Mutations In Different Exons Research Articles

247P Efficacy of therapy directed against PI3K and cyclin inhibitors based on the location of PI3K mutations

Activation of the phosphatidylinositol-3-kinase (PI3K) pathway is responsible of activating the phosphorylation cascade that regulates cell survival and metabolism. Mutations in this pathway may occur in 28-46% of HR+/HER2- advanced breast cancers. It is known that the presence of this mutation confers a worse prognosis. We set out to analyze if PI3K mutations in different exons confer resistance to treatments directed against this target and to cyclin inhibitors (inh-CDK4/6). We have analyzed 96 patients with advanced breast cancer HR+/HER2- subsidiaries of treatment with PI3K-targeted therapy in the hospital of Jaén from February/20 to January/23. To detect PIK3CA mutations we used the cobas ® diagnostic kit that detects: exons 1 (R88Q), 4, 7 (C420R), 9 (E542K, E545K/A/D/G, Q546E/R) and 20 (H1047R/Y/L). We obtained 26% mutations in PI3K (5% exon 1, 40% exon 9, 55% exon 20). All in women with a mean age at diagnosis of 48 years and 56 at diagnosis of metastases, located at bone 65%, lung 35%, liver 23% and CNS 4% All patients have been treated with inh-CDK4/6. The median progression-free survival (mSLP) is 17 months (1-60). In patients with exon 1 mutation, mSLP is 25 months, in exon 9, 13 (2-43) and in exon 20, 19 (1-60) 60% of the patients have progressed to inh-CDK4/6 and as next treatment we used alpelisib. In 58% corresponds to 2 line in metastatic disease. The mSLP is 5 months (1-22) with a median follow-up of 14 months (1-34) and 69% of events. In patients with mutation in exon 1 the mSLP is 2 months, in exon 9, 5 (1-13) and in exon 20, 9 (2-22). Treatment was associated with an antiestrogen (77%) with a mSLP 7 months (1-22) or an aromatase inhibitor with a mSLP 3 months (2-10). 53% suffered grade 3 adverse effects (G3 AE): hyperglycemia (80% exon 9, 16% exon 20), diarrhea (20% exon 9) and skin rash (16% exon 20). 14% discontinued treatment due to toxicity. The PFS with alpelisib is lower than reported in the SOLAR1 study and is similar to Bylieve study, whose population is most similar to our sample. Exon 20 carriers are those who obtain a higher PFS, as well as those who receive it with an antiestrogen. Exon 9 carriers have a higher rate of G3 AE With respect to inh-CDK4/6, the PFS corresponds to the literature, with exon 20 carriers obtaining a higher PFS.

Abstract 5403: Genetic, immunologic and prognostic heterogeneity in CRC patients with KRAS mutations

Abstract Genetic, immunologic and prognostic heterogeneity in CRC patients with KRAS mutations Background Currently, most patients with mCRC are treated with a combination of a cytotoxic and a biologic agent. Several factors should be put into account which influencing the treatment strategy for patients with mCRC. including tumor resectability, prior systemic therapy; tumor location (left or right), and molecular characteristics such as mutation in gene Kirsten rat sarcoma viral oncogene homolog (KRAS). It has been demonstrated to be a tightly associated with poor response to anti-EGFR treatment, and even poor response to immune checkpoint inhibitors for patients with colorectal cancer. Therefore, KRAS mutational status testing has been highlighted in recent years. Methods This study queried the data from Formalin-Fixed Paraffin-Embedded (FFPE) tissues of 2,294 CRC patients which underwent a targeted next-generation sequencing assay performed by 3DMed Clinical Laboratory Inc., a College of American Pathologists (CAP) certified and Clinical Laboratory Improvement Amendments (CLIA) certified laboratory of 3D Medicines Inc. between January, 2017 and June, 2020 in China, to obtain a comprehensive molecular profile of KRAS mutations. Co-occurring genomic alterations, tumor mutational burden (TMB), microsatellite instability (MSI) status and PD-L1 expression (Dako 22C3 or VentanaSP263, TPS score) were analyzed. High TMB was defined as TMB ≥10 muts/Mb. Postoperative follow-up data for 27 patients were collected and analyzed. Results KRAS mutation account for 48.7% Chinese CRC patients. In addition, proportions of KRAS mutation in different exons were counted. KRAS mutation in CRC mainly occurred in exon2 (84.0%), exon3 (6.1%), exon4 (7.4%), and exon5 (0.7%). KRAS mutation in exon2, the main mutations were G12D (35.4%), G12V (20.8%), G13D (21.9%), G12C (5.9%) ad G12S (5.8%). while in exon3 and exon4, the main mutations were Q61H (50.7%) and A146T (73.2%), respectively. The proportion of MSI-H in CRC patients with and without KRAS mutation were very close. While it seemed that there were more MSI-H patients in KRAS exon3 and exon4 mutation. In addition, PD-L1 expression was detected in 1220 tumor samples by means of IHC. Among these available tumor samples, level of PD-L1expression were similar among patients without KRAS mutation, KRAS mutation in different exons, but it reveled that there were very small part of patient with strong positive PD-L1 level (TPS≥50). In addition, different prognosis were found among different mutations of KRAS exon2, in detail, worse PFS was showed in CRC patients with KRAS mutation in exon2 G12D than G12V and G13D. Conclusions CRC patients with KRAS mutations in different exons and different site have different genetic and prognostic characteristics, these patients should be managed differently in clinical practice. Citation Format: Zhengqi Wen, Hushan Zhang, Ruize Zhou, Xihong Liu, Wenliang Li. Genetic, immunologic and prognostic heterogeneity in CRC patients with KRAS mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5403.

A comprehensive molecular analysis and genotype–phenotype correlation in patients with familial mediterranean fever

Familial Mediterranean fever is an auto inflammatory genetic disease involving especially Turks, Armenians, Arabs and non-Ashkenazi Jews and caused by variants in the MEFV gene. In this study, we aimed to evaluate the distribution and frequency of clinical, MEFV gene variants in FMF patients and the relationship between mutations in different exons and phenotype-genotype and clinical findings. 1028 patients diagnosed as FMF were included. The most common genotypes were M694V / R202Q heterozygous (10.4%), M694V homozygous (7.5%), M694V / E148Q / R202Q heterozygous (4.6%), V726A heterozygous (4.5%), M680I heterozygous (4.2%). c.1611-1 G > C, G152R, S104C, R116S, E336K, R461Q mutations were detected in the literature for the first time in FMF patients. We also divided the patients into 4 groups according to whether the MEFV mutations were exon 10 or non-exon 10. The first group consisted of non-exon 10 homozygous or compound heterozygous (n = 180) patients, Group 2 consisted of exon 10- non-exon 10 compound heterozygous (n = 318) patients, Group 3 consisted of exon 10 homozygous or compound heterozygous (n = 256) patients, while Group 4 consisted of heterozygous (n = 227) patients at any exon. There was no significant difference between the groups in terms of abdominal pain, arthritis, arthralgia, vomiting diarrhea, erysipelas like rash, amyloidosis, renal failure family history. There was no difference in fever between Group 1 (55.6%) and 2 (62.3%); however, these two groups were different from Group 3 (75.8%) and 4 (76.7%). Group 3 (18.8%) had the highest rate of appendectomy. In addition, allele frequencies of all mutations detected in the analyses were compared with allele frequencies of healthy people in the gnomad database. It is useful to analyse all exons in the MEFV gene with the next generation sequence analysis in the detection of FMF disease. S104C, R116S, G152R, E336K, R461Q, L508Q and c.1611-1 G > C mutations are also new variants in literature. c.1611-1 G > C is a possible pathogenic variant.

A novel FLCN mutation in family members diagnosed with primary spontaneous pneumothorax.

BackgroundPrimary spontaneous pneumothorax (PSP) is a disease characterized by the accumulation of air in the pleural space between the lung and thoracic wall. It is more common in young, tall, thin, and asthenic men. A family history was reported for approximately 11.5% of individuals admitted with PSP. The literature has reported cases diagnosed with familial PSP, who have no manifestations of Birt–Hogg–Dubé (BHD) syndrome but mutations in different exons of the Folliculin (FLCN) gene. The aim of this study is to present a Turkish family in which 13 members from three generations of the same family developed recurrent isolated spontaneous pneumothorax with a novel mutation in the FLCN.MethodsA male proband was diagnosed with spontaneous pneumothorax in the emergency department of the University of Health Sciences Haydarpasa Numune Training and Research Center, Istanbul, Turkey. His 12 relatives from three generations diagnosed with PSP, as revealed by his family history, were invited to the hospital to give blood samples for mutation analysis. The Sanger sequence data of FLCN were analyzed on the ENSEMBL website using SeqScape 3 and Codon Aligner software.ResultsA novel heterozygous mutation c. 1273C>T (p.Gln425Ter) was detected in exon 11 of the FLCN, which caused PSP in the proband and his 12 relatives tested using Sanger sequencing.ConclusionWe found that a heterozygous mutation in exon 11 of FLCN c. 1273C>T (p.Gln425Ter), which was identified for the first time in our study, might cause isolated familial spontaneous pneumothorax.

Mutational analysis of ARSB gene in mucopolysaccharidosis type VI: identification of three novel mutations in Iranian patients.

Objective(s):Mucopolysaccharidosis VI (MPS VI) or Maroteaux-Lamy syndrome is a rare metabolic disorder, resulting from the deficient activity of the lysosomal enzyme arylsulfatase B (ARSB). The enzymatic defect of ARSB leads to progressive lysosomal storage disorder and accumulation of glycosaminoglycan (GAG) dermatan sulfate (DS), which causes harmful effects on various organs and tissues and short stature. To date, more than 160 different mutations have been reported in the ARSB gene.Materials and Methods:Here, we analyzed 4 Iranian and 2 Afghan patients, with dysmorphism indicating MPS VI from North-east Iran. To validate the patients’ type of MPS VI, urine mucopolysaccharide and leukocyte ARSB activity were determined. Meanwhile, genomic DNA was amplified for all 8 exons and flanking intron sequences of the ARSB gene to analyze the spectrum of mutations responsible for the disorder in all patients.Results:Abnormal excretion of DS and low leukocyte ARSB activity were observed in the urine samples of all 6 studied patients. In direct DNA sequencing, we detected four different homozygous mutations in different exons, three of which seem not to have been reported previously: p.H178N, p.H242R, and p.*534W. All three novel substitutions were found in patients with Iranian breed. We further detected the IVS5+2T>C mutation in Afghan siblings and four different homozygous polymorphisms, which have all been observed in other populations. Conclusion:results indicated that missense mutations were the most common mutations in the ARSB gene, most of them being distributed throughout the ARSB gene and restricted to individual families, reflecting consanguineous marriages.

P1.02-026 Detection of Low-Abundant EGFR Somatic Mutations by PNA Clamping-Assisted Fluorescence Melting Curve Analysis

Detecting mutations is becoming important for both predicting disease progression and drug responses to treatment of cancer patients. Current mutation detection methods for cancer diagnosis are mainly based on the invasive sampling technique such as a tissue biopsy, but some patients may not available for this invasive procedure. Therefore, circulating tumor DNA (ctDNA) would be a good alternative for those patients. However, testing methods for tissue biopsy sample are not applicable to ctDNA samples due to relatively lower sensitivity. A highly sensitive assay method is required for detecting mutations in liquid biopsy samples. We have developed a highly sensitive and simple method to detect somatic mutation from ctDNA in patient’s plasma. This new real-time PCR-based testing method (PANAMutyper™) has maximized unique properties of peptide nucleic acid (PNA). It contains a PNA clamp and PNA detection probes in the each reaction tubes. A optimized PNA clamp can tightly bind to only wild-type DNA sequences, and then suppress amplification during the PCR reaction. Meanwhile, a PNA detection probe that conjugated with a fluorescent dye and a quencher, can detect a specific target mutant-type DNA and each mutation can be genotyped by melting peak analysis. PANAMutyper™ is able to detect 47 different mutations in exon 18, 19, 20 and 21 of EGFR gene with detection limits as low as 0.01%. In order to confirm the validity in real condition, we conducted spiking test. Ten of mutant clones DNA were used as standard materials. (G719A, G719S, G719S, S768I, Exon19 deletion, two Exon20 insertion, T790M, L858R, L861Q). Mix the each mutant clone DNA and human plasma to 1, 10, 100, 1000 copies per microliter concentration. And then we extracted ctDNA from prepared sample. As a result, all of ten mutant clone DNA was detected 1 copy/㎕. This result suggest that PANAMutyper™ is applicable to ctDNA derived from patient’s plasma sample. The high concordance, specificity, and sensitivity of this test have demonstrated that EGFR mutation status can be accurately assessed by PANAMutyper™ using ctDNA. Therefore, PANAMutyperTM can be used in various clinical areas including companion diagnostics and monitoring acquired mutations.

Oncogenic KIT mutations in different exons lead to specific changes in melanocyte phospho-proteome

Mutations in the proto-oncogene c-KIT (KIT) are found in several cancers, and the site of these mutations differs markedly between cancer types. We used site directed mutagenesis to induce KIT559, KIT642 and KIT816 mutations in primary human melanocytes (PHM) and we investigated the impact of each mutation on KIT function. We studied canonical KIT-signaling pathways by immunoblotting, and we used stable isotope labeling by amino acids in cell culture (SILAC) and kinase prediction models to identify kinases differently activated in respective mutants. We validated our results with the analysis of phosphorylation levels of selected substrates for each kinase. We concluded that CK1 ε and δ are more active in cell clones harboring KIT559 and KIT642 mutations, whereas PAK4 is more active in clones with KIT816 mutation. Our findings might help to develop further therapeutic options for tumors with specific KIT mutations in different domains. Biological significanceDifferent types of cancers harbor mutations in the oncogene KIT. The use of small molecules inhibitors directly targeting KIT had a limited success in the treatment of patients with KIT mutant cancers. Our study describes specific phospho-proteome changes due to different KIT mutations, and provides targets of further therapeutic options.

RET gene mutations spectrum in patients with medullary thyroid carcinoma (MTC) from Great Poland region

Medullary thyroid carcinoma (MTC) is an epithelial tumor of the thyroid gland, derived from thyroid parafollicular C cells and represents approximately 4% of thyroid carcinomas. In 50 % of patients, MTC is diagnosed at an advanced stage. Due to a lack of TSH receptors on C-cells surface MTC does not respond to suppression therapy with TSH and radio-iodine treatment, and thereby it conferring worse outcomes overall. 5-year survival rates range from 75% for patients with stage III disease to 40% for patients with stage IV disease. MTC is associated with one of three clinically different, inherited endocrine syndromes caused by germline mutations in the RET gene. It encodes a transmembrane receptor tyrosine kinase which is expressed in cells derived from the neural crest. Activating mutations in the RET gene are detected in the majority (95%) patients with an inherited medullary thyroid carcinoma and approximately 30-50% of cases without family history. Familial medullary thyroid carcinomas (FMTCs) are associated with mutations in codons 609, 611, 618, and 620 (exon 10), as well as codon 768 (exon 13) and codon 804 (exon 14). In FMTC patients, MTC is often the only clinical symptom. Multiple endocrine neoplasia (MEN) 2A is associated in the most cases, with RET gene mutations in codons 609, 611, 618, and 620, as well as in codon 634. Patients with MEN2A typically have MTC, pheochromocytoma, and primary hyperparathyroidism (PHPT). Patients diagnosed with MEN 2B present the most aggressive type of MTC, pheochromocytoma but not PHPT, musculoskeletal abnormalities and other developmental defects. This syndrome is associated mostly with mutation in codon 918 in exon 16. Here we report the RET gene mutations spectrum in a group from Great Poland (Wielkopolska) region. Molecular analysis was performed for 304 individuals: patients with diagnosed MTC or apparent MEN 2A syndrome and first and second degree relatives. Studied group included also one family with MEN 2B syndrome. All patients were diagnosed in the Department of Endocrinology, Metabolism, and Internal Diseases of the Poznan University of Medical Sciences. We screened RET gene exons: 10, 11, 13-16 using pyrosequencing for specific codons and HRMA technique followed by Sanger sequencing. Identification of RET gene mutations was possible for 50 patients (32%). This group included 12 family cases and in 6 subjected individuals diagnosis were possible before cancer occurring. In a group of 106 (68%) MTC patients we did not detect mutations in analyzed RET gene regions. The most frequent was missense substitutions occurring in codon 634 (72%): C634R (58%), C634G (10%) and C634Y (4%). Surprisingly, in our group of patients we did not detect mutations in exon 10, as they are frequent in other populations. However, we observed 3 different mutations in exon 13 (18%) and in exon 14 (6%). In four unrelated patients with negative family history we detected missense change Y791F, which is associated with late onset MTC, but interestingly one of patients was homozygous and presented more aggressive course of the disease (characterized with multifocality and onset in the moment of diagnosis at 47 years of age). In MEN 2B family, M918A mutation was detected. Due to the fact, that we observe a different mutational spectrum in our patients, we consider more extensive analysis of the RET gene as reasonable.

CYP1A1 and GSTP1 gene variations in breast cancer: a systematic review and case-control study.

In first part of this study, a systematic review was designed to explore the involvement of CYP1A1 and GSTP1 genes in breast cancerogenesis. Based on systematic review, we designed a study to screen CYP1A1 and GSTP1 genes for mutation and their possible association with breast carcinogenesis. A total of 400 individuals were collected and analyzed by PCR-SSCP. After sequence analysis of coding region of CYP1A1 we identified eleven mutations in different exons of respective gene. Among these eleven mutations, ~3 folds increased breast cancer risk was found associated with Asp82Glu mutation (OR 2.99; 95% CI 1.26-7.09), with Ser83Thr mutation (OR 2.99; 95% CI 1.26-7.09) and with Glu86Ala mutation (OR 3.18; 95% CI 1.27-7.93) in cancer patients compared to controls. Furthermore, ~4 folds increase in breast cancer risk was found associated with Asp347Glu, Phe398Tyr and 5178delT mutations (OR 3.92; 95% CI 1.35-11.3) in patients compared to controls. The sequence analysis of GSTP1 resulted in identification of total five mutations. Among these five mutations, ~3 folds increase in breast cancer risk was observed associated with 1860G>A mutation, with 1861-1876delCAGCCCTCTGGAGTGG mutation (OR 2.70; 95% CI 1.10-6.62) and with 1861C>A mutation (OR 2.97; 95% CI 1.01-8.45) in cancer patients compared to controls. Furthermore, ~5 folds increase in breast cancer risk was associated with 1883G>T mutation (OR 4.75; 95% CI 1.46-15.3) and ~6 folds increase in breast cancer risk was found associated with Iso105Val mutation (OR 6.43; 95% CI 1.41-29.3) in cancer patients compared to controls. Our finding, based on systematic review and experimental data suggest that the polymorphic CYP1A1 and GSTP1 genes may contribute to risk of developing breast cancer.

Abstract 344: Finding the Achilles’ heel of Muscle Giant---TALEN-mediated Gene-editing in Zebrafish Titin

Background: TITIN (TTN) has more than 300 exons and encodes a gigantic protein that is crucial for heart and muscle development. Mutations in TTN caused a variety of human diseases including cardiomyopathy and muscular dystrophy. Recently, dilated cardiomyopathy-associated mutations on TTN have been found more frequently in exons encoding A-band domains but less in exons encoding the N-terminal Z-disc domains, suggesting that mutations in different exons of TTN cause distinct consequences. To elucidate the underlying mechanisms, we leveraged the Transcription Activator-Like Effects Nuclease (TALEN) technology in zebrafish to introduce truncating mutations in different exons of ttn, and then study their effects on heart and somites. Results: We generated truncational mutations in different exons of zebrafish titins encoding Z-disc, N2B, Novex-3, and A domains, respectively. Because zebrafish contains two titin homologues, ttna and ttnb, we introduced mutations in both genes at the corresponding loci. While both Z-disc and A band mutations on ttna disrupted sarcomere assembly in heart and somites, Z-disc or A band mutations on ttnb only affect somites without affecting the heart. Interestingly, a Z-disc mutation on ttna resulted in milder phenotypes than an A-band mutation, while a Z-disc mutation on ttnb generated severer phenotypes than an A-band mutation. No phenotype was observed in the homozygous fish in either ttna-novex-3 or ttnb-N2B mutant fish. Conclusions: A spectrum of truncational mutations in ttna and ttnb has been generated in zebrafish using the TALEN technology. Mutations in different exons result in different phenotypes. Detailed characterization of these mutants and double mutants will be presented, which shall elicit distinct contribution of alternative splicing and exon skipping as two candidate mechanisms during pathogenesis of Titinopathies.

Hereditary myopathy with early respiratory failure: occurrence in various populations

ObjectiveSeveral families with characteristic features of hereditary myopathy with early respiratory failure (HMERF) have remained without genetic cause. This international study was initiated to clarify epidemiology and the genetic underlying...

The first hundred families diagnosed with familial hypercholesterolaemia in two lipid clinics in lothian

As in other areas of Western Europe, familial hypercholesterolaemia (FH) is one of the most frequent genetic disorders in the UK. Genetic testing for this condition is available in Scotland. This observational study describes the genetic variants found when the databases of the two principal designated lipid clinics in South East Scotland were amalgamated. The genetic mutations were identified by the Molecular Genetics Department in Aberdeen Royal Infirmary by sequencing all 18 exons of the LDLR gene and using multiplex ligation probe amplification (MLPA) to identify major deletions and duplications in the gene. Variants of the APOB gene at codon 3527 and the common PCSK9 mutations were also investigated. The first 100 genetically positive families (as opposed to individuals) were studied and unidentified variants included. Care was taken not to include patients who could be related. Sixty six different genetic mutations were found, including point mutations, major rearrangements, deletions or duplications or unidentified variants. No PCSK9 mutations were found. The frequency of mutations in different exons of the LDLR gene was explored. There was a higher frequency than expected of unclassified variants. Our results were compared with two commercial kits and analysed with the pick-up rate, in this population, disappointing.

Analysis of polymorphism within POU1F1 gene in relation to milk production traits in dairy Sarda sheep breed

The ovine POU1F1 gene is localized on chromosome 1 and it contains five introns and six exons. In different mammalian species some mutations in different exons are associated with different production traits. The aim of our research was to study the POU1F1 gene nucleotide sequence to detect possible polymorphisms and their relationships with milk productive traits in Sarda breed sheep. The study had been conducted on 140 ewes, 4 or 5 years old coming from a farm located in Sardinia. All the animals were multiparous, lactating and in their third to fifth lactation. Individual milk yield had been recorded monthly and for each sample fat, protein, casein, lactose, and somatic cell count values were analysed. A jugular blood sample was collected from each ewe to perform genomic DNA extraction. PCR, SSCP and sequencing analysis were carried out to examine the six exons to highlight possible SNPs. One-way ANOVA was used to analyse association of variants with milk yield and/or its composition. Two novel SNP were found: 121 C>T in the 5'UTR of the fourth intron fragment and 249 G>A in the 3'UTR of the sixth exon fragment. The statistical analysis did not shown association between milk productive traits and the found polymorphisms. However, further investigations about the promoter region or the prophet genes, like the PROP-1, could clarify its exact role in regulating the productive traits in sheep.

Prognostic Significance of DNA Methyltransferase 3A Mutations in Cytogenetically Normal Acute Myeloid Leukemia: A Study by the Acute Leukemia French Association

Abstract 2506 Introduction:The development of massively parallel sequencing technologies has led to the identification of somatic DNA methyltransferase 3A (DNMT3A) gene mutations in acute myeloid leukemia (AML), with the highest frequency being found in cytogenetically normal (CN) AML. DNMT3A mutations have been suggested to predict poor clinical outcome in AML, but only few data are available on their prognostic significance within CN-AML. The aim of this study was to determine the frequency, the main associated features, and the prognostic significance of DNMT3A mutations in CN-AML. Patients and methods:This retrospective study was performed in 123 young adult patients (16–60 years) with previously untreated primary CN-AML and enrolled on two concomitant protocols of the Acute French Leukemia Association (ALFA), the ALFA-9801 and ALFA-9802 trials. DNMT3A mutations were screened on genomic DNA by PCR and direct Sanger sequencing. We focused our screening on the 3 conserved domains of DNMT3A (the proline-tryptophane-tryptophane-proline (PWWP) domain, the ADD-type zinc finger domain, and the C5-methyltransferase domain), corresponding to exons 8–9 and 11–23. The patients were also assessed for the presence of FLT3 internal tandem duplication (FLT3-ITD), FLT3 tyrosine kinase domain (FLT3-TKD), NPM1, CEBPA, WT1, IDH1, and IDH2 mutations. Results:Thirty-eight DNMT3A mutations were identified in 36 of the 123 (29%) patients. These alterations consisted of 36 nucleotide substitutions and 2 frameshift deletions. Thirty out of 36 (83%) nucleotide substitutions affected the amino acid residue R882 (R882H, n = 21; R882C, n = 7; R882P, n = 2), 5 represented other missense alterations, and 1 was a nonsense mutation. Two patients exhibited 2 heterozygous missense mutations in different exons, and one patient had a homozygous missense mutation. DNMT3A mutated and wild-type cases did not differ in terms of age, gender, and white blood cell (WBC) count at presentation. DNMT3A mutations were strongly associated with the French-American-British (FAB) subtypes M4/M5 (P =.0002) and the presence of NPM1 mutations (P =.0006), and tended to often co-occur with IDH1R132 mutations (P = .09). In the entire cohort, complete remission rate was found lower in DNMT3A mutated patients than in DNMT3A wild-type patients, but without reaching statistical significance (80% vs 90%, P =.24). DNMT3A mutated patients had a shorter event-free survival (5-year EFS: 13% vs 32%, P =.02) and overall survival (5-year OS: 23% vs 45%, P =.02) compared to DNMT3A wild-type patients. We next performed subgroup analysis according to the NPM1/FLT3-ITD genotypes. In patients with the non-favorable genotypes, that is NPM1 mutated/FLT3-ITD positive, NPM1 wild-type/FLT3-ITD positive, NPM1 wild-type/FLT3-ITD negative (n = 86), 18 (21%) had a concomitant DNMT3A mutation. In this high-risk subgroup of CN-AML, DNMT3A mutations conferred a worse clinical outcome (5-year EFS: 0% vs 23%, P =.02; 5-year OS: 14% vs 37%, P =.06). In patients with the favorable genotype NPM1 mutated/FLT3-ITD negative (n = 37), 18 (49%) were found to display a concomitant DNMT3A mutation. Within this favorable subgroup, patients carrying a DNMT3A mutation had a significantly inferior EFS and OS compared to DNMT3A wild-type patients (5-year EFS: 25% vs 65%, P =.01; 5-year OS: 29% vs 76%, P =.02). Furthermore, in multivariate analysis including age, WBC count, NPM1/FLT3-ITD genotypes, and DNMT3A mutational status, the presence of a DNMT3A mutation remained an independent adverse prognostic factor for EFS (hazard ratio = 2.29; 95% CI, 1.42 to 3.70; P =.0007) and OS (hazard ratio = 2.34; 95% CI, 1.37 to 4.00; P =.002). Conclusion:DNMT3A mutations are one of the most common gene mutations in CN-AML and independently predict poor clinical outcome. Testing for DNMT3A mutations could help further improve risk stratification in CN-AML. Disclosures:No relevant conflicts of interest to declare.

Low frequency of common LRRK2 mutations in Mexican patients with Parkinson's disease

Mutations in leucine-rich repeat kinase 2 gene ( LRRK2) account for as much as 5–6% of familial Parkinson's disease (PD) and 1–2% of sporadic PD. These mutations represent the most frequent cause of autosomal dominant PD, particularly in certain ethnic groups. In this first report concerning LRRK2 mutations in Mexican-mestizos, we screened 319 consecutive PD patients (186 males; 133 females; mean age at onset: 52.4 years) for LRRK2 mutations in exons 31 and 41 and for the mutation in exon 35, which produces the Y1699C substitution. Three (0.94%) patients, two with sporadic PD and one with familial PD (disease mean age at onset, 53.3 years), were heterozygous for LRRK2 mutations. Of these three, two patients had one of two different mutations in exon 31 (R1441G and R1441H, respectively); the other patient carried the G2019S mutation in exon 41. The Y1699C mutation was absent from this PD sample. Four additional subjects, unaffected relatives of one PD patient with a mutation in LRRK2, were subsequently genetically tested. None of the three LRRK2 mutations identified was present in 200 neurologically healthy Mexican control individuals. These findings have important implications for molecular testing of LRRK2 mutations in Mexican PD patients.

Congenital factor X deficiencies with a defect only or predominantly in the extrinsic or in the intrinsic system: A critical evaluation

Congenital Factor X deficiency is commonly classified as type I, in which there is a concomitant decrease of activity and antigen (CRM negative), and in type II, in which activity is low but antigen is normal or near normal (CRM positive). During the past decades it was shown that type II was by itself very heterogeneous. It was shown in fact that some forms showed a defect in all three assay systems (extrinsic, intrinsic, and RVV dependent), whereas others showed a defect only in two of the three systems. Molecular biology analysis, whenever available, has failed so far to supply clear explanations for these discrepancies. The purpose of the present article was an attempt to correlate the clotting activities seen in these two defects with other clotting, chromogenic, immunological assays, and molecular biology results. There are in the literature 10 families that show a predominant defect in the extrinsic system, and four families that show a predominant defect in the intrinsic system. All patients showed a normal, near normal, or reduced level of antigen that is always definitively higher than the clotting counterpart. Molecular biology studies revealed mutations in different exons, namely 2, 4, 5, 6, and 8. These mutations in different exons do not allow any clear genotype-phenotype conclusions, but indicate that mutations in different exons may give rise to the same phenotype. The study underlines the importance of a multipronged evaluation of all cases with Factor X deficiency. In fact only by this approach can an acceptable classification of the defect be reached.

Colorectal carcinoma prognosis can be predicted by alterations in gene p53 exons 5 and 8

Gene p53 alteration is a genetic event described in the progression from adenoma to colorectal carcinoma. Most of the p53 mutations occur in exons 5 to 8 in highly preserved regions and in the three main structural domains of the p53 protein. It is possible that mutations affecting different structural regions may present different effects on the p53 protein function and, due to this, they may have different prognostic meaning. The study population consisted of 353 patients diagnosed with sporadic colorectal cancer. Mutations in 5-8 exons of p53 gene were detected by means of single strand conformation polymorphism (SSCP). All samples that showed different migration bands in SSCP were confirmed by sequencing. A total of 69 patients (19.7%) showed alterations of the gene p53. It was observed that mutation in codon 175 in exon 5 was related to tumors located in the colon (p = 0.01) and the mutation in the codon 288 in exon 8 was related to rectal tumors (p = 0.02). In the study of overall survival, mutation in codon 175 of exon 5 conferred a better prognosis and alterations of exon 8 were related to a worse prognosis in different population subgroups: in men, in patients younger than 71 years old, in the tumors located in the proximal colon, the ones moderately differentiated, and those that are mucinous. According to this study, mutations in different exons of p53 are related to different phenotypes in colorectal cancer. These phenotypes could mean differences in the clinical evolution of the patients.

Different Prognostic Roles of Mutations in the Helical and Kinase Domains of the PIK3CA Gene in Breast Carcinomas

In breast cancer, the PIK3CA gene is frequently mutated at "hotspots" in exons 9 and 20, corresponding to the helical and kinase domains, respectively. We decided to investigate the association of PIK3CA mutations with pathologic features and clinical outcome in a large series of patients with breast cancer. Frozen samples from 163 consecutive patients were analyzed for PIK3CA mutations using PCR single-strand conformation polymorphism and sequence analyses. We identified 46 missense mutations, 24 (53%) in exon 9, and 21 (47%) in exon 20. Twelve (50%) of the 24 mutations in exon 9 were of the E542K type and 11 (46%) were of the E545K type. Twenty (95%) of the 21 mutations in exon 20 were H1047R substitutions. Mutations in exon 9 were more frequent in lobular carcinomas (42% of cases) than in ductal carcinoma (11% of cases; P = 0.002). At univariate survival analysis, PIK3CA exon 20 mutations were associated with prolonged overall and disease-free survival, whereas mutations in exon 9 were associated with significantly worse prognosis. At multivariate analysis, exon 9 PIK3CA mutations were the strongest independent factor to predict poor prognosis for disease-free survival (P = 0.0003) and overall survival (P = 0.001). Our data show that exon 9 PIK3CA mutations are typical of infiltrating lobular carcinomas. In addition, they indicate that PIK3CA mutations in different exons are of different prognostic value: exon 9 mutations are independently associated with early recurrence and death, whereas exon 20 PIK3CA mutations are associated with optimal prognosis.

Three novel missense germline mutations in different exons ofMSH6gene in Chinese hereditary non-polyposis colorectal cancer families

To investigate the germline mutations of MSH6 gene in probands of Chinese hereditary non-polyposis colorectal cancer (HNPCC) families fulfilling different clinical criteria. Germline mutations of MSH6 gene were detected by PCR-based DNA sequencing in 39 unrelated HNPCC probands fulfilling different clinical criteria in which MSH2 and MLH1 mutations were excluded. To further investigate the pathological effects of detected missense mutations, we analyzed the above related MSH6 exons using PCR-based sequencing in 137 healthy persons with no family history. The clinicopathological features were collected from the Archive Library of Cancer Hospital, Fudan University and analyzed. Four germline missense mutations distributed in the 4(th), 6(th) and 9(th) exons were observed. Of them, three were not found in international HNPCC databases and did not occur in 137 healthy controls, indicating that they were novel missense mutations. The remaining mutation which is consistent with the case H14 at c.3488A>T of exon 6 of MSH6 gene was also found in the controls, the rate was approximately 3.65% (5/137) and the type of mutation was not found in the international HNPCC mutational and SNP databases, suggesting that this missense mutation was a new SNP unreported up to date. Three novel missense mutations and a new SNP observed in the probands of Chinese HNPCC families, may play an important role in the development of HNPCC.

Differences in methylation patterns in the methylation boundary region of IDS gene in hunter syndrome patients: implications for CpG hot spot mutations

Hunter syndrome, an X-linked disorder, results from deficiency of iduronate-2-sulfatase (IDS). Around 40% of independent point mutations at IDS were found at CpG sites as transitional events. The 15 CpG sites in the coding sequences of exons 1 and 2, which are normally hypomethylated, account for very few of transitional mutations. By contrast, the CpG sites in the coding sequences of exon 3, though also normally hypomethylated, account for much higher fraction of transitional mutations. To better understand relationship between methylation status and CpG transitional mutations in this region, the methylation patterns of 11 Hunter patients with transitional mutations at CpG sites were investigated using bisulfite genomic sequencing. The patient cohort mutation spectrum is composed of one mutation in exon 1 (one patient) and three different mutations in exon 3 (10 patients). We confirmed that in normal males, cytosines at the CpG sites from the promoter region to a portion of intron 3 were hypomethylated. However, specific CpG sites in this area were more highly methylated in patients. The patients with p.R8X (exon 1), p.P86L (exon 3), and p.R88H (exon 3) mutations had a hypermethylated condition in exon 2 to intron 3 but retained hypomethylation in exon 1. The same trend was found in four patients with p.A85T (exon 3), although the degree of hypermethylation was less. These findings suggest methylation patterns in the beginning of IDS genomic region are polymorphic in humans and that hypermethylation in this region in some individuals predisposes them to CpG mutations resulting in Hunter syndrome.