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SUPPLEMENTAL DATA

Clinical relevance of systematic phenotyping and exome sequencing in patients with short stature

Short Running Title: Genetic evaluation of patients with short stature

Nadine N Hauer1, M.Sc., Bernt Popp1, M.D., Eva Schoeller1, M.Sc., Sarah Schuhmann1, M.D., Karen E Heath2, Ph.D., Alfonso Hisado-Oliva2, B.Sc., Patricia Klinger3, Ph.D., Cornelia Kraus1, Ph.D., Udo Trautmann1, Ph.D., Martin Zenker4, M.D., Christiane Zweier1, M.D., Ph.D., Antje Wiesener1, M.D., Rami Abou Jamra5, M.D., Erdmute Kunstmann6, M.D., Dagmar Wieczorek7,8, M.D., Steffen Uebe1, Ph.D., Fulvia Ferrazzi1, Ph.D., Christian Büttner1, M.Sc., Arif B Ekici1, Ph.D., Anita Rauch10, M.D., Heinrich Sticht11, Ph.D., Helmuth-Günther Dörr12, M.D., André Reis1, M.D., Christian T Thiel1, M.D.

1Institute of Human Genetics, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany2Institute of Medical and Molecular Genetics (INGEMM) and Skeletal dysplasia Multidisciplinary Unit (UMDE), Hospital Universitario La Paz, Universidad Autónoma de Madrid, IdiPAZ, and CIBERER, Madrid, Spain

3Department of Orthopaedic Rheumatology, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany4Institute of Human Genetics, Otto-von-Guericke University Magdeburg, Magdeburg, Germany5Institute of Human Genetics, University of Leipzig, Leipzig, Germany6Institute of Human Genetics, University of Wuerzburg, Wuerzburg, Germany7Institute of Human Genetics, University of Essen, Essen, Germany8Institute of Human-Genetics, University Duesseldorf, Duesseldorf, Germany9Institute of Human Genetics, Helmholtz Zentrum München, Neuherberg, Germany10Institute of Medical Genetics, University of Zurich, Zurich, Switzerland11Institute of Biochemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen, Germany12Department of Pediatrics and Adolescent Medicine, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Erlangen,

Germany

1

Table of Content

Supplementary Methods............................................................................................................................................4

Recruitment and systematic phenotyping of 565 patients.....................................................................................4

Selection and clinical description of the 200 individuals for exome analysis.........................................................5

Exome Sequencing................................................................................................................................................5

Library preparation & Sequencing......................................................................................................................5

Mapping & Variant calling...................................................................................................................................5

Variant priorization strategy & bioinformatics pipeline........................................................................................6

Variant validation................................................................................................................................................6

Interpretation of variants in known short stature associated genes.......................................................................6

Application of ACMG criteria..............................................................................................................................6

Predictive model for subgrouping based on phenotypic information..................................................................8

Phenotype re-evaluation of the identified variants.............................................................................................8

Supplementary Figures..............................................................................................................................................9

Figure S1. Height distribution of included individuals............................................................................................9

Figure S2. Coverage distribution of exome samples. 20x coverage was achieved in 95.5 % of the target (red) and the median coverage was 125.3x (blue).........................................................................................................9

Figure S3. Schematic representation of ACAN with its domain structure based on Uniprot...............................10

Figure S4. Schematic representation of ANKRD11 with its domain structure based on Uniprot.........................10

Figure S5. Schematic representation of CASK with its domain structure based on Uniprot................................10

Figure S6. Schematic representation of CLCN5 with its domain structure based on Uniprot..............................11

Figure S7. Schematic representation of COL2A1 with its domain structure based on Uniprot............................11

Figure S8. Schematic representation of CUL7 with its domain structure based on Uniprot................................11

Figure S9. Schematic representation of FGD1 with its domain structure based on Uniprot................................12

Figure S10. Schematic representation of FGFR3 with its domain structure base on Uniprot..............................12

Figure S11. Schematic representation of FLNB with its domain structure based on Uniprot..............................12

Figure S12. Schematic representation of GHSR with its domain structure based on Uniprot.............................13

Figure S13. Schematic representation of HDAC6 with its domain structure based on Uniprot...........................13

Figure S14. Schematic representation of IFT140 with its domain structure based on Uniprot............................13

Figure S15. Schematic representation of IGF1R with its domain structure based on Uniprot.............................14

Figure S16. Schematic representation of IHH with its domain structure based on Uniprot.................................14

Figure S17. Schematic representation of KAT6B with its domain structure based on Uniprot............................14

Figure S18. Schematic representation of KDM6A with its domain structure based on Uniprot...........................15

Figure S19. Schematic representation of KRAS with its domain structure based on Uniprot..............................15

Figure S20. Schematic representation of MAP2K1 with its domain structure based on Uniprot.........................15

Figure S21. Schematic representation of MATN3 with its domain structure based on Uniprot...........................16

Figure S22. Schematic representation of NPR2 with its domain structure based on Uniprot..............................16

Figure S23. Schematic representation of PDE3A with its domain structure based on Uniprot............................16

Figure S24. Schematic representation of PDE4D with its domain structure based on Uniprot...........................17

Figure S25. Schematic representation of SLC26A2 with its domain structure based on Uniprot........................17

Figure S26. Schematic representation of TRIM37 with its domain structure based on Uniprot...........................17

Supplementary Tables.............................................................................................................................................18

Table S1. Details of targeted diagnoses of 565 patients with short stature.........................................................18

2

Table S3. Overview of mutations in known short stature associated genes and patients' phenotypes in 200 exome individuals.................................................................................................................................................25

Table S4. Details of mutations identified in known short stature associated genes of 200 exome individuals....30

Table S5. Phenotypes of 200 exome individuals with potential causal mutations in known short stature genes 32

Table S6. Symptoms of identified mutations in known short stature associated genes......................................35

Supplementary References.....................................................................................................................................36

3

Supplementary Methods

Recruitment and systematic phenotyping of 565 patients

The study was approved by the ethics committee of the Friedrich-Alexander-Universität Erlangen-Nürnberg

(FAU). The parents designated legal guardian or the affected individual provided written informed consent. Here,

the risks and benefits of a research-based whole exome analysis were discussed and an option for disclosure of

medically actionable incidental findings provided.

We aimed to enroll patients and at least the parents (trios) in our study. All patients were examined by local

medical specialists prior to enrollment. All patients have been evaluated by an expert in pediatric endocrinology

(Prof. Dörr and his team) as well as further specialists (ophthalmologist, ENT, gastroenterologist) where

applicable. This panel of experts made the decision whether growth hormone testing and further specific clinical

evaluation was necessary. The standard serology included IGF1, IGFBP3, DHEAS, TSH, fT4, and fT3

parameters and growth hormone stimulation tests when prior tests indicated a growth hormone deficiency. 474 of

the 565 patients (84 %) had endocrine testing for growth hormone deficiency. The remaining patients did not

receive serology testing as dysmorphic features or a disproportionate growth were indicative of a genetic cause

and a endocrine cause highly unlikely. In 29 patients (5.13 %) an Arginin growth hormone stimulation test was

performed as initial serology parameters suggested a growth hormone deficiency, which could further be

excluded. Therefore, these patients were enrolled in the study. 398 patients (70.44 %) were tested for thyroid

disease and only in 3 patients (0.53 %) fT4 and TSH levels suggested a subclinical hypothyroidism. As these

patients received treatment and did not show any further clinical signs of hypothyroidism we considered this not

be the obvious cause of their short stature and they were likewise included in the study. Antibody screening for

celiac disease was followed by gastroscopy when elevated antibody parameters were detected. This applied to

one patient (0.18 %), where celiac disease was ruled out by further endoscopy and biopsy histology. No patient

had any obvious chronic diseases as potential underlying cause of short stature.

Patients with a disproportionate form of short stature received a radiographic evaluation of the spine, pelvis and

knee for metaphyseal-, epiphyseal- or spondylo-dysplasia. Overall, radiographic evaluation was performed in 52

patients (9.2%) where further clinical and dysmorphological evaluation suggested a skeletal dysplasia. X-rays

were evaluated by the same clinician and discussed in a panel of experts. As these results were not conclusive

and were not clearly attributable to one of the known forms of skeletal dysplasias by the panel of experts, these

patients were scored as idiopathic short stature and included in the study.

Bone age was measured as a means to propose the patients’ final adult height. Based on the age of the patients

and available results, we were able to obtain the bone age for 30 % of our patients.

The psychomotor development of our patients was based on the patients’ history. Patients, who were attending

regular school or were proposed to attend regular school without any known history of developmental issues have

been classified as “IQ normal”. Patients with learning disabilities requiring additional attention but without global

developmental issues have been classified as “IQ 70-85”. Patients with moderate to severe developmental issues

have not been included in the study.

Only patients with short stature of unknown origin were than referred to our genetic clinic for further genetic

evaluation. Enrollment in the study was dependent on informed consent to this study. About 95 % of the patients

presenting with short stature, who are suitable for the study, have been enrolled. Systematic phenotyping1 was

performed according to a standardized questionnaire by the same clinician. The standardized questionnaire

included an exhaustive examination and documentation of the growth pattern of the patient’s history, a family

history of at least 3 generations for evaluation of a potential mode of inheritance, all medical issues and

assessment of the developmental status. For all patients the ethnic differences in height were attributed and the 4

age correlated standard deviation score (SDS) for comparison calculated. In addition, we included WHO scores

for international comparison.

The received phenotype information was included in a database and compared with clinical information from the

London Dysmorphology database and published data. The integrated information was than discussed with a

panel of experts in clinical genetics and dysmorphology. The following diagnostic workup includes the common

causes of short stature. Further targeted analysis was based on recognizable phenotypic patterns. A karyotype

was performed for 96 % of the patients, followed by SHOX-Deletion and mutation screening (58.5 %) and FGFR

analysis (49 %). In 29.5 % of the patients imprinting alterations (UPD7, UPD14, Silver-Russel syndrome) were

tested. Molecular karyotyping (array analysis) was performed according to our approach2 taking into account copy

number variants above 100kb (Supplementary Table 1). The most common analysis for monogenic disorders was

performed for Noonan syndrome in 25 % of the patients where phenotypic features were suggestive of a

rasopathy.

Selection and clinical description of the 200 individuals for exome analysis

From the group of 565 patients 200 patients were selected where no diagnosis was made by previous targeted

analysis (491 patients). To exclude a selection bias a group of 200 patients were randomly selected and tested in

regards of age, height distribution, prenatal growth retardation (small for gestational age), stature type, head

circumference, development, sex, and bone age. A Chi-square p-value was calculated for age (P-value 0.70),

stature type (P-value 0.87), development (P-value 0.99), sex (P-value 0.91), bone age (P-value 0.68 excluding

not available values), and prenatal growth retardation (P-value 0.68). A Kolmogorov-Smirnov test was performed

to achieve a p-value for height (P-value 0.18, Supplementary Figure S1) and head circumference (P-value 0.18).

Based on these results the 200 selected patients were representative compared to the group of 565 patients.

A detailed overview of all parameters is listed in the file “Clinical Characteristics 200 Exome Individuals.pdf”.

For the 100 patients who were analysed as trios and all affected only analyzed patients harboring de novo

variants in known short stature genes, we confirmed maternity and paternity.

Exome Sequencing

Library preparation & Sequencing

We collected blood samples for DNA extraction from 200 patients, their parents and, where available, further

family members. All DNAs passed our quality control and were then enriched using the SureSelect Human All

Exon Kits (Agilent, Santa Clara, CA) and sequenced on a SOLiD 5500 XL (12 patients) (Thermo Fisher Scientific,

Waltham, MA, USA) and Illumina HiSeq 2500 instrument (188 patients) (Illumina, San Diego, CA). Image analysis

and base calling was performed using the SOLiD and HiSeq instrument control software with default parameters.

Mapping & Variant calling

After demultiplexing with bcl2fastq v1.8.4 from Illumina, read alignment was performed with BWA3 version 0.7.8

using the bwa mem algorithm with the human genome assembly hg19 (GRCh37) as reference. Duplicate reads

were marked with Picard (http://broadinstitute.github.io/picard) (version 1.111). Furthermore, local re-alignment

around potential InDel sites was performed with the Genome Analysis Toolkit4 version 3.1. Single-nucleotide

variants and small insertions and deletions (indels) were detected using five different callers: HaplotypeCaller and

UnifiedGenotyper of the aforementioned Genome Analysis Toolkit, SNVer5, freeBayes6 and Platypus7. Variant

annotation was performed using ANNOVAR8 in conjunction with a variety of open and proprietary annotation

5

database files such as mutalyzer.9 We achieved an average coverage of 160x. On average, 95.5 % of the target

sequence was covered at least 20x (Supplementary Figure S2).

Variant priorization strategy & bioinformatics pipeline

Variants were assessed for all 200 individuals and family members based on different modes of inheritance

(autosomal dominant de-novo, autosomal recessive, and X-linked recessive) facilitated by using our in-house

Next Generation Sequencing Variant Analyzer tool.

Only Variants called with GATKHap, GATKUG4,10 or SNVer5 were analyzed. We included variants which were

covered by at least 10 % of the average coverage of the patient’s exome and for which at least 5 novel alleles

were detected. We included only variants with a frequency of 10 -3 or below from the 1000 Genomes Project11, the

Exome Variant Server (http://evs.gs.washington.edu/EVS/), the Exome Aggregation Consortium (ExAC)12 server

or 0.15 % in our in-house variant database (1 out of 639 samples without growth retardation).

Different cut-off values for the ratio of novel allele to reference allele reads were used. For analyses of

homozygous variants, we included variants with at least 80 % novel allele reads. Heterozygous variants were

included only when called in at least 20 % and at most 70 % of all reads. For analyses of hemizygous variants, we

included only variants on the X-chromosome in males for which the novel allele was called in at least 20 % of all

reads. As we consider a stronger impact of protein altering variants, we restricted our analyses to exonic variants

and variants in intronic canonical splice sites ranging from -12 to +5. Furthermore, we excluded synonymous

variants from our analyses. Considering missense variants, we only included variants with a CADD-value of at

least 10 (other variants are considered benign). The remaining variants were inspected with the Integrative

Genomics Viewer to exclude sequencing errors in repetitive regions.

Variants of all 200 individuals were analyzed for 1000 known growth related genes compiled from MedGen (NCBI;

search term: short stature) and Human Phenotype ontology13 (search term: growth delay) regarding the

inheritance mode and the phenotype of each individual (Supplementary Table S2). The gene list was designed to

include all growth relevant genes in order not to miss any disease causing variant in an already known short

stature gene. Whether the patient’s phenotype fits the so far reported clinical spectrum was then carefully re-

evaluated by a clinical geneticist. Segregation of the variant in the family, including further available affected and

unaffected family members, was confirmed by Sanger sequencing and compared with the parental phenotype

and the affected status of further available family members.

Variant validation

All remaining variants were validated by Sanger sequencing. Splice site variants have been evaluated using in

silico prediction (Alamut software (ColdSpringHarborLaboratories)) and rtPCR where RNA available and

expression in lymphocytes. NPR2 variants were confirmed to be pathogenic by their inability to synthesize cGMP

and/or their retention in the endoplasmic reticulum, determined by a cGMP ELISA and immunohistochemistry

respectively. Methods were as described in Hisado-Oliva et al, 2015.14

Interpretation of variants in known short stature associated genes

Application of ACMG criteria

For the interpretation of the pathogenicity of variants in known short stature associated genes, we applied the

recommendation for the interpretation of sequence variants of the American College of Medical Genetics and

Genomics and the Association for Molecular Pathology from Richards et al. (Table on next page).15 Thereby, we

defined a null variant as a nonsense variant, a frameshift variant, a canonical ±1 or 2 splice site variant or a splice

site variant with an effect on splicing as confirmed by rtPCR. As a database for previously reported disease 6

causing mutations, we used ClinVar and the Human Gene Mutation Database (HGMD)16 updated 2015 (PS1,

PS3, PM5, PP2, BP1). PS2 was fulfilled for all our de novo mutations as we confirmed maternity and paternity

based on exome data from trios (see also section “Selection and clinical description of the 200 individuals for

exome analysis”) or in case of de novo mutations in patients with affected only exomes based on genotypes by

microsatellite analyses. PS4 was interpreted as fulfilled, if the Odds Ratio was higher than 5 when comparing the

frequency in our 200 patients to the frequency in a control cohort of 1187 controls (i.e. BS2 was not fulfilled). PM2

was interpreted according to the frequency of the variant in ExAC. PM2 was fulfilled for a frequency below 10-3 for

recessive and below 10-4 for dominant inherited variants. These cut-off values were chosen based on the

observation of similar frequencies for pathogenic mutations in known short stature genes. PP1 was fulfilled, if at

least one further family member was affected of the disease and carried the mutation. BA1, BP2 and BP7 were

never fulfilled according to our variant selection criteria. PP3 was fulfilled, if the Combined Annotation Dependent

Depletion score (CADD)17 was higher than 15 and at least two out of four further in silico prediction programmes

(SIFT18,19, PolyPhen-220, MutationTaster21 and MutationAssessor22,23) predicted deleterious (i.e. BP4 was not

fulfilled). As variants were excluded, if an equally affected family member did not carry the same mutation or if an

unaffected family member also carries the same mutation, BS4 was never fulfilled. This information is also

summarized in the following table. The final categorization based on these criteria was then performed using the

Genetic Variant Interpretation Tool24.

7

Table ACMG criteria for interpretation of variants in known short stature associated genes

Criterion remarkPVS1 Nonsense, frameshift variant, canonical ±1 or 2 splice site, splice site with effect on splicing (confirmed by

rtPCR)PS1 According to ClinVar and HGMD (2015)PS2 Applies to all of our de novo mutationsPS3 According to ClinVar and HGMD (2015)PS4 for very rare variants, threshold of statistical significance not reachable => PM7PM1 naPM2 According to ExAC (recessive <10-4; dominant <10-5)PM3 Applies to all of our compound heterozygous mutationsPM4 naPM5 According to ClinVar and HGMD (2015)PM6 Applies to all our de novo mutations in patients with affected only exomesPM7 Adapted category from PS4; Odds Ratio > 5PP1 ≥2 affected individuals in the family who carry the mutationPP2 According to ClinVar and HGMD (2015)PP3 CADD≥15 and ≥2 of 4 further in silico prediction toolsPP4 Applies to all of our identified mutationsPP5 naBA1 naBS1 naBS2 Odds Ratio < 5BS3 naBS4 naBP1 According to ClinVar and HGMD (2015)BP2 naBP3 naBP4 CADD<15 and <2 of 4 further in silico prediction toolsBP5 naBP6 naBP7 nana: not attributable

Predictive model for subgrouping based on phenotypic information

A support vector machine approach was used to examine the predictive value of the combination of height, OFC,

intellectual disability, syndromic vs. isolated phenotype, prenatal growth retardation and accelerated/decelerated

bone age. From a collective of 210 patients, 100 were randomly selected as a training sample and used to train

an SVM with SVMlight.25 Utilizing first a linear, than a polynomial kernel, a model was generated for predicting the

mutation status in the remaining 110 patients.

Phenotype re-evaluation of the identified variants

All patients where we identified mutations in known short stature associated genes were thoroughly re-evaluated

for associated clinical features reported in the respective instances by a board of clinical experts. This included

the evaluation of the parental phenotype based on the segregation of the identified mutation. We considered the

segregation to be confirmed when either both parents showed a height above -2 SDS and no obvious phenotypic

features associated with the phenotype in the patient in case of a de novo, homozygous, compound heterozygous

or X-linked recessive inherited variant, or the parent carrying the heterozygous mutation showed convincing

clinical features in case of an autosomal dominant inheritance. As we considered incomplete penetrance in an

affected parent, a parent carrying the mutation was considered affected even when the height was above -2 SDS,

but additional clinical signs and further family history was indicative of the inheritance pattern. As available, further

affected and unaffected family members were analyzed in regards to the identified mutation.

8

Supplementary Figures

Figure S1. Height distribution of included individuals. The height of each individual is presented as standard deviation (SD) for age and sex. When more measurements are available the lowest SD is used. No significant difference in height SDS is observed for samples with a known mutation compared to all exome samples.

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150

010

2030

4050

6070

8090

100

Fold Coverage

Per

cent

of T

arge

ts

95.5% of targetcovered at least 20x

median coverage 125.3x

Figure S2. Coverage distribution of exome samples. 20x coverage was achieved in 95.5 % of the target (red) and the median coverage was 125.3x (blue).

9

Figure S3. Schematic representation of ACAN with its domain structure based on Uniprot. Known missense (blue), known nonsense (green) and in this study identified mutations (red) are marked. Autosomal recessive mutations in ACAN are associated with the Spondylometaphyseal dysplasia, aggrecan type [MIM612813], autosomal dominant mutations in ACAN are associated with Osteochondritis dissecans, short stature, and early-onset osteoarthritis [MIM165800] and with Spondyloepiphyseal dysplasia, Kimberley type [MIM608361].

Figure S4. Schematic representation of ANKRD11 with its domain structure based on Uniprot. Known missense (blue), known nonsense (green) and in this study identified mutations (red) are marked. Autosomal dominant mutations in ANKRD11 are associated with KBG syndrome [MIM148050].

Figure S5. Schematic representation of CASK with its domain structure based on Uniprot. Known missense (blue), known nonsense (green) and in this study identified mutations (red) are marked. X-linked mutations in CASK are associated with FG syndrome 4 [MIM300422] and with Mental retardation and microcephaly with pontine and cerebellar hypoplasia [MIM30749].

10

Figure S6. Schematic representation of CLCN5 with its domain structure based on Uniprot. Known missense (blue), known nonsense (green) and in this study identified mutations (red) are marked. X-linked recessive mutations in CLCN5 are associated with Dent disease [MIM300009], with Hypophosphatemic rickets [MIM300554], with Nephrolithiasis, type I [MIM310468] and with Proteinuria, low molecular weight, with hypercalciuric nephrocalcinosis [MIM308990].

Figure S7. Schematic representation of COL2A1 with its domain structure based on Uniprot. Known missense (blue), known nonsense (green) and in this study identified mutations (red) are marked. Mainly autosomal dominant mutations in COL2A1 are associated with various forms of skeletal dysplasia.

Figure S8. Schematic representation of CUL7 with its domain structure based on Uniprot. Known missense (blue), known nonsense (green) and in this study identified mutations (red) are marked. Autosomal recessive mutations in CUL7 are associated with 3-M syndrome [MIM273750].

11

Figure S9. Schematic representation of FGD1 with its domain structure based on Uniprot. Known missense (blue), known nonsense (green) and in this study identified mutations (red) are marked. X-linked recessive mutations in FGD1 are associated Aarskog-Scott syndrome [MIM305400].

Figure S10. Schematic representation of FGFR3 with its domain structure base on Uniprot. Known missense (blue), known nonsense (green) and in this study identified mutations (red) are marked. Autosomal dominant mutations in FGFR3 are associated with several forms of skeletal dysplasias.

Figure S11. Schematic representation of FLNB with its domain structure based on Uniprot. Known missense (blue), known nonsense (green) and in this study identified mutations (red) are marked. Autosomal dominant mutations in FLNB are associated with Atelosteogenesis, type I [MIM108720], with Atelosteogenesis, type III [MIM108721], with Boomerang dysplasia [MIM112310] and with Larsen syndrome [MIM150250], autosomal recessive mutations are associated with Spondylocarpotarsal synostosis syndrome [MIM272460].

12

Figure S12. Schematic representation of GHSR with its domain structure based on Uniprot. Known missense (blue), known nonsense (green) and in this study identified mutations (red) are marked. Autosomal recessive and dominant mutations in GHSR are associated with Growth hormone deficiency, isolated partial [MIM615925].

Figure S13. Schematic representation of HDAC6 with its domain structure based on Uniprot. Known missense (blue), known nonsense (green) and in this study identified mutations (red) are marked. X-linked mutations in HDAC6 are associated with Chondrodysplasia with platyspondyly, distinctive brachydactyly, hydrocephaly, and microphthalmia [MIM300863].

Figure S14. Schematic representation of IFT140 with its domain structure based on Uniprot. Known missense (blue), known nonsense (green) and in this study identified mutations (red) are marked. Autosomal recessive mutations in IFT140 are associated with Short-rib thoracic dysplasia 9 with or without polydactyly [MIM266920].

13

Figure S15. Schematic representation of IGF1R with its domain structure based on Uniprot. Known missense (blue), known nonsense (green) and in this study identified mutations (red) are marked. Autosomal dominant and recessive mutations in IGF1R are associated with resistance to Insulin-like growth factor I [MIM270450].

Figure S16. Schematic representation of IHH with its domain structure based on Uniprot. Known missense (blue), known nonsense (green) and in this study identified mutations (red) are marked. Autosomal recessive mutations in IHH are associated with Acrocapitofemoral dysplasia [MIM607778], autosomal dominant mutations with Brachydactyly type A1 [MIM112500].

Figure S17. Schematic representation of KAT6B with its domain structure based on Uniprot. Known missense (blue), known nonsense (green) and in this study identified mutations (red) are marked. Autosomal dominant mutations in KAT6B are associated with Genitopatellar syndrome [MIM606170] and with SBBYSS syndrome [MIM603736].

14

Figure S18. Schematic representation of KDM6A with its domain structure based on Uniprot. Known missense (blue), known nonsense (green) and in this study identified mutations (red) are marked. X-linked dominant mutations in KDM6A are associated with Kabuki syndrome 2 [MIM300867].

Figure S19. Schematic representation of KRAS with its domain structure based on Uniprot. Known missense (blue), known nonsense (green) and in this study identified mutations (red) are marked. Autosomal dominant mutations in KRAS are associated with Noonan syndrome [MIM609942].

Figure S20. Schematic representation of MAP2K1 with its domain structure based on Uniprot. Known missense (blue), known nonsense (green) and in this study identified mutations (red) are marked. Autosomal dominant mutations in MAP2K1 are associated with Cardiofaciocutaneous syndrome [MIM615279].

15

Figure S21. Schematic representation of MATN3 with its domain structure based on Uniprot. Known missense (blue), known nonsense (green) and in this study identified mutations (red) are marked. Autosomal dominant mutations in MATN3 are associated with multiple epiphyseal dysplasia [MIM607078], autosomal recessive mutations with Spondyloepimetaphyseal dysplasia [MIM608728].

Figure S22. Schematic representation of NPR2 with its domain structure based on Uniprot. Known missense (blue), known nonsense (green) and in this study identified mutations (red) are marked. Autosomal dominant mutations in NPR2 are associated with Epiphyseal chondrodysplasia, Miura type [MIM615923] and with Short stature with nonspecific skeletal abnormalities [MIM616255], autosomal recessive mutations with Acromesomelic dysplasia, Maroteaux type [MIM602875].

Figure S23. Schematic representation of PDE3A with its domain structure based on Uniprot. Known missense (blue), known nonsense (green) and in this study identified mutations (red) are marked. Autosomal dominant mutations in PDE3A are associated with Hypertension and brachydactyly syndrome [MIM112410].

16

Figure S24. Schematic representation of PDE4D with its domain structure based on Uniprot. Known missense (blue), known nonsense (green) and in this study identified mutations (red) are marked. Autosomal dominant mutations in PDE4D are associated with Acrodysostosis 2, with or without hormone resistance [MIM614613].

Figure S25. Schematic representation of SLC26A2 with its domain structure based on Uniprot. Known missense (blue), known nonsense (green) and in this study identified mutations (red) are marked. Autosomal recessive mutations in SLC26A2 are associated with Achondrogenesis Ib [MIM600972], Atelosteogenesis II [MIM256050] and Diastrophic dysplasia [MIM222600].

Figure S26. Schematic representation of TRIM37 with its domain structure based on Uniprot. Known missense (blue), known nonsense (green) and in this study identified mutations (red) are marked. Autosomal recessive mutations in TRIM37 are associated with Mulibrey nanism [MIM253250].

17

Supplementary Tables

Table S1. Details of targeted diagnoses of 565 patients with short stature

Category Details Affected gene(s) MIM No. PatientsCNVs Del 1q21.1 6 genes 612474 3 (0.53 %)

Dup 5q22.1 59 genes na 3 (0.53 %)

Del 13q22.1 1 gene na 2 (0.35 %)

Del 14q21.1 1 gene na 2 (0.35 %)

Del 17p13 13 - 46 genes 613776 3 (0.53 %)

Del 22q11.22 1 gene na 2 (0.35 %)

Dup 16p12.2-p11.2 74 genes na 2 (0.35 %)

Dup 17q11.2 2 genes 613675 2 (0.35 %)

Dup 1q21.1 6 genes 612475 2 (0.35 %)

Dup 2q21.2 3 genes na 2 (0.35 %)

Dup 2q33.2 3 genes na 2 (0.35 %)

Dup 7q36.3 1 gene na 2 (0.35 %)

Del 14q23.1 8 genes na 1 (0.18 %)

Del 15q25.2-q25.3 29 genes 614294 1 (0.18 %)

Del 17q12 19 genes 614527 1 (0.18 %)

Del 1q32.1 27 genes na 1 (0.18 %)

Del 22q11.21 23 genes na 1 (0.18 %)

Del 2q36.1 31 genes na 1 (0.18 %)

Del 5p15.33 1 gene na 1 (0.18 %)

Del 7q36.1-q36.2 29 genes na 1 (0.18 %)

Dup 17q12 20 genes 614526 1 (0.18 %)

Dup 19q13.43 13 genes na 1 (0.18 %)

Dup 2p23.3 77 genes na 1 (0.18 %)

Dup 3q29 29 genes 611936 1 (0.18 %)

Syndromic short stature Leri-Weill dyschondrosteosis SHOX 127300 8 (1.41 %)

Silver-Russel syndrome imprinting defect 180860 6 (1.06 %)

Trichorhinophalangeal syndrome TRPS1 190350 4 (0.70 %)Noonan syndrome SHOC2

PTPN11RAF1

607721163950611553

3 (0.53 %)

Atypical SHOX-Deletion SHOX 127300 1 (0.18 %)

Charge syndrome CHD7 214800 1 (0.18 %)

DiGeorge syndrome 30 - 40 genes 188400 1 (0.18 %)

DYRK1A-related disorder DYRK1A 614104 1 (0.18 %)

Short-rib thoracic dysplasia DYNC2H1 613091 1 (0.18 %)

Large chrom. aberration Turner syndrome na 5 (0.88 %)

Turner syndrome mosaicism na 2 (0.35 %)

Marker chromosomes na 1 (0.18 %)

Isochromosome Xq na 1 (0.18 %)

Skeletal dysplasia A-/Hypochondroplasia FGFR3 134934 2 (0.35 %)

Spondyloperipheral dysplasia COL2A1 271700 1 (0.18 %)

18

Table S2. 1000 growth associated genes from MedGen and OMIM

Gene HGNC ID Gene HGNC

ID Gene HGNC ID Gene HGNC

IDAAAS 13666 EFNB1 3226 MCPH1 6954 RNF213 14539

AARS 20 EFTUD2 30858 MCTP2 25636 RNF216 21698

AASS 17366 EHHADH 3247 MECP2 6990 RNU4ATAC 34016

ABCA12 14637 EHMT1 24650 MED12 11957 ROGDI 29478

ABCB11 42 EIF2AK3 3255 MEF2C 6996 ROR2 10257

ABCB4 45 EIF4A3 18683 MESP2 29659 RPGRIP1L 29168

ABCB6 47 ELAC2 14198 MGAT2 7045 RPL11 10301

ABCB7 48 ELN 3327 MGP 7060 RPL15 10306

ABCC8 59 ELOVL4 14415 MIR17HG 23564 RPL26 10327

ABCD4 68 EMG1 16912 MKKS 7108 RPL35A 10345

ABCG5 13886 ENPP1 3356 MKRN3 7114 RPL5 10360

ABCG8 13887 EP300 3373 MKS1 7121 RPS10 10383

ABHD5 21396 EPB42 3381 MLXIPL 12744 RPS17 10397

ACACA 84 EPCAM 11529 MLYCD 7150 RPS19 10402

ACAN 319 EPG5 29331 MMP13 7159 RPS24 10411

ACP5 124 EPHX1 3401 MMP14 7160 RPS26 10414

ACTB 132 ERBB2 3430 MMP2 7166 RPS29 10419

ACTG1 144 ERCC1 3433 MMP9 7176 RPS6KA3 10432

ADAMTS10 13201 ERCC2 3434 MOCS1 7190 RPS7 10440

ADAMTS17 17109 ERCC3 3435 MOCS2 7193 RSPH4A 21558

ADAMTS2 218 ERCC4 3436 MPL 7217 RSPH9 21057

ADAMTSL2 14631 ERCC5 3437 MPLKIP 16002 RTEL1 15888

ADNP 15766 ERCC6 3438 MPV17 7224 RTTN 18654

ADRB2 286 ERCC8 3439 MPZ 7225 RUNX2 10472

ADSL 291 ESCO2 27230 MRPS22 14508 RYR1 10483

AFF2 3776 EVC 3497 MT-CO1 7419 SALL1 10524

AGA 318 EVC2 19747 MT-CO2 7421 SALL4 15924

AGK 21869 EXOSC3 17944 MT-CO3 7422 SAR1B 10535

AGL 321 EXT1 3512 MT-CYB 7427 SAT1 10540

AGPS 327 EXT2 3513 MTFMT 29666 SATB2 21637

AGRP 330 EYA1 3519 MT-ND1 7455 SBDS 19440

AKT1 391 FAM111A 24725 MT-ND5 7461 SC5D 10547

ALDH18A1 9722 FAM111B 24200 MT-ND6 7462 SCN4A 10591

ALDH3A2 403 FAM20C 22140 MTR 7468 SCN9A 10597

ALDOA 414 FAM58A 28434 MT-RNR1 7470 SCO1 10603

ALG1 18294 FANCA 3582 MT-TF 7481 SCRIB 30377

ALMS1 428 FANCB 3583 MT-TK 7489 SDCCAG8 10671

ALOX12B 430 FANCC 3584 MT-TL1 7490 SDHA 10680

ALOXE3 13743 FANCD2 3585 MT-TQ 7495 SDHAF1 33867

ALPL 438 FANCE 3586 MT-TS1 7497 SDHB 10681

ALX3 449 FANCF 3587 MT-TS2 7498 SDHC 10682

ALX4 450 FANCG 3588 MT-TV 7500 SDHD 10683

AMER1 26837 FANCI 25568 MT-TW 7501 SEC23A 10701

AMPD2 469 FANCL 20748 MVK 7530 SECISBP2 30972

ANK1 492 FANCM 23168 MYCN 7559 SEMA3E 10727

ANKRD11 21316 FAR1 26222 MYH3 7573 SEPN1 15999

ANTXR1 21014 FARS2 21062 MYH7 7577 Sep 09 7323

19

AP1S1 559 FASTKD2 29160 MYH8 7578 SERPINF1 8824

AP4B1 572 FAT4 23109 MYO5B 7603 SERPINH1 1546

AP4E1 573 FBN1 3603 NAA10 18704 SETBP1 15573

AP4S1 575 FBXL4 13601 NALCN 19082 SF3B4 10771

APOPT1 20492 FCGR2B 3618 NBAS 15625 SFXN4 16088

AQP2 634 FERMT1 15889 NBN 7652 SGCE 10808

ARG1 663 FGD1 3663 NDE1 17619 SH3PXD2B 29242

ARHGAP26 17073 FGF10 3666 NDN 7675 SHH 10848

ARID1A 11110 FGF17 3673 NDST1 7680 SHOC2 15454

ARID1B 18040 FGF23 3680 NDUFA1 7683 SHOX 10853

ARL6 13210 FGF8 3686 NDUFA10 7684 SHROOM4 29215

ARSB 714 FGFR1 3688 NDUFA11 20371 SIL1 24624

ARSE 719 FGFR2 3689 NDUFA12 23987 SIM1 10882

ARVCF 728 FGFR3 3690 NDUFA2 7685 SIX6 10892

ARX 18060 FIG4 16873 NDUFA9 7693 SKIV2L 10898

ASAH1 735 FKBP10 18169 NDUFAF1 18828 SLC10A2 10906

ASCL1 738 FLNA 3754 NDUFAF2 28086 SLC12A1 10910

ASL 746 FLNB 3755 NDUFAF3 29918 SLC16A1 10922

ASPM 19048 FLVCR2 20105 NDUFAF4 21034 SLC17A5 10933

ASXL1 18318 FOXE1 3806 NDUFAF5 15899 SLC19A2 10938

ATM 795 FOXRED1 26927 NDUFAF6 28625 SLC20A2 10947

ATP5A1 823 FSHR 3969 NDUFB3 7698 SLC25A13 10983

ATP6V0A2 18481 FTCD 3974 NDUFB9 7704 SLC26A2 10994

ATP6V0A4 866 FTO 24678 NDUFS1 7707 SLC26A3 3018

ATP6V1B1 853 FUCA1 4006 NDUFS2 7708 SLC26A4 8818

ATP7A 869 G6PC 4056 NDUFS3 7710 SLC29A3 23096

ATP8A2 13533 G6PC3 24861 NDUFS4 7711 SLC2A2 11006

ATP8B1 3706 G6PD 4057 NDUFS6 7713 SLC34A1 11019

ATPAF2 18802 GALNS 4122 NDUFS7 7714 SLC34A3 20305

ATR 882 GAN 4137 NDUFS8 7715 SLC35C1 20197

ATRIP 33499 GATA1 4170 NDUFV1 7716 SLC35D1 20800

ATRX 886 GATA4 4173 NDUFV2 7717 SLC37A4 4061

AUH 890 GATA6 4174 NEU1 7758 SLC39A13 20859

AUTS2 14262 GBA 4177 NF1 7765 SLC39A4 17129

AVPR2 897 GCK 4195 NFIX 7788 SLC4A1 11027

B3GALT6 17978 GDF1 4214 NHP2 14377 SLC4A4 11030

B3GALTL 20207 GDF5 4220 NIN 14906 SLC5A2 11037

B3GAT3 923 GDF6 4221 NIPAL4 28018 SLC5A5 11040

B4GALT7 930 GDNF 4232 NIPBL 28862 SLC6A19 27960

BANF1 17397 GFM1 13780 NKX2-1 11825 SLC6A8 11055

BBIP1 28093 GH1 4261 NKX2-5 2488 SLC7A7 11065

BBS1 966 GHR 4263 NKX2-6 32940 SLX4 23845

BBS10 26291 GHRHR 4266 NKX3-2 951 SMAD4 6770

BBS12 26648 GHSR 4267 NLRC4 16412 SMARCA2 11098

BBS2 967 GJA1 4274 NLRP3 16400 SMARCA4 11100

BBS4 969 GJB2 4284 NODAL 7865 SMARCAL1 11102

BBS5 970 GJB3 4285 NOG 7866 SMARCB1 11103

BBS7 18758 GJB4 4286 NOP10 14378 SMARCE1 11109

BBS9 30000 GJB6 4288 NOTCH1 7881 SMC1A 11111

20

BCOR 20893 GK 4289 NOTCH2 7882 SMC3 2468

BCS1L 1020 GLA 4296 NOTCH3 7883 SMOC1 20318

BDNF 1033 GLB1 4298 NPHP1 7905 SMPD1 11120

BLM 1058 GLE1 4315 NPHP3 7907 SMS 11123

BMP1 1067 GLI2 4318 NPHP4 19104 SNAI2 11094

BMP15 1068 GLI3 4319 NPHS1 7908 SNAP29 11133

BMP2 1069 GLIS3 28510 NPR2 7944 SNRPB 11153

BMPER 24154 GLYCTK 24247 NR0B1 7960 SNRPN 11164

BMPR1A 1076 GMPPA 22923 NR0B2 7961 SOS1 11187

BMPR1B 1077 GNA11 4379 NR1I3 7969 SOST 13771

BRAF 1097 GNAS 4392 NR3C2 7979 SOX11 11191

BRCA1 1100 GNPAT 4416 NR5A1 7983 SOX2 11195

BRCA2 1101 GNPTAB 29670 NRAS 7989 SOX3 11199

BRCC3 24185 GNPTG 23026 NRTN 8007 SOX9 11204

BRF1 11551 GNRH1 4419 NSD1 14234 SP7 17321

BRIP1 20473 GORAB 25676 NSDHL 13398 SPG20 18514

BTK 1133 GP1BB 4440 NSUN2 25994 SPINK5 15464

BUB1 1148 GPC6 4454 NT5C2 8022 SPINT2 11247

BUB1B 1149 GPD1 4455 NUBPL 20278 SPR 11257

BUB3 1151 GPR143 20145 OBSL1 29092 SPRTN 25356

C10orf2 1160 GRHL2 2799 OCRL 8108 SPTA1 11272

C12orf57 29521 GRIA3 4573 OFD1 2567 SRCAP 16974

C15orf41 26929 GRM1 4593 OGDH 8124 SRY 11311

C5orf42 25801 GSC 4612 OPA3 8142 STAC3 28423

CA2 1373 GTF2H5 21157 ORC1 8487 STAMBP 16950

CA8 1382 GTPBP3 14880 ORC4 8490 STAT1 11362

CANT1 19721 GUCY2D 4689 ORC6 17151 STAT3 11364

CASC5 24054 GUSB 4696 OSTM1 21652 STAT5B 11367

CASK 1497 GYS2 4707 OTX2 8522 STEAP3 24592

CASP8 1509 H19 4713 PALB2 26144 STIL 10879

CASR 1514 HADH 4799 PAPSS2 8604 STIM1 11386

CAV1 1527 HARS2 4817 PAX1 8615 STK11 11389

CBL 1541 HCCS 4837 PAX2 8616 STRA6 30650

CC2D2A 29253 HCFC1 4839 PAX6 8620 STS 11425

CCBE1 29426 HDAC4 14063 PAX8 8622 STT3B 30611

CCDC28B 28163 HDAC6 14064 PCCA 8653 STX16 11431

CCDC8 25367 HDAC8 13315 PCCB 8654 SUCLA2 11448

CD96 16892 HERC2 4868 PCNA 8729 SUCLG1 11449

CDAN1 1713 HES7 15977 PCNT 16068 SUMF1 20376

CDC6 1744 HESX1 4877 PCYT1A 8754 SURF1 11474

CDH3 1762 HFE 4886 PDE11A 8773 TAB2 17075

CDK5RAP2 18672 HIRA 4916 PDE3A 8778 TACO1 24316

CDKN1C 1786 HLA-DQB1 4944 PDE4D 8783 TAT 11573

CDSN 1802 HMGA2 5009 PDE6D 8788 TAZ 11577

CDT1 24576 HMGB3 5004 PDE8B 8794 TBC1D20 16133

CENPE 1856 HMOX1 5013 PDGFB 8800 TBCE 11582

CENPJ 17272 HNF1B 11630 PDGFRB 8804 TBX1 11592

CEP135 29086 HNF4A 5024 PDX1 6107 TBX15 11594

CEP152 29298 HOXD13 5136 PET100 40038 TBX22 11600

21

CEP164 29182 HPRT1 5157 PEX1 8850 TBX3 11602

CEP290 29021 HRAS 5173 PEX10 8851 TBX5 11604

CEP57 30794 HSD11B2 5209 PEX11B 8853 TBX6 11605

CEP63 25815 HSD17B4 5213 PEX12 8854 TCTN3 24519

CERS3 23752 HSPG2 5273 PEX13 8855 TECPR2 19957

CFC1 18292 HYAL1 5320 PEX14 8856 TERT 11730

CHCHD10 15559 HYLS1 26558 PEX16 8857 TFAP2A 11742

CHD7 20626 IARS2 29685 PEX19 9713 TG 11764

CHRNA1 1955 IBA57 27302 PEX2 9717 TGDS 20324

CHRNB1 1961 IDH1 5382 PEX26 22965 TGFB1 11766

CHRND 1965 IDS 5389 PEX3 8858 TGFB3 11769

CHRNG 1967 IDUA 5391 PEX5 9719 TGM1 11777

CHST14 24464 IFIH1 18873 PEX6 8859 THRA 11796

CHST3 1971 IFITM5 16644 PEX7 8860 THRB 11799

CHSY1 17198 IFT122 13556 PGAP3 23719 TINF2 11824

CKAP2L 26877 IFT140 29077 PGK1 8896 TMCO1 18188

CLCN5 2023 IFT172 30391 PGM1 8905 TMEM165 30760

CLCN7 2025 IFT27 18626 PGM3 8907 TMEM173 27962

CLCNKB 2027 IFT43 29669 PHC1 3182 TMEM216 25018

CLMP 24039 IFT80 29262 PHEX 8918 TMEM237 14432

CLP1 16999 IGBP1 5461 PHF6 18145 TMEM67 28396

CLPP 2084 IGF1 5464 PHGDH 8923 TMEM70 26050

CNGB3 2153 IGF1R 5465 PHKA2 8926 TNFRSF11A 11908

CNTNAP2 13830 IGF2 5466 PHKB 8927 TNFRSF11B 11909

COG1 6545 IGHMBP2 5542 PHKG2 8931 TNFSF11 11926

COL10A1 2185 IHH 5956 PHOX2B 9143 TNNI2 11946

COL11A1 2186 IKBKAP 5959 PIEZO2 26270 TNNT3 11950

COL11A2 2187 IKBKG 5961 PIGL 8966 TP53 11998

COL1A1 2197 IL21 6005 PIGO 23215 TP63 15979

COL1A2 2198 IL2RG 6010 PIK3CA 8975 TPM2 12011

COL27A1 22986 IMPAD1 26019 PIK3R1 8979 TPO 12015

COL2A1 2200 INPPL1 6080 PITX1 9004 TRAPPC11 25751

COL3A1 2201 INS 6081 PITX2 9005 TRAPPC2 23068

COL5A1 2209 INSR 6091 PKLR 9020 TREX1 12269

COL5A2 2210 INVS 17870 PLEC 9069 TRH 12298

COL6A2 2212 IQCB1 28949 PLEKHM1 29017 TRIM32 16380

COL7A1 2214 IYD 21071 PLK4 11397 TRIM37 7523

COL9A1 2217 JAG1 6188 PLOD2 9082 TRIP11 12305

COL9A2 2218 JAGN1 26926 PLOD3 9083 TRMT10A 28403

COL9A3 2219 KANSL1 24565 PLP1 9086 TRNT1 17341

COLEC11 17213 KAT6B 17582 PMM2 9115 TRPS1 12340

COMP 2227 KCNJ1 6255 PNKP 9154 TRPV4 18083

COMT 2228 KCNJ10 6256 PNPLA1 21246 TSFM 12367

COX10 2260 KCNJ11 6257 PNPLA2 30802 TSHB 12372

COX14 28216 KCNJ2 6263 PNPLA6 16268 TSHR 12373

COX15 2263 KDM5C 11114 PNPT1 23166 TSPYL1 12382

COX20 26970 KDM6A 12637 POC1A 24488 TTC21B 25660

COX6B1 2280 KIAA0196 28984 POLE 9177 TTC37 23639

COX7B 2291 KIAA1033 29174 POLG 9179 TTC7A 19750

22

CREBBP 2348 KIAA1279 23419 POLR3A 30074 TTC8 20087

CRIPT 14312 KIAA2022 29433 POLR3B 30348 TTI2 26262

CRTAP 2379 KIF11 6388 POMC 9201 TUBB4A 20774

CTC1 26169 KIF1A 888 PORCN 17652 TUBGCP6 18127

CTCF 13723 KIF22 6391 POU1F1 9210 TWIST1 12428

CTDP1 2498 KIF2A 6318 PPARG 9236 TXNL4A 30551

CTLA4 2505 KIF5C 6325 PPIB 9255 UBR1 16808

CTNNB1 2514 KIF7 30497 PPOX 9280 UCP3 12519

CTNS 2518 KISS1R 4510 PQBP1 9330 UFD1L 12520

CTSA 9251 KIT 6342 PRKAR1A 9388 UQCC2 21237

CTSK 2536 KLF1 6345 PRKDC 9413 UQCC3 34399

CUL4B 2555 KLLN 37212 PROP1 9455 UROC1 26444

CUL7 21024 KMT2A 7132 PRPS1 9462 UROD 12591

CYB5R3 2873 KMT2D 7133 PSAT1 19129 UROS 12592

CYP11B1 2591 KRAS 6407 PSMB8 9545 USB1 25792

CYP11B2 2592 KRT14 6416 PSMC3IP 17928 USP9X 12632

CYP19A1 2594 KRT5 6442 PSPH 9577 VDR 12679

CYP21A2 2600 L1CAM 6470 PTDSS1 9587 VLDLR 12698

CYP27B1 2606 LAMTOR2 29796 PTEN 9588 VPS13A 1908

CYP2R1 20580 LARGE 6511 PTF1A 23734 VPS13B 2183

CYP4F22 26820 LARP7 24912 PTH1R 9608 VPS53 25608

DCHS1 13681 LARS2 17095 PTHLH 9607 WDPCP 28027

DCX 2714 LBR 6518 PTPN11 9644 WDR19 18340

DDHD2 29106 LEMD3 28887 PTPN22 9652 WDR34 28296

DDR2 2731 LEPR 6554 PUF60 17042 WDR35 29250

DDX11 2736 LEPRE1 19316 PUS1 15508 WDR60 21862

DGUOK 2858 LFNG 6560 PYCR1 9721 WDR62 24502

DHCR24 2859 LHX3 6595 PYGL 9725 WDR73 25928

DHCR7 2860 LHX4 21734 RAB18 14244 WDR81 26600

DHODH 2867 LIAS 16429 RAB23 14263 WFS1 12762

DKC1 2890 LIFR 6597 RAB33B 16075 WHSC1 12766

DLL3 2909 LINS 30922 RAB3GAP1 17063 WISP3 12771

DLX5 2918 LIPN 23452 RAB3GAP2 17168 WNT1 12774

DMXL2 2938 LMBR1 13243 RAB40AL 25410 WNT5A 12784

DNA2 2939 LMBRD1 23038 RAD21 9811 WNT7A 12786

DNAJC19 30528 LMNA 6636 RAD50 9816 WRAP53 25522

DNAJC3 9439 LMX1B 6654 RAD51C 9820 WRN 12791

DNASE1 2956 LRBA 1742 RAF1 9829 WT1 12796

DNMT3B 2979 LRP5 6697 RAI1 9834 WWOX 12799

DOK7 26594 LTBP2 6715 RAPSN 9863 XYLT1 15516

DOLK 23406 LTBP3 6716 RARB 9865 YARS2 24249

DPP6 3010 LZTFL1 6741 RBBP8 9891 ZBTB16 12930

DPYD 3012 MAF 6776 RBCK1 15864 ZBTB18 13030

DPYS 3013 MAGEL2 6814 RBM10 9896 ZBTB20 13503

DSG1 3048 MALT1 6819 RBM28 21863 ZBTB24 21143

DST 1090 MAN2B1 6826 RBM8A 9905 ZC4H2 24931

DUOX2 13273 MAP2K1 6840 RDH11 17964 ZDHHC15 20342

DUOXA2 32698 MAP2K2 6842 RECQL4 9949 ZEB2 14881

DYM 21317 MARS2 25133 RET 9967 ZFP57 18791

23

DYNC1H1 2961 MASP1 6901 RFT1 30220 ZFPM2 16700

DYNC2H1 2962 MATN3 6909 RFX6 21478 ZMPSTE24 12877

EBP 3133 MBD5 20444 RIN2 18750 ZMYND11 16966

ECE1 3146 MBTPS2 15455 RIPK4 496 ZNF335 15807

ECEL1 3147 MC4R 6932 RIT1 10023 ZNF592 28986

EDN3 3178 MCM4 6947 RNASEH2A 18518 ZNF674 17625

EDNRB 3180 MCM9 21484 RNF168 26661 ZNF81 13156

24

Table S3. Overview of mutations in known short stature associated genes and patients' phenotypes in 200 exome individuals

Trio 28 AffOnly 26 AffOnly 47 AffOnly 62 AffOnly 89 Trio 11 Trio 58

Gene ACAN ANKRD11 CASKMutation(s)

c.1180C>T, p.(Arg394*) c.515del, p.(Gln172Argfs*59) c.1774C>T, p.(Gln592*)

c.5597C>A,p.(Ser1866*)

c.151T>G, p.(Cys51Gly)c.1770_1776del,

p.(Pro591Glyfs*60)c.979G>A, p.(Glu327Lys)

Inheritance autosomal dominant autosomal dominant x-linked

maternal maternal maternal de novo paternal de novo maternal

Diagnosis Osteochondritis dissecans with short stature KBG syndrome FG syndrome

Gender female female male male female male male

SGA no no no no yes yes no

Height (SDS)

Patient -3.8 -3.6 -3.2 -2.0 -3.5 -2.3 -2.6

Mother -1.9 -3.8 -1.8 -0.3 -1.1 -0.6 0.1

Father -2.3 -2.4 0.7 -1.2 -3.2 0.3 -0.6

Weight (SDS) -1.8 -1.7 -1.3 -0.04 -1.6 -2.9 0.4

OFC (SDS) -1.7 -1.6 1.6 2.7 0.5 -3.0 -2.2

Dyspropotionate SOS no no no no no no no

Syndromic SOS yes no no no no yes yes

Bone age delayed delayed na accelerated accelerated delayed delayed

IGF-1 (SDS) -0.8 -1.4 na -0.5 -0.06 0.2 na

IGFBP3 (SDS) -1.7 -0.3 na 0.3 1.0 -0.6 na

IQ normal normal normal normal normal 70-85 normal

Syndromic features

Eyes Epicantal fold,sparse eyebrows

Deeply set, hypotelorism

Ears Macrotia

Mouth Thin upper vermilion border

Long philtrum, thin vermilion, high palate,

macrodontia

Thorax Barrel-shaped Barrel-shaped Barrel-shaped Barrel-shaped Barrel-shaped Gynecomastia

Hand/Feet Brachydactyly,Broad thumb

Brachydactyly,broad thumb

Brachydactyly Brachydactyly, joint laxity, fingertip pads

Genitalia Hypospadia

HeadProminent forehead Prominent forehead

Dolichocephaly,Prominent forehead

Frontal bossing

25

Trio 67 Trio 38 Trio 62 AffOnly 4 Trio 27 AffOnly 97 AffOnly 95

Gene CLCN5 COL2A1 CUL7 FGD1 FGFR3 FLNBMutation(s)

c.298C>T, p.(Arg100Trp)

c.1043G>T, p.(Gly348Val)

c.3419G>C, p.(Gly1140Ala) c.3425-1G>C, p.? c.1546C>G, p.

(Pro516Ala)c.1612A>G, p.

(Ile538Val)

c.7276G>A, p.(Glu2426Lys)c.1640C>T, p.(Ala547Val)

Inheritance x-linked autosomal dominant autosomal recessive x-linked autosomal dominant autosomal recessive

maternal paternal de novo maternal & paternal maternal maternal maternal & paternal

Diagnosis Hypophosphatemic rickets Stickler syndrome 3-M syndrome Aarskog syndrome Hypochondroplasia Spondylocarpotarsal

synostosis syndrome

Gender male male male male male female female

SGA No na yes yes no yes yes

Height (SDS)

Patient -3.2 -5.4 -9.7 -4.3 -2.2 -2.8 -2.6

Mother -0.1 -1.8 0.7 -1.1 -1.6 -2.2 1.3

Father 0.3 -3.3 0.7 -2.0 -1.3 -0.7 -1.5

Weight (SDS) -2.6 -2.4 -5.6 -2.3 -0.7 -1.1 -3.6

OFC (SDS) -0.8 na -1.3 0.6 -1.5 0.2 -5.1

Dyspropotionate SOS no no yes yes yes no no

Syndromic SOS no no yes no yes no yes

Bone age delayed na na delayed na na na

IGF-1 (SDS) -0.8 -1.5 na 1.2 na na -3.7

IGFBP3 (SDS) 0.6 -0.9 na 1.3 na na -2.5

IQ normal normal normal normal normal normal normal

Syndromic features

Eyes Sparse eyebrows Hypertelorism Epicanthal fold

Ears Thick, arched eyebrow

Mouth Thin vermilion border Thin upper lip vermilion

Thorax Wide intermamillary distance Pectus carinatum Barrel-shaped chest

Hand/Feet Brachydactyly Brachydactyly Hyperextensibility Pes planus, elbow contracture Camptodactyly

Genitalia

Head Hearing impairment

26

Trio 18 AffOnly 77 AffOnly 37 AffOnly 65 AffOnly 84 AffOnly 68

Gene GHSR HDAC6 IFT140 IGF1R IHH KAT6BMutation(s)

c.1049C>G, p.(Thr350Ser)

c.2371A>G,p.(Met791Val)

c.3245A>T, p.(Asp1082Val)c.410G>A, p.(Arg137Gln)

c.3812A>G, p.(Glu1271Gly)

c.811C>T, p.(Leu271Phe)

c.5646del, p.(Asn1883Thrfs*2)

Inheritance autosomal dominant x-linked autosomal recessive autosomal dominant autosomal dominant autosomal dominant

paternal maternal maternal & paternal maternal maternal de novo

Diagnosis Isolated partial growth hormone deficiency

Chondrodysplasia with platyspondyly

Mainzer-Saldino syndrome

Resistance to insulin-like growth factor I Brachydactyly, type A1 Genitopatellar syndrome

Gender female male male male male male

SGA no no no no no no

Height (SDS)

Patient -2.7 -2.0 -2.1 -2.5 -2.1 -3.4

Mother -1.8 1.3 0.6 -2.2 -2.8 -1.0

Father -2.6 -1.2 0 -1.5 -1.2 0

Weight (SDS) -0.5 -1.6 -1.2 -1.4 -0.8 -3.3

OFC (SDS) -0.4 -0.4 -0.8 -0.2 1.2 -2.0

Dyspropotionate SOS no no no no yes no

Syndromic SOS no yes yes no yes yes

Bone age delayed delayed na normal delayed na

IGF-1 (SDS) -0.3 -3.0 -0.3 -1.1 -1.6 -1.6

IGFBP3 (SDS) -1.7 -1.9 -1.1 -0.4 -1.1 -1.0

IQ normal normal 70-85 normal normal normal

Eyes Downslanted palpebral fissures Proptosis Hypertelorism Proptosis

Ears Macrotia Microtia

Mouth

Thorax Absent nipples Pectus excavatum Pectus excavatum

Hand/FeetShort toes Fingertip pads Hyperextensibility at

elbow BrachydactylyLimited

pronation/supination of forearm

Genitalia Hypospadia

Head Narrow forehead, trigonocephaly

27

Trio 2 AffOnly 96 Trio 10 Trio 5 AffOnly 44 AffOnly 50 Trio 28

Gene KDM6A KRAS MAP2K1 MATN3 NF1 NPR2Mutation(s) c.1834C>T, p.(Arg612*) c.565-1G>A, p.? c.458A>G, p.

(Asp153Gly)c.383G>C, p.(Gly128Ala)

c.1322C>T, p.(Ser441Phe)

c.2320A>C, p.(Thr774Pro)

c.941T>A, p.(Leu314Gln)

Inheritance autosomal dominant autosomal dominant autosomal dominant autosomal dominant autosomal dominant autosomal dominant

de novo de novo de novo de novo de novo de novo paternal

DiagnosisKabuki syndrome 2 Noonan syndrome

spectrumNoonan syndrome

spectrumMultiple epiphyseal

dysplasia NeurofibromatosisShort stature with

nonspecific skeletal abnormalities

Gender female female male male male female female

SGA no no no no yes no no

Height (SDS)

Patient -2.6 -1.9 -2.6 -2.3 -3.9 -0.8 -3.8

Mother 0.1 0.4 0.6 1.6 -1.6 1.4 -1.9

Father -0.9 1.0 -0.6 -0.7 0 0 -2.3

Weight (SDS) -2.6 -2.2 -3.2 -2.5 -3.8 -0.5 -1.9

OFC (SDS) -1.8 0.9 -0.5 0.45 -2.4 2.6 -1.7

Dyspropotionate SOS no no no no no no no

Syndromic SOS yes no no yes no no yes

Bone age na delayed delayed na delayed normal delayed

IGF-1 (SDS) na -0.7 -3.3 na -3.0 0.7 -0.8

IGFBP3 (SDS) na -0.3 -3.6 na -2.2 0.7 -1.7

IQ 70-85 normal 70-85 normal normal normal normal

Syndromic features

Eyes Epicanthal fold, hypertelorism, myopia

Epicanthal fold, periorbital fullness

Ears Low-set ears,prominent helix

Preauricular pit

Mouth Thin vermilion Widely spaced teeth

Thorax Narrow chest Barrel-shaped chest Barrel-shaped chest

Hand/Feet Cubitus valgus Short nails Pes planus Broad thumb, elbow contracture

Genitalia

Head

28

AffOnly 17 AffOnly 85 Trio 77 AffOnly 72 AffOnly 74 AffOnly 23 AffOnly 57

Gene NPR2 PDE3A PDE4D PTPN11 SLC26A2 TRIM37Mutation(s)

c.2794C>T, p.(Arg932Cys)

c.1669C>T, p.(Arg557Cys)

c.1346G>A, p.(Gly449Asp) c.896C>, p.(Ser299Tyr) c.922A>G, p.

(Asn308Asp)

c.1957T>A, p.(Cys653Ser)c.353T>A, p.(Val118Glu)

c.2461dup, p.(Ile821Asnfs*6);

c.2377_2378del, p.(Leu793Valfs*2)

Inheritance autosomal dominant autosomal dominant autosomal dominant autosomal dominant autosomal recessive autosomal recessive

paternal de novo de novo de novo de novo maternal & paternal maternal & paternal

Diagnosis Short stature with nonspecific skeletal abnormalities

Hypertension and brachydactyly syndrome Acrodysostosis 2 Noonan syndrome

spectrumMultiple epiphyseal

dysplasia 4 Mulibrey nanism

Gender female female female female male male male

SGA no yes no yes no no no

Height (SDS)

Patient -3.0 -4.7 -2.9 -2.0 -3.5 -3.6 -4.0

Mother -0.7 0 0 -1.1 -0.3 1.1 0.4

Father -2.6 -0.7 -0.3 -0.4 -0.3 0.3 -0.4

Weight (SDS) -1.1 -2.8 1.2 -0.4 -2.5 0.1 -5.4

OFC (SDS) -1.1 -3.3 -0.4 1.1 -2.5 0.1 -1.0

Dyspropotionate SOS no no yes yes no yes no

Syndromic SOS no no yes no yes no no

Bone age na na na na na na na

IGF-1 (SDS) na na na na -5.0 na na

IGFBP3 (SDS) na na na na -2.5 na na

IQ nomal normal normal normal 70-85 normal normal

Syndromic features

Eyes Epicanthal fold Long eyelashes

Ears

Mouth

Thorax Narrow chest Supernumerary nipples Atrial septal defect, barrel-shaped chest

Scoliosis, pectus excavatum

Hand/Feet Brachydactyly, elbow contracture

Acromelia, Hyperextensibility at

wristsShort limbs Pes planus, coxa vara

Genitalia Cryptorchidism

HeadSparse hair Cow lick

Pointed chin, dolichocephaly, frontal

bossing

29

Table S4. Details of mutations identified in known short stature associated genes of 200 exome individuals

Sample No. Gene ACMG Category Genomic position (GRCh37) RefSeq number cDNA level Protein level Disease

autosomal dominantTrio 2 KDM6A Pathogenic (Ia) chrX:g.44922973C>T NM_021140.2 c.1834C>T p.(Arg612*) Kabuki syndrome 2

Trio 5 MAP2K1 Pathogenic (IIIA) chr15:g.66729175G>C NM_002755.3 c.383G>C p.(Gly128Ala) Noonan syndrome spectrum

Trio 10 KRAS Pathogenic (II) chr12:g.25362838T>C NM_004985.3 c.458A>G p.(Asp153Gly) Noonan syndrome spectrum

Trio 11 ANKRD11 Pathogenic (Ia) chr16:g.89351174_89351180del NM_001256182.1 c.1770_1776del

p.(Pro591Glyfs*60) KBG syndrome

Trio 18 GHSR Likely pathogenic (V) chr3:g.172163003G>C NM_198407.1 c.1049C>G p.(Thr350Ser) Isolated partial growth hormone deficiency

Trio 28 ACAN Pathogenic (Ib) chr15:g.89388864C>T NM_013227.3 c.1180C>T p.(Arg394*) Osteochondritis dissecans with short stature

Trio 28 NPR2 Likely pathogenic (V) chr9:g.35799682T>A NM_003995.3 c.941T>A p.(Leu314Gln) Short stature with nonspecific skeletal abnormalities

Trio 38 COL2A1 Likely pathogenic (V) chr12:g.48383569C>A NM_001844.4 c.1043G>T p.(Gly348Val) Stickler syndrome

Trio 62 COL2A1 Pathogenic (IIIb) chr12:g.48370611C>G NM_001844.4 c.3419G>C p.(Gly1140Ala) Stickler syndrome

Trio 77 PDE3A Pathogenic (IIIa) chr12:g.20769240G>A NM_000921.4 c.1346G>A p.(Gly449Asp) Hypertension and brachydactyly syndrome

AffOnly 17 NPR2 Likely Pathogenic (V) chr9:g.35808587C>T NM_003995.3 c.2794C>T p.(Arg932Cys) Short stature with nonspecific skeletal abnormalities

AffOnly 26 ACAN Pathogenic (Ib) chr15:g.89383303del NM_013227.3 c.515del p.(Gln172Argfs*59) Osteochondritis dissecans with short stature

AffOnly 44 MATN3 Pathogenic (IIIb) chr2:g.20194143G>A NM_002381.4 c.1322C>T p.(Ser441Phe) Multiple epiphyseal dysplasia

AffOnly 47 ACAN Pathogenic (Ib) chr15:g.89392710C>T NM_013227.3 c.1774C>T p.(Gln592*) Osteochondritis dissecans with short stature

AffOnly 50 NF1 Pathogenic (IIIb) chr17:g.29554304A>C NM_001042492.2 c.2320A>C p.(Thr774Pro) Neurofirbomatosis type 1

AffOnly 62 ACAN Pathogenic (Ia) chr15:g.89401413C>A NM_013227.3 c.5597C>A p.(Ser1866*) Osteochondritis dissecans with short stature

AffOnly 65 IGF1R Likely pathogenic (V) chr15:g.99500379A>G NM_000875.3 c.3812A>G p.(Glu1271Gly) Resistance to insulin-like growth factor I

AffOnly 68 KAT6B Pathogenic (Ia) chr10:g.76790228del NM_012330.3 c.5646del p.(Asn1883Thrfs*2) Genitopatellar syndrome

AffOnly 72 PDE4D Pathogenic (IIIb) chr5:g.58334711G>T NM_001104631.1 c.896C>A p.(Ser299Tyr) Acrodysostosis 2

AffOnly 74 PTPN11 Pathogenic (II) chr12:g.112915523A>G NM_002834.3 c.922A>G p.(Asn308Asp) Noonan syndrome spectrum

AffOnly 84 IHH Likely pathogenic (V) chr2:g.219920354G>A NM_002181.3 c.811C>T p.(Leu271Phe) Brachydactyly, type A1

AffOnly 85 NPR2 Pathogenic (IIIb) chr9:g.35802239C>T NM_003995.3 c.1669C>T p.(Arg557Cys) Short stature with nonspecific skeletal abnormalities

AffOnly 89 ACAN Likely pathogenic (V) chr15:g.89381974T>G NM_013227.3 c.151T>G p.(Cys51Gly) Osteochondritis dissecans with short stature

AffOnly 96 KDM6A Pathogenic (Ia) chrX:g.44894175G>A NM_021140.2 c.565-1G>A p.? Kabuki syndrome 2

AffOnly 97 FGFR3 Pathogenic (IIIb) chr4:g.1807363A>G NM_000142.4 c.1612A>G p.(Ile538Val) Hypochondroplasia

autosomal recessiveAffOnly 4 CUL7 Pathogenic (Ib) chr6:g.43011369C>G NM_001168370.1 c.3425-1G>C p.? 3-M syndrome

AffOnly 23 SLC26A2 Pathogenic (IIIa) chr5:g.149361113T>A NM_000112.3 c.1957T>A p.(Cys653Ser) Multiple epiphyseal dysplasia 4

Likely pathogenic (IV) chr5:g.149357568T>A NM_000112.3 c.353T>A p.(Val118Glu)

AffOnly 37 IFT140 Likely pathogenic (IV) chr16:g.1573854T>A NM_014714.3 c.3245A>T p.(Asp1082Val) Mainzer-Saldino syndrome

Likely pathogenic (V) chr16:g.1642549C>T NM_014714.3 c.410G>A p.(Arg137Gln)

30

AffOnly 57 TRIM37 Pathogenic (Ib) chr17:g.57093086dup NM_001005207.2 c.2461dup p.(Ile821Asnfs*6) Mulibrey nanism

Pathogenic (Ib) chr17:g.57094665_57094666del NM_001005207.2 c.2377_2378del

p.(Leu793Valfs*2)

AffOnly 95 FLNB Likely pathogenic (V) chr3:g.58149042G>A NM_001164317.1 c.7276G>A p.(Glu2426Lys) Spondylocarpotarsal synostosis syndrome

Likely pathogenic (V) chr3:g.58090836C>T NM_001164317.1 c.1640C>T p.(Ala547Val)

X-linkedTrio 27 FGD1 Likely pathogenic (V) chrX:g.54491974G>C NM_004463.2 c.1546C>G p.(Pro516Ala) Aarskog syndrome

Trio 58 CASK Likely pathogenic (V) chrX:g.41485893C>T NM_003688.3 c.979G>A p.(Glu327Lys) FG syndrome

Trio 67 CLCN5 Likely pathogenic (V) chrX:g.49834668C>T NM_001127899.1 c.298C>T p.(Arg100Trp) Hypophosphatemic rickets

AffOnly 77 HDAC6 Likely pathogenic (V) chrX:g.48681063A>G NM_006044.2 c.2371A>G p.(Met791Val) Chondrodysplasia with platyspondyly

31

Table S5. Phenotypes of 200 exome individuals with potential causal mutations in known short stature genes

Patient Phenotype Gene Disease association ReferenceTrio 2 Postnatal growth retardation, mild

learning impairment, moderate short stature, normocephaly.

KDM6A Kabuki syndrome 2 [MIM300867]. The phenotype is less typical in patients with KDM6A mutations compared to MML2 mutation patients. Short stature and postnatal growth retardation is observed in the majority of KDM6A patients.

26,27

Trio 5 Postnatal growth retardation, normal intellectual development, mild short stature, muscular hypotonia.

MAP2K1 Cardiofaciocutaneous syndrome [MIM615279]. The phenotype includes congenital heart defects, short stature and a distinct facial gestalt with macrostomia and horizontal shaped palpepral fissures.

28,29

Trio 10 Postnatal growth retardation, mild learning impairment, moderate short stature.

KRAS Noonan syndrome 3 [MIM609942]. The phenotype includes congenital heart defects, short stature and mild to moderate ID.

30,31

Trio 11 Primordial growth retardation, mild learning impairment, macrodontia.

ANKRD11 KBG syndrome [MIM148050]. The phenotype includes dental and skeletal anomalies and postnatal short stature.

32,33

Trio 18 Postnatal growth retardation, normal intellectual development, mild growth retardation.

GHSR Isolated partial Growth hormone deficiency [MIM615925]. Mild growth retardation of 1 SD below the mean.

34,35

Trio 27 Postnatal growth retardation, normal intellectual development, mild to profound growth retardation with short limbs.

FGD1 Aarskog-Scott syndrome [MIM305400]. The phenotype shows a high variability with ID, short stature, hyperextensibility.

36

Trio 28 Postnatal growth retardation, normal intellectual development, profound short stature.

ACAN Spondylometaphyseal dysplasia, aggrecan type [MIM612813], Osteochondritis dissecans, short stature, and early-onset osteoarthritis [MIM165800] and Spondyloepiphyseal dysplasia, Kimberley type [MIM608361]. Heterozygous mutations lead to mild form of short stature with osteochondritis dissecans and early-onset osteoarthritis.

37-39

NPR2 Epiphyseal chondrodysplasia, Miura type [MIM615923], Short stature with nonspecific skeletal abnormalities [MIM616255], autosomal recessive mutations with Acromesomelic dysplasia, Maroteaux type [MIM602875]. Heterozygous mutations lead to mild short stature.

40,41

Trio 38 Isolated, proportionate growth retardation, normal intellectual development, wide intermamillary distance

COL2A1 Stickler syndrome [MIM108300], Kniest dysplasia [MIM156550], and varous forms of skeletal dysplasias. Common feature is short stature. The severity in clinical presentation can be variable.

42,43

Trio 58 Postnatal growth retardation, normal intellectual development, mild short stature, brachydactyly, finger pads, joint laxity.

CASK FG syndrome 4 [MIM300422], Mental retardation and microcephaly with pontine and cerebellar hypoplasia [MIM30749]. The phenotype includes ID, poor growth and hypotonia caused by nonsense mutations.

44

Trio 62 Severe primordial growth retardation, mild rhizomelic shortening of the extremities, pectus carinatum.

COL2A1 Stickler syndrome [MIM108300], Kniest dysplasia [MIM156550], and various forms of skeletal dysplasias. Common feature is short stature. The severity in clinical presentation can be variable.

42,43

Trio 67 Postnatal growth retardation, normal intellectual development, profound short stature, hearing impairment.

CLCN5 Dent disease [MIM300009], Hypophosphatemic rickets [MIM300554], Nephrolithiasis, type I [MIM310468], Proteinuria, low molecular weight, with hypercalciuric nephrocalcinosis [MIM308990].

45,46

Trio 77 Mild growth retardation with height below the estimated family target height, severe brachydactyly.

PDE3A Hypertension and brachydactyly syndrome [MIM112410]. Severe brachydactyly with proportionate short stature, hypertension in adult age.

47,48

32

AffOnly 4 Primordial growth retardation, profound short stature, barrel-shaped chest.

CUL7 3-M syndrome [MIM273750]. The phenotype includes short stature, triangular face, flat thorax and hypospadias.

49,50

AffOnly 17 Postnatal growth retardation, normal intellectual development, profound short stature.


40,41

AffOnly 23 Postnatal growth retardation with growth below the average familial height, skeletal anomalies.

SLC26A2 Achondrogenesis Ib [MIM600972], Atelosteogenesis II [MIM256050], Diastrophic dysplasia [MIM222600]. The phenotype includes dysproportionate stature with skeletal anomalies.

51



37-39

AffOnly 37 Postnatal growth retardation, mild learning impairment, mild short stature.

IFT140 Short-rib thoracic dysplasia 9 with or without polydactyly [MIM266920]. Phenotypic features include short stature, early-onset retinitis pigmentosa, chronic renal failure and skeletal anomalies.

52


MATN3 Multiple epiphyseal dysplasia [MIM607078], Spondyloepimetaphyseal dysplasia [MIM608728]. Mild to moderate short stature, early-onset osteoarthritis.

53,54



37-39

AffOnly 50 Postnatal growth retardation, normal intellectual development, moderate proportionate short stature, absent nipples, brachydactyly, epicantus.

NF1 Neurofibromatosis type 1 [MIM162200], Neurofibromatosis-Noonan syndrome [MIM601321]. The phenotype is highly variable and include short stature, Café-au-lait spots, fibromata.

55,56

AffOnly 57 Postnatal growth retardation, normal intellectual development, profound short stature, dolichocephaly.

TRIM37 Mulibrey nanism [MIM253250]. The phenotype includes prenatal growth retardation, cardiomyopathy, facial features and increases risk for Wilms tumor.

57,58

AffOnly 62 Postnatal growth retardation, normal intellectual development, mild short stature.


37-39

AffOnly 65 Slow primordial growth with moderate short stature.

IGF1R Resistance to Insulin-like growth factor [MIM270450]. The phenotype includes intrauterine growth retardation with poor postnatal growth. The severity in clinical presentation can be variable.

59,60

33


KAT6B Genitopatellar syndrome [MIM606170] and SBBYSS syndrome [MIM603736]. The phenotype includes absent patellae, genital und renal anomalies, dysmorphic features, ID and short stature. Might be highly variable.

61-63

AffOnly 72 Postnatal growth retardation, mild learning impairment, mild short stature, short nose with bulbous tip, brachydactyly.

PDE4D Acrodysostosis 2, with or without hormone resistance [MIM614613]. The phenotype includes short stature, maxillary hypoplasia, short nose, brachydactyly and ID.

64,65

AffOnly 74 Postnatal growth retardation, mild learning impairment, moderate proportionate short stature, sparse hair, barrel-shaped thorax, pulmonic stenosis.

PTPN11 Noonan syndrome 1 [MIM 163950], LEOPARD syndrome 1 [MIM151100]. The phenotype includes short stature, developmental delay, congenital cardia defects.

56,66

AffOnly 77 Postnatal growth retardation normal intellectual development, mild short stature, hypospadias, trigonocephaly.

HDAC6 Chondrodysplasia with platyspondyly, distinctive brachydactyly, hydrocephaly, and microphthalmia [MIM300863].

67,68

AffOnly 84 Postnatal growth retardation normal intellectual development, mild short stature, brachydactyly.

IHH Acrocapitofemoral dysplasia [MIM607778], Brachydactyly type A1 [MIM112500]. The phenotype varies and includes brachydactyly, short stature and other skeletal anomalies.

69



40,41

AffOnly 89 Primordial growth retardation, normal intellectual development, profound short stature.


37-39

AffOnly 95 Primordial growth retardation, normal intellectual development, profound short stature, microcephaly.

FLNB Atelosteogenesis, type I [MIM108720], Atelosteogenesis, type III [MIM108721], Boomerang dysplasia [MIM112310], Larsen syndrome [MIM150250], Spondylocarpotarsal synostosis syndrome [MIM272460]

70

AffOnly 96 Postnatal growth retardation, mild learning impairment, mild short stature, normocephaly.

KDM6A Kabuki syndrome 2 [MIM300867]. The phenotype is less typical in patients with KDM6A mutations compared to MML2 mutation patients. Short stature and postnatal growth retardation is observed in the majority of KDM6A patients.

26,27

AffOnly 97 Primordial growth retardation, mild to profound short stature, normal intellectual development.

FGFR3 Thanatophoric dysplasia [MIM187600 & MIM187601], Achondroplasia [MIM100800], Hypochondroplasia [MIM146000]. Variable phenotype from lethal to mild short stature.

71,72

34

Table S6. Symptoms of identified mutations in known short stature associated genes

Symptom Genes*osteoarthritis ACAN73, CUL7, MATN374

hearing loss COL2A175

orthopedic symptoms COL2A175, FGFR3, IHH, SLC26A2, HDAC6, FLNB70

developmental issues KDM6A27, ANKRD11, PDE4D27, CASK27, FGD136, PTPN1176, NF177

bleeding diathesis PTPN1178

neoplasia TRIM3757, PTPN1176, NF179

hearing loss COL2A175, FLNB70

multiple malformations KAT6B61, KRAS30, MAP2K129, PTPN1176

chronic kidney disease IFT14080, CLCN581

GH signalling pathway defects GHSR, IGF1R, PTPN1182

severe hypertension PDE3A47

*: Information derived from publications and GeneReviews

35

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