Unique X-linked familial FSGS with co-segregating heart block disorder is associated with a mutation in the NXF5 gene
Human Molecular Genetics
Unique X-linked familial FSGS with co-segregating heart block disorder is associated with a mutation in the NXF5 gene
Teresa Esposito 2
Rod A. Lea 1
Bridget H. Maher 1
Dianne Moses 1
Hannah C. Cox 1
Sara Magliocca 2
Andrea Angius 0
Dale R. Nyholt 6
Thomas Titus 5
Troy Kay 5
Nicholas A. Gray 4
Maria P. Rastaldi 3
Alan Parnham 5
Fernando Gianfrancesco 2
Lyn R. Griffiths 1
0 Institute of Genetic and Biomedical Research, National Research Council of Italy , Cagliari , Italy
1 Genomics Research Centre, Griffith Health Institute, Griffith University , Gold Coast, QLD 4222 , Australia
2 Institute of Genetics and Biophysics 'Adriano Buzzati-Traverso', National Research Council of Italy , Naples , Italy
3 Renal Research Laboratory, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico & Fondazione D'Amico per la Ricerca sulle Malattie Renali , Milano , Italy
4 Renal Services, Nambour Hospital , Nambour, QLD , Australia
5 Renal Department, Gold Coast Hospital , QLD , Australia
6 Queensland Institute of Medical Research , Herston Road, Herston, Brisbane 4006 , Australia
Focal segmental glomerulosclerosis (FSGS) is the consequence of a disease process that attacks the kidney's filtering system, causing serious scarring. More than half of FSGS patients develop chronic kidney failure within 10 years, ultimately requiring dialysis or renal transplantation. There are currently several genes known to cause the hereditary forms of FSGS (ACTN4, TRPC6, CD2AP, INF2, MYO1E and NPHS2). This study involves a large, unique, multigenerational Australian pedigree in which FSGS co-segregates with progressive heart block with apparent X-linked recessive inheritance. Through a classical combined approach of linkage and haplotype analysis, we identified a 21.19 cM interval implicated on the X chromosome. We then used a whole exome sequencing approach to identify two mutated genes, NXF5 and ALG13, which are located within this linkage interval. The two mutations NXF5-R113W and ALG13-T141L segregated perfectly with the disease phenotype in the pedigree and were not found in a large healthy control cohort. Analysis using bioinformatics tools predicted the R113W mutation in the NXF5 gene to be deleterious and cellular studies support a role in the stability and localization of the protein suggesting a causative role of this mutation in these co-morbid disorders. Further studies are now required to determine the functional consequence of these novel mutations to development of FSGS and heart block in this pedigree and to determine whether these mutations have implications for more common forms of these diseases in the general population.
Focal segmental glomerulosclerosis (FSGS) is a disease
affecting the glomerular podocyte where the pathology is focal (not
all glomeruli are involved), segmental (only part of a glomerulus
is involved) with the presence of sclerosis of the glomerular
capillary tuft that become more widespread with disease
progression. It is clinically characterized by proteinuria and a
progressive decline in renal function, eventually requiring renal
replacement therapy (1 – 6). Onset may be congenital, usually
in childhood, or may present in adulthood. FSGS accounts for
35% of all cases of idiopathic nephrotic syndrome and represents
∗To whom correspondence should be addressed at: Genomics Research Centre, Griffith Health Institute, Griffith University, QLD 4222, Australia.
Tel: +61 755528664; Fax: +61 755529081; Email:
†The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
‡The authors wish it to be known that, in their opinion, the final two authors should be regarded as joint Senior Authors.
a final common pathway for a variety of other renal and systemic
diseases (e.g. diabetic nephropathy) (1,3,5).
Familial forms of FSGS are linked to a number of genetic loci
(Supplementary Material, Table S1). Mutations in some of these
genes, for example NPHS1 and NPHS2, are also found in
sporadic cases of FSGS (7). Many of the FSGS mutations
have also been associated with syndromes involving non-renal
tissues, for example nail-patella syndrome and Denys-Drash/
Frasier syndrome. Genetic variation at the MYH9/APOL1
locus, which encodes the non-muscle myosin heavy chain and
apoprotein L-1 (chromosome 22), has also been associated
with an increased risk of hypertension and FSGS in
AfricanAmericans, but is not associated with familial FSGS (8 – 11).
The mutated genes in the familial forms of FSGS encode
podocyte proteins involved in the maintenance of glomerular
slit diaphragms, which in turn, are responsible for holding
back macromolecular structures like proteins within the vascular
bed. The affected proteins include nephrin (NPHS1), a cation
channel (TRPC6), a-actinin 4 (ACTN4), podocin (NPHS2),
formin (INF2), an adaptor protein (CD2AP), a non-muscle
class I myosin (Myo1E), a RhoA-activated Rac1
GTPase-activating protein (ARHGAP24) and a phospholipase C (PLCE1)
(12 – 20) (Supplementary Material, Table S1). Some of the
genes identified for familial cases of non-syndromal FSGS
may also be important in the more common, so-called, ‘sporadic’
versions of the disease (7 – 10).
To date, there are no reports in the literature of FSGS linkage
to the X chromosome. We have identified a large Australian
pedigree with FSGS and associated heart block which tends to
transmit these two diseases in an X-linked recessive manner.
FSGS and co-morbid heart block have not been previously
observed for familial FSGS and there is no clinical or
histological evidence of Fabry disease in this family.
To date, three genetic loci have been associated with
progressive familial heart block (PFHB1A, PFHB1B and PFHB2)
located on chromosomes 3p21, 19q13.2 – q13.3 and 1q32.2 –
q32.3, respectively. Mutations causing cardiac conduction
defects were found in the genes SCN5A (PFHB1A) SCN1B
and TRPM4 (PFHB1B); however, no association with FSGS
has been described at these loci (13,21 – 23).
We hypothesize that a novel mutation(s) in a gene(s) residing
on the X chromosome is responsible for causing FSGS and
comorbid heart block in this affected pedigree. Here, we provide
a full description of this new phenotype and describe a combined
approach of linkage and whole exome sequencing to identify the
genetic mutation associated with these co-segregating diseases
in this Australian pedigree.
Clinical characteristics of FSGS pedigree
The pedigree (Supplementary Material, Fig. S1) came to our
attention in 1998 after consultation with the proband (V:5(ID75)),
who was diagnosed to have FSGS at the age of 32. At the age of
42, he developed complete heart block and required permanent
pacemaker insertion. He was noted to be hypertensive at the
age of 48 in the context of a family history of hypertension.
Repeat renal biopsy at the age of 48 showed global sclerosis in
3/10 glomeruli. A further 3/10 glomeruli showed segmental
sclerosing lesions (Fig. 1). There was moderate tubular
atrophy and interstitial fibrosis and a mild lymphocytic
interstitial infiltrate. The interlobular arteries showed moderate
arteriosclerosis. Immunofluorescence showed finely granular IgA
deposition in the mesangium. Electron microscopy confirmed
frequent small mesangial immune deposits. Renal function
deteriorated and he started peritoneal dialysis at age 56. An
echocardiogram at age 57 was reported showing mild concentric
hypertrophy and a coronary angiogram was unremarkable. He
was diagnosed with prostatic carcinoma at age 57 and
obstructive sleep apnoea consequent to obesity at the age of 59. He is
currently maintained on haemodialysis.
In the six generations starting from the mid-1800s, with the
migration of a male from Britain, early death was a curse that
followed the males of this family. A total of 12 males are known to
have developed progressive decline in renal function. The causes
of premature death in three males (ID1, ID8 and ID12) from three
earlier generations who died in their second to fourth decade
cannot be confirmed; however, family histories suggest renal
failure. Four of the affected males in more recent generations
have died, one after pacemaker failure (IV:19(ID50)) and one
5 years after renal transplant (IV:26(ID62)). Two others had
presumed renal failure (III:5(ID14) and III:7(ID16)) and one of
these also had heart block (III:5(ID14)) (history from family,
death certificate or medical records). A histological diagnosis
of the renal pathology is unavailable for these four affected
males as they died prior to the development of renal biopsy as
a routine clinical investigation in the regions they were residing.
At present, there are five living males (V:1(ID69), V:5(ID75),
V:14(ID85), V:24(ID105), VI:7(ID136)) with FSGS. Biopsy
FSGS or renal Cardiac conduction
RF, renal failure; KT, kidney transplant; FSGS, confirmed by biopsy;
P, proteinuria; U, unknown; PM, pacemaker; HB, progressive heart block.
data were available for three of the five living individuals for this
study (V:1(ID69), V:5(ID75) and VI:7(ID136)).
At least six males suffering from kidney failure are also known
to have developed progressive cardiac conduction disorder
(ID14, ID16, ID50, ID69, ID75 and ID85) which started with
first degree heart block and eventually led to complete heart
block. Three of these individuals are deceased and the surviving
three have pacemakers. The two remaining individuals
diagnosed with FSGS (ID105 and ID136) have not shown symptoms
of heart block to date; however, these individuals are the
youngest affected males and may therefore still present with this
An additional living male (V:30(ID113)) presented with
firstdegree heart block and mild proteinuria at 20 years of age. This
individual may either have a less severe phenotype or may be
destined to proceed to the full syndrome with advancing age.
The male-only presentation of FSGS traits and associated
cardiac conduction disorder implies that an X-linked recessive
mode of transmission is acting in this pedigree. A summary of
clinical characteristics of affected males is shown in Table 1.
While female relatives are unaffected for the combined
phenotypes of progressive FSGS and cardiac conduction disease,
other potentially related clinical disorders have been observed
in females of this pedigree. Two daughters (IV:7(ID34),
IV:16(ID47)) of affected males have had proteinuria with mild
histological evidence of FSGS on renal biopsy, but have not
developed progressive renal disease by their sixth decade. An
additional two females (IV:23(ID55) and V:8(ID78)) have had
proteinuria and haematuria for three decades without
deterioration of renal function. Furthermore, pre-eclampsia has been
reported in 10 women (IV:7(ID34), IV:16(ID47), IV:23
(ID55), IV:24(ID59), V:10(ID80), V:11(ID82), V:13(ID84),
V:16(ID88), VI:9(ID139), VI:12(ID142)), including the two
presenting with mild non-progressive FSGS, with no residual
loss of renal function.
Other conditions noted in this family include clear cell renal
carcinoma in a male, transitional cell carcinoma of the renal
pelvis in a female who is a putative obligate carrier under the
X-linked model (she also has an affected son), a malignant
renal tumour in a young female and a renal ‘cyst’ in a putative
carrier who had mild FSGS on renal biopsy in her third
decade. A summary of the clinical phenotypes observed in
living relatives are detailed in Supplementary Material,
This family appears to be unique. There have been no reports
of any pedigree co-inheriting FSGS and progressive cardiac
conduction disorder, nor have renal tumours been associated with
either condition, other than a known increased risk of renal
malignancy in patients with established renal failure. Moreover,
there has not been a previous report of FSGS or associated
disease being transmitted in an X-linked fashion.
Only males exhibit the full form of both FSGS and progressive
heart block in this large multigenerational pedigree. The
transmission pattern is suggestive of an X-linked mode of inheritance.
To test this hypothesis, linkage analysis was performed using a
high density (3.8 cM) X-chromosomal scan. To prevent
model misspecification, non-parametric linkage methods were
employed. Two linkage scans were performed using subtly
different phenotypic models. The results of multipoint linkage
analysis are detailed in Figure 2A.
The first model included only those males who exhibited
complete FSGS (Model 1). Significant evidence of linkage was
detected at Xq markers, with a maximum LOD score of 2.72
(P ¼ 2 × 1024) observed at marker DXS1106. When the male
individual presenting with a milder phenotype was included as
affected (Model 2), the peak linkage signal at marker
DXS1106 increased to a LOD score of 3.32 (P ¼ 5 × 1025).
The upper and lower limits of the 95% confidence interval
were at 118 and 100 cM, respectively. This result suggests the
disease gene(s) must reside between markers DXS8077 and
DXS8064, encompassing a 21.19 cM interval spanning
Xq21.33 to Xq24.
Co-segregation of the FSGS phenotype with the Xq haplotype
is depicted in Figure 2B. A predisposing haplotype has been
inherited in all affected individuals. One unaffected individual
(VI:2(ID137) also shares the predisposing haplotype.
Subsequent to this analysis, clinical information has become available
showing that proteinuria has developed in this individual.
Recombination events in four affected individuals (IV:13(ID50),
V:1(ID69), V:2(ID75) and V:12(ID113)) reveal a shared
disease haplotype comprised of five markers, spanning
Xq22.1 – 23.
In order to exclude linkage of autosomal loci to the phenotypes
under investigation, 20 individuals (6 men from the youngest
generations, with 3 affected and 3 unaffected, and 14 females,
including 11 obligate carriers and 3 unaffected) from the
Australian pedigree were genotyped using the Affymetrix
GenomeWide Human SNP Array 6.0. While known FSGS-associated
genes have been previously sequenced in this family, this
approach enabled exclusion of potentially causative insertions
and deletions in these genes. Furthermore, this approach
confirmed the X-chromosome disease locus at the region between
markers rs394127 and rs10521569, spanning a genomic region
of 16 Mb.
This genomic region houses 145 genes (Supplementary
Material, Fig. S2) including interesting candidates that are
reported in Supplementary Material, Table S3. Recently, the
phenylalanine at codon 222 of the COL4A5 gene located within
this linkage interval was found mutated in a family showing
X-linked glomerulopathy (24). Direct sequencing of all coding
regions, including exon – intron boundaries, of the COL4A5
gene (and other candidates listed in Supplementary Material,
Table S3) did not identify any mutations in affected members
of the pedigree, therefore excluding these genes as causative.
FSGS gene identification strategy
We performed whole-exome sequencing of two affected males
from the Australian FSGS pedigree (ID69, and ID85). We
adopted a prioritization scheme, well detailed in Table 2 to
identify the pathogenic mutation in each individual, similar to the
approach taken by other recent studies (25 – 27). In a first step, we
performed a thorough survey of all previously identified FSGS
genes in order to definitively exclude their involvement in the
disease phenotype. After filtering (Table 2), we matched data
from both individuals to obtain shared variants. Eighty
autosomal non-synonymous coding variants were shared between
the two samples. Only 40 of these variants were not found in
the 1000 Genomes database and were confirmed by direct
sequencing in the two patient samples.
None of the autosomal variants showed complete segregation
with the disease in our family, adding support to the X-linked
inheritance of the disease as demonstrated by linkage analysis
Assuming that FSGS segregates as an X-linked disorder in our
family, we focused on X-linked hemizygous variants that were
not registered in dbSNPs or were not identified in our six
in-house exomes. We surveyed all genes of the X chromosome
in each individual with particular attention on the 20 Mb
region detected by linkage analysis (see Fig. 2A and
Supplementary Material, Fig. S2). We obtained coverage of more than 40×
for almost all exons, including exon – intron boundary and 5′ and
3′ untranslated regions (Supplementary Material, Fig. S3).
However, we found that the first exons in 12 different genes
located in the linkage interval were not covered by a suitable
number of sequences. To ensure that these regions were fully
interrogated, we also analysed by direct Sanger sequencing to
exclude the presence of variants in these exons. This sequencing
approach identified four X-linked candidate gene mutations for
ID69 and six X-linked candidate gene mutations for ID85.
ID69 + ID85
SNV: single nucleotide variation; ∗Non-synonymous variants obtained after to filter
with six in house genome (one healthy individual and five individuals with unrelated
diseases); Aut + 1000 genomes ¼ autosomal variants remaining after filtering with
1000 genome database; Aut + segregation ¼ Autosomal variants remaining
after segregation analysis in FSGS family; X + segregation ¼ X variants
remaining after segregation analysis; X + segr. + delet ¼ X variants remaining
after segregation analysis and after analysis of deleterious effect of the change
with SIFT and Polyphen 2.
Among them, only mutations in two candidate genes, the
Nuclear RNA Export Factor 5 (NXF5) gene and the
AsparagineLinked Glycosylation 13 (ALG13) gene, were common in the
two individuals and were located in the linkage candidate
region (Table 2).
The inheritance of the R113W variant in the NXF5 gene and
the T141L/ N121I variant in the ALG13 gene (T141L, isoform
2 NM_018466 and N121I isoform 3 NM_001039210) were
examined by Sanger sequencing in DNA of 56 individuals of
the family. This analysis confirmed that both variants segregated
with the disease phenotype in the pedigree. We then checked
these variants in the 1000 Genome database, and did not find
any observations of them. In addition, we analysed 598
chromosomes from unrelated healthy controls from the same
geographical origin. Both the R113W variant of the NXF5 gene and the
T141L/N121I variant in the ALG13 gene were absent in this
cohort, suggesting a role of either of these variants in the
complex phenotype observed in this family.
We then checked the linkage interval between the two
candidate genes (NXF5 and ALG13) for SNP alleles that were not
shared between the two affected samples. Approximately 1000
SNPs were tested between the two genes using an Affymetrix
chip assay and a deep sequencing approach; for these SNPs, all
alleles were shared between the two affected samples, providing
evidence of a single haplotype spanning these two genes in the
Nuclear RNA export factor 5 (NXF5) gene
Mutation analysis confirmed that the c.337C.T change in exon
7 of the NXF5 gene causes the R113W amino acid change
(Fig. 3A). Most variants underlying rare Mendelian diseases
either affect highly conserved sequences and/or are predicted
to be deleterious. For this reason, we analysed the R113W
variant with SIFT, PolyPhen2, PMut and Align GVGD
html, http://genetics.bwh.harvard.edu/pph2/, http://mmb2.pcb.
ub.es:8080/PMut/; http://agvgd.iarc.fr/agvgd_input.php) to
confirm a deleterious effect of this variant. At this position
(R113), only arginine (R), lysine (K) or glutamine (Q) is
tolerated (Supplementary Material, Fig. S4). PolyPhen-2, PMut and
Align GVGD analysis supported the notion that this variant is
‘damaging’ with a probability score of 0.67 (PolyPhen2) and
prediction Class C65 (GV: 0,00; GD: 101,29 Align GVGD).
Three transcripts (ENST00000473265, ENST00000361708
and ENST00000537026) are described in the Ensembl genome
browser (http://www.ensembl.org) for the human NXF5 gene,
so to perform the expression profiling of these three transcripts
of the NXF5 gene, we designed four sets of primers specific for
each isoform. The expression profile was analysed using a
panel of adult human tissues using a real-time PCR assay. A
different expression profile was observed for the different isoforms;
significant expression was detected in the brain, pancreas,
kidney, testes, ovary and lung. A low expression level was
detected in heart and no expression was detected in muscle
(Fig. 3B). No difference of expression of the NXF5 gene was
observed in cDNA samples derived from patients blood
(Fig. 3C). Direct sequencing of the PCR fragments showed
that the mutated allele is expressed also in female carriers,
suggesting that there is not skewed X inactivation (Fig. 3C). These
data could explain the mild phenotype that we observed in
To assess the expression of this gene in podocytes, an assay
was designed to detect the Nxf7 gene (the mouse homologue of
NXF5) using RNA obtained from mouse podocytes as human
podocyte tissue was unavailable. Comparable levels of
expression were observed for all tissues examined (Fig. 3D).
The NXF5 gene belongs to the NXF gene family which, in
humans, also includes the NXF1 located on chromosome 11,
as well as NXF2 and NXF3 genes clustered on Xq22.1.
Evolutionary conservation analysis shows high similarity for these
proteins. NXF3 seems to be the most ancient gene and, together
with NXF1 and NXF2, is conserved in all mammals (Fig. 3E).
Nxf7 is present only in mice and rats and was lost in primates,
and NXF5 is a recently acquired gene present only in
chimpanzees and humans. Some authors propose that the human NXF5
gene is the analogue of the mouse Nxf7 gene (28). Interestingly,
the R113W amino acid change is conserved in all genes and all
species except the NXF3 gene, in which the arginine (R) is
replaced with glutamine (Q) (Fig. 3E). However, glutamine is
one of the three amino acids that SIFT predicts to be tolerated
in this protein position.
To assess if the NXF5-R113W mutation affects the stability
and/or the subcellular localization of the protein, Human
embryonic kidney (HEK)-293 and Human kidney-2 (HK-2) human cell
lines were transiently transfected with wt or mutant constructs
pEGFP-NXF5-C1-113R; pEGFP-NXF5-C1-113W). The GFP
constructs gave a non-specific pattern of expression resembling
the empty pGFP-C1 vector, suggesting that this protein could be
modified by digestion. Preliminary data suggest that the
Histagged NXF5 wt (113R) protein is localized in the nucleus
and in the cytoplasm, including in the cellular processes and
shows a higher level of expression than the mutated protein.
The NXF5-113W protein localized mainly at the level of the
nuclear membrane, suggesting that this mutation could affect the
stability and the localization of the protein (Fig. 4A and B).
Rhodamine phalloidin treatment of the cells, which stains the
F-actin filaments, did not show differences in actin organization
when the cells were transfected with wt or mutant proteins
Asparagine-linked glycosylation 13 (ALG13) gene
In Ensembl, at least six transcripts of the ALG13 gene are
reported to be protein coding. The AC/TT nucleotide change
identified in the examined family is located in an intronic
region in isoform 1 (long isoform). In isoform 2
(NM_018466), the AC/TT variant is located in exon 6 at position
421 – 422 and causes the amino acid change T141L, while in
isoform 3 (NM_001039210), the AC/TT variant is located in
exon 6 at position 362 – 363 and causes the amino acid change
N121I. Mutation analysis confirmed the segregation of this
variant in all affected members of the FSGS family (Fig. 5A).
The missense change affects highly evolutionarily conserved
residues of ALG13 (Fig. 5B); however, SIFT software predicted
a tolerated effect for the T141L ALG13 variant, but the software
was not able to predict any effect for the N121I variant.
Polyphen-2 and PMut analyses supported this by showing a
very low probability of a damaging effect at this variant (P ,
0.05). However, the Align GVGD software predicts a damaging
effect of the ALG13-T141L variant (GV:0.00; GD: 92.35; Class
To perform expression profiling of the ALG13 gene, we
analysed a panel of adult human tissues using a real-time PCR
assay designed to specifically amplify the three ALG13 isoforms.
All isoforms were highly expressed in the ovary, kidney,
pancreas, brain, testes, lung and heart. The short isoforms 2 and 3
were also highly expressed in liver, and low expression levels
were detected in muscle (Fig. 5C). No difference of expression
was observed for both isoforms in cDNA samples derived
from patients’ blood (Fig. 5D). Podocyte expression was
performed using RNAs from mouse tissue. Alg13 showed highest
expression in the mouse podocyte from the panel (Fig. 5E).
To assess any functional effect of the ALG13-T141L
mutation, human HEK-293 and HK-2 cell lines were transiently
transfected with wt or mutant constructs. Preliminary data do not
show any difference between wt and mutant proteins in the
level of expression and cytoplasm subcellular localization,
suggesting that this mutation does not impair the stability or the
localization of the protein and does not affect actin organization
within the cell (data not shown).
Overall, the complete absence of the R113W mutation in the
NXF5 gene in the healthy control cohort coupled with the in
silico prediction of a deleterious effect provides strong evidence
that the R113W mutation in the NXF5 gene could be the
causative disease mutation. In addition, however, the T141L/N121I
mutation in the ALG13 gene cannot be ruled out completely
despite three in silico predictions (SIFT PMut and PolyPhen2)
that this mutation has a low probability of causing a damaging
effect. Consequently, this mutation may also contribute a
small effect on disease phenotype.
FSGS, one of the most common glomerulopathies, is typically
marked by massive proteinuria and often associated with
unremitting nephrotic syndrome and inexorable progression to
endstage kidney disease (2,4,6). Unfortunately, immunosuppressive
therapy is frequently unsuccessful in the inherited forms of
Podocyte dysfunction is critical to the development of FSGS.
Previously identified causal mutations in FSGS have shown the
importance of a number of cytosolic genes encoding proteins
that influence the podocyte cytoskeleton (e.g. ACTN4, which
encodes alpha-actinin-4; MYH9, which encodes myosin heavy
chain 9; INF2, which encodes inverted formin 2; and MYO1E,
a gene that encodes myosin 1E, a non-muscle class I myosin).
Through the exome sequencing strategy, we identified a novel
causative mutation (R113W) in the NXF5 gene. In eukaryotes,
the nuclear export of mRNA is mediated by nuclear export
factor 1 (NXF1) receptors. Based on the extensive structural
similarity of NXF family receptors, both within and across
species, they are thought, like TAP/NXF1, to be involved in
mRNA metabolism. For some of them, such activity has been
confirmed by mRNA export assays (human NXF2), whereas
the others (such as human NXF3) were inactive in these assays
In mice, Nxf2 and Nxf7 are analogues of human NXF2 and
NXF5, respectively. Tretyakova et al. (28) showed that Nxf2
has properties similar to those of TAP/NXF1; in contrast, Nxf7
has cytoplasmic RNA transport factor properties. The study
further showed that Nxf7 binds to the light chain (L chain) of
the brain-specific microtubule-associated protein MAP1B,
which interacts with cytoplasmic microtubules and actin
filaments. Subcellular localization of mouse Nxf factors showed
that GFP-Nxf7 was found only in small cytoplasmic foci, both
in the cell body and in the neurites; it was excluded from the
nucleus. GFP-Nxf7 formed mobile particles in the neurites,
and a fraction of them moved unidirectionally. In both cell
types, both anterograde and retrograde translocations were
observed. The involvement of cytoskeletal components in
Nxf7 particle motility was analysed with a short-duration
colchicine treatment. This completely abolished the motility,
indicating the involvement of microtubules. Our preliminary cellular
studies show that the wt human NXF5 protein is localized in
the nucleus and in the cytoplasm, including in the cellular
processes, likes those observed for NXF2 (28). Moreover, the
R113W mutation was observed to potentially affect the stability
and the localization of the protein. Additional functional studies
will be necessary to establish its role in the disease phenotype.
A number of papers show that deletion or duplication in the
NXF gene cluster, in particular, loss or overexpression of the
NXF5 gene, cause severe mental retardation in men (31 – 34).
Mutation analysis of the NXF5 gene and its neighbouring
homologue, the NXF2 gene, did not identify causative
mutations in 45 men with various forms of syndromic X-linked
MR (XLMR) or in 70 patients with non-specific XLMR
(32). Our patients do not show any cognitive impairment;
however, it is not rare that different mutations in the same
gene cause different phenotype manifestations. Furthermore,
genes expressed in different tissues, when mutated, show
alteration only in specific tissues. Mutations involving WT1, a
tumour suppressor gene, lead to FSGS with multi-faceted
clinical expressions such as Frasier syndrome (FSGS, XY
hermaphroditism and high risk of gonadoblastoma) (35) and
Bilateral Wilm’s tumour associated with FSGS (36). Abnormal
splice variants of WT1 have also been associated with
nonsyndromic FSGS. Again, in an analogous manner, mutations
affecting the Chromatin bundling protein (SMARCAL1)
cause Schimke syndrome (immuno-osseous dysplasia) (37)
which has FSGS as one of the components.
It is interesting to note that a number of women within this
pedigree also reported pre-eclampsia. There are a number of
publications in the literature that link pre-eclampsia with FSGS. Some
have suggested that the occurrence of pre-eclampsia in families
with FSGS may be indicative of being a carrier of FSGS genetic
variants (38). Other detailed investigations of pre-eclampsia
often identify FSGS lesions in the biopsies of patients. In this
case, there is significant debate regarding whether pre-eclampsia
causes FSGS or whether FSGS is a predating condition (39).
Nagai suggests that FSGS-like lesions occur in pre-eclampsia
but may not be progressive as proteinuria resolves after pregnancy
(40). Similarly, Unverdi et al. (41) recently concluded that
persistent proteinuria after delivery is a predictor of an underlying renal
disease. The information we have available on the family shows
a common occurrence of pre-eclampsia suggesting a genetic
contributor. Thus while the occurrence of pre-eclampsia in this family
may support the ‘carrier’ theory, it is difficult to conclusively
determine as not all women with pre-eclampsia have had sons and
some women in earlier generations do not report pre-eclampsia.
The significance of the mutation in the ALG13 gene, which is
an N-linked glycosylase is unclear. It is tempting to postulate a
role for this mutation with the presence of IgA mesangial
deposits, as defective glycosylation of IgA molecule is postulated to be
one of the pathogenetic factors underlying IgA nephropathy
(42). Again defective glycosylation of ion channel conductance
proteins has been shown to underlie inherited cardiac conduction
defects (43). However, preliminary studies in our lab do not
suggest a functional significance for this mutation.
The pathogenesis of the underlying cardiac conduction defect
is a matter of speculation. Clinical observation suggests that the
initial manifestation is first degree atrioventricular block with
prolongation of P-R interval on the electrocardiogram. This
appears to progress to third degree atrioventricular block with
atrioventricular dissociation leading either to death in the
earlier generations or needing cardiac pacemaker insertion in
the current generations. One has to speculate that the same
defects in the podocyte are likely to be the mechanisms
leading to dysfunctional cardiac conduction, consequent to
abnormalities in protein trafficking from the NXF5 gene
Our expression studies, performed on RNA derived from
patients’ blood, show that the mutation does not alter the
expression or stability of the NXF5 transcript. In addition, the
expression of the mutated allele in the female carriers demonstrates that
there is no skewing of X inactivation, this may therefore explain
the mild phenotype observed in the female carriers.
We propose that the R113W mutation in the NXF5 gene
identified in the examined FSGS family alters the biological function
of the protein specifically in podocytes and in cardiac
HISPurkinje system, producing an alteration in mRNA transport
through cellular body and along the extensions.
MATERIALS AND METHODS
The study protocol was approved by the Griffith University
Human Research Ethics Committee. The study complies with
the Australian Health Ethics Committee (AHEC) and the
National Statement on Ethical Conduct in Research Involving
Humans (National Health and Medical and Medical Research
Council) guidelines. All subjects gave informed consent prior
to participation. In the case of minors, parental consent was
obtained. A single, large, multigenerational Australian pedigree
was utilized in this investigation and is depicted in
Supplementary Material, Figure S1. Clinical details were gathered by
screening medical records (where available), patient interviews
and telephone interviews with treating physicians. Cause of
death of deceased individuals was obtained either through
interview or examination of family records and death certificates
where available. Histological details were obtained from
hospital records where available. Phenotypic data were available
for 89 individuals (26 males and 63 females) ranging in age
from 4 to 94 years old. All individuals were of Caucasian
ancestry. DNA was available for 75 individuals (20 males; 56
females). The sampled individuals included four severely
affected males and an additional mildly affected male with
firstdegree heart block.
DNA and genotyping
For all participants over the age of 18 years, DNA was extracted
from peripheral blood by means of a QIAmpw DNA Maxi Kit
(Qiagen). For participants aged under 18 years, DNA was
collected from buccal swaps and isolated using a QIAmpw DNA
Mini Kit (Qiagen). Genotyping was performed at the Australian
Genome Research Facility (AGRF), Melbourne. A total of 47
highly polymorphic microsatellite markers with an average
density of 3.8 cM (minimum gap between markers 0.26 cM;
maximum gap between markers 11.586) were genotyped
across the entire X chromosome. Markers were amplified by
PCR using fluorescently labelled primer pairs. All PCRs were
performed in a total volume of 6 ml, using a PTC-225 DNA
Engine Tetra (MJ Research Inc., Waltham, MA, USA) under
standard conditions. PCR products were multiplexed and
electrophoresed on a 3730 DNA Analyzer (Applied Biosystems).
Data were analysed using Applied Biosystems Genescan
version 3.1 and Genotyper version 2.1 software.
We also used Affymetrix Genome-Wide Human SNP Array
6.0, containing more than 1.8 million genetic markers, including
more than 906 600 single nucleotide polymorphisms (SNPs) and
more than 946 000 probes for the detection of copy number
variation (CNV). Genotypes of each SNP were generated with
Birdseed v2. Quantile normalization was performed at probe level on
the whole data set (sample + 240 references). For each single
marker (SNP or CNV), the ratio in log 2 scale between the
sample and reference set was then calculated.
Sex-averaged map positions were generated via locally
weighted linear regression from NCBI build 35.1 physical map
positions and the Rutgers genetic map (45). Pedstats version
0.6.8 was used to detect typing errors (46). Erroneous genotypes
were removed prior to analysis. Multipoint non-parametric
linkage analysis was undertaken using MINX version
1.1-alpha3 at 1 cM increments (46). LOD scores were calculated
according to the Kong and Cox exponential model (47).
Haplotyping was performed in MINX and results were graphically
displayed using Progeny version 5 (http://www.progenygenetics.
com/clinical/pedigree.html). MINX has a bit size limit of 24.
Due to this computational constraint, the original pedigree was
trimmed to facilitate analysis. The final trimmed pedigree
structure was of 22 bit complexity (41 individuals; 22 males; 19
females) and contained all necessary individuals to examine
the hypothesis of an X-linked recessive disease locus. DNA
was available for 26 (12 males; 14 females) pedigree members
individuals. Two genetic models were examined. In model 1, a
total of 10 males (5 with DNA available) were classed as
affected. In model 2, the male individual presenting with a
milder version of the phenotype (ID113) was also classed as
affected (11 affected individuals; 6 with DNA available).
Whole-exome sequencing strategy
Genomic DNA samples from two affected males belonging to
the Australian FSGS pedigree (ID69, and ID85) were assayed
using the NimbleGen SeqCap EZ ExomeTM capture kits
(Roche) and resultant fragments sequenced with one lane per
sample on an Illumina GAIIx (Illumina, San Diego, CA, USA)
with 90 bp paired-end reads. A total of 44 363 000 (ID69) and
53 937 532 (ID85) paired-end reads were obtained and aligned
to the human reference genome sequence (GRCh37/hg19)
with MAQ7 and NextGENe software v2.00 with sequence
condensation by consolidation (SoftGenetics, State College, PA,
USA). This approach resulted in a coverage of all exons
(including exon – intron boundaries) and 5′ and 3′ untranslated regions
of more than ×40. Supplementary Material, Figure S3 depicts
coverage of part of the COL4A5 gene located in the linkage
interval. Single nucleotide variants (SNVs) were called with MAQ
and NextGENe. Small insertions and deletions were detected
with NextGENe. Called SNVs were annotated with SeattleSeq
Annotation and were filtered with dbSNP131.
Validation and segregation analysis of the selected variants was
performed by Sanger sequencing. PCR amplification and direct
sequencing protocols have been previously described (48).
To examine the tissue-specific expression pattern of the NXF5
gene, real-time PCR was performed on total RNAs from
human adult tissues purchased from Stratagene, with the
LightCycler system DNA Engine Opticon 2 (MJ Research). One
microgram of total RNA was reverse transcribed with the
Transcriptor HiFi cDNA Synthesis kit (Roche). qPCRs were
performed as described in previous paper (49). The expressions
were normalized versus glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) to account for differences in starting material
and cDNA reaction efficiency. Agarose gel electrophoresis was
performed to further confirm the specific PCR products.
NXF5 transcript primers were:
NXF5-1F: 5′ TTACTGCGCCCTACTCTGTG 3′
NXF5-1R: 5′ CCATGCAGTTTCTTGATT 3′
NXF5-2F: 5′ CCCAGAGAGCCAGAGCCTAA 3′
NXF5-2R: 5′ TGATCATGAGTGGGGCAAAT 3′
NXF5-3F: 5′ AAGGATGGTCTCAGGGTTCT.3′
NXF5-3R: 5′ AAGCCTGCACCATTTCCTGT 3′
NXF5-4F: 5′ AAGGAAGGTGGTGGTGTTTG 3′
NXF5-3R: 5′ AAGCCTGCACCATTTCCTGT 3′
The last primer was used both with the primer NXF5-3F and with
the primer NXF5-4F.
The GAPDH primers were forward primer 5′
AGCCACATCGCTCAGACAC 3′ and reverse primer 5′
Twenty-two transcripts are reported in Ensembl database (http://
www.ensembl.org/Homo_sapiens/Gene/Summary) for ALG13
gene, 6 of which are reported as protein coding. The AC/TT
nucleotide change identified in our family is located in the intronic
region in transcript 1 (long isoform) and in exonic region in
transcripts 2 and 3 (short isoforms). We designed three different
primer pairs, specific for the three isoforms to test their
expression profile. The expression pattern was examined by real-time
Kidneys were taken from mice aged between 7 and 10 days (max
15 days), and immediately immersed in culture medium
(DMEM F12 medium supplemented with 10% FCS, 5 ug/ml
transferrin, 10-7M hydrocortisone, 5 ng/ml sodium selenite,
0.12 U/ml insulin, 100 U/ml penicillin, 100 ug/ml streptomycin,
2 mM L-glutamine). The kidney was decapsulated, before
passing through a series of sieves of decreasing mesh size:
100, 75, 50 and 36 mm. Glomeruli were collected from the top
of the 36 mm sieves, centrifuged at 280g for 10 min at 48C.
The pellet was re-suspended in the medium and further manually
purified under a stereomicroscope by pipetting not only debris,
but also the remaining glomeruli still surrounded by the
Bowman’s capsule. The glomeruli were then seeded at 378C,
in 5% CO2 humidified atmosphere, in culture flasks precoated
with collagen type IV. After 7 – 10 days, glomeruli were ready
to be detached by treatment with trypsin-EDTA, followed by
filtering through the 36 mm mesh sieve to separate glomeruli from
primary podocytes cells. When cultures were passaged, 90% of
cells were identified as podocytes based on microscopy. These
cells were seeded onto thermonax plastic collagen cover slips
and characterized by immunocytochemistry (we used nephrin,
podocin, cytokeratin, CD31, alpha-SMA, CD45).
RNA preparation and RT-PCR assay were performed as
already described (49), using the following mouse specific
Supplementary Material is available at HMG online.
Conflict of Interest statement. None declared.
Alg13F-mouse: 5′ AGTCTGGAGAAAGGCAAACC 3′
Alg13R-mouse: 5′AATCCAACAACTTTATCCAA 3′
Nxf7F: 5′ TGTCCCAGACTTTCGCATTG 3′
Nxf7R: 5′ GCAGCTTCTGTGATTTAGGA 3′
Gapdh-F: 5′ TCCCTCAAGATTGTCAGCAA 3′
Gapdh-R: 5′ AGATCCACAACGGATACATT 3′
Plasmids preparation, cell culture and transfection
The NXF5-337C (wt) construct was purchased from
LifeSciences and the ALG13 cDNA clone (NM_018466) was
synthesized at GeneCust Europe. These clones were used as template
for the PCR reaction to generate the full-length cDNA fragments
to introduce in pcDNA3.1D/V5-His-Topo vector (Invitrogen)
and pEGFP-C1 and pEGFP-N2 vectors (Clontech). The
R113W (NXF5) and T141L (ALG13) mutagenesis were
carried out by the Quick Change II XL site-directed mutagenesis
technique according to the manufacturer’s instructions
(Stratagene). The whole coding sequences (NXF5 plus GFP, NXF5
plus His-Tag, ALG13 plus GFP and ALG13 plus His-Tag) of
This work was supported by the Short-term Mobility Program
from the Italian National Research Council to T.E., a Corbett
Fellowship to R.A.L., a National Health and Medical Research
Council Postgraduate scholarship to H.C.C., a National Health
and Medical Research Council Fellowship (339462 and
613674) and an Australian Research Council Future Fellowship
(FT0991022) to D.R.N. Research was also supported by a
Griffith University Research Grant.
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