Whole Genome Analysis As a Tool for Diagnosis in Silver-Russell

BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) J Med Genet

Whole genome analysis as a tool for diagnosis in Silver-Russell

syndrome.

Ahmed SN Alhendi1, Derek Lim2, Shane McKee3, Meriel McEntagart4, Katriona

Tatton-Brown4, Genomics England Research Consortium5, I Karen Temple1,6,

Justin H Davies1,6, Deborah JG Mackay1,7,8

Supplementary Information

Alhendi ASN, et al. J Med Genet 2021;0:1–10. doi: 10.1136/jmedgenet-2021-107699 BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) J Med Genet

Supplementary Table1. Lists of genes and regions used for SNV analysis. Supplementary Table 2A: number of NH-CSS features given for participants in the 100KGP SRS cohort. Supplementary Table 2B: features of the Netchine-Harbison Clinical Scoring System (NH-CSS) Supplementary Table 3: SNVs plausibly contributing to participant phenotype. Supplementary Table 4: Structural variants identified in participants. Supplementary Text 1: the Genomics England Research Consortium

Supplementary Figure 1: SNVs in CDKN1C and IGF2 in participants with SRS. A: pedigree information for families of participants 18, 53 and 64. Grey filled symbols indicate participant affected by SRS; black dot indicates individual carrying the variant specified. B: ideogram of chr11; the red bar to the left of the ideogram marks chr11p15.5, where CDKN1C and IGF2 are located. C: IGV visualisation of the relevant sequences in participant, mother and father, showing the variant in the affected participant and unaffected parent.

Supplementary Figure 2: Compound heterozygosity for SNVs in ORC1 in Participant 66. A: pedigree information for participant 66. B: ideogram of chr1; the red bar marks location of ORC1. C: IGV visualisation of the relevant sequences in participant, mother and father, showing both variants in the affected participant and one in each unaffected parent.

Supplementary Figure 3: SNVs in IGF1R in Participants 22 and 31. A: pedigree information for participant 22; B: pedigree information for participant 31. C: ideogram of chr15; the red bar marks location of IGF1R. D: IGV visualisation of the relevant sequences in participant 22, mother and father, showing both variants in the affected participant and one in each unaffected parent. E: IGV visualisation showing the SNV in participant 31 and the affected father.

Supplementary Figure 4: Deletion in chr17q24 in Participant 61. A: pedigree information for family 61. B: Ideogram of chromosome 17; red bar marks chr17q24. C: visualisation of read depth around the 185kb deletion in the participant and the mother affected by intellectual disability. D: IGV visualisation showing the breakpoints of the deletion in participant and mother.

Supplementary Figure 5: region of homozygosity affecting 7q21 in participant 25. A: Ideogram of chromosome 7; red oblong shows region of 7q21 with homozygosity. B: visualisations of allele fraction and depth ratio in proband and mother, spanning 45Mb in chr7q. The allele fraction visualisation of the participant (top panel) shows homozygosity of SNPs in a 16.6Mb region that encompasses the imprinted PEG10/SGCE locus region, while read depth ratio (second panel) is normal, showing no loss or gain of genetic material. C: IGV visualisation encompassing the PEG10 TSS-DMR, showing homozygous SNPs in the participant co-located with heterozygous SNPs in the mother, indicating isodisomy of maternal origin with no paternal contribution.

Supplementary Figure 6: paternally-inherited variant within IC1 in participant 34. A: pedigree information for participant 34. B: Ideogram of chromosome 11; red bar marks chr11p15. C: IGV visualisation of the relevant sequences in participant, mother and father, showing paternally-inherited variant within IC1: chr11:2000298G>A; note that the maternally-inherited variant is rs148619931, MAF 0.006. D: Sanger traces (reverse-complement strand) in participant 34 and unrelated normal control (DNA from participant 34’s parents was unavailable).

Supplementary Figure 7: Illustration for sample stratification and association tests. (A) Flow chart for burden and SKAT-O tests. (B) Flow chart for Ethnicity PCA analysis. (C) visitation of first two principal components of stratified samples of 48 SRS cases (coloured blue), 8204 control genomes (coloured in red). (D) Scree plot of the first ten PCs from the PCA ancestry analysis. Frist five PCs used as covariates for SKAT- O association test.

Supplementary Text 1: the Genomics England Research Consortium

Supplementary Table1. Lists of genes and regions used for SNV analysis.

Gene Chr Missense Z-score pLI Mode of inheritance Disorder or clinical association LHX4 1 0.703632 0.401094 BIALLELIC, AD/AR, not imprinted Combined GH deficiency NRAS 1 2.004148 0.912773 BIALLELIC, AD, not imprinted Noonan syndrome PADI6 1 NA NA BIALLELIC, MEG, not imprinted Multi-locus imprinting disorder TBX19 1 0.57612 2.80E-05 BIALLELIC, AR, not imprinted Isolated adrenocortical deficiency; Congenital adrenal hypoplasia; GH deficiency TSHB 1 0.42056 0.005758 BIALLELIC, AR, not imprinted Isolated TSH deficiency; Congenital hypothyroidism; Intellectual disability PAPPA2 1 0.125958 0.00125 BIALLELIC, AR, not imprinted IUGR and IGF abnormalities ORC1 1 -0.11451 3.39E-05 BIALLELIC, AR, not imprinted Meier-Gorlin Syndrome GPR161 1 1.078227 0.029579 BIALLELIC, AR, not imprinted Pituitary Stalk Interruption Syndrome TCF7L1 2 1.297827 0.753752 BIALLELIC, AD, not imprinted Combined GH deficiency SIX3 2 3.951477 0.348879 BIALLELIC, AD, not imprinted Holoprosencephaly GLI2 2 2.168623 0.99894 BIALLELIC, AD, not imprinted Isolated adrenocortical deficiency; holoprosencephaly SOS1 2 2.179027 1 BIALLELIC, AD, not imprinted Noonan syndrome OBSL1 2 1.274362 6.64E-16 BIALLELIC, AR, not imprinted 3-M syndrome IFT172 2 0.21688 2.29E-18 BIALLELIC, AR, not imprinted GH deficiency POMC 2 0.752798 0.000689 BIALLELIC, AR, not imprinted Isolated adrenocortical deficiency; Congenital adrenal hypoplasia ORC4 2 -0.5099 2.29E-06 BIALLELIC, AR, not imprinted Meier-Gorlin Syndrome IHH 2 2.790678 0.349924 BIALLELIC, AR, not imprinted Skeletal dysplasia HDAC4 2 3.505028 0.99999 BIALLELIC, NK, not imprinted Skeletal dysplasia;Intellectual disability ZDBF2 2 -2.00831 1.38E-07 imprinted, pat allele expressed Nasopalpebral Lipoma-Coloboma Syndrome POU1F1 3 -0.83494 0.618818 BIALLELIC, AD/AR, not imprinted GH deficiency HESX1 3 -0.60547 0.008163 BIALLELIC, AD/AR, not imprinted Isolated GH deficiency; combined GH deficiency SOX2 3 4.37597 0.396697 BIALLELIC, AD, not imprinted Hypogonadotropic hypogonadism PROK2 3 -0.2408 0.270454 BIALLELIC, AD, not imprinted Kallmann Syndrome; Hypogonadotropic hypogonadism RAF1 3 2.819856 0.999777 BIALLELIC, AD, not imprinted Noonan syndrome RASA2 3 1.320378 5.32E-17 BIALLELIC, AD, not imprinted Noonan syndrome ROBO1 3 -0.2718 3.04E-08 BIALLELIC, AD, not imprinted Pituitary Stalk Interruption Syndrome FANCD2 3 -0.24982 2.48E-14 BIALLELIC, AR, not imprinted Fanconi Anemia; Congenital Anomalies

MRAS 3 2.730697 0.859947 BIALLELIC, AD, not imprinted Noonan syndrome PITX2 4 3.073633 0.672789 BIALLELIC, AD, not imprinted GH deficiency; Glaucoma (developmental) FGFR3 4 1.390999 0.002909 BIALLELIC, AD, not imprinted Hypogonadotropic hypogonadism GNRHR 4 -0.84015 0.013968 BIALLELIC, AR, not imprinted GH deficiency; Hypogonadotropic hypogonadism PCSK1 5 0.808795 0.08964 BIALLELIC, AD/AR, not imprinted Isolated adrenocortical deficiency; TSH deficiency PIK3R1 5 2.416686 0.99824 BIALLELIC, AD, not imprinted SHORT syndrome PROP1 5 -0.4043 0.299127 BIALLELIC, AR, not imprinted GH deficiency GHR 5 -1.29022 0.000232 BIALLELIC, AR, not imprinted Isolated GH deficiency RHOBTB3 5 1.583368 0.000534 imprinted, pat allele expressed placental imprinting KHDC3L 6 1.425771 0.241205 BIALLELIC, MEG, not imprinted Multi-locus imprinting disorder CUL7 6 0.481975 2.24E-11 BIALLELIC, AR, not imprinted 3-M syndrome ZFP57 6 1.512658 0.001816 BIALLELIC, AR, not imprinted Transient neonatal diabetes PLAGL1 6 1.082015 0.881694 imprinted, pat allele expressed Transient neonatal diabetes CRYBG1 6 NA NA imprinted, pat allele expressed placental imprinting GCK 7 4.390419 0.204306 BIALLELIC, AD/AR, not imprinted Diabetes - neonatal onset; Congenital hyperinsulinism GHRHR 7 -0.55935 0.002679 BIALLELIC, AD/AR, not imprinted Isolated GH deficiency DLX5 7 1.512352 0.67598 BIALLELIC, AD/AR, not imprinted Skeletal dysplasia GLI3 7 1.522202 0.999995 BIALLELIC, AD, not imprinted GH deficiency, hypopituitarism SHH 7 4.419256 0.912961 BIALLELIC, AD, not imprinted Holoprosencephaly BRAF 7 3.991339 0.999978 BIALLELIC, AD, not imprinted Noonan syndrome CPA1 7 1.227588 1.10E-05 BIALLELIC, NK, not imprinted SRS - potential candidate CPA4 7 -0.35596 3.55E-07 BIALLELIC, NK, not imprinted SRS - potential candidate DDC 7 0.838084 4.61E-05 BIALLELIC, NK, not imprinted Not imprinted, chr7p12 FOXP2 7 1.566146 0.996179 not imprinted, NK Not Imprinted chr7q; Intellectual disability IGFBP1 7 0.827032 0.001783 BIALLELIC, NK, not imprinted SRS - potential candidate IGFBP3 7 0.680124 0.362252 BIALLELIC, NK, not imprinted SRS - potential candidate CPA5 7 0.079416 0.047489 BIALLELIC, NK, not imprinted Not imprinted, chr7q32 COPG2 7 0.625655 0.056037 not imprinted, NK Not imprinted, chr7q32 COPG2IT1 7 NA NA not imprinted, NK Not imprinted, chr7q32 GRB10 7 1.854174 0.367424 imprinted, mat allele expressed SRS candidate gene

PHKG1 7 -0.10563 3.58E-10 imprinted, mat allele expressed Neuromuscular disorders; Undiagnosed metabolic disorders KLF14 7 2.321079 0.218114 imprinted, mat allele expressed placental imprinting SGCE 7 0.200519 0.001386 imprinted, pat allele expressed myoclonic dystonia PEG10 7 2.501583 0.894179 imprinted, pat allele expressed myoclonic dystonia KLHDC10 7 3.802972 0.996684 imprinted, pat allele expressed placental imprinting MESTIT 7 NA NA imprinted, pat allele expressed SRS candidate gene MEST 7 2.784847 0.997527 imprinted, pat allele expressed SRS candidate gene FGFR1 8 2.798366 0.988744 BIALLELIC, AD, not imprinted Hypogonadotropic hypogonadism; GH deficiency; Hydrocephalus TRHR 8 0.360101 0.265022 BIALLELIC, AR, not imprinted Isolated TSH deficiency; Congenital hypothyroidism NBN 8 -0.99176 1.74E-10 BIALLELIC, AR, not imprinted Nijmegen syndrome PLAG1 8 2.033229 0.829917 BIALLELIC, AD, not imprinted SRS ZFAT 8 0.964071 0.002929 imprinted, pat allele expressed placental imprinting NPR2 9 4.219216 0.991661 BIALLELIC, AD/AR, not imprinted Skeletal dysplasia PTCH1 9 2.859042 0.999999 BIALLELIC, AD, not imprinted Holoprosencephaly LHX3 9 1.580221 0.158418 BIALLELIC, AR, not imprinted Combined GH deficiency FANCC 9 -1.15141 5.88E-10 BIALLELIC, AR, not imprinted Fanconi Anemia; Congenital Anomalies FANCG 9 0.38046 9.75E-09 BIALLELIC, AR, not imprinted Fanconi Anemia; Congenital Anomalies ROR2 9 0.500189 0.464719 BIALLELIC, AR, not imprinted Robinow syndrome; Skeletal dysplasia;Limb disorders; Clefting FGF8 10 2.444696 0.927195 BIALLELIC, AD, not imprinted Hypogonadotropic hypogonadism SHOC2 10 2.56787 0.991705 BIALLELIC, AD, not imprinted Noonan syndrome CDON 11 -0.89652 3.05E-12 BIALLELIC, AD, not imprinted GH deficiency; Holoprosencephaly; Intellectual disability CBL 11 1.731347 0.006787 BIALLELIC, AD, not imprinted Noonan syndrome RRAS2 11 2.046811 0.674254 BIALLELIC, NK, not imprinted Noonan syndrome HRAS 11 2.687401 0.007941 BIALLELIC, AD, not imprinted Noonan syndrome RIT1 11 2.203705 0.66681 BIALLELIC, AD, not imprinted Noonan syndrome PHLDA2 11 2.242643 0.02426 imprinted, mat allele expressed growth restriction CDKN1C 11 3.623878 0.522822 imprinted, mat allele expressed IMAGe syndrome H19 11 NA NA imprinted, mat allele expressed SRS/Beckwith-Wiedemann syndrome KCNQ1 11 2.725929 2.45E-05 imprinted, mat allele expressed SRS/Beckwith-Wiedemann syndrome KCNQ1OT1 11 NA NA imprinted, mat allele expressed SRS/Beckwith-Wiedemann syndrome

IGF2 11 2.308109 0.025303 imprinted, pat allele expressed SRS/Beckwith-Wiedemann syndrome TRPV4 12 3.119985 7.32E-06 BIALLELIC, AD, not imprinted Neuromuscular disorders; Skeletal dysplasia A2ML1 12 -0.79124 1.33E-22 BIALLELIC, AD, not imprinted Noonan syndrome HMGA2 12 1.544619 0.733852 BIALLELIC, AD, not imprinted SRS KRAS 12 1.356303 0.001061 BIALLELIC, AD, not imprinted Noonan syndrome PTPN11 12 3.433309 0.999877 BIALLELIC, AD, not imprinted Noonan syndrome IGF1 12 1.937295 0.469523 BIALLELIC, AR, not imprinted SRS; Growth failure in early childhood ZIC2 13 NA NA BIALLELIC, AD, not imprinted Holoprosencephaly OTX2 14 1.020326 0.7446 BIALLELIC, AD, not imprinted Isolated GH deficiency; combined GH deficiency SOS2 14 1.974677 0.999428 BIALLELIC, AD, not imprinted Noonan syndrome YY1 14 4.902073 0.967831 BIALLELIC, AD, not imprinted Skeletal dysplasia; Early onset dystonia; Intellectual disability MEG3 14 NA NA imprinted, mat allele expressed Temple Syndrome RTL1 14 3.372282 0.008166 imprinted, pat allele expressed Temple syndrome DLK1 14 1.313621 0.400971 imprinted, pat allele expressed Temple syndrome IGF1R 15 3.636006 0.552974 BIALLELIC, AD/AR, not imprinted SRS; Growth failure in early childhood MAP2K1 15 3.425931 0.994501 BIALLELIC, AD, not imprinted Noonan syndrome BLM 15 -0.99758 4.14E-11 BIALLELIC, AR, not imprinted Bloom syndrome ARNT2 15 2.230844 0.95902 BIALLELIC, AR, not imprinted Combined GH deficiency CEP152 15 -1.59462 4.42E-21 BIALLELIC, AR, not imprinted Seckel syndrome ACAN 15 1.02597 0.999994 BIALLELIC, NK, not imprinted Sponyloepimetaphseal dysplasia - Extracellular matrix regulation SRCAP 16 2.229441 1 BIALLELIC, AD/AR, not imprinted Floating Harbour syndrome ANKRD11 16 2.761927 1 BIALLELIC, AD, not imprinted KBG Syndrome CREBBP 16 5.583523 1 BIALLELIC, AD, not imprinted Rubinstein-Taybi syndrome; Skeletal dysplasia FANCA 16 -5.80625 2.08E-34 BIALLELIC, AR, not imprinted Fanconi Anemia; Congenital Anomalies IGFALS 16 -1.47941 2.80E-05 BIALLELIC, AR, not imprinted IUGR and IGF abnormalities CDT1 16 -1.70346 7.76E-09 BIALLELIC, AR, not imprinted Meier-Gorlin Syndrome ORC6 16 -0.53077 0.000734 BIALLELIC, AR, not imprinted Meier-Gorlin Syndrome CTCF 16 4.864774 0.997925 BIALLELIC, NK, not imprinted Fetal anomalies; Intellectual disability; Clefting ZNF597 16 -1.18498 0.134236 imprinted, mat allele expressed Prenatal growth retardation and dysmorphic features GH1 17 -0.35189 0.017432 BIALLELIC, AD/AR, not imprinted Isolated growth hormone deficiency

KANSL1 17 0.49284 0.999734 BIALLELIC, AD, not imprinted Koolen-de Vries syndrome;17q21.31 microdeletion syndrome NF1 17 6.218942 1 BIALLELIC, AD, not imprinted Neurofibromatosis-Noonan Syndrome COL1A1 17 3.969277 1 BIALLELIC, AD, not imprinted Skeletal dysplasia; Growth failure in early childhood RNPC3 17 -0.23012 0.525298 BIALLELIC, AR, not imprinted Isolated growth hormone deficiency STAT5B 17 5.145115 0.999953 BIALLELIC, AR, not imprinted IUGR and IGF abnormalities CDC6 17 0.187357 1.81E-06 BIALLELIC, AR, not imprinted Meier-Gorlin Syndrome Mulibrey Nanism; Growth failure in early childhood; Undiagnosed metabolic TRIM37 17 1.424747 0.172389 BIALLELIC, AR, not imprinted disorders TGIF1 18 1.264691 0.090759 BIALLELIC, AD, not imprinted Holoprosencephaly RAX 18 2.673702 0.841215 BIALLELIC, AR, not imprinted Combined Growth hormone deficiency MC2R 18 0.163208 0.057447 BIALLELIC, AR, not imprinted Isolated adrenocortical deficiency; Congenital adrenal hypoplasia RRAS 19 1.305584 0.287257 BIALLELIC, NK, not imprinted Noonan syndrome MAP2K2 19 1.4812 0.863261 BIALLELIC, AD, not imprinted Noonan syndrome DNMT1 19 6.422047 1 BIALLELIC, AD, not imprinted SRS - potential candidate NLRP2 19 -1.9835 1.88E-20 BIALLELIC, MEG, not imprinted Multi-locus imprinting disorder NLRP5 19 -3.13153 1.24E-07 BIALLELIC, MEG, not imprinted Multi-locus imprinting disorder NLRP7 19 0.880506 1.10E-12 BIALLELIC, MEG, not imprinted Multi-locus imprinting disorder CCDC8 19 0.61642 0.167397 BIALLELIC, AR, not imprinted 3-M syndrome Oliver–McFarlane and Laurence–Moon syndrome; GH and gonadotrophin PNPLA6 19 5.992573 1.59E-08 BIALLELIC, AR, not imprinted deficiencies ZNF331 19 2.281628 0.680598 imprinted, pat allele expressed growth restriction FOXA2 20 NA NA BIALLELIC, AD, not imprinted Combined GH deficiency; congenital hyperinsulinism; childhood-onset diabetes GHRH 20 0.531293 0.455313 BIALLELIC, AR, not imprinted GH deficiency PROKR2 20 0.233776 0.000669 BIALLELIC, AR, not imprinted Kallmann Syndrome; GH deficiency KCNE1 21 -0.46485 0.004186 BIALLELIC, AR, not imprinted Long QT syndrome PCNT 21 -2.00613 4.18E-38 BIALLELIC, AR, not imprinted MOPDII LZTR1 22 0.835664 3.38E-52 BIALLELIC, AD, not imprinted Noonan syndrome SOX3 X NA NA X-linked, XLR Combined GH deficiency BTK X 4.381168 0.999563 X-linked, XLR GH deficiency EIF2S3 X 3.813165 0.916324 X-linked, XLR GH deficiency; TSH deficiency; Diabetes - neonatal onset; Clefting KAL1 X 0.818184 0.938574 X-linked, XLR Hypogonadotropic hypogonadism

TBL1X X 2.536938 0.9985 X-linked, XLR Isolated TSH deficiency; Congenital hypothyroidism IGSF1 X 0.219482 0.99686 X-linked, XLR Isolated TSH deficiency; GH deficiency; Congenital hypothyroidism KDM6A X 2.197903 0.999987 X-linked, XLR Kabuki syndrome; Congenital hyperinsulinism SHOX X 1.914339 0.738458 X-linked, XLR Skeletal dysplasia; Limb disorders

AD: autosomal dominant; AR: autosomal recessive; Chr: chromosome; GH: Growth Hormone; IGF: insulin-like growth factor; IUGR: intrauterine growth restriction; MEG: maternal-effect gene; NK: not known; TSH: thyroid-stimulating hormone; XLR: X-linked recessive; Missense Z-score: intolerance to missense variants; pLI: probability of loss of function intolerance.

Supplementary Table 2

Table 2A: number of NH-CSS features given for participants in the 100KGP SRS cohort. NH-CSS Number of Number of participants features participants with IC1 LOM 0 3 0 1 15 0 2 18 0 3 9 1 4 17 1 5 10 3 6 0 0 ≤2 36 (50%) ≥3/6 36 (50%) 1 ≥4 27 (37.5%) 4 ≥4* 12 (16.7%) 2 *meeting full definition of clinical SRS: ≥4/6 NH-CSS criteria, including prominent forehead and relative macrocephaly

Table 2B: features of the Netchine-Harbison Clinical Scoring System (NH-CSS)

Clinical criterion Definition SGA (birth weight and/or birth length) ≤−2 SDS for gestational age Postnatal growth failure Height at 24 ± 1 months ≤−2 SDS or height ≤−2 SDS below mid-parental target height Relative macrocephaly Head circumference at birth ≥1.5 SDS above birth weight at birth and/or length SDS Protruding forehead Forehead projecting beyond the facial plane on a side view as a toddler (1–3 years) Body asymmetry LLD of ≥0.5 cm or arm asymmetry or LLD <0.5 cm with at least two other asymmetrical body parts (one non-face) Feeding difficulties BMI ≤−2 SDS at 24 months or current use of a feeding and/or low BMI tube or cyproheptadine for appetite stimulation

Taken from Wakeling et al, 2017. Individuals scoring three or more warrant molecular testing for SRS. A clinical diagnosis of SRS is considered if a patient scores at least four of six from these criteria. If all molecular tests are normal and differential diagnoses have been ruled out, patients scoring at least four of six criteria, including both prominent forehead and relative macrocephaly, should be diagnosed as clinical SRS. LLD, leg length discrepancy; SDS, SD score; SGA, small for gestational age.

Supplementary Table 3: SNVs plausibly contributing to participant phenotype. The table lists SNVs that have GNOMAD frequency <0.01 and internal 100KGP frequency ≤0.01, CADD≥15, that alter the coding sequence of genes listed in Supplementary Table 1, and are scored by neither SIFT nor Polyphen-2 as benign. Those that plausibly account for the presentation of the participant are mentioned in the main text and marked with an asterisk against the Participant number. Others are potentially contributing to the phenotype of the participant.

Partici Disorder Gene Variant (HGVS) GT Transmis GnomAD CADD_ SIFT(score) Polyphen- pant sion Phred 2(score) 4* SHORT syndrome PIK3R1 NM_181523.2:c.1456G>T:p.A486S het unknown - 27.8 D (0) PrD(0.993) 5 MENTAL RETARDATION, X-LINKED, HUWE1 NM_031407.6:c.718T>C.p.L573P het X-linked - 27.2 D (0) - SYNDROMIC, TURNER TYPE 6 STX4 NM_001272095:c.169G>A:p.V57M het mat 0.00003490 28.9 D (0) PrD 8* MLID NLRP2 NM_001174082:c.T1823C:p.L608P het mat 0.0003 23.1 D (0.001) PrD(0.999) 9 GH-IGF CHD7 NM_017780:c.4894C>T:p.R1632C het de novo 0.00004434 32 D (0.015) PrD(0.91) or pat 11* Noonan syndrome LZTR1 NM_006767.4:c.1354A>G:p.K452E§ het pat 0.00004465 22.4 T (0.7) B (0.138) 11 Microcephaly, progressive, seizures, and QARS NM_001272073.1:c.2131G>A:p.V711M het de novo 0.00001193 23.2 D (0.005) PrD(0.953) cerebral and cerebellar atrophy 12 GH-IGF GHRHR NM_000823.3:c.1268G>A:p.C423Y het pat 0.0001473 27 D (0) PrD(0.994) 12 GH-IGF FGF22 NM_020637:331G>A:pV111M het pat 0.00006196 22.8 D (0.083) PrD(0.806) 13 Sponyloepimetaphseal dysplasia ACAN NM_013227.3:c.4525C>T:p.L1509F het mat 0.002187 13.62 T (0.59) PrD(0.998) 14 GH-IGF GH1 NM_000515.3:c.332C>T:p.S71L het pat 0.00001193 24 D (0.025) PrD(0.644) 18* IMAGe syndrome CDKN1C NM_001122631.1:c.809G>C:p.R281T het mat - 27 D (0) PrD(0.950) 21 GH-IGF IGFALS NM_001146006.1:c.1644G>A:p.Y548X het mat 0.00000543 54 - - 8 22* GH-IGF IGF1R NM_001291858.1:c.476T>C:p.L159P het pat - 28.7 D (0.022) PrD(0.957) 22* GH-IGF IGF1R NM_001291858.1:c.3770C>T:p.P1257L het mat 0.0000279 28.8 D (0.018) PrD(0.985) 31* GH-IGF IGF1R NM_001291858.1:c.1597A>G:p.K533E het pat 0.00002791 25 D (0.046) B(0.034) 34* MLID NLRP7 NM_001127255.1:c.930G>T:p.Q310H het mat 0.001719 15.8 T (0.21) PD(0.666) 39 ACTH-independent macronodular GNAS NM_001077489.2:c.819C>G:p.I273M het de novo - 21.9 D (0) PrD(0.963) adrenal hyperplasia 39 Kleefstra syndrome 2 KMT2C NM_170606:c.2534G>A:p.G845E het de novo - 28.9 D (0.001) PrD(0.999) 40* SCN8A NM_001330260.2:c.3502C>T:p.R1168W het de novo 0.0000131 26.0 D (0) B(0.061) 40* SON NM_138927.4:c.1342C>T:p.448X het de novo - 36 - - 41* MLID NLRP2 NM_001174081.3:c.1469G>A:p.R490H het mat 7.97E-06 20.9 D (0.05) PD(0.693) 42 STX4 NM_001272095:c.477G>A:p.M159I het mat 0.00004886 30 D PrD 43* MLID NLRP7 NM_001127255.1:c701T>C:p.L234S het mat 0.0008668 18.59 D (0.01) PD(0.609) 44 GH-IGF GH1 NM_000515.3:c.610C>T:p.R204C het pat 0.00000697 24.4 D (0.01) B(0.214) 8 44 GH-IGF GRB7 NM_001242443.1:c.1055G>A:p.R352H het mat 0.002656 23.5 D (0.024) PrD(0.858)

45 GH-IGF, Diabetes Mellitus, Noninsulin- IGF2BP2 NM_001291875:c.94C>T:p.R32X het pat 0.00000398 36 - - Dependent 45 Sponyloepimetaphseal dysplasia ACAN NM_013227.3:c.185C>A:p.T62N het mat 0.0004494 22.3 T (0.185) PrD(0.939) 49 GH-IGF FGFR1 NM_001174065.1:c.374C>T:p.S125L het mat 1.22E-05 24.6 D (0.04) B(0.057) 51 GH-IGF IGFALS NM_001146006.1:c.1237C>T:p.R413W het mat 0.0002329 25.6 D (0.007) PrD(0.761) 51 Bartter syndrome, type 1 antenatal SLC12A1 NM_000338.2:c.3209A>G:p.Y1070C het mat - 32 D (0) PrD(0.999) 52 MLID NLRP2 NM_001174081.1:c.1885T>C:p.S629P het de novo 0.000998 22.2 D (0.02) PD(0.857) or pat 53* SRS IGF2 NM_001291861.3:c.443C>T:p.A146V het pat 0.0000082 15.39 D (0.07) B(0.011) 53 X-LINKED, SYNDROMIC FGD1 NM_004463.2:c.1132G>T:p.A378S het X-linked - 25.3 D (0.007) PD(0.934) 56* Kabuki syndrome KDM6A NM_001291418.1:c.3462G>A:p.W1154X hom_alt X-linked - 45 - - 57 STX4 NM_001272095:c.477G>A:p.M159I het pat 0.00004886 30 D PrD 58 Geleophysic dysplasia 1 ADAMTSL2 NM_001145320:976T>G:p.F326V hom_alt mat - 25 D (0.003) B(0.253) 62* MLID PADI6 NM_207421.3:c.1709G>A:p.R570H het mat 1.46E-05 24 D (0.01) PD(0.418) 63 Short-rib thoracic dysplasia 7 with or WDR35 NM_020779:c.3279G>C:p.Q1093H het mat 0.002049 20.4 D (0.021) PD(0.735) without polydactyly 63 Short-rib thoracic dysplasia 7 with or WDR35 NM_001006657:c145G>T:p.D49Y het pat 0.00001999 23.7 D (0.014) PD(0.988) without polydactyly 63* MLID NLRP2 NM_001174081.1:c.1889T>C:p.L630P het mat 0.0001871 22.3 D (0.001) PD(0.968) 64* SRS IGF2 NM_001291861.3:c.245C>T:p.T82M het pat 0.00000405 23.1 T (0.566) PD(0.472) 64 Hypochondroplasia FGF3 NM_001163213.1:c.779C>G:p.P260R het mat - 24 D (0.005) PrD(1) 64 Lysyl hydroxylase 3 (LH3) deficiency PLOD3 NM_001048:c.668A>G:p.N223S het pat 0.00000795 26.2 D (0.012) PrD(0.998) 2 66 GH-IGF GH1 NM_000515.3:c.114C>G:p.N38K het pat 0.00001193 16.97 D (0.028) B(0.412) 66* Meier-Gorlin Syndrome ORC1 NM_001190818.1:c.806C>T:p.S269L het mat 0.00636 22.7 D (0.002) PD(0.525) 66* Meier-Gorlin Syndrome ORC1 NM_001190818.1:c.1945G>A:p.V649I het pat - 19.06 T (0.32) PrD(0.874) GH-IGF, growth hormone or insulin-like growth factor pathway defects; D, deleterious; T, tolerated; PrD, probably damaging; PD, possibly damaging; B, benign § The LZTR1 variant listed for participant 11 is included in this table because, despite being annotated as benign by both SIFT and Polyphen2, it affects the first base of a coding exon and therefore may affect splicing.

Supplementary Table 4: Structural variants identified in participants. The table lists CNVs larger than 10kb, overlapping exonic regions of protein-coding genes or imprinted regions within our gene panel, and not found in apparently healthy individuals. Those that plausibly contribute to the presentation of the participant are marked with an asterisk against the Participant number.

Participant SV transmission genes of note pathogenicity

1 chr4:147,233-194,027 mat likely benign 16 chr14:104,134,695-104,198,744 NK KIF26A likely benign

17 dup chr19:42,813,147-42,982,813 pat likely benign

17 dup chr12:5,468,896-5,519,482 pat likely benign 19 dup chr19:6,889,320-7,107,348 pat MBD3L2, MBD3L3, MBD3L4, likely benign MBD3L5, MBD3L2B 23 dup chr14:42,966,021-44,939,245 mat likely benign 24 dup, chr14:65,820,804-66,643,528 mat GPHN, CCDC196 likely benign

26 del chr7:6,052,554-6,104,548 mat likely benign 28 dup chr2:36,103,442-36,247,691 mat DOP1B, DOPEY2 likely benign 30 dup chr12: 95,150,524-95,267,376 pat FGD6 likely benign 32 dup chr2:219,516,425-219,641,518 mat OBSL1 likely benign 34 chr19:51,768,084-52,114,366 pat Several ZNF genes likely benign 43 dup chr1:223,365,094-224,143,498 mat COL27A1 likely benign 43 Inv dup chr9:114,286,051-114,344,724 pat COL27A1 likely benign

45 del chr13:55,212,134-57,480,701 pat likely benign 46 dup chr12:33,370,643-34,654,887 mat SYT10 likely benign 49 del chr8:27,988,737-28,089,991 mat NUGGC likely benign 50* del chr12:102,347,498-102,758,174 mat IGF1 plausibly pathogenic 52 dup chr12:309,905-629,370 mat KDM5A, CCDC77 likely benign 53 dup chr1:13,329,000-13,431,000 pat PRAMEF17/19/20 likely benign

55 dup chr14:20,241,359-21,539,680 mat likely benign

55 del chr19:42,786,164-43,043,068 pat likely benign

55 inv chr16:1,516,497-2,069,902 pat likely benign

57 dup chr1:88,650,000-88,863,000 mat likely benign 61* del chr17:67,334,824-67,519,712 mat PSMD12 potentially contributing to phenotype 65 dup chr3:186,524,382-186,653,400 pat likely benign

68 dup chr19:36,687,564-36,878,358 mat likely benign

68 dup chr21:46,019,932-46,125,142 pat likely benign 69 inv chr19:53,839,087-54,950,787 pat NLRP7 likely benign 70* Del chr6:130,000,000-150,000,000 pat PLAGL1 plausibly pathogenic

Supplementary Text 1: the Genomics England Research Consortium Ambrose, J. C. 1; Arumugam, P.1; Bleda, M. 1; Boardman-Pretty, F. 1,2; Boustred, C. R. 1; Brittain, H.1; Caulfield, M. J.1,2; Chan, G. C. 1; Fowler, T. 1; Giess A. 1; Hamblin, A.1; Henderson, S.1,2’ Hubbard, T. J. P. 1; Jackson, R. 1; Jones, L. J. 1,2; Kasperaviciute, D. 1,2; Kayikci, M.1; Kousathanas, A.1; Lahnstein, L.1; Leigh, S. E. A.1; Leong, I. U. S.1; Lopez, F. J.1; Maleady-Crowe, F. 1; Moutsianas, L. 1,2; Mueller, M. 1,2; Murugaesu, N. 1; Need, A. C. 1,2; O‘Donovan P. 1; Odhams, C. A. 1; Patch, C. 1,2; Perez-Gil, D. 1; Pereira, M. B.1; Pullinger, J. 1; Rahim, T.1; Rendon, A.1; Rogers, T.1; Savage, K.1; Sawant, K. 1; Scott, R. H.1; Siddiq, A.1; Sieghart, A. 1; Smith, S. C. 1; Sosinsky, A. 1,2; Stuckey, A. 1; Tanguy M. 1; Thomas, E. R. A.1,2; Thompson, S.R.1; Tucci, A.1,2; Walsh, E.1; Welland, M. J.1; Williams, E.1 ; Witkowska, K.1,2; Wood, S.M.1,2.

1. Genomics England, London, UK 2. William Harvey Research Institute, Queen Mary University of London, London, EC1M 6BQ, UK.

Alhendi ASN, et al. J Med Genet 2021;0:1–10. doi: 10.1136/jmedgenet-2021-107699