Tom Hearn

Mutation of ALMS1, a large gene with a tandem repeat encoding 47 amino acids, causes Alström syndrome

Alström syndrome (OMIM 203800) is an autosomal recessive disease, characterized by cone–rod retinal dystrophy, cardiomyopathy and type 2 diabetes mellitus, that has been mapped to chromosome 2p13 (refs 1–5). We have studied an individual with Alström syndrome carrying a familial balanced reciprocal chromosome translocation (46, XY,t(2;11)(p13;q21)mat) involving the previously implicated critical region. We postulated that this individual was a compound heterozygote, carrying one copy of a gene disrupted by the translocation and the other copy disrupted by an intragenic mutation. We mapped the 2p13 breakpoint on the maternal allele to a genomic fragment of 1.7 kb which contains exon 4 and the start of exon 5 of a newly discovered gene (ALMS1); we detected a frameshift mutation in the paternal copy of the gene. The 12.9-kb transcript of ALMS1 encodes a protein of 4,169 amino acids whose function is unknown. The protein contains a large tandem-repeat domain comprising 34 imperfect repetitions of 47 amino acids. We have detected six different mutations (two nonsense and four frameshift mutations causing premature stop codons) in seven families, confirming that ALMS1 is the gene underlying Alström syndrome. We believe that ALMS1 is the first human disease gene characterized by autosomal recessive inheritance to be identified as a result of a balanced reciprocal translocation.

Wolcott-Rallison syndrome: pathogenic insights into neonatal diabetes from new mutation and expression studies of EIF2AK3

Wolcott-Rallison syndrome (OMIM 226980) is a rare autosomal recessive disorder characterised by permanent insulin requiring diabetes developing in the newborn period or early infancy, an early tendency to skeletal fractures, and spondyloepiphyseal dysplasia.1–8 The syndrome results from mutations in the gene encoding the eukaryotic translation initiation factor 2-α kinase 3 ( EIF2AK3 , also called PERK or PEK ).9 This enzyme phosphorylates EIF2A at Ser51 to regulate the synthesis of unfolded proteins in the endoplasmic reticulum.10 Targeted disruption of the Eif2ak3 gene in mice also causes diabetes because of the accumulation of unfolded proteins triggering β cell apoptosis.1112 Although these murine models have provided significant insight into the pathogenesis of Wolcott-Rallison syndrome, only three human cases have been characterised genetically.89 Here, we report genetic analysis of two further cases, and demonstrate new features of the expression pattern of human EIF2AK3 that offer possible explanations for important clinical features of the syndrome that are not apparent in the transgenic mouse models. Primers were designed to amplify all EIF2AK3 exons and splice site sequences from genomic DNA (table 1). Sequences were amplified by 35 cycles of polymerase chain reaction (PCR) using a proof-reading DNA polymerase in 50 μl reactions and purified (Qiaquick, Qiagen, Crawley, Sussex, UK). Products were sequenced using the BigDye terminator cycle sequencing kit according to the manufacturer’s instructions (Perkin-Elmer, Foster City, California, USA) and an ABI 377 sequencer (Applied Biosystems, city, county, UK). Sequences were compared to the published EIF2AK3 gene by BLAST analysis (http://www.ncbi.nlm.nih.gov/BLAST/). For potential mutations, the PCR and sequencing was repeated to confirm the result. Restriction digest analysis was also used to confirm the mutation in case 2, where the G→A substitution destroyed an Hph I site. View this table: Table 1 Primers used to amplify the EIF2AK3 gene Optimal conditions were determined for …

Nek2 localises to the distal portion of the mother centriole/basal body and is required for timely cilium disassembly at the G2/M transition

The NIMA-related kinase Nek2 promotes centrosome separation at the G2/M transition and, consistent with this role, is known to be concentrated at the proximal ends of centrioles. Here, we show by immunofluorescence microscopy that Nek2 also localises to the distal portion of the mother centriole. Its accumulation at this site is cell cycle-dependent and appears to peak in late G2. These findings are consistent with previous data implicating Nek2 in promoting reorganisation of centrosome-anchored microtubules at the G2/M transition, given that microtubules are anchored at the subdistal appendages of the mother centriole in interphase. In addition, we report that siRNA-mediated depletion of Nek2 compromises the ability of cells to resorb primary cilia before the onset of mitosis, while overexpression of catalytically active Nek2A reduces ciliation and cilium length in serum-starved cells. Based on these findings, we propose that Nek2 has a role in promoting cilium disassembly at the onset of mitosis. We also present evidence that recruitment of Nek2 to the proximal ends of centrioles is dependent on one of its substrates, the centrosome cohesion protein C-Nap1.

In vitro expression of NGN3 identifies RAB3B as the predominant Ras-associated GTP-binding protein 3 family member in human islets.

Neurogenin 3 (NGN3) commits pancreatic progenitors to an islet cell fate. We have induced NGN3 expression and identified upregulation of the gene encoding the Ras-associated small molecular mass GTP-binding protein, RAB3B. RAB3B localised to the cytoplasm of human β-cells, both during the foetal period and post natally. Genes encoding alternative RAB3 proteins and RAB27A were unaltered by NGN3 expression and in human adult islets their transcripts were many fold less prevalent than those of RAB3B. The regulation of insulin exocytosis in rodent β-cells and responsiveness to incretins are reliant on Rab family members, notably Rab3a and Rab27a, but not Rab3b. Our results support an important inter-species difference in regulating insulin exocytosis where RAB3B is the most expressed isoform in human islets.

Evidence for reciliation of RPE1 cells in late G1 phase, and ciliary localisation of cyclin B1

The primary cilium, an organelle that transduces extracellular signals important for development and tissue homeostasis, is typically assembled upon cell cycle exit and disassembled upon cell cycle re‐entry. Cilium assembly is thought to be suppressed in cycling cells, however the extent of suppression is not clear. For example, primary cilia are present in certain proliferating cells during development, and a period of reciliation has been reported to occur in late G1 in murine 3T3 cells released from serum starvation‐induced quiescence. Human retinal pigmented epithelial (hTERT‐RPE1; herein, RPE1) cells are commonly used to investigate pathways regulating cilium disassembly, however the ciliary disassembly profile of these cells remains uncertain. A period of reciliation has not been observed. Here, we analyse the ciliary disassembly profile of RPE1 cells by immunofluorescence microscopy. The results suggest a profile similar to 3T3 cells, including a period of reciliation in late G1 and a second wave of deciliation in S phase. We present evidence that arresting cells in early S phase with hydroxyurea or excess thymidine prevents the second wave of deciliation, and that deciliation is initiated shortly after release from a thymidine block, consistent with coupling to DNA replication. These findings support the often overlooked notion that cilium formation can occur in late G1, and suggest that RPE1 cells could serve as a model system for studying the molecular pathways that direct this process, in addition to those that stimulate cilium disassembly. We also present immunofluorescence data indicating that cyclin B1 localises to primary cilia.

Evidence for centriolar satellite localization of CDK1 and cyclin B2.

Centriolar satellites are 70-100 nm non-membranous particles implicated in the trafficking and folding of many centrosomal proteins, including the products of several disease genes. Centriolar satellites are often focused around the centrosome in interphase and either scattered throughout the cell or mostly undetectable in mitosis. The molecular mechanism underlying their disassembly/dispersal in mitosis remains unknown. Here, we present immunofluorescence microscopy data suggesting that CDK1 and cyclin B2 localise to centriolar satellites. These findings support recent biochemical data suggesting that a major component of centriolar satellites (PCM1) is a CDK1 substrate, and thus point to a role for this kinase in promoting disassembly of centriolar satellites in mitosis.

Glucagon-Like Peptide-1 (GLP1) signalling stimulates human beta-cell differentiation: support for increasing beta-cell numbers in Type 2 diabetes

withdrawn GlaxoSmithKline Young Diabetologist Travel Award

The long acting GLP-1 analogue, liraglutide, induces beta cell differentiation during normal human pancreas development

Background and Aims: Glucagon-like peptide-1 (GLP-1) stimulates glucose-dependent insulin secretion; the primary reason long-acting analogues have been developed for use in Type 2 diabetes. In rodents and tumour cell lines, GLP-1 signaling has also increased beta cell Fmass_ by promoting beta cell neogenesis, proliferation and inhibiting apoptosis. However, little is known about the role of GLP-1 during normal human beta cell differentiation. We have investigated GLP-1 signaling, via the long-acting analogue Liraglutide (Novo Nordisk A/S) in the developing human pancreas. Materials and Methods: With ethical approval and informed consent, human fetal material was collected from first trimester termination and processed for fixed tissue, RNA and protein analysis. In vitro culture models were established to interrogate beta cell differentiation. Insulinpositive cells were expressed as a percentage of total epithelial cell number. Results: Human pancreas was isolated at 8 weeks postconception (wpc), immediately prior to significant beta cell differentiation (Piper et al, J Endocrinology, 181, 11–23, 2004). GLP-1, along with its receptor, was expressed in the developing pancreas and duodenum. In 7-day explant culture, Liraglutide increased insulinpositive cell number by 68% (n=6), but had no effect on beta cell proliferation or apoptosis. Consistent with this implication of de novo differentiation, exposure to Liraglutide decreased epithelial progenitor cell proliferation by 24%. The effects of Liraglutide on beta cell number were abrogated by co-incubation with 10-fold molar excess of the GLP-1 receptor antagonist, Exendin 9–39, restoring insulin-positive cell number to just below 100% of control. The use of Exendin 9–39 alone reduced insulin-positive cells by 26%. Conclusion: Liraglutide increases human primary beta cell number, via GLP-1 receptor signaling that most likely enhances beta cell differentiation. For Exendin 9– 39 to decrease insulin-positive cell number by itself suggests that native GLP-1 signaling is very important during early human development. Furthermore, our data suggest that altering the cell cycle status of progenitor cells may be an underlying mechanism that regulates human beta cell differentiation. Clinically, Liraglutide may increase human beta cell mass in Type 2 diabetes. In addition, modulating the GLP-1 pathway may be a potent mechanism for manipulating stem cells to beta cells as an ambitious transplantation therapy in Type 1 diabetes. Supported by Novo Nordisk