clinical investigation www.kidney-international.

org

A mutation in transcription factor MAFB causes

Focal Segmental Glomerulosclerosis with Duane
Retraction Syndrome
Yoshinori Sato1,15, Hiroyasu Tsukaguchi2,15, Hiroyuki Morita3, Koichiro Higasa4,17, Mai Thi Nhu Tran5,
Michito Hamada5, Toshiaki Usui5,6, Naoki Morito6, Shoichiro Horita7, Takao Hayashi8, Junko Takagi3,
Izumi Yamaguchi4, Huan Thanh Nguyen2, Masayo Harada9, Kiyoko Inui1, Yuichi Maruta1,
Yoshihiko Inoue1, Fumihiko Koiwa1, Hiroshi Sato10, Fumihiko Matsuda4, Shinya Ayabe11, Seiya Mizuno12,
Fumihiro Sugiyama12, Satoru Takahashi5,12,13,14 and Ashio Yoshimura1,16
1
Division of Nephrology, Department of Medicine, Showa University Fujigaoka Hospital, Fujigaoka, Japan; 2Second Department of
Internal Medicine, Kansai Medical University, Hirakata, Japan; 3Division of Endocrinology and Metabolism, Department of Internal
Medicine, Aichi Medical University School of Medicine, Nagakute, Japan; 4Center for Genomic Medicine, Kyoto University Graduate School
of Medicine, Kyoto, Japan; 5Department of Anatomy and Embryology, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan;
6
Department of Nephrology, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan; 7Department of Cell Biology, Graduate School of
Medicine, Kyoto University, Kyoto, Japan; 8Department of Ophthalmology, School of Medicine, Teikyo University, Tokyo, Japan;
9
Department of Clinical Anatomy, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan;
10
Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan; 11Experimental Animal Division, RIKEN BioResource
Center, Tsukuba, Japan; 12Laboratory Animal Resource Center, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan;
13
International Institute for Integrative Sleep Medicine (WPI-IIIS), University of Tsukuba, Tsukuba, Japan; and 14Life Science Center of
Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba, Japan

Focal segmental glomerulosclerosis (FSGS) is a leading
cause of end-stage renal disease in children and adults.
Genetic factors significantly contribute to early-onset FSGS,
but the etiologies of most adult cases remain unknown.
Genetic studies of monogenic syndromic FSGS exhibiting
extra-renal manifestations have uncovered an unexpected
biological role for genes in the development of both
podocytes and other cellular lineages. To help define these
roles, we studied two unrelated families with FSGS
associated with Duane Retraction Syndrome, characterized
by impaired horizontal eye movement due to cranial nerve
malformation. All four affected individuals developed FSGS
and Duane Retraction Syndrome in their first to second
decade of life, manifested as restricted abduction together
with globe retraction and narrowed palpebral fissure on
attempted adduction. Hypoplasia of the abducens nerves
and hearing impairment occurred in severely affected
individuals. Genetic analyses revealed that affected
individuals harbor a rare heterozygous substitution
Correspondence: Hiroyasu Tsukaguchi, Second Department of Internal
Medicine, Kansai Medical University, 2-5-1 Shinmachi, Hirakata, Osaka 573-
1010, Japan. E-mail: tsukaguh@hirakata.kmu.ac.jp
15
(p.Leu239Pro) in MAFB, a leucine zipper transcription
factor. Luciferase assays with cultured monocytes indicated
that the substitution significantly reduced transactivation
of the F4/80 promoter, the known MAFB recognition
element. Additionally, immunohistochemistry indicated
reduced MAFB expression in the podocytes of patients.
Structural modeling suggested that the p.Leu239Pro
substitution in the DNA-binding domain possibly interferes
with the stability of the adjacent zinc finger. Lastly,
podocytes in neonatal mice with p.Leu239Pro displayed
impaired differentiation. Thus, MAFB mutations impair
development and/or maintenance of podocytes, abducens
neurons and the inner ear. The interactions between MAFB
and regulatory elements in these developing organs are
likely highly specific based on spatiotemporal
requirements.
Kidney International (2018) 94, 396–407; https://doi.org/10.1016/
j.kint.2018.02.025
KEYWORDS: FSGS; kidney development; nephrotic syndrome; podocyte;
transcriptional regulation
Copyright ª 2018, Published by Elsevier, Inc., on behalf of the International
Society of Nephrology.

These authors contributed equally.
16
Present address: Shin-yokohama Daiichi Clinic, 3-6-4 Shinyokohama
Kouhoku, Yokohama City, Kanagawa, 222-0033 Japan.
17
Present address: Department of Genome Analysis, Institute of Biomed-
ical Science, Kansai Medical University, 2-5-1 Shinmachi, Hirakata, Osaka,
573-1010, Japan.
Received 13 June 2017; revised 24 January 2018; accepted 8 February
2018; published online 14 June 2018
F
ocal segmental glomerulosclerosis (FSGS) is a clinico-
pathologic entity of nephrotic syndrome that is char-
acterized by a typical histological pattern of injury,
steroid resistance, and progression to end-stage renal disease
(ESRD).1,2 Recent studies regarding familial FSGS demon-
strated that most of the genes associated with the disease
encode podocyte proteins. Those observations highlighted

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Y Sato et al.: MAFB DNA-binding mutations in FSGS-DRS
the significant role of podocytes in the development and
maintenance of structural integrity of the glomerular filtra-
tion barrier.3,4 FSGS genes can be functionally classified
into several categories4; these include (i) slit membrane com-
ponents, (ii) the actin cytoskeleton of the foot processes, (iii)
nuclear envelope stability and transcription factors, (iv) mito-
chondrial function, and (v) glomerular basement membrane
and/or extracellular matrices. Genetic FSGS is mostly mani-
fested as a single-organ disorder, while a small fraction is
recognized as syndromic FSGS and involves extrarenal organs
in addition to the kidneys.
FSGS may be accompanied by ophthalmologic complica-
tions. However, the mechanistic link between FSGS and eye
movement disorders, such as Duane retraction syndrome
(DRS, MIM 126800),5 remains elusive. DRS is characterized
by restriction of horizontal outward eye movements (abduc-
tion), with eyeball retraction upon adduction. DRS is a
congenital form of strabismus with an incidence of 0.1%.5
Studies of individuals with DRS have suggested the presence
of aberrant innervation of the extraocular muscles secondary
to the developmental failure of cranial nerve VI.5 In rare
cases, syndromic DRS may be accompanied by other mal-
formations, including those of the kidneys and limbs.5 For
example, Okihiro syndrome (MIM 607323) is caused by
mutations in SALL4, a zinc finger transcriptional factor
expressed in neuron and kidney progenitors. In Okihiro
syndrome, radial forearm malformation is the major feature
(w90%), and other defects including DRS and renal anom-
alies may occasionally coexist.6,7
Studies of genetic FSGS with unique extrarenal features
have allowed serendipitous discoveries of key factors in the
development of the kidneys and other various organs.8 In this
context, we aimed to explore the molecular cause of familial
FSGS accompanying DRS (FSGS-DRS). Given that a fraction
of DRS cases are caused by a single gene mutation in Sall4 or
CHN1,6,7,9 we hypothesized that there might be as-yet-
unidentified genes that lead to FSGS-DRS. Here, by
applying next-generation sequencing, we identified a novel
missense variant (p.Leu239Pro) of MAFB, a member of the
large MAF family of basic leucine zipper transcription factors.
MAFB is specifically expressed in glomerular podocytes,
macrophages, and osteoclasts, and plays key roles in their dif-
ferentiation, development, and functional maintenance.10–13
MAFB consists of 3 parts: the N-terminal activation domain,
the DNA binding domain, and a leucine zipper required for
dimerization.13 MAFB binds its 2 half-site palindromic recog-
nition elements through homo- or heterodimerization via the
DNA binding domain and leucine zipper with other
coactivating partners such as c-Fos, c-Jun, etc.12,13 Previously
reported MAFB mutations remarkably cluster into the
N-terminal transactivation domain, and typically cause
multicentric carpotarsal osteolysis syndrome (MCTO; MIM
166300).14 The pathognomonic feature is the excessive reab-
sorption of carpotarsal bones due to osteoclast activation. In
MCTO, FSGS is not a ubiquitous feature and, if present, the
severity of any associated renal disease varies widely among
Kidney International (2018) 94, 396–407
clinical investigation
affected individuals, thereby making it difficult to fully
understand the role of MAFB in podocytes. MCTO patients
generally lack abnormalities in eye or auditory function.14
In the present study, we first demonstrate that the MAFB
DNA-binding domain variant causes FSGS-DRS in 2 unrelated
families. One affected individual also had hearing impairment.
These distinctive clinical subtypes suggest that the DNA-
binding domain of MAFB regulates transcription in a manner
that is temporally and spatially relevant to development and
maintenance of homeostasis in podocytes and cranial nerves.
RESULTS
Clinical phenotypes
FSGS-DRS Family-S. This case was originally reported
elsewhere.15 The proband was born to healthy, unrelated
parents. There was no family history of renal or other
inherited diseases (Supplementary Table S1). The patient
displayed appropriate growth during the perinatal period and
childhood, with the exception of preaxial polydactyly on the
left foot. At the age of 5, he was diagnosed with DRS and
subsequently had ocular motion corrected by surgery (Table 1
and Supplementary Figure S1). At the age of 7, he first
developed mild proteinuria without hematuria and noticed
increasing proteinuria with gradual deteriorating renal func-
tion after 12 years of age. FSGS was diagnosed by renal biopsy
at the age of 17 (Supplementary Figure S2). Despite steroid
therapy, he progressed to ESRD at the age of 19. He received a
kidney from his mother when he was 22 years old, and the
allograft maintained favorable renal function for more than
20 years. He noticed bilateral hearing loss at the age of 40, and
audiometry indicated bilateral sensory deafness, particularly
in the high-frequency range.
FSGS-DRS Family-Y. This is an unrelated Japanese family,
in which the mother (II-2) and 2 children (III-1 and III-2)
were affected (Table 1). The parents were not consanguin-
eous, and the father was healthy. The mother (II-2) experi-
enced nephrotic syndrome with renal dysfunction before
giving birth to the first child at the age of 24. No renal biopsy
had been performed. She developed ESRD and started to
undergo hemodialysis at the age of 29. The reduced ability to
turn her left eye outward resulted in a diagnosis of DRS type 1
(mainly defective abduction with normal or minimally
defective adduction).5
Both children (III-1 and III-2) were born full-term and
developed normally with appropriate growth during the
perinatal period and childhood. Affected sister III-1 man-
ifested nephrotic proteinuria at the age of 10, with mild
microscopic hematuria. Initial renal biopsy at age 11 revealed
minor glomerular abnormalities with only minimal (w5%)
global sclerosis and tubulointerstitial lesions or arterioscle-
rosis (Supplementary Figure S3). She did not respond to
steroid therapy, and showed gradual deterioration of renal
function. A second renal biopsy was performed when she was
23, which showed FSGS with severe interstitial fibrosis and
tubular atrophy (Figure 1). Immunofluorescence revealed
only segmental nonspecific deposition of IgM and C3. There
397
clinical investigation Y Sato et al.: MAFB DNA-binding mutations in FSGS-DRS

Table 1 | Clinical characteristics of 2 families with FSGS-DRS limited abduction with globe retraction into the left eye
Family-S Family-Y (Supplementary Figure S1).
II-2 III-1 III-2
In summary, all 4 affected individuals developed adolescent-
Patient ID II-2 onset FSGS with DRS. Patient II-2 from Family-S developed
Sex M F F M
hearing loss (Table 1), whereas other members did not notice
Kidney phenotypes any hearing problems. All affected individuals lacked any evi-
Age onset 7 15 10 12 dence of developmental delay, intellectual disability, or upper
proteinuria (yr)
Age onset 19 29 27 NA limb deformities and carpo-tarsal joint involvement.
ESRD (yr)
Renal histology FSGS (17) NA FSGS (11, 23) FSGS (18) Whole-genome sequence analysis
(age, yr)
We performed whole-genome sequencing for 3 affected and 4
Immunofluorescence IgM þþ (seg) Negative (1st) IgM þ (mes)
deposit a unaffected individuals from the 2 FSGS-DRS families. Based
IgA þ/-(mes) IgM þ Seg (2nd) upon the pedigree structure, rare variants were selected
C3 þ/- (mes) assuming a dominant (de novo) or recessive model for
Urinalysis
Proteinuria (g/d) >3.5 >3.5 >3.5 >3.5
Family-S and a dominant model for Family-Y (Figure 3,
Steroid response No No No No Supplementary Figures S4–S8, and Supplementary Table S1).
Hematuria 1þ NA 1þ 1þ After filtering procedures, only 2 variants, heterozygous
Hypertension Yes Yes Yes Yes p.Leu239Pro in MAFB (NM_005461.4) and homozygous
Eye phenotypes
Age onset aberrant 5 27 11 22
p.Gly116Arg in FGF20 (NM_019851.2), co-segregated with
motion (yr) FSGS-DRS phenotypes in both families (Figure 3,
DRS subclass Type1 Type1 Type1 Type1 Supplementary Figure S5, and Supplementary Tables S2 and
(laterality) (R and L) (L) (L) (L) S3). The FGF20 p.Gly116Arg variant (rs3793405) was
Extra-ocular NA NA No Yes
muscle atrophyb excluded because of its population allele frequency over 0.03.
Cranial nerve NA NA Yes No The MAFB p.Leu239Pro was a rare missense substitution,
VI hypoplasiac which is absent in both ethnic-matched healthy controls
Other manifestations Hearing loss (in-house n ¼ 2063, public n ¼ 3235) and healthy controls
Polydactylyd
from the world-wide consortium (ExAC). However, a rare
DRS, Duane retraction syndrome; ESRD, end-stage renal disease; FSGS, focal
segmental glomerulosclerosis; L, left; mes, global mesangium; NA, not applicable; R,
variant of a different substitution at 239, p.Leu239Val, has
right; seg, segmental pattern. been reported in dbSNP at a minor allele frequency of 0.001
The clinical subtypes of DRS have been described in prior studies5,9,16,17 Type I is in populations of European descendent (c.715 C>G,
characterized by (i) the inability to move an eye outward toward the ear (abduction),
(ii) narrowing of the palpebral fissure (pseudo-ptosis), and (iii) retraction of the rs6029274), of which the significance remains unknown.
eyeball when attempting to look toward the nose (adduction).
a
The p.Leu239Pro variant was classified as a deleterious
Immunofluorescent deposits observed in renal biopsy. Patient III-1 underwent 2
biopsies (first at age 11 and second at age 23). variant by in silico prediction algorithms. It should be noted
b
Morphologic changes in extraocular muscles and oculomotor nerves were evalu- that our 2 unrelated FSGS-DRS families share an identical
ated by magnetic resonance imaging.
c
Cranial nerve VI functions as abducens motor neuron.
MABF variant, p.Leu239Pro. These patients therefore repre-
d
Preaxial polydactyly was observed in the left foot, which was surgically corrected in sent extremely rare examples in which the same T-to-C
early childhood. transition in the second base of codon 239 (CTG to CCG)
occurred in 2 distinct families. The p.Leu239Pro variant occurs
de novo in Family-S (Figure 3 and Supplementary Figure S9),
while dominantly transmitting from the mother to 2 offspring
was 85% global glomerulosclerosis present, an increase of in Family-Y. Detailed haplotype analysis revealed that there
w80% from the previous biopsy. She was diagnosed with was no extra-paternity between the parents of Family-Y
DRS type 1 in her left eye at the age of 11 (Figure 2), similar (Supplementary Figure S10). The affected individuals from
to her mother, but currently has no hearing impairment. Family-Y and Family-S do not share any common haplotypes,
Brain magnetic resonance imaging showed the absence of the suggesting that the p.Leu239Pro mutation arose de novo in
left abducens nerve. Family-S, independent of that in Family-Y.
Affected brother III-2 first manifested proteinuria at the The Leucine 239 residue is evolutionally conserved among
age of 12. He developed nephrotic proteinuria (>3.5 g/d), species. Some DRS cases that exhibit inner ear defects and/or
along with moderate microscopic hematuria, when he was 17. urinary tract anomalies are reportedly associated with copy
Renal biopsy performed at age 18 revealed FSGS number variations (CNVs) such as deletions and/or dupli-
(Supplementary Figure S3). His histological findings and cations.18,19 However, our re-evaluation for CNVs using
renal impairment were milder than those of his sister, III-2. whole-genome sequencing data did not find any significant
He did not notice any disabilities in sight or hearing. Based structural genome variations, including at the SALL4 locus on
on magnetic resonance imaging, there was mild but diffuse chromosome 20q13, in our patients. Combined, these results
external ocular muscle atrophy (Supplementary Figure S1). indicate that the MAFB p.Leu239Pro is the most likely causal
Ophthalmological examination at age 22 revealed subtle variant of the FSGS-DRS phenotype.

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Y Sato et al.: MAFB DNA-binding mutations in FSGS-DRS clinical investigation

Figure 1 | Renal pathology of a patient with focal segmental glomerulosclerosis accompanying Duane retraction syndrome (FSGS-DRS)
with an MAFB mutation. (a) Light micrographs of a kidney biopsy. Specimens from FSGS-DRS Family-Y (affected individual III-1) at age 23 are
shown. A low-magnification view (periodic acid–Schiff stain) shows 85% global glomerulosclerosis and w50% tubular atrophy and cellular
infiltration in the surrounding interstitium. Mild arteriolar thickening is observed (arrows). Bar ¼ 200 mm (x7). (b,c) Enlarged image. Glomerulus
displays segmental sclerosis (arrowhead) with tuft adhesion (arrow). Bar ¼ 100 mm in b. (d,e) Electron micrographs. The same specimens from
FSGS-DRS Family-Y (patient III-1) were used. (c) Effacement of podocyte foot processes and some mesangial expansion with subendothelial
electron dense deposits are apparent. The thickness of the glomerular basement membrane (GBM; 234 nm, indicated by an arrow) is normal.
Bar ¼ 50 mm. (d) There is no evidence of splitting, lamellation, or fragmentation, thereby excluding the possibility of a primary basement
membrane defect. No accumulation of storage materials nor dysmorphic mitochondria in the podocyte cytoplasm are evident. Bar ¼ 2 mm
(x2000). (e) Approximately 60% of the foot processes exhibit a flattened pattern (arrowheads). Bar ¼ 500 nm (x10,000). E, endothelial cell; M,
mesangial cell; P, podocyte. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.

Structural modeling of the MAFB p.Leu239Pro variant
We next evaluated the structural effects of p.Leu239Pro via 3-
dimensional modeling based on the crystallized murine Mafb
structure.13 Leucine 239 is located in helix 3 at the distal end
of an extended homology region. Helix 3 is situated imme-
diately adjacent to other helices 1 and 2 within the same
extended homology region, all of which are adjacent to the
basic region, a direct DNA-binding segment (Figure 4). This
portion does not appear to be directly involved in either
heterodimerization between MAFB and coactivating partners
(e.g., c-Fos and c-Jun) or DNA binding of MAFB to the
MAFB-responsive element (MARE). Instead, the side chains

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clinical investigation Y Sato et al.: MAFB DNA-binding mutations in FSGS-DRS

Figure 2 | Eye movements and magnetic resonance imaging scans of patients with focal segmental glomerulosclerosis accompanying
Duane retraction syndrome with an MAFB variant. (a,b) Ocular motion at various diagnostic positions of gaze. Affected individual III-1 of
Family-Y is shown. (a) Frontal view. In the left eye, there was limited outward movement (abduction) with narrowing of the palpebral fissure on
inward gaze (adduction). (b) Lateral view of the left eye. Compared with the primary position (upper panel), retraction of the globe into the orbit
and up-shoot were observed upon attempted adduction (lower panel). (c,d) Axial magnetic resonance imaging. T2-weighted (c) and fluid-
attenuated inversion recovery (d) images showed overall normal brain parenchyma and arterial structures. (e) Magnified T2-weighted image.
The left abducens nerve (arrowhead) was absent, while the right one was preserved (arrow). (f,g) Coronal T1-weighted images revealed no
apparent atrophy of the extraocular muscles. IR, inferior rectus; LR, lateral rectus; MR, medial rectus; SR/LPS, superior rectus/levator palpebrae
superioris; VB, vitreous body. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.

of Leu 239 are predicted to form a hydrophobic core that
gathers the neighboring leucine residues (Leu216, Leu 224,
and Leu228) together. Thus, Leu239 is necessary for correct
folding of the basic DNA-binding domain (amino acids 240-
259, Figures 4 and 5). A new proline incorporation at Leu 239
likely affects this helical folding, thus destabilizing the domain
structure and function. This could hinder the transactivation
of MAFB by altering the conformation suitable for DNA
binding, involving Lys 240 and Arg 243, which are located in
close proximity to Leu 239. This structural analysis supports
the idea that p.Leu239Pro is pathogenic.
MAFB increased reporter activity more than 11-fold, the
p.Leu239Pro variant was unable to activate transcription
(Supplementary Figure S11). Next, we co-transfected wild-
type and variant MAFB into HEK 293T cells in varying ratios
(Figure 6a). Transfection with the p.Leu239Pro variant sup-
pressed wild-type transcriptional activity in a dose-dependent
manner. Western analysis showed that p.Leu239Pro is
expressed similar to the wild-type (Figure 6b). These data
indicate that p.Leu239Pro could exert a dominant-negative
effect and/or simply reduce the gene-dosage (haplo-
insufficiency).

Transactivation reporter assay for p.Leu239Pro
We next assessed the transactivation of the p.Leu239Pro
variant by measuring luciferase reporter activity under the
regulation of the monocyte-derived F4/80 promoter, the
functioning MAFB recognition element.11 While wild-type
MAFB and podocyte marker expression in glomeruli
Immunohistochemistry for MAFB and podocyte markers
(WT1, nephrin and podocin) using biopsied renal tissues
demonstrated that these proteins were exclusively expressed
in control glomerular podocytes. In the glomeruli from three

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Y Sato et al.: MAFB DNA-binding mutations in FSGS-DRS clinical investigation

Figure 3 | MAFB sequence analysis in 2 families with focal segmental glomerulosclerosis accompanying Duane retraction syndrome
(FSGS-DRS). (a) Family-S. (b) Family-Y. Polymerase chain reaction direct sequencing revealed that affected individuals (II-2 in Family-S and III-1
and III-2 in Family-Y) have a heterozygous T-to-C transition in codon 239 (c.716T>C, CTG to CCG), leading to the p.Leu239Pro substitution
(arrows). Clinically healthy family members are homozygous for the wild-type sequence, suggesting co-segregation of p.Leu239Pro with the
disease phenotype. The cDNA numbering of human MAFB is based on GenBank reference NM_005461.4. Squares represent males, and circles
represent females. Closed symbols indicate affected individuals, while opened symbols indicate unaffected individuals. A DNA sample was not
available from the deceased mother (II-2, diagonal line).

affected individuals (Family-S II-2 and Family-Y III-1 and III-
2), the number of MAFB- or podocin-positive cells was lower
than in controls (Figure 7, Supplementary Figures S12 and
S13, and Supplementary Table S4). Moreover, podocin
staining intensity in the glomeruli of affected patients was
diffusely weaker than in controls (Figure 7), suggesting that
podocytes are gradually depleted.
Glomeruli in mice harboring a p.Leu239Pro variant
To verify the pathogenicity of the Leu239Pro variant, we
generated mice carrying an Mafb p.Leu239Pro variant by use
of CRISPR/Cas9-mediated genome editing. Sixteen newborn
mice, which were homozygous for the Leu239Pro variant,
died within 48 hours after birth. The podocytes of homozy-
gous neonatal mice displayed incomplete differentiation with
failure to project their elaborate foot processes, suggesting
that the p.Leu239Pro variant impairs podocyte maturation
(Figure 8 and Supplementary Figure S14). In contrast, the
remaining 18 mice, which were heterozygous for p.Leu239-
Pro, were born healthy and survived without renal manifes-
tations up to 2 months of age. The podocyte abnormalities
observed in homozygous p.Leu239Pro neonates support our
conclusion that p.Leu239Pro could alter podocyte structure
and function and account for the glomerulosclerosis in FSGS-
DRS patients.
DISCUSSION
Relevance of MAFB p.Leu239Pro variant in FSGS-DRS
In the present study, we identified a dominant missense MAFB
variant as causing FSGS with DRS. Whole-genome sequencing
combined with kinship, haplotype, and CNV analyses revealed
a very rare example in which an identical MAFB p.Leu239Pro
substitution arose independently in 2 separate families. Func-
tional characterization with a luciferase assay and murine

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Figure 4 | Structural effects of the p.Leu239Pro variant on transcriptional regulation. Schematic models for the binding of the heteromeric
MafB/c-Fos complex to MARE are shown. Structures are depicted by a ribbon presentation (left panel) and magnified images of the
DNA-binding sites (right panel). (a) Frontal view versus 120 horizontal rotation. (b) Frontal view versus 45 vertical rotation. The heteromeric
complex of murine MAFB (light green) and c-Fos (blue) bound to the TRE-type MAFB regulatory element (T-MARE[b], 10 bp) of the target DNA
(gray, combined stick and semitransparent surface) is modeled according to PDB:2WT713. MABF and c-Fos dimerize through the leucine zipper
domains (Zip) and bind together into the 2 half-sites of T-MARE. Helix 3 (H3) of the basic region (BR) of MAFB binds to T-MARE. Lys240 and
Arg243 situated within helix 3 of the BR (blue letters) interact with the thymine bases (T) on the minus strand at positions 7 (T-7) and 6 (T-6),
respectively. The proximal helix-turn-helix motif encoded by the extended homology region (EHR) is thought to modulate the affinity and
selectivity of DNA binding of MAFB. Four leucine residues, 216 (in helix 1, H1), 224, 228 (in helix 2, H2), and 239 (in helix 3, H3), comprise the core
structure for correct folding of this ancillary DNA binding region. A Leu-to-Pro substitution at 239 is predicted to disrupt the van-der-Waals
forces among the side chains of the 4 leucine residues, likely impairing proper folding of the EHR. Figures were produced with PyMOL (http://
www.pymol.org).

model supports the hypothesis that a missense substitution in
the DNA binding domain impairs podocyte structure and/or
function through alteration in MAFB-mediated transcriptional
regulation. The clinical spectrum of FSGS-DRS and the
genotype-phenotype correlation caused by MAFB mutations
are consistent with previous observations both from human
and murine models.11,14,17
We concluded that a DNA-binding domain missense
MAFB p.Leu239Pro variant is responsible for the FSGS-DRS
phenotype for the following reasons. First, the p.Leu239Pro
variant is the only rare variant that fulfilled our criteria. It
affected a conserved amino acid position. Another candidate,
FGF20 p.Gly116Arg, was excluded because of its high minor
allele frequency in the normal population. Moreover, we did
not find any expression of FGF20 in developing abducens
nerves in the hindbrain (data not shown). The known DRS
gene SALL4 was also ruled out by both sequence and copy
number variant analyses. Second, the p.Leu239Pro variant co-
segregated with both FSGS and DRS phenotypes in 2 unre-
lated families. Third, structural modeling pointed to the
essential role of leucine 239, and the substitution to proline
may cause deleterious misfolding of the basic and/or DNA-
binding domain.13
Fourth, functional luciferase reporter assays using cultured
monocytes indicated that the p.Leu239Pro variant impairs the
transactivation of MAFB onto the F4/80 MAF-responsive
element through haploinsufficiency or a dominant negative
mechanism. Fifth, mutations in the DNA-binding domain

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Figure 5 | Domain structure and location of mutations in the DNA-binding domains. (a) Domain structure of MAFB. MAFB comprises 2
main domains, an N-terminal transactivation domain (TAD, orange) and a C-terminal DNA-binding domain (DBD). The latter consists of an
extended homology region (EHR, green), basic region (BR, purple), and leucine zipper (Zip, red). The MAFB mutations reported to date have
been exclusively clustered in the hot spot of TAD (arrows) and predominantly cause an osteolytic bone disease phenotype (multi-centric
carpotarsal osteolysis, MCTO). Amino acid numbering is based on NP_005452.2. (b) Sequence alignment of large MAF (MAFB and MAF) and the
locations of disease-causing mutations in the DBD. Leu 239 is located in the last residues within the C terminus of the EHR (arrow) and is
conserved among vertebrates (box). This lies immediately adjacent to the basic region, where boxed residues (blue) are involved in the DNA
binding of MAFB to its responsive element (MARE). Besides our MAFB p.L239P variant, 4 disease-causing DBD variants (p.Arg288Pro,
p.Lys297Arg, p.Arg299Ser, and p.Gln303Pro) are reported in the DBD for another large MAF paralog, MAF, in familial or isolated cataracts and/or
anterior segment dysgenesis of the eye20 and sensory touch defects.21 The mutated residues are indicated with boxes. The asterisk indicates
the position of p.Asn248Ser in krenu mice.23 Alignment was created using ClustalW. “:” indicates fully conserved, while “.” indicates partially
conserved. Chimp: Pan troglodytes; Cattle: Bos taurus; Mouse: Mus musculus; Frog: Xenopus tropicalis; Zebrafish: Danio rerio.

have been reported in MAF (formerly known as c-Maf),
another member of the large MAF family; and mutations in
the DNA-binding domain cause juvenile-onset cataracts20 and
touch insensitivity,21 while mutations in the transactivation
domain lead to multisystem developmental delay.22 Sixth,
previous mouse studies have demonstrated that MAFB is
expressed in affected tissues, including the glomeruli,11,23
developing rhombomeres 5 and 6,24,25 and the cochlea of
the inner ear.25 Moreover, genetically manipulated mouse
models with null and hypomorphic Mafb mutations showed
developmental defects in both kidney and hindbrain,11,23
thereby recapitulating the clinical features of FSGS-DRS.
Our observations support the idea that the MAFB DNA-
binding domain plays a key role in the development of
various tissues including the renal glomeruli and hindbrain,
from which the abducens and auditory nerves are derived.17,24
Mouse model of the Mafb p.Leu239Pro variant
To verify the pathogenic significance, we generated mice with a
p.Leu239Pro variant using CRIPSR-Cas9–mediated gene
editing. The podocytes of homozygous p.Leu239Pro neonates
were unable to complete the final step of differentiation, failing
to project cellular processes to the outer surface of the
glomerular capillaries.11,23 Instead, the glomerular capillaries
were lined with immature podocytes of columnar shape and
were obliterated by mesangial matrix expansion, thereby
exhibiting a diffuse pattern of mesangial sclerosis resembling
mice lacking slit membrane proteins.26,27 The perinatal
lethality is seemingly attributed to insufficient suckling and
food intake, similar to that reported in homozygous null or
p.Asn248Ser mice.11,23 In contrast, heterozygous p.Leu239Pro
neonates did not show any gross abnormalities, including renal
problems, up to 2 months of age. Further extended follow-up
will be necessary, however, to clarify whether the heterozygous
p.Leu239Pro variant could give rise to FSGS with age.
These results from genetically modified mice helped to
understand the effect of gene dosage on the phenotype. The
krenu mice, generated by chemical mutagenesis, harbor a
p.Asn248Ser mutation in the DNA-binding domain, and
display renal and hindbrain developmental defects.23 The
heterozygous krenu mutation only leads to subtle atrophy of
the cranial nerve nuclei, suggesting that p.Asn248Ser repre-
sents a mild hypomorphic allele.23 In contrast, another classic
Kreisler mouse (kr) which harbors an X-ray-induced Mafb
inversion, displays a total disruption of abducens nerve
nuclei.24,25 The phenotypes of our reported patients closely
resemble that of the krenu (missense) rather than classic kr
(null allele) mice in that renal involvement is more severe
than the hindbrain defects (abducens nerve VI and inner
ear).25 Moreover, similar dosage effects were also observed for

Kidney International (2018) 94, 396–407 403

clinical investigation
Figure 6 | Transactivation profiles and protein stability of the
p.Leu239Pro MAFB variant. (a) Co-transfection reporter assay. Wild-
type (WT) and p.Leu239Pro MAFB variant were transfected with the
luciferase reporter pGL-F4/80, containing a macrophage-derived
MAFB responsive element, into HEK 293 cells under varying molar
ratios. WT activation of the Luc reporter was significantly suppressed
in the presence of the p.Leu239Pro variant in a dose-dependent
manner, suggesting a mild dominant-negative inhibition and/or a
simple dosage-reduction effect (haplo-insufficiency) by p.Leu239Pro.
*P < 0.05, **P < 0.01 (Student t-test). Data from 3 independent ex-
periments in duplicate are shown as the mean  SD. (b) Western
analysis. HEK 293T cells were transfected with the same WT or
p.Leu239Pro MAFB expression vectors used in the luciferase assays
above. Cell lysates were transferred onto membranes, and protein
was detected using anti-human MAFB or b-actin antibodies. Both
wild-type and mutant MAFB were expressed at equivalent levels. To
optimize viewing of this image, please see the online version of this
article at www.kidney-international.org.
the kidney phenotypes. Classic heterozygous kr (kr/þ) mice
as well as homozygous krenu (kenu/kenu) mice exhibited effaced
podocyte foot processes and reduced podocin expression.11,23
In contrast, the classic homozygous kr (kr/kr) mice also dis-
played more severe developmental defects in renal agenesis.11
Mechanisms of FSGS and DRS phenotypes caused by MAFB
mutations
Most previously reported pathogenic variants causing DRS are
different from that identified in our 2 families. The disease-
causing MAFB mutations reported to date are of only 2
types: either C-terminal large deletions truncating the leucine
zipper17 or missense changes in the N-terminal transactivation
domain.14,28 The C-terminal deletions cause loss-of-function
404
Y Sato et al.: MAFB DNA-binding mutations in FSGS-DRS
and dominant negative effects, while missense variants are
thought to mostly act through a gain-of-function effect,
thereby generating unique phenotypes, including multicentric
carpotarsal osteolysis syndrome (MCTO).
Our expression experiments suggest that the p.Leu239pro
variant may simply lose its transcriptional activity (hap-
loinsufficiency) or may interfere with the wild-type in a
dominant negative fashion. However, our functional analysis
is limited to the luciferase assay experiments, and therefore it
remains speculative as to whether the phenotypic differences
among these patients arise from the distinct type or location
(domain) of the mutations. Other functional studies will be
necessary to elucidate the underlying pathomechanism and
biochemical properties of p.Leu239Pro MAFB, such as DNA
binding ability and affinity to the target-responsive DNA
elements.
Clinical spectrum of MAFB mutations
Recent studies of genetic DRS individuals in the presence or
absence of deafness showed that the development of hearing
loss is related to the type of MAFB C-terminal deletion: smaller
truncations that preserve the leucine-zipper dimerization ac-
tivity cause deafness, while larger truncations lacking the entire
DNA binding domain do not result in auditory deficits.17
These observations suggest that deafness may manifest only
when the gene dosage of MAFB is markedly reduced by
dominant-negative inhibition. However, the kidney disease
status was unknown in these patients with MAFB deletions.
The mechanisms by which MAFB mutations lead to FSGS
remain to be elucidated. Previously reported disease-causing
MAFB mutations exclusively clustered in the amino-
terminal region and caused MCTO, a rare autosomal domi-
nant skeletal disorder in which the cardinal feature is
osteolysis.14 Moreover, MCTO can occur with or without
FSGS. When FSGS coexists, renal diseases manifest with
variable expressivity in approximately 50% of all cases, likely
reflecting an incomplete penetrance of the disease allele.14,28
FSGS typically appears several years after the osteolysis has
manifested. Unlike MCTO patients, all of our patients dis-
played FSGS-DRS but lacked any bone disease. A question
that remains is how the p.Leu239Pro MAFB variant specif-
ically affects kidney and cranial nerve development, but not
monocyte-lineage differentiation (osteoclastogenesis).
Further study will be needed to clarify tissue-specific (e.g.,
renal) cofactors of MAFB and their combinatorial control of
gene expression.
Conclusion
In the present study, we demonstrated that a novel MAFB
DNA-binding domain missense variant causes unique FSGS-
DRS (with the addition of deafness in 1 patient). Transcrip-
tional activity of MAFB through the DNA-binding domain
appears to be essential for early development of the glomeruli
as well as the hindbrain from which cranial nerves VII and
VIII originate. Further study with MAFB coactivating part-
ners and their recognition elements will aid our
Kidney International (2018) 94, 396–407
Y Sato et al.: MAFB DNA-binding mutations in FSGS-DRS clinical investigation

Figure 7 | Immunohistochemistry for MAFB and podocin in glomeruli of patients with the MAFB p.Leu239Pro variant. Sections of
biopsied renal specimens from members of FSGS-DRS Family-Y (III-1 and III-2) and Family-S (II-2) were stained using antibodies against MAFB
and podocin, a known podocyte marker. Signals were detected using a horseradish peroxidase-conjugated secondary antibody and
3,30 -diaminobenzidine (DAB) reagent. As controls, glomerular staining for 3 unrelated patients with minimal change nephrotic syndrome are
shown. Compared with controls, the number of nuclei (indicated by MAFB) as well as capillary staining intensity along the slit membrane
(visualized by podocin) was reduced in podocytes of patients with the p.Leu239Pro variant, suggesting the presence of podocytopenia
and disorganized slit membrane complex. Bar ¼ 50 mm. To optimize viewing of this image, please see the online version of this article at
www.kidney-international.org.

understanding of the molecular mechanisms underlying
FSGS and congenital cranial dysinnervation disorders.29
METHODS
Whole-genome analysis and Sanger sequencing
This study was approved by the Ethics Committee on Human
Genome Analysis of Showa University, and informed written consent
was obtained from all patients and their family members. Genomic
DNA was extracted from peripheral blood or saliva by use of the
QIAamp DNA blood kit (Qiagen, Hilden, Germany) or the ORAgene
DNA OG-575 (DNA Genotek Inc., Ontario, Canada). Samples were
subjected to whole-genome analysis (HiSeq X Illumina) at Macrogen
(Seoul, Korea). A total of 761.7 Gb with a quality of $Q30 was
produced. On average, 670 million paired-end reads were obtained for
Family-S (33.55x coverage on average) and 722 million for Family-Y
(36.37x coverage; Supplementary Figure S4). Paired-end reads were
aligned to the hg19 GRCh38.p7 reference human genome using the
Burrows-Wheeler Aligner tool. The resulting SAM files were realigned
and recalibrated using the Genome Analysis Toolkit framework.
Variant calling was performed using the Unified Genotyper tools from
the Genome Analysis Toolkit. All identified variants were annotated
using ANNOVAR. Candidate pathogenic variations were screened
according to the public databases: dbSNP (Build 142), 1000 Genomes
(November 2010 data release), 10Gen Data Set (version 1.04), NHLBI
GO Exome Sequencing Project (ESP6500SI), or the Human Genetic
Variation Database.30 Segregation of MAFB variants was confirmed
using the Sanger method (Supplementary Table S3).

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clinical investigation Y Sato et al.: MAFB DNA-binding mutations in FSGS-DRS

Figure 8 | Histology of glomeruli in MafbL239P/L239P mice. Hematoxylin and eosin staining showing renal histology of (a) wild-type
(MafbWT/WT) and (b) homozygous p.Leu239Pro (L239P) neonatal mice (MafbL239P/L239P). The glomerular capillaries of homozygous L239P mice
were covered by a corona of immature, columnar-shaped podocytes and were obliterated upon mesangial matrix expansion. Bar ¼ 40 mm.
Transmission electron microscopy ultrastructure of capillary tufts is shown in (c) wild-type (MafbWT/WT) and (d) homozygous L239P neonates
(MafbL239P/L239P). The arrangement of podocyte foot processes in homozygous L239P mice is seriously distorted with nearly 100% showing
a flattened pattern, while those in wild-type mice are regularly interspaced. Bar ¼ 5 mm. Arrows indicate effaced foot processes. E, endothelial
cell; P, podocyte. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.

Transcriptional analysis of mutant MAFB
See Supplementary Methods.
Immunohistochemistry of renal specimens
See Supplementary Methods.
Generation of p. Leu239Pro Mafb mutant mice using CRISPR/
Cas9 technology
To create mutant mice carrying the p.Leu239Pro variant, we intro-
duced a c.716 T>C point mutation into the Mafb gene according to
our previously reported method31 with some modification. The
sequence (5’-TCCGCCGCTTCTGCTTCAGG-3’) was selected as the
guide RNA (gRNA) target. The gRNA was synthesized and purified
using the GeneArt Precision gRNA Synthesis Kit (Thermo Fisher
Scientific, Waltham, MA) and dissolved in Opti-MEM (Thermo
Fisher Scientific). In addition, we designed a 101-nt single-stranded
DNA oligonucleotide (ssODN) donor for inducing c.716 T>C; the
nucleotide C was placed between the 50-nt 50 - and 30 -homology
arms derived from positions 666 to 715 and 717 to 766 of the Mafb
gene, respectively. This ssODN was ordered as Ultramer DNA oligos
from Integrated DNA Technologies (Coralville, IA) and dissolved in
Opti-MEM.
Pregnant mare serum gonadotropin (5 units) and human chori-
onic gonadotropin (5 units) were i.p. injected into female C57BL/6J
mice (Charles River Laboratories, Kanagawa, Japan) with a 48-hour
interval, and unfertilized oocytes were collected from their oviducts.
We then performed in vitro fertilization with these oocytes and sperm
from male C57BL/6J mice (Charles River Laboratories) according to
standard protocols. Five hours later, gRNA (25 ng/ml), ssODN (100 ng/
ml) and GeneArt Platinum Cas9 Nuclease (100 ng/ml; Thermo Fisher
Scientific) were electroplated into 229 zygotes using the NEPA 21
electroplater (Nepa Gene, Chiba, Japan). The poring pulse was set to:
225 V, 2 ms pulse width, 50 ms pulse interval, and þ4 pulse number.
The transfer pulse was set to: 20 V, 50 ms pulse width, 50 ms pulse
interval, and 5 pulse number (attenuation rate was set to 40%). After
electroporation, 187 of the embryos were developed to the 2-cell stage
and all 2-cell embryos were transferred into the oviducts of pseudo-
pregnant ICR females; 50 neonates were born. Thirty-two of the
pups died within 48 hrs. We collected genomic DNA from the 32 dead
and 18 surviving founder mice.
To confirm the c.716 T>C point mutation induced by CRISPR/
Cas9, we amplified the genomic region including the target sites by
polymerase chain reaction with the following primers; MafBL239P
QC primer F: 5’-GCAACAGCTACCCACTAGCC-3’, and MafBL239P
QC primer R: 5’-GCCCTCTTCTTAGCCAAGGT-3’. The polymerase

406 Kidney International (2018) 94, 396–407

Y Sato et al.: MAFB DNA-binding mutations in FSGS-DRS
chain reaction products were sequenced using the BigDye Termi-
nator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific) and
MafBL239P QC primer F.
DISCLOSURE
All the authors declared no competing interests.
ACKNOWLEDGMENTS
We are grateful to all of the patients and their family members who
participated in our study. We would like to thank Prof. So Iwata
(Department of Cell Biology, Graduate School of Medicine, Kyoto
University) for advice and review of the manuscript. We thank Drs.
Kosuke Higashida (Department of Pediatrics, Yamanashi University,
Yamanashi, Japan) and Hiroaki Yamanaka (Tokyo Renal Pathology
Institute, Tokyo, Japan) for assistance in collecting clinical information
and for pathological review, respectively. This work was supported by
Grants-in-Aid for Scientific Research (C) (26461246, 17K09719 to HT)
from the Japan Society for the Promotion of Science.
SUPPLEMENTARY MATERIAL
Supplementary Methods.
Figure S1. Ocular phenotype in affected individuals from 2 FSGS-DRS
families.
Figure S2. Renal pathology in a FSGS-DRS patient from Family-S.
Figure S3. Renal histology of FSGS-DRS patients from Family-Y.
Figure S4. Mapping statistics and coverage of WGS.
Figure S5. Filtering strategy for WGS variants in 2 families.
Figure S6. Filtering process for WGS variants in Family-S under the
recessive model.
Figure S7. Filtering process for WGS variants in Family-S under the
dominant de novo model.
Figure S8. Filtering process for WGS variants in Family-Y under the
dominant transmission model.
Figure S9. Kinship analysis of FSGS-DRS families.
Figure S10. Haplotype analysis around the MAFB p.Leu239Pro variant
in Family-S.
Figure S11. Transactivation profiles of the p.Leu239Pro MAFB variant.
Figure S12. Immunohistochemistry for WT1 and nephrin in glomeruli
of patients with the MAFB p.Leu239Pro variant.
Figure S13. Co-immunofluorescence of MAFB with WT1 in human
glomeruli.
Figure S14. Ultrastructure and immunofluorescence staining of
glomeruli in MafbL239P/L239P mice.
Table S1. Clinical phenotypes of members from FSGS-DRS Family-S
and Family-Y.
Table S2. Lists of candidate genes identified by filtering process.
Table S3. Primer design for PCR direct sequencing of human MAFB
gene.
Table S4. Density of MAFB-positive nuclei per glomerulus between
control and affected individuals with FSGS-DRS.
Supplementary material is linked to the online version of the paper at
www.kidney-international.org.
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