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Introduction: Large Granular Lymphocyte (LGL) leukemia is a rare lymphoproliferative disorder usually manifested in elderly individuals, with mean age of onset at 65. However, LGL leukemia cases in pediatric and young adult (age 50) cohort. Methods: We retrospectively collected clinical data from 23 young adult (age 50) LGL leukemia patients. All participants signed informed consent to provide biospecimens and clinical records to the LGL Leukemia Registry. Whole exome (PBMC and saliva) and RNA sequencing (PBMC) was conducted on 115 patients. Sanger sequencing was utilized to determine STAT3 mutational status in the remaining 102 patients. Results: STAT3 is the most frequently mutated gene in LGL leukemia, and the mutation is associated with lower absolute neutrophil counts (ANC). We observed no differences in the frequency of STAT3 somatic mutations in young adult, adult, and older adult LGL leukemia (p=0.143). However, we detected a significant positive linear relationship between age of the patient and ANC values (p=0.002) for STAT3 mutant patients (Figure 1A). STAT3 wild-type (WT) patients did not display this relationship (p=0.756). No age associated trends were detected for hemoglobin values, although we detected decreasing red blood cell (RBC) values with increasing age for both STAT3 mutated and WT patients (p= 0.031, 0.010, respectively). No difference in sex (male vs female) balance was found within the young adult cohort (p=0.190). As rheumatoid arthritis (RA) is observed in ~30% of LGL leukemia patients, we asked if this prevalence also holds in the younger cohort. RA was observed in similar proportion in all young, adult, and older adult groups (p=1). To compare the severity of disease manifestation between the age groups, we first asked how many patients ever initiated LGL-related treatments in each age group. We found that patients under 50 are more likely to be on LGL related treatments compared to patients over the age of 50 (p=0.016) (Figure 1B). Next, we examined length of survival between age groups. Despite increased initiation of LGL related treatments, no statistically significant difference in overall survival (OS) was observed between patients under 50 and patients over the age of 50 (p=0.33) (Figure 1C). Therefore, we asked if the treatment response in younger patients is better compared to older patients. We observed no statistically significant differences in the routine LGL treatment responses between patients under 50 and patients over the age of 50 (p=0.732). Interestingly, epigenetic and chromatin modifier mutations are equally common in patients 50 years old and under relative to older adult patients (p= 0.271) (Figure 1D). Transcriptomic analysis of the PBMC data revealed enrichment of TNFα signaling in patients older than 50, while IFNα and IFNγ signaling was enriched in patients 50 years old and younger. Conclusions: We report the clinical and molecular analysis of LGL leukemia in pediatric/young adult and adult patients compared to the older adult patients that comprise the majority of LGL cases. We show that while STAT3 mutation frequency is similar across all age groups, younger patients with STAT3 mutation show lower ANC values compared to older patients. Importantly, we show that younger patients exhibit a more severe disease, as indicated by increased initiation of LGL treatment. However, overall survival in younger groups compared to older patients is not impacted. Finally, we show that younger patients display similar proportions of epigenetic and chromatin modifying mutations that are a prominent feature of older patients, and feature enrichment of IFNα and IFNγ signaling distinct from older patients. Figure 1 Figure 1. Disclosures Feith: Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees. Loughran: Dren Bio: Membership on an entity's Board of Directors or advisory committees; Keystone Nano: Membership on an entity's Board of Directors or advisory committees; Bioniz Therapeutics: Membership on an entity's Board of Directors or advisory committees; Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Introduction The hemoglobin molecule is a tetramer consisting of 2 pairs of globin chains, each of which contains a heme group. During fetal development, the major hemoglobin is Hb F (α2γ2). In a normal adult, the major hemoglobin is Hb A (α2β2). The α-globin gene cluster is located on

Abstract 1013 More than 3% of Chinese in Hong Kong are heterozygous carriers of β-thalassemia. Homozygotes or compound heterozygotes for β-thalassemia are usually severely ill and require monthly transfusions. Increased production of fetal hemoglobin (HbF) can modulate the disease severity by compensating for the shortfall of HbA caused by the β-thalassemia mutations. HbF level in adults varies and is regulated as a multigenic trait. Three major HbF quantitative trait loci (QTL) have been identified: the C/T SNP also known as the Xmn I site at the Gγ-globin gene promoter, the BCL11A polymorphism on chromosome 2p16, and the HBS1L-MYB intergenic polymorphism (HMIP) on chromosome 6q23. The functional motif for each of these 3 QTLs responsible for their effects upon HbF is not known. We undertook a genome-wide association study (GWAS), using Illumina Human 610-Quad BeadChip array, on 619 Chinese β-thalassemia heterozygotes from Hong Kong. In this population, the variance in HbF due to HMIP is 13.5%, significantly higher than that due to BCL11A polymorphism (6.4%). We used 1,000 Genomes Project data, SNP imputation, comparisons of association results across populations, predicted binding of transcription factors, and phylogenetic conservation to identify the functional variant in HMIP. Based on these lines of evidence, a hitherto unreported association between HbF expression and a 3-bp deletion on chromosome 6q23 was found. In 335 Chinese β-thalassemia heterozygotes, the 3-bp deletion polymorphism is in complete linkage disequilibrium with rs9399137, the SNP found in multiple GWAS to be most significantly associated with HbF (P=1.4E-24 in the Chinese cohort GWAS). Flanking this deletion are conserved binding sites for TAL1/SCL1, E47, GATA, and RUNX1/AML1, which are essential erythropoiesis-related transcription factors. The 3-bp deletion changes the normal DNA binding configuration of these transcription factors and spatial configuration for DNA-protein binding and/or protein-protein interactions. Furthermore, this 3-bp deletion polymorphism resides within a likely erythroid distal regulatory region manifested by DNase I hypersensitivity and GATA-1 binding (Wahlberg et al, Blood 114:1254, 2009). We hypothesized that a 61-bp fragment of DNA that encompasses the site of the 3-bp deletion polymorphism might have enhancer-like activity. When ligated to the Gγ-globin gene 1.4 kb proximal promoter linked to a luciferase reporter gene, the 61-bp fragment of DNA enhances the Gγ-globin gene promoter activity by more than 3-fold after transient transfection into K562 cells. A 58-bp fragment of DNA that includes the 3-bp deletion has 60% more enhancer-like activity than the 61-bp fragment without the deletion. These findings suggest that this 3-bp deletion polymorphism is most likely the functional motif accounting for HMIP modulation of HbF. Further studies are needed to identify target genes for this enhancer-like activity mediated by the DNA sequences encompassing the 3-bp deletion polymorphism in HMIP. These studies also suggest that this experimental approach could be used to identify functional motifs in other genotype-phenotype association studies. Disclosures: No relevant conflicts of interest to declare.

The occurrence of Hb H hydrops fetalis is reported for the first time. The mother has zeta-alpha thalassemia 1 (zeta zeta alpha alpha/----) and the father has non-deletion alpha thalassemia [zeta zeta alpha alpha/zeta zeta (alpha alpha)T]. The complete deletion of the zeta alpha cluster on one chromosome was confirmed by quantitation of alpha and zeta gene numbers, the normal alpha and zeta gene patterns arising from the remaining normal chromosome, and the decreased alpha/beta globin chain ratio of 0.57. The non-deletion alpha thalassemia defect could only be identified by the imbalanced alpha/beta globin chain ratio of 0.65 in the presence of normal gene numbers and patterns. The newborn was markedly anemic, unlike those with classical Hb H disease, because the non-deletion alpha thalassemia defect is more severe than alpha thalassemia 2. The decreased zeta genes during fetal life might have additional deleterious effects. In this family, the distinct BamHI restriction fragment length polymorphism in the hypervariable region of the zeta genes may be used for future prenatal diagnosis.

A new point mutation due to C----T transition at codon 189 (TCA) of the factor VIII:C gene was found in a Chinese patient with moderately severe hemophilia A. This mutation abolishes the EcoRI site (GAATTC) in exon 4 and can be directly detected by polyacrylamide gel electrophoresis of amplified genomic DNA.

A new frameshift mutation due to an insertion of G between codon 14/15 of the beta-globin gene was found in two unrelated Chinese patients with Cooley's anemia. The first patient (W.S.) was homozygous for haplotype 5 (Chinese) and carried a codon 41/42 (four base pair deletion) mutant, while the second patient (C.K.) was homozygous for haplotype 2 (Chinese), and also had a codon 17 (A----T) nonsense mutation. Molecular cloning and M13 sequencing of the beta gene in patient W.S. revealed that the new mutant was found in a beta-globin gene framework type 3 (Asian). Direct sequencing was performed on polymerase chain reaction-amplified genomic DNA from patient C.K. With the new mutation, an additional BstNI or EcoRII recognition site is generated and the abnormal restriction fragment (134 basepair) can be directly visualized on polyacrylamide gel electrophoresis of the amplified genomic DNA.