Massively parallel sequencing of patients with intellectual disability, congenital anomalies and/or autism spectrum disorders with a targeted gene panel

10.1371/journal.pone.0093409PLoS ONE94-POLN

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

<i>L1CAM</i> splice site mutation in Patient 1.

<p>(A) IGV snapshot of c.3458-1G>A variant in the <i>L1CAM</i> gene (Chr X:153129005, hg19). (B) Sanger sequencing confirmation of c.3458-1G>A variant (NM_000425.3) (C) Partial cDNA sequence showing the mutant allele with the 5 bp deletion.</p

<i>USP9X</i> splice site mutation in Patient 3.

<p>(A) IGV snapshot of c.1986-1G>T variant in <i>USP9X</i> (Chr X:41025124, hg19). (B) Sanger sequencing confirmation of c.1986-1G>T variant (NM_001039590.2) in Patient 3. (C) Partial cDNA sequence showing expression of both the wild type and low level mutant allele with the 13 bp deletion. (D) Partial cDNA sequence of control patient. (E) Partial genomic DNA sequence of exon 15 (uppercase, blue) and intron 14 (lowercase, red) of <i>USP9X</i> gene showing the c.1986-1G>T variant (arrow) and the 13 bp deletion (r.1986_1998delATTTTTATTGAAG) which is underlined.</p

Summary of MPS data for the eight patients.

<p>Summary of MPS data for the eight patients.</p

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