ORIGINAL RESEARCH published: 29 August 2019 doi: 10.3389/fgene.2019.00746 Broad Phenotypes of Disorders/ Differences of Sex Development in MAMLD1 Patients Through Oligogenic Disease Christa E. Flück 1, Laura Audí 2, Mónica Fernández-Cancio 2, Kay-Sara Sauter 1, Idoia Martinez de LaPiscina 3, Luis Castaño 4, Isabel Esteva 5 and Núria Camats 2* 1 Pediatric Endocrinology, Diabetology and Metabolism, Department of Pediatrics and Department of BioMedical Research, Bern University Hospital and University of Bern, Bern, Switzerland, 2 Growth and Development Research Unit, Vall d’Hebron Research Institute (VHIR), Center for Biomedical Research on Rare Diseases (CIBERER), Instituto de Salud Carlos III, Barcelona, Spain, 3 Endocrinology and Diabetes Research Group, BioCruces Bizkaia Health Research Institute, Cruces University Hospital, CIBERDEM, CIBERER, University of the Basque Country (UPV-EHU), Barakaldo, Spain, 4 Pediatric Endocrinology Section, Cruces University Hospital, Endocrinology and Diabetes Research Group, BioCruces Bizkaia Health Research Institute, CIBERDEM, CIBERER, University of the Basque Country (UPV-EHU), Barakaldo, Spain, 5 Endocrinology Section, Gender Identity Unit, Regional University Hospital of Malaga, Málaga, Spain Disorders/differences of sex development (DSD) are the result of a discordance between Edited by: Jeff Schwartz, chromosomal, gonadal, and genital sex. DSD may be due to mutations in any of the genes Griffith University, Australia involved in sex determination and development in general, as well as gonadal and/or genital Reviewed by: development specifically. MAMLD1 is one of the recognized DSD genes. However, its role is Maki Fukami, controversial as some MAMLD1 variants are present in normal individuals, several MAMLD1 National Center for Child Health and Development (NCCHD), Japan mutations have wild-type activity in functional studies, and the Mamld1-knockout male mouse Vincent Russel Harley, presents with normal genitalia and reproduction. We previously tested nine MAMLD1 variants Hudson Institute of Medical Research, Australia detected in nine 46,XY DSD patients with broad phenotypes for their functional activity, but *Correspondence: none of the mutants, except truncated L210X, had diminished transcriptional activity on known Núria Camats target promoters CYP17A1 and HES3. In addition, protein expression of MAMLD1 variants was nuria.camats@vhir.org similar to wild-type, except for the truncated L210X. We hypothesized that MAMLD1 variants may not be sufficient to explain the phenotype in 46,XY DSD individuals, and that further genetic Specialty section: This article was submitted to studies should be performed to search for additional hits explaining the broad phenotypes. We Systems Endocrinology, therefore performed whole exome sequencing (WES) in seven of these 46,XY patients with a section of the journal Frontiers in Genetics DSD and in one 46,XX patient with ovarian insufficiency, who all carried MAMLD1 variants. Received: 21 March 2019 WES data were filtered by an algorithm including disease-tailored lists of MAMLD1-related and Accepted: 16 July 2019 DSD-related genes. Fifty-five potentially deleterious variants in 41 genes were identified; 16/55 Published: 29 August 2019 variants were reported in genes in association with hypospadias, 8/55 with cryptorchidism, Citation: 5/55 with micropenis, and 13/55 were described in relation with female sex development. Flück CE, Audí L, Fernández-Cancio M, Sauter K-S, Patients carried 1-16 variants in 1-16 genes together with their MAMLD1 variation. Network Martinez de LaPiscina I, Castaño L, analysis of the identified genes revealed that 23 genes presented gene/protein interactions with Esteva I and Camats N (2019) Broad Phenotypes of Disorders/Differences MAMLD1. Thus, our study shows that the broad phenotypes of individual DSD might involve of Sex Development in MAMLD1 multiple genetic variations contributing towards the complex network of sexual development. Patients Through Oligogenic Disease. Front. Genet. 10:746. Keywords: whole exome sequencing, MAMLD1, disorders/differences of sex development, hypospadias, doi: 10.3389/fgene.2019.00746 phenotype variability, oligogenic disorder Frontiers in Genetics | www.frontiersin.org 1 August 2019 | Volume 10 | Article 746 Flück et al. Oligogenic MAMLD1 DSD Phenotypes INTRODUCTION to wild-type, except for the truncated L210X. We therefore hypothesized that MAMLD1 variants may not be sufficient to Disorders/differences of sex development (DSD) occur when explain the phenotype in 46,XY DSD carriers, and that further there is a discordance between chromosomal, gonadal, and genetic studies should be performed to search for additional hits genital sex (Ostrer, 2014). DSD may be due to mutations in any explaining the broad variability. of the genes involved in sex determination and development In the past decade, High throughput sequencing (HTS) in general, as well as gonadal and/or genital development has changed the genetic approach in research and diagnostics. specifically (Ostrer, 2014). Whole-exome sequencing (WES) has led to the discovery of many MAMLD1 (Xq28, OMIM 300120) is one of the recognized new genes and has given insight into complex traits. Oligogenic DSD-related genes (Fukami et al., 2006; Baxter et al., 2015). inheritance is currently discovered for several disorders by Variations in MAMLD1 sequence have been described mainly HTS. In the field of sex development, digenic inheritance has in 46,XY DSD individuals, mostly associated with hypospadias recently been suggested in a 46,XY DSD patient with gonadal (Fukami et al., 2006; Kalfa et al., 2008; Chen et al., 2010; Kalfa dysgenesis (NR5A1 and MAP3K1 variants) (Mazen et al., 2016); et al., 2012; Metwalley and Farghaly, 2012; Camats et al., 2015; in a family with 46,XY DSD males (NR5A1 variants) and 46,XX Igarashi et al., 2015; Eggers et al., 2016), but also with other POF females (NR5A1 and TBX2) (Werner et al., 2017); as well DSD phenotypes, including micropenis (Chen et al., 2010; as in a DSD patient with ambiguous genitalia, micropenis, Kalfa et al., 2012; Camats et al., 2015; Liu et al., 2017), and/ and inguinal testes (SEMA3A and AKR1C4) (Fan et al., 2017). or crypthorchidism (Fukami et al., 2006; Kalfa et al., 2008; Similarly, we found oligogenic origin of disease in heterozygous Kalfa et al., 2012; Camats et al., 2015), 46,XY with female NR5A1 46,XY DSD patients by performing WES (Camats et al., external genitalia (Fukami et al., 2006; Camats et al., 2015) 2018). In addition, in patients with hypospadias, an oligogenic and 46,XY with complete gonadal dysgenesis (Ruiz-Arana et origin was suggested by two other NGS studies (Kon et al., 2015; al., 2015). Furthermore, one homozygous MAMLD1 variant Eggers et al., 2016). was also reported in a 46,XX patient with gonadal dysgenesis, Therefore, in this study, we performed WES in seven 46,XY primary amenorrhea, bilateral streak gonads and clitoromegaly patients with DSD (Camats et al., 2015) and one 46,XX patient (Brandao et al., 2011). with ovarian insufficiency, who all carried MAMLD1 variants. However, the role of MAMLD1 in sex development is WES data were filtered by common tools and a disease-tailored controversial for several reasons: a) some MAMLD1 variants algorithm including MAMLD1-related and DSD-related are present in the normal population (Fukami et al., 2006; Chen known and candidate genes. Additional hits in likely disease- et al., 2010; Gaspari et al., 2011; Kalfa et al., 2011); b) the same causing genes were detected in all eight MAMLD1 carriers. Our MAMLD1 variant may be present in patients with different results suggest that oligogenic origin of disease may contribute phenotypes (Camats et al., 2015); c) MAMLD1 variants are towards the broad phenotypes of human MAMLD1. not present in all DSD individuals of the same family (Fukami et al., 2006); d) several MAMLD1 mutations present wild-type activity in functional studies (Camats et al., 2015); and e) the PATIENTS AND METHODS Mamld1-knockout male mouse presents with normal genitalia and reproduction (Miyado et al., 2012; Miyado et al., 2017). Patients MAMLD1 is expressed in human fetal and adult testis and in The study was approved by the Ethics Committee of Hospital human ovaries (Fukami et al., 2006; O’Shaughnessy et al., 2007; Universitari Vall d'Hebron (Barcelona, Spain) (CEIC: Camats et al., 2015), and seems to be involved in sex development PR(IR)23/2016). Written informed consent was obtained in fetal life and in adult reproductive function. Yet its exact role from the patients for the publication of their cases. Eight is not clear. It is expressed in mice gonadal cells during start of DSD patients (seven 46,XY and one 46,XX) each carrying androgen biosynthesis up to male external genitalia formation one MAMLD1 variant were analyzed using WES. Clinical and is therefore thought to be involved in the expression of and genetic characteristics of 46,XY patients were previously Leydig-cell genes (Miyado et al., 2012), as well as supporting reported in detail in Camats et al., (2015) and are summarized testosterone production in critical periods of male development in Table 1 together with the 46,XX patient. (Fukami et al., 2008; Nakamura et al., 2011). In contrast, Mamld1-KO mice present normal external genitalia (but small testes and reduced seminiferous tubule size and proliferating DNA Extraction, WES and Bioinformatic germ cells) and reproduce similarly to wild-type mice (Miyado Analysis et al., 2012; Miyado et al., 2017). These findings challenge the role DNA was extracted from blood leukocytes using QiaCube of MAMLD1 in sex development. (Qiagen, Hilden, Germany) or manually using a DNA isolation In a previous study, we tested functional activity of nine kit (Qiagen). WES was performed by CNAG (Centre Nacional MAMLD1 variants detected in nine 46,XY DSD patients with d’Anàlisi Genòmica, Barcelona, Spain). Libraries were prepared broad phenotypes (Camats et al., 2015). None of the MAMLD1 with a SureSelect Human All Exon V5 capture kit (Agilent, Santa mutants, except truncated L210X, had diminished transcriptional Clara, CA, USA) and sequenced with a HiSeq™ 2000 sequencing activity on known target promoters CYP17A1 and HES3. In system (v3, 2x100, Illumina, San Diego, CA, USA). Putative addition, protein expression of MAMLD1 variants was similar candidate variants were confirmed by Sanger sequencing. Frontiers in Genetics | www.frontiersin.org 2 August 2019 | Volume 10 | Article 746 TABLE 1 | Clinical, biochemical and genetic characteristics of the studied MAMLD1 patients. Frontiers in Genetics | www.frontiersin.org Flück et al. Patient Karyotype Phenotype and origin Gonadal function Adrenal function MAMLD1 variant Variants after Candidate Candidate assigned sex (age) (age) filtering by variants genes (C) gene list (A) (B) 1 46,XY Penoscrotal hypospadias. Normal hCG test. Normal Synacthen test. p.V505A NM_005491:c.1514T>C 547 9 7 (7) Female Small penis. Unilateral cryptorchidia. Histology: normal for age (2y). Venezuelan origin. 2 46,XY Penoscrotal hypospadias. Normal baseline T (3m). Normal baseline p.A503E NM_005491:c.1508C>A 492 1 1 (6) Male Small penis. Normal hCG test (9m). (3d). Testes: 2 ml. Spanish origin. 3 46,XY Penoscrotal hypospadias. Normal baseline (12m). NA p.S730S NM_005491:c.2190G>A 570 2 2 (9) Female Small penis. No hCG test. Histology: normal for age (nests of normal Leydig cells; normal fertility index (1y) Müllerian ducts. Spanish origin 4 46,XY Female genitalia. Normal hCG test (2y). Normal baseline p.H347Q NM_005491:c.1041C>A 633 4 4 (3) Female Gonads in labia. (2y). rs62641609 Spanish origin. 5 46,XY Penoscrotal hypospadias. Normal hCG test. Normal baseline p.H347Q NM_005491:c.1041C>A 929 6 6 (4) Male Testes 2 ml. Normal AMH (2.5y). (2.5y). rs62641609 Spanish origin. 3 6 46,XY Penoscrotal hypospadias. Normal prepubertal Normal baseline p.L724V NM_005491:c.2170C>G 710 16 16 (8) Male Small penis. baseline T (15 m). (15 m). Testes 1 ml. Normal AMH. Esophageal atresia. Right aortic arch. North African origin. 7 46,XY Hypospadias. Normal baseline Normal baseline p.Q501Q502 429 5 5 (5) Male Short penis. T and gonadotropins (70y). NM_005491:c.1503_1504dupCAGCAG Testes 8 ml. (70y). Delayed puberty. Fathered a boy. Gynaecomastia. Fathered a boy. Swiss origin. 8 46,XX Female external genitalia. High gonadotropins Normal (27y). NM_005491:c.*126C>MIT 574 14 13 Female Small ovaries and uterus, with fallopian and low/normal Oligogenic MAMLD1 DSD Phenotypes Month 2019 | Volume 10 | Article 746 tubes. Primary amenorrhea (15y). estradiol (27y). Histology: large amount of primordial follicles (no evidence of maturation), atresic follicles. Delayed growth. Spanish origin. All patients presented one hemizygous/heterozygous variant in MAMLD1. In parentheses: patients in Camats et al. (2015); NA, not analyzed; d, day(s); m, month(s); y, year(s). (A) Filtered by DSD-related and MAMLD1-related gene list. (B) Number of candidate variants per patient: related to sex development, DSD phenotypes, and/or in MAMLD1-related genes, and with MAF ≤ 0.01. (C) Number of candidate genes per patient: genes containing at least one candidate variant per patient. Flück et al. Oligogenic MAMLD1 DSD Phenotypes The genomic datasets were annotated (alignment with human predictors from dbscSNV ADA and RF and SPIDEX splicing genome hg19/grch37) and filtered with the functional annotation predictor (DPSI), and those splicing predictors from Alamut of genetic variants from HTS data (ANNOVAR; http://annovar. Visual software were: SSF, MaxEnt, NNSPLICE, GeneSplicer openbioinformatics.org/) (Wang et al., 2010), visualized and and Ex-Skip. explored in Integrative Genomics Viewer (IGV, Broad Institute, The following bioinformatics software tools were used for Cambridge, MA, USA; https://www.broadinstitute.org/igv/ the interpretation and classification of variants: InterVar (http:// (Robinson et al., 2011). Frequencies of variants of relevant wintervar.wglab.org/, clinical interpretation of genetic variants candidate genes were obtained from the Genome Aggregation by the ACMG/AMP 2015 guideline), VarSome (The Human Database (gnomAD; http://gnomad.broadinstitute.org/about) Genomics Search Engine; https://varsome.com/), ClinVar (Lek et al., 2016) and the Collaborative Spanish Variant Server (https://www.ncbi.nlm.nih.gov/clinvar/) and Alamut Visual 2.11 (CSVS; CIBERER BIER, Valencia, Spain; http://csvs.babelomics. (https://www.interactive-biosoftware.com/es/alamut-visual/). org/; August 2018) (Dopazo et al., 2016). gnomAD includes We searched for reported (potentially) disease-causing variants gene variants from exome and genome sequencing data: 123,136 with the Human Gene Mutation Database (HGMD® Professional exomes and 15,496 genomes from unrelated individuals (from 2018.2, http://www.biobase-international.com/product/hgmd; population and disease-specific studies). CSVS database includes Biobase) and dbSNP (http://www.ncbi.nlm.nih.gov/snp/). We (among others) exomes from a population of 267 healthy used STRING for the search for interactions within genes carriers unrelated subjects. of interesting variants (DSD-related and/or MAMLD1-related). WES data were filtered by a disease-tailored list of MAMLD1- Data from STRING are extracted from known interactions related and DSD-related known and candidate genes (n = 606) (curated databases, experimentally determined interactions), similar to the algorithm previously set up for Camats et al., predicted interactions (gene neighborhood, gene fusions, (2018). We generated a project-specific filter for DSD-related gene co-occurrence) and other inferred evidences such as text and MAMLD1-related genes by searching in published literature mining, co-expression and protein homology. We used Pubmed and databases. DSD-related genes are part of our DSD-gene (https://www.ncbi.nlm.nih.gov/pubmed/) and OMIM (https:// database and tools (Camats et al., 2018), which have been www.omim.org) to build our DSD gene list and for further currently updated. The DSD-related gene list included genes data analysis. The datasets generated for this study are publicy with reported (potentially) deleterious variants in patients available in dbSNP (Sherry, 2001): https://www.ncbi.nlm.nih. with 46,XY and 46,XX DSD, genes with reported (potentially) gov/projects/SNP/snp_viewBatch.cgi?sbid=1063030. disease-causing variants in syndromic patients with involvement of sex development, those “related” to DSD conditions in KO/ mutant animal models (mice and rats), and also overexpressed, Variant Analysis Per Patient upregulated or downregulated genes in rodent embryonic After annotation, variant analysis was performed by the gonadal cells (Camats et al., 2018). For the search for functional following steps. A) Each patient’s WES data were first filtered by human partners of MAMLD1 and for possible interactions within our MAMLD1- and DSD-related known and candidate genes. interesting genes, the Search Tool for the Retrieval of Interacting B) We kept variants with MAF (minor allele frequency) ≤0.015 Genes/Proteins (STRING, http://string-db.org/) (Jensen et al., or not detected in gnomAD, and variants with the following 2009) and the Biological General Repository for Interaction predicted type, consequences and locations: splicing (intronic Datasets (BioGRID, thebiogrid.org) (Stark, 2006) were used. or exonic), exonic, intergenic, regulatory. C) We confirmed the We used 30 pathogenic predictors to predict possible impact correct annotation and location of variants by checking their of amino acid substitutions on the structure, function and alignment data in IGV (alignment with human genome hg19/ evolutionary conservation of corresponding human proteins grch37) (data not shown). D) We excluded variants that were and to predict impact on splicing. These in-silico predictors were considered non-relevant for our study: E.g. 1) variants found in accessed through ANNOVAR (Wang et al., 2010) annotation more than two patients, 2) variants in repeat regions, 3) variants and run through Alamut Visual 2.11 (https://www.interactive- in genes or gene regions with high variability, 4) variants with low biosoftware.com/es/alamut-visual/). Functional exonic predictors coverage and/or low quality, 5) variants with non-similar allelic were CADD (Combined Annotation Dependent Depletion depths. E) We revised variants with the annotated pathogenic of single-nucleotide and insertion/deletion variants, http:// predictors: functional exonic, evolutionary-conservation and cadd.gs.washington.edu/) (Kircher et al., 2014), SIFT (Scale- splicing predictors (ANNOVAR and Alamut Visual software), as invariant feature transform; http://sift.jcvi.org/), PolyPhen-2 previously described. F) We run InterVar and VarSome to classify (Polymorphism Phenotyping v2: HumDiv, HumVar; http:// the variants, searched for reported (potentially) human disease- genetics.bwh.harvard.edu/pph2/index.shtml), Provean (http:// causing variants with the HGMD, and revised evidences of provean.jcvi.org), MutationAssessor (http://mutationassessor. relationship with DSD, sex development and clinical phenotype org/r3/), Mutation Taster (http://www.mutationtaster.org/), LRT, of each patient with literature and database search. G) We FATHEMM, Fathmm-MKL, PROVEAN, VEST3 (Variant Effect used STRING to find out interactions among genes carriers of Scoring Tool), MetaSVM, MetaLR, MCAP, DANN and fitCons. interesting variants (DSD-related and/or MAMLD1-related) Exonic predictors on evolutionary conservation were: GERP++, (Figures 1 and 2). H) We checked MAF in a healthy cohort of phyloP (vertebrate and mammalians), phastCons (vertebrate Spanish population (CSVS: 267 unrelated healthy controls). and mammalians) and SiPhy. Splicing predictors were: splicing I) We rejected variants with MAF ≥ 0.01 (gnomAD, CVSV, Frontiers in Genetics | www.frontiersin.org 4 August 2019 | Volume 10 | Article 746 Flück et al. Oligogenic MAMLD1 DSD Phenotypes FIGURE 1 | Interaction network of DSD- and MAMLD1-related genes identified in DSD individuals harboring genetic variants in MAMLD1. The scheme depicts an overview of detected genes and their interrelationship. For the search for functional human partners, the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING, http://string-db.org/) was used. Nodes represent proteins. Filled nodes show proteins with known or predicted 3D structure. Empty nodes depict proteins with unknown 3D structure. Candidate genes are underlined. Known interactions correspond to curated databases (turquoise lines) and experimentally determined interactions (pink lines). Predicted interactions correspond to gene neighborhood (green lines), gene fusions (red lines) and gene co-occurrence (blue lines). Other interactions correspond to text mining (yellow lines), co-expression (black lines) and protein homology (violet lines). August 2018), thus less plausible to be a DSD-causing variant. genes: GLI2 (associated to gonadal/genital anomalies) and Importantly, synonymous variants were not rejected because it RECQL4 (associated to syndromic hypospadias) (Table 2). has been shown that they may affect splicing. Patient 4 had four variants in four genes: CDH23, COL9A3, MAML1 and NOTCH1; all, except MAML1, have been proposed to be associated with gonadal development (Table 2). In patient RESULTS 5, six variants in six genes were found: BNC2, FGF10, HSD3B2, IRX5, MAML2 and NOTCH2; all, except MAML2, have also been WES performed in eight unrelated subjects (seven 46,XY and associated with hypospadias or gonadal development (Table 2). one 46,XX) harboring hemizygous/heterozygous MAMDL1 Patient 6 carried 16 variants in 16 genes: ATF3, BNC2, CYP1A1, variants revealed several candidate gene variants that potentially EYA1, FLNA, FRAS1, GLI3, HOXA13, IRX5, IRX6, MAML1, contribute to each patient’s phenotype. A detailed summary NRP1, MAML3, PROP1, PTPN11 and WDR11 (Table 2). Thirteen of patients’ characteristics and number of variants and genes of these genes are associated with risk of hypospadias and/or is shown in Table 1. A list of identified candidate variants and syndromes that include abnormal gonadal/genital development, corresponding information from literature is given in Table 2. whereas MAML1 is unrelated, MAML3 has been proposed to We identified a total of 55 potentially deleterious/candidate be associated with female gonadal development and PROP1 heterozygous/hemizygous variants in 41 genes in the eight has only been associated with anterior pituitary insufficiency/ hemizygous/heterozygous MAMLD1 patients (Tables 1 and 2). hypogonadotropic hypogonadism. In addition, six of these genes In the seven 46,XY patients 1–16 variants were found in a total have previously been described in patients with aortic diseases of 1–16 genes, while the 46,XX MAMLD1 patient revealed 14 and cardiopathies (Table 2). Patient 7 presented five variants in additional variants in 13 genes. (Tables 1 and 2). five genes: EVC, MAML3, NOTCH2, PPARGC1B and WDR11; Patient 1 harbored nine variants in seven genes: CYP1A1, four of them associated with hypospadias or male gonadal EVC, GRID1, NOTCH1, RET, RIPK4 and ZBTB16, all of them development and one, MAML3, with female gonadal development associated to gonadal/genital anomalies (Table 2). Patient 2 (Table 2). Finally, patient 8 harbored 14 variants in 13 genes: carried one variant in RECQL4, associated with syndromic CUL4B, DAPK1, EMX2, FREM2, IGFBP2, MAML2, MAML3, hypospadias (Table 2). Patient 3 presented two variants in two MYO7A, NOTCH1, PIK3R3, TGFBI, WNT9A and WNT9B. Frontiers in Genetics | www.frontiersin.org 5 August 2019 | Volume 10 | Article 746 Flück et al. Oligogenic MAMLD1 DSD Phenotypes FIGURE 2 | Interaction networks of DSD- and MAMLD1-related genes identified per single MAMLD1 individual. (A) to (F) correspond to the interaction networks per patient. For the search for functional human partner, the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING, http://string-db.org/) was used. Nodes represent proteins. Filled nodes show proteins with known or predicted 3D structure. Empty nodes depict proteins with unknown 3D structure. Candidate genes are underlined. Known interactions correspond to curated databases (turquoise lines) and experimentally determined interactions (pink lines). Predicted interactions correspond to gene neighborhood (green lines), gene fusions (red lines) and gene co-occurrence (blue lines). Other interactions correspond to text mining (yellow lines), co-expression (black lines) and protein homology (violet lines). Genes with no interactions are on the right side of each network. Frontiers in Genetics | www.frontiersin.org 6 August 2019 | Volume 10 | Article 746 TABLE 2 | Identified genes and variants per patient after specific filtering. Frontiers in Genetics | www.frontiersin.org Flück et al. Patient Gene Chromosome: Type/ HGVSc,HGVSp dbSNP ID gnomAD: CSVS: Predictors Interpretation/classification (6) Evidence Coordinates consequence MAF MAF Exonic Exonic Exonic Splicing Splicing InterVar ClinVar VarSome ACMG HGMD: Gene characteristics: predictor: predictors: predictors: predictor predictors variant (7) evidences for genotype- CADD (1) Functional conservation (4) (Alamut) phenotype correlation (8) impact (2) (3) (5) 1 CYP1A1 15:75013544 snv/missense NM_000499.5:c.1162C>G:p. – ND ND 24.9 13 6 0 5 VUS ND – VUS: PM2, No Association to hypospadias (His388Asp) PP3 (van der Zanden et al., 2012) 1 EVC 4:5798754 snv/missense NM_153717.2:c.1892C>T:p. rs139481521 0.0003 ND 23.5 5 3 0 3 VUS VUS – VUS: BP4 No Syndromic (Ellis–van (Thr631Met) Creveld syndrome) micropenis (D’Asdia et al., 2013; Ibarra-Ramirez et al., 2017), gonadal development? (Beverdam and Koopman, 2006; Jameson et al., 2012) 1 EVC 4:5785368 snv/ NM_153717.2:c.1653G>A:p. rs151293705 0.0003 ND 2.382 NA NA 1 3 Likely other VUS VUS: BP7 No Syndromic (Ellis–van synonymous (Pro551=) benign Creveld syndrome) micropenis (D’Asdia et al., 2013; Ibarra-Ramirez et al., 2017), gonadal development? (Beverdam and Koopman, 2006; Jameson et al., 2012) 1 GRID1 10:87966142 snv/missense NM_017551.2:c.499A>G:p. rs956188880 0.00004 ND 10.04 1 5 0 NA VUS ND – VUS: BP4 No Candidate to hypospadias (Met167Val) (van der Zanden et al., 2012) 1 NOTCH1 9:139399213 snv/ NM_017617.5:c.4930C>T:p. rs568700183 0.0003 ND 0.018 NA NA 0 4 Likely Likely Likely Likely benign: No Related to SHH and FGF10 7 synonymous (Leu1644=) benign benign|Likely benign BP6, BP7 (Grinspon and Rey, 2014) benign 1 RET 10:43609955 snv/missense NM_020975.5:c.1907C>T:p. rs1035958105 0.00001 0 23.0 6 6 0 NA VUS VUS VUS Likely DM; thyroid Syndromic (CAKUT (Thr636Met) pathogenic: carcinoma syndrome) cryptorchidism PM1, PM2, (Chatterjee et al., 2012), PP2, PP3 gonadal development? (Jameson et al., 2012; Li et al., 2014) 1 RIPK4 21:43161468 snv/missense NM_020639.2:c.1885G>A:p. rs199669994 0.00004 ND 7.899 4 4 NA NA VUS ND – VUS: PM2 BP1 No Syndromic (Popliteal (Asp629Asn) pterygium syndrome) genital hypoplasia (Mitchell et al., 2012), micropenis, hypoplastic scrotum, inguinal hernia (Kalay et al., 2012), gonadal development? (Beverdam and Koopman, 2006; Jameson et al., 2012) 1 RIPK4 21:43176830 snv/missense NM_020639.2:c.329C>T:p. rs200823657 0.00005 ND 22.7 3 5 0 NA VUS NDw – VUS: PM2 BP1 No Syndromic (Popliteal Oligogenic MAMLD1 DSD Phenotypes Month 2019 | Volume 10 | Article 746 (Ser110Leu) pterygium syndrome) genital hypoplasia (Mitchell et al., 2012), micropenis, hypoplastic scrotum, inguinal hernia (Kalay et al., 2012), gonadal development? (Beverdam and Koopman, 2006; Jameson et al., 2012) 1 ZBTB16 11:114027160 snv/intronic NM_006006.5:c.1366+4G>C – ND ND 13.81 NA NA 1 4 ND ND – VUS: PM2 BP1 No Syndromic (deletion 11q23) cryptorchidism and micropenis (Fischer et al., 2008) (Continued) TABLE 2 | Continued Frontiers in Genetics | www.frontiersin.org Flück et al. Patient Gene Chromosome: Type/ HGVSc,HGVSp dbSNP ID gnomAD: CSVS: Predictors Interpretation/classification (6) Evidence Coordinates consequence MAF MAF Exonic Exonic Exonic Splicing Splicing InterVar ClinVar VarSome ACMG HGMD: Gene characteristics: predictor: predictors: predictors: predictor predictors variant (7) evidences for genotype- CADD (1) Functional conservation (4) (Alamut) phenotype correlation (8) impact (2) (3) (5) 2 RECQL4 8:145738828 snv/missense NM_004260.3:c.2237C>T:(p. rs201883228 0.0002 ND 26.8 6 5 NA NA VUS VUS VUs VUS: BP1 No Syndromic (Rothmund– Ala746Val) Thomson syndrome) hypospadias, bilateral inguinal hernia (Kellermayer et al., 2005) 3 GLI2 2:121747688 snv/missense NM_005270.4:c.4198G>T:p. rs143914758 0.0001 ND 23.4 7 2 NA NA Likely ND VUS VUS: - No Increased risk of (Gly1400Cys) benign hypospadias (Carmichael et al., 2013), male gonadal development (Jameson et al., 2012), masculinization of male external genitalia (Rey et al., 2000) 3 RECQL4 8:145737701 snv,missense NM_004260.3:c.3062G>A:p. rs34666647 0.004 2.375 2 0 0 4 Likely other|Benign Benign Likely benign: Cancer Syndromic (Rothmund– (Arg1021Gln) benign PM2, PM5, Thomson Syndrome) BP1, BP4 hypospadias, bilateral inguinal hernia (Kellermayer et al., 2005) 4 CDH23 10:73559034 snv/nonsense NM_022124.5:c.7221C>A:p. rs779038178 ND 0.002 35 5 6 1 3 Likely ND – Pathogenic: No Gonadal development? (Tyr2407*) pathogenic PVS1, PM2, (Jameson et al., 2012; Li PP3 et al., 2014) 4 COL9A3 20:61448956 snv/missense NM_001853.4:c.116C>G:(p. rs1028982816 0.00001 ND 22.7 12 5 0 NA VUS ND – VUS: PP3 No Male gonadal development (Pro39Arg) (Nef et al., 2005; Beverdam and Koopman, 2006; Jameson et al., 2012) 8 4 MAML1 5:179193385 snv/ NM_014757.4:c.1374C>T:p. rs61748799 0.003 0.004 0.089 NA NA 0 1 Likely ND – VUS: BP7 No No (MAMLD1-related) synonymous (Asp458=) benign 4 NOTCH1 9:139401803 snv/ NM_017617.5:c.3597C>T:p. rs150666307 0.00009 ND 11.70 NA NA 0 3 Likely Likely benign Likely Likely benign: No Related to SHH and FGF10 synonymous (Leu1199=) benign benign BP6, BP7 (Grinspon and Rey, 2014) 5 BNC2 9:16436324 snv/missense NM_017637.5:c.1868C>A:p. rs114596065 0.0022 0.002 5.290 8 6 NA NA Benign Benign Benign VUS: BP6 No Hypospadias (Bhoj et al., (Pro623His) 2011; van der Zanden et al., 2012; Baxter et al., 2015; Kon et al., 2015), gonadal development? (Jameson et al, 2012) 5 FGF10 5:44388817 snv/upstream/ NG_011446.1:c.-33G>A rs17233910 0.005 0.002 19.99 NA NA NA NA ND ND – VUS: - No Increased risk hypospadias missense in human (van der Zanden et al., 2012; Carmichael et al., 2013; Svechnikov et al., 2014); development of the glans penis (Rey et al., 2000) 5 HSD3B2 1:119964831 snv/missense NM_000198.3:c.707T>C:p. rs35887327 0.003788 ND 8.277 3 3 NA NA Likely VUS VUS Likely DM?; Hypospadias (Codner et al., Oligogenic MAMLD1 DSD Phenotypes Month 2019 | Volume 10 | Article 746 (Leu236Ser) benign Pathogenic: HSD3B2 2004; Kon et al., 2015; PS1, PP2, deficiency Eggers et al., 2016), sex PP5, BP4 development (Baxter and Vilain, 2013; Baxter et al., 2015), hormone synthesis (McCartin et al., 2000) 5 IRX5 16:54967040 snv/missense NM_005853.5:c.707C>T:p. rs115549200 0.0088 ND 11.97 3 4 NA NA Benign ND – VUS: BP4 No Association to hypospadias (Pro236Leu) (Geller et al., 2014; Grinspon and Rey, 2014), female gonadal development? (Nef et al., 2005) (Continued) TABLE 2 | Continued Frontiers in Genetics | www.frontiersin.org Flück et al. Patient Gene Chromosome: Type/ HGVSc,HGVSp dbSNP ID gnomAD: CSVS: Predictors Interpretation/classification (6) Evidence Coordinates consequence MAF MAF Exonic Exonic Exonic Splicing Splicing InterVar ClinVar VarSome ACMG HGMD: Gene characteristics: predictor: predictors: predictors: predictor predictors variant (7) evidences for genotype- CADD (1) Functional conservation (4) (Alamut) phenotype correlation (8) impact (2) (3) (5) 5 MAML2 11:95826473 snv/missense NM_032427.3:c.722G>A:p. rs111958464 0.005 ND 32 6 6 0 NA VUS ND – VUs: - No No (MAMLD1-related) (Arg241Gln) 5 NOTCH2 1:120469147 snv/ NM_024408.3:c.3980A>G:p. rs61752484 0.0037 0.004 20.4 6 6 0 NA Likely Benign Benign VUS: BP6 DM?; Primary ovarian failure nonsynonymous (Asp1327Gly) benign cardiopathy (Patiño et al., 2017); male gonadal development? (Jameson et al., 2012) 6 ATF3 1:212788544 snv/missense NM_001674.3:c.181G>T:p. – ND ND 7.826 3 5 1 NA Benign ND – VUS: PM2, No Hypospadias (Beleza- (Ala61Ser) BP4 Meireles et al., 2008; van der Zanden et al., 2012), female gonadal development? (Jameson et al., 2012) 6 BNC2 9:16436324 snv/missense NM_017637.5:c.1868C>A:p. rs114596065 0.0022 0.002 5.290 8 6 NA NA Benign Benign Benign VUS: BP6 No Hypospadias (Bhoj et al., (Pro623His) 2011; van der Zanden et al., 2012; Baxter et al., 2015; Kon et al., 2015), gonadal development? (Jameson et al., 2012) 6 CYP1A1 15:75012979 snv/missense NM_001319216.2:c.1303C> rs41279188 0.0047 ND 33 11 5 NA NA VUS ND Benign VUS: PP3, BP6 No Association to hypospadias A:p.(Arg435Ser) (van der Zanden et al., 2012) 6 EYA1 8:72211882 snv/ NM_000503.5:c.630T>C:p. rs373102227 0.00008 ND 10.56 NA NA 1 4 Likely VUS VUS VUS: PP3, BP7 No Associated to hypospadias synonymous (Ser210=) benign (Grinspon and Rey, 2014; Hwang et al., 2014), male 9 gonadal development? (Jameson et al., 2012) 6 FLNA X:153596078 snv/ NM_001456.3:c.651C>T:p. rs34644500 0.0002 ND 5.473 NA NA 1 3 Likely Likely benign Likely Likely benign: No Hypospadias, synonymous (Asp217=) benign benign BP4, BP6, cryptorchidism, diminished BP7 androgen receptor (Carrera- García et al., 2017), female gonadal development? (Jameson et al., 2012) 6 FRAS1 4:79334181 snv/missense NM_025074.6:c.4367T>C:p. rs560902495 0.00003 ND 24.6 12 5 NA NA VUS ND – VUS: PM2, No Syndromic (Fraser (Ile1456Thr) PP3, BP1 syndrome) abnormal genitourinary system (Retterer et al., 2016; Kornacki et al., 2017); female gonadal development? (Jameson et al., 2012) 6 GLI3 7:42066017 snv/intronic NM_000168.5:c.1029-6G>A rs748670269 0.00002 ND 0.004 NA NA 0 3 NA ND – VUS: BP4 No Increased risk of hypospadias (Carmichael Oligogenic MAMLD1 DSD Phenotypes Month 2019 | Volume 10 | Article 746 et al., 2013), early genital primordia (Rey and Grinspon, 2011), female gonadal development? (Jameson et al., 2012) (Continued) TABLE 2 | Continued Frontiers in Genetics | www.frontiersin.org Flück et al. Patient Gene Chromosome: Type/ HGVSc,HGVSp dbSNP ID gnomAD: CSVS: Predictors Interpretation/classification (6) Evidence Coordinates consequence MAF MAF Exonic Exonic Exonic Splicing Splicing InterVar ClinVar VarSome ACMG HGMD: Gene characteristics: predictor: predictors: predictors: predictor predictors variant (7) evidences for genotype- CADD (1) Functional conservation (4) (Alamut) phenotype correlation (8) impact (2) (3) (5) 6 HOXA13 7:27239079 snv/missense NM_000522.4:c.618C>G:p. rs774388075 0.00002 ND 22.3 5 5 NA NA VUS ND – VUS: - No Associated to hypospadias (Phe206Leu) (Beleza-Meireles et al., 2007; van der Zanden et al., 2012; syndromic, Hand- foot-genital)/Guttmacher syndrome) hypospadias (Innis et al., 2002), small penis (Goodman et al., 2000), genital tubercle development (Grinspon and Rey, 2014) 6 IRX5 16:54965347 deletion/inframe NM_005853.5:​ rs1057518726 ND ND – NA NA NA NA NA VUS VUS VUS: PM4: No Association to hypospadias c.240_242delCTC:p.(Ser81del) nonframeshift (Geller et al., 2014; deletion Grinspon and Rey, 2014), female gonadal development? (Nef et al., 2005) 6 IRX6 16:55362842 snv/missense NM_024335.2:c.952T>A:p. rs61743419 0.0014 ND 5.599 3 4 0 NA VUS ND – VUS: - No entry for Associated to hypospadias (Phe318Ile) this gene (Grinspon and Rey, 2014) 6 MAML1 5:179193168 snv/missense NM_014757.4:c.1157G>T:p. rs777367230 0.0001 0.003 22.9 9 5 0 NA VUS ND – VUS: - No No (MAMLD1-related) (Gly386Val) 6 MAML3 4:140811687 snv/ NM_018717.5:c.903C>T:p. rs76066862 0.0015 0.002 2.445 NA NA NA 2 Likely ND – VUS: - No Female gonadal NOT synonymous (Asp301=) benign development? (Jameson 10 CONFIRMED et al., 2012) 6 NRP1 10:33469272 snv/missense NM_003873.5:c.2504G>A:p. – ND ND 5.556 6 4 NA NA VUS ND – VUS: PM2 No DSD (Baxter et al., 2015), (Gly835Asp) gonadal development? (Jameson et al., 2012) 6 PROP1 5:177421299 deletion/ NM_006261.4:c.150delA:p. rs587776683 ND ND – NA NA NA NA NA Pathogenic Likely VUS: PVS1, DM; No, hypogonadotropic frameshift (Arg53Aspfs*112) pathogenic PP5 pituitary hipogonadism (Reynaud hormone et al., 2005; Reynaud et al., deficiency 2005; Baxter and Vilain, 2013; Baxter et al., 2015; Eggers et al., 2016) 6 PTPN11 12:112856827 snv/upstream NM_002834.4:c.-89G>A – ND ND 16.23 NA NA NA NA NA ND – VUS: PM2, No Syndromic (Noonan PP3 syndrome) cryptorchidism (Tartaglia et al., 2002) 6 WDR11 10:122668121 snv/missense NM_018117.11:c.3571G>A:p. rs149486212 0.0001 0.004 34 14 5 NA NA VUS ND – VUS : PP2, No Hypospadias (Eggers et al., (Gly1191Ser) PP3 2016, Fan et al., 2017), small testes (Fan et al., 2017) 7 EVC 4:5800455 snv/missense NM_153717.2:c.2240C>T:p. rs151091776 0.0002 ND 18.37 5 4 0 NA VUS ND – VUS: BP4 No Syndromic (Ellis–van (Ala747Val) Creveld syndrome) Oligogenic MAMLD1 DSD Phenotypes Month 2019 | Volume 10 | Article 746 hypospadias (D’Asdia et al., 2013) and micropenis (Ibarra-Ramirez et al., 2017), gonadal development? (Beverdam and Koopman, 2006; Jameson et al., 2012) 7 MAML3 4:140811709 snv/missense NM_018717.5:c.881A>G:p. rs115966590 0.0028 ND 13.26 7 6 NA NA VUS ND – VUS: - No Female gonadal (Asn294Ser) development? (Jameson et al., 2012) 7 NOTCH2 1:120458982 snv/missense NM_024408.3:c.6363G>C:p. rs144047610 0.0004 0.002 23.4 9 5 NA NA VUS VUS VUS VUS: - DM?, Primary ovarian failure (Lys2121Asn) Bicuspid (Patiño et al., 2017); male aortic valve gonadal development? (Jameson et al., 2012) (Continued) TABLE 2 | Continued Frontiers in Genetics | www.frontiersin.org Flück et al. Patient Gene Chromosome: Type/ HGVSc,HGVSp dbSNP ID gnomAD: CSVS: Predictors Interpretation/classification (6) Evidence Coordinates consequence MAF MAF Exonic Exonic Exonic Splicing Splicing InterVar ClinVar VarSome ACMG HGMD: Gene characteristics: predictor: predictors: predictors: predictor predictors variant (7) evidences for genotype- CADD (1) Functional conservation (4) (Alamut) phenotype correlation (8) impact (2) (3) (5) 7 PPARGC1B 5:149219653 snv/missense NM_133263.3:c.2668G>A:p. rs150637009 0.0056 ND 19.13 5 5 0 NA VUS ND – VUS: BP4 No Candidate to hypospadias (Ala890Thr) (van der Zanden et al., 2012) 7 WDR11 10:122637900 snv/missense NM_018117.11:c.1592C>G:p. rs775506715 0.00004 ND 24.1 14 6 1 NA VUS ND – VUS: PP2, PP3 No Hypospadias (Eggers et al., (Ser531Cys) 2016, Fan et al., 2017), small testes (Fan et al., 2017) 8 CUL4B X:119708447 snv/missense NM_003588.3:c.26G>A:p. rs149016283 0.0002 ND 18.73 1 NA 1 NA Likely Likely benign Likely Likely benign: No Abnormal genitourinary (Gly9Glu) benign benign BP4, BP6 system (Retterer et al., 2016) 8 DAPK1 9:90321476 snv/missense NM_004938.3:c.3490G>A:p. rs937952689 0.00007 ND 24.3 8 5 NA NA VUS ND – VUS: - No Female gonadal (Asp1164Asn) development? (Jameson et al., 2012) 8 EMX2 10:119305133 snv/intronic NM_004098.3:c.407-10C >T – 0.000004 ND 9.098 NA NA 1 4 ND ND – VUS: BP4 No 46,XX DSD (Liu et al., 2015), sex determination (Biason-Lauber, 2010; Jakob and Lovell-Badge, 2011; Eggers and Sinclair, 2012), (female) gonadal development (Rey et al., 2000; Jameson et al., 2012; Grinspon and Rey, 2014), (Jameson et al., 11 2012); 46,XY DSD (Piard et al., 2014) 8 FREM2 13:39454885 insertion/ NM_207361.5:c.9472dupC:p. – ND ND – NA NA NA NA ND ND – Likely No Syndromic (Fraser frameshift (Gln3160Thrfs*6) pathogenic: syndrome) abnormal PVS1, PM2 genitalia (De Bernardo et al., 2015), female gonadal development (Jameson et al., 2012) 8 IGFBP2 2:217526593 snv/missense NM_000597.3:c.685C>A:p. – ND ND 23.7 8 6 0 NA VUS ND – VUS: PM2, No entry for Candidate gene in ovary (Gln229Lys) PP3 this gene development (Clement et al., 2007), female gonadal development? (Jameson et al., 2012; Munger et al., 2013). 8 MAML2 11:95826575 snv/missense NM_032427.3:c.620G>A:p. rs191391876 0.0002 ND 24.4 9 5 0 NA VUS ND – VUS: - No No (MAMLD1-related) (Arg207His) 8 MAML3 4:140811709 snv/missense NM_018717.5:c.881A>G:p. rs115966590 0.0028 0.004 13.26 7 6 NA NA VUS ND – VUS: - No Female gonadal (Asn294Ser) development? (Jameson et al., 2012) Oligogenic MAMLD1 DSD Phenotypes Month 2019 | Volume 10 | Article 746 8 MYO7A 11:76883787 delins/intronic NM_000260:c.1798-7_1798- – ND ND – NA NA NA NA ND ND – – No Male gonadal development? 6delCCinsAT (Jameson et al., 2012; Li et al., 2014) 8 MYO7A 11:76883790 delins/ NM_000260: c.1798- – ND ND – NA NA NA NA ND ND – – No Male gonadal development? intronic-exonic 4_1801delinsGGCTGCT (Jameson et al., 2012; Li et al., 2014) 8 NOTCH1 9:139405111 snv/missense NM_017617.5:c.2734C>T:p. rs201620358 0.002 ND 31 12 6 0 3 Likely VUS VUS VUS: PP3 No Related to SHH and FGF10 (Arg912Trp) benign (Grinspon and Rey, 2014) 8 PIK3R3 1:46521570 snv/missense NM_003629.3:c.838G>A:p. rs186728731 0.0001 ND 25.5 6 6 0 2 VUS ND – VUS: PP3 No Female gonadal (Asp280Asn) development? (Beverdam and Koopman, 2006; Jameson et al., 2012) (Continued) Flück et al. Oligogenic MAMLD1 DSD Phenotypes Among them, only MAML2 has not been related to gonadal or uterus (Waschk et al., 2016), phenotype correlation (8) system (Carroll et al., 2005; evidences for genotype- Mayer–Rokitansky–Küster– Hauser syndrome (Waschk All patients were heterozygote for these variants and were checked and confirmed through IGV software (alignment with human genome hg19/grch37; https://www.broadinstitute.org/igv/, Broad Institute, Cambridge, MA, USA). (1) Combined exonic predictor CADD: Combined Annotation Dependent Depletion (cutoff score: consensus Beverdam and Koopman, development? (Nef et al., organogenesis urogenital Grinspon and Rey, 2014) Gene Mutation Database Biobase (HGMD® Professional 2018.2, http://www.biobase-international.com/product/hgmd): reported variant: variant class, reference, clinical association Human Gene Mutation Database Biobase. (8) Gene characteristics: evidences for genotype-phenotype correlation. Data obtained from pubmed, HGMD genitourinary system development (Table 2). NNSPLICE, GeneSplicer, Ex-Skip. (6) InterVar: Clinical Interpretation of genetic Variants by ACMG/AMP guideline (http://wintervar.wglab.org/); ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/); VarSome: The Human Genomics Search Engine (https://varsome.com/); ACMG: ACMG classification from VarSome search engine. (7) Human Gene characteristics: et al., 2016); bicornuate Gonadal development? predictors (evolutionary conservation) (ANNOVAR, 6): GERP++, phyloP (vertebrate and mammalians), phastCons (vertebrate and mammalians), SiPhy. (4) Splicing predictors (ANNOVAR, 3): splicing predictor from dbscSNV ADA and RF, and SPIDEX splicing predictor (DPSI). (5) Splicing predictors (Alamut software, 5): SSF, MaxEnt, (Jameson et al., 2012, 2005; Beverdam and Clement et al., 2007, ≥20; deleterious for DSD ≥18; contained in ANNOVAR). (2) Exonic predictors (funcional impact) (ANNOVAR, 15): SIFT, PolyPhen2 HumDiv, PolyPhen2 HumVar, LRT, MutationTaster, MutationAssessor, FATHEMM, Fathmm-MKL, PROVEAN, VEST3 (Variant Effect Scoring Tool), MetaSVM, MetaLR, MCAP, DANN, fitCons. (3) Exonic The following genes showed variants in two patients: CYP1A1 Koopman, 2006) Female gonadal in patients 1 and 6; EVC in patients 1 (2 variants) and 7; IRX5 in Evidence patients 5 and 6; MAML1 in patients 4 and 6; MAML2 in patients 2006) 5 and 8; NOTCH2 in patients 5 and 7; RECQL4 in patients 2 and variant (7) 3 and WDR11 in patients 6 and 7 (Table 2). In addition, 2 genes HGMD: No No No presented variants in 3 patients: MAML3 (patients 6, 7 and 8) and NOTCH1 (patients 1, 4 and 8). Furthermore, RIPK4 presented VUS: PM2 2 variants in patient 1. Finally, BNC2 variant c.1868C>A:p. ACMG VUS: - VUS: - (Pro623His) (MAF = 0.002) was detected in 2 patients (patient Interpretation/classification (6) 1 and 7) and MAML3 variant c.881A>G:p.(Asn294Ser) (MAF = VarSome 0.0028) in patients 7 and 8 (Table 2). – – – We performed interactome analysis for the identified DSD genes using bioinformatic tools for the analysis of possible gene- ClinVar protein interactions. The network comprising all genes identified ND ND ND is shown in Figure 1. Overall, a connection was found for 27 of the 41 genes. MAMLD1 connects directly to MAML1/2/3. Via InterVar VUS VUS ND NOTCH1/2 8 genes are in connection with MAMLD1, namely WNT9A/9B, GLI2/3, FGF10, RET, PROP1 and NRP1. Some of predictor: predictors: predictors: predictor predictors (Alamut) Splicing these genes are also central nodes for further connections; e.g. NA NA (5) 4 GLI3 for EVC, FGF10, GLI2, RIPK4 and EYA1; and RET for PIK3R3 with PTPN11, which also is connected with RIPK4. Splicing NA (4) 0 0 RIPK4 itself is a central node for ZBTB16, CUL4B, GLI3 and PTPN11. NRP1 is connected to FLNA and EYA1 connects CADD (1) Functional conservation Predictors Exonic with FRAS1 and FREM2. In addition, 2 isolated gene couples NA (3) 5 4 have been revealed by our analysis: CYP1A1-HSD3B2 and MYO7A-CDH23. These observations give an idea of the complex impact (2) Exonic interactions among genes related to sex development. NA 11 5 The specific interactome of identified genes in patients 1 and 4 to 8 is shown in Figure 2. In patients 1, 4, 5, 7 and 8, MAMLD1 Exonic 4.531 21.1 34 and MAMLD1-related genes (MAML1, MAML2 or MAML3) are directly related to NOTCH1/2 (Figures 2A–C, E, F). In patient 1, CSVS: 0.007 0.004 MAF ND there are 2 networks: ZBTB16-RIPK4 and MAMLD1-NOTCH1- RET (Figure 2A). In patient 6, GLI3, EYA1 and FRAS1 as well as gnomAD: 0.00002 0.0010 0.004 MAF FLNA and NRP1 seem directly related (Figure 2D). In patient 8, NOTCH1 plays a central role connecting to WNT9A, WNT9B rs147650812 rs145836311 rs530502749 dbSNP ID and MAMLD1 network (Figure 2F). DISCUSSION NM_003395.3:c.1070G>A:p. NM_000358.2:c.2012-5T>C NM_003396.2:c.134C>T:p. and String. ND, not detected; -, not shown; NA, not analysed; no, not previously related to DSD. Sex development is a very complex biological event which requires HGVSc,HGVSp the concerted collaboration of a large network of genes in a spatial (Arg357His) (Pro45Leu) and temporal correct fashion. In the past, much has been learned about human sex development from monogenic DSD, but the broad spectrum of phenotypes in numerous DSD individuals remains consequence snv/missense snv/missense snv/intronic a conundrum. Oligogenic disease has been proposed. In fact, Type/ multiple genetic hits, which might not be deleterious by themselves, have been found in several individuals with DSD (Kon et al., 2015; Chromosome: 5:135398870 1:228109247 17:44949939 Coordinates Eggers et al., 2016; Mazen et al., 2016; Werner et al., 2017; Camats et al., 2018). In a previous study of 46,XY DSD patients carrying MAMLD1 variants, we showed that none of the variants were functionally pathogenic except for a stop variant (Camats et al., TABLE 2 | Continued WNT9B WNT9A TGFBI Gene 2015). In the present study, we searched for additional genetic hits in DSD patients harboring MAMLD1 mutations and manifesting with Patient unexplained broad phenotypes. Using HTS and a custom-made 8 8 8 Frontiers in Genetics | www.frontiersin.org 12 August 2019 | Volume 10 | Article 746 Flück et al. Oligogenic MAMLD1 DSD Phenotypes algorithm including DSD- and MAMLD1-related genes from Table 2). Five of these genes (DAPK1, IGFBP2, MAML3, PIK3R3 literature and databases, we identified potentially deleterious and WNT9A) have so far only been related to female gonadal genetic variants in additional genes in all MAMLD1 individuals. development (Table 2). Thus, we believe that the broad phenotype of individuals carrying Overall, the genes detected in our eight studied patients with MAMLD1 variants is due to additional genetic hits. MAMLD1 variants have been previously reported in humans In our study, we identified 55 additional heterozygous/ with hypospadias, cryptorchidism, micropenis, and other hemizygous variants in 41 genes in seven 46,XY DSD hemizygous urogenital abnormalities; or they have been found involved in and one 46,XX DSD heterozygous MAMLD1 patients. Among sexual and gonadal development. Also, some of them have been the 41 genes, 16 have been previously reported in humans with associated with specific syndromes in patients with genitourinary hypospadias (ATF3, BNC2, CYP1A1, EMX2, EYA1, FLNA, anomalies: CAKUT syndrome, Ellis–van Creveld syndrome, GLI3, GRID1, GLI2,, FGF10, HOXA13, HSD3B2, IRX5, IRX6, Fraser syndrome 1, Fraser syndrome 2, hand–foot–genital/ PPARGC1B and WDR11 (Table 2); 8 have been related to Guttmacher syndrome, Noonan syndrome, Mayer–Rokitansky– cryptorchidism (BNC2, FLNA, RET, RECQL4, NRP1, PTPN11, Küster–Hauser syndrome, Popliteal pterygium syndrome and RIPK4 and ZBTB16), and 5 genes have been found in patients with Rothmund–Thomson syndrome (see Table 2). However, none of micropenis (BNC2, EVC, FGF10, RIPK4 and ZBTB16). Also, 15 the present patients presented a complete phenotype for any of genes have been described in other types of DSD (CUL4B, EMX2, these syndromes, maybe because none of the variants completely FRAS1, FREM2, HSD3B2, NOTCH2 and NRP1) (Table 2) and/ impairs gene expression and protein function, as inferred by or were reported in different syndromes (CYP1A1, EVC, FRAS1, the in silico analyses. Detailed information on these genes from HOXA13, PTPN11, RECQL4, RET, RIPK4 and ZBTB16) (Table 2). current literature is given in Supplementary Materials (S1). In addition, 27 genes had been previously described in the context A search for an underlying network comprising variants in the of sex or gonadal development (ATF3, BNC2, CDH23, COL9A3, identified genes related to MAMLD1 revealed a considerable number DAPK1, EMX2, EVC, EYA1, FLNA, FRAS1, FREM2, GLI2, GLI3, of genes which showed gene-gene, gene-protein or protein-protein HOXA13, IGFBP2, IRX5, MAML3, MYO7A, NOTCH1, NOTCH2, interactions (Figures 1 and 2) suggesting that genetic variations in NRP1, PIK3R3, RET, RIPK4, TGFBI, WNT9A and WNT9B). these genes may affect sex development. In addition, MAML3 was Thirteen of these genes have been found involved in female found in a network related to female gonadal development (Jameson gonadal development and 46,XX DSD (ATF3, DAPK1, EMX2, et al., 2012). Accordingly, one variant in MAML3 was present in our FLNA, FRAS1, FREM2, GLI3, IGFBP2, IRX5, MAML3, PIK3R3, 46,XX patient. The analysis of gene/protein network interactions WNT9A and WNT9B), 8 of which in patient 8 (Table 2). per patient gives an idea of the complexity of the interactions among According to OMIM, almost all of our patients presented at genes related to sex development. The more variants detected least one variant in a gene with autosomal dominant inheritance in DSD-related genes, the better to build an interaction network (AD) (COL9A3, GLI2, FGF10, FLNA, EYA1, GLI3, HOXA13, searching for clues on genetic relationship(s) for sex development. NOTCH1, NOTCH2, PTPN11, RET, TGFB and WDR11), while In our DSD individuals carrying MAMLD1 variants, three genes other genes (CDH23, MYO7A and PPARGC1B) may have both seemed prominent in the network analysis, NOTCH1, NOTCH2 AD and autosomal recessive (AR) inheritance. FLNA and CUL4B and GLI3. NOTCH signaling is a highly conserved signaling are X-linked (XLR), while CYP1A1, FREM2, EVC, HSD3B2, IRX5, pathway and comprises 4 transmembrane receptors. It is essential PROP1, RAS1, RECQL4, RIPK4 and ZBTB16 are known for AR for the regulation of embryonic development of multiple organ inheritance. No information on inheritance is currently available systems including gonadal development (Windley and Wilhelm, for the remaining genes including ATF3, BNC2, GRID1, DAPK1, 2016). NOTCH signaling is implicated in Leydig cell differentiation IRX6, IGFBP2, MAML1, MAML2, MAML3, NRP1, PIK3R3, in an inhibitory regulatory fashion (Windley and Wilhelm, 2016). WNT9A and WNT9B. Autosomal dominant mutations in NOTCH1 cause the Adams– The seven MAMLD1 patients with 46,XY DSD presented Oliver syndrome (OMIM 616028), while autosomal dominant phenotypes from female external genitalia (patient 4) to variable mutations in NOTCH2 are reported in the Alagille syndrome 2 degrees of hypospadias, cryptorchidism and small penis (Table 1). (OMIM 610205) and in the Hajdu–Cheney syndrome (OMIM Interestingly, patient 4 with female external genitalia had normal 102500). By contrast, GLI3 is a zinc-finger transcription factor T secretion. Similarly, patient 5 carrying a heterozygous HSD3B2 belonging to the desert hedgehog (DHH) signal transduction variant, had normal levels of 17OH-pregnenolone, DHEA and pathway. DHH signaling is essential for driving Leydig cell DHEA-S (data not shown). Patient 6, who presented with a right differentiation (Windley and Wilhelm, 2016). Thus, NOTCH and aortic arch, was found to carry variants in five genes (BNC2, DHH signaling work together to regulate Leydig cell development FLNA, MAML1, NRP1 and PTPN11) that have been previously (Windley and Wilhelm, 2016). Autosomal dominant mutations in described in patients with heart and/or vascular anomalies GLI3 are described in the Pallister–Hall syndrome (OMIM 146510) (Tartaglia et al., 2002; Bhoj et al., 2011; Lee et al., 2014; Shaheen or in the Greig cephalopolysyndactyly syndrome (OMIM 175700). et al., 2015; Preuss et al., 2016; Chen et al., 2018). The 46,XX Taken together, our results expand the landscape of genes patient (patient 8), with primary amenorrhea, hypergonadotropic possibly involved in DSD by revealing both new and old players. hypogonadism, normal female external genitalia and small Genetic platforms for DSD diagnostics currently consider about uterus harbored gene variants involved in gonadal development 270 genes that have been identified with monogenetic forms of and DSD (CUL4B, DAPK1, EMX2, FREM2, IGFBP2, MAML3, DSD in (mostly) several independent individuals (Cools et al., MYO7A, NOTCH1, PIK3R3, TGFBI, WNT9A and WNT9B; 2018). Our eight MAMLD1 individuals share variants in 19 genes Frontiers in Genetics | www.frontiersin.org 13 August 2019 | Volume 10 | Article 746 Flück et al. Oligogenic MAMLD1 DSD Phenotypes comprised in such DSD panels, including ATF3, BNC2, CUL4B, writing – review and editing. MF-C: Investigation, writing – review EVC, FLNA, FRAS1, FREM2, GLI3, HOXA13, HSD3B2, IRX5, and editing. K-SS: Validation, interpretation, writing – review NOTCH2, PROP1, PTPN11, RECQL4, RET, RIPK4, WDR11 and and editing. IM: Resources, interpretation, writing – review and ZBTB16. By contrast, through our work 22 new genes are now editing. LC: Resources, writing – review and editing. IE: Resources, added for considering with differences in sex development: CDH23, writing – review and editing. NC: Conceptualization, data-curation, COL9A3, CYP1A1, DAPK1, EMX2, EYA1, FGF10, GLI2, GRID1, formal analysis, investigation, methodology, interpretation, project IGFBP2, IRX6, MAML1, MAML2, MAML3, MYO7A, NOTCH1, administration, supervision, visualization, writing – original draft NRP1, PIK3R3, PPARGC1B, TGFBI, WNT9A and WNT9B. preparation, writing – review and editing. Ideally, genetic variants are tested functionally for proof of their disease-causing effect in model systems. However, when finding multiple variants, which may all contribute only partially, FUNDING such testing is no longer feasible. Therefore, the likelihood of disease-causing effect of identified variants was assessed in our This work was supported by grants of the Swiss National Science study by established bioinformatic tools for genetics and by Foundation (http://www.snf.ch) (320030-146127) to CF, the assessing the genotype-phenotype correlation in each patient Instituto de Salud Carlos III (www.isciii.es/; Madrid, Spain) with current knowledge from literature and databases in the Centro de Investigación Biomédica en Red de Enfermedades field. In future studies with bigger sample size, next-generation Raras (CIBERER, http://www.ciberer.es/) U-712 to MF-C, the statistical genetic analyses may be employed to identify Agency for Management of University and Research Grants associations between a group of variants and the complex trait of (AGAUR; agaur.gencat.cat), Barcelona, Spain (2009SGR31) to sex development (Weissenkampen et al., 2019). LA, and by the Beatriu de Pinós Fellowship 2014 BP-B 00145 In summary, HTS analysis indicates that the broad DSD (AGAUR, Catalonia, Spain), the Instituto de Salud Carlos phenotypes of MAMLD1 patients may be due to additional III (www.isciii.es/; Madrid, Spain) Centro de Investigación variants in other DSD-related genes. We found up to 55 additional Biomédica en Red de Enfermedades Raras (CIBERER; http:// genetic hits that may contribute to the DSD phenotype making www.ciberer.es/) U-712 to NC. an oligogenic causation plausible. Bioinformatic network analysis can help in interpreting complex genetic data and put identified ACKNOWLEDGMENTS single candidate genes into a greater perspective to understand their possible role in DSD biology. We acknowledge the patients, families, and their primary physicians for sharing their data for our study. We also thank DATA AVAILABILITY Ida Paramonov for her help in the bioinformatic analysis. This work was supported by grants of the Swiss National Science The datasets generated for this study are publicly available in Foundation (http://www.snf.ch) (320030-146127) to CF, the dbSNP (Sherry, 2001): https://www.ncbi.nlm.nih.gov/projects/ Instituto de Salud Carlos III (www.isciii.es/; Madrid, Spain) SNP/snp_viewBatch.cgi?sbid=1063030. Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER, http://www.ciberer.es/) U-712 to MF-C, the Agency for Management of University and Research Grants ETHICS STATEMENT (AGAUR; http://agaur.gencat.cat/en/inici/), Barcelona, Spain The study was approved by the Ethics Committee of (2009SGR31) to LA, and by the Beatriu de Pinós Fellowship Hospital Universitari Vall d'Hebron (Barcelona, Spain) (CEIC: 2014 BP-B 00145 (AGAUR, Catalonia, Spain) and the Instituto PR(IR)23/2016). Written informed consent was obtained de Salud Carlos III (www.isciii.es/; Madrid, Spain) Centro from the patients for the publication of their cases. de Investigación Biomédica en Red de Enfermedades Raras (CIBERER; http://www.ciberer.es/) U-712 to NC. AUTHOR CONTRIBUTIONS SUPPLEMENTARY MATERIAL CF: Conceptualization, funding acquisition, investigation, methodology, interpretation, project administration, resources, The Supplementary Material for this article can be found online at: supervision, writing – original draft preparation, writing – review https://www.frontiersin.org/articles/10.3389/fgene.2019.00746/ and editing. LA: Interpretation, supervision, resources, visualization, full#supplementary-material REFERENCES development. J. Clin. Endocrinol. Metab. 100, E333–E344. doi: 10.1210/ jc.2014-2605 Baxter, R. M., and Vilain, E. (2013). 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Mutations in WNT9B are associated with Mayer-Rokitansky- Küster-Hauser syndrome. Clin. Genet. 89 (5), 590–596. doi: 10.1111/cge.12701 Copyright © 2019 Flück, Audí, Fernández-Cancio, Sauter, Martinez de LaPiscina, Weissenkampen, J. D., Jiang, Y., Eckert, S., Jiang, B., Li, B., and Liu, D. J. (2019). Castaño, Esteva and Camats. This is an open-access article distributed under the Methods for the analysis and interpretation for rare variants associated with terms of the Creative Commons Attribution License (CC BY). The use, distribution complex traits. Curr. Protoc. Hum. Genet. 101 (1), e83. doi: 10.1002/cphg.83 or reproduction in other forums is permitted, provided the original author(s) and Werner, R., Mönig, I., Lünstedt, R., Wünsch, L., Thorns, C., Reiz, B., et al. (2017). the copyright owner(s) are credited and that the original publication in this journal New NR5A1 mutations and phenotypic variations of gonadal dysgenesis. PLoS is cited, in accordance with accepted academic practice. No use, distribution or One 12 (5), e0176720. doi: 10.1371/journal.pone.0176720 reproduction is permitted which does not comply with these terms. Frontiers in Genetics | www.frontiersin.org 17 August 2019 | Volume 10 | Article 746