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Characterization of 35 novel NR5A1/SF-1 variants identified in individuals with

atypical sexual development: The SF1next study

Rawda Naamneh Elzenaty

1,2,3,*

, Idoia Martinez de Lapiscina

1,2,4,5,6,7,*

, Chrysanthi

Kouri

1,2,3

, Kay-Sara Sauter

1,2

, Grit Sommer

1,2

, Luis Castaño

4,5,6,7,8,9

, Christa E. Flück

1,2, #

on behalf of the

SF1next

study group.

Pediatric Endocrinology, Diabetology and Metabolism, Department of Pediatrics,

Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland.

Department

of BioMedical Research, University of Bern, Bern, Switzerland.

Graduate School for

Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland.

Research into

the genetics and control of diabetes and other endocrine disorders, Biobizkaia Health

Research Institute, Cruces University Hospital, Barakaldo, Spain.

CIBER de Diabetes y

Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III,

Madrid, Spain.

CIBER de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III,

Madrid, Spain.

Endo-ERN, Amsterdam, The Netherlands.

Department of Pediatric

Endocrinology, Cruces University Hospital, Barakaldo Spain.

University of the Basque

Country (UPV-EHU), Leioa, Spain.

These authors contributed equally to this work

Corresponding author: Christa E. Flück, Pediatric Endocrinology, Diabetology and

Metabolism;

University Children’s Hospital Bern; Freiburgstrasse 65 / C845; 3010 Bern;

Switzerland

E-mail: christa.flueck@unibe.ch

ORCID: 0000-0002-4568-5504

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Conflict of interest: all authors declare no conflict of interest.

Keywords: Steroidogenic factor 1 (SF-1/

NR5A1

), Differences of sex development (DSD),

broad phenotype, genotype-phenotype correlation

Abstract

Context

Steroidogenic factor 1 (

NR5A1

/SF-1) is a nuclear receptor that regulates sex

development, steroidogenesis and reproduction. Genetic variants in

NR5A1

/SF-1 are

common among differences of sex development (DSD) and associate with a wide range

of phenotypes, but their pathogenic mechanisms remain unclear.

Objective

Novel, likely disease-causing

NR5A1

/SF-1 variants from the SF1next cohort of

individuals with DSD were characterized to elucidate their pathogenic effect.

Methods

Different

in silico

tools were used to predict the impact of novel

NR5A1

/SF-1 variants on

protein function. An extensive literature review was conducted to compare and select the

best functional studies for testing the pathogenic effect of the variants in a classic cell

culture model. The missense

NR5A1

/SF-1 variants were tested on the promoter

luciferase reporter vector

-152CYP11A1

_pGL3 in HEK293T cells and assessed for their

cytoplasmic/nuclear localization by Western blot.

Results

Thirty-five novel

NR5A1

/SF-1 variants were identified in the SF1next cohort. Seventeen

missense

NR5A1

/SF-1 variants were functionally tested. Transactivation assays showed

reduced activity for 40% of the variants located in the DNA binding domain and variable

activity for variants located elsewhere. Translocation assessment revealed three variants

(3/17) with affected nuclear translocation. No clear genotype-phenotype, structure-

function correlation was found.

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Conclusions

Genetic analyses and functional assays do not explain the observed wide phenotype of

individuals with these novel

NR5A1

/SF-1 variants. In nine individuals, additional likely

disease-causing variants in other genes were found, strengthening the hypothesis that

the broad phenotype of DSD associated with

NR5A1

/SF-1 variants may be caused by an

oligogenic mechanism.

Introduction

Differences of sex development (DSD) are rare, mostly genetic disorders and comprise a

group of heterogenous conditions that lead to atypical chromosomal, gonadal, and/or

anatomic sex development and related function (1). Since the Chicago consensus in

2005 (1), DSD are grouped into main categories of chromosomal, 46,XY and 46,XX DSD

that are further divided in different subgroups. Still, genotypic and phenotypic

characteristics of DSD are very broad and variable, and they may or may not be more

specific for certain subgroups. For people with DSD, it is important to have an exact

diagnosis at the molecular level for receiving specific information on health outcomes and

treatment options as well as for genetic counselling (2, 3). Although advancements in

genetics have enhanced the knowledge in the field of DSD significantly, current genetic

approaches still fail to find the underlying molecular diagnosis in about half of individuals

with a DSD. Chromosomal and monogenic DSD with a characteristic genotype-

phenotype correlation such as Turner or Klinefelter syndrome and DSD associated with

congenital adrenal hyperplasia or complete androgen insensitivity seem easy to diagnose

(2-5). DSD caused by variants in genes manifesting with a broad phenotype like

NR5A1

/SF-1,

SOX9, SOX8

and

DHH

are more difficult to diagnose

(6-9), and those that

are most difficult to diagnose are when next-generation sequencing (NGS) approaches

reveal multiple candidate gene variants classified as variants of unknown significance

(VUS) by current guidelines (10).

Variants in

NR5A1

/SF-1 are reported causative in approximately 15% of all cases of

46,XY DSD (11).

NR5A1/

SF-1 is a transcription factor that regulates expression of

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multiple genes and interacts with many proteins involved in sex and adrenal

development, steroidogenesis and reproduction (12). The first human

NR5A1/

SF-1 gene

variant was reported in a 46,XY DSD individual with adrenal failure and complete gonadal

dysgenesis (13). Thereafter, the gonadal and reproductive phenotype associated with

human

NR5A1/

SF-1 variants became predominant and encompassed a broad spectrum

including 46,XY and 46,XX individuals with DSD, spermatogenic failure, primary ovarian

insufficiency (POI) and even healthy carriers (14, 15). But an explanation for this broad

phenotypic manifestation is still missing.

Reported

NR5A1/

SF-1 disease-causing variants are found throughout the whole gene

without obvious hot spots and can be missense, nonsense, small insertions

–deletions

(indels), complete gene deletions or splice-site variants. They are mostly found in

heterozygosis and only a few are compound heterozygous or homozygous (16).

To confirm pathogenicity, many

NR5A1/

SF-1 variants found in individuals with a DSD

have been tested by

in vitro

cell-based studies.

NR5A1/

SF-1 variants located in the DNA

binding domain (DBD) of the SF-1 protein revealed consistently impaired transactivation

activity when studied on different gene promoters, whereas promoter studies testing

variants located in the hinge region (HR) and the ligand binding domain (LBD) showed

variable results (16, 17). For heterozygous

NR5A1/

SF-1 variants, a dominant negative

effect where the mutated protein disrupts the function of the normal protein, even when

present in only one copy, has never been found (16, 18-30), and also haploinsufficiency

seems unlikely to explain the highly variable phenotype between individuals with the

same

NR5A1/

SF-1 variant and even between family members (31).

Similarly, protein modelling and structure-function prediction attempts failed to explain

pathogenicity of variants consistently (16, 19, 32, 33).

Thus, all these studies did not find a phenotype-genotype-function correlation.

More recently, oligogenic inheritance (2, 7, 17, 31, 34-38), genetic variants in non -coding

regulatory elements (39), variable allelic expression (7, 35), epigenetic regulation and

environmental factors (40) have been suggested as possible explanations for the broad

manifestation of DSD associated with

NR5A1/

SF-1 variants.

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Therefore, to gain further insight into DSD related to

NR5A1/

SF-1, we set up a large

international collaboration in the

SF1next

study where we collected existing data on

phenotype and genotype of the largest cohort to date of 197 individuals harbouring a

NR5A1/

SF-1 variant (41). In this cohort, 35 novel

NR5A1/

SF-1 variants were reported

that had not been characterized previously. Here we provide the clinical, genetic and

functional characterization of these novel variants. We used various bioinformatic

methods and performed classic cell-based functional studies aiming at elucidating their

disease-causing effects.

Materials and Methods

Literature search for functional studies of NR5A1/SF-1 variants

We used the Human Gene Mutation Database (HGMD, by April 2022) to search for

publications, in which functional studies were performed to assess the pathogenicity of

missense

NR5A1/

SF-1 variants; these included transactivation studies with promoter

reporters in classic cell models (Supplementary table 1)(42) and other studies such as

protein expression, nuclear transfer and DNA binding (Supplementary table 2)(42). In

Supplementary table 3 we also collected clinical and genetic data from patients

harbouring the corresponding variants(42).

Ethical approval

Written informed consent was obtained from all participants and/or their parents. The

study was approved by the I-DSD registry (UKCRN ID12729) and the local ethical

committees responsible for the participating clinicians, for Switzerland Swiss Ethics

(BASEC ID 2016-01210).

Case reports and genetic analyses

The 39 patients with a DSD carrying 35 novel

NR5A1/

SF-1 variants included in this work

are part of the

SF1next

study cohort (41). Clinical and genetic data were provided

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anonymized by the responsible clinicians through REDCap (Research Electronic Data

Capture). To classify the severity of the DSD phenotype of the patients, we used a

modified external genitalia score (EGS) based on the karyotype and characteristics of the

external genitalia at birth or before genital surgery (41). We considered the identified

NR5A1/

SF-1 variants as novel when not reported before and/or when

in vitro

functional

studies hadn’t been performed. In these patients we also assessed the possible

pathogenicity of additional gene variants reported through REDCap.

In silico analyses and variant classification

We searched for previously reported clinical associations in ClinVar and HGMD

databases and the literature (e.g. PubMed). Among the variants considered as novel for

this study, 29/35 had not been reported before and 6/35 had been reported in the

literature but no

in vitro

functional testing was done.

We predicted the possible effect of identified novel nonsynonymous genetic variants on

the structure and function of the protein using Polyphen -2, (Polymorphism Phenotyping

v2, http://genetics.bwh.harvard.edu/pph2/), Panther (Protein ANalysis THrough

Evolutionary Relationships, http://www.pantherdb.org/tools/csnpScore.do), SNPs and Go

(https://snps-and-go.biocomp.unibo.it/snps-and-go/), CADD (Combined Annotation

Dependent Depletion, https://cadd.gs.washington.edu/) and the calibrated scores given

by VarSome (43) for Revel (Rare Exome Variant Ensemble Learner), SIFT (Scale-

invariant feature transform), Provean (Protein Variation Effect Analyzer), Mutation taster

and M-CAP (Mendelian Clinically Applicable Pathogenicity) (see supplementary table

4)(42). Variants were classified for pathogenicity according to the standards and

guidelines of the American College of Medical Genetics and Genomics (ACMG) (10)

using VarSome (43).

In vitro testing of transactivation activity

Promoter luciferase reporter vector of human

-152CYP11A1

_pGL3, HA-tagged wild-type

(WT) cDNA of

NR5A1/

SF-1 in pcDNA3, empty control vector pcDNA3, and

Renilla

-TK

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(pRL-

) were all available from previous work (16). The HA-tagged human

NR5A1/

SF-1

cDNA (NM 004959.5) containing pcDNA3 vector was used as a template to generate the

novel

NR5A1/

SF-1 variant expression vectors by PCR-based site directed mutagenesis

using specific primers (Supplementary table 5)(42) and the QuickChange protocol by

Stratagene (Agilent Technologies Inc., Santa Clara, CA, USA). Only the variant

NR5A1/

SF-1 expression vector containing c.977G>T was custom made (GenScript,

Piscataway, NJ, USA). The coding sequences of all mutant expression vectors were

confirmed by direct sequencing.

Non-steroidogenic, human embryonic kidney HEK293T cells were cultured as previously

described (16). For promoter activity experiments, cells were cultured on 12-well plates

and transiently transfected with 200 ng WT or mutant

NR5A1/

SF-1 expression vectors,

800 ng of the promoter luciferase reporter construct

-152CYP11A1

_pGL3, and 30 ng of

the pRL-TK

vector as an endogenous control using Lipofectamine 2000

(Invitrogen,

Glasgow, UK) in Opti-MEM (1X)-reduced serum medium (Gibco, Thermo Fisher

Scientific, US). Forty-eight hours after transfection, cells were washed with PBS, lysed

and assayed for luciferase activity with a dual- luciferase assay using a microplate

Luminometer reader (Fluoroskan Ascent

FL & Fluoroskan Ascent

, Thermo Fisher).

Specific

Firefly

luciferase readings were standardized against

Renilla

luciferase control

readings. Experiments were repeated two to four times in duplicates and data were

summarized giving the mean ± standard error of the mean (SEM). Statistical significance

was examined by the Student’s t-test (GraphPad Prism, GraphPad Software, Boston,

MA, USA).

Assessment of nuclear transfer of wild-type and variant NR5A1/SF-1

HEK293T cells were cultured on 6-well plates and transiently transfected with WT or

mutant

NR5A1/

SF-1 expression vectors using Lipofectamine 2000

(Invitrogen) in Opti-

MEM (1X)-reduced serum medium (Gibco). 48 hours after transfection, cells were

collected with trypsin and washed with PBS, and then immediately collected for preparing

cytoplasmic and nuclear extracts using the NE-PER

™ nuclear and cytoplasmic extraction

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reagents according to the manufacturer’s instructions (Thermo Fisher Scientific). Protein

concentrations were measured by the DC protein assay kit (Bio-Rad, Hercules, CA,

USA). Nuclear and cytoplasmic protein fractions of WT and variant SF1 cell extracts were

then analysed by Western blot with an antibody against HA-tag (RRID: AB_390918) for

HA tagged-NR5A1/SF-1, Lamin B1 (RRID: AB_11002649) and Rab11 (RRID:

AB_397984) as nuclear and cytoplasmatic markers, respectively. Expression of β-actin

protein (RRID: AB_476692) was used as control. HA tagged-

NR5A1/

SF-1 and



-Actin

band intensity on Western blots were quantified by the FUSION FX6 software program of

the FUSION FX EDGE Imaging System (Witec AG, Sursee, Switzerland). For exact

information on antibodies used, see Supplementary table 6(42).

Results

Review of reported promoter transactivation studies of NR5A1/SF-1

variants in cell

models

To find the most successful functional assay system in a cell model for assessing

pathogenicity of novel

NR5A1/

SF-1 variants, we reviewed the corresponding literature.

Overall, we found 313 experiments performed on 98 different missense

NR5A1/

SF-1

variants (Supplementary table 1)(42). Non-steroidogenic cells were used in 280/313

(89.4%) experiments, with the HEK293T cell line being used most often (181/313,

57.8%). In promoter transactivation assays, we found that the

CYP11A1

promoter

reporter was employed in 108/313 (34.5%) experiments, followed by promoter reporters

AMH

(45/313, 14.3%),

CYP17A1

(39/313, 12.4%) and TESCO

(40/313, 12.8%)

(Supplementary table 1) (42). In total, 63 transactivation experiments using a

CYP11A1

promoter reporter in HEK293T cells were performed for 57 different

NR5A1/

SF-1

variants. In 38 out of 63 (60.3%) experiments performed on

NR5A1/

SF-1 variants

located

in the DBD, a significantly reduced activity was found. By contrast, variants located in the

HR or LBD of the SF-1 protein showed reduced activity in only 22% or 22.4%,

respectively (Supplementary table 1)(42). No dominant-negative effect was observed in

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26 studies that tested the combined impact of the variant together with the WT human

NR5A1/

SF-1 expression vector (Supplementary table 1)(42).

In addition to transcriptional activation experiments, other

in vitro

studies were performed

using different methods and techniques (Supplementary table 2)(42). SF-1 protein

expression was assessed by Western -blot (WB) for 50

NR5A1/

SF-1 variants, and most

variants (66%) showed similar protein expression to WT. Furthermore, 62

NR5A1/

SF-1

variants were tested for nuclear translocation using immunofluorescence (IF). Generally,

variants located in the DBD impaired nuclear translocation more likely compared to

variants located elsewhere. In addition,

NR5A1/

SF-

1 variants’ binding to target gene

promoters such as steroidogenic enzymes, was tested with Electrophoretic Mobility-Shift

Assays (EMSA) in 18 studies (Supplementary table 2)(42).

NR5A1/

SF-1 variants located

in the DBD and the LBD showed 75% and 67% reduced binding to their responsive

elements, respectively (Supplementary table 2)(42). Finally, structure predictions for 44

NR5A1/

SF-1 variants were performed using different

in silico

tools, and almost all studies

(89%) showed structural defects indicating that amino acid substitutions might affect

DNA, ligand and/or cofactor interactions (Supplementary table 2)(42).

Taken together, a correlation between genotypes and phenotypes has not been found so

far. For illustration: The DBD located

NR5A1/

SF-1 variant c.43G>A, (p.Val15Met),

classified as pathogenic, was described in a patient with a severe 46,XY DSD phenotype

but also in a 46,XX female with typical genitalia and POI (20, 33). Similarly, variant

c.634G>A (p.Gly212Ser), also classified as pathogenic, was found in a 46,XY male

without DSD but with a low sperm count, and a 46,XY female with sex reversal (38, 44).

Clinical characteristics of patients harbouring novel NR5A1/SF-1

variants

A summary of the clinical features of the 39 subjects harbouring novel

NR5A1/

SF-1

variants is given in Table 1. Most of the patients had a 46,XY karyotype (36/39, 92.3%).

None of the subjects had an adrenal phenotype. Concerning DSD, 66.7% (26/39) were

classified as having a severe DSD phenotype. An opposite sex phenotype was found in

10/39 (25.6%), a mild in 2/39 (5.1%) and a typical phenotype for karyotype in 2/39

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(5.1%). Subjects classified as severe manifested with ambiguous genitalia at birth or in

early infancy, and were registered either as male (17/26, 65.4%) or female (8/26, 30.8%).

Patients 33 and 38, who were initially registered as female were reassigned to male at

age ten years and less than one year, respectively. Patient 7 was registered male few

months after birth. All patients classified as opposite sex presented with typical female

external genitalia and were registered female at birth. Two males (patients 13 and 20)

were classified as having mild DSD with micropenis, mild hypospadias and scrotal

gonads. Patient 14 presented with typical male external genitalia but developed

gynecomastia at age 11 years; he was classified as typical.

Two patients had a 46,XX karyotype (2/39). Patient 25 was referred at 12 years as a

typical female with amenorrhea and abnormal uterus on magnetic resonance imaging

(MRI), while patient 38 presented with ambiguous genitalia and small ovotestes at age 34

years. Patient 4 was a 47,XXY phenotypic female and presented with amenorrhea at the

age of 16 years; she had a normal uterus, but gonadal biopsy revealed testicular tissue.

Fourteen patients (33.3%) had anomalies in other organs, half of whom had spleen and

associated blood system anomalies (7/14, 50.0%) (Table 1). So far, none of the patients

has had any kind of cancer reported, but the median age of the study group was only 10

years (range 0-32 years).

Family history of our studied individuals revealed DSD or reproductive disorders in 11

individuals from 10 unrelated families (Table 1). These were mostly 46,XY males with

either isolated hypospadias, hypospadias and cryptorchidism or micropenis (4/11,

36.4%). A 46,XY female with complete gonadal dysgenesis was also reported. Two

affected 46,XX females presented with POI at the age of 39 years or needed assisted

reproductive technology (ART) to achieve pregnancy. Genetic testing was not performed

in four relatives who presented with POI, menstrual irregularities, hypospadias and

cryptorchidism or isolated hypospadias. Abnormalities such as hyperextensibility, T1D

(type 1 diabetes), left ventricular non compaction and developmental delay were reported

in relatives of four index cases from four different families.

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Genetic characteristics of patients harbouring novel NR5A1/SF-1

variants

Thirty-five novel

NR5A1/

SF-1 variants were reported in 39 patients from the

SF1ne

study cohort (Table 2, Figure 1) (41). These were mostly missense variants (19/35,

54.3%), followed by frameshift insertions or deletions (8/35, 22.8%), nonsense or intronic

variants (3/35, 8.6% each), synonymous and non -frameshift (2/35, 5.7% each) and one

big deletion (1/35, 2.8%).

We classified the identified novel variants according to the ACMG guidelines (10) (Figure

1). Among the novel

NR5A1/

SF-1 variants, 64.1% (25/39) classified as (likely)

pathogenic, the rest were either VUS (10/39, 25.6%) or (likely) benign (4/39, 10.3%). All

novel

NR5A1/

SF-1 variants were found in heterozygosis, and only patient 16 was a

compound heterozygote for two variants. Genetic analysis had been performed by NGS

in 27 patients (27/39, 69.2%) (Table 2), either by targeted gene panels (16/39, 41.0%) or

whole exome sequencing (WES) (11/39, 28.2%) (Figure 1). In 16 patients (15/39, 38.5%)

a single gene analysis was performed for the molecular diagnosis, together with an array

in two patients.

Results of genetic testing of relatives was available from 21 families (21/39, 53.8%).

NR5A1/

SF-1 variants were found

de novo

in six patients (patients 9, 10, 14, 18, 26 and

28; 6/21, 28.6%); in the other 15, a heterozygous carrier was identified in the family,

although genetic analysis of both parents was only performed in 14 families (Table 2).

In nine (23%) individuals with a

NR5A1/

SF-1 variant, additional genetic variants were

reported in a total of 28 different genes, with one to 16 additional variants per individual

(Table 2, Figure 1). The majority of these additional variants were classified as VUS

(11/28, 39.3%) and likely benign (LB) (8/28, 28.6%), followed by benign (B) (6/28,

21.4%), likely pathogenic (LP) (1/28, 3.6%), pathogenic (P) (1/28, 3.6%) and one

undetermined variant (1/28, 3.6%). Pathogenicity prediction of these with respect to the

associated DSD phenotype was similarly poor as for the related, specific

NR5A1

/SF-1

variants. Of the eight individuals (46,XY or 47,XXY) with an opposite sex or severe DSD

phenotype, two presented with at least one (likely) pathogenic additional variant, three

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individuals had VUS and three had (likely) benign variants. Only patient 25 with a typical

phenotype carried a VUS and a likely benign additional variant.

Protein structure prediction and in vitro functional testing of novel NR5A1/SF-1

variants

We tested 17 novel missense

NR5A1/

SF-1 variants originating from 18 DSD patients of

the

SF1next

cohort for their impact on protein structure and function (Table 2). Identified

variants were located throughout the SF-1 protein; ten were located in the DBD, six in the

LBD and one at the C-terminus. Comparison of SF-1 protein similarity across species

revealed that all 17 variants and the surrounding regions are highly conserved (Figure 2).

Structure prediction programs suggested structural defects in all. Novel

NR5A1/

SF-1

gene variants were thus classified as (likely) pathogenic or VUS (Table 2).

After literature review (Supplementary table 1)(42), we decided to use HEK293T cells

transfected with WT or mutant

NR5A1/

SF-1 expression vectors and with the

CYP11A1

promoter reporter for the functional studies of our 17 novel missense

NR5A1/

SF-1

variants (Figure 3). Four out of ten

NR5A1/

SF-1 variants located in the DBD showed

severely impaired reporter activity (p.Cys13Ser, p.Arg39Leu, p.Cys73Tyr and

p.Cys73Trp), while the other variants had similar activity as WT (Figure 3A). Six out of

seven variants located in the LBD and C-terminus showed 50% or more transactivation

activity on the

CYP11A1

promoter reporter compared to WT, except Ala280Glu (Figure

3A).

We also assessed nuclear translocation of WT and variant

NR5A1/

SF-1 in transfected

HEK293T cells. Only three variants located in the DBD (p.Cys13Ser, p.Cys73Tyr and

p.Cys73Trp) affected nuclear translocation compared to the WT protein, which showed

about 80% nuclear localization. None of the variants contained in the LBD or the C -

terminus differed from the WT (Figure 4A).

Relating our functional study results to the clinical phenotype of the patients, only two out

of nine variants of patients with a severe phenotype showed impaired transactivation

activity (p.Cys13Ser and p.Arg39Leu), and only one (p.Cys13Ser) affected the nuclear

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translocation. Similarly, only two of six variants of patients with opposite sex had severely

impaired transactivation activity (Figure 3B) and affected nuclear translocation

(p.Cys73Tyr and p.Cys73Trp) (Figure 4B). By contrast,

NR5A1/

SF-1 variants of

individuals with typical female or mild phenotypes showed similar transactivation activity

and nuclear translocation as WT (Figure 3B and 4B).

Taken together, we found that the DSD phenotypes of the individuals, pathogenicity

prediction and ACMG classification of the related

NR5A1/

SF-1 variants and results of the

in vitro

functional assessments aligned only in four out of the 17 studied variants.

NR5A1

/SF-1 variants p.Cys13Ser, p.Arg39Leu, p.Cys73Tyr and Cys73Trp harboured by

patients with a severe or an opposite sex phenotype were all classified as either

pathogenic or likely pathogenic (Table 2), and

in silico

tools predicted them as either

pathogenic, probably damaging or disease causing (Supplementary table 4)(42). In

addition, these predictions were confirmed by both functional

in vitro

assays. By contrast,

variants p.Pro14Ser, p.Gly17Val, p.Phe70Leu and p.Cys73Ser, also found in patients

with a severe or an opposite sex phenotype, were also all classified and predicted as

(likely) pathogenic (Table 2 and Supplementary table 4)(42), but in these cases functional

assays failed to confirm a disease-causing effect. Furthermore, variants located in the

LBD were almost all classified as VUS and

in silico

as well as

in vitro

studies showed

diverse results not aligning to each other (Figures 3A and 3B).

Discussion

NR5A1

/SF-1 variants are associated with unexplained broad DSD phenotypes (14, 15,

17, 31). This is also reflected in the

SF1next

study cohort comprising 197 individuals with

novel and known

NR5A1

/SF-1 variants (41). Here, we characterized the novel

NR5A1

/SF-1 variants identified in this cohort by established

in silico

and

in vitro

methods

and reviewed the corresponding literature for known variants. Our review revealed that

although most reported variants were classified as (likely) pathogenic and were predicted

to disrupt SF-1 protein structure, only some variants, mostly located in the DBD, had

impaired transactivation activity on different promoter reporters in several cell models

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(Supplementary tables 1 and 2) (16, 19, 32, 42, 45). Similarly, only a few of the 17 novel

NR5A1

/SF-1 variants tested in our study showed impaired transcriptional activation

activity and affected SF-1 nuclear translocation. These few variants were located in the

DBD and the corresponding phenotype was severe or opposite sex. However, for most of

the individuals with DSD who had

NR5A1

/SF-1 variants,

in silico

predictions and results

from

in vitro

testing did not align with the phenotype. Thus, a clear genotype-phenotype,

structure-function correlation remains elusive for

NR5A1

/SF-1 variants.

So far more than 260

NR5A1/

SF-1 variants located in all regions of the SF-1 protein have

been described in 46,XY and 46,XX individuals, presenting healthy or with variable

severity of DSD (15). In our study, clinical characteristics of the individuals with novel

heterozygous

NR5A1

/SF-1 variants were also variable, but most had a 46,XY karyotype

and a severe DSD. In line with other reports (15, 16), severity of the phenotype did not

correlate with specific

NR5A1/

SF-1 variants. Missense, frameshift, or synonymous

NR5A1

/SF-1 variants were observed in individuals with a severe DSD phenotype (Figure

1). It is important to realize that the reported

NR5A1

/SF-1 variants in our studied patients

may not explain the DSD phenotype at all or only in combination with other genetic

variants. Thus, further genetic testing in such patients is advised.

ACMG classification (10) of the novel

NR5A1

/SF-1 variants identified in the

SF1next

study cohort suggested a pathogenic or likely pathogenic impact for 2 out of 3 variants

(23/35). Novel variants located in the DBD were predicted (likely) pathogenic, while

variants located in the LBD were mostly predicted VUS and B. However, corresponding

functional experiments revealed mixed results and an alignment of data was only found

for variants p.Cys13Ser, p.Arg39Leu, p.Cys73Tyr and p.Cys73Trp located in the DBD.

Similar results have been reported in the literature indicating that there is no clear

genotype-phenotype correlation for

NR5A1

/SF-1 variants (19, 20, 32, 38, 46). Guidelines

recommend assessing pathogenicity of missense variants by

in silico

prediction methods

and functional tests (10), but currently used test methods may not reveal a clear answer.

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In our study, prediction software programs for gene variants classified most

NR5A1

/SF-1

variants more accurately as (likely) pathogenic, while functional assays were less

predictive.

Similar results were obtained by protein structure-based prediction of pathogenicity for

the 17 novel missense

NR5A1/

SF-1 variants where almost all variants located in the DBD

were suggested pathogenic or VUS, but aligned with the

in vitro

functional assays in less

than 1 in 3 cases (5/17).

Functional testing is recommended for variant classification (10), but after reviewing the

related literature originating from numerous research groups, we conclude that

established

in vitro

assays for assessing the activity of

NR5A1

/SF-1 variants are in doubt

(Supplementary table 1 and 2) (19, 20, 25, 32, 38, 42, 46-48). In some studies, functional

studies were able to provide clear experimental evidence for a disease-causing effect of

tested

NR5A1

/SF-1 variants, while others were inconclusive showing mixed correlative

results between and within studies for different variants for no obvious experimental

reasons. Thus, false-negative or false-positive results could be suspected for maybe

missing factors in the experimental models used. Overall, in reported studies and in our

study, functional tests were most predictive for

NR5A1

/SF-1 variants located in the DBD

of the SF-1 protein (Supplementary tables 1 and 2)(42). The DBD of SF-1 is a highly

conserved domain among species which comprises two zinc finger (ZNI and ZNII)

domains essential for the recognition of the DNA target sequences (16, 49). In our study,

the novel

NR5A1/

SF-1 variants p.Cys13Ser, p.Cys73Tyr and p.Cys73Trp located in the

ZN finger domains had reduced activity and these variants affect important cysteine

residues involved in the

NR5A1/

SF-1 binding to the recognition sites. The p.Arg39Leu

variant also showed activity loss and is located in the hinge region that links the zinc

fingers and is involved in stabilizing the non -specific contacts across the DNA minor

groove (16, 49). However, the novel

NR5A1

/SF-1 p.Pro14Ser, p.Gly17Val and

p.Cys73Ser variants, which are also located in the zinc finger domains and also

manifested with a severe or opposite sex phenotype, showed unaffected transactivation

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activity and nuclear translocation, and these results remain unexplained. In our study, we

only used the

-152CYP11A1

promoter luciferase reporter construct in non -steroidogenic

HEK293 cells. SF-1 targets many genes during sex determination and differentiation,

therefore using additional promoters, such as SOX9 or AMH, and different cell lines,

might be helpful to explain a particular phenotype caused by a concrete

NR5A1

/SF-1

variant. The appropriate functional study should be chosen based on the phenotype of

the patient to obtain an accurate genotype-phenotype correlation. However, previous

studies including several promoters and cell lines also show heterogeneous results (16).

Thus, something is clearly missing when it comes to understanding the mechanism of

disease related to

NR5A1/

SF-1 variants and the broad spectrum of DSD. Several

hypotheses have been put forward over the years including genetic and environmental

factors affecting

NR5A1/

SF-1 expression, activity and degradation, as well as overlooked

co-factors of SF-1 or other genes working in networks together with

NR5A1/

SF-1 to

reveal a DSD phenotype by oligogenic mechanisms (15, 40, 50, 51). Evidence for

involvement of several of these factors in SF-1-related DSD has been reported by many

studies. Here we mention only few. Recently, variants in non -coding promoter regions of

NR5A1/

SF-1 have been reported in 3 patients with 46,XY DSD (39).

In vitro

analyses

showed that promoter activity was affected in all cases. WES in two of the patients also

revealed additional variants in

SRA1, WWOX

, and

WDR11

genes with potential impact

on the DSD phenotype (39). Dominant negative effect was presumed initially, as most

DSD individuals carry heterozygous

NR5A1/

SF-1 variants, but corresponding

in vitro

experiments did not confirm a dominant negative effect (16, 18). Variable allelic

expression due to imbalanced cis-regulation of mutant versus wild-type alleles could also

explain variable expressivity of a phenotype when mutations are present in a

heterozygous state (15). In fact, it has been shown that complex modes of allelic

expression are implied in development and pathologies, including autosomal dominant

disorders (52) but to our knowledge, this gene-specific theory has not been tested in

NR5A1

/SF-1 variants so far.

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By contrast, many studies have reported oligogenicity as a mechanism to explain the

broad and inter-individual and intra-familial variable DSD phenotypes associated with

NR5A1/

SF-1 variants (2, 31, 50). Newer parallel sequencing strategies have facilitated

the identification of gene variants in individuals with DSD, but they have also revealed

that healthy individuals carry many variants and informed that the genetics explaining an

atypical sex development might be complex (2, 3, 50). While some DSD may be

explained by monogenic variants, others may be caused by oligogenic variants in

interacting genes. Accordingly, we and others have described several patients with a

DSD who have

NR5A1

/SF-1 variants in whom other likely disease-causing additional

gene variants were found (17, 53-55). However, so far mechanistic confirmation in these

cases is missing, as it is very difficult to assess the disease-contribution of each variant

contained in a complex, multi-gene network where the effect of the single variant might

be mild. Thus, identification of additional genetic hits in individuals with a DSD poses

large challenges for distinguishing between disease-causing variants and variants that do

not contribute to the phenotype. In the future, human tissue-derived models such as

organoids or

in vitro

cellular reprogramming of pluripotent stem cells (iPSC) may enable

studies of oligogenic mechanism as the patient-derived material contains the individual

genetic background (56-58).

In this study, nine patients were reported to have additional gene variants (Table 2 and

Methods). Five patients (patients 4, 8, 21, 35 and 39) harboured additional genetic hits in

genes related to sex development and differentiation (e.g.

AMH, SRD5A2

DHX37

steroidogenesis (e.g.

R) and hypogonadotropic hypogonadism (e.g.

SOX10

) (8, 17,

59-62). On the other hand, patients 10 and 25 were found to have 16 and 2 additional

variants, respectively, in genes that have not been related to DSD so far. The clinical

relevance of copy number variants in patients 15 and 16 has been described before (63).

Additional gene variants reported in this study have been achieved through different NGS

methods, in different laboratories using different algorithms to annotate the variants of

interest. Therefore, subsequent data analysis depended on the criteria of the researcher

from the corresponding laboratory and clarification of the role of some of these gene

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variants in the pathogenesis of DSD is missing. With the increasing use of NGS methods

for the molecular diagnosis of individuals with a DSD, it is expected that more patients

with multiple gene variants will be identified, which may not be deleterious alone but may

contribute to the observed DSD phenotype when occurring in combination with a

heterozygous

NR5A1

/SF-

1 variant. If so, the genotype−phenotype correlation will depend

on the specific combinatory effect of the involved genetic variants and will be unique in

many cases. In addition, this will not only be true in DSD patients carrying

NR5A1

/SF-1

variants but also for DSD related to other genetic variants.

In conclusion, we characterized 35 novel

NR5A1

/SF-1 variants identified in individuals

with a DSD and their family members of the international

SF1next

study cohort. Protein

structure analyses and functional studies were performed for 17 novel missense variants.

We found that current genetic analyses and functional assays for studying novel variants

NR5A1/

SF-1 frequently do not explain the observed phenotype. In nine individuals,

additional likely disease-causing variants in other genes were found, strengthening the

hypothesis that the broad phenotype of DSD with

NR5A1

/SF-1 variants may be caused

by an oligogenic mechanism.

Funding

This study was supported by a project grant of the Swiss National Science Foundation

(320030-197725). IM is supported by a Postdoctoral Fellowship Grant from the Education

Department of Basque Government (Spain).

Acknowledgments

We thank all the patients for participating in the study.

Members of

SF1next

team are: S. Abali, Acibadem Mehmet Ali Aydinlar University,

School of Medicine, Istanbul (Turkey); ZY. Abali, Marmara University, Department of

Pediatric Endocrinology and Diabetes, Pendik, Istanbul (Turkey); F. Ahmed,

Developmental Endocrinology Research Group, School of Medicine, Dentistry & Nursing,

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University of Glasgow, Glasgow (UK), Office for Rare Conditions, University of Glasgow,

Glasgow (UK); L. Akin, Department of Pediatric Endocrinology and Diabetes, Ondokuz

Mayis University, Samsun (Turkey); MC. Almaraz, Department of Endocrinology and

Nutrition, Hospital Regional Universitario de Málaga, Instituto de Investigación Biomédica

de Málaga, Málaga (Spain); L. Audí, Growth and Development Group, Vall d’Hebron

Research Institute, CIBERER, Barcelona (Spain); M. Aydin, Department of Pediatric

Endocrinology and Diabetes, Ondokuz Mayis University, Samsun (Turkey); A. Balsamo,

Researcher of Alma Mater Studiorum, S.Orsola-Malpighi, University Hospital, Bologna

(Italy); F. Baronio, Department of Medical and Surgical Sciences, IRCCS S.Orsola-

Malpighi, University Hospital, Bologna (Italy); J. Bryce, Office for Rare Conditions,

University of Glasgow, Glasgow (UK); K. Busiah, Paediatric Endocrinology, Diabetology

and Obesity Unit, Lausanne University Hospital, University of Lausanne, Lausanne

(Switzerland); M. Caimari, Hospital Universitario Son Espases, Palma de Mallorca

(Spain); N. Camats-

Tarruella, Growth and Development Group, Vall d’Hebron Research

Institute, CIBERER, Barcelona (Spain); A. Campos-Martorell, Hospital Universitario Vall

d'Hebron, Barcelona (Spain); A. Casteràs, Department of Endocrinology, Hospital

Universitario Vall d'Hebron, Barcelona (Spain); S. Çetinkaya, University of Health

Sciences Turkey, Dr. Sami Ulus Obstetrics and Pediatrics Training and Research

Hospital, Clinic of Pediatric Endocrinology, Ankara (Turkey); YM. Chan, Division of

Endocrinology, Boston Children's Hospital, Boston (USA), Department of Pediatrics,

Harvard Medical School, Boston (USA); HL. Claahsen -van der Grinten, Department of

Paediatric Endocrinology, Radboud University Nijmegen Medical Centre, Nijmegen

(Netherlands); I. Costa, Pediatric Department, Manises Hospital, Manises (Spain); M.

Cools, Department of Internal Medicine and Paediatrics, Division of Paediatric

Endocrinology, Ghent University Hospital, Ghent University, Ghent (Belgium); FF.

Darendeliler Istanbul University, Istanbul Faculty of Medicine, Pediatric Endocrinology

Unit, Istanbul (Turkey); JH. Davies, Southampton Children's Hospital, University Hospital

Southampton, Southampton (UK); I. Esteva, Endocrinology Department, Gender Identity

Unit, Regional University Hospital of Malaga, Málaga (Spain); H. Fabbri -Scallet,

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Laboratory of Human Molecular Genetics, Center of Molecular Biology and Genetic

Engineering (CBMEG)/Unicamp, Sao Paulo (Brazil); CA. Finlayson, Division of

Endocrinology, Ann & Robert H. Lurie Children's Hospital of Chicago, Chicago (USA); E.

Garcia, Hospital Virgen del Rocío, Sevilla (Spain); B. Garcia-Cuartero Pediatric

Endocrinology Department, Ramon y Cajal University Hospital, Madrid (Spain); A.

German, Haemek Medical Center, Afula (Israel), Technion Israel Institute of Technology,

Haifa (Israel); E. Globa, Ukrainian Research Center of Endocrine Surgery, Endocrine

Organs and Tissue Transplantation, MoH of Ukraine, Kyiv (Ukraine); G. Guerra-Junior,

Interdisciplinary Group for the Study of Sex Determination and Differentiation (GIEDDS),

Department of Pediatrics, School of Medical Sciences, State University of Campinas, Sao

Paulo (Brazil); J. Guerrero, Hospital Infantil La Paz, Madrid (Spain); T. Guran, Marmara

University, Department of Pediatric Endocrinology and Diabetes, Pendik, Istanbul

(Turkey); SE. Hannema, Erasmus Medical Centre, Sophia Children’s Hospital,

Department of Pediatric Endocrinology, Rotterdam (Netherlands), Leiden University

Medical Centre, Department of Pediatrics, Leiden (Netherlands); O. Hiort, Division of

Pediatric Endocrinology and Diabetes, Department of Pediatrics, University of Lübeck,

Lübeck (Germany); J. Hirsch, Division of Urology, Ann & Robert H. Lurie Children's

Hospital of Chicago, Chicago (USA); I. Hughes, Department of Paediatrics, University of

Cambridge, Cambridge (UK); M. Janner, Pediatric Endocrinology, Diabetology and

Metabolism, Department of Pediatrics, Inselspital, Bern University Hospital, University of

Bern, Bern (Switzerland); Z. Kolesinska, Department of Pediatric Endocrinology and

Rheumatology, Poznan University of Medical Sciences, Poznan (Poland); K. Lachlan,

Wessex Clinical Genetics Service, University Hospital Southampton, Southampton (UK);

D. L'Allemand, Department of Endocrinology, Children's Hospital of Eastern Switzerland,

St.Gallen (Switzerland); JK. Malikova, Department of Paediatrics, Second Faculty of

Medicine of Charles University and University Hospital Motol, Prague (Czech Republic);

M. Lang-Muritano, Division of Pediatric Endocrinology and Diabetology and Children's

Research Centre, University Children's Hospital, University of Zurich, Zurich

(Switzerland); A. Lucas-Herald, Developmental Endocrinology Research Group, School

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of Medicine, Dentistry & Nursing, University of Glasgow, Glasgow (UK); J. Mammadova,

Department of Pediatric Endocrinology and Diabetes, Ondokuz Mayis University,

Samsun (Turkey); K. MсElreavey, Human Developmental Genetics, Institute Pasteur,

Paris (France); V. Mericq, Institute of Maternal and Child Research, University of Chile,

Santiago (Chile); I. Mönig, Division of Pediatric Endocrinology and Diabetes, Department

of Pediatrics, University of Lübeck, Lübeck (Germany); F. Moreno, Hospital Infantil La Fe,

Valencia, Spain; J. Mührer, Division of Pediatric Endocrinology and Diabetology and

Children's Research Centre, University Children's Hospital, University of Zurich, Zurich

(Switzerland); M. Niedziela, Department of Pediatric Endocrinology and Rheumatology,

Poznan University of Medical Sciences, Poznan (Poland); A. Nordenstrom, Pediatric

Endocrinology, Karolinska University Hospital, Department of Women’s and Children’s

Health, Karolinska Institutet, Stockholm (Sweden); B. Orman, University of Health

Sciences Turkey, Ankara (Turkey), Dr. Sami Ulus Obstetrics and Pediatrics Training and

Research Hospital, Clinic of Pediatric Endocrinology, Ankara (Turkey); S. Poyrazoglu,

Istanbul University, Istanbul Faculty of Medicine, Pediatric Endocrinology Unit, Istanbul

(Turkey); JM. Rial, Pediatric Endocrinology Department, Hospitem Rambla, Santa Cruz

de Tenerife (Spain); MM. Rutter, Division of Endocrinology, Cincinnati Children’s Hospital

Medical Center, Department of Pediatrics, University of Cincinnati, Cincinnati (USA),

DSD Translational Research Network (USA); A. Rodríguez, Biocruces Bizkaia Health

Research Institute, Cruces University Hospital, UPV-EHU, CIBERDEM, CIBERER, Endo-

ERN, Barakaldo (Spain); T. Schafer-Kalkhoff, Division of Endocrinology, Cincinnati

Children’s Hospital, Cincinnati (USA); S. Seneviratne Department of Paediatrics,

University of Colombo, Colombo (Sri Lanka); M. Sredkova-Ruskova University Pediatrics

Hospital, Medical University, Department of Clinical Genetics, Sofia (Bulgaria); L. Tack,

Department of Internal Medicine and Paediatrics, Division of Paediatric Endocrinology,

Ghent University Hospital, Ghent University, Ghent (Belgium); R. Tadokoro-Cuccaro,

Department of Paediatrics, University of Cambridge, Cambridge (UK); A. Thankamony,

Department of Paediatrics, Addenbrooke’s Hospital, Cambridge University Hospitals NHS

Foundation Trust, Cambridge (UK); M. Tomé, Department of Endocrinology and Nutrition,

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Hospital Regional Universitario de Málaga, Instituto de Investigación Biomédica de

Málaga, Málaga (Spain); A. Vela, Biocruces Bizkaia Health Research Institute, Cruces

University Hospital, UPV-EHU, CIBERDEM, CIBERER, Endo-ERN, Barakaldo (Spain);

M. Wasniewska, University of Messina, Department of Human Pathology of Adulthood

and Childhood, Messina (Italy); D. Zangen, Faculty of Medicine, Hebrew University of

Jerusalem, Jerusalem (Israel); Pediatric Endocrinology Unit, Hadassah -Hebrew

University Medical Center, Jerusalem (Israel); N. Zelinska, Ukrainian Research Center of

Endocrine Surgery, Endocrine Organs and Tissue Transplantation, MoH of Ukraine, Kyiv

(Ukraine).

Data Availability Statement

Access to basic data is possible through the international I-DSD registry; general rules

apply (https://sdmregistries.org/about/). Additional data were collected in a project-

specific REDCap database governed by the Clinical Trials Unit (CTU) at University of

Bern, Switzerland. Genetic data are also stored on servers of the University of Bern.

These data can also be accessed upon reasonable request according ethical and

informed consent.

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Tables

Table 1.

Clinical characterization of the DSD patients harbouring novel

NR5A1/

SF-1 variants

identified by the SF1next study.

Table 2.

Genetic characterization of the novel

NR5A1/

SF-1 gene variants and of

additional variants identified in related genes in combination.

Figure legends

Figure 1.

Novel

NR5A1/

SF-1 variants identified in 39 DSD patients included in the

SF1next

study. For each patient, pathogenicity prediction of the novel

NR5A1/

SF-1

variant, method of genetic workup and information on additional gene variants that have

been identified, is represented. Severity of the DSD phenotype of each patient harbouring

a novel

NR5A1/

SF-1 variant is also indicated on the y-axis.

Figure 2

. Protein localization and conservation across species of the newly identified

human

NR5A1/

SF-1 missense variants. Shown is a multiple alignment of parts of the SF-

1 protein sequences across different species. Localization of the newly identified human

amino acid variants are given in bold and seem highly conserved across different

species.

Figure 3.

Transcriptional activity studies of 17 novel missense

NR5A1

/SF-1 variants. The

ability of wild-type (WT) and mutant

NR5A1/

SF-1 to activate the promoter of the

CYP11A1

gene was tested in non-steroidogenic HEK293T cells. Cells were transiently

transfected with

NR5A1/

SF-1 expression vectors and the

-152CYP11A1

promoter

luciferase reporter construct.

Activity of

NR5A1/

SF-1 variants is shown with respect to

the ACMG pathogenicity classification (43) and the localization of the variants in the

protein structure.

Activity of the

NR5A1/

SF-1 variants is shown with respect to the

ACMG pathogenicity classification (43) and according to the phenotype of affected

individuals. Luciferase activity was measured with the Dual Luciferase assay system

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(Promega). Results are shown as the mean ± standard error of the mean (SEM) of three

to four independent experiments, all performed in duplicate. *, p<0.05; **, p<0.005; ***,

p<0.001. RLU, relative light units.

Figure 4.

Nuclear translocation studies of 17 novel missense

NR5A1

/SF-1 variants.

Western blot showing cytoplasmic and nuclear localization of wild-type (WT) and mutant

HA tagged-SF-1 proteins. HEK293T cells were transiently transfected with WT and

mutant

NR5A1/

SF-1 expression vectors for 48 hours. Cytoplasmic and nuclear protein

fractions were separated and probed by Western blots. An anti-HA antibody was used

to detect the

NR5A1/

SF-

1 protein at 53kDa. β-actin (42 kDa) was used as loading

control, Rab11(24 kDa) was used as a cytoplasmic protein marker and Lamin B1 (66

kDa) as a nuclear protein marker. The intensity of the HA tagged-

NR5A1/

SF-

1 and β-

actin bands was quantified by using the FUSION FX6 EDGE Imaging System. Data are

expressed as percentage of HA tagged-

NR5A1/

SF-1 cytoplasmic (in white) and nuclear

(coloured) fraction of total protein, normalized to β-actin. Results from two independent

experiments are presented as mean ± SEM.

Results are shown according to

localization of variants in the

NR5A1/

SF-1 protein.

Results shown with respect to the

corresponding DSD phenotypes.

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Table 1. Clinical characterization of the DSD patients harbouring novel

NR5A1/

SF-1 variants identified by the SF1next study.

Patient

Karyotype

DSD
phenotype
classification

Sex
assignment/
reassignment

Current or last age of assessment/Clinical
description

Other organ
anomalies/Cancer

Family history

46,XY

Severe

M/No

1y, micropenis (10-20mm), perineal hypospadias,
labioscrotal testes, posterior fused labioscrotum.
Cytoscopy: Mullerian remnants. Masculinizing
genitoplasty and orchidopexy. 6y, penis (>30mm),
coronal hypospadias. 10y, testes 4mL, gynecomastia,
Tanner: Genitals II, pubic hair II.

No/No

46,XY

Severe

1y, perineal hypospadias, labioscrotal gonads,
posterior fused labioscrotum.

Flat nasal root;
epicanthus/No

46,XY

Severe

1y, micropenis (10-20mm), scrotal hypospadias,
labioscrotal testes, posterior labioscrotum fusion.
Masculinizying genitoplasty (1y, 1y, 5y, 6y).

No/No

Mother

: pregnancy

achieved by ART

47,XXY

Opposite sex

16y, female external genitalia. MRI: normal uterus and
gonads. Spontaneous start of puberty, no menarche,
Tanner: genitals III, breast IV, pubic hair IV. 17y,
biopsy of right gonad, at histology: atrophic
seminiferous tubules and testicular tissue, Leydig cell
proliferation.

No/No

Father

azoospermia;
Cousin: menstrual
irregularities.

46,XY

Severe

6m, perineal hypospadias, inguinoscrotal gonads. 3y,
orchidopexy and masculinizing genitoplasty. 14y, penis
(21-30mm), coronal hypospadias, testes (4-5mL),
Tanner: genitals I, pubarche I. Spontaneous start of
puberty.

No/No

46,XY

Severe

At birth, micropenis (<10mm), penoscrotal
hypospadias, labioscrotal gonads. 2y, coronal
hypospadias. Masculinizing genitoplasty (1y, 2y).

No/No

Mother

: POI (39y)

46,XY

Severe

Other/M

1m, penis (<10mm), penoscrotal hypospadias,
labioscrotal testes. US: rudimentary uterus.
Reassigned sex to male. 2y, orchidopexy. 5y, typical
male meatal opening and 10-20mm penis. 12y, testes
2-4mL, Tanner: genitals IV and pubarche IV. US:
uterus is absent.

Brachymetatarsia
and
brachymetacarpia;
long-jointed hands
and feet;
decreased muscle
mass/No

Mother

hyperextensibility;
sister

: CGD.

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46,XY

Severe

2m, micropenis (10-20mm), penoscrotal hypospadias,
labioscrotal testes. Masculinizing genitoplasty (2m, 2y).
8y, penis >30mm. 11y, Testes 6-8mL, Tanner: genitals
II and pubarche I. 16y, Tanner: genitals V and
pubarche V. Testes 15-20mL. Spontaneous start of
puberty.

No/No

46,XY

Opposite sex

7y, inguinoscrotal gonads. US: normal uterus and
streak gonads. 10y, bilateral gonadectomy.

No/No

46,XY

Severe

2y, perineal hypospadias, unfused labioscrotum. US:
rudimentary uterus.

Urogenital sinus;
2y, development
delay; 4y, epilepsy,
severe mental
retardation/No

46,XY

Severe

2y, perineal hypospadias, impalpable and inguinal
testes. Masculinizing genitoplasty.

No/No

Father

: urogenital

sinus, hypospadias,
cryptorchidism;
brother

hypospadias,
unilateral
cryptorchidism;
grandfather:
hypospadias.

46,XY

Severe

3y, micropenis (21-30mm), penoscrotal hypospadias,
labioscrotal and inguinal testes. 3y, masculinizing
genitoplasty. 9y, penis >30mm, coronal hypospadias.
12y, testes 2mL, Tanner: genitals III, pubarche III.

Hb and
reticulocytes above
normal range,
thrombocytosis/No

46,XY

Mild

2y, coronal hypospadias, labioscrotal testes, 21-30mm
penis, fused labioscrotum. US: uterus is absent.
Masculinizing genitoplasty (1y, 2y). 7y, typical male
meatal opening, inguinoscrotal gonads, penis >30mm.

No/No

Father

hypospadias

46,XY

Typical

11y, penis (>30mm), scrotal testes (2mL),
gynecomastia, Tanner: genital I, pubic hair II.

Elevated insulin/No

46,XY

Severe

9m, micropenis (21-30mm), penoscrotal hypospadias,
inguinal testes, unfused labioscrotum. Masculinizing
genitoplasty (1y, 2y, 3y). 6y, penis (>30mm),
labioscrotal gonads.

No/No

46,XY

Severe

1y, micropenis (10-20mm), penoscrotal hypospadias,
labioscrotal testes, unfused labioscrotum.
Masculinizing genitoplasty (1y, 2y).

Ventricular septum
defect/No

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46,XY

Severe

11m, micropenis (21-30mm), penoscrotal hypospadias,
labioscrotal testes. Masculinizing genitoplasty (1y, 2y).
8y, penis >30mm. Masculinizing genitoplasty.
Orchidopexy. 10y, testes 3mL, Tanner: genital III, pubic
hair IV.

No/No

46,XY

Severe

1y, micropenis (10-20mm), perineal hypospadias,
labioscrotal testes, posterior labioscrotal fusion.
Feminizing genitoplasty. Bilateral gonadectomy, at
histology: normal for Karyotype. 16y, Tanner: breast V.
Induction of puberty.

No/No

46,XY

Severe

11m, micropenis (21-30mm), perineal hypospadias,
labioscrotal and inguinoscrotal testes. Orchidopexy and
masculinizing genitoplasty (2y).

No/No

46,XY

Mild

At infancy, orchidopexy and masculinizing surgeries for
hypospadias, cryptorchidism and micropenis. 4y, penis
(21-30mm), labioscrotal testes. Masculinizing
genitoplasty. 11y, penis (>30mm), testes 4.5mL,
Tanner: genital II and pubic hair II.

No/No

46,XY

Opposite sex

16y, typical female external genitalia MRI: hypoplastic
uterus and testes. Tanner: Breast II; pubarche V.
Sponaneous start of puberty: Bilateral gonadectomy, at
histology: testis with primitive seminiferous tubules.
Sertoli cells only and occasional spermatogonia.
Vaginal hypoplasia.

No/No

46,XY

Opposite sex

15y, external female genitalia, clitoromegalia (>30mm).
Tanner: breast I, pubarche III. At imaging: hypoplastic
uterus and small testes. Vaginal hypoplasia. 17y,
induction of puberty. Bilateral gonadectomy, at
histology: bilateral testicular structures and fallopian
tubes.

No/No

Father

micropenis,
hypospadias, left
ventricular non
compaction.

46,XY

Opposite sex

1y, female external genitalia. US: normal uterus.

No/No

Father

hypospadias,
oligospermia, ART
for conception.

46,XY

Severe

3y, micropenis (10-20mm), penoscrotal hypospadias,
labioscrotal testes, posterior labioscrotal fusion.
Feminizing genitoplasty.

No/No

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46,XX

Typical

12y, typical female external genitalia. MRI: abnormal
uterus. Tanner: breast III; pubic hair IV. 20y,
spontaneous start of puberty. Tanner: breast V; pubic
hair V. No menarche. US: normal uterus and gonads.

Anemia; skoliosis,
dislocation of the
hip; muscle
weakness, spastic
tetraparesis;
wheelchair bound,
ataxia; pachygyria;
epilepsy,
tetraparesis/No

46,XY

Opposite sex

1y, penis (21-30mm), typical female meatal opening,
impalpable testes, unfused labioscrotum. Laparoscopy:
mullerian remnants.

No/No

46,XY

Severe

10m, micropenis (21-30mm), perineal hypospadias,
inguinal gonads, posterior labioscrotal fusion.

No/No

46,XY

Severe

8m, micropenis (10-20mm), perineal hypospadias,
labioscrotal testes, posterior labioscrotal fusion.

Abnormal
morphology spleen,
poikilocytes, giant
thrombocytes,
thrombocytosis, no
HJB, no PRBC/No

46,XY

Severe

At birth, micropenis (10-20mm), penoscrotal
hypospadias, labioscrotal testes. Masculinizing
genitoplasty.

No/No

46,XY

Opposite sex

12y, typical female external genitalia. US: hypoplastic
uterus and abnormal testes. Bilateral gonadectomy, at
histology: Left gonad with atrophic testis tissue and
small amount of Sertoli cells; right gonad only
epididymis. Induction of puberty. 17y, Tanner: breast V
and pubic hair V.

No/No

46,XY

Severe

F/M

10y, penoscrotal hypospadias, impalpable gonads. US:
right teste 5mL; left is unknown. Spontaneous start of
puberty, Tanner: genitals II; pubic hair IV. Biopsy (right
gonad): Leydig cell hyperplasia, focal testes atrophy,
mature spermatogenesis. Masculinizing genitoplasty
(1y, 2y). Reassigned sex to male. 13y, labisocrotal
testes (7-9mL) , Tanner: breast III; pubic hair V.

Accelerated bone
age; pain while
micturation/No

46,XY

Severe

3y, micropenis (21-30mm), scrotal hypospadias,
labioscrotal testes. US: Mullerian remnants. 5y, penis
(>30mm), perineal hypospadias. Masculinizing
genitoplasty (5y, 6y). 12y, testes 4-5mL, Tanner: breast

Abnormal
morphology spleen,
mild
thrombocytosis,

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I, genital III, pubic hair I. Spontaneous start of puberty.

elevated
reticulocytes/No

46,XY

Severe

1y, micropenis (10-20mm), typical female meatal
opening, inguinoscrotal gonads, posterior labioscrotal
fusion. Feminizing genitoplasty and bilateral
gonadectomy. 11y, vaginal hypoplasia, Tanner: breast
III, pubic hair III. Spontaneous start of puberty.

Abnormal
morphology
spleen/No

46,XY

Severe

3y, penoscrotal hypospadias, inguinoscrotal testes.
Masculinizing genitoplasty (1y, 2y, 3y).

No/No

46,XY

Opposite sex

12y, typical female meatal opening, inguinal testes,
penis (>30mm), hirsutism. Tanner: pubic hair V.
Spontaneous start of puberty.

Accessory
spleen/No

46,XY

Opposite sex

13y, typical female external genitalia. MRI: hypoplastic
uterus and streak ovaries. Spontaneous start of
puberty. Tanner: breast III; pubic hair V.

No/No

Mother: POI (25y),
premature
menopause (37y).

46,XY

Severe

4m, micropenis (10-20mm), scrotal hypospadias,
labioscrotal testes. Orchidopexy and masculinizing
genitoplasty (1y, 2y).

No/No

Father

: micropenis

46,XX

Severe

34y, penoscrotal hypospadias, inguinal (left, 20mL) and
labioscrotal (right, 4mL) testes, posterior labioscrotal
fusion, gynecomastia, Tanner: breast III, genitals V.
MRI: abnormal uterus, US: abnormal/small testes.
Genitoplasty. Left gonad biopsy, at histology: Sertoli
only, Leydig cell hyperplasia, ovarian tissue with
corpora albicantia and follicles in epididymis.

No/No

46,XY

Severe

1y, micropenis (10-20mm), penoscrotal hypospadias,
inguinal gonads. Masculinizing genitoplasty (3y) and
orchidopexy (4y). 5y, micropenis (21-30mm), coronal
hypospadias, labioscrotal gonads, .

Intracranial cyst/No

Father

: T1D;

brother:
penoscrotal
hypospadias,
labioscrotal/inguinal
gonads,
developmental
delay, quadriplegic
cerebral palsy

M, male: F, female; ART, assisted reproductive technology; CGD, complete gonadal dysgenesis; m, month; MRI, magnetic resonanc e imaging; POI, primary

ovarian insufficiency; T1D, type 1 diabetes; US, ultrasound; y, years.

DSD classification according to the severity of the phenotype of external genitalia

(41)

Relatives with a confirmed

NR5A1/

SF-1 gene variant.

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Table 2. Genetic characterization of the novel

NR5A1/

SF-1 gene variants and of additional variants identified in related genes in combination.

Patient

Chromosome
position

Gene

Variant

Zygosity

Previously reported

ACMG classification
(criteria) (43)

Family studies

Method

9:127265637

NR5A1

c.35_38dup; p.Pro14Valfs*19

Het

LP (PVS1, PM2)

F: ND; M: wt

WES

9:127265638

NR5A1

c.37T>A; p.Cys13Ser

Het

LP (PP3, PM1, PM2)

SGA

9:127265635

NR5A1

c.40C>T; p.Pro14Ser

Het

VUS (PM1, PM2, PP3)

F: ND; M: het

SGA

9:127265625

NR5A1

c.50G>T; p.Gly17Val

Het

LP (PP3, PM1, PM2)

F: het; M: wt

TGP

19:2249631

AMH

c.300C>T; p.Phe100=

Het

B (BS1, BS2, BP4, BP6, BP7) F: wt; M: het

9:127265486

NR5A1

c.116G>T; p.Arg39Leu

Het

46,XY DSD (64)

LP (PP3, PM1, PM5, PM2)

F: ND; M: het

TGP

9:127265394

NR5A1

c.208T>C; p.Phe70Leu

Het

VUS (PM1, PM2, PP3)

F: ND; M: het

WES

9:127265385

NR5A1

c.217T>A; p.Cys73Ser

Het

P (PM5, PP3, PM1, PM2)

F: wt; M: het; S: het SGA

2:31805706

SRD5A2

c.265C>G; p.Leu89Val

Hom

Prostate cancer (65) B (BA1, BP6, BP4)

TGP

9:127265384

NR5A1

c.218G>C; p.Cys73Ser

Het

P (PM5, PP3, PM1, PM2)

F: het; M: wt

9:127265384

NR5A1

c.218G>A; p.Cys73Tyr

Het

P (PP3, PM1, PM5, PM2,
PP5)

F: wt; M: wt

WES

10*

1:43895417

SZT2

c.4039C>T; p.Arg1347Cys

Het

LB (BP4, BP1, PM2)

WES

2:73677990

ALMS1

c.4207A>G; p.Thr1403Ala

Het

LB (BP4, BP1, PM2)

3:15686753

BTD

c.1330G>C; p.Asp444His

Het

Biotinidase activity
(66)

VUS (PP5, PM2)

3:48508260

TREX1

c.206T>C; p.Leu69Pro

Het

VUS (PP3, PM2)

3:58415529

PDHB

c.701-3C>T

Het

LB (BP4, PM2)

5:82815537

VCAN

c.1412C>T; p.Thr471Met

Het

B (BS1, BS2, BP1, BP4)

6:152650875

SYNE1

c.14732G>A; p.Arg4911His

Het

LB (BP4, BP1, PM2)

6:157099607

ARID1B

c.369_392dup;
p.Gln124_Gln131dup

Het

B (BS1, BS2)

7:103341394

RELN

c.865A>G; p.Asn289Asp

Het

VUS (PM2, BP1)

9:127265383

NR5A1

c.219C>G; p.Cys73Trp

Het

P (PP3, PM1, PM5, PP5,
PM2)

F: wt; M: wt

10:28250492

ARMC4

c.1386+5G>A

Het

VUS (PP3, PM2)

11:103029673

DYNC2H1

c.4295T>C; p.Ile1432Thr

Het

VUS (PP3, PM2)

11:124794912

HEPACAM

c.139G>A; p.Val47Met

Het

VUS (PM1, PM2, BP4)

14:88407888

GALC

c.1685T>C; p.Ile562Thr

Het

Krabbe disease (67) B (BA1, BP6, BP4)

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15:28259941

OCA2

c.1025A>G; p.Tyr342Cys

Het

Ocular albinism (68) LP (PP3, PM2, PP5)

17:78087041

GAA

c.2065G>A; p.Glu689Lys

Het

Alpha-glucosidase
activity (69)

B (BA1, BP6, BP4)

21:44837615

SIK1

c.1784G>A; p.Arg595Gln

Het

LB (BP1, BP4, PM2)

9:127265357

NR5A1

c.244+1G>T

Het

P (PVS1, PM2, PP5)

F: het; M: wt; Br:
Het

WES

12*

9:127262992

NR5A1

c.247G>A; p.Val83Met

Het

46,XY DSD (70, 71)

VUS (PM1, PP3, PM2)

TGP

13*

9:127262992

NR5A1

c.247G>T; p.Val83Leu

Het

46,XY DSD (72)

VUS (PM1, PP3, PM2)

F: het; M: wt

TGP

9:127262866

NR5A1

c.370_373del; p.Pro124Argfs*171 Het

LP (PVS1, PM2)

F: wt; M: wt

SGA

2p16.3p16.3 (50732444-
50894316)x1

46,XY DSD (63)

VUS (PM2)

SGA/Arr
ay

9:127262846

NR5A1

c.393G>A; p.Pro131=

Het

LB (BP4, BP7, PM2)

16p13.11p13.11(15830681-
16270149)x3

46,XY DSD (63)

VUS (PM2)

9:127262846

NR5A1

c.393G>A; p.Pro131=

Het

LB (BP4, BP7, PM2)

SGA/Arr
ay

9:127262802

NR5A1

c.437G>C; p.Gly146Ala

Het

Adrenal disease (26)

B (BA1, BP6, BS3, BP4,
PM1)

Xq13.3.3q13.3(74380482-
74567915)x2

Het

46,XY DSD (63)

VUS (PM2)

F: wt; M: het

9:127262846

NR5A1

c.393G>A; p.Pro131=

Het

LB (BP4, BP7, PM2)

SGA

9:127262687

NR5A1

c.552del; p.Tyr185Thrfs*111

Het

LP (PVS1, PM2)

F: wt; M: wt

WES

9:127262684

NR5A1

c.555C>A; p.Tyr185*

Het

LP (PVS1, PM2)

SGA

9:127262607

NR5A1

c.632_668del; p.Tyr211Cysfs*73

Het

LP (PVS1, PM2)

F: ND; M: het

SGA

21*

7:75612866

POR

c.859G>C; p.Ala287Pro

Het

Disordered
steroidogenesis (73)

P (PS3, PP5, PP3, PM2,
BP1)

TGP

9:127262559

NR5A1

c.680T>C; p.Ile227Thr

Het

46,XY DSD (72)

VUS (PM1, PM2)

F:het; M: ND

22*

9:127262400

NR5A1

c.839C>A; p.Ala280Glu

Het

46,XY DSD (72, 74)

LP (PP3, PM1, PM2)

F: mosaic; M: ND;
S: het

TGP

9:127255373

NR5A1

c.926A>T; p.Asp309Val

Het

LP (PP3, PM1, PM2)

F: het; M: wt

WES

9:127255353

NR5A1

c.946del; p.Gln316Serfs*18

Het

LP (PVS1, PM2)

WES

25*

4:126329821

FAT4

c.686A>G; p.Tyr229Cys

Het

VUS (PM2, BP1)

WES

4:126372555

FAT4

c.5278A>G; p.Ile1760Val

Het

LB (BP4, BP1, PM2)

9:127255322

NR5A1

c.977T>C; p.Val326Ala

Het

VUS (PM1, PM2, PP3)

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9:127255314

NR5A1

c.985C>T; p.Gln329*

Het

LP (PVS1, PM2)

F: wt; M: wt

SGA

9:127255314

NR5A1

c.985C>T; p.Gln329*

Het

LP (PVS1, PM2)

TGP

9:127253508

NR5A1

c.991-1G>A

Het

LP (PVS1, PM2)

F: wt; M: wt

WES

29*

9:127253506

NR5A1

c.992T>G; p.Val331Gly

Het

VUS (PM1, PP3, PM2)

TGP

9:127253415

NR5A1

c.1065_1066insTGCTGCAGCTGC
TTGCGCTGG;p.Val355_Leu356in
sCysCysSerCysLeuArgTrp

Het

LP (PM1, PM4, PM2)

SGA

31*

9:127253393

NR5A1

c.1105G>T; p.Val369Phe

Het

VUS (PM1, PP3, PM2)

SGA

9:127253389

NR5A1

c.1106_1109del; p.Val369Alafs*12 Het

LP (PVS1, PM2)

SGA

9:127245036

NR5A1

c.(1138+1_1139-
1)_(1386+1_1387-1)del;
p.Asp380_Thr461del

Het

P (PVS1, PM2)

SGA

9:127245286

NR5A1

c.1139-2A>G

Het

LP (PVS1, PM2)

TGP

9:127245211

NR5A1

c.1157_1211dup; p.Tyr404*

Het

LP (PVS1, PM2)

TGP

12:125460041

DHX37

c.904G>A; p.Gly302Ser

Het

VUS (PP3, PM1, PM2)

36*

9:127245212

NR5A1

c.1211A>G; p.Tyr404Cys

Het

LP (PP3, PM1, PM5, PM2)

TGP

9:127245116

NR5A1

c.1307A>G; p.Tyr436Cys

Het

VUS (PM1, PP3, PM2 )

F: het; M: wt

TGP

9:127245070

NR5A1

c.1353G>A; p.Leu451=

Het

B (BS1, BS2, BP6, BP6, BP7) ND

TGP

39*

2p16.3 dup

F: wt; M: het; Br:
het

TGP

9:127245044

NR5A1

c.1379A>T; p.Gln460Leu

Het

46,XY DSD (75)

LB (BP4, PM1, PM2)

F: het; M: wt; Br:
het

22:38369662

SOX10

c.1241A>C; p.His414Pro

Het

LB (BS2, PP3)

22:38369619

SOX10

c.1284G>T; p.Met428lIe

Het

LB (BS2)

B, benign; Br, brother; F, father; Het, heterozygous; Hom, homozygous; LB, likely benign; LP, likely pathogenic; M, mother; ND, not determined; P, pathogenic; S,

sister; SGA, single gene analysis; TGP, targeted-gene panel; VUS, variant of unknown significance; WES, whole -exome sequencing. Individuals in which next-

generation sequencing (either TGP or WES) was used as the genetic approach are highlighted in bold. *SF-1/

NR5A1

variants tested for functionality in this study.

Sequence information is based on the following reference sequences or transcripts: ALMS1: ENST00000409009; AMH: NM_000479.3; ARID1B: NM_020732.3;

ARMC4: NM_018076.2; BTD: NM_001281723.3; DHX37: NM_032656.3; DYNC2H1: ENST00000398093; FAT4: ENST00000335110; GAA: NM_000152. 3; GALC:

NM_001201401.1; HEPACAM: NM_152722.4; NR5A1: NM_004959.4; OCA2: NM_000275.2; PDHB: NM_001173468.1; POR: NM_000941.2; RELN:

NM_005045.3; SIK1: NM_173354.3; SOX10: NM_006941.3; SRD5A2: NM_000348.4; SYNE1: ENST00000448038.1; SZT2: NM_015284.3; TREX1:

NM_033629.3; VCAN: NM_004385. ACMG criteria for classification of variants pathogenicity: PVS1, very strong evidence of patho genicity; PS1/2, strong evidence

of pathogenicity; PM1-6, moderate evidence of pathogenicity; BA1, stand-alone evidence of benign impact; BP1/2; supporting evidence of benign impact; BS1 -4;

strong

evidence

benign

impact.

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