Number Of Mutant Alleles Research Articles

An update on clinical presentation and responses to therapy of patients with hereditary hypophosphatemic rickets with hypercalciuria (HHRH)

Pathogenic variants in solute carrier family 34, member 3 (SLC34A3), the gene encoding the sodium-dependent phosphate cotransporter 2c (NPT2c), cause hereditary hypophosphatemic rickets with hypercalciuria (HHRH). Here, we report a pooled analysis of clinical and laboratory records of 304 individuals from 145 kindreds, including 20 previously unreported HHRH kindreds, in which two novel SLC34A3 pathogenic variants were identified. Compound heterozygous/homozygous carriers show above 90% penetrance for kidney and bone phenotypes. The biochemical phenotype for heterozygous carriers is intermediate with decreased serum phosphate, tubular reabsorption of phosphate (TRP (%)), fibroblast growth factor 23, and intact parathyroid hormone, but increased serum 1,25-dihydroxy vitamin D, and urine calcium excretion causing idiopathic hypercalciuria in 38%, with bone phenotypes still observed in 23% of patients. Oral phosphate supplementation is the current standard of care, which typically normalizes serum phosphate. However, although in more than half of individuals this therapy achieves correction of hypophosphatemia it fails to resolve the other outcomes. The American College of Medical Genetics score correlated with functional analysis of frequent SLC34A3 pathogenic variants in vitro and baseline disease severity. The number of mutant alleles and baseline TRP (%) were identified as predictors for kidney and bone phenotypes, baseline TRP (%) furthermore predicted response to therapy. Certain SLC34A3/NPT2c pathogenic variants can be identified with partial responses to therapy, whereas with some overlap, others present only with kidney phenotypes and a third group present only with bone phenotypes. Thus, our report highlights important novel clinical aspects of HHRH and heterozygous carriers, raises awareness to this rare group of disorders and can be a foundation for future studies urgently needed to guide therapy of HHRH.

Mutant DHFR Prevalence and Pyrimethamine Resistance among Asymptomatic Adolescents in Kwara State

Plasmodium falciparum malaria remains a leading public health problem in sub-Saharan Africa and its control is seriously challenged by drug resistance. Resistance to sulfadoxine-pyrimethamine is mediated by point mutations in genes encoding the target enzymes dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps). Blood from a total of 176 subjects aged 11-19 years from Ilorin East and Irepodun Local Government areas (LGAs) was examined for mutant alleles of the Plasmodium falciparum dihydrofolate reductase gene by polymerase chain reaction (PCR) at codon 51 and 108 respectively. Rapid Diagnostic Test kits were used for the malaria test. Of the 176 participants, 73 (41.5%) subjects tested positive for malaria parasite while 95 (54.0%) tested negative. For both N51I and S108N SNPscreening, mutant alleles were dominant over the wild type. The Iponrin community (Ilorin East LGA) recorded the highest percentage of mutant alleles 14 (33.33%) of the N51I SNP, while the Igbonla community (Irepodun LGA) had the least number of mutant alleles 8 (19.04%). On the other hand, the Alakuko community recorded the highest number of alleles 14 (31.80%) for S108N mutant while the Igbonla community (Irepodun LGA) had the least number of mutant alleles 8 (18.18%). There was widespread pyrimethamine resistance among the studied population and the malaria parasite remains persistent among the studied population. Therefore, there is a need for monitoring antimalarial drug resistance in Nigeria for prompt management of the antimalarial drug resistance menace.

PS1436 IFN‐Α TREATMENT NORMALIZES THE PLATELET COUNTS AND REDUCES QUIESCENT STEM CELLS IN A CALR‐DEL52 TRANSGENIC MOUSE MODEL, IN VIVO

Background:Treatment options for Myeloproliferative Neoplasms (MPN) have mainly been limited to disease managing risk of thromboembolic events and long‐term transformation to acute myelogenous leukemia and myelofibrosis. To date, type I interferons (IFNs) are the only class of drugs with curative potential in MPN as they can actually reduce the allelic burden of mutant clones in patients. The mechanism of action of IFN in MPNs remains elusive. This is mainly because modelling IFN treatment in mouse models has proven to be difficult due to the short half‐life of murine non‐pegylated IFNs. This shortcoming was solved by the murine pegylated IFN‐α (murine ropeginterferon‐a, mRopeg) developed by PharmaEssentia (Taipei, Taiwan) which can be used to model IFN treatment in mouse models of MPN.Aims:Generation of a conditional transgenic mouse model for CALR‐del52 induced MPN and modelling IFN treatment in these mice using murine pegylated IFN‐α (mRopeg).Methods:The CALR‐del52 transgenic mice (with human mutant sequence) were generated on a C57BL/6 background by homologous recombination of embryonic stem cells at Ozgene (Perth, Australia). The CALR‐del52 floxed mice were bred with vavCre transgenics, thereby allowing the expression of CALR‐del52 specifically in the hematopoietic cells. MPN phenotype in the mice was characterized by peripheral blood counts and flow cytometry. IFN treated mice received mRopeg weekly (600 ng) by subcutaneous injection for 4 weeks. Control mice received the same volume of vehicle (in this case, phosphate buffered saline – PBS).Results:Heterozygous CALR‐del52fl/+;vavCre mice displayed elevated platelet counts starting from 6 weeks of age, with normal white blood cell and red blood cell counts. However, homozygous CALR‐del52fl/fl;vavCre mice developed much higher platelet counts associated with increased white blood cell and decreased red blood cell counts. Homozygous CALR‐del52 mice also developed splenomegaly starting from 6 months of age. Also at 6 months, there was significant increase in the megakaryocyte population in the bone marrow, along with significant increase in LSK cells (Lin− Sca+ kit+) and the quiescent (Fraction A) stem cell population. IFN‐α treatment of CALR‐del52 transgenic mice for 4 weeks resulted in the normalization of the platelet counts to wild type levels. Interestingly, the total stem cell population (LSK cells) in the bone marrow increased upon IFN‐α treatment. However, the percentage of quiescent stem cells (Fraction A) reduced, while the percentage of cycling stem cells (Fraction B) and multipotent stem cells (Fraction C) was increased.Summary/Conclusion:Expression of CALR‐del52 in hematopoietic cells leads to the development of MPN phenotype in mice, and the severity positively correlated with the number of mutant alleles. Heterozygous CALR‐del52 mice developed an Essential Thrombocythemia (ET) like phenotype, with high platelet counts but normal white blood and red blood cell counts. However, the homozygous CALR‐del52 mice developed signs of myelofibrosis with age, including reduced red blood cell counts and splenomegaly. IFN‐α treatment resulted in normalization of platelet numbers to wild type levels within 4 weeks of treatment. Importantly, it also resulted in significant reduction of the quiescent hematopoietic stem cells, while cycling and multipotent stem cells were increased. This suggests that IFN‐α induces proliferation of quiescent stem cells into cycling and multipotent stem cells in CALR‐del52 mutant cells.image

Abstract 5650: Functional proteomics for precise cancer diagnosis and therapy using the MSK Quantitative Cancer Proteomics Atlas

Abstract The advent of molecular biology and molecular profiling in clinical medicine has transformed our understanding of the molecular basis of human cancer. As a result, we are increasingly improving the classification of human tumors based on their specific genetic and molecular mechanisms of pathogenesis. However, currently only a small number of mutant alleles guide treatment decisions, while most observed mutations remain of unknown pathologic and clinical significance. In addition, even for recently approved drugs, such as those targeting activated kinase signaling, clinical efficacy is highly varied, with no currently satisfactory means to identify molecular markers of response and resistance. To address this need, and broadly enable transformative future advances in precision oncology and patient outcomes, we have now developed the Quantitative Cancer Proteomics Atlas (QCPA). This technology permits highly multiplexed, quantitative analysis of the expression and biochemical activity of hundreds of proteins, covering most recurrently mutated and known pathogenic pathways in cancer cells, and designed to be applied to clinically accessible patient specimens. QCPA enables concomitant measurement of cellular concentration and stoichiometry of regulatory post-translational modification of key proteins mediating aberrant survival and proliferation. The activation status of these proteins is then used as a proxy to infer regulatory status of relevant biochemical pathways. These sentinel proteins include 467 known effectors of kinase regulatory signaling, apoptosis, stress response, and proliferation. QCPA measurements can be made using conventional parallel reaction monitoring mass spectrometry, or using an improved method termed accumulated ion monitoring (Cifani & Kentsis 2017). We established analytical metrics of QCPA assays in cancer cell lines, and demonstrated sensitivity and quantification accuracy amenable to measure endogenous chemically modified proteins from microgram specimens. We then demonstrated the ability of QCPA profiling to detect differential biochemical activities in response to defined stimuli in vitro. Next, we deployed QCPA profiling to reveal functional differences between primary human acute myeloid leukemia specimens and CD34+ myeloid progenitors, measuring specific activation of biochemical pathways in cancer cells. Finally, we tested the clinical utility of functional proteomic profiling by determining molecular markers of response and resistance to ERK pathway inhibitors in patient specimens. Thus, QCPA functional proteomic profiling provides a tool to achieve precise diagnosis and rational targeted cancer therapies. Citation Format: Paolo Cifani, Ahmet Dogan, David B. Solit, Alex Kentsis. Functional proteomics for precise cancer diagnosis and therapy using the MSK Quantitative Cancer Proteomics Atlas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5650.

T786C Mutation in the Endothelial Nitric Oxide Synthase Gene in Patients With Primary Osteonecrosis.

Mutations in the T786C endothelial nitric oxide synthase gene (eNOS) are associated with osteonecrosis and Prinzmetal's angina. Nitric oxide is necessary for bone health and ameliorates Prinzmetal's angina. This study compared mutations of T786C eNOS in 146 patients with primary osteonecrosis, 114 patients with Prinzmetal's angina, and 83 normal control subjects. Patients with osteonecrosis had more mutant eNOS alleles than control subjects (42% vs 22%, respectively; P<.0001) but had the same number of mutant alleles as patients with Prinzmetal's angina (42% vs 41%, respectively; P=.7), who in turn had more mutant eNOS alleles than control subjects (41% vs 22%, respectively; P=.0001). Of 146 patients with primary osteonecrosis, 65 (45%) had none of the 5 thrombophilias (Factor V Leiden heterozygosity, high levels of Factors VIII and XI, anticardiolipin antibody immunoglobulin M, and homocysteine) that otherwise distinguished patients with osteonecrosis from control subjects (P<.05). No associations were found between eNOS hetero-homozygosity and the 5 major thrombophilias in primary osteonecrosis. Of the 65 patients who had osteonecrosis but no major thrombophilias, for 41 (28% of the total sample of 146), eNOS hetero-homozygosity was the only abnormality. Normalization of nitric oxide levels with l-arginine 9 g/d or l-citrulline 800 mg/d, both of which relieve vasospastic angina in Prinzmetal's angina, which has the same eNOS genotype as primary osteonecrosis, may slow or stop the progression of osteonecrosis. Placebo-controlled trials of patients with primary osteonecrosis who are hetero-homozygous for the T786C eNOS mutation and have no major thrombophilias are needed to assess the safety and efficacy of this treatment. [Orthopedics. 2017; 40(5):e898-e903.].

The influence of genetic drift on the formation and stability of polymorphisms arising from negative frequency-dependent selection

We consider the simplest form of negative frequency-dependent selection in a biallelic haploid population, where the selection coefficient of a mutant allele is a linear function of the allele׳s frequency, and changes from positive to negative as the frequency is increased. In an effectively infinite population this behaviour leads to a stable polymorphism. We present a theoretical investigation of what occurs in a finite population, where a long-lived polymorphism may be formed, but which fluctuates and ultimately disappears due to random genetic drift. We model the dynamics as a branching process and explicitly take into account differences between the census population size and the effective population size, which play different roles in the dynamics. We characterise the behaviour of the population in terms of three distinct timescales associated with: (i) early loss of mutant alleles, (ii) achievement of the long-lived polymorphism, (iii) disappearance of the polymorphism. Timescales (i) and (iii) depend on the effective population size and are, as a consequence, affected by random genetic drift, while timescale (ii) depends primarily on the census size and is relatively insensitive to genetic drift. Analysis and simulations of the branching process clarify the different influences of the census and effective population sizes. One substantial quantitative difference, between populations where the effective and census population sizes coincide and where they differ, lies in the number of mutant alleles in the long-lived polymorphism. This number is approximately proportional to the census size. Thus assuming the census size equals a much smaller effective population size predicts a much smaller number of mutants in the long-lived polymorphism.

Phenotype and molecular characteristics in 45 Chinese children with 5α-reductase type 2 deficiency from South China.

Affected by steroid 5α-reductase type 2 deficiency (5α-RD2), 46, XY individuals present divergent phenotypes characterized by undervirilization of male external genitalia. To identify the disorder, mutational analysis of 5α-reductase type 2 gene (SRD5A2) is a reliable approach. The genotype-phenotype relationship has not been elucidated. To improve the diagnosis and expand the knowledge of the disease, we collected and analysed relevant data of clinical diagnosis, biological investigation and molecular determination in 45 children with the SRD5A2 gene mutations from South China in our centre. We studied a cohort of 45 Chinese children with SRD5A2 gene mutations. Isolated microphallus (35·6%) and microphallus associated with various degrees of hypospadias (55·6%) were frequent phenotype. Female external genitalia with clitoromegaly (8·9%) were rare. 16 of 18 (88·9%) cases had hCG-stimulated T/DHT ratio above 10. In 45 patients, we identified 15 different mutations. Five have never been described: p.His90ThrfsX31, p.Gly21Arg, p.Gly149Asp, p.Arg145Leu and p.Gly66Arg. The p.Arg227Gln mutation was detected in 41 (91·1%) patients. The p.Leu89Val polymorphism was found in 38 (84·4%) patients. Homozygous mutations were presented in 16 (35·6%) patients, compound heterozygous mutations in 20 (44·4%) patients, compound heterozygous mutations alone with the p.Leu89Val polymorphism in nine (20·0%) patients. Exons 1 and 4 were most affected, and the number of mutant alleles per exon was 78·1% and 12·2%, respectively. The study demonstrated a wide spectrum of phenotypes, biological profiles and genotypes in the children with 5α-RD2 from South China. The heterozygous mutation p.Arg227Gln is presumably a hot spot mutation and suggests a founder effect in the population of South China that may explain a moderate phenotype among our patients. Our report provides new insights into the molecular mechanism of 5α-RD2 and help to the diagnosis and treatment of this disease.

Common breast cancer risk variants in the post-COGS era: a comprehensive review

Breast cancer has a strong heritable component, with approximately 15% of cases exhibiting a family history of the disease. Mutations in genes such as BRCA1, BRCA2 and TP53 lead to autosomal dominant inherited cancer susceptibility and confer a high lifetime risk of breast cancers. Identification of mutations in these genes through clinical genetic testing enables patients to undergo screening and prevention strategies, some of which provide overall survival benefit. In addition, a number of mutant alleles have been identified in genes such as CHEK2, PALB2, ATM and BRIP1, which often display incomplete penetrance and confer moderate lifetime risks of breast cancer. Studies are underway to determine how to use the identification of mutations in these genes to guide clinical practice. Altogether, however, mutations in high and moderate penetrance genes probably account for approximately 25% of familial breast cancer risk; the remainder may be due to mutations in as yet unidentified genes or lower penetrance variants. Common low penetrance alleles, which have been mainly identified through genome-wide association studies (GWAS), are generally present at 10 to 50% population frequencies and confer less than 1.5-fold increases in breast cancer risk. A number of single nucleotide polymorphisms (SNPs) have been identified and risk associations extensively replicated in populations of European ancestry, the number of which has substantially increased as a result of GWAS performed by the Collaborative Oncological Gene–environment Study consortium. It is now estimated that 28% of familial breast cancer risk is explained by common breast cancer susceptibility loci. In some cases, SNP associations may be specific to different subsets of women with breast cancer, as defined by ethnicity or estrogen receptor status. Although not yet clinically established, it is hoped that identification of common risk variants may eventually allow identification of women at higher risk of breast cancer and enable implementation of breast cancer screening, prevention or treatment strategies that provide clinical benefit.

Abstract SY11-02: Detection of tumor-associated mutations in circulating DNA: clinical applications and experiences.

Abstract The detection of tumor-associated mutations is of paramount importance in the era of personalized medicine. Mutational testing is now a prerequisite for the use of some approved therapies (e.g., KRAS for cetuximab in colorectal cancer [CRC]; BRAF for vemurafenib in melanoma), and these clearly established correlations between tumor mutational status and drug response elevate the importance and urgency of evaluating such associations in clinical trials of investigational drugs. While archival primary tumor tissue is often used for mutational evaluation, such material has inherent limitations, which may be overcome by recent technological developments enabling the detection of tumor-associated mutations using plasma-derived DNA. For example, when a tumor tissue specimen is unavailable, use of plasma DNA would allow mutational status to be ascertained without the need for an invasive procedure to obtain a new tumor sample. In addition, it is now apparent that most patients treated with targeted therapies will eventually develop drug resistance, often via the acquisition of new tumor-associated mutations; these mutations may vary not only between patients but also between metastases within an individual patient. As such, the mutational status of an archival primary tumor specimen may not be relevant to guide the selection of subsequent therapies, and obtaining fresh tumor tissue from each metastasis that arises following the development of drug resistance is impractical. In such instances, mutational analysis of DNA derived from a real-time plasma sample obtained after the onset of drug resistance may offer advantages in terms of both availability and biological relevance, since new mutations acquired in response to a particular targeted therapy may be detectable in plasma DNA. Finally, mutational analysis of plasma DNA may be useful in clinical trials to evaluate potential correlations between mutational status and clinical outcome. For such exploratory analyses, the collection of archival tumor specimens from a high proportion of enrolled patients can be logistically and ethically difficult to achieve, not to mention of questionable relevance given that acquired mutations would not be detectable in these specimens. Collection of fresh tumor tissue samples at study entry would provide biologically relevant material, but can be problematic and costly to obtain in large, global clinical trials. Thus, the utility of plasma DNA for real-time mutational analysis in the clinical-trial setting offers several distinct advantages. Since DNA derived from both normal and tumor cells exists in the circulation, the detection of tumor-associated mutations in plasma DNA requires the ability to identify a relatively small number of mutant alleles among an excess of wild-type alleles. With the goal of identifying the most suitable technology for this purpose, we conducted a comparison of available methodologies and found that BEAMing technology (Beads, Emulsions, Amplification, and Magnetics) offered very sensitive detection of known tumor-associated mutations using plasma DNA, although this technique is not well suited for the discovery of previously unknown mutations. We have now used BEAMing to analyze more than 2,000 patient samples collected from oncology clinical trials, allowing us to evaluate a number of genes (e.g., KRAS, NRAS, HRAS, BRAF, PIK3CA, AKT1, EGFR, KIT, and PDGFRA) in different cancer types (e.g., CRC, gastrointestinal stromal tumors, hepatocellular carcinoma, non-small-cell lung cancer, and breast cancer). We have used BEAMing of plasma DNA both prospectively, to enroll patients into a phase I trial based on a molecular profile of interest, and retrospectively, to evaluate potential associations between mutational status and clinical outcome in phase II and III trials. In many of these trials, collection of both fresh plasma and archival tumor tissue from a subset of patients has enabled us to compare mutational status in patient-matched plasma and tumor samples. Our experiences with BEAMing of plasma DNA to determine tumor-associated mutational status will be discussed. Citation Format: Michael Jeffers, Chetan D. Lathia, Scott M. Wilhelm, Dimitris Voliotis, Dirk Laurent, Carol E. Pena. Detection of tumor-associated mutations in circulating DNA: clinical applications and experiences. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr SY11-02. doi:10.1158/1538-7445.AM2013-SY11-02

Single nucleotide polymorphisms in the phosphatidylethanolamine N‐methyltransferase gene may influence choline requirement

Humans derive the choline they require from their diet and from de novo biosynthesis catalyzed by the enzyme phosphatidylethanolamine N‐methyltransferase (PEMT). In previous studies, we found that pre‐menopausal women were resistant to developing organ dysfunction when consuming a low choline diet as compared to post‐menopausal women and men. Also, we showed that the gene PEMT is estrogen responsive, allowing pre‐menopausal women to form more of their required choline via PEMT. However, some pre‐menopausal women (44%) did develop choline deficiency and we found that they had a haplotype containing an allele for a single nucleotide polymorphism (SNP) in PEMT (rs12325817). To determine whether this effect of the SNP increased with the number of mutant alleles, we recruited 27 pre‐menopausal women with 0, 1 or 2 of the alleles. They were fed a baseline diet containing 550 mg choline/day for 10 days, followed by a choline deficient diet for up to 42 days, and a repletion diet with 850 mg choline for 10 days. Subjects were deemed clinically choline deficient if they developed hepatic steatosis or muscle dysfunction on the low choline diet and if the organ dysfunction disappeared when choline was returned to the diet. We found that 80% of the subjects who were homozygous for the SNP became choline deficient compared to 43% with 1 variant allele, and 13% without the SNP (p=0.02). We conclude that the risk of a pre‐menopausal woman becoming choline deficient is proportional to the allele dose for the SNP rs12325817.This research is supported by grants from NIH‐ DK55865, DK56350 and RR00046.

A novel mutation in Hr causes abnormal hair follicle morphogenesis in hairpoor mouse, an animal model for Marie Unna Hereditary Hypotrichosis

Hairpoor mice (Hr(Hp)) were derived through N-ethyl-N-nitrosourea (ENU) mutagenesis. These mice display sparse and short hair in the Hr(Hp)/+ heterozygous state and complete baldness in the Hr(Hp)/Hr(Hp) homozygous state. This phenotype was irreversible and was inherited in an autosomal semidominant manner. Hair follicles (HFs) of Hr(Hp)/+ mice underwent normal cycling and appeared normal, although smaller than those of the wild-type mice. In contrast, HFs of Hr(Hp)/Hr(Hp) mice became cyst-like structures by postnatal day (P) 21. The number and length of vibrissae decreased in a dose-dependent manner as the number of mutant alleles increased. A positional candidate gene approach was used to identify the gene responsible for the hairpoor phenotype. Genetic linkage analysis determined that the hairpoor locus is 2 cm from D14Mit34 on chromosome 14. Sequence analysis of the exons of the candidate gene hairless revealed a T-to-A transversion mutation at nucleotide position 403 (exon 2), presumably resulting in abolishment of an upstream open reading frame (uORF). In addition, we also found that the near-naked mouse (Hr(N)), a spontaneously arising mutant, harbors a A402G transition in its genome. Both mutations were in the uATG codon of the second uORF in the 5' UTR and corresponded to the mutations identified in Marie Unna Hereditary Hypotrichosis (MUHH) patients. In the present study we describe the phenotype, histological morphology, and molecular etiology of an animal model of MUHH, the hairpoor mouse.

Plastid chaperonin proteins Cpn60α and Cpn60β are required for plastid division in Arabidopsis thaliana

BackgroundPlastids arose from a free-living cyanobacterial endosymbiont and multiply by binary division as do cyanobacteria. Plastid division involves nucleus-encoded homologs of cyanobacterial division proteins such as FtsZ, MinD, MinE, and ARC6. However, homologs of many other cyanobacterial division genes are missing in plant genomes and proteins of host eukaryotic origin, such as a dynamin-related protein, PDV1 and PDV2 are involved in the division process. Recent identification of plastid division proteins has started to elucidate the similarities and differences between plastid division and cyanobacterial cell division. To further identify new proteins that are required for plastid division, we characterized previously and newly isolated plastid division mutants of Arabidopsis thaliana.ResultsLeaf cells of two mutants, br04 and arc2, contain fewer, larger chloroplasts than those of wild type. We found that ARC2 and BR04 are identical to nuclear genes encoding the plastid chaperonin 60α (ptCpn60α) and chaperonin 60β (ptCpn60β) proteins, respectively. In both mutants, plastid division FtsZ ring formation was partially perturbed though the level of FtsZ2-1 protein in plastids of ptcpn60β mutants was similar to that in wild type. Phylogenetic analyses showed that both ptCpn60 proteins are derived from ancestral cyanobacterial proteins. The A. thaliana genome encodes two members of ptCpn60α family and four members of ptCpn60β family respectively. We found that a null mutation in ptCpn60α abolished greening of plastids and resulted in an albino phenotype while a weaker mutation impairs plastid division and reduced chlorophyll levels. The functions of at least two ptCpn60β proteins are redundant and the appearance of chloroplast division defects is dependent on the number of mutant alleles.ConclusionOur results suggest that both ptCpn60α and ptCpn60β are required for the formation of a normal plastid division apparatus, as the prokaryotic counterparts are required for assembly of the cell division apparatus. Since moderate reduction of ptCpn60 levels impaired normal FtsZ ring formation but not import of FtsZ into plastids, it is suggested that the proper levels of ptCpn60 are required for folding of stromal plastid division proteins and/or regulation of FtsZ polymer dynamics.

Response: Re: Pharmacogenomic Variation of CYP2D6 and the Choice of Optimal Adjuvant Endocrine Therapy for Postmenopausal Breast Cancer: A Modeling Analysis

Adjuvant endocrine treatment with aromatase inhibitors improves disease-free survival compared with tamoxifen in postmenopausal women with estrogen receptor-positive breast cancer. This difference could be due to differences in tamoxifen metabolism because levels of endoxifen, the active tamoxifen metabolite, vary with the number of mutant alleles, including the *4 allele, of the gene encoding cytochrome P450 2D6 (CYP2D6).We created a Markov model to determine whether tamoxifen or aromatase inhibitor monotherapy maximized 5-year disease-free survival for patients with the wild-type CYP2D6 genotype (wt/wt). Annual risks of recurrence with aromatase inhibitors and tamoxifen in breast cancer patients who were not selected by CYP2D6 genotype were derived from the Breast International Group 1-98 trial. Genotype frequencies and the hazard ratio for cancer recurrence on tamoxifen among patients with the *4/*4 genotype relative to the wt/wt or wt/*4 genotypes (HR(*4/*4) = 1.86) were based on data from an analysis of the North Central Cancer Treatment Group trial of adjuvant tamoxifen. We explored the impact of CYP2D6(*4) heterozygosity on disease-free survival for wt/wt patients by studying a range of effect (ie, recurrence on tamoxifen) estimates, from no effect of the single mutation (Eff(wt/*4) = 0, recurrence rate in wt/*4 patients same as that in wt/wt patients) to complete effect (Eff(wt/*4) = 1 recurrence rate in wt/*4 patients same as that in *4/*4 patients).With HR(*4/*4) = 1.86 and Eff(wt/*4) = 0.5, the 5-year disease-free survival of tamoxifen-treated patients with no mutations (wt/wt) was 83.9%, that is, essentially the same as that (84.0%) for genotypically unselected patients who were treated with aromatase inhibitors. With greater HR(*4/*4) estimates, disease-free survival with tamoxifen exceed that with aromatase inhibitors in wt/wt patients, even at lower assumed Eff(wt/*4) ratios.Modeling suggests that among patients who are wild type for CYP2D6, 5-year disease-free survival outcomes are similar to or perhaps even superior with tamoxifen than with aromatase inhibitors. Endocrine therapy tailored to CYP2D6 genotype could be considered for women who are newly diagnosed with breast cancer, particularly those who have with concerns about either the relative toxicity or the increased cost of aromatase inhibitors.

Pharmacogenomic Variation of CYP2D6 and the Choice of Optimal Adjuvant Endocrine Therapy for Postmenopausal Breast Cancer: A Modeling Analysis

Adjuvant endocrine treatment with aromatase inhibitors improves disease-free survival compared with tamoxifen in postmenopausal women with estrogen receptor-positive breast cancer. This difference could be due to differences in tamoxifen metabolism because levels of endoxifen, the active tamoxifen metabolite, vary with the number of mutant alleles, including the *4 allele, of the gene encoding cytochrome P450 2D6 (CYP2D6). We created a Markov model to determine whether tamoxifen or aromatase inhibitor monotherapy maximized 5-year disease-free survival for patients with the wild-type CYP2D6 genotype (wt/wt). Annual risks of recurrence with aromatase inhibitors and tamoxifen in breast cancer patients who were not selected by CYP2D6 genotype were derived from the Breast International Group 1-98 trial. Genotype frequencies and the hazard ratio for cancer recurrence on tamoxifen among patients with the *4/*4 genotype relative to the wt/wt or wt/*4 genotypes (HR(*4/*4) = 1.86) were based on data from an analysis of the North Central Cancer Treatment Group trial of adjuvant tamoxifen. We explored the impact of CYP2D6(*4) heterozygosity on disease-free survival for wt/wt patients by studying a range of effect (ie, recurrence on tamoxifen) estimates, from no effect of the single mutation (Eff(wt/*4) = 0, recurrence rate in wt/*4 patients same as that in wt/wt patients) to complete effect (Eff(wt/*4) = 1 recurrence rate in wt/*4 patients same as that in *4/*4 patients). With HR(*4/*4) = 1.86 and Eff(wt/*4) = 0.5, the 5-year disease-free survival of tamoxifen-treated patients with no mutations (wt/wt) was 83.9%, that is, essentially the same as that (84.0%) for genotypically unselected patients who were treated with aromatase inhibitors. With greater HR(*4/*4) estimates, disease-free survival with tamoxifen exceed that with aromatase inhibitors in wt/wt patients, even at lower assumed Eff(wt/*4) ratios. Modeling suggests that among patients who are wild type for CYP2D6, 5-year disease-free survival outcomes are similar to or perhaps even superior with tamoxifen than with aromatase inhibitors. Endocrine therapy tailored to CYP2D6 genotype could be considered for women who are newly diagnosed with breast cancer, particularly those who have with concerns about either the relative toxicity or the increased cost of aromatase inhibitors.

Could treatment with tamoxifen be superior to aromatase inhibitors in early-stage breast cancer after pharmacogenomic testing? A modeling analysis

502 Background: Adjuvant endocrine treatment with upfront aromatase inhibitors yields improved disease-free survival compared to tamoxifen in unselected women with estrogen receptor-positive breast cancer. However, levels of endoxifen, the active tamoxifen metabolite, are known to vary with the number of mutant alleles of the cytochrome P450 CYP2D6 enzyme. We created a Markov model to examine whether the optimal treatment strategy for patients with wild type genetics (wt/wt) might differ from that for patients with homozygous (*4/*4) or heterozygous (wt/*4) mutations. Methods: Annual recurrence risks with aromatase inhibitors and tamoxifen in unselected women were taken from the BIG 1–98 trial. Based on data from Goetz et al., we assumed that the percent of patients who are homozygous and heterozygous for mutations are 6.8% and 21.1%, respectively, and that the hazard ratio (HR) for increased cancer recurrence on tamoxifen among *4/*4 carriers is 1.86 relative to patients with wt/wt or wt/*4 genotypes. Because the efficacy of tamoxifen among wt/*4 patients is not known, we tested the full possible range, from efficacy same as wt/wt patients (Ehet=0) to that of *4/*4 mutation carriers (Ehet=1). Results: In the unselected group, 5-year disease-free survival (5-DFS) with aromatase inhibitors and tamoxifen was 0.840 and 0.813, respectively. Under baseline assumptions, tamoxifen is more effective than aromatase inhibitors among wt/wt patients as long as the effect in heterozygotes is at least 54% (Ehet=0.54) of that in *4/*4 patients. With increasing HR for *4/*4 patients, tamoxifen estimates exceed those of aromatase inhibitors in the wt/wt cohort even at lower assumed Ehet ratios (see table ). Conclusion: In patients without CYP2D6 mutations, modeling suggests that initial treatment with tamoxifen could be superior to treatment with aromatase inhibitors, supporting the use of genetic testing to determine the optimal treatment strategy. [Table: see text] No significant financial relationships to disclose.

Phalaris canariensis is a domesticated form of P. brachystachys

Three hybrid plants were obtained by hot-water emasculation of Phalaris canariensis L. panicles immediately before anthesis, followed by the daily application of pollen from P. brachystachys Link. The hybrids were fully fertile, and the seed retention characteristic of P. canariensis was inherited in the F1, F2 and F3 generations as a single recessive trait, showing that P. canariensis is a cultigen derived from P. brachystachys. The dominant allele for seed shedding is designated Ssh. All 1008 plants examined in segregating F3 families had either short sterile florets and shed their seeds, or long sterile florets and retained their seeds, i.e. there were no recombinants. No seed retaining BC1F2 plants could be found in the progeny of (P. truncata Guss.×P. canariensis)×P. truncata, suggesting that the ssh'allele' may be a number of mutant alleles in a segment of chromosome in which recombination is suppressed in hybrids of P. canariensis with P. brachystachys, but in not those with P. truncata. Compared with P. brachystachys, P. canariensis has heavier seeds, stronger panicle branches and thicker peduncles. These traits were polygenically inherited in F2 and thus appear to have been developed by the accumulation of alleles during cultivation of P. canariensis as a grain crop. Modified seeds with much reduced sterile florets were found in the terminal spikelets of most panicle branches of P. brachystachys. Unlike the typical seeds, the modified seeds were often retained in the outer glumes when the panicle fragmented. It seems that this system allows these seeds to be dispersed much further by animals, wind or water than typical seeds. This mechanism has been lost in P. canariensis.

A large scale screen for genes (3rd chromosome) related to Wingless signaling pathway.

A wing specific F1 genetic screen was carried out using the powerful Drosophila genetic system, combined with yeast FRT/FLP and GAL4/UAS system. Form the wing phenotypes and germline clone embryonic cuticle phenotypes observed in these mutant alleles, a number of mutant alleles of known or unknown genes were isolated. Among them, fifteen mutant alleles related to Wingless signal transduction were further isolated; the arm of these mutations located were determined, and their location in the chromosome were roughly mapped.

Enlarged endolymphatic duct and sac syndrome: relationship between MR findings and genotype of mutation in Pendred syndrome gene.

Pendred syndrome (PDS) is characterized by profound deafness in childhood, positive perchlorate challenge, and goiter. PDS is often associated with enlarged endolymphatic duct and sac (EEDS), and recently, PDS gene mutations have been reported even in those patients with EEDS without classic Pendred syndrome. In a previous report, the number of mutant alleles was correlated with the degree of subclinical thyroid abnormality, but not with hearing loss, in patients with missense mutation H723R. It also has been reported that the hearing loss in EEDS was not correlated with the EEDS volume, cochlear modiolar area, or signal intensity of the endolymphatic sac. We evaluated the correlations between the number of mutant alleles and these parameters in patients with EEDS to investigate the mechanisms underlying this condition. The study group was comprised of 16 Japanese patients with EEDS diagnosed by MR imaging. The H723R mutation was homozygous in six patients and heterozygous in six patients, with no mutation found in four patients. The modiolar area, EEDS volume, and signal intensity ratio (sac signal/cerebrospinal fluid signal) were not significantly correlated with the number of mutant alleles. PDS gene mutations may not be the only cause of EEDS, and the mechanisms underlying EEDS remain unclear.

The striatal dopaminergic deficit is dependent on the number of mutant alleles in a family with mutations in the parkin gene: evidence for enzymatic parkin function in humans

Autosomal recessive parkinsonism associated with mutations in the parkin gene represents a monogenic form of hereditary parkinsonism. We performed [ 18F]6-fluorodopa (FDOPA) positron emission tomography as a measurement of the nigrostriatal dopaminergic system as well as extensive haplotype analysis of the PARK 2 gene locus in 14 subjects with parkin mutations. In parkin subjects, the reduction of striatal FDOPA uptake increased with the number of mutated alleles and was also slightly obvious in asymptomatic parkin gene carriers in the heterozygous state. The abnormal FDOPA uptake pattern in parkin patients did not significantly differ from that of sporadic Parkinson's disease. Our data are in agreement with an enzymatic dysfunction of the gene's translational product, which has been shown to promote protein degradation as an ubiquitin-protein ligase. Thus, parkinsonism in parkin gene carriers may be related to abnormal nigral protein accumulation in the presence of a suprathreshold enzyme dysfunction.

Prevalence of the methylenetetrahydrofolate reductase 677C > T mutation in the Mediterranean Spanish population. Association with cardiovascular risk factors.

Methylenetetrahydrofolate reductase (MT-HFR) is a key enzyme involved in folate metabolism. A common cytosine (C) to a thymine (T) mutation at nucleotide 677 (677C > T) in the MTHFR gene which converts an alanine residue to a valine, has been related with several biochemical phenotypes and with cardiovascular risk, depending on the population studied. Our objective was to estimate the prevalence of the 677C > T mutation in a large and randomly selected sample (289 men and 427 women) from the Mediterranean Spanish population, and to test the association between this genetic variant and some cardiovascular risk factors. For both genders, the prevalence of CC, CT and TT subjects was 32.0, 52.2 and 15.8%, respectively. The frequency (95% confidence interval) of the 677T allele was 0.44 (0.40-0.48) in men and 0.40 (0.37-0.44) in women. This prevalence was significantly different from other European countries, and among the highest reported in the world for any healthy population. We found no association between the 677C > T gene variants and age, body mass index (BMI), total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglycerides or diastolic blood pressure in men and women. However, in men, a statistically significant increase of systolic blood pressure with the number of mutant alleles was found (122.2 mmHg in CC, 125.1 mmHg in CT and 128.5 mmHg in TT subjects; p for trend = 0.030). This association remained significant (p = 0.047) even after adjustment for age, BMI, alcohol consumption, tobacco smoking, education and physical activity.