A Familial Disorder of Altered DNA-Methylation
A Familial Disorder of Altered DNA-Methylation
We report on two fetuses (III-1, III-3) and one child (III-2) of a healthy non-consanguineous Turkish couple (II-3, II-4). Family histories were unremarkable on both sides. In the first pregnancy (III-1), omphalocele and shortened femora were noticed at 21 weeks of gestation. At 33 weeks, hypoplastic thorax and clover leaf skull were noted and the pregnancy was terminated. On pathological examination marked lung hypoplasia was confirmed and abnormal lung lobulation, gall bladder agenesis, hydronephrosis and further abnormalities were noted. As X-rays showed no signs of skeletal dysplasia or craniosynostosis, the tentative diagnosis of SRS was suggested. At that time only maternal UPD7 was known as the cause for SRS, but microsatellite analyses on cultured amniocytes ruled this out. The second pregnancy (III-2) was complicated by early diagnosis of molar changes in the placenta, asymmetrical fetal growth restriction, omphalocele and massively elevated β-human chorionic gonadotropin (β-HCG). The child was born at 32 weeks. After birth coarse facial features, facial haemangioma, omphalocele and asymmetric growth restriction were suggestive both of SRS and BWS, which prompted methylation studies. Postnatally body proportions harmonised. The child is developmentally delayed. In the third pregnancy (III-3), early asymmetric growth retardation, elevated β-HCG and molar changes of the placenta were noted. The pregnancy ended by spontaneous fetal demise 1 week after chorionic villi sampling at 12 weeks. For further details, see Figure 1A, Online Supplementary Results, Table S1 and Figure S1.
(Enlarge Image)
Figure 1.
Pedigree of the family and results of NLRP7 mutation and DNA-methylation analyses. (A) Pedigree of the family. Presence of the NLRP7 variant (c.2156C>T), segregation of chromosome 19 determined by microsatellite (D19S927, D19S926 and D19S418) and SNP analysis of the NLRP7/NLRP2 genes are shown. (B) Enrichment analysis of the genes differentially methylated in affected individuals independent of the tissue. Genes (n=87) differentially methylated between patient samples and tissue-matched controls were significantly enriched for promoters with low CpG content (p<0.001, χ test), as well as for imprinted genes (p<0.001). In contrast, differentially methylated genes were depleted for promoters with high CpG content (p<0.001) and CpG island (p<0.001). The enrichment was calculated comparing the proportion of the respective group of the array with the differentially methylated genes. Grey bars indicate the proportion present on the array and black bars the proportion present on the 87 differentially methylated genes. HCP, promoters of high CpG content; ICP, promoters of intermediate CpG content; LCP, promoters of low CpG content; *** p<0.001 (χ test), ns, not significant (p>0.05). (C) NLRP7 mutation analysis in the parents identifies a heterozygous c.2156C>T (p.A719 V) variant in the mother (II-4) of the affected individuals. The father (II-3) carries only the wildtype allele.
Various specimens of the affected individuals (III-1, III-2, III-3) and peripheral blood samples of further family members were investigated (see online Supplementary Table S2). Detailed information on analysed materials and used methods (including PCR conditions and primer sequences) are provided in the online Supplementary Appendix. The study has been approved by the Institutional Review Board of the Medical Faculty of the Christian-Albrechts University Kiel (AZ B305/08). All family members investigated and for minors the respective parents gave written informed consent for their participation.
Karyotyping, FISH and molecular karyotyping were performed according to standard techniques and manufacturers' instructions.
Mutation analysis of NLRP2 (NM_001174081), NLRP7 (NM_001127255), ZFP57 (NM_001109809), KHDC3L (NM_001017361) and of the oocyte-specific variant of DNMT1 (NM_001130823) was performed by Sanger sequencing. Segregation of NLRP7/NLRP2 alleles was verified by microsatellite analysis on chromosome 19.
Exome enrichment using the NimbleGen Human SeqCap EZ V.3.0 Kit followed by sequencing on an Illumina HiSeq 2000 system and data analysis was carried out on DNA from two affected children (III-1 and III-2), the parents (II-3 and II-4) and the maternal grandparents (I-3, I-4) as detailed in the online Supplementary Appendix.
Locus-specific DNA-methylation analysis was performed using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA), methylation-specific PCR, sequence-based quantitative methylation analysis and bisulfite pyrosequencing. Global DNA-methylation was analysed using LUminometric Methylation Assay (LUMA). For array-based DNA-methylation quantification of 27 578 CpG sites, the HumanMethylation27 DNA Analysis BeadChip (Illumina) was applied. Raw hybridisation signals were analysed using GenomeStudio software (GSE47879). A detailed description of the bioinformatic analysis of array-based methylation data is provided in the online Supplementary Appendix.
Patients and Methods
Case Reports
We report on two fetuses (III-1, III-3) and one child (III-2) of a healthy non-consanguineous Turkish couple (II-3, II-4). Family histories were unremarkable on both sides. In the first pregnancy (III-1), omphalocele and shortened femora were noticed at 21 weeks of gestation. At 33 weeks, hypoplastic thorax and clover leaf skull were noted and the pregnancy was terminated. On pathological examination marked lung hypoplasia was confirmed and abnormal lung lobulation, gall bladder agenesis, hydronephrosis and further abnormalities were noted. As X-rays showed no signs of skeletal dysplasia or craniosynostosis, the tentative diagnosis of SRS was suggested. At that time only maternal UPD7 was known as the cause for SRS, but microsatellite analyses on cultured amniocytes ruled this out. The second pregnancy (III-2) was complicated by early diagnosis of molar changes in the placenta, asymmetrical fetal growth restriction, omphalocele and massively elevated β-human chorionic gonadotropin (β-HCG). The child was born at 32 weeks. After birth coarse facial features, facial haemangioma, omphalocele and asymmetric growth restriction were suggestive both of SRS and BWS, which prompted methylation studies. Postnatally body proportions harmonised. The child is developmentally delayed. In the third pregnancy (III-3), early asymmetric growth retardation, elevated β-HCG and molar changes of the placenta were noted. The pregnancy ended by spontaneous fetal demise 1 week after chorionic villi sampling at 12 weeks. For further details, see Figure 1A, Online Supplementary Results, Table S1 and Figure S1.
(Enlarge Image)
Figure 1.
Pedigree of the family and results of NLRP7 mutation and DNA-methylation analyses. (A) Pedigree of the family. Presence of the NLRP7 variant (c.2156C>T), segregation of chromosome 19 determined by microsatellite (D19S927, D19S926 and D19S418) and SNP analysis of the NLRP7/NLRP2 genes are shown. (B) Enrichment analysis of the genes differentially methylated in affected individuals independent of the tissue. Genes (n=87) differentially methylated between patient samples and tissue-matched controls were significantly enriched for promoters with low CpG content (p<0.001, χ test), as well as for imprinted genes (p<0.001). In contrast, differentially methylated genes were depleted for promoters with high CpG content (p<0.001) and CpG island (p<0.001). The enrichment was calculated comparing the proportion of the respective group of the array with the differentially methylated genes. Grey bars indicate the proportion present on the array and black bars the proportion present on the 87 differentially methylated genes. HCP, promoters of high CpG content; ICP, promoters of intermediate CpG content; LCP, promoters of low CpG content; *** p<0.001 (χ test), ns, not significant (p>0.05). (C) NLRP7 mutation analysis in the parents identifies a heterozygous c.2156C>T (p.A719 V) variant in the mother (II-4) of the affected individuals. The father (II-3) carries only the wildtype allele.
Materials
Various specimens of the affected individuals (III-1, III-2, III-3) and peripheral blood samples of further family members were investigated (see online Supplementary Table S2). Detailed information on analysed materials and used methods (including PCR conditions and primer sequences) are provided in the online Supplementary Appendix. The study has been approved by the Institutional Review Board of the Medical Faculty of the Christian-Albrechts University Kiel (AZ B305/08). All family members investigated and for minors the respective parents gave written informed consent for their participation.
Cytogenetic Analyses
Karyotyping, FISH and molecular karyotyping were performed according to standard techniques and manufacturers' instructions.
Molecular Studies
Mutation analysis of NLRP2 (NM_001174081), NLRP7 (NM_001127255), ZFP57 (NM_001109809), KHDC3L (NM_001017361) and of the oocyte-specific variant of DNMT1 (NM_001130823) was performed by Sanger sequencing. Segregation of NLRP7/NLRP2 alleles was verified by microsatellite analysis on chromosome 19.
Exome enrichment using the NimbleGen Human SeqCap EZ V.3.0 Kit followed by sequencing on an Illumina HiSeq 2000 system and data analysis was carried out on DNA from two affected children (III-1 and III-2), the parents (II-3 and II-4) and the maternal grandparents (I-3, I-4) as detailed in the online Supplementary Appendix.
DNA-Methylation Analysis
Locus-specific DNA-methylation analysis was performed using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA), methylation-specific PCR, sequence-based quantitative methylation analysis and bisulfite pyrosequencing. Global DNA-methylation was analysed using LUminometric Methylation Assay (LUMA). For array-based DNA-methylation quantification of 27 578 CpG sites, the HumanMethylation27 DNA Analysis BeadChip (Illumina) was applied. Raw hybridisation signals were analysed using GenomeStudio software (GSE47879). A detailed description of the bioinformatic analysis of array-based methylation data is provided in the online Supplementary Appendix.
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