Original Article Diagnostic Genetics Ann Lab Med 2014;34:134-138 http://dx.doi.org/10.3343/alm.2014.34.2.134 ISSN 2234-3806 • eISSN 2234-3814 Identification of a De Novo Heterozygous Missense FLNB Mutation in Lethal Atelosteogenesis Type I by Exome Sequencing Ga Won Jeon, M.D.1,*, Mi-Na Lee, M.D.2,*, Ji Mi Jung, M.D.1, Seong Yeon Hong, M.D.3, Young Nam Kim, M.D.4, Jong Beom Sin, M.D.1, and Chang-Seok Ki, M.D.2 Department of Pediatrics1, Inje University College of Medicine, Busan Paik Hospital, Busan; Department of Laboratory Medicine and Genetics2, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul; Department of Obstetrics and Gynecology3, Catholic University of Daegu, Daegu Catholic University Medical Center, Daegu; Department of Obstetrics and Gynecology4, Inje University College of Medicine, Busan Paik Hospital, Busan, Korea Background: Atelosteogenesis type I (AO-I) is a rare lethal skeletal dysplastic disorder Received: June 28, 2013 characterized by severe short-limbed dwarfism and dislocated hips, knees, and elbows. Revision received: September 10, 2013 Accepted: December 26, 2013 AO-I is caused by mutations in the filamin B (FLNB) gene; however, several other genes can cause AO-like lethal skeletal dysplasias. Corresponding author: Chang-Seok Ki Department of Laboratory Medicine and Methods: In order to screen all possible genes associated with AO-like lethal skeletal dys- Genetics, Samsung Medical Center, plasias simultaneously, we performed whole-exome sequencing in a female newborn hav- Sungkyunkwan University School of Medicine, 81 Irwon-ro, Gangnam-gu, ing clinical features of AO-I. Seoul 135-710, Korea Results: Exome sequencing identified a novel missense variant (c.517G>A; p.Ala173Thr) Tel: +82-2-3410-2709 Fax: +82-2-3410-2719 in exon 2 of the FLNB gene in the patient. Sanger sequencing validated this variant, and E-mail: [email protected] genetic analysis of the patient’s parents suggested a de novo occurrence of the variant. *The first two authors contributed equally Conclusions: This study shows that exome sequencing can be a useful tool for the identi- to the work. fication of causative mutations in lethal skeletal dysplasia patients. © The Korean Society for Laboratory Medicine This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecom- mons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the Key Words: Atelosteogenesis type I, FLNB, Mutation, Exome sequencing original work is properly cited. INTRODUCTION rately owing to their clinical heterogeneity [3, 4]. AO is subdivided into types I (AO-I), II (AO-II), and III (AO-III). Lethal skeletal dysplasias commonly include thanatophoric dys- AO-I and AO-III are associated with FLNB mutations [5], while plasia, achondrogenesis, osteogenesis imperfecta type 2, con- AO-II is caused by SLC26A2 (DTDST) mutations [6]. The FLNB- genital hypophosphatasia, and atelosteogenesis (AO), and can related disorders constitute a spectrum of phenotypes ranging be distinguished by four key features: bone mineralization, frac- from spondylocarpotarsal synostosis syndrome and Larsen syn- tures, macrocranium, and trunk length [1]. AO is a rare lethal drome at the mild end, to AO-I, AO-III, and boomerang dyspla- skeletal dysplasia. It represents a heterogeneous group of disor- sia at the severe end [7, 8]. Besides FLNB, many genes, includ- ders with the following features: incomplete ossification of long ing ALPL, ARSE, COL1A1/2, COL2A1, COL11A1/2, CRTAP, bones and markedly short, hypoplastic distal femora and hu- DYNC2H1, EBP, EVC, EVC2, FGFR3, IFT80, IFT122, LEPRE1, meri resulting in rhizomelic dwarfism [2]. Rare types of lethal NEK1, PEX7, PPIB, SLC26A2, SLC35D1, SOX, TRIP11, and skeletal dysplasias, including AO, are difficult to diagnose accu- WDR35, have been identified to be involved in AO-like lethal 134 www.annlabmed.org http://dx.doi.org/10.3343/alm.2014.34.2.134 Jeon GW, et al. Exome sequencing for atelosteogenesis type I skeletal dysplasias [9]. 2. Whole-exome sequencing Several recent studies have described how exome sequenc- Genomic DNA was extracted from peripheral blood leukocytes ing has allowed the identification of causative mutations in many by using a Wizard Genomic DNA purification kit (Promega, Mad- heterogeneous and complicated disorders [10, 11]. Confirming ison, WI, USA), according to the manufacturer’s instructions. a definitive molecular diagnosis is often difficult and costly in Exome sequencing was performed by DNA Link (DNA Link Inc., skeletal dysplasia because patients may be in a critical condi- Seoul, Korea), and the bioinformatics services were provided by tion, in addition to having a phenotypically complex presenta- Samsung SDS (Samsung SDS, Ltd., Seoul, Korea). The SureSe- tion. This clinical and genetic diversity reflects the number of lect Human All Exon 50 Mb kit (Agilent Technologies, Santa causative genes, and molecular diagnosis of such disorders by Clara, CA, USA) was used for in-solution enrichment of coding conventional Sanger sequencing, usually an expensive and exons and flanking intronic sequences, following the manufac- time-consuming gene-by-gene screening process, is sometimes turer’s standard protocol. Adapted sequences for the Illumina impractical [10]. Exome sequencing allows simultaneous analy- HiSeq2000 sequencing system (Illumina Inc., San Diego, CA, sis of all coding sequences of genes. The advantage of exome USA) were ligated, and the enriched DNA samples were sub- sequencing is that it does not require a prior knowledge of the jected to standard sample preparation for the HiSeq2000 instru- genes responsible for a disorder. In this study, we performed ment. The Burrows-Wheeler alignment (BWA) was used to align exome sequencing of a newborn patient with clinical features of sequence reads to the human reference genome (hg19), and AO-I and successfully identified a de novo FLNB mutation. variants were called using the GATK software package [12-14]. The single nucleotide polymorphism (SNP) and short indel can- METHODS didates were identified at nucleotide resolution. These variants were annotated by ANNOVAR (version 2011 Jun 18) [15] to fil- 1. Patient ter SNPs reported in the dbSNP database (build 135) [16] and A female baby was born as the first of twins of non-consanguin- the 1000 Genomes Project (http://1000genomes.org). eous healthy parents at 27 weeks of gestation via emergent ce- Polyphen-2 (http://genetics.bwh.harvard.edu/pph2/) and sarean section due to fetal distress. Both her parents were 31 yr SIFT (Sorting Intolerant From Tolerant) (http://sift.jcvi.org/) were old, and her mother’s obstetric history was gravida 1, para 0. used to predict the consequences of the missense variants on She was impregnated with twins through in vitro fertilization due protein function. The candidate variant identified by exome se- to infertility. Prenatal ultrasonography at 25 weeks of gestation quencing was confirmed by using conventional sequencing. revealed that one fetus had polyhydramnios and multiple skele- tal anomalies consistent with skeletal dysplasia, such as very RESULTS poorly ossified femora and humeri with severe shortening of all extremities. 1. Patient The patient had respiratory difficulty from birth and required The patient’s weight was 685 g (<10th percentile), height was mechanical ventilation support. It was very difficult to advance 22 cm (<10th percentile), and head circumference was 24 cm the endotracheal tube into the trachea after passing through the (10-25th percentile) at birth. This is in contrast to the second in- larynx. A pulmonary surfactant was instilled to treat the respira- fant twin, who had a weight of 925 g (25-50th percentile), height tory distress syndrome of prematurity. Oxygenation and ventila- of 36 cm (50th percentile), and head circumference of 26 cm tion were not effective even though high-frequency oscillatory (50-75th percentile). The affected infant also had a depressed ventilation was applied. She died of respiratory failure at three nasal bridge, hypertelorism, micrognathia, and low-set ears at hours after birth, possibly secondary of laryngeal stenosis and birth. Her thoracic cage was small, and the abdomen was protu- pulmonary hypoplasia. berant. She had markedly short limbs, with talipes equinovarus The clinical features of the patient indicated AO. However, deformities and spatulated short fingers. Radiographs showed other AO-like skeletal dysplasias could not be excluded. As con- incomplete ossification and hypoplasia of the vertebrae, humeri, ventional gene-by-gene sequencing is too costly and time-con- femora, tarsals, phalanges, and pelvis. Radiographs also re- suming, exome sequencing, allowing simultaneous analysis of vealed multiple skeletal abnormalities. The clavicles were rela- multiple genes, was performed as a post-mortem genetic study tively elongated, the thorax was small and bell-shaped, and sev- with the written informed consent of the parents. eral vertebrae had scoliosis and coronal clefts. The humeri were http://dx.doi.org/10.3343/alm.2014.34.2.134 www.annlabmed.org 135 Jeon GW, et al. Exome sequencing for atelosteogenesis type I A D D B E C E E E chr3:58062997 FLBN NM_001457.3 exon 2 c.517G>A (p.Ala173Thr) Fig. 3. Integrative Genomics Viewer snapshot of the novel FLNB E mutation (NM_001457.3: c.517G>A; p.Ala173Thr) identified by E exome sequencing. Fig. 1. Radiograph showing incomplete ossification and hypoplasia of the vertebrae, humeri, femora, tarsals, phalanges, and pelvis. The variants, 23 genes identified through literature survey, including clavicles are relatively elongated (A), the thorax is small and bell- PubMed, UpToDate, and GeneReviews, were screened for de- shaped (B), and the vertebrae have scoliosis and coronal clefts (C). termining the relevance of the remained variants (Fig. 2). Conse- The humeri are severely shortened and distally hypoplastic (D), the fibulae are completely absent, and both ankle joints, knee joints, quently, we found one non-synonymous variant (NM_001457.3: and elbow joints are dislocated (E).