👤 Cristy Meyer

Small intestinal neuroendocrine tumors (SINET) are the commonest malignancy of the small intestine; however, underlying pathogenic mechanisms remain poorly characterized. Whole-genome and -exome sequencing has demonstrated that SINETs are mutationally quiet, with the most frequent known mutation in the cyclin-dependent kinase inhibitor 1B gene (CDKN1B) occurring in only ∼8% of tumors, suggesting that alternative mechanisms may drive tumorigenesis. The aim of this study is to perform genome-wide molecular profiling of SINETs in order to identify pathogenic drivers based on molecular profiling. This study represents the largest unbiased integrated genomic, epigenomic, and transcriptomic analysis undertaken in this tumor type. Here, we present data from integrated molecular analysis of SINETs (n = 97), including whole-exome or targeted CDKN1B sequencing (n = 29), HumanMethylation450 BeadChip (Illumina) array profiling (n = 69), methylated DNA immunoprecipitation sequencing (n = 16), copy-number variance analysis (n = 47), and Whole-Genome DASL (Illumina) expression array profiling (n = 43). Based on molecular profiling, SINETs can be classified into three groups, which demonstrate significantly different progression-free survival after resection of primary tumor (not reached at 10 years vs. 56 months vs. 21 months, P = 0.04). Epimutations were found at a recurrence rate of up to 85%, and 21 epigenetically dysregulated genes were identified, including CDX1 (86%), CELSR3 (84%), FBP1 (84%), and GIPR (74%). This is the first comprehensive integrated molecular analysis of SINETs. We have demonstrated that these tumors are highly epigenetically dysregulated. Furthermore, we have identified novel molecular subtypes with significant impact on progression-free survival. Show less

Women with epithelial ovarian cancer (EOC) are usually treated with platinum/taxane therapy after cytoreductive surgery but there is considerable inter-individual variation in response. To identify germline single-nucleotide polymorphisms (SNPs) that contribute to variations in individual responses to chemotherapy, we carried out a multi-phase genome-wide association study (GWAS) in 1,244 women diagnosed with serous EOC who were treated with the same first-line chemotherapy, carboplatin and paclitaxel. We identified two SNPs (rs7874043 and rs72700653) in TTC39B (best P=7x10-5, HR=1.90, for rs7874043) associated with progression-free survival (PFS). Functional analyses show that both SNPs lie in a putative regulatory element (PRE) that physically interacts with the promoters of PSIP1, CCDC171 and an alternative promoter of TTC39B. The C allele of rs7874043 is associated with poor PFS and showed increased binding of the Sp1 transcription factor, which is critical for chromatin interactions with PSIP1. Silencing of PSIP1 significantly impaired DNA damage-induced Rad51 nuclear foci and reduced cell viability in ovarian cancer lines. PSIP1 (PC4 and SFRS1 Interacting Protein 1) is known to protect cells from stress-induced apoptosis, and high expression is associated with poor PFS in EOC patients. We therefore suggest that the minor allele of rs7874043 confers poor PFS by increasing PSIP1 expression. Show less

Voltage-gated potassium channels are highly diverse proteins representing the most complex class of voltage-gated ion channels from structural and functional perspectives. Deficiency of these channels usually results in various human disorders. To describe a novel autosomal recessive syndrome associated with We used SNP arrays, linkage analyses, autozygosity mapping, whole-exome sequencing, RT-PCR and two-electrode voltage-clamp recording. We identified a missense variant (p.Arg89Gln) in Show less

Acute myeloid leukemia (AML) is a heterogeneous disease characterized by the malignant transformation of hematopoietic precursors to a pathogenic cell clone. Chromosomal band 11q23 harboring MLL (=mixed lineage leukemia) gene is known to be involved in rearrangements with variety of genes as activating partners of MLL in different AML subtypes. Overall, an unfavorable prognosis is associated with MLL abnormalities. Here we investigated an 11-month-old male presenting with hyperleukocytosis being diagnosed with AML subtype FAB-M5b. In banding cytogenetics a der(19)t(19;?)(q13.3;?) and del(Y)(q11.23) were found as sole aberrations. Molecular cytogenetics revealed that the MLL gene was disrupted and even partially lost due to a t(10;19;11)(p12.31;q13.31;q23.3), an MLL/MLLT10 fusion appeared, and the der(Y) was an asymmetric inverted duplication with breakpoints in Yp11.2 and Yq11.23. The patient got hematopoietic stem cell transplantation from his haploidentical mother. Still three months afterwards 15% of blasts were detected in bone marrow and later the patient was lost during follow-up. The present case highlights the necessity to exclude MLL rearrangements, even when there seems to be no actual hint from banding cytogenetics. Show less

Metabolic disorders and hypersensitivities affect tolerability and impact adherence to highly active antiretroviral therapy (HAART). The aim of this study was to determine the prevalence of C-482T/T-455C variants in the Apolipoprotein C3 (APOC3) promoter gene and Human leukocyte antigen (HLA)-B*57:01, known to impact lipid metabolic disorders and hypersensitivity respectively; and to correlate genotypes with gender, CD4+ cell count and viral load in an HIV infected cohort in northern South Africa. Frequencies of C-482 and T-455 polymorphisms in APOC3 were determined by restriction fragment length polymorphism analysis. Allele determination for HLA-B was performed with Assign SBT software in an HLA library. Analysis of APOC3 C-482 site revealed a prevalence of 196/199 (98.5%) for CC, 1/199 (0.5%) for CT and 2/199 (1.0%) for TT genotype (p = 0.000 with 1° of freedom; χ2 = 126.551). For the T-455 site, prevalences were: 69/199 (35%) for TT and 130/199 (65%) for the CC genotype (p = 0.000 with 1° of freedom; χ2 = 199). There was no association between gender and the presence of -482 (p = 1; χ2 = 0.00001) or -455 genotypes (p = 0.1628; χ2 = 1.9842). There was no significant difference in the increase in CD4+ cell count irrespective of genotypes. Significant increases in CD4+ cell count were observed in males and females considering the -455C genotype, but not in males for the -455T genotype. Viral load decreases were significant with the -455C and -482C genotypes irrespective of gender. HLA-B*57:01 was not identified in the study cohort. The apparently high prevalence of APOC3 T-455CC genotype needs confirmation with a larger samples size and triglyceride measurements to support screening of patients to pre-empt HAART associated lipid disorders. Show less

There is no published data comparing dietary management of urea cycle disorders (UCD) in different countries. Cross-sectional data from 41 European Inherited Metabolic Disorder (IMD) centres (17 UK, 6 France, 5 Germany, 4 Belgium, 4 Portugal, 2 Netherlands, 1 Denmark, 1 Italy, 1 Sweden) was collected by questionnaire describing management of patients with UCD on prescribed protein restricted diets. Data for 464 patients: N-acetylglutamate synthase (NAGS) deficiency, n=10; carbamoyl phosphate synthetase (CPS1) deficiency, n=29; ornithine transcarbamoylase (OTC) deficiency, n=214; citrullinaemia, n=108; argininosuccinic aciduria (ASA), n=80; arginase deficiency, n=23 was reported. The majority of patients (70%; n=327) were aged 0-16y and 30% (n=137) >16y. Prescribed median protein intake/kg body weight decreased with age with little variation between disorders. The UK tended to give more total protein than other European countries particularly in infancy. Supplements of essential amino acids (EAA) were prescribed for 38% [n=174] of the patients overall, but were given more commonly in arginase deficiency (74%), CPS (48%) and citrullinaemia (46%). Patients in Germany (64%), Portugal (67%) and Sweden (100%) were the most frequent users of EAA. Only 18% [n=84] of patients were prescribed tube feeds, most commonly for CPS (41%); and 21% [n=97] were prescribed oral energy supplements. Dietary treatment for UCD varies significantly between different conditions, and between and within European IMD centres. Further studies examining the outcome of treatment compared with the type of dietary therapy and nutritional support received are required. Show less

Chromosomal rearrangements of the human MLL (mixed lineage leukemia) gene are associated with high-risk infant, pediatric, adult and therapy-induced acute leukemias. We used long-distance inverse-polymerase chain reaction to characterize the chromosomal rearrangement of individual acute leukemia patients. We present data of the molecular characterization of 1590 MLL-rearranged biopsy samples obtained from acute leukemia patients. The precise localization of genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and novel TPGs identified. All patients were classified according to their gender (852 females and 745 males), age at diagnosis (558 infant, 416 pediatric and 616 adult leukemia patients) and other clinical criteria. Combined data of our study and recently published data revealed a total of 121 different MLL rearrangements, of which 79 TPGs are now characterized at the molecular level. However, only seven rearrangements seem to be predominantly associated with illegitimate recombinations of the MLL gene (≈ 90%): AFF1/AF4, MLLT3/AF9, MLLT1/ENL, MLLT10/AF10, ELL, partial tandem duplications (MLL PTDs) and MLLT4/AF6, respectively. The MLL breakpoint distributions for all clinical relevant subtypes (gender, disease type, age at diagnosis, reciprocal, complex and therapy-induced translocations) are presented. Finally, we present the extending network of reciprocal MLL fusions deriving from complex rearrangements. Show less

Hypertrophic cardiomyopathy (HCM) is a cardiovascular disease with autosomal dominant inheritance caused by mutations in genes coding for sarcomeric and/or regulatory proteins expressed in cardiomyocytes. In a small cohort of HCM patients (n=8), we searched for mutations in the two most common genes responsible for HCM and found four missense mutations in the MYH7 gene encoding cardiac β-myosin heavy chain (R204H, M493V, R719W, and R870H) and three mutations in the myosin-binding protein C3 gene (MYBPC3) including one missense (A848V) and two frameshift mutations (c.3713delTG and c.702ins26bp). The c.702ins26bp insertion resulted from the duplication of a 26-bp fragment in a 54-year-old female HCM patient presenting with clinical signs of heart failure due to diastolic dysfunction. Although such large duplications (>10 bp) in the MYBPC3 gene are very rare and have been identified only in 4 families reported so far, the identical duplication mutation was found earlier in a Dutch patient, demonstrating that it may constitute a hitherto unknown founder mutation in central European populations. This observation underscores the significance of insertions into the coding sequence of the MYBPC3 gene for the development and pathogenesis of HCM. Show less

Recurrent deletions have been associated with numerous diseases and genomic disorders. Few, however, have been resolved at the molecular level because their breakpoints often occur in highly copy-number-polymorphic duplicated sequences. We present an approach that uses a combination of somatic cell hybrids, array comparative genomic hybridization, and the specificity of next-generation sequencing to determine breakpoints that occur within segmental duplications. Applying our technique to the 17q21.31 microdeletion syndrome, we used genome sequencing to determine copy-number-variant breakpoints in three deletion-bearing individuals with molecular resolution. For two cases, we observed breakpoints consistent with nonallelic homologous recombination involving only H2 chromosomal haplotypes, as expected. Molecular resolution revealed that the breakpoints occurred at different locations within a 145 kbp segment of >99% identity and disrupt KANSL1 (previously known as KANSL1). In the remaining case, we found that unequal crossover occurred interchromosomally between the H1 and H2 haplotypes and that this event was mediated by a homologous sequence that was once again missing from the human reference. Interestingly, the breakpoints mapped preferentially to gaps in the current reference genome assembly, which we resolved in this study. Our method provides a strategy for the identification of breakpoints within complex regions of the genome harboring high-identity and copy-number-polymorphic segmental duplication. The approach should become particularly useful as high-quality alternate reference sequences become available and genome sequencing of individuals' DNA becomes more routine. Show less

Chromosomal rearrangements affecting the MLL gene are associated with high-risk pediatric, adult and therapy-associated acute leukemia. In this study, conventional cytogenetic, fluorescence in situ hybridization, and molecular genetic studies were used to characterize the type and frequency of MLL rearrangements in a consecutive series of 45 Portuguese patients with MLL-related leukemia treated in a single institution between 1998 and 2011. In the group of patients with acute lymphoblastic leukemia and an identified MLL fusion partner, 47% showed the presence of an MLL-AFF1 fusion, as a result of a t(4;11). In the remaining cases, a MLL-MLLT3 (27%), a MLL-MLLT1 (20%), or MLL-MLLT4 (7%) rearrangement was found. The most frequent rearrangement found in patients with acute myeloid leukemia was the MLL-MLLT3 fusion (42%), followed by MLL-MLLT10 (23%), MLL-MLLT1 (8%), MLL-ELL (8%), MLL-MLLT4 (4%), and MLL-MLLT11 (4%). In three patients, fusions involving MLL and a septin family gene (SEPT2, SEPT6, and SEPT9), were identified. The most frequently identified chromosomal rearrangements were reciprocal translocations, but insertions and deletions, some cryptic, were also observed. In our series, patients with MLL rearrangements were shown to have a poor prognosis, regardless of leukemia subtype. Interestingly, children with 1 year or less showed a statistically significant better overall survival when compared with both older children and adults. The use of a combined strategy in the initial genetic evaluation of acute leukemia patients allowed us to characterize the pattern of MLL rearrangements in our institution, including our previous discovery of two novel MLL fusion partners, the SEPT2 and CT45A2 genes, and a very rare MLL-MLLT4 fusion variant. Show less

Genetic aberrations involving the mixed lineage leukemia (MLL) gene are frequently diagnosed in infant acute lymphoblastic and acute myeloid leukemia. More than 60 fusion partner genes have been described at the molecular level, 31 of which have been characterized solely in infant leukemia cases. Here we describe a new MLL fusion partner gene, NEBL, which was identified in a case of acute myeloid leukemia in an infant. The chromosomal breakpoints of the MLL-NEBL and NEBL-MLL fusion genes were cloned by long-distance inverse polymerase chain reaction. The chromosomal breakpoints were located at 10p12, approximately 570 kb telomic of the MLLT10 (AF10) gene. AF10 and NEBL are localized in such close vicinity that they cannot be distinguished cytogenetically by G banding. Therefore, the combination of cytogenetic and independent molecular techniques such as long-distance inverse polymerase chain reaction are indispensable for the rapid identification and characterization of rare MLL rearrangements. Show less

Distal arthrogryposis type I (DA1) is a disorder characterized by congenital contractures of the hands and feet for which few genes have been identified. Here we describe a five-generation family with DA1 segregating as an autosomal dominant disorder with complete penetrance. Genome-wide linkage analysis using Affymetrix GeneChip Mapping 10K data from 12 affected members of this family revealed a multipoint LOD(max) of 3.27 on chromosome 12q. Sequencing of the slow-twitch skeletal muscle myosin binding protein C1 (MYBPC1), located within the linkage interval, revealed a missense mutation (c.706T>C) that segregated with disease in this family and causes a W236R amino acid substitution. A second MYBPC1 missense mutation was identified (c.2566T>C)(Y856H) in another family with DA1, accounting for an MYBPC1 mutation frequency of 13% (two of 15). Skeletal muscle biopsies from affected patients showed type I (slow-twitch) fibers were smaller than type II fibers. Expression of a green fluorescent protein (GFP)-tagged MYBPC1 construct containing WT and DA1 mutations in mouse skeletal muscle revealed robust sarcomeric localization. In contrast, a more diffuse localization was seen when non-fused GFP and MYBPC1 proteins containing corresponding MYBPC3 amino acid substitutions (R326Q, E334K) that cause hypertrophic cardiomyopathy were expressed. These findings reveal that the MYBPC1 is a novel gene responsible for DA1, though the mechanism of disease may differ from how some cardiac MYBPC3 mutations cause hypertrophic cardiomyopathy. Show less

Autosomal dominant polycystic kidney disease (ADPKD) is the most frequent monogenic cause of kidney failure, characterized by the development of renal cysts. ADPKD is caused by mutations of the polycystin-1 (PC1) or polycystin-2 (PC2) genes. PC2 encodes a Ca(2+)-permeable cation channel, and its dysfunction has been implicated in cyst development. The transcriptional coactivator with PDZ binding motif (TAZ) is required for the integrity of renal cilia. Its absence results in the development of renal cysts in a knock-out mouse model. TAZ directly interacts with PC2, and it has been suggested that another yet unidentified PDZ domain protein may be involved in the TAZ/PC2 interaction. Here we describe a novel interaction of TAZ with the multi-PDZ-containing PALS1-associated tight junction protein (PATJ). TAZ interacts with both the N-terminal PDZ domains 1-3 and the C-terminal PDZ domains 8-10 of PATJ, suggesting two distinct TAZ binding domains. We also show that the C terminus of PC2 strongly interacts with PDZ domains 8-10 and to a weaker extent with PDZ domains 1-3 of PATJ. Finally, we demonstrate that both TAZ and PATJ impair PC2 channel activity when co-expressed with PC2 in oocytes of Xenopus laevis. These results implicate TAZ and PATJ as novel regulatory elements of the PC2 channel and might thus be involved in ADPKD pathology. Show less

Attention deficit/hyperactivity disorder (ADHD) is a highly heritable neurodevelopmental disorder of childhood onset. Clinical and biological evidence points to shared common central nervous system (CNS) pathology of ADHD and restless legs syndrome (RLS). It was hypothesized that variants previously found to be associated with RLS in two large genome-wide association studies (GWA), will also be associated with ADHD. SNPs located in MEIS1 (rs2300478), BTBD9 (rs9296249, rs3923809, rs6923737), and MAP2K5 (rs12593813, rs4489954) as well as three SNPs tagging the identified haplotype in MEIS1 (rs6710341, rs12469063, rs4544423) were genotyped in a well characterized German sample of 224 families comprising one or more affected sibs (386 children) and both parents. We found no evidence for preferential transmission of the hypothesized variants to ADHD. Subsequent analyses elicited nominal significant association with haplotypes consisting of the three SNPs in BTBD9 (chi2 = 14.8, df = 7, nominal p = 0.039). According to exploratory post hoc analyses, the major contribution to this finding came from the A-A-A-haplotype with a haplotype-wise nominal p-value of 0.009. However, this result did not withstand correction for multiple testing. In view of our results, RLS risk alleles may have a lower effect on ADHD than on RLS or may not be involved in ADHD. The negative findings may additionally result from genetic heterogeneity of ADHD, i.e. risk alleles for RLS may only be relevant for certain subtypes of ADHD. Genes relevant to RLS remain interesting candidates for ADHD; particularly BTBD9 needs further study, as it has been related to iron storage, a potential pathophysiological link between RLS and certain subtypes of ADHD. Show less

Chromosomal rearrangements of the human MLL gene are associated with high-risk pediatric, adult and therapy-associated acute leukemias. These patients need to be identified, treated appropriately and minimal residual disease was monitored by quantitative PCR techniques. Genomic DNA was isolated from individual acute leukemia patients to identify and characterize chromosomal rearrangements involving the human MLL gene. A total of 760 MLL-rearranged biopsy samples obtained from 384 pediatric and 376 adult leukemia patients were characterized at the molecular level. The distribution of MLL breakpoints for clinical subtypes (acute lymphoblastic leukemia, acute myeloid leukemia, pediatric and adult) and fused translocation partner genes (TPGs) will be presented, including novel MLL fusion genes. Combined data of our study and recently published data revealed 104 different MLL rearrangements of which 64 TPGs are now characterized on the molecular level. Nine TPGs seem to be predominantly involved in genetic recombinations of MLL: AFF1/AF4, MLLT3/AF9, MLLT1/ENL, MLLT10/AF10, MLLT4/AF6, ELL, EPS15/AF1P, MLLT6/AF17 and SEPT6, respectively. Moreover, we describe for the first time the genetic network of reciprocal MLL gene fusions deriving from complex rearrangements. Show less

Adherent cells undergo remarkable changes in shape during cell division. However, the functional interplay between cell adhesion turnover and the mitotic machinery is poorly understood. The endo/exocytic trafficking of integrins is regulated by the small GTPase Rab21, which associates with several integrin alpha subunits. Here, we show that targeted trafficking of integrins to and from the cleavage furrow is required for successful cytokinesis, and that this is regulated by Rab21. Rab21 activity, integrin-Rab21 association, and integrin endocytosis are all necessary for normal cytokinesis, which becomes impaired when integrin-mediated adhesion at the cleavage furrow fails. We also describe a chromosomal deletion and loss of Rab21 gene expression in human cancer, which leads to the accumulation of multinucleate cells. Importantly, reintroduction of Rab21 rescued this phenotype. In conclusion, Rab21-regulated integrin trafficking is essential for normal cell division, and its defects may contribute to multinucleation and genomic instability, which are hallmarks of cancer. Show less

The PSD-95 family of membrane- associated guanylate kinases (MAGUKs) are thought to act as molecular scaffolds that regulate the assembly and function of the multiprotein signaling complex found at the postsynaptic density of excitatory synapses. Genetic analysis of PSD-95 family members in the mammalian nervous system has so far been difficult, but the zebrafish is emerging as an ideal vertebrate system for studying the role of particular genes in the developing and mature nervous system. Here we describe the cloning of the zebrafish orthologs of PSD-95, PSD-93, and two isoforms of SAP-97. Using in situ hybridization analysis we show that these zebrafish MAGUKs have overlapping but distinct patterns of expression in the developing nervous system and craniofacial skeleton. Using a pan-MAGUK antibody we show that MAGUK proteins localize to neurons within the developing hindbrain, cerebellum, visual and olfactory systems, and to skin epithelial cells. In the olfactory and visual systems MAGUK proteins are expressed strongly in synaptic regions, and the onset of expression in these areas coincides with periods of synapse formation. These data are consistent with the idea that PSD-95 family members are involved in synapse assembly and function, and provide a platform for future functional studies in vivo in a highly tractable model organism. Show less

We postulated that dose-responsive satiety after oil premeals varies with the number of gut sensors stimulated by lipolytic products along intestine. These experiments in fasted rats on satiety after oil premeals were performed to 1) determine whether satiety was induced by lipolytic products but not triglycerides; 2) confirm that oil empties from the stomach at rates that vary with oil loads; 3) ascertain that increasing rates of oil entry into duodenum extend the length of gut contacted by lipolytic products; and 4) judge whether length of gut contacted correlated with dose-responsive satieties to dietary oils. 5) Using specific antagonists, we attempted to define how satiety was signalled by gut sensors. Timing and degrees of satiety did not correlate with timing and extent of gastric distensions but, rather, with the timing and extent of spread of lipolytic products along small bowel. Satiety after the highest premeal load of oil was blocked by Pluronic L-81, an inhibitor of intestinal secretion of apolipoprotein A-IV, but was unaffected by MK-329 (a specific antagonist of cholecystokinin) or by capsaicin blockade of chemosensory nerves. Show less