Diabetes Volume 63, October 2014 3557 Deimante Simaite,1,2 Julia Kofent,3 Maolian Gong,1,2 Franz Rüschendorf,2 Shiqi Jia,4 Pamela Arn,5 Kristi Bentler,6 Carolyn Ellaway,7 Peter Kühnen,8 Georg F. Hoffmann,9 Nenad Blau,9,10 Francesca M. Spagnoli,3 Norbert Hübner,2 and Klemens Raile1 Recessive Mutations in PCBD1 Cause a New Type of Early-Onset Diabetes Diabetes 2014;63:3557–3564 | DOI: 10.2337/db13-1784 GENETICS/GENOMES/PROTEOMICS/METABOLOMICS Mutations in several genes cause nonautoimmune di- This condition responds to and can be treated with oral abetes, but numerous patients still have unclear genetic drugs instead of insulin, which is important clinical informa- defects, hampering our understanding of the develop- tion for these patients. Finally, patients at risk can be ment of the disease and preventing pathogenesis- detected through a newborn screening for phenylketonuria. oriented treatment. We used whole-genome sequencing with linkage analysis to study a consanguineous family with early-onset antibody-negative diabetes and identi- Diabetes is classified into type 1 diabetes (T1D) caused by fied a novel deletion in PCBD1 (pterin-4 a-carbinolamine autoimmune b-cell destruction; type 2 diabetes (T2D), dehydratase/dimerization cofactor of hepatocyte nuclear caused by relative insulin deficiency in face of insulin re- a factor 1 ), a gene that was recently proposed as a likely sistance; gestational diabetes mellitus; and other specific cause of diabetes. A subsequent reevaluation of patients types, including monogenic diabetes (1). T2D is a poly- with mild neonatal hyperphenylalaninemia due to muta- genic disease with over 60 susceptibility loci and numer- tions in PCBD1 from the BIODEF database identified ous risk variants colocated with genes, causing monogenic three additional patients who had developed HNF1A-like diabetes (2). Monogenic diabetes is defined by neonatal, diabetes in puberty, indicating early b-cell failure. We found that Pcbd1 is expressed in the developing pancreas childhood, or a postpubertal age of onset, variable clinical of both mouse and Xenopus embryos from early specifi- presentation, a lack of autoimmunity, and acanthosis nig- cation onward showing colocalization with insulin. Impor- ricans as well as uncommon obesity and ketoacidosis ; tantly, a morpholino-mediated knockdown in Xenopus outside the neonatal period. It affects 1% of diabetic revealed that pcbd1 activity is required for the proper es- patients (1,3). Interestingly, monogenic cases are often tablishment of early pancreatic fate within the endoderm. accompanied by endogenous insulin production and lack We provide the first genetic evidence that PCBD1 muta- of insulin resistance. Heterozygous GCK, HNF1A, and tions can cause early-onset nonautoimmune diabetes with HNF4A mutations are the most common cause, although features similar to dominantly inherited HNF1A-diabetes. more than 20 other genes have been described (3,4). 1Experimental and Clinical Research Center, Charité Medical Faculty and Max- 9Division of Inborn Metabolic Diseases, Department of General Pediatrics, Uni- Delbrück Center for Molecular Medicine, Berlin, Germany versity Children’s Hospital, Heidelberg, Germany 2Cardiovascular and Metabolic Diseases, Max-Delbrück Center for Molecular 10Division of Metabolism, University Children’s Hospital, Zürich, Switzerland Medicine, Berlin, Germany Corresponding author: Klemens Raile, [email protected]. 3Laboratory of Molecular and Cellular Basis of Embryonic Development, Max- Received 21 November 2013 and accepted 5 May 2014. Delbrück Center for Molecular Medicine, Berlin, Germany 4Department of Neuroscience, Max-Delbrück Center for Molecular Medicine, This article contains Supplementary Data online at http://diabetes Berlin, Germany .diabetesjournals.org/lookup/suppl/doi:10.2337/db13-1784/-/DC1. 5Division of Genetics, Department of Pediatrics, Nemours Children’sClinic, N.H. and K.R. contributed equally as senior authors. Jacksonville, FL © 2014 by the American Diabetes Association. Readers may use this article as 6Division of Pediatric Genetics and Metabolism, Department of Pediatrics, Uni- long as the work is properly cited, the use is educational and not for profit, and the versity of Minnesota Amplatz Children’s Hospital, Minneapolis, MN work is not altered. 7Western Sydney Genetics Program, Royal Alexandra Hospital for Children, Westmead, New South Wales, Australia 8Institute for Experimental Pediatric Endocrinology, Charité Medical Faculty, Ber- lin, Germany 3558 PCBD1 Mutations Cause Diabetes Diabetes Volume 63, October 2014 However, the disease-causing variants in numerous fam- nuclear factor 1 a (DCoH) (5), for further analysis. Our ilies remain obscure. Identifying novel genes would pro- decision was based on the strong expression of PCBD1 in vide insights into pathogenesis and suggest new treatment pancreatic islets (T1DBase and ref. 6), mouse pancreatic strategies for rare monogenic diabetes as well as common progenitors (7), and its interaction with HNF1A and polygenic T1D and T2D. HNF1B transcription factors, essential for proper pancre- atic b-cell function (5,8,9). Moreover, PCBD1 lay within the RESEARCH DESIGN AND METHODS suggestive linkage region (Fig. 1C) and was the only gene We sought to identify additional genes by combining linkage containing a frameshift deletion. Subsequently, we vali- analysis with the whole-genome sequencing of a consanguin- dated the deletion in all diabetic members of family 1 by eous family 1 (Fig. 1A and Table 1) with nonautoimmune Sanger sequencing (Fig. 1B), using exon-intron spanning diabetes and no pathogenic mutations in HNF1A, HNF1B, primers (Supplementary Table 1). HNF4A, INS, ABCC8,orKCNJ11 genes. Our institutional review boards approved the studies and written consent RESULTS was obtained from the participants or their guardians. PCBD1 is a bifunctional protein that acts as an enzyme in To pinpoint suggestive linkage regions, we performed the regeneration of cofactor tetrahydrobiopterin (BH4) a haplotype mapping and parametric linkage analysis of (10), crucial for the function of aromatic amino acid seven members of family 1 (Fig. 1A, red borders) using hydroxylases, and as a dimerization cofactor of transcrip- HumanCytoSNP-12 v2.1 BeadChip (Illumina) and Merlin tion factors HNF1A and HNF1B (5), which are important software, assuming recessive inheritance, complete pene- in liver and pancreas development and function. The en- trance, and a disease allele frequency of 0.001. We zymatic function of PCBD1 is defective in newborns with obtained 24 genomic regions with positive logarithm of mild transient hyperphenylalaninemia (HPA) and high odds (LOD) scores, including 13 regions on 10 chromo- urinary levels of primapterin caused by recessive muta- somes with maximal LOD score of 1.3 (Supplementary tions (11,12). Recently, a PCBD1 defect was suggested to Fig. 1). We next performed a whole-genome sequencing cause hypomagnesemia and diabetes (13). The novel ho- of five individuals from family 1 (Fig. 1A, asterisks) on the mozygous deletion c.46del in family 1 results in a premature Complete Genomics platform (Mountain View, CA). The stop codon p.[(Lys16Cys*5)];[(Lys16Cys*5)] that abolishes Complete Genomics pipeline was used for read mapping the transcription factor–binding and enzymatic functions and allele calling. On average, both alleles were called for of PCBD1 (Fig. 1G). ;96% genomic and ;98% exonic positions, whereas ;97% As III-2 developed diabetes in puberty, we reeval- and ;99% of the called ones were covered by at least 10 uated patients with neonatal HPA caused by PCBD1 reads, respectively (Supplementary Table 1). We found ;4 who appeared in the BIODEF database (14). Three out million small variations (small indels and single nucleotide of seven children from six families, exhibiting three polymorphisms [SNPs]) per individual genome including different biallelic PCBD1 defects of p.[(Glu87*)];[(Glu87*)], more than 23,000 variants in each exome. p.[(Glu97Lys)];[(Gln98*)], and p.[(Gln98*)];[(Gln98*)], had To distinguish relevant SNPs and small indels from already developed antibody-negative diabetes with normal other variations, we used Complete Genomics Analysis pancreatic morphology (Fig. 1D–F and Table 1). Taken Tools, ANNOVAR, and custom scripts. We first removed together, our analyses of seven independent families pro- intergenic variants and anticipated recessive inheritance vide strong genetic evidence that mutations in PCBD1 (Supplementary Fig. 2). Therefore, homozygous variants cause puberty-onset diabetes. were required to be present in index case III-2 and het- We tested the in vitro effects of a transient Pcbd1 erozygousinII-1,II-2,andI-4,butnotinI-3,whoisnot knockdown in glucose-sensitive mouse insulinoma cells related to I-4. As monogenic diabetes is uncommon, we using small interfering RNA and found no obvious defects predicted the disease-causing variant to be rare and in insulin production or glucose-stimulated insulin secre- likely not yet identified. Thus, we removed all the var- tion after an 80% inactivation of Pcbd1 (data not shown). iants present in dbSNP 137; 1000 Genomes Project; Endocrine pancreas dysfunction not only may arise from National Heart, Lung, and Blood Institute Exome b-cell inability to produce and/or secrete insulin but also Sequencing Project; 69 sequenced individuals from Com- from impaired b-cell development, proliferation, and ad- plete Genomics; and our in-house database of non- aptation during fetal, neonatal, and pubertal age.