We have read the entire paper with great care. We totally agree with
the findings of the respected authors in this respective article. The
article seems to be used by many researchers as a reference article so we
need to update the status of SMA diagnosis for non deleted SMA patients.
Many new approaches have been employed to diagnose non deleted SMA. One
of such methods is long range PCR method (LR-PCR) (Clemont et al....
We have read the entire paper with great care. We totally agree with
the findings of the respected authors in this respective article. The
article seems to be used by many researchers as a reference article so we
need to update the status of SMA diagnosis for non deleted SMA patients.
Many new approaches have been employed to diagnose non deleted SMA. One
of such methods is long range PCR method (LR-PCR) (Clemont et al., 2004).
Secondly, we can not just focus on the mutations described in this
article. We need to focus on all known mutations in the SMN1 gene in non
deleted SMA patients. Although Clermont and colleagues claimed that 90% of
the mutations in the SMN1 gene can be found by their method (Clermont et
al.,2004), yet many other groups have reported individual mutations. We
would like to enumerate the list of all these mutations. All the mutations
are divided into four main groups; missence mutation, Nonsense mutation,
Frame-shift mutation and Splice site mutations.
The missence mutations (total 25) have been reported in Ex.1 in type
II and III SMA patients (Parsons et al., 1998), Ex. 2a in type II and III
SMA patients (Sun et al., 2005), Ex.3 in type I (Cusco et al., 2004,
Clermont et al., 2004, Prior et al., 2007, Sun et al., 2005, Koatni et
al., 2007), Ex.3 in type II (Clermont et al., 2004, Sun et al., 2005),
Ex.3 in type III SMA patient (Sun et al., 2005, Ex.4 in type I SMA patient
(Zapletalova et al., 2007), Ex. 6 in type III SMA patients (Rochette et
al., 1997, Clermont et al., 2004, Sun et al., 2005, Hahnen et al., 1997,
Parson et al., 1998, Wirth et al., 1999), Ex.6 in type I SMA patient
(Clermont et al., 2004), Ex.6 in type II SMA patient (Alias et al., 2009,
Prior et al., 2007, Lefebvre et al., 1995, Wirth et al., 1999, Rochette et
al., 1997, Clermont et al., 2004, Zapletalova et al., 2007, Hahnen et al.,
1997, Parson et al., 1998, Sun et al., 2005, Ex.6 in type III SMA patients
(Hahnen et al., 1997, Parsons et al., 1998, Wirth et al., 1999, Sun et
al., 2005, Zapletalova et al., 2007, Alias et al., 2009, Burglen et al.,
1996, Skordiset et al., 2001), Ex.7 in type I SMA patient (Talbot et al.,
1997) and Ex.7 in type II and III SMA patients (Wang et al., 1998).
The nonsense mutations (total 5) were curated to be included in Ex.1 in
type I SMA patient (Wirth et al., 1999), Ex.1 in type III SMA patient (Sun
et al., 2005), Ex.3 in type II SMA patient (Sossi et al., 2001, Prior et
al., 2007), Ex.3 in type III SMA patient (Wirth et al., 1999, Sun et al.,
2005, Sossi et al., 2001, Prior et al., 2007), Ex.3 in type I SMA patient
(Bricchta et al., 2008), Ex.4 in type I SMA patient (Alias et al., 2009),
Ex.5 in type I SMA patient (Tsai et al., 2001).
Considering the fram-shift mutations (total 18), it included mutation in
Ex.1 of type I SMA patient (Wirth et al., 1999), Ex.1 in type III SMA
patient (Wirth et al., 1999), Ex.2a in type I SMA patient (Prior et al.,
2007, Zapletalova et al., 2007), Ex.2a in type I SMA patient (Wirth et
al., 1999, Alias et al., 2008), Ex.2b in type I SMA patient (Clermont et
al., 2004), Ex. 2b in type III SMA patient (Wirth et al., 1999), Ex.3 in
type I SMA patient (Brahe et al., 1996, Sossi et al., 2001, Alias et al.,
2008), Ex.3 in type II SMA patient (Bussaglia et al., 1995, Martin et al.,
2002, Cusco et al., 2003, Alias et al., 2008), Ex.3 in type III SMA
patient (Alias et al., 2008, Clermont et al., 2004, Bussaglia et al.,
1995, Martin et al., 2002, Cusco et al., 2003), Ex.4 in type I SMA patient
(Clermont et al., 1997, Clermont et al., 2004), Ex.4 in type II SMA
patient (Parons et al., 1998, Wirth et al., 1999), Ex.4 in type III SMA
patient (Parson et al., 1998), Ex.5 in type I, II and III SMA patients
(Wirth et al., 1999, Clermont et al., 2004), Ex.6 in type I, II and III
SMA patients (Wirth et al., 1999, Clermont et al., 2004, Parsons et al.,
1996, Parsons et al., 1998, Martin et al 2002, Clermont et al., 2004,
Alias et al., 2008, Martin et al., 2002).
The splice site mutations in the SMN1 gene in non deleted SMA patients
include 40Int.4 in type I SMA patient (Brichta et al., 2008), 41Int.6 in
type I SMA patient (Martin et al., 2002), 42Int.6 in type I SMA patient
(Eggermann et al., 2008), 43Int.6 in type I SMA patient (Lefebvre et al.,
1995), 44Int.7 in type I SMA patient (Wirth et al., 1999) and 45Int.7 in
type II SMA patient (Lefebvre et al., 1995).
The copy number analysis of the SMN1 gene is not enough as the SMA
modified genes; SMN2 and NAIP are reported to modify the disease severity
(Watihayati et al., 2009). We will emphasize that the title of the article
is making the readers confusing as considering the article to be the
diagnostic protocol for the spinal muscular atrophy in non deleted SMA
patients. The severity of the disease could be estimated by many
parameters even in the non deleted SMA patients and recently published
report has prooven that (Watihayati et al., 2009) beside the report of
Teguh and colleagues which states that only PCR detection of exon7 of the
SMN1 is enough to diagnose SMA (Teguh et al., 2011) but yet the authors
did not explain the diagnosis of non deleted SMA patients. Very humbly we
would like to add this response with a request to the respected authors to
consider the findings of SMN2 copy number and the NAIP gene deletion with
the available data of this patient.
It was with great interest that we have read the recent article published by Kamath et al. [1] dealing with NOTCH2 mutations in patients affected by Alagille syndrome (ALGS) negative for JAG1 gene mutations and rearrangements. This original article brings relevant information about the mutation frequencies and genotype-phenotype data regarding NOTCH2 gene. Previously only two mutations, one missense and one splicing, have been rep...
It was with great interest that we have read the recent article published by Kamath et al. [1] dealing with NOTCH2 mutations in patients affected by Alagille syndrome (ALGS) negative for JAG1 gene mutations and rearrangements. This original article brings relevant information about the mutation frequencies and genotype-phenotype data regarding NOTCH2 gene. Previously only two mutations, one missense and one splicing, have been reported in two families with ALGS and renal disease [2]. Here we report a family with two siblings affected by a severe cholestasis carrying a maternal inherited frameshift mutation in exon 18 (NM_024408.2:c.2765del;p.Asn922MetfsX9) of NOTCH2 gene. The novel unreported variant was predicted to activate the nonsense mediated mRNA decay and is localized on EGF-like 24 domain of the protein. Moreover, it was absent in the unaffected father and in the third unaffected sibling such as in a cohort of 566 healthy control alleles. Subsequent analysis of genomic DNA from maternal healthy grandparents indicated that the mutation had occurred de novo in the mother. After a deep clinical and instrumental re-evaluation, our patients did not show any cardiac disease, skeletal, ocular abnormalities or facies typical of ALGS. NOTCH2 sequencing analysis has been performed in other 9 cases negatives for JAG1 mutations. No other patient with NOTCH2 mutations has been identified.
The two siblings underwent liver transplantation due to the severe liver dysfunction, while the mother presented only a mild hypercholesterolemia, suggesting a wide variable expressivity of the disease in the family. Also the patient described by Kamath et al. [1], carrying a frameshift mutation in exon 16 (p.Ser856fs16X), had only liver involvement further confirming the phenotypic variability associated with this inherited disease. The variable expressivity is typical of patients affected by ALGS carrying mutations in JAG1 gene. Classic diagnostic criteria for ALGS combine the presence of bile duct paucity on liver biopsy with three of five systems affected: liver, heart, skeleton, eye and dysmorphic facies. Recently, it has been shown that JAG1 mutations are present also in patients having only one or two ALGS criteria [3]. This finding is not surprising, considering that even family members sharing the same mutation can have a wide spectrum of phenotypic manifestations ranging from life-threatening cardiac or liver disease to subtle or null clinical features. In our experience, JAG1 mutations are identified in 93% of patients with three or more diagnostic criteria, while they are rarely identified (4 mutated out of 55 indexes analyzed) in patients having cholestasis alone or associated to another ALGS diagnostic feature. The low mutational detection rate in JAG1 and NOTCH2 genes leads to question the real utility to perform the sequencing analysis of such large genes in a clinical context in this cohort of patients. The "cascade" screening of these genes is expensive and time consuming in isolated cholestasis associated or not with others minor clinical features creating anxiety and false expectations in families. For this reasons, we believe that a new strategy of analysis, based on targeted next-generation sequencing (NGS) of all genes causative of liver diseases, may be more appropriate for characterizing atypical ALGS patients. A comprehensive NGS analysis of liver genes may be more useful for clinicians, patients and families decreasing time and costs by ending the diagnostic odyssey as already demonstrated for other genetic conditions.
References:
1. Kamath BM, Bauer RC, Loomes KM, Chao G, Gerfen J, Hutchinson A, Hardikar W, Hirschfield G, Jara P, Krantz ID, Lapunzina P, Leonard L, Ling S, Ng VL, Hoang PL, Piccoli DA, Spinner NB. NOTCH2 mutations in Alagille syndrome. J Med Genet 2012;49:138-44.
2. McDaniell R, Warthen DM, Sanchez-Lara PA, Pai A, Krantz ID, Piccoli DA, Spinner NB. NOTCH2 mutations cause Alagille syndrome, a heterogeneous disorder of the notch signaling pathway. Am J Hum Genet. 2006;79:169-73.
3. Guegan K, Stals K, Day M, Turnpenny P, Ellard S. JAG1 mutations are found in approximately one third of patients presenting with only one or two clinical features of Alagille syndrome. Clin Genet 2011. doi: 10.1111/j.1399-0004.2011.01749.x. [Epub ahead of print].
It was with great interest that I read the study by Tang and co-
authors [1], in which they discovered twenty-five novel mutations in DNA
polymerase gamma. Based on the presence of p.G268A substitution in
heterozygosis in 19 subjects from a cohort of 2697 unrelated patients,
they proposed to reclassify this mutation as a neutral polymorphism or a
polymorphic modifier rather than a pathological mutation. Smith and co-
auth...
It was with great interest that I read the study by Tang and co-
authors [1], in which they discovered twenty-five novel mutations in DNA
polymerase gamma. Based on the presence of p.G268A substitution in
heterozygosis in 19 subjects from a cohort of 2697 unrelated patients,
they proposed to reclassify this mutation as a neutral polymorphism or a
polymorphic modifier rather than a pathological mutation. Smith and co-
authors [2] confirmed that POLG G268A is not pathogenic but represent
neutral polymorphism on the basis of the presence of this mutations in
three healthy subjects and on the basis of the frequencies of the allele
p.G268A in their patient cohort, which were similar to those found in a
control population of European origin by the NHLBI exome sequencing
project [3].
I and co-authors previously demonstrated that in yeast the mip1G224A
allele, corresponding to the human p.G268A substitution, was associated
with a 2.2-fold increase in petite frequency, which is the frequency of
mutants with large deletions and/or loss of mtDNA [4]. A further analysis
made in our laboratory on yeast mip1 mutations corresponding to human
substitutions classified as neutral SNPs [5] conserved from yeasts to
mammals (E. Baruffini and T. Lodi, unpublished results), suggested the
existence of a class of substitutions, that also includes G224A, which are
not neutral. These mutations caused a 1.5 to 2.5-fold increase in petite
frequency, which was lower compared to the petite frequency caused by
mutations recognized as pathological, for which at least a 10-fold
increase in petite frequency or the total loss of mtDNA were observed in
our laboratory [4, 6-9]. This class includes also the mip1E900G and
mip1Q766C alleles, corresponding to the human p.E1143G and p.R964C
substitutions, respectively. The former (2-fold increase in petite
frequency) is a substitution classified in humans as a phenotypic modifier
that can modulate disease mutations [10, 11]. The latter (2-fold increase)
seems to be pathological in humans when in heterozygosis with p.A862T
[8,12] or with p.A962T [1] but not pathological in homozygosis [13];
however it is associated with mitochondrial toxicity susceptibility to
stavudine both in humans [13] and in yeast [14] and the mutant DNA
polymerase gamma showed an impaired polymerase activity [15].
Conclusion: analysis in yeast suggests that p.G268A is neither a
pathological mutation nor a neutral SNP. It can be classified, together
with R964C and E1143G, as a "phenotypic modifier", as suggested by
Copeland [10], as an "unclassified variant", as suggested by Tang and co-
authors [1] or as an "Ecogenetic Single Nucleotide Variant (ESNV)", as
suggested by Saneto and Naviaux for genetic variations whose phenotypic
expression is determined by interaction with genetic, epigenetic and
environmental factors [16]. In general, these observations suggest that
the validation of the pathological significance of novel mutations, whose
discovery is exponentially increasing thanks to the next generation
sequencing, can take advantage, when possible, of experimental analysis in
model systems to confirm or disavow their role in the disease.
References
1. Tang S, Wang J, Lee NC, Milone M, Halberg MC, Schmitt ES, Craigen WJ,
Zhang W, Wong LJ. Mitochondrial DNA polymerase gamma mutations: an ever
expanding molecular and clinical spectrum. J Med Genet 2011;48:669-681.
2. Smith C. POLG p.G268A and p.G517V are not pathogenic mutations. J
MedGenet 2011; eletter
3. NHLBI exome sequencing project, https://esp.gs.washington.edu/drupal/
4. Baruffini E, Lodi T, Dallabona C, Puglisi A, Zeviani M, Ferrero I.
Genetic and chemical rescue of the Saccharomyces cerevisiae phenotype
induced by mitochondrial DNA polymerase mutations associated with
progressive external ophthalmoplegia in humans. Hum Mol Genet 2006;15:2846
-2855.
5. dbSNP, http://www.ncbi.nlm.nih.gov/snp/
6. Baruffini E, Ferrero I, Foury F. Mitochondrial DNA defects in
Saccharomyces cerevisiae caused by functional interactions between DNA
polymerase gamma mutations associated with disease in human. Biochim
Biophys Acta 2007;1772:1225-1235.
7. Spinazzola A, Invernizzi F, Carrara F, Lamantea E, Donati A, Dirocco M,
Giordano I, Meznaric-Petrusa M, Baruffini E, Ferrero I, Zeviani M.
Clinical and molecular features of mitochondrial DNA depletion syndromes.
J Inherit Metab Dis 2009;32:143-158.
8. Stricker S, Pr?ss H, Horvath R, Baruffini E, Lodi T, Siebert E, Endres
M, Zschenderlein R, Meisel A.J. A variable neurodegenerative phenotype
with polymerase gamma mutation. J Neurol Neurosurg Psychiatry 2009;80:1181
-1182.
9. Baruffini E, Horvath R, Dallabona C, Czermin B, Lamantea E, Bindoff L,
Invernizzi F, Ferrero I, Zeviani M, Lodi T. Predicting the contribution of
novel POLG mutations to human disease through analysis in yeast model.
Mitochondrion 2011;11:182-190.
10. Human DNA Polymerase Gamma Mutation Database,
http://tools.niehs.nih.gov/polg/
11. Chan SS, Longley MJ, Copeland WC. Modulation of the W748S mutation in
DNA polymerase gamma by the E1143G polymorphismin mitochondrial disorders.
Hum Mol Genet 2006;15:3473-3483.
12. Wong LJ, Naviaux RK, Brunetti-Pierri N, Zhang Q, Schmitt ES, Truong C,
Milone M, Cohen BH, Wical B, Ganesh J, Basinger AA, Burton BK, Swoboda K,
Gilbert DL, Vanderver A, Saneto RP, Maranda B, Arnold G, Abdenur JE,
Waters PJ, Copeland WC. Molecular and clinical genetics of mitochondrial
diseases due to POLG mutations. Hum Mutat 2008;29:E150-E172.
13. Yamanaka H, Gatanaga H, Kosalaraksa P, Matsuoka-Aizawa S, Takahashi T,
Kimura S, Oka S. Novel mutation of human DNA polymerase gamma associated
with mitochondrial toxicity induced by anti-HIV treatment. J Infect Dis
2007;195:1419-1425.
14. Baruffini E, Lodi T. Construction and validation of a yeast model
system for studying in vivo the susceptibility to nucleoside analogues of
DNA polymerase gamma allelic variants. Mitochondrion 2010;10:183-187.
15. Bailey CM, Kasiviswanathan R, Copeland WC, Anderson KS. R964C mutation
of DNA polymerase gamma imparts increased stavudine toxicity by decreasing
nucleoside analog discrimination and impairing polymerase activity.
Antimicrob Agents Chemother 2009;53:2610-2612.
16. Saneto RP, Naviaux RK. Polymerase gamma disease through the ages. Dev
Disabil Res Rev 2010;16:163-174.
The respective article was well read by us. We agree with the
precious scientific findings by the authors but at the same time we would
like to recall the two very basic fundamental functions of CBP, which
involve CBP as a bridging molecule and a cofactor (Montmini et al., 1986)
for CREB modulated gene expression and histone acetyltransferase activity
of CBP on CREB modulated gene expression (Lu et al., 2003) specificall...
The respective article was well read by us. We agree with the
precious scientific findings by the authors but at the same time we would
like to recall the two very basic fundamental functions of CBP, which
involve CBP as a bridging molecule and a cofactor (Montmini et al., 1986)
for CREB modulated gene expression and histone acetyltransferase activity
of CBP on CREB modulated gene expression (Lu et al., 2003) specifically
and on histone acetylation in general.
This study specifically targeted lymphoid cell lines from patients
with Rubinstein-Taybi syndrome but it has provided a new horizon towards
defining some crucial event as the respective authors have tried to
correlate the general post-transcriptional events (via histone acetylation
level) with the transcriptional events (by CBP and p300 as a co-
activators/cofactors). The study has given the overall status of the
histone acetylation level and mutations within the CBP gene but yet unable
to describe the role of these mutations in CBP specifically to any
specific genes/motifs within the whole genome or in the CRE elements in
the promoter region of different genes, which could play a vital role in
circumscribing the clinical severity of the disease as has been in seen in
a report from Sarmila et al., 2004; which stated the role in
overexpressing the SMN genes associated with Spinal Muscular Atrophy. The
disease shared quite similar clinical features with Rubinstein-Taybi
syndrome with a vast heterogeneity.
The transcription factors are reported to be phosphorylated by
Protein Kinase K (PKA) which is dependent on increase level of cAMP. CREB
has been reported to be the best linked between PKA activation and gene
transcription (Montmini et al., 1986). Authors also studied p300 which has
been reported to interact with many transcription factors, reflecting the
role of p300 and CBP as co-activators more generally in signal integration
(Goodman et al., 2000).
Deficit in histone acetylation in cell lines in this study can be
correlated to the defect in histone acetyltransferase activity of CBP in
general on the whole genome but the specific effect through the invitro
CREB acetylation must be considered by the respective authors by atleast
the linkage analysis of the CREB induced genes in the described nine
patients in this study. We assume, by doing so, the role of CREB or CRE
modulated disease severity could be ruled out. We have done the same for
Spinal Muscular Atrophy (data not published yet).
Similarly, the use of histone deacetylases (HDACi)followed by the the
acetylation status is too general for the entire genome. For sure, HDACi
will recover the "acetyltransferase activity" of CBP but the effect of
HDACi on the coactivation function of the CBP is not well explained. Any
of the histone deacetylases, will increase the whole genome expression but
defining the specific HDACi molecule for a specific gene is the need for
decreasing and circumscribing the clinical severity of Rubinstein-Taybi
syndrome. Some studies have been reported explaining the effect of HDACi
in SMA which make use of several molecules (Sumner et al., 2006 and
Brichta et al., 2003, Andreassi et al., 2004).
There is a need to explore the specific genes for their epigentic
control and effect in Rubinstein-Taybi syndrome therefore, further studies
are needed to confirm the effect of these mutation and histone
acetylation; towards defining specific genes in circumscribing the
clinical severity of Rubinstein-Taybi syndrome and to develop a strategy
for gene therapy.
References:
1. Montminy MR, Bilezikjian LM, (1987). Binding of a nuclear protein
to the cyclic-AMP response element of the somatostatin gene. Nature.
15;328(6126):175-8.
2. Lu Q, Hutchins AE, Doyle CM, Lundblad JR, Kwok RP, (2003).
Acetylation of cAMP-responsive element-binding protein (CREB) by CREB-
binding protein enhances
CREB-dependent transcription. J Biol Chem. 2;278(18):15727-34.
3. Sarmila Majumder, Saradhadevi Varadharaj, Kalpana Ghoshal, Umrao
Monani, Arthur H. M. Burghes, Samson T. Jacob, (2004). Identification of a
Novel Cyclic AMP-response Element (CRE-II) and the Role of CREB-1 in the
cAMP-induced Expression of the Survival Motor Neuron (SMN) Gene. The
journal of biological chemistry: 279, 15: 14803-1481.
4.Goodman RH, Smolik S, (2000). CBP/p300 in cell growth,
transformation, and development. Genes Dev. 1;14(13):1553-77.
5. Sumner CJ, (2006). Therapeutics development for spinal muscular
atrophy. NeuroRx: 3:235-245.
6. Brichta L, Hofmann Y, Hahnen E, Siebzehnrubl FA, Raschke H,
Blumcke I, Eyupoglu IY, Wirth B, (2003). Valproic acid increases the SMN2
protein level: a well-known drug as a potential therapy for spinal muscula
In their recently published paper describing mutations in
mitochondrial DNA polymerase gamma, Tang et al.(1) propose that the POLG
p.G268A (c.803G>C) and p.G517V (c.1550G>T) variants which have
previously been reported as pathogenic mutations should be considered as
unclassified variants that may represent rare neutral polymorphisms or
polymorphic modifiers.
In their recently published paper describing mutations in
mitochondrial DNA polymerase gamma, Tang et al.(1) propose that the POLG
p.G268A (c.803G>C) and p.G517V (c.1550G>T) variants which have
previously been reported as pathogenic mutations should be considered as
unclassified variants that may represent rare neutral polymorphisms or
polymorphic modifiers.
We have also identified these variants in our own cohort of 627
patients that were referred with a suspected disorder of mitochondrial DNA
maintenance. We detected p.G268A in 9 individuals (allele frequency
0.72%) of ages from 3 months to 63 years. Symptoms were variable ranging
from severe early onset mitochondrial DNA depletion syndrome to milder
late onset neuropathy/ataxia/ophthalmoplegia. In all 9 cases the variant
occurred as a heterozygous change with no other pathogenic POLG mutation,
suggesting that p.G268A is not a recessive mutation. Parental samples
were available in 3 cases, and for each of these we found the variant in
an unaffected parent, suggesting that p.G268A is very unlikely to be a
dominant mutation. Furthermore, one of the 9 index cases was later found
to be compound heterozygous for pathogenic mutations in another gene
associated with mtDNA maintenance disorders, DGUOK. In another case the
proband had a similarly affected sibling who did not have p.G268A.
Similarly, we have identified p.G517V 8 times (allele frequency
0.64%), always as a heterozygous change with no other pathogenic POLG
mutation. The age of the individuals ranged from 1-81 yrs and the
phenotype varied from infantile epilepsy/failure to thrive to adult onset
ophthalmoplegia/ataxia/myopathy. The unaffected parent of 1 of these
individuals was also heterozygous for p.G517V. A second affected
individual was later found to have pathogenic mutations in RRM2B.
Furthermore, the allele frequencies of p.G268A and p.G517V in our
patient cohort are remarkably similar to those found in a large control
population of European origin (2700 alleles) by the NHLBI exome sequencing
project (2); 0.59% and 0.86% respectively.
Therefore, our data, taken together with that for Tang et al. and the
NHLBI exome sequencing project, confirm that POLG p.G268A and p.G517V are
not pathogenic and are highly likely to represent neutral polymorphisms.
References.
(1) Tang S, Wang J, Lee NC, Milone M, Halberg MC, Schmitt ES, Craigen WJ,
Zhang W, Wong LJ. Mitochondrial DNA polymerase gamma mutations: an ever
expanding molecular and clinical spectrum. J Med Genet 2011;48:669-681.
(2) https://esp.gs.washington.edu/drupal/
Re: Genetic variant in the promoter of connective tissue growth
factor gene confers susceptibility to nephropathy in type 1 diabetes. Wang
et al., J Med Genet 2010; 47:391-397. Doi:10,1136/jmg.2009.073098
It was with great interest that we read the recent study by Wang et
al. on a novel C/G single nucleotide polymorphism (SNP) at position -20 in
the promoter of the connective tissue growth factor (CTGF) gene confe...
Re: Genetic variant in the promoter of connective tissue growth
factor gene confers susceptibility to nephropathy in type 1 diabetes. Wang
et al., J Med Genet 2010; 47:391-397. Doi:10,1136/jmg.2009.073098
It was with great interest that we read the recent study by Wang et
al. on a novel C/G single nucleotide polymorphism (SNP) at position -20 in
the promoter of the connective tissue growth factor (CTGF) gene confers
susceptibility to diabetic nephropathy in patients with type 1 diabetes
(T1D).[1] Based on these findings we studied this SNP in our cohort of T1D
to determine its association with the development of diabetic nephropathy.
This SNP was genotyped in 932 European Caucasoid subjects and failed to
detect the polymorphism.
Connective tissue growth factor (CTGF) is a secreted protein with a
molecular weight at 36-38 kDa and plays an important role in the balance
of degradation and synthesis of extracellular matrix although its
physiological functions are not limited to this.[2] Several studies have
demonstrated that CTGF plays a fundamental role in the histopathological
changes seen in diabetic nephropathy. It has been demonstrated that low
levels of glomerular CTGF are found in normal human glomeruli, but both
mRNA and protein levels of CTGF increase during the early stages of
diabetic nephropathy and continue to increase with disease progressionand
these increases also correlate with the degree of albuminuria.[3]
The CTGF gene is located on Chromosome 6q23 and has 5 exons. Several
SNPs have been identified in the promoter, introns, exons and 3'UTR
regions of the gene.[1,4-6] Some of these SNPs have been studied and shown
to be associated with various conditions including systemic sclerosis and
cardiovascular diseases.[5-6] Addition, Wang et al. report has described a
novel C/G SNP at position -20 in the promoter of the CTGF gene that is
associated with nephropathy in T1D.[1] In this report, 862 subjects from
the DCCT/EDIC cohort of T1D were genotyped. The frequencies of the CC, CG
and GG genotypes were 62.76% (541/862), 31.90% (275/862) and 5.34%
(46/862) in this cohort respectively. The frequency of GG genotype in
patients with microalbuminuia (albumin excretion rate (AER) >40mg/24h)
was significantly higher than patients with AER <40mg/24h, p<0.0001.
The GG genotype was also shown to have greater transcriptional activity
than that of the CG and CC genotypes.
A total of 739 Caucasoid patients with T1D (Female: 402; Male: 347)
and with or without microvascular complications and 193 normal ethnically
matched controls (Female: 101; Male: 92) were recruited in our study.
Patients with T1D had an average age of 30.83 years (range: 1-76 years)
and the average age of onset was 16.86 years (range: <1-52 years) with
an average duration of T1D at 13.98 years (range: 0-55 years). The study
was approved by the Local Research Ethical Committee and informed consent
was obtained from all subjects. All patients have T1D as defined by The
Expert Committee on the Diagnosis and Classification of Diabetes
Mellitus.[7] Normal controls were obtained from cord blood samples
following normal healthy obstetric delivery in Derriford Hospital,
Plymouth.
Genomic DNA was prepared from peripheral and cord blood samples using
the Nucleon II extraction kit (Scotlab, Lanarkshire, UK) following the
manufacture's instruction. DNA samples were sent in 96-well plates to
KBioscioences together with the SNP information published in Wang's
study.[1] The sequence which covers the location of the SNP in bracket:
GTATAAAAGC[C/G]TCGGGCCGCC has been checked and confirmed with the
published sequence of the CTGF gene.[4,8] Genotyping for this SNP was
performed using KASPar assays which are a proprietary in-house system
(KBiosciences, Herts, UK). Unexpectedly, our results showed there was no
SNP at the position -20 of the CTGF gene in our entire population; all
subjects had the CC genotype.
We tried to understand why there were discrepancies between our
findings and those of Wang et al.[1] Wang's study is the only publication
regarding this SNP. There are a number of possible reasons for the
discrepancy. Firstly, with respect of the ethnicity, our studied
population was 100% Caucasian, Wang's subjects from the DCCT/EDIC cohort
of T1D contained 96-97% of Caucasian.[9] The genetic backgrounds might be
different even within Caucasian populations between different geographic
locations.[5,10] but we would expect that the differences in the genetic
backgrounds would cause the differences in the frequency of each genotype
rather than no genetic variants at the position -20 of the CTGF gene.
Secondly, sample size could be an issue, according to Wang's results: the
frequencies of GG and GC were 5.34% and 31.90% respectively in their
population, we should be able to detect about 39 subjects with GG and 235
subjects with GC genotypes out of our 739 subjects with T1D. Therefore, it
is unlikely that our sample size was too small to allow the detection of
the minor genotypes GG or GC. Thirdly, genotyping techniques may give rise
to false positives or negatives. We used a highly reputable commercial
genotyping facility-KBiosciences. Furthermore, our samples have been
extensively genotyped including another SNP (rs9399005) in the CTGF gene
that were typed in parallel to this one (Our unpublished data, 2011).
Wang's study used PCR in their-own laboratory and confirmed this SNP by bi
-directional sequencing. Sequences were detected on a Megabase N500
sequencer and results were analysed with sequencer software (Gene Codes
Corporation, Ann Arbour, Michigan, USA). Consequently, it is unlikely that
technical issues could explain the discrepancy between the sets of
results. Finally, we don't think that the disparate results in this SNP
between the two groups are due to the gender, age, duration of diabetes or
age at onset of diabetes of patients either as the CTGF gene is not
located in the X or Y chromosomes and the age, duration of diabetes and
age at onset of diabetes of patients in both groups were similar. In
conclusion, the reason for this discrepancy is unclear but is probably a
reflection of the heterogeneity of populations. Therefore, further studies
are needed to confirm this SNP from different groups or independent
populations.
References:
1. Wang B, Cater RE, Jaffa MA, et al. Genetic variant in the promoter of
connective tissue growth factor gene confers susceptibility to nephropathy
in type 1 diabetes. J Med Genet 2010;47:391-397.
2. Mason RM. Connective tissue growth factor (CCN2), a pathogenic factor
in diabetic nephropathy. What does it do? How does it do it? J Cell Commun
Signal 2009;3:95-104.
3. Wahab NA, Schaefer L, Weston BS, et al. Glomerular expression of
thrombospondin-1, transforming growth factor beta and connective tissue
growth factor at different stages of diabetic nephropathy and their
interdependent roles in mesangial response to diabetic stimuli.
Diabetologia 2005;48:2650-2660.
4. Blom IE, van Diji AJ, de Weger RA, et al. Identification of human ccn2
(connective tissue growth factor) promoter polymorphisms. J Clin Pathol
Mol Pathol 2001;54:192-196.
5. Granel B, Agriro L, Hachulla E, et al. Association between a CTGF gene
polymorphism and system sclerosis in a French population. J Rheumatol
2010;37:351-358.
6. Cozzolino M, Biondi ML, Banfi E, et al. CCN2 (CTGF) gene polymorphism
is a novel prognostic risk factor for cardiovascular outcomes in
hemodialysis patients. Blood Purif 2010;30:272-276.
7. The Expert Committee on the diagnosis and classification of diabetes
mellitus. Report of the Expert Committee on the diagnosis and
classification of diabetes mellitus. Diabetes Care 2003;26:S5-S20.
8. Homo sapiens chromosome 6, GRCh37.p2 primary reference assembly.
Retrieved on 23rd June 2011 from http://www.ncbi.nlm.nih.gov
9. The Diabetes Control and Complications Trial Research Group. The effect
of intensive treatment of diabetes on the development and progression of
long-term complications in insulin-dependent diabetes mellitus. N Eng J
Med 1993;329:977-986.
10. Rueda B, Simeon C, Hesselstrand R, et al. A large multicenter analysis
of CTGF-945 promoter polymorphism does not confirm association with
systemic sclerosis susceptibility or phenotype. Ann Rheum Dis 2009;68:1618
-1620.
It was with great interest that I read Casasnovas et al article
"Phenotypic Spectrum of MFN2 Mutations in the Spanish Population". The
authors mention the Gly298Arg mutation in one of their families, with 2
affected individuals, and state that this has previously been described.
They refer to Lawson's 2005 article (Ref#10, Lawson VH, Graham BV,
Flanigan KM. Clinical and electrophysiologic features of
CMT2A with mutation...
It was with great interest that I read Casasnovas et al article
"Phenotypic Spectrum of MFN2 Mutations in the Spanish Population". The
authors mention the Gly298Arg mutation in one of their families, with 2
affected individuals, and state that this has previously been described.
They refer to Lawson's 2005 article (Ref#10, Lawson VH, Graham BV,
Flanigan KM. Clinical and electrophysiologic features of
CMT2A with mutations in the mitofusin 2 gene. Neurology 2005;65:197) as
having described this mutation previously.
However, upon careful review of Lawson's article, I only found
mention of this mutation as a SNP found by the Utah researchers in 3% of
their control chromosomes.
Would the authors please comment on what evidence they have that this
is a disease causing mutation, or is this a benign polymorphism? As the
authors mention, this point mutation is in a highly conserved region of
the dynamin like GTPase region of mitofusin 2, and results in a non-
synonymous amino acid change, therefore one would suspect it as disease
causing.
It was with great interest that we read the recent article by
Schrader et al. on the low frequency of CDH1 mutations in early-onset and
familial lobular breast cancer (1). As detailed by Schrader et al., the
cancer syndrome hereditary diffuse gastric cancer (HDGC), in addition to a
high risk of diffuse gastric cancer (DGC), is associated with an increased
risk of lobular breast carcinoma, a specific histological subtype of...
It was with great interest that we read the recent article by
Schrader et al. on the low frequency of CDH1 mutations in early-onset and
familial lobular breast cancer (1). As detailed by Schrader et al., the
cancer syndrome hereditary diffuse gastric cancer (HDGC), in addition to a
high risk of diffuse gastric cancer (DGC), is associated with an increased
risk of lobular breast carcinoma, a specific histological subtype of the
disease (2,3). Germline mutations in CDH1, encoding E-cadherin have been
identified as the underlying cause of HGDC in 30-50% of families (4). We
have identified a deletion of exon 16 occurring in an individual with
lobular breast cancer with an associated family history of gastric cancer.
In addition, several groups have reported infrequent CDH1 inactivating
mutations in sporadic and familial cases of lobular breast carcinoma that
are not associated with HGDC (5-8).
An increased incidence of breast cancer has also been reported in
families with Saethre-Chotzen syndrome (SCS) (9), an autosomal dominant
craniosynostosis syndrome, which is characterised by premature fusion of
coronal sutures and limb abnormalities. SCS is caused by mutations in the
basic helix-loop-helix transcription factor TWIST1. Interestingly, TWIST1
over-expression has been associated with breast cancer progression and
metastasis through loss of E-cadherin mediated cell-cell adhesion (10).
As part of our continuing study on factors contributing to the risk
of breast cancer, we have recently undertaken a similar study to
investigate the contribution of variants in CDH1 and TWIST1 to lobular
breast cancer in a familial setting. We selected 104 unrelated individuals
with lobular breast cancer, all with a family history of breast cancer
fulfilling NICE criteria for BRCA1 and BRCA2 screening (>20% risk of
mutation) (11). Sequence analysis and multiplex ligation-dependent probe
amplification (MLPA) of BRCA1 and BRCA2 identified no mutations in this
group. The age at first presentation of the probands ranged from 28-68
years and 14 patients had bilateral breast cancer. All 104 were screened
for germline mutations in the coding regions of CDH1 and TWIST1 and 86
were successfully analysed for large deletions/amplifications in CDH1
using MLPA.
Like Schrader et al., we found no truncating point mutations in CDH1
however, a heterozygous deletion of CDH1 exons 1 and 2 was observed in one
patient. Three of her sisters were also affected with lobular disease, two
with invasive breast cancer. In one of these, with lobular carcinoma in
situ, DNA was available for testing and confirmed the presence of the
mutation. The four sisters were aged 50, 49, 51 and 53 years at diagnosis
of lobular breast cancer, the proband eventually dying from primary
pancreatic cancer aged 60 years. There was no history of gastric cancer in
up to third degree relatives in the family. Oliveira et al. previously
reported deletion of CDH1 exons 1 and 2 segregating in three families with
familial gastric cancer (4), one of which was associated with lobular
breast cancer. However, our finding is the first report of a CDH1 deletion
in the context of lobular breast cancer without a history of gastric
cancer.
A novel non-synonymous change in exon 5 of CDH1, c.670C>T,
p.R224C, was identified in one patient. DNA was unavailable from an
affected family member to test for segregation. The variant was not
identified in a panel of 180 ethnically matched healthy controls. However,
in-silico analysis (Polyphen) predicted this missense change to be benign
and the residue is not conserved across species. Therefore, it is
inconclusive if this variant predisposes to breast cancer in this family.
A novel synonymous change c.2451G>A in CDH1 exon 16 was observed
in two patients and four patients had the intronic variant c.532-18C>T
which has not been described in SNP databases. There is no evidence to
support pathogenicity for these variants.
No mutations were identified in TWIST1.
In agreement with Schrader et al., we conclude that germline
mutations in CDH1 are not common in familial lobular breast cancer.
Although large single or multiple exon deletions can be occasionally
identified in association with a highly penetrant phenotype for lobular
breast cancer. We cannot however rule out the possibility that
hypermethylation of promoter and regulatory regions of both CDH1 and
TWIST1 contribute to the altered expression of these genes frequently
observed in breast tumours. Additional studies are needed to provide more
insight into the aetiology of lobular breast carcinoma and to identify
further causal variants.
Acknowledgements.
This work was funded through support of the NIHR Manchester Biomedical
Research Centre and Central Manchester Foundation Trust Research Grant.
References.
1. Schrader KA, Masciari S, Boyd N, Salamanca C, Senz J, Saunders DN,
Yorida E, Maines-Bandiera S, Kaurah P, Tung N, Robson ME, Ryan PD, Olopade
OI, Domchek SM, Ford J, Isaacs C, Brown P, Balmana J, Razzak AR, Miron P,
Coffey K, Terry MB, John EM, Andrulis IL, Knight JA, O'Malley FP, Daly M,
Bender P; kConFab, Moore R, Southey MC, Hopper JL, Garber JE, Huntsman DG.
Germline mutations in CDH1 are infrequent in women with early-onset or
familial lobular breast cancers.
J Med Genet 2011;48:64-8.
2. Keller G, Vogelsang H, Becker I. Diffuse type gastric and lobular
breast carcinoma in familial gastric cancer patient with an E-Cadherin
germline mutation. Am J Pathol 1999;155:337-42.
3. Brooks-Wilson AR, Kaurah P, Suriano G, Leach S, Senz J, Grehan N,
Butterfield YS, Jeyes J, Schinas J, Bacani J, Kelsey M, Ferreira P,
MacGillivray B, MacLeod P, Micek M, Ford J, Foulkes W, Australie K,
Greenberg C, LaPointe M, Gilpin C, Nikkel S, Gilchrist D, Hughes R,
Jackson CE, Monaghan KG, Oliveira MJ, Seruca R, Gallinger S, Caldas C,
Huntsman D. Germline E-Cadherin mutations in hereditary diffuse gastric
cancer: assessment of 42 families and review of genetic screening
criteria. J Med Genet 2004;41:508-17.
4. Oliveira C, Senz J, Kaurah P, Pinheiro H, Sanges R, Haegert A, Corso G,
Schouten J, Fitzgerald R, Vogelsang H, Keller G, Dwerryhouse S, Grimmer D,
Chin SF, Yang HK, Jackson CE, Seruca R, Roviello F, Stupka E, Caldas C,
Huntsman D. Germline CDH1 deletions in hereditary diffuse gastric cancer
families. Hum Mol Genet 2009;18:1545-1555.
5. Berx G., Cleton-Jansen AM, Strumane K, de Leeuw WJ, Nollet F, van Roy
F, Cornelisse C. E-Cadherin is inactivated in a majority of invasive human
lobular breast cancers by truncation mutations throughout its
extracellular domain. Oncogene 1996; 13:1919-25.
6. Vos CB, Cleton-Jansen AM, Berx G, de Leeuw WJ, ter Haar NT, van Roy F,
Cornelisse CJ, Peterse JL, van de Vijver MJ. E-cadherin inactivation in
lobular carcinoma in situ of the breast: an early event in tumorigenesis.
Br J Cancer 1997;76:1131-1133.
7. Masciari S, Larsson N, Senz J, Boyd N, Kaurah P, Kandel MJ, Harris LN,
Pinheiro HC, Troussard A, Miron P, Tung N, Oliveira C, Collins L, Schnitt
S, Garber JE, Huntsman D. Germline E-Cadherin mutations in familial
lobular breast cancer. J Med Genet 2007;44:726-731.
8. Salahshor S, Haixin L, Hou H, Kristensen VN, Loman N, Sj?berg-Margolin
S, Borg A, B?rresen-Dale AL, Vorechovsky I, Lindblom A. Low frequency of E
-cadherin alterations in familial breast cancer. Breast Cancer Res
2001;3:199-207.
9. Sahlin P, Windh P, Lauritzen C, Emanuelsson M, Gr?nberg H, Stenman G.
Women with Saethre-Chotzen syndrome are at increased risk of breast
cancer. Genes Chr Can 2007;46:656-660.
10. Yang J, Mani SA, Donaher JL, Ramaswamy S, Itzykson RA, Come C,
Savagner P, Gitelman I, Richardson A, Weinberg RA. Twist, a master
regulator of morphogenesis, plays an essential role in tumour metastasis.
Cell 2004;117:927-939.
11. NICE clinical guideline 41. Familial breast cancer. The classification
and care of women at risk of familial breast cancer in primary, secondary
and tertiary care. 2006.
We have read the entire paper with great care. We totally agree with the findings of the respected authors in this respective article. The article seems to be used by many researchers as a reference article so we need to update the status of SMA diagnosis for non deleted SMA patients. Many new approaches have been employed to diagnose non deleted SMA. One of such methods is long range PCR method (LR-PCR) (Clemont et al....
It was with great interest that I read the study by Tang and co- authors [1], in which they discovered twenty-five novel mutations in DNA polymerase gamma. Based on the presence of p.G268A substitution in heterozygosis in 19 subjects from a cohort of 2697 unrelated patients, they proposed to reclassify this mutation as a neutral polymorphism or a polymorphic modifier rather than a pathological mutation. Smith and co- auth...
The respective article was well read by us. We agree with the precious scientific findings by the authors but at the same time we would like to recall the two very basic fundamental functions of CBP, which involve CBP as a bridging molecule and a cofactor (Montmini et al., 1986) for CREB modulated gene expression and histone acetyltransferase activity of CBP on CREB modulated gene expression (Lu et al., 2003) specificall...
In their recently published paper describing mutations in mitochondrial DNA polymerase gamma, Tang et al.(1) propose that the POLG p.G268A (c.803G>C) and p.G517V (c.1550G>T) variants which have previously been reported as pathogenic mutations should be considered as unclassified variants that may represent rare neutral polymorphisms or polymorphic modifiers.
We have also identified these variants in our...
Re: Genetic variant in the promoter of connective tissue growth factor gene confers susceptibility to nephropathy in type 1 diabetes. Wang et al., J Med Genet 2010; 47:391-397. Doi:10,1136/jmg.2009.073098
It was with great interest that we read the recent study by Wang et al. on a novel C/G single nucleotide polymorphism (SNP) at position -20 in the promoter of the connective tissue growth factor (CTGF) gene confe...
It was with great interest that I read Casasnovas et al article "Phenotypic Spectrum of MFN2 Mutations in the Spanish Population". The authors mention the Gly298Arg mutation in one of their families, with 2 affected individuals, and state that this has previously been described. They refer to Lawson's 2005 article (Ref#10, Lawson VH, Graham BV, Flanigan KM. Clinical and electrophysiologic features of CMT2A with mutation...
It was with great interest that we read the recent article by Schrader et al. on the low frequency of CDH1 mutations in early-onset and familial lobular breast cancer (1). As detailed by Schrader et al., the cancer syndrome hereditary diffuse gastric cancer (HDGC), in addition to a high risk of diffuse gastric cancer (DGC), is associated with an increased risk of lobular breast carcinoma, a specific histological subtype of...
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