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In their interesting paper (1) A. Smith and colleagues postulate, that after a negative test for BRCA1 and BRCA2 women are still at increased risk. They therefore recommend to continue screening. There are several reasons why surveillance recommendations, after a negative test for the family mutation, are premature I think.
1. It should be clear how high the rest risk is: This is not really cl...
In their interesting paper (1) A. Smith and colleagues postulate, that after a negative test for BRCA1 and BRCA2 women are still at increased risk. They therefore recommend to continue screening. There are several reasons why surveillance recommendations, after a negative test for the family mutation, are premature I think.
1. It should be clear how high the rest risk is: This is not really clear from this paper as DNA-testing bias of breast cancer cases has played a part. When testing first degree relatives of a BRCA1/2 mutation carrier one would expect about 50% to test positive. However table 1 shows that 67% (376/ 560) tested positive. This strongly
suggests preferential testing of breast cancer cases which will certainly also increase the percentage breast cancers in the negative tested group, without any modifying genetic factor. We have previously shown that such a DNA testing bias can wrongfully suggest increased risks in non-BRCA1/2
patients (2,3). To exclude this bias one should only take the cancer incidence after the DNA test into account.
2. In order to recommend screening one should know at which age the possible rest risk may be expected. Certainly one shouldn’t continue screening from age 25-30 onwards like in carriers if the increased risk possibly occurs mainly above age 50. Screening with mammography at age 25-
40 yrs. has too low a sensitivity, may cause too many false-positive results and possibly induce breast cancer, so is not justified for only slightly increased risk (4).
3. As the results came from families with a high caseload, one does not know whether such modifying genes will be present in BRCA1/ BRCA2 families with fewer cancers.
When many breast and ovarian cases test negative for the family mutation, a second gene mutation or risk from the untested side of the family may need to be investigated.
References:
1. Phenocopies in BRCA1 and BRCA2 families: evidence for modifier genes and implications for screening. A Smith, A Moran, MC Boyd, Bulman M, Shenton A, Smith L et al. JMed Genet 2006:doi:10.1136/jmg.2006.043091
2. Selection bias influences reported contralateral breast cancer incidence and survival in high risk non-BRCA1/BRCA2 patients. Tilanus-Linthorst MMA, Bartels K, Alves C, Bakri B, Crepin E, van den Ouweland A et al. Breast Ca Res Treat 2006;95(2):117-23.
3. Contralateral recurrence and prognostic factors in familial non-BRCA1/2 associated breast cancer. Tilanus-Linthorst MMA, Alves C, Seynaeve C,
Menke-Pluymers MBE, Eggermont AMM and Brekelmans CTM. Br J Surg 2006; 93 (8):961-968.
4. Griebsch I, Brown J, Dixon A, Dixon M, Easton D, Eeles R et al. Cost-effectiveness of screening with contrast enhanced magnetic resonance imaging vs. X-ray mammography of women at a high familial risk of breast cancer. Br J Cancer. 2006 Oct 9;95(7):801-10.
Bonapace et al. (1) analyzed the IGF-1 gene in one SGA child that presented clinical and laboratory findings compatible with isolated IGF-1 deficiency. Molecular study of the IGF-1 gene in this patient disclosed a homozygous nucleotide substitution (AATATA > AAAATA) in the upstream
core polyadenylation signal (UCPAS) located in IGF-1 exon 6 3’ UTR. The fact that this was the only mutation found within...
Bonapace et al. (1) analyzed the IGF-1 gene in one SGA child that presented clinical and laboratory findings compatible with isolated IGF-1 deficiency. Molecular study of the IGF-1 gene in this patient disclosed a homozygous nucleotide substitution (AATATA > AAAATA) in the upstream
core polyadenylation signal (UCPAS) located in IGF-1 exon 6 3’ UTR. The fact that this was the only mutation found within the entire coding region and the absence of this finding in 100 unrelated healthy controls led the
authors to conclude that this mutation was responsible for the patient’s clinical findings.
We recently sequenced the IGF-1 gene of 118 SGA children and identified 4 children (approximately 2% of alleles) who carried the same mutation described by Bonapace et al. in heterozygous state. To assess this mutation in our control population, we sequenced 90 healthy controls
and surprisingly identified 5 individuals who carried this same mutation, 2 in homozygous and 3 in heterozygous state, which corresponds to 4% of studied alleles. The heights of the 2 male homozygous individuals were 191 and 177 cm, respectively (Height SDS of 2.5 and 0.3).
It is noteworthy that the AATATA > AAAATA mutation yields an alternative AATATA motif 2 bp downstream from the physiological UCPAS hexamer (2). Bonapace et al. attempted to assess the mutation effect on mRNA transcription and maturation by RT-PCR. Although the sequences for
the two RT-PCR primers were not provided in the original publication, it may be inferred that the 2 primers must have targeted the 5’ end of exon 6 and sequences downstream of the pre-mRNA cleavage site of IGF-1 gene. For this reason, the RT-PCR performed was unable to amplify products from 1.1
kb mRNA and assess the effect of AATATA > AAAATA mutation on mRNA processing. In the experiment depicted by Bonapace et al., 2 distinct amplification products (340 and 450 bp) were obtained. As discussed by Chen et al., in a recent review about variants in the 3’ regulatory regions, the 340 bp RT-PCR product obtained by Bonapace et al. probably
derived from non-specific PCR amplifications and the 450 bp product corresponds to a cDNA sequence amplified from the normally expressed 7.6 kb IGF-1 mRNA species (2).
In conclusion, our finding of the AATATA > AAAATA alteration in normal height controls implies that this mutation in the IGF-1 UCPAS region is not responsible for the clinical and laboratory findings described in IGF-1 deficiency children, and actually represents a
polymorphism. In view of these findings, the molecular basis of the IGF-1 deficiency etiology in the patient described by Bonapace et al. remains to be elucidated.
References
1. Bonapace G, Concolino D, Formicola S, Strisciuglio P 2003 A novel mutation in a patient with insulin-like growth factor 1 (IGF1) deficiency. J Med Genet 40:913-7
2. Chen JM, Ferec C, Cooper DN 2006 A systematic analysis of disease-associated variants in the 3' regulatory regions of human protein-coding genes I: general principles and overview. Hum Genet 120:1-21
We read with interest the article by Scott et al. in which the authors
review the syndromes and chromosomal abnormalities associated with Wilms
tumor (WT) [1].
Although the research criteria described by the Authors were meant to
ascertain comprehensively the conditions reported in association with WT,
we would like to signal an unusual association that was not reported in an
otherwise extreme...
We read with interest the article by Scott et al. in which the authors
review the syndromes and chromosomal abnormalities associated with Wilms
tumor (WT) [1].
Although the research criteria described by the Authors were meant to
ascertain comprehensively the conditions reported in association with WT,
we would like to signal an unusual association that was not reported in an
otherwise extremely exhaustive paper.
In 1986 Malpuech et al. [2], reported a patient with a chromosomal
deletion in the region 11p13, associated with the WAGR syndrome, and a
severe phenotype including toes polydactyly. Following this report, in May
2005 two articles published in the American Journal of Medical Genetics,
one by us and the other by a French group, added the description of four
WAGR patients showing hallucal polydactyly [3] [4]. Three cases, a girl
described in our study and the twin girls of the French study, showed
allux bilateral polydactyly, while the remaining WAGR boy, reported by
Bremond-Gignac and collegues, had monolateral polydactyly. In all four
cases cytogenetic analyses revealed the presence of an interstitial
deletion involving chromosome 11p13. In addition, two cases, our patient
and one of the twin sisters, were diagnosed with WT.
Assuming a frequency of allux polydactily and of WAGR in the
population of approximately 1:40.000 and 1:1.250.000, respectively [5]
[6], the expected chance occurrence of individuals with both phenotypes
would be approximately 1:5x10e10.
The observation of polydactyly in at least five WAGR patients with
verified 11p interstitial deletion, indicates a non-random association and
the possibility should be considered that polydactyly is another feature
related to WAGR and then to an increased risk of WT. The occurrence of
polydactily in WAGR patients could be due either to a very low penetrant
trait associated with haploinsufficiency of one of the genes present in
the critical WAGR region, or to a positional effect of the deletion on a
gene mapped close to it.
References
[1] Scott RH, Stiller CA, Walker L, Rahman N. Syndromes and
constitutional chromosomal abnormalities associated with Wilms tumour. J
Med Genet 2006.
[2] Malpuech G, Sultan C, Bertheas MF, Loire C, Renaud H, Francannet
C, Vanlieferinghen P. Male pseudohermaphroditism, partial androgen
receptors defect, 11p13 deletion: indication of gene localization. Am J
Med Genet 1986; 24(4):679-684.
[3] Manoukian S, Crolla JA, Mammoliti P, Testi MA, Zanini R,
Carpanelli M, Piozzi E, Sozzi G, De Vecchi G, Terenziani M, Spreafico C,
Collini P, Radice P, Perotti D. Bilateral prexial polydactyly in a WAGR syndrome
patient. Am J Med Genet A 2005; 134(4):426-429.
[4] Bremond-Gignac D, Gerard-Blanluet M, Copin H, Bitoun P, Baumann
C, Crolla JA, Benzacken B, Verloes A. Three patients with hallucal
polydactyly and WAGR syndrome, including discordant expression of Wilms
tumor in MZ twins. Am J Med Genet A 2005; 134(4):422-425.
[5] Orioli IM, Castilla EE. Thumb/hallux duplication and preaxial
polydactyly type I. Am J Med Genet 1999; 82(3):219-224.
[6] Breslow NE, Norris R, Norkool PA, Kang T, Beckwith JB, Perlman
EJ, Ritchey ML, Green DM, Nichols KE. Characteristics and outcomes of
children with the Wilms tumor-Aniridia syndrome: a report from the
National Wilms Tumor Study Group. J Clin Oncol 2003; 21(24):4579-4585.
Hoffmann et al. have done a quite remarkable study on the assessment of response following treatment of the disorder like fabry disease which does not have any reliable biochemical marker.
Since the author has assessed three times, it could be better to use repeated measures analysis by mixed model instead of using wilcoxon's rank sum test or student's t test.
In response to the comments of Helms et al.: We recognize that in
the
presence of substantial heterogeneity, much larger sample sizes may be
required to replicate an effect. Thus, we cannot rule out the possibility
that
the variants in the RUNX binding site and the RAPTOR gene make a
substantial contribution to psoriasis susceptibility in some settings.
Nevertheless, in our sample, which includes...
In response to the comments of Helms et al.: We recognize that in
the
presence of substantial heterogeneity, much larger sample sizes may be
required to replicate an effect. Thus, we cannot rule out the possibility
that
the variants in the RUNX binding site and the RAPTOR gene make a
substantial contribution to psoriasis susceptibility in some settings.
Nevertheless, in our sample, which includes 2,261 genotyped individuals
and
1,143 psoriatics appropriate for FBAT association analysis, we see no
significant evidence (p<.05) of association between psoriasis and
these
SNPs. To our knowledge, this is the largest sample yet examined for these
markers.
In accordance with the findings reported by Kadakol et al. (1) we
found in a population study,(2) done in Malays, two cases of neonatal
jaundice (NNJ)with a combination of a mutation in the encoding region of
the UGT1A1 gene and the classical Gilbert syndrome mutation.
These two patients had severe early onset NNJ, only slowly responding
to intensive phototherapy. Both babies had a normal G...
In accordance with the findings reported by Kadakol et al. (1) we
found in a population study,(2) done in Malays, two cases of neonatal
jaundice (NNJ)with a combination of a mutation in the encoding region of
the UGT1A1 gene and the classical Gilbert syndrome mutation.
These two patients had severe early onset NNJ, only slowly responding
to intensive phototherapy. Both babies had a normal G6PD status and were
feeding well. Their respective mothers had each blood group O positive and
each baby had blood group B positive with a negative direct Coombs test.
Liver tests other than bilirubin levels were normal. Three mutations in
the UGT1A1 gene were tested (the A(TA)7TAA mutation, the G71R mutation
which is commonly causing Gilbert Syndrome in Taiwanese and Japanese and
the G493R mutation which was recently found in 2 related Malay patients
with NNJ)(2). Each baby was found to have two mutations: the A(TA)7TAA
mutation and the G71R mutation. None had the G493R mutation.
The cases reported here underline the importance of the findings
reported by Kadakol et al.(1). The two patients would have been labeled as
having ABO-incompatibility if the genetic testing would have not been
performed. Especially in Southeast Asian populations where the incidence
of neonatal jaundice is very high, routine screening for mutations in the
UGT1A1 gene may be useful. It may help in genetic counseling of the
parents and in prevention of kernicterus in coutries with an early
neonatal discharge policy.
References:
1. Kadakol A, Sappal BS, Ghosh SS, Lowenheim M, Chowdhury A, Chowdhury S,
Santra A, Arias IM, Chowdhury JR, and Chowdhury NR. Interaction of coding
region mutations and the Gilbert-type promoter abnormality of the UGT1A1
gene causes moderate degrees of unconjugated hyperbilirubinaemia and may
lead to neonatal kernicterus. J Med Genet 2001; 38: 244-249
2.Yusoff S, Van Rostenberghe H, Yusoff NM, Talib NA, Ramli N, Ismail
NZ, Ismail WP, Matsuo M, Nishio H. Frequencies of A(TA)(7)TAA, G71R, and
G493R Mutations of the UGT1A1 Gene in the Malaysian Population. Biol
Neonate. 2005 Oct 6;89(3):171-176 [Epub ahead of print]
This paper by Ingles et al. has once again highlighted the role of
compound and double mutations in patients with hypertrophic cardiomyopathy
(HCM).[1] They detected compound or double mutations in 4 of 23 (17%) of
probands found to have mutations in the genes that were screened. This
large percentage of compound mutations has serious implications for any
HCM mutation screening programme. Mutations in...
This paper by Ingles et al. has once again highlighted the role of
compound and double mutations in patients with hypertrophic cardiomyopathy
(HCM).[1] They detected compound or double mutations in 4 of 23 (17%) of
probands found to have mutations in the genes that were screened. This
large percentage of compound mutations has serious implications for any
HCM mutation screening programme. Mutations in HCM are spread across
several genes and because of the logistics and cost involved, mutation
screening is limited to a few labs and is time consuming. This reduces the
clinical availability of the screening process. Such a high incidence of
compound and double mutations would further add to the complexity and
costs of the screening process.
This data on compound and double mutations should however be
interpreted with caution. Firstly, all four families were small, only one
genotype positive individual in three families and three in the fourth, thus
proving co-segregation of sequence variation with disease phenotype is not
possible. Secondly, in none of the four families were the two mutations
shown to independently give rise to the HCM phenotype. Thirdly, three of the eight
compound mutations were novel and no functional studies were carried out.
Fourthly, in all four families MyBP-C was involved.
The three criteria chosen by the authors to consider a sequence
variation to be disease causing are inadequate. The first criteria of co-
segregation of the sequence variation with affected members obviously does
not apply to such small families. The second criteria of absence of
mutation in 300 unrelated chromosomes from healthy adult controls also has
a rider. Some of the sequence variations are only found in specific ethnic
groups for example a deletion in intron 32 of MyBP-C. On testing healthy
controls, this deletion was not found in 270 Caucasians but was present in
7% of healthy controls from South India.[2] We have also found this
deletion in 4.2% of healthy North Indian controls. Thus some sequence
variations are restricted to and even common in particular ethnic groups.
Therefore screening 150 healthy controls as the authors have done is not
adequate. The ethnicity of each individual proband found to have a novel
sequence variation should be determined and healthy controls from that
particular ethnicity studied. In case a patient has ancestors from multiple
ethnicities, healthy controls should be studied in all groups to render
the data scientifically valid. The third criteria mentioned is
conservation of mutated residue among species and isoforms. The degree of
conservation has not been discussed at all in the manuscript. Thus it is
not possible to conclude that the novel sequence variations found by the
authors are disease causing. These could be at best labelled as probable
mutations. They could also be disease modifying variations or just simple
polymorhisms. Similar issues also arise in earlier reports of compound
mutations.[3][4]
Vast amount of data on genotyping of HCM patients is now available
and a number of sequence variations have been identified. The significance
of these is often not clear. As in other fields of cardiology, there is an
urgent need to sort out these sequence variations. A group or a consensus
committee needs to be formed that can go through all the sequence
variations reported and classify them as either definite mutations,
probable mutations, disease modifying variations or just polymorhisms.
This then has to be continued as an ongoing process to include the data
generated in the future.
References
1. Ingles J, Doolan A, Chiu C, Seidman J, Seidman C, Semsarian C. Compound
and double mutations in patients with hypertrophic cardiomyopathy:
implications for genetic testing and counseling. J Med Genet 2005;42:e59.
2. Waldmuller S, Sakthivel S, Saadi AV et al. Novel deletions in MYH7 and
MYBPC3 identified in Indian families with familial hypertrophic
cardiomyopathy. J Mol Cell Cardiol 2003;35:623-36.
3. Richard P, Charron P, Carrier L et al. Hypertrophic cardiomyopathy
distribution of disease genes, spectrum of mutations, and implications for
a molecular diagnosis strategy. Circulation 2003;107:2227-32.
4. Van Driest SL, Vasile VC, Ommen SR et al. Myosin binding protein C
mutations and compound heterozygosity in hypertrophic cardiomyopathy. J Am
Coll Cardiol 2004;44:1903-10.
We are studying the genetic basis of non-syndromic hearing loss in North Indian population and we performed the PCR-RFLP assay described by authors for the detection of W24X mutation in this article. The assay was carried out using the primers (1F and 1R) and the restriction enzyme Alu1, as described by the authors. However, we have observed a distinctly different RFLP pattern for this mutation as compared...
We are studying the genetic basis of non-syndromic hearing loss in North Indian population and we performed the PCR-RFLP assay described by authors for the detection of W24X mutation in this article. The assay was carried out using the primers (1F and 1R) and the restriction enzyme Alu1, as described by the authors. However, we have observed a distinctly different RFLP pattern for this mutation as compared to that reported by Ramshankar et al, 2003.
The authors have reported that the presence of W24X mutation introduces an Alu1 restriction site. According to them, Alu1 digestion of 286bp PCR amplified product from unaffected subjects produces a single fragment of 286bp, whereas the presence of W24X mutation in homozygous genotype would produce two fragments of 182bp and 104bp and in W24X heterozygotes, it would generate three fragments of 286bp, 182bp and 104bp.
However, we observed that the presence of W24X mutation in homozygous state produced three fragments of 168bp 104bp and 14bp, whereas in W24X heterozygotes we found four fragments of 272bp, 168bp 104bp and 14bp, which suggested that there may be an additional Alu1 site in the 286bp PCR product. An in-silico PCR amplification followed by restriction digestion using REBASE (http://rebase.neb.com) showed that the 286bp fragment amplified by the given primers carries another Alu1 restriction site independent of the one created by the presence of W24X mutation (Fig.1).
Hence, even in the absence of W24X mutation, the Alu1 restriction digestion of 286 amplicon gives two fragments of 272bp and 14bp (Fig.2). Indeed, we have observed these RFLP patterns in control and W24X positive samples. Thus, our results confirm the presence of an additional Alu1 site in the 286bp amplified PCR product giving RFLP pattern different from that reported by these authors.
Figure 1. Alu1 restriction sites in 286bp amplicon (Part of NC_004004).
We read with interest the recent article in the J Med Genet by
Tessier et al.[1] confirming our report[2] of association between
type 1 diabetes and the 2´5´-oligoadenylate synthetase OAS1
antiviral gene. However, their conclusion differs from ours
concerning which single nucleotide polymorphism (SNP) in OAS1
is most likely to produce the functional effect on diabetes
predisposition – the exon 3 non-...
We read with interest the recent article in the J Med Genet by
Tessier et al.[1] confirming our report[2] of association between
type 1 diabetes and the 2´5´-oligoadenylate synthetase OAS1
antiviral gene. However, their conclusion differs from ours
concerning which single nucleotide polymorphism (SNP) in OAS1
is most likely to produce the functional effect on diabetes
predisposition – the exon 3 non-synonymous SNP rs3741981 or
the intron5/exon7 splice site SNP rs10774671.
Since these SNPs are in linkage disequilibrium, determining which is more likely to
be functional is not a simple exercise. We postulated that the splice
site SNP, which as we previously showed[3] creates different
isoforms of the enzyme and is highly significantly associated with
OAS enzyme activity, is the best functional candidate, rather than
the exon 3 SNP which Tessier et al. champion. There were two
reasons for our conclusion:
1) the splice site SNP was more
strongly associated with diabetes in our data than the exon 3 SNP;
2) we clearly demonstrated in our previous study[3] that while
controlling for genetic variation at the exon 3 SNP, the splice site
SNP was still significantly associated with enzyme activity, but the
reverse was not true – there was no association of enzyme activity
with the exon 3 SNP while controlling for genetic variation at the
splice site SNP.
Since the strong effect on antiviral enzyme activity
is a plausible functional link to diabetes susceptibility, these two
observations led us to suggest that the splice site SNP is the most
likely determinant of diabetes predisposition. Tessier et al. found
similar strength of diabetes association for the exon 3 and splice
site SNPs, but further analysis (see below) convinced them that
the
exon 3 SNP was more important. They suggested that the exon 3
SNP could alter dsRNA binding, which is required for enzyme
activation. Hartmann et al.[4] reported that dsRNA binds to OAS1
along an extensive positively-charged groove. There is no
evidence that the GGC(Glycine) to AGC(Serine) substitution
encoded by the exon 3 SNP is within this groove or alters dsRNA
binding.
Nevertheless, it is theoretically possible that some other effect of
OAS1 genetic variation (other than changing enzyme activity)
could also predispose to type 1 diabetes. The question of which
SNPs create functional effects becomes even more pertinent as
further studies reveal new OAS1 associations -- for example, it was
recently reported[5] that susceptibility to SARS virus infection is
significantly associated with both the exon 3 SNP and exon 7 SNP
rs2660 (the latter is in tight linkage disequilibrium with the exon 7
splice site SNP).
Therefore, to directly address the question of
possible effects of the exon 3 SNP on diabetes susceptibility, we
have re-analyzed our data in the same manner as Tessier et al.
They argued that the critical test was to examine transmission of
the haplotype G-A (G allele at the exon 3 SNP and A allele at the
splice site SNP) to diabetic children from parents who were
heterozygous for haplotypes G-A and A-A. Since the G allele is
associated with diabetes at both SNPs, this particular transmission
test would be expected to show overtransmission of G-A to diabetic
children if the exon 3 SNP is functional (as they suggested), but no
overtransmission of G-A if the exon 3 SNP is not functional (as we
postulated). [Note -- in Tessier et al., the major and minor alleles at
the exon 3 SNP are incorrectly called T and C, rather than A and G
as read off the plus strand.]
Tessier et al. observed 50
transmissions and 27 non-transmissions of the G-A haplotype from
G-A/A-A parents, with a corrected p value of 0.03. In our families,
there were 64 parents who were heterozygous G-A/A-A. The G-A
haplotype was transmitted 46 times and not transmitted 52 times to
their 98 diabetic children (difference not significant). These results
do not replicate those of Tessier et al. In fact, when data from the
two studies are combined, 96 transmissions and 79
non-transmissions no longer constitutes significant
overtransmission of the G-A haplotype (chi-squared = 1.65,
uncorrected p = 0.20).
Since this critical test for a functional effect
at the exon 3 SNP fails, we again conclude that the splice site SNP
is the best candidate for the effect on predisposition to type 1
diabetes. Ultimately, biological rather than statistical tests will be
needed to conclusively establish the effects of these OAS1 SNPs
on enzyme activity and other processes such as apoptosis; we are
currently performing such experiments.
In their discussion, Tessier et al. stated they were "puzzled" by the
fact that we found the diabetes relative risk of the OAS1 gene to be
comparable to that of the insulin (INS) gene region. However, we
clearly stated that our relative risks were obtained by comparisons
with unaffected siblings, not with unrelated persons in the general
population, which could alter the relative risks.[2] We suggested
that our OAS1 data supports not only a predisposing effect of
splice site allele G (as also found by Tessier et. al.) but a protective
effect of splice site allele A, particularly in the context of other
diabetes-predisposing genes, since the A allele was found
significantly more often in unaffected siblings than in parental
non-transmitted alleles.[2]
Finally, in their discussion Tessier et al. suggested that there may
be parental-sex-specific OAS1 effects, since the overtransmission
of OAS1 susceptibility alleles was statistically significant from
mothers but not statistically significant from fathers for all three
SNPs examined (note that due to linkage disequilibrium, these are
not independent tests).
However, they did not report whether these
differences between maternal and paternal transmission rates
were statistically significant. Data in their Table 4 shows that there
is also overtransmission of predisposing alleles from fathers but
the effect is not as strong as from mothers; our calculations indicate
that the differences between their maternal and paternal
transmission rates are not significant (data not shown). They also
suggested that mothers of diabetic children may have lower
frequencies of diabetes-predisposing genotypes (GG + GA) than
fathers (p = 0.045 for one of the three SNPs). In our families, we do
not see any significant differences between mothers and fathers in
frequencies of alleles transmitted/non-transmitted to diabetic or
unaffected children, nor any significant differences between
mothers and fathers in frequencies of predisposing genotypes or
alleles (data not shown). Thus, given the available data, we
conclude that there is currently little evidence for OAS1
parental-sex-specific effects on diabetes susceptibility.
Conflicting interests: none declared
References
1. Tessier MC, Qu HQ, Frechette R, Bacot F, Grabs R, Taback SP,
Lawson ML, Kirsch SE, Hudson TJ, Polychronakos C. Type 1
diabetes and the OAS gene cluster: association with splicing
polymorphism or haplotype? J Med Genet Published Online First
13 Jul 2005; doi:10.1136/jmg.2005.035212 [Epub ahead of print]
2. Field LL, Bonnevie-Nielsen V, Pociot F, Lu S, Nielsen TB,
Beck-Nielsen H. OAS1 splice site polymorphism controlling
antiviral enzyme activity influences susceptibility to type 1
diabetes. Diabetes 2005; 54(5):1588-1591.
3. Bonnevie-Nielsen V, Field LL, Lu S, Zheng DJ, Li M, Martensen
PM, Nielsen TB, Beck-Nielsen H, Lau YL, Pociot F. Variation in
antiviral 2',5'-oligoadenylate synthetase (2'5'AS) enzyme activity is
controlled by a single-nucleotide polymorphism at a
splice-acceptor site in the OAS1 gene. Am J Hum Genet 2005;
76(4):623-633.
4. Hartmann R, Justesen J, Sarkar SN, Sen GC, Yee VC. Crystal
structure of the 2-specific and double-stranded RNA-activated
interferon-induced antiviral protein 2-5-oligoadenylate synthetase.
Mol Cell 2003; 12(5):1173-1185.
5. Hamano E, Hijikata M, Itoyama S, Quy T, Phi NC, Long HT, Ha le
D, Ban VV, Matsushita I, Yanai H, Kirikae F, Kirikae T, Kuratsuji T,
Sasazuki T, Keicho N. Polymorphisms of interferon-inducible
genes OAS-1 and MxA associated with SARS in the Vietnamese
population. Biochem Biophys Res Commun 2005;
22;329(4):1234-1239.
In the recently published report "Analysis of RUNX1 Binding Site
and RAPTOR Polymorphisms in Psoriasis: No Evidence for
Association Despite Adequate Power and Evidence for Linkage"[1]
the authors report failure to see association of psoriasis
susceptibility to a region of chromosome 17q25. This region was
first identified with linkage analysis by our group[2], and we recently
reported evidence for a...
In the recently published report "Analysis of RUNX1 Binding Site
and RAPTOR Polymorphisms in Psoriasis: No Evidence for
Association Despite Adequate Power and Evidence for Linkage"[1]
the authors report failure to see association of psoriasis
susceptibility to a region of chromosome 17q25. This region was
first identified with linkage analysis by our group[2], and we recently
reported evidence for association to two unlinked loci from this
chromosomal region.[3] In this instance one associated SNP allele
leads to loss of a putative RUNX binding site. It is important to
address the approach and findings of Stuart et al. (2005)[1] since
they omit important factors that may be required for replicating
associations between genetic loci and complex traits:
1) The statement about adequate power in the title, and the
discussion in the text is misleading. The power these authors
report is only accurate for the parameters they considered. They
fail to consider the inherent heterogeneity of a disease such as
psoriasis and do not incorporate a number of critical parameters
into their power analyses. These include: (i) locus heterogeneity,
(ii) disequilibrium (r2) less than 1.0, (iii) phenotype and/or
genotype misclassification and (iv) the effects and interactions of
environmental risk factors with genetic factors (currently unknown
for complex traits). All are realistic factors and all can substantially
reduce power to detect either linkage and/or association.[4-15]
2) The issue of genetic heterogeneity is of great importance in
complex human disease genetics. It has been demonstrated that
sample size needed to replicate a positive linkage study exceeds,
by a considerable margin, the original sample size, particularly
when factors such as heterogeneity are involved.[16] Effect sizes in
published positive studies tend to be inflated over what one gets
after much more data collection.[17] Such studies are simply more
fortunate with respect to the component of the study that leads to
publication, but it does not mean that they are false positive
results. Since Stuart et al. (2005) did not include heterogeneity into
their power calculations they cannot have addressed this issue.
3) Genotype misclassification seems particularly relevant in this
work. The authors comment that they reconstructed haplotypes
using the methods implemented in Merlin and PHASE (with the
exception of FBAT). This reconstruction was done to address
issues about inflation in type I error due to SNP markers that are
not in linkage equilibrium. However, it has been well documented
that haplotype reconstruction methods such as these are prone to
misclassification18, and that misclassification probabilities may be
large for nuclear families like those studied in this work.[19]
4) It is not clear why the authors did not perform a TDT analysis
using all the affected individuals in the pedigrees, i.e. perform a
linkage analysis with the tightly linked markers? The authors report
that they had 1,285 affected individuals in total. Yet, they chose to
perform an association analysis with TDT, therefore using a
maximum of 351 trios (Table 1), or less than 1/3 of the available
data. The TDT linkage analysis using all affecteds was the
analysis performed in our study[3] and so without a corresponding
analysis on their part, a direct comparison is not possible. It is also
interesting that although the ratios of transmitted to untransmitted
alleles are by no means significant in the study of Stuart et al.
(2005) there is a trend in all pedigrees in the direction we report:
(for SNP9/rs734232 the ratio of the associated A allele in
transmitted versus non-transmitted chromosomes is 235:219).
5) Another issue that could be relevant is the pedigree
ascertainment. Although the ratio of sampled affected to unaffected
individuals in the pedigrees in our study[3] does not appear to be
very different from the same ratio in the pedigrees of Stuart et al.
(2005)[1], there could also be an effect if there is a difference in the
way the pedigrees come to attention in the two studies. It is
possible that the complete pedigrees (including unsampled
individuals) are larger with more affected individuals in the former
study[3] than in the latter.[1] The two samples could then end up with
different fractions of a particular risk haplotype. A comparison of
haplotype frequencies obtained by Stuart et al. (2005)[1] and
Helms et al. (2003)[3] does indeed suggest some systematic
difference between the data sets and/or analyses. Stuart et al.
(2005)[1] state that the combined frequency for the two most common
haplotypes was between .992-.999 for the two regions (depending
somewhat on methods of reconstruction). In the study from Helms
et al. (2003)[3] , the combined frequency of the two most common
haplotypes on non-transmitted chromosomes was 0.94-0.95. This
suggests either that the reconstructions are flawed, or that the
samples differ. The difference could either lead to difference in
power, since if one has more different haplotypes there may be
more informative transmissions. Alternatively it could indicate a
fundamental sampling difference.
6) Given that we report five SNPs driving association in our
families, it is surprising that Stuart et al. (2005)[1] did not attempt to
examine the haplotypes determined by more than just three
associated SNPs. As we report, in a case/control study, evidence
for association with associated haplotypes demarcated by these
five driving SNPs was greater than with single SNPs.[3]
7) Stuart et al. (2005)[1] also state that "only one marker in this
peak
exceeded the p=0.05 level of significance after the most stringent
level of correction for multiple testing", making independent
confirmation critical. However, our level of correction for multiple
testing employed the FDR method[20,21], because employing a
Bonferroni correction for association studies with multiple SNPs is
considered excessively conservative, generally obliterating any
signal in large-scale associations studies. With the FDR method,
nine markers in the SLC9A3R1/NAT9 remained significant. There
are several other studies reporting that adjusting for multiple
testing in studies is less important than other concerns.[22,23]
8) A final general comment is warranted. We believe that
chromosome 17q25 may harbor a number of loci for psoriasis
susceptibility. The issue of clustering of genes for complex traits
has been reported by others[24,25], and is thought to facilitate
identification of susceptibility loci with linkage analyses. Stuart et
al. (2005) also see linkage of psoriasis to 17q25, in a region
harboring the putative RUNX site downstream from SLC9A3R1/
NAT9 and in a region distinct from that seen in two independent
studies of large multiplex families[2,26], but where we also see
linkage in our large set of nuclear families.[3] Previous studies with
simulation data suggest that single major gene analysis of
complex traits has good power to localize genes to a specific
chromosome, but power to localize beyond the chromosomal level
may be significantly compromised.[27] A full understanding of the
contribution of different susceptibility factors from this region will be
necessary as we seek to understand the genetic complexity of this
disease.
C Helms1 L Cao1 JG Krueger2 EM Wijsman3 F Chamian2 D
Gordon4 M Heffernan5 JA Wright-Daw1 J Robarge1 J Ott4 P-Y
Kwok6 A Menter7 AM Bowcock1
1Department of Genetics Washington University School of
Medicine St. Louis Missouri, 63110, USA.
2Laboratory for Investigative Dermatology The Rockefeller
University New York NY 10021, USA.
3Div. of Medical Genetics and Dept. Biostatistics University of
Washington Seattle Washington, 98195, USA.
4Lab of Statistical Genetics The Rockefeller University New York
NY 10021, USA.
5Div. Of Dermatology Washington University School of Medicine St. Louis Missouri, 6310, USA.
6Department of Dermatology Cardiovascular Research Institute,
and Center for Human Genetics University of California San
Francisco CA 94143, USA.
7Department of Internal Medicine Division of Dermatology Baylor
University Medical Center Dallas Texas, 75246 USA.
*Correspondence should be addressed to A.M.B. (email:
bowcock@genetics.wustl.edu).
References
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3. Helms C, Cao L, Krueger JG, Wijsman EM, Chamian F, Gordon
D, Heffernan M, Daw J, Robarge J, Ott J, Kwok P-Y, Menter A,
Bowcock AM. A putative RUNX1 binding site variant between
SLC9A3R1 and NAT9 is associated with susceptibility to psoriasis.
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4. Sillanpaa MJ, Auranen K. Replication in genetic studies of
complex traits. Ann Hum Genet 2004;68:646-57.
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J. A transmission disequilibrium test for general pedigrees that is
robust to the presence of random genotyping errors and any
number of untyped parents. Eur J Hum Genet 2004;12:752-61.
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16. Suarez BK, Hampe CL, van Eerdewegh P. Problems of
replicating linkage claims in psychiatry. Washington: American
Psychiatric Press, 1994.
17 Goring HHH, Terwilliger JD, Blangero J (2001) Large upward
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