Greg Whittemore, MS4 MD/MS Candidate Columbia University Vagelos College of Physicians and Surgeons Mentors: Cathy Mendelsohn and Simone Sanna-Cherchi

Never in the Urinary Tract – Causing Urinary Tract Malformations: the case of Tbx6 Greg Whittemore, MS4 MD/MS Candidate Columbia University Vagelos College of Physicians and Surgeons Mentors: Cathy Mendelsohn and Simone Sanna-Cherchi

Columbia University P20 Developmental Center for Benign Urology Research Congenital anomalies of the kidney and urinary tract (CAKUT) : Conditions and Epidemiology

Liu & Ahram, Kidney Int. in preparation Genetics of CAKUT

• Point mutations: 6–20% of CAKUT caused by single-gene defects with over 50 genes identified thus far (most commonly HNF1B, PAX2, EYA1, SALL1 and others)

• Structural variants / Copy number variations (CNVs; i.e. deletions or duplications of germline DNA often affecting multiple genes): additional 2-10% of CAKUT caused by large CNVs associated to genomic disorders (ex. 22q11.2, 17q12, and others)

Verbitsky & Westland, Nat Genet. 2019 Sanna-Cherchi & Westland, J Clin Invest. 2018 van der Ven et al, J Am Soc Nephrol. 2018 Standard paradigm for gene identification in humans

• Step 1: unbiased, hypothesis-free genetic study to localize a gene or region of the genome associated with the phenotype (linkage studies in families, GWAS, exome or genome sequencing…)

• Step 2: candidate gene selection / prioritization. Classically it has been recognized that, if a gene causes a phenotype when mutated, it should be expressed in the tissue where the phenotype occurs

• Step 3: generation of a vertebrate model that recapitulates the human phenotype 1q21.1 4p (Wolf−Hirschhorn)

) 0.75 ) 0.50

% %

( (

y Deletion y

c c

n Duplication n e 0.50 e

u u

q q

e e

r r 0.25

f f

n n

o o

i 0.25 i

t t

a a

l l

u u

p p

o o CNVP study0.00 in 2,824 CAKUT cases andP 0.00 21,498 controls identifies the T A U V S R K M ls T A U V S R K M ls U K O U C U H T o U K O U C U H T o K P D V − U tr K P D V − U tr chromosomeA 16p11.2 microdeletionK L n syndromeA as a causeK Lofn CAKUT C E o C E o C C

16p11.2 Common phenotypes16p13.11 with 16p11.2

) 1.50 ) 0.75microdeletion: -6 % P = 4.4 x 10 %

( (

y 1.25 OR = 13.7 y c c • Congenital scoliosis

n n

e 1.00 e 0.50• Spondylocostal dysostosis

u u

q q • Autism spectrum disorder

e e

r 0.75 r

f f

n n

o o

i 0.50 i 0.25Classic association between congenital

t t

a a l l scoliosis and CAKUT u 0.25 u

p p

o o

P 0.00 P 0.00

T A U V S R K M ls T A U V S R K M ls U K O U C U H T o U K O U C U H T o K P D V − U tr ElK-Kateb et al, AmP JD MedV Genet− A.U 2014tr A K L n A K L n C E o C Hanson et al., Biol Psychiatry.E 2015o C Vitko, Cass & Winter, J Urol. 1972C Verbitsky & Westland, Nat Genet. 2019 MacEwen, Winter & Hardy, J Bone Joint Surg. 1972 17q12 (RCAD) 22q11 (DiGeorge)

) 2.00 ) 1.25

% %

( (

1.75 y y 1.00 c 1.50 c

n n

e e

u 1.25 u 0.75

q q

e e

r 1.00 r

f f

n 0.75 n 0.50

o o

i i

t t

a 0.50 a l l 0.25

u u

p 0.25 p

o o

P 0.00 P 0.00

T A U V S R K M ls T A U V S R K M ls U K O U C U H T o U K O U C U H T o K P D V − U tr K P D V − U tr A K L n A K L n C E o C E o C C Defining a driver for CAKUT at the 16p11.2 locus: deletion mapping

Chromosome 16 p11.2

Verbitsky & Westland, Nat Genet. 2019 PlausibilityPlausibility forfor TBX6TBX6 asas aa CAKUTCAKUT genegene

• IInvolvednvolved iinn eearlyarly mmouseouse ddevelopmentevelopment

• HHumanuman ttruncatingruncating mmutationsutations associatedassociated w withith ccongenitalongenital sscoliosiscoliosis

• HHeterozygouseterozygous mmutationsutations aarere veryvery rrareare inin humanshumans

Yang,Yang, Wu,Wu, etet al.al. KidneyKidney International.International. 20202020 Mouse Tbx6 allelic series

Alejandra Perez Tbx6– null allele Tbx6rv spontaneous hypomorphic allele

Qingxue Liu Genotype Phenotype Tbx6+/+ wild type Tbx6rv/+ wild type Tbx6+/– minor vertebral abnormalities Tbx6rv/rv fused ribs and vertebrae Tbx6rv/– severe fusion of ribs and vertebrae Tbx6–/– lethal E9.5

In collaboration with Mendelsohn and Papaioannou labs Severe reduction in Tbx6 gene dosage causes CAKUT with complete penetrance  supporting causality

Wild type Tbx6rv/-

A B C

D E F

Verbitsky & Westland, Nat Genet. 2019

100 µm 100 µm H I J

K L M

100 µm 100 µm N Pax2 Ecad O V regressed nd

cnd nd

1 cnd ub R C 1 ub not regressed

E np np D Milder mutations recapitulate CAKUT phenotypes in 16p11.2 deletion including obstructive uropathy and duplicated ureters supporting pleiotropic effect

Wild type Tbx6rv/rv

A B C

D E F Verbitsky & Westland, Nat Genet. 2019

5 E15 wt

E Analysis of the lower urinary tract for CAKUT phenotypes identifies anorectal malformations  further pleiotropy

Wild Type Tbx6rv/rv Tbx6rv/– Bladder Hindgut Hindgut Bladder Hindgut Bladder

E15.5 Urethra

Anus Additional lower urinary tract defects that correlate with urethra malformations and PUV

Wild Type Tbx6rv/rv

Vagina Bladder Vagina Bladder Urethra

Urethra E18.5 Initial conclusions and questions

• TBX6 reduced gene dosage is the cause of CAKUT in patients with the chromosome 16p11.2 microdeletion

• Tbx6 reduced gene dosage has a profound pleiotropic effect similar to the 16p11.2 microdeletion causing upper and lower urinary tract defects

• Open question: How do TBX6 mutations cause CAKUT and what are the mechanisms for such pleiotropic effect? Tbx6 is expressed in the intermediate mesoderm surrounding the cloaca at E9.5 but NEVER in the developing urinary tract

Wild Type Wild Type

Tbx6 DAPI cloaca

E9.5 E10.5

tail mesenchyme neural tube

How is it possible? Some hint: Does the nephric duct insert into the cloaca?

Wild Type Tbx6rv/rv E11.5 Cl ND

ND Tbx6 gene dose reduction affects the position & angle of ureteric bud formation and disrupts the normal interactions between the ureteric bud and nephric progenitors

Wild Type Tbx6rv/– Tbx6rv/–

ECTOPIC NEURAL TUBE Mendelsohn, Organogenesis.2009 Verbitsky & Westland, Nat Genet. 2019 Rationale for urinary tract defects in Tbx6 mutants

• Tbx6 appears to be critical for insertion of CND into the cloaca • Expressed at the right place at the right time and gene dose reduction results in failure of CND insertion • CND insertion is critical for normal UB induction and insertion of ureter into bladder  mechanism for VUR, OU, DCS, KA (Mackie Stephens hypothesis)

• Tbx6 dose reduction results in ectopic neural tubes in place of posterior somites • Grobstein et al in the 1950’s demonstrated that neural tube is capable of attracting and inducing mesenchyme in vitro • Abnormal position of kidney mesenchyme  mechanism of DCS, KA Conclusions and Future Directions • Tbx6 gene-dose reduction is sufficient to cause all categories of human CAKUT observed in the chromosome 16p11.2 microdeletion syndrome

• Tbx6 is expressed in peri-cloacal mesenchyme at E9.5, but, surprisingly, never in the developing ureter or kidney

• We propose different mechanisms and explanations for the TBX6 mutations causal role and the observed pleiotropy: - Failure of nephric duct insertion - Ectopic neural tubes - Failed or incomplete cloacal septation • Future directions: studies aimed at understanding the transcriptional dysregulation resulting from TBX6 mutations and identification of potential intervention targets Acknowledgements Columbia University Miguel Verbitsky, Ali Gharavi, Krzysztof Kiryluk, Jonathan Barasch, Shumyle Alam, Pasquale Casale, James McKiernan, Fangming Lin, Priya Krithivasan, Byum Hee Kil, David Goldstein, Cathy Mendelsohn, Virginia Papaioannou Bari, Italy Loreto Gesualdo, Milena Gigante Patients and family Belo Horizonte, Brazil Ana Cristina Simoes Silva members Brescia, Italy Francesco Scolari, Claudia Izzi Cagliari, Italy Giuseppe Masnata Sanna-Cherchi Lab CHOP, Philadelphia Hakon Hakonarson, Donna McDonald-McGinn, Elaine Zackai Jerry Martino CKiD Study Bradley A. Warady, Susan L. Furth, Craig Wong Dina Ahram Dublin, Ireland David Barton, John Darlow Duke University Nicholas Katsanis, Erica Davis Qingxue Liu Genoa, Italy Gian Marco Ghiggeri, Gianluca Caridi, Monica Bodria, Alba Juntao Ke Carrea Yask Gupta Harvard University Iain Drummond, Friedhelm Hildebrandt, Asaf Vivante Tze Yin Lim Milan, Italy Daniele Cusi, The Hypergenes Consortium, Gina Jin Messina, Italy Giovanni Conti, Domenico Santoro Alejandra Perez Olomouc, Czech Republic Hana Flogelova Palermo, Italy Silvio Maringhini Parma, Italy Landino Allegri Mendelsohn Lab Paris, France Cecile Jeanpierre PRCM, Poland Anna Materna-Kiryluk, Anna Latos-Bielenska, Marcin Zaniew Skopje, Macedonia Velibor Tasic, Zoran Gucev Split, Croatia Marijan Saraga, Mirna Saraga Babic, Kristina Drnasin U. Michigan, Ann Arbor Edgar Otto, Matthew Sampson VUMC, Amsterdam Rik Westland, Johanna van Wijk Yale University Richard Lifton Funding: NIDDK: R01DK103184, R01DK115574, R21DK098531, U54DK104309, P20DK116191 AHA Grant-in-Aid Irving Institute Joint NIH MOH Finalized Research Grant Paul Marks Scholar Columbia CTSA Precision Medicine Pilot Award