ETO Kaoru

IMAIZUMI Taichi

NAGATA Satoru

YAMAMOTO Toshiyuki

YAMAMOTO-SHIMOJIMA Keiko

YANAGISHITA Tomoe

Tokyo Women’s Medical University

A Novel PAFAH1B1 Splicing Variant Identifiedin a Patient with Classical Lissencephaly著者名 YANAGISHITA Tomoe, ETO Kaoru,YAMAMOTO-SHIMOJIMA Keiko, IMAIZUMI Taichi,NAGATA Satoru, YAMAMOTO Toshiyukijournal orpublication titleTokyo Women's Medical University Journalvolume 4page range 85-89year 2020-12-25URL http://hdl.handle.net/10470/00032751―85―Case ReportTWMUJ 4: 85-89, 2020A Novel PAFAH1B1 Splicing Variant Identified in a Patient withClassical LissencephalyTomoe Yanagishita,１,2 Kaoru Eto,１Keiko Yamamoto-Shimojima,１,2Taichi Imaizumi,２,3 Satoru Nagata,１ and Toshiyuki Yamamoto１,2１Department of Pediatrics, Tokyo Women’s Medical University, Tokyo, Japan２Institute of Medical Genetics, Tokyo Women’s Medical University, Tokyo, Japan３Department of Pediatrics, St. Marianna University School of Medicine, Kawasaki, Japan(Accepted June 30, 2020)(Advance Publication by J-STAGE August 27, 2020)Lissencephaly is a severe brain malformation associated with abnormal formation of cerebral sulci. Until now, many genesrelated to lissencephaly have been identified. Among them, classical lissencephaly has been often related to the platelet acti-vating factor acetylhydrolase 1B regulatory subunit 1 gene (PAFAH1B1). We identified a novel PAFAH1B1 splicing variant(NM_000430.3:c.118-2A>G) in a female patient with classical lissencephaly manifesting as developmental delay, growthfailure, and epilepsy. This variant was not detected in her parents, indicating de novo occurrence of the variant in the patient.To confirm whether this variant is in fact related to splicing error, RNA expression was analyzed. As suspected, an abnormalshort band excluding exon 4 was additionally detected. This evidence confirmed that the novel variant is definitely patho-genic to cause lissencephaly in the patient.Key Words: agyria, West syndrome, Miller-Dieker syndromeIntroductionLissencephaly is one of the malformations of the brain,which manifests as abnormal formation of cerebral sulci.Through radiological examination using magnetic reso-nance imaging (MRI), lissencephaly has been classifiedinto several types, including agyria, pachygyria, and sub-cortical band heterotopia.１These radiological classifica-tions are related to genetic classifications. Miller-Diekersyndrome has been established as one of the congenitalmalformation syndromes and it has been related to thesubtelomeric deletion of 17 p.２Patients with Miller-Dieker syndrome (MIM #247200) show classical lissen-cephaly and facial dysmorphism. Thus, genes related tothese clinical features had been suspected to be localizedin 17p. In 1993, the platelet activating factor acetylhydro-lase 1B regulatory subunit 1 gene (PAFAH1B1; formername LIS1) located on 17p was isolated as the gene re-sponsible for lissencephaly.３Later, the tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activationprotein epsilon gene (YWHAE), which is located more to-wards the telomeric region and at a distance from PA-Corresponding Author: Toshiyuki Yamamoto, Institute of Medical Genetics, Tokyo Women’s Medical University, 8-1 Kawada-cho,Shinjuku-ku, Tokyo 162-8666, Japan. yamamoto.toshiyuki@twmu.ac.jpdoi: 10.24488/twmuj.2020003CopyrightⒸ 2020 Society of Tokyo Women’s Medical University. This is an open access article distributed under the terms of CreativeCommons Attribution License (CC BY), which permits unrestricted use, distribution, and reproduction in any medium, provided theoriginal source is properly credited.―86―Figure　1　Brain magnetic resonance imaging of the patient. (A) An axial T1-weight image shows agyria and thick cortical surface. (B) A sagittal T1-weighted image shows reduced volume of the brain.A BFAH1B1, was identified as the gene related to facial dys-morphism.４Recently developed equipments for genomic analysishave promoted the understanding of the genomic basis oflissencephaly. Many genes, including the reelin (RELN),the tubulin alpha 1a (TUBA1A) , and the doublecortin(DCX) genes, have been identified as the genes responsi-ble for brain malformation. Intriguingly, PAFAH1 B 1-associated lissencephaly, included in Online MendelianInheritance in ManⓇ(https://omim.org/) as an autosomaldominant disease of Lissencephaly 1 (MIM #607432),accounts for majority of the cases (40%) in lissencephalycohorts.５，６Consequently, many disease-causingnucleotide variants of PAFAH1B1 have been reported.７，８Here, we have identified a novel disease-causingvariant of PAFAH 1 B 1 in a patient with classicallissencephaly. As the identified variant was located onthe splicing acceptor site, abnormal splicing pattern wasconfirmed by molecular analysis.Case ReportA 4-month-old girl was born at 39 weeks and 5 days ofgestation with birth weight of 2,370 g, height of 42.6 cm,and occipitofrontal circumference (OFC) of 31.7 cm.Apgar score was 7 / 9, indicating that there was noasphyxia at birth. Because dilatation of bilateralventricles had been suggested after fetal ultrasound at 36weeks of gestation, the girl was admitted to neonatalintensive care unit at birth. On 6th day after delivery,brain magnetic resonance imaging ( MRI ) revealedlissencephaly associated with no gyrus formation on thesurface of the cerebrum (Figure 1). After discharge fromthe hospital, she started to show myoclonus-likemovements. Although phenobarbital was prescribed forseveral weeks, she began to suffer from series ofepileptic spasms and admitted to our hospital when shewas months old.At the time of admission, her height was 55.5 cm(－4.0 SD), weight was 4.9 kg (－2.4 SD), and OFC was37.0 cm (－ 3.1 SD ) , indicating growth failure andmicrocephaly. There was no dysmorphic finding. Vitalsigns were within normal limit. On neurologicalexamination, generalized hypotonia was noted because ofwhich she had poor head control. Routine laboratoryexamination showed no abnormality ; however,electroencephalogram revealed atypical hypsarrhythmia(Figure 2), indicating West syndrome. Hence, the girlwas started on adrenocorticotropic hormone therapy.For precise diagnosis, genetic diagnosis wasperformed. Fluorescence in-situ hybridization ( FISH )showed no deletion in the 17 p 13.3 region, which isresponsible for Miller-Dieker syndrome. Thus,comprehensive genomic analysis was performed. Afterobtaining written informed consent from her parents,peripheral blood samples were collected from the patientand her parents. This study was performed in accordancewith the Declaration of Helsinki and was approved by the―87―Figure　2　Electroencephalogram. Atypical hypsarrhythmia with high voltage wave bursts is shown.secFp1-F7F7-T3T3-T5T5-O1Fp2-F8F8-T4T4-T6T6-O2Fp1-F3F3-C3C3-P3P3-O1Fp2-F4F4-C4C4-P4P4-O2Fz-CzCz-PzPG1-PG2X1-X2-Gene Analysis Research Ethics Committee of TokyoWomen’s Medical University ( No. 341 B ) . GenomicDNA was extracted from the blood samples using aQIAamp DNA extraction kit (Qiagen, Hilden, Germany).Next-generation sequencing ( NGS ) was performed toscreen single-nucleotide variants using TruSight One ver1.0 sequencing panel (Illumina, San Diego, CA, USA) asdescribed previously.９The extracted data were mapped toa reference genome (GRCh37/hg19) using the BWAEnrichment ver 1.0 cloud software ( Illumina ) , andannotated and filtered using the Variant Studio software(Illumina).After filtering the data, a novel heterozygous variantwas detected in the splicing acceptor site of exon 4 ofPAFAH1B1 (NM_000430.3:c.118-2A>G). There was nopossible candidate variant in other genes related tolissencephaly. Sanger sequencing confirmed theexistence of this variant in the patient but not in herparents (Figure 3A). This indicated de novo occurrenceof the variant. To further confirm whether this variant didin fact cause the splicing abnormality, expressed RNAwas examined. Total RNA extracted from Epstein-Barrvirus-transformed leukocytes was reverse-transcribed(RT) into complimentary DNA (cDNA). The cDNA wasamplified by RT-PCR as described previously.１０Theresult showed the presence of an abnormal short band inaddition to the normal band (Figure 3B). By use of DNAextraction kit (Qiagen), DNA amplicons were extractedthrough separated bands of both a normal- and anabnormal short band. Sanger sequencing performed usingthe extracted DNA samples confirmed exon 4 skipping(Figure 3C).DiscussionIn this study, we reported a patient with classicallissencephaly. The patient presented with developmentaldelay, hypotonia, and epileptic spasms. These clinicalfindings were typical of the clinical course oflissencephaly. According to the previously proposedclassification of classic lissencephaly, brain MRI findingsof this patient were considered as grade 1.１１From thisradiological pattern, PAFAH1 B 1 located on 17 p wassuspected as the most possible candidate gene.１２However, chromosomal deletion involving 17 p wasdismissed based on the results of FISH. PAFAH 1 B 1―88―Figure　3　Results of molecular analyses. (A) Genomic sequence for the patient shows a nucleotide alteration (a>g) in the splicing acceptor site of exon 4, whereas control shows the wild type sequence. (B) Electrophoresis of the amplicons obtained after reverse transcribed (RT)-PCR revealed an addi-tional short band (Band 2) in addition to the normal band (Band 1) in the patient. (C) Sanger sequence for Band 2 confirmed exon 4 skipping, whereas Band 1 (expected to be derived from the normal allele) showed normal pattern.Exon 4A A T G A A G A Ac t c t a gN E EgWild typePatientMarkerControlPatientBand 1Band 2Exon 5Exon 3Exon 3 Exon 4L D V V M EL D V N E EBand 2Band 1T T A G A T G T G A A T G A A G A AT T A G A T G T G G T T A T G G A AIntron 3ACBconsists of as many as 10 exons and there are more than10 possible candidate genes of lissencephaly other thanPAFAH 1 B 1. Thus, comprehensive genomic analysisusing TruSight One was considered as a more efficientway for examining the status of candidate genes thantargeted Sanger sequencing.As suspected, a de novo variant was identified inPAFAH1B1. Splice variants are rare in PAFAH1B1.１３ Weconfirmed exon skipping by RT-PCR and subsequentSanger sequencing. The exon 4 that was deleted in thepatient consists of multiples of three nucleotides. Thus,exon 4 skipping was predicted to cause in-frame deletionand not frame-shift deletion. Generally, frameshift leadsto premature termination and subsequent nonsense-mediated mRNA decay. Thus, frameshift variants areusually related to loss-of-function and most of thevariants identified in PAFAH1B1 are related to loss-of-function.１３In comparison, in-frame deletions aregenerally related to milder phenotypes in geneticdisorders ; however, in this case, exon 4 encodes animportant coiled-coil domain, which binds tomicrotubule-associated binding proteins.１４，１５Therefore,exon 4 skipping was considered to be related to loss-of-function mechanism.AcknowledgementsThe authors would like to express gratitude for the patient andher family for their cooperation. This work was supported byJSPS KAKENHI JP18K07803 (TY).Conflicts of Interest: There is no conflict of interest forany of the authors.References1．Di Donato N, Chiari S, Mirzaa GM et al: Lissencephaly:―89―Expanded imaging and clinical classification. Am J MedGenet A 173: 1473―1488, 20172．Shimojima K, Sugiura C, Takahashi H et al: Genomiccopy number variations at 17p13.3 and epileptogenesis.Epilepsy Res 89: 303―309, 20103．Reiner O, Carrozzo R, Shen Y et al: Isolation of aMiller-Dieker lissencephaly gene containing G proteinbeta-subunit-like repeats. Nature 364: 717―721, 19934．Schiff M, Delahaye A, Andrieux J et al: Furtherdelineation of the 17 p 13.3 microdeletion involvingYWHAE but distal to PAFAH 1 B 1 : four additionalpatients. Eur J Med Genet 53: 303―308, 20105．Di Donato N, Timms AE, Aldinger KA et al: Analysis of17 genes detects mutations in 81% of 811 patients withlissencephaly. Genet Med 20: 1354―1364, 20186．Parrini E, Conti V, Dobyns WB et al: Genetic Basis ofBrain Malformations. 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A Novel PAFAH1B1 Splicing Variant Identified in a Patient with Classical Lissencephaly

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A Novel PAFAH1B1 Splicing Variant Identified in a Patient with Classical Lissencephaly

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