Somatic SF3B1 mutation in myelodysplasia with ring sideroblasts.

BACKGROUND: Myelodysplastic syndromes are a diverse and common group of chronic hematologic cancers. The identification of new genetic lesions could facilitate new diagnostic and therapeutic strategies. METHODS: We used massively parallel sequencing technology to identify somatically acquired point mutations across all protein-coding exons in the genome in 9 patients with low-grade myelodysplasia. Targeted resequencing of the gene encoding RNA splicing factor 3B, subunit 1 (SF3B1), was also performed in a cohort of 2087 patients with myeloid or other cancers. RESULTS: We identified 64 point mutations in the 9 patients. Recurrent somatically acquired mutations were identified in SF3B1. Follow-up revealed SF3B1 mutations in 72 of 354 patients (20%) with myelodysplastic syndromes, with particularly high frequency among patients whose disease was characterized by ring sideroblasts (53 of 82 [65%]). The gene was also mutated in 1 to 5% of patients with a variety of other tumor types. The observed mutations were less deleterious than was expected on the basis of chance, suggesting that the mutated protein retains structural integrity with altered function. SF3B1 mutations were associated with down-regulation of key gene networks, including core mitochondrial pathways. Clinically, patients with SF3B1 mutations had fewer cytopenias and longer event-free survival than patients without SF3B1 mutations. CONCLUSIONS: Mutations in SF3B1 implicate abnormalities of messenger RNA splicing in the pathogenesis of myelodysplastic syndromes. (Funded by the Wellcome Trust and others.).

Missense glucokinase mutation in maturity-onset diabetes of the young and mutation screening in late-onset diabetes.

We describe a codon 299 mutation in the glucokinase gene in a British pedigree with maturity-onset diabetes of the young (MODY) resulting in a substitution of glycine to arginine. One out of fifty patients diagnosed with classical late-onset type 2 diabetes mellitus was also found to have this mutation. All nine relatives of this patient who have inherited the mutation have type 2 diabetes, although six others without the mutation are also present with diabetes. The discovery that glucokinase mutations can cause MODY and was also found in ten affected members of a pedigree with type 2 diabetes in which MODY had not previously been considered indicates that diagnosis based on molecular pathology will be helpful in understanding the aetiology of type 2 diabetes.

Assessment of clonality in gastrointestinal cancer by DNA fingerprinting.

DNA fingerprinting with three different probes (33.15, 33.6, and alpha-globin 3'HVR) was investigated as a method for the determination of clonality in gastrointestinal tumors. In 29/44 carcinomas the tumor DNA showed clonal somatic mutations that were not seen in the corresponding peripheral blood and normal mucosa samples. The changes consisted of either novel fingerprint bands, losses of bands, or both. The probe 33.15 yielded the highest rate of abnormal DNA fingerprints (21/44 carcinomas). Sequential use of the probes increased the number of cases where clonal fingerprint markers could be detected. One out of five colorectal adenomas also showed a clonal loss of a fingerprint band. In two cases of gastric cancer, DNA from the metastatic tumor had a different DNA fingerprint from that found in the primary carcinoma. DNA fingerprinting offers a novel approach to determining clonality in tumors and may prove useful for the study of tumor progression.

hTERT, the catalytic component of telomerase, is downregulated in the haematopoietic stem cells of patients with chronic myeloid leukaemia.

Telomere shortening is associated with disease progression in chronic myeloid leukaemia (CML). To investigate the biology and regulation of telomerase in CML, we evaluated expression of the telomerase components, its regulators and several telomeric-associated proteins. Quantitative real-time-polymerase chain reaction (PCR) was used to compare gene expression in the CD34+/leukaemic blast cells of 22 CML patient samples to the CD34+ cell population of healthy individuals. hTERT, the catalytic component of telomerase, was downregulated in eight of 12 chronic phase (CP) patients (P = 0.0387). Furthermore, hTERT was significantly downregulated in two of three patients in accelerated phase (AP) and seven of seven patients in blast crisis (BC), P = 0.0017. Expression of hTR and telomeric-associated proteins TEP1, TRF1, TRF2, tankyrase and PinX1 was high in the majority of CP and AP patients. With the exceptions of TEP1 and hTR, expression of these factors was highest in CP and decreased during disease progression. Expression of c-Myc, a positive regulator of hTERT transcription, correlated with hTERT expression and decreased with disease progression, falling below control levels in BC. hTERT levels were increased in CP patients following successful treatment with imatinib, relative to untreated CP patients. We suggest that reduced hTERT expression directly causes the shortened telomeres observed in CML.

Assessment of clonality in human tumors: a review.

The various methods of determination of the clonality of human tumors are described. There are three major approaches based on X-chromosome inactivation analysis, lymphocyte analysis, and somatic mutation analysis. For each of these approaches there are established methods and more recent methods based on DNA analysis. The increasing number of methods available increases the scope of clonality determination to most tumors. All the methods have inherent advantages and disadvantages, and these are discussed in relation to their clinical application.

Availability of type II diabetic families for detection of diabetes susceptibility genes.

Type II diabetes is a familial disorder, as evidenced by the increased prevalence in monozygotic cotwins and first-degree relatives of affected subjects; however, its genetic etiology is largely unknown. Well-characterized pedigrees are an essential resource for the study of susceptibility genes for type II diabetes. This study describes a 5-yr search for type II diabetic families in Oxfordshire, U.K. We interviewed 950 type II diabetic subjects concerning the availability of first-degree relatives; 127 Caucasian families ascertained through a proband with type II diabetes were studied, and 589 first-degree relatives were characterized. Three large pedigrees with maturity-onset diabetes of the young, and 8 multiplex multigenerational type II diabetic pedigrees were identified. We identified 12 sib-pairs in which both siblings had type II diabetes; however, only 7 sib-pairs had both parents alive, and 2 of these had both parents affected. If one also considers one sib having diabetes and one sib having glucose intolerance as being an affected sib-pair, we identified 30 sib-pairs of which 7 had both parents affected and probably had bilineal inheritance. We identified 76 complete nuclear families with both parents and offspring available for study, but only 6 were of optimal structure for linkage analysis. In conclusion, multiplex pedigrees and type II diabetic sib-pairs with living parents are uncommon, and their ascertainment requires a substantial investment of resources. Large-scale collaborative multicenter initiatives would be needed to collect a large resource of family material for the study of susceptibility genes for type II diabetes.

Distribution of type II diabetes in nuclear families.

Type II diabetes has a substantial genetic component, but the mode of inheritance and the molecular basis of this inheritance are uncertain. This study documents the familial distribution of the disease in the parents and siblings of a consecutive series of type II diabetic subjects. We studied 66 first-degree relatives of 20 white subjects with type II diabetes and both parents alive. They were tested with a continuous infusion of glucose (5 mg.kg IBW-1.min-1) (n = 49) or FPG and hemoglobin A1c (n = 17). Seven probands had neither parent affected with diabetes or IGT, 10 had one parent affected (6 with diabetes and 4 with IGT), and 3 had both parents affected. The probands with affected and those with unaffected parents were phenotypically similar. These findings indicate that a sizable subgroup of type II diabetic subjects may have neither parent affected with a demonstrable abnormality of glucose tolerance. The assumption of autosomal dominance with complete penetrance is not supported, although it remains possible that a dominant gene of low penetrance may play a role in some pedigrees. Polygenic inheritance would appear likely, and genetic heterogeneity may occur. The inheritance of diabetic traits from phenotypically normal parents needs to be considered in the analysis of genetic linkage with type II diabetes.

Linkage analysis of maturity-onset diabetes of the young with microsatellite polymorphisms. No linkage to ADA or GLUT2 genes in two families.

MODY is a form of NIDDM inherited as an autosomal dominant condition. We studied the linkage of MODY to two loci: ADA and GLUT2 in two large pedigrees with nonradioactive microsatellite polymorphic systems. A positive linkage of ADA to MODY was recently demonstrated in the large RW pedigree. Formal linkage analysis excluded a tight linkage between ADA and MODY with a LOD score of -5.82 and -2.24 at a recombination fraction of 0.01 in the two families. This result suggests genetic heterogeneity in the molecular basis of MODY. GLUT2 is a candidate gene that is expressed in the liver and beta-cells of pancreatic islets. In the two families studied, the disease did not cosegregate with GLUT2 alleles. The LOD scores for GLUT2 were -7.79 and -1.9 at a recombination fraction of 0.001 in the two families, thus providing evidence against the involvement of GLUT2 in MODY.

Pulsed field gel electrophoresis on single murine hemopoietic colonies.

We report a technique which allows for the direct molecular analysis of single whole murine hemopoietic colonies by pulsed field gel electrophoresis (PFGE). Murine bone marrow cells were plated out in semi-solid agarose and gave rise to macroscopic colonies after 11 days in culture. Single colonies were excised from the agarose using a sterile blade and embedded without further manipulation in molten low-melting-temperature agarose. The leucocyte DNA contained within the agarose plug was subjected to restriction enzyme digestion and PFGE. Sufficient high molecular weight DNA is afforded by this method to achieve a hybridization signal with a single copy probe. This method will make PFGE directly applicable to the clonal analysis of chromosomal aberrations in hemopoietic stem and progenitor cells.

Molecular diagnosis of haematological neoplasms.

DNA analysis has become of practical value in the diagnosis and classification of leukaemias and lymphomas. This is exemplified by the study of lymphoproliferative disorders using immunoglobulin and T-cell receptor gene probes for the determination of clonality and cell lineage. Chromosomal analysis with DNA probes is now a useful complementary approach to cytogenetics. For example, the study of particular lymphomas or chronic myelogenous leukaemia with DNA probes hybridising to specific chromosomal breakpoints allows the detection of chromosomal translocations at a genomic level. Chromosomal loss in neoplastic cells can be detected by DNA probes in individual heterozygous for particular restriction fragment length polymorphisms, most efficiently by locus-specific hypervariable region probles. These techniques will enable progress to be made in the understanding of the biology of remission and disease progression in haematological malignancies.

Thalassaemia intermedia.

Most patients homozygous for beta thalassaemia have beta thalassaemia major, a severe illness requiring regular blood transfusions. However, some homozygotes remain well without regular transfusions and are described by the term thalassaemia intermedia. Three factors have now been identified which may result in beta thalassaemia intermedia: the inheritance of mild beta+ thalassaemia mutations, the co-inheritance of alpha thalassaemia and the inheritance of factors enhancing gamma-globin gene expression. In addition other less common genetic interactions also result in thalassaemia intermedia such as the compound heterozygous state for beta and delta beta thalassaemia. These patients need careful clinical follow up, especially since the complications of hypersplenism and iron overload (even in the absence of blood transfusion) can occur.

Factor V Leiden and thermolabile methylenetetrahydrofolate reductase in extreme old age.

Both factor V Leiden and the C677T methylenetetrahydrofolate reductase (MTHFR) gene mutation are associated with premature vascular disease, and yet are found at surprisingly high allele frequencies in European populations, 2.7% and 35% respectively. We have investigated the prevalence of these mutations in 87 UK residents over the age of ninety, to look for any evidence of their association with premature death. Five factor V Leiden heterozygotes were found, giving an allele frequency of 2.9%, similar to that in the general UK population. The frequency of the thermolabile C677T MTHFR mutation was 36% with 11% homozygotes, again similar to that in the UK population; these genotypes are in Hardy-Weinberg equilibrium, suggesting that there is not strong selection against the homozygous state. One person was both heterozygous for factor V Leiden and homozygous for the C677T mutation. This study suggests that neither factor V Leiden nor thermolabile MTHFR are risk factors for premature death.

DNA polymorphism 5' to the JH region of the human immunoglobulin heavy chain gene and immunoglobulin gene rearrangements in leukemia.

DNA samples of two patients with acute myeloblastic leukemia were noted to have a variant band on digestion with HindIII and hybridization to a probe for the joining region of the immunoglobulin (Ig) heavy chain gene (JH). Further analysis showed that the variant band represented a constitutional DNA polymorphism rather than an Ig gene rearrangement. The HindIII fragment detected by the JH probe includes a highly polymorphic region 5' to the Ig heavy chain locus, and hence variant germline bands may be seen on autoradiography that can be mistaken for Ig gene rearrangements. The use of several different restriction enzymes and the analysis of the constitutional DNA allows a clear distinction to be made between somatic gene rearrangements and DNA polymorphisms.

Epstein-Barr virus-associated anaplastic large cell lymphoma in renal transplant patients.

A novel association, Epstein-Barr virus-positive Ki-1+/CD30+ anaplastic large cell non-Hodgkin's lymphoma of B-cell phenotype in immunosuppressed renal transplant recipients is reported. Case 1 involved an aggressive clinical evolution, whereas case 2 followed a more "benign" clinical course. Both lymphomas were Epstein-Barr virus-positive as assessed by in situ hybridization, Southern blot, polymerase chain reaction, and immunohistochemical analysis. Both lymphomas contained a single clonal Epstein-Barr virus terminal-repeat fragment. In case 1, clonality was confirmed by the detection of bi-allelic immunoglobulin (Ig) heavy chain gene rearrangement. Case 2 showed germline Ig genes at presentation and oligoclonal Ig heavy chain gene rearrangements at relapse. These results are consistent with the notion that anaplastic large cell lymphoma might arise in a B cell transformed by Epstein-Barr virus at a very early stage, before Ig gene rearrangement. The latter may occur later in the course of clonal evolution, thus permitting investigators to trace intermediate and late stages within a process of multistep lymphomagenesis and/or tumor progression.

Rapid HLA typing by multiplex amplification refractory mutation system.

AIMS: To detect HLA susceptibility and protective alleles associated with insulin dependent diabetes mellitus (IDDM) using a multiplex amplification refractory mutation system (ARMS). These include DR3 and DR4 alleles at the DRB1 locus, presence or absence of aspartic acid at position 57 (Asp-57) of the DQB1 locus, and presence or absence of arginine at position 52 (Arg-52) of the DQA1 locus. METHODS: The ARMS approach was used to design allele specific primers for the detection of the major susceptibility and protective alleles for IDDM. These include DR3 and DR4 alleles at the DRB1 locus, Asp-57 and non-Asp-57 at the DQB1 locus, and Arg-52 and non-Arg-52 alleles at the DQA1 locus. The allele specificity of each set of primers was first tested separately using DNA samples from 15 individuals previously typed for the DRB1, DQB1, and DQA1 loci using the sequence specific oligonucleotide (SSO) technique. The possibility of using multiplex ARMS for typing multiple susceptibility/protective alleles for IDDM was further investigated by testing various combinations of allele specific primers, thereby reducing the number of separate polymerase chain reactions required to type all these alleles. RESULTS: A "three-tube" system worked well and gave accurate results. Tube 1 contained ARMS primers for the detection of IDDM susceptibility alleles DR3 and DR4; tube 2 contained ARMS primers for the detection of susceptibility alleles non-Asp-57 and Arg-52; and tube 3 contained ARMS primers for the detection of the protective alleles Asp-57 and non-Arg-52. DNA samples typed with this ARMS method were in complete agreement with those obtained using the SSO technique. CONCLUSION: This method is rapid and has no requirement for radioactivity. It is an efficient method for population screening.

Pulsed field gel electrophoresis on frozen tumour tissue sections.

The application of pulsed field gel electrophoresis (PFGE) to the molecular genetic analysis of solid tumours has been restricted by the requirement for whole single cells as a DNA source. A simple technique which allows for the direct analysis of histologically characterised solid tumour material by pulsed field gel electrophoresis was developed. Single frozen tissue sections obtained from colonic carcinoma specimens were embedded without further manipulation in molten, low melting temperature agarose. The tumour DNA contained within the agarose plug was subjected to restriction enzyme digestion and PFGE. Sufficient high molecular weight DNA is yielded by this method to obtain a hybridisation signal with a single copy probe. Histological examination of adjacent tissue sections may also be carried out, permitting correlation between molecular analysis and tumour histology.

Chromosome 11 allele loss in sporadic insulinoma.

DNA was extracted from tissue samples of three unrelated cases of insulinoma. Chromosome 11 allele loss was investigated using several chromosome 11 specific probes which detect restriction fragment length polymorphisms. In one case, which proved informative for many of the chromosome 11 markers, allele loss was shown on both 11p and 11q. This finding is of considerable interest as the allele loss closely corresponds to that recently reported in insulinomas occurring in the familial multiple endocrine neoplasia type 1 (MEN-1) syndrome.

Evidence for monoclonal T lymphocyte proliferation in angioimmunoblastic lymphadenopathy.

The arrangement of the T cell receptor and immunoglobulin genes was analysed in lymphoid tissue biopsy specimens from 25 cases of angioimmunoblastic lymphadenopathy. Nineteen cases showed a rearrangement of the gene coding for the beta chain of the T cell receptor, and in one case a clonal rearrangement of immunoglobulin genes was shown (in which the T cell receptor gene was in a germline configuration). These findings indicate that a monoclonal T cell proliferation is present in most cases of angioimmunoblastic lymphadenopathy, and they also correlate with the fact that some patients who present with this disorder subsequently develop a T cell lymphoma.

Practical value of genotypic analysis for diagnosing lymphoproliferative disorders.

The value of DNA hybridisation (using immunoglobulin and T cell receptor gene probes) was assessed during the diagnosis of problematical lymphoid tissue biopsy specimens. In 14 of 18 specimens (78%), which contained a malignant lymphoproliferation of uncertain aetiology, this technique permitted the demonstration of a monoclonal proliferation of B cells (nine cases) or T cells (five cases). In five further lymph node biopsy specimens, in which the differential diagnosis lay between a reactive or malignant process, a clonal proliferation was shown in three cases. DNA analysis is, therefore, of practical value in resolving many of the diagnostic problems that arise in the assessment of lymphoid tissue biopsy specimens.

Technical optimization of RhD zygosity determination by real-time quantitative polymerase chain reaction: implication for fetal RhD status determination by maternal plasma.

We have recently reported the development of a multiplex, real-time quantitative polymerase chain reaction (PCR) assay for RhD zygosity determination based on the coamplification and quantification of RHD-specific sequences in relation to a reference gene, albumin. This paper discusses the optimization procedure and technical parameters of this multiplex assay.