In this study, we evaluated eighteen probands with PS deficiency in whom mutations were not found by exon targeted sequencing. Ten out of eighteen probands were type I deficient and eight were type III deficient. Nine of the probands had family members that could be studied. In six out of eighteen probands (33%) we discovered deletions or duplications in PROS1 with the MLPA method. Of these six, one had type III deficiency (isolated proband) and the others had type I deficiency (two isolated probands and three probands with family members).
To confirm the deletions found by MLPA, we performed a qPCR analysis using primers that were designed for selected introns and we chose three genes for normalization (GPR15
on chromosome 3 and PRR15
on chromosome 7). Surprisingly, four members from one of the families possessed a very large deletion that also involved GPR15
, 5 Mb upstream from exon 1 of PROS1.
Evidence that PROS1
deletions might involve nearby genes up to at least 6.5 kb upstream was found before (Johansson et al. 2005
; Yin et al. 2007
; Yoo et al. 2009
), but deletions larger than that were not reported. On the basis of the available data it is not possible to delineate how far the deletion extends beyond GPR15
, but this result illustrates that some PS deficient families may have quite large deletions of chromosome 3. We do not have access to further clinical information from this family but it is possible that, because the deleted genes are all in a haploinsufficiency state, no other inherited diseases are apparent. It may be worthwhile in the future to evaluate selected PS deficient patients for the presence of other inherited disorders.
Although more than 200 mutations have been described in PS deficient patients, less than 5% are gross deletions or duplications [Gandrille et al. 2000
; Stenson et al. 2009
at HGMD (http://www.hgmd.cf.ac.uk/ac
. Accessed 12 May 2009)]. Until 2005, only two large deletions were described in PROS1
(Ploos van Amstel et al. 1989
; Schmidel et al. 1991
). One of the likely reasons for this is that the detection technique, i.e. Southern blotting, is laborious and time-consuming. Moreover, Southern blots of PROS1
can not be easily interpreted because of the presence of the PS pseudogene.
Johansson et al. (2005
) found large deletions in three out of eight investigated PS deficient families (38%, all type I and mixed type I/III deficiency), who were PROS1
mutation-negative, suggesting that CNVs are an important factor in PS deficiency. These authors performed a segregation analysis using a dense set of SNPs and microsatellite markers and this approach proved to be efficient in some families. In other families the markers were not informative, showing the limitations of the use of segregation analysis. Furthermore, PROS1
is not directly targeted and deletions/duplications of exons that are not covered by the microsatellite markers could easily be missed.
Three recent case-reports document two large deletions and one large duplication in PROS1
(Choung et al. 2008
; Yin et al. 2007
; Yoo et al. 2009
). In these three studies, MLPA was used to screen for CNVs, underlining the usefulness of this technique for the examination of large gene rearrangements, even in a single patient. In the present study, we provided further evidence that gross CNVs are an important cause of PS deficiency in both families and isolated cases. We found deletions or duplications in 50% of the type I (5 out of 10), and 12.5% of the type III deficient (1 out of 8) point mutation-negative probands, suggesting that gene deletions are less common in type III families. This is, of course, not surprising as type III individuals have (near) normal levels of total PS, which is not easily reconciled with a gene deletion. It is also interesting to note that the only type III deficient patient with a CNV was an isolated proband, which does not exclude that it comes from a family with mixed type I/III deficiency.
The present panel of patients was drawn from a large set of French PS deficient individuals. Eighteen probands with a high probability of having a hereditary PS deficiency were selected. In 33% of the probands we found a CNV, showing that deletions/duplications are common in patients with putative hereditary PS deficiency. However, one should take care in extrapolating those numbers to consecutive patients with PS deficiency. Together, it seems to be worthwhile to include MLPA as a screening tool when investigating the molecular bases of hereditary PS deficiency.
We can only speculate on the reasons why CNVs occur quite frequently in the PROS1
gene. Out of the five deletions and one duplication, three involved the complete gene, and one of these extends at least 5 Mb to the 5′(telomeric)-direction. Previous studies have also shown large deletions of PROS1
involving exon 1 and nearby genes (Johansson et al. 2005
; Yin et al. 2007
; Yoo et al. 2009
) which gave rise to the idea that there could be a rearrangement focus somewhere telomeric to the NSUN3
gene (Yoo et al. 2009
), but this claim was never objectively analyzed. In the remaining cases, exons 4, 9, and 11 where always involved. Thus, a hotspot for rearrangement might exist somewhere in these regions of PROS1
. Further, the first case of PS deficiency due to a translocation [t(3;21)(q11.2;q22)] was recently described (Hurtado et al. 2009
), reinforcing the idea that the PROS1
locus may be prone to structural changes. It has been shown that highly homologous low copy repeat structures, as well as AT-rich palindromes and peri-centromeric repeats are located at breakpoints of rearrangement leading to both homologous recombination and non-homologous end joining rearrangements mechanisms (Shaw and Lupski 2004
Despite that this paper and those that precede it now report that CNVs are quite common in PS deficiency, there remains a large part of PS deficiency with unknown genetic causes. There are several possible reasons for this. First, the MLPA technique that was used did cover most, but still not all of the exons, so it remains possible that small deletions that hit these exons are often present in the remaining cases. The reason that some exons are not covered has to do with interference by the PS pseudogene in the MLPA reactions or with difficulty in examining AT rich regions. Secondly, the promoter region of PROS1
was only recently characterized and perhaps mutations in this region are quite common. Mutation analysis in the past has only poorly covered this region of the gene (de Wolf et al. 2006
; Tatewaki et al. 2003
). Thirdly, inversions of parts of the gene can not be ruled out by the MLPA technique as this does not change the copy numbers of the exons. Finally, recombination between gene and pseudogene may go unnoticed with the currently available detection techniques.
In conclusion, this study confirms that gross gene abnormalities in PROS1 are common in PS deficient patients and it appears that MLPA is a useful tool in direct screening for copy number changes in PROS1. The qPCR confirmed the results, which underlines the accuracy of MLPA as a screening tool.