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 would like to thank Dr. Hennekam for his comments but would like
to reply to several points made by him. We agree with Dr. Hennekam that
there is a good correlation between the current nosology and the FBN1
mutation uptake, but an important goal for the new nosology is to make it
simpler and more easily applicable (which is not always true for the
current one). There is also an important focus on the cardiovascular
a...
We would like to thank Dr. Hennekam for his comments but would like
to reply to several points made by him. We agree with Dr. Hennekam that
there is a good correlation between the current nosology and the FBN1
mutation uptake, but an important goal for the new nosology is to make it
simpler and more easily applicable (which is not always true for the
current one). There is also an important focus on the cardiovascular
aspect of the disease which remains the most important for the life
expectancy.
Dr. Hennekam suggests that the five major changes in the revised
nosology have severe shortcomings. However, we believe there are many more
reasons mentioned in the paper that played a role in the revision. Since
physicians associate the diagnosis of "Marfan syndrome", above all else,
with risk for aortic aneurysm/dissection, it can be detrimental to
diagnose MFS in patients without tangible evidence of such risk. Avoidable
consequences associated with misdiagnosis of MFS include: restriction of
career aspirations or access to insurance benefits; additional financial
burden associated with frequent medical care; anxiety or situational
depression; unfounded marital or reproductive decisions; loss of health
benefits or psychosocial stigmatization associated with exercise
restriction, a particularly important issue during childhood. The
challenge is to balance such concerns with the paramount need to maintain
good health through proper counseling and application of sound
anticipatory medical practices. Toward this objective, it is also
important to avoid the diagnosis of MFS when clinical or molecular
observations could reveal alternative (and often more severe) diagnoses
that mandate specialized counseling or management protocols.
We agree that the focus of the current nosology is on the
cardiovascular and the ocular complications, which from a medical
perspective are still the most objective and have the greatest medical
implications. As such it is logical to start from those two symptoms. We
agree with Dr Hennekam that according to the patient survey the skeletal
features may be subjectively more important but we believe this very
important for the management aspect but not so much for the diagnostic
decision making process. Individual skeletal findings do not bear great
sensitivity but as a whole they are indeed important. As such, we did not
abandon the skeletal findings but these are put more in the context of all
systemic findings and the more important skeletal findings do get a
greater weight in this systemic score. Other major criteria that have not
been validated such as dural ectasia do get lower weight. For eg do we
really want a patient with skeletal findings and dural ectasia and striae
be called MFS ? We believe there is a major difference between clinical
characteristics to make a diagnosis and on the other side characteristics
that are important in the clinical management. Skeletal features in
themselves rarely allow to make a MFS diagnosis.
We agree that there will always be patients with TGFBR1/2 mutations
that resemble Marfan syndrome but we prefer to distinguish two categories
based on the gene (FBN1 versus TGFBR1/2) because of the different medical
implications. Again this is a choice made from a practical viewpoint to
create a clear situation even though we recognize that also within the
TGFBR1/2 patients there is a spectrum of disease.
We agree that there is poor genotype (FBN1)-phenotype correlation and
this also reflected in the current nosology. For eg. we do stress that
certain FBN1 mutations do not have cardiovascular implications and that
ectopia lentis syndrome is possible with FBN1 mutations. For eg we also
abandon the use of neonatal MFS; this represents the severe end of a
phenotypic spectrum. With the current nosology, it is very important to
distinguish TGFBR1/2 mutants because they have a more severe natural
history. A nosology should also be made for the great majority of
patients, not for the exceptions and with the current technology an FBN1
mutation is found in close to 95%
The combination of aortic root dilation and ectopia lentis is very
specific; there is no other diagnosis than Marfan syndrome. In the current
literature there is no consensus on the definition and most optimal
technique to determine dural ectasia. Until there is better data on the
specificity of dural ectasia, we have opted to include it in the systemic
score. We had hours of discussion with ophthalmologists on the methods and
cut-offs that could be used. We believe the majority of the
ophthalmologist do no not measure axial globe length or corneal
flattening. Although we agree that these might be interesting to study the
different ocular findings for their specificity. Again there are no good
data in the literature about sensitivity and specificity of the ocular
findings. The pulmonary artery dilation, again has no standard measurement
method or cut-off; moreover the clinical relevance of this finding is very
low.
The key point is that physicians should not only look for the
features specific for Marfan syndrome but also for other relevant
differential diagnostics; this is specifically true for Loeys-Dietz
syndrome, Shprintzen-Goldberg syndrome and vascular Ehlers-Danlos
syndrome. There are criteria for vascular Ehlers-Danlos syndrome (although
they also need better validation) and for Loeys-Dietz syndrome it is too
early in the discovery to define good criteria, but we will continue to
work on that.
There is many other points that are made in the introduction of the
paper; eg children, psychosocial burden,... that also call for changes
not only in the five sections that are now quoted by Dr Hennekam. We also
do now define specific diagnostic categories for children and define
alternative diagnoses such as MASS, MVPS (mitral valve prolapse) and ELS
(ectopia lentis syndrome) in individuals older than 20 years.
Prof Dr Bart Loeys
Prof Dr Harry Dietz
Prof Dr Anne De Paepe
Bancroft et al. describe the successful establishment of a novel
specialist clinic for BRCA1/2 mutation carriers. (1) The authors should be
applauded for the introduction of this specialized, multi-disciplinary
clinic. However, although their study provides elaborate data on numbers
of patients followed in this clinic, it remains unclear what the
information provision and guidance for decision-making in the multi-
discipl...
Bancroft et al. describe the successful establishment of a novel
specialist clinic for BRCA1/2 mutation carriers. (1) The authors should be
applauded for the introduction of this specialized, multi-disciplinary
clinic. However, although their study provides elaborate data on numbers
of patients followed in this clinic, it remains unclear what the
information provision and guidance for decision-making in the multi-
disciplinary team consisted of. This is of special importance with respect
to the counselling of BRCA1/2 carriers whether or not to undergo
prophylactic mastectomy. In the article of Bancroft the effect of
prophylactic mastectomy is emphasized. In contrast with surveillance for
ovarian cancer that appeared to be ineffective, either annual
mammography plus MRI screening or prophylactic mastectomy seem to offer
comparable results with respect to survival (2); in our opinion there is
no need to direct patients towards a decision for prophylactic mastectomy.
Of note, although the rate of false-positive MRI results in this
population is high, the impact of a false-positive MRI on the choice for
prophylactic mastectomy is limited and is determined by the woman's
preference before the establishment of a BRCA mutation. (3) In our
multidisciplinary clinic for BRCA1/2 carriers, which we started in 1999,
27% had an initial preference for prophylactic mastectomy. After a median
observation period of 2 years, 30% had undergone prophylactic mastectomy.
(4) We believe that a careful counselling of the pros and cons of
prophylactic mastectomy and an open discussion on real and perceived risk
reduction by prophylactic mastectomy is crucial. For carriers of a BRCA1/2
mutation and their family members such counselling may be the surplus
value of a specialized multidisciplinary clinic.
References
1. Bancroft EK, Locke I, Ardern-Jones A et al. The carrier clinic: an
evaluation of a novel clinic dedicated to the follow-up of BRCA1 and BRCA2
carriers--implications for oncogenetics practice. J Med Genet. 2010;47:486
-491.
2. Kurian AW, Sigal BM, Plevritis SK. Survival analysis of cancer risk
reduction strategies for BRCA1/2 mutation carriers. J Clin Oncol.
2010;28:222-231.
3. Hoogerbrugge N, Kamm YJ, Bult P et al. The impact of a false-positive
MRI on the choice for mastectomy in BRCA mutation carriers is limited. Ann
Oncol. 2008;19:655-659.
4. Landsbergen KM, Prins JB, Kamm YJ et al. Female BRCA mutation carriers
with a preference for prophylactic mastectomy are more likely to
participate an educational-support group and to proceed with the preferred
intervention within 2 years. Fam Cancer. 2010;9:213-220.
The Ghent criteria as proposed in 1996 are world-wide well accepted
to define the diagnostic criteria for Marfan syndrome. The criteria are
easy to use and work extremely well, shown by finding causative FBN1
Mutations in 97% of cases. Indeed this specificity of diagnostic criteria
is amongst the highest reported in any syndromic entity.
A large group of superb Marfan specialists have now suggested a revision
of these cr...
The Ghent criteria as proposed in 1996 are world-wide well accepted
to define the diagnostic criteria for Marfan syndrome. The criteria are
easy to use and work extremely well, shown by finding causative FBN1
Mutations in 97% of cases. Indeed this specificity of diagnostic criteria
is amongst the highest reported in any syndromic entity.
A large group of superb Marfan specialists have now suggested a revision
of these criteria (1). Unfortunately the suggested five major changes in
the revision have severe shortcomings:
1. More weight to aortic root aneurysm/dissection and ectopia lentis.
Marfan syndrome is a systemic entity, in which many body parts are
involved. There is no ratio to increase some of these manifestations
(aorta; eye) or decrease others (skeleton). None has a complete
sensitivity, none is 100% specific. The authors may argue that the eye and
cardiovascular manifestations have the most significant implications.
However, the use of new management strategies may well change this in the
near future. More importantly, medical implications should not be of
importance in establishing diagnostic criteria: sensitivity and
specificity should be leading here. In this respect it is useful
mentioning that the specificity of aortic root aneurysm/dissection is
likely to be lower than the specificity of dural ectasia. One may also
consider what individuals having Marfan syndrome themselves consider as
the most important manifestations. Indeed, in a questionnaire study of 857
European Marfan patients it was found that not the vascular or visual
problems were the most important issues for the patients themselves but
this was their body build (2). In fact from a patient perspective this
argues to tag more importance to the skeletal findings than any other
manifestation.
2. More prominent role of FNB1 and TGFB1/2 testing. The genotype-
phenotype correlation of Marfan syndrome with FBN1 mutations is weak.
There are patients with a FBN1 mutation who do not have Marfan syndrome
and there are patients who have all manifestations of Marfan syndrome who
do not have a (detectable) FBN1 or TGFB1/2 mutation. As has been shown in
many other entities it may still be that Marfan syndrome will be
genetically heterogeneous. Therefore it is doubtful whether it is wise to
put more emphasis on any molecular results than was already done in the
existing Ghent criteria.
3. Removal or downgrading of less specific manifestations. Dural
ectasia is downgraded as it may be ..sensitive but is not specific.. .
However, as stated above it may well be that it is more specific than
enlargement of the aortic root or ectopia lentis, and it is easily and
reliably determinable (3, 4). Increased globe length and corneal
flattening are removed as ..they have an unclear specificity and are not
routinely measured by ophthalmologists.. . No one doubts these eye
manifestations form part of the multisystemic manifestations of Marfan
syndrome. It seems more logical to perform studies reliably determining
the specificity of global length and corneal flattening in Marfan
syndrome, and to instruct ophthalmologist to perform these (not difficult
and cheap) investigations than to remove the manifestations from the
criteria. Pulmonic artery dilatation is removed as ..it is not specific to
this diagnosis. In addition complications ... occur rarely.. . Beyond
doubt pulmonic manifestations are however a manifestation of the vascular
system in Marfan syndrome and therefore can be useful. In addition,
whether or not a manifestation provides complications is not influencing
its usefulness as a diagnostic criterion. Surely there are still
subjective qualifiers in the present proposal (such as mitral valve
prolaps or pectus carinatum) but this remains inescapable in describing
the human phenotype.
4. Differentiation from other connective tissue disorders. This is
very valuable. However, the diagnostic criteria of one entity should not
be influenced by diagnostic criteria of other entities. The similarity of
Marfan syndrome to entities such as Loeys-Dietz syndrome and Shprintzen-
Goldberg syndrome would simply urge for reliable diagnostic criteria of
each of these entities, as comparing the criteria with those in Marfan
syndrome would help best in allowing such differentiation.
5. Providing context-specific recommendations for patient counselling
and follow-up. This is an excellent point and surely needs attention.
However in itself such recommendations are no reason to change the
diagnostic criteria, and can easily be published separately.
I conclude that the reasoning to change the existing diagnostic
criteria contains significant flaws, and suggest keeping using the
existing Ghent criteria. The points mentioned by the authors do not ask
for adaptation of the Ghent criteria: they ask for amendments regarding
entities from which Marfan syndrome should be differentiated by defining
these entities better; they ask for separate recommendations for
counselling and follow-up. Specific attention to the age dependent nature
of some manifestations can be added too, as this is now added only for the
aortic root enlargement. Suggestions for additional studies of specific
manifestations can be made or the studies can be initiated by the authors
themselves.
The present Ghent criteria function extremely well and are well
incorporated in the care for Marfan patients: with limited amendments, the
criteria can remain unchanged and even function better.
References
1. Loeys B, Harry C Dietz, Braverman AC, Callewaert BL, De Backer J,
Devereux RB, Hilhorst-Hofstee Y, Jondeau G, Faivre L, Milewicz DM, Pyeritz
RE, Sponseller PD, Wordsworth P, De Paepe AM. The revised Ghent nosology
for the Marfan syndrome. J Med Genet 2010;47:476-485.
2. De Bie S, De Paepe A, Delvaux I, Davies S, Hennekam RC. Marfan syndrome
in Europe. Community Genet 2004;7:216-225.
3. Lundby R, Rand-Hendriksen S, Hald JK, Lilleas FG, Pripp AH, Skaar S,
Paus B, Geiran O, Smith HJ. Dural ectasia in Marfan syndrome: a case
control study. AJNR 2009;30:1534-1540.
4. Sznajder M, Krug P, Taylor M, Moura B, Leparc JM, Boileau C, Jondeau G,
Chevallier B, Pelage JP, Stheneur C. Spinal imaging contributes to the
diagnosis of Marfan syndrome. Joint Bone Spine 2010 May 7. [Epub ahead of
print]
In the July issue, Loeys and colleagues present new diagnostic
criteria for Marfan Syndrome (MFS) in their manuscript "The revised Ghent
nosology for the Marfan Syndrome"[1]. After publication of these Revised
Ghent Marfan criteria, a manuscript was published which in part supports
their opinions[2]. After complimenting Loeys et.al. with the result of
their multidisciplinary effort, we would like...
In the July issue, Loeys and colleagues present new diagnostic
criteria for Marfan Syndrome (MFS) in their manuscript "The revised Ghent
nosology for the Marfan Syndrome"[1]. After publication of these Revised
Ghent Marfan criteria, a manuscript was published which in part supports
their opinions[2]. After complimenting Loeys et.al. with the result of
their multidisciplinary effort, we would like to comment on two issues:
First, the authors allocate more importance to genetic criteria for
making a diagnosis of MFS and much less importance to the clinical feature
"dural ectasia", compared to the 1996 "Revised diagnostic criteria for
Marfan Syndrome". In our opinion this is a prudent decision, as in
clinical practice a group of patients emerged after 1996, who had one
major criterium for the diagnosis of Marfan Syndrome (usually aortic
dilatation) as well as dural ectasia. Combined with even the slightest
involvement of other organ systems (usually the skeletal system), patients
in this group fulfilled the former diagnostic criteria for MFS[3], while
in our hospitals most of these cases are non-familial and without FBN1 or
TGFBR1/2 mutation. Although dural ectasia was once seen as an important
and discriminating feature of MFS[4], different measurement methods of
dural ectasia have been the subject of debate[5], and a recent study
corroborated the role of dural ectasia as a non-specific marker for
connective tissue disease[2]. In the now presented Revised Ghent criteria,
cases with dural ectasia need substantial more other symptomatology to
fulfil the criteria for Marfan Syndrome, which is in line with one of the
aims of the paper, preventing overdiagnosis of Marfan Syndrome.
Second, a maybe minor point, is that the authors propose to label the
term "potential Marfan Syndrome" to young individuals in whom a FBN1
mutation is identified, but in whom "aortic measurements are still below
Z=3". This seems contradictory with the rest of their manuscript, as the
fulfilment of criteria needed for an unequivocal diagnosis of Marfan
Syndrome, does not depend on the aortic root diameter alone, but on other
developing symptoms as well. Furthermore, the term "potential" means
"possible"[6] and as such is confusing in this context, as the penetrance
of FBN1 mutations/Marfan Syndrome is 100% at adult age. Terminology like
"latent MFS" or "subclinical MFS" seems more appropriate, but in our
opinion there is no need to make things more complicated then they already
are: In our experience, children with a (pathogenic) FBN1 mutation but
with no or only minimal clinical symptoms, are labelled by their families
and doctors as having "MFS" or "carrier of MFS", without problems or
confusion.
Reference List
(1) Loeys BL, Dietz HC, Braverman AC, Callewaert BL, De BJ, Devereux
RB, Hilhorst-Hofstee Y, Jondeau G, Faivre L, Milewicz DM, Pyeritz RE,
Sponseller PD, Wordsworth P, De Paepe AM. The revised Ghent nosology for
the Marfan syndrome. J Med Genet 2010 Jul;47(7):476-85.
(2) Sheikzadeh S, Rybczynski M, Habermann CR, Bernhardt AMJ, Arslan-
Kirchner M, Keyser B, Kaemerrer H, Mir TS, Staebler A, Oezdal N, Robinson
PN, Berger J, Meinertz T, von Kodolitsch Y. Dural ectasia in individuals
with Marfan-like features but exclusion of mutations in the genes FBN1,
TGFBR1 and TGFBR2. Clin Genet 2010 Jun 23;doi:10.1111/j.1399-
0004.2010.0194.x.
(3) De PA, Devereux RB, Dietz HC, Hennekam RC, Pyeritz RE. Revised
diagnostic criteria for the Marfan syndrome. Am J Med Genet 1996 Apr
24;62(4):417-26.
(4) Fattori R, Nienaber CA, Descovich B, Ambrosetto P, Reggiani LB,
Pepe G, Kaufmann U, Negrini E, von KY, Gensini GF. Importance of dural
ectasia in phenotypic assessment of Marfan's syndrome. Lancet 1999 Sep
11;354(9182):910-3.
(5) Weigang E, Ghanem N, Chang XC, Richter H, Frydrychowicz A, Szabo
G, Dudeck O, Knirsch W, von SP, Langer M, Beyersdorf F. Evaluation of
three different measurement methods for dural ectasia in Marfan syndrome.
Clin Radiol 2006 Nov;61(11):971-8.
(6) Internet Communication, 03-08-2010.
http://dictionary.reference.com/browse/potential
Dr. Constantin Polychronakos, Editor Journal of Medical Genetics
Dear Dr. Polychronakos
We read with great interest the recent publication from your journal
entitled "SOD1, ANG, VAPB, TARDBP, and FUS mutations in familial
amyotrophic lateral: genotype - phenotype correlations" Millecamps S.,
Salachas F., Cazeneuve C., et al. J Med Genet published online June 24,
2010 doi: 10.1136/jmg.2010.077180. This manuscript brings re...
Dr. Constantin Polychronakos, Editor Journal of Medical Genetics
Dear Dr. Polychronakos
We read with great interest the recent publication from your journal
entitled "SOD1, ANG, VAPB, TARDBP, and FUS mutations in familial
amyotrophic lateral: genotype - phenotype correlations" Millecamps S.,
Salachas F., Cazeneuve C., et al. J Med Genet published online June 24,
2010 doi: 10.1136/jmg.2010.077180. This manuscript brings relevant
information about the mutation frequencies and genotype-phenotype data
regarding the main genes responsible for the familial forms of ALS (FALS).
Additionally, as well pointed by the authors, this study is the first
large scale evaluation of FALS in the French population.
However, we would like to call the authors attention for two important
aspects: 1) According to the authors "...The P56S carrier was of Japanese
descent, and represents
the first non-Brazilian patient carrying this mutation...".
The ALS8 form was first mapped at 20q13 in a large Brazilian family in
2004, which was published in the Journal of Medical Genetics (Nishimura et
al., 2004A). Later in the same year, the c.166C-T / p.Pro56Ser mutation in
the VAPB gene was identified in this and six other families (Nishimura et
al., 2004B) as responsible for this disorder. Additionally,
microsatellites analyses around VAPB showed that these families share a
common haplotype. It suggests a founder effect estimated to have occurred
about 500 years ago, concomitant with Brazilian colonization by Portugal
(Nishimura et al., 2005). Recently, in collaboration with our group, Funke
et al. (2010) identified a German ALS8 patient, with no family record of
Brazilian or Portuguese ancestry, carrying the c.166C-T mutation in VAPB,
which showed that ALS8 is no more restricted to Brazil. Therefore, this
German patient comprises the first case of an ALS8 patient out of Brazil
and this should be corrected in Millecamps et al., MS.
2) The German patient, identified by Funke et al. (2010), carries an
haplotype which is different from the one found in the Brazilian ALS8
families. This suggests that the c.166C-T (P56S) mutation is not due to
the same founder and that, probably, it happened at least in two
independent events. Millecamps et al., (2010) claims that "...The finding
of a P56S VAPB mutation in a patient of Japanese origin presumably
reflects the Portuguese trading connection with the Far East and Brazil in
the mid 16th century..." suggesting that the Japanese and the Brazilian
families could share the same genomic region. However, they do not show
such haplotype analysis or present any historical evidence for this
supposition.
We believe that such information is relevant for the researchers in the
ALS field and for this reason an erratum should be considered in order to
ensure the best information to the readers of the Journal of Medical
Genetics .
We appreciate your attention Sincerely
Miguel Mitne-Neto PhD Candidate Human Genome Research Center University of
Sao Paulo
References
Mayana Zatz Professor of Genetics
Director- Human Genome Research Center University of Sao Paulo
President-Brazilian Muscular Dystrophy Association
[A] Nishimura A.L., Mitne-Neto M., Silva H.C.A., Oliveira J.R.M., Vainzof
M., Zatz M. (2004) "A novel locus for late onset amyotrophic lateral
sclerosis/motor neurone disease variant at 20q13" J Med Genet. 41, 315-320
[B] Nishimura A.L., Mitne-Neto M., Silva H.C., Richieri-Costa A.,
Middleton S., Cascio D., Kok F., Oliveira J.R., Gillingwater T., Webb J.,
Skehel P., Zatz M. (2004). A mutation in the vesicle- trafficking protein
VAPB causes late-onset spinal muscular atrophy and amyotrophic lateral
sclerosis. Am J Hum Genet 75, 822-31
Nishimura A.L., Al-Chalabi A., Zatz M. (2005). A common founder for
amyotrophic lateral sclerosis type 8 (ALS8) in the Brazilian population.
Hum Genet 118, 499- 500
Funke A.D., Esser M., Kruttgen A., Weis J., Mitne-Neto M., Lazar M.,
Nishimura A.L., Sperfeld A.D., Trillenberg P., Senderek J., Krasnianski
M., Zatz M., Zierz S., Deschauer M. (2010) The p.P56S mutation in the VAPB
gene is not due to a single founder: the first European case. Clin Genet
77, 302-303
Millecamps S., Salachas F., Cazeneuve C., et al. "SOD1, ANG, VAPB, TARDBP,
and FUS mutations in familial amyotrophic lateral: genotype - phenotype
correlations" (2010) J Med Genet published online June 24, 2010 doi:
10.1136/jmg.2010.077180.
-----------------------------------------------------------------
"Commentary on investigations of somatic NKX2-5 mutations in congenital
heart disease (CHD) "
-----------------------------------------------------------------
Somatic mutations in transcription factor genes pertinent to cardiac
tissue development have been put forward as a molecular rationale of CHD.
Nkx2-5 is a homeodomain-containing transcription...
-----------------------------------------------------------------
"Commentary on investigations of somatic NKX2-5 mutations in congenital
heart disease (CHD) "
-----------------------------------------------------------------
Somatic mutations in transcription factor genes pertinent to cardiac
tissue development have been put forward as a molecular rationale of CHD.
Nkx2-5 is a homeodomain-containing transcription factor and is conserved
in many organisms from flies to humans. It is an important transcriptional
regulator of mammalian heart development. Absence of Nkx2-5 in animal
models results in lethality due to impaired heart tube looping (for
instance, see review [1]). In this regard, Draus et al. [2] reported that
somatic mutations in NKX2-5 do not represent an important aetiologic
pathway in pathologic cardiac development in patients with cardiac septal
defects. This conclusion was based on the genetic analysis of frozen
cardiac tissue samples of 28 patients with septal defects (ASD, n=13),
ventricular septal defects (VSD, n=5), and atrioventricular canal defects
(AVCD, n=10). Cardiac tissue samples were collected from diseased tissue
located immediately adjacent to the defect and from anatomically normal
tissue located at a site remote from the defect (right atrial appendage).
Except for one nonsynonymous germline sequence variant in 1 patient, two
synonymous germline sequence variants in 2 separate patients, and a common
single nucleotide polymorphism (SNP) in 16 patients, no NKX2-5 somatic
mutations were obtained.
The motivation behind Draus et al. study [2] was to replicate in
frozen cardiac tissues our previous findings of somatic mutations in the
NKX2-5 gene that we analyzed in the Leipzig collection of malformed hearts
[3;4]. We investigated 68 malformed hearts from unrelated individuals
collected from 1954 to 1982 and stored in formalin at the Institute of
Anatomy, University of Leipzig, Germany. After detailed morpho-
pathological characterization, the malformed hearts were classified by
sequential segmental analysis [5] according to their septal defects, e.g.
29 VSDs, 16 ASDs and 23 AVSDs. But besides their septal defects, the
patients had complex cardiac malformations, and many were affected by
patent foramen ovale (PFO), patent ductus arteriosus (PDA) or both.
Indeed, at least 42 different cardiac malformations were represented in
this patients' cohort. Most of the patients died during early infancy from
the disease and four patients within VSDs and 14 within AVSDs had Down
syndrome. As control, we also investigated 10 formalin-fixed, normal
hearts from the same collection, as well as five frozen normal hearts.
To summarize briefly our previous findings on NKX2-5 [3;4], direct
sequencing in 'affected' tissues (i.e. near septal defect) identified 35
different nonsynonymous mutations which were mainly absent in 'unaffected'
tissues (i.e. away septal defect) in the same patient's heart. Certain
mutations were frequent yet specific to a particular septal defect.
Several of the frequent nonsynonymous mutations impaired protein function
by reporter assays [6] or by bioinformatic prediction. Most important,
detected mutations were absent in control hearts from the same collection.
The NKX2-5 dbSNPs rs2277923 (c.63A>G, p.E21) and rs703752 (c.*61T>G)
were also found indicating integrity of the archival DNA even after many
years of formalin storage. Surprisingly, many patients had several NKX2-5
mutations, and after cloning of PCR fragments with several closely-spaced
heterozygous mutations, more than the two expected haplotypes were found.
We proposed that somatic NKX2-5 mutations as a possible mechanism of
disease in complex congenital heart disease. We tried to explain the
simultaneous occurrence of multiple NKX2-5 mutations as a reflection of
the complexity of the observed cardiac malformations, with patients
carrying multiple defects in the heart. We also suggested that a likely
explanation for the multiple haplotypes would be a mixed population of
cardiomyocytes carrying different mutations or de novo chromosomal
rearrangements and gene duplications in the tissues of patients affected
by various mutations.
We wish to comment on the Draus et al. study on the following points:
Draus et al. stated: "Six hundred and five somatic sequence variants
were reported in the coding and flanking intron regions of NKX2-5. When we
analysed the spectrum of these sequence variants found after amplification
from DNA extracted from formalin fixed tissue, we were surprised to
discover that A>G substitutions dominated these studies (58% of these
sequence variants were A>G substitutions) and only 31% were G>A
substitutions."
1) Comment: We find such representation of our results inaccurate. For
NKX2- 5, we reported 35 different nonsynonymous mutations, several of
which are common, specific to septal defects, and affect protein
function. Among these 35 nonsynonymous mutations, 8 are A>G
substitutions and include the loss-of-function mutation p.K183E which is
located in the third helix of homeodomain, and detected in 22 of 23 of
AVSDs, but not in VSDs.
Furthermore, 4 different G>A substitutions were found. We also found 14
synonymous mutations in the coding region of NKX2-5, 5 of these are
A>G including dbSNPs rs2277923 and 3 of these A>G were frequently
detected. Consequently, there needs to be an erratum in the publication of
Draus et al. that it incorrectly quoted our findings.
Response: While we appreciate the scientific discussion, we believe
no erratum is required. The same "35 different nonsynonymous mutations"
from the comment above, are represented by 336 independent, mutational
events , according to Table 1 (J Med Genetics report by Reamon-Buettner
and Borlak), and by 337 independent, mutational events, according to
Table 2 (Am J of Pathology report by Reamon-Buettner et al), out of 68 CHD
hearts. The paragraph which contains the quoted sentence from Draus et
al comes from a discussion on mutational spectra comparisons. Mutational
spectra analysis is the study of mutational events with regard to
frequency and kind (Keohavong and Thilly, PNAS , 1992, 89 (10):4623-4627).
The Reamon-Buettner and Borlak reports from the archived hearts suggest
that the NKX2.5 mutations are somatic in origin, therefore each mutation
arose via an independent event. Multiple mutational events were often
observed in the NKX2.5 gene within the same individual, and were often the
same mutation by NKX2.5 sequence position, suggesting numerous mutational
hotspot sites. Draus et al. noted that the spectrum of these
independent events are different from what would have been expected based
on the mutational spectra observed in major databases such as the HGMD
database, which represents inherited mutations causing or associated with
human disease, and the TP53 somatic database, which represents somatic
mutations found in the P53 gene from tumors. This form of analysis is of
interest because different spectra would suggest different mutational
pathways. While the HGMD database reports their mutational spectra by
counting different mutations only once, this is across greater than 2500
genes and was done to avoid confusion between recurrent and identical-by-
descent lesions. The TP53 mutation database, a more analogous comparison
as it targets a single gene and also somatic events, represents the same
mutation multiple times as long as they arose independently. G>A
mutations are represented significantly more frequently than A>G
mutations in both HGMD and TP53 databases. However, A>G mutations
occur more frequently than G>A mutations in the reports from the
Liepzig collection, suggesting a different mutational mechanism, and is
true EVEN if one only counts different, nonsynonymous NKX2.5 mutations.
Furthermore, mutational spectra analysis does not assume causality
therefore greater than 600 nonsynonymous, synonymous, and intronic
sequence variants published from the Liepzig collection were included in
the mutational spectra analysis of Draus et al.
Finally, we note that in our study of 28 subjects, we did not find a
single somatic sequence variant in NKX2.5 by direct Sanger sequencing.
Draus et al. stated: "Similarly, the same group found somatic
sequence variants in the TBX5 gene from the same archival collection of
hearts. TBX5 encodes a transcription factor that interacts with NKX2-5 and
is important in vertebrate cardiac development. One hundred and thirty-
five somatic sequence variations have been reported by the same authors in
the coding and flanking intron regions of TBX5. Of the single nucleotide
substitutions reported, 87 percent were adenine-to-guanine substitutions.
Only 11 percent were guanine-to-adenine substitutions."
Comment: Again, the representation of our results is incorrect. For TBX5,
we obtained only 9 single base substitutions in the coding region of TBX5
resulting in nonsynonymous change that were absent in matched 'unaffected'
tissues [7]. Six mutations would affect amino acids in the T-domain. No
frequent mutation was obtained except c.236C>T (p.A79V) which was
detected in five cases. Only 11 of 68 patients had nonsynonymous TBX5
mutations and none were found in VSDs.
Response: Again, mutational spectra analysis does not assume
causality therefore nonsynonymous, synonymous, and intronic sequence
variants published from the 68 CHD hearts (Liepzig collection), this time
in the TBX5 gene (Human Mutation, Reamon-Buettner and Borlak), were
included in the mutational spectra analysis of Draus et al. Again, A>G
mutations occur more frequently than G>A mutations in the reports from
the Liepzig collection, EVEN if one only counts the different TBX5
mutations.
Draus et al. stated: "In our study, an average of 1.3 microgram of
genomic DNA (gDNA) was isolated from 5 mg of tissue obtained as surgical
discards that were immediately frozen. In comparison, yields from the
Leipzig collection were reportedly 0.5 to 1 microgram of genomic DNA from
25 mg DNA obtained from archival tissue. Therefore, the recovery of gDNA
from our fresh frozen samples was more than five times that of the
formalin-fixed samples. The relatively poor gDNA yield from the Leipzig
collection likely reflects the poor quality of DNA taken from samples that
had been fixed in formalin between 22 to 50 years."
Comment: We wish to point out that the average size of genomic DNA
isolated from the Leipzig collection was about 2 kb, and 20-50 ng was used
to amplify three NKX2-5 fragments of sizes 489, 472, and 573 bp for
sequence analysis. Although the quantity of DNA from fixed material was
not comparable to that of fresh tissue, the quality of the genomic DNA as
shown by the amplified NKX2-5 fragments was amenable for genetic analysis.
Response: The average size of genomic DNA extracted from the Leipzig
collection is shorter than what was found from fresh frozen tissue
(greater than 10 kb from fresh frozen tissue).
Draus et al. stated: "The Reamon-Buettner studies are significantly
different from germline NKX2-5 mutations as well as significantly
different from inherited disease associated mutations, tumour derived
somatic mutations, and mitochondrial mutations. While these differences
may reflect true differences between the patients at the mutational
pathway level (germline versus somatic versus mitochondrial mutations),
geographic and/or patient level (Germany and/or CHD patients versus an
international collections of cancer databases), or the temporal level
(over 22 years ago versus the current era), it is also possible that the
previously reported somatic variants reflect postmortem artefacts of
fixation or low quality DNA template. Recent studies also show that lower
starting yield of genomic DNA from archival samples can result in higher
misincorporation rates during PCR in a sequence dependent manner"
Comment: Assuming the NKX2-5 mutations identified by us are postmortem
artefacts of fixation, then why are the unaffected tissues or same
formalin-fixed normal hearts did not contain the mutations? Why do
patients carry common pathogenic mutations?
Response: The Reamon-Buettner et al, AJP article states "we found
that the mutations in diseased heart tissues were mainly absent in matched
normal heart tissue". They later state that three nucleotide alterations
(G833A, T984A, A1205T) were found in 10 formalin-fixed normal hearts. How
do the authors differentiate these nucleotide alterations from mutations
(i.e. how do they know that the 35 non-synonymous mutations are common
"pathogenic" mutations? Pathogenic mutations suggest causality).
Draus et al. stated: " Recent studies also show that lower starting
yield of genomic DNA from archival samples can result in higher
misincorporation rates during PCR in a sequence dependent manner 8"
Comment: As already mentioned earlier, the average size of genomic DNA
isolated from the Leipzig collection was about 2 kb, and 20-50 ng was used
to amplify three NKX2-5 fragments of sizes 489, 472, and 573 bp for
sequence analysis. The paper by Akbari et al. [8] used paraffin-embedded
tissues and the source for genomic DNA isolation was few microdissected
cells.
Response: The average size and yield from the archived tissue show
that the average size is shorter, and the average yield is five times less
than that from fresh frozen specimens. This suggests that the nucleic
acid template from archived formalin fixed tissue is of poorer quality
than fresh frozen specimens, which is not surprising and has been
previously described. In addition to a high frequency of sequence
alterations arising due to formalin fixation of archival specimens
(Williams et al, AJP, 1999 and Quach et al, BMC clin pathol, 2004), Akbari
et al demonstrates that from formalin-fixed, paraffin-embedded tissue
specimens, PCR-generated artifacts can also occur in a sequence specific
manner.
Taken collectively, the study of Draus et al. provided erroneous
information that requires an erratum and clarification. While the
pathogenesis of CHD is complex, in which majority of CHD being sporadic,
it is an acknowledged fact that patients have no family history of the
disease and therefore does not follow a typical Mendelian trait. There is
clinical or phenotype heterogeneity within affected individuals, as well
as within and between families. Disease association is difficult owing to
lack of clear genotype-phenotype correlation of mutations. Furthermore, at
the molecular and cellular levels, heart development can practically go
wrong in many directions, i.e. transcriptional regulators, signaling
pathways and chromatin remodeling, leading to diverse cardiac
malformations observed in CHD.
Thus, somatic mutations in NKX2-5 may represent just one among the many
causes of CHD, and their non-detection in another patients' cohort is,
therefore, not totally surprising. Indeed, since NKX2-5 gene mutations
have been implicated in human CHD [9], the coding region of NKX2-5 has
been consistently analyzed for additional disease-associated sequence
alterations. As of today, we documented 41 different NKX2-5 germline
mutations most of which lead to amino acid change (nonsynonymous
mutations). Most of these mutations would affect protein function, yet
there is lack of genotype-phenotype correlation of NKX2-5 mutations in CHD
(see review [10]). Notably, the detection frequency of NKX2-5 mutations in
sporadic cases of CHD is about 2 % and in several studies none was found.
Many identified NKX2-5 mutations are familial cases, and families have
their private mutations. Thus, a single germline NKX2-5 mutation as the
direct cause of disease or a simple genetic analysis does not reveal
causation.
With this letter, we wish to highlight the erroneous quotation and
interpretation of our studies.
Response: While we agree that it is possible that somatic events
could represent one cause of CHD, our conclusion, that no evidence
supporting somatic NKX2.5 sequence variants as being causative was found
in our study of fresh frozen tissue from subjects with congenital heart
disease, remains the same.
The findings by Reamon-Buettner could be due to differences in the
cohort, differences in the population (Germany versus US), or differences
in DNA integrity.
As in our discussion,
"The Reamon-Buettner studies are significantly different from
germline NKX2-5 mutations as well as significantly different from
inherited disease associated mutations, tumour derived somatic mutations,
and mitochondrial mutations. While these differences may reflect true
differences between the patients at the mutational pathway level (germline
versus somatic versus mitochondrial mutations), geographic and/or patient
level (Germany and/or CHD patients versus an international collections of
cancer databases), or the temporal level (over 22 years ago versus the
current era), it is also possible that the previously reported somatic
variants reflect postmortem artefacts of fixation or low quality DNA
template".
Caution should be utilized when interpreting somatic mutation data
from formalin-fixed archival samples. If possible, a second method such as
RFLP analysis on genomic DNA, not cloned DNA, is recommended.
Respectfully,
Draus et al.
References
1. Bartlett H, Veenstra GJ, Weeks DL. Examining the cardiac NK-2
genes in early heart development. Pediatr Cardiol 2010; 31:335-41.
2. Draus JM, Jr, Hauck MA, Goetsch M, Austin EH, III, Tomita-Mitchell
A, Mitchell ME. Investigation of somatic NKX2-5 mutations in congenital
heart disease. J Med Genet 2009; 46:115-22.
3. Reamon-Buettner SM, Hecker H, Spanel-Borowski K, Craatz S, Kuenzel
E, Borlak J. Novel NKX2-5 mutations in diseased heart tissues of patients
with cardiac malformations. Am J Pathol 2004; 164:2117-25.
4. Reamon-Buettner SM, Borlak J. Somatic NKX2-5 mutations as a novel
mechanism of disease in complex congenital heart disease. J Med Genet
2004; 41:684-90.
5. Craatz S, Kunzel E, Spanel-Borowski K. Classification of a
collection of malformed human hearts: practical experience in the use of
sequential segmental analysis. Pediatr Cardiol 2002; 23:483-90.
6. Inga A, Reamon-Buettner SM, Borlak J, Resnick MA. Functional
dissection of sequence-specific NKX2-5 DNA binding domain mutations
associated with human heart septation defects using a yeast-based system.
Hum Mol Genet 2005; 14:1965-75.
7. Reamon-Buettner SM, Borlak J. TBX5 mutations in Non-Holt-Oram
Syndrome (HOS) malformed hearts. Hum Mutat 2004; 24:104.
8. Akbari M, Hansen MD, Halgunset J, Skorpen F, Krokan HE. Low copy
number DNA template can render polymerase chain reaction error prone in a
sequence-dependent manner. J Mol Diagn 2005; 7:36-9.
This letter was submitted by the JMG editorial staff on behalf of Dr. Michael Mitchell, author of Investigation of somatic NKX2-5 mutations in congenital heart disease.
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----------------------------------------------------------------- "Commentary on investigations of somatic NKX2-5 mutations in congenital heart disease (CHD) " -----------------------------------------------------------------
Somatic mutations in transcription factor genes pertinent to cardiac tissue development have been put forward as a molecular rationale of CHD. Nkx2-5 is a homeodomain-containing transcription...
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