ARTICLE © American College of Medical Genetics and Genomics Pathogenic variants in CDC45 on the remaining allele in patients with a chromosome 22q11.2 deletion result in a novel autosomal recessive condition Marta Unolt, MD 1,2, Molka Kammoun, PhD 3, Beata Nowakowska, PhD 4, Gail E. Graham, MD 5, T. Blaine Crowley 1, Matthew S. Hestand, PhD3, Wolfram Demaerel, PhD 3, Maciej Geremek, MD, PhD 4, Beverly S. Emanuel, PhD1,6, Elaine H. Zackai, MD 1,6, Joris R. Vermeesch, PhD3 and Donna McDonald-McGinn, MS, LCGC 1,6 Purpose: The 22q11.2 deletion syndrome (22q11.2DS) is the most or other atypical findings. common microdeletion in humans, with highly variable phenotypic Conclusion: This study supports CDC45 as a causative gene in expression. Whereas congenital heart defects, palatal anomalies, craniosynostosis, as well as a number of other anomalies. We immunodeficiency, hypoparathyroidism, and neuropsychiatric suggest that this association results in a condition independent of conditions are observed in over 50% of patients with 22q11DS, a – “ ” Meier Gorlin syndrome, perhaps representing a novel condition subset of patients present with additional atypical findings such as and/or a cause of features associated with Baller–Gerold syndrome. craniosynostosis and anorectal malformations. Recently, pathogenic In addition, this work confirms that the phenotypic variability variants in the CDC45 (Cell Division Cycle protein 45) gene, – observed in a subset of patients with 22q11.2DS is due to located within the LCR22A LCR22B region of chromosome pathogenic variants on the nondeleted chromosome. 22q11.2, were noted to be involved in the pathogenesis of craniosynostosis. Genetics in Medicine (2020) 22:326–335; https://doi.org/10.1038/s41436- Methods: We performed next-generation sequencing on DNA 019-0645-4 from 15 patients with 22q11.2DS and atypical phenotypic features such as craniosynostosis, short stature, skeletal differences, and Keywords: 22q11.2 deletion syndrome; CDC45 gene; anorectal malformations. next-generation sequencing; rare nonsynonymous variants; Results: We identified four novel rare nonsynonymous variants in craniosynostosis CDC45 in 5/15 patients with 22q11.2DS and craniosynostosis and/ INTRODUCTION ~30%. Furthermore, developmental delay, cognitive deficits, The 22q11.2 deletion syndrome (22q11.2DS) is the most and psychiatric illness such as schizophrenia are important common microdeletion in humans, with an estimated features of this condition. However, none of these features prevalence of 1 in 2000–4000 live births and 1 in 1000 appear to be fully penetrant, and each exhibits variable unselected fetuses.1 expression. Moreover, a subset of patients presents with The 22q11.2 deletion is most frequently a de novo event additional “atypical” findings, such as craniosynostosis, that results from nonallelic homologous recombination congenital diaphragmatic hernia, anorectal malformations, between specific low copy repeats (LCRs), LCR22A and and airway anomalies.1 LCR22D, resulting in a ~3 MB deletion.2 It has been previously suggested that large heterozygous The phenotypic expression of 22q11.2DS is highly variable. deletions may unmask recessive variants on the remaining In general, the associated clinical findings include congenital allele, resulting in pathogenic phenotypes. For example, it has cardiac abnormalities, palatal anomalies, and immunodefi- already been demonstrated that, in patients with 22q11.2 ciency in ~75% of patients; hypoparathyroidism and gastro- deletion, hemizygous pathogenic variants in SNAP29 can intestinal differences in ~50%; genitourinary anomalies in unmask the autosomal recessive conditions CEDNIK 1Division of Human Genetics, Children’s Hospital of Philadelphia, Philadelphia, PA, USA; 2Department of Pediatrics, Sapienza University of Rome, Rome, Italy; 3Center for Human Genetics, KU Leuven, Leuven, Belgium; 4Department of Medical Genetics, Institute of Mother and Child, Warsaw, Poland; 5Department of Genetics, Children’s Hospital of Eastern Ontario, Ottawa, Canada; 6Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA. Correspondence: Marta Unolt (unolt. [email protected]) Submitted 31 January 2019; accepted: 15 August 2019 Published online: 2 September 2019 326 Volume 22 | Number 2 | February 2020 | GENETICS in MEDICINE UNOLT et al ARTICLE (cerebral dysgenesis, neuropathy, ichthyosis, and kerato- spectrum, to include rare gastrointestinal anomalies, such as derma) and potentially Kousseff syndromes.3 Likewise, intestinal malrotation, imperforate/anteriorly displaced anus hemizygous pathogenic variants in GP1BB cause and congenital diaphragmatic hernia and short stature (in Bernard–Soulier syndrome.4 Thus, there is increasing evi- absence of any endocrine or metabolic cause) and patellar dence that our ability to identify these pathogenic variants will anomalies. Six additional patients were identified with these help in understanding the function of previously unchar- features and available DNA (Table S1). acterized genes that contribute to the unusual phenotypes Finally, in thinking about CDC45 as a putative cause for observed in a subset of patients with 22q11.2DS. Conversely, craniosynostosis in these patients we contacted authors who – and perhaps as importantly, it will have a great impact on had previously reported such patients.9 12 The Children’s understanding the causative genes underlying human auto- Hospital of Eastern Ontario and University of Ottawa somal recessive conditions. (Canada) had DNA available for their previously reported Recently, pathogenic variants in the CDC45 (Cell Division patient9 (Table S1). Cycle protein 45) gene were suggested to be involved in the As the control group, we enrolled 85 patients with 22q11 pathogenesis of both Meier–Gorlin syndrome (MGS) and deletion and without atypical clinical features described craniosynostosis.5,6 MGS (MIM 224690) is a rare autosomal above, for whom exome sequencing data were received recessive primordial dwarfism disorder, characterized by earlier. In addition, CDC45 targeted resequencing was microtia, short stature, and absent or hypoplastic patellae performed in 48 more 22q11.2 patients without atypical (Table 1).7 In 1998, Saha et al. mapped the CDC45 gene to findings. chromosome 22q11.2, within the DiGeorge syndrome critical region (LCR22A–B).8 Exome sequencing and variant analysis Here we identified CDC45 variants in a cohort of patients Exome sequencing (ES) was performed on eight patients. 1234567890():,; with a 22q11.2 deletion and craniosynostosis and/or other Because the initial hypothesis was that a pathogenic variant atypical developmental anomalies. in the remaining allele causes the observed atypical findings, we focused only on the 22q11 region for ES data analysis. MATERIALS AND METHODS Following genome library preparation using the TruSeq As we had previously identified pathogenic variants on the DNA Library Preparation Kit (Illumina), exome capture remaining intact chromosome 22q11.2 allele in patients was performed with SeqCap EZ Human Exome Library v3.0 with atypical phenotypic features, such as Bernard–Soulier (Roche, NimbleGen) for eight patients (Table S1). Subse- syndrome due to GP1BB pathogenic variants and poly- quently, 100-bp paired-end reads were generated on Illumina microgyria and keratoderma due to pathogenic variants in HiSeq2000 according to the manufacturer’s instructions. SNAP29, following the recent publication by Fenwick et al.5 Reads were mapped to the reference human genome (hg19) we hypothesized that similar pathogenic variants in CDC45 using Burrows–Wheeler Aligner 0.7.8. The number of gene might explain craniosynostosis in patients with uniquely mapped reads ranged from 48 million to 108 22q11.2DS. Thus, we retrospectively analyzed records on million. The mean coverage at each target base varied from 1305 patients, from the 22q and You Center at The 54x to 139×, with a minimum of 80% of the bases covered by Children’s Hospital of Philadelphia (CHOP), a large at least 30 reads in all the patients. SAMtools v 0.1.19 was comprehensive multidisciplinary program for patients with used to sort and merge the data. Indel containing reads were 22q11.2DS, founded following the identification of the N25 realigned using RealignerTargetCreator and IndelRealigner of fluorescence in situ hybridization (FISH) probe at CHOP in GATK 3.4.46. Variants were called using HaplotypeCaller, the early 1990s. The study was approved by the Institu- LeftAlignAndTrimVariants, and GenotypeGVCFs of GATK tional Review Board of CHOP (07–005352) with appro- 3.5. Variants were annotated with ANNOVAR (version 2015) priate informed consent obtained on all subjects. In including for dbSNP (dbSNP137), 1000 Genomes data, and addition, the study data were handled in compliance with the ESP6500 panel (NHLBI GO Exome Sequencing Project the Health Insurance Portability and Accountability Act of [ESP], Seattle, WA), (http://evs.gs.washington.edu/EVS/). 1996 (HIPAA) regulations. All patients had a laboratory Functional predictions for the amino acid changes according confirmed 22q11.2 deletion and presented with “atypical” to different models (SIFT, PolyPhen-2, LRT, and Mutation- clinical features (found in <10% of overall cohort). These Taster) were retrieved from dbNSFP (a database of human findings initially included craniofacial anomalies, such as nonsynonymous single-nucleotide polymorphisms [SNPs] craniosynostosis, choanal atresia or cleft lip, and limb