate leukemogenesis. Mutation assessment of 177 adults with CBF-AML, including 68 with t(8;21) and 109 with inv(16)/t(16;16), identified not only mutations well known in CBF-AML but also mutations in the CCND1 and CCND2 genes, which represent novel frequent molecular alterations in AML with t(8;21). Altogether, CCND1 (n=2) and CCND2 (n=8) mutations were detected in 10 (15%) patients with t(8;21) in our cohort. A single CCND2 mutation was also found in 1 (0.9%) patient with inv(16). In contrast, CCND1 and CCND2 mutations were detected in only 11 (0.77%) of 1426 non-CBF-AML patients. All CCND2 mutations cluster around the highly conserved amino-acid residue threonine 280 (Thr280). We show that Thr280Ala-mutated CCND2 leads to increased phosphorylation of the retinoblastoma protein, thereby causing significant cell cycle changes and increased proliferation of AML cell lines. The identification of CCND1 and CCND2 mutations as frequent mutational events in t(8;21) AML may provide further justification for cell cycle-directed therapy in this disease.

MicroRNA-related sequence variations in human cancers.

MicroRNAs are emerging as a most promising field in basic and translational research, explaining the pathogenesis of numerous human diseases and providing excellent tools for their management. This review considers the effects of microRNA sequence variations and their implication in pathogenesis and predisposition to human cancers. Although the role of microRNAs still remains to be elucidated, functional, and populational studies indicate that microRNA variants are important factors underlying the process of carcinogenesis. Further understanding of the cellular and molecular basis of microRNA action will lead to the identification of their new target genes and microRNA-regulated pathways. As a consequence, novel models of cancer pathogenesis can be proposed, and serve as a basis for elucidation of new prognostic and diagnostic tools for human cancers.

Recurrent and founder mutations in the PMS2 gene.

Germline mutations in PMS2 are associated with Lynch syndrome (LS), the most common known cause of hereditary colorectal cancer. Mutation detection in PMS2 has been difficult due to the presence of several pseudogenes, but a custom-designed long-range PCR strategy now allows adequate mutation detection. Many mutations are unique. However, some mutations are observed repeatedly across individuals not known to be related due to the mutation being either recurrent, arising multiple times de novo at hot spots for mutations, or of founder origin, having occurred once in an ancestor. Previously, we observed 36 distinct mutations in a sample of 61 independently ascertained Caucasian probands of mixed European background with PMS2 mutations. Eleven of these mutations were detected in more than one individual not known to be related and of these, six were detected more than twice. These six mutations accounted for 31 (51%) ostensibly unrelated probands. Here, we performed genotyping and haplotype analysis in four mutations observed in multiple probands and found two (c.137G>T and exon 10 deletion) to be founder mutations and one (c.903G>T) a probable founder. One (c.1A>G) could not be evaluated for founder mutation status. We discuss possible explanations for the frequent occurrence of founder mutations in PMS2.

Microcephalic osteodysplastic primordial dwarfism type I with biallelic mutations in the RNU4ATAC gene.

Microcephalic osteodysplastic primordial dwarfism type I (MOPD I) is a rare autosomal recessive developmental disorder characterized by extreme intrauterine growth retardation, severe microcephaly, central nervous system abnormalities, dysmorphic facial features, skin abnormalities, skeletal changes, limb deformations, and early death. Recently, mutations in the RNU4ATAC gene, which encodes U4atac, a small nuclear RNA that is a crucial component of the minor spliceosome, were found to cause MOPD I. MOPD I is the first disease known to be associated with a defect in small nuclear RNAs. We describe here the clinical and molecular data for 17 cases of MOPD I, including 15 previously unreported cases, all carrying biallelic mutations in the RNU4ATAC gene.

The genetic basis of colorectal cancer in a population-based incident cohort with a high rate of familial disease.

BACKGROUND AND AIMS: Colorectal cancer (CRC) is the second most frequent cancer in developed countries. Newfoundland has the highest incidence of CRC in Canada and the highest rate of familial CRC yet reported in the world. To determine the impact of mutations in known CRC susceptibility genes and the contribution of the known pathways to the development of hereditary CRC, an incident cohort of 750 patients with CRC (708 different families) from the Newfoundland population was studied. METHODS: Microsatellite instability (MSI) testing was performed on tumours, together with immunohistochemistry analysis for mismatch repair (MMR) genes. Where indicated, DNA sequencing and multiplex ligation-dependent probe amplifications of MMR genes and APC was undertaken. DNA from all patients was screened for MUTYH mutations. The presence of the BRAF variant, p.V600E, and of MLH1 promoter methylation was also tested in tumours. RESULTS: 4.6% of patients fulfilled the Amsterdam criteria (AC), and an additional 44.6% fulfilled the revised Bethesda criteria. MSI-high (MSI-H) was observed in 10.7% (n=78) of 732 tumours. In 3.6% (n=27) of patients, CRC was attributed to 12 different inherited mutations in six known CRC-related genes associated with chromosomal instability or MSI pathways. Seven patients (0.9%) carried a mutation in APC or biallelic mutations in MUTYH. Of 20 patients (2.7%) with mutations in MMR genes, 14 (70%) had one of two MSH2 founder mutations. 17 of 28 (61%) AC families did not have a genetic cause identified, of which 15 kindreds fulfilled the criteria for familial CRC type X (FCCTX). CONCLUSIONS: Founder mutations accounted for only 2.1% of cases and this was insufficient to explain the high rate of familial CRC. Many of the families classified as FCCTX may have highly penetrant mutations segregating in a Mendelian-like manner. These families will be important for identifying additional CRC susceptibility loci.

Genetic predisposition to human disease: allele-specific expression and low-penetrance regulatory loci.

The two alleles of a gene can be expressed at different levels, the extreme example being imprinting, a condition in which one allele is totally suppressed. Recently, subtle differences in the expression of the two alleles have been detected in numerous human genes and in a few cases, have been associated with a genetic predisposition to disease. The underlying mechanisms are largely unexplored.

A novel mutation in the sulfate transporter gene SLC26A2 (DTDST) specific to the Finnish population causes de la Chapelle dysplasia.

BACKGROUND: Mutations in the sulfate transporter gene SLC26A2 (DTDST) cause a continuum of skeletal dysplasia phenotypes that includes achondrogenesis type 1B (ACG1B), atelosteogenesis type 2 (AO2), diastrophic dysplasia (DTD), and recessive multiple epiphyseal dysplasia (rMED). In 1972, de la Chapelle et al reported two siblings with a lethal skeletal dysplasia, which was denoted "neonatal osseous dysplasia" and "de la Chapelle dysplasia" (DLCD). It was suggested that DLCD might be part of the SLC26A2 spectrum of phenotypes, both because of the Finnish origin of the original family and of radiographic similarities to ACG1B and AO2. OBJECTIVE: To test the hypothesis whether SLC26A2 mutations are responsible for DLCD. METHODS: We studied the DNA from the original DLCD family and from seven Finnish DTD patients in whom we had identified only one copy of IVS1+2T>C, the common Finnish mutation. A novel SLC26A2 mutation was found in all subjects, inserted by site-directed mutagenesis in a vector harbouring the SLC26A2 cDNA, and expressed in sulfate transport deficient Chinese hamster ovary (CHO) cells to measure sulfate uptake activity. RESULTS: We identified a hitherto undescribed SLC26A2 mutation, T512K, homozygous in the affected subjects and heterozygous in both parents and in the unaffected sister. T512K was then identified as second pathogenic allele in the seven Finnish DTD subjects. Expression studies confirmed pathogenicity. CONCLUSIONS: DLCD is indeed allelic to the other SLC26A2 disorders. T512K is a second rare "Finnish" mutation that results in DLCD at homozygosity and in DTD when compounded with the milder, common Finnish mutation.

Amnionless (AMN) mutations in Imerslund-Gräsbeck syndrome may be associated with disturbed vitamin B12 transport into the CNS.

Familial selective vitamin B12 (cobalamin, Cbl) malabsorption (Imerslund-Gräsbeck syndrome, IGS, OMIM 261100) is a group of autosomal recessive disorders characterized by selective malabsorption of Cbl from the terminal ileum in the presence of normal histology. Mutations in the amnionless (AMN) and cubilin (CUBN) genes are known to be causes of IGS. Their gene products combine to form a receptor complex (cubam), which is instrumental in the binding and transport of Cbl in the gut. As opposed to Cbl transport in the terminal ileum, normal transport of Cbl into the CNS is poorly understood and little is known regarding its molecular basis. Studies in adults with neuropsychiatric disease have suggested the presence of an active transport mechanism into the central nervous system constituting a blood-brain barrier (BBB) for Cbl. A child with IGS, compound heterozygous for a missense and a nonsense mutation in the amnionless (AMN) protein gene, was noted to have a high daily cobalamin (Cbl) requirement for neuropsychiatric, but not for systemic metabolic and haematological, remission. Measurements of CSF Cbl revealed evidence that the transport of Cbl into the central nervous system was impaired, and a standard Schilling test was consistent with a dose response of cobalamin transport across the terminal ileum. Amnionless protein is known to be expressed in the fetal and postnatal central nervous system, and is known to be involved in Cbl transport in other tissues such as kidney as well as the gut. It is possible that an active Cbl transport mechanism at the BBB exists, and that the amnionless (AMN) protein may be part of this mechanism, as it is in cobalamin transport in the terminal ileum.

A frame-shift mutation of PMS2 is a widespread cause of Lynch syndrome.

BACKGROUND: When compared to the other mismatch repair genes involved in Lynch syndrome, the identification of mutations within PMS2 has been limited (<2% of all identified mutations), yet the immunohistochemical analysis of tumour samples indicates that approximately 5% of Lynch syndrome cases are caused by PMS2. This disparity is primarily due to complications in the study of this gene caused by interference from pseudogene sequences. METHODS: Using a recently developed method for detecting PMS2 specific mutations, we have screened 99 patients who are likely candidates for PMS2 mutations based on immunohistochemical analysis. RESULTS: We have identified a frequently occurring frame-shift mutation (c.736_741del6ins11) in 12 ostensibly unrelated Lynch syndrome patients (20% of patients we have identified with a deleterious mutation in PMS2, n = 61). These individuals all display the rare allele (population frequency <0.05) at a single nucleotide polymorphism (SNP) in exon 11, and have been shown to possess a short common haplotype, allowing us to calculate that the mutation arose around 1625 years ago (65 generations; 95% confidence interval 22 to 120). CONCLUSION: Ancestral analysis indicates that this mutation is enriched in individuals with British and Swedish ancestry. We estimate that there are >10 000 carriers of this mutation in the USA alone. The identification of both the mutation and the common haplotype in one Swedish control sample (n = 225), along with evidence that Lynch syndrome associated cancers are rarer than expected in the probands' families, would suggest that this is a prevalent mutation with reduced penetrance.

Impact of walking epidural analgesia on obstetric outcome of nulliparous women in spontaneous labour.

BACKGROUND: To explore the effects of walking epidural analgesia on obstetric and neonatal outcomes, we performed a case-control study. METHOD: Each nulliparous woman receiving walking epidural analgesia using 0.0625% bupivacaine (n = 44) was matched to two nulliparous historical controls receiving 0.125% or 0.25% bupivacaine (n = 88 each) for epidural analgesia while recumbent. RESULTS: Maternal and obstetric parameters, fetal status and presentation, and oxytocin use were comparable among groups. Those receiving walking epidural analgesia walked for a mean of 60 min (range: 20-75 min). In the control groups the mean total durations of labour were shorter (58 min in the 0.125% group and 99 min in the 0.25% group, P < 0.05). Significantly fewer walking epidural analgesia cases than controls required instrumental vaginal delivery (P < 0.05). No other differences in obstetric or fetal outcome were observed and no mother fell or stumbled while walking. CONCLUSION: Although it was associated with a prolonged first stage of labour, walking epidural analgesia appeared safe for nulliparous women and their babies.

MSH6 missense mutations are often associated with no or low cancer susceptibility.

Mismatch repair (MMR) deficiency in tumours from patients with the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome is mainly caused by mutations in the MLH1, MSH2, and MSH6 genes. A major challenge in the clinical management of patients with suspected HNPCC is the frequent occurrence of missense mutations in MSH6. These can be considered neither deleterious nor clinically innocent a priori. To assess their significance we studied five novel MSH6 missense mutations in six patients derived from a series of consecutive endometrial and colorectal cancer patients selected for study after their tumours were determined to be microsatellite unstable. We tested each mutated protein for heterodimerisation with MSH2 and for in vitro MMR capability. Four mutations (R128L, P623L, K728T, G881K+S) showed no impairment of these functions while the fifth (E1193K) displayed marked impairment of both functions. These results, taken together with our previous similar findings concerning six other missense mutations in MSH6, allow us to conclude that many or most missense changes in MSH6 likely are clinically innocent, whereas some missense changes such as E1193K, which lead to impaired MMR, are likely to be clinically significant, but have low penetrance.

A diagnostic biochip for the comprehensive analysis of MLL translocations in acute leukemia.

Reciprocal rearrangements of the MLL gene are among the most common chromosomal abnormalities in both Acute Lymphoblastic and Myeloid Leukemia. The MLL gene, located on the 11q23 chromosomal band, is involved in more than 40 recurrent translocations. In the present study, we describe the development and validation of a biochip-based assay designed to provide a comprehensive molecular analysis of MLL rearrangements when used in a standard clinical pathology laboratory. A retrospective blind study was run with cell lines (n=5), and MLL positive and negative patient samples (n=31), to evaluate assay performance. The limits of detection determined on cell line data were 10(-1), and the precision studies yielded 100% repeatability and 98% reproducibility. The study shows that the device can detect frequent (AF4, AF6, AF10, ELL or ENL) as well as rare partner genes (AF17, MSF). The identified fusion transcripts can then be used as molecular phenotypic markers of disease for the precise evaluation of minimal residual disease by RQ-PCR. This biochip-based molecular diagnostic tool allows, in a single experiment, rapid and accurate identification of MLL gene rearrangements among 32 different fusion gene (FG) partners, precise breakpoint positioning and comprehensive screening of all currently characterized MLL FGs.

Hashimoto's thyroiditis with papillary thyroid carcinoma (PTC)-like nuclear alterations express molecular markers of PTC.

AIMS: Focal papillary thyroid carcinoma (PTC)-like nuclear alterations have been documented in Hashimoto's thyroiditis; however, the molecular association between PTC and Hashimoto's thyroiditis is poorly understood. The aim of this study was to determine whether molecular expression patterns of PTC are present in association with PTC-like nuclear alterations in Hashimoto's thyroiditis. METHODS AND RESULTS: The expression of four genes known to be up-regulated in PTC [LGALS3 (galectin3), CITED1, KRT19 (cytokeratin 19) and FN1 (fibronectin-1)] and the human mesothelial cell protein identified by monoclonal antibody HBME1 was evaluated. Immunohistochemistry was performed on 23 cases of Hashimoto's thyroiditis with focal or diffuse Hürthle cell change and PTC-like nuclear alterations, 37 PTC and 18 normal thyroids. Focal expression of galectin3 (GAL3), CITED1, cytokeratin 19 (CK19), HBME1 and fibronectin-1 (FN1) was seen in 87%, 65%, 43%, 26% and 17% of Hashimoto's thyroiditis, respectively, only in thyrocytes showing PTC-like nuclear alterations. In contrast, diffuse expression of GAL3, CITED1, CK19, HBME1 and FN1 was seen in 100%, 95%, 70%, 87% and 89% of PTC, respectively. Normal thyroid tissues did not express any of these proteins. Following immunohistochemistry, four Hashimoto's thyroiditis cases were found to contain foci of PTC. These foci were highlighted by the diffuse and strong expression of PTC-associated proteins, which prompted additional retrospective scrutiny of the haematoxylin and eosin-stained sections leading to appreciation of complete PTC-type nuclear atypia. CONCLUSIONS: Focal PTC-like immunophenotypic changes in Hashimoto's thyroiditis suggest the possibility of early, focal premalignant transformation in some cases of Hashimoto's thyroiditis.

Identification of a deletion in the mismatch repair gene, MSH2, using mouse-human cell hybrids monosomal for chromosome 2.

Hereditary non-polyposis colorectal cancer is characterized by mutations in one of the DNA mismatch repair genes, primarily MLH1, MSH2, or MSH6. We report here the identification of a genomic deletion of approximately 11.4 kb encompassing the first two exons of the MSH2 gene in two generations of an Ohio family. By Southern blot analysis, using a cDNA probe spanning the first seven exons of MSH2, an alteration in each of three different enzyme digests (including a unique 13-kb band on HindIII digests) was observed, which suggested the presence of a large alteration in the 5' region of this gene. Mouse-human cell hybrids from a mutation carrier were then generated which contained a single copy each of human chromosome 2 on which the MSH2 gene resides. Southern blots on DNA from the cell hybrids demonstrated the same, unique 13-kb band from one MSH2 allele, as seen in the diploid DNA. DNA from this same monosomal cell hybrid failed to amplify in polymerase chain reactions (PCRs) using primers to exons 1 and 2, demonstrating the deletion of these sequences in one MSH2 allele, and the breakpoints involving Alu repeats were identified by PCR amplification and sequence analysis.

Clonidine premedication improves metabolic control in type 2 diabetic patients during ophthalmic surgery.

BACKGROUND: In stressful conditions, increasing blood glucose concentrations are closely related to an increase in catecholamines and cortisol release. Clonidine, a centrally acting alpha(2)-adrenoceptor agonist, has neuroendocrine effects, including inhibition of sympathoadrenal activity. We therefore evaluated the effect of clonidine on blood glucose control and insulin requirements during ophthalmic surgery when given as premedication in type 2 diabetic patients. METHODS: After randomization, patients were premedicated with clonidine or flunitrazepam (control). Patients were given insulin by continuous i.v. infusion to maintain blood glucose in the range 5.5-11.1 mmol litre(-1). Blood glucose concentrations were measured every 15 min during surgery, and hourly for 6 h after surgery. Plasma C-peptide and counter-regulatory hormones were also measured. RESULTS: Glycaemia was significantly lower in the clonidine group (P<0.01) and the median amount of insulin administered was significantly reduced: clonidine group 9.0 (interquartile range 5.1) units; control 18.6 (10.2) units; P<0.01). Plasma catecholamine concentrations were lower in patients given clonidine (P<0.05) but there was no difference in cortisol concentrations. CONCLUSION: Premedication of type 2 diabetic patients with clonidine 90 min before surgery improves blood glucose control and decreases insulin requirements during ophthalmic surgery.

The founder mutation MSH2*1906G-->C is an important cause of hereditary nonpolyposis colorectal cancer in the Ashkenazi Jewish population.

Hereditary nonpolyposis colorectal cancer (HNPCC) is caused by mutations in the mismatch-repair genes. We report here the identification and characterization of a founder mutation in MSH2 in the Ashkenazi Jewish population. We identified a nucleotide substitution, MSH2*1906G-->C, which results in a substitution of proline for alanine at codon 636 in the MSH2 protein. This allele was identified in 15 unrelated Ashkenazi Jewish families with HNPCC, most of which meet the Amsterdam criteria. Genotype analysis of 18 polymorphic loci within and flanking MSH2 suggested a single origin for the mutation. All colorectal cancers tested showed microsatellite instability and absence of MSH2 protein, by immunohistochemical analysis. In an analysis of a population-based incident series of 686 Ashkenazi Jews from Israel who have colorectal cancer, we identified 3 (0.44%) mutation carriers. Persons with a family history of colorectal or endometrial cancer were more likely to carry the mutation than were those without such a family history (P=.042), and those with colorectal cancer who carried the mutation were, on average, younger than affected individuals who did not carry it (P=.033). The mutation was not detected in either 566 unaffected Ashkenazi Jews from Israel or 1,022 control individuals from New York. In hospital-based series, the 1906C allele was identified in 5/463 Ashkenazi Jews with colorectal cancer, in 2/197 with endometrial cancer, and in 0/83 with ovarian cancer. When families identified by family history and in case series are included, 25 apparently unrelated Ashkenazi Jewish families have been found to harbor this mutation. Although this pathogenic mutation is not frequent in the Ashkenazi Jewish population (accounting for 2%-3% of colorectal cancer in those whose age at diagnosis is <60 years), it is highly penetrant and accounts for approximately one-third of HNPCC in Ashkenazi Jewish families that fulfill the Amsterdam criteria.