Assertion: Recommendations for following patients with Riata silicone-only leads for implantable cardioverter-defibrillators (ICDs) are not based on meaningful data, and SJM has not instituted clinical studies.Facts: We have conducted extensive bench and short-term studies, and on the basis of the scientific data and clinical experience, in which 85% of externalized conductors in returned leads functioned normally, we have worked with our independent medical advisory board to develop guidelines regarding care management. These guidelines have received support from both an expert panel of the Heart Rhythm Society and the Food and Drug Administration. No reports of a failure to pace or deliver a shock have been attributed to the presence of an externalized conductor.We recognize that externalized conductors still present complex care-management issues for physicians. To help inform future care-management recommendations, SJM initiated a 500-patient, multicenter study to evaluate the long-term performance of Riata silicone-only leads. SJM communicated all available information to physicians and regulatory bodies around the world, beginning in 2010. We updated physicians again in November 2011 and participated in a recent public physician forum on the topic, hosted by the Minneapolis Heart Institute Foundation.Assertion: There is no surveillance system in place to detect adverse events involving SJM's next-generation Durata leads.Facts: SJM created prospective, actively monitored registries to assess the performance of its Optim/Durata leads more than 5 years ago. 2,3 We feel that SJM has effective postmarketing surveillance to protect patient safety. We maintain three postmarketing-surveillance registries that include data on 10,836 leads that were implanted at 292 medical centers with more than 24,000 patient-years of experience representing the true clinical performance of Optim/Durata leads. To date, there have been no reports of externalization of conductors and a very low (0.09%) incidence of mechanical failure from any cause.Assertion: The Durata lead is similar in design to the Riata silicone-only lead.Facts: The Durata lead incorporates substantial design changes that considerably reduce the risk of externalization of conductors and improve overall reliability. These changes include increasing the insulation thickness by 50% with Optim (a material 50 times more abrasion-resistant than silicone) and repositioning the conductors in the lead to reduce cable tension. Furthermore, there have been no reports of externalization of conductors during the more than 5 years that the Durata lead has been on the market, with approximately 250,000 leads implanted worldwide.

No abstract

Key Points• Germline JAK2V617I mutation as a sole genetic event does not suppress hematopoietic stem cells.• JAK2V617I induces weaker constitutive activation than JAK2V617F but considerable cytokine hyperresponsiveness.The association between somatic JAK2 mutation and myeloproliferative neoplasms (MPNs) is now well established. However, because JAK2 mutations are associated with heterogeneous clinical phenotypes and often occur as secondary genetic events, some aspects of JAK2 mutation biology remain to be understood. We recently described a germline JAK2V617I mutation in a family with hereditary thrombocytosis and herein characterize the hematopoietic and signaling impact of JAK2V617I. Through targeted sequencing of MPN-associated mutations, exome sequencing, and clonality analysis, we demonstrate that JAK2V617I is likely to be the sole driver mutation in JAK2V617I-positive individuals with thrombocytosis. Phenotypic hematopoietic stem cells (HSCs) were increased in the blood and bone marrow of JAK2V617I-positive individuals and were sustained at higher levels than controls after xenotransplantation. In signaling and transcriptional assays, JAK2V617I demonstrated more activity than wild-type JAK2 but substantially less than JAK2V617F. After cytokine stimulation, JAK2V617I resulted in markedly increased downstream signaling compared with wild-type JAK2 and comparable with JAK2V617F. These findings demonstrate that JAK2V617I induces sufficient cytokine hyperresponsiveness in the absence of other molecular events to induce a homogeneous MPN-like phenotype. We also provide evidence that the JAK2V617I mutation may expand the HSC pool, providing insights into both JAK2 mutation biology and MPN disease pathogenesis. (Blood. 2013;121(20):4156-4165)

1738 Since the initial description of V617F somatic mutation in patients with Philadelphia chromosome negative myeloproliferative neoplasms (MPNs), a remarkable association between alterations in the JAK2 gene and MPNs has emerged. In addition to V617F, a number of other mutations have been detected in exons 12–15 of the JAK2 gene. Furthermore, a specific JAK2 haplotype predisposes to somatic V617F mutation and MPN. However, the link between JAK mutation and MPNs is not straightforward. For example, in familial cases of MPNs, occurrence of V617F is heterogeneous and occurs as a somatic rather than germline mutation. The underlying inherited genetic abnormality in many of these cases remains unknown. Similarly, occurrence of JAK2 V617F in sporadic MPNs is also heterogeneous and is associated with variable disease characteristics. Thus, understanding of the relationship between JAK2 mutation and MPN disease phenotype remains far from complete. We herein report a family with germline V617I mutation (Figure 1), associated with mild/moderate thrombocythaemia and thrombosis. All patients had normal haematocrit, WBC and peripheral blood (PB) morphology without splenomegaly or other abnormality on physical examination. P1: Presented in 2006 at the age of 53 with a significant ischaemic cerebrovascular event and a platelet count of 750 × 109/l, with a history of longstanding thrombocythaemia (>10 years; 700–970 × 109/l). Bone marrow examination showed normal architecture with increased numbers of morphologically normal megakaryocytes and no fibrosis. She was commenced on aspirin and hydroxycarbamide resulting in good control of the platelet count. Aspirin was not tolerated due to recurrent epistaxis. Subsequent JAK2 mutation screening by pyrosequencing demonstrated an abnormal pyrogram pattern subsequently identified to be V617I by Sanger sequencing. Quantification of allelic level by a pyrosequencing assay designed to detect V617I confirmed heterozygous (≈50%) V617I in PB mononuclear cells (MNCs), CD3+ cells, CD66+ myeloid cells, buccal swab DNA and hair follicle DNA. P2: Daughter of P1. 34 years. Asymptomatic. Persistent thrombocythemia (470–604 × 109/l). Heterozygous (≈50%) V617I in PB MNCs, CD3+ T cells, CD66+ myeloid cells, buccal swab DNA and hair follicle DNA. P3: Son of P1. 36 years. Asymptomatic. Persistent thrombocythemia (606–648 × 109/l). Heterozygous (≈50%) V617I in PB MNCs, CD3+ T cells, CD66+ myeloid cells and buccal swab DNA. P4: Son of P1. 38 years. Asymptomatic. Persistent thrombocythemia (456–526 × 109/l). Heterozygous (≈50%) V617I in PB MNCs, CD3+ T cells, CD66+ myeloid cells and buccal swab DNA. P5: Daughter of P1. 40 years. Platelet count 294 × 109/l. V617I negative in all tissues. V617I has been previously reported to occur rarely in MPN (PMID: 19074595) and to be constitutively-activating in cell line models (PMID: 18326042) and molecular dynamic simulations (PMID: 19744331). Single cell intracellular quantitative pSTAT3 FACS analysis of PB cells from P1-4 demonstrated GCSF hyper-responsiveness of V617I positive PB CD33+ and CD34+ cells. For example, in comparison with normal controls (NC; n=6) V617I CD33+ myeloid cells (n=4) showed a 14-fold increased pSTAT3 mean fluorescent intensity relative to unstimulated cells in response to 15 minutes stimulation with 0.8 ng/ml GCSF (11% vs 156%; P<.001). BFU-E and CFU-MK in PB were not significantly different between NCs (n=4) and V617I (n=4). CFU-GM were increased in the PB of V617I patients (25 vs 46 colonies/200,000 PB MNCs; P<.05). Cytokine independent colonies were not observed. Single nucleotide polymorphism array did not reveal any additional acquired abnormalities in P1-4. None of the patients carried a JAK2 46/1 haplotype. Patients 2–4 are currently under observation and receiving low-dose aspirin only. Germline activating JAK2 mutation is a previously unreported cause of inherited thrombocythaemia (autosomal dominant) and should be considered in familial cases or when rare JAK2 mutations are detected. Importantly, V617F allele specific PCR, would not detect such mutations and, consequently, it is possible that similar cases may have been missed. Finally, the mild/moderate thrombocytosis (without evidence of fibrosis over 50 years) observed with human germline V617I provides fundamental insights into the role of JAK2 mutations in the pathophysiology of human MPNs. Disclosures: No relevant conflicts of interest to declare.

We review and report here the genotypes and phenotypes of 60 novel thalassemia and abnormal hemoglobin (Hb) mutations discovered following the adoption of routine DNA sequencing of both α- and β-globin genes for all UK hemoglobinopathy samples referred for molecular investigation. This screening strategy over the last 10 years has revealed a total of 11 new β chain variants, 15 α chain variants, 19 β-thalassemia (β-thal) mutations and 15 α(+)-thalassemia (α(+)-thal) mutations. The large number of new thalassemia alleles confirms the wide racial heterogeneity of mutations in the UK immigrant population. Eleven of the new variants ran with Hb A on high performance liquid chromatography (HPLC), demonstrating the value of routine sequencing of both α- and β-globin genes for all hemoglobinopathy investigations. The new β chain variants are: Hb Bury [β22(B4)Glu  →  Asp (HBB: c.69A > T)], Hb Fulwood [β35(C1)Tyr → His (HBB: c.106T > C)], Hb Little Venice [β42(CD1)Phe → Cys (HBB: c.128T > G)], Hb Cork [β57(E1)Asn → Ser (HBB: c.173A > G), Hb Basingstoke [β118(GH1)Phe → Ser (HBB: c.356T > C)], Hb Howden [β20(B2)Val → Ala (HBB: c.62T > C)], Hb Wilton [β41(C7)Phe → Leu (HBB: c.126C > A)], Hb Belsize Park [β120(GH3)Lys → Asn (HBB: c.363A > T)], Hb Hampstead Heath [β2(NA2)His → Gln;β26(B8)Glu → Lys (HBB: c.[6C > G;79G > A])], Hb Grantham [β85(F1)Phe → Cys (HBB: c.257T > G)] and Hb Calgary [β64(E8)Gly → Val (HBB: c.194G > T). The new α chain variants are: Hb Edinburgh [α70(E19)Val → Gly (HBA2: c.212T > G)], Hb Walsgrave [α116(GH4)Glu → Val (HBA2: c.350A > T)], Hb Wexham [α117(GH5) and 118(H1) insertion Ser (HBA1: c.354-355insTCA)], Hb Coombe Park [α127(H10)Lys → Glu (HBA2: c.382A > G)], Hb Oxford [α17(A15)Val → Asp (HBA2: c.53T > A)], Hb Bridlington [α32(B13)Met → Thr (HBA1: c.98T > C), Hb Wolverhampton [α81(F2)Ser → Tyr (HBA2: c.9245C > A)], Hb Little Waltham [α13(A11)Ala → Asp (HBA2: c.41C > A)], Hb Derby [α61(E10)Lys → Arg (HBA1: c.185A > G)], Hb Uttoxter [α74(EF3)Tyr → Asp (HBA2: c.223G > T)], Hb Harehills [α124(H7)Ser → Cys (HBA1: c.374C > G)], Hb Hekinan II [α27(B8)Glu → Asp (HBA1: c.84G > T)], Hb Manitoba IV [α102(G9)Ser → Arg (HBA1: c.307A > C), Hb Witham [α139(HC1)Lys → Arg (HBA2: c.419A > G) and Hb Farnborough [α9(A7)Asn → Asp (HBA1: c.28A > G). In addition, 10 more paralogous α-globin chain variants have been discovered. The novel β-thal alleles are: HBB: c.-138C > G, HBB: c.-121C > T, HBB: c.-80T > G, HBB: c.18_19delTG, HBB: c.219_220insT, HBB: c.315 + 2_315 + 13delTGAGTCTATGGG, HBB: c.316-70C > G, HBB: c.345_346insTGTGCTG, HBB: c.354delC, HBB: c.376-381delCCAGTG, HBB: c.393T > A, HBB: c.394_395insA, HBB: c.375_376insA, HBB: c.*+95_*+107delTGGATTCTinsC, HBB: c.* + 111_*+112delAA, HBB: c.*+112A > T, HBB: c.394C > T, HBB: c.271delG and HBB: c.316-3C > T. The novel α (+ )-thal alleles are: HBA1: c.95+1G > C, HBA1: c.315C > G [Hb Donnington, α104(G11)Cys → Trp], HBA1: c.327delC, HBA1: c.333_345del, HBA1: c.*+96G > A, HBA2: c.2T > G, HBA2: c.112delC, HBA2: c.143delA, HBA2: c.143_146delACCT, HBA2: c.156_157insG, HBA2: c.22...

Approximately one third of thalassaemia patients on record in Lebanon have thalassaemia intermedia. We have analysed three factors in a panel of 73 patients with this less severe form of the disease in our population: mild beta-globin gene mutations, deletions in the alpha-globin gene and the presence of a polymorphism for the enzyme Xmn I in the Ggamma-promoter region. The results show that the most important contributing factor is the beta-genotype: 68% of patients have a mild beta+ mutation (IVSI-6, cd29, -88 or -87), while 26% of patients are positive for the Xmn I polymorphism associated with increased production of HbF, which showed strong linkage to particular mutations (IVSII-1, cd8 and cd30). However, the genotype phenotype correlation is difficult, because many patients were initially misdiagnosed as thalassaemia major and were started early on regular blood transfusions, which was stopped later on. This illustrates well the importance of an early accurate diagnosis of thalassaemia intermedia for appropriate clinical management.

We quantified Hb Bart's (gamma4) levels by high performance liquid chromatography (HPLC) in 103 fresh cord blood samples from Homerton Hospital, East London, UK. The alpha-globin gene arrangement was determined by Southern blot hybridization and genomic sequence analysis of the alpha-globin genes. The cord blood Hb Bart's levels ranged from 0.5 to 11.9% of total hemoglobin (Hb) and were arranged into three categories: i) levels below 1.5%; ii) levels between 1.5 and 5.7%; iii) levels above 6.1%. These corresponded to a normal alpha-globin genotype, a single deleted/inactivated alpha-globin gene and two deleted/inactivated alpha-globin genes, respectively. The study identified the 3.7 kb and 20.5 kb alpha-thalassemia (thal) deletions, three non deletional alpha-thal mutations and a novel alpha-globin gene rearrangement. Hb Bart's screening of fresh umbilical cord blood is an effective method to evaluate globin chain imbalance. This strategy could be utilized to screen populations for the incidence of alpha-thal and also to identify rare or new molecular lesions that reduce alpha-globin gene expression.

We have characterized a newly identified 16.6 kb deletion which removes a significant proportion of the human alpha-globin cluster including the psizeta1, alpha(D), psialpha1 and alpha2-globin genes but leaves the duplicated alpha1 gene intact. This complicated rearrangement results from a combination of slippage and strand switching at sites of microhomology during replication. Functional analysis shows that expression of the remaining alpha1 gene is increased, rather than down-regulated by this deletion. This could be related to its proximity to the remote upstream alpha-globin regulatory elements or reduced competition for these elements in the absence of the dominant alpha2-globin gene. The finding of a very mild phenotype associated with such an extensive deletion in the alpha-globin cluster implies that much of the DNA removed by the deletion is likely to be functionally unimportant. These findings suggest that other than the upstream regulatory elements and promoter proximal elements there are unlikely to be additional positive cis-acting sequences in the alpha-globin cluster.