Maggie Brett

Massively Parallel Sequencing of Patients with Intellectual Disability, Congenital Anomalies and/or Autism Spectrum Disorders with a Targeted Gene Panel

Developmental delay and/or intellectual disability (DD/ID) affects 1–3% of all children. At least half of these are thought to have a genetic etiology. Recent studies have shown that massively parallel sequencing (MPS) using a targeted gene panel is particularly suited for diagnostic testing for genetically heterogeneous conditions. We report on our experiences with using massively parallel sequencing of a targeted gene panel of 355 genes for investigating the genetic etiology of eight patients with a wide range of phenotypes including DD/ID, congenital anomalies and/or autism spectrum disorder. Targeted sequence enrichment was performed using the Agilent SureSelect Target Enrichment Kit and sequenced on the Illumina HiSeq2000 using paired-end reads. For all eight patients, 81–84% of the targeted regions achieved read depths of at least 20×, with average read depths overlapping targets ranging from 322× to 798×. Causative variants were successfully identified in two of the eight patients: a nonsense mutation in the ATRX gene and a canonical splice site mutation in the L1CAM gene. In a third patient, a canonical splice site variant in the USP9X gene could likely explain all or some of her clinical phenotypes. These results confirm the value of targeted MPS for investigating DD/ID in children for diagnostic purposes. However, targeted gene MPS was less likely to provide a genetic diagnosis for children whose phenotype includes autism.

Incidentalome from Genomic Sequencing: A Barrier to Personalized Medicine?

Background In Western cohorts, the prevalence of incidental findings (IFs) or incidentalome, referring to variants in genes that are unrelated to the patients primary condition, is between 0.86% and 8.8%. However, data on prevalence and type of IFs in Asian population is lacking. Methods In 2 cohorts of individuals with genomic sequencing performed in Singapore (total n = 377), we extracted and annotated variants in the 56 ACMG-recommended genes and filtered these variants based on the level of pathogenicity. We then analyzed the precise distribution of IFs, class of genes, related medical conditions, and potential clinical impact. Results We found a total of 41,607 variants in the 56 genes in our cohort of 377 individuals. After filtering for rare and coding variants, we identified 14 potential variants. After reviewing primary literature, only 4 out of the 14 variants were classified to be pathogenic, while an additional two variants were classified as likely pathogenic. Overall, the cumulative prevalence of IFs (pathogenic and likely pathogenic variants) in our cohort was 1.6%. Conclusion The cumulative prevalence of IFs through genomic sequencing is low and the incidentalome may not be a significant barrier to implementation of genomics for personalized medicine.

ARCN1 Mutations Cause a Recognizable Craniofacial Syndrome Due to COPI-Mediated Transport Defects

Cellular homeostasis is maintained by the highly organized cooperation of intracellular trafficking systems, including COPI, COPII, and clathrin complexes. COPI is a coatomer protein complex responsible for intracellular protein transport between the endoplasmic reticulum and the Golgi apparatus. The importance of such intracellular transport mechanisms is underscored by the various disorders, including skeletal disorders such as cranio-lenticulo-sutural dysplasia and osteogenesis imperfect, caused by mutations in the COPII coatomer complex. In this article, we report a clinically recognizable craniofacial disorder characterized by facial dysmorphisms, severe micrognathia, rhizomelic shortening, microcephalic dwarfism, and mild developmental delay due to loss-of-function heterozygous mutations in ARCN1, which encodes the coatomer subunit delta of COPI. ARCN1 mutant cell lines were revealed to have endoplasmic reticulum stress, suggesting the involvement of ER stress response in the pathogenesis of this disorder. Given that ARCN1 deficiency causes defective type I collagen transport, reduction of collagen secretion represents the likely mechanism underlying the skeletal phenotype that characterizes this condition. Our findings demonstrate the importance of COPI-mediated transport in human development, including skeletogenesis and brain growth.

A submicroscopic deletion involving part of the CREBBP gene detected by array-CGH in a patient with Rubinstein-Taybi syndrome

We report a girl with Rubinstein-Taybi syndrome (RSTS) who was found to have copy number loss on 16p13.3 by array-CGH. She has developmental delay and other features of RSTS including downslanting palpebral fissures, a prominent nose with the nasal septum extending below the alae nasi, broad thumbs and big toes, postaxial polydactyly of the right foot and constipation from birth. We report the junction sequence across the breakpoint region for a microdeletion in RSTS. The sequencing results also showed that the deletion was 81.4kb involving three genes DNASE 1, TRAP 1, and CREBBP.

DICER1 deletion and 14q32 microdeletion syndrome: an additional case and a review of the literature.

Department of Paediatrics, KK Research Centre, DNA Diagnostic and Research Laboratory, KK Women’s and Children’s Hospital and Paediatric Academic Clinical Programme, Singhealth Duke-NUS Graduate School of Medicine, Singapore, Singapore Correspondence to Saumya S. Jamuar, MBBS, MRCPCH, Department of Paediatrics, KK Women’s and Children’s Hospital, 100 Bukit Timah Road, Singapore 229899, Singapore Tel: + 65 639 41129; fax: + 65 629 17923; e-mail: saumya.s.jamuar@kkh.com.sg

De novo 3q22.1 q24 deletion associated with multiple congenital anomalies, growth retardation and intellectual disability.

We describe a boy with a de novo deletion of 15.67 Mb spanning 3q22.1q24. He has bilateral micropthalmia, ptosis, cleft palate, global developmental delay and brain, skeletal and cardiac abnormalities. In addition, he has bilateral inguinal hernia and his right kidney is absent. We compare his phenotype with seven other patients with overlapping and molecularly defined interstitial 3q deletions. This patient has some phenotypic features that are not shared by the other patients. More cases with smaller deletions defined by high resolution aCGH will enable better genotype-phenotype correlations and prioritizing of candidate genes for the identification of pathways and disease mechanisms.

Acute lymphoblastic leukemia in a child with a de novo germline gnb1 mutation.

Acute Lymphoblastic Leukemia in a Child with a De Novo Germline GNB1 Mutation Maggie Brett, Angeline H. M. Lai, Teck-Wah Ting, Ah-Moy Tan, Roger Foo, Saumya Jamuar, and Ene-Choo Tan* Research Laboratory, KK Women’s and Children’s Hospital, Singapore Genetics Service, KK Women’s and Children’s Hospital, Singapore Paediatrics Academic Clinical Programme, SingHealth Duke-NUS Medical School, Singapore Haematology/Oncology Service, KK Women’s and Children’s Hospital, Singapore Genome Institute of Singapore, Singapore

Cockayne Syndrome due to a maternally-inherited whole gene deletion of ERCC8 and a paternally-inherited ERCC8 exon 4 deletion.

Cockayne Syndrome (CS) is an autosomal recessive disorder that causes neurological regression, growth failure and dysmorphic features. We describe a Chinese female child with CS caused by deletions of exon 4 of ERCC8 on one chromosome and exons 1-12 on the other chromosome. By using chromosomal microarray, multiplex ligation-dependant probe analysis and long range PCR, we showed that she inherited a 277 kb deletion affecting the whole ERCC8 gene from the mother and a complex rearrangement resulting in deletion of exon 4 together with a 1,656 bp inversion of intron 4 from the father. A similar complex rearrangement has been reported in four unrelated Japanese CS patients. Analysis of the deletion involving exon 4 identified LINE and other repeat elements that may predispose the region to deletions, insertions and inversions. The patient also had insulin-dependent diabetes mellitus, a rare co-existing feature in patients with CS. More research will be needed to further understand the endocrine manifestations in CS patients.

Intragenic multi-exon deletion in the FBN1 gene in a child with mildly dilated aortic sinus: a retrotransposal event

Marfan syndrome is an autosomal dominant disorder affecting mainly the skeletal, ocular and cardiovascular systems. Most cases are caused by mutations in the fibrillin-1 gene (FBN1), although there are some reports on deletions involving FBN1 and other additional genes. We report a male patient who was first evaluated at 4 years of age. Echocardiogram showed a mildly dilated aortic sinus. He also had a history of muscular ventral septal defect which was closed spontaneously and trivial mitral regurgitation. Other phenotypic features include frontal bossing, anteverted ears, joint hyperlaxity, learning disability, skin striae, and height and weight in the >97th centile but no other diagnostic findings of MFS and does not fulfill the revised Ghent criteria. Chromosomal microarray analysis showed a deletion of approximately 36.8 kb at 15q21.1, which starts in intron 6 and ends in intron 9 and includes three FBN1 exons. Sequence analysis of the breakpoint region confirmed the deletion and revealed a concomitant insertion of a retrotransposon within the intron 6/intron 9 region. The intragenic deletion of exons 7–9 was likely the result of a retrotransposition event by a MAST2-SVA element mediated by repetitive sequences.

Piebaldism with multiple café-au-lait–like hyperpigmented macules and inguinal freckling caused by a novel KIT mutation

CALM: caf e-au-lait macules NF1: neurofibromatosis type 1 TK: tyrosine kinase INTRODUCTION Piebaldism is a rare autosomal dominant disorder of pigmentation characterised by patches of leukoderma and white forelock. It is most often caused by mutations in the KIT proto-oncogene receptor tyrosine kinase (TK), which result in the defective migration and differentiation of melanoblasts and melanocytes. Other mutations that have been described include a deletion and a double nucleotide variant in the gene encoding snail family zinc finger 2 (SNAI2/SLUG). The occasional coexistence of multiple caf e-aulait macules (CALMs) in piebaldism and also piebaldism in association with CALMs and intertriginous freckling may lead to diagnostic confusion with neurofibromatosis type 1 (NF1). Another differential diagnosis is Legius syndrome, which is caused by loss-of-function mutations in the SPRED1 gene. The syndrome is characterised by CALMs and intertriginous freckling with macrocephaly, lipomas, and learning disability. Unlike NF1, patients with Legius syndrome do not have cutaneous or plexiform neurofibromas, skeletal dysostosis, or optic pathway gliomas. The overlapping presentations of these related pigmentary disorders make it difficult to distinguish between them during the early stages and present a challenge for initial diagnosis.