Ferrochelatase Mutation Screening by Denaturing High Performance Liquid Chomatography in Idiopathic Acquired Sideroblastic Anemia (Refractory Anemia with Ringed Sideroblasts) and Erythrocytic Protoporphyria.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3735-3735
Author(s):  
Kathleen A. Hecksel ◽  
Gordon W. Dewald ◽  
David P. Steensma
Keyword(s):  
High Performance ◽  
Mutation Screening ◽  
Point Mutations ◽  
Refractory Anemia ◽  
Coding Region ◽  
Sideroblastic Anemia ◽  
Ringed Sideroblasts ◽  
Proper Form ◽  
Molecular Etiology ◽  
Normal Urine

Abstract BACKGROUND: The molecular etiology of idiopathic acquired sideroblastic anemia (IASA), now considered a form of myelodysplastic syndrome, is currently unknown. Romslo et al (Blood 1982) reported a patient with IASA who had moderately elevated free erythrocytic protoporphyrin (FEP); this observation is now frequently made in IASA, helping distinguish IASA (high FEP) from inherited sideroblastosis (low FEP). In addition, rare patients with erythropoietic protoporphyria (EPP)—defined by cutaneous photosensitivity, dramatically elevated FEP levels, and germline point mutations in the ferrochelatase (FECH) gene at 18q21.3—have ringed sideroblasts in their marrow. We hypothesized that patients with IASA might have acquired somatic FECH mutations. METHODS AND RESULTS: We designed a denaturing high performance liquid chromatography (DHPLC) assay to explore this possibility and to avoid problems related to mutation screening in the setting of mixed clonality. To validate the DHPLC assay, the coding region of the FECH gene from 2 molecularly undiagnosed EPP patients without liver disease was analyzed. In one patient, both a heterozygous 69delG mutation (GenBank Accession NM_000140) and heterozygosity for the FECH expression-modulating IVS3-48C/T polymorphism were detected. In the other patient, no FECH coding mutations or polymorphisms were detected, either by DHPLC or conventional dye sequencing. FEP measurements were then obtained on 2 IASA patients and were elevated (65 and 115 mcg/dL; normal 1–10 mcg/dL) with normal urine and fecal porphyrins. Genomic DNA obtained from these 2 patients as well as archival DNA from 30 other patients with IASA was amplified and tested for FECH mutations. No coding region mutations were detected. Synonymous polymorphisms in exon 7 (rs536765) and 9 (rs536560) were found in 6/32 (19%; normal heterozygosity 0.398) and 14/32 (44%; normal heterozygosity 0.402) samples, respectively. The IVS3-48C/T polymorphism (rs2272783) was found in 3/32 (9%) (prevalence in the general population is 11%, Gouya Nat Genet 2002). In addition, 3 intronic polymorphisms not in the refSNP database were detected: IVS8+34 C/T (2/32), IVS8-61delG (5/32), and IVS9-59delA (2/32). CONCLUSION: IASA is not associated with coding mutations in FECH. Elevation of FEP in ASA must instead be due to the lack of mitochondrial iron in the proper form for incorporation into the porphyrin ring; attention should instead focus on factors responsible for maintaining the appropriate redox state and compartmentalization of iron. DHPLC can detect FECH mutations and polymorphisms, but because of the high frequency of the latter and the consequent need to sequence multiple exons, it is not a practical screening tool for studying undiagnosed EPP patients.

Mutations in the AML1 Gene Define an Unfavorable Subgroup in Acute Myeloid Leukemia with FAB M0.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4340-4340
Author(s):  
Frank Dicker ◽  
Mirjam Klaus ◽  
Torsten Haferlach ◽  
Wolfgang Kern ◽  
Wolfgang Hiddemann ◽  
...  
Keyword(s):  
High Performance ◽  
De Novo ◽  
Mutation Screening ◽  
Point Mutations ◽  
Gene Mutations ◽  
Normal Karyotype ◽  
Runt Domain ◽  
Screening Efficiency ◽  
Genetic Aberration ◽  
Aml1 Gene

Abstract The AML1/RUNX1 gene is the most frequent target for chromosomal translocations in leukemia. Recently point mutations in the AML1 gene have been demonstrated as another mode of genetic aberration. AML1 mutations have been reported in de novo MDS and AML, as well as in therapy related MDS and AML. The AML M0 subtype has been found to be most frequently affected by sporadic AML1 gene mutations. We analysed AML1 gene mutations in a cohort of 49 M0 patients. Mutation screening was performed either with SSCP (n=21) and/or denaturating High Performance Liquid Chromatography (dHPLC) (n=33), 5 cases were analyzed by both methods. SSCP screening of exons 3–5 of the AML1 gene was carried out at the genomic level. These exons cover the socalled Runt domain, which is most frequently mutated. Fragments with aberrant mobility were sequenced. With this method 5 cases were found to be mutated. Subsequently, to improve the screening efficiency an assay using dHPLC was established. Hereby, we screened the cDNA of patient samples for mutations in amino acid codons 1–277 of the AML1b transcript, where the Runt domain is located between codons 49 and 178. All 5 cases detected by SSCP were confirmed by dHPLC. Nine mutations were detected in the cohort of 28 cases (32%) which had not been analyzed by SSCP. In total, 14 of the 49 samples (29%) tested were identified to be mutated, which is a slightly higher frequency than previously reported. In the cohort of 35 AML1 non-mutated cases 20 (57%) had a normal karyotype and 15 (43%) an aberrant karyotypes, whereas only 6 of the 14 AML1 mutated cases (43%) had a normal karyotype (p=0.001). Three of the AML1 mutated cases (21%) also had FLT3 mutations. One had an FLT3-LM, one an FLT3-TKD mutation, and one case both LM and TKD mutations. Clinical follow up data were available for 33 patients (22 AML1 non- mutated, 11 AML1 mutated). The median OS and EFS of the AML1 non-mutated versus the mutated group was 276 days versus 63 days (p = 0.0679) and 276 vs. 63 days (p=0.0630) respectively. Thus the AML1 mutated cases tend to have a worse clinical outcome. When other AML subtypes were screened for AML1 mutations, i.e. M1 (n=26), M2 (n=21) and M4 (n=3), only 1 additional AML1 mutation was detected, confirming the highest prevalence of AML1 mutations in M0. In conclusion, 1) we established a new assay to screen for AML1 mutations. 2) We confirmed the high incidence of AML1 gene mutations in AML M0, both in cases with normal and aberrant karyotype. 3) For the first time we demonstrated that AML1 mutations define an unfavorable subentity in AML M0.

Mitochondrial Ferritin Expression and Clonality of Hematopoiesis in Patients with Refractory Anemia with Ringed Sideroblasts.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3444-3444 ◽  
Author(s):  
Matteo G. Della Porta ◽  
Luca Malcovati ◽  
Anna Galli ◽  
Sabrina Boggi ◽  
Erica Travaglino ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Early Stage ◽  
Refractory Anemia ◽  
Specific Marker ◽  
Erythroid Cells ◽  
Prognostic Information ◽  
Sideroblastic Anemia ◽  
Erythroid Progenitor ◽  
Ringed Sideroblasts

Abstract Sideroblastic anemias are a heterogeneous group of disorders that have in common the presence of erythroblasts with iron-loaded mitochondria defined as ringed sideroblasts. We have previously demonstrated that mitochondrial iron deposition in these disorders is in the form of mitochondrial ferritin (MtF), suggesting that this latter may be a specific marker of sideroblastic anemia (Cazzola et al, Blood2003;101:1996–2000). The most common type of acquired sideroblastic anemia is the myelodysplastic syndrome (MDS) defined as refractory anemia with ringed sideroblasts, which is generally associated with a relatively benign clinical course. In the present work, we studied the relationship between MtF expression and clonality of hematopoiesis in 55 consecutive female patients with low-risk MDS, including 20 cases with ringed sideroblasts and 35 cases without ringed sideroblasts. The expression of MtF, as well as that of cytosolic ferritin (H and L subunits) and of transferrin receptor (CD71), was evaluated by flow cytometry in bone marrow erythroid cells; in selected cases, these immunophenotypic investigations were also performed on liquid cultures of purified CD34-positive cells. X-chromosome inactivation patterns (XCIPs) were assessed in peripheral blood granulocytes and in bone marrow CD34-positive cells by analysis of both DNA methylation at the HUMARA and PGK loci and of IDS gene expression. Within informative females, 11 out of 12 patients with ringed sideroblasts displayed clonal XCIPs in granulocytes; by contrast, only 9 out of 22 patients without ringed sideroblasts displayed clonal XCIPs. Purified CD34-positive cells showed clonal XCIPs in 6 out of 7 patients with ringed sideroblasts but had polyclonal XCIPs in 4 out of 5 individuals without ringed sideroblasts. Flow cytometry evaluation of bone marrow erythroid cells showed that MtF expression was restricted to MDS patients with ringed sideroblasts. A close positive relationship was found between MtF and CD71 expression (r=.49, P=.001); this association may reflect the cytosolic iron deprivation induced by MtF overexpression. Analysis of cultured erythroid progenitor cells showed that MtF was detectable at a very early stage of differentiation from CD34-positive cells. Addition of erythropoietin to the culture system sustained the appearance of a polyclonal erythroid population in 2 out of 4 patients with ringed sideroblasts and clonal CD34-positive cells. These observations suggest that refractory anemia with ringed sideroblasts is a truly clonal stem cell disorder, while more than 50% of patients with refractory anemia without ringed sideroblasts have evidence of polyclonal hematopoiesis. The clonal pattern of CD34-positive cells and the early appearance of MtF during erythroid differentiation suggest that - despite be benign natural history of refractory anemia with ringed sideroblasts - the initial pathogenetic event in this condition occurs in multipotent stem cells. Although the mechanisms responsible for overexpression of MtF are still unclear, flow cytometry evaluation of this protein is a useful diagnostic tool that also provides helpful prognostic information.

scholarly journals 126.A repository of ENU mutant mouse lines and their potential for male fertility research

2004 ◽  
Vol 16 (9) ◽  
pp. 126
Author(s):  
C. L. Kennedy ◽  
A. E. O’Connor ◽  
L. G. Sanchez-Partida ◽  
C. C. Goodnow ◽  
D. M. De Kretser ◽  
...  
Keyword(s):  
Male Fertility ◽  
High Performance ◽  
Large Scale ◽  
Mutation Screening ◽  
Point Mutations ◽  
Serum Levels ◽  
Reproductive Technologies ◽  
Specific Gene ◽  
Contraceptive Agents ◽  
Wide Range

One in 25 western men are infertile and the causal factor is frequently unknown, although it is expected that many are genetic in origin. My project aims to identify genes critical to mouse spermatogenesis using ENU mutagenesis. A further aim was to develop a repository of mutant mice and data on their fertility parameters for use by the reproductive biology community. This research will aid the diagnosis and development of specific treatments for human infertility and the development of contraceptive agents. The potent mutagen N-ethyl-N-nitrosourea (ENU) was utilized to generate libraries of C57BL/6 mice with random point mutations throughout their genomes. A 3 generational breeding program produced mice that were homozygous for a number of mutations. I subsequently performed a number of large scale screens on 3rd generation males, identifying lines carrying recessive mutations specifically affecting male fertility. Thus far we have observed a wide range of abnormal testis phenotypes including Sertoli Cell only, hypospermatogenesis, meiosis arrest, abnormal sperm morphology and abnormal hormone levels. From these analyses a repository including all data and tissues collected from 1200 3rd generation male mice from 122 different lines has been developed and will become publicly available. This includes testis and epididymal histology and serum levels of FSH, LH, activin A and inhibin. Further, I have stored gDNA long term and cryopreserved sperm to enable regeneration of lines in the future. In addition, I have developed a high throughput mutation screening protocol for the detection of mutations within genes of interest using denaturing high performance liquid chromatography (DHPLC). Collectively, our repository and gene screening techniques can be used in conjunction with artificial reproductive technologies to generate mouse models reflective of human conditions and altered specific gene function.

scholarly journals Cytogenetic and Molecular Genetic Characterization of Children with Short Stature / Citogenetska in Molekularno Genetska Opredelitev Nizke Rasti Pri Otrocih

2015 ◽  
Vol 54 (2) ◽  
pp. 98-102 ◽  
Author(s):  
Tinka Hovnik ◽  
Darja Šmigoc Schweiger ◽  
Primož Kotnik ◽  
Jernej Kovač ◽  
Tadej Battelino ◽  
...  
Keyword(s):  
Short Stature ◽  
Gene Deletion ◽  
Mutation Screening ◽  
Molecular Genetic ◽  
Point Mutations ◽  
Idiopathic Short Stature ◽  
Fish Analysis ◽  
Coding Region ◽  
Exon 2 ◽  
Shox Gene

Abstract Background. The deficiency of SHOX gene (short stature homeobox-containing gene) has been recognized as the most frequent monogenetic cause of short stature. SHOX gene has been associated with short stature in Turner syndrome and Leri Weill dyschondrosteosis as well with non-syndromic idiopathic short stature. The aim of this study was to determine the frequency of SHOX deletions and mutations in a cohort of Slovenian children with short stature, and to delineate indications for routine SHOX gene mutation screening. Methods and results. 40 selected subjects with idiopathic short stature were screened for entire SHOX gene deletion and for mutations in the SHOX gene coding region (exon 2 to 6), together with sequences flanking the exon-intron boundaries. FISH analysis on metaphase and interphase spreads revealed no entire gene deletion. Additionally, no pathogenic point mutations or smaller deletion/duplications were identified in this study group. Conclusions. SHOX gene deletions and point mutations are not a common cause of idiopathic short stature in a cohort of Slovenian children with short stature. Therefore, the frequency of SHOX mutations must be much lower as expected based on the reported data.

Late-onset X-linked sideroblastic anemia following hemodialysis

Blood ◽  
2003 ◽  
Vol 101 (11) ◽  
pp. 4623-4624 ◽  
Author(s):  
Kazumichi Furuyama ◽  
Hideo Harigae ◽  
Chiharu Kinoshita ◽  
Toshihiko Shimada ◽  
Kazuko Miyaoka ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
Late Onset ◽  
The Elderly ◽  
Nutritional Deficiencies ◽  
Maintenance Hemodialysis ◽  
Sideroblastic Anemia ◽  
Ringed Sideroblasts ◽  
Aminolevulinate Synthase ◽  
Deficient Activity

Abstract X-linked sideroblastic anemia (XLSA) is due to deficient activity of erythroid-specific 5-aminolevulinate synthase (ALAS2). We report here a patient who developed sideroblastic anemia at the age of 81 years while undergoing hemodialysis. The diagnosis of sideroblastic anemia was established by the presence of ringed sideroblasts in the bone marrow, and treatment with oral pyridoxine completely eliminated the ringed sideroblasts. We identified a novel point mutation in the fifth exon of this patient's ALAS2 gene, which resulted in an amino acid change at residue 159 from aspartic acid to asparagine (Asp159Asn). In vitro analyses of recombinant Asp159Asn ALAS2 revealed that this mutation accounted for the pyridoxine-responsiveness of this disease. The very late onset in this case of XLSA emphasizes that nutritional deficiencies caused either by dietary irregularities in the elderly or, as in this case, by maintenance hemodialysis therapy, may uncover occult inherited enzymatic deficiencies in the heme biosynthetic pathway.

scholarly journals Heteroplasmic Point Mutations of Mitochondrial DNA Affecting Subunit I of Cytochrome c Oxidase in Two Patients With Acquired Idiopathic Sideroblastic Anemia

Blood ◽  
1997 ◽  
Vol 90 (12) ◽  
pp. 4961-4972 ◽  
Author(s):  
Norbert Gattermann ◽  
Stefan Retzlaff ◽  
Yan-Ling Wang ◽  
Götz Hofhaus ◽  
Jürgen Heinisch ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mitochondrial Dna ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Respiratory Chain ◽  
Ferric Iron ◽  
Point Mutations ◽  
Polymorphism Analysis ◽  
Sideroblastic Anemia ◽  
Wide Range

Mitochondrial iron overload in acquired idiopathic sideroblastic anemia (AISA) may be attributable to mutations of mitochondrial DNA (mtDNA), because these can cause respiratory chain dysfunction, thereby impairing reduction of ferric iron (Fe3+) to ferrous iron (Fe2+). The reduced form of iron is essential to the last step of mitochondrial heme biosynthesis. It is not yet understood to which part of the respiratory chain the reduction of ferric iron is linked. In two patients with AISA we identified point mutations of mtDNA affecting the same transmembrane helix within subunit I of cytochrome c oxidase (COX I; ie, complex IV of the respiratory chain). The mutations were detected by restriction fragment length polymorphism analysis and temperature gradient gel electrophoresis. One of the mutations involves a T → C transition in nucleotide position 6742, causing an amino acid change from methionine to threonine. The other mutation is a T → C transition at nt 6721, changing isoleucine to threonine. Both amino acids are highly conserved in a wide range of species. Both mutations are heteroplasmic, ie, they establish a mixture of normal and mutated mitochondrial genomes, which is typical of disorders of mtDNA. The mutations were present in bone marrow and whole blood samples, in isolated platelets, and in granulocytes, but appeared to be absent from T and B lymphocytes purified by immunomagnetic bead separation. They were not detected in buccal mucosa cells obtained by mouthwashes and in cultured skin fibroblasts examined in one of the patients. In both patients, this pattern of involvement suggests that the mtDNA mutation occurred in a self-renewing bone marrow stem cell with myeloid determination. Identification of two point mutations with very similar location suggests that cytochrome c oxidase plays an important role in the pathogenesis of AISA. COX may be the physiologic site of iron reduction and transport through the inner mitochondrial membrane.

Mutation Profiling in Haemophilia A

2001 ◽  
Vol 85 (04) ◽  
pp. 577-579 ◽  
Author(s):  
J. Oldenburg
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
Mutation Screening ◽  
Screening Method ◽  
Coagulation Factor ◽  
Causative Mutation ◽  
Haemophilia A ◽  
Coding Region ◽  
Systematic Analysis ◽  
Intron 22 Inversion ◽  
Gradient Gel Electrophoresis

SummaryHaemophilia A is a X-linked recessive bleeding disorder caused by deficiency or absence of coagulation factor VIII (FVIII) due to heterogeneous defects in the FVIII gene. The large size of the FVIII gene (26 exons spanning 186 kb) has hampered mutation analysis for many years. In 1991 the first systematic analysis of the complete coding region of the FVIII gene was performed by Higuchi et al. using Denaturing Gradient Gel Electrophoresis (DGGE) as a mutation screening method (1, 2). Notably, the causative mutation was not found in about half of the severely affected patients (1). This mystery was solved in 1993, when the intron 22 inversion was discovered (3, 4) that accounts for about 50% of the severe haemophilia A cases. The inversion mutation can be easily detected by Southern Blot. A recently described PCR-based method is more sophisticated, however once established, it allows rapid and convenient detection of the intron 22 inversion (5).

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