Delineation of phenotypes and genotypes related to cohesin structural protein RAD21

Vol.:(0123456789)
1 3
Human Gene ics (2020) 139:575–592
h ps://doi.o g/10.1007/s00439-020-02138-2
ORIGINAL INVESTIGATION
Delinea ion o pheno ypes andgeno ypes ela ed ocohesin
s uc u al p o ein RAD21
LianneC.K ab1,2,3 · IñigoMa cos‑Alcalde4,5 · MelissaAssa 6· MeenaBalasub amanian7 ·
JanneBaye Ande sen8· Anne‑Ma ieBisgaa d9 · Da idR.Fi zpa ick10· SannaGudmundsson11 ·
Syl iaA.Huisman1,12 · TugbaKalayci13· SaskiaM.Maas1,14· F anciscoMa inez15 · ShaneMcKee16 ·
LeonieA.Menke1 · PaulA.Mulde 17 · Oli e D.Mu ch18· MichaelPa ke 19· JuanPie20· FelicianoJ.Ramos21 ·
ClaudineRieubland22· JillA.Rosen eldMok y23 · EmanuelaSca ano24· Ma wanShinawi25 ·
PaulinoGómez‑Pue as4 · ZeynepTüme 8,26 · RaoulC.Hennekam1
Recei ed: 7 Janua y 2020 / Accep ed: 10 Feb ua y 2020 / Published online: 19 Ma ch 2020
© The Au ho (s) 2020
Abs ac
RAD21 encodes a key componen o he cohesin complex, and a ian s in RAD21 ha e been associa ed wi h Co nelia de
Lange Synd ome (CdLS). Limi ed in o ma ion on pheno ypes a ibu able o RAD21 a ian s and geno ype–pheno ype
ela ionships is cu en ly published. We ga he ed a se ies o 49 indi iduals om 33 amilies wi h RAD21 al e a ions [24
di e en in agenic sequence a ian s (2 ecu en ), 7 unique mic odele ions], including 24 hi he o unpublished cases.
We e alua ed consequences o 12 in agenic a ian s by p o ein modelling and molecula dynamic s udies. Full clinical
in o ma ion was a ailable o 29 indi iduals. Thei pheno ype is an a enua ed CdLS pheno ype compa ed o ha caused by
a ian s in NIPBL o SMC1A o acial mo phology, limb anomalies, and especially o cogni ion and beha io . In he 20
indi iduals wi h limi ed clinical in o ma ion, addi ional pheno ypes include Mungan synd ome (in pa ien s wi h biallelic
a ian s) and holop osencephaly, wi h o wi hou CdLS cha ac e is ics. We desc ibe se e al addi ional cases wi h pheno-
ypes including scle oco nea, in which in ol emen o he RAD21 a ian is unce ain. Va ian s we e equen ly amilial,
and geno ype–pheno ype analyses demons a ed s iking in e amilial and in a amilial a iabili y. Ca e ul pheno yping is
essen ial in in e p e ing consequences o RAD21 a ian s, and p o ein modeling and dynamics can be help ul in de e mining
pa hogenici y. The cu en s udy should be help ul when counseling amilies wi h a RAD21 a ia ion.
In oduc ion
RAD21 (ENSG00000164754; OMIM *606462) is a key
componen o he cohesin complex and i o ms a i-pa i e
ing oge he wi h SMC1A and SMC3 (Fig.1 and Suppl.
Fig. S1). The cohesin complex is a majo modula o o ch o-
mosome s uc u e, is in ol ed in egula ing ch omosome
seg ega ion du ing mi osis, DNA epai and ch oma in con-
densa ion, and plays an impo an ole in gene ansc ip ion
du ing in e phase and cellula homeos asis (Kamada and
Ba illa 2018; Mullende s e al. 2015; Wa in e al. 2016).
RAD21 has been implica ed in addi ional p ocesses includ-
ing media ion o epigene ic silencing and induc ion o apop-
osis (Fishe e al. 2017; Pa i e al. 2002). Va ian s in genes
encoding a ious s uc u al o unc ional componen s o he
cohesin complex, including RAD21, SMC1A, SMC3, BRD4,
STAG1/2, NIPBL, HDAC8, WAPL, ANKRD11 and in single
indi iduals PDS5A and ESPL1, ha e been implica ed in Co -
nelia de Lange Synd ome (CdLS) (Ansa i e al. 2014; Kline
e al. 2018; Woods e al. 2014; Yuan e al. 2019). RAD21
spans ~ 29Kb and has 14 exons (13 coding, 1 noncoding)
ha oge he encode a p o ein o 631 amino acids (McKay
e al. 1996).
Zeynep Tüme and Raoul C. Hennekam con ibu ed equally.
Elec onic supplemen a y ma e ial The online e sion o his
a icle (h ps ://doi.o g/10.1007/s0043 9-020-02138 -2) con ains
supplemen a y ma e ial, which is a ailable o au ho ized use s.
* Lianne C. K ab
[email p o ec ed]
* Zeynep Tüme
Ze[email p o ec ed]
* Raoul C. Hennekam
[email p o ec ed]
Ex ended au ho in o ma ion a ailable on he las page o he a icle
576 Human Gene ics (2020) 139:575–592
1 3
RAD21 a ian s a e ound in a mino i y o CdLS
pa ien s. To da e, nine missense a ian s and 5 mic ode-
le ions ha e been epo ed in CdLS pa ien s (Kline e al.
2018). CdLS is cha ac e ized by dis inc acial ea u es,
g ow h delay, mic ocephaly, limb educ ion de ec s, in el-
lec ual disabili y (ID) and beha io al p oblems, especially
sel -inju ious beha io (SIB) and au ism spec um diso -
de (ASD) (Kline e al. 2018). RAD21 a ian s ha e also
been associa ed wi h scle oco nea (Zhang e al. 2019) and
Mungan synd ome (Ch onic Idiopa ic In es inal Pseudoo-
bs uc ion; OMIM #611376, in pa ien s wi h biallelic
RAD21 a ian s) (Bono a e al. 2015; Mungan e al. 2003),
each in a single amily in which no ema ks on CdLS ea-
u es we e made in he epo . Loss o unc ion- a ian s in
cohesin genes including RAD21 we e ound in indi idu-
als wi h holop osencephaly o whom some demons a ed
CdLS ea u es as well (K uszka e al. 2019).
RAD21 is posi ioned on ch omosome 8q24.11, be ween
TRPS1 (T icho-Rhino-Phalangeal synd ome ype 1;
OMIM *604386) and EXT1 (Mul iple Exos oses ype
1; OMIM *608177). Se e al mic odele ions in ol ing
RAD21 encompass genes nex o RAD21 (con iguous gene
synd ome), complica ing a ibu ion o RAD21 o he phe-
no ype (Dea do e al. 2012; Pe eza e al. 2012; Wuy s
e al. 2002). TRPS ype 2 o Lange -Giedion synd ome
(OMIM #150230) in ol es TPRS1, RAD21 and EXT1,
and he acial pheno ype is mainly de e mined by loss o
TRPS1, whe eas he bony abno mali ies a ise om he loss
o EXT1 (Maas e al. 2015).
Based on he small case se ies o CdLS pa ien s wi h
RAD21 a ian s epo ed so a , ace and limb mani es a ions
o CdLS seem o be less p onounced compa ed o indi idu-
als wi h a ian s in he o he cohesin complex genes, and he
impac on cogni i e unc ioning seems a enua ed, wi hou
clea geno ype–pheno ype co ela ion (Kline e al. 2018;
Mino e al. 2014). He e, we epo on a case se ies o 49
pa ien s om 33 amilies wi h RAD21 al e a ions, including
all p e iously published cases wi h sequence a ian s, mos
o which wi h upda ed clinical da a. We included 24 hi h-
e o unpublished cases. We p esen geno ype da a, e alua e
he pa hogenici y o in agenic a ian s by a combina ion o
pheno ype, p o ein modelling, and molecula dynamic s ud-
ies, and p o ide in o ma ion on clinical pheno ype, includ-
ing cogni i e and beha io al unc ioning, in e amilial and
in a amilial a iabili y, and geno ype–pheno ype associa-
ions. We compa e he RAD21 pheno ype o ha o pa ien s
wi h NIPBL and SMC1A a ian s.
Resul s
We iden i ied 219 cases wi h RAD21 a ian s, o which
49 pa ien s om 33 amilies we e included in his s udy
(Tables1 andS1). We desc ibe in TableS6 hose excluded
cases ha s ill may be o in e es such as published cases
117
,
000
,
000 117
,
500
,
000 118
,
000
,
000 118
,
500
,
000 119
,
000
,
000
8
q
23.3 8
q
24.11 8
q
24.12
F1
9
F31
F20
F32
F21
F3
3
F22
TRPS1 EIF3H
UTP2
3
RAD21
A
ARD
SLC30A
8
MED30 EXT1
Ch 8
p.(T p23*) – F1
p.(Phe6Val) – F23
p.(A g65Gln) – F2
p.(Se 345P o) – F26
p.(P o355Leu) – ClinVa
p.(P o376A g) – F8
p.(Th 461Ile) – F10
p.(Asp541_Gln568del) – F11
p.(Gly575Ala) – ClinVa
p.(Cys585A g) – F13; F14
p.(A g586Gln) – ClinVa
p.(Gln592del) – F17
p.(Phe600del) – F18
p.(Leu603P o) – F29
p.(Se 618Gly) – ClinVa
p.(Ala622Th ) – F30
p.(Lys29P o s*10) – F24
c.274+1G>A – F3
p.(Se 198A g s*6) – F4
p.(Ile206Th s*3) – F5
p.(Se 235Ile s*19) – F6
p.(Glu315GIn s*9) – F7
p.(Ala318P o s*7) – F25
c.1161+1G>A – F27
p.(Lys406A g s*4) – F9
p.(Glu518A g s*19) – F2
8
p.(Gly547Ala s*65) – F12
p.(A g586*) – F15; F16
SMC1A2/1GATS3CMS
1368268553042633011
a
b
Fig. 1 P esen ly epo ed RAD21 a ian s. a RAD21 (ho izon al ba )
has h ee binding domains: SMC3 (p.1–103), STAG1/2 (p.362–403)
and SMC1A (p.558–628). Sizes o he binding domains a e no
shown o scale. T unca ing RAD21 a ian s a e shown abo e, and
missense mu a ions and in- ame dele ions a e shown below he p o-
ein ep esen a ion. Va ian s o which p o ein modelling is a ailable,
a e ma ked in bold. F amily numbe . The ho izon al black line ep-
esen s he in ame dele ion p.(Asp541_Gln568del). ClinVa a i-
an s which a e epo ed in he ClinVa da abase and could be in es-
iga ed o pa hogenei y wi h p o ein modelling (see supplemen a y
Table S6). b Genomic egion showing he mic odele ions including
RAD21
577Human Gene ics (2020) 139:575–592
1 3
wi h in ol emen o o he mo bid genes (Dea do e al.
2012; Maas e al. 2015; Pe eza e al. 2012; Wuy s e al.
2002; Yuen e al. 2015), a ian s epo ed as a ian o
unknown signi icance (VUS) ha emained wi h unknown
signi icance subsequen o e-e alua ion, and cases o
whom he ela ionship be ween pheno ype and RAD21
a ian could no be con i med(K uszka e al. 2019; Zhang
e al. 2019).
The 49 pa ien s can be di ided in o wo g oups: coho
A includes 29 pa ien s (22 amilies) wi h su icien clinical
da a; and coho B includes 20 pa ien s (11 amilies) wi h
incomple e da a. O he 49 cases, 24 a e new. Twen y-
i e we e p e iously published (Ansa i e al. 2014; Bono a
e al. 2015; Boyle e al. 2017; Dea do e al. 2012; Do -
al e al. 2019; Gudmundsson e al. 2018; K uszka e al.
2019; Lee e al. 2014; Ma inez e al. 2017; McB ien e al.
2008; Mino e al. 2014; Yuan e al. 2019), and o 19 o
hese clinical da a could be upda ed (Table1). Pa ien s
o igina ed om Aus alia, Belgium, Canada, Denma k,
Ge many, I aly, Ne he lands, Spain, Sweden, Swi ze land,
Tu key, Uni ed Kingdom and Uni ed S a es.
Geno ype
The 33 amilies ha bo 31 di e en a ian s: se en
unique copy numbe a ia ions (CNVs) and 24 in a-
genic sequence a ian s. Two o he la e we e ecu en
[p.(Cys585A g) and p.(A g586*), each ound in 2 ami-
lies (Table1, Fig.1)]. A ela i ely la ge p opo ion o he
cases a e amilial (nine ou o 21 index cases o whom
inhe i ance could be es ablished). The se en CNVs we e
all dele ions, six o which included o he genes in addi ion
o RAD21. O he 24 di e en sequence a ian s, 13 a e
p edic ed be unca ing (2 nonsense, 2 splice si e and 9
ameshi a ian s), and hese a e sca e ed h oughou he
gene. Th ee o he a ian s a e in- ame dele ions, wo o
which a ec a single amino acid, while he 665bp dele ion
includes he whole exon 13. The missense a ian s end
o clus e a he unc ional domains o he p o ein. Some
a ian s in coho B may be ecu en bu su icien da a
a e lacking o con i m his (TableS6).
E alua ion o pa hogenici y o RAD21 a ian s using
molecula dynamic analyses
Fo 12 in agenic a ian s ( en missense a ian s and wo
3bp in- ame dele ions, om indi iduals in coho A,
B and TableS6) i was possible o ca y ou s uc u al
analysis, as hei subs i u ed esidues a e loca ed in one
o he domains o which 3D a angemen can be mod-
eled (RAD21-SMC3 domain, RAD21-STAG domain and
RAD21-SMC1A domain, Fig.2; Figs. S2-3). In e ac ions
be ween RAD21 and i s binding pa ne s a e shown in
Fig. S1.
Modeled missense a ian s wi hin heRAD21‑SMC3
domain ( esidues 18–87 ha bo ing A g65Gln),
andRAD21‑STAG domain ( esidues 321–392 ha bo ing
Se 345P o, P o355Leu andP o376A g)
Subs i u ion o A g65 wi h Gln (A g65Gln) is a semi-con-
se a i e a ia ion ha did no p omo e de ec able s uc u al
o dynamic changes in he complex. The Se 345P o a ian
impai s RAD21 and STAG1/2 in e ac ions due o p omo ion
o a de no o cu ed small alpha-helix segmen ha binds
o he p e-exis ing alpha helix, which sepa a es om he
su ace o STAG2. No s uc u al o dynamic e ec s o P o-
355Leu o P o367A g on RAD21 i sel could be obse ed.
Ne e heless, P o376A g does p omo e he o ma ion o a
new sal b idge be ween RAD21 and STAG2, which is p e-
dic ed o cause o e -s abiliza ion o he in e ac ion be ween
he wo p o eins.
Modeled missense a ian s wi hin heRAD21‑SMC1A
domain ( esidues 543–628 ha bo ing Gly575Ala,
Cys585A g, A g586Gln, Gln592del, Phe600del, Leu603P o,
Se 618Gly, andAla622Th )
Fou o he eigh a ian s in his domain (Cys585A g; A g-
586Gln; Gln592del; Leu603P o) a e p edic ed o cause
a s uc u al e ec . A g586Gln des abilizes he RAD21-
SMC1A domain by loss o a sal b idge be ween A g586
and Glu577, and he al e ed posi ion o Glu577 adds
an addi ional nega i e cha ge o he RAD21 su ace o
RAD21-SMC1A. Cys585A g has a simila e ec , in e -
ac ing wi h Glu583 and causing A g586 o lose i s con ac
wi h Glu577. The MD simula ion shows ha bo h Gln592del
and Leu603P o, bu no Phe600del, a ec he posi ioning o
SMC1A-Asn35 a he ATPase si e 1 by changing he posi-
ion o Lys605.
Pheno ype
Physical ea u es
Indi idual CdLS sco es and majo and mino anomalies
in coho A a e p o ided in TableS2-3. Clinical ea u es
o coho A a e compa ed o hose o NIPBL and SMC1A
coho s in Table2 and illus a ed in Fig.3 and Fig. S4. Clini-
cal in o ma ion o coho B is a ailable in supplemen al
ma e ials S5 and will no be discussed u he in he ex , as
clinical da a a e limi ed. We men ion da a in he ex only i
no ep esen ed in he ables.
578 Human Gene ics (2020) 139:575–592
1 3
Table 1 Molecula indings o he p esen ly epo ed se ies o indi iduals wi h RAD21 a ian s
PID Re e ence Sou ce CdLS
sco ea
Exon/
in on
Nucleo ide change P edic ed amino
acid change
Type Inhe i ance
Coho A—su icien clinical da a
F1 Ma inez 2017 Upda ed 9 Exon 2 c.68G > A p.(T p23*) Nonsense De no o
F2 Clin a New ≥ 7 Exon 2 c.194G > A p.(A g65Gln) Missenseb
F3a Ansa i 2014 P1 Upda ed ≥ 10 In on 3 c.274 + 1G > A Splice si e Familial
(pa e nal)
F4 Mino 2014 P2 Upda ed 12 Exon 6 c.592_593dupAG p.(Se 198A g s*6) F ameshi
F5 Unpublished New 9 Exon 6 c.617_620del p.(Ile206Th s*3) F ameshi De no o
F6a Boyle 2017 IV.16 Upda ed 12 Exon 7 c.704delG p.(Se 235Ile s*19) F ameshi Familial
(ma e nal)
F6b Boyle 2017 III.1 Upda ed 10 Exon 7 c.704delG p.(Se 235Ile s*19) F ameshi Familial
(pa en s no
es ed)
F6c Boyle 2017 III.2 Upda ed 9 Exon 7 c.704delG p.(Se 235Ile s*19) F ameshi Familial
(pa en s no
es ed)
F6d Boyle 2017 III.5 Upda ed 9 Exon 7 c.704delG p.(Se 235Ile s*19) F ameshi Familial
(pa en s no
es ed)
F6e Unpublished New 12 Exon 7 c.704delG p.(Se 235Ile s*19) F ameshi Familial
(ma e nal)
F7 Do al 2019 O iginal
da a
≥ 11 Exon 9 c.943_946del p.(Glu315Gln s*9) F ameshi De no o
F8 Dea do 2012 P5 O iginal
da a
≥ 10 Exon 9 c.1127C > G p.(P o376A g) MissensebDe no o
F9 K uszka 2019 P14 Upda ed 13 Exon 10 c.1217_1224del p.(Lys406A g s*4) F ameshi De no o
F10 Unpublished New 10 Exon 11 c.1382C > T p.(Th 461Ile) Missense Familial
(pa e nal)
F11a Mino 2014 P1 Upda ed 8 Exon 13 c.1621-
388_1704 + 193del
p.(Asp541_
Gln568del)
In ame
dele ion
Familial
(ma e nal)
F11b Mino 2014 mo he P1 Upda ed ≥ 5 Exon 13 c.1621-
388_1704 + 193del
p.(Asp541_
Gln568del)
665bp
in ame
dele ion
F12 Unpublished New 13 Exon 13 c.1635del p.(Gly-
547Ala s*65)
F ameshi De no o
F13 Dea do 2012, P6 O ginal da a ≥ 12 Exon 14 c.1753T > C p.(Cys585A g) MissensebDe no o
F14a Unpublished New 12 Exon 14 c.1753T > C p.(Cys585A g) MissensebFamilial
(pa en s no
es ed)
F14b Unpublished New ≥ 10 Exon 14 c.1753T > C p.(Cys585A g) Missense Familial
(pa en s no
es ed)
F15 Unpublished New ≥ 12 Exon 14 c.1756C > T p.(A g586*) Nonsense
F16a Unpublished New 10 Exon 14 c.1756C > T p.(A g586*) Nonsense Familial
(pa e nal)
F16b Fa he , unpublished New ≥ 10 Exon 14 c.1756C > T p.(A g586*) Nonsense
F17 Gudmunsson 2019 Upda ed 8 Exon 14 c.1774_1776del p.(Gln592del) In ame
dele ionb
De no o
F18 Unpublished New 9 Exon 14 c.1800_1802del p.(Phe600del) In ame
dele ionb
F19 Dea do 2012 P4 O iginal
da a
≥ 12 Whole
gene
a [hg19] 8q23
.3q24.11(116880827–118875305)x1
2Mb dele-
ion
F20 Unpublished New ≥ 12 Whole
gene
a [hg19] 8q23
.3q24.11(116915114–119171074)x1
2.3Mb
dele ion
De no o
579Human Gene ics (2020) 139:575–592
1 3
Table 1 (con inued)
PID Re e ence Sou ce CdLS
sco ea
Exon/
in on
Nucleo ide change P edic ed amino
acid change
Type Inhe i ance
F21 Dea do 2012 P2,
McB ein 2008
O iginal
da a
≥ 12 Whole
gene
a [hg19] 8q23
.3q24.12(117571728–119260904)x1
1.7Mb
dele ion
De no o
F22 Unpublished New 12 Exons
1–9
a [hg19] 8q24.11(117866471–
117893495)x1
27kb dele-
ion
Coho B—insu icien clinical da a
F3b Ansa i 2014 Upda ed In on 3 c.274 + 1G > A n/a Splice si e
F23 Deciphe 271431 New Exon 2 c.16T > G p.(Phe6Val) Missense De no o
F24 Unpublished New Exon 2 c.85delinsCCT p.(Lys29P o s*10) F ameshi
F25a Deciphe 272901 New Exon 9 c.951del p.(Ala318P o s*7) F ameshi Familial
(pa e nal)
F25b Deciphe 272901 a he New Exon 9 c.951del p.(Ala318P o s*7) F ameshi
F26 Deciphe 275402 New Exon 9 c.1033T > C p.(Se 345P o) MissensebDe no o
F27a Yuan 2018 P2 Upda ed In on
10
c.1161 + 1G > A Splice si e Familial
(ma e nal)
F27b Yuan 2018 mo he P2 Upda ed In on
10
c.1161 + 1G > A Splice si e
F28a K uszka 2019 P12/Yuan
2019 P1
Upda ed Exon 12 c.1550dupC p.(Glu-
518A g s*19)
F ameshi Familial
(pa e nal)
F28b K uszka 2019 P12
a he /Yuan 2019 P1
a he
Upda ed Exon 12 c.1550dupC p.(Glu-
518A g s*19)
F ameshi
F29 Lee 2014 P76 O iginal
da a
Exon 14 c.1808T > C p.(Leu603P o) MissensebDe no o
F30a Bono a 2015 IV.9 Upda ed Exon 14 c.[1864G > A];
[1864G > A]
p.(Ala622Th ) MissensebFamilial (bo h
pa en s)
F30b Bono a 2015 IV.10 Upda ed Exon 14 c.[1864G > A];
[1864G > A]
p.(Ala622Th ) MissensebFamilial (bo h
pa en s)
F30c Bono a 2015 IV.11 Upda ed Exon 14 c.[1864G > A];
[1864G > A]
p.(Ala622Th ) MissensebFamilial (bo h
pa en s)
F30d Unpublished New Exon 14 c.[1864G > A] p.(Ala622Th ) MissensebFamilial (nos)
F30e Unpublished New Exon 14 c.[1864G > A] p.(Ala622Th ) MissensebFamilial (nos)
F30 Unpublished New Exon 14 c.[1864G > A] p.(Ala622Th ) MissensebFamilial (nos)
F31 ClinVa New Whole
gene
a [hg19] 8q23.3-24.11(116902507–
118942698)x1
2Mb
dele ion;
includes
se e al
genes
F32 ClinVa New Whole
gene
a [hg19] 8q23.3-24.11(117509968–
118391406)x1
880kb
dele ion;
includes
se e al
genes
F33 ClinVa New Whole
gene
a [hg19] 8q24.11(117714768–
119072307)x1
1.4Mb
dele ion;
includes
se e al
genes
Coho A: de ailed clinicalda a a ailable, including in o ma ion on all ca dinal CdLS ea u es; coho B: insu icien clinicalda a a ailable
F amily numbe , P pa ien numbe in he espec i e publica ion, nos no o he wise speci ied
a Based on (Kline e al. 2018); ≥ de ines a leas (mino c i e ia missing). Sco e < 4 is insu icien o indica e molecula es ing o CdLS; sco e
4–8 indica es molecula es ing o CdLS indica ed; sco e 9–10 indica es non-classic CdLS; sco e 11 o highe indica es classic CdLS
b Va ian s in es iga ed wi h p o ein modelling

580 Human Gene ics (2020) 139:575–592
1 3
All pa ien s in coho A (age ange 0–61yea s, median 9
yea s, mean 18yea s; 15 males) had CdLS sco es o a leas
i e, su icien o wa an molecula gene ic es ing o CdLS.
In abou 60% o index cases (13/21 index cases in which his
was speci ied) CdLS was suspec ed p io o es ing. The e
was no gende di e ence in CdLS sco es. No RAD21 a ian
would ha e been missed using he CdLS consensus c i e ia
o molecula s udies (Kline e al. 2018). Clinical sco es o
pa ien s wi h CdLS suspec ed p io o es ing (median 11.5;
ange 8–13) we e highe han hose no suspec ed o ha e
CdLS (median 9.5; ange 5–13).
Cogni ion, de elopmen andbeha io
Cogni i e unc ioning, de elopmen al miles ones and beha -
io al unc ioning in he RAD21 g oup a e a enua ed com-
pa ed o he NIPBL and SMC1A g oups (Tables3 and S4).
The majo i y o RAD21 pa ien s (16/29, 55%) ha e no mal
o mildly impai ed cogni i e unc ioning (SMC1A g oup
32%; NIPBL g oup 7%) (Huisman e al., 2017; Mulde
e al., 2019). In all h ee g oups, he e is a end owa ds
mo e language-based p oblems han mo o -based p oblems
in de elopmen . S ill, all RAD21 pa ien s aged 3yea s and
abo e we e able o use some wo ds. The e was no co ela-
ion be ween he se e i y o cogni i e impai men in RAD21
pa ien s and p esence o mic ocephaly (p ena al, pos na al,
o bo h; da a no shown).
14/25 RAD21 pa ien s (56%) wi h su icien ly a ailable
in o ma ion on beha io had p oblems, mainly ea u es o
anxie y, ADHD, ASD, and obsessi e–compulsi e beha io .
ASD ela ed p oblems, agg ession and SIB we e less p e a-
len compa ed o he SMCIA and NIPBL g oups.
Geno ype–pheno ype compa isons incoho A
Mic odele ions e susin agenic a ian s
The e was a end owa ds highe CdLS sco es and mo e
equen ly impai ed g ow h pa ame e s in pa ien s wi h
mic odele ions compa ed o hose wi h in agenic a ian s,
bu no di e ences we e appa en in equency o majo
mal o ma ions o cogni i e and beha io al p oblems. We
e ained om s a is ical analyses as small numbe s would
make esul s oo un eliable and less use ul. Exos oses,
ela ed o EXT1 haploinsu iciency, likely caused he uppe
limb anomalies.
T unca ing e susnon‑ unca ing sequence a ian s
The e was no di e ence in CdLS sco es o g ow h pa am-
e e s be ween indi iduals wi h unca ing and hose wi h
non- unca ing sequence a ian s (median 10; ange 9–13
and median 9.5; ange 5–12, espec i ely).
Mal o ma ions andgeno ype
Fo 12/15 pa ien s wi h in agenic a ian s and majo mal-
o ma ions o heal h p oblems, he a ian was loca ed in
a p o ein-binding domain (F2, F3a, F8, F9, F11a, F11b,
F12, F14a, F14b, F16a, F17, F18). As numbe s a e small i
emains unce ain whe he his is uly an associa ion. The
ypes o majo mal o ma ions did no di e .
In a amilial a ia ion
The in a amilial a ia ion can be conside able (Tables S1,
S3-4; Fig.3), especially in cogni ion and beha io . Th ough
ob ious asce ainmen bias cogni ion is mo e equen ly
impai ed in index cases. Se e al amilies include pa ien s
wi h ID and pa ien s wi h appa en ly no mal cogni i e unc-
ioning. The in a amilial a ia ion canno be explained by
mosaicism in mos amilies.
Discussion
We epo on RAD21 a ian s in 49 indi iduals, some wi h
su icien clinical da a (coho A), o he s wi h limi ed clini-
cal da a (coho B). RAD21 a ian s a e equen ly amilial,
o en unique, and wi hou ob ious ho spo s o a ian s o
mic odele ions b eakpoin s, al hough missense a ian s end
o clus e a ound p o ein binding domains.
RAD21 missense a ian s and hei p edic ed e ec
onp o ein unc ion
The s uc u al and unc ional analysis indica ed ha a leas
six ou o wel e modeled RAD21 missense a ian s a e
likely pa hogenic (Se 345P o, P o367A g, Cys585A g, A g-
586Gln ( epo ed as a VUS), Gln592del and Leu603P o).
I pheno ype da a and li e a u e/da abase in o ma ion a e
aken in o accoun , h ee mo e RAD21 modeled missense
a ian s a e likely pa hogenic (A g65Gln ( epo ed as VUS),
Phe600del, Ala622Th ).
The A g65 is loca ed wi hin he RAS21-SMC3 domain
in he close p oximi y o Ty 67, and al e ing he kinase/
phospha ase ecogni ion mo i A g-X-Ty a ound Ty 67
may a ec he phospho yla ion-based egula ion o RAD21
(Amanchy e al. 2011; Hoque and Ishikawa 2001; Ho nbeck
e al. 2015; Li e al. 2009). In addi ion, a con ac be ween
he PDS5 p o ein and he RAD21-SMC3/SMC3-head com-
plex is in ol ed in he opological en apmen o DNA by
cohesin (Guacci e al. 2019). As A g65 is loca ed owa ds
581Human Gene ics (2020) 139:575–592
1 3
he sol en , A g65Gln may impac he RAD21-PDS5 ec-
ogni ion and, hus, dis u b hei in e ac ion.
The in e ac ion be ween RAD21 and STAG1/2 is c u-
cial o he p ope unc ioning o he cohesin complex
(Guacci e al. 2019), and bo h impai ing (Se 345P o) o
o e -s abilizing (P o367A g) a ian s wi hin he RAD21-
STAG domain a e p edic ed o cause dys unc ion o he
complex, p esumably h ough a ec ing he con inuous
cycle o o ma ion and disengagemen o he cohesin ing
(Ma cos-Alcalde e al. 2017).
The s uc u al model o he RAD21-SMC1A domain
a ionalizes he key unc ion o RAD21 in he ATPase
eac ion a he SMC1A/SMC3 head, which is pi o al o
he opening o he cohesin ing, and hus he cyclic p ocess
(Ma cos-Alcalde e al. 2017). The Cys585A g and A g-
586Gln a ian s des abilize he RAD21-SMC1A domain;
and Gln592del and Leu603P o (bu no Phe600del) dis-
u b he cyclic p ocess h ough he disloca ion o Lys605.
Al hough he Phe600del a ian does no seem o a ec
RAD21 s uc u e, i leads o a classical CdLS pheno ype
wi hou a ian s in addi ional known CdLS genes (using
a a ge ed gene panel). Thus, i does seem likely pa ho-
genic. Un o una ely, he c ys al s uc u e o RAD21 is
no a ailable o o he domains o in e ac ing pa ne s such
as WAPL and PDS5, bu ea lie molecula s udies p o ide
addi ional in o ma ion o o he missense a ian s.
The impo ance o he egula ion o he in e ac ion
be ween RAD21-SMC1A and SMC1A/SMC3 head is
demons a ed by he se e al esidues in ol ed in phospho-
yla ion and ubiqui ina ion in he RAD21-SMC1A domain
(Hegemann e al. 2011; Hoque and Ishikawa 2001; Ho n-
beck e al. 2015). Ala622 is posi ioned nex o Th 623, a
subs a e o p o ein phospho yla ion by PLK1 (Ho nbeck
e al. 2015; Tsai e al. 2015). A pa hogenic e ec o a i-
an Ala622Th is suppo ed by s udies showing dec eased
bowel ansi and loss o en e ic neu ons in zeb a ish
wi h Ala622Th knockdown h ough mo pholinos and by
pa ien s wi h biallelic Ala622Th a ian s andMungan
synd ome wi h CIPO (ch onic in es inal pseudo-obs uc-
ion) (Bono a e al. 2015). The he e ozygous membe s o
his amily had some clinical ea u es o he CdLS spec-
um, bu as i was no possible o e ie e u he clinical
da a, i emains unce ain whe he hey ha e a ull CdLS
pheno ype, and whe he his a ian can lead o a pheno-
ype in he e ozygous o m.
Fo wo addi ional a ian s ha could no be modeled,
he li e a u e suppo s ha hey a e likely pa hogenic. Phe6
is ound close o Se 9, a phospho yla ion si e desc ibed
in he human p o eome (Gauci e al. 2009; Guacci e al.
2019). The Phe6Val a ian ( epo ed as aVUS) would
modi y he kinase/phospha ase ecogni ion mo i , hus
a ec ing he p o ein beha io . Simila ly, as esidue Th 461
is lanked by Se esidues (Se 459 and Se 466), bo h
implica ed in phospho yla ion- egula ed dissocia ion o
cohesin om ch omosome a ms (Hau e al. 2005; Ho n-
beck e al. 2015), i may modi y he kinase/phospha ase
ecogni ion mo i .
Clinical pheno ype
Physical pheno ype
RAD21 a ian s can lead o a CdLS pheno ype (RAD21-
CdLS). The (limi ed) a ailable in o ma ion o indi iduals
om coho B sugges s ha biallelic RAD21 a ian s can
also lead o Mungan synd ome and monoallelic RAD21
a ian s o holop osencephaly (like one case in coho A)
and possibly schizoph enia, al hough in he la e he asso-
cia ion may be a spu ious coincidence. In TableS6 we
desc ibe se e al addi ional cases wi h pheno ypes includ-
ing scle oco nea and schizoph enia, in which pa hogenic-
i y o he RAD21 a ian is deba able. Due o incomple e
in o ma ion i emains unce ain whe he hese indi iduals
a e also showing CdLS cha ac e is ics. Indeed, when we
succeeded in ob aining u he clinical in o ma ion, se -
e al indi iduals u ned ou o show CdLS cha ac e is ics
no men ioned in he publica ion ( o ins ance in he amily
wi h Mungan synd ome). Addi ionally, one may specula e
ha pheno ypes a e also a ibu able (possibly in addi ion
o he RAD21 a ian ) o a ian s in o he genes.
Compa ison opheno ypes o NIPBL andSMC1A a ian s
In pa ien s wi h su icien clinical da a a ailable (coho A)
mos ea u es associa ed wi h CdLS a e p esen . Howe e ,
he p e alence o ea u es is lowe compa ed o hose in he
SMC1A and NIPBL coho , and he deg ee o se e i y is yp-
ically less. Se e e isual impai men and diaph agma ic he -
nias a e a e in RAD21 pa ien s, and eeding di icul ies a e
uncommon. RAD21 pa ien s less equen ly ha e inc eased
body hai (hi su ism, bushy eyeb ows, low scalp hai lines),
majo limb mal o ma ions a e no epo ed, and hands and
ee a e gene ally o no mal size. S ill, mino anomalies o
hands and ee a e common, such as e al pads, abno mal
lexion c ease pa e ns, and camp odac yly. Pa ien s wi h
RAD21 a ian s ha e gene ally less impai ed g ow h a bi h,
and sho s a u e and mic ocephaly de elop pos na ally. P e-
na al mic ocephaly has been demons a ed o be a p edic o
o mo e se e e cogni i e impai men in CdLS in he p e-
molecula e a (Hawley e al. 1985) bu his does no hold
o RAD21 pa ien s. F equency and se e i y o congeni al
hea de ec s a e simila o hose in he NIPBL and SMC1A
coho s. Gas o-esophageal e lux is simila in equency bu
in RAD21 i is ypically mild and es ic ed o ea ly child-
hood. No RAD21 pa ien s exhibi a Re -like pheno ype as
can occu in a subg oup o pa ien s wi h SMC1A a ian s
582 Human Gene ics (2020) 139:575–592
1 3
(Huisman e al. 2017). The CdLS sco e emains a eliable
ool, and he p esen s udy does no call o an adjus men
o he diagnos ic ad ice om he CdLS guidelines (Kline
e al. 2018).
Unusual anomalies in he RAD21 cases a e e eb al
anomalies (cle s and hemi e eb ae). The e is a single
indi idual wi h a NIPBL a ian and Klippel–Feil anomaly
(pe sonal obse a ion RCH), and uppe ce ical spine mal-
o ma ions ha e been epo ed in o he pa ien s wi h NIPBL
a ian s as well (Be ini e al. 2014). Mal o ma ions o
s uc u es de i ed om he emb yonic o egu a e ela i ely
equen in RAD21 pa ien s and ha e only a ely been
desc ibed in CdLS (Hamil on e al. 2014; Kang e al. 2018;
Mende e al. 2012). Holop osencephaly spec um anomalies
ha e been linked o se e al cohesin genes (K uszka e al.
2019), including RAD21, al hough in one indi idual his
emains unce ain (TableS6). The p e alence o holop os-
encephaly spec um in RAD21-CdLS mus emain unce ain
as b ain MRIs a e ypically no indica ed in indi iduals wi h
CdLS due o he bu den o he p ocedu e and lack o conse-
quences o indings o ca e (Kline e al. 2018).
583Human Gene ics (2020) 139:575–592
1 3
De elopmen , cogni ion andbeha io
Mos da a on cogni ion and beha io in he p esen coho
a e based on subjec i e in o ma ion p o ided by physicians
and no on o mal es ing. The e o e, eliabili y emains
unce ain. S ill, all da a poin o a lowe p e alence and
dec eased se e i y o ID in RAD21 pa ien s compa ed o
NIPBL and SMC1A g oups: de elopmen al miles ones a e
mo e equen ly a ained, he cogni i e le el is es ima ed
highe ,and agg ession and au ism a e less equen . SIB,
a hallma k o CdLS in gene al (Kline e al. 2018), is in e-
quen in RAD21 indi iduals.
E en i an IQ is no mal, sub le di icul ies in neu opsy-
chological domains known o be a ec ed in CdLS (Kline
e al. 2018) may in luence cogni i e pe o mance. Pe iodic
o mal sc eening o neu opsychological and beha io al
p oblems is s ill wa an ed in all indi iduals wi h RAD21
a ian s, o allow o ea ly ecogni ion o p oblems and
access o ele an suppo sys ems. In addi ion, o mal (in-
pe son) assessmen s can p e en misdiagnoses, such as
au ism, by pu ing beha io al cha ac e is ics in o he pe -
spec i e o he de elopmen al le el o pa ien s (Mulde e al.
2019).
Na u al his o y
The na u al his o y da a om he p esen s udy indica e ha
p egnancies and bi h end o p og ess no mally, p ena al
g ow h e a da ion being p esen in a small mino i y. Abou
hal o he pa ien s ha e congeni al anomalies (cle pal-
a e; ca diac anomalies). Majo limb de ec s ha e no been
ound; diaph agma ic he nia, anal a esia o choanal a esia
occu occasionally. Pa ien s ha e ypically mild acial dys-
mo phisms, no small hands o ee , and inc eased body hai
is less appa en compa ed o SMC1A and NIPBL pa ien s.
The clinical diagnosis o CdLS may, he e o e, be di icul .
Neona al eeding is usually no p oblema ic. Re lux is
common bu no se e e. Typical de elopmen is somewha
slow, mainly in speech de elopmen , and physical he apy
o speech he apy may be indica ed. As hey g ow up, chil-
d en only occasionally de elop new medical p oblems. Hal
o he child en show a p og essi e bu s ill mild g ow h
delay in head ci cum e ence and heigh . Vision is mos ly
no mal; hea ing loss is ound in a hi d o indi iduals and
may equi e hea ing de ices. Mos o he pa ien s a e able
o a end egula educa ion o educa ion o child en wi h
mild cogni i e disabili ies. Mos ha e some beha io al p ob-
lems (mainly anxie y, ADHD o ASD) o limi ed se e i y,
and agg ession and SIB a e uncommon. No uncommonly,
RAD21 pa ien s a e able o s a a amily, and some a e only
diagnosed when mo e se e ely a ec ed o sp ing is ecog-
nized. This indica es ha ca e ul amily analysis is pa a-
moun in each amily in which someone is diagnosed wi h
a RAD21 a ian .
Geno ype–pheno ype associa ions
The ela i ely mild pheno ype o pa ien s wi h RAD21 a i-
an s seems o indica e ha RAD21 is no highly in ole an o
loss-o - unc ion, in con as o o he CdLS-associa ed genes
(NIPBL, SMC1A, PDS5, WAPL, STAG2) (Gause e al. 2010).
Suppo ing his, Dea do e al. ound haploinsu iciency o
RAD21 led o app oxima ely hal ed RAD21 RNA in a cell
line om a pa ien wi h classical CdLS, while haploinsu -
iciency o NIPBL is o en associa ed wi h a compensa o y
up egula ion o RNA le els, p esumably om he in ac
allele (Bo ck e al. 2006; Dea do e al. 2012; Newki k
Fig. 2 S uc u al modeling o RAD21-SMC1A domain bound o he
head domain o SMC1A/SMC3 complex. a Model o he RAD21-
SMC1A domain ( esidues 543–628, g een) associa ed o he head
domains o SMC1A (g ey) and SMC3 (o ange), close o he ATP
molecule in ATPase si e 1 (ATP-1) o he SMC1A/SMC3 dime .
Posi ion o a ec ed esidues (Gly575, Cys585, A g586, Gln592,
Phe600, Leu603, Se 618 and Ala622) is indica ed as ed sphe es.
Loca ions o o he impo an esidues (Lys573, Gly575, Lys605,
and Th 623) a e indica ed. Residue Cys585 is loca ed nex o esi-
due A g586. Residue A g586 in e ac s h ough a sal b idge wi h
RAD21 esidue Glu577, s abilizing RAD21-SMC1A s uc u e. Th ee
mu a ed esidues (Gln592, Phe600, Leu603) a e loca ed in he same
alpha-helix as key esidue Lys605, p edic ed o main ain he co ec
posi ioning o SMC1A-Asn35 a ATPase si e 1, pu ing i in o con-
ac wi h a ca aly ic wa e molecule and, hus, allowing p og ession
o he ATPase eac ion, pi o al o opening o he cohesin ing and he
cyclic p ocess (Ma cos-Alcalde e al. 2017). Va ian s Se 618Gly and
Ala622Th do no cause s uc u al al e a ions. b Roo mean squa e
de ia ion (RMSD, in Angs oms) o modeled s uc u es (WT, wild-
ype, blue line; Gly575Ala, ed; Cys585A g, ligh pu ple; A g586Gln,
da k g een; Gln592del, ligh blue; Phe600del, o ange; Leu603P o,
cyan; Se 618Gly, da k pu ple; Ala622Th , ligh g een. No ele an
di e ences in RMSD alues demons able in he ajec o ies o he
mu a ed models when compa ed wi h wild- ype model and wi h one
ano he . c Va ian Cys585A g causes he adjacen A g586 o lose
in e ac ion wi h Glu577, and bo h he A g586 and Glu577 esidues
change hei posi ion in he mu an p o ein by poin ing owa ds he
sol en , which modi ies he dis ibu ion o cha ges in he su ace o
RAD21-SMC1A, while he new A g585 esidue is s abilized in a
no el in e ac ion wi h Glu583. d Model o a ian Gln592del a e
100 ns o MD. New posi ions o A g590, Lys591 and Lys605 due
o he absence o Gln592 a e indica ed. Dele ion o Gln592 esidue
causes adjacen Lys591 o be loca ed in he same posi ion as he
missing amino acid. This si ua ion p omo es a con o ma ional change
in he alpha helix, causing he Lys605, which is placed in he same
alpha helix, o mo e away om si e 1 o he ATPase. e Model o
a ian Phe600del (g een) compa ed o wild- ype model (pink) a e
100 ns o MD. Despi e he local ea angemen in he alpha helix,
dis o ions o he alpha helix a e no ele an as esidue Leu601 is
placed spa ially in he posi ion equi alen o he dele ed Phe600 du -
ing he MD ajec o y, allowing Lys605 o emain in he same posi-
ion. S uc u e o wild- ype RAD21-SMC1A (le ) and a ian
Leu603P o ( igh ) models a e 100ns o MD. P esence o mu a ed
P o603 ins ead o wild- ype Leu603 p omo es a local change in he
bending angle o he alpha-helix in which i is loca ed, esul ing in a
con o ma ional change in he alpha helix ha mo es Lys605 ou o i s
ini ial posi ion close o ATPase si e 1
◂
590 Human Gene ics (2020) 139:575–592
1 3
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Publishe ’s No e Sp inge Na u e emains neu al wi h ega d o
ju isdic ional claims in published maps and ins i u ional a ilia ions.
A ilia ions
LianneC.K ab1,2,3 · IñigoMa cos‑Alcalde4,5 · MelissaAssa 6· MeenaBalasub amanian7 ·
JanneBaye Ande sen8· Anne‑Ma ieBisgaa d9 · Da idR.Fi zpa ick10· SannaGudmundsson11 ·
Syl iaA.Huisman1,12 · TugbaKalayci13· SaskiaM.Maas1,14· F anciscoMa inez15 · ShaneMcKee16 ·
LeonieA.Menke1 · PaulA.Mulde 17 · Oli e D.Mu ch18· MichaelPa ke 19· JuanPie20· FelicianoJ.Ramos21 ·
ClaudineRieubland22· JillA.Rosen eldMok y23 · EmanuelaSca ano24· Ma wanShinawi25 ·
PaulinoGómez‑Pue as4 · ZeynepTüme 8,26 · RaoulC.Hennekam1
1 Depa men o Pedia ics, Ams e dam UMC, Uni e si y
o Ams e dam, Meibe gd ee 9, 1105AZAms e dam,
TheNe he lands
2 Co daan, Ou pa ien Clinic o ID Medicine, Klinke weg 75,
1033PKAms e dam, TheNe he lands
3 Odion, Ou pa ien Clinic o ID Medicine, Pu me end,
TheNe he lands
4 Molecula Modelling G oup, Cen o de Biología Molecula
Se e o Ochoa, CBMSO (CSIC-UAM), Mad id, Spain
5 School o Expe imen al Sciences-IIB, Uni e sidad F ancisco
de Vi o ia, UFV, PozuelodeAla cón, Spain
6 Banne Child ens Specialis s Neu ology Clinic, Glendale,
AZ, USA
7 Clinical Gene ics Se ice, She ield Child en’s Hospi al,
Academic Uni o Child Heal h, Uni e si y o She ield,
She ield, UK
8 Depa men o Clinical Gene ics, Kennedy Cen e ,
Copenhagen Uni e si y Hospi al, Rigshospi ale ,
Gl. Lande ej 7, 2600Glos up, Denma k
9 Depa men o Pedia ics andAdolescen Medicine,
Copenhagen Uni e si y Hospi al, Rigshospi ale , Glos up,
Denma k
10 MRC Human Gene ics Uni , Uni e si y o Edinbu gh,
Edinbu gh, UK
11 Depa men o Immunology, Gene ics andPa hology,
Uppsala Uni e si y, Uppsala, Sweden
592 Human Gene ics (2020) 139:575–592
1 3
12 P insens ich ing, Pu me end, TheNe he lands
13 Di ision o Medical Gene ics, Depa men o In e nal
Medicine, Is anbul Uni e si y, Is anbul, Tu key
14 Depa men o Clinical Gene ics, Ams e dam UMC,
Uni e si y o Ams e dam, Ams e dam, TheNe he lands
15 Unidad de Gené ica, Hospi al Uni e si a io y Poli écnico La
Fe, Valencia, Spain
16 No he n I eland Regional Gene ics Se ice, Bel as Ci y
Hospi al, Bel as , UK
17 Au ism Team No he n-Ne he lands, Jonx Depa men
o You h Men al Heal h andAu ism, Len is Psychia ic
Ins i u e, G oningen, TheNe he lands
18 Ins i u e o Medical Gene ics, Uni e si y Hospi al o Wales,
Ca di , UK
19 Clinical Gene ic Se ice, No he n Gene al Hospi al,
She ield, UK
20 Uni o Clinical Gene ics Uni , Se ice o Pedia ics,
Uni e si y Hospi al “Lozano Blesa”, Uni e si y o Za agoza
School o Medicine, Sa agossa, Spain
21 Uni o Clinical Gene ics Uni andFunc ional Genomics,
Depa men o Pha macology andPhysiology, Uni e si y
o Za agoza School o Medicine, Sa agossa, Spain
22 Depa men o Pedia ics, Di ision o Human Gene ics,
Inselspi al, Uni e si y o Be n, Be n, Swi ze land
23 Depa men o Molecula andHuman Gene ics, Baylo
College o Medicine, Baylo Gene ics Labo a o ies, Hous on,
TX, USA
24 Ra e Disease Uni , Depa men o Pedia ics, S . O sola
Hospi al, Bologna, I aly
25 Depa men o Pedia ics, Di ision o Gene ics andGenomic
Medicine, Washing on Uni e si y School o Medicine,
S .Louis, MO, USA
26 Depa men o Clinical Medicine, Uni e si y o Copenhagen,
Copenhagen, Denma k