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
Letter to Editor
Shaheen et al. 1 recently reported a homozygous 5 bps deletion mutation
(c.1299_1303delTAAG; p.Phe433Leufs*6) in the human polo-like kinase 4
(PLK4,MIM 605031) gene and proposed that it is a compelling candidate for
primordial dwarfism (PD). Autozygosity mapping and LOD score, method of
estimating linkage distances, are adopted for the novel locus
identification. Candidate gene hunting among 144 in the r...
Letter to Editor
Shaheen et al. 1 recently reported a homozygous 5 bps deletion mutation
(c.1299_1303delTAAG; p.Phe433Leufs*6) in the human polo-like kinase 4
(PLK4,MIM 605031) gene and proposed that it is a compelling candidate for
primordial dwarfism (PD). Autozygosity mapping and LOD score, method of
estimating linkage distances, are adopted for the novel locus
identification. Candidate gene hunting among 144 in the region is based on
filtering through ToppGene suite 2. Mutation screening through Sanger
sequencing of PLK4 gene is only based on ToppGene suite ranking. In this
study authors have not mentioned how many and which samples were selected
for genotyping and linkage analysis so the predicted maximum LOD score
cannot be calculated. Maximum multipoint LOD score (2.5) obtained by the
authors is quite lower than the established threshold values (3.0). In
article it has been stated that all PD genes in training set in comparison
to all 144 genes of the locus in test set were enrolled in ToppGene
candidate prioritization. These genes lists must be provided along with
test parameters. In our opinion these informations are essential for data
reproducibility and validation.
Only PLK4 gene has been selected for Sanger sequencing but The candidate
region (Chr4:112904466-129392060, GRCh37/hg19) includes some of the well-
known syndromes characterized by autosomal recessive intellectual
disabilities, microcephaly, short stature, and overlapping phenotypes.
Alazami syndrome caused by mutations in LARP7, autosomal recessive type 1
mental retardation caused by mutations in PRSS12, Bardet-Biedl syndrome
caused by mutations in BBS7 and BBS12, Van Maldergem syndrome 2 caused by
mutations in FAT4, neuronal ceroid lipofuscinosis 7 caused by mutations in
MFSD8, a multisystem disorder including brain function caused by mutation
in SCLT1, as well as genes involved in mitosis and brain development e.g.
MAD2L1, FGF2, INTU, have not been discussed throughout the manuscript.
Furthermore, chromatograms for obligate carriers have not been presented
nor the Saudi population screening for minor allele frequency has been
performed. Similarly a homozygous variant (NM_014264.4, c.1556G>C;
p.Trp519Ser) reported in ESP6500 (http://evs.gs.washington.edu/EVS/)
alters a highly conserved amino acid of PLK4 however it has not been
assigned to any of primordial dwarfism phenotype and not discussed by the
authors. In fact, phenotypic overlaps among the study mutants and the
above syndromes would be of far more value instead of paying an
exaggerated attention to PLK4 biology.
References
1 Shaheen R, Al Tala S, Almoisheer A et al. Mutation in PLK4,
encoding a master regulator of centriole formation, defines a novel locus
for primordial dwarfism. Journal of medical genetics 2014.
2 Chen J, Bardes EE, Aronow BJ et al. ToppGene Suite for gene list
enrichment analysis and candidate gene prioritization. Nucleic acids
research 2009; 37: W305-11.
I have read all the respective article and I agreed to most of the
findings. Recently, our group has been working with metabolomics towards
lipodystropy and we did found similar findings. Recently it has been
reported (Abael et al., 2010) that the changes in local thyroid hormone
(TH) metabolism may occur in areas with lipoatrophy. These changes could
be because of different molecular mechanisms (for example; levels of...
I have read all the respective article and I agreed to most of the
findings. Recently, our group has been working with metabolomics towards
lipodystropy and we did found similar findings. Recently it has been
reported (Abael et al., 2010) that the changes in local thyroid hormone
(TH) metabolism may occur in areas with lipoatrophy. These changes could
be because of different molecular mechanisms (for example; levels of
monocarboxylate transporter 8 and iodothyronine deiodinases). Recently, it
has been reported that the prelamin A accumulating mesenchymal stem cells
(hMSCs) have a premature aging phenotype which affects their functional
competence in vivo. The combination of prelamin A accumulation and stress
conditions enhance the aging phenotype by dysregulating the activity of
the octamer binding protein Oct-1(Infante et al., 2014). There is a need
to evaluate Oct-1 in type 2 familial partial lipodystrophy. Stress could
be in the form of hypoxia and oxygen concentration and PO2 is documented
to be involved in the activity of nucleoside reverse transcriptase
inhibitors (NRTIs) to characterised the metabolic effect on the targeted
adipose tissues (Gentil et al., 2006). There could be a relation between
hypoxia inducible factors (HIF1 & HIF2) to be defined for
lipodystrophy.
Hypoxia Inducible Factor 1 (HIF-1) was firstly identified to be a
nuclear factor involved in activation of gene expression under the
response of reduced cellular O2 during Erythropoietin expression and
regulation (Semenza & Wang, 1992). Following this invention, HIF-1 was
reported to be involved in expression and regulation of Vascular
Endothelial Growth Factor (VEGF), glycolytic enzymes, nitric oxide
synthetase and glucose transporter 1 (Glut 1) (Wenger & Gassmann.
1997; Semenza, 1998). Structurally, HIF-1 is a heterodimer composed of two
basic helix-loop-helix PAS domains (bHLH-PAS) protein, called HIF-1? and
HIF-1? subunits (Wang et al., 1995).
Tian and colleagues (Tian et al., 1997) identified a novel bHLH-PAS
protein called Endothelial PAS domain protein (EPAS1) with 48% sequence
similarity with HIF-1 ?. Further studies termed it as HIF-like factor
(HLF) (Ema et al., 1997) followed by HIF-related factor (HRF) (Flamme et
al., 1997) followed by current name given to this factor to be HIF-2
(Wenger & Gassmann, 1997). Recently, it has been shown that HIF-2 ?
forms a functional heterodimer with HIF-1?, resulting in the HIF-2 complex
and this complex has been found to bind the same hypoxia response element
(HRE) of Epo and VEGF gene to which HIF-1 complex binds. Both of these HRE
and bindings (binding of HIF-1 and HIF-2 complex) are activated under
hypoxic conditions (Tian et al., 1997, Ema et al., 1997).
The role of HIF-2 as compared to HIF-1 has been much less studied.
Patel and colleagues (Patel et al., 2008) reported HIF-2 complex to be
more tissue specific than HIF-1 by suggesting its response to different
levels of O2 and this justified strongly the role of HIF-2 on expression
of genes in obesity and inflammation. Hyperplasia and hypertrophy in
adipocytes can lead to many morphological and molecular changes in
adipocytes resulting in hypoxia, metabolic dysregulation and increased
expression and secretion of cytokines (Hosogai et al., 2007, Ye et al.,
2007). Different vital genes involved in metabolic and diet induced
obesity have been reported to be expressed under the action of HIF-1.
Recently, NF-KB has been reported to be induced through hypoxia (Culver et
al., 2010). It has been suggested that the role of HIF-2 and NF-?B can be
considered because of a strong evidence of former (HIF-2) been reported to
be linked with hypoxia inducible inhibition of insulin signaling
(Regazzetti et al., 2009). There is a strong evidence of hypoxia in
obesity (Ye et al., 1997), with a reported finding of HIF-2 to be tissue
specific rather than HIF-1 (Patel et al., 2008).
The potential role of HIF-2 and NF-KB in pathways involving vital
genes and proteins in response of adipocytes to hypoxia needs to be
explored at molecular level to get a better picture and role of hypoxia in
inducing obesity genes expression in this respective report beside
inflammation as per induced.
References:
Culver C, Sundqvist A, Mudie S, Melvin A, Xirodimas D, Rocha S.
Mechanism of hypoxia-induced NF-kappaB. Mol Cell Biol. 2010
Oct;30(20):4901-21. doi: 10.1128/MCB.00409-10.
Ema M, Taya S, Yokotani N, Sogawa K, Matsuda Y, and Fujii-Kuriyama Y
(1997) A novel bHLH-PAS factor with close sequence similarity to hypoxia-
inducible factor 1alpha regulates the VEGF expression and is potentially
involved in lung and vascular development. Proc Natl Acad Sci USA 94: 4273
-4278.
Gentil C, Le Jan S, Philippe J, Leibowitch J, Sonigo P, Germain S,
Pi?tri-Rouxel F. Is oxygen a key factor in the lipodystrophy phenotype?
Lipids Health Dis. 2006 Oct 18;5:27.
Hosogai, N., Fukuhara, A., Oshima, K., Miyata, Y., Tanaka, S.,
Segawa, K., Furukawa, S., Tochino, Y., Komuro, R., Matsuda, M., and
Shimomura, I. (2007) Adipose tissue hypoxia in obesity and its impact on
adipocytokine dysregulation. Diabetes 56, 901-911.
Infante A, Gago A, de Eguino GR, Calvo-Fern?ndez T, G?mez-Vallejo V,
Llop J, Schlangen K, Fullaondo A, Aransay AM, Mart?n A, Rodr?guez CI.
Prelamin A accumulation and stress conditions induce impaired Oct-1
activity and autophagy in prematurely aged human mesenchymal stem cell.
Aging (Albany NY). 2014 Apr;6(4):264-80.
Lado-Abeal J, Calvo RM, Victoria B, Castro I, Obregon MJ, Araujo-
Vilar D.Regional decrease of subcutaneous adipose tissue in patients with
type 2 familial partial lipodystrophy is associated with changes in
thyroid hormone metabolism. Thyroid. 2010 Apr;20(4):419-24. doi:
10.1089/thy.2009.0267.
Patel SA, Simon MC. Biology of hypoxia-inducible factor-2alpha in
development and disease. Cell Death Differ. 2008 Apr;15(4):628-34. doi:
10.1038/cdd.2008.17. Epub 2008 Feb 15. Review. PubMed PMID: 18259197;
PubMed Central PMCID:PMC2882207.
Regazzetti C, Peraldi P, Gr?meaux T, Najem-Lendom R, Ben-Sahra I,
Cormont M, Bost F, Le Marchand-Brustel Y, Tanti JF, Giorgetti-Peraldi S.
Hypoxia decreases insulin signaling pathways in adipocytes. Diabetes. 2009
Jan;58(1):95-103. doi: 10.2337/db08-0457.
Tian H, McKnight SL, and Russell DW (1997) Endothelial PAS domain
protein 1 (EPAS1), a transcription factor selectively expressed in
endothelial cells. Genes Dev 11: 72-82.
Semenza, G. L. and Wang, G. L. (1992). A nuclear factor induced by
hypoxia via de novo protein synthesis binds to the human erythropoietin
gene enhancer at a site required for transcriptional activation. Molec.
cell. Biol. 12, 5447-5454.
Semenza, G. L. (1998). Hypoxia-inducible factor 1: master regulator
of O2 homeostasis. Curr. Opin. Genet. Dev. 8, 588-594.
Wang, G.L., Jiang, B.H., Rue, E.A., Semenza, G.L., 1995.
Hypoxia?inducible factor?1 is a basic? helix?loop?helix?pas heterodimer
regulated by cellular O2 tension. Proc Natl Acad Sci USA 92, 5510?5514.
Wenger, R. H. and Gassmann, M. (1997). Oxygen(es) and the hypoxia-
inducible factor-1. Biol. Chem. 378, 609-616.
Ye, J., Gao, Z., Yin, J., and He, Q. (2007) Hypoxia is a potential
risk factor for chronic inflammation and adiponectin reduction in adipose
tissue of ob/ob and dietary obese mice. Am J Physiol Endocrinol Metab 293,
E1118-1128.
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...
Letter to Editor Shaheen et al. 1 recently reported a homozygous 5 bps deletion mutation (c.1299_1303delTAAG; p.Phe433Leufs*6) in the human polo-like kinase 4 (PLK4,MIM 605031) gene and proposed that it is a compelling candidate for primordial dwarfism (PD). Autozygosity mapping and LOD score, method of estimating linkage distances, are adopted for the novel locus identification. Candidate gene hunting among 144 in the r...
I have read all the respective article and I agreed to most of the findings. Recently, our group has been working with metabolomics towards lipodystropy and we did found similar findings. Recently it has been reported (Abael et al., 2010) that the changes in local thyroid hormone (TH) metabolism may occur in areas with lipoatrophy. These changes could be because of different molecular mechanisms (for example; levels of...
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