Mutations in SETD2 Cause a Novel Overgrowth Condition
Mutations in SETD2 Cause a Novel Overgrowth Condition
A total of 16 patients were included in the study, namely, 3 SoS, 11 'Sotos-like' patients and 2 WS patients. All presented overgrowth, macrocephaly and learning disability. They were regularly followed (once a year) and had repeated bone age assessment at various ages. Three patients were considered as typical Sotos patients as they fulfilled the diagnostic criteria defined by Cole and Hughes: facial gestalt, overgrowth >2 SD, advanced bone age and macrocephaly >2 SD. Eleven patients were considered as Sotos-like patients as they presented with less specific or absence of facial gestalt but fulfilled the other diagnostic criteria. Finally, two patients were considered as Weaver syndrome patients. They both presented with the suggestive facial gestalt, overgrowth, macrocephaly, deep set nails, camptodactyly and accelerated carpal maturation. All patients were regularly followed in the French Department of Medical Genetics and phenotypically scored by clinical geneticists. The study was approved by the local ethics committee. Informed consent was obtained from all patients and/or parents. In all cases, routine G banding and R banding chromosome analyses showed a normal karyotype. A high-resolution array-comparative genomic hybridisation (array-CGH; Agilent technologies, Palo Alto, California, USA) was also performed in all index cases and excluded any large genomic rearrangements. Blood samples from probands and their parents were obtained and genomic DNA was isolated from EDTA anticoagulated using a Nucleon kit (Amersham, UK) according to the manufacturer's instructions. The NSD1 gene was previously screened in all patients, and no NSD1 mutation was found.
The coding sequences of 14 H3K27 methylation-related genes (AEBP2, EED, EZH2, EZH1, HDAC2, JARID2, PCL1, PCL2, PCL3, RBBP4, RBBP7, SIRT1, SUZ12, and UTX) and 8 H3K36 methylation-related genes (NSD1, NDS2, NSD3, SETD2, SETD3, ASH1L, SETMAR and SMYD2) were analysed using a targeted NGS approach. Experiments were performed in the NGS platform of the Cochin hospital, Paris (Assistance Publique—Hôpitaux de Paris, France). The custom primers panel targeting the 22 genes was designed using the AmpliSeq Designer (Lifetechnologies, Saint-Aubin, France). Twenty nanograms of genomic DNA are amplified to generate the library using the Ion AmpliSeq Library Kit V.2.0 (Lifetechnologies). NGS libraries preparation was performed using the Ion AmpliSeq Library Kit V.2.0 (Lifetechnologies) according to the manufacturer's instructions (Ion AmpliSeq Library Preparation, Publication Part Number MAN0006735, Revision 5.0, July 2013, Lifetechnologies). The amplified libraries were purified using Agencourt AMPure XP beads (Beckman Coulter, Brea, California, USA). Prior to library pooling and sequencing sample preparation, amplified libraries were validated and quantified using the 2100 Bioanalyzer microfluidic platform (Agilent Technologies, Santa Clara, California, USA). Emulsion PCR was performed using the Ion OneTouch Instrument (Lifetechnologies). Enrichment of the template-positive Ion OneTouch 200 ion sphere particles (ISPs, containing clonally amplified DNA) PCR was performed using the Ion OneTouch ES (Lifetechnologies) according to the manufacturer's procedures. An ISP quality control was then performed using a QubitR 2.0 Fluorometer. The ISP Quality Control assay on the Qubit 2.0 Fluorometer labelled the ISPs 200 with two different fluorophores: Alexa Fluor 488 and Alexa Fluor 647. The probe labelled Alexa Fluor 488 annealed to all of the ISPs present while the probe labelled Alexa Fluor 647 to only the ISPs with extended templates. The ratio of the Alexa Fluor 488 fluorescence (all ISPs present) to the Alexa Fluor 647 fluorescence (templated ISPs) yielded the % templated ISPs. The template-positive ISPs were loaded on Ion 316 chips and sequenced with an Ion Personal Genome Machine (PGM) System (Lifetechnologies). Data Ion Torrent reads were collected by the Ion Torrent Suite software V.3.6.2, which also sorted the data according to the barcodes. Data collected on the PGM were collated and reanalysed using the Torrent Suite 3.6.2 using FASTQ files from the Ion Torrent Browser.
Sequence alignment and extraction of single-nucleotide polymorphisms (SNPs) and short insertions/deletions (indels) were performed using the Variant Caller plugin on the Ion Torrent Browser and DNA sequences visualised using the Integrated Genomics Viewer (IGV, V.2.3) from Broad Institute (Cambridge, Massachusetts, USA). The NextGENe software V.2.3.3 (Softgenetics, State College, Pennsylvania, USA) was also used for sequence alignment, extraction of SNPs and short indels, and their visualisation and annotation. In brief, major calling parameters were chosen as follows: minimum allele frequency ≥10% for both SNPs and In/Del (short insertions/deletions), minimum sequencing depth ≥6X for SNPs and ≥15X for In/Del, and minimum sequencing depth on either strand ≥5X for In/Del. A NGS bioinformatics analysis was also performed for single and multiexon deletions/duplications identification. In this method, quantitative values were obtained from the number of reads for each amplicon of each sample, extracted using the Coverage Analysis plugin on the Ion Torrent Browser V.3.6.2 (Lifetechnologies). Read number for each separated amplicon was normalised by dividing each amplicon read number by the total of amplicon read numbers of a control gene from the same sample. Normalised read number obtained for each amplicon of a sample was then divided by the average normalised read number of control samples for the corresponding amplicon. Copy number ratios of <0.7 and >1.3 were considered deleted and duplicated, respectively.
Point mutations detected by targeted NGS were confirmed using Sanger DNA sequencing analysis performed on the corresponding exon only. Mutational screening was performed using bidirectional DNA sequencing of the purified PCR products with the ABI Big Dye terminator sequencing kit (Applied Biosystems) on an ABI Prism 3130 automatic DNA sequencer (Applied Biosystems). Sequences were aligned with Seqscape analysis software V.2.5 (Applied Biosystems). The primer oligonucleotide sequences and PCR conditions are available upon request.
Subjects and Methods
Subjects
A total of 16 patients were included in the study, namely, 3 SoS, 11 'Sotos-like' patients and 2 WS patients. All presented overgrowth, macrocephaly and learning disability. They were regularly followed (once a year) and had repeated bone age assessment at various ages. Three patients were considered as typical Sotos patients as they fulfilled the diagnostic criteria defined by Cole and Hughes: facial gestalt, overgrowth >2 SD, advanced bone age and macrocephaly >2 SD. Eleven patients were considered as Sotos-like patients as they presented with less specific or absence of facial gestalt but fulfilled the other diagnostic criteria. Finally, two patients were considered as Weaver syndrome patients. They both presented with the suggestive facial gestalt, overgrowth, macrocephaly, deep set nails, camptodactyly and accelerated carpal maturation. All patients were regularly followed in the French Department of Medical Genetics and phenotypically scored by clinical geneticists. The study was approved by the local ethics committee. Informed consent was obtained from all patients and/or parents. In all cases, routine G banding and R banding chromosome analyses showed a normal karyotype. A high-resolution array-comparative genomic hybridisation (array-CGH; Agilent technologies, Palo Alto, California, USA) was also performed in all index cases and excluded any large genomic rearrangements. Blood samples from probands and their parents were obtained and genomic DNA was isolated from EDTA anticoagulated using a Nucleon kit (Amersham, UK) according to the manufacturer's instructions. The NSD1 gene was previously screened in all patients, and no NSD1 mutation was found.
Targeted Next-generation Sequencing
The coding sequences of 14 H3K27 methylation-related genes (AEBP2, EED, EZH2, EZH1, HDAC2, JARID2, PCL1, PCL2, PCL3, RBBP4, RBBP7, SIRT1, SUZ12, and UTX) and 8 H3K36 methylation-related genes (NSD1, NDS2, NSD3, SETD2, SETD3, ASH1L, SETMAR and SMYD2) were analysed using a targeted NGS approach. Experiments were performed in the NGS platform of the Cochin hospital, Paris (Assistance Publique—Hôpitaux de Paris, France). The custom primers panel targeting the 22 genes was designed using the AmpliSeq Designer (Lifetechnologies, Saint-Aubin, France). Twenty nanograms of genomic DNA are amplified to generate the library using the Ion AmpliSeq Library Kit V.2.0 (Lifetechnologies). NGS libraries preparation was performed using the Ion AmpliSeq Library Kit V.2.0 (Lifetechnologies) according to the manufacturer's instructions (Ion AmpliSeq Library Preparation, Publication Part Number MAN0006735, Revision 5.0, July 2013, Lifetechnologies). The amplified libraries were purified using Agencourt AMPure XP beads (Beckman Coulter, Brea, California, USA). Prior to library pooling and sequencing sample preparation, amplified libraries were validated and quantified using the 2100 Bioanalyzer microfluidic platform (Agilent Technologies, Santa Clara, California, USA). Emulsion PCR was performed using the Ion OneTouch Instrument (Lifetechnologies). Enrichment of the template-positive Ion OneTouch 200 ion sphere particles (ISPs, containing clonally amplified DNA) PCR was performed using the Ion OneTouch ES (Lifetechnologies) according to the manufacturer's procedures. An ISP quality control was then performed using a QubitR 2.0 Fluorometer. The ISP Quality Control assay on the Qubit 2.0 Fluorometer labelled the ISPs 200 with two different fluorophores: Alexa Fluor 488 and Alexa Fluor 647. The probe labelled Alexa Fluor 488 annealed to all of the ISPs present while the probe labelled Alexa Fluor 647 to only the ISPs with extended templates. The ratio of the Alexa Fluor 488 fluorescence (all ISPs present) to the Alexa Fluor 647 fluorescence (templated ISPs) yielded the % templated ISPs. The template-positive ISPs were loaded on Ion 316 chips and sequenced with an Ion Personal Genome Machine (PGM) System (Lifetechnologies). Data Ion Torrent reads were collected by the Ion Torrent Suite software V.3.6.2, which also sorted the data according to the barcodes. Data collected on the PGM were collated and reanalysed using the Torrent Suite 3.6.2 using FASTQ files from the Ion Torrent Browser.
Sequence alignment and extraction of single-nucleotide polymorphisms (SNPs) and short insertions/deletions (indels) were performed using the Variant Caller plugin on the Ion Torrent Browser and DNA sequences visualised using the Integrated Genomics Viewer (IGV, V.2.3) from Broad Institute (Cambridge, Massachusetts, USA). The NextGENe software V.2.3.3 (Softgenetics, State College, Pennsylvania, USA) was also used for sequence alignment, extraction of SNPs and short indels, and their visualisation and annotation. In brief, major calling parameters were chosen as follows: minimum allele frequency ≥10% for both SNPs and In/Del (short insertions/deletions), minimum sequencing depth ≥6X for SNPs and ≥15X for In/Del, and minimum sequencing depth on either strand ≥5X for In/Del. A NGS bioinformatics analysis was also performed for single and multiexon deletions/duplications identification. In this method, quantitative values were obtained from the number of reads for each amplicon of each sample, extracted using the Coverage Analysis plugin on the Ion Torrent Browser V.3.6.2 (Lifetechnologies). Read number for each separated amplicon was normalised by dividing each amplicon read number by the total of amplicon read numbers of a control gene from the same sample. Normalised read number obtained for each amplicon of a sample was then divided by the average normalised read number of control samples for the corresponding amplicon. Copy number ratios of <0.7 and >1.3 were considered deleted and duplicated, respectively.
Sanger Sequencing
Point mutations detected by targeted NGS were confirmed using Sanger DNA sequencing analysis performed on the corresponding exon only. Mutational screening was performed using bidirectional DNA sequencing of the purified PCR products with the ABI Big Dye terminator sequencing kit (Applied Biosystems) on an ABI Prism 3130 automatic DNA sequencer (Applied Biosystems). Sequences were aligned with Seqscape analysis software V.2.5 (Applied Biosystems). The primer oligonucleotide sequences and PCR conditions are available upon request.