Mutations in PTF1A cause pancreatic and cerebellar agenesis Gabrielle S Sellick 1 , Karen T Barker 1 , Irene Stolte-Dijkstra 2 , Christina Fleischmann 1 , Richard J Coleman 1 , Christine Garrett 3 , Anna L Gloyn 4 , Emma L Edghill 4 , Andrew T Hattersley 4 , Peter K Wellauer 5 , Graham Goodwin 6 & Richard S Houlston 1 Individuals with permanent neonatal diabetes mellitus usually present within the first three months of life and require insulin treatment 1,2 . We recently identified a locus on chromosome 10p13–p12.1 involved in permanent neonatal diabetes mellitus associated with pancreatic and cerebellar agenesis in a genome-wide linkage search of a consanguineous Pakistani family 3 . Here we report the further linkage analysis of this family and a second family of Northern European descent segregating an identical phenotype. Positional cloning identified the mutations 705insG and C886T in the gene PTF1A, encoding pancreas transcription factor 1a, as disease- causing sequence changes. Both mutations cause truncation of the expressed PTF1A protein C-terminal to the basic-helix-loop-helix domain. Reporter-gene studies using a minimal PTF1A deletion mutant indicate that the deleted region defines a new domain that is crucial for the function of this protein. PTF1A is known to have a role in mammalian pancreatic development 4,5 , and the clinical phenotype of the affected individuals implicated the protein as a key regulator of cerebellar neurogenesis. The essential role of PTF1A in normal cerebellar development was confirmed by detailed neuropathological analysis of Ptf1a À/À mice. Linkage to our previously identified locus on 10p13–p12.1 associated with permanent neonatal diabetes mellitus (PNDM) defined by the single-nucleotide polymorphism (SNP) markers rs1398431 and rs2149639 in the Pakistani family (Family 1; Fig. 1a) was confirmed in both families using microsatellite markers (Fig. 1a,b). Marker data allowed further positional refinement of the linked region to the 7.7- Mb interval between rs1398431 and D10S586. The combined lod score for both families between markers D10S548 and D10S586 was 4.4. We selected three genes mapping to the region of linkage, PIP5K2A, PTF1A and CACNB2, for candidate gene screening because of their expression in human or mouse pancreatic and cerebellar tissue and implied biological function. We sequenced coding regions including all exon-intron boundaries of each gene using genomic DNA from all available members of both families. In addition to a common polymorphism detected in family 1 (787T-C, dbSNP identifier rs7918487, control C allele frequency 0.45), which did not segregate with the disease phenotype, we identified two different mutations in PTF1A, both of which resulted in truncation of the expressed protein (Fig. 2a). In family 1, affected individuals (V:4, V:8 and V:9; Fig. 1a) were homozygous with respect to a 886C-T transition in exon 2 causing a nonsense mutation (R296X). In family 2, the affected individual (V:1; Fig. 2a) was homozygous with respect to a single– base pair insertion (705insG) in exon 1, which caused a frameshift mutation (P236fsX270) and truncation of the PTF1A protein at codon 270 (Fig. 2a,b). The mutations segregated with the disease phenotype and carrier status in both families. Neither mutation was present in 190 alleles from healthy, unrelated individuals or had been previously documented by either dbSNP or the Celera Human SNP databases. The 705insG and 886C-T mutations in PTF1A cause truncation of the C-terminal 32 amino acids of the expressed protein, which is a functional domain a priori. To examine the effect of the deletion on transcriptional activity, we compared the abilities of full-length and truncated PTF1A (PTF1ADC) to activate a luciferase reporter con- struct containing Ptf1-E47 recognition motifs. The availability of a mouse Ptf1a clone and literature describing the binding motif recognized by murine Ptf1 led us to work with the mouse clone, constructing a truncated version at the position equivalent to the most C-terminal deletion in the human sequence. The Ptf1-E47 recognition motif of the rat chymotrypsin gene com- prises two conserved sequences: an A box that is unique to Ptf1 binding sites 6 and an E box recognized by ordinary basic helix-loop- helix (bHLH) proteins 7 . E47 is a protein that heterodimerizes with bHLH proteins to stimulate transcription from E box–containing promoters. Using a promoter containing four repeats of the A/E motif of the rat chymotrypsinogen B gene, Obata et al. 8 showed that Published online 14 November 2004; doi:10.1038/ng1475 1 Section of Cancer Genetics, Institute of Cancer Research, Surrey SM2 5NG, UK. 2 Department of Clinical Genetics, University Hospital Groningen, Groningen 9700 RB, The Netherlands. 3 North West Thames Regional Genetics Service, Kennedy-Galton Centre, North West London Hospitals NHS Trust, Harrow HA1 3UJ, UK. 4 Diabetes and Vascular Medicine, Peninsula Medical School, Exeter EX2 5AX, UK. 5 Swiss Institute for Experimental Cancer Research (ISREC), CH-1066 Epalinges, Switzerland. 6 Section of Molecular Carcinogenesis, Institute of Cancer Research, Surrey SM2 5NG, UK. Correspondence should be addressed to R.S.H. (Richard.Houlston@icr.ac.uk). NATURE GENETICS VOLUME 36 [ NUMBER 12 [ DECEMBER 2004 1301 LETTERS © 2004 Nature Publishing Group http://www.nature.com/naturegenetics