The rates of de novo meiotic deletions and duplications causing several genomic disorders in the male germline

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1 Supplementary information for: The rates of de novo meiotic deletions and duplications causing several genomic disorders in the male germline Daniel J. Turner 1, Marcos Miretti 1, Diana Rajan 1, Heike Fiegler 1, Nigel P. Carter 1, Martyn L Blayney 2, Stephan Beck 3 and Matthew E. Hurles 1

2 Supplementary Note: 1. Estimating the prevalence of de novo WBS deletions from our spermbased NAHR assays To scale our sperm-based estimates of NAHR rates in the assayed hotspot in the male germline to estimate the population prevalence of WBS, we need to estimate the proportion of male NAHR events that are not captured by or assay, due to them falling outside the hotspot, and then we need to take into account the relative rate at which WBS deletions arise in the paternal and maternal germlines. Predicted prevalence = µ/h + Sµ/H Where µ = the average rate assayed in our experiments on 5 sperm donors, H is the proportion of paternal NAHR events that fall in the assayed hotspot and S is the ratio of maternal/paternal NAHR events. It has previously been noted that there are two hotspots of NAHR activity in parents with WBS offspring, one found in parents that are heterozygous for an inversion encompassing the deleted region and one for parents that are not heterozygous for the inversion 1,2. The hotspot we have assayed is that found in parents not heterozygous for the inversion. Only males who are not heterozygous carriers of the inversion should have NAHR activity in the assayed hotspot. We assume that our sperm donors are similarly not heterozygous for the inversion, which exists at a frequency of ~5% in the population. Bayes et al report the proportion of WBS deletion breakpoints that fall within the assayed hotspots specifically for parents not heterozygous for the inversion, these estimates of the proportion of breakpoints that fall within the hotspot are valid for our sperm donor. Due to difficulties with their assay Bayes et al report this proportion as a range, with between 4 and 8 out of 19 WBS deletions having breakpoints within the assayed hotspot. Therefore we similarly report the predicted prevalence as a range, using 4/19 and 8/19 as alternative estimates for H. Thomas et al report a meta-analysis of WBS patients in which they add their data to previous findings to generate a total of 148 maternal WBS deletions and 130 paternal WBS deletions 3. They report that this is not a significant difference and therefore it is not possible to reject the null hypothesis that the rate of NAHR is equal in the male and female germlines. Consequently we estimate S as 1. Inputing the estimates of S and H, together with the upper and lower 95% estimates of for µ (which are calculated from the standard deviation of estimates from individual sperm donors assuming that they are normally-distributed), into the equation above gives an estimate of the population prevalence of the WBS deletion of 1/7,161 to 1/47,416.

3 2. Comparing sperm-based and disease-based estimates of the prevalence of de novo CMT1A duplications Using the equation described above, we used Lopes et al estimates 4 of the proportion of paternal duplication breakpoints occurring in the CMT1A-REP NAHR hotspot (22/34) and the ratio of maternal/paternal NAHR events (2/34) together with our upper and lower 95% estimates of µ to estimate the population prevalence of de novo duplications based on our sperm assays to be 1/22,718 to 1/78,955. Estimating the population prevalence of de novo CMT1A duplications from disease prevalence is complicated by the observation that due to the absence of reproductive lethality, most duplications are inherited. Skre et al estimated the prevalence of autosomal dominant CMT to be 36/100, CMT1 is thought to comprise 78% of autosomal dominant CMT 6, and PMP22 duplications are thought to account for 70.7% of CMT1 7. Of these duplications it has been estimated that 22% are de novo 8. Multiplying these parameters together gives a rough estimate of the prevalence of de novo duplications of 1/22,896. Other epidemiological data support a lower rate for the de novo CMT1A duplication. An alternative estimate of the prevalence of CMT1 is 1/5,000 9, multiplying this by the proportion that are de novo duplications from above gives estimates of the de novo rate of 1/32,000. An alternative estimate of the prevalence of the CMT1A duplication is 11.2/100,000 10, which when multiplied by the proportion that are de novo from above gives an estimate of the de novo rate of 1/40,584. These lower rates are supported by a lower estimate of the overall prevalence of CMT of 10/100, Therefore, a range of 1/23,000 to 1/41,000 seems to capture the uncertainty present in current estimates of the de novo rate of CMT1A duplication from disease prevalence. 3. Estimating the rates of intra-chromatidal, inter-chromatidal and interchromosomal NAHR at the CMT1A-REP and WBS-LCR hotspots. If I = rate of intra-chromosomal /rate of inter-chromosomal rearrangements, then: I (deletions) = (α+β)/γ I (duplications) = β/γ Rate of deletions = α+β+γ Rate of duplications = β+γ

4 I (duplications) can be estimated for the CMT1A duplication in the paternal germline from published information (I = 1/49) 12. Solving the simultaneous equations above gives the rate estimates quoted in the main text. To estimate α, β and γ for the WBS-LCR hotspot we need an estimate of I (intrachromosomal NAHR/ inter-chromosomal NAHR) for WBS deletions occurring in the paternal germline. This estimate is complicated by the fact that if the deletion occurs in the germline of a parent heterozygous for the inversion, then the resultant NAHR event is always inter-chromosomal 1. As we have assayed NAHR rates in an individual who does not appear to be heterozygous for the inversion (see above), we need to correct the known estimates of I, for the contribution of parents carrying the inversion. In a meta-analysis, Thomas et al report that I is 23/61 3. The proportion of these that are likely to result from parents heterozygous for the inversion was estimated by Osborne et al to be 33% (4/12 families tested). If we remove 33% of the observations of I from above, all of them inter-chromosomal, we end up with a corrected value of I of 23/33. These observations are, however, sex-averaged, and are not specific to the male germline. The only non-inverted paternal-specific estimates of I come from Bayes et al 1, who report that of 6 paternal germline WBS deletions from parents not heterozygously inverted, 3 are intrachromosomal and 3 are inter-chromosomal, giving I =3/3. If we calculate α, β and γ from the simultaneous equations and estimate I = 23/33 we get: α = 5.01x10-6 β = -1.09x10-6 γ = 5.63x10-6 If we calculate α, β and γ from the simultaneous equations and estimate I = 3/3 we get: α = 5.01x10-6 β = -2.35x10-7 γ = 4.78x10-6 Clearly a negative mutation rate, as is the case for both estimates of β above, is not biologically meaningful. This is presumably partly due to the imprecision with which the various parameters, and especially I are estimated. We note that there is an additional potential complication with estimating α, β and γ for the WBS-LCR hotspot. The complex nature of the genomic architecture at the WBS-LCR locus means that in addition to deletions and duplications resulting from NAHR between centromeric and medial low copy repeats, it is possible that our assays might also detect inversions caused by NAHR between medial and telomeric repeats, but only if the breakpoint fell within the same short hotspot region. Such inversions necessarily produce both recombinant haplotypes in a

5 single event and so contribute equally to deletion and duplication frequencies. Consequently, if the rate of detected inversions was so high that it dwarfed the rates of deletion and duplication we would observe these latter rates to be approximately equal, which is clearly not the case (table 1); the rate of deletions is ~2X higher than the rate of duplications. If inversion NAHR events were contaminating our estimates of deletion and duplication rates it would cause the absolute NAHR rates to be over-estimated, but it would cause the rate of α relative to β+γ to be under-estimated. Accounting for potential contamination by inversion events cannot account for the apparent negative rates of β, as these become even more negative if inversions are incorporated. Notwithstanding these caveats, the inference that at the WBS-LCRs, in common with the CMT1A-REP duplication, the rates of intra-chromatidal NAHR and interchromosomal NAHR are relatively similar and the rate of inter-chromatidal NAHR is much lower, appear to be robust. References 1. Bayes, M., Magano, L.F., Rivera, N., Flores, R. & Perez Jurado, L.A. Mutational mechanisms of Williams-Beuren syndrome deletions. Am J Hum Genet 73, (2003). 2. Osborne, L.R. et al. A 1.5 million-base pair inversion polymorphism in families with Williams-Beuren syndrome. Nat Genet 29, (2001). 3. Thomas, N.S. et al. Parental and chromosomal origins of microdeletion and duplication syndromes involving 7q11.23, 15q11-q13 and 22q11. Eur J Hum Genet 14, (2006). 4. Lopes, J. et al. Fine mapping of de novo CMT1A and HNPP rearrangements within CMT1A-REPs evidences two distinct sexdependent mechanisms and candidate sequences involved in recombination. Human Molecular Genetics 7, (1998). 5. Skre, H. Genetic and clinical aspects of Charcot-Marie-Tooth's disease. Clincal Genetics 6, (1974). 6. Harding, A.E. & Thomas, P.K. The clinical features of hereditary motor and sensory neuropathy types I and II. Brain 103, (1980). 7. Nelis, E. et al. Estimation of the mutation frequencies in Charcot-Marie- Tooth disease type 1 and hereditary neuropathy with liability to pressure palsies: a European collaborative study. Eur J Hum Genet 4, (1996). 8. Marques, W., Jr. et al. 17p duplicated Charcot-Marie-Tooth 1A: characteristics of a new population. J Neurol 252, (2005). 9. Harding, A.E. & Reilly, M. Molecular genetics of inherited neuropathies. in Peripheral Nerve Disorders 2 (eds. Ashbury, A.K. & Thomas, P.K.) (Butterworth-Heinmann, London, 1995).

6 10. Morocutti, C. et al. Charcot-Marie-Tooth disease in Molise, a centralsouthern region of Italy: an epidemiological study. Neuroepidemiology 21, (2002). 11. Emery, A.E. Population frequencies of inherited neuromuscular diseases-- a world survey. Neuromuscul Disord 1, (1991). 12. Lopes, J. et al. Homologous DNA exchanges in humans can be explained by the yeast double-strand break repair model: a study of 17p11.2 rearrangements associated with CMT1A and HNPP. Human Molecular Genetics 8, (1999).

7 Supplementary Methods DNA extraction from semen We transferred 2ml of semen to a 50ml Falcon Tube (BD Biosciences) and added 900ml resuspension buffer (1x SSC, 0.2% SDS), before mixing gently and centrifuging at 3,000rpm for 2 minutes. We discarded supernatants into Virkon disinfectant (Day Impex LTD) and resuspended pellets in 10ml 1x SSC, mixing and centrifuging as before. We then resuspended pellets in 10ml buffer K (0.2x SSC, 0.1% SDS, 1M b-mercaptoethanol), added 10µl 800units/ml proteinase K solution (Sigma) and incubated tubes for 1 hr at 37 with occasional mixing. To the contents of each Falcon Tube we added an equal volume of phenol / chloroform / isoamyl alcohol (25:24:1), mixed the tubes gently until emulsified and centrifuged as before. We transferred the upper, aqueous layer into a clean 50ml Falcon Tube, discarded the lower, phenolic layer, added 5ml TE to the interphase and repeated the phenol extraction. We combined the aqueous layers from the two phenol extractions and performed an ethanol precipitation: to the 15ml aqueous phase we added 1.5ml 3M sodium acetate ph5.4 and 37.5ml 100% ethanol, mixed tubes gently and centrifuged as before. We washed pellets with 70% ethanol and resuspended them in 10ml T0.1E buffer (10mM Tris-Cl ph8.0, 0.1mM EDTA, ph8.0) by incubating at 50 C overnight with gentle shaking. We performed a second ethanol precipitation, by adding 1ml 3M sodium acetate ph5.4 and 25ml 100% ethanol. We washed pellets in 70% ethanol and resuspended them in 10ml 5mM Tris-HCl ph7.5 be incubating overnight at 50 C, as before.

8 a) WBS-LCR 1.55Mb cen 143kb 143kb Cc Ac Bc Cm Bm Am 143kb Bt At Ct tel Centromeric Medial Telomeric b) LCR17p 5Mb 124kb cen proximal SMS-REP middle SMS-REP distal SMS-REP 124kb tel LCR17pD 200kb 200kb 200kb LCR17pA c) AZFa-HERV 780kb cen 10kb 11.5kb LINE tel Proximal HERV Distal HERV d) CMT1A-REP 1.5Mb cen 24kb 24kb tel Proximal CMT1A-REP Distal CMT1A-REP Supplementary Figure 1. Schematic genomic structure of four genomic disorder regions. Regions that undergo NAHR and for which our assays were designed are shown in blue. Other regions of homology are shown in grey / black. a) 7q11.23 (Williams-Beuren). There are three large segmental duplications: centromeric, medial and telomeric, each of which is composed of three distinct blocks: A, B and C. The 1.55Mb major deletion / duplication arises from NAHR between the directly orientated 143kb Bc and Bm blocks (shown in blue). Inversion of the region, caused by NAHR between inverted duplications is also possible. b) 17p11.2 (LCR17 Smith Magenis). 80% of cases of Smith Magenis Syndrome are caused by NAHR between the directly orientated 200kb proximal and distal SMS-REPs, resulting in a 4Mb deletion. The corresponding duplication has been reported. A rarer 5Mb deletion arises from NAHR between two directly orientated 124kb LCR17 segmental duplications (shown in blue) that flank the proximal and distal SMS-REPs. c) Yq11.21(AZFa). NAHR between two HERV15 proviruses that lie in direct orientation lead to deletion / duplication of approximately 800kb. The repeats are homologous over 10kb, but the distal repeat also contains a 1.5kb LINE insertion. d) 17p12 (Charcot Marie Tooth / HNPP). The two CMT1A-REPs are 24kb long and 1.5Mb apart, in direct orientation. NAHR between the CMT1A-REPs causes duplication / deletion of the 1.5Mb region.

9 Amplification efficiency of duplication assay (%) CMT1A-REP AZFa-HERV WBS-LCR LCR17p 95% confidence interval of amplification efficiency from control experiments Efficiency needed to fully account for difference in rate between deletions and duplications Supplementary Figure 2. Amplification efficiency of duplication assays. To exclude the possibility that lower amplification efficiency of duplication assays accounts for the observed differences in deletion and duplication rates we estimated the amplification efficiency of each duplication assay (shown in blue) and compared it to the amplification efficiency that would be needed to fully account for the difference between deletion and duplication rates (shown in red), assuming that the deletion assay was itself 100% efficient a conservative assumption. The experimental details of this experiment are detailed in the Methods section.

10 Supplementary Figure 3. Array Comparative Genome Hybridisation results on 8 sperm donors. Each plot shows log 2 ratio against chromosomal coordinate. A. Whole genome profiles; B. Chromosome 7 profiles, blue line shows the WBS region; C. Chromosome 17 profiles, blue lines show the LCRp and CMT1A regions; D. Chromosome Y profiles, blue line shows the AZFa region; E. WBS-LCR region profiles, blue lines show the duplicated sequences sponsoring deletions and duplications; F. CMT1A-REP region profiles, blue lines show the duplicated sequences sponsoring deletions and duplications; G. LCR17p region profiles, blue lines show the duplicated sequences sponsoring deletions and duplications; H. AZFa-HERV region profiles, blue lines show the duplicated sequences sponsoring deletions and duplications.

11 A

12 B

13 C

14 D

15 E

16 F

17 G

18 H

19 Supplementary Table 1. Gene conversion events accompanying crossovers CMT1A duplication Min start gene conv Min stop gene conv Min GC length Max start GC position Max stop GC position Max GC length position position CMT1A deletion Min start gene conv Min stop gene conv Min GC length Max start GC position Max stop GC position Max GC length position position LCR17 duplication Min start gene conv Min stop gene conv Min GC length Max start GC position Max stop GC position Max GC length position position

20 Supplementary Table 2. PCR primer sequences used in NAHR assays Primers used for cloning and paralog-specific PCRs. Mismatched bases are shown in lower case. CMT1A ComF1 CMT1A ComR1 CMT1A PF1 CMT1A PR1 CMT1A DF1 CMT1A DR1 CMT1A PF2 CMT1A PR2 CMT1A DF2 CMT1A DR2 CMT1A Tqm HERVD1 proxf1 HERVD1 distf1 HERVD1 ComR1 HERVD1 PF1 HERVD1 DF1 HERVD1 PR1 HERVD1 DR1 HERVD1 DF2 HERVD1 PF2 HERVD1 DR2 HERVD1 PR2 HERVD1 Tqm LCR17 ComF1 LCR17 ComR1 LCR17 AF1 LCR17 DF1 LCR17 AF2 LCR17 DF2 LCR17 AR2 LCR17 DR2 LCR17 AR1 LCR17 DR1 LCR17 Tqm WBS ComF1 WBS ComR1 WBS CTF1 WBS MF1 WBS CTR1 WBS MR1 WBS CTR2 WBS MR2 WBS CTF2 WBS MF2 WBS Tqm TTACATCTCCTATATACCCAGAAAGGAAAC TCTTGATTAAAACAGCTAAACTTCAACAAC TTGGATTCAAAGATATTAGTGTTcT TGATATTTAAAGATTTCATGtC GGATTCAGAGACATTAGTGTTcC CATGATATTTAAAGATTTCATGtT GAAACATACTAGTTGATATCTTCTgA CATGTCATTAGACCAAAGAaC AGAAACATACTAGTTGATATCTTCTaT CATGTCATTAGACCAAAGAaT 6FAM-AAGAAGAATCGTGGGCACACCACCA-TAMRA CAGGACATGAGAGATGCTCTTTTC CAGGACATGAGAGAGGATTGTTTTATC CCTGACCTGCTGCCTTGTAAC GAAAGGGAATTAATTGGCAGTtT ACTGGAAATTAATTGGCCATtC GCATAGATAGTGACAGTGGCCTaG TGGCATAGATAGTGACAGTAGCCTaC CTGCAAGGTATTAGTGGCaTG GATAGTGCAAGGTATTAATGGaTCA CTCTTTTCTTTGGCCTCTGaG CTCTTTTCTTTGGCCTCTCaA 6FAM-TGCGGTGTTAGGTTGCTAGCAGTGGTACTTC-TAMRA TGGATCCTGCTCAGTTCCAC CCACAAAACCTCCACACATTATCTC AGTGCTTAGGATCTTATGACTGAAtC AGTGCTTAGGATCTTATGACTGAAtT AATAGAGGGACAAACTTCTCTTAGATaCT ATAGAGGGACAAACTTCTCTTAGATaTG CTTTGTTATATTAGAGAACAGAGTCTTCAtT TGTTATATTAGGGAACAGAGTCTTCAtC GTTGTGCACAAGTGTTCTGaA CTGTTGTGCACAAGTGTTCTAaG 6FAM-TGAAGGTAGCAGTGGTGCCATGCAAC-TAMRA CCACTGCATTTGTCAGCAATC GGGCAAATCTGAAGAACGAGC GGTCACAGCATCCAGGAGGtA GTCACAGCATCCAGGAGGtG TTTAAACAACGAACTTGATTTTATTACTCCaGT CAACGAACTTGATTTTATTACTCCaGC TGCTGGCCTTTGTGTTATCtAG GCTGGCCTTTGTGTTATCtTC CTGGCCAGCAACAAaGC ACCTGGCCAGCAACAAaGT 6FAM-TGGTCAAGAGCAGCAGTCAAAAATAATTGGTTTC-TAMRA

21 Supplementary Table 3. PCR cycling conditions for NAHR assays Cycling conditions and MgCl 2 concentrations for paralog-specific PCR reactions. 1 PCR CMT1A 3mM MgCl 2 HD1 3mM MgCl 2 LCR17 dup 2mM MgCl 2 LCR17 del 2mM MgCl 2 WBS 2.5mM MgCl 2 94 C 2 min 94 C 2 min 94 C 2 min 94 C 2 min 94 C 2 min 94 C 30 sec 94 C 30 sec 94 C 30 sec 94 C 30 sec 94 C 30 sec 56.5 C 30 sec x C 15 sec x C 15 sec x C 15 sec x C 15 sec x5-0.5 C -0.5 C -0.5 C -0.5 C 72 C 70 sec 72 C 65 sec 72 C 50 sec 72 C 50 sec 72 C 92 sec 72 C 10 min 94 C 30 sec 94 C 30 sec 94 C 30 sec 94 C 30 sec 4 C 58 C 15 sec x31 58 C 15 sec x31 58 C 15 sec x32 63 C 15 sec x31 72 C 65 sec 72 C 50 sec 72 C 50 sec 72 C 92 sec 72 C 10 min 72 C 10 min 72 C 10 min 72 C 10 min 4 C 4 C 4 C 4 C

22 2 PCR CMT1A 3mM MgCl 2 HD1 3.5mM MgCl 2 LCR17 3mM MgCl 2 WBS 2.5mM MgCl 2 94 C 2 min 94 C 2 min 94 C 2 min 94 C 2 min 94 C 30 sec 94 C 30 sec 94 C 30 sec 94 C 30 sec 58 C 30 sec x50 61 C 30 sec x40 64 C 32 sec x50 63 C 30 sec x50 72 C 40 sec 72 C 40 sec 72 C 15 sec 72 C 63 sec 72 C 10 min 72 C 10 min 72 C 10 min 72 C 10 min 4 C 4 C 4 C 4 C