Recurrent Point Mutations Research Articles

Experimental therapeutics for patients with myeloproliferative neoplasias.

Philadelphia chromosome (Ph)-negative myeloproliferative neoplasms (MPNs) are characterized by stem cell-derived, unrestrained clonal myeloproliferation. The World Health Organization classification system, proposed in 2008, identifies 7 distinct categories of Ph-negative MPNs including essential thrombocythemia (ET); polycythemia vera (PV); primary myelofibrosis (PMF); mastocytosis; chronic eosinophilic leukemia; chronic neutrophilic leukemia; and MPN, unclassifiable. For many years, the treatment of ET, PV, and PMF, the most frequently diagnosed Ph-negative MPNs, has been largely supportive. In recent years, that paradigm has been challenged because of the discovery of a recurrent point mutation in the Janus kinase 2 (JAK2) gene (JAK2(V617F)). This mutation can be detected in the vast majority of patients with PV and approximately half of patients with ET or PMF and serves as both a diagnostic marker as well as representing a putative molecular target for drug development. Several putative targeted agents with significant in vitro JAK2 inhibitory activity and various degrees of JAK2 specificity are currently undergoing clinical evaluation. Furthermore, other investigational non-tyrosine kinase inhibitor approaches such as immunomodulatory agents and pegylated interferon- have also shown promising results in MPNs.

Spectrum of BRCA1/2 point mutations and genomic rearrangements in high-risk breast/ovarian cancer Chilean families

The distribution of BRCA1/2 germline mutations in breast/ovarian cancer (BC/OC) families varies among different populations. In the Chilean population, there are only two reports of mutation analysis of BRCA1/2, and these included a low number of BC and/or OC patients. Moreover, the prevalence of BRCA1/2 genomic rearrangements in Chilean and in other South American populations is unknown. In this article, we present the mutation-detection data corresponding to a set of 326 high-risk families analyzed by conformation-sensitive gel electrophoresis and heteroduplex analysis. To determine the contribution of BRCA1/2 LGRs in Chilean BC patients, we analyzed 56 high-risk subjects with no pathogenic BRCA1/2 point mutations. Germline BRCA1/2 point mutations were found in 23 (7.1%) of the 326 Chilean families. Families which had at least three BC and/or OC cases showed the highest frequency of mutations (15.9%). We identified 14 point pathogenic mutations. Three recurrent mutations in BRCA1 (c.187_188delAG, c.2605_2606delTT, and c.3450_3453delCAAG) and three in BRCA2 (c.4969_4970insTG, c.5374_5377delTATG, and c.6503_6504delTT) contributed to 63.6 and 66.7% of all the deleterious mutations of each gene, which may reflect the presence of region-specific founder effects. Taken together BRCA1/2 recurrent point mutations account for 65.2% (15/23) of the BRCA1/2 (+) families. No large deletions or duplications involving BRCA1/2 were identified in a subgroup of 56 index cases negative for BRCA1/2 point mutations. Our study, which is the largest conducted to date in a South American population, provides a comprehensive analysis on the type and distribution of BRCA1/2 mutations and allelic variants.

Comprehensive Analysis of PTEN Allelic Status Confirms Gross Genomic Rearrangement as the Main Mechanism of PTEN Inactivation in Sporadic Breast Carcinomas.

Abstract BackgroundThe PTEN tumour suppressor gene is frequently inactivated in a wide range of cancers and encodes a nonredundant phosphatase which antagonizes the PI3K signalling pathway. Previous IHC analysis and genomic characterization (through BAC CGH array) of a series of 135 sporadic breast carcinomas identified some tumours with decreased or absent PTEN levels and/or loss at 10q23.3, including the PTEN locus. Comprehensive molecular genetic analysis of PTEN, in addition to PI3KCA and TP53 point mutation screening were performed on this large homogenous series of breast carcinomas, giving a molecular portrait of key players of sporadic breast carcinogenesis.Materials and methodsA series of 135 sporadic invasive ductal carcinomas resected between 1989 and 1992 at Institut Bergonie underwent PTEN molecular genetic screening through QMPSF to detect gross genomic rearrangements and the recently developed Enhanced Mismatch Mutation Analysis (EMMA) to detect point mutations and to confirm gross genomic rearrangements. Tumours with a suspected PTEN large deletion identified using these techniques, along with any additional tumours previously shown to have decreased or absent PTEN levels and/or loss at 10q23.3, were analyzed using Agilent 4x44K high density oligo CGH arrays. The TP53 status of all tumours was assessed through IHC and direct sequencing. In addition, molecular analysis of the recurrent point mutations of PI3KCA, the catalytic subunit of the PI3 kinase, was performed.ResultsGross PTEN gene rearrangements were detected in 48 tumours through QMPSF ; 19 biallelic large deletions, 21 monoallelic large deletions and 8 large duplications. Variant EMMA profiles indicating a PTEN point mutation have been identified in approximately 15% of tumours which are confirmed by direct sequencing. High density oligo CGH array performed on 54 tumours demonstrates 7 monoallelic large deletions and 9 biallelic large deletions to date (analysis to be completed). A total of 39 tumours were shown to have mutant TP53 on IHC and/or a TP53 mutation, which interestingly only includes 8/19 tumours with a biallelic PTEN large deletion. Finally, an overall assessment of the above gene status, including results of the PI3KCA point mutation screen will be presented.ConclusionThis study shows that the predominant mechanism of PTEN inactivation in sporadic breast carcinomas is through gross genomic rearrangements rather than deleterious point mutations, and that this is not restricted to BRCA1-mutated tumours. Furthermore, previous reports have suggested that biallelic inactivation of PTEN in tumours with wild-type TP53 would result in cell death and therefore not be advantageous for tumour growth. In this series, we did not observe a correlation between biallelic inactivation of PTEN and TP53 mutation status (p=0.31, chi-squared test). Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3151.

Chin-on-Chest Deformity in Patients with Fibrodysplasia Ossificans Progressiva

Fibrodysplasia ossificans progressiva is a rare and disabling autosomal dominant disorder of joint malformations and progressive heterotopic ossification1-4 caused by a recurrent missense point mutation in activin receptor IA (ACVR1, also called ALK2), a bone morphogenetic protein (BMP) type-I receptor2,3,5. Joints become permanently ankylosed in positions that drastically impair activities of daily living and even life itself6. Progressive heterotopic ossification usually begins in the first decade of life, typically appearing first in the dorsal, axial, cranial, and proximal regions of the body, and then later in the ventral, appendicular, caudal, and distal regions1-4,7. Lesions are characterized by the rapid appearance of painful, highly vascular fibroproliferative swellings that progress through an endochondral process to form mature ossicles of heterotopic bone3,8. Lesions may develop spontaneously, although falls9, soft-tissue injury2,3,10, mandibular blocks11, intramuscular immunizations12, viral illnesses13, and surgical trauma10 can precipitate new flare-ups resulting in heterotopic ossification. Although the classic clinical features of fibrodysplasia ossificans progressiva have been well described, manifestations of the disease affecting the cervical spine are less well known. Neck stiffness is an early finding in most patients and can precede the onset of heterotopic ossification2,4,10. Characteristic malformations of the cervical spine include large posterior elements; tall, narrow vertebral bodies; and fusion of the facet joints from C2 to C72,14,15. In the present report, we describe a rare, but potentially life-threatening, chin-on-chest deformity that may develop in patients with fibrodysplasia ossificans progressiva and a surgical strategy that has been effective for achieving substantial correction of the deformity. The families were informed that …

RUNX1 DNA-binding mutations and RUNX1-PRDM16 cryptic fusions in BCR-ABL+ leukemias are frequently associated with secondary trisomy 21 and may contribute to clonal evolution and imatinib resistance

AbstractAcquired molecular abnormalities (mutations or chromosomal translocations) of the RUNX1 transcription factor gene are frequent in acute myeloblastic leukemias (AMLs) and in therapy-related myelodysplastic syndromes, but rarely in acute lymphoblastic leukemias (ALLs) and chronic myelogenous leukemias (CMLs). Among 18 BCR-ABL+ leukemias presenting acquired trisomy of chromosome 21, we report a high frequency (33%) of recurrent point mutations (4 in myeloid blast crisis [BC] CML and one in chronic phase CML) within the DNA-binding region of RUNX1. We did not found any mutation in de novo BCR-ABL+ ALLs or lymphoid BC CML. Emergence of the RUNX1 mutations was detected at diagnosis or before the acquisition of trisomy 21 during disease progression. In addition, we also report a high frequency of cryptic chromosomal RUNX1 translocation to a novel recently described gene partner, PRDM16 on chromosome 1p36, for 3 (21.4%) of 14 investigated patients: 2 myeloid BC CMLs and, for the first time, 1 therapy-related BCR-ABL+ ALL. Two patients presented both RUNX1 mutations and RUNX1-PRDM16 fusion. These events are associated with a short survival and support the concept of a cooperative effect of BCR-ABL with molecular RUNX1 abnormalities on the differentiation arrest phenotype observed during progression of CML and in BCR-ABL+ ALL.

Myoclonus-dystonia: significance of largeSGCEdeletions

Myoclonus-dystonia (M-D) is an autosomal-dominant movement disorder caused by mutations in SGCE. We investigated the frequency and type of SGCE mutations with emphasis on gene dosage alterations and explored the associated phenotypes. We tested 35 M-D index patients by multiplex ligation-dependent probe amplification (MLPA) and genomic sequencing. Mutations were found in 26% (9/35) of the cases, all but three with definite M-D. Two heterozygous deletions of the entire SGCE gene and flanking DNA and a heterozygous deletion of exon 2 only were detected, accounting for 33% (3/9) of the mutations found. Both large deletions contained COL1A2 and were additionally associated with joint problems. Further, we discovered one novel small deletion (c.771_772delAT, p.C258X) and four recurrent point mutations (c.289C>T, p.R97X; c.304C>T, p.R102X; c.709C>T, p.R237X; c.1114C>T, p.R372X). A Medline search identified 22 articles on SGCE mutational screening. Sixty-four unrelated M-D patients were described with 41 different mutations. No genotype-phenotype association was found, except in patients with deletions encompassing additional genes. In conclusion, a rigorous clinical preselection of patients and careful accounting for non-motor signs should precede mutational tests. Gene dosage studies should be included in routine SGCE genetic testing.

RUNX1 DNA-Binding Mutations and RUNX1-PRDM16 Cryptic Fusion in BCR/ABL+ Leukemias Are Frequently Associated with Secondary Trisomy 21 and May Contribute to Clonal Evolution and Imatinib Resistance.

Background: Besides the extensively studied point mutations in the BCR/ABL TK domain as the clinically most important cause of imatinib (IM) resistance either in CML and Philadelphia-positive (Ph+) ALL, additional acquired genetic events contributing to resistance/disease progression are not fully understood. Many evidences suggest that the enhanced survival and differentiation arrest of the CML blast crisis (BC) cells depends on the cooperation of BCR/ABL with other genes deregulated during disease progression. A recent study has identified RUNX1/AML1 transcription factor gene as a modulator of the cellular response towards IM in vitro and in vivo in mice, suggesting its possible involvement in disease persistence in IM-resistant CML patients.Purpose: We investigated RUNX1 molecular abnormalities (point mutations or cryptic chromosomal RUNX1 translocation to a novel recently described gene partner PRDM16 at 1p36) in 18 Ph+ leukemias. The observation that RUNX1 mutated allele is frequently duplicated by acquired trisomy of the altered chromosome 21 in acute myeloblastic leukemias (AML) prompt us to focus our analysis on a selected cohort (1 CP-CML, 3 AP-CML, 8 myeloid BC, 1 lymphoid BC, 2 de novo Ph+ ALL-B, 1 de novo AML and 1 therapy-related- Ph+ ALL) presenting acquired trisomy 21 along disease course.Methods: From peripheral blood leucocytes, recurrent mutations were investigated by sequencing DNA PCR fragments corresponding to exon 3 to 8 of RUNX1. RUNX1-PRDM16 fusion was investigated by RT-PCR (i.e. RUNX1 exon 5-PRDM16 exon 2 junction) and by FISH using the TEL/AML1 dual color/dual fusion translocation probes (Vysis, Downer's grove, IL, USA).Results: We report a high frequency (33%) of recurrent point mutations (4 in myeloid BC CML and 1 in CP-CML) within the DNA-binding region of RUNX1. We did not find any mutation in de novo BCR/ABL+ ALLs or lymphoid BC CMLs. Onset of RUNX1 mutations was detected at diagnosis or before the acquisition of trisomy 21. We also report a high frequency of RUNX1-PRDM16 fusion for 3 out of 7 investigated patients: 2 CMLs and, for the first time reported to our knowledge, 1 therapy-related BCR/ABL+ ALL. RUNX1-PRDM16 is probably transcribed at low levels since only few bone marrow metaphases with trisomy 21 were t(1;21)(p36;q22) positive. Two patients presented both RUNX1 mutations and RUNX1-PRDM16 fusion. All these events are associated with a poor survival: 14 out of 18 patients died with a median survival of 3 months after the diagnosis of the acquired clonal trisomy 21. Furthermore, none of the patients in our study raised durable optimal responses to IM.Conclusion: Our data support the concept of a cooperative effect of BCR/ABL with molecular RUNX1 abnormalities on the differentiation arrest phenotype observed during progression of CML and in BCR/ABL+ ALL. Our findings also support that it may exist a heterogeneous genetic status at diagnosis of CML, underlying the need of further investigations able to define more precisely the molecular disease characteristics at diagnosis for better therapeutic decision adjustments.

JAK2 V617F is a rare finding in de novo acute myeloid leukemia, but STAT3 activation is common and remains unexplained

Signal transducer and activator of transcription (STAT) proteins are phosphorylated and activated by Janus kinases (JAKs). Recently, several groups identified a recurrent somatic point mutation constitutively activating the hematopoietic growth factor receptor-associated JAK2 tyrosine kinase in diverse chronic myeloid disorders - most commonly classic myeloproliferative disorders (MPD), especially polycythemia vera. We hypothesized that the JAK2 V617F mutation might also be present in samples from patients with acute myeloid leukemia (AML), especially erythroleukemia (AML-M6) or megakaryoblastic leukemia (AML-M7), where it might mimic erythropoietin or thrombopoietin signaling. First, we documented STAT3 activation by immunoblotting in AML-M6 and other AML subtypes. Immunoperoxidase staining confirmed phosphorylated STAT3 in malignant myeloblasts (21% of cases, including all AML-M3 samples tested). We then analyzed genomic DNA from 162 AML, 30 B-cell lymphoma, and 10 chronic lymphocytic leukemia (CLL) samples for JAK2 mutations, and assayed a subset for SOCS1 and FLT3 mutations. Janus kinase2 V617F was present in 13/162 AML samples (8%): 10/13 transformed MPD, and three apparent de novo AML (one of 12 AML-M6, one of 24 AML-M7, and one AML-M2 - all mixed clonality). FLT3 mutations were present in 5/32 (16%), while SOCS1 mutations were totally absent. Lymphoproliferative disorder samples were both JAK2 and SOCS1 wild type. Thus, while JAK2 V617F is uncommon in de novo AML and probably does not occur in lymphoid malignancy, unexplained STAT3 activation is common in AML. Janus kinase2 extrinsic regulators and other proteins in the JAK-STAT pathway should be interrogated to explain frequent STAT activation in AML.

A Simple and Rapid Quantitative Method of Detection of the Common Achondroplasia Mutation: Analysis in Mismatch Repair Deficient Cells

Achondroplasia is the most common form of dwarfism and has an incidence of approximately 1/7500. In more than 97% of cases, it is caused by a recurrent point mutation, a G to A substitution at nucleotide position 1138 (G1138A) of the fibroblast growth factor receptor 3 gene. Although this is an autosomal dominant condition, more than 90% of all mutations occur sporadically making this one of the most mutagenic sites in the human genome. The reasons for the high spontaneous G1138A mutation rate are not known. This investigation was performed by developing a simple and rapid semi-quantitative allele specific PCR based assay capable of reliably detecting more than 25 mutant G1138A copies in a pool of 300 000 wild type molecules. Using this assay, the G1138A mutation frequency was measured in cell lines deficient in mismatch repair (LoVo, SW48) and comparing it with controls. No differences were found in the frequency of this point mutation between the mismatch repair deficient and wild type cell lines.

MEIS 1 expression is downregulated through promoter hypermethylation in AML1-ETO acute myeloid leukemias.

Retroviral insertional mutagenesis in BXH2 mice commonly induces myeloid leukemias. One of the most frequently involved genes in experimental studies is Meis 1. In contrast to other genes in murine models, Meis 1 has not been affected by recurrent chromosomal translocations or point mutations in human leukemias. We found a constant downregulation of the Meis 1 gene mRNA in AML1-ETO acute myeloid leukemias and in those cases harboring in frame mutations in the bZIP domain of CEBPalpha. The absence of the Meis 1 mRNA was not caused by inactivating point mutations in the coding sequence. Promoter hypermethylation was present in more than half of the cases (9/14), including samples obtained from the widely employed Kasumi-1 cell line. Double treatment with 5-Aza-2'-deoxycytidine and trichostatin A of the Kasumi-1 cell line partially reverses Meis 1 inhibition. HoxA9 levels were also low. In a cell line model (U937 Tet AML1-ETO), AML1-ETO expression was not associated with Meis 1 suppression at 72 h. Nevertheless, Meis 1 repression is dependent on the AML1-ETO transcript levels in treated leukemic patients. Chimeric products that arise from chromosomal translocations may be associated with locus-specific epigenetic inactivation. It remains to be investigated when this methylation process is acquired and which are the basic mechanisms underlying these molecular events in AML1-ETO and CEBPalpha-mutated AML.

Remarkable infidelity of polymerase gammaA associated with mutations in POLG1 exonuclease domain.

To better understand the still unknown pathologic mechanism involved in the accumulation of multiple mtDNA deletions in stable tissues. A large-scale screening of mtDNA molecules from skeletal muscle was performed in 14 patients with progressive external ophthalmoplegia (PEO) and 2 patients with mitochondrial neurogastrointestinal encephalomyopathy carrying mutations on ANT1, C10ORF2 or POLG1, and TP genes. Patients with at least one mutation in the exonuclease domain of POLG1 showed the highest frequency of individually rare point mutations only in the mtDNA control region; in addition, high levels, in terms of frequency and heteroplasmy, of recurrent mutations (A189G, T408A, and T414G) and alterations affecting the (HT)D310 region were detectable in many of the patients. Two homozygous POLG1 mutations, within the exonuclease domain, were able to induce an increased mutational burden also in fibroblasts from patients with PEO. Specific POLG1 mutations directly affect the integrity of the mtDNA by reducing its proof-reading exonuclease activity, resulting in the accumulation of heteroplasmic levels of both randomly rare and recurrent point mutations in the skeletal muscle tissue and fibroblasts.

Five novel and four recurrent point mutations in the antithrombin gene causing venous thrombosis.

We analyzed the antithrombin (AT) gene in 9 unrelated Japanese patients with thrombotic disease. All 7 exons, the splice junctions, and the 5'-flanking region of the AT gene were amplified by polymerase chain reaction and sequenced directly. Nine different point mutations, all in the heterozygous state, were identified. Five novel (M-32T, M89K, L146H, Q159X, and L409P) and 2 previously reported (R132X and R359X) point mutations were identified in patients with type 1 deficiency. Two different missense mutations, R393C and R393H, located in the protease reactive site were detected in patients with type 2 deficiency. No other sequence abnormalities in the AT gene were detected by direct sequencing. None of the mutations was present in 100 alleles from 50 unrelated Japanese control subjects Although type 1 deficiency was diagnosed in patient 7 on the basis of approximately 50% AT antigen and activity levels, the data indicated that the novel L409P mutation is a type 2 pleiotropic effects (PE) deficiency because its location in the C-terminal portion of the reactive site is similar to the locations of reported PE type mutations, and it is highly conserved among other serpins.

High mutational burden in the mtDNA control region from aged muscles: a single-fiber study

The ageing process is associated with the accumulation of somatic mutations of mitochondrial DNA (mtDNA). The aged human skeletal muscle tissue presents a mosaic of fibers when stained histochemically for cytochrome c oxidase (COX) activity with a proportion of COX negative fibers. Given the potential relevance of any alteration in the mtDNA control region for replication, we analysed the correlation between the presence of mutations and their degree of heteroplasmy and the COX phenotype in individual muscle fibers of aged healthy donors. A region of the mtDNA D-loop was cloned from single fiber-derived DNA and multiple clones were analysed. This strategy showed that a high level of mutational burden is present in all fibers and that several types of mtDNA rearrangements are detectable: recurrent (A189G, T408A and T414G) and rare point mutations, length variations affecting the homopolymeric tract and the (CA) n repeat and macrodeletions. The aggregate mutational load in the D-loop region correlated with the single fiber COX phenotype, suggesting that the cumulative burden of multiple, individually rare, mtDNA alterations might functionally impair the mitochondrial genetic machinery.

Sensitive and Efficient Detection of RB1 Gene Mutations Enhances Care for Families with Retinoblastoma

Timely molecular diagnosis of RB1 mutations enables earlier treatment, lower risk, and better health outcomes for patients with retinoblastoma; empowers families to make informed family-planning decisions; and costs less than conventional surveillance. However, complexity has hindered clinical implementation of molecular diagnosis. The majority of RB1 mutations are unique and distributed throughout the RB1 gene, with no real hot spots. We devised a sensitive and efficient strategy to identify RB1 mutations that combines quantitative multiplex polymerase chain reaction (QM-PCR), double-exon sequencing, and promoter-targeted methylation-sensitive PCR. Optimization of test order by stochastic dynamic programming and the development of allele-specific PCR for four recurrent point mutations decreased the estimated turnaround time to <3 wk and decreased direct costs by one-third. The multistep method reported here detected 89% (199/224) of mutations in bilaterally affected probands and both mutant alleles in 84% (112/134) of tumors from unilaterally affected probands. For 23 of 27 exons and the promoter region, QM-PCR was a highly accurate measure of deletions and insertions (accuracy 95%). By revealing those family members who did not carry the mutation found in the related proband, molecular analysis enabled 97 at-risk children from 20 representative families to avoid 313 surveillance examinations under anesthetic and 852 clinic visits. The average savings in direct costs from clinical examinations avoided by children in these families substantially exceeded the cost of molecular testing. Moreover, health care savings continue to accrue, as children in succeeding generations avoid unnecessary repeated anaesthetics and examinations.

A TWIST in the fate of human osteoblasts identifies signaling molecules involved in skull development.

The twist gene was first recognized by the Nobel Prize–winning laboratory of Nusslein-Volhard (1) and described in Drosophila as a zygotic gene required for dorsoventral patterning (2). Along the dorsoventral axis during the blastoderm stages of Drosophila, the most ventral nuclei constitute the mesodermal anlage which gives rise to cells that invaginate at gastrulation. With the loss of twist expression, homozygous mutant embryos fail to develop a mesoderm layer, resulting in few if any mesodermally derived internal organs. The head segments appear everted due to abnormal head involution, and the anterior end of the embryo is “twisted” in the egg. In mice, Twist expression also is initially observed along the dorsoventral gradient and then in the mesoderm and neural crest cell derivatives (3). Later, Twist is found in the somites, head mesenchyme, aortic arches, branchial arches, limb buds, and mesenchyme underneath the epidermis (4). Twist is expressed in the undifferentiated cells committed to muscle and cartilage development and inhibits their differentiation. Its transcripts are present in primary osteoblastic cells of the newborn mouse calvaria (5). Mouse embryos homozygous for the disruption of the Twist gene exhibit defects in the head mesenchyme, failure of neural tube closure in the cranial region, and pyknotic nuclei found in cranial nerves (6). Heterozygous mice display skull defects of poorly developed squamosal bones and of overdeveloped interparietal bones and limb abnormalities (7). The conservation of the Twist mutant phenotype also extends to humans. The heterozygous loss of the TWIST gene or its function results in a common autosomal dominant syndrome, Saethre-Chotzen, which is characterized by craniosynostosis, facial dysmorphisms, and hand and foot abnormalities (8, 9). Of these developmental abnormalities, craniosynostosis — the premature fusion of cranial sutures — is the most notable. Among the cranial sutures, the coronal suture across the width of the skull is most often prematurely synostosed, leading to a brachycephalic skull — one that is box-shaped and of shortened length. Mineralization of the cranium normally occurs directly from membrane-derived paraxial mesoderm. Ossification centers coalesce to form bones, and their osteogenic fronts meet to induce sutures along their lines of approximation. The skull continues to grow by appositional growth at the suture, with deposition of premineralized bone matrix along the suture margins. Several hypotheses have been proposed that premature synostosis is due to the abnormal development of the mesodermal blastema, accelerated bone growth, and/or increased differentiation. The discovery of the new disease gene TWIST generates an important tool that potentially could help in elucidating the signaling pathway and pathogenesis involved in craniosynostosis. A study reported in this issue of the JCI is evidence that this potential is being realized. Yousfi and colleagues from the Marie laboratory (10) identified TWIST target genes using human mutant calvaria osteoblastic cells from a child with Saethre-Chotzen syndrome. This individual carries a previously reported and apparently recurrent point mutation in TWIST that introduces a premature stop codon (8, 9, 11). The product of this Y103X mutant allele would be predicted to be truncated upstream of the basic helix-loop-helix (bHLH) domain, which is normally required for dimerization and for DNA binding. However, since the patient’s osteoblasts showed decreased TWIST mRNA and protein levels, it seems likely that mRNAs carrying this mutation do not survive nonsense-mediated decay and that the Y103X allele is a true null. In vitro and in vivo experiments with TWIST mutant cells showed increased cell growth and ability to form bonelike nodular structures. These cells and aggregates exhibited increased expression of osteoblastic markers, alkaline phosphatase, and type I collagen, independent of cell growth. However, TWIST mutant cells showed reduction of osteocalcin mRNA expression during osteogenesis. The altered expression of these osteoblastic genes with craniosynostosis is consistent with previously published observations (5, 12, 13). Overexpression of TWIST by transfection studies in osteosarcoma cells may inhibit osteoblast differentiation (12), and reduction of murine Twist expression occurs with osteoblastic maturation (5). Osteocalcin-deficient mice have increased bone formation (13). Therefore, the significance of the findings from the Marie laboratory (10) is that the premature ossification of cranial sutures in Saethre-Chotzen syndrome patients with TWIST mutations is caused by increased osteoblastic cell proliferation and differentiation (Figure ​(Figure11). Figure 1 Elements involved in the molecular pathway of sutural osteoblast differentiation (modified from ref. 15). Top: normal cellular differentiation and relationship of TWIST and FGFR signaling. Bottom: altered gene expression in TWIST Y103X mutant osteoblasts ...

Seven novel and four recurrent point mutations in the factor VIII (F8C) gene.

Haemophilia A is a X-linked bleeding disorder, caused by deficiency in the activity of coagulation factor VIII due to mutations in the corresponding gene. The most common defect in patients is an inversion of the factor VIII gene that accounts for nearly 45% of individuals with severe hemophilia A. Point mutations and small deletions/insertions are responsible for the majority of cases with moderate to mild clinical course and for half of the severe hemophilia A occurrences. The majority of these mutations are "private", because of the high mutation rate for this particular gene. We report on eleven pathological changes in the factor VIII sequence detected in male patients with haemophilia A or in female obligate carriers. Seven of these mutations are novel [E204N, E265X, M320T, F436C, S535C, N2129M and R2307P] and four have been previously identified [V162M, R527W, R1966X, and R2159C]. Genotype-phenotype correlations and computer prediction analysis on the effect of missense mutations on the secondary structure of the factor VIII protein are performed and the relationships evaluated.

Sex related expressivity of the phenotype in coronal craniosynostosis caused by the recurrent P250R FGFR3 mutation

A recurrent point mutation in the fibroblast growth factor receptor 3 (FGFR3) gene that converts proline 250 into arginine is commonly associated with coronal craniosynostosis and has allowed definition of...

Yield of mtDNA mutation analysis in 2,000 patients

The multiplex polymerase chain reaction-allele specific oligonucleotides (PCR/ASO) dot blot hybridization method was used to detect 44 mitochondrial DNA point mutations in 2,000 patients suspected as having mitochondrial DNA disorders. These point mutations are classified into four categories. Category I consists of primary disease-causing, heteroplasmic point mutations. Homoplasmic nucleotide substitutions that have been reported to be possibly disease associated are in Category II. Homoplasmic nucleotide substitutions that are thought to be benign polymorphism are included in category III. The novel nucleotide substitutions recently discovered in our laboratory by single strand conformation polymorphism analysis are in category IV. Frequencies of these 44 nucleotide substitutions in 2,000 patients and 262 control individuals were studied. The results indicated that analysis of 12 recurrent disease-causing point mutations in category I identified 5.4% of the patients suspected as having mitochondrial DNA disorders. Since the mitochondrial disorders are a group of complex, heterogeneous, and multisystemic diseases, it is often difficult to confirm clinical diagnosis without molecular studies. Thus, the multiplex PCR/ASO method is an effective approach for initial screening of mtDNA mutations in patients suspected as having mitochondrial DNA disorders.

Consequences of factor IX mutations in 26 families with haemophilia B.

Haemophilia B is due to a variety of mutations within the factor IX gene. In the Seattle series, 26 additional unrelated families have had a mutation identified within the past 2 years. Of these, 11 were common recurrent point mutations identifiable by rapid restriction digest screening; eight of these probably represent founder mutations. 15 others were identified by sequencing amplified coding region fragments; eight are novel. Two each had frameshift and donor splice mutations and 11 had missense mutations. Five of these mutations associated with normal levels of circulating dysfunctional factor IX were computer modelled into coordinates for factor IXa.

1.P.266 Detection of unknown and recurrent point mutations in hypercholesterolemic patients

1.P.266 Detection of unknown and recurrent point mutations in hypercholesterolemic patients