We have found a statistically significant (P
= 0. 007) threefold overrepresentation of duplications in the chromosome 16p13.1 region in schizophrenia cases compared with controls. We found a similar threefold overrepresentation of deletions in cases, but this was not significant (P
> 0.05). The great majority of duplications and deletions we found using Illumina microarrays are identical to those reported by two groups using array comparative genomic hybridization.12,13
They span the same 1.5 Mb region that includes intervals that we refer to as intervals I and II; we have also identified novel duplications and a single deletion involving interval II only.
The distinction between CNVs involving intervals I and II and interval II alone is dependent on a group of seven SNP probes at 15.0–15.1 Mb (). However, we are confident that our classifications are correct; every CNV detected by the independent Dosage Miner and QuantiSNP algorithms was then inspected by eye, and in all cases, we could make a clear distinction between those involving both intervals I and II (Dup/Del I + II) and interval II only (Dup/Del II). In addition, genealogical analysis of the Icelandic data set, performed post hoc, showed a clear separation of CNV types. In total, 36 out of 54 carriers were clustered in 11 families (ancestral clustering, max depth three generations), including seven families with Dup I + II (nine, three and five times two carriers), two families with Del I + II (six and two carriers), one family with Dup II (four carriers) and one family with Dup II + III (two carriers). In no instance did we observe two different CNVs segregating within the same family.
The breakpoints for all three types of deletion/duplication are located in areas with high low copy repeat content, reflecting apparent genomic instability of the region.25
The repeats are in the same orientation, and non-allelic homologous recombination (NaHR) between these low copy repeats seems to be the most likely explanation for the recurrence of the rearrangements and for their identical size. Three inversion polymorphisms have previously been described in the 16p13.1 region.26,27
A large duplication in a patient with MR has also been reported,28
as has a smaller de novo
A much larger duplication (8 Mb) of the region has also been reported in two unrelated patients with autistic features.30
Our most striking finding is the increased risk of schizophrenia associated with duplications at the 16p13.1 locus. Recurrent deletions at several loci have now been reported to be significantly associated with schizophrenia, but, to date, duplications associated with schizophrenia have mostly been isolated case reports. It is more difficult to decide whether the duplications are genuinely pathogenic or benign. The problem has already been encountered by Hannes et al
with duplications in MR at this locus. They found duplications in five out of 1027 cases, three of whom had striking behavioral problems similar to those reported by Ullmann et al
However, because the duplication was present in 5 out of 2000 controls, they could not decide whether it was genuinely pathogenic. The different sizes of duplications and deletions at the 16p13.1 locus also present difficulties when it comes to assessing statistical association. Statistically, we have used the straightforward approach of counting all duplications and then deletions as equivalent events, and only then tried to condition on those duplications or deletions that have the same breakpoints as those reported previously in autism and MR/multiple congenital anomalies.12,13
Although caution must be exercised when interpreting results from such a small number of cases, there are several grounds supporting that our findings are indeed genuine. First, given the rarity of the duplications, the overall association with schizophrenia is statistically (P
= 0.0071) significant, and increases (P
= 0.00010) when the Dup I + II is considered separately. In addition, identical duplications at the 16p13.1 locus have already been found associated with autism.12
Second, four of the schizophrenia duplication cases had an early onset of illness (12, 17, 19 and 19 years) and in this respect resembled the 16p13.1 deletion cases in which three of the five schizophrenia cases also had early onset of illness (15, 17 and 18 years, see Supplementary Information
, part 2). With a mean age at onset of 24 years, this reflects an overrepresentation of early onset cases, although not reaching significance. Third, in an Icelandic family, the duplication co-segregates with neuropsychiatric disorders, including two cases of schizophrenia, and individual cases of attention deficit hyperactivity disorder, dyslexia and alcoholism. Owing to the small size of the family and the diversity of the neuropsychiatric phenotypes, calculating a logarithm of the odds (LOD) score did not seem appropriate. This range of phenotypes we observe in a single family is not unexpected, as an overlap of phenotypic features between autism and attention deficit hyperactivity disorder has been extensively reported, and individuals with attention deficit hyperactivity disorder are at increased risk of schizophrenia.31–33
Fourth, the duplications at this locus seem to be under negative selection. Cluster analysis of the Dup I + II events in the Icelandic population finds that the carriers cluster less than expected in families, that is, the genealogical clusters are smaller, and sporadic carriers more numerous than would be expected if the duplications were selectively neutral. The arguments and methods are laid out in Supplementary Information
, part 4 and are also given in Stefansson et al
These clustering results, however, are informal, and so any conclusions must be provisional. Fifth, Kirov et al
have identified three chromosome 16p13.11 duplications in 471 schizophrenia cases and 6 out of 2792 controls. In addition, the International Schizophrenia Consortium finds overrepresentation of large 16p13.11 duplications (13 of 3391 cases versus 7 of 3181 controls have duplications encompassing interval II, see at http://pngu.mgh.harvard.edu/isc/isc-r1.cnv.bed
but this overrepresentation is mostly explained by the Aberdeen sample, which overlaps between our study and the International Schizophrenia Consortium study (although separately genotyped and analyzed), and therefore, does not provide further support to our finding.
Finally, with regard to the Dup I II duplications, although the Scottish sample seems+to be driving the association observed, as 6 out of 12 carriers among affected cases belong to that sample, the association of Dup I + II in this study remains significant even after removing the Scottish samples from the analysis (P = 0.0033, OR = 5.94).
The duplicated region contains two strong candidate genes (NTAN1 and NDE1), over- or underexpression of either or both of which at key stages of neurodevelopment could predispose to autism, MR and/or schizophrenia.
gene is located in the small island of non-repeated sequence called interval one. It encodes an N-terminal asparagine amidase that has been implicated in social behavior and memory. Over-expression of NTAN1
leads to reduction in the MAP2 (microtubule-associated protein 2) expression through the ubiquitin proteasome pathway. Reduced expression of MAP2
may be a useful marker for diagnosis of schizophrenia and bipolar disorder in vivo35,36
and in vitro
Mice with a disrupted NTAN1 gene show less locomotion in an open field and impairment of several spatial memory tasks.39,40
are highly homologous genes involved in brain development, neuronal proliferation, migration and synapse formation. They encode proteins that biologically interact with DISC1 and LISI proteins, with NDE1
appearing to be interchangeable with its homolog NDEL
, except that NDE1
is expressed earlier in development. nde1
-null mice are viable and display microcephaly with thinning cortical layer and reduced numbers of neurons. Interestingly, two out of three reported autism cases with duplication had increased head circumference.12
-null mice display defects in neuronal proliferation and neuronal migration. The NDE1 protein directly interacts with the DISC1 protein at the C-terminal end that is distal to a truncating mutation that is reported to segregate with schizophrenia and other forms of major mental illness in a large Scottish DISC1
As truncated DISC1 is known to alter NDE1/NDEL function, the duplications we report here may have a similar biological effect as the truncating mutation associated with schizophrenia in the Scottish family.
All duplications and deletions in our study involve interval II that harbors the NDE1
gene and this makes dysregulation of NDE1 expression the most parsimonious explanation for the increased risk of the phenotypes reported by Ullmann et al
and by the authors of this paper. On the other hand, the strongest association is with duplications that also involve interval I. It is possible that combined changes in expression of NTAN1
increase susceptibility over changes in expression of NDE1
alone. No clear-cut findings emerged from our examination of the 16p13.1 region for allelic association for schizophrenia. The findings are discussed along with association studies following conditioning for DISC1
Ser704Cys in Supplementary Information
, part 3.
Further work is required before the clinical implications of our findings become clear. On the one hand, the data strongly suggest that recurrent duplications at 16p13.1 locus increase the risk of schizophrenia. They also strengthen the hypothesis that there are shared genetic risk factors between schizophrenia, MR and autism. However, the odds ratios, even for the Dup I + II, are substantially less than the increased risks we have observed for three out of four recurrent deletions on chromosomes 1, 15 and 22.6
Whether the smaller odds ratio we observe for duplications is a feature of the 16p13.1 locus itself, or it is part of a broader rule that recurrent duplications are generally less penetrant than recurrent deletions, remains to be determined. The 16p13.1 duplications we observe are rare, at a rate of about 3 or 4 per 1000 schizophrenia cases, and, estimating from the control population in the present study, about 0.08% in the general population. This makes it difficult to obtain precise measurements of overall risk or risk for individual neuropsychiatric disorders. Analysis of CNV data from sets of cases and controls considerably larger than the sets we report in this paper, which itself to date is one of the largest assembled, will be required. These and many other questions will need to be answered before the exciting findings that arise from CNV analysis can be used in clinical practice for diagnostics, disease classification or genetic testing.