Michael Zeschnigk

Exome sequencing identifies recurrent somatic mutations in EIF1AX and SF3B1 in uveal melanoma with disomy 3

Gene expression profiles and chromosome 3 copy number divide uveal melanomas into two distinct classes correlating with prognosis. Using exome sequencing, we identified recurrent somatic mutations in EIF1AX and SF3B1, specifically occurring in uveal melanomas with disomy 3, which rarely metastasize. Targeted resequencing showed that 24 of 31 tumors with disomy 3 (77%) had mutations in either EIF1AX (15; 48%) or SF3B1 (9; 29%). Mutations were infrequent (2/35; 5.7%) in uveal melanomas with monosomy 3, which are associated with poor prognosis. Resequencing of 13 uveal melanomas with partial monosomy 3 identified 8 tumors with a mutation in either SF3B1 (7; 54%) or EIF1AX (1; 8%). All EIF1AX mutations caused in-frame changes affecting the N terminus of the protein, whereas 17 of 19 SF3B1 mutations encoded an alteration of Arg625. Resequencing of ten uveal melanomas with disomy 3 that developed metastases identified SF3B1 mutations in three tumors, none of which targeted Arg625.

Identification of chromosomes 3, 6, and 8 aberrations in uveal melanoma by microsatellite analysis in comparison to comparative genomic hybridization

In uveal melanoma, monosomy 3 is strongly associated with metastic disease and poor prognosis. Cytogenetic analysis and comparative genomic hybridization (CGH) have been used to identify chromosomal aberrations in uveal melanoma. As these methods are costly and time consuming in routine diagnostic settings, we evaluated whether tumors with monosomy 3 can be reliably identified by microsatellite analysis (MSA). In addition, we also tested if aberrations of chromosomes 6 and 8, which have also been associated with the course of the disease, can be detected by MSA. We established a protocol for MSA of 23 markers, 3-4 on each arm of chromosomes 3, 6, and 8. Twenty tumors were analyzed by CGH and MSA, and 10 tumors were analyzed by MSA only. For chromosome 3, the results of CGH and MSA were concordant, thus indicating that the dosage of this chromosome can reliably be determined by MSA. However, MSA failed to detect copy number gains at 6p in some tumors. Moreover, despite quantitative evaluation of allele ratios, it was not possible to discern 8p losses and gains reliably. We thus conclude that while MSA can be used to determine monosomy 3 in uveal melanoma, careful interpretation of results for chromosomes 6 and 8 is recommended.

A comprehensive molecular study on Coffin–Siris and Nicolaides–Baraitser syndromes identifies a broad molecular and clinical spectrum converging on altered chromatin remodeling

Chromatin remodeling complexes are known to modify chemical marks on histones or to induce conformational changes in the chromatin in order to regulate transcription. De novo dominant mutations in different members of the SWI/SNF chromatin remodeling complex have recently been described in individuals with Coffin-Siris (CSS) and Nicolaides-Baraitser (NCBRS) syndromes. Using a combination of whole-exome sequencing, NGS-based sequencing of 23 SWI/SNF complex genes, and molecular karyotyping in 46 previously undescribed individuals with CSS and NCBRS, we identified a de novo 1-bp deletion (c.677delG, p.Gly226Glufs*53) and a de novo missense mutation (c.914G>T, p.Cys305Phe) in PHF6 in two individuals diagnosed with CSS. PHF6 interacts with the nucleosome remodeling and deacetylation (NuRD) complex implicating dysfunction of a second chromatin remodeling complex in the pathogenesis of CSS-like phenotypes. Altogether, we identified mutations in 60% of the studied individuals (28/46), located in the genes ARID1A, ARID1B, SMARCB1, SMARCE1, SMARCA2, and PHF6. We show that mutations in ARID1B are the main cause of CSS, accounting for 76% of identified mutations. ARID1B and SMARCB1 mutations were also found in individuals with the initial diagnosis of NCBRS. These individuals apparently belong to a small subset who display an intermediate CSS/NCBRS phenotype. Our proposed genotype-phenotype correlations are important for molecular screening strategies.

TERT promoter mutations in ocular melanoma distinguish between conjunctival and uveal tumours

Background:Recently, activating mutations in the TERT promoter were identified in cutaneous melanoma. We tested a cohort of ocular melanoma samples for similar mutations.Methods:The TERT promoter region was analysed by Sanger sequencing in 47 uveal (ciliary body or choroidal) melanomas and 38 conjunctival melanomas.Results:Mutations of the TERT promoter were not identified in uveal melanomas, but were detected in 12 (32%) conjunctival melanomas. Mutations had a UV signature and were identical to those found in cutaneous melanoma.Conclusion:Mutations of TERT promoter with UV signatures are frequent in conjunctival melanomas and favour a pathogenetic kinship with cutaneous melanomas. Absence of these mutations in uveal melanomas emphasises their genetic distinction from cutaneous and conjunctival melanomas.

Loss of heterozygosity of 1p in uveal melanomas with monosomy 3

Gains and losses of chromosomes 1, 3, 6 and 8 are nonrandom chromosomal aberrations in uveal melanoma. Monosomy 3 is the most frequent abnormality and is associated with poor prognosis. To identify regions of allelic loss on the short arm of chromosome 1 and to investigate if these alterations contribute to uveal melanoma progression, we performed microsatellite analysis of 10 loci in 70 uveal melanomas. A total of 51 tumors were obtained from patients with clinical follow‐up data, 19 tumors were from recent patients without follow‐up. Loss of heterozygosity (LOH) of at least 1 marker was more frequent in tumors with monosomy 3 (40%) than in tumors with disomy 3 (10%). In particular, loss of the entire short arm of chromosome 1 was only observed in tumors with monosomy 3 (p = 0.0001). By comparing the extent of 1p LOH in all tumors with monosomy 3, we were able to define a smallest region of overlap (SRO) of approximately 55 Mb, which is flanked by markers D1S507 and D1S198. On the basis of our data and published cytogenetic data, we propose that 1p31 harbors genes involved in the progression of uveal melanoma with monosomy 3.

Prognostic significance of chromosome 3 alterations determined by microsatellite analysis in uveal melanoma: a long-term follow-up study.

Background:In uveal melanoma (UM), the most frequent primary intraocular tumour in adults, loss of one entire chromosome 3 (monosomy 3 (M3)) is observed in ∼50% of tumours and is significantly associated with metastatic disease. The strong association of metastatic disease with M3 offers the opportunity for molecular prognostic testing of UM patients.Methods:To re-evaluate M3 as prognostic marker in our clinical and laboratory setting and to determine the metastatic potential of rare tumours with partial M3, we performed a comprehensive study on 374 UM patients treated by enucleation in our clinic within 10 consecutive years, starting in 1998. Genotyping of all tumours was performed by microsatellite analysis.Results:Median follow-up time was 5.2 years. The disease-specific mortality rates (death by UM metastases) for tumours with disomy 3 (D3) and M3 were 13.2% and 75.1%, respectively. The disease-specific survival was worse when M3 was observed together with chromosome 8 alterations (P=0.020). Death of UM metastases was also observed in 12 patients (9%) with D3 tumours. The metastasising D3 tumours showed a larger basal tumour diameter (P=0.007), and were more frequently of mixed or epitheloid cell type (P<0.0001) than D3 tumours that did not metastasise. Mortality rate of tumours showing partial M3 (8.3%) was as low as that for tumours with D3.Conclusion:This shows that large tumours with disomy 3 have an increased risk to develop metastases. On the basis of these results, our clinic offers routine prognostic testing of UM patients by chromosome 3 typing.

IGF2/H19 hypomethylation in Silver–Russell syndrome and isolated hemihypoplasia

Silver–Russell syndrome (SRS) is a clinically and genetically heterogeneous syndrome characterized by severe pre and postnatal growth retardation, body asymmetry and a typical facial phenotype with a triangular face and relative macrocephaly. In 30% of patients, the differentially methylated IGF2/H19 imprinting center region (ICR1) on chromosome 11p15 was found to be hypomethylated, as determined by Southern blot analysis of an HpaII restriction site close to the third CTCF-binding site (CTS3) within ICR1. Using bisulfite treatment and a real-time PCR-based methylation assay (QAMA), we analyzed the third and sixth CTCF-binding sites (CTS3, CTS6) in 5 patients with CTS3 hypomethylation, in 14 patients who were suspected to have SRS but were normal by Southern blot analysis, and in 1 patient with body asymmetry without any other features of SRS or Beckwith–Wiedemann syndrome (BWS). In all 5 patients with CTS3 hypomethylation, in 5 of 14 patients who were judged to be normal at CTS3 by Southern blot analysis and in the patient with isolated body asymmetry, we found CTS3 and CTS6 hypomethylation by QAMA. Using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA), we obtained similar results at four additional ICR1 sites in the CTS6 region. These results show that ICR1 hypomethylation occurs more often in SRS patients than as previously thought as well as in isolated hemihypoplasia. Furthermore, we show that methylation analysis by QAMA and MLPA is more sensitive in detecting ICR1 hypomethylation than Southern blot analysis of CTS3.

Massive parallel bisulfite sequencing of CG-rich DNA fragments reveals that methylation of many X-chromosomal CpG islands in female blood DNA is incomplete

Methylation of CpG islands (CGIs) plays an important role in gene silencing. For genome-wide methylation analysis of CGIs in female white blood cells and in sperm, we used four restriction enzymes and a size selection step to prepare DNA libraries enriched with CGIs. The DNA libraries were treated with sodium bisulfite and subjected to a modified 454/Roche Genome Sequencer protocol. We obtained 163 034 and 129 620 reads from blood and sperm, respectively, with an average read length of 133 bp. Bioinformatic analysis revealed that 12 358 (7.6%) blood library reads and 10 216 (7.9%) sperm library reads map to 6167 and 5796 different CGIs, respectively. In blood and sperm DNA, we identified 824 (13.7%) and 482 (8.5%) fully methylated autosomal CGIs, respectively. Differential methylation, which is characterized by the presence of methylated and unmethylated reads of the same CGI, was observed in 53 and 52 autosomal CGIs in blood and sperm DNA, respectively. Remarkably, methylation of X-chromosomal CGIs in female blood cells was most often incomplete (25-75%). Such incomplete methylation was mainly found on the X-chromosome, suggesting that it is linked to X-chromosome inactivation.

Low frequency of imprinting defects in ICSI children born small for gestational age

Although there is an increased frequency of low birth weight after assisted reproduction, the mechanisms underlying this association are unclear. We have proposed that some of the children conceived by intracytoplasmic sperm injection (ICSI) with low birth weight might have an epimutation (faulty methylation pattern) in one of the imprinted genes involved in fetal growth control, eg, KCNQ1OT1, PEG1, PEG3, GTL2, IGF2/H19 and PLAGL1. Using bisulfite DNA sequencing and sequence-based quantitative methylation analysis (SeQMA), we determined the methylation pattern of these genes in buccal smears from 19 ICSI children born small for gestational age (SGA, birth weight <3rd percentile) and from 29 term-born normal weight children after spontaneous conception. We detected clear hypermethylation of KCNQ1OT1 and borderline hypermethylation of PEG1 in one and the same ICSI child. The other children and the parents of the affected child have normal methylation patterns. Imprinting defects appear to be a rare finding in ICSI children born SGA. Methylation of the paternal KCNQ1OT1 and PEG1 alleles may be a previously unrecognized cause of SGA. The epimutations found in the SGA child, whose father had oligozoospermia, probably result from an imprint erasure defect in the paternal germ line and therefore appear to be linked to the fertility problem of the father and not to in vitro fertilization/ICSI.

Aberrant hypomethylation of the cancer–testis antigen PRAME correlates with PRAME expression in acute myeloid leukemia

PRAME is a tumor-associated antigen, which belongs to the family of cancer–testis antigens (CTA). The expression of CTA is mainly restricted to the testis and various tumors. In contrast to other CTA, PRAME expression is also frequently detected in acute and chronic leukemias. Due to this expression pattern, PRAME has attracted great interest as a prognostic tumor marker that can be used for the detection of minimal residual disease and as a potential target for immunotherapy. In acute myeloid leukemia (AML), PRAME expression has been observed in 30–64% of cases. To evaluate whether epigenetic mechanisms contribute to PRAME activation in AML, we studied DNA methylation of 15 CpG dinucleotides within a CpG-rich region located in the intron 1 of the PRAME gene. DNA methylation was determined by sequence analysis of cloned PCR products generated from bisulfite-treated genomic DNA. Methylation patterns were correlated with PRAME mRNA levels as determined by microarray analysis and real-time PCR. We found almost complete methylation in mononuclear blood cells from two healthy donors and in bone marrow cells of four PRAME-negative AML patients. In contrast, the degree of PRAME methylation was clearly reduced in four PRAME-positive AML bone marrow samples. In particular, these samples were characterized by the presence of clones, which were completely devoid of methylation. The significant inverse correlation between the degree of methylation and PRAME expression suggests a causal role of DNA methylation in PRAME regulation. Such a role is further supported by the observation that treatment of PRAME-negative cell lines U-937 and THP-1 with the demethylating agent 5′-Aza-2′dC resulted in a dose-related upregulation of PRAME expression.