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BACKGROUND: Germline mutations in telomerase and other telomere maintenance genes manifest in the premature aging short telomere syndromes. Myelodysplastic syndromes and acute myeloid leukemia (MDS/AML) account for 75% of associated malignancies, but how these cancers overcome the inherited telomere defect is unknown. METHODS: We used ultra-deep targeted sequencing to detect somatic reversion mutations in 17 candidate telomere lengthening genes among controls and patients with short telomere syndromes with and without MDS/AML, and we tested the functional significance of these mutations. RESULTS: While no controls carried somatic mutations in telomere maintenance genes, 29% (16 of 56) of adults with germline telomere maintenance defects carried at least 1 (P < 0.001), and 13% (7 of 56) had 2 or more. In addition to TERT promoter mutations, which were present in 19%, another 13% of patients carried a mutation in POT1 or TERF2IP. POT1 mutations impaired telomere binding in vitro and some mutations were identical to ones seen in familial melanoma associated with longer telomere length. Exclusively in patients with germline defects in telomerase RNA (TR), we identified somatic mutations in nuclear RNA exosome genes RBM7, SKIV2L2, and DIS3, where loss-of-function upregulates mature TR levels. Somatic reversion events in 6 telomere-related genes were more prevalent in patients who were MDS/AML-free (P = 0.02, RR 4.4, 95% CI 1.2–16.7), and no patient with MDS/AML had more than 1 reversion mutation. CONCLUSION: Our data indicate that diverse adaptive somatic mutations arise in the short telomere syndromes. Their presence may alleviate the telomere crisis that promotes transformation to MDS/AML. FUNDING: This work was supported by the NIH, the Commonwealth Foundation, the S&R Foundation Kuno Award, the Williams Foundation, the Vera and Joseph Dresner Foundation, the MacMillan Pathway to Independence Award, the American Society of Hematology Scholar Award, the Johns Hopkins Research Program for Medical Students, and the Turock Scholars Fund.

The melanocortin system has been implicated in the regulation of various physiological functions including melanogenesis, steroidogenesis, energy homeostasis, and feeding behavior. Five melanocortin receptors have been identified to date and belong to the family of G protein-coupled receptors (GPCR). Post-translational modification of the proopiomelanocortin (POMC) prohormone leads to the biosynthesis of the endogenous melanocortin agonists, including α-melanocyte stimulating hormone (α-MSH), β-MSH, γ-MSH, and adrenocorticotropic hormone (ACTH). All the melanocortin agonists derived from the POMC prohormone contain a His-Phe-Arg-Trp tetrapeptide sequence that has been implicated in eliciting the pharmacological responses at the melanocortin receptors. Herein, an alanine (Ala) positional scan is reported for the endogenous α-MSH ligand and the synthetic, more potent, NDP-MSH peptide (Ac-Ser(1)-Tyr(2)-Ser(3)-Nle(4)-Glu(5)-His(6)-DPhe(7)-Arg(8)-Trp(9)-Gly(10)-Lys(11)-Pro(12)-Val(13)-NH2) at the cloned mouse melanocortin receptors to test the assumption that the structure-activity relationships of one ligand would apply to the other. Several residues outside of the postulated pharmacophore altered potency at the melanocortin receptors, most notably the 1560-, 37-, and 15-fold potency loss when the Glu(5) position of α-MSH was substituted with Ala at the mMC1R, mMC3R, and mMC4R, respectively. Importantly, the altered potencies due to Ala substitutions in α-MSH did not necessarily correlate with equivalent Ala substitutions in NDP-MSH, indicating that structural modifications and corresponding biological activities in one of these melanocortin ligands may not be predictive for the other agonist.

Agouti-related protein (AGRP) is a potent orexigenic peptide that antagonizes the melanocortin-3 and -4 receptors (MC3R and MC4R). While the C-terminal domain of AGRP, AGRP(87-132), is equipotent to the full-length peptide, further truncation decreases potency at the MC3R and MC4R. Herein, we report AGRP-derived peptides designed to mimic the active β-hairpin secondary structure that contains the hypothesized Arg-Phe-Phe pharmacophore. The most potent scaffold, c[Pro-Arg-Phe-Phe-Asn-Ala-Phe-DPro], comprised the hexa-peptide β-hairpin loop from AGRP cyclized through a DPro-Pro motif. A 20 compound library was synthesized from this scaffold for further structure-activity relationship studies. The most potent peptide from this library was an asparagine to diaminopropionic acid substitution that possessed sub-nanomolar antagonist activity at the mMC4R and was greater than 160-fold selective for the mMC4R versus the mMC3R. The reported ligands may serve as probes to characterize the melanocortin receptors in vivo and leads in the development of novel therapeutics.

Aim Donor HLA-specific antibody represents a risk for rejection or engraftment failure following HLA mismatched solid organ or hematopoietic stem cell transplantation. We have examined the immunogenicity of Bw4 subtype mismatches in 211 renal recipients post-transplant. Methods HLA typing was performed using Luminex based rSSOP (LAB Type, One Lambda). HLA single antigen panels (LABScreen, One Lambda) were used to identify HLA-Bw4 sensitization. Results Bw4 epitopes present on HLA-B and HLA-A antigens can be divided into 8 subtypes based on amino acid differences at positions 77–83 (Voorter et al. Tissue Antigens, 2000). Furthermore, residues 80–83 have been shown to impact the electrostatic charge and explain observed serological Bw4 patterns (Kosmoliaptsis et al. Human Immunol. 2011). Bw4 motifs TALR and TLLR (expressed on HLA-B∗44:02, B∗13:01 and B∗37:01, B∗27:05, B47:01, respectively) have similar electrostatic potentials, but are uniquely different from the IALR Bw4 motif (expressed on HLA-B∗57:01, 58:01, 49:01, 51:01, 53:01, 15:16, 15:13, 38:01 59:01 and HLA-A23:01, 24:02, 24:03, 25:01,32:01). Previously, using serum/cell adsorption experiments, we have shown that sensitization to the IALR can occur in recipients possessing TALR or TLLR. The incidence of sensitization to these different Bw4 motifs is shown in Table 1.The IALR motif evoked the highest incidence of sensitization in recipients possessing Bw6, Bw4-TALR, or Bw4-TLLR in their own phenotype. In contrast, mismatches involving TALR or TLLR provoked little sensitization in recipients possessing IALR. Conclusions Structural and electrostatic potential differences between Bw4 subtypes appear sufficient to elicit alloimmune responses. Exposure to IALR, the most common Bw4 motif, can provoke a substantial alloantibody barrier to prospective donors (CPRA of 54%). Moreover, Bw4 sensitization may be relevant when selecting HLA allele mismatched donors for antigens that contain multiple Bw4 subtypes (i.e. HLA-B27, B38, B44, and B53). Download : Download full-size image