Nat. Genet. 36, 497–501 (2004).

In the original report, methylation of the MLH1 promoter was detected in a small proportion of the FACS-sorted spermatozoa from male individual TT, who harbored a soma-wide MLH1 epimutation. To determine whether the MLH1 methylation detected was, in fact, due to residual somatically derived DNA, we reassessed the original spermatozoa sample using two methodologies, but this time, we included methylation analysis of the imprinted control gene SNRPN, which is unmethylated in spermatozoa cells1.

We employed the same colony hybridization technique used in the original report for the detection of methylation at both the MLH1 and SNRPN promoters. Primers (Supplementary Table 1 online) were designed to amplify both the methylated and the unmethylated bisulfite-converted DNA templates with equal efficiency. Amplicons were cloned using the pGEMTeasy vector system (Promega), and 500 colonies for each gene were arrayed onto gridded filters. These were then hybridized sequentially with γ-32P-end-labeled probes (Supplementary Table 1) complementary to methylated and unmethylated templates of the respective genes. For MLH1, 4 of 423 (0.95%) clones were derived from methylated alleles (4 had a positive hybridization signal with the methylation-specific probe and 419 with the unmethylated probe), a finding consistent with the original report of 5 of 526 (0.95%) methylated colonies. However, for SNRPN, 5 of 434 clones containing target inserts also gave a positive signal with the methylation-specific probe, indicating that 1.15% SNRPN alleles in the sample were methylated. These findings were confirmed using fluorescence-based real-time methylation-specific PCR (f-MSP) of the MLH1 promoter2 as well as the SNURF-SNRPN promoter3, using the MYOD1 gene to control for DNA input4. The levels of methylation were measured with reference to an in vitro 100% methylated human DNA control (CpGenome Universal Methylated DNA, Chemicon) to obtain a percentage methylation reference (PMR) value, as previously described5. The peripheral blood of individual TT showed PMR values consistent with monoallelic methylation of SNRPN and MLH1, as expected (Fig. 1). However, the PMR values of the DNA from his spermatozoa were 0.32% ± 0.25 for MLH1 and 0.66% ± 0.09 for SNRPN (Fig. 1). In addition, we analyzed a new sample of motile spermatozoa from individual TT by f-MSP, which was isolated by 'swim-up' followed by FACS. This sample was devoid of MLH1 methylation, with a trace of SNRPN methylation (0.16% ± 0.16). The spermatozoa samples from the normal control male, whether isolated by FACS or swim-up plus FACS, showed low levels of SNRPN methylation alone (0.35–1%), illustrating the difficulty in acquiring a pure sample of spermatozoa from semen.

Figure 1: Equivalent levels of MLH1 and SNRPN methylation in spermatozoa as determined by fluorescence real-time methylation specific PCR.
figure 1

Bar chart shows the percentage methylation reference (PMR) values for peripheral blood lymphocytes (PBL) and sperm from individual TT and a normal control following methylation-specific PCR of MLH1 and the SNURF-SNRPN locus. The mean PMR values are provided numerically above each bar. Spermatozoa were isolated from diploid cells either by FACS or by 'swim-up' (SU) followed by FACS, as labeled. Error bars, s.d.

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Irrespective of molecular technique, the level of allelic methylation of SNRPN marginally exceeded that of MLH1 in the original spermatozoa sample from individual TT. These new data indicate that the MLH1 methylation previously reported in the spermatozoa of subject TT is most likely an artifact, attributable to a low level of contamination of the sample with either somatic cells or free DNA derived from somatic cells. In a second spermatozoa sample, no MLH1 methylation was detected at all. These new data alter the original interpretation that incomplete resetting of the epigenetic mark on MLH1 had occurred in a proportion of TT's spermatozoa and suggest instead that reversal is complete in the actual gametes.

Editor's note: These new data are reported by Megan P. Hitchins and Robyn L. Ward. Authors David I.K. Martin and Catherine M. Suter do not agree with the content of this Addendum.

Note: Supplementary information is available on the Nature Genetics website.