ALBERT

Description: We performed a retrospective analysis of our Spanish database of patients with ET in order to assess the role of different response predictive potential factors to anagrelide treatment. 411 ET patients from 54 sites from February 2005 to August 2006 were included in a retrospective chart review. ET was diagnosed according to PVSG criteria (1997). All patients had started treatment with anagrelide before December 2004, either as a first line or as a second line therapy. The population was divided itself in three groups of risk at diagnosis: of high Risk: those patients older than 60 years or with previous history of thrombosis (40.6%); of low Risk: patients younger than 60 years with thrombocytosis lower than 1.5· 109/L, without cardiovascular risk factors and previous history of thrombosis (35.8%); intermediate risk: those patients who did not reunite the criteria of previously mentioned groups (23.6%). The response to the treatment was defined as complete remission (CR), when a reduction of platelets′ count were equal or less than 400×109/L, or a reduction higher than 50% with respect to the basal numbers; partial remission (PR), when the platelets′ count was between 400× 109/L and 600×109/L with respect to the basal numbers; and no response, those with a lower reduction than PR or increasing their platelets′count. CR was obtained in 219 patients (53.6%; 95%CI = 48.6–58.5) and PR in 113 (27.6%; 95%CI = 23.4–32.2), giving an overall response (OR) rate of 81.2% [95%CI = 77.0–84.9]. The influence of certain factors (such as age, gender, risk group, platelets levels before treatment with anagrelide, previous cytoreductive untreated patients and maximum anagrelide dose), in the objective response was explored. A chi-square test and a multivariate analysis (logistic regression) were performed. A worse response (p = 0.021) was associated with a higher dose (≥ 2.5 mg per day). A trend (p = 0.103) to better response in patients without previous treatment (anagrelide as a first line) was detected. Additionally a significant association (p = 0.02) between the previously treated with hydroxyurea and the presence of evolutive hematological transformations (myelofibrosis and acute myeloid leukemia/myelodysplastic syndrome) was observed. It is difficult to set a cause effect relation in all these findings due to the retrospective design of the analysis. As a conclusion, the response to anagrelide in ET patients was demonstrated to be independent of the age, gender, platelets′ count and level of previous risk.

Description: The balance between actions of procoagulant and anticoagulant factors protects organisms from bleeding and thrombosis. Thus, antithrombin deficiency increases the risk of thrombosis, and complete quantitative deficiency results in intrauterine lethality. However, patients homozygous for L99F or R47C antithrombin mutations are viable. These mutations do not modify the folding or secretion of the protein, but abolish the glycosaminoglycan-induced activation of antithrombin by affecting the heparin-binding domain. We speculated that the natural β-glycoform of antithrombin might compensate for the effect of heparin-binding mutations. We purified α- and β-antithrombin glycoforms from plasma of 2 homozygous L99F patients. Heparin affinity chromatography and intrinsic fluorescence kinetic analyses demonstrated that the reduced heparin affinity of the α-L99F glycoform (KD, 107.9 ± 3nM) was restored in the β-L99F glycoform (KD, 53.9 ± 5nM) to values close to the activity of α-wild type (KD, 43.9 ± 0.4nM). Accordingly, the β-L99F glycoform was fully activated by heparin. Similar results were observed for recombinant R47C and P41L, other heparin-binding antithrombin mutants. In conclusion, we identified a new type of mosaicism associated with mutations causing heparin-binding defects in antithrombin. The presence of a fully functional β-glycoform together with the activity retained by these variants helps to explain the viability of homozygous and the milder thrombotic risk of heterozygous patients with these specific antithrombin mutations.

Description: Background: De-ubiquitinating enzyme BAP1, a fundamental deubiquitinase in the epigenetic regulation of transcription factors and functionally related to ASXL1, is mutated in a hereditary cancer syndrome with increased risk of mesothelioma and uveal melanoma. In a recent murine study, absolute BAP1 depletion generated specimens with similar characteristics to myelodysplastic / myeloproliferative syndromes in humans (ineffective hematopoiesis and myeloproliferation), mainly to chronic myelomonocytic leukemia (CMML) (Dey, et al. Science 2012). Aim: The aim of this study was to quantify BAP1 gene expression in patients diagnosed with a variety of myeloid neoplasms, and compared it with healthy donors. We furthermore explored the possible association of BAP1 low expression level and the presence of ASXL1 mutations or BRCA1 protein levels. In addition, a regression analysis to determine the possible correlation of peripheral blood and bone marrow expression levels was performed. Methods: We included patients diagnosed between 2008-2014 of CMML, myelodysplastic sydrome (MDS) chronic myeloid leukemia (CML) and acute myeloid leukemia (AML), of whom bone marrow DNA and RNA were available at diagnosis. As controls, 6 healthy bone marrow donors were used. BAP1 and BRCA1 expressions levels were quantified by RT-qPCR, using the same healthy bone marrow donor sample as an inter-assay normalizing- calibrator. The study of somatic ASXL1 mutations was carried out by the Sanger method. For statistical studies, the T-Student, Pearson correlation and/or U Mann-Whitney test, were used when needed. For survival analysis COX regression and the ROC curves were used. A two-side P

Description: Annexin V has phospholipid-binding capacity and plays a potent antithrombotic role. Recently, a C to T transition has been described in the Kozak region of this gene, affecting the nucleotide preceding the initiation ATG codon. We have developed a simple method to detect this genetic change, showing by analysis of 580 Mediterranean white subjects that the −1C to T transition (−1C〉T) is a common polymorphism (allele frequency, 0.121). This polymorphism is in linkage disequilibrium with a new C〉G polymorphism located 27 bp downstream in intron 2. We show that −1C/C carriers presented significantly lower plasma levels of annexin V than −1C/T subjects (0.45 ± 0.20 ng/mL versus 0.73 ± 0.28 ng/mL, respectively;P = .02). In vitro transcription/translation experiments support that the −1T allele increases translation efficiency. The clinical relevance of the −1C〉T change was investigated in consecutive patients with nontraumatic spontaneous intracranial hemorrhage (n = 225), deep venous thrombosis (n = 151), and coronary heart disease (n = 101). Finally, we also studied 166 survivors of an acute myocardial infarction occurring at age of 45 or less. This polymorphism seems to have a minor effect in bleeding disorders, but to play a protective role against early myocardial infarction, reducing by 2-fold the risk of developing the disease (P = .006; odds ratio, 0.51; 95% confidence interval, 0.30-0.85).

Description: Thymic-independent peripheral expansion of CD8+ cells derived from the graft in the initial stage of post-HSCT immune recovery is a well-known physiological event. Nevertheless, the description of symptomatic LGL leukemias and aggressive malignant cases in this setting may generate uncertainty, mostly in those cases in which the cytotoxic T lymphocyte expansion CTLe persists beyond the early transplantation period. We aimed to assess the nature of CTLe in adults during the post-alloHSCT period in a series of 154 patients with a long term surveillance. We studied the longitudinal kinetics of those expansions, their relation to clinical events, and their phenotypic and molecular features, including recently reported CTL leukemia-STAT3 mutations. In our study, trying to adhere to the WHO annotation of T-LGL, we considered two definitions for a CTL expansion: an absolute increase (≥ 2000 x109/L), and a relative expansion (a CD8/CD4 ratio ≥ 1.5), persisting more than six months in both cases. Persistent relative CTLe cases are frequent (49%) and related with timoglobulin prophylaxis (p≤0.001), acute graft versus host disease (GVHD, p=0.02), reduced intensity conditioning (p=0.04) and fungal and viral infections in the early post-HSCT. No differences in the number of serious infectious events from day 180 was found. Absolute CTLe are scarce (9%), related with chronic GVHD and absence of relapses. TCR rearrangement was reported as clonal and oligoclonal in the majority of patients with CTLe. We studied in a cross sectional manner with an extended immunophenotypic panel 17 patients: 5 patients with an absolute CTLe and 12 cases with a relative CTLe. A similar cytotoxic T αβ-effector phenotype was observed in all cases, with slight differences in the expression of CD25, CD16 and 1a. One patient with a relative CTLe expressed CD56 intensely: his ratio normalized at day 730 and no immune-related events were recorded. DNA stored during the post-alloHSCT setting was available from 68/75 relative CTLe patients (14/14 absolute CTLe cases). All of them went through molecular TCR rearrangement and STAT3 exon 21 mutations determination. In the relative CTLe cohort, TCR rearrangement was described as clonal, oligoclonal or polyclonal in 77%, 16% and 7%, respectively. Regarding absolute CTLe patients, TCR rearrangement was described as clonal in all the patients (n=14) of this subset. To increase the sensibility of the Sanger PCR, it was performed on DNA from CD3+ sorted cells in 54 out of 68 cases. No STAT3 mutation could be found in the CD3+ sorted fraction of relative or absolute defined CTLe. Not using an absolute threshold would establish a diagnosis of a persistent CTL expansion in 49% of our cohort of allo-transplanted patients. Additional diagnostic tools, as an effector phenotype, the presence of a NK marker or a monoclonal TCR rearrangement would not reduce significantly that percentage: CD57 was invariably expressed in CTLe cases, and 80% of our patients with expansions showed a TCR monoclonal pattern. STAT3 mutations resulting in persistent proliferation of CTL clones are a frequent event in large granular lymphocytic leukemia, and those clones have also been described in autoimmunity-driven disorders as acquired aplastic anemia and hypocellular myelodysplastic syndromes. We establish in this study the absence of exon 21 STAT3 mutations in the persistent CTL expansions found in a large series of patients with a long-term post-alloHSCT surveillance. The absence of STAT3 mutations and the CD8/CD4 declining longitudinal kinetics in the late period, supports its benign nature, expressed clinically by the null detrimental impact of these expansions on post-transplant outcome and/or serious infectious events. Figure 1. Relative and absolute CTLe kinetics. A) Linear representation over time of the CD8/CD4 ratio in the 75 patients with relative CTLe. B) Trend linear plot of 25 patients with a relative CTLe and a follow up of, at least, 1440 days from transplantation. Each line depicts a patientxs longitudinally measured CD8/CD4 ratio. Patients are grouped by similar pattern of ratio behaviour through follow up. The line "slope" depicts the magnitude of the change between time points. C) Linear representation over time of the CD3/CD8 count in PB in the 14 patients with an absolute CTLe. D) Trend linear plot illustrating the CD8/CD4 ratio behaviour of the 14 patients with an absolute CTLe. Figure 1. Relative and absolute CTLe kinetics. A) Linear representation over time of the CD8/CD4 ratio in the 75 patients with relative CTLe. B) Trend linear plot of 25 patients with a relative CTLe and a follow up of, at least, 1440 days from transplantation. Each line depicts a patientxs longitudinally measured CD8/CD4 ratio. Patients are grouped by similar pattern of ratio behaviour through follow up. The line "slope" depicts the magnitude of the change between time points. C) Linear representation over time of the CD3/CD8 count in PB in the 14 patients with an absolute CTLe. D) Trend linear plot illustrating the CD8/CD4 ratio behaviour of the 14 patients with an absolute CTLe. Disclosures No relevant conflicts of interest to declare.

Description: Background: We and others have reported on the impact of recurrent somatic mutations not only in the multistep pathogenetic process, but also in the clinical heterogeneity of chronic lymphocytic leukemia (CLL) patients. Immunophenotyping, as part of the diagnostic workout, is used for assessing clonality, as a differential diagnosis tool, and to examine the expression of molecules associated with a worse prognosis. Recently, NOTCH1 mutations have been linked to low CD20 levels in CLL and with a relative resistance to anti-CD20 immunotherapy in vitro. But to date, there is limited information on the correlation between cell surface marker expression and the presence of somatic mutations in CLL. The aim of this study was to evaluate potential associations between an extended phenotypic panel and the mutational status of 13 recurrently mutated genes in CLL detected by deep sequencing. Patients and Methods: To this end, we performed targeted NGS sequencing of blood samples, collected at diagnosis, from 131 CLL patients. Every patient underwent, at baseline, a flow cytometry characterization with a panel including (sIg)λ, (sIg)κ, CD19, CD5, CD11b, CD81, CD10, CD79b, CD29, CD38, FMC7, CD22, CD45, CD103, CD11c, CD25, ZAP70, CD11a, and CD24. We designed a TruSeq Custom Amplicon panel (Illumina, Inc. San Diego, CA, USA) containing 13 genes and covering 28.099 bases. The average amplicon size was 238 base pairs and ~ 99.1% of the regions were covered on both strands. Paired-end sequencing (2x250 bp) was performed with MiSeq v2.2 chemistry, and a mean depth of 998 reads/base within the regions of interest was obtained. Raw data were analyzed with IlluminaonJboard Real Time Analysis (RTA v.2.4.60.8) software and MiSeq Reporter. Results: With a median age of 68 y.o. (range, 33-95) and a slight male predominance, the median follow up time of our cohort was 43 months (24-104). We found that 47/131 (35%) patients harbored at least one mutation, with NOTCH1 (n = 13, 10%), ATM (n = 10, %), TP53 (n = 8, %), and SF3B1 (n = 8, 5.5%), as the most frequently mutated genes. Those patients with a NOTCH1 mutation showed a lower CD25 expression (25 mean fluorescence intensity units (MFIu)) than those without a mutation (45 MFIu), p=0.001. In addition, a higher expression of CD5 (265 vs. 219 MFIu, p= 0.02), of the monoclonal light chain (90.5 vs. 58.6 MFIu, p=0.03), and a higher percentage of CD38+ cells in the CD19+CD5+ compartment (37% vs. 19%, p=0.006) were significantly associated with the presence of, at least, one somatic mutation. We could not validate the recently reported association between the presence of NOTCH1 mutations and a low expression of CD20. In our cohort, the MFI expression in NOTCH1 mutated and non-mutated patients was 176 and 135 units, respectively (p=0.2) In the multivariate Cox analysis, the presence of a somatic variant in TP53 and a higher percentage of positive CD38 cells in the tumour population showed both a worse overall survival and shorter time to first treatment. The independence of these two variables was also supported by not finding a significative difference percentage of CD38 positive cells between TP53 mutated and non mutated cases (p=0.5). Conclusions: The associations described herein suggest potential pathogenic pathways in CLL, in particular the CD25-NOTCH1 axis, with a significative inferior expression of CD25 when activating NOTCH1 mutations are present. The relationship found between these two variables, with an inversed direction to that found in physiological conditions, has also been shown in the setting of NOTCH1-mutated acute lymphoblastic leukemia, emerging as a potential targetable pathway in this subset of CLL patients. Disclosures Maciejewski: Apellis Pharmaceuticals Inc: Membership on an entity's Board of Directors or advisory committees; Alexion Pharmaceuticals Inc: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau.

Description: Background and aims: Significant efforts have been made in international guidelines since 2009 to establish recommendations for the initial work-up of patients with suspected immune thrombocytopenia (ITP), and to define who should be treated and how. A previous retrospective study of 101 ITP patients identified important areas of inappropriateness in the diagnostic and therapeutic management of ITP (Lozano et al, 2016), which could negatively impact patient outcomes. This study was aimed to further analyze the level of implementation of current recommendations in the standard practice of adult ITP patients in an independent validation cohort, and compare it to the pre-guideline period. Methods: We collected retrospectively the clinical data of 146 primary adult ITP patients who initiated treatment with thrombopoietin receptor agonists (TPO-RA) between January 2012 and December 2014. Data on patient characteristics were obtained from medical records from November 2016 to January 2018 in a multicenter study from 19 secondary and tertiary Spanish hospitals. To evaluate the laboratory diagnosis and the appropriateness of treatment according to guidelines, two cohorts of patients were considered: "pre-group" and "post-group" depending on the date of diagnosis (before or after January 2010). Results: Patients in pre-group (n=71) and post-group (n=75) had a median follow-up from diagnosis of 13.6 years (7.5-54.2 years), and 4.6 years (2.2-7.6 years), respectively. The level of compliance of general diagnostic tests was analyzed and compared. Peripheral blood smear, an examination recommended by all recent guidelines, was performed in 84% of patients in the post-group, and in only 64% of those in the pre-group (P=0.007). Bone marrow assessment at diagnosis of the disorder was ordinarily performed in around half of the patients regardless of the period (54% and 47% in the pre- and post- groups, respectively; P=0.408). Remarkably, in 49% of the patients in the pre-group, bone marrow evaluation was primarily performed due to the department policy, whereas that reason decreased to 25% in the post-group (P=0.027). Moreover, in 21% and 9% of patients in the pre-group and post-group that underwent a bone marrow assessment at diagnosis, the peripheral blood film had not been previously examined (P=0.192). In our cohort, differences in the treatment patterns were analyzed. Prednisone was used as first line-therapy in 89% and 84% of pre- and post- groups, respectively (P=0.406). There was a significant decrease in the duration of first-line therapy with prednisone from start until withdrawal in the post-group compared with the pre-group (median 77 vs. 122 days, respectively; P=0.004). Following first-line treatment, more patients in the pre-group were exposed to further prednisone (45% vs. 25%, P=0.003), and also the number of second or subsequent lines of therapies with this corticosteroid were significantly higher (61 courses of prednisone retreatment in 32 patients vs. 22 courses in 19 patients in the pre- and post- groups, respectively; P=0.008). As expected, a higher proportion of patients in the pre-group underwent splenectomy, and also received more immunosuppressant and immunomodulatory drugs than those in the post-group prior to TPO-RA therapy (P

Description: Background and Aim: Azacitidine have shown clinical activity in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), particularly at low, non-cytotoxic doses favoring hypomethylation over cytotoxicity. Cancer/testis antigens (CTAs, encoding for immunogenic proteins which are normally expressed in the testicles and trophoblastic cells of the ovary, have been shown to undergo derepression after the use of demethylating agents in cancer cell line models. We took advantage of the unique model of Aza-treated high risk MDS to in vivocharacterize candidates for immunotherapy following hypometilating therapy. Methods:Targeted RNA-Seq (tRNA-Seq) designed to capture 214 CTA genes was performed in 19 MDS or CMML patients at day 0 and at day +28 after first AZA cycle. In 10 patients the analysis was extended to third and/or four cycles. Sequencing, coverage, mapping and alignment was performed using the Ion Torrent Browser Suite. We used LIMMA package or empirical analysis of digital gene expression data in R (edgeR) to identify differential expressed genes. A homogeneous treatment schedule with AZA (75 mg/m2/day x 7 days) was received by each patient.The response criteria were from IWG 2006, including a bone marrow and cytogenetic reevaluation after 6 cycles. The main candidates selected from de tRNA-seq experiment were characterized at protein level in cell lysate and/or plasma by Western Blot. Primary antibodies used from Thermo Fisher were: ADAM29, DDX53, PRAME, B-TUBULINA, FAM46D, B-ACT, TMPRSS12, MAGEB4, GAPDH, MAGEA1, MAGEA2, TSPY2, Cxorf48, DNAJB8, NY-ES0-1, CT83/Cxorf61 and TFDP3 from Santa Cruz Biotechnology. We used either cell lysate or plasma to study the protein levels in non-reducing SDS-PAGE at different acrylamide concentration depending on the protein size. ImageJ was used to quantify protein levels. Results:The median age of the cohort was 69 (range 48-81 years). The cohort consisted of 16 MDS and 3 CMML patients. MDS Patients were stratified based on IPSS as low or intermediate-1 (n=14) and intermediate-2 or high (n=2) risk groups and CMML patients based on a CPSS of intermediate-2 (n=3). Five patients were classified as complete responders achieving a complete cytogenetic and/or marrow remission (CR). Regarding the tRNA-Seq, on average, we achieved 3.888.308 mapped reads per sample (p25-p75, 1.2x106 - 5.9x106) which represents a sufficient depth for digital gene expression profiling of 214 genes. CTAs re-expressed significantly after one AZA cycle in complete responders were TFDP3 (FC=6,4), ZNF645 (FC=2), MAGEB4 (FC=3,1), MAGEA5 (FC=2), DDX53 (FC=2), VENTPX1 (FC=5,5), TSPY2(FC=3,4) and TSYP3 (FC=3,5). Other potential candidates with significantly re-expression in any responders vs. non responder were:SLCO6A1, ADAM29, DDX53, PRAME, FAM46D, TMPRSS12, MAGEA1, MAGEA2, Cxorf48, DANJB8, NYESO-1 and CT83. In the western-blot experiments, protein expression, longitudinally measured in peripheral blood lysate and/or plasma showed a parallel dynamic to gene expression in the case of TFDP3and DDX53. Conclusions:In this study, targeted RNA-seq is shown as effective and accurate tool to detect weakly expressed transcripts as CTAs. In this work, TFDP3 and DDX53, validated at proteomic level, emerge as most promising candidates for immunotherapy in combination with hypomethylating agents in MDS patients. Disclosures No relevant conflicts of interest to declare.

Description: The effectiveness of antiplatelet therapy as primary prophylaxis for thrombosis in low-risk essential thrombocythemia (ET) is not proven. In this study, the incidence rates of arterial and venous thrombosis were retrospectively analyzed in 300 low-risk patients with ET treated with antiplatelet drugs as monotherapy (n = 198) or followed with careful observation (n = 102). Follow-up was 802 and 848 person-years for antiplatelet therapy and observation, respectively. Rates of thrombotic events were 21.2 and 17.7 per 1000 person-years for antiplatelet therapy and observation, respectively (P = .6). JAK2 V617F–positive patients not receiving antiplatelet medication showed an increased risk of venous thrombosis (incidence rate ratio [IRR]: 4.0; 95% CI: 1.2-12.9; P = .02). Patients with cardiovascular risk factors had increased rates of arterial thrombosis while on observation (IRR: 2.5; 95% CI: 1.02-6.1; P = .047). An increased risk of major bleeding was observed in patients with platelet count greater than 1000 × 109/L under antiplatelet therapy (IRR: 5.4; 95% CI: 1.7-17.2; P = .004). In conclusion, antiplatelet therapy reduces the incidence of venous thrombosis in patients with JAK2-positive ET and the rate of arterial thrombosis in patients with associated cardiovascular risk factors. In the remaining low-risk patients, this therapy is not effective as primary prophylaxis of thrombosis, and observation may be an adequate option.

Description: Introduction Hermansky-Pudlak syndrome (HPS) is an inherited platelet disorder characterized by bleeding diathesis, oculocutaneous albinism (OCA) and, sometimes, serious clinical complicationssuch as immunodeficiency, granulomatous colitis, and/or pulmonary fibrosis. Heterogeneous clinical symptoms and a large number of possible genetic culprits (10 HPS genes, 〉120 exons) complicate an unequivocal diagnosis of HPS. This study aimed to assess the clinical and platelet phenotype in ten patients with suspected HPS, and to identify the underlying genetic defects. Methods Ten patients from six families (F1 and F3 were Spanish, F2 was Turkish and F4, F5 and F6 were Portuguese) presenting with OCA (confirmed by skin biopsy) and bleeding diathesiswere included. Bleeding was evaluated by ISTH-BAT score. Phenotyping included, in patients with fresh blood samples available, platelet aggregation and ATP release, flow cytometry (FC), 14C-serotonin uptake and whole-mount electron microscopy (EM). Patients DNA was analyzed using two different targeted panels by high throughput sequencing (HTS). Sequence variants classification was performed according to ACMP recommendations. Results Patient characteristics are summarized in table 1. In F1, that had no history of consanguinity, there were two affected sisters. Patients 1 (P1) had several episodes of gastrointestinal bleeding (GI), which was attributed to granulomatous colitis. F2 is a consanguineous Turkish family, were P3 had severe rectal bleeding, requiring colectomy combined with ileostomy surgery. Pathological examination of the colon was reported as non-granulomatous colitis. Her older sister (P4) had exhibited dyspnea and shortness according to diffuse bilateral pulmonary fibrosis (BPF) diagnosis. In F3, P5 had been referred with acute GI bleeding secondary to angiodysplasia. In the non-consanguineous F4, HPS was first confirmed in P6, who showed blonde hair, nystagmus and low visual acuity; his older sister was diagnosed with HPS later, at the age of 56 years old (P7), because her OCA was masked using dark brown hair-coloring products. In P8, born from a non-consanguineous family (F5), HPS was suspected early in life, four months of age, upon recognition of OCA, nystagmus, deep visual deficiency and exotropia with compensatory torticollis. Lastly, in the consanguineous Portuguese family (F6), the two affected children (P9 and P10) had also showed a horizontal and torsional nystagmus and reduced visual activity. P10 also suffered from epilepsy and mild development delay. In phenotyping studies, the Spanish patients (P1, P2, P5) showed impaired platelet aggregation to mild agonists and reduced platelet dense granules by FC and EM. No platelet studies could be performed in F2. In Portuguese patients (F4, F5 and F6), the ATP release studies demonstrated a dense granule deficiency (Table 1). Molecular diagnosis was achieved, as a first-line approach, by means of HTS gene panels that revealed: a) F1 (P1 & P2) a homozygous deletion c.2054delC (p.P685L fs17*) in exon 13 of the HPS4, which had been previously reported in one Asian patient who showed BPF; b) F2 (P3 & P4): anovel missense homozygousvariant c.272T〉C (p.L91P) in exon 4 of the HPS4. Remarkably, the phenotype of the two Turkish sisters was different, with one having had severe GI bleeding requiring colectomy, and the other had developed BPF. C) F3 (P5): a novel heterozygous variant c.2464C〉T (p.R822*) in exon 13 of the HPS3 was detected; d) F4 (P6 & P7) and F5 (P8): here a nonsense variant c.307C〉T (p.Q103*) was identified in exon 5 of the DTNBP1, which was previously reported in a Portuguese patient. E) F6 (P9 & P10): these patients carried a novel five base pair duplication in the single exon of HPS6, c.60_64dup (p.L22R fs*33). Conclusions This study reports 10 new HPS patients, which demonstrates the heterogeneous nature of this syndrome and the complex phenotype-genotype correlations. The novel HTStechnology has facilitated the molecular diagnosis of HPS in these patients. Among the underlying molecular pathology, we identified a novel p.L91P variant in HPS4 that is associated with a severe clinical phenotype. Funding Gerencia Regional de Salud (GRS 1647/A/17), Fundación Séneca (19873/GERM/15), Instituto de Salud Carlos III (ISCIII, PI17/01966, PI17/01311,CB15/00055), Grupo de trabajo SETH and Instituto de Investigación Biomédica de Salamanca (IBSAL, IBY17/00006). Table Table. Disclosures No relevant conflicts of interest to declare.