Familial Mediterranean fever (FMF) is an autoinflammatory disorder generally caused by recessively inherited mutations in the MEFV gene. FMF is quite prevalent in Armenian population in which majority of patients have two mutated alleles, yet in 18% of symptomatic patients just one mutation has been detected. To explain this finding, we analyzed the symptoms and genotypes of 1299 patients, including 236 affected heterozygous patients with definite diagnosis of FMF. We selected a subset of 63 heterozygous, homozygous and asymptomatic normal individuals and completely sequenced their MEFV genes (exons) to discover any other mutations potentially missed by currently used screening method. Besides four synonymous polymorphisms in exon two and five, we found a T267I mutation in one heterozygous patient with a severe case of FMF who should have been designated as compound heterozygous, yet the other genotypes were all accurate. We used binomial probability distribution of symptoms in homozygous FMF patients to estimate the likelihood of their occurrences in heterozygous patients and demonstrated the assemblage of patients into groups with similar clinical criteria using statistical clustering. We found extremely high probabilities for the presence of FMF symptoms in heterozygous individuals and determined that symptoms were equally likely to occur in both analyzed genotypes. Therefore, our study supports the rising evidence that a single MEFV mutation could be associated with mild FMF symptoms. However, heterozygous patients presenting with severe phenotype should be further analyzed for less common second MEFV mutation using gene sequencing.

The ciliate Tetrahymena, a model organism, contains divergent mitochondrial (Mt) genome with unusual properties, where half of its 44 genes still remain without a definitive function. These genes could be categorized into two major groups of KPC (known protein coding) and Ymf (genes without an identified function). To gain insights into the mechanisms underlying gene divergence and molecular evolution of Tetrahymena (T.) Mt genomes, we sequenced three Mt genomes of T.paravorax, T.pigmentosa, and T.malaccensis. These genomes were aligned and the analyses were carried out using several programs that calculate distance, nucleotide substitution (dn/ds), and their rate ratios (ω) on individual codon sites and via a sliding window approach. Comparative genomic analysis indicated a conserved putative transcription control sequence, a GC box, in a region where presumably transcription and replication initiate. We also found distinct features in Mt genome of T.paravorax despite similar genome organization among these ∼47 kb long linear genomes. Another significant finding was the presence of at least one or more highly variable regions in Ymf genes where majority of substitutions were concentrated. These regions were mutation hotspots where elevated distances and the dn/ds ratios were primarily due to an increase in the number of nonsynonymous substitutions, suggesting relaxed selective constraint. However, in a few Ymf genes, accelerated rates of nonsynonymous substitutions may be due to positive selection. Similarly, on protein level the majority of amino acid replacements occurred in these regions. Ymf genes comprise half of the genes in Tetrahymena Mt genomes, so understanding why they have not been assigned definitive functions is an important aspect of molecular evolution. Importantly, nucleotide substitution types and rates suggest possible reasons for not being able to find homologues for Ymf genes. Additionally, comparative genomic analysis of complete Mt genomes is essential in identifying biologically significant motifs such as control regions.

BackgroundFamilial Mediterranean Fever (FMF) is an autoinflammatory disorder caused by mutations in the MEFV gene. These mutations appear in different populations with different frequencies and their caused symptom severities vary from mild to moderate to severe depending on the mutation type.MethodsIn this study, we analyzed the mutations that have been reported in the MEFV gene from symptomatic FMF patients and compared their frequencies in different populations from the 1000 Genome and the Exome databases, using statistical clustering. We also analyzed the nucleotide and amino acid substitution patterns across the MEFV gene.ResultsWe found 16 (8%) nonsynonymous mutations outside exon 10 that did not cluster with known disease‐causing mutations (DCMs), due to their high frequencies in other populations. We also studied the substitution patterns for nucleotides and amino acids to determine the conserved and variable regions in the MEFV gene. In general more nonsynonymous substitutions were reported in exons 2, 3, and 10 from the FMF database (symptomatic FMF patients) compared to the 1000 Genome and the Exome databases. The same was true for amino acid (AA) substitutions where there were 1.5 times more radical (RAD) to conservative (CON) changes. However, when it came to AA substitutions exon 10 was quite conserved with a RAD/CON ratio of 0.9. In fact, we report that the most severe FMF symptoms are caused by conservative mutations in two highly conserved exon 10 regions.ConclusionWe found presumptive FMF‐causing mutations that did not cluster with DCMs based on their allele frequencies. We also observed that the type of mutation is less likely to determine the severity of the FMF symptoms; rather it was the location of the mutations that was the determining factor.

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3542 Background: Achieving major molecular response (MMR) is an important milestone in chronic myeloid leukemia (CML) therapy. MMR has been defined as a 3-log reduction in BCR-ABL transcript levels from a standardized baseline (BL) established in the IRIS trial (Hughes TP, N Engl J Med. 2003). Standardization has been achieved through the development of an IS, which defines MMR as BCR-ABLIS = 0.1%. In contrast, the NCCN defines MMR as a 3-log reduction in BCR-ABL transcript levels but is indefinite on the definition of BL. Here, using reconstructed samples emulating CML patient BCR-ABL levels, the pairwise concordance of MMR determination was examined within and between 3 labs using the IS-standardized GeneXpert® (GX) system and 3 labs using laboratory-developed tests (LDTs). For comparative purposes, this analysis assumes BL is established at the time of diagnosis. Methods: 100 virtual patients (VPs) were emulated based on data from the REVEAL BCR-ABL Methods Comparison Study, in which 8 discrete levels of blinded K562 cell–spiked blood corresponding to BCR-ABLIS ratios ranging from ∼10% to ∼0.01% were analyzed by 3 labs using the IS-standardized GX system and 3 labs using non-IS LDTs. VP emulations were guided by actual patient outcomes in landmark analyses of 7- treatment response (Hughes TP, Blood. 2010). Treatment response profiles over an 18-month time horizon were modeled by assigning one of the 8 BCR-ABL levels ranging from approximately 10%-0.01% IS sampled in the REVEAL study to each of 4 virtual time points (eg, 3, 6, 12, and 18 months). BL levels were selected from quartiles representing pretreatment BCR- ABL ratios between 50–150%; results based on BL levels observed in the IRIS clinical trial will also be presented. 600 VP transcript profiles (VTPs) were then reconstructed using data from each of the 6 laboratories for all 100 VPs. The final 18-month time point in each VTP provided the BCR-ABL level against which the IS or NCCN objective criterion was applied to make MMR determinations. MMR concordance was evaluated by inspecting all possible inter-lab pairwise comparisons among the 100 VPs. Results: Pairwise concordance in MMR as determined by NCCN criterion among all 6 labs is shown in Fig 1A. MMR determinations among the 3 GX labs were concordant in 88% to 93% of VPs. In contrast, MMR determinations among the LDTs were concordant in 43% to 80% of VPs, and MMR determinations were concordant in 53% to 91% of VPs when compared between GX labs and LDTs. When MMR determination based on IS criterion for GX was considered, MMR concordance improved to 93% to 96% among the GX labs in contrast to 51% to 92% concordance observed between the GX and LDT sites (Fig 1B). It is noteworthy that Lab D results more closely approximated the IS than results from the other LDTs examined in the REVEAL study (data not shown). Although Lab D does not report results per the IS, it does report results relative to a median diagnostic BL, similar to the approach used in the IRIS trial. A healthcare system based on LDTs without any attempted IS standardization resulted in MMR concordance of only 43%. Potential sources of discordance among tests will be discussed in detail. Conclusions: These results illustrate that the NCCN criterion for MMR determination is not adequate for inter-lab comparisons of BCR-ABL transcript levels near the clinically important level of MMR. In contrast, standardization to the IS improves inter-lab concordance in MMR determination. Taken together, these results highlight the discrepancies that may result when comparing molecular responses between labs not standardized to the IS. As attainment of MMR is a critical milestone of CML therapy, errors in MMR determination may have an adverse impact on CML disease management. Disclosures: Reddy: Novartis: Research Funding, as Presenting Author, sponsorship to attend ASH. Höfling:Novartis: Employment. Manning:Novartis: Employment. Mignault:Novartis: Employment. Mullaney:Novartis: Employment. Ossa:Novartis: Employment. Stein:Novartis: Employment. Wang:Novartis: Employment. Yang:Novartis: Employment.

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