REPORT

Mainzer-Saldino Syndrome Is a CiliopathyCaused by IFT140 Mutations

Isabelle Perrault,1,16 Sophie Saunier,2,16 Sylvain Hanein,1 Emilie Filhol,2 Albane A. Bizet,2

Felicity Collins,3 Mustafa A.M. Salih,4 Sylvie Gerber,1 Nathalie Delphin,1 Karine Bigot,5

Christophe Orssaud,6 Eduardo Silva,7 Veronique Baudouin,8 Machteld M. Oud,9 Nora Shannon,10

Martine Le Merrer,1 Olivier Roche,6 Christine Pietrement,11 Jamal Goumid,12 Clarisse Baumann,12

Christine Bole-Feysot,13 Patrick Nitschke,14 Mohammed Zahrate,13 Philip Beales,15 Heleen H. Arts,9

Arnold Munnich,1 Josseline Kaplan,1,* Corinne Antignac,2 Valerie Cormier-Daire,1

and Jean-Michel Rozet1,*

Mainzer-Saldino syndrome (MSS) is a rare disorder characterized by phalangeal cone-shaped epiphyses, chronic renal failure, and early-

onset, severe retinal dystrophy. Through a combination of ciliome resequencing and Sanger sequencing, we identified IFT140mutations

in sixMSS families and in a family with the clinically overlapping Jeune syndrome. IFT140 is one of the six currently known components

of the intraflagellar transport complex A (IFT-A) that regulates retrograde protein transport in ciliated cells. Ciliary abundance and local-

ization of anterograde IFTs were altered in fibroblasts of affected individuals, a result that supports the pivotal role of IFT140 in proper

development and function of ciliated cells.

Ciliopathies are an emerging class of genetic disorders

caused by altered cilia assembly, maintenance, or func-

tion.1,2 They comprise a broad range of phenotypes that

result from developmental or functional defects of unique

or multiple systems. Syndromic ciliopathies that affect

bone development are classified as skeletal ciliopathies.1,2

Mutations in genes that encode components of the intra-

flagellar transport complex A (IFT-A), which drives

retrograde ciliary transport,3 are a major cause of skeletal

ciliopathies.4–11 Alterations of all IFT-A components but

one (IFT140) have been previously reported to cause Sense-

nbrenner, Jeune, and/or short-rib polydactyly syndromes

(IFT43 [MIM 614068], IFT122 [MIM 606045], IFT139

[MIM 612014], IFT144 [MIM 608151], WDR35 [MIM

613602]).4–11

Mainzer-Saldino syndrome (MSS [MIM 266920]), or con-

orenal syndrome (CRS), is a rare autosomal recessive

disease defined by phalangeal cone-shaped epiphyses

(PCSE), chronic renal disease, nearly constant retinal

dystrophy, andmild radiographic abnormality of the prox-

imal femur.12 Occasional features include short stature,

cerebellar ataxia, and hepatic fibrosis.12 MSS shares retinal

dystrophy with Leber congenital amaurosis (LCA [MIM

204000]) and nephronophthisis (NPHP [MIM 256100]),

and skeletal features with asphyxiating thoracic dystrophy

(ATD [MIM 208500]), or Jeune syndrome, and cranioecto-

1INSERM, U781 & Department of Genetics, Paris Descartes University, 75015 P3Department of Clinical Genetics, Children’s Hospital at Westmead, Sydney, N

King Saud University, Riyadh 11461, Saudi Arabia; 5Centre d’Exploration et de6Department of Ophthalmology, Paris Descartes University, 75015 Paris, Franc

Coimbra, Portugal; 8Department of Nephrology, Centre Hospitalier Universita

boud University Nijmegen Medical Centre, 6525 GA, Nijmegen, The Netherla11Department of Pediatrics, American Memorial Hospital, CHU Reims, 510

75019 Paris, France; 13Genomics Platform, Imagine Foundation and Paris Desc

cartes University, 75015 Paris, France; 15Molecular Medicine Unit, University16These authors contributed equally to this work

*Correspondence: josseline.kaplan@inserm.fr (J.K.), jean-michel.rozet@inserm

DOI 10.1016/j.ajhg.2012.03.006. �2012 by The American Society of Human

864 The American Journal of Human Genetics 90, 864–870, May 4, 2

dermal dysplasia (CED [MIM 218330]), or Sensenbrenner

syndrome, suggesting that it is a ciliopathy.

We collected 15 families presenting three diagnostic

criteria of MSS, namely early-onset retinal dystrophy,

PCSE, and renal disease, and two families with nonovert

renal disease (Tables S1 and S2). Informed consent was

obtained for each individual participating in this study,

which was approved by the Comite de Protection des Per-

sonnes ‘‘Ile-de-France II.’’ To identify the genetic mutations

responsible for MSS, we subjected the genomic DNA of an

affected individual born to unrelated parents (FI1; Figure 1

and Table S1) to ciliome resequencing, using a 5.3 Mb

customized Agilent SureSelect Target Enrichment library

to capture 32,146 exons of 1,666 genes. We first focused

our analysis on consensus splice-site changes, nonsynony-

mous variants, and insertion and/or deletion in coding

regions. Considering that MSS-causing mutations are

rare, we assumed that the affected individual was likely

compound heterozygous for variants absent in the

dbSNP132, 1000Genome, and in-house databases. This

pointed to one candidate gene only, IFT140 (intraflagellar

transport protein 140 homolog (chalydomonas);

[NM_014714.3]), which encodes the last IFT-A component

not yet shown to be related to skeletal ciliopathies (Table

S3). We found both a missense and a donor splice-site

mutation (Figure 2 and Table S1). The missense mutation

aris, France; 2INSERM, U983, Paris Descartes University, 75015 Paris, France;

SW 2145, Australia; 4Division of Pediatric Neurology, College of Medicine,

Ressources Therapeutiques en Ophtalmologie (CERTO), 75015 Paris, France;

e; 7Department of Ophthalmology, Coimbra University Hospital, 3000-548

ire (CHU) Robert Debre, 75019 Paris, France; 9Department of Genetics, Rad-

nds; 10Clinical Genetics Service, City Hospital, Nottingham NG5 1PB, UK;

92 Reims Cedex, France; 12Department of Genetics, CHU Robert Debre,

artes University, 75015 Paris, France; 14Bioinformatics Platform, Paris Des-

College London (UCL) Institute of Child Health, London WC1N 1EH, UK

.fr (J.-M.R.)

Genetics. All rights reserved.

012

Figure 1. Clinical and Radiological Manifestations of MSS-Affected Individuals(A and B) Photograph and thorax X-rays of affected individual FI1 at 17 years of age showing narrow chest.(C–E) Hip X-rays of affected individuals FI1 (17 years), FIII1 (9 years), and FIII2 (5 years) showing flattened femoral epiphyses (C–D) andwide femoral neck with areas of sclerosis in the metaphyseal region (arrows).(F–J) Hand X-ray of affected individuals FI1, FIII1, FIII2, FII1, and FV1 showing PCSE (arrows).(K–N) Renal biopsy from affected individual FVI1 at 2 years (periodic acid-Schiff [PAS] in K and L; trichrome staining in M and N)showing severe tubulointerstitial lesions characterized by dedifferentiated and dilated tubules as well as atrophic tubules surroundedby marked thickening of the tubular basement membrane (arrow) and interstitial fibrosis with infiltrates (asterisks). Scale bar: 100 mm.

altered an acidic residue conserved across species

(c.1990G>A [p.Glu664Lys]), whereas we expected the

splice-site mutation to result in the skipping of exon 18

and/or the use of a surrounding cryptic splice site (c.2399þ1G>T). Sanger sequencing confirmed these results, and

segregation analysis excluded allelism of the variants.

Additional ciliome resequencing and/or Sanger

sequencing of the 29 IFT140 coding exons (3 to 31, Table

S4) and intron-exon boundaries detected homozygous or

compound-heterozygous disease-causing mutations in

five other MSS families (Figures 1 and 2; Table 1, Tables

S1 and S3) and single heterozygote mutations in four

additional families (Table 1 and Table S2). All changes

were absent from 200 control chromosomes and were pre-

dicted to be deleterious with the use of the Alamut Muta-

tion Interpretation Software, a decision support system

for mutation interpretation based on Align DGVD,

Polyphen-2, SIFT, SpliceSiteFinder-like, MaxEntScan,

NNSPLICE and Human Splicing Finder (Table 1). Segrega-

tion of compound-heterozygous and homozygous muta-

tions with the disease was confirmed.

Affected individuals of the nonconsanguineous simplex

FII and multiplex FV families were compound heterozy-

gous for missense and truncating mutations, whereas

those of the multiplex consanguineous families III and

IV from Saudi Arabia were homozygous for missense muta-

tions (Table 1 and Figure 2). Affected cases of these latter

two families harbored the c.1990G>A (p.Glu664Lys)

change identified in affected individual FI1 (Table 1 and

Figure 2). Linkage analysis at the IFT140 locus detected

an intragenic IFT140 recombination, precluding the search

for linkage disequilibrium in families segregating this latter

change (Figure S1). Affected individuals FVIII1, FIX1, and

FX1 were single heterozygous for missense mutations.

FXI1 harbored a conservative change that we predicted

The Am

would create 4 bp upstream of the intron 5 donor splice

site, a competing donor site, the use of which would

result in the appearance of a premature stop codon

(c.489C>T [p.Gly163Gly and p.Glu164Thrfs*10]; Table 1

and Table S2).

The effect of the c.634G>A (p.Gly212Arg), c.699T>G

(p.Ile233Met), c.932A>G (p.Tyr311Cys), and c.1990G>A

(p.Glu664Lys) changes on the IFT140 localization was as-

sessed in the telomerase-immortalized retinal pigment

epithelial cell line (RPE1). Flag-tagged IFT140 mutant

proteins showed a partial to nearly complete loss of basal

body localization associated with an increase of cytoplasm

staining, whereas the wild-type Flag-tagged IFT140 protein

predominantly localized to the basal bodies in RPE1

cells (Figure 3A). The c.1990G>A (p.Glu664Lys) change

displayed the most severe disorganization (IFT140 misloc-

alization in 80% of the cells; Figure 3A).

To assess the impact of IFT140 mutations on ciliogene-

sis, abundance and morphology of primary cilia were

studied in cultured fibroblasts of affected individuals FII1

and FIV3. By staining the cilia axonemes with acetylated

alpha-tubulin, we detected absent cilia in a high propor-

tion of cells of affected cases compared to controls (mean

affected cases versus mean controls: 55.10% versus

83.61%, p < .0001), supporting a defect in ciliogenesis

and/or cilia maintenance (Figure 3B).

To determine the effect of the IFT140 mutations on

retrograde intraflagellar transport, we analyzed the endog-

enous subcellular localization of IFT140. The fibroblasts of

affected individuals FII1 (compound heterozygous for a

splice-site mutation and the c.932A>G [p.Tyr311Cys]

change) and FIV3 (homozygous for the c.1990G>A

[p.Glu664Lys] change) exhibited an unaltered IFT140

localization along the cilia axoneme (Figure 3C). However,

two components of the anterograde transport IFT-B

erican Journal of Human Genetics 90, 864–870, May 4, 2012 865

Figure 2. IFT140 Organization, Protein Structure, and Mutations Identified in Homozygous and Compound-HeterozygousIndividuals Affected with Skeletal Ciliopathies

complex, IFT88 and IFT46, were evenly distributed along

the cilium of both affected individual fibroblasts, whereas

they were predominantly detected at the base and the tip

of the cilium in control fibroblasts (p< 0.0001), suggesting

an alteration in retrograde ciliary transport (Figure 3C).

Table 1. IFT140 Mutations Identified in Ten Individuals Affected with

Family Pedigree Diagnosis

Allele 1

Mutation Predicted Effe

FI simplex,nonconsanguineous

MSS c.2399þ1G>T loss of intron 1splice-site

FI simplex,nonconsanguineous

MSS c.2399þ1G>T loss of intron 1splice-site

FII simplex,nonconsanguineous

MSS c.932A>G p.Tyr311Cys (d

FIII multiplex,consanguineous

MSS c.1990G>A p.Glu664Lys (d

FIV multiplex,consanguineous

MSS c.1990G>A p.Glu664Lys (d

FV multiplex,nonconsanguineous

MSS c.634G>A p.Gly212Arg (dand/or alteratio6 donor-splice s

FVI simplex,consanguineous

MSS c.699T>G p.Ile233Met (de

FVII simplex,nonconsanguineous

Jeunesyndrome

c.2399þ1G>T loss of intron 1splice-site

FVIII simplex,nonconsanguineous

MSS c.1565G>A p.Gly522Glu (d

FIX simplex,nonconsanguineous

MSS c.874G>A p.Val292Met (d

FX simplex,nonconsanguineous

MSS c.1727G>A p.Arg576Gln (d

FXI simplex,nonconsanguineous

MSS c.489C>T p.Gly163Gly andonor splice sit5 and predicted(p.Glu164Thrfs

aVariant pathogenicity according to Align DGVD, Polyphen-2, SIFT, SpliceSiteFindthe Alamut Interpretation Software 2.0.

866 The American Journal of Human Genetics 90, 864–870, May 4, 2

The defect in ciliogenesis and/or ciliary maintenance

and the aberrant distribution of IFT88 and IFT46 in cells

of affected individuals is consistent with the report of short

cilia and aberrant distribution of IFT88 in mutant reduced

mechanoreceptor potential A (rempA), the Drosophila

Mainzer-Saldino Syndrome and a Child with Jeune Syndrome

Allele 2

cta Mutation Predicted Effecta

9 donor c.1990G>A p.Glu664Lys (deleterious)

9 donor c.1990G>A p.Glu664Lys (deleterious)

eleterious) c.857_860del p.Ile286Lysfs*6

eleterious) c.1990G>A p.Glu664Lys (deleterious)

eleterious) c.1990G>A p.Glu664Lys (deleterious)

eleterious)n exonite

c.3916dup p.Ala1306Glyfs*56

leterious) c.699T>G p.Ile233Met (deleterious)

9 donor c.634G>A p.Gly212Arg (deleterious)and/or alteration exon6 donor splice site

eleterious) no mutationidentified

eleterious) no mutationidentified

eleterious) no mutationidentified

d creation of an additionale 4 bp upstream of intronto result in c.488_491del*10)

no mutationidentified

er-like, MaxEntScan, NNSPLICE and Human Splicing Finder available through

012

ortholog of IFT140.13 The ternary IFT-B subcomplex, made

up of IFT52, IFT88, and IFT46, is crucial for the stabiliza-

tion of the IFT-B particle.14–16 Therefore, the data we

report further support the view that alterations of IFT-A

components disorganize assembly and/or maintenance

of ciliary structure by impairing retrograde IFT through

the redistribution of ciliary proteins (notably IFT-B com-

plex components).5,7,8,17,18

Genetic and clinical heterogeneity are hallmarks of

multisystemic ciliopathies.1,2 In addition, several exam-

ples have been reported which demonstrate that different

mutations in a same gene, e.g., WDR35,6,11 WDR19 [MIM

608151],8 CEP290 [MIM 610142],19 and IQCB1 [MIM

609237],20–22 can give rise to a broad range of phenotypes,

from isolated nephronophtisis or LCA to multisystemic

and sometimes embryonically lethal conditions. Geno-

type-phenotype correlations and global mutation load in

ciliary genes have been suggested to account for this clin-

ical variability.1,2 Here we report that IFT140 mutations

consistently caused PCSE as well as retinal dystrophy and

occasional chronic renal failure, hepatic fibrosis, addi-

tional skeletal abnormalities, or neurological symptoms.

Interestingly, although retinal dystrophy is an occasional

feature of skeletal ciliopathies,1,2 it is very uncommon in

patients with IFT-Amutations.5–8,10 Conversely, all affected

individuals with IFT140mutations and full ophthalmolog-

ical examination (n ¼ 9/10; six families) had LCA or early-

onset, severe retinal dystrophy between birth and 4 years

of age (Table S1). With regard to individual FVI1, who had

no reported retinal disease at the age of 2 years, electrophys-

iological recordings were not available to assess the func-

tion of photoreceptors, the alteration of which typically

precedes fundus changes. Retinal dystrophy appears there-

fore to be a very stringent clinical manifestation of IFT140

alterations. Although there is no clear-cut correlation

between tissue expression of IFTs and clinical features, the

recent report of the role of IFT140 in photoreceptor cell

ciliogenesis24 is consistent with the retinal disease of

MSS-affected individuals harboring IFT140 mutations.

Chronic renal failure is an inclusion criterion in MSS.

However, age-at-onset and outcome of the dysfunction

vary, even within families.12 Therefore, it is possible that

renal failure occurs in affected individuals in families III

and IV (n ¼ 5, age range 4–17 years) late in the course of

the disease. This would be consistent with the recent

report of pronounced renal cystic disease in mice resulting

from HoxB7-Cre-driven loss of Ift140 in the renal collect-

ing duct.23 Nevertheless, affected individuals from families

III and IV were homozygous for a missense mutation

(c.1990G>A [p.Glu664Lys]), whereas all affected persons

with renal failure but one (FVI1) harbored a severe trun-

cating mutation (in addition to a missense mutation),

raising the question of whether some genotype-phenotype

correlations may exist. From this point of view, it is worth

noting that affected individual FVI1 harbored a homozy-

gous missense mutation (c.699T>G [p.Ile233Met]) and

heterozygous mutations in other ciliopathy genes,

The Am

TMEM67 (MIM 609884; data not shown) and XPNPEP3

(MIM 613553; Table S3B), which could contribute to

kidney failure.

Cerebral MRIs were consistently normal, but siblings

in two families exhibited neurological symptoms that

included mild intellectual impairment or autistic features,

seizures, and epilepsy (Families III and IV, respectively;

Table S1). Considering the high degree of consanguinity

of the two families, it is difficult to determine whether

some or all these traits are accounted for by IFT140

mutations, or if these disabilities are independently

inherited.

Some of the skeletal abnormalities of individuals with

IFT140mutations overlap with clinical symptoms of Jeune

and Sensenbrenner syndromes—namely, short hands, hip

and cranial abnormalities, narrow chest, and/or short

stature (Table S1). This overlap opens a debate as to

whether some affected individuals with IFT140 mutations

are affected with complex MSS, or incomplete Jeune or

Sensenbrenner syndromes (e.g., FI1, who had a narrow

chest but no respiratory deficiency and no trident acetab-

ular roof, or FVI1, who had trident acetabular roof,

a narrow chest, and short ribs; Table S1). Additionally,

this clinical overlap raises the question of whether other

skeletal ciliopathies are accounted for by IFT140 alter-

ations. So far the screening of large cohorts of individuals

affected with Sensenbrenner of Jeune syndromes failed to

detect IFT140 mutations.8 However, during this study, we

had the opportunity to identify IFT140 mutations in

a child affected with Jeune syndrome (FVII1, Table S1).

The child was compound heterozygous for a splice-site

mutation and the c.634G>A (p.Gly212Arg) change identi-

fied in Family V. These data suggest that mutations in

IFT140 may be a rare cause of Jeune syndrome and that

there is no clear correlation between the IFT140 genotype

and the severity of the disease.

The clinical presentation of the disease did not differ

significantly in MSS-affected individuals with two IFT140

disease alleles compared to single-heterozygous patients

(4/17) or affected individuals with no mutation (7/18;

Tables S1 and S2). It is possible that some of the latter

10/17 affected individuals may harbor undetected IFT140

mutations lying in unscreened regions, such as untrans-

lated regions or introns. Furthermore, following the

example of the deep intronic CEP290 c.1991þ1655A>G

mutation, which accounts for approximately 60% of

CEP290 disease alleles in LCA, it is possible that a common

undetected IFT140 mutation may exist.19 However, like

most other ciliopathies, MSS may be genetically heteroge-

neouswith someor all 10/17 affected individuals, including

single heterozygotes, harboring mutations in other genes.

In summary, we here report on compound heterozy-

gosity or homozygosity for IFT140 mutations in six

families affected with MSS and an individual affected

with Jeune syndrome. After Sensenbrenner and Jeune

syndromes, MSS is the latest skeletal ciliopathy ascribed

to IFT disorganization.

erican Journal of Human Genetics 90, 864–870, May 4, 2012 867

Figure 3. Distribution of Wild-Type and Mutant Endogenous or Flag-Tagged IFT140 in Fibroblasts and RPE1 Cells(A) Aberrant distribution of Flag-tagged mutant IFT140 proteins in RPE1 cells. Cells were transfected with pCMV-IFT140-WT-Flag,pCMV-IFT140-Glu212Arg-Flag, pCMV-IFT140-Ile233Met-Flag, pCMV-IFT140-Tyr311Cys-Flag, and pCMV-IFT140-Glu664Lys-Flagplasmids, respectively. Flag-tagged proteins were stained with the use of rabbit anti-Flag (1:200, Sigma-Aldrich) and Alexa Fluor555-conjugated anti-rabbit (1:200, Molecular Probes; red) primary and secondary antibodies, respectively. Basal bodies were stainedwith the use ofmousemonoclonal anti-g-tubulin (1:1000, Sigma-Aldrich) and Alexa Fluor 488-conjugated anti-mouse (1:200, MolecularProbes) primary and secondary antibodies, respectively. Images were recorded with a Leica SP5 confocal microsocope (Leica). Scalebars 5 mm. Wild-type (WT) IFT140 is clearly visible at the basal body (white arrow), whereas mutant proteins exhibit a significantcytoplasmic staining with decreased basal body labeling. The graph shows the percentage of transfected cells with localization of theFlag-tagged IFT140 protein at the basal body calculated from two independent experiments, and n > 100 cells for each transfectioncondition (***: p < 0.001 calculated via Dunn’s Multiple Comparison Test following the analysis of variance [ANOVA] test, GraphPadPrism Software).

868 The American Journal of Human Genetics 90, 864–870, May 4, 2012

Supplemental Data

Supplemental Data include one figure and four tables and can be

found with this article online at http://www.cell.com/AJHG/.

Acknowledgments

We are grateful to all affected individuals and their families for

their participation in the study. We thank R. Devaux for confocal

microscopy and C. Masson and S. Pruvost for technical assistance.

This work was supported by grants from Retina France, the Fonda-

tion pour la Recherche Medicale (FRM DEQ20071210558 to S.S.),

the Agence Nationale de la Recherche (grants R09087KS and

RPV11012KK to S.S.), and the Dutch Kidney Foundation

(KJPB09.009 and IP11.58 to H.H.A.).

Received: January 4, 2012

Revised: February 1, 2012

Accepted: March 9, 2012

Published online: April 12, 2012

Web Resources

The URLs for data presented herein are as follows:

Alamut Interpretation Software 2.0, http://alamut.interactive-

biosoftware.com

UCSC Genome Browser, http://genome.ucsc.edu

Online Mendelian Inheritance in Man (OMIM), http://www.

omim.org

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ts of MSS-affected individuals with IFT140mutations compared toversus 83.61% calculated from two independent experiments andsignificant difference [PLSD] test according to the significance ofwere stained with the use of rabbit polyclonal anti-pericentrin:1000, Sigma-Aldrich; green) primary and secondary antibodies,al anti-acetylated alpha-tubulin (1:1000, Sigma-Aldrich) and Alexand secondary antibodies, respectively. Images were recorded with

dividuals and controls. IFTs were stained with the use of IFT140F. Mallin-Guerin), and IFT88 (1:100, rabbit polyclonal; gift ofgoat anti-rabbit or rabbit anti-goat (1:1000; green) secondarywere labeled with the use of DAPI (Southern Biotech). Images

ss S.A.S.). Scale bars 5 mm. Acetylated alpha-tubulin and IFT140, and patient FIV3 cells (Pearson’s coefficients calculated via theression and localization of IFT140, IFT46 and IFT88 were mislocal-ong the axoneme versus predominant staining at the base and thentage of cilia exhibiting both basal and apical staining in controlsents and nR 40 cells for each cell line and each IFT, ***: p< 0.0001

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