Mild Recessive Mutations in Six Fraser Syndrome– Related Genes Cause Isolated Congenital Anomalies of the Kidney and Urinary Tract

BRIEF COMMUNICATION www.jasn.org

† ‡ ‡ Stefan Kohl,* Daw-Yang Hwang,* Gabriel C. Dworschak,* Alina C. Hilger, § Pawaree Saisawat,§ | | ‡ †† Asaf Vivante,* Natasa Stajic, ¶ Radovan Bogdanovic, ¶ Heiko M. Reutter, ** Elijah O. Kehinde, ‡‡ Velibor Tasic, and Friedhelm Hildebrandt*§§

†

*Department of Medicine, Boston Children’s Hospital, Harvard Medical School, Boston, Massachusetts; Division of BRIEF COMMUNICATION Nephrology, Department of Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan; ‡Institute of Human Genetics, and **Department of Neonatology, Children’s Hospital, University of Bonn, Bonn, Germany; §Department of Pediatrics, University of Michigan, Ann Arbor, Michigan; |Medical Faculty, University of Belgrade, Belgrade, Serbia; ¶Institute of Mother and Child Healthcare of Serbia, Belgrade, Serbia; ††Department of Surgery, Kuwait University, Safat, Kuwait; ‡‡Department of Pediatric Nephrology, University Children’s Hospital, Skopje, Macedonia; and §§Howard Hughes Medical Institute, Chevy Chase, Maryland

ABSTRACT Congenital anomalies of the kidney and urinary tract (CAKUT) account for approx- different forms of syndromic CAKUT imately 40% of children with ESRD in the United States. Hitherto, mutations in 23 have been described,4 isolated CAKUT genes have been described as causing autosomal dominant isolated CAKUT in account for the majority of cases.5,6 Twenty- humans. However, .90% of cases of isolated CAKUT still remain without a molec- three autosomal dominant genes have ular diagnosis. Here, we hypothesized that genes mutated in recessive mouse mod- been identified to cause isolated CAKUT, els with the specific CAKUT phenotype of unilateral renal agenesis may also be with TNXB, WNT4,andDSTYK being mutated in humans with isolated CAKUT. We applied next-generation sequencing the most recent ones.7–10 According to technology for targeted exon sequencing of 12 recessive murine candidate genes in the Mouse Genome Informatics database 574 individuals with isolated CAKUT from 590 families. In 15 of 590 families, we (http://www.informatics.jax.org), 1768 identified recessive mutations in the genes FRAS1, FREM2, GRIP1, FREM1, ITGA8, monogenic mouse models for CAKUT and GREM1, all of which function in the interaction of the ureteric bud and the have been described, many of which are metanephric mesenchyme. We show that isolated CAKUT may be caused partially recessive and do not have a human dis- by mutations in recessive genes. Our results also indicate that biallelic missense ease correlate. mutations in the Fraser/MOTA/BNAR spectrum genes cause isolated CAKUT, In this study, we hypothesized that whereas truncating mutations are found in the multiorgan form of Fraser syndrome. targeted genes in recessive mouse models The newly identified recessive biallelic mutations in these six genes represent the with CAKUT may also be mutated in molecular cause of isolated CAKUT in 2.5% of the 590 affected families in this study. humans with isolated CAKUT. Candi- date genes were selected based on re- J Am Soc Nephrol 25: 1917–1922, 2014. doi: 10.1681/ASN.2013101103

Received October 22, 2013. Accepted January 16, Congenital anomalies of the kidney and CAKUT comprise a broad spectrum of 2014. urinary tract (CAKUT) are one of the structural malformations and functional S.K. and D.-Y.H. contributed equally to this work. most frequent congenital abnormalities anomalies, including unilateral renal Published online ahead of print. Publication date in humans, taking a high toll on affected agenesis, renal hypodysplasia, ureteropelvic available at www.jasn.org. individuals, their families, and health care. junction obstruction, and vesicoureteral The estimated occurrence of CAKUT is reﬂux. The clinically distinct CAKUT Correspondence: Dr. Friedhelm Hildebrandt, Di- vision of Nephrology, Boston Children’sHospital, – 1 3 6 per 1000 live births. They represent phenotypes have in common a disturbed 300 Longwood Avenue, Boston, MA 02115. Email: the most frequent cause of CKD and embryonic codevelopment of tissues de- [email protected]

ESRD in children in the United States, rived from the ureteric bud and the meta- Copyright © 2014 by the American Society of accounting for .40% of all cases.2 nephric mesenchyme.3 Although .200 Nephrology

J Am Soc Nephrol 25: 1917–1922, 2014 ISSN : 1046-6673/2509-1917 1917 BRIEF COMMUNICATION www.jasn.org cessive murine models for CAKUT with (n=6, 4, and 1, respectively) (Table 1). splice-site mutation in ITGA8 (Table 1). the distinct CAKUT phenotype of unilat- Mutations in FRAS1, FREM2,orGRIP1 ThenucleotidechangeITGA8 c.2982 eral renal agenesis in homozygous null were detected in individuals with differ- +2T.C is predicted to abrogate the animals. We reckoned that mouse models ent isolated CAKUT phenotypes (Table splice donor site of exon 28 (59 splicing with unilateral renal agenesis represented 1). Ten of 11 individuals had no extrare- site) by five publically available splice-site the most promising CAKUT candidate nal manifestations characteristic for prediction algorithms (SpliceSiteFinder- genes, because unilateral renal agenesis is a Fraser syndrome (e.g., cryptophthalmos, like, MaxEntScan, NNSPLICE, GeneSplicer, severe and specificCAKUTphenotype.To syndactyly, or genital malformations). and Human Splicing Finder). test this hypothesis, we performed bar- One individual, A3455-21, had CAKUT In individual A3573-21, we detected a coded next-generation sequencing (NGS)– and anal atresia, which is a diagnostic homozygous missense mutation in the based exon sequencing of these candidate criterion of Fraser syndrome (Table 1). BMP4-antagonist GREM1 (Table 1). genesaspreviouslydescribedbyour However, this was not sufficient to make GREM1 p.P35A segregated from the het- group.11,12 the clinical diagnosis.13 Interestingly, we erozygous parents to the affected child We analyzed the coding sequences of here discovered recessive missense mu- with unilateral renal agenesis. The unaf- 12 recessive murine candidate genes tations in FRAS1, FREM2,andGRIP1 as fected sibling A3579-22 was heterozy- (Supplemental Table 1) in 672 individuals novel causes of isolated CAKUT, gous for the mutation (Table 1). from 590 families with isolated CAKUT whereas biallelic truncating mutations In this study, we identified mutations (Supplemental Table 2). Mutations in 17 areknowntocauseFrasersyndrome in six recessive murine “single kidney” known CAKUT-causing genes were ex- with multiorgan involvement (Supple- candidate genes in 672 individuals cluded before this study (see Concise mental Table 3). from 590 families as novel single-gene Methods). For individuals with renal hy- In addition to the Fraser syndrome causes of isolated CAKUT. These six re- podysplasia (n=101), we also excluded genes, we detected recessive mutations in cessive genes account for mutations in the presence of an HNF1B deletion by the gene FREM1 that cause the closely 2.5% of 590 families with isolated CAKUT. quantitative PCR. Fraser-related phenotypes Manitoba Together with heterozygous mutations in In 15 of 672 individuals from 590 Oculotrichoanal (MOTA) syndrome14 17 known autosomal dominant CAKUT- families (2.5%) with isolated CAKUT,we (OMIM 248450) or Bifid Nose with or causing genes, which account for another identified 21 different mutations in six without Anorectal and Renal Anomalies 6.3%,16 we are now able to molecularly different recessive genes (Table 1). These (BNAR) syndrome15 (OMIM 608980) in “solve” almost 10% of cases with isolated genes were FRAS1, FREM2, GRIP1, an allele-dependent manner. In this CAKUT in our cohort. FREM1, ITGA8,andGREM1.Themost study, we identified two unrelated indi- We detected recessive biallelic mis- frequently mutated gene in this study viduals with isolated CAKUT with the sense mutations in the four Fraser spec- was FRAS1, accounting for six unrelated same homozygous mutation in FREM1 trum genes in individuals with isolated individuals with isolated CAKUT. In the (FREM1 p.A1627S) (Table 1). Individual CAKUT. The proteins encoded by the six murine “single kidney” genes BAG6, A688-21 had isolated CAKUT, whereas Fraser genes FRAS1, FREM2,andFREM1 CTNNBIP1, DACT1, ILK, LIN7C,and individual A3369-21 had CAKUT with form a tertiary protein complex lining LRP4, there were no biallelic mutations bilateral syndactyly of toes II-III, which the extracellular epithelial-mesenchymal present in humans with isolated CAKUT. is a feature of MOTA syndrome. How- interface.17,18 Loss of function of any of We considered recessive alleles as likely ever, MOTA/BNAR-characteristic facial these proteins disrupts the Fraser-protein disease causing if they either were protein- dysmorphism was absent from both in- complex and leads to the severe multior- truncating (n=2) or missense mutations dividuals. Similarly to the detected muta- gan developmental phenotype of Fraser affecting an evolutionary conserved tions in the Fraser genes, our data support syndrome in humans and mice (Supple- amino acid residue and with a minor al- that biallelic missense mutations in mental Table 1).14,19–21 In this study, 11 lele frequency of ,1% in 13,000 control FREM1 cause isolated CAKUT, whereas of 13 individuals with recessive mutation chromosomes of the National Heart truncating mutations are only reported in in the Fraser genes had no extrarenal Lung and Blood Institute Exome Se- individuals with the severe multiorgan phenotype. We observed almost exclu- quencing Project (n=17). All detected phenotype of MOTA/BNAR syndrome sively biallelic missense mutations in in- alleles were confirmed by Sanger sequenc- (Supplemental Table 3). dividuals with isolated CAKUT, whereas ing in genomic DNA of the affected In addition to Fraser spectrum genes, individuals with Fraser syndrome gener- individuals. we detected homozygous recessive mu- ally are reported to have protein-truncating In the genes FRAS1, FREM2,and tations in ITGA8 and GREM1,which alleles (Supplemental Table 3). This may GRIP1, which cause Fraser syndrome if have not previously been implicated in represent an example of allelism in a mutated (Online Mendelian Inheritance human CAKUT. In individual A876-21 recessive disease, which has been de- in Man [OMIM] 219000), we detected with a left duplicated collecting system scribed and discussed more extensively recessive missense mutations in 11 un- and left high-grade vesicoureteral reflux, in nephronophthisis, a rare form of cystic related individuals with isolated CAKUT we detected a homozygous obligatory kidney disease.22 Hence, we propose the

1918 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 1917–1922, 2014 mScNephrol Soc Am J Table 1. Recessive mutations in 6 murine candidate genes detected in 15 unrelated individuals with isolated CAKUT Nucleotide Amino Segregation/ Family - Geographic Renal Extrarenal Gene Sex Change Exon Acid State of Mm Gg Xt Dr EVS SIFT MT PP2 Individual Origin Phenotype Phenotype (Zygosity) Change Alleles FRAS1 A1250-21 Male Kuwait Hypospadias None c.4579C.T (H) 34 R1527W Hom R R P R 24 of 12,416 Deleterious Disease 0.724 25: causing 1917 FRAS1 A1402-21 Male Arabia Right duplex None c.4579C.T (h) 34 R1527W transa R R P R 24 of 12,416 Deleterious Disease 0.724 –

92 04RcsieMttosi sltdCAKUT Isolated in Mutations Recessive 2014 1922, causing c.9364C.T (h) 62 R3122W transa R R R R 0 of 13,000 Deleterious Disease 1 causing FRAS1 A2381-22b Female Germany Left agenesis None c.7867C.T (h) 55 R2623* Paternal 0 of 13,000 N/A N/A N/A inheritance c.9821G.A (h) 64 R3274H Maternal R R R R 0 of 13,000 Deleterious Disease 0.999 inheritance causing FRAS1 A3455-21 Male Macedonia Right agenesis Anal c.4579C.T (h) 34 R1527W Maternal R R P R 24 of 12,416 Deleterious Disease 0.724 atresia inheritance causing c.7622A.G (h) 53 N2541S Paternal N N N N 28 of 12,274 Deleterious Disease 0.997 inheritance causing FRAS1 A3975-21 Male Germany Right ectopic None c.4159_4161 31 A1387L trans in A A A A 13 of 12,487 N/A N/A N/A medullary delinsTTA (h) 1000Gc cystic kidney disease c.9806G.A (h) 64 R3269Q trans in R R R R 88 of 12,570 Deleterious Disease 0.999 1000Gc causing FRAS1 A4175-21 Male UK VUR None c.9806G.A (H) 64 R3269Q Hom R R R R 88 of 12,570 Deleterious Disease 0.999 causing c.9959C.T (h) 64 S3320F n/a S S S S 0 of 13,000 Deleterious Disease 0.999 causing FREM2 A1023-21d Male India Right agenesis None c.4031G.A (h) 1 R1344H trans in R R R R 25 of 12,981 Deleterious Disease 0.085 1000Gc causing c.7535G.A (h) 15 R2512H trans in R R R R 10 of 12,996 Deleterious Disease 0.929 www.jasn.org 1000Gc causing c.7013C.T (h) 12 T2338I n/a T T T T 0 of 13,000 Deleterious Disease 0.999 causing FREM2 A1232-21 Male Kuwait PUV, right VUR, None c.649C.T (h) 1 R217C transe R R R E 0 of 13,000 Deleterious Disease 0.836 left UPJO, causing

CKD COMMUNICATION BRIEF c.4031G.A (h) 1 R1344H transe R R R R 25 of 12,981 Deleterious Disease 0.085 causing FREM2 A1548-21 Male India B UPJO CM c.685C.T (h) 1 R229C transe R R R R 0 of 13,000 Deleterious Disease 0.95 causing c.4820A.G (h) 1 D1607G transe D D D D 0 of 13,000 Deleterious Disease 0.983 causing 1919 BRIEF COMMUNICATION www.jasn.org

hypothesis that the detected missense 0.075 0.606 0.087 0.893 0.893 0.015 mutations causing isolated CAKUT represent hypomorphic alleles, which can be compensated for in the development of causing causing causing causing causing causing extrarenal tissues and consequently cause

V according to the the mildest phenotype in the Fraser/ – MOTA/BNAR/CAKUT spectrum.23

(NM_021150.3); duplex, Phenotypic heterogeneity has been reported in Fraser/MOTA syndrome, GRIP1 even within affected families.24 Simi- larly, the unrelated individuals A688-21

EVS SIFT MT PP2 and A3369-21 have the same homozygous mutation (Table 1); however, only 0 of 13,000 N/A N/A N/A individual A3369-21 has syndactyly of ux graded in Roman numerals I ﬂ toe II/III, which represents a “mild-

(NM_013372.6). end” feature of the Fraser spectrum. In a previous study, we hypothesized I V V V 0 of 13,000 Deleterious Disease GREM1 P P P P 34 of 12,952 Tolerated Disease R R R R 0 of 13,000 Tolerated Disease A A A A 1 of 12,307 Deleterious Disease A A A A 1 of 12,307 Deleterious Disease G G G G 0 of 13,000 Deleterious Disease that heterozygous mutations in FRAS1 Mm Gg Xt Dr and FREM2 may cause autosomal dominant isolated CAKUT in humans.25 How-

meaning they are on different chromosomes; Agenesis, renal agenesis; N/A, ever, this hypothesis is not supported by ” , Alleles

State of the data of the current more extensive inheritance inheritance inheritance, maternal inheritance (NM_003638.1); trans Segregation/ Hom study and insight from growing public in-

“ variant databases (Supplemental Table 4). ITGA8 FRAS1 has been shown to be ex- Acid Amino Change — pressed in glomerular podocytes and po- alleles are docyte precursors.26 In this study, we did 25

Exon not observe nephrotic syndrome in any trans, of the participants with mutations in the C (H) 28 Fraser genes. This is in line with individ- . A (h) 16 G616R Paternal T (h) 22 R917L Maternal T (H) 28 A1627S Hom T (H) 28 A1627S Hom C (H) 13 I2404T Hom G (H) 2c P35A Paternal . . . . 13 . uals with Fraser syndrome. Hence, we . A2381-21 had bilateral renal agenesis with absent urinary bladder (termination of pregnancy). Change 25 conclude that FRAS1 is not essential for mutation p.T2338I. (Zygosity) Nucleotide glomerulogenesis. c.2750G Besides mutations in the Fraser genes, FREM2 we identiﬁed two individuals with ho-

; EVS, allele frequency in EVS database; SIFT, sorting intolerant from tolerant prediction class; MT, mutation taster prediction class; PP2, mozygous mutations in ITGA8 and None c.4879G None c.2982+2T rmed by subcloning of genomic DNA and Sanger sequencing of mono-molecular clones. Extrarenal Phenotype

ﬁ GREM1, which previously have not been implicated in human CAKUT. In- terestingly, all of the six genes found mu- Dr, danio rerio because individuals of the 1000 Genomes Project show absence of linkage disequilibrium (Supplemental Table 6). ; because of uneven distribution (24 of 12,416 versus 0 of 13,000) of counts in the National Heart Lung and Blood Institute Exome Sequencing Project tated in this study functionally converge Renal trans trans on two signaling pathways essential for V°, right RHD left VUR III° Phenotype kidney development: Integrin-a8inter- acts indirectly with the Fraser-protein

(NM_207361.4); PUV, posterior urethral valves; UPJO, ureteropelvic junction obstruction; CM, cardiomyopathy of unknown etiology; complex, with this interaction being essential for GDNF expression in the meta- Origin 17,27 FREM2

Geographic nephric mesenchyme. GDNF-RET (NM_144966.5); B, bilateral; SD II/III, syndactyly toes II/III; RHD, renal hypodysplasia; (NM_025074.6); H, homozygous; Hom, homozygous; h, heterozygous; signaling at the ureteric bud/metanephric mesenchyme interface is counterbal- Sex FREM1 FRAS1 anced by BMP4 signaling. GREM1 is an in 1000G, both alleles are present in the 1000G cohort but never in the same individual (Supplemental Table 6); VUR, vesicoureteral re cation of VUR;

ﬁ antagonist of BMP4, a known CAKUT-

trans causing gene, and thus acts synergisti- Continued

Family - cally with the Fraser-protein complex/ Individual A3390-21 Female Macedonia Right duplex None c.1846G A358-21 Female Kuwait B VUR, CKD None c.7211T A3369-21 Male Macedonia B VUR III° B SD II/III c.4879G A688-21 Male Macedonia Right VUR A876-21 Female Macedonia Left duplex, A3573-21 Female Macedonia Left agenesis None c.103C integrin-a8/GDNF axis.28 On the basis of the well deﬁned mouse Gene A2381-22 was published in a previous study of ourA1023-21 group was reporting published identical in mutations. a previous study of our group for the heterozygous No parental DNA available. Compound heterozygous state was con No parental DNA available. Alleles are most likely in No parental DNA available. Alleles are most likely in GRIP1 FREM2 FREM1 FREM1 not applicable; International Classi ITGA8 cohort of 6500 control individuals (http://evs.gs.washington.edu/EVS/). PolyPhen2 humvar score; Table 1. Mm, mus musculus; Gg, gallus gallus; Xt, xenopus tropicalis a b c d duplex collecting system; e GREM1 phenotype of unilateral renal agenesis, we

1920 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 1917–1922, 2014 www.jasn.org BRIEF COMMUNICATION identified six recessive monogenic causes with isolated CAKUT (n=5), or were reported This work was supported by grants from ofisolatedCAKUTinhumans,twoofthem in mouse models with conditional/complex the National Institutes of Health (R01- as novel CAKUT genes. We thereby iden- knock-out alleles, or were not purely autosomal DK045345 and R01-DK088767 to F.H.). F.H. tified the first recessive isolated CAKUT recessive. FREM2 was included in this study is an investigator of the Howard Hughes genes in humans. The six genes found to be because three of four Fraser spectrum genes Medical Institute,a Doris Duke Distinguished mutated encode for proteins that func- (FRAS1, GRIP1, and FREM1)mettheinclusion Clinical Scientist, and a Warren E. Grupe tionallyconverge on the GDNF-RET/BMP criteria. The selected candidate genes were Professor. signaling pathways at the interface of the BAG6, CTNNBIP1, DACT1, FRAS1, FREM1, ureteric bud and the metanephric mesen- FREM2, GREM1, GRIP1, ILK, ITGA8, LIN7C, chyme. We showed genetic evidence that and LRP4 (Supplemental Table 1). DISCLOSURES mild mutation in the Fraser spectrum None. genes (FRAS1, FREM2, GRIP1,and Targeted Exon Sequencing FREM1) causes isolated CAKUT as the For 12 genes, 355 target-specificprimerpairs mildest phenotype of the clinical spec- were designed covering 273 coding exons REFERENCES trum. These six genes contribute to 2.5% (Supplemental Table 5). The amplicon size of all cases of isolated CAKUT; thus, up to ranged from 150 to 290 bp. Targeted ampli- 1. Harambat J, van Stralen KJ, Kim JJ, Tizard EJ: 20% of individuals with CAKUT could fication and NGS was done as described pre- Epidemiology of chronic kidney disease in children. Pediatr Nephrol 27: 363–373, 2012 “ ” viously by our group11,12 with the following now be molecularly solved by exon se- 2. North American Pediatric Renal Trials and quencing in 29 genes and copy number alterations: 12 primer pairs were multiplexed Collaborative Studies: NAPRTCS 2011 An- – variation analysis.9,29 31 Rapidly develop- in 48 pools to allow for amplification of 576 nual Report, Rockville, MD, The EMMES ing sequencing technology will continue PCR products per sample simultaneously us- Corporation, 2011 to facilitate identification of rare single- ing the Fluidigm 48.48 Access Array IFC Sys- 3. Ichikawa I, Kuwayama F, Pope JC 4th, Stephens FD, Miyazaki Y: Paradigm shift from tem. NGS was carried out using an Illumina gene causes of human CAKUTand enables classic anatomic theories to contemporary 3 us to conduct large-scale mutation analy- HiSeq2000 instrument (1 150 bp single cell biological views of CAKUT. Kidney Int 61: ses providing families with a genetic diag- reads) or Illumina MiSeq V2 instrument 889–898, 2002 nosisinanincreasingnumberofcases. (23250 bp paired reads). Our results showed 4. Limwongse C: Syndromes and malforma- that 326 amplicons (91.8%) had a coverage tions of the urinary tract. In: Pediatric Ne- phrology, 6th Ed., edited by Avner ED, .1003 and 11 amplicons (3.0%) were cov- Harmon WE, Niaudet P, Yoshikawa N, Berlin, , 3 CONCISE METHODS ered 10 (Supplemental Table 3). All Springer, 2009, pp 122–138 identified mutations were confirmed by 5. Weber S: Novel genetic aspects of congeni- Sanger sequencing in genomic DNA. Segre- tal anomalies of kidney and urinary tract. Curr Human Participants – After informed consent, we obtained clinical gation analysis was performed if parental Opin Pediatr 24: 212 218, 2012 6. Yosypiv IV: Congenital anomalies of the kid- DNA was available. Alternatively, fragments data, blood samples, and pedigrees from ney and urinary tract: A genetic disorder? Int individuals with CAKUT. Approval for re- of genomic DNA harboring two alleles were JNephrol2012: 909083, 2012 search on humans was obtained from the TA cloned into pGEM Easy-vector (Promega). 7. Gbadegesin RA, Brophy PD, Adeyemo A, University of Michigan Institutional Review Six clones were Sanger sequenced in order Hall G, Gupta IR, Hains D, Bartkowiak B, Board. Mutations in the following genes to confirm the compound heterozygous Rabinovich CE, Chandrasekharappa S, Homstad A, Westreich K, Wu G, Liu Y, state. known to be mutated in isolated CAKUT in Holanda D, Clarke J, Lavin P, Selim A, Miller humans were excluded before this study: S, Wiener JS, Ross SS, Foreman J, Rotimi C, BMP4, BMP7, CDC5L, CHD1L, EYA1, Bioinformatics Winn MP: TNXB mutations can cause ves- GATA3, HNF1B, PAX2, RET, ROBO2, NGS data alignment and variant detection icoureteral reflux. JAmSocNephrol24: – SALL1, SIX1, SIX2, SIX5, SOX17, UMOD, were performed using CLC Genomics Work- 1313 1322, 2013 8. Vivante A, Mark-Danieli M, Davidovits M, bench 4.9 software. Variant filtering was and UPK3A. HNF1B deletions were excluded Harari-Steinberg O, Omer D, Gnatek Y, by quantitative PCR in individuals with the carried out as published previously by our Cleper R, Landau D, Kovalski Y, Weissman I, CAKUT phenotype of renal hypodysplasia. group.11,12 Homozygous or $2 heterozygous Eisenstein I, Soudack M, Wolf HR, Issler N, alleles in one gene were considered for sub- Lotan D, Anikster Y, Dekel B: Renal hypo- sequent confirmation by Sanger sequencing dysplasia associates with a WNT4 variant that Candidate Gene Selection causes aberrant canonical WNT signaling. J (Supplemental Table 5). Candidate genes were selected from the Am Soc Nephrol 24: 550–558, 2013 Mouse Genome Informatics database based 9. Sanna-Cherchi S, Sampogna RV, Papeta N, on the presence of the CAKUT phenotype of Burgess KE, Nees SN, Perry BJ, Choi M, unilateral renal agenesis (phenotype ID MP: ACKNOWLEDGMENTS Bodria M, Liu Y, Weng PL, Lozanovski VJ, 0003604) in homozygotes. Eighty-one geno- Verbitsky M, Lugani F, Sterken R, Paragas N, Caridi G, Carrea A, Dagnino M, Materna- types involving 39genes matchedthiscriterion. The authors thank the affected individuals, Kiryluk A, Santamaria G, Murtas C, Ristoska- Twenty-eight genes were excluded because their families, and their physicians who con- Bojkovska N, Izzi C, Kacak N, Bianco B, they either were known to be associated tributed to this study. Giberti S, Gigante M, Piaggio G, Gesualdo L,

J Am Soc Nephrol 25: 1917–1922, 2014 Recessive Mutations in Isolated CAKUT 1921 BRIEF COMMUNICATION www.jasn.org

Kosuljandic Vukic D, Vukojevic K, Saraga- 17. Kiyozumi D, Takeichi M, Nakano I, Sato Y, candidate genes in patients with unilateral Babic M, Saraga M, Gucev Z, Allegri L, Latos- Fukuda T, Sekiguchi K: Basement mem- renal agenesis. Kidney Int 81: 196–200, 2012 Bielenska A, Casu D, State M, Scolari F, braneassemblyoftheintegrina8b1 ligand 26. Pitera JE, Scambler PJ, Woolf AS: Fras1, a Ravazzolo R, Kiryluk K, Al-Awqati Q, D’Agati nephronectin requires Fraser syndrome- basement membrane-associated protein VD, Drummond IA, Tasic V, Lifton RP, associated proteins. JCellBiol197: 677– mutated in Fraser syndrome, mediates both Ghiggeri GM, Gharavi AG: Mutations in 689, 2012 the initiation of the mammalian kidney and DSTYK and dominant urinary tract malfor- 18. Kiyozumi D, Sugimoto N, Sekiguchi K: the integrity of renal glomeruli. Hum Mol mations. N Engl J Med 369: 621–629, 2013 Breakdown of the reciprocal stabilization of Genet 17: 3953–3964, 2008 10. Vivante A, Kohl S, Hwang DY, Dworschak QBRICK/Frem1, Fras1, and Frem2 at the 27. Pitera JE, Woolf AS, Basson MA, Scambler GC, Hildebrandt F: Single-gene causes of basement membrane provokes Fraser syn- PJ: Sprouty1 haploinsufficiency prevents re- congenital anomalies of the kidney and uri- drome-like defects. Proc Natl Acad Sci U S A nal agenesis in a model of Fraser syndrome. J nary tract (CAKUT) in humans. Pediatr 103: 11981–11986, 2006 Am Soc Nephrol 23: 1790–1796, 2012 Nephrol 29: 695–704, 2014 19. Jadeja S, Smyth I, Pitera JE, Taylor MS, van 28. Michos O, Panman L, Vintersten K, Beier K, 11. Halbritter J, Porath JD, Diaz KA, Braun DA, Haelst M, Bentley E, McGregor L, Hopkins J, Zeller R, Zuniga A: Gremlin-mediated BMP an- Kohl S, Chaki M, Allen SJ, Soliman NA, Chalepakis G, Philip N, Perez Aytes A, Watt tagonism induces the epithelial-mesenchymal Hildebrandt F, Otto EA; GPN Study Group: FM, Darling SM, Jackson I, Woolf AS, feedback signaling controlling metanephric Identification of 99 novel mutations in a Scambler PJ: Identification of a new gene kidney and limb organogenesis. Development worldwide cohort of 1,056 patients with a mutated in Fraser syndrome and mouse 131: 3401–3410, 2004 nephronophthisis-related ciliopathy. Hum myelencephalic blebs. Nat Genet 37: 520– 29. Sanna-Cherchi S, Kiryluk K, Burgess KE, Genet 132: 865–884, 2013 525, 2005 Bodria M, Sampson MG, Hadley D, Nees SN, 12. Halbritter J, Diaz K, Chaki M, Porath JD, 20. Vogel MJ, van Zon P, Brueton L, Gijzen M, Verbitsky M, Perry BJ, Sterken R, Lozanovski Tarrier B, Fu C, Innis JL, Allen SJ, Lyons RH, van Tuil MC, Cox P, Schanze D, Kariminejad VJ, Materna-Kiryluk A, Barlassina C, Kini A, Stefanidis CJ, Omran H, Soliman NA, Otto EA: A, Ghaderi-Sohi S, Blair E, Zenker M, Corbani V, Carrea A, Somenzi D, Murtas C, High-throughput mutation analysis in patients Scambler PJ, Ploos van Amstel HK, van Ristoska-Bojkovska N, Izzi C, Bianco B, with a nephronophthisis-associated ciliopathy Haelst MM: Mutations in GRIP1 cause Fraser Zaniew M, Flogelova H, Weng PL, Kacak N, applying multiplexed barcoded array-based syndrome. J Med Genet 49: 303–306, 2012 Giberti S, Gigante M, Arapovic A, Drnasin K, PCR amplification and next-generation se- 21. McGregor L, Makela V, Darling SM, Vrontou Caridi G, Curioni S, Allegri F, Ammenti A, quencing. JMedGenet49: 756–767, 2012 S, Chalepakis G, Roberts C, Smart N, Rutland Ferretti S, Goj V, Bernardo L, Jobanputra V, 13. van Haelst MM, Scambler PJ, Hennekam RC; P, Prescott N, Hopkins J, Bentley E, Shaw A, Chung WK, Lifton RP, Sanders S, State M, Fraser Syndrome Collaboration Group: Roberts E, Mueller R, Jadeja S, Philip N, Nelson Clark LN, Saraga M, Padmanabhan S, Fraser syndrome: A clinical study of 59 cases J, Francannet C, Perez-Aytes A, Megarbane A, Dominiczak AF, Foroud T, Gesualdo L, and evaluation of diagnostic criteria. Am J Kerr B, Wainwright B, Woolf AS, Winter RM, Gucev Z, Allegri L, Latos-Bielenska A, Cusi D, Med Genet A 143A: 3194–3203, 2007 Scambler PJ: Fraser syndrome and mouse Scolari F, Tasic V, Hakonarson H, Ghiggeri 14. Slavotinek AM, Baranzini SE, Schanze D, blebbed phenotype caused by mutations in GM, Gharavi AG: Copy-number disorders Labelle-Dumais C, Short KM, Chao R, Yahyavi FRAS1/Fras1 encoding a putative extracellular are a common cause of congenital kidney M, Bijlsma EK, Chu C, Musone S, Wheatley A, matrix protein. Nat Genet 34: 203–208, 2003 malformations. Am J Hum Genet 91: 987– Kwok PY, Marles S, Fryns JP, Maga AM, 22. Chaki M, Hoefele J, Allen SJ, Ramaswami G, 997, 2012 Hassan MG, Gould DB, Madireddy L, Li C, Janssen S, Bergmann C, Heckenlively JR, Otto 30. Weber S, Moriniere V, Knüppel T, Charbit M, Cox TC, Smyth I, Chudley AE, Zenker M: EA, Hildebrandt F: Genotype-phenotype cor- Dusek J, Ghiggeri GM, Jankauskiené A, Mir Manitoba-oculo-tricho-anal (MOTA) syndrome relation in 440 patients with NPHP-related S, Montini G, Peco-Antic A, Wühl E, is caused by mutations in FREM1. J Med ciliopathies. Kidney Int 80: 1239–1245, 2011 Zurowska AM, Mehls O, Antignac C, Genet 48: 375–382, 2011 23. Nathanson J, Swarr DT, Singer A, Liu M, Schaefer F, Salomon R: Prevalence of muta- 15. Alazami AM, Shaheen R, Alzahrani F, Snape Chinn A, Jones W, Hurst J, Khalek N, Zackai tions in renal developmental genes in chil- K, Saggar A, Brinkmann B, Bavi P, Al-Gazali E, Slavotinek A: Novel FREM1 mutations dren with renal hypodysplasia: Results of the LI, Alkuraya FS: FREM1 mutations cause bifid expand the phenotypic spectrum associated ESCAPE study. JAmSocNephrol17: 2864– nose, renal agenesis, and anorectal malfor- with Manitoba-oculo-tricho-anal (MOTA) 2870, 2006 mations syndrome. Am J Hum Genet 85: syndrome and bifid nose renal agenesis 31. Thomas R, Sanna-Cherchi S, Warady BA, 414–418, 2009 anorectal malformations (BNAR) syndrome. Furth SL, Kaskel FJ, Gharavi AG: HNF1B and 16. Hwang DY, Dworschak GC, Kohl S, Saisawat Am J Med Genet A 161A: 473–478, 2013 PAX2 mutations are a common cause of renal P, Vivante A, Hilger AC, Reutter HM, Soliman 24. Prasun P, Pradhan M, Goel H: Intrafamilial hypodysplasia in the CKiD cohort. Pediatr NA, Bogdanovic R, Kehinde EO, Tasic V, variability in Fraser syndrome. Prenat Diagn Nephrol 26: 897–903, 2011 Hildebrandt F: Mutations in 12 known dom- 27: 778–782, 2007 inant disease-causing genes clarify many 25. Saisawat P, Tasic V, Vega-Warner V, Kehinde congenital anomalies of the kidney and uri- EO, Günther B, Airik R, Innis JW, Hoskins BE, nary tract [published online ahead of print Hoefele J, Otto EA, Hildebrandt F: Identifi- This article contains supplemental material online January 15, 2014]. Kidney Int doi:10.1038/ cation of two novel CAKUT-causing genes by at http://jasn.asnjournals.org/lookup/suppl/doi:10. ki.2013.508 PubMed massively parallel exon resequencing of 1681/ASN.2013101103/-/DCSupplemental.

1922 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 1917–1922, 2014 Supplemental Table 1. Targeted recessive genes in murine models with unilateral renal agenesis and respective phenotypes caused by recessive mutations in humans. Gene Mouse Model with Inh. in Inh. in Phenotype if mutated in Renal Involvement MGI short description of murine phenotype2 Lit. Symbol unilateral renal agenesis1 Mice Humans Humans [OMIM#]

“Targeted disruption of this gene results in either embryonic lethality Bag6tm1Pmc/Bag6tm1Pmc following abnormal brain development or neonatal death associated with BAG6 AR n/a none no severe developmental defects in the lung and kidney. These developmental 1 defects are associated with widespread aberrant apoptosis and proliferation.” “Homozygous null mice display neonatal lethality associated with rupture of tm1Taki Ctnnbip1 / the gut, posteriorized neural cell fate within the neural plate, abnormal CTNNBIP1 tm1Taki AR n/a none no 2 Ctnnbip1 craniofacial morphology, and renal agenesis due to arrest of ureteric bud branching.” “Mice homozygous for a knock-out allele exhibit neonatal lethality, abnormal DACT1 Dact1tm1Yegc/Dact1tm1Yegc AR n/a none no embryogenesis, blind-ended colons, and abnormal renal/urinary system.” 3, 4

“Mice homozygous for mutations at this locus display a significant amount of bl bl Fraser syndrome 1 embryonic lethality due to hemorrhaging of embryonic blisters. Survival is FRAS1 Fras1 /Fras1 AR AR yes 5, 6 [219000] variable on genetic backgrounds. Kidney development is severely affected and syndactyly is common.” Bifid nose with or without “Homozygous mutation of this gene results in subepidermal blistering, cryptophthalmos, syndactyly, and renal agenesis.” anorectal and renal 7, 8 FREM1 Frem1eyes2/Frem1eyes2 AR AR anomalies [608980] yes

Manitoba oculotrichoanal syndrome [248450] “Mice homozygous for mutations at this locus display a significant amount of No targeted allele caused Fraser syndrome 2 embryonic lethality due to hemorrhaging of embryonic blisters. Kidney FREM2 AR AR yes development is severely affected and syndactyly is common. Phenotypes of 9 unilateral renal agenesis. [219000] homozygous mutants are indistinguishable from those of Fras1 homozygous mutant.” “Homozygous null mice display neonatal lethality with bilateral agenesis of the ld ld GREM1 Grem1 /Grem1 AR n/a none no kidneys and ureters, oligodactyly, limb skeletal malformations, cyanosis, 10-12 dyspnea, and abnormal lung morphology.” eb eb Fraser syndrome 3 “Homozygous ablation of gene function results in embryonic lethality and GRIP1 Grip1 /Grip1 AR AR yes 13 [219000] blistering skin lesions.” “Homozygous disruptions of this gene result in embryonic lethality. Homozygous mutant embryos fail to form a mature epiblast and die around tm9.1Ref tm9.1Ref ILK Ilk /Ilk AR n/a none no time of implantation. Conditional deletion targeted specifically to 14 chondrocytes lead to reduced cell proliferation, dwarfism, and shortened limbs.” “Mice homozygous for disruptions in this gene usually die by the end of the tm1Lfr tm1Lfr ITGA8 Itga8 /Itga8 AR n/a none no second day after birth. Those that do survive have reduced kidneys and 15 abnormal stereocilia in the inner ear.” tm1Dsb tm1Dsb “Targeted disruption of this gene appears to have no phenotype, but when LIN7C Lin7c /Lin7c AR n/a none no combined with Lin7a or Lin7a and Lin7b results in early postnatal lethality.” 16, 17 Cenani-Lenz syndactyly “Homozygous mice have malformed digits on all 4 feet, some exhibiting LRP4 Lrp4mitt/Lrp4mitt AR AR syndrome [212780] no brachydactyly, some syndactyly.” 18, 19 Sclerosteosis 2 [614305] 1Genotypes of mouse models (MGI allele symbols). 2Obtained from http://www.informatics.jax.org/ AR, autosomal recessive; Inh; mode of inheritance; Lit, literature.

LITERATURE

1. Desmots, F, Russell, HR, Lee, Y, Boyd, K, McKinnon, PJ: The reaper-binding protein scythe modulates apoptosis and proliferation during mammalian development. Mol Cell Biol, 25: 10329-10337, 2005. 2. Satoh, K, Kasai, M, Ishidao, T, Tago, K, Ohwada, S, Hasegawa, Y, Senda, T, Takada, S, Nada, S, Nakamura, T, Akiyama, T: Anteriorization of neural fate by inhibitor of beta-catenin and T cell factor (ICAT), a negative regulator of Wnt signaling. Proc Natl Acad Sci U S A, 101: 8017-8021, 2004. 3. Wen, J, Chiang, YJ, Gao, C, Xue, H, Xu, J, Ning, Y, Hodes, RJ, Gao, X, Chen, YG: Loss of Dact1 disrupts planar cell polarity signaling by altering dishevelled activity and leads to posterior malformation in mice. J Biol Chem, 285: 11023-11030, 2010. 4. Suriben, R, Kivimae, S, Fisher, DA, Moon, RT, Cheyette, BN: Posterior malformations in Dact1 mutant mice arise through misregulated Vangl2 at the primitive streak. Nat Genet, 41: 977-985, 2009. 5. McGregor, L, Makela, V, Darling, SM, Vrontou, S, Chalepakis, G, Roberts, C, Smart, N, Rutland, P, Prescott, N, Hopkins, J, Bentley, E, Shaw, A, Roberts, E, Mueller, R, Jadeja, S, Philip, N, Nelson, J, Francannet, C, Perez-Aytes, A, Megarbane, A, Kerr, B, Wainwright, B, Woolf, AS, Winter, RM, Scambler, PJ: Fraser syndrome and mouse blebbed phenotype caused by mutations in FRAS1/Fras1 encoding a putative extracellular matrix protein. Nat Genet, 34: 203-208, 2003. 6. Vrontou, S, Petrou, P, Meyer, BI, Galanopoulos, VK, Imai, K, Yanagi, M, Chowdhury, K, Scambler, PJ, Chalepakis, G: Fras1 deficiency results in cryptophthalmos, renal agenesis and blebbed phenotype in mice. Nat Genet, 34: 209-214, 2003. 7. Beck, TF, Veenma, D, Shchelochkov, OA, Yu, Z, Kim, BJ, Zaveri, HP, van Bever, Y, Choi, S, Douben, H, Bertin, TK, Patel, PI, Lee, B, Tibboel, D, de Klein, A, Stockton, DW, Justice, MJ, Scott, DA: Deficiency of FRAS1-related extracellular matrix 1 (FREM1) causes congenital diaphragmatic hernia in humans and mice. Hum Mol Genet, 22: 1026-1038, 2013. 8. Al-Gazali, LI, Bakir, M, Hamud, OA, Gerami, S: An autosomal recessive syndrome of nasal anomalies associated with renal and anorectal malformations. Clin Dysmorphol, 11: 33-38, 2002. 9. Jadeja, S, Smyth, I, Pitera, JE, Taylor, MS, van Haelst, M, Bentley, E, McGregor, L, Hopkins, J, Chalepakis, G, Philip, N, Perez Aytes, A, Watt, FM, Darling, SM, Jackson, I, Woolf, AS, Scambler, PJ: Identification of a new gene mutated in Fraser syndrome and mouse myelencephalic blebs. Nat Genet, 37: 520-525, 2005. 10. Woychik, RP, Stewart, TA, Davis, LG, D'Eustachio, P, Leder, P: An inherited limb deformity created by insertional mutagenesis in a transgenic mouse. Nature, 318: 36-40, 1985. 11. Khokha, MK, Hsu, D, Brunet, LJ, Dionne, MS, Harland, RM: Gremlin is the BMP antagonist required for maintenance of Shh and Fgf signals during limb patterning. Nat Genet, 34: 303-307, 2003. 12. Kleinebrecht, J, Selow, J, Winkler, W: The mouse mutant limb-deformity (ld). Anat Anz, 152: 313-324, 1982. 13. Takamiya, K, Kostourou, V, Adams, S, Jadeja, S, Chalepakis, G, Scambler, PJ, Huganir, RL, Adams, RH: A direct functional link between the multi-PDZ domain protein GRIP1 and the Fraser syndrome protein Fras1. Nat Genet, 36: 172-177, 2004. 14. Lange, A, Wickstrom, SA, Jakobson, M, Zent, R, Sainio, K, Fassler, R: Integrin-linked kinase is an adaptor with essential functions during mouse development. Nature, 461: 1002-1006, 2009. 15. Muller, U, Wang, D, Denda, S, Meneses, JJ, Pedersen, RA, Reichardt, LF: Integrin alpha8beta1 is critically important for epithelial-mesenchymal interactions during kidney morphogenesis. Cell, 88: 603-613, 1997. 16. Olsen, O, Funke, L, Long, JF, Fukata, M, Kazuta, T, Trinidad, JC, Moore, KA, Misawa, H, Welling, PA, Burlingame, AL, Zhang, M, Bredt, DS: Renal defects associated with improper polarization of the CRB and DLG polarity complexes in MALS-3 knockout mice. J Cell Biol, 179: 151-164, 2007. 17. Olsen, O, Moore, KA, Fukata, M, Kazuta, T, Trinidad, JC, Kauer, FW, Streuli, M, Misawa, H, Burlingame, AL, Nicoll, RA, Bredt, DS: Neurotransmitter release regulated by a MALS-liprin-alpha presynaptic complex. J Cell Biol, 170: 1127-1134, 2005. 18. Li, Y, Pawlik, B, Elcioglu, N, Aglan, M, Kayserili, H, Yigit, G, Percin, F, Goodman, F, Nurnberg, G, Cenani, A, Urquhart, J, Chung, BD, Ismail, S, Amr, K, Aslanger, AD, Becker, C, Netzer, C, Scambler, P, Eyaid, W, Hamamy, H, Clayton-Smith, J, Hennekam, R, Nurnberg, P, Herz, J, Temtamy, SA, Wollnik, B: LRP4 mutations alter Wnt/beta-catenin signaling and cause limb and kidney malformations in Cenani-Lenz syndrome. Am J Hum Genet, 86: 696-706, 2010. 19. Weatherbee, SD, Anderson, KV, Niswander, LA: LDL-receptor-related protein 4 is crucial for formation of the neuromuscular junction. Development, 133: 4993-5000, 2006.

Supplemental Table 2: Demographics and epidemiologic characterization of 590 families with CAKUT.

Number of families 590 Number of affected individuals 672

CAKUT phenotype Count VUR 269 Renal hypodysplasia 101 Unilateral renal agenesis 80 Duplex system 80 Ureteropelvic junction obstruction 77 Renal ectopia 34 Multicystic dysplastic kidney 33 Posterior urethral valves 30 Ureterovesical junction obstruction 30 Hydronephrosis 25 Horseshoe kidney 18 “Multiple cysts” 17 Suma 794

Origin of affected individuals Count Macedonian 350 German 61 Kuwaiti 50 Indian 49 Albanian 48 Serbian 30 UK 16 Egyptian 9 USA 8 Romani 8 Hungarian 7 Somalia 6 Arabic 6 Turkish 6 Hispanic 4 Taiwanese 4 Jordanian 2 Caucasian 2 Europe 2 Swedish 1 Syrian 1 Asian 1 Bosnian 1 Sum 672 a Sum of observed CAKUT-phenotypes exceeds the number of affected individuals due to presence of >1 phenotypes in 195/672 individuals. Supplemental Table 3. Comparison of biallelic missense mutations in individuals with isolated CAKUT versus protein-truncating mutations in individuals with Fraser syndrome.

Isolated CAKUT Fraser Syndrome M/M M/T T/T M/M M/T T/T Ref. FRAS1 5 (7 a.) 1 (2 a.) n/a 3 (4 a.) n/a 15 (19 a.) 1-6 FREM2 4 (5 a.) n/a n/a 3 (1 a.) n/a 1 (1 a.) 7, 8 GRIP1 1 (2 a.) n/a n/a n/a n/a 3 (2 a.) 9 FREM1 2 (1 a.) n/a n/a 3 (3 a.) 1 (2 a.) 11 (12 a.) 10-13 a., alleles; M/M, number of families with biallelic missense mutations; M/T, number of families with biallelic mutations including one protein-truncating allele; T/T, number of families with biallelic protein-truncating mutations; Ref., References.

REFERENCES

1. McGregor, L, Makela, V, Darling, SM, Vrontou, S, Chalepakis, G, Roberts, C, Smart, N, Rutland, P, Prescott, N, Hopkins, J, Bentley, E, Shaw, A, Roberts, E, Mueller, R, Jadeja, S, Philip, N, Nelson, J, Francannet, C, Perez-Aytes, A, Megarbane, A, Kerr, B, Wainwright, B, Woolf, AS, Winter, RM, Scambler, PJ: Fraser syndrome and mouse blebbed phenotype caused by mutations in FRAS1/Fras1 encoding a putative extracellular matrix protein. Nat Genet, 34: 203-208, 2003. 2. Slavotinek, A, Li, C, Sherr, EH, Chudley, AE: Mutation analysis of the FRAS1 gene demonstrates new mutations in a propositus with Fraser syndrome. American journal of medical genetics Part A, 140: 1909-1914, 2006. 3. Cavalcanti, DP, Matejas, V, Luquetti, D, Mello, MF, Zenker, M: Fraser and Ablepharon macrostomia phenotypes: concurrence in one family and association with mutated FRAS1. American journal of medical genetics Part A, 143: 241-247, 2007. 4. van Haelst, MM, Maiburg, M, Baujat, G, Jadeja, S, Monti, E, Bland, E, Pearce, K, Hennekam, RC, Scambler, PJ: Molecular study of 33 families with Fraser syndrome new data and mutation review. American journal of medical genetics Part A, 146A: 2252-2257, 2008. 5. Ogur, G, Zenker, M, Tosun, M, Ekici, F, Schanze, D, Ozyilmaz, B, Malatyalioglu, E: Clinical and molecular studies in two families with Fraser syndrome: a new FRAS1 gene mutation, prenatal ultrasound findings and implications for genetic counselling. Genetic counseling, 22: 233-244, 2011. 6. Ng, WY, Pasutto, F, Bardakjian, TM, Wilson, MJ, Watson, G, Schneider, A, Mackey, DA, Grigg, JR, Zenker, M, Jamieson, RV: A puzzle over several decades: eye anomalies with FRAS1 and STRA6 mutations in the same family. Clinical genetics, 83: 162-168, 2013. 7. Jadeja, S, Smyth, I, Pitera, JE, Taylor, MS, van Haelst, M, Bentley, E, McGregor, L, Hopkins, J, Chalepakis, G, Philip, N, Perez Aytes, A, Watt, FM, Darling, SM, Jackson, I, Woolf, AS, Scambler, PJ: Identification of a new gene mutated in Fraser syndrome and mouse myelencephalic blebs. Nat Genet, 37: 520-525, 2005. 8. Shafeghati, Y, Kniepert, A, Vakili, G, Zenker, M: Fraser syndrome due to homozygosity for a splice site mutation of FREM2. American journal of medical genetics Part A, 146A: 529-531, 2008. 9. Vogel, MJ, van Zon, P, Brueton, L, Gijzen, M, van Tuil, MC, Cox, P, Schanze, D, Kariminejad, A, Ghaderi-Sohi, S, Blair, E, Zenker, M, Scambler, PJ, Ploos van Amstel, HK, van Haelst, MM: Mutations in GRIP1 cause Fraser syndrome. J Med Genet, 49: 303-306, 2012. 10. Alazami, AM, Shaheen, R, Alzahrani, F, Snape, K, Saggar, A, Brinkmann, B, Bavi, P, Al-Gazali, LI, Alkuraya, FS: FREM1 mutations cause bifid nose, renal agenesis, and anorectal malformations syndrome. Am J Hum Genet, 85: 414-418, 2009. 11. Slavotinek, AM, Baranzini, SE, Schanze, D, Labelle-Dumais, C, Short, KM, Chao, R, Yahyavi, M, Bijlsma, EK, Chu, C, Musone, S, Wheatley, A, Kwok, PY, Marles, S, Fryns, JP, Maga, AM, Hassan, MG, Gould, DB, Madireddy, L, Li, C, Cox, TC, Smyth, I, Chudley, AE, Zenker, M: Manitoba-oculo-tricho-anal (MOTA) syndrome is caused by mutations in FREM1. J Med Genet, 48: 375-382, 2011. 12. Mitter, D, Schanze, D, Sterker, I, Muller, D, Till, H, Zenker, M: MOTA Syndrome: Molecular Genetic Confirmation of the Diagnosis in a Newborn with Previously Unreported Clinical Features. Molecular Syndromology, 3: 136-139, 2012. 13. Nathanson, J, Swarr, DT, Singer, A, Liu, M, Chinn, A, Jones, W, Hurst, J, Khalek, N, Zackai, E, Slavotinek, A: Novel FREM1 mutations expand the phenotypic spectrum associated with Manitoba-oculo-tricho-anal (MOTA) syndrome and bifid nose renal agenesis anorectal malformations (BNAR) syndrome. American journal of medical genetics Part A, 161A: 473-478, 2013. Supplemental Table 4: Comparison of truncating variants in 6,500 healthy individuals in the EVS Server in recessive genes mutated in individuals with isolated CAKUT versus autosomal recessive and autosomal dominant control genes. Note that 6,500 healthy control individuals have heterozygous truncating alleles in the genes recessively mutated in individuals with isolated CAKUT (upper panel). Note that the number of truncating alleles in these genes compares to the number of truncating alleles in other genes mutated in known recessive disorders (middle panel) whereas truncating alleles in genes mutated in autosomal dominant disorders are unusual (lower panel).

AR genes in isolated FRAS1* FREM1 FREM2* GREM1 GRIP1 ITGA8 CAKUT Human Disorder Fraser 1 MOTA Syndr. Fraser 2 none Fraser 3 none

# Var. # Ind. # Var. # Ind. # Var. # Ind. Var. Ind. # Var. # Ind. # Var. # Ind.

Stop gained 4 5 8 10 4 4 0 0 1 1 1 1

Stop lost 0 0 0 0 0 0 0 0 0 0 0 0

Frameshift 3 3 3 4 5 5 0 0 0 0 1 1

Sum Trunc. 7 8 11 14 9 9 0 0 1 1 2 2

AAs #1 4012 2179 3169 184 1076 1063

Control AR NPHP1 NPHS1 NPHS2 CFTR ATP7B GALT IDUA FANCA Known Human Cystic Nephronophthisis 1 Cong. NS 1 Cong. NS 2 Wilson Disease Galactosemia MPS I (Hurler) Fanconi Anemia A Disorder Fibrosis

# Var. # Ind. # Var. # Ind. # Var. # Ind. Var. Ind. # Var. # Ind. # Var. # Ind. # Var. # Ind. # Var. # Ind. Stop gained 2 2 5 5 0 0 12 41 7 12 0 0 2 13 3 3 Stop lost 0 0 3 3 0 0 0 0 0 0 0 0 0 0 0 0 Frameshift 0 0 0 0 1 234 16 60 3 5 1 1 2 54 4 33 Sum Trunc. 2 2 8 8 1 234 28 101 10 17 1 1 4 67 7 36 AAs # 733 1241 383 1480 1465 379 653 1455

Control AD EYA1 SALL1 PAX2 ROBO2 HNF1B CREBBP WT1 APC Known Human Renal Cysts + Familial Aden. BOR Townes-Brocks Papillorenal VUR 2 Rubinstein Taybi Denys-Drash Disorder Diabetes Poliposis

# Var. # Ind. # Var. # Ind. # Var. # Ind. Var. Ind. # Var. # Ind. # Var. # Ind. # Var. # Ind. # Var. # Ind. Stop gained 0 0 0 0 0 0 0 0 0 0 1 1 0 0 1 1 Stop lost 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Frameshift 0 0 0 0 3 1742 1 1 0 0 1 27 0 0 1 2 Sum Trunc. 0 0 0 0 3 174 1 1 0 0 2 28 0 0 2 3 AAs # 592 1324 431 1394 557 2442 517 2843 1 Amino acid number derived from the transcript mentioned in Table 1 or longest transcript/isoform. 2 3 of 3 frameshift variants in PAX2 are C-terminal after AA 397. Trunc., truncating variant; AD, autosomal dominant; AR, autosomal recessive; cong., congenital; Ind., individuals with truncating variant in the EVS Server; Var., number of different truncating variants in the EVS Server.

*Note that in 6,500 individuals of the EVS-database, there are 8 and 9 individuals with protein-truncating variants in FRAS1 and FREM2, respectively. The presence of rare protein-truncating variants in a large cohort of healthy individuals is acceptable for recessive genes but highly unusual in autosomal dominant genes. Hence, FRAS1 and FREM2 are unlikely to be autosomal dominant genes. Supplemental Table 5. Exon-flanking oligo nucleotides primers for 12 CAKUT candidate genes and median coverage of targeted regions. Primers highlighted in red failed to generate PCR-products.

Median Gene Amplicon Sequence Forwar Primer Sequence Reverse Primer Length Coverage BAG6 BAG6_E25 CCTTGTGTGCTTGCTGGG GTCCCCTGATGAGGAAGGG 200 2771.19 BAG6 BAG6_E24 TTGATCCTCTTTTCCCTCCC ACCCCTCCCACTAGACCATC 227 1424.92 BAG6 BAG6_E23 GGCAGCTGAAAGTCAAGGAC TTGATTCCAGGCATGACG 219 2966.93 BAG6 BAG6_E22 TCTGAAGCCTGGCTCTTCC GCCATGACCCACTGGATTAC 214 3488.00 BAG6 BAG6_E21 ATGGGGTCAGGGTACTTGAG GTGTCAGATGGCGGGAAG 240 1630.87 BAG6 BAG6_E20 AGGTGGTTGAGGGTTTTGTG AAGTACCCTGACCCCATCG 229 3128.62 BAG6 BAG6_E19 CATTCCCTCCTTGTATTTTGC GCTAAGGATCTGGGGCTAGG 182 3617.43 BAG6 BAG6_E18 TTTTGGGATCTTTGTGTTATCTTG CCTGCACATGCAACAGGC 184 3604.84 BAG6 BAG6_E17 TGGAAGTCTTGGGAGGACTG AAGACTGGGAGTGGAGGAGG 183 4891.03 BAG6 BAG6_E16 TGCTAACATCTGCCCTTGTC TGCAAAAGAGGAGAGTTCTGG 237 1898.34 BAG6 BAG6_E15.2 GGTCTTGAGAGCCTGTCACC GGAGCAAGCCAAAGCATC 202 3972.00 BAG6 BAG6_E15.1 TGTGTCTTCATTCTCACCTCAC ACACCCTGCACCACTGAG 224 587.61 BAG6 BAG6_E14.2 TCAACCCTCCATGGCTGATC GTGAATGGCAAGCCAGC 196 5501.52 BAG6 BAG6_E14.1 ATGCTCAGCCCAGCTTTC CCCTAGCAGGTTCCCCAG 219 3764.07 BAG6 BAG6_E13 GCATGGAAGGGCAGGTC AAACTCTGAGTGGGGAAGAATG 163 5991.61 BAG6 BAG6_E12 TTCGGTCTGTCTCTTCTGCC TCTCTCCCTAGACTGTTACGCAC 188 3968.48 BAG6 BAG6_E11 TACCTCAGGGCCACTGTCTC CAGCCCTTCCCTTTCTCTAC 169 4737.66 BAG6 BAG6_E10.1 ATGACAGGAAATGGGACTCG ATGAACCTCCCTCATCATGC 283 1384.16 BAG6 BAG6_E10.2 GGAGGAGACTGGAAACATGG GGAGGTGCCTCTGCATTG 195 4515.49 BAG6 BAG6_E09 GACTCCAGCTTGATATTTTCTGC TCTCCCTCAGGCCAGACTC 280 1491.86 BAG6 BAG6_E08 AATCTGAGGAAGGCTTTGGC CTGAGGAGAAAGGGCAGGG 211 3651.19 BAG6 BAG6_E07.2 GGAGCCAGTAGCCTTGAGC ACTTAGGATTCCCCACCCAC 217 3757.24 BAG6 BAG6_E07.1 GCAGTCTTACCCTGCCTTTG CGCTCCTCCACTTCTTCTGC 201 3890.79 BAG6 BAG6_E06 ATGGCTGGGACCTGATTTAG TATGAAGGCCAAACCCTGTG 264 1805.94 BAG6 BAG6_E05 CCAGAACAACTTCCCTTACCC ATCTCTCAGCCAGGTCCACC 164 5309.60 BAG6 BAG6_E04 ACCTGTTCATACTATGTTTTGGTG CTACCCACAAAAGCCCTCC 279 1382.85 BAG6 BAG6_E03 ATTCCTGTGCATGCCTCTTC TAGCCCCAACCTCTGAACTG 183 3363.69 BAG6 BAG6_E02 TTTCGGGTACACTCTGGTCTG AAAAGCTAAAGGTGTCTTCCAG 211 2771.87 CTNNBIP1 CTNNBIP1_E06 TCGGAAGTACTTTGTCCTCAG CAGATCTCTTGGCCCTCAAC 246 2200.64 CTNNBIP1 CTNNBIP1_E05 AGGTGCCTTTCAGGGAACAG GCTACGCTTTCTAAGGGAACC 229 2281.28 CTNNBIP1 CTNNBIP1_E04 TTTGATGCTGTTGCTTGCTC GCCTGACACCCCACAGG 169 4551.61 DACT1 DACT1_E01.1 GGTGACGGCTCTCGCTG ACCAGCAGCTCTTGGCG 237 0.00 DACT1 DACT1_E01.2 GCGAGGCAGACACCGAG AGCCGTGCTCCACCTTC 272 0.00 DACT1 DACT1_E02 CACCCCTGTATGTTCATGTTTC GTTAACCACAAGGGGCTGAG 242 1898.44 DACT1 DACT1_E03 GACATGATGTTAATTCCAACGG GCCAAATAACCTTTTAAGGGC 263 1243.78 DACT1 DACT1_E04.1 TTCATTTCTTCAGAGTGTACCTTTAAC CCCGTCAGACAAAGGAGAAAC 269 1592.39 DACT1 DACT1_E04.2 TCGCTATCCCAGTCCACTTC ATGGGAAGCAGACCACAGAC 193 924.03 DACT1 DACT1_E04.3 AGTTCCTTACCGTCCCCAAG GTTCACACTGTTGCCCTGTG 222 631.57 DACT1 DACT1_E04.4 AACCAAGAACCAGCGTGAAC TGGCAGGTGCTTACTCTGAAG 290 1289.17 DACT1 DACT1_E04.5 CCTCAGGCGCTGCCTCC TAGACAGCAGGGGAGGAGTG 253 1917.14 DACT1 DACT1_E04.6 CGCCGCAGGAGAACAAAG AGGCTGGAGTTCTTCACGAC 265 2268.66 DACT1 DACT1_E04.7 TGGTCAAGGCCCAGTTTATC CGGGAACACCAGCCTCGG 270 991.79 DACT1 DACT1_E04.8 AGTGTCGCTTCCCAGATGAC GGGCCTCTTCGTAGGAAATC 240 556.22 DACT1 DACT1_E04.9 CACAAGCGAACTGACTACCG CTCACACTCGACTCGCTGTC 275 1704.55 DACT1 DACT1_E04.10 TTACACCACCAACTGCTTCG TCACTCAAACCGTCGTCATC 272 1960.18 DACT1 DACT1_E04.11 CCGCTTTCGGTCTGGCTC AAAGGCACTAGCATCCATACG 216 2240.32 FRAS1 FRAS1_E01 TCTTGGATGCTGAAGGCTG AAACTGCACGATCACTCACAC 202 573.47 FRAS1 FRAS1_E02 TGGAAGAAGCTCATTTTCCTG TCACTCAGGAACTTCAATCAAGC 226 707.60 FRAS1 FRAS1_E03 TGGGACTATTGATGGTGCAG AACCTGACCCTTCACTGACTTTAC 246 569.29 FRAS1 FRAS1_E04 TCCCATCTGTTGTTGTCACC TCTGCAGACACAGAATCAACC 264 331.09 FRAS1 FRAS1_E05 ATGAAAGCCTGTGTTTTGGC GGGTCCCCTTCTACAAGCTC 245 393.50 FRAS1 FRAS1_E06 AGTCAGCCCTGGGATCAAC TGCCTGCCATAATCTCAGAAG 274 339.98 FRAS1 FRAS1_E07 GATGGATTGTTCCTGTTCTGC TGGCAGAGTCAGACATGAGG 221 867.52 FRAS1 FRAS1_E08 GACATTTGATTTTCCATGTTCTTC CCCAGAAAAGAGCTTAAACAAG 188 859.51 FRAS1 FRAS1_E09 CTTGGGTGTTTCACCGTCTC AACGTTCCAAATGGAAATGC 282 179.22 FRAS1 FRAS1_E10 TAACAGTGTGCCAGCGTTTC AATGTTCCCTCATTAGATTAATTTTG 273 93.26 FRAS1 FRAS1_E11 AAAGTTGCATGTTCCTTGGG AGTGCCATCAAATGTGGCTG 268 209.00 FRAS1 FRAS1_E12 TTCTTCAGCAGCAAGCTCTC GGGGAAGAGGATCTCTGAGC 290 225.69 FRAS1 FRAS1_E13 GGAGTTCACAGAGTTCTCCCC ACCTCTGGGTTGTCCACTTG 214 1010.63 FRAS1 FRAS1_E14 TAAGCACTGGCATGGGG GGGAGCTGTCTTGGTAGCTG 261 579.62 FRAS1 FRAS1_E15 ACCAGTCCTTTCTGCCGTAG AAAGGACACGTCAGCATCG 229 669.14 FRAS1 FRAS1_E16 TCTTTTGCTCACTATTGCCTTTC GATGATGATGATGGTGACCC 220 876.71 FRAS1 FRAS1_E17 GCACTTTATTCACCCATGTCC TTGTGGGATAGCCTCTCAAG 236 562.07 FRAS1 FRAS1_E18 TGATCCTTGGATTATTTTCTGC AGATAAGCCCAGAGGCTACC 263 86.00 FRAS1 FRAS1_E19 TTTCCTGATTGTCTCCTTTGC ATAGGCAATGTGCCTCAGC 212 548.99 1

FRAS1 FRAS1_E20 CATGCTGAGCTGCTAATTCC CCAGTGATTTCCGAGGAGTC 228 986.87 FRAS1 FRAS1_E21 CTTCTCTGTGCTCCCCTTCC AAGAGAACCTCAGGTGGCAG 231 624.03 FRAS1 FRAS1_E22 GACATCATGGTTTCTGTTGTGTC GAAAGGAGAGAAGGTTCATTGG 218 583.90 FRAS1 FRAS1_E23 CATCTGTCTAGGTTTGATCAGTGC AAAGTGGTATCACTGTGTAAAAGAATC 277 556.39 FRAS1 FRAS1_E24 TCTGAGGGTGGACAGAAAGG ACAAATCAGAGGTGGCCAAG 249 312.02 FRAS1 FRAS1_E25 AAGCCACACAGATTCCTGATG CTGCCCTCACTTCTTTCCAG 226 287.74 FRAS1 FRAS1_E26 CTGGTGTTGCCTTGATTGAG TTCACCAGACCATCTGAGG 220 516.80 FRAS1 FRAS1_E27.01 CATAAGCTTGACCTTTGGATTC TCCAGCTGAACCTCTTTTCC 231 1009.33 FRAS1 FRAS1_E27.02 CCTCCTGAATGTCCAAGACC GATGGAAAGGAGGCAGGAAG 235 958.21 FRAS1 FRAS1_E28 TTTTCTTATTCCTTTCTTTGGGAC AAACCTAGATTTCGTGCTTGAG 186 175.39 FRAS1 FRAS1_E29.01 TGTCCTTTCTTTCCCTCTTAACTG GCCTCTAGAGAGTTCATCCAGC 262 256.92 FRAS1 FRAS1_E29.02 CCGGCAACCCCTATCTATC TGACTACCCATATCTTAACTTTGTGAG 264 382.21 FRAS1 FRAS1_E30 CAACAACAGAACTGGATAACTTATGTG GCTGAATGCAACGATGAAAC 287 160.77 FRAS1 FRAS1_E31 CACTAACTATGTGGCCTGTGC GAGCGATTGGATTATTGGATTC 265 433.48 FRAS1 FRAS1_E32 CCACGTACTAACATGCTCTGC TGCTGTGTGCCTCCTCTAAG 231 843.69 FRAS1 FRAS1_E33 TGACTAATTTAAAAGCCCAGAGC TCACTCAAAAGATCATTTACTGCAC 258 75.00 FRAS1 FRAS1_E34 CCCACCAACAAGAAGAGCAG TGCCTCAAAGCAGAGAGGAG 259 369.69 FRAS1 FRAS1_E35 TTCCTAACCTCTGCCCTTTG TGATGGGAACAAAAAGAAAGAAC 200 427.91 FRAS1 FRAS1_E36 AAGAGGACCAGACTACAAGTTG ATCCCATTTCCAAAGCCATG 272 162.65 FRAS1 FRAS1_E37 TCTCACCACGCTTCCTTCTC TCCAAGCAGGTCAGTGACAG 197 336.00 FRAS1 FRAS1_E38.01 GAAAAGCATTATTCCCAGAAGC CGGTCTTCTGACAGGGACAC 211 1372.25 FRAS1 FRAS1_E38.02 CATGATGGTTCCTCTACCCG CATCAGAAGATGCGGCG 249 1341.42 FRAS1 FRAS1_E39 CATGTCCTGCCTCATGTTTG AACAGAGCTACAACCAGTTGCTG 258 317.21 FRAS1 FRAS1_E40 TTACCTGGCTTGTCATCGTG CATTACTACGGATTCCAGCTCTC 278 173.20 FRAS1 FRAS1_E41 TGTGAAATCACCAAGGACTCTC CCCTCCCTTCTCTCAAGGTC 224 270.91 FRAS1 FRAS1_E42.01 TTCAGCCTAGTAAGCAGGAATTG TCCAACTTCCAAAGTCTTTACCTC 274 245.60 FRAS1 FRAS1_E42.02 ATGAAAGCATTGAGCCAACC ACTCAAGCCTTCCCCAAATG 283 211.80 FRAS1 FRAS1_E43 TGCATTTTCTTTCTTCTTAAATATCTG TGTTTATTCTAACAGCCCATCG 277 44.30 FRAS1 FRAS1_E44.01 CCTCTGAGGCACTTGACATTC AGGCCATCAGTGAGGGAGAAC 290 224.56 FRAS1 FRAS1_E44.02 CTGGCATGGTCGTGGATG TGGAAGAAAGTACTGCATACTGG 221 821.22 FRAS1 FRAS1_E45 GGAAATGTTGGGGAATAACC GTAGGTGCAGTCCACGGAAG 281 355.38 FRAS1 FRAS1_E46 CAGAAAGCACTAATAGCAAATCTTG GAGCCTCGAGGTGAACAAAG 250 271.00 FRAS1 FRAS1_E47 GAGAAGATCCCATTTCAAATCAC TGGCTGCCCTCAATACTTAC 287 193.77 FRAS1 FRAS1_E48 GAGAGTATGACACTTCCCTAGCC ACTCTGCCTACTCCACTCAAAG 283 198.54 FRAS1 FRAS1_E49 TGGGTAGGTGCTAGGGACAG AGAGAACCCATGGGAAGAAG 221 532.57 FRAS1 FRAS1_E50.01 CAATTGGGCATCACTCACTC GGGTTTGTCCCATCAGAAAC 271 705.58 FRAS1 FRAS1_E50.02 CCATCGAGCGAACCAGC GTGCTAGGCTACCTCCTCCAG 219 921.07 FRAS1 FRAS1_E51 CTTCAGGTACTTGTGAATGATGAG TGGTGTGAACATGACAGAGG 195 801.54 FRAS1 FRAS1_E52 TCCTGCTTTATTCCCACCTC TCTCCCATTTCTAAGTTCCCAG 235 417.97 FRAS1 FRAS1_E53 CATCCCTGGGTTAATTCACAG TGATTTCTCAAGAAGGAAAGCAC 237 441.66 FRAS1 FRAS1_E54 TTTCATAACCATGGGGCATAG ATTGCTTGCAGCCATGTCAC 282 64.76 FRAS1 FRAS1_E55.01 CTATGATGCCCTGGGGAAG TAATGTGGGTTCATCCTCGG 251 733.33 FRAS1 FRAS1_E55.02 GAATTCACCCAGGCGAAG TGGGAACCAAATGAGTTAATAAGTG 263 583.39 FRAS1 FRAS1_E56.01 GGAGGGACCAAGCTCATTAAC GACATCACAGGTGGACATGG 257 1303.41 FRAS1 FRAS1_E56.02 TCCCTAAGTCAGCTATGGGAAG GCCAAGGCAATCTCAAACTC 184 2155.25 FRAS1 FRAS1_E56.03 TGTCCACCTGTGATGTCATG CAGGAGACAGGTGAAGGAAAC 236 1002.94 FRAS1 FRAS1_E57 AAACCTCGCTGATTTTCTGG TCACAAACCGACCTGTCAAC 272 295.73 FRAS1 FRAS1_E58 CACCAAAGACAGGCAAGTTG GACCTACCAGCTGGCATCC 276 269.76 FRAS1 FRAS1_E59.01 AACCATGCTTTGGAATCTGC TCACTGATGACTCATAGCTCAGG 271 387.44 FRAS1 FRAS1_E59.02 CTAGTGCCCAGCATGCAG TCTCATCTTTCAGCCACCAC 242 545.70 FRAS1 FRAS1_E60 AGCAAATCACTGATATCTTGCTG TGTCCCCTCAACCTAAGAGTAAC 259 437.56 FRAS1 FRAS1_E61 ACTGGCTCTTGTTTTCCTAACC AACCCACAGATTGAGGTTGG 266 363.22 FRAS1 FRAS1_E62 AAGCTGCACTCTCTTGGGTG AAACAGCAATCCTTTGGGTC 287 211.72 FRAS1 FRAS1_E63.01 GCTTAGTTCTCTGACCTTGCC ACTTCCCAGCTGAACTGCAC 252 591.63 FRAS1 FRAS1_E63.02 CCCTGCGACCCTCATTTC GCATGGACCATAAGGAATTTG 275 586.78 FRAS1 FRAS1_E64.01 TGAACCAGATCATGTAAGGAGC CCTGGAAGCCTCTGGATGTC 242 1316.65 FRAS1 FRAS1_E64.02 GACAAGGTGGGCCATGTG TCCTGGGAAACACACAAAGG 246 1257.62 FRAS1 FRAS1_E65 TTGACCATTTATAGCAGTCAGC CTGGGCTAGCATGGAAAGAC 258 283.33 FRAS1 FRAS1_E66.01 AGTAGGGTCTCAGGTGATTGAAAG ACATAATAAGCATCAAAGGTCCAC 281 347.97 FRAS1 FRAS1_E66.02 TTCCTGGATGATGTGGTCTATG TTCAGGAACAAACAAGGTTGAC 276 313.07 FRAS1 FRAS1_E67 AAATGGAACCAAGATGTGGTG ATCTTGCTTGAGTGAAGCCC 289 163.20 FRAS1 FRAS1_E68 CCATGCCCTAGCATTTTCTC CATGGATGTATAGCAAAATGTTAATC 270 41.89 FRAS1 FRAS1_E69 TTTTGTGGGGCTCTACCAG TGGAAAACCATTCAGCTCTACC 290 95.65 FRAS1 FRAS1_E70 GGGTAAGCCAGCAACCTATC CCATGTTGATTTGGGGGTTAC 274 87.45 FRAS1 FRAS1_E71 AGCTGACCCTGCTCTCAAAG AGAGCCTCTTTCTGCAGGTG 248 460.06 FRAS1 FRAS1_E72 AAGGGAGCATGTTTAACCCC GAAGTCATAGCTGAGGAAATCTG 256 36.93 FRAS1 FRAS1_E73 CACCTACCAATATAGTGGAACCATC TTGGTCTTAAGCTTCTGTTCTTCTC 245 159.92 FRAS1 FRAS1_E74.01 TCTTCAGAGGTGGTCTGTGG GCTCTACCAGGTCCCTTCG 181 2098.43 FRAS1 FRAS1_E74.02 GTCCAGCGCTCTCTCACAG TGTGACAGGGACGCAGCTAC 185 851.42 FRAS1 FRAS1_E74.03 AATGGCACCAATATGAAGTCC CAGTGCAGTAGTGTCTATTCACG 283 388.72 FRAS1 FRAS1_E74.04 GAAGAAGAAGCCCGCAGAG AAAAAAATACACATAGGTCTCCTCC 264 496.51 2

FRAS1 FRAS1_E74.05 TGCTAAAGTCAAAAGACTGAATC GCTGTCTGCCACTCAAAGC 190 445.48 FREM1 FREM1_E38 ACGCAATAAAAGTAATTGGTACATTC TGACAAACTCCAGGTGGC 281 123.90 FREM1 FREM1_E37 GCAGAAATGTTAACTGCTTGTTG AAGGAAGTAGCAGGGTGGTG 220 487.41 FREM1 FREM1_E36 TCCTTATGCTGAACCAACTCC CCGCTTCCATGAGCAAATAG 254 279.89 FREM1 FREM1_E35 TTGCTAAATTGCCTTTCCTTC GAATAAAGGTGCTTGGCTGC 192 645.01 FREM1 FREM1_E34 GAATTGCTCATTTTTTTGTTTG GCAAACAACTTGTTATAAGGTGAG 289 90.77 FREM1 FREM1_E33 TTGGTGGTTTTACCCTATTGC AATAGCTTCATTAAATACTTGCATCC 187 425.62 FREM1 FREM1_E32 CTGCCATCTTGCATGCC TCATTTCCCAGAAGGTCCC 289 180.31 FREM1 FREM1_E31 CCTAGGTGATTGAAATAGCAATTAAAG AGCACGTTGGCTGTTGAAG 280 196.44 FREM1 FREM1_E30 TTTAAGAGCATGAGTTTAGTCCAG TCTACAATCTCCAGACATGCCTAC 250 116.70 FREM1 FREM1_E29 CCCTATAGGCATATACTATGCATTCTC TGAAATTTCCTTGGTCACTCTG 290 58.58 FREM1 FREM1_E28 TATTTAATGTTTTGTTTGATTACCC CATTTCACTTTCCTAATATTCAGATAC 271 5.40 FREM1 FREM1_E27 GAGGGGCCTTTGGACAAC GGCAATTGTAAGGATTAAGGAGG 276 192.23 FREM1 FREM1_E26.02 CACCTTCCTCTTGGTTCAGC TTCACATCTCTGGCAAATGAAC 272 368.58 FREM1 FREM1_E26.01 AGAATCCATGCTGAAGGCTG CCCACAGGTAGAGCTGGC 272 418.48 FREM1 FREM1_E25.02 TGCTCTATGTCATCACCTCCC CCAAAGAAGGGGTTTGAAAAG 194 981.65 FREM1 FREM1_E25.01 TCTGCCTTTGCCTTGGAC TTGGCTGAAGTTTGTAATGGG 263 569.63 FREM1 FREM1_E24 GCAGCTGACTAACTCTGGCTC CCCAGTAAACCTTGGTAGACG 254 493.93 FREM1 FREM1_E23 TCAATATAAGTGTCCCCTACTAAAGG ATTGAGAAGATTTATACCTGCTACG 274 62.00 FREM1 FREM1_E22 TTAAGACATTATCTGTGACATATGTC GAATTGTATAGAAATCTGAAAGGG 266 14.00 FREM1 FREM1_E21 TATGCATTGTTGACTTTTCTCATG ACAAATGCATGGAAGTGGAAC 290 192.59 FREM1 FREM1_E20 GGAAAAATTTTTTTTTTATTTATCTG GATAAAAGAGAAATGGAACATTTG 282 0.00 FREM1 FREM1_E19 GGATTTCATGCTAAGTTGTAATTGTG AGGGAGTCATTGCTGGTGAC 264 220.39 FREM1 FREM1_E18 GGCAGGTTTCAGCTATAGTTGTG TGAACCACAGGTATGACACTAGG 287 215.98 FREM1 FREM1_E17.02 TGCTACTGATGTGGACAGCG TCTCATGTTGCTTGCATTCC 196 1441.11 FREM1 FREM1_E17.01 GAGCAATGGATTACCTGTCAAAG TGACTCCAGCTCTCCTCACC 268 858.73 FREM1 FREM1_E16 AGTGAACACATGGCCAAAGC TATGGCAGCACAAAACAGAC 252 510.75 FREM1 FREM1_E15 GGCTTTGAATGTTTCCTTCC CTAAAGCATGTAAATAAAAAAACCATG 282 113.93 FREM1 FREM1_E14 CAAGTCAGCACCCACATATCC AGTTACCATTTCTAGTAGGTCTTCAAG 287 234.21 FREM1 FREM1_E13 TTCTTCTCACAATGAAGTCTTTCAG TCTAAGGGCACACTTGCATC 245 234.66 FREM1 FREM1_E12 TTTGGTATTTCAAATTAATGGTACAG GAACTAGTAGCCCAAATGGTGAC 272 60.98 FREM1 FREM1_E11 CAAAAGTATGTGGTGTTTTATGGAC ACCTGTGCACAAGACTTTGG 221 582.86 FREM1 FREM1_E10.02 AATTCCCCATCAACGTCTTG CATTCAAATGATCTTAACTGCCC 245 842.37 FREM1 FREM1_E10.01 TGCTCAATCTGCTCCATCTC CCCTCCTCCAGTTCAATCAC 246 716.93 FREM1 FREM1_E09 GTGTATATGTGCCTCATTTTCTTTATC CTTTTGGATTCTTTGCTAGGTTACC 241 353.46 FREM1 FREM1_E08 TTCAGTGTGTGGCTCCTTTG TTCCCTTCAGGGCTATGTTG 191 490.69 FREM1 FREM1_E07.02 TCCTTGACTACATCAGTTCTGGAC GCTGGAAGCTTTGTCTCTCC 240 452.94 FREM1 FREM1_E07.01 TTAGCCCTGTATGATATCACCTC GAGTCACATAGCCCTGGAGC 278 274.86 FREM1 FREM1_E06 GTGACTCTCTTAATCCAGTGCTG GGGCACCCACACATAACC 259 321.83 FREM1 FREM1_E05.02 TGAATTCAATGGCTTGTCCC CCCAGAAAGGCATTAAAAGAC 225 746.51 FREM1 FREM1_E05.01 CCAATTAATATTGCTTTTGCCC TCCAGGCTAGCCATCCTATC 216 916.71 FREM1 FREM1_E04 TCTCCTGGCAAAATGTTAAATC TGGTTACTTGGCAGCAAATG 209 803.42 FREM1 FREM1_E03 GAGTTGGGCCCTGTCAGC TGAACAGAAAATCGCAGATAGG 280 193.19 FREM2 FREM2_E01.01 CTCAGGCTGACCTGTCCAAG CCAGCACTATGGCCTCCTC 259 400.06 FREM2 FREM2_E01.02 GCTGTCCCCTGGTCTCG GCCCAGGTGAGAGTAGCG 262 303.56 FREM2 FREM2_E01.03 CTTCCCGTGCGACTTTG ACTCCTCTGTCTCGGGCTG 269 347.95 FREM2 FREM2_E01.04 TTGTGACTCGGAACTTGCC CGGCTGTGTGGCGATAG 270 301.61 FREM2 FREM2_E01.05 AGCTTTCCAGGAACTAGGCG AGCATGTCTGGGGTCAGG 258 560.05 FREM2 FREM2_E01.06 AAGCCCAGTTTCGTGGC AAAGAGGCGCTCCTGGTC 270 402.62 FREM2 FREM2_E01.07 GGCTCCTGAAGATTGCCTAC TCGCTGATGACCAAGTTTTG 250 142.86 FREM2 FREM2_E01.08 CTATGAGGGTCAGTCTCGGC GCGAAGCACCAGGTTGTC 259 229.17 FREM2 FREM2_E01.09 CAGCATGATGACAGAGACGG ATGCCCCGTAGTTAAGAAGG 270 521.57 FREM2 FREM2_E01.10 CCTGAGTGCAACTGACATGG TGTGAACTGGTCTGTGACTGG 265 431.93 FREM2 FREM2_E01.11 AACAGAGGGCAGGCTGTTC TCTCGGTCATCTGTGTCCAG 267 436.41 FREM2 FREM2_E01.12 AATCCCAGCTCACACCACTG AACTGGAACTGGGCCACTC 268 237.34 FREM2 FREM2_E01.13 GACCCCCGGGTCAAGAAC CTCTCATGTGGCCATGTTTG 276 268.45 FREM2 FREM2_E01.14 AGAGTTGCACGTGAATGATG CTGGCCGGACATTTACTCTG 257 410.73 FREM2 FREM2_E01.15 CCCTGACTGATAGCTGCTCC CTGCTGGAATGCCATTGAC 279 260.81 FREM2 FREM2_E01.16 TGACTTTCCTCTTGGAAGATCC GCTATCAACAGGCAGGATGG 251 395.93 FREM2 FREM2_E01.17 TGGTGGCAATACTATCCAAGG TATGGCAATCCCTGCTCTTG 265 310.06 FREM2 FREM2_E01.18 TTGAAAACATTTCTCCAGCAC GACTCATGCCTTCCATCACC 273 263.63 FREM2 FREM2_E01.19 TTCCCACCAATGATGAACAG AACTGTCTTCCTGGGTCTCG 279 247.68 FREM2 FREM2_E01.20 TGGTCGAAAGCTTCACCTTG AAACCAAAGATTTGTCTTCTGAATC 279 256.01 FREM2 FREM2_E01.21 GGACTAGAAATAGAAATTGGGGATAC ATTAGGTCCCGAATGCCCTC 251 387.20 FREM2 FREM2_E01.22 CCCAGGATGAAGTAGACAGAAAC GCCCTGGTGATGGTAAAAAC 277 262.41 FREM2 FREM2_E01.23 AGCACTAGTGACTTGAACAGTCC TGTCCACATCGCTAATGGAG 259 486.20 FREM2 FREM2_E01.24 AGTCACCGATGGACGTAACC TGCTTGGTAAAAACCATGACAG 255 510.29 FREM2 FREM2_E01.25 ACCCAGGTGCCTATTCATGG TTATTCACTGCGATTTGGGG 269 405.34 FREM2 FREM2_E01.26 GATCCAGGTCTTGGCTGTTG AACAAGGATAACCAAGAAAAGAAAC 275 258.74 FREM2 FREM2_E02 CATAATGAATCATCAATTTTACTCTGG CTCTCCTTGCGGTCACTACC 190 1550.14 FREM2 FREM2_E03 TGAAGATCACTCATTCAAGAAAGC AATCACTGTAGAATTATAAACCTTTGC 275 6.00 3

FREM2 FREM2_E04.01 TCCAAGCGAAAATGAAACTAAG TCAGACTGCTCATGCTCCC 189 1445.04 FREM2 FREM2_E04.02 AGTTCAACCCAGGCCAGAC CATAAACCAGTTGACGATGGC 189 1695.20 FREM2 FREM2_E05 AATGCTTGCAATTGTGTTTTC TCATGGTAAAATGTTATGGCTCC 243 243.23 FREM2 FREM2_E06.01 TGCTGTGTATGAAAATAATTGTGTC GCTCAGAAGGACATGGAAGG 263 386.77 FREM2 FREM2_E06.02 GGGAGAAACTGTGTCGGATAG TCCTGATGTTGATCTTCCCG 203 904.91 FREM2 FREM2_E07 TGGAAGCATGTCAATAGAGTTTG AAAAGAGCCGAGTGGGAAAC 250 363.06 FREM2 FREM2_E08 CCAACTGAATATTTTTTTG TATGATTTCAATTATCATTG 288 0.00 FREM2 FREM2_E09 GAAATTTCTGTTCACTTTAATCCTTTG TGAGTAAAAATTCGGCTTCTTC 290 191.38 FREM2 FREM2_E10 TCCTATGGGATGTGATTGACC TTGGCTGGATTTTAGTTTCTAATTTAC 262 430.05 FREM2 FREM2_E11 TTTGTCTTTGTTTCCCAC GGCATGATCCATCAGGAGAC 249 218.74 FREM2 FREM2_E12 TTAAATCTGTGATGTTAC CATGAATTTACATACACATTAC 188 0.00 FREM2 FREM2_E13 GCCAGGGATCGTGAGTATTG TTTCCTCAATTTTGTGAATCTCC 277 192.73 FREM2 FREM2_E14.01 AGAATGCGTTCCACCTGTTC GCCAGGCTGAAAGTATATGGAG 268 677.37 FREM2 FREM2_E14.02 CCAGCCCTATGAGAGAAGTGG TGATCTTCAGGGGAAAATGG 211 982.12 FREM2 FREM2_E15 CCTTCTTTTCGCATAAATATGGTC TTCCATGGATTTGCTTACCC 202 480.87 FREM2 FREM2_E16.01 TTTAAAGTGTTCAAAATTCTTGGC AGCGCAAGGTAGTGGAACC 289 256.29 FREM2 FREM2_E16.02 GTGAAGACTCATTATGGTTTCTTG CCAATTGGTTTATCCAGTGACC 266 396.51 FREM2 FREM2_E17 TTAGGCCCACTTAGTTAACACTG AAACTATAGAATTTTCCAAGTCTTCTG 284 162.31 FREM2 FREM2_E18 GGAAGGAATCTGTACATAAGGGG CGCTGAAATGTGTGAAGATG 250 164.55 FREM2 FREM2_E19 ATGCACTCTTTTAATTGAACCAC TAATCAGATGGCAGCAGCAG 237 249.72 FREM2 FREM2_E20 GCTGCTGCCATCTGATTACAC ACAAAGTGGGATTCTGCAAG 248 312.21 FREM2 FREM2_E21 TTGTGCACCATCAGAACCAC CAGAGCATCCCTCTCATTTG 212 340.81 FREM2 FREM2_E22 ATAACATTTCCACATCTC GCTCATTTTTTGGGTCAAAG 241 8.00 FREM2 FREM2_E23 GAGAAACATGCTGTCCTGGC GGCATATAAATGTTCCATAACTTGC 287 138.58 FREM2 FREM2_E24.01 TCTCTTGCAAAGAGTCGTGG GTTTTCAGCACCAATCTCCC 275 367.42 FREM2 FREM2_E24.02 CGGAAGAAGAGAGAGATCAGG AGGCAGATGGTGAGTAACCC 233 921.05 FREM2 FREM2_E24.03 CTGCAGTCAGCCTGGTCAC GGCACTTACGGAAAAGGTTG 228 782.03 GREM1 GREM1_E02.01 GTGTCTTCCCCTCTCTGTGC TGGACTCCAGCACCTCCTC 263 238.17 GREM1 GREM1_E02.02 CAGACTCAGTCGCCCCAG GATGTGCCTGGGGATGTAG 258 460.66 GREM1 GREM1_E02.03 ACAGTCGCACCATCATCAAC TTCCTAGGACATGCTGGGTG 264 492.68 GRIP1 GRIP1_E25 ACCCACTGGCTTCACAGAAG CCTGTGCTTGCAGTTAATGCC 255 2715.51 GRIP1 GRIP1_E24 CGGGTTTTGGGAATTATGG TGTTCTGTTCACTCCAATCTCC 219 3950.93 GRIP1 GRIP1_E23 GCTAAATATTCACATCCCATTTTC GCTCCCAGTGAGAAATTACTTG 233 2097.47 GRIP1 GRIP1_E22.2 AGCTTCCAGGAGCGCAG ATGCTATGCAGAATGGCTGCC 215 4435.68 GRIP1 GRIP1_E22.1 TTAGTCCATGGCTTAATTGTTTC CACATCTGAAGGCAGGGTG 197 5250.27 GRIP1 GRIP1_E21 GGTAGCCTTGGTCCTGCTAC TTGCTGTCCAGAAGGCAAG 230 1706.03 GRIP1 GRIP1_E20 CTGGCAAGTTCCTAACCCAG GAAACAATAAAAGTAAGTAGCAGGACC 187 4707.71 GRIP1 GRIP1_E19 GGGCCCAATGCTAACAC TGACCATGCAGTCATCTTGG 238 2356.17 GRIP1 GRIP1_E18 TTTACTCTGATACTTAAACTCCTTTC TGGGCTTCAACAATGACAAG 281 895.07 GRIP1 GRIP1_E17 AGGTATGTCAGGGAATTTATATTCAG TCAAGGAAGAAAATGCACTGG 272 860.55 GRIP1 GRIP1_E16 TGAATCTATGCATCTTACTCTTTTGC GTTGTGAGGAAGGAAGCCC 276 16.39 GRIP1 GRIP1_E15 TCATCATTAGTCATGTGTTACTTTGC ATCTTTGGTGACAGATGGGC 244 1973.34 GRIP1 GRIP1_E14 GGGAAATGTACAGTAAGGGGTG CTTTGGGTGTGGAAGGTGTC 214 2197.20 GRIP1 GRIP1_E13 TGCTCCAGTCCTCAGTGTTC CCTGCAAACCATTTGACAAC 205 1108.43 GRIP1 GRIP1_E12 ACAACCTCTCACATGTGGTTC CCAATATAACTCACATGCAGTCC 222 8.81 GRIP1 GRIP1_E11 TGCTTGCTCCAACCTAAAGTC ACGCAGAGCAGAAAATGATG 273 1501.33 GRIP1 GRIP1_E10 CACCTGAGAGTGCTCTGTGC CCAAATGCCATTGGCTGTC 227 3055.19 GRIP1 GRIP1_E9b CTTATTTTTCATCATCACTTCATATG ACCAGGAGGAGAAAGCATGG 215 2094.48 GRIP1 GRIP1_E09 GGCACTGCCTCTTACCGAG TGCTAGAGGGAGCAACACTG 269 2144.42 GRIP1 GRIP1_E08 TTCATCTCTTTCTTTTGTCTCTTTG CGGAAAAGTAGGCACTTTCTAC 249 858.47 GRIP1 GRIP1_E07 GACTATTAATACTAAGCCCCATTATGC AGACAGTGACGTTCTGGTGC 262 1865.84 GRIP1 GRIP1_E06 CAATGAAAGCAGTTTTAAGCTCTG TCAACTTCTATGTTAAAGTCCCCAG 189 3516.80 GRIP1 GRIP1_E05 TTTTATGCCACATTGAATGATTTAG AACCAAATACCCAAATACCACC 245 1083.07 GRIP1 GRIP1_E04 GGTCTGTTGAAACAACATTGTC CACTGGGGGTCTTGGAATG 232 1807.61 GRIP1 GRIP1_E03 AGGAGAAATGACATTGGTGATTC TGTTCCTTTCAGTAGATTTCCCC 235 1765.73 GRIP1 GRIP1_E02 TGGAAATTAACCAAACGCTTC TTTAGAATCACCAAATAACAGGATTC 183 3980.81 GRIP1 GRIP1_E01 CACTGGGACTACCTTTCTCCTG TCGCTGAGGGAATAACAAGG 202 4091.27 ILK ILK_E02 CCACAGTCCTCAGGCTTCC CTTCCTCTCATCCACCAACC 173 4746.99 ILK ILK_E03 TTCAGGAATCAAAACCTTTGC TGATGTGGATGACGAAGGAG 262 1552.36 ILK ILK_E04 CTGACTGTACTTTCTGCCTCTTC CCATCTCAGGAATTAAGGGC 174 5730.83 ILK ILK_E05 AGTGACTGCCAGCGAGGTAG TGAGATTCTGGCCCATCTTC 276 2487.87 ILK ILK_E06 CCCTAGCTTGTGTCCTCTCG TACCCAGCTTCACCCTAACC 243 3402.81 ILK ILK_E07 AATAATCCTGGCCTCTTGGG CCTTGCCCACTAACTGGAGG 247 2142.15 ILK ILK_E8-13.1 CAAGCCTCCTAACCCCTACC ACTGGGAGCACATTTGGATG 273 1958.29 ILK ILK_E8-13.2 CAACCACTCCCTCCCTCTTC CTCTGGTCCACGACGAAATC 254 2537.07 ILK ILK_E8-13.3 GGGAGCCTCTCTGAACTATTTG TAATTCGGGCAGTCATGTCC 280 1393.54 ILK ILK_E8-13.4 TCTGTTTTCTCTTCCTCAGATTG ACATGTCTGCTGAGCGTCTG 279 1407.31 ILK ILK_E8-13.5 CCCTATCTCTCCAGCTCTGC GCCCTTGGGACAGGATTAC 265 1830.13 ILK ILK_E8-13.6 TTGGCTCCTCACATATTTGTTC TGTCCTGCATCTTCTCAAGG 258 2169.26 ILK ILK_E8-13.7 GAATGAAGACCCTGCAAAGC GGCTGGGGTAGTACCATGAC 286 4075.45 ITGA8 ITGA8_E30 TTCACAGGTTCCCAGAATGAC GAACAGGACCAGTGTTTGAGG 185 4712.22 4

ITGA8 ITGA8_E29 GATCCTTTCAGATCAACTTCCG TAAATTCCCTCAACAGCCCC 280 1204.59 ITGA8 ITGA8_E28 TGCCTTGAAATGCAAAACAC GCCTAGCACAAGCTAGACAGTG 282 23.56 ITGA8 ITGA8_E27 TCTGACGTGTTCCCACTGC AGCAGTGTGTATGTGCTTCTG 177 5027.83 ITGA8 ITGA8_E26 GAAAATGTAGCAACTGTTCATGTG AAGCCACTCATTTCCCTCAG 233 1841.75 ITGA8 ITGA8_E25 GCCTTCTGCTGGGTCATTAG TCAACAAAGCCACTGATTAGAC 234 1898.57 ITGA8 ITGA8_E24 TTACCTGGGCCATTGCTAAC CCCCTAACACAACTTTAACACAG 186 4097.25 ITGA8 ITGA8_E23 TGTAATAATACTCCTGAGCAGATAG AATGCATCAGGAAAATTCCAC 218 954.04 ITGA8 ITGA8_E22 TTACAGTGATCCAGGGTGGG GTGAGCTTTTAAGGCCAAGG 278 681.25 ITGA8 ITGA8_E21 GATGTCATTTATCCTGCCCC TCCATTTGTTCCCTGTGAAG 205 3439.02 ITGA8 ITGA8_E20 AAGAAGTAGAGTACCTAATCCTTC GCTCTTGACTTCAAACAATTTGC 224 1391.12 ITGA8 ITGA8_E19 AGAGGAAAGCTCTGGTTCCG TGTAATTCCATTAGATAGAAAGTACAC 221 1560.36 ITGA8 ITGA8_E18 TTGCAGCTATTTCATGGAGC TGCTTGAGAAAATGCCGTC 227 2812.87 ITGA8 ITGA8_E17 AGAGCACTGTGTTCACTGAATAC AGGCCAGAGAAGTCTTTCTGG 237 503.99 ITGA8 ITGA8_E16 TCTGGGATTAGGTAAAGGAAGG TGGTCTGTGCAGAGAAATGG 210 2004.49 ITGA8 ITGA8_E15 AGTGAAGTGGGCTTCTGCC GAGTTTGAGAAGCACCGGAC 208 3392.24 ITGA8 ITGA8_E14 GCATGTAGGGGTATTCTATGTGC TCCCCAGAATAAATCGTTGG 244 1614.44 ITGA8 ITGA8_E13 TACACATAAAACATGTTTTATGTCTG CTCTCACGTGGGAAAAGAAAG 256 486.76 ITGA8 ITGA8_E12.2 GAATTTGAGAGCAACCCCAG AAATGACTTCCACGCTTTGG 193 5482.62 ITGA8 ITGA8_E12.1 GTGCTGAGCTGCTTTCTTTG TACCGAATCTCCCAAACGTC 191 6140.17 ITGA8 ITGA8_E11 TGTACCACAATGAGATGAATCTTTC GTTGAGGCAATAAGGAAGGG 250 1142.57 ITGA8 ITGA8_E10 AATAAGATCATTCCGTGGGC GTCTACTGGTAACCCAGAGTG 266 764.21 ITGA8 ITGA8_E09 AGTAATTCCCCTTCTAAATGGC TGTAATTCCATCAAGGAGCAC 150 5301.76 ITGA8 ITGA8_E08 ACCATGGTAGCCCTTTCAC ATGCCATTTTACCATTTCCC 195 1757.62 ITGA8 ITGA8_E07 CCTGCCTCTTGCTTTCATTC AAACCACAAATGTCTTGG 207 1256.75 ITGA8 ITGA8_E06 GGTTGCTGTTGCTGTTTCTG AACGCTTCATTTGAGAAACTATG 209 2264.00 ITGA8 ITGA8_E05 TTCTAGAGAAAATAACATAATATCCTG TCAGATTAAATATTAGGAACTGCGAG 197 34.45 ITGA8 ITGA8_E04 CAATTAATTCACCCAATAATTTAACAG ACTTGAGCTAATGTCAGTTTCAAG 276 317.52 ITGA8 ITGA8_E03 CCACATTTTACAAATTATTAACTCTTC AAATGCCAACAGCCTTATCG 277 725.85 ITGA8 ITGA8_E02 GCCCAAAGAGTGACTTTCTCC TGGTTCTTCCAAACCCAGG 218 1438.13 ITGA8 ITGA8_E01.2 GGGGATGTTGCTGTGGTC AGCCGCTGGGACCTGAC 196 1383.70 ITGA8 ITGA8_E01.1 GGTAGCAGCCACCCACC CTGTGAGCTTTTCCACGTCC 172 527.77 LIN7C LIN7C_E05 AACTTAGATATTAGTGTATGGTTGGTTG TTCTCTAGCTAAAACGCAAAATG 245 1240.99 LIN7C LIN7C_E04 AGTCATATTTTTAATATGTAAAACTTTC CACTACAAATAGAAATAAAATTCATCC 275 1.10 LIN7C LIN7C_E03 AAACCAGTCCCTCTCATTTTTG TAATTTATGAAGAATTCTTACTTTTGG 247 87.20 LIN7C LIN7C_E02 AGGCATTGAGAAAACCCTTG TTGAACCCAAATTTAGAGTATTATCTG 245 640.29 LIN7C LIN7C_E01 TACTCACTTTTCCGGCTTCC GATCTCAGAGCCTGGGTCAC 221 273.95 LRP4 LRP4_E38.2 ATGCATGAAGACAGACACGG AAGGAGAAGGAACAGGCAGG 237 3421.08 LRP4 LRP4_E38.1 TGACCATATCTGCCCACTCTC TGCTCCGTCTCTGTGTCATC 235 2546.54 LRP4 LRP4_E37 TTTCTCATCACCTTCCCTCC CTCGCCAAAAAGACCCTTG 207 2728.07 LRP4 LRP4_E36 GCTAGAAAGTTGTCGGTGGC GTGGCCCCTGTAGCAAGAC 166 6108.98 LRP4 LRP4_E35 CCTGAAAGCCCCCACTTAC GTTCATCAACCCCAGAGTCC 273 1855.05 LRP4 LRP4_E34 GTTTATGGTTCCAGTGGCCC CCATGATCCTGCATTGAACC 207 3920.26 LRP4 LRP4_E33 TGGGAGACCTGATTCTGTCC GTGGCTCCAGCCATACAGTC 193 5077.53 LRP4 LRP4_E32 TTGGGCTTCCCCTGTTG TCCCACAGATGTTAAGGAAGC 211 2576.18 LRP4 LRP4_E31 CTGGGCTCCCTGGCAAG CATCCCAGTCAAGGAGGTTTAG 207 4196.53 LRP4 LRP4_E30 CTTCCCATGCCTTGATGATG GATTTCCAGGAGGCTGTTTG 210 3121.57 LRP4 LRP4_E29 ATCCAACACTGGGCTCTCC TCTCTTCTGCTGGCCATAAAC 268 1631.63 LRP4 LRP4_E28.2 GACCTGTGATCCCTCTCCTG TCTGGAGGTTTCAGTGTTGC 218 2215.68 LRP4 LRP4_E28.1 GATGTTCCAGGCTAGGTGTG CAGTGAGATACGCCGGATG 226 3601.35 LRP4 LRP4_E27 ACCCTCTCCTTCCTCTGC CAGAGGCTCTGACTCACAG 286 1247.11 LRP4 LRP4_E26 TGTGGTAGCTGCTGGAATAAC GAAGCAGCAGGGACACG 242 2506.62 LRP4 LRP4_E25 CCAGTGTGCTCTTTTGACTTC CCTTTACCCCGTCATAACCC 269 1521.45 LRP4 LRP4_E24 CGGTGAGGGTCTAGGTTGAG CAGACAGGTGGTCCCTGG 162 4298.15 LRP4 LRP4_E23 TTAATGGCTGTGCTCGAGTG AAGGCCAGGTGGGAAGAG 221 2990.82 LRP4 LRP4_E22 CATTGAACATCCCCTCCTCTC ATTGCCCCCTCCCAGAG 211 3573.09 LRP4 LRP4_E21 GTTGAAGATGACACAGTGTTGG TAGGGCATAGGAGGGCCAC 274 1630.08 LRP4 LRP4_E20 GTAAGACCTGCCTTGCCTTG AGATGTTTTAGTGCCACCCTTACC 279 1852.53 LRP4 LRP4_E19 CAAGACTAGAATTAATCTCTGTATCCC ATGGAGCTCATTCCCAAGG 175 4900.62 LRP4 LRP4_E18 TGGTGATACAGCTTTGCTTCC GAATCCCAGGGAGCCAG 153 3712.14 LRP4 LRP4_E17 TAAAAGAAGACCCCTTTCGGC TTCAACCTCCCCACGCTG 266 2064.37 LRP4 LRP4_E16 GACCTTCGCTGATCCTCTTG CCATGGAGGCTGGTGTTG 193 1158.33 LRP4 LRP4_E15 TCCAGACTGAGGTCTTCCTG GAGGCTACTTTGGCTCACCC 246 2374.93 LRP4 LRP4_E14 CTGCCCACTTCCCAATTTAC ATGAAGGAGACTGAAGGAAGG 280 1499.09 LRP4 LRP4_E13 GCCTGGGTTGACTCCTTG AGAAAGTTCGGGAGCCTGAG 233 2899.83 LRP4 LRP4_E12.2 TGCTGCTTAACAACCTGGAG TCCATGGGGCCTTGTTC 178 5836.93 LRP4 LRP4_E12.1 CGGCTCTGAAACCCAGTG GACCAGAAGACAAGCTCGC 191 6371.07 LRP4 LRP4_E11 GAGTGGGAGGACGACAGAAG AGCCAAGTGCCAACAGCC 200 3922.73 LRP4 LRP4_E10 CAACTCCCAACTCTGGCTTG CTCAGAACCCCGACTCTGC 206 904.30 LRP4 LRP4_E09 TTGCTATGACCTGACCCTCC GGGGTTCGGCCACAAAC 206 3767.51 LRP4 LRP4_E08 CACCTTTCATTAAGCCTTTGC CTATAGGCTAAGGGTTCTGG 196 3206.88 LRP4 LRP4_E07 GGGACACTGCACAGACTGG GCCCAACCTAATTCCATTTC 197 3802.13 5

LRP4 LRP4_E06 ATCCAGGCCTGAGTGTGTG ACCCAAGCAGTTCTTCCCAG 234 336.44 LRP4 LRP4_E05 CCCTCAGAACTGCTGTCCC AACCCAACAGCCTGAGGTC 183 4256.40 LRP4 LRP4_E04 GCAGCCTGACTGCTCTGTG ACCCACTGGCCACCTTG 185 1652.85 LRP4 LRP4_E03 CTGGCCTAGTTGAGCCTGAG CTTTCCCAGTGGAACTCAATG 236 1369.94 LRP4 LRP4_E02 ACTGATTCCTTTTCCCTCCG CAGGTCCCTCTCCACCAC 218 2327.22 LRP4 LRP4_E01 TGCACCCGGGACGCTTC CGGACCCAGGGACAAAC 193 0.00

Supplemental Table 6: Genotype comparison of compound heterozygous alleles identified in three individuals with CAKUT in individuals of the 1000 Genomes Project (http://browser.1000genomes.org/) demonstrates that these alleles are in trans.

Individual, Sex, Origin 1st allele 2nd allele

A3975-2.1, M, GER FRAS1 A1387L (EVS: 13/12487) FRAS1 R3269Q (EVS: 88/12570) NA19700, M, ASW WT G/A (het) NA12413, M, CEU WT G/A (het) HG00177, F, FIN WT G/A (het) HG00104, F, GBR WT G/A (het) HG00123, F, GBR WT G/A (het) HG00134, F, GBR WT G/A (het) NA20533, F, TSI WT G/A (het) NA20757, F, TSI WT G/A (het)

A1023-2.1, M, IND FREM2 R1344H (EVS: 25/12981) FREM2 R2512H (EVS: 10/12996) NA18635, M, CHB A/G (het) WT HG00280, M, FIN A/G (het) WT HG00284, M, FIN A/G (het) WT ASW, Americans of African ancestry in Southwestern US; CEU, Utah residents with Northern or Western European ancestry; CHB, Hang Chinese in Beijing, China; CLM, Columbians from Medellin, Columbia; EVS, Exome Variant Server; FIN, Finnish in Finland; GBR, British in England and Scotland; GER, German; IND, Indian; M, male; MAC, Macedonian; TSI, Toscani in Italy. Supplemental Table 7. Evaluation workflow applied to Next Generation Sequencing- based mutation-analysis in 12 murine candidate genes in 672 individuals with CAKUT.

Applied Quality Control Filters  Exclude variant if variant frequency in NGS-reads ≤ 20%  Exclude variant if coverage ≤ 10x

Applied Variant Filters  Exclude variant if present in common dnSNP132 (MAF >1%)  Exclude variant if present in ≥ 5% of subjects  Exclude variant if synonymous or not splice-site affecting  Exclude single heterozygous variants in one gene

Variants were considered disease-causing and reported in Table 1 if:  Confirmed in Sanger sequencing  Positive segregation analysis  Protein-truncating variants  Affected amino acid residue is evolutionary conserved in vertebrates  Variant is not present in a homozygous state in the EVS server and its minor allele frequency (MAF) is less than 1% in 13,000 control chromosomes (EVS).  Variant prediction software do not unanimously predict the variant to be benign (PolyPhen2 Hum Var, SIFT, Mutation Taster).