Citations

Antibody secreting plasma cells (PCs) play an important role in effective humoral immune responses. The low frequency of bone marrow PCs in humans makes it challenging to obtain sufficient numbers of PCs for biologic studies. Previous studies have employed in vitro model systems to generate cells that morphologically, phenotypically, and functionally resemble normal polyclonal PCs. Gene expression profiles of in vitro generated PCs (IVPCs) mirror their normal counterparts, however to date extensive immunoglobulin (Ig) repertoire analysis of IVPCs is lacking. Here, we used a modified 3-step protocol to generate IVPCs and used RNA-seq to explore the transcriptome with emphasis on the Ig repertoire of plasmablasts and PCs. Total B cells were isolated from 3 normal donors and cultured with various cytokines and the B cell activators CpG ODN and CD40L. RNA was obtained from freshly isolated B cells (Day 0; D0) as well as from Day 4 (D4) plasmablasts, and Day 10 (D10) IVPCs. Morphologically, D10 cells exhibited typical PC morphology, including an eccentric nucleus and perinuclear hof. RNA-seq was performed on total RNA from all 3 donors and time points using the Standard TRuSeq v2 library prep and with paired end sequencing on the Illumina HiSeq 4000 platform. Principle component analysis of gene expression data showed that D0, D4 and D10 cells could be clearly segregated across all 3 normal donors. Of importance, transcripts previously described as distinguishing B cells from PCs were found to be differentially expressed including overexpression of CXCR5, CD19, EBF, CD83, PAX5, IRF8 in D0 B cells and overexpression of IRF4, Blimp-1, XBP1, BCMA, SLAMF7, Syndecan-1, CD38 and CD27 in IVPCs, thus validating our in vitro model for generating PCs. Furthermore, expression of cell cycle related transcripts such as CKS1, CDK1, and CCDN2 followed the pattern of low expression in resting B cells, increased expression in plasmablasts, and decreased expression in IVPCs confirming the cells are actively cycling in a manner comparable to cells in vivo. D10 IVPCs also overexpressed transcripts known to be upregulated during the unfolded protein response. As expected from Ig secreting cells, D10 IVPCs had an over-representation of Ig transcripts. At D0, resting B cells had high levels of IgD and IgM heavy chain (HC) transcripts. At D10, IgM transcripts modestly increased with Log2 fold change (FC) = 3 and as expected, IgD levels decreased significantly (Log2 FC = -2.2). IgA and IgG isotype transcripts significantly increased at D10 (Log2 FC > 6.0) with the IgG4 subtype having the greatest Log2 FC at 8.4. Next we focused on the Ig repertoire of D0, D4, and D10 cells. By aligning to known germline Ig sequences in IMGT/V-Quest (www.imgt.org) and then assembling the paired ends of D0, D4 and D10 Ig transcripts, we were able to analyze the Ig repertoire. Since the Ig HC variable (V) region is encoded by V, diversity (D) and joining (J) segments, only fragments that could be confidently determined were considered. All but 3 IGHV transcripts (IGHV3-35, IGHV3-47 and IGHV7-8) and 2 IGHD transcripts (IGHD4-4 and IGHD5-5) were found and all IGHJ segments were represented across the differentiation spectrum. In D0 cells, the number of unique VDJ combinations ranged from 643 to 863 across all 3 normal samples and increased to a range of 2524 to 2867 in D10 IVPCs. When looking at the differential expression of each VDJ combination from D0 to D10, a pairwise t-test for relative frequency showed that there was no significant change greater than 1%, suggesting the repertoire diversity was not skewed, thus proving the conditions for stimulation were not targeting any one starting B cell. Our data also allowed us to track clonal expansions during differentiation as defined by the increasing frequency of sequences with identical nucleotide sequence in the V region and CDR3 (including D and J regions). Hence, a single sequence could be tracked from D0 to D10. Of interest, in a small sampling of the total available sequences, only those B cells with a mutated IGHV region, characteristic of a memory B cell, went on to expand in this system whereas B cells with an unmutated IGHV did not. Our analysis of the Ig repertoire of IVPCs suggests this system provides a functional model to study Ig repertoire along the B cell differentiation process and further delineate the conditions that may result in a clonal expansion, a hallmark of many hematologic malignancies including multiple myeloma. Disclosures No relevant conflicts of interest to declare.

This article presents three cases of cranial nerve palsy following shoulder surgery with general anesthesia in the beach chair position. All patients underwent preoperative ultrasound-guided interscalene nerve block. Two cases of postoperative hypoglossal and one case of combined hypoglossal and recurrent laryngeal nerve palsies (Tapia’s syndrome) were identified. Through this case series, we provide a literature review identifying postoperative cranial nerve palsies in addition to the discussion of possible etiologies. We suggest that intraoperative patient positioning and/or airway instrumentation is most likely causative. We conclude that the beach chair position is a risk factor for postoperative hypoglossal nerve palsy and Tapia’s syndrome.

44 Background: A noninvasive cfDNA blood test detecting multiple cancers at earlier stages could decrease cancer mortality. In earlier discovery work, whole-genome bisulfite sequencing outperformed whole-genome and targeted sequencing approaches for multi-cancer detection across stages at high specificity. Here, multi-cancer detection and TOO localization using bisulfite sequencing of plasma cfDNA to identify methylomic signatures was evaluated in preparation for clinical validation, utility, and implementation studies. Methods: 2301 analyzable participants (1422 cancer [ > 20 tumor types, all stages], 879 non-cancer) were included in this prespecified substudy from the Circulating Cell-free Genome Atlas (CCGA) (NCT02889978) study - a prospective, multi-center, observational, case-control study with longitudinal follow-up. Plasma cfDNA was subjected to a targeted methylation sequencing assay using high-efficiency methylation chemistry to enrich for methylation targets, and a machine learning classifier determined cancer status and tissue of origin (TOO). Observed methylation fragments characteristic of cancer and TOO were combined across targeted regions and assigned a relative probability of cancer and of a specific TOO. Results: Performance is reported at 99% specificity (ie, a combined false positive rate across all cancer types of 1%), a level required for population-level screening. Across cancer types, sensitivity ranged from 59 to 86%. Combined cancer detection (sensitivity [95% CI]) was 34% (27-43%) in stage I (n = 151), 77% (70-83%) in stage II (n = 171), 84% (79-89%) in stage III (n = 204), and 92% (88-95%) in stage IV (n = 281). TOO was provided for 94% of all cancers detected; of these, TOO was correct in > 90% of cases. Conclusions: Detection of multiple deadly cancers across stages using methylation signatures in plasma cfDNA was achieved with a single, fixed, low false positive rate, and simultaneously provided accurate TOO localization. This targeted methylation assay is undergoing validation in preparation for prospective clinical investigation as a cancer detection diagnostic. Clinical trial information: NCT02889978.

Abstract Despite the remarkable outcomes and recent FDA approval of CD19 directed chimeric antigen receptor T (CART19) cell therapy in B cell malignancies, the durable responses in diffuse large B cell lymphoma are less than 40% and CART activity in chronic lymphocytic leukemia (CLL) is further limited. This is thought to be related to loss of CART persistence, poor trafficking to lymph nodes and inhibition by the leukemic microenvironment. Therefore, strategies to enhance CART cell function to overcome these limitations are needed. Recent studies have shown that abnormal expression of the receptor tyrosine kinase (RTK) AXL is associated with poor prognosis in human cancers. AXL signaling is associated with tumor proliferation, survival, metastasis, and drug resistance. Inhibition of AXL RTK with TP-0903, a high affinity AXL inhibitor has been found to induce robust apoptosis of CLL B cells. Based on the significant modulation of T cell functions observed with BTK inhibitor, we examined the role of AXL RTK inhibition with TP-0903 on T cell function in CLL and other B cell malignancies. First, we investigated the effect of AXL inhibition on T cell phenotype in normal donors. When naïve T cells were stimulated with PMA/Ionomycin and cultured with low dose TP0903, cytokine production was favorably altered through the promotion of Th1 and reduction of Th2 cytokines. This was associated with a significant reduction of inhibitory receptors (Fig 1a). Western blot of T cell lysates suggests low dose TP-0903 results in inhibition of LCK. When effector T cells and regulatory T cells (Treg) were treated with TP-0903 for 3 days, there was a preferential reduction of Treg (Fig 1b). Next, we investigated the influence of TP-0903 on CART19 cell phenotype and functions. Here, we used 41BB costimulated, lentiviral-transduced CART cells. Similar to our findings on naïve T cells, TP-0903 treatment led to polarization of CART cells into a Th1 phenotype when T cells were stimulated with the CD19+ mantle cell lymphoma (MCL) cell line JeKo or with leukemic B cells isolated from CLL patients (Fig 1c). TP-0903 treatment also significantly downregulated inhibitory receptors on activated CART cells, including a reduction of canonical cytokines known to be associated with the development of cytokine release syndrome (CRS) (Fig 1c). The combination of CART19 cells and TP-0903 yielded a synergistic antitumor activity against JeKo in vitro, at low E:T ratios (Fig 1d). Western blot of T cell lysates revealed phosphorylation of LCK was remarkably reduced in the presence of TP-0903, suggesting a mechanism for the observed Th1 polarization. We compared the transcriptome of activated CART cells treated with TP-0903 and more than 100 genes were differentially expressed compared to non-treated cells. Among these genes, immune synapse related genes such as cell junction and cell migration related genes were significantly increased in activated CART cells treated with TP-0903. To investigate the effect of AXL RTK inhibition of CART cells with TP-0903 in vivo, we established MCL xenografts through the injection of 1.0x106 of JeKo into NSG mice. A week after the injection of JeKo, mice were treated with either vehicle alone, TP-0903 (20mg/kg/day) alone, 0.5x106 of CART19 alone, or TP-0903 (20mg/kg/day)+0.5x106 of CART19. Three weeks after the treatment, mice were rechallenged with 1.0x106 of JeKo. Mice treated with CART19 and TP-0903 rejected the JeKo tumor challenge while mice previously treated with CART19 alone redeveloped JeKo, suggesting that AXL inhibition enhanced CART cell persistence (Fig 1e). Finally, we validated our preclinical findings in a correlative analyses of Phase I clinical trial of TP-0903 for patients with solid tumors (NCT02729298). Blood T cells from 3 patients were isolated and analyzed before and a week after treatment with TP-0903. Similar to our findings, there was a significant reduction in Tregs, reduction of inhibitory receptors and polarization to a Th1 phenotype. These findings will be further investigated in a planned Phase I clinical trial of TP-0903 in relapsed/refractory CLL (NCT03572634). In summary, we demonstrated for the first time that AXL inhibitior is capable of polarizing T cells into a Th1 phenotype, downregulates inhibitory receptors, reduces CRS associated cytokines and synergizes with CART cells in B cell malignancies. These findings encourage further study of TP-0903 as an enhancer of T cell immunotherapies. Disclosures Mouritsen: Tolero Pharmaceuticals: Employment. Foulks:Tolero Pharmaceuticals: Employment. Warner:Tolero Pharmaceuticals: Employment. Parikh:Janssen: Research Funding; Abbvie: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding; MorphoSys: Research Funding; Pharmacyclics: Honoraria, Research Funding; Gilead: Honoraria. Ding:Merck: Research Funding. Kay:Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta: Research Funding; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees. Kenderian:Tolero Pharmaceuticals: Research Funding; Humanigen: Research Funding; Novartis: Patents & Royalties.

Abstract Aberrant over production of monoclonal immunoglobulin is a classic feature of the plasma cell (PC) malignancy multiple myeloma (MM). At diagnosis and more frequently upon relapse, some MM patients present with increased production of monoclonal free light chain (FLC) without a corresponding increase in intact immunoglobulin. This phenomenon is commonly referred to as light chain escape (LCE). As previously described (Walters et al. 2018, Experimental Hematology, Vol 57 p42-49), we have established a monoclonal IgA kappa MM cell line, designated as MC-B11/14, from the bone marrow (BM) aspirate of a 46 year old man diagnosed with MM. This cell line is non-hyperdiploid and possesses an 11;14 chromosomal translocation. Given our long standing interest in immunoglobulin function and secretion, we used CRISPR technology to knock out IgA heavy chain production in an effort to create a model of LCE. Successful knockout (KO) of IgA expression was demonstrated by immunofluorescence, flow cytometry, and western blot. As expected, secretion of intact IgA was undetectable in the MC-B11/14IgA- cells. Notably, no significant difference in morphology, phenotypic markers, or growth patterns was observed between the MC-B11/14WT and MC-B11/14IgA- cells. Examination of drug sensitivity between the MC-B11/14WT and MC-B11/14IgA- cells revealed that the response to pomalidomide treatment was similar between the MC-B11/14WT and MC-B11/14IgA- cells; however, the MC-B11/14IgA- cells were found to be more resistant to treatment with bortezomib. Given that increased bortezomib resistance was the only significant difference detected between the MC-B11/14WT and MC-B11/14IgA- cell lines in our initial study, we decided to take a metabolomics approach to examine whether loss of IgA heavy chain expression may alter cellular metabolism. Metabolomics or the profiling of small molecules is a powerful tool to characterize the metabolome of cells/biological systems and has proven to be useful in identifying alterations that occur in malignancy. In this regard, we employed pathway-specific targeted LC-MS/MS protocols in order to examine over 330 metabolites in 35 metabolic pathways in MC-B11/14WT and MC-B11/14IgA- methanolmetabolite extracts using an Agilent 1290 UPLC-6490 QQQ-MS liquid chromatography mass-spectrometer. Of the 330 metabolites examined, 28 were found to be significantly more abundant in the MC-B11/14IgA- cells compared to MC-B11/14 WT cells while 79 were found to be significantly less abundant. In general, the majority of significant metabolic differences between the two cell lines were found to involve metabolites from various amino acid synthesis pathways. More specifically, the MC-B11/14 WT cell line was found to possess significantly more citraconic acid, anthranilic acid, and homovanillic acid. By contrast, further analysis revealed that the MC-B11/14IgA- cell line was found to possess an 11.5 fold increase of the metabolite 2-hydroxyglutarate (2-HG). 2-HG has previously been shown to act as a competitive antagonist of α-ketoglutarate (α-KG), which results in the inhibition of α-KG-dependent dioxygenases including the JmjC domain-containing histone demethylases (KDMs) and the ten-eleven translocation (TET) family of DNA hydroxylases. Notably, these proteins are known to play a role in histone and DNA demethylation, respectively. Taken together, these data suggest that MM cells that have undergone LCE may also possess an accumulation of 2-HG which could lead to altered patterns of histone and DNA methylation. Given that MM patients who relapse with LCE typically have a poorer prognosis, further investigation of metabolomics in light-chain only expressing cell lines and patients who have relapsed with LCE is warranted. Disclosures No relevant conflicts of interest to declare.

Abstract Intercellular communication between multiple myeloma (MM) cells and the normal bone marrow stroma leads to a modification of the bone marrow microenvironment, which favors tumor progression. New developments in extracellular vesicle (EV) research suggest that this diverse population of vesicles, released by cancer cells including MM cells, express transmembrane proteins and carry cargo that can modify recipient cells in myriad ways. In particular, tumor-derived EVs have been shown to create permissive microenvironments that lead to metastatic colonization by circulating tumor cells. As we have previously shown that MM EVs can enhance proliferation of recipient MM cells, we hypothesize that MM EVs can potentially play a much larger role in MM development and progression. In order to understand the scope of potential EV roles in MM, we executed a comprehensive proteomic analysis of the cargo of MM EVs. We isolated EVs from patient derived MM cell lines that represent the most common genetic variants of this tumor, and used in vitro generated plasma cells (IVPCs) as a reference population. In a first pass analysis, we selected proteins that were expressed ≥3 fold higher in MM EVs than in IVPC EVs, and this analysis identified 306 proteins. Of interest, included in the 306 differentially proteins were several involved in the regulation of cell adhesion such as members of the a disintegrin and metalloprotease domain (ADAM8, 9, 10, 13, 15 and 22) family, which are also associated with inhibition of cell proliferation. Moreover, CEACAM1, PTPRK, and CDH2, which are also linked to cellular adhesion, were also expressed at a higher level in MM EVs. Various proteins linked to myeloma cell biology were likewise found to be over-represented in MM EVs, and these include BCMA, ITGAV, ITGB5, IL6ST (gp130), CD276 (B7-H3), and CD28. As we are most interested in biologically relevant and actionable proteins present uniquely in MM derived EVs, we filtered from our data proteins that were also present in EVs from IVPCs. We further filtered out proteins known to be present in EVs from normal cells. This filtering strategy reduced the number of interesting candidates to 8, which included CD28, MET, TRKC, and ADAM15. Thus far, we have confirmed the presence of these 4 proteins in a panel of MM EVs, and we are currently in the process of validating additional candidate cargo proteins for their biological role in enhancing tumor cell proliferation and/or protection from apoptosis. In summary, EV proteomic analysis of cell lines representing MM genetic subtypes can lead to the identification of biologically relevant proteins transported systemically by EVs as well as suggest novel biomarkers that are easily detectable in plasma and may permit earlier recognition of disease progression in patients with MM. Disclosures No relevant conflicts of interest to declare.

Abstract Backgrounds B cell receptor (BCR) signals and microenvironment protection have been recognized to have key influences on CLL survival and activation. Akt activation is down-stream of the BCR signaling cascade and is critical in the mediation of the bi-directional interactions between CLL B-cells and bone marrow stroma. MK2206 is an allosteric Akt inhibitor previously tested for safety in solid tumor patients. Our preclinical data has demonstrated the synergy of MK2206 with bendamustine (B) to induce CLL apoptosis in vitro. MK2206 also abolished the signal activation and cytokine production induced by BCR ligation in CLL leukemic cells (Ding, ASH 2012). Therefore, we conducted a phase 1 trial testing the safety and efficacy of MK2206 in combination with bendamustine and rituximab (BR) for patients with relapsed/refractory CLL. Methods A phase 1 dose-escalation study was designed to define the maximum tolerated dose (MTD) as well as the safety and efficacy of 3 dose levels (90 mg or 135 mg or 200 mg weekly) of the oral Akt inhibitor MK2206 in combination with bendamustine (70 mg/m2 daily for 2 days in each cycle) and rituximab (cycle 1: 375 mg/m2, cycle 2-6: 500 mg/m2) therapy in relapse/refractory CLL patients. To test the targeting efficacy of MK2206 on Akt and downstream signals, we designed a one-week “run-in” of single agent MK2206 before the initiation of BR during cycle 1. MK2206 was then given along with BR on day 1 of cycles 2-6. Tumor response was evaluated 2 months after the 6th/last cycle of therapy based on IWCLL 2008 criteria. Correlative laboratory studies included assessment of Akt signaling and the plasma cytokine levels (multiplex beads assay (Invitrogen)) at baseline and after one week of first dose of single agent MK2206. Results Nine patients (median age 68) were enrolled in the phase I portion of this trial. All 9 patients had unmutated immunoglobulin heavy chain variable region genes (IGHV) and 4 had del(11q). Six had received prior chemoimmunotherapy (CIT: FCR or PCR or PCO) and 5 had high risk disease defined as early progression within 26 months of the last CIT. Three had received alemtuzumab containing regimen. We have observed potent lymphocyte mobilization in all evaluated patients (n=7) after one-dose of MK2206 during week 1 (mean increase ALC: 35%) as well as reductions of plasma CCL3, CCL4 and CCL2 in the majority of the patients. A single dose-limiting toxicity (DLT) (febrile neutropenia and hemolytic anemia) was observed at dose level 1 (90 mg weekly) among 6 patients treated at this dose. Two patients with dose-limiting toxicities at dose level 2 (135 mg weekly) were observed where one patient had a rash and one patient had dehydration and febrile neutropenia among three patients treated at this dose. Accordingly, the 90 mg once weekly dose was determined to be the MTD for phase 2 trial testing. The most frequent grade 3 or 4 adverse events observed to date were neutropenia (44%) including febrile neutropenia (22%), rash (22%), diarrhea (22%), nausea and vomiting (11%), dehydration (11%), hemolytic anemia (11%) and thrombocytopenia (11%). The frequency of cytopenias was similar to the published German experience with BR (50% grade 3 or 4 hematological toxicities compared to 44% in N1087) in relapsed CLL patients (Fischer, JCO 2011). The median number of cycles received was 6 (range 1-6). Using an intent to treat analysis, two patients achieved complete response (CR, n= 2, 22%), one patient achieved nodular partial response (nPR, n=1, 11%) and five patients achieved a partial response (PR, n=5, 56%). The overall response rate (ORR) is 89%. Conclusion The Akt inhibitor MK2206 in combination with BR is well tolerated in patients with relapsed/refractory CLL. Single dose of MK2206 inhibits Akt phosphorylation, mobilizes leukemic cells and decreases plasma cytokines involved in BCR signals. The preliminary efficacy of this combination demonstrated promising results with a 89% ORR and 22% CR albeit in the small phase 1 cohort. These results compare favorably to BR alone (9% CR and 59% ORR) in relapsed/refractory CLL patients. Phase 2 testing of this combination is now underway. Acknowledgment This work was supported by the funding from NCI grant K23CA160345 (WD), NCI grant CA95241 (NEK) and Alliance for Clinical Trials in Oncology (CA025224 and CA33601). Disclosures: Off Label Use: MK2206, used in a phase 1 trial of relapsed CLL disease. Shanafelt:Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding. Zent:Novartis: Research Funding; GlaxSmithKline: Research Funding; Biothera: Research Funding; Genzyme: Research Funding; Genentech : Research Funding. Reeder:Celgene: Research Funding; Novartis: Research Funding; Millenium: Research Funding.

Abstract The introduction of novel immunomodulatory drugs (IMiDs), such as lenalidomide, has drastically improved the survival of patients with multiple myeloma (MM). While it has been shown that patients with specific cytogenetic subtypes, namely t(4;14), have the best outcomes when treated with bortezomib-based regimens, the relationship between cytogenetic subtypes and response to IMiDs remains unclear. Using DNA synthesis and apoptosis assays, we investigated the relationship between cytogenetic subtype and lenalidomide response in a representative panel of human myeloma cell lines (HMCLs). Additionally, we examined HMCL protein expression levels of the lenalidomide target cereblon (CRBN) and its putative downstream target interferon regulatory factor 4 (IRF4), both of which have previously been shown to be predictive of lenalidomide response in HMCLs. Our results reveal that response to lenalidomide did not correlate with specific cytogenetic translocations. There were, however, distinct groups of lenalidomide-responsive and non-responsive HMCLs, as defined by inhibition of cellular proliferation; notably, all of the hyperdiploid HMCLs fell into the latter category. Repeated dosing of lenalidomide significantly lowered the IC50 of the responsive HMCL ALMC-1 (IC50 = 2.6 μM versus 0.005 μM, p=0.004), but did not have an effect on the IC50 of the non-responsive HMCL DP-6 (p>0.05). Moreover, no association was found between lenalidomide responsiveness and CRBN or IRF4 expression levels. In summary, our data indicate that lenalidomide sensitivity is independent of cytogenetic subtype in HMCLs. Additionally, while CRBN and IRF-4 have been shown to be associated with response to lenalidomide in patients, these findings do not translate back to HMCLs, which could be attributable to factors present in the bone marrow microenvironment. These results suggest that the development of a new treatment paradigm based on cytogenetic subtype of MM alone may be insufficient, and that additional markers may need to be incorporated. Furthermore, the disconnect between our findings of expression of CRBN and IRF-4 in lenalidomide-treated HMCLs and patient data highlights the need for further investigation into the role of the bone marrow microenvironment on the regulation of these previously demonstrated markers of lenalidomide response. Disclosures: No relevant conflicts of interest to declare.

Abstract Hereditary atransferrinemia is a rare but instructive disorder that has previously been reported in only 8 patients in 6 families. It is characterized by microcytic anemia and by iron loading, and can be treated effectively by plasma infusions. We now report the first case known in the United States. We determined the sequences flanking the exons of the human transferrin gene and sequenced all of the exons and some of the flanking regions of the patient's DNA and that of her parents. The patient's DNA revealed a 10-base pair (bp) deletion, followed by a 9-bp insertion of a duplicated sequence. There was also a G→C transversion at complementary DNA (cDNA) nt 1429, predicting that a proline was substituted for the alanine in amino acid position 477 (Ala 477 Pro). The latter mutation occurs at an evolutionarily highly conserved site; 704 control alleles were screened and this point mutation was not found. Each of the patient's transferrin genes contains one mutation, ie, the patient is a compound heterozygote for these mutations, because one was found in each of her parents. In addition to these mutations, which we regard to be causative in the patient's atransferrinemia, a silent polymorphism at cDNA 1572 G→C was found in exon 13 as well as 2 previously unreported polymorphisms at IVS8 + 62 c→t and IVS14-4 c→a. The mutation in nt 1572 and that in intron 8 were common in the general population; the intron 14 mutation is rare.