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Yuval Ramot1, Abraham Zlotogorski1, Maurice van Steensel2,3,4
1 Department of Dermatology, Hadassah - Hebrew University Medical
Center, Jerusalem, Israel
2 School of Medicine and School of Life Sciences, University of
Dundee, United Kingdom
3 Institute of Medical Biology, Singapore
4 Lee Kong Chian School of Medicine, Nanyang Technological
University, Singapore
In their recently published article, Shah et al. claim that a
homozygous loss-of-function mutation in KRT83 leads to recessive
progressive symmetric erythrokeratoderma.1 However, since KRT83 encodes a
hair-specific keratin, we believe it is highly unlikely that a mutation in
this gene would cause a strictly epidermal phenotype.
Keratins are intermediate filaments that provide structural support
to epithelial cells, in addition to other biological properties.2-4 They
are unique in that each has a very specific expression pattern, which has
been studied extensively.5 To date, mutations in hair keratins have all
been associated with phenotypes restricted to the hairs and nails.6
Specifically, mutations in KRT83 had been linked exclusively to
monilethrix.7,8
Progressive symmetric erythrokeratoderma and erythrokeratoderma
variabilis do not present with a hair phenotype. These strictly epidermal
disorders are presently grouped together as erythrokeratodermia variabilis
et progressiva (OMIM #133200) because they have the same molecular basis -
mostly autosomal dominant mutations in GJB3 and GJB4.9 The encoded
proteins are highly expressed in the epidermis.9,10
In their discussion, the authors state that keratin K83 is also
expressed outside of the hair follicle. They claim that Kb23, which is the
rat ortholog for K83, is expressed in the whole skin, but the reference
that they cite has only demonstrated expression in the hair follicle, and
not the epidermis.11 The same is true for the claimed expression in the
sheep wool follicles, where the reference cited shows expression only in
the wool follicle, and not the epidermis.12 Shah et al. also claim that
according to the human protein atlas, K83 is expressed in the skin, but
actually, it was found only in the hair and not in the epidermis
(http://www.proteinatlas.org/ENSG00000170523-KRT83/tissue). Staining as
shown in the atlas is highly restricted, even though the antibody used in
the human protein atlas is likely to recognize additional hair keratins.
The expression atlas that is also cited in the article gives information
on expression patterns of genes (http://www.ebi.ac.uk/gxa/home), and is
not based on immunohistochemistry as stated by the authors. Since the skin
samples used for expression profiling in this database are of whole skin
and not the epidermis alone, they are likely to also include parts of hair
follicles. Obviously, expression of KRT83 could be observed in these
samples. In contrast, a large number of studies demonstrate robust,
specific and reproducible expression of K83 in the hair.5,13-15 Thus, we
feel that it is safe to conclude that K83 is hair-specific and is not
expressed in human epidermis.
We consider it unlikely that mutations in a keratin that is
exclusively expressed in the hair would cause a severe epidermal phenotype
that also involves the palms and soles, areas that are devoid of hair
follicles. If they did, there would be profound implications for our
understanding of keratins, and of skin biology in general. We believe that
such a provocative claim should have been accompanied by proper
demonstration that K83 is indeed expressed in the epidermis, and
functional evidence that its loss can lead to an epidermal-specific
phenotype.
References
1 Shah K, Ansar M, Mughal ZU et al. Recessive progressive symmetric
erythrokeratoderma results from a homozygous loss-of-function mutation of
KRT83 and is allelic with dominant monilethrix. J Med Genet 2016, Dec 13,
Epub ahead of print.
2 Ramot Y, Zlotogorski A. Keratins: the hair shaft's backbone
revealed. Exp Dermatol 2015; 24: 416-7.
3 Ramot Y, Paus R. Harnessing neuroendocrine controls of keratin
expression: a new therapeutic strategy for skin diseases? Bioessays 2014;
36: 672-86.
4 Ramot Y, Paus R, Tiede S et al. Endocrine controls of keratin
expression. Bioessays 2009; 31: 389-99.
5 Moll R, Divo M, Langbein L. The human keratins: biology and
pathology. Histochem Cell Biol 2008; 129: 705-33.
6 Ramot Y, Zlotogorski A. Molecular genetics of alopecias. Curr Probl
Dermatol 2015; 47: 87-96.
7 van Steensel M, Vreeburg M, Urbina MT et al. Novel KRT83 and KRT86
mutations associated with monilethrix. Exp Dermatol 2015; 24: 222-4.
8 van Steensel MA, Steijlen PM, Bladergroen RS et al. A missense
mutation in the type II hair keratin hHb3 is associated with monilethrix.
J Med Genet 2005; 42: e19.
9 van Steensel MA, Oranje AP, van der Schroeff JG et al. The missense
mutation G12D in connexin30.3 can cause both erythrokeratodermia
variabilis of Mendes da Costa and progressive symmetric
erythrokeratodermia of Gottron. Am J Med Genet A 2009; 149A: 657-61.
10 Ishida-Yamamoto A, McGrath JA, Lam H et al. The molecular
pathology of progressive symmetric erythrokeratoderma: a frameshift
mutation in the loricrin gene and perturbations in the cornified cell
envelope. Am J Hum Genet 1997; 61: 581-9.
11 Nanashima N, Akita M, Yamada T et al. The hairless phenotype of
the Hirosaki hairless rat is due to the deletion of an 80-kb genomic DNA
containing five basic keratin genes. J Biol Chem 2008; 283: 16868-75.
12 Yu Z, Wildermoth JE, Wallace OA et al. Annotation of sheep keratin
intermediate filament genes and their patterns of expression. Exp Dermatol
2011; 20: 582-8.
13 Langbein L, Yoshida H, Praetzel-Wunder S et al. The keratins of
the human beard hair medulla: the riddle in the middle. J Invest Dermatol
2010; 130: 55-73.
14 Langbein L, Schweizer J. Keratins of the human hair follicle. Int
Rev Cytol 2005; 243: 1-78.
15 Langbein L, Rogers MA, Winter H et al. The catalog of human hair
keratins. II. Expression of the six type II members in the hair follicle
and the combined catalog of human type I and II keratins. J Biol Chem
2001; 276: 35123-32.
We only became aware of the paper by Reinstein et al. after our
manuscript was published online. It is gratifying to know that we are not
the only group who has identified left ventricular non-compaction (LVNC)
in males with loss-of-function mutations in NONO.
Enabled by recent advances in sequencing technologies, genotypes from thousands of individuals are now available in online databases. While most of them aim to be the reference source of genotypes from healthy individuals, however, due to the lack of accompanying clinical data, geneticists now face the challenge of separating pathogenic mutations and rare polymorphisms. The fr...
Enabled by recent advances in sequencing technologies, genotypes from thousands of individuals are now available in online databases. While most of them aim to be the reference source of genotypes from healthy individuals, however, due to the lack of accompanying clinical data, geneticists now face the challenge of separating pathogenic mutations and rare polymorphisms. The frequency of pathogenic variants within a population database could be inflated by subjects who are not yet diagnosed or misdiagnosed. In particular, it is more challenging for geneticists working on late on-set neurodegenerative diseases, such as SCA40 (1), where pathogenic variants may lurk within the genome for decades, until gradual deterioration of symptoms could be observed. In the case of SCA34, only 63% of ELOVL4 L168F carriers demonstrated ataxia (2). This is possibly due to the relatively young age of carriers (less than 48 years old), and thus the ataxia symptoms take time to develop (2).
The ExAC database (3) is one of the largest genotype databases to date, which combines sequencing data from 60,706 unrelated individuals in cohorts such as National Institute of Mental Health Controls, schizophrenia, bipolar disorder, Tourette's syndrome, and The Cancer Genome Atlas (TCGA). This clearly shows that pathogenic variants can be found in ExAC, and thus it might not be suitable to be treated as the genetic background of healthy population. MacArthur et. al. (3) have clearly stated that ExAC was designed to be the reference data set for childhood-onset Mendelian diseases, and the subjects are free of known severe pediatric diseases only[1]. Therefore, it is questionable to claim a variant as rare polymorphism based on ExAC alone.
To illustrate the prevalence of neurodegenerative diseases markers in population genetics databases, we have screened all known markers of autosomal dominant Spinocerebellar Ataxia (SCA) and Spastic Paraplegia (SPG) other than SCA40 (Table 1). Altogether, 12 SCA markers and 6 SPG markers were found in the analysis with allele frequency ranging from 8.236e-06 to 0.0262. This reaffirms the notion that subjects with neurodegenerative diseases may exist in population genetics databases. It is known that the pathogenicity of a disease marker can change in different ethnicity background (4, 5). Ethnic specific modifiers may alter the penetrance of these markers, accounting for higher frequency of these markers in certain populations (5, 6). Since SCA40 marker induces apoptosis through JNK pathway activation (1), the relative activity of JNK1/2/3 and c-Jun could modulate the levels of apoptosis (7-9), regulating the penetrance of SCA40 marker as a result.
Besides, we believe the eLetter authors misinterpreted 1000 genome phase III frequency for CCDC88C R464H, since only R464C and R464S (rs371123543) could be found in the data according to NCBI 1000 Genome browser (Figure 1). To date, CCDC88C R464H is still unreported in 1000 Genomes phase III, 1KJPN (10), ESP6500, GoNL (11), SGVP (12), and UK10K ALSPAC/TWINSUK cohort (13). The absence of CCDC88C R464H in 1KJPN database, which curates the whole-genome sequences of 1,070 healthy Japanese individuals, clearly contradicts with the eLetter authors' claim that the variant is relatively common in Japanese control alleles. In our original study, we screened 199 local healthy subjects, but none of them harbours R464H. The eLetter authors also independently screened 24 local healthy subjects and 85 disease controls, yet the variant was only found in one patient with SOD1-associated autosomal dominant amyotrophic lateral sclerosis (ALS). Given the extremely low frequency of CCDC88C R464H variant in the population, it is unlikely that R464H represents a rare polymorphism. Of note, cerebellar ataxia was previously reported in an ALS case associated with SOD1 variants (14), so the cerebellar features of the patient containing both SOD1 and CCDC88C R464H variants merits further investigation.
To explore the possibility of finding additional disease markers in our patient samples after the publication of our study in 2014, we now reanalyzed the sequencing data using the latest human genome reference (GRCh38), updated BWA alignment tool (version 0.7.15) (15), GATK haplotypecaller variant caller (Version 3.5) (16), Clinvar database (build 20160831), dbSNP 147, and 1000 genome database phase 3, yet no other known pathogenic marker was further identified. In addition to our in-house variant prioritization method as outlined in our original study, we did not observe any discrepancy after cross-checking our results against KGGSeq results (17). Together with the support of multipoint parametric genetic linkage analysis, gene expression profile mining and functional impact predictions as described in our study, we believe we have gathered substantial in silico evidence to support CCDC88C R464H as a disease marker.
In addition to the in silico predictions that the CCDC88C R464H is a pathogenic mutation, here we provide additional functional evidence that the CCDC88C R464H activated the JNK-caspase pathway in the primary neuronal cells derived from the mammalian brain. We used the day-18 rat embryonic cortical neurons as our experimental cell model. Similar to the results we previously obtained from the human HEK293 cells, overexpression of the CCDC88C R464H induced the JNK hyperphosphorylation and caspase-3 cleavage in rat primary cortical neurons (Figure 2). This result therefore strengthened our hypothesis that CCDC88C R464H triggers apoptosis and contributes to the cerebellar atrophy in SCA40 patients.
Table 1--Screening of autosomal dominant Spinocerebellar ataxia and Spastic paraplegia markers in population genetics databases.
Figure 1--CCDC88C variants around Arg-464 position in 1000 Genome Phase III.
Figure 2--Overexpression of the CCDC88C R464H DNA construct induced JNK hyperphosphorylation and caspase-3 cleavage in rat primary cortical neuronal cells. Two independent trials (Set1 and Set2) were performed and the results are consistent.
References
1. Tsoi,H., Yu,A.C.S., Chen,Z.S., Ng,N.K.N., Chan,A.Y.Y., Yuen,L.Y.P., Abrigo,J.M., Tsang,S.Y., Tsui,S.K.W., Tong,T.M.F., et al. (2014) A novel missense mutation in CCDC88C activates the JNK pathway and causes a dominant form of spinocerebellar ataxia. J. Med. Genet., 51, 590-5.
2. Cadieux-Dion,M., Turcotte-Gauthier,M., Noreau,A., Martin,C., Meloche,C., Gravel,M., Drouin,C.A., Rouleau,G.A., Nguyen,D.K. and Cossette,P. (2014) Expanding the clinical phenotype associated with ELOVL4 mutation: study of a large French-Canadian family with autosomal dominant spinocerebellar ataxia and erythrokeratodermia. JAMA Neurol., 71, 470-5.
3. Lek,M., Karczewski,K.J., Minikel,E. V., Samocha,K.E., Banks,E., Fennell,T., O'Donnell-Luria,A.H., Ware,J.S., Hill,A.J., Cummings,B.B., et al. (2016) Analysis of protein-coding genetic variation in 60,706 humans. Nature, 536, 285-291.
4. Pollak,A., Skorka,A., Mueller-Malesi?ska,M., Kostrzewa,G., Kisiel,B., Waligora,J., Krajewski,P., O?dak,M., Korniszewski,L., Skarzy?ski,H., et al. (2007) M34T and V37I mutations in GJB2 associated hearing impairment: evidence for pathogenicity and reduced penetrance. Am. J. Med. Genet. A, 143A, 2534-43.
5. Mannan,A.U. (2008) Response to Martignoni et al. Am. J. Hum. Genet., 83, 128-130.
6. Smeets,C.J.L.M. and Verbeek,D.S. (2016) Reply: SCA23 and prodynorphin: is it time for gene retraction? Brain, 139, e43.
7. Ham,J., Babij,C., Whitfield,J., Pfarr,C.M., Lallemand,D., Yaniv,M. and Rubin,L.L. (1995) A c-Jun dominant negative mutant protects sympathetic neurons against programmed cell death. Neuron, 14, 927-39.
8. Yang,D.D., Kuan,C.Y., Whitmarsh,A.J., Rincon,M., Zheng,T.S., Davis,R.J., Rakic,P. and Flavell,R.A. (1997) Absence of excitotoxicity-induced apoptosis in the hippocampus of mice lacking the Jnk3 gene. Nature, 389, 865-70.
9. Kuan,C.Y., Yang,D.D., Samanta Roy,D.R., Davis,R.J., Rakic,P. and Flavell,R.A. (1999) The Jnk1 and Jnk2 protein kinases are required for regional specific apoptosis during early brain development. Neuron, 22, 667-76.
10. Nagasaki,M., Yasuda,J., Katsuoka,F., Nariai,N., Kojima,K., Kawai,Y., Yamaguchi-Kabata,Y., Yokozawa,J., Danjoh,I., Saito,S., et al. (2015) Rare variant discovery by deep whole-genome sequencing of 1,070 Japanese individuals. Nat. Commun., 6, 8018.
11. Francioli,L.C., Menelaou,A., Pulit,S.L., van Dijk,F., Palamara,P.F., Elbers,C.C., Neerincx,P.B.T., Ye,K., Guryev,V., Kloosterman,W.P., et al. (2014) Whole-genome sequence variation, population structure and demographic history of the Dutch population. Nat. Genet., 46, 818-825.
12. Teo,Y.-Y., Sim,X., Ong,R.T.H., Tan,A.K.S., Chen,J., Tantoso,E., Small,K.S., Ku,C.-S., Lee,E.J.D., Seielstad,M., et al. (2009) Singapore Genome Variation Project: a haplotype map of three Southeast Asian populations. Genome Res., 19, 2154-62.
13. Walter,K., Min,J.L., Huang,J., Crooks,L., Memari,Y., McCarthy,S., Perry,J.R.B., Xu,C., Futema,M., Lawson,D., et al. (2015) The UK10K project identifies rare variants in health and disease. Nature, 526, 82-90.
14. Yasser,S., Fecto,F., Siddique,T., Sheikh,K.A. and Athar,P. (2010) An unusual case of familial ALS and cerebellar ataxia. Amyotroph. Lateral Scler., 11, 568-70.
15. Li,H. and Durbin,R. (2009) Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics, 25, 1754-60.
16. McKenna,A., Hanna,M., Banks,E., Sivachenko,A., Cibulskis,K., Kernytsky,A., Garimella,K., Altshuler,D., Gabriel,S., Daly,M., et al. (2010) The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res., 20, 1297-303.
17. Li,M.-X., Gui,H.-S., Kwan,J.S.H., Bao,S.-Y. and Sham,P.C. (2012) A comprehensive framework for prioritizing variants in exome sequencing studies of Mendelian diseases. Nucleic Acids Res., 40, e53.
The report that a novel missense mutation in CCDC88C activates the
JNK pathway and causes a dominant form of spinocerebellar ataxia that
appeared in your Journal (1) is of great interest. Although we identified
the same heterozygous missense variation (c.1391G>A, p.R464H) as that
reported (1) in a Japanese patient with autosomal dominant cerebellar
ataxia (ADCA), we report here that this varia...
The report that a novel missense mutation in CCDC88C activates the
JNK pathway and causes a dominant form of spinocerebellar ataxia that
appeared in your Journal (1) is of great interest. Although we identified
the same heterozygous missense variation (c.1391G>A, p.R464H) as that
reported (1) in a Japanese patient with autosomal dominant cerebellar
ataxia (ADCA), we report here that this variation in the CCDC88C gene may
not cause ADCA.
The proband in our family is a 55-year-old female. She visited our
hospital because of unsteadiness of gait at age 49. Neurological
examination revealed mild limb and truncal cerebellar ataxia. Brain MRI
revealed cerebellar atrophy. Molecular analysis of the patient excluded
SCA1, SCA2, MJD, SCA6, SCA7, SCA8, SCA12, SCA17, SCA31, SCA36 and DRPLA.
We have followed her as an outpatient and the symptoms have progressed
mildly over the past six years.
The father noted unsteadiness of gait at age 50. His cerebellar
ataxia progressed and he died due to aspiration pneumonia at age 87. The
mother at age 85 and two sisters at ages 57 and 53 showed no neurological
deficits. Unfortunately, we could not get information on the father's
grandparents.
On whole exome sequencing, we identified a heterozygous variation
(c.1391G>A , p.R464H) in the CCDC88C gene as the most likely candidate
causative mutation because this location is highly conserved among species
(1), and bioinformatic analyses including Mutation Taster, PROVEN, and
SIFT predicted it to be disease-causing (1). Then we continued to examine
whether or not this mutation cosegregates with the disease in our
pedigree. Unexpectedly, both affected and unaffected individuals in our
family exhibited the same heterozygous variation on Sanger sequencing.
Moreover, there is a relatively high rate of this variation among Japanese
control alleles, and ExAC revealed 21 A alleles in a total of 119,910
chromosomes. In addition, the minor allele counts in 1,000 genomes and
HGVD were 0.05% and 0.3%, respectively. We checked whether or not
c.1391G>A could be a common variant in the local population in 24 local
healthy subjects and 85 disease controls. Although the variant was not
found in the former, it was found in a 48-year-old patient with a SOD1
mutation with autosomal dominant amyotrophic lateral sclerosis in the
latter. Thus, this heterozygous variation (c. 1391G>A, p.464H) in the
CCDC88C gene may not cause ADCA.
This study was approved by the institutional review board of
Yamanashi University, and informed consent was obtained from all
participating individuals.
Reference
1. Tsoi H, Yu AC, Chen ZS, Ng NK, Chan AY, Yuen LY, Abrigo JM, Tsang
SY, Tsui SK, Tong TM, Lo IF, Lam ST, Mok VC, Wong LK, Ngo JC, Lau KF, Chan
TF, Chan HY. A novel missense mutation in CCDC88C activates the JNK
pathway and causes a dominant form of spinocerebellar ataxia. J Med Genet
2014; 51: 590-595.
Response to e letter ID jmedgenet el; 2826 by Refaeli et al., dated
June14, 2016
A considerable amount of literature on the role of podocalyxin-like
(PODXL) protein in normal mammalian kidney functions and to a lesser
extent in mouse brain development is available. We agree that these
studies particularly, "Anuria, Omphalocele, and Perinatal Lethality in
Mice Lacking the Cd34-Related Protein Podocalyxin" by Doyonnas et...
Response to e letter ID jmedgenet el; 2826 by Refaeli et al., dated
June14, 2016
A considerable amount of literature on the role of podocalyxin-like
(PODXL) protein in normal mammalian kidney functions and to a lesser
extent in mouse brain development is available. We agree that these
studies particularly, "Anuria, Omphalocele, and Perinatal Lethality in
Mice Lacking the Cd34-Related Protein Podocalyxin" by Doyonnas et al (1)
and "Podocalyxin is a Novel Polysialylated Neural Adhesion Protein with
Multiple Roles in Neural Development and Synapse Formation" by Vitureira
et al (2) using podxl -/- mouse embryos and newborns have unequivocally
demonstrated the role of this gene in renal function and brain development
respectively. Doyonnas et al., (1) in their paper do mention that i)
anuria and renal failure is evidenced only postnatally as there is
possible maternal clearance in the embryos; ii) there is no evidence of
extensive proteinuria, which is characteristic of leaky podocyte
filtration in human nephrotic syndromes; and iii) more importantly, that
there were no developmental anomalies in the hematopoietic and vascular
endothelial cells which also express high levels of podocalyxin. The last
observation, authors hypothesized, may have been enabled through
functional compensation by other sialomucins such as CD34. Data on the
status of neural development in their newborn mice with identical genetic
background, which would have proved the effect of podxl deficiency in
these two vital processes is however missing in their paper. Overall, the
results from these mouse studies suggest that i) podxl protein is almost
ubiquitous in expression, multifunctional, may have varying interacting
partners and that functional/genetic redundancy exists.
In our opinion, these features of PODXL are exactly what the two mutation
studies in this gene one on autosomal dominant familial focal and
segmental glomerulosclerosis (FSGS) (3) and our study on autosomal
recessive juvenile parkinsonism (4) reflect. This is not unexpected
considering the different genetic backgrounds in different families.
Notably, in the paper by Barua et al, (3) i) the incomplete penetrance of
the private variant p.L442R (i.e., one individual carrying the mutation
but asymptomatic even at 53 years of age); ii) varying age of onset
ranging from early teenage to adulthood but developing end stage renal
disease between 2nd to 6th decade of life; iii) varying disease severity
across affected individuals in the family but certainly not post-
natal/newborn mortality (unlike in mice); and iv) rarity of mutations in
PODXL gene in 176 additional FSGS families; together with mutant PODXL
protein characterization in vitro which showed that the index mutation
results in dimerization but does not alter protein stability,
extracellular domain glycosylation, cell surface expression, global
subcellular localisation and interactions with its intracellular binding
partner ezrin are all the features of PODXL gene that need to be kept in
mind prior to assigning it an indispensable role in kidney function and
survival in humans. It is indeed appropriate that Barua et al (3) conclude
that rare variants with low statistical genetic evidence such as observed
in their study do not contribute significantly to glomerular disease in
humans.
Based on these observations, it is not unlikely that the index family
with ARJP with homozygous mutations predicted to result in loss of protein
did not manifest any symptoms of FSGS or any other kidney dysfunction,
based on the data of last clinical examination in 2009. As for the two
other concerns of Refaeli et al on the index mutation being unlikely of
germline origin and NGS errors, both can be disregarded as both the
parents are heterozygous for the index mutation (data not shown), and all
informative NGS data are validated by Sanger sequencing. Further, their
concern that mutations identified in unrelated PD cohort are just common
protein altering variants is also ruled out as these were not observed in
either our population or reported in any publically available large
databases and therefore, were functionally characterized in our study. As
for the demonstration of absence of PODXL RNA or protein, we have not been
able to do that due to non-availability of tissues. It may please be noted
that in the absence of clinical indications, it is difficult to obtain
ethical committee clearance for collection of target tissues.
In summary, from careful interpretation of the findings in the above
mentioned studies as well as abundant data in literature [for eg. See Liao
and Zhang, PNAS, 2008 (5)], it is clear that there are notable
differences between mice and humans in i) the developmental phenomena; ii)
functional compensatory mechanisms/genetic redundancy; iii) gene-gene
interactions; iv) tissue specific transcript expression etc. Though
experimental evidence for most of these aspects with specific reference to
PODXL is currently unavailable, such differences may be believed to be
useful to explain pleiotropy in PODXL [for eg. similar to that documented
in cystic fibrosis (6)], possible tolerance to null alleles enabling
apparently normal kidney function as witnessed in the ARJP family in our
study (4) and comparatively late age of onset of FSGS, incomplete
penetrance of the gene mutation etc. (3) and several other associated
features.
Finally, we would like to thank Rafaeli et al for their interest in our
work and giving us an opportunity to highlight some of the differences
which may exist between humans and mice in general and for role of
podocalyxin in particular. We firmly believe that findings from animal
models of human diseases are powerful tools but are not always true
representatives for understanding complete human health and disease
biology. Additional data as and when available on PODXL associated human
phenotypes would be most insightful for our enhanced understanding of the
role of PODXL in humans.
References:
1. Doyonnas R, Kershaw DB, Duhme C, et al. Anuria, omphalocele, and
perinatal lethality in mice lacking the CD34-related protein podocalyxin.
J Exp Med 2001;194(1):13-27.
2. Vitureira N, Andres R, Perez-Martinez E, et al. Podocalyxin is a novel
polysialylated neural adhesion protein with multiple roles in neural
development and synapse formation. PLoS One 2010;5(8):e12003.?
3. Barua M, Shieh E, Schlondorff J, et al. Exome sequencing and in vitro
studies identified podocalyxin as a candidate gene for focal and segmental
glomerulosclerosis. Kidney Int 2014;85(1):124-33.
4. Sudhaman S, Prasad K, Behari M, et al. Discovery of a frameshift
mutation in podocalyxin-like (PODXL) gene, coding for a neural adhesion
molecule, as causal for autosomal-recessive juvenile Parkinsonism. J Med
Genet 2016; 53(7):450-6.
5. Liao B, Zhang J. Null mutations in human and mouse orthologs frequently
result in different phenotypes. PNAS 2008;105(19):6987-92.
6. Sing C, Risser D, Howatt W, Erickson R. Phenotypic heterogeneity in
cystic fibrosis. American Journal of Medical Genetics 1982;13:179-195.
Conflict of Interest:
None declared
Dr. Sumedha Sudhaman1 Prof. Kameshwar Prasad2 Prof. Madhuri Behari2
Dr. Uday B Muthane3 Dr. Ramesh C Juyal4 Prof. BK Thelma1 1Department of
Genetics, University of Delhi South Campus, New Delhi, India 2Department
of Neurology, All India Institute of Medical Sciences, New Delhi, India
3Parkinson's and Ageing Research Foundation, Bengaluru, Karnataka, India
4Regional Center for Biotechnology, Faridabad, Haryana, India
Sudhaman, et al. Discovery of a frameshift mutation in podocalyxin-
like (PODXL) gene, coding for a neural adhesion molecule, as causal for
autosomal-recessive juvenile Parkinsonism. J Med Genet. 0:1-7, 2016.
In the February 2016 edition of the Journal of Medical Genetics,
Sudhaman et al(1) report the identification of a PODXL variant
(c.89_90insGTCGCCCC) as the causative mutation in an Indian fam...
Sudhaman, et al. Discovery of a frameshift mutation in podocalyxin-
like (PODXL) gene, coding for a neural adhesion molecule, as causal for
autosomal-recessive juvenile Parkinsonism. J Med Genet. 0:1-7, 2016.
In the February 2016 edition of the Journal of Medical Genetics,
Sudhaman et al(1) report the identification of a PODXL variant
(c.89_90insGTCGCCCC) as the causative mutation in an Indian family with
autosomal recessive juvenile parkinsonism (ARJP). They further claim that
the insertion mutation in exon 1 results in a frameshift and loss of
podocalyxin protein expression in disease-afflicted family members
(homozygous individuals). They subsequently identify three novel PODXL
variants (heterozygous missense mutations) in unrelated individuals with
Parkinson's disease (PD).
As the authors note in their report, we previously demonstrated that
Podxl-deficient neurons (derived from Podxl-/- mouse embryos) display
defective neurite branching in vitro(2). Thus, we concur with Sudhaman et
al that the loss of PODXL expression from neurons or other functionally
disruptive mutations in PODXL may alter neurite growth and branching.
However, we have also shown that germline loss of Podxl in mice leads to
anuria and death less than 24 hours after birth(3). This lethal Podxl-null
phenotype is the result of a failure to produce podocyte foot processes
and filtration slits during kidney morphogenesis. In 2014, Barua et al(4)
reported a missense mutation in an exon encoding the transmembrane domain
of podocalyxin as a cause for autosomal dominant focal segmental
glomerulosclerosis (FSGS). Affected individuals presented with renal
symptoms in the second decade of life eventually progressing to end-stage
renal disease requiring dialysis or renal transplant(4). Thus, these
previous studies in human and mouse have underscored the indispensible
role for PODXL in normal mammalian kidney function and survival.
From that vantage point we find it difficult to reconcile the late
age of onset and phenotype observed in the Sudhaman manuscript. The
phenotype was restricted to Parkinsonian symptoms with an onset of 17, 16
and 13 years. The fact that these individuals displayed normal birth and
adolescent milestones prior to disease onset, and specifically normal
renal function at age of examination (22, 20 and 17 years, respectively),
is incompatible with the known function for this protein in kidney and
leaves us skeptical of the conclusion that the ARJP-afflicted individuals
in the family documented in the Sudhaman manuscript are indeed germline
and functionally null mutations in PODXL.
We note that the DNA sequence reported in the Sudhaman et al
manuscript maps to a known in-frame Pro-Ser expansion polymorphism
(rs759639123) (or similar dbSNPs) following Ser31 (duplication of GTCGCC).
As this sequence is particularly GC rich, it is prone to sequencing errors
that could have been misinterpreted as a null mutation.
Although the authors suggested the possibility of nonsense mediated
decay, they did not provide any data suggesting complete absence of PODXL
RNA or podocalyxin protein, further raising concern as to whether the
variant generates a truly null allele. While this locus and polymorphism
could well be linked to the observed ARJP phenotype we would argue that,
based on the published literature and the known mouse Podxl null
phenotypes, it is unlikely that these afflicted individuals are carrying
two germline null alleles.
We are also concerned with the authors' interpretation of the
replication study, which involved screening for any PODXL variants in 280
PD patients. Due to the high frequency of rare protein-altering variants
in the human genome, there is a distinct possibility that these missense
PODXL variants are not directly associated with PD. The three reported
PODXL rare variant carriers consist of one young-onset, one old-onset and
one familial PD patient where the variant functionality and segregation
studies were not described in the report. The presence of such variants,
in the absence of confirmatory biochemical or phenotypic evidence, should
not be interpreted as causal. Furthermore, two (p.294Q and p.S373N) of the
three variants display poor evolutionary conservation and exhibit
discordant amino acids between human and rodent proteins.
Finally, because podocalyxin is highly expressed by vascular
endothelia and kidney podocytes, immunohistologic evaluation of
podocalyxin expression on vessel-containing skin biopsies (or better
still, renal biopsies) from the ARJP-afflicted patients described in the
manuscript would likely resolve this issue. In lieu of that data, we would
argue that the main conclusions of this manuscript remain speculative.
Respectfully,
Mr. Ido Refaeli1
Dr. Michael R. Hughes1
Mr. Moses Lee2
Prof. Murim Choi2
Prof. Kelly M. McNagny1
1Department of Medical Genetics, University of British Columbia,
Vancouver, BC CANADA and 2Department of Biomedical Sciences, Seoul
National University, Seoul, REPUBLIC OF KOREA
REFERENCES
1. Sudhaman S, Prasad K, Behari M, et al. Discovery of a frameshift
mutation in podocalyxin-like (PODXL) gene, coding for a neural adhesion
molecule, as causal for autosomal-recessive juvenile Parkinsonism. J Med
Genet 2016.
2. Vitureira N, Andres R, Perez-Martinez E, et al. Podocalyxin is a
novel polysialylated neural adhesion protein with multiple roles in neural
development and synapse formation. PLoS One 2010;5(8):e12003.
3. Doyonnas R, Kershaw DB, Duhme C, et al. Anuria, omphalocele, and
perinatal lethality in mice lacking the CD34-related protein podocalyxin.
J Exp Med 2001;194(1):13-27.
4. Barua M, Shieh E, Schlondorff J, et al. Exome sequencing and in
vitro studies identified podocalyxin as a candidate gene for focal and
segmental glomerulosclerosis. Kidney Int 2014;85(1):124-33.
I just read the articles on SMA1, SMA2, SMA3. I felt it important to
tell you of my son, born 1/26/70. When I took him to his first checkup at
3 months old, I voiced my concern for his floppy head. Again at 6 months
old, when he couldn't sit up, roll over, kick against resistance.
Finally, the Pediatrician agreed to get an appointment with Yale Hospital
in CT at 8 mos. The biopsy was shown to be SMA 1. We weren't given...
I just read the articles on SMA1, SMA2, SMA3. I felt it important to
tell you of my son, born 1/26/70. When I took him to his first checkup at
3 months old, I voiced my concern for his floppy head. Again at 6 months
old, when he couldn't sit up, roll over, kick against resistance.
Finally, the Pediatrician agreed to get an appointment with Yale Hospital
in CT at 8 mos. The biopsy was shown to be SMA 1. We weren't given any
chance of survival. He was subsequently re-diagnosed at 3 years old, and
again the results were, SMA1. He is now 46 years old. Graduated UCONN with
honors and is still living in Litchfield CT. He is not on a ventilator but
uses a bi-pap, 6 to 8% lung capacity. We have been told over and over
there is no medication and there is no trials he could be involved with.
However, with the social media being his way to communicate with the mass
population he has discovered the Salbutamol available now through a client
with SMA2. She has given us hope that this new drug will give him even
the slightest increase in strength to keep the two fingers he uses to
control his fiber optic wheelchair. Unfortunately its too expensive. It is
heartbreaking to come so close to possibly gaining enough strength to stay
mobile. If there is any possible way he could be helpful to your programs
via computer please let us know. Respectfully,
June Lajoie Strada
I wish to thank the authors of the review (1) of my book on
scientific writing (2). Here are further free documents for authors: the
Micro-Article, described in the book, is a template allowing to identify
the main discovery before starting to write an article (3). Writing a
Review Article in 7 Steps is a short note providing guidelines to write a
review or a book chapter (4). This note proposes in particular a
convenie...
I wish to thank the authors of the review (1) of my book on
scientific writing (2). Here are further free documents for authors: the
Micro-Article, described in the book, is a template allowing to identify
the main discovery before starting to write an article (3). Writing a
Review Article in 7 Steps is a short note providing guidelines to write a
review or a book chapter (4). This note proposes in particular a
convenient method to build the abstract by writing first short conclusions
at the end of the manuscript sections. The golden rules of scientific
publishing are outlined in a brief presentation (5). Scientific Writing
and Communication is the presentation of my workshop given to Masters,
PhDs and research scientists (6).
References: 1. J. Med. Genet. 2015 0:jmedgenet-2015-103182v1-
jmedgenet-2015-103182; doi:10.1136/jmedgenet-2015-103182, 2. Scientific
Writing for Impact Factor Journals. Nova Science Publishers. 2013.
https://www.novapublishers.com/catalog/product_info.php?products_id=42211,
3. Micro-article. http://fr.slideshare.net/lichtfouse/micro-arten, 4.
Writing a review article in 7 steps.
http://fr.slideshare.net/lichtfouse/writea-review, 5.
http://fr.slideshare.net/lichtfouse/scientific-writing-for-impact-factor-
journals, 6. Scientific Writing and Communication.
http://fr.slideshare.net/lichtfouse/scientific-writing-and-communication
I wish to thank the authors of the review (1) of my book on
scientific writing (2). Here are further free documents for authors: the
Micro-Article, described in the book, is a template allowing to identify
the main discovery before starting to write an article (3). Writing a
Review Article in 7 Steps is a short note providing guidelines to write a
review or a book chapter (4). This note proposes in particular a
convenie...
I wish to thank the authors of the review (1) of my book on
scientific writing (2). Here are further free documents for authors: the
Micro-Article, described in the book, is a template allowing to identify
the main discovery before starting to write an article (3). Writing a
Review Article in 7 Steps is a short note providing guidelines to write a
review or a book chapter (4). This note proposes in particular a
convenient method to build the abstract by writing first short conclusions
at the end of the manuscript sections. The golden rules of scientific
publishing are outlined in a brief presentation (5). Scientific Writing
and Communication is the presentation of my workshop given to Masters,
PhDs and research scientists (6).
References: 1. J. Med. Genet. 2015 0:jmedgenet-2015-103182v1-
jmedgenet-2015-103182; doi:10.1136/jmedgenet-2015-103182, 2. Scientific
Writing for Impact Factor Journals. Nova Science Publishers. 2013.
https://www.novapublishers.com/catalog/product_info.php?products_id=42211,
3. Micro-article. http://fr.slideshare.net/lichtfouse/micro-arten, 4.
Writing a review article in 7 steps.
http://fr.slideshare.net/lichtfouse/writea-review, 5.
http://fr.slideshare.net/lichtfouse/scientific-writing-for-impact-factor-
journals, 6. Scientific Writing and Communication.
http://fr.slideshare.net/lichtfouse/scientific-writing-and-communication
Yuval Ramot1, Abraham Zlotogorski1, Maurice van Steensel2,3,4
1 Department of Dermatology, Hadassah - Hebrew University Medical Center, Jerusalem, Israel
2 School of Medicine and School of Life Sciences, University of Dundee, United Kingdom
3 Institute of Medical Biology, Singapore
4 Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore
In their rec...
We only became aware of the paper by Reinstein et al. after our manuscript was published online. It is gratifying to know that we are not the only group who has identified left ventricular non-compaction (LVNC) in males with loss-of-function mutations in NONO.
Conflict of Interest:
None declared
The association has been described before but is not cited in the JMG manuscript
Eur J Hum Genet. 2016 Jun 22. doi: 10.1038/ejhg.2016.72.
Intellectual disability and non-compaction cardiomyopathy with a de novo NONO mutation identified by exome sequencing.
Conflict of Interest:
None declared
To the Editor of Journal of Medical Genetics:
Enabled by recent advances in sequencing technologies, genotypes from thousands of individuals are now available in online databases. While most of them aim to be the reference source of genotypes from healthy individuals, however, due to the lack of accompanying clinical data, geneticists now face the challenge of separating pathogenic mutations and rare polymorphisms. The fr...
To the editor:
The report that a novel missense mutation in CCDC88C activates the JNK pathway and causes a dominant form of spinocerebellar ataxia that appeared in your Journal (1) is of great interest. Although we identified the same heterozygous missense variation (c.1391G>A, p.R464H) as that reported (1) in a Japanese patient with autosomal dominant cerebellar ataxia (ADCA), we report here that this varia...
Response to e letter ID jmedgenet el; 2826 by Refaeli et al., dated June14, 2016 A considerable amount of literature on the role of podocalyxin-like (PODXL) protein in normal mammalian kidney functions and to a lesser extent in mouse brain development is available. We agree that these studies particularly, "Anuria, Omphalocele, and Perinatal Lethality in Mice Lacking the Cd34-Related Protein Podocalyxin" by Doyonnas et...
Re:
Sudhaman, et al. Discovery of a frameshift mutation in podocalyxin- like (PODXL) gene, coding for a neural adhesion molecule, as causal for autosomal-recessive juvenile Parkinsonism. J Med Genet. 0:1-7, 2016.
In the February 2016 edition of the Journal of Medical Genetics, Sudhaman et al(1) report the identification of a PODXL variant (c.89_90insGTCGCCCC) as the causative mutation in an Indian fam...
I just read the articles on SMA1, SMA2, SMA3. I felt it important to tell you of my son, born 1/26/70. When I took him to his first checkup at 3 months old, I voiced my concern for his floppy head. Again at 6 months old, when he couldn't sit up, roll over, kick against resistance. Finally, the Pediatrician agreed to get an appointment with Yale Hospital in CT at 8 mos. The biopsy was shown to be SMA 1. We weren't given...
I wish to thank the authors of the review (1) of my book on scientific writing (2). Here are further free documents for authors: the Micro-Article, described in the book, is a template allowing to identify the main discovery before starting to write an article (3). Writing a Review Article in 7 Steps is a short note providing guidelines to write a review or a book chapter (4). This note proposes in particular a convenie...
I wish to thank the authors of the review (1) of my book on scientific writing (2). Here are further free documents for authors: the Micro-Article, described in the book, is a template allowing to identify the main discovery before starting to write an article (3). Writing a Review Article in 7 Steps is a short note providing guidelines to write a review or a book chapter (4). This note proposes in particular a convenie...
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