Capítulo I. Definición del problema: justificación, metodología y teoría
6. Metodología: fases y técnicas
6.2. Fuentes y establecimiento de líneas de trabajo
Comparing the rate of null genotype SNPs identified in 22q11.2DS patients with and
without psychiatric disorders sought to establish whether additional secondary CNVs
occurring within the non-deleted region of chromosome 22q11.2 could potentially
play a role in the increased risk of psychiatric disorders.
Association analysis identified no evidence for significantly increased rate of null
genotype SNPs, Bonferroni threshold= 0.017, in either the 29 22q11.2DS patients with
ADHD (uncorrected Fishers exact test p-value= 0.56), the 18 patients with ASD
(uncorrected Fishers exact test p-value= 1.0), or the 37 patients with a childhood
psychiatric disorder (uncorrected Fishers exact test p-value= 1.0) when compared to
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A
22q11.2DS+ADHD 22q11.2DS-PSYCH Fisher
exact two- tailed p-
value
Carrying a Null SNP Not carrying a Null SNP Total Carrying a Null SNP Not carrying a Null SNP Total
Number of 22q11.2DS patients Frequency Number of 22q11.2DS patients Frequency - Number of 22q11.2DS patients Frequency Number of 22q11.2DS patients Frequency - 2 6.9% 27 93.1% 29 1 2.6% 34 97.1% 35 0.59 B
22q11.2DS+ASD 22q11.2DS-PSYCH Fisher
exact two- tailed p-
value
Carrying a Null SNP Not carrying a Null SNP Total Carrying a Null SNP Not carrying a Null SNP Total
Number of 22q11.2DS patients Frequency Number of 22q11.2DS patients Frequency - Number of 22q11.2DS patients Frequency Number of 22q11.2DS patients Frequency - 0 0.0% 18 100% 18 1 2.6% 34 97.1% 35 1.0 C
22q11.2DS+PSYCH 22q11.2DS-PSYCH Fisher
exact two- tailed p-
value
Carrying a Null SNP Not carrying a Null SNP Total Carrying a Null SNP Not carrying a Null SNP Total
Number of 22q11.2DS patients Frequency Number of 22q11.2DS patients Frequency - Number of 22q11.2DS patients Frequency Number of 22q11.2DS patients Frequency - 2 5.7% 35 94.6% 37 1 2.6% 34 97.1% 35 1.0
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5.6. Discussion
It is widely known that 22q11.2DS is associated with a wide spectrum of
neuropsychiatric phenotypes. This chapter set out to identify whether additional
genetic variants present on the non-deleted 22q11.2 chromosome influenced the
increased risk to psychiatric disorders seen in 22q11.2DS patients. A number of
studies have tested SNPs at 22q11.2 for association with psychiatric disease in
22q11.2DS patients, but these have been selected because of their relevance to
candidate genes and have generated inconclusive results.
This study systematically tested 5,027 SNPs spanning the deletion at 22q11.2 for
association with childhood psychiatric disorders in patients with 22q11.2DS. The
results described in this chapter revealed no evidence for a significant association for
common SNPs with neuropsychiatric symptoms in 22q11.2DS children (ADHD
and/or ASD) (Bonferroni p-values >9.9x10-6, FDR >0.05).
A reasonable explanation for the failure of this study to provide a positive finding is
the small sample size involved in the analyses. Only 76 samples for well-characterized
22q11.2DS patients with available psychiatric phenotypes information were analysed.
While this is comparable to previous candidate based studies, due to the large number
of SNPs (n= 5,027) analysed, the corrected significance threshold of this study was
more stringent. A larger sample size would be required to achieve adequate statistical
power (Klein 2007). It is however worth noting that while more samples were
originally ascertained for this study, the rigorous quality control procedures resulted
~19 recruited samples being excluded.
The Genetic Power Calculator (Purcell, Cherny & Sham 2003) was used to calculate
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unaffected) had an 80% power to detect an association at p-value= 0.05 for a risk allele
with a genotype relative risk (GRR) of 2.03 and allele frequency of 0.06. It is however
possible that as 22q11.2DS patients already carry a primary insult (i.e. the large
deletion) then the effect size of an additional risk allele at chromosome 22q11.2 could
be larger in these patients than is present in studies of other psychiatric disease cohorts.
For example, it has been shown that all 22q11.2DS children have a lower IQ when
compared to their non-deleted siblings (Niarchou et al. 2014). It is therefore plausible
that this compromised cognition could result in risk alleles for psychiatric disease
having a greater relative risk in children carrying the deletion. While unlikely, it is
potentially possible that the samples used in this thesis had sufficient power to detect
an additional risk allele whose disease penetrance was amplified in this way. An
estimate of the sample sizes required to obtain an 80% power to detect lower GRR are
given in the Table 5- 4.
GRR
Sample size
*P-value of 0.05 **P-value of 9.9x10-6
Cases Controls Cases Controls
1.05 13,807 13,117 48,689 46,255 1.1 3,483 3,309 12,284 11,670 1.2 1,022 971 3,607 3,427 1.3 461 438 1,626 1,545 1.4 263 250 927 881 1.5 170 162 601 571 1.6 120 114 432 410 1.7 89 85 315 300 1.8 69 66 245 233 1.9 55 52 196 186 2.0 45 43 161 153 2.01 44 42 158 150
Table 5- 4: The required sample sizes to obtain an 80% power to detect a common risk variant (allele frequency = 0.05) with a range of GRR.
* Uncorrected p-value threshold.
** Bonferroni corrected p-value threshold in our study.
Figure 6- 1* Uncorrected p-value threshold.
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From Table 5-, it can be seen that a larger sample size is required to detect risk alleles
with decreased effect size.
While this study has focussed on testing risk alleles for association with psychiatric
disease in patients with 22q11.2DS, any true risk allele present at 22q11.2 would also
be identified (albeit with a reduced GRR) in association studies of non-deleted patients
with psychiatric disease compared to controls. Several GWA studies of ASD have now
suggested a number of common genetic variants associated with the risk of ASD. They yielded ~112 genome-wide significant loci spanned by 200 ASD candidate genes (Weiss et al. 2009; Wang et al. 2009; Anney et al. 2010; Salyakina et al. 2010). From
all the reported findings for ASD and autism, none of these risk loci were found within
the typical deleted region of 22q11.2 chromosome. Moreover, GWAS analysis was
performed in a large case-control cohort for five psychiatric disorders; ASD, ADHD,
bipolar disorder, major depressive disorder, and schizophrenia including 33,332 cases
and 27,888 controls of European ancestry. The study identified SNPs at four
significant risk loci at genome-wide significance threshold (p-value <5x10-8). These regions are on chromosomes 3p21 and 10q24, and SNPs mapped to two L-type
voltage-gated calcium channel subunits, CACNA1C and CACNB2 (Cross-Disorder
Group of the Psychiatric Genomics Consortium 2013). Again none of these reported
psychiatric risk loci mapped to the 22q11.2 region. The results of the largest and very
recent GWAS study conducted for schizophrenia identified 108 distinct genome-wide
significant loci being associated with schizophrenia. Three loci of the 108 are found
in chromosome 22 (chr22_39987017_D at 39,975,317-40,016,817; rs9607782 at
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al. 2014), however none of the three loci was located in the common 22q11.2 deleted
region.
Taken as a whole, it is therefore possible that the findings of these GWA studies
support those presented in this chapter, and that no psychiatric disease risk variants
are present in the region of 22q11.2.
The second part of this chapter attempted to identify evidence for small potential
deletions within the remaining 22q11.2 chromosome of 22q11.2DS patients. These
were identified by detecting SNPs that had null genotypes and this revealed 3 potential
secondary deletions present in three 22q11.2DS patients. Association analysis
indicated that these potential deletions were not significantly enriched in the
22q11.2DS patients with psychiatric phenotypes (p-value >0.017). Nevertheless, it is
of potential interest that the Null SNP (and a potential secondary deletion) carried by
two 22q11.2DS patients with ADHD are located in the genes THAP7 and PRODH, as
previous studies have identified nominally significant evidence for association to
ADHD with common variants spanned by both genes (Neale et al. 2010; Stergiakouli
et al. 2012; Hinney et al. 2011; Yang et al. 2013; Mick et al. 2010; Neale et al. 2008).
In addition, transcripts for THAP7 were found to be significantly downregulated in
22q11.2DS patients compared to non-deleted controls.
This analysis of potential secondary deletions was intended as a preliminary
investigation, however, the approach of identifying null SNPs was limited by the
distribution of probes on the genotyping platform. In addition, a single marker with
null genotypes is not a perfect representation of a small deletion, while this study did
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sample genotyping. A future study should utilise a next generation sequencing (NGS)
in order to obtain a more detailed and precise understanding of the background variants
in the non-deleted 22q11.2 chromosome in 22q11.2DS patients (McDonald-McGinn
et al. 2013). This approach would allow a more systematic analysis and would not be
dependant on the distribution of pre-selected SNPs that are present on the genotyping
microarray. Future NGS based studies would also benefit from the increased power of
studying larger numbers of individuals with 22q11.2DS in order to reduce both the
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