View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Online Research @ Cardiff CASE REPORT published: 13 September 2019 doi: 10.3389/fgene.2019.00835 Compound Heterozygosity for Novel Truncating Variants in the LMOD3 Gene as the Cause of Polyhydramnios in Two Successive Fetuses Ye Wang 1, Caixia Zhu 1, Liu Du 2, Qiaoer Li 3, Mei-Fang Lin 2, Claude Férec 4,5, David N. Cooper 6, Jian-Min Chen 4† and Yi Zhou 1*† 1 Fetal Medicine Center, Department of Obstetrics and Gynecology, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China, 2 Department of Ultrasonic Medicine, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China, 3 Jiangmen Central Hospital, Affiliated Jiangmen Hospital of Sun Yat-Sen University, Jiangmen, China, 4 EFS, Univ Brest, Inserm, UMR 1078, GGB, Brest, France, 5 CHU Brest, Service de Génétique, Brest, France, 6Institute of Medical Genetics, School of Medicine, Cardiff University, Cardiff, United Kingdom Polyhydramnios is sometimes associated with genetic defects. However, establishing an Edited by: Fan Jin, accurate diagnosis and pinpointing the precise genetic cause of polyhydramnios in any Zhejiang University, China given case represents a major challenge because it is known to occur in association with Reviewed by: over 200 different conditions. Whole exome sequencing (WES) is now a routine part of Liang-Liang Fan, Central South University, China the clinical workup, particularly with diseases characterized by atypical manifestations and Wenbin Zou, significant genetic heterogeneity. Here we describe the identification, by means of WES, Changhai Hospital, China of novel compound heterozygous truncating variants in the LMOD3 gene [i.e., c.1412delA *Correspondence: (p.Lys471Serfs*18) and c.1283dupC (p.Gly429Trpfs*35)] in a Chinese family with two Yi Zhou [email protected] successive fetuses affected with polyhydramnios, thereby potentiating the prenatal diagnosis of nemaline myopathy (NM) in the proband. LMOD3 encodes leiomodin-3, which is localized †These authors share senior authorship to the pointed ends of thin filaments and acts as a catalyst of actin nucleation in skeletal and cardiac muscle. This is the first study to describe the prenatal and postnatal manifestations Specialty section: of LMOD3-related NM in the Chinese population. Of all the currently reported NM-causing This article was submitted to Genetic Disorders, LMOD3 nonsense and frameshifting variants, c.1412delA generates the shortest truncation a section of the journal at the C-terminal end of the protein, underscoring the critical role of the WH2 domain in Frontiers in Genetics LMOD3 structure and function. Survey of the prenatal phenotypes of all known LMOD3- Received: 24 May 2019 Accepted: 13 August 2019 related severe NM cases served to identify fetal edema as a novel presenting feature that Published: 13 September 2019 may provide an early clue to facilitate prenatal diagnosis of the disease. Citation: Keywords: LMOD3, nemaline myopathy, polyhydramnios, prenatal diagnosis, truncating variant, whole exome sequencing Wang Y, Zhu C, Du L, Li Q, Lin M-F, Férec C, Cooper DN, Chen J-M and Zhou Y (2019) Compound Heterozygosity for Novel Truncating INTRODUCTION Variants in the LMOD3 Gene as the Cause of Polyhydramnios Polyhydramnios, an excess of amniotic fluid in the amniotic sac, is diagnosed if the single in Two Successive Fetuses. deepest pocket (SDP) is of ≥ 8 cm or the amniotic fluid index (AFI) is of ≥ 25 cm (Moore and Front. Genet. 10:835. Cayle, 1990; Magann et al., 2007). It is present in 1–3% of pregnancies (Maymon et al., 1998; doi: 10.3389/fgene.2019.00835 Biggio et al., 1999; Magann et al., 2007) and may occur as a consequence of both environmental Frontiers in Genetics | www.frontiersin.org 1 September 2019 | Volume 10 | Article 835 Wang et al. Novel Truncating Variants in LMOD3 (e.g., perinatal exposure to TORCH infections, maternal MATERIALS AND METHODS gestational and pregestational diabetes) and genetic factors (Bartha et al., 2003; Keshavarz et al., 2005; Touboul et al., 2007; Subjects Kishore et al., 2011; Kollmann et al., 2014). Indeed, a search for This study was carried out in accordance with the polyhydramnios in the Human Phenotype Ontology database recommendations of “ethical regulations of biomedical research (https://hpo.jax.org/app/browse/term/HP:0001561) yielded involving humans, Ethics Committee of the First Hospital 204 different conditions and 143 different genes. Nearly one affiliated to Sun Yat-sen University” with written informed half of all pregnancies with polyhydramnios are associated consent from all subjects. All subjects gave written informed with varying degrees of fetal abnormality, including fetal consent in accordance with the Declaration of Helsinki. The death (Kollmann et al., 2014). Therefore, it is extremely protocol was approved by the “Ethics Committee of the First important to identify the genetic causes of polyhydramnios in Hospital affiliated to Sun Yat-sen University”. Prenatal diagnosis affected families with a view to providing genetic counselling was undertaken through ultrasound-guided umbilical cord and prenatal diagnosis for subsequent pregnancies. blood puncture in accordance with standard practice. With the decreasing cost of next generation sequencing, whole exome sequencing (WES) has been increasingly employed as an Karyotype and Chromosomal Microarray important diagnostic tool for clinical purposes (Boycott et al., Analysis 2013; Lee et al., 2014; Posey et al., 2017), especially in the case of Standard G-banding karyotyping was performed according to diseases characterized by atypical manifestations and significant standard laboratory procedures. The chromosomal microarray levels of genetic heterogeneity (Ku et al., 2012). Here we describe experiments were conducted using the high-resolution the use of WES to identify the genetic cause of polyhydramnios Affymetrix CytoScan HD arrays (Affymetrix Inc., Santa Clara, in two successive fetuses in a Chinese family. CA) according to the manufacturer’s protocols. Detected copy number variants were evaluated for clinical significance by comparing them with data in the scientific literature and CASE PRESENTATION publicly available databases: UCSC (Ku et al., 2012), DECIPHER database (https://decipher.sanger.ac.uk/), Database of Genomic A 35-year-old woman was referred to our centre at the First Variants (DGV, http://dgv.tcag.ca/dgv/app/home), the Affiliated Hospital of Sun Yat-Sen University after her third International Standards for Cytogenomic Arrays (ISCA, https:// fetus (II:3; Figure 1A) had been found to have polyhydramnios www.iscaconsortium.org/), and Online Mendelian Inheritance (SDP of 9.5 cm and an AFI of 30.7 cm) and hydrocele of testis at in Man (OMIM, https://www.omim.org/). The results were then 29 gestational weeks (GW). Prior to this, her second fetus (II:2) analyzed by the Chromosome Analysis Suite software version had presented with polyhydramnios and generalized edema 3.3.0; the reporting threshold of copy number variants was set (pleural effusion, ascites, skin edema of the chest, abdomen, at 10 kb, with marker count at over 50, as previously reported and scalp) at 34 GW (Figures 2A–D) and was then terminated (Wang et al., 2015). at 36 GW. Her first child (II:1) is a healthy girl, now aged 6 years. Both parents were of south Chinese origin, healthy and non- consanguineous. Exposure to known mutagenic or teratogenic Whole Exome Sequencing (WES) agents during pregnancy was not reported. WES was performed in fetus II:3 and its parents. Genomic In the case of II:2, only standard G-banding karyotyping DNA was randomly fragmented and then purified by means (using cord blood cells taken at 35 GW) followed by of the magnetic particle method. Sequences were captured chromosomal microarray analysis were performed at the by Agilent SureSelect version 4 (Agilent Technologies, Santa time, yielding negative findings. In the case of fetus II:3, Clara, CA) according to the manufacturer’s protocols. After molecular genetic analysis including WES was performed at enrichment and purification, the DNA libraries were sequenced 31 GW. The WES results were returned to us 6 weeks later, on a NextSeq500 sequencer following the manufacturer’s confirming a diagnosis of nemaline myopathy (NM) (see instructions (Illumina, San Diego). The reads were aligned below). Genetic counselling was provided to the couple, who to hg19/GRCh37.p10 using the Burrows-Wheeler Aligner opted to continue the pregnancy. The boy was born by full- (version 0.59) (Li and Durbin, 2009). Base quality recalibration term vaginal delivery at 37 GW; his Apgar scores were 5 and and local realignment of the Burrows-Wheeler aligned reads 6 (normal, 10) at 1 and 5 min of life, respectively. He showed were then processed by means of GATK IndelRealigner stiffness of limbs, little movement and scrotal swelling, and (https://software.broadinstitute.org/gatk/documentation/ died of respiratory failure 2 days after birth. tooldocs/current/org_broadinstitute_gatk_tools_walkers_ Clinical findings in the two affected fetuses (II:2 and II:3) are indels_IndelRealigner.php) and GATK BaseRecalibrator illustrated in Figure 2 and summarized in Table 1. (https://software.broadinstitute.org/gatk/documentation/ tooldocs/current/org_broadinstitute_gatk_tools_walkers_ bqsr_BaseRecalibrator.php), respectively. Single nucleotide Abbreviations: AFI, amniotic fluid index; GW, gestational weeks; NM, nemaline variations