Katharina Wimmer

Diagnostic criteria for constitutional mismatch repair deficiency syndrome: suggestions of the European consortium ‘Care for CMMRD’ (C4CMMRD)

Constitutional mismatch repair deficiency (CMMRD) syndrome is a distinct childhood cancer predisposition syndrome that results from biallelic germline mutations in one of the four MMR genes, MLH1, MSH2, MSH6 or PMS2. The tumour spectrum is very broad, including mainly haematological, brain and intestinal tract tumours. Patients show a variety of non-malignant features that are indicative of CMMRD. However, currently no criteria that should entail diagnostic evaluation of CMMRD exist. We present a three-point scoring system for the suspected diagnosis CMMRD in a paediatric/young adult cancer patient. Tumours highly specific for CMMRD syndrome are assigned three points, malignancies overrepresented in CMMRD two points and all other malignancies one point. According to their specificity for CMMRD and their frequency in the general population, additional features are weighted with 1–2 points. They include multiple hyperpigmented and hypopigmented skin areas, brain malformations, pilomatricomas, a second childhood malignancy, a Lynch syndrome (LS)-associated tumour in a relative and parental consanguinity. According to the scoring system, CMMRD should be suspected in any cancer patient who reaches a minimum of three points by adding the points of the malignancy and the additional features. The diagnostic steps to confirm or refute the suspected diagnosis are outlined. We expect that application of the suggested strategy for CMMRD diagnosis will increase the number of patients being identified at the time when they develop their first tumour. This will allow adjustment of the treatment modalities, offering surveillance strategies for second malignancies and appropriate counselling of the entire family.

Guidelines for surveillance of individuals with constitutional mismatch repair-deficiency proposed by the European Consortium “Care for CMMR-D” (C4CMMR-D)

Lynch syndrome (LS) is an autosomal dominant disorder caused by a defect in one of the DNA mismatch repair genes: MLH1, MSH2, MSH6 and PMS2. In the last 15 years, an increasing number of patients have been described with biallelic mismatch repair gene mutations causing a syndrome referred to as ‘constitutional mismatch repair-deficiency’ (CMMR-D). The spectrum of cancers observed in this syndrome differs from that found in LS, as about half develop brain tumours, around half develop digestive tract cancers and a third develop haematological malignancies. Brain tumours and haematological malignancies are mainly diagnosed in the first decade of life, and colorectal cancer (CRC) and small bowel cancer in the second and third decades of life. Surveillance for CRC in patients with LS is very effective. Therefore, an important question is whether surveillance for the most common CMMR-D-associated cancers will also be effective. Recently, a new European consortium was established with the aim of improving care for patients with CMMR-D. At a workshop of this group held in Paris in June 2013, one of the issues addressed was the development of surveillance guidelines. In 1968, criteria were proposed by WHO that should be met prior to the implementation of screening programmes. These criteria were used to assess surveillance in CMMR-D. The evaluation showed that surveillance for CRC is the only part of the programme that largely complies with the WHO criteria. The values of all other suggested screening protocols are unknown. In particular, it is questionable whether surveillance for haematological malignancies improves the already favourable outcome for patients with these tumours. Based on the available knowledge and the discussions at the workshop, the European consortium proposed a surveillance protocol. Prospective collection of all results of the surveillance is needed to evaluate the effectiveness of the programme.

Amplification and overexpression of the IGF2 regulator PLAG1 in hepatoblastoma

There is evidence that 8q amplification is associated with poor prognosis in hepatoblastoma. A previous comparative genomic hybridization analysis identified a critical region in chromosomal bands 8q11.2–q13. Using restriction landmark genomic scanning in combination with a virtual genome scan, we showed that this region is delineated by sequences within contig NT_008183 of chromosomal subbands 8q11.22–q11.23. A real‐time PCR–based genomic copy number assay of 20 hepatoblastomas revealed gain or amplification in this critical chromosomal region in eight tumors. The expression of four genes and expressed sequence tags (ESTs) within this newly defined region was assayed by real‐time reverse transcriptase polymerase chain reaction (RT‐PCR) in four tumors with and six tumors without gain or amplification. The PLAG1 oncogene was found to be highly expressed in all but one tumor compared to normal liver tissue. Furthermore, quantitative RT‐PCR revealed that the expression level of the developmentally regulated transcription factor PLAG1 was 3–12 times greater in hepatoblastoma tumors and cell lines compared to age‐matched normal liver and comparable to the expression in fetal liver tissue. PLAG1 has been shown be a transcriptional activator of IGF2 in other tumor types. Using luciferase reporter assays, we demonstrated that PLAG1 transactivates transcription from the embryonic IGF2 promoter P3, also in hepatoblastoma cell lines. Thus, our results provide evidence that PLAG1 overexpression may be responsible for the frequently observed up‐regulation of IGF2 in hepatoblastoma and therefore may be implicated in the molecular pathogenesis of this childhood neoplasia.

Conservation of hotspots for recombination in low-copy repeats associated with the NF1 microdeletion

Several large-scale studies of human genetic variation have provided insights into processes such as recombination that have shaped human diversity. However, regions such as low-copy repeats (LCRs) have proven difficult to characterize, hindering efforts to understand the processes operating in these regions. We present a detailed study of genetic variation and underlying recombination processes in two copies of an LCR (NF1REPa and NF1REPc) on chromosome 17 involved in the generation of NF1 microdeletions and in a third copy (REP19) on chromosome 19 from which the others originated over 6.7 million years ago. We find evidence for shared hotspots of recombination among the LCRs. REP19 seems to contain hotspots in the same place as the nonallelic recombination hotspots in NF1REPa and NF1REPc. This apparent conservation of patterns of recombination hotspots in moderately diverged paralogous regions contrasts with recent evidence that these patterns are not conserved in less-diverged orthologous regions of chimpanzees.

Type 2 NF1 Deletions Are Highly Unusual by Virtue of the Absence of Nonallelic Homologous Recombination Hotspots and an Apparent Preference for Female Mitotic Recombination

Approximately 5% of patients with neurofibromatosis type 1 (NF1) exhibit gross deletions that encompass the NF1 gene and its flanking regions. The breakpoints of the common 1.4-Mb (type 1) deletions are located within low-copy repeats (NF1-REPs) and cluster within a 3.4-kb hotspot of nonallelic homologous recombination (NAHR). Here, we present the first comprehensive breakpoint analysis of type 2 deletions, which are a second type of recurring NF1 gene deletion. Type 2 deletions span 1.2 Mb and are characterized by breakpoints located within the SUZ12 gene and its pseudogene, which closely flank the NF1-REPs. Breakpoint analysis of 13 independent type 2 deletions did not reveal any obvious hotspots of NAHR. However, an overrepresentation of polypyrimidine/polypurine tracts and triplex-forming sequences was noted in the breakpoint regions that could have facilitated NAHR. Intriguingly, all 13 type 2 deletions identified so far are characterized by somatic mosaicism, which indicates a positional preference for mitotic NAHR within the NF1 gene region. Indeed, whereas interchromosomal meiotic NAHR occurs between the NF1-REPs giving rise to type 1 deletions, NAHR during mitosis appears to occur intrachromosomally between the SUZ12 gene and its pseudogene, thereby generating type 2 deletions. Such a clear distinction between the preferred sites of mitotic versus meiotic NAHR is unprecedented in any other genomic disorder induced by the local genomic architecture. Additionally, 12 of the 13 mosaic type 2 deletions were found in females. The marked female preponderance among mosaic type 2 deletions contrasts with the equal sex distribution noted for type 1 and/or atypical NF1 deletions. Although an influence of chromatin structure was strongly suspected, no sex-specific differences in the methylation pattern exhibited by the SUZ12 gene were apparent that could explain the higher rate of mitotic recombination in females.

The NF1 gene contains hotspots for L1 endonuclease-dependent de novo insertion.

Long interspersed (L1) and Alu elements are actively amplified in the human genome through retrotransposition of their RNA intermediates by the ∼100 still retrotranspositionally fully competent L1 elements. Retrotransposition can cause inherited disease if such an element is inserted near or within a functional gene. Using direct cDNA sequencing as the primary assay for comprehensive NF1 mutation analysis, we uncovered in 18 unrelated index patients splicing alterations not readily explained at the genomic level by an underlying point-mutation or deletion. Improved PCR protocols avoiding allelic drop-out of the mutant alleles uncovered insertions of fourteen Alu elements, three L1 elements, and one poly(T) stretch to cause these splicing defects. Taken together, the 18 pathogenic L1 endonuclease-mediated de novo insertions represent the largest number of this type of mutations characterized in a single human gene. Our findings show that retrotransposon insertions account for as many as ∼0.4% of all NF1 mutations. Since altered splicing was the main effect of the inserted elements, the current finding was facilitated by the use of RNA–based mutation analysis protocols, resulting in improved detection compared to gDNA–based approaches. Six different insertions clustered in a relatively small 1.5-kb region (NF1 exons 21(16)–23(18)) within the 280-kb NF1 gene. Furthermore, three different specific integration sites, one of them located in this cluster region, were each used twice, i.e. NM_000267.3(NF1):c.1642-1_1642 in intron 14(10c), NM_000267.3(NF1):c.2835_2836 in exon 21(16), and NM_000267.3(NF1):c.4319_4320 in exon 33(25). Identification of three loci that each served twice as integration site for independent retrotransposition events as well as 1.5-kb cluster region harboring six independent insertions supports the notion of non-random insertion of retrotransposons in the human genome. Currently, little is known about which features make sites particularly vulnerable to L1 EN-mediated insertions. The here identified integration sites may serve to elucidate these features in future studies.

Review and update of SPRED1 mutations causing Legius syndrome.

Legius syndrome presents as a mild neurofibromatosis type 1 (NF1) phenotype. Multiple café‐au‐lait spots and macrocephaly are present with or without axillary or inguinal freckling. Other typical NF1‐associated features (Lisch nodules, bone abnormalities, neurofibromas, optic pathway gliomas, and malignant peripheral nerve sheath tumors) are systematically absent. Legius syndrome is caused by germline loss‐of‐function SPRED1 mutations, resulting in overactivation of the RAS–MAPK signal transduction cascade. The first families were identified in 2007. Here, we review all identified SPRED1 mutations and summarize molecular, clinical, and functional data. All mutations have been deposited in a database created using the Leiden Open Variation Database software and accessible at http://www.lovd.nl/SPRED1. At present, the database contains 89 different mutations identified in 146 unrelated probands, including 16 new variants described for the first time. The database contains a spectrum of mutations: 29 missense, 28 frameshift, 19 nonsense, eight copy number changes, two splicing, one silent, one in‐frame deletion and a mutation affecting the initiation codon. Sixty‐three mutations and deletions are definitely pathogenic or most likely pathogenic, eight SPRED1 mutations are probably benign rare variants, and 17 SPRED1 missense mutations are still unclassified and need further family and functional studies to help with the interpretation. Hum Mutat 33:1538–1546, 2012.

Rhabdomyosarcoma in patients with constitutional mismatch-repair-deficiency syndrome

Background: Biallelic germline mutations in the mismatch repair genes MLH1, MSH2, MSH6 or PMS2 cause a recessive childhood cancer syndrome characterised by early-onset malignancies and signs reminiscent of neurofibromatosis type 1 (NF1). Alluding to the underlying genetic defect, we refer to this syndrome as constitutional mismatch repair-deficiency (CMMR-D) syndrome. The tumour spectrum of CMMR-D syndrome includes haematological neoplasias, brain tumours and Lynch syndrome-associated tumours. Other tumours, such as neuroblastoma, Wilm tumour, ovarian neuroectodermal tumour or infantile myofibromatosis, have so far been found only in individual cases. Results: We analysed two consanguineous families that had members with suspected CMMR-D syndrome who developed rhabdomyosarcoma among other neoplasias. In the first family, we identified a pathogenic PMS2 mutation for which the affected patient was homozygous. In family 2, immunohistochemistry analysis showed isolated loss of PMS2 expression in all tumours in the affected patients, including rhabdomyosarcoma itself and the surrounding normal tissue. Together with the family history and microsatellite instability observed in one tumour this strongly suggests an underlying PMS2 alteration in family 2 also. Conclusion: Together, these two new cases show that rhabdomyosarcoma and possibly other embryonic tumours, such as neuroblastoma and Wilm tumour, belong to the tumour spectrum of CMMR-D syndrome. Given the clinical overlap of CMMR-D syndrome with NF1, we suggest careful examination of the family history in patients with embryonic tumours and signs of NF1 as well as analysis of the tumours for loss of one of the mismatch repair genes and microsatellite instability. Subsequent mutation analysis will lead to a definitive diagnosis of the underlying disorder.

GAB2 Is a Novel Target of 11q Amplification in AML/MDS

Chromosome arm 11q amplifications involving the mixed lineage leukemia gene (MLL) locus are rare but recurrent aberrations in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). We have recently shown that in addition to the MLL core amplicon, independent sequences in 11q23–24 and/or 11q13.5 are coamplified within the same cytogenetic markers in 90% and 60% of patients, respectively. Here we further narrow down the minimal amplicon in 11q13.5 to 1.17 Mb by means of semi‐quantitative PCR and FISH analyses. The newly defined amplicon contains seven genes, including the GRB2‐associated binding protein 2 (GAB2). Using real‐time RT‐PCR we show a significant transcriptional upregulation of GAB2 in the patients who have GAB2 coamplified with MLL. Thus, the adaptor molecule GAB2 that has already been shown to enhance oncogenic signaling in other neoplasias appears as a novel target of 11q amplification in AML/MDS.

A novel third type of recurrent NF1 microdeletion mediated by nonallelic homologous recombination between LRRC37B-containing low-copy repeats in 17q11.2†

Large microdeletions encompassing the neurofibromatosis type‐1 (NF1) gene and its flanking regions at 17q11.2 belong to the group of genomic disorders caused by aberrant recombination between segmental duplications. The most common NF1 microdeletions (type‐1) span 1.4‐Mb and have breakpoints located within NF1‐REPs A and C, low‐copy repeats (LCRs) containing LRRC37‐core duplicons. We have identified a novel type of recurrent NF1 deletion mediated by nonallelic homologous recombination (NAHR) between the highly homologous NF1‐REPs B and C. The breakpoints of these ∼1.0‐Mb (“type‐3”) NF1 deletions were characterized at the DNA sequence level in three unrelated patients. Recombination regions, spanning 275, 180, and 109‐bp, respectively, were identified within the LRRC37B‐P paralogues of NF1‐REPs B and C, and were found to contain sequences capable of non‐B DNA formation. Both LCRs contain LRRC37‐core duplicons, abundant and highly dynamic sequences in the human genome. NAHR between LRRC37‐containing LCRs at 17q21.31 is known to have mediated the 970‐kb polymorphic inversions of the MAPT‐locus that occurred independently in different primate species, but also underlies the syndromes associated with recurrent 17q21.31 microdeletions and reciprocal microduplications. The novel NF1 microdeletions reported here provide further evidence for the unusually high recombinogenic potential of LRRC37‐containing LCRs in the human genome. Hum Mutat 31:742–751, 2010.