Michael Cullen

An integrated haplotype map of the human major histocompatibility complex.

Numerous studies have clearly indicated a role for the major histocompatibility complex (MHC) in susceptibility to autoimmune diseases. Such studies have focused on the genetic variation of a small number of classical human-leukocyte-antigen (HLA) genes in the region. Although these genes represent good candidates, given their immunological roles, linkage disequilibrium (LD) surrounding these genes has made it difficult to rule out neighboring genes, many with immune function, as influencing disease susceptibility. It is likely that a comprehensive analysis of the patterns of LD and variation, by using a high-density map of single-nucleotide polymorphisms (SNPs), would enable a greater understanding of the nature of the observed associations, as well as lead to the identification of causal variation. We present herein an initial analysis of this region, using 201 SNPs, nine classical HLA loci, two TAP genes, and 18 microsatellites. This analysis suggests that LD and variation in the MHC, aside from the classical HLA loci, are essentially no different from those in the rest of the genome. Furthermore, these data show that multi-SNP haplotypes will likely be a valuable means for refining association signals in this region.

Rare missense variants in POT1 predispose to familial cutaneous malignant melanoma

Although CDKN2A is the most frequent high-risk melanoma susceptibility gene, the underlying genetic factors for most melanoma-prone families remain unknown. Using whole-exome sequencing, we identified a rare variant that arose as a founder mutation in the telomere shelterin gene POT1 (chromosome 7, g.124493086C>T; p.Ser270Asn) in five unrelated melanoma-prone families from Romagna, Italy. Carriers of this variant had increased telomere lengths and numbers of fragile telomeres, suggesting that this variant perturbs telomere maintenance. Two additional rare POT1 variants were identified in all cases sequenced in two separate Italian families, one variant per family, yielding a frequency for POT1 variants comparable to that for CDKN2A mutations in this population. These variants were not found in public databases or in 2,038 genotyped Italian controls. We also identified two rare recurrent POT1 variants in US and French familial melanoma cases. Our findings suggest that POT1 is a major susceptibility gene for familial melanoma in several populations.

Fine Mapping of IGAD1 in IgA Deficiency and Common Variable Immunodeficiency: Identification and Characterization of Haplotypes Shared by Affected Members of 101 Multiple-Case Families

To limit the region containing a mutation predisposing to selective IgA deficiency (IgAD) and common variable immunodeficiency (CVID), 554 informative members of 101 multiple-case families were haplotyped at the IGAD1 candidate locus in the MHC. Microsatellite markers were placed onto the physical map of IGAD1 to establish their order and permit rapid haplotype analyses. Linkage analysis of this extended family set provided additional support for a strong susceptibility locus at IGAD1 with a maximum multipoint nonparametric linkage score in excess of 3. Although the transmission of maternal IGAD1 haplotypes from unaffected heterozygous parents to the affected offspring was in excess, this was not apparent in multiple-case families with a predominance of affected mothers, suggesting that this parental bias is influenced by the affection status of transmitting parents and supporting a maternal effect in disease susceptibility. Of 110 haplotypes shared by 258 affected family members, a single haplotype (H1) was found in 44 pairs of affected relatives, accounting for the majority of the IGAD1 contribution to the development of IgAD/CVID in our families. The H1 allelic variability was higher in the telomeric part of the class III region than in the distal part of the class II region in both single- and multiple-case families. Incomplete H1 haplotypes had most variant alleles in the telomeric part of the analyzed region in homozygous IgAD/CVID patients, whereas this was not observed in unaffected homozygotes. These data suggest that a telomeric part of the class II region or centromeric part of the class III region is the most likely location of IGAD1. This study was supported by the Swedish and British Medical Research Councils, the Swedish Strategic Research Foundation, the Primary Immunodeficiency Association of the United Kingdom MSMT VS96097, the Karolinska Institute, and federal funds from the National Cancer Institute, National Institutes of Health, under Contract NO1-CO-56000. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. Address correspondence and reprint requests to Dr. Igor Vořechovský, Department of Biosciences at NOVUM, Karolinska Institute, CBT Hälsovägen 7, SE-14157 Huddinge, Sweden. E-mail address: igvo@smtp.biosci.ki.se Abbreviations used in this paper: IgAD, selective IgA deficiency; CVID, common variable immunodeficiency; TDT, transmission disequilibrium test; NPL, nonparametric linkage; PCR-SSP, PCR using sequence-specific oligonucleotide primers.

Characterization of 12 microsatellite loci of the human MHC in a panel of reference cell lines

The human genome contains a large number of interspersed microsatellite repeats which exhibit a high degree of polymorphism and are inherited in a Mendelian fashion, making them extremely useful genetic markers. Several microsatellites have been described in the HLA region, but allele nomenclature, a set of broadly distributed controls, and typing methods have not been standardized, which has resulted in discrepant microsatellite data between laboratories. In this report we present a detailed protocol for genotyping microsatellites using a semi-automated fluorescence-based method. Twelve microsatellites within or near the major histocompatibility complex (MHC) were typed in the 10th International Histocompatibility Workshop homozygous typing cell lines (HTCs) and alleles were designated based on size. All loci were sequenced in two HTCs providing some information on the level of complexity of the repeat sequence. A comparison of allele size obtained by genotyping versus that obtained by direct sequencing showed minor discrepancies in some cases, but these were not unexpected given the technical differences in the methodologies. Fluorescence-based typing of microsatellites in the MHC described herein is highly efficient, accurate, and reproducible, and will allow comparison of results between laboratories.

Abstract 3128: Efficacy of diethyldihydroxyhomospermine against human pancreatic adenocarcinoma using orthotopic implantation of human pancreatic L3.6pl cells into the pancreas of nude mice

Purpose: To determine the anti-neoplastic effects of (S,S)N1,N14-diethyl-3,12-dihydroxyhomospermine ([HO]2DEHSPM, SUN-101) after subcutaneous (s.c.) administration after orthotopic transplantation of human pancreatic cancer cells (L3.6pl) in the pancreas of nude mice. Methods: L3.6pl cells were injected into the pancreas of nude mice and seven to ten days later the treatment groups were: Study 1: mice (n=10/group) with saline (control), SUN-101 (25 mg/kg), SUN-101 (50 mg/kg), and SUN-101 (100 mg/kg) QD for 4 to 6 weeks. Study 2: mice (n=10/group) with saline (control), SUN-101 (25 mg/kg, QD), SUN-101 (25 mg/kg three times a week, QOD), SUN-101 (15 mg/kg, QOD), and SUN-101 (5 mg/kg, QOD) for 4 to 6 weeks Study 3: mice (n=10/group) with saline (control), Gemzar (100 mg/kg, intraperitoneal (i.p.), twice a week), SUN-101 (25 mg/kg, QOD), and Gemzar plus SUN-101 for 4 to 6 weeks. Results: In study 1, the optimal dose of SUN-101 was determined to be 25 mg/kg QD. This dose reduced up to 82.9% the weight of human pancreatic tumors in mice. Treatment with 50 and 100 mg/kg doses resulted in decreased body weight and proved to be toxic in mice. Histologic changes in the liver included hepatocyte reparative change and in the exocrine pancreas included a mild decrease of cytoplasmic granules in the epithelium of the pancreatic acini. In study 2, the optimal dose of 25 mg/kg administrated QOD was less toxic than daily 25 mg/kg administrations. The pancreatic weight and volume were decreased (47.8% and 66.6%, respectively) with 25 mg/kg administrated QOD and dose related decreases were observed at the 15 mg/kg QOD (20.1% and 52.6%, respectively). No decrease in tumor weight or volume was noted with 5 mg/kg administrated QOD. In study 3, the treatment of Gemzar, SUN-101, and Gemzar plus SUN-101 resulted in 18.7%, 35.6%, and 42.4% decreases in the body weight, respectively. Compared with tumor-bearing control, the treatment of Gemzar, SUN-101, and Gemzar plus SUN-101 resulted in 24.7%, 58.8%, and 67.2% decreases in the pancreas weight, respectively, and 37.8%, 58.4%, and 72.9% decreases in the tumor volumes, respectively. The incidence of liver metastasis was also decreased with SUN-101, Gemzar, and combination of Gemzar plus SUN-101. Conclusions: SUN-101 administered QD or QOD 25 mg/kg inhibited the growth of human pancreatic carcinoma in mice. SUN-101 demonstrated higher toxicity including disruption of the digestive process at daily 50 and 100 mg/kg doses. Co-administration of SUN-101 with gemcitabine appeared to have an additive or synergistic effect on reduction in the pancreatic tumor. Citation Format: Ajit K. Shah, Michael T. Cullen, Cheryl H. Baker. Efficacy of diethyldihydroxyhomospermine against human pancreatic adenocarcinoma using orthotopic implantation of human pancreatic L3.6pl cells into the pancreas of nude mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3128. doi:10.1158/1538-7445.AM2014-3128

Abstract 2778: Fine mapping the KLK3 locus on chromosome 19q13.33 associated with prostate cancer susceptibility and PSA levels

Prostate cancer is the most commonly diagnosed non-cutaneous cancer in men in the United States with over 200,000 new cases and 30,000 deaths estimated in 2010. Measurements of serum prostate specific antigen (PSA) protein levels form the basis for a widely used test to screen men for prostate cancer. Germline variants in the gene that encodes the PSA protein (KLK3) have been shown to be associated with serum PSA levels. In genome-wide association studies (GWAS) of prostate cancer, KLK3 variants have been suggested to be prostate cancer risk factors. Based on a resequence analysis of a 56 kb region on chromosome 19q13.33, centered on the KLK3 gene, that characterized common germline variations in the region, we fine mapped this locus by genotyping tag SNPs in 3,522 prostate cancer cases and 3,338 controls from five cohort and case-control studies. The cohort studies are: the Prostate, Lung, Colon and Ovarian (PLCO) Cancer Screening Trial, the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study (ATBC), the American Cancer Society Prevention Study II Nutrition Cohort (CPS-II) and the Cohort of Norway (CONOR). The case-control study is the French Prostate Case-Control Study (CeRePP) which is a hospital based case-control study. This study has > 80% power to detect an association with an odds ratio of 1.25 (assuming a MAF of 0.05, a prevalence of prostate cancer = 1.5067% and alpha = 0.05). We did not observe a strong association for KLK3 variants, previously reported to confer risk for prostate cancer (rs2735839; P = 0.20). However, three highly correlated SNPs (rs17632542, rs62113212 and rs62113214) demonstrated an association with prostate cancer (P = 3.41×10 −4 , per-allele odds ratio (OR) = 0.77, 95% CI = 0.67-0.89). In particular, the signal is apparent in nonaggressive prostate cancer cases with Gleason score −5 , per-allele trend OR = 0.67, 95% CI = 0.56-0.81) but not in advanced cases with Gleason score > 8 or stage ≥ III (P = 0.31, per-allele OR = 1.12, 95% CI = 0.90-1.40). One of the three highly correlated SNPs, rs17632542, introduces a non-synonymous amino acid change in the KLK3 protein with a predicted benign or neutral functional impact. Baseline PSA levels were lower in control subjects with one or more minor alleles at any one of the three SNPs (1.12 ng/ml, 95% CI = 0.96-1.28) as compared to those with no minor alleles (1.61 ng/ml, 95% CI = 1.49-1.72) (P = 9.70×10 −5 ). Together our results suggest that germline KLK3 variants could influence the diagnosis of nonaggressive prostate cancer by influencing the likelihood of biopsy. It is possible that the KLK3 locus also contributes to prostate cancer risk, but additional studies will be needed to dissect the contribution of KLK3 to PSA and prostate risk separately. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2778. doi:10.1158/1538-7445.AM2011-2778