\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[ngerman,greek,english]{babel}



\usepackage{float}








\begin{document}

\title{Involvement of MAP3K7 in FMD2 and CSCF, delineation of
genotype/phenotype correlations.}



\author[1]{Geeske van Woerden}%
\author[1]{Richelle Senden}%
\author[1]{Charlotte de Konink}%
\author[1]{Rossella Avagliano Trezza}%
\author[2]{anwar baban}%
\author[3]{Jennifer Bassetti}%
\author[1]{Yolande Van Bever}%
\author[4]{Lynne Bird}%
\author[5]{Bregje van Bon}%
\author[1]{Alice Brooks}%
\author[6]{Qiaoning Guan}%
\author[7]{Eric Klee}%
\author[8]{Carlo Marcelis}%
\author[7]{Joel Morales-Rosado}%
\author[7]{Lisa Schimmenti}%
\author[6]{Amy Shikany}%
\author[9]{Paulien Terhal}%
\author[6]{Kathryn Weaver}%
\author[10]{Marja Wessels}%
\author[11]{Hester van Wieringen}%
\author[12]{Anna Hurst}%
\author[12]{Catherine  Gooch}%
\author[13]{Marco Tartaglia}%
\author[13]{Marcello Niceta}%
\author[1]{Ype Elgersma}%
\author[1]{Serwet Demirdas}%
\affil[1]{Erasmus MC}%
\affil[2]{Bambino Gesù Children's Hospital}%
\affil[3]{Weill Cornell Medicine}%
\affil[4]{UCSD}%
\affil[5]{Nijmegen}%
\affil[6]{Cincinnatti Children's Hospital Medical Center Division of Infectious Diseases}%
\affil[7]{Mayo Clinic}%
\affil[8]{Radboudumc}%
\affil[9]{University Medical Center Utrecht}%
\affil[10]{Erasmus University Rotterdam}%
\affil[11]{Sint Antonius Hospital Surgery}%
\affil[12]{Department of Genetics, University of Alabama at Birmingham, Birmingham, Alabama, USA}%
\affil[13]{Bambino Gesù Children’s Hospital, IRCCS}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
Mitogen-Activated Protein 3 Kinase 7 (MAP3K7, MIM 602614) encodes the
ubiquitously expressed transforming growth factor \selectlanguage{greek}β \selectlanguage{english}(TGF-\selectlanguage{greek}β\selectlanguage{english})--activated
kinase 1 (TAK1), which plays a crucial role in many cellular processes.
Variants in the MAP3K7 gene have been linked to 2 distinct disorders:
frontometaphyseal dysplasia type 2 (FMD2, MIM \#617137) and
cardiospondylocarpofacial syndrome (CSCF, MIM \#157800). The fact that
different variants can induce 2 distinct phenotypes suggests a
phenotype/genotype correlation, but no side-by-side comparison has been
done thus far to confirm this. Here we significantly expand the cohort
and the description of clinical phenotypes for individuals with CSCF and
FMD2 who carry variants in MAP3K7. We show that in contrast to
FMD2-causing variants, CSCF-causing variants in MAP3K7 have a
loss-of-function effect. Additionally, patients with pathogenic variants
in MAP3K7 are at risk for cardiac disease, have symptoms associated with
connective tissue disease and we show overlap in clinical phenotypes of
CSCF with Noonan syndrome. Together, we provide evidence for a molecular
fingerprint of FMD2- versus CSCF-causing MAP3K7 variants and conclude
that variants in MAP3K7 should be considered in the differential
diagnosis of patients with syndromic congenital cardiac defects and/or
cardiomyopathy, syndromic connective tissue disorders and in the
differential diagnosis of Noonan syndrome.%
\end{abstract}%



\sloppy


\textbf{Involvement of MAP3K7 in FMD2 and CSCF, delineation of
genotype/phenotype correlations.}

Geeske M. van Woerden\textsuperscript{1,2,3\#}*, Richelle
Senden\textsuperscript{2\#}, Charlotte de Konink\textsuperscript{1,3},
Rossella Avagliano Trezza\textsuperscript{1,3}, Anwar
Baban\textsuperscript{4}, Jennifer Alisha Bassetti\textsuperscript{5},
Yolande van Bever\textsuperscript{2,3}, Lynne M.
Bird\textsuperscript{6}, Bregje W. van Bon\textsuperscript{7}, Alice S.
Brooks\textsuperscript{2,3}, Qiaoning Guan\textsuperscript{8,9}, Eric W.
Klee\textsuperscript{10,11}, Carlo Marcelis\textsuperscript{7}, Joel
Morales Rosado\textsuperscript{10,11,12}, Lisa A.
Schimmenti\textsuperscript{13}, Amy R Shikany\textsuperscript{14},
Paulien A Terhal\textsuperscript{15}, K. Nicole
Weaver\textsuperscript{8,9}, Marja W. Wessels\textsuperscript{2}, Hester
van Wieringen\textsuperscript{16}, Anna C Hurst\textsuperscript{17},
Catherine F Gooch\textsuperscript{17}, Marco
Tartaglia\textsuperscript{o}, Marcello Niceta\textsuperscript{os}, Ype
Elgersma\textsuperscript{abc} and Serwet Demirdas\textsuperscript{b}*

\textsuperscript{1}Department of Neuroscience, Erasmus Medical Center,
Rotterdam, the Netherlands.

\textsuperscript{2}Department of Clinical Genetics, Erasmus Medical
Centre, Rotterdam, the Netherlands.

\textsuperscript{3}The \emph{ENCORE} Expertise Center for
Neurodevelopmental Disorders, Erasmus Medical Center, Rotterdam, the
Netherlands.

\textsuperscript{4}Pediatric Cardiology and Cardiac Arrhythmias Unit,
Bambino Ges\selectlanguage{ngerman}ù Children's Hospital, IRCCS, Rome, Italy.

\textsuperscript{5}Division of Medical Genetics, Department of
Pediatrics, Weill Cornell Medicine, New York, NY, USA.

\textsuperscript{6}Department of Pediatrics, University of California,
San Diego; Division of Genetics/Dysmorphology, Rady Children's Hospital
San Diego, San Diego, California, USA.

\textsuperscript{7}Department of Human Genetics, Radboud University
Medical Center, Nijmegen, The Netherlands.

\textsuperscript{8}Division of Human Genetics, Cincinnati Children's
Hospital Medical Center, Cincinnati, OH, USA.

\textsuperscript{9}Department of Pediatrics, University of Cincinnati
College of Medicine, Cincinnati, Ohio, USA.

\textsuperscript{10}Center for Individualized Medicine, Mayo Clinic,
Rochester, MN, USA.

\textsuperscript{11}Department of Quantitative Health Sciences, Mayo
Clinic, Rochester, MN, USA

\textsuperscript{12}Department of Clinical Genomics, Mayo Clinic,
Rochester, MN, USA.

\textsuperscript{13}Department of Otorhinolaryngology Head and Neck
Surgery, Ophthalmology, Clinical Genomics, and Biochemistry and
Molecular Biology, Mayo Clinic, Rochester, MN, USA.

\textsuperscript{14}The Heart Institute, Cincinnati Children's Hospital
Medical Center, Cincinnati, Ohio, USA.

\textsuperscript{15}Department of Genetics, University Medical Centre
Utrecht, Utrecht, the Netherlands.

\textsuperscript{16}Department of Pediatrics, St. Antonius hospital,
Nieuwegein, the Netherlands.

\textsuperscript{17}Department of Genetics, University of Alabama at
Birmingham, Birmingham, Alabama, USA.

\textsuperscript{18}Genetics and Rare Diseases Research Division,
Ospedale Pediatrico Bambino Gesù, IRCCS, Rome, Italy.

\textsuperscript{19}Department of Pediatrics, Sapienza University, Rome,
Italy.

\# These authors contributed equally

\textbf{*Corresponding authors}

S. Demirdas, MD PhD (phone: +31 10 7036915; email:
s.demirdas@erasmusmc.nl) and G.M. van Woerden, PhD (phone: +31 10
7032695; email: g.vanwoerden@erasmusmc.nl)

\textbf{Funding}

EWK, JMR, LAS are supported by the Center for Individualized Medicine,
Mayo Clinic.

Part of the work of AB, MN and MT was supported by funding from the
Italian Ministry of Health (CCR-2017-23669081, RCR-2020-23670068\_001 to
MT) and Italian Ministry of Research (FOE 2019, 2020 \emph{Sviluppo di
protocolli innovativi e applicazione di nuovi strumenti -omici nei
pazienti orfani di diagnosi} to MT). GMvW is funded by NWO-VIDI
(016.Vidi.188.014)



\textbf{Abstract}

Mitogen-Activated Protein 3 Kinase 7 (\emph{MAP3K7} , MIM 602614)
encodes the ubiquitously expressed transforming growth factor \selectlanguage{greek}β
\selectlanguage{english}(TGF-\selectlanguage{greek}β\selectlanguage{english})--activated kinase 1 (TAK1), which plays a crucial role in many
cellular processes. Variants in the \emph{MAP3K7} gene have been linked
to 2 distinct disorders: frontometaphyseal dysplasia type 2 (FMD2, MIM
\#617137) and cardiospondylocarpofacial syndrome (CSCF, MIM \#157800).
The fact that different variants can induce 2 distinct phenotypes
suggests a phenotype/genotype correlation, but no side-by-side
comparison has been done thus far to confirm this. Here we significantly
expand the cohort and the description of clinical phenotypes for
individuals with CSCF and FMD2 who carry variants in \emph{MAP3K7} . We
show that in contrast to FMD2-causing variants, CSCF-causing variants
in\emph{MAP3K7} have a loss-of-function effect. Additionally, patients
with pathogenic variants in \emph{MAP3K7} are at risk for cardiac
disease, have symptoms associated with connective tissue disease and we
show overlap in clinical phenotypes of CSCF with Noonan syndrome.
Together, we provide evidence for a molecular fingerprint of FMD2-
versus CSCF-causing \emph{MAP3K7} variants and conclude that variants
in\emph{MAP3K7} should be considered in the differential diagnosis of
patients with syndromic congenital cardiac defects and/or
cardiomyopathy, syndromic connective tissue disorders and in the
differential diagnosis of Noonan syndrome.

\textbf{Keywords:} MAP3K7, frontometaphyseal dysplasia type 2,
cardiospondylocarpofacial syndrome , Noonan Syndrome

\textbf{Introduction}

The \emph{MAP3K7} gene (Mitogen-Activated Protein 3 Kinase 7) encodes
transforming growth factor \selectlanguage{greek}β \selectlanguage{english}(TGF-\selectlanguage{greek}β\selectlanguage{english})--activated kinase 1 (TAK1), which
plays a vital role in innate and adaptive immunity by regulating
inflammatory responses and regulating cell differentiation, cell
survival and apoptosis

(Dai et al., 2012; Yu et al., 2014). Variants in \emph{MAP3K7} have been
associated with two autosomal dominant conditions; frontometaphyseal
dysplasia type 2 (FMD2) caused by recurrent gain-of-function variants

(Wade et al., 2016), and cardiospondylocarpofacial syndrome (CSCF)
caused by individual variants most often considered to induce
loss-of-function

(Le Goff et al., 2016).

FMD is a progressive skeletal dysplasia characterized by sclerosis of
the skull, joint contractures and under-modeled long bones, scoliosis
and prominent supraorbital ridges. Other features include hypertelorism,
down-slanting palpebral fissures, broad nasal bridge, full cheeks,
micrognathia, hydronephrosis, cleft palate, hearing loss, ulnar
deviation of the hands, camptodactyly, wrist contractures, long fingers,
structural cardiac defect and keloid scars (Costantini et al., 2018).
FMD is caused by gain-of-function variants in \emph{FLNA} ,
\emph{TAB2}and \emph{MAP3K7}

(Robertson, 2004; Giuliano et al., 2005; Wade et al., 2016; 2017). Most
individuals have normal intellect, although intellectual disability has
been reported

(Basart et al., 2015; Wade et al., 2016). In total, 19 FMD2 patients
carrying variants in \emph{MAP3K7} have been described in literature
(Wade et al., 2017; Costantini et al., 2018). A recurrent variant was
identified in 16 patients (c.1454C\textgreater{}T (p.Pro485Leu)), and
the other three described variants causing the FMD2 phenotype are
c.208G\textgreater{}C (p.Glu70Gln), c.299T\textgreater{}A (p.Val100Glu)
and c.502G\textgreater{}C (p.Gly168Arg). This recurrent variant leads to
a severe skeletal phenotype, keloid scarring, and (in 3 out of 16
patients) intellectual disability (ID). The three patients with the
infrequent missense variants show a milder phenotype consistent with
FMD, without ID (Costantini et al., 2018).

CSCF was first described in 1966 as a new syndrome and autosomal
dominant inheritance was suggested (Forney et al., 1966). The initial
report describes a mother and two of her four daughters with congenital
mitral valve insufficiency, skeletal malformations (fusion of cervical
vertebrae/tarsal bones/carpal bones, phalangeal shortening), conductive
deafness and short stature. The hearing loss resulted from fusion of the
stapes to the round window and was improved by surgical correction. In
2010, two additional patients with an overlapping phenotype were
reported introducing the name CSCF for the first time (Sousa et al.,
2010). However, it was not until 2016 that the gene \emph{MAP3K7} was
identified as the cause of CSCF by Le Goff et al.

(Le Goff et al., 2016) who described 6 patients with CSCF phenotype and
a causative heterozygous missense variant in the gene: three sporadic
cases and one family (a father and two sons). Morlino et al. summarize
these patients in their publication, adding a twelfth novel patient with
CSCF due to a \emph{de} \emph{novo} variant in the \emph{MAP3K7} gene
(Morlino et al., 2018). In 2020, the same group published functional
studies on patient fibroblasts demonstrating that CSCF is caused by
loss-of-function variants in the gene through the TGF\selectlanguage{greek}β-\selectlanguage{english}pathway (Micale
et al., 2020). Interestingly, intellectual disability (ID) has been
described in FMD2, but not in CSCF.

Here, we expand the cohort of individuals with CSCF and FMD2 carrying
variants in \emph{MAP3K7} , describing 14 novel patients with CSCF and 2
novel patients with FMD2 (one with the recurrent variant and severe ID).
Based on the phenotype of the patients we suggest that both CSCF and
FMD2 are connective tissue diseases. We describe severe congenital left
sided cardiac defects in CSCF patients and we hypothesize an overlap of
CSCF patients with the Noonan syndrome (NS) (OMIM 163950) phenotype.
Additionally, we provide for the first time a side-by-side comparison of
the biochemical effects of missense variants in \emph{MAP3K7} for CSCF
and FMD2, aiming to find a molecular fingerprint to distinguish between
the two disorders.

\textbf{Methods}

\emph{Patients}Sixteen patients with novel variants in the \emph{MAP3K7}
gene were included in this clinical observation with functional
laboratory analysis of the variants. Patients were identified in the
clinical setting with trio whole-exome sequencing. Patients from five
countries (7 medical centers in total) were identified using Genematcher
(Sobreira et al., 2015). Written consent was obtained from adult
patients and parents of pediatric patients for participation in the
study and for publishing photographs.

\emph{Clinical observations}

Cross-sectional clinical assessment (medical history, physical
examination, variant classification and (if consented) photography) was
performed by the treating physician to complete a detailed chart of
phenotypic features. If available, radiologic, and cardiac analysis
outcomes were collected from the medical file. All data were entered in
a pseudo-anonymized digital database. The descriptive data were analyzed
and the results are presented in the manuscript text and \textbf{Table
1.}

\emph{Ethical considerations}

The study was conducted according to the principles of the Declaration
of Helsinki (10th version, October 2013, see for the most recent
version: www.wma.net (World Medical Association 2013)) and in accordance
with the Dutch Medical Research Involving Human Subjects Act (WMO).
Approval from the medical ethics comity of the Erasmus Medical Center
was given for conducting our retrospective case-series study in these
patients.

\emph{Laboratory assessments}

\emph{Constructs}

The \emph{MAP3K7\textsuperscript{WT}} (NM\_003188.4) cDNA was obtained
from the human brain library by PCR (Phusion high fidelity, Thermo
Fisher) using the following primers: FW 5'-GTCTCACCCGGATTGTCC; RV
5'-AATGTAACGGTCCCAGAG. To be able to clone this sequence into our dual
promotor expression vector (Proietti Onori et al., 2018), the fragment
was tagged with restriction enzymes AscI; PacI by PCR (Phusion high
fidelity, Thermo Fisher) using primers: FW 5'-
GAATTGCGGCGCGCCACCATGTCTACAGCCTCTGCC; RV 5'-
GAATCCTTAATTAATCATGAAGTGCCTTGTCG. The point mutations were introduced
with site-directed mutagenesis (Phusion high fidelity, Thermo Fisher)
using the following primers: \emph{MAP3K7} c.143G\textgreater{}A
(p.Gly48Glu), FW 5'-GGAAGAGGAGCCTTTGAAGTTGTTTGCAAAGCT and RV
5'-AGCTTTGCAAACAACTTCAAAGGCTCCTCTTCC; \emph{MAP3K7} c.
208G\textgreater{}C (p.Glu70Gln), FW 5'-
TAAACAAATAGAAAGTGAATCTCAGAGGAAAGCGTTTATTGTAGA and RV 5'-
TCTACAATAAACGCTTTCCTCTGAGATTCACTTTCTATTTGTTTA;
\emph{MAP3K7}c.248G\textgreater{}A (p.Arg83His), FW 5'-
GCTTCGGCAGTTATCCCATGTGAACCATCCTAATA and RV 5'-
TATTAGGATGGTTCACATGGGATAACTGCCGAAGC; \emph{MAP3K7} c.
299T\textgreater{}A (p.Val100Glu), FW 5'-
GGAGCCTGCTTGAATCCAGAGTGTCTTGTGATGGAATAT and RV 5'-
ATATTCCATCACAAGACACTCTGGATTCAAGCAGGCTCC;
\emph{MAP3K7}c.329G\textgreater{}A (p.Gly110Asp), FW 5'-
GATGGAATATGCTGAAGGGGACTCTTTATATAATGTGCTGC and RV
5'-GCAGCACATTATATAAAGAGTCCCCTTCAGCATATTCCATC;
\emph{MAP3K7}c.337T\textgreater{}G (p.Tyr113Asp), FW
5'-TGAAGGGGGCTCTTTAGATAATGTGCTGCATGG and RV
5-CCATGCAGCACATTATCTAAAGAGCCCCCTTCA; \emph{MAP3K7} c.502G\textgreater{}C
(p.Gly168Arg), FW 5'- CTTACTGCTGGTTGCAGGGCGGACAGTTCTAAAAATTTG and RV 5'-
CAAATTTTTAGAACTGTCCGCCCTGCAACCAGCAGTAAG;
\emph{MAP3K7}c.586A\textgreater{}G (p.Met196Val), FW 5'-
GGGAGTGCTGCTTGGGTGGCACCTGAAGTTT and RV
5'-AAACTTCAGGTGCCACCCAAGCAGCACTCCC; \emph{MAP3K7} c.617A\textgreater{}G
(p.Tyr206Cys), FW 5'- GAAGTTTTTGAAGGTAGTAATTGCAGTGAAAAATGTGACGTCTTC and
RV 5'- GAAGACGTCACATTTTTCACTGCAATTACTACCTTCAAAAACTTC;
\emph{MAP3K7}c.616T\textgreater{}G (p.Tyr206Asp), FW 5'-
GTTTTTGAAGGTAGTAATGACAGTGAAAAATGTGACG and RV 5'-
CGTCACATTTTTCACTGTCATTACTACCTTCAAAAAC;
\emph{MAP3K7}c.721T\textgreater{}G (p.Trp241Gly), FW 5'-
CCCAGCTTTCCGAATCATGGGGGCTGTTCATAATGGTAC and RV 5'-
GTACCATTATGAACAGCCCCCATGATTCGGAAAGCTGGG;
\emph{MAP3K7}c.1454C\textgreater{}T (p.Pro485Leu), FW
5'-ACTACAGCCTCTAGCACTGTGCCCAAACTCCAAAG and RV
5'-CTTTGGAGTTTGGGCACAGTGCTAGAGGCTGTAGT. For the \emph{in
vivo}experiments the `empty vector' serves as control, consisting of the
dual promotor expression vector only (no gene insertion in MCS).

The \emph{TAB1\textsuperscript{wt}} (NM\_006116.3) cDNA was obtained
from human brain library by PCR (Phusion high fidelity, Thermo Fisher)
using the following primers: FW 5'-ATGGCGGCGCAGAGGAG and RV 5'-
CTACGGTGCTGTCACCAC. This was then also cloned in our dual promotor
expression vector, tagging the fragment with restriction enzymes AscI;
PacI by PCR (Phusion high fidelity, Thermo Fisher) using primers: FW
5'-GGCGCGCCACCATGGCGGCGCAGAGGAG and RV 5'-TTAATTAACTACGGTGCTGTCACCAC.

\emph{HEK293T transfection}

To assess the functional effects of the different \emph{MAP3K7}variants,
HEK293T cells were cultured in DMEM/10\% Fetal Calf Serum (FCS)/1\%
penicillin/streptomycin in 6-wells plates and transfected with the
following constructs: Control vector;\emph{MAP3K7\textsuperscript{WT};
MAP3K7\textsuperscript{G48E}; MAP3K7\textsuperscript{E70Q};
MAP3K7\textsuperscript{R83H}; MAP3K7\textsuperscript{V100E};
MAP3K7\textsuperscript{G110D}; MAP3K7\textsuperscript{Y113D};
MAP3K7\textsuperscript{G168R}; MAP3K7\textsuperscript{M196V};
MAP3K7\textsuperscript{Y206C}; MAP3K7\textsuperscript{Y206D};
MAP3K7\textsuperscript{W241G}; MAP3K7\textsuperscript{P485L};
TAB1\textsuperscript{WT}.} In single construct transfections, 3ug
construct per well was transfected. When in co-transfection of the
different \emph{MAP3K7} constructs with\emph{TAB1\textsuperscript{WT}} ,
1.5 \selectlanguage{greek}µ\selectlanguage{english}g/construct/well was transfected. Constructs for co-transfection
were mixed before actual transfection, allowing both constructs to be
transfected at the same time. At subconfluency (60-70\%), cells were
transfected with PEI according to the manufacturer instructions (Sigma).
To avoid toxicity of prolonged exposure to PEI, the medium was refreshed
after 4-6 hours. Transfected cells were used to prepare protein lysates
for Western blot.

\emph{Western blot}

The cells were harvested in ice cold PBS, 48-72 hours after
transfection. Lysate samples were prepared by homogenizing the harvested
HEK293Tcells in lysate buffer (10mM Tris-HCl 6.8, 2.5\% SDS, 2mM EDTA)
containing protease inhibitor cocktail 2 (\#P5726, Sigma) and 3
(\#P0044, Sigma) and protease inhibitor (\#P8340, Sigma). Protein
concentrations were determined using the BCA kit (Pierce). Final working
protein concentrations were adjusted to 1 mg/ml. Western blots were
probed with primary antibodies against MAP3K7 (sc-7967, 1:1000, Santa
Cruz), phospho-MAP3K7 (Thr187; \#4536, 1:1000, Cel Signalling), ERK1/2
(\#9102, 1:2000, Cell Signaling), phospho-ERK1/2 (\#9101, 1:2000; Cell
Signaling), Actin (MAB1501R, 1:20.000, Chemicon), RFP (\#600401379,
1:2000, Rockland), NFKB (sc-514451, 1:1000, Santa Cruz), phospho-NFkb
(sc-136548, 1:1000, Santa Cruz), GAPDH (2118S, 1:2000, Cell Signaling),
TAB1 (67020-1-Ig, 1:10.000, Proteintech) and secondary antibodies (goat
anti-mouse (\#926-32210) and goat anti-rabbit (\#926-68021), all
1:15.000, LI-COR). Blots were quantified using LI-COR Odyssey Scanner
and Odyssey 3.0 software.

\emph{Statistical analysis}

All data were assumed to be normally distributed. Statistical difference
between the conditions for the \emph{in vitro} overexpression
experiments was determined using one-way analysis of variance (ANOVA)
followed by Bonferroni's post-hoc test for multiple comparisons. For the
Western blot analysis, two-tailed unpaired t-test was used (dual
comparison). Neuronal migration was analyzed based on the proportion of
electroporated cells targeted to the cortical plate at P1 (defined as
the most proximal 40\% of dorsoventral distance between the pia and
ventricle (first 4 of 10 equally-spaced bins)) or to layer 2/3 of the
somatosensory cortex, defined as the proximal 30\% of dorsoventral
distance between the proximal 10\% of the cortex (assumed to represent
layer 1) and ventricle (bin 2-4 of equally-spaced bins). For neuronal
migration analyses, we used a minimum of 3 targeted pups per condition.



\textbf{Results}

A combined overview of the variants in our cohort and reported in
literature are shown in \textbf{Figure 1} . Patient characteristics and
detailed phenotypic description are shown in \textbf{Table 1} . If a
specific feature was not clearly reported as positive or negative for a
patient, this feature was considered as missing data for the respective
patient. For this reason, the total number of patients per feature may
differ in our results.

\emph{Clinical phenotype of cardiospondylocarpofacial syndrome}

Of the fourteen patients with the CSCF phenotype, twelve carried\emph{de
novo} variants in \emph{MAP3K7} , one female inherited the variant from
her father and one female did not inherit the variant from her mother,
but father was unavailable for testing. The clinical phenotype and
patient characteristics are presented in detail in\textbf{Table 1} . Two
adult CSCF patients were included. \textbf{Figure 2} shows photographs
of the patients with CSCF who consented for publication.

\emph{Dysmorphic features}

The most frequently recorded dysmorphic features are hypotonic face
(6/12 patients), full cheeks (9/13), low set ears (5/10), posteriorly
rotated ears (7/12), hypertelorism (8/12), ptosis (8/13), epicanthal
folds (6/12), peri-orbital fullness (7/11), anteverted nares (7/11),
round tipped nose (9/11), long philtrum (7/9) and widely spaced nipples
(7/9). Short stature (\textless{}-2 SDS for age and gender) was observed
in 9/14 CSCF patients, ranging from -2.1 SDS to -4 SDS. Other less
frequently reported features are listed in \textbf{Table 1} .

\emph{Cardiac features}

Interestingly, CSCF patients showed a wide range of cardiac anomalies.
Congenital heart defects that mostly seem to be left sided are reported
in 7/13 patients ranging from mitral valve prolapse to hypoplastic left
heart syndrome spectrum (Y206C variant, patient 11; \textbf{Table 1} ).
Cardiomyopathy was observed in 4/12 patients (1 hypertrophic and 3
dilated), the most severe in a neonate with the 143G\textgreater{}A
variant (patient 2), who is on the waiting list for heart
transplantation.

\emph{Musculoskeletal features}

Carpal fusion was assessed in seven patients and found in two of them.
Brachydactyly was frequently observed (6/9). Pectus excavatum was seen
in 3/11 patients. Joint laxity was a consistent feature in the pediatric
patients and reported in 8/10 patients. Scoliosis was reported in two
patients and for one of the patients this was described as a Hobbs curve
of 35 degrees. Vertebral anomalies were identified in 4/6 patients and
involved fusion of vertebrae (also of the odontoid process and anterior
arch of C1), small intervertebral distance (thoracic), loss of height of
vertebrae and dysmorphic vertebrae. No contractures were described in
our CSCF patients.

\emph{Neurologic features}

No ID was described in any of the CSCF patients. Autism spectrum
disorder, however, was present in 3/7 patients. Other neurologic
features that drew attention are hypotonia (7/10) and hypoplasia of
muscles; right pectoralis hypoplasia in one patient and hypoplasia of
the adductor pollicis (3/6).

\emph{Hearing loss}

Five out of eleven patients are reported to have hearing loss: 1
sensorineural with defective inner ear formations, and the other 4
conductive/mixed.

\emph{Other features}

One male patient had cryptorchidism. Ten out of 11 patients had feeding
difficulties at a young age, frequently needing nasogastric tube feeding
for a period of time.

\emph{Clinical phenotype frontometaphyseal dysplasia type 2}

Only two patients in this cohort have FMD2. Both patients have a\emph{de
novo} variant (one having the recurrent variant). The clinical phenotype
and patient characteristics are presented in detail in

\textbf{Table 1.}

\emph{Dysmorphic features}

The reported dysmorphic features in our two patients are consistent with
the FMD2 patients reported in medical literature, including full cheeks,
hypertelorism, epicanthal folds and micrognathia (\textbf{Figure 3} ).

\emph{Cardiac features}

Both FMD2 patients had a persistent ductus arteriosus, which was part of
a more complex cardiac defect (ventricular septal defect, pulmonary
atresia, bicuspid aortic valve with mild stenosis, progressive aortic
root dilatation (\textgreater{} 3 --z-score), persistent left superior
vena cava draining into the coronary sinus and ectopic atrial
tachycardia) in the patient born at 35 weeks of gestation with the
recurrent P485L variant. The congenital heart defect in this patient was
a major reason for failure to thrive and recurrent admissions in her
early months of life.

\emph{Musculoskeletal features}

Both FMD2 patients had scoliosis and short stature (-2.2 SDS and -3.1
SDS). Both patients show camptodactyly of their fingers, flexion
contractures (elbow with dislocation of the radial head) and carpal
fusion. The older male patient has joint laxity. The younger female
patient shows flared metaphyses, broad thumbs/fingers osteosclerosis,
and partially fused with dysmorphic cervical vertebrae.

\emph{Neurologic features}

The patient with the recurrent variant has severe ID. Her brain MRI
shows cerebral atrophy, thin corpus callosum and Chiari 1 malformation.
She also has hypotonia and is suspected to possibly have muscular
atrophy. The other patient has normal intellect and tone, as well as
muscular hypoplasia of his hands.

\emph{Hearing loss}

The adult patient (with the non-recurrent variant) was shown to have
mixed conductive/sensorineural hearing loss. The pediatric female
patient with the recurrent variant was reported to also have hearing
loss (type not specified). No temporal bone imaging is available for
these patients.

\emph{Other features}

The adult patient (with the non-recurrent variant) had cryptorchidism
and was consulted for ambiguous genitalia as a newborn. The young female
patient (with the recurrent variant) had congenital stridor due to
laryngo -- trachea-bronchomalacia that improved with time and without
the need for tracheostomy. Feeding problems are managed by PEG. Both
patients showed excessive keloid scarring.

\emph{Functional assessment of the missense variants identified in
MAP3K7}

The differences in clinical features seen in FMD2 versus CSCF patients
are thought to be caused by different underlying molecular
mechanisms.\emph{MAP3K7} variants giving rise to FMD2 are most often
recurrent, gain-of-function variants

(Wade et al., 2016), whereas CSCF is frequently caused by non-recurrent
missense variants, where for some variants a loss-of-function effect was
shown, but the functional effect of other variants remains unknown. Our
cohort, in combination with already published patients, allowed us to
assess whether there is indeed a molecular fingerprint that can
distinguish between FMD2 and CSCF. Since our cohort has only 2 FMD2
patients, we included the previously published FMD2-causing missense
variants in \emph{MAP3K7}

(Wade et al., 2016).

In searching for a molecular fingerprint, we first assessed the
expression levels of the different \emph{MAP3K7} variants upon
overexpression in HEK-293T cells. Whereas the variants causing FMD2
showed similar or even a significantly higher expression level compared
to MAP3K7\textsuperscript{WT}, all CSCF variants showed significantly
lower expression levels compared to MAP3K7\textsuperscript{WT} (FMD2
variants: one-way ANOVA F{[}5,25{]}=6.16, p=0.0008;
MAP3K7\textsuperscript{WT} versus MAP3K7\textsuperscript{E70Q}: p=0.39;
MAP3K7\textsuperscript{WT} versus MAP3K7\textsuperscript{V100E}:
p=0.087; MAP3K7\textsuperscript{WT} versus
MAP3K7\textsuperscript{Y113D}: p=0.42; MAP3K7\textsuperscript{WT} versus
MAP3K7\textsuperscript{G168R}: p=0.40; MAP3K7\textsuperscript{WT} versus
MAP3K7\textsuperscript{P485L}: p=0.0079, Dunnett's multiple comparison
test; CSCF variants: one-way ANOVA F{[}7,23{]}=6.39, p=0.0003;
MAP3K7\textsuperscript{WT} versus MAP3K7\textsuperscript{G48E}: p=0.004;
MAP3K7\textsuperscript{WT} versus MAP3K7\textsuperscript{R83H}:
p=0.0017; MAP3K7\textsuperscript{WT} versus
MAP3K7\textsuperscript{G110D}: p=0.041; MAP3K7\textsuperscript{WT}versus
MAP3K7\textsuperscript{M196V}: p=0.0013; MAP3K7\textsuperscript{WT}
versus MAP3K7\textsuperscript{Y206C}: p=0.01; MAP3K7\textsuperscript{WT}
versus MAP3K7\textsuperscript{Y206D}: p=0.0025;
MAP3K7\textsuperscript{WT} versus MAP3K7\textsuperscript{W241G}:
p\textless{}0.0001, Dunnett's multiple comparison test ; \textbf{Figure
4A} ). With the exception of MAP3K7\textsuperscript{W241G}, reduced
expression levels of the CSCF variants could be normalized to control
levels upon co-transfection with TAB1 (one-way ANOVA F{[}7,54{]}=4.3,
p\textless{}0.001; MAP3K7\textsuperscript{WT} versus
MAP3K7\textsuperscript{G48E}: p\textless{}0.0001;
MAP3K7\textsuperscript{WT} versus MAP3K7\textsuperscript{R83H}: p=0.86;
MAP3K7\textsuperscript{WT} versus MAP3K7\textsuperscript{G110D}: p=0.9;
MAP3K7\textsuperscript{WT} versus MAP3K7\textsuperscript{M196V}: p=0.25;
MAP3K7\textsuperscript{WT} versus MAP3K7\textsuperscript{Y206C}: p0.9;
MAP3K7\textsuperscript{WT} versus MAP3K7\textsuperscript{Y206D}: p=0.06;
MAP3K7\textsuperscript{WT} versus MAP3K7\textsuperscript{W241G}: p=0.02,
Dunnett's multiple comparison test; \textbf{Figure 4B} ), indicating
that these variants cause instability of the MAP3K7 protein in the
absence of TAB1.

Co-expression of \emph{MAP3K7} with \emph{TAB1} has been shown to result
in \emph{MAP3K7} autophosphorylation at Thr187, as well as a slower
migration of the TAB1 band on Western blot (Sakurai et al., 2000).
Therefore, we used this assay as a readout for the kinase activity
of\emph{MAP3K7} . Consistent with literature, co-expression of
MAP3K7\textsuperscript{WT} with TAB1 resulted in slower migration of the
MAP3K7, as well as the TAB1 band on Western blot (\textbf{Figure 4B} ),
which corresponded with MAP3K7 autophosphorylation of MAP3K7 at Thr187
(\textbf{Figure 4C} ). In support of previous findings that FMD2 is
caused mainly by gain-of-function variants, co-expression of the
FMD2-related \emph{MAP3K7} variants with TAB1, all resulted in equal or
(a trend towards) increased levels of autophosphorylation
of\emph{MAP3K7} at Thr187 compared to MAP3K7\textsuperscript{WT}
(one-way ANOVA F{[}5,42{]}=5.43, p=0.0006; MAP3K7\textsuperscript{WT}
versus MAP3K7\textsuperscript{E70Q}: p=0.9; MAP3K7\textsuperscript{WT}
versus MAP3K7\textsuperscript{V100E}: p=0.4; MAP3K7\textsuperscript{WT}
versus MAP3K7\textsuperscript{Y113D}: p=0.03; MAP3K7\textsuperscript{WT}
versus MAP3K7\textsuperscript{G168R}: p=0.0004;
MAP3K7\textsuperscript{WT}versus MAP3K7\textsuperscript{P485L}: p=0.9,
Dunnett's multiple comparison test (\textbf{Figure 4C} )). In contrast,
upon co-transfection with TAB1 the majority of the CSCF-related MAP3K7
variants showed significantly reduced pThr187 autophosphorylation levels
compared to MAP3K7\textsuperscript{WT} (one-way ANOVA F{[}7,54{]}=15.9,
p\textless{}0.0001; MAP3K7\textsuperscript{WT} versus
MAP3K7\textsuperscript{G48E}: p\textless{}0.0001;
MAP3K7\textsuperscript{WT} versus MAP3K7\textsuperscript{G110D}:
p\textless{}0.0001; MAP3K7\textsuperscript{WT} versus
MAP3K7\textsuperscript{M196V}: p\textless{}0.0001;
MAP3K7\textsuperscript{WT} versus MAP3K7\textsuperscript{Y206C}:
p=0.0002;MAP3K7\textsuperscript{WT} versus
MAP3K7\textsuperscript{Y206D}: p\textless{}0.0001;
MAP3K7\textsuperscript{WT} versus MAP3K7\textsuperscript{W241G}:
p\textless{}0.0001, Dunnett's multiple comparison test (\textbf{Figure
4C} )). Interestingly, one CSCF-related \emph{MAP3K7} variant
(MAP3K7\textsuperscript{R83H}), did not show reduced pThr187
autophosphorylation levels compared to
MAP3K7\textsuperscript{WT}(MAP3K7\textsuperscript{WT} versus
MAP3K7\textsuperscript{R83H}: p=0.48, Dunnett's multiple comparison test
(\textbf{Figure 4C} )).

MAP3K7 is known to have several downstream substrates, through which it
affects different pathways in the cell (Xu and Lei, 2020; Aashaq et al.,
2019). Finding that the stability and the autophosphorylation levels
seem to distinguish between CSCF and FMD2-related \emph{MAP3K7}variants,
we next sought to understand whether there is a difference in substrate
regulation between the CSCF and FMD2-related \emph{MAP3K7}variants.
Consistent with the reduced autophosphorylation, we found that the
CSCF-related \emph{MAP3K7} variants resulted in reduced phosphorylated
NFkB compared to MAP3K7\textsuperscript{WT}, again with the exception of
MAP3K7\textsuperscript{R83H} (one-way ANOVA F{[}7,51{]}=23.73,
p\textless{}0.0001; MAP3K7\textsuperscript{WT} versus
MAP3K7\textsuperscript{G48E}: p\textless{}0.0001;
MAP3K7\textsuperscript{WT} versus MAP3K7\textsuperscript{R83H}: p=0.66;
MAP3K7\textsuperscript{WT} versus MAP3K7\textsuperscript{G110D}:
p\textless{}0.0001; MAP3K7\textsuperscript{WT} versus
MAP3K7\textsuperscript{M196V}: p\textless{}0.0001;
MAP3K7\textsuperscript{WT} versus MAP3K7\textsuperscript{Y206C}:
p\textless{}0.0001; MAP3K7\textsuperscript{WT} versus
MAP3K7\textsuperscript{Y206D}: p\textless{}0.0001;
MAP3K7\textsuperscript{WT} versus MAP3K7\textsuperscript{W241G}:
p\textless{}0.0001, Dunnett's multiple comparison test (\textbf{Figure
5A} )).

As for the FMD2-related \emph{MAP3K7} variants, the results were less
straightforward. Overall, the variants did not cause increased levels of
phosphorylated NFkB, instead most variants showed a trend or significant
reduction in phosphorylated NFkB (one-way ANOVA F{[}5,49{]}=4.11,
p=0.0034; MAP3K7\textsuperscript{WT} versus
MAP3K7\textsuperscript{E70Q}: p=0.04; MAP3K7\textsuperscript{WT} versus
MAP3K7\textsuperscript{V100E}: p=0.4; MAP3K7\textsuperscript{WT} versus
MAP3K7\textsuperscript{Y113D}: p=0.54; MAP3K7\textsuperscript{WT} versus
MAP3K7\textsuperscript{G168R}: p=0.018; MAP3K7\textsuperscript{WT}versus
MAP3K7\textsuperscript{P485L}: p=0.74, Dunnett's multiple comparison
test (\textbf{Figure 5A} )). These results indicate that whereas
stability and autophosphorylation of \emph{MAP3K7} at Thr187 can
potentially be used as molecular fingerprint for distinguishing FMD2 and
CSCF variants, not all downstream pathways of MAP3K7 are necessarily
differentially affected.

The majority of the CSCF patients showed clinical features in part
similar to NS, where the RAS-MAPK pathway is hyperactivated (van der
Burgt, 2007; Jorge et al., 2009). We therefore assessed whether the
CSCF-related \emph{MAP3K7} variants upregulated the RAS-MAPK pathway.
However, in contrast to what is normally seen in NS, we found that most
of the CSCF-related \emph{MAP3K7} variants resulted in reduced
phosho-ERK levels compared to MAP3K7\textsuperscript{WT} (one-way ANOVA
F{[}7,24{]}=6.78, p=0.0002; MAP3K7\textsuperscript{WT} versus
MAP3K7\textsuperscript{G48E}: p=0.016; MAP3K7\textsuperscript{WT} versus
MAP3K7\textsuperscript{G110D}: p=0.007; MAP3K7\textsuperscript{WT}versus
MAP3K7\textsuperscript{M196V}: p=0.005; MAP3K7\textsuperscript{WT}
versus MAP3K7\textsuperscript{Y206C}: p=0.008;
MAP3K7\textsuperscript{WT} versus MAP3K7\textsuperscript{Y206D}:
p=0.009; MAP3K7\textsuperscript{WT}versus MAP3K7\textsuperscript{W241G}:
p=0.021, Dunnett's multiple comparison test (\textbf{Figure 5B} )).
Consistent with our previous results, only MAP3K7\textsuperscript{R83H}
behaved similar to MAP3K7\textsuperscript{WT}
(MAP3K7\textsuperscript{WT} versus MAP3K7\textsuperscript{R83H}: p=0.9,
Dunnett's multiple comparison test (\textbf{Figure 5B} )).

\textbf{Discussion}

In this study, we significantly expand the published cohort of patients
with variants in the \emph{MAP3K7} gene, causing either CSCF or FMD2 and
assess for the first time the genotype-phenotype correlation
for\emph{MAP3K7} variants. We show that missense variants in
\emph{MAP3K7}causing CSCF reduce MAP3K7 protein stability and
autophosphorylation at Thr187, whereas \emph{MAP3K7} variants causing
FMD2 show normal or enhanced MAP3K7 protein expression and
autophosphorylation levels. However, when assessing one of the
downstream targets, NFkB, no difference was observed between the
variants causing CSCF or FMD2, indicating that further research is
required to assess what downstream targets are differentially affected,
to further elucidate the precise molecular mechanism underlying CSCF and
FMD2.

Using whole exome sequencing (WES) in 5 independent Dutch pediatric
patients (from two separate medical centers) suspected to have NS,
variants of unknown significance were found in \emph{MAP3K7} . Further
clinical assessment (detailed physical examination and radiologic
assessment of the skeleton) of these patients confirmed the diagnosis of
CSCF. To further characterize genotype/phenotype correlations in these
patients, we studied 13 children (12 CSCF, 1 FMD2) and 3 adults (1 FMD2)
with variants in \emph{MAP3K7.} Features of our CSCF patients
overlapping NS included short stature (n=9; 64\%), congenital cardiac
abnormalities (n=7, 54\%), cardiomyopathy (n=4, 33\%) posteriorly
rotated and/or low set ears (n=7; 58\%), hypertelorism (n=8; 67\%),
ptosis (n=8, 62\%), widely spaced nipples (n=7, 78\%) and epicanthal
folds (n=6; 50\%). Downslanting palpebral fissures (n=3; 23\%),
triangular face (n=4; 33\%), short or webbed neck (n=3, 27\%) and
scoliosis (n=2, 22\%) were less often present. Distinguishing features
of the CSCF patients are the wide nose with bulbous tip (n=9; 82\%),
peri-orbital fullness (n=7, 64\%) and the full cheeks (n=9; 69\%).
Information about the neurological phenotype was available for 7
patients, who showed normal intelligence; three had autism spectrum
disorder. Hypotonia was found in 7 patients (70\%), and joint laxity in
8 (80\%). The resemblance of CSCF to NS made us wonder if CSCF should
also be considered a RASopathy; however, the RAS/MAPK pathway is
downregulated instead of upregulated, suggesting an alternative
explanation for the resemblance of CSCF to NS. Despite these molecular
results, CSCF should be considered in the differential diagnosis of
NS-patients based on the described clinical features.

Both our FMD2 patients showed phenotypes compatible with previously
reported patients (Wade et al., 2017). Interestingly, our adult male
FMD2 patient initially presented with ambiguous genitalia and he has a
mild skeletal phenotype with normal/high intelligence. The pediatric
patient with the recurrent variant for FMD2 has a severe cardiac,
neurological and skeletal phenotype. Distinguishing features of the FMD2
patients in comparison to NS and CSCF were prominent supraorbital
ridges, flexion contractures of the elbows, ulnar deviation of the
hands, interphalangeal joint contractures, camptodactyly and keloid
scarring (all present in both patients). Overlapping features with NS
were short stature, congenital cardiac defects, hypertelorism,
epicanthal folds, ptosis (n=1) and failure to thrive in infancy. Neither
patient had a triangular face, short or webbed neck. FMD2 patients have
more severe dysmorphism than CSCF patients (\textbf{Figure 3} ).

Regarding the occurrence of left sided heart lesions and the potential
progressive aspect in our patient of aortic dilatation, these features
go along with previous reports that genes which belong to TGF\selectlanguage{greek}β \selectlanguage{english}cascade
are potentially prone to this subcategory of cardiac involvement (Baban
et al., 2018). It might be wise to establish a specific screening
program through serial electrocardiography and echocardiography detect
progressive aortic dilatation or arrhythmias.

Our cohort in combination with the \emph{MAP3K7} mutations causing FMD2
described in literature allowed for the first time to search for
genotype/phenotype correlations, i.e. side-by-side comparison of the
different missense mutations causing either FMD2 or CSCF. Confirming
previous reports, we found that the FMD2-causing mutations
in\emph{MAP3K7} produce a gain-of-function effect on MAP3K7.
Additionally, we now provide clear evidence that the CSCF-causing
variants in\emph{MAP3K7} have a loss-of-function effect. This is most
clearly seen when looking at the expression and pThr187 levels of
MAP3K7. Interestingly, when assessing one of the downstream targets
of\emph{MAP3K7} , NFkB (Xu and Lei, 2020), this distinction between the
FMD2- or CSCF-causing variants is not evident, suggesting other
molecular pathways are involved in the pathogenesis of these two
disorders. Indeed, we have only assessed the effect on this pathway in
co-expression of TAB1 and MAP3K7. Possibly there is a more distinct
phenotype when assessing the function of MAP3K7 when in complex with
TAB2/3. Additionally, other downstream pathways remain to be studied.

In this paper we significantly expanded the cohort of patients with
either CSCF or FMD2 and performed for the first time a side-by-side
comparison of the different missense variants related to either FMD2 or
CSCF. We have shown that both clinical phenotypes exhibit a clear
overlap with other syndromic connective tissue disorders with respect to
bone deformities, short stature, hypermobile joints, contractures and
cardiac anomalies. This fits what is known about \emph{MAP3K7} 's effect
on the TGF\selectlanguage{greek}β \selectlanguage{english}pathway

(Le Goff et al., 2016; Yu et al., 2014). Additionally, we have
identified that cardiac anomalies are common in patients with
pathogenic\emph{MAP3K7} variants, which may be severe. The cardiac
phenotype ranged from left sided congenital heart defects to
cardiomyopathies (both hypertrophic and dilated) and arrhythmias. The
latter subgroup is infrequent in NS (Pierpont and Digilio, 2018; Gelb et
al., 2015). In conclusion, we show that expression and
autophosphorylation levels of\emph{MAP3K7} can serve as a molecular
fingerprint to distinguish between FMD2- or CSCF-causing \emph{MAP3K7}
variants. Additionally, we show that \emph{MAP3K7} variants should be
considered in the differential diagnosis of patients with syndromic
congenital cardiac (valve) anomalies and/or cardiomyopathy, syndromic
connective tissue disorders as well as NS.

\textbf{Acknowledgements}

We thank our patients for their willingness to share their information
to further our understanding of CSCF and FMD2. We thank dr. Ineke van
der Burgt (Division of Human Genetics, Radboud medical center, Radboud
University, Nijmegen the Netherlands) for her valuable insights and help
setting up the study.

\textbf{Conflict of interest}

None of the co-authors have any conflict of interest to declare.

\textbf{Data availability}

All data is available upon reasonable request.

\textbf{Bibliography}

Aashaq, S, Batool, A, Andrabi, KI. 2019. TAK1 mediates convergence of
cellular signals for death and survival. Apoptosis 24: 3--20.

Baban, A, Magliozzi, M, Loeys, B, Adorisio, R, Alesi, V, Secinaro, A,
Corica, B, Vricella, L, Dietz, HC, Drago, F, Novelli, A, Amodeo, A.
2018. First evidence of maternally inherited mosaicism in TGFBR1 and
subtle primary myocardial changes in Loeys-Dietz syndrome: a case
report. BMC Med. Genet. 19: 170--7.

Basart, H, van de Kar, A, Ad\selectlanguage{ngerman}ès, L, Cho, T-J, Carter, E, Maas, SM,
Wilson, LC, van der Horst, CMAM, Wade, EM, Robertson, SP, Hennekam, RC.
2015. Frontometaphyseal dysplasia and keloid formation without FLNA
mutations. Am. J. Med. Genet. 167: 1215--1222.

Costantini, A, Wallgren-Pettersson, C, Mäkitie, O. 2018. Expansion of
the clinical spectrum of frontometaphyseal dysplasia 2 caused by the
recurrent mutation p.Pro485Leu in MAP3K7. European Journal of Medical
Genetics 61: 612--615.

Dai, L, Aye Thu, C, Liu, X-Y, Xi, J, Cheung, PCF. 2012. TAK1, more than
just innate immunity. IUBMB Life 64: 825--834.

Forney, WR, Robinson, SJ, Pascoe, DJ. 1966. Congenital heart disease,
deafness, and skeletal malformations: a new syndrome? J Pediatr 68:
14--26.

Gelb, BD, Roberts, AE, Tartaglia, M. 2015. Cardiomyopathies in Noonan
syndrome and the other RASopathies. Prog Pediatr Cardiol 39: 13--19.

Giuliano, F, Collignon, P, Paquis-Flucklinger, V, Bardot, J, Philip, N.
2005. A new three-generational family with frontometaphyseal dysplasia,
male-to-female transmission, and a previously reported FLNA mutation.
Am. J. Med. Genet. 132A: 222--222.

Jorge, AAL, Malaquias, AC, Arnhold, IJP, Mendonca, BB. 2009. Noonan
syndrome and related disorders: a review of clinical features and
mutations in genes of the RAS/MAPK pathway. Horm Res 71: 185--193.

Le Goff, C, Rogers, C, Le Goff, W, Pinto, G, Bonnet, D, Chrabieh, M,
Alibeu, O, Nistchke, P, Munnich, A, Picard, C, Cormier-Daire, V. 2016.
Heterozygous Mutations in MAP3K7, Encoding TGF-\selectlanguage{greek}β-\selectlanguage{english}Activated Kinase 1,
Cause Cardiospondylocarpofacial Syndrome. Am. J. Hum. Genet. 99:
407--413.

Micale, L, Morlino, S, Biagini, T, Carbone, A, Fusco, C, Ritelli, M,
Giambra, V, Zoppi, N, Nardella, G, Notarangelo, A, Schirizzi, A,
Mazzoccoli, G, Grammatico, P, Wade, EM, Mazza, T, Colombi, M, Castori,
M. 2020. Insights into the molecular pathogenesis of
cardiospondylocarpofacial syndrome: MAP3K7 c.737-7A~\textgreater{}~G
variant alters the TGF\selectlanguage{greek}β-\selectlanguage{english}mediated \selectlanguage{greek}α-\selectlanguage{english}SMA cytoskeleton assembly and
autophagy. Biochim Biophys Acta Mol Basis Dis 1866: 165742.

Morlino, S, Castori, M, Dordoni, C, Cinquina, V, Santoro, G, Grammatico,
P, Venturini, M, Colombi, M, Ritelli, M. 2018. A novel MAP3K7 splice
mutation causes cardiospondylocarpofacial syndrome with features of
hereditary connective tissue disorder. Eur J Hum Genet: 1--5.

Pierpont, ME, Digilio, MC. 2018. Cardiovascular disease in Noonan
syndrome. Current Opinion in Pediatrics 30: 601--608.

Proietti Onori, M, Koopal, B, Everman, DB, Worthington, JD, Jones, JR,
Ploeg, MA, Mientjes, E, van Bon, BW, Kleefstra, T, Schulman, H, Kushner,
SA, K\selectlanguage{ngerman}üry, S, Elgersma, Y, van Woerden, GM. 2018. The intellectual
disability-associated CAMK2G p.Arg292Pro mutation acts as a pathogenic
gain-of-function. Hum. Mutat. 39: 2008--2024.

Robertson, SP. 2004. Molecular pathology of filamin A: diverse
phenotypes, many functions. Clinical Dysmorphology 13: 123--131.

Sakurai, H, Miyoshi, H, Mizukami, J, Sugita, T. 2000.
Phosphorylation-dependent activation of TAK1 mitogen-activated protein
kinase kinase kinase by TAB1. FEBS Lett. 474: 141--145.

Sobreira, N, Schiettecatte, F, Valle, D, Hamosh, A. 2015. GeneMatcher: a
matching tool for connecting investigators with an interest in the same
gene. Hum. Mutat. 36: 928--930.

Sousa, SB, Baujat, G, Abadie, V, Bonnet, D, Sidi, D, Munnich, A, Krakow,
D, Cormier-Daire, V. 2010. Postnatal growth retardation, facial
dysmorphism, spondylocarpal synostosis, cardiac defect, and inner ear
malformation (cardiospondylocarpofacial syndrome?)--a distinct syndrome?
Am. J. Med. Genet. 152A: 539--546.

van der Burgt, I. 2007. Noonan syndrome. Orphanet Journal of Rare
Diseases 2: 4--6.

Wade, EM, Daniel, PB, Jenkins, ZA, McInerney-Leo, A, Leo, P, Morgan, T,
Addor, MC, Adès, LC, Bertola, D, Bohring, A, Carter, E, Cho, T-J, Duba,
H-C, Fletcher, E, Kim, CA, Krakow, D, Morava, E, Neuhann, T,
Superti-Furga, A, Veenstra-Knol, I, Wieczorek, D, Wilson, LC, Hennekam,
RCM, Sutherland-Smith, AJ, Strom, TM, Wilkie, AOM, Brown, MA, Duncan,
EL, Markie, DM, Robertson, SP. 2016. Mutations in MAP3K7 that Alter the
Activity of the TAK1 Signaling Complex Cause Frontometaphyseal
Dysplasia. Am. J. Hum. Genet. 99: 392--406.

Wade, EM, Jenkins, ZA, Daniel, PB, Morgan, T, Addor, MC, Adès, LC,
Bertola, D, Bohring, A, Carter, E, Cho, T-J, de Geus, CM, Duba, H-C,
Fletcher, E, Hadzsiev, K, Hennekam, RCM, Kim, CA, Krakow, D, Morava, E,
Neuhann, T, Sillence, D, Superti-Furga, A, Veenstra-Knol, HE, Wieczorek,
D, Wilson, LC, Markie, DM, Robertson, SP. 2017. Autosomal dominant
frontometaphyseal dysplasia: Delineation of the clinical phenotype. Am.
J. Med. Genet.

Xu, Y-R, Lei, C-Q. 2020. TAK1-TABs Complex: A Central Signalosome in
Inflammatory Responses. Front Immunol 11: 608976.

Yu, J, Zhang, F, Wang, S, Zhang, Y, Fan, M, Xu, Z. 2014. TAK1 is
activated by TGF-\selectlanguage{greek}β \selectlanguage{english}signaling and controls axonal growth during brain
development. J Mol Cell Biol 6: 349--351.

\textbf{Table 1. Patient characteristics and detailed phenotypic
description of all included patients.}

\textbf{Figure legends}

\textbf{Figure 1. Schematic overview of MAP3K7 showing the localization
of the different variants.} Schematic overview of \emph{MAP3K7} showing
the Kinase and TAB1 binding domain at the N-terminal and the TAB2/3
binding domain at the C-terminal of \emph{MAP3K7} . In black the
CSCF-related \emph{MAP3K7} variants are indicated and in blue the
FMD2-related \emph{MAP3K7} variants. The underlined variants are the
novel variants identified in our cohort. MAP3K7\textsuperscript{P485L}is
the most recurrent FMD2-causing variant.

\textbf{Figure 2. Light photographs of patients with CSCF}

Five patients with CSCF: patients show hypertelorism, full eyelids, full
cheeks, round nasal tip, low set ears and almond shaped eyes.

\textbf{Figure 3. Clinical description and light
photography/radiological pictures of the patient with the recurrent
P485L variant causing FMD2 (patient 16).}

Cranio-facial dysmorphisms at 2 months (panels 1-3): bi-temporal
narrowing, hypotelorism, down-slanting palpebral fissures, short nose
with flat nasal bridge and depressed root, long and deeply grooved
filter, microretrognathia. Radiological findings (panels 4-7) at 7
months: a partial fusion of cervical vertebrae (C2-C3) and reduced bone
length in relation to age. Magnetic resonance imaging (MRI) at 7 months:
T2-weighted hyperintensity of presumed vascular lesion in the coronal
and transverse planes (panels 8-9); T1 weighed scans in the sagittal
plane: cerebral atrophy, reduced corpus callosum, and Chiari 1
malformation (panel 10). Cranial time-of-flight (TOF) MR angiogram (3T)
at 7 months: flow reduction of the M2 branch of left middle cerebral
artery (MCA) (panel 11). Facial appearance at 3 y.o. (panel 12).

\textbf{Figure 4. Differential expression and autophosphorylation of
variants in \emph{MAP3K7} causing CSCF or FMD2. A)} Western blot
analysis showing reduced expression levels of CSCF-causing
\emph{MAP3K7}variants (black circles), compared to
MAP3K7\textsuperscript{WT}, but normal or even increased expression
levels of FMD2-causing \emph{MAP3K7}variants (blue circles), compared to
MAP3K7\textsuperscript{WT}, when overexpressed in HEK-293T cells. N=3
for all CSCF-causing variants, except MAP3K7\textsuperscript{W241G}
(N=6). N=6 for all FMD2-causing variants, except
MAP3K7\textsuperscript{Y113D} and MAP3K7\textsuperscript{G168R} (N=3).
WT N=7. \textbf{B)} Western blot analysis showing that expression levels
of CSCF-causing \emph{MAP3K7}variants is normalized upon TAB1
co-expression, except for MAP3K7\textsuperscript{W241G}. N=7 for all
conditions, except for WT (N=14) and MAP3K7\textsuperscript{W241G}
(N=6). \textbf{C)} Western blot analysis of the autophosphorylation of
\emph{MAP3K7} at Thr187 showing that except for
MAP3K7\textsuperscript{R83H}, all CSCF-causing\emph{MAP3K7} variants
show reduced pThr187 compared to MAP3K7\textsuperscript{WT}, whereas
FMD2-causing variants show normal or increased levels of pThr187
compared to MAP3K7\textsuperscript{WT}. N=7 for all conditions, except
for WT (N=14) and MAP3K7\textsuperscript{Y206C} and
MAP3K7\textsuperscript{Y113D} (N=6). Error bars represent standard error
of the mean (SEM). * p\textless{}0.05; ** p\textless{}0.01; ***
p\textless{}0.0001.

\textbf{Figure 5. Western blot analysis of downstream targets
of\emph{MAP3K7}. A)} Western blot analysis reveals reduced
phosphorylation of NFkB upon overexpression of most CSCF-causing (black
circles) and some FMD2-causing (blue circles) \emph{MAP3K7} variants in
HEK-293T cells, compared to MAP3K7\textsuperscript{WT}. N=7 for all
conditions except for WT (N=10). \textbf{B)} Western blot analysis of
phosphoERK1/2 reveals reduced phosphorylation of ERK1/2 upon
overexpression of most CSCF-causing \emph{MAP3K7} variants compared to
MAP3K7\textsuperscript{WT}, except for MAP3K7\textsuperscript{R83H}. N=4
for all conditions, except for WT (N=8). Error bars represent standard
error of the mean (SEM). * p\textless{}0.05; *** p\textless{}0.0001.

\textbf{Hosted file}

\verb`Table 1.docx`  available at \url{https://authorea.com/users/447164/articles/546219-involvement-of-map3k7-in-fmd2-and-cscf-delineation-of-genotype-phenotype-correlations}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/figure-1-MAP3K7/figure-1-MAP3K7}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-2/Figure-2}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-3/Figure-3}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/figure-4/figure-4}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/figure-5/figure-5}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

