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Comprehensive genetic analysis by whole exome sequencing in 352

Korean pediatric patients with unknown neurodevelopmental disorders

Youngha Lee, MS 1,8; Jin Sook Lee, MD, PhD2,3,4,8; Soo Yeon Kim, MD2,8; Jaeso

Cho, MD1; Yongjin Yoo, PhD 1; Sangmoon Lee, MD, PhD1; Taekyeong Yoo, BS1;

Moses Lee, MS1; Jieun Seo, BS1; Jeongeun Lee, MS1,5; Jana Kneissl, BS1;

Eunha Kim, BS1; Hyuna Kim, MD2; Woo Joong Kim, MD2; Jon Soo Kim, MD6;

Jung Min Ko, MD, PhD2; Anna Cho, MD7; Byeong Chan Lim, MD, PhD2; Won

Seop Kim, MD, PhD6; Murim Choi, PhD1,2*; Jong-Hee Chae, MD, PhD2*

1Department of Biomedical Sciences, Seoul National University College of

Medicine, Seoul, Republic of Korea. 2Department of Pediatrics, Seoul National

University College of Medicine, Seoul, Republic of Korea. 3Department of

Pediatrics, Gil Medical Center, Gachon University College of Medicine, Incheon,

Republic of Korea. 4Department of Genome Medicine and Science, Gil Medical

Center, Gachon University College of Medicine, Incheon, Republic of Korea.

5Interdisciplinary Program for Bioengineering, Graduate School, Seoul National

Universty, Seoul, Republic of Korea. 6Department of Pediatrics, College of

Medicine, Chungbuk National University, Cheongju, Republic of Korea.

7Department of Pediatrics, Ewha Womans University School of Medicine,

Seoul, Republic of Korea. 8Equal contributions

*Corresponding authors

Online-Only Figures eFigure 1. Patients categorized to clinical diagnosis (n = 352 patients). eFigure 2. Functional implication of the novel during brain development. eFigure 3. The Age distribution of all patients when their symptom began and the distance between age of onset to WES analysis (n = 352 patients). eFigure 4. Identification of an inherited deletion at 2p22.3 in the family with hereditary spastic paraplegia (HSP). eFigure 5. Pathogenic variants divided by inheritance patterns (n = 186 patients). eFigure 6. Pathogenic variants categorized by function (n = 244 variants).

Online-Only Tables eTable 1. List of copy number variations discovered in this study. eTable 2. List of known neurologic disorder associated genes. eTable 3. Examples of research collaboration through data sharing program such as GeneMatcher.

Online-Only Videos

Video 1. A boy with SLC2A1 mutation suffering from abnormal gait and dystonia.

Video 2. The same boy from Video 1 after identification of genetic cause and subsequent ketogenic diet.

eFigure 1. Patients categorized to clinical diagnosis (n = 352 patients).

LS, Leigh syndrome; LD, Leukodystrophy; HSP, Hereditary spastic paraplegia

eFigure 2. Functional implication of the novel genes during brain development.

(A) The eight periods of human brain devolopment1,2. Numbers of available samples from the BrainSpan transcriptome database are shown. PCW, post-conceptual weeks. (B) Statistical evaluations of the novel network strength compared to the simulated networks using the known gene co-expression network in whole brain of each developmental period as a background. The observed number of edges that were formed between novel and known networks were shown in red against 105 randomly permutated values. FDR-adjusted P-values are shown in blue. (C)

Whole brain divided into four anatomical regions based on brain transcriptome similarity3. (D) Same statistical analysis with (A) in 32 spatio-temporal windows (four brain regions and eight developmental periods)2. FDR-adjusted P-values of each spatio-temporal window are displayed with color intensity. The numbers of RNA-seq samples in each window are shown in each cell. NA denotes sample size < 5.

eFigure 3. The Age distribution of all patients when their symptom began and the distance between age of onset to WES analysis (n = 352 patients).

(A) The age of onset of all patients (year). (B) The age of onset of patients whose symptoms began before 24 months

(The first bar in the (A)). (C) The Age distribution of all patients when the WES analysis was performed. (D) The distance between patient’s age of onset and the time of WES analysis.

eFigure 4. Identification of an inherited deletion at 2p22.3 in the family with hereditary spastic paraplegia (HSP).

(A) Pedigree of an HSP family with 8 affected individuals across four generations. (B) WES-based log2-based copy- number values of subject HSP-9 compared with an unrelated normal subject on 2 showing the presence of heterozygous microdeletion. Captured intervals of copy-number loss are indicated by red dots. (C) Enlarged view of deletion-bearing region at 2p22.3 encompassing SPAST. Blue solid bars represent the deleted intervals. Alu repeat elements at the deletion breakpoints are indicated by red solid bars and aligned with the UCSC Genome Browser

RepeatMasker, represented by dotted lines. (D) Validation of the deleted regions by quantitative PCR of genomic DNA.

The red bars represent an average copy-number of five patients and the gray bars represent an average copy-number of five normal individuals from the family. Error bars represent standard error. CN: copy-neutral region. (E) DNA sequence analysis of the deleted regions. The DNA fragments containing the deletion was amplified by the deletion-specific primer pairs. The deletion-specific PCR products of 3.8 kb (Del 1 & 2) and 4.0 kb (Del 3) are observed in each affected individual.

Reference sequences surrounding the breaking points are indicated in blue and red color.

eFigure 5. Pathogenic variants divided by inheritance patterns (n = 186 patients).

eFigure 6. Pathogenic variants categorized by function (n = 244 variants).

eTable 1. List of copy number variations discovered in this study.

CNV CNV inheritanc No. of Notable gene(s) No. Simple Diagnosis interval Del/Dup Ref length e pattern genes* In the interval (Mb, hg19) chr1:107.0- 1 Rett syndrome-like 6.1 Mb Del de novo 76 WNT2B, NTNG1 4 113.1 chr1:0.5- 2 Rett syndrome-like 2.5 Mb Del de novo 66 KCNAB2, CHD5 5 3.1 Hereditary chr2:32.3- 165.5 3 Del Inherited 3 SPAST 6 spastic paraplegia 32.5 kb HDAC4, PRLH, PER2, Unknown chr2:234.8- TWIST2, CAPN10, 4 8.0 Mb Del de novo 68 7 encephalopathy 242.8 KIF1A, FARP2, D2HGDH, PDCD1 chr3:9.0- SETD5, SLC6A1, 5 Rett syndrome-like 4.0 Mb Del de novo 50 8,9 13.0 SLC6A11 PIGG, CPLX2, FGFRL1, 6 Rett syndrome-like chr4:0-2.8 2.8 Mb Del de novo 45 CTBP1, SLBP, LETM1, 10 WHSC1/2 PRKG2, RASGEF1B, chr4:71.6- 7 Multiple anomaly 16.9 Mb Del de novo 103 HNRNPD, HNRNPDL, 11 88.5 ENOPH1, SCD5 8 Congenital hypotonia chr5:139.0- 567.7 Del de novo 8 NRG2, PURA 12 139.6 kb Epileptic chr7:119.7- 9 170 kb Dup de novo 0 KCND2 upstream - encephalopathy 119.9 Epileptic 10 chr9:0-47.3 47.3 Mb Dup de novo 218 n.d. 13 encephalopathy Developmental delay, chr15:20.3- TUBGCP5, NIPA1, 11 3.4 Mb Del de novo 12 14 Joubert syndrome 23.7 NIPA2, CYFIP1 Unknown chr22:42.8- 12 8.6 Mb Del de novo 95 SHANK3, IGF1 15 severe retardation 51.3 chr22:43.2- 13 Multiple anomaly 8.1 Mb Del de novo 87 SHANK3, IGF1 15 51.3 Developmental delay, chrX::41.4- 299.5 14 Del de novo 1 CASK 16 microcephaly 41.7 kb Unknown chrX:100.1 hemizygou 15 7.2 Mb Dup 74 PLP1 17 encephalopathy -107.3 s

Neurodegeneration chrX:102.5 692.2 16 with severe motor Dup de novo 15 PLP1 17 -103.2 kb developmental arrest

* Based on UCSC genes (knownGene) track. Only -coding genes were counted. eTable 2. List of known neurologic disorder associated genes

Inheritance pattern Gene No.

ANKRD11, ANO3, ASXL3(2), ATL1(3), BICD2, C19orf12, CHD7, CLTC, COL1A1(2), CTNNB1(3), DNM1(2),

DYNC1H1, FOXG1, GABBR2, GABRB1, GNAO1(2), GRIA2, GRIN1, GRIN2A, GRIN2B(2), KCNC1,

Autosomal-dominant KIAA0196, KIF1A(2), KMT2A(2), MAGEL2, MAP2K1, MRAS, NALCN, NIPBL, PMP22, PPP2R5D, RAB11B, 47

RHOBTB2, RYR1, SATB2, SCN1A, SCN2A(2), SLC2A1, SLC6A1, SLC6A5, SOX10, SPAST, STXBP1,

TCF4(2), TUBB4A(2), UBE3A, ZEB2

ABAT, ALDH7A1, APTX, ASAH1, ATAD3A, ATP6V0A2, BRAT1, CLN6(2), CNTNAP1, ECHS1(2), EIF2B2,

EIF2B3, ERCC5, FIG4, GDAP1, GLB1(2), GMPPB(2), HADHA, HADHB, HSD17B4, HSPA9, IARS2(2),

Autosomal-recessive IGHMBP2, KIAA1109, KLHL40, LONP1, NARS2(2), NDUFAF6(2), NDUFS1, NDUFV1, PLA2G6, PNPT1(2), 45

POC1A, POLR1C, PPT1, PRUNE, SACS, SLC19A3(2), SLC25A15, ST3GAL5, SURF1, TMEM173, TUBA8,

WDR62, WDR81

ATRX(3), CASK, CDKL5(3), EDA, GRIA3, HDAC8(2), HPRT1, IQSEC2(2), MECP2(3), NAA10, NSDHL, X-linked 17 OPHN1(2), PAK3, PLP1(3), SMC1A, USP9X, ZC4H2

eTable 3. Examples of international research collaboration through data sharing program such as GeneMatcher.

Gene No. of patients Ref

GABBR2 4 18

RAB11B 5 19

SLC6A1 34 20

CHD3 35 21

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