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STUDY QUESTIONCan long-read amplicon sequencing be beneficial for preclinical preimplantation genetic testing (PGT) workup in couples with a de novo pathogenic variant in one of the prospective parents?SUMMARY ANSWERLong-read amplicon sequencing represents a simple, rapid and cost-effective preclinical PGT workup strategy that provides couples with de novo pathogenic variants access to universal genome-wide haplotyping-based PGT programs.WHAT IS KNOWN ALREADYUniversal PGT combines genome-wide haplotyping and copy number profiling to select embryos devoid of both familial pathogenic variants and aneuploidies. However, it cannot be directly applied in couples with a de novo pathogenic variant in one of the partners due to the absence of affected family members required for phasing the disease-associated haplotype.STUDY DESIGN, SIZE, DURATIONThis is a prospective study, which includes 32 families that were enrolled in the universal PGT program at the University Hospital of Leuven between 2018 and 2022. We implemented long-read amplicon sequencing during the preclinical PGT workup to deduce the parental origin of the disease-associated allele in the affected partner, which can then be traced in embryos during clinical universal PGT cycles.PARTICIPANTS/MATERIALS, SETTING, METHODSTo identify the parental origin of the disease-associated allele, genomic DNA from the carrier of the de novo pathogenic variant and his/her parent(s) was used for preclinical PGT workup. Primers flanking the de novo variant upstream and downstream were designed for each family. Following long-range PCR, amplicons that ranged 5–10 kb in size, were sequenced using Pacific Bioscience and/or Oxford Nanopore platforms. Next, targeted variant calling and haplotyping were performed to identify parental informative single-nucleotide variants (iSNVs) linked to the de novo mutation. Following the preclinical PGT workup, universal PGT via genome-wide haplotyping was performed for couples who proceeded with clinical PGT cycle. In parallel, 13 trophectoderm (TE) biopsies from three families that were analyzed by universal PGT, were also used for long-read amplicon sequencing to explore this approach for embryo direct mutation detection coupled with targeted long-read haplotyping.MAIN RESULTS AND THE ROLE OF CHANCEThe parental origin of the mutant allele was identified in 24/32 affected individuals during the preclinical PGT workup stage, resulting in a 75% success rate. On average, 5.95 iSNVs (SD = 4.5) were detected per locus of interest, and the average distance of closest iSNV to the de novo variant was ∼1750 bp. In 75% of those cases (18/24), the de novo mutation occurred on the paternal allele. In the remaining eight families, the risk haplotype could not be established due to the absence of iSNVs linked to the mutation or inability to successfully target the region of interest. During the time of the study, 12/24 successfully analyzed couples entered the universal PGT program, and three disease-free children have been born. In parallel to universal PGT analysis, long-read amplicon sequencing of 13 TE biopsies was also performed, confirming the segregation of parental alleles in the embryo and the results of the universal PGT.LIMITATIONS, REASONS FOR CAUTIONThe main limitation of this approach is that it remains targeted with the need to design locus-specific primers. Because of the restricted size of target amplicons, the region of interest may also remain non-informative in the absence of iSNVs.WIDER IMPLICATIONS OF THE FINDINGSTargeted haplotyping via long-read amplicon sequencing, particularly using Oxford Nanopore Technologies, provides a valuable alternative for couples with de novo pathogenic variants that allows access to universal PGT. Moreover, the same approach can be used for direct mutation analysis in embryos, as a second line confirmation of the preclinical PGT result or as a potential alternative PGT procedure in couples, where additional family members are not available.STUDY FUNDING/COMPETING INTEREST(S)This work was supported by KU Leuven funding (no. C1/018 to J.R.V.) and Fonds Wetenschappelijk Onderzoek (1241121N to O.T.). J.R.V. is co-inventor of a patent ZL910050-PCT/EP2011/060211-WO/2011/157846 ‘Methods for haplotyping single-cells’ and ZL913096-PCT/EP2014/068315-WO/2015/028576 ‘Haplotyping and copy number typing using polymorphic variant allelic frequencies’ licensed to Agilent Technologies. All other authors have no conflict of interest to declare.TRIAL REGISTRATION NUMBERN/A.

Supplementary Figure 1 from Glomus Tumors in Neurofibromatosis Type 1: Genetic, Functional, and Clinical Evidence of a Novel Association

Neurofibromatosis type 1 (NF1) is a common disorder that arises secondary to mutations in the tumor suppressor gene NF1. Glomus tumors are small, benign but painful tumors that originate from the glomus body, a thermoregulatory shunt concentrated in the fingers and toes. We report 11 individuals with NF1 who harbored 20 glomus tumors of the fingers and 1 in the toe; 5 individuals had multiple glomus tumors. We hypothesized that biallelic inactivation of NF1 underlies the pathogenesis of these tumors. In 12 NF1-associated glomus tumors, we used cell culture and laser capture microdissection to isolate DNA. We also analyzed two sporadic (not NF1-associated) glomus tumors. Genetic analysis showed germ line and somatic NF1 mutations in seven tumors. RAS mitogen-activated protein kinase hyperactivation was observed in cultured NF1−/− glomus cells, reflecting a lack of inhibition of the pathway by functional neurofibromin, the protein product of NF1. No abnormalities in NF1 or RAS mitogen-activated protein kinase activation were found in sporadic glomus tumors. By comparative genomic hybridization, we observed amplification of the 3′-end of CRTAC1 and a deletion of the 5′-end of WASF1 in two NF1-associated glomus tumors. For the first time, we show that loss of neurofibromin function is crucial in the pathogenesis of glomus tumors in NF1. Glomus tumors of the fingers or toes should be considered as part of the tumor spectrum of NF1. [Cancer Res 2009;69(18):7393–401]

Supplementary Figure Legend from Glomus Tumors in Neurofibromatosis Type 1: Genetic, Functional, and Clinical Evidence of a Novel Association

Supplementary Table 1 from Glomus Tumors in Neurofibromatosis Type 1: Genetic, Functional, and Clinical Evidence of a Novel Association

Supplementary Table 2 from Glomus Tumors in Neurofibromatosis Type 1: Genetic, Functional, and Clinical Evidence of a Novel Association

Genotype; Cancer phenotypes; Genetic tumour Genotip; Fenotips del càncer; Tumor genètic Genotipo; Fenotipos del cáncer; Tumor genético Background Truncating pathogenic or likely pathogenic variants of CDH1 cause hereditary diffuse gastric cancer (HDGC), a tumour risk syndrome that predisposes carrier individuals to diffuse gastric and lobular breast cancer. Rare CDH1 missense variants are often classified as variants of unknown significance. We conducted a genotype–phenotype analysis in families carrying rare CDH1 variants, comparing cancer spectrum in carriers of pathogenic or likely pathogenic variants (PV/LPV; analysed jointly) or missense variants of unknown significance, assessing the frequency of families with lobular breast cancer among PV/LPV carrier families, and testing the performance of lobular breast cancer-expanded criteria for CDH1 testing. Methods This genotype-first study used retrospective diagnostic and clinical data from 854 carriers of 398 rare CDH1 variants and 1021 relatives, irrespective of HDGC clinical criteria, from 29 institutions in ten member-countries of the European Reference Network on Tumour Risk Syndromes (ERN GENTURIS). Data were collected from Oct 1, 2018, to Sept 20, 2022. Variants were classified by molecular type and clinical actionability with the American College of Medical Genetics and Association for Molecular Pathology CDH1 guidelines (version 2). Families were categorised by whether they fulfilled the 2015 and 2020 HDGC clinical criteria. Genotype–phenotype associations were analysed by Student's t test, Kruskal-Wallis, χ2, and multivariable logistic regression models. Performance of HDGC clinical criteria sets were assessed with an equivalence test and Youden index, and the areas under the receiver operating characteristic curves were compared by Z test. Findings From 1971 phenotypes (contributed by 854 probands and 1021 relatives aged 1–93 years), 460 had gastric and breast cancer histology available. CDH1 truncating PV/LPVs occurred in 176 (21%) of 854 families and missense variants of unknown significance in 169 (20%) families. Multivariable logistic regression comparing phenotypes occurring in families carrying PV/LPVs or missense variants of unknown significance showed that lobular breast cancer had the greatest positive association with the presence of PV/LPVs (odds ratio 12·39 [95% CI 2·66–57·74], p=0·0014), followed by diffuse gastric cancer (8·00 [2·18–29·39], p=0·0017) and gastric cancer (7·81 [2·03–29·96], p=0·0027). 136 (77%) of 176 families carrying PV/LPVs fulfilled the 2015 HDGC criteria. Of the remaining 40 (23%) families, who did not fulfil the 2015 criteria, 11 fulfilled the 2020 HDGC criteria, and 18 had lobular breast cancer only or lobular breast cancer and gastric cancer, but did not meet the 2020 criteria. No specific CDH1 variant was found to predispose individuals specifically to lobular breast cancer, although 12 (7%) of 176 PV/LPV carrier families had lobular breast cancer only. Addition of three new lobular breast cancer-centred criteria improved testing sensitivity while retaining high specificity. The probability of finding CDH1 PV/LPVs in patients fulfilling the lobular breast cancer-expanded criteria, compared with the 2020 criteria, increased significantly (AUC 0·92 vs 0·88; Z score 3·54; p=0·0004). Interpretation CDH1 PV/LPVs were positively associated with HDGC-related phenotypes (lobular breast cancer, diffuse gastric cancer, and gastric cancer), and no evidence for a positive association with these phenotypes was found for CDH1 missense variants of unknown significance. CDH1 PV/LPVs occurred often in families with lobular breast cancer who did not fulfil the 2020 HDGC criteria, supporting the expansion of lobular breast cancer-centred criteria. Funding European Reference Network on Genetic Tumour Risk Syndromes, European Regional Development Fund, Fundação para a Ciência e a Tecnologia (Portugal), Cancer Research UK, and European Union's Horizon 2020 research and innovation programme. European Reference Network on Genetic Tumour Risk Syndromes, European Regional Development Fund, Fundação para a Ciência e a Tecnologia (Portugal), Cancer Research UK, and European Union's Horizon 2020 research and innovation programme.

In recent years, immune checkpoint inhibitors (ICPI) have become widely used for multiple solid malignancies. Reliable predictive biomarkers for selection of patients who would benefit most are lacking. Several tumor types with somatic or germline alterations in genes involved in the DNA damage response (DDR) pathway harbor a higher tumor mutational burden, possibly associated with an increased tumoral neoantigen load. These neoantigens are thought to lead to stronger immune activation and enhanced response to ICPIs. We present a series of seven patients with different malignancies with germline disease-associated variants in DDR genes (BRCA1, BRCA2, CHEK2) responding favorably to ICPIs. ispartof: Curr Oncol vol:28 issue:5 pages:3227-3239 ispartof: location:Switzerland status: published

Keratinocytic epidermal nevi (KEN) are characterized clinically by permanent hyperkeratosis in the distribution of Blaschko's lines and histologically by hyperplasia of epidermal keratinocytes. KEN with underlying RAS mutations have been associated with hypophosphatemic rickets and dysplastic bone lesions described as congenital cutaneous skeletal hypophosphatemia syndrome. Here, we describe two patients with keratinocytic epidermal nevi, in one associated with a papular nevus spilus, who presented with distinct localized congenital fibro-osseous lesions in the lower leg, diagnosed on both radiology and histology as osteofibrous dysplasia, in the absence of hypophosphatemia or rickets, or significantly raised FGF23 levels but with distinct mosaic HRAS mutations. This expands the spectrum of cutaneous/skeletal mosaic RASopathies and alerts clinicians to the importance of evaluating for bony disease even in the absence of bone profile abnormalities. ispartof: PEDIATRIC DERMATOLOGY vol:37 issue:5 pages:890-895 ispartof: location:United States status: published

BACKGROUND: Pathogenic PTEN germline variants cause PTEN Hamartoma Tumor Syndrome (PHTS), a rare disease with a variable genotype and phenotype. Knowledge about these spectra and genotype-phenotype associations could help diagnostics and potentially lead to personalized care. Therefore, we assessed the PHTS genotype and phenotype spectrum in a large cohort study. METHODS: Information was collected of 510 index patients with pathogenic or likely pathogenic (LP/P) PTEN variants (n = 467) or variants of uncertain significance. Genotype-phenotype associations were assessed using logistic regression analyses adjusted for sex and age. RESULTS: At time of genetic testing, the majority of children (n = 229) had macrocephaly (81%) or developmental delay (DD, 61%), and about half of the adults (n = 238) had cancer (51%), macrocephaly (61%), or cutaneous pathology (49%). Across PTEN, 268 LP/P variants were identified, with exon 5 as hotspot. Missense variants (n = 161) were mainly located in the phosphatase domain (PD, 90%) and truncating variants (n = 306) across all domains. A trend towards 2 times more often truncating variants was observed in adults (OR = 2.3, 95%CI = 1.5-3.4) and patients with cutaneous pathology (OR = 1.6, 95%CI = 1.1-2.5) or benign thyroid pathology (OR = 2.0, 95%CI = 1.1-3.5), with trends up to 2-4 times more variants in PD. Whereas patients with DD (OR = 0.5, 95%CI = 0.3-0.9) or macrocephaly (OR = 0.6, 95%CI = 0.4-0.9) had about 2 times less often truncating variants compared to missense variants. In DD patients these missense variants were often located in domain C2. CONCLUSION: The PHTS phenotypic diversity may partly be explained by the PTEN variant coding effect and the combination of coding effect and domain. PHTS patients with early-onset disease often had missense variants, and those with later-onset disease often truncating variants. ispartof: EUROPEAN JOURNAL OF MEDICAL GENETICS vol:65 issue:12 ispartof: location:Netherlands status: published

Background RASopathies are a group of disorders that result from mutations in genes coding for proteins involved in regulating the Ras-MAPK signaling pathway, and have an increased incidence of autism spectrum disorder (ASD). Legius syndrome is a rare RASopathy caused by loss-of-function mutations in the SPRED1 gene. The patient phenotype is similar to, but milder than, Neurofibromatosis type 1—another RASopathy caused by loss-of-function mutations in the NF1 gene. RASopathies exhibit increased activation of Ras-MAPK signaling and commonly manifest with cognitive impairments and ASD. Here, we investigated if a Spred1-/- mouse model for Legius syndrome recapitulates ASD-like symptoms, and whether targeting the Ras-MAPK pathway has therapeutic potential in this RASopathy mouse model. Methods We investigated social and communicative behaviors in Spred1-/- mice and probed therapeutic mechanisms underlying the observed behavioral phenotypes by pharmacological targeting of the Ras-MAPK pathway with the MEK inhibitor PD325901. Results Spred1-/- mice have robust increases in social dominance in the automated tube test and reduced adult ultrasonic vocalizations during social communication. Neonatal ultrasonic vocalization was also altered, with significant differences in spectral properties. Spred1-/- mice also exhibit impaired nesting behavior. Acute MEK inhibitor treatment in adulthood with PD325901 reversed the enhanced social dominance in Spred1-/- mice to normal levels, and improved nesting behavior in adult Spred1-/- mice. Limitations This study used an acute treatment protocol to administer the drug. It is not known what the effects of longer-term treatment would be on behavior. Further studies titrating the lowest dose of this drug that is required to alter Spred1-/- social behavior are still required. Finally, our findings are in a homozygous mouse model, whereas patients carry heterozygous mutations. These factors should be considered before any translational conclusions are drawn. Conclusions These results demonstrate for the first time that social behavior phenotypes in a mouse model for RASopathies (Spred1-/-) can be acutely reversed. This highlights a key role for Ras-MAPK dysregulation in mediating social behavior phenotypes in mouse models for ASD, suggesting that proper regulation of Ras-MAPK signaling is important for social behavior.

Congenital melanocytic nevus syndrome (CMNS) is a rare condition characterized by pigmented skin lesions that is usually present at birth and is associated with an increased risk of neurological abnormalities and malignant melanoma. It mostly results from a post-zygotic NRAS mutation of neural-derived crest cells, leading to an uncontrolled cell growth. Due to the increased knowledge of the genetics underlying CMNS, targeted therapy becomes a promising treatment option. We present a case of CMNS in a newborn. Physical examination at birth showed a giant congenital melanocytic nevus, extending from the occipital to the lower lumbar region. An MRI-scan revealed multiple cerebral and cerebellar parenchymal lesions. Genetic analysis of the cutaneous lesions showed the presence of a NRAS Q61R mutation. The patient was treated with dermabrasion to reduce the color intensity of the nevus. However, this was complicated with recurrent wound infections and laborious wound healing. At the age of 1 year, the patient had an age-appropriate psychomotor development, without neurological deficits. This article is protected by copyright. All rights reserved. ispartof: Journal Of Cutaneous Pathology vol:48 issue:12 pages:1497-1503 ispartof: location:United States status: Published online

Background: The introduction of multi-gene panel testing and improved awareness under patients and physicians has led to an increase of individuals with known germline pathogenic variants in hereditary breast and ovarian cancer (HBOC) genes. Significant regional differences exist in germline mutational landscape. We aimed to report the findings from multi-gene panel testing in a large Belgian cohort of individuals at risk for HBOC. Methods: All individuals who underwent multi-gene panel testing for HBOC at the Center for Human Genetics of the University Hospitals Leuven since the introduction of the panel were included (March 2016-April 2019). All included individuals were considered candidates for HBOC-panel testing by the requesting physician based on a personal or familial history of breast and/or ovarian cancer. Testing criteria from the Belgian Society of Human Genetics (www.beshg.be/download/guidelines/Guidelines_HBOC_2018.pdf) were met in the vast majority. The panel used was the BRCA Hereditary Cancer MASTR Plus® (Agilent, Belgium), with sequencing of BARD1, BRCA1, BRCA2, BRIP1, RAD51C, RAD51D, TP53, MRE11A, RAD50, NBN, FAM175A, ATM, PALB2, STK11, MEN1, PTEN, CDH1, MUTYH, CHEK2, BLM, XRCC2, EPCAM, MLH1, MSH6, PMS2 and MSH2. Sequencing was performed by NGS on a Miseq platform (Illumina). Genomic deletions and duplications in BRCA1 and BRCA2 were investigated with multiplex ligation-dependent probe amplification. We hereby report on the frequency of pathogenic and likely pathogenic germline variants in this population. Results: In 5422 individuals who underwent multi-gene panel testing, we detected 665 pathogenic or likely pathogenic variants in 639 patients (11,7%). In 25 patients (0.46%), more than one relevant alteration was detected with double heterozygosity in 24 individuals and triple heterozygosity in one. Germline variants in BRCA1 and BRCA2 were detected in 178 (3.3%) and 144 (2.7%) patients, resulting in a fraction of 26,4% and 21,4% of detected variants respectively. Relevant alterations in CHEK2, ATM, PALB2 and TP53 were observed in 135 (2.5%), 93 (1.7%), 26 (0.5%) and 11 (0.2%) patients respectively, accounting together for 39.3% of detected variants. Alterations in BRIP1/RAD51C/RAD51D were retrieved in 64 patients (1.2%) and alterations in mismatch-repair genes MSH6/MLH1/PMS2/MSH2 were detected in 0.3% of patients. These patients where dominantly referred for familial history of ovarian cancer. Furthermore, germline alterations in PTEN, CDH1 and BLM were observed in 3, 2 and 1 cases respectively. Double heterozygosity for ATM+CHEK2 and for ATM+BRCA2 were both observed in 3 cases. In the patient with triple heterozygosity, co-occurrence of pathogenic variants in BRCA2, ATM and CHEK2 was detected. Conclusions: In a large Belgian cohort of 5422 individuals at risk for HBOC who underwent multi-gene panel testing, a pathogenic or likely pathogenic germline variant was detected in 11,7% of patients, and in 0,46% of patients double or triple heterozygosity for HBOC-variants was observed. Almost 40% of detected variants were alterations beyond BRCA correlated with hereditary breast cancer (CHEK2, ATM, PALB2 and TP53). Given the time-lag to predictive testing in families, a significant rise in healthy carriers with these non-BRCA alterations is expected in the upcoming years. Citation Format: Kevin Punie, Griet Hoste, Griet Van Buggenhout, Ellen Denayer, Hilde Brems, Hilde Peeters, Ann Smeets, Ines Nevelsteen, Patrick Neven, Jan Ardui, Renate Prevos, Machteld Keupers, Chantal Van Ongeval, Giuseppe Floris, Christine Desmedt, Hans Wildiers, Geneviève Michils, Hilde Van Esch, Eric Legius. Germline mutational landscape in 5422 individuals at risk for hereditary breast and ovarian cancer who underwent multi-gene panel testing [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-08-03.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder affecting approximately 1 in 2,000 newborns. Up to 5% of NF1 patients suffer from pseudarthrosis of a long bone (NF1-PA). Current treatments are often unsatisfactory, potentially leading to amputation. To gain more insight into the pathogenesis we cultured cells from PA tissue and normal-appearing periosteum of the affected bone for NF1 mutation analysis. PA cells were available from 13 individuals with NF1. Biallelic NF1 inactivation was identified in all investigated PA cells obtained during the first surgery. Three of five cases sampled during a later intervention showed biallelic NF1 inactivation. Also, in three individuals, we examined periosteum-derived cells from normal-appearing periosteum proximal and distal to the PA. We identified the same biallelic NF1 inactivation in the periosteal cells outside the PA region. These results indicate that NF1 inactivation is required but not sufficient for the development of NF1-PA. We observed that late-onset NF1-PA occurs and is not always preceded by congenital bowing. Furthermore, the failure to identify biallelic inactivation in two of five later interventions and one reintervention with a known somatic mutation indicates that NF1-PA can persist after the removal of most NF1 negative cells.

The patient, a boy born in 1991, showed pronounced polyostotic fibrous dysplasia due to McCune–Albright syndrome, as well as Gilbert syndrome and Charcot–Marie–Tooth neuropathy caused by a DNM2 mutation. In addition, the patient, his sister, mother and maternal grandfather had intermittently increased plasma arginine and lysine levels, most probably due to heterozygosity for a novel pathogenic SLC7A2 variant.

RASopathies are neuro-cardio-facio-cutaneous disorders stemming from mutations in genes regulating the RAS-MAPK pathway. Legius syndrome is a rare RASopathy disorder caused by mutations in the SPRED1 gene. SPRED1 protein negatively regulates activation of Ras by inhibiting RAS/RAF and by its interaction with neurofibromin, a Ras GTPase-activating protein (RAS-GAP). Cognitive impairments have been reported in Legius syndrome as well as in other RASopathy disorders. Modelling these cognitive deficits in a Spred1 mouse model for Legius syndrome has demonstrated spatial learning and memory deficits, but other cognitive domains remained unexplored. Here, we attempted to utilize a cognitive touchscreen battery to investigate if Spred1-/- mice exhibit deficits in other cognitive domains. We show that Spred1-/- mice had heterogeneous performance in instrumental operant learning, with a large subgroup (n = 9/20) failing to reach the standard criterion on touchscreen operant pretraining, precluding further cognitive testing. To examine whether targeting the RAS-MAPK signalling pathway could rescue these cognitive impairments, Spred1-/- mice were acutely treated with the clinically relevant mitogen-activated protein kinase (MEK) inhibitor PD325901. However, MEK inhibition did not improve their instrumental learning. We conclude that Spred1-/- mice can model severe cognitive impairments that cannot be reversed in adulthood.

OBJECTIVES: To apply the Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE) to a consecutive series of endometrial cancer (EC) patients diagnosed at a tertiary referral center and assign EC specimens to one of four molecular subgroups using immunohistochemistry (IHC) for p53/mismatch repair protein expression and sequencing for Polymerase Epsilon Exonuclease Domain Mutations (POLE-EDM). Mismatch Repair Deficient (MMR-D) cases were more thoroughly investigated to identify underlying somatic or germline genetic defects. METHODS: Hundred-and eight consecutive endometrial cancer patients, diagnosed between March 2017 and April 2019, were subjected to immunohistochemical and molecular analysis, according to ProMisE. IHC for p53 and the mismatch repair proteins (MLH1, PMS2, MSH6 and PMS2) was performed. All patients were also tested for POLE-EDM by Sanger sequencing. In addition, tumor and corresponding normal tissue of cases with abnormal MMR IHC were tested by PCR for microsatellite instability (MSI) (MSI analysis system, Promega). Hypermethylation of MLH1 promotor was tested with (methylation specific) multiplex ligation dependent probe amplification. MMR-D cases were subjected to germline mutation analysis of the mismatch repair genes, using next generation sequencing on MiSeq (Illumina) with the BRCA Hereditary Cancer MASTR Plus, (Multiplicom/Agilent), RNA mutation analysis and MLPA. RESULTS: FIGO classification was stage IA (n = 54), IB (n = 22) II(n = 8), III(n = 18) and IV(n = 6). Of the 33 patients with MMR-D on IHC (31%), 26 showed MLH1 promotor hypermethylation as the probable cause of MMR-D. The remaining 7 patients without MLH1 promotor hypermethylation were referred for germline analysis of Lynch syndrome. Six patients carried a pathogenic germline mutation in one of the mismatch repair genes: MSH6(n = 3), PMS2(n = 1), MLH1(n = 1) and MSH2 (n = 1). Pathogenic POLE-EDM were identified in 7 (6%) patients. Multiple molecular features (POLE-EDM + MMR-D or POLE-EDM + p53 abnormal) were observed in 4 patients (4%). A high concordance between MMR-D and microsatellite instability was observed in our cohort. In cases of a genetic defect in the MMR genes, we do note a large proportion of cases exhibiting microsatellite instability. On the contrary a hypermutation state, as seen in POLE EDM, does not result in accompanied phenotypic changes in MSI status. CONCLUSION: The ProMisE classification proved to be an efficient and easily implementable system. Future research should elucidate the precise biological and prognostic meaning of the cases with multiple molecular markers. ispartof: GYNECOLOGIC ONCOLOGY vol:157 issue:1 pages:245-251 ispartof: location:United States status: published

PURPOSE: Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder associated with cognitive deficits. The NF1 cognitive phenotype is generally considered to be highly variable, possibly due to the observed T2-weighted hyperintensities, loss of heterozygosity, NF1-specific genetic modifiers, or allelic imbalance. METHODS: We investigated cognitive variability and assessed the contribution of genetic factors by performing a retrospective cohort study and a monozygotic twin case series. We included data of 497 children with genetically confirmed NF1 and an IQ assessment, including 12 monozygotic twin and 17 sibling sets. RESULTS: Individuals carrying an NF1 chromosomal microdeletion showed significant lower full-scale IQ (FSIQ) scores than individuals carrying intragenic pathogenic NF1 variants. For the intragenic subgroup, the variability in cognitive ability and the correlation of IQ between monozygotic NF1 twin pairs or between NF1 siblings is similar to the general population. CONCLUSIONS: The variance and heritability of IQ in individuals with NF1 are similar to that of the general population, and hence mostly driven by genetic background differences. The only factor that significantly attenuates IQ in NF1 individuals is the NF1 chromosomal microdeletion genotype. Implications for clinical management are that individuals with intragenic NF1 variants that score

The mechanical properties of the microenvironment have been increasingly accepted as potent modulators of cell behavior and function in physiological and pathological conditions. In tumors, tissue rigidity has been recognized as a potent driver of both neoplastic initiation from healthy tissue and progression toward malignancy. Several benign or malignant tumors are fibrotic and characterized by increased deposition, turnover, and posttranslational modifications of the extracellular matrix (ECM) that progressively stiffens the stroma. Likewise, neurofibromas are complex tumors composed of transformed Schwann cells, immersed in a heterogeneous fibrotic matrix. The molecular crosstalks that govern neurofibroma onset, growth, invasiveness, and progression toward malignancy remain poorly understood. In this chapter we propose that neurofibroma development requires the integration of potent pro-tumorigenic circuitries, such as those mastered by hyperactive Ras, and altered mechanosignaling axes conveyed by a stiff ECM, and that these pathways could be orchestrated by a pro-inflammatory extracellular milieu.

Children with neurofibromatosis type 1 (NF1) may exhibit an incomplete clinical presentation, making difficult to reach a clinical diagnosis. A phenotypic overlap may exist in children with other RASopathies or with other genetic conditions if only multiple café-au-lait macules (CALMs) are present. The syndromes that can converge in these inconclusive phenotypes have different clinical courses. In this context, an early genetic testing has been proposed to be clinically useful to manage these patients. We present the validation and implementation into diagnostics of a custom NGS panel (I2HCP, ICO-IMPPC Hereditary Cancer Panel) for testing patients with a clinical suspicion of a RASopathy (n = 48) and children presenting multiple CALMs (n = 102). We describe the mutational spectrum and the detection rates identified in these two groups of individuals. We identified pathogenic variants in 21 out of 48 patients with clinical suspicion of RASopathy, with mutations in NF1 accounting for 10% of cases. Furthermore, we identified pathogenic mutations mainly in the NF1 gene, but also in SPRED1, in more than 50% of children with multiple CALMs, exhibiting an NF1 mutational spectrum different from a group of clinically diagnosed NF1 patients (n = 80). An NGS panel strategy for the genetic testing of these two phenotype-defined groups outperforms previous strategies.

Background Neurofibromatosis 1 (NF1) leads to the development of benign and malignant peripheral nerve sheath tumors (MPNST). MPNST have been described to develop in preexisting benign plexiform neurofibromas (PN) and have a poor prognosis. Atypical neurofibromas (ANF) were recently described as precursor lesions for MPNST, making early detection and management of ANF a possible strategy to prevent MPNST. We aimed to clinically characterize ANF and identify management approaches. Methods We analyzed clinical, imaging, and pathology findings of all patients with NF1 and ANF at 3 institutions. Results Sixty-three patients had 76 ANF (32M/31F; median age 27.1 y). On MRI, most ANF appeared as distinct nodular lesions and were 18F-fluorodeoxyglucose (FDG) avid. Forty-six ANF were associated with pain, 19 with motor weakness, 45 were palpable or visible, and 13 had no clinical signs. Completely resected ANF (N = 57) have not recurred (median follow-up, 4.1 y; range, 0-14 y). Four ANF transformed into MPNST and 17 patients had a history of MPNST in a different location than was their ANF. Conclusions Growth of distinct nodular lesions, pain, and FDG-PET avidity should raise concern for ANF in NF1. Patients with ANF are at greater risk for development of MPNST. Complete resection of ANF may prevent development of MPNST.

The best known cafe-au-lait syndrome is neurofibromatosis type 1 (NF1). Legius syndrome (LS) is another, rarer syndrome with cafe-au-lait macules (CALMs). In young patients their clinical picture is often indistinguishable. We investigated the presence of choroidal abnormalities in syndromes with CALMs as a candidate tool for a more efficient diagnosis. Thirty-four patients with NF1 (14 with a truncating mutation, 14 with a non-truncating mutation and 6 with unknown mutation) and 11 patients with LS. All patients underwent an ophthalmological examination. Infrared images were performed. Choroidal nodules were diagnosed in 65% of the NF1 group. About 71% of NF1 patients with a truncating mutation and 50% of patients with a non-truncating mutation were found to have nodules. Choroidal nodules were seen in 18% of the LS patients, never more than one nodule/eye was detected in this group. Choroidal nodules are more abundantly present in NF1 genotypes with truncating mutations. In contrast, the number of choroidal nodules in LS is comparable with their presence in healthy individuals. Especially at an early age, when the clinical picture is incomplete, the detection of choroidal nodules is of diagnostic value, and helps in an appropriate genetic counselling and follow-up. These results support the suggestion to include choroidal nodules to the diagnostic criteria for NF1.

In Neurofibromatosis type 1, NF1 gene mutations in Schwann cells (SC) drive benign plexiform neurofibroma (PNF), and no additional SC changes explain patient-to-patient variability in tumor number. Evidence from twin studies suggests that variable expressivity might be caused by unidentified modifier genes. Whole exome sequencing of SC and fibroblast DNA from the same resected PNFs confirmed biallelic SC NF1 mutations; non-NF1 somatic SC variants were variable and present at low read number. We identified frequent germline variants as possible neurofibroma modifier genes. Genes harboring variants were validated in two additional cohorts of NF1 patients and by variant burden test. Genes including CUBN, CELSR2, COL14A1, ATR and ATM also showed decreased gene expression in some neurofibromas. ATM-relevant DNA repair defects were also present in a subset of neurofibromas with ATM variants, and in some neurofibroma SC. Heterozygous ATM G2023R or homozygous S707P variants reduced ATM protein expression in heterologous cells. In mice, genetic Atm heterozygosity promoted Schwann cell precursor self-renewal and increased tumor formation in vivo, suggesting that ATM variants contribute to neurofibroma initiation. We identify germline variants, rare in the general population, overrepresented in NF1 patients with neurofibromas. ATM and other identified genes are candidate modifiers of PNF pathogenesis.

Constitutional heterozygous loss-of-function mutations in the SPRED1 gene cause a phenotype known as Legius syndrome, which consists of symptoms of multiple cafe-au-lait macules, axillary freckling, learning disabilities and macrocephaly. Legius syndrome resembles a mild neurofibromatosis type 1 (NF1) phenotype. It has been demonstrated that SPRED1 functions as a negative regulator of the RAS-ERK pathway and interacts with neurofibromin, the NF1 gene product. However, the molecular details of this interaction and the effects of the mutations identified in Legius syndrome and NF1 on this interaction have not yet been investigated. In this study, using a yeast two-hybrid system and an immunoprecipitation assay in HEK293 cells, we found that the SPRED1 EVH1 domain interacts with the N-terminal 16 amino acids (aa) and the C-terminal 20 aa of the GTPase-Activating Protein (GAP)-related domain (GRD) of neurofibromin, which form two crossing α-helix coils outside the GAP domain. These regions have been shown to be dispensable for GAP activity and are not present in p120GAP. Several mutations in these N- and C-terminal regions of the GRD in NF1 patients and pathogenic missense mutations in the EVH1 domain of SPRED1 in Legius syndrome reduced the binding affinity between the EVH1 domain and the GRD. EVH1 domain mutations with reduced binding to the GRD also disrupted the ERK suppression activity of SPRED1. These data clearly demonstrate that SPRED1 inhibits the Ras-ERK pathway by recruiting neurofibromin to Ras through the EVH1-GRD interaction, and this study also provides molecular basis for the pathogenic mutations of NF1 and Legius syndrome. ispartof: Journal of Biological Chemistry vol:291 issue:7 pages:3124-34 ispartof: location:United States status: published

Regulation of RAS by ubiquitination The protein LZTR1 is mutated in human cancers and developmental diseases. Work from two groups now converges to implicate the protein in regulating signaling by the small guanosine triphosphatase RAS. Steklov et al. showed that mice haploinsufficient for LZTR1 recapitulated aspects of the human disease Noonan syndrome. Their biochemical studies showed that LZTR1 associated with RAS. LZTR1 appears to function as an adaptor that promotes ubiquitination of RAS, thus inhibiting its signaling functions. Bigenzahn et al. found LZTR1 in a screen for proteins whose absence led to resistance to the tyrosine kinase inhibitors used to treat cancers caused by the BCR-ABL oncogene product. Their biochemical studies and genetic studies in fruitflies also showed that loss of LZTR1 led to increased activity of RAS and signaling through the mitogen-activated protein kinase pathway. Science , this issue p. 1177 , p. 1171

“Jaffe–Campanacci syndrome” describes the complex of multiple nonossifying fibromas of the long bones, mandibular giant cell lesions, and cafe-au-lait macules in individuals without neurofibromas. We sought to determine whether Jaffe–Campanacci syndrome is a distinct genetic entity or a variant of neurofibromatosis type 1. We performed germline NF1, SPRED1, and GNAS1 (exon 8) mutation testing on patients with Jaffe–Campanacci syndrome or Jaffe–Campanacci syndrome–related features. We also performed somatic NF1 mutation testing on nonossifying fibromas and giant cell lesions. Pathogenic germline NF1 mutations were identified in 13 of 14 patients with multiple cafe-au-lait macules and multiple nonossifying fibromas or giant cell lesions (“classical” Jaffe–Campanacci syndrome); all 13 also fulfilled the National Institutes of Health diagnostic criteria for neurofibromatosis type 1. Somatic NF1 mutations were detected in two giant cell lesions but not in two nonossifying fibromas. No SPRED1 or GNAS1 (exon 8) mutations were detected in the seven NF1-negative patients with Jaffe–Campanacci syndrome, nonossifying fibromas, or giant cell lesions. In this study, the majority of patients with cafe-au-lait macules and nonossifying fibromas or giant cell lesions harbored a pathogenic germline NF1 mutation, suggesting that many Jaffe–Campanacci syndrome cases may actually have neurofibromatosis type 1. We provide the first proof of specific somatic second-hit mutations affecting NF1 in two giant cell lesions from two unrelated patients, establishing these as neurofibromatosis type 1–associated tumors. Genet Med 16 6, 448–459.

Multiple café-au-lait macules (CALMs) are the hallmark of Von Recklinghausen disease, or neurofibromatosis type 1 (NF1). In 2007 we reported that some individuals with multiple CALMs have a heterozygous mutation in the SPRED1 gene and have NF1-like syndrome, or Legius syndrome. Individuals with Legius syndrome have multiple CALMs with or without freckling, but they do not show the typical NF1-associated tumors such as neurofibromas or optic pathway gliomas. NF1-associated bone abnormalities and Lisch nodules are also not reported in patients with Legius syndrome. Consequently, individuals with Legius syndrome require less intense medical surveillance than those with NF1. The SPRED1 gene was identified in 2001 and codes for a protein that downregulates the RAS-mitogen activated protein kinase (RAS-MAPK) pathway; as does neurofibromin, the protein encoded by the NF1 gene. It is estimated that about 1-4% of individuals with multiple CALMs have a heterozygous SPRED1 mutation. Mutational and clinical data on 209 patients with Legius syndrome are tabulated in an online database (http://www.lovd.nl/SPRED1). Mice with homozygous knockout of the Spred1 gene show learning deficits and decreased synaptic plasticity in hippocampal neurons similar to those seen in Nf1 heterozygous mice, underlining the importance of the RAS-MAPK pathway for learning and memory. Recently, specific binding between neurofibromin and SPRED1 was demonstrated. SPRED1 seems to play an important role in recruiting neurofibromin to the plasma membrane.

Congenital pseudarthrosis of the tibia (CPT) is a rare disease which normally presents itself during early childhood by anterolateral bowing of the tibia and spontaneous tibial fractures. Although the exact etiology of CPT is highly debated, 40–80% of CPT patients are carriers of a mutation in the Neurofibromatosis Type 1 (NF1) gene, which can potentially result in an altered phenotype of the skeletal cells and impaired bone healing. In this study we use a computational model of bone regeneration to examine the effect of the Nf1 mutation on bone fracture healing by altering the parameter values of eight key factors which describe the aberrant cellular behaviour of Nf1 haploinsufficient and Nf1 bi-allelically inactivated cells. We show that the computational model is able to predict the formation of a hamartoma as well as a wide variety of CPT phenotypes through different combinations of altered parameter values. A sensitivity analysis by “Design of Experiments” identified the impaired endochondral ossification process and increased infiltration of fibroblastic cells as key contributors to the degree of severity of CPT. Hence, the computational model results have added credibility to the experimental hypothesis of a genetic cause (i.e. Nf1 mutation) for CPT.

Neurofibromatosis type 1 (NF1) is a common, autosomal dominant, tumor-predisposition syndrome that arises secondary to mutations in NF1. Glomus tumors are painful benign tumors that originate from the glomus body in the fingers and toes due to biallelic inactivation of NF1. We karyotyped cultures from four previously reported and one new glomus tumor and hybridized tumor (and matching germline) DNA on Illumina HumanOmni1-Quad SNP arrays (∼1 × 10(6) SNPs). Two tumors displayed evidence of copy-neutral loss of heterozygosity of chromosome arm 17q not observed in the germline sample, consistent with a mitotic recombination event. One of these two tumors, NF1-G12, featured extreme polyploidy (near-tetraploidy, near-hexaploidy, or near-septaploidy) across all chromosomes. In the remaining four tumors, there were few cytogenetic abnormalities observed, and copy-number analysis was consistent with diploidy in all chromosomes. This is the first study of glomus tumors cytogenetics, to our knowledge, and the first to report biallelic inactivation of NF1 secondary to mitotic recombination of chromosome arm 17q in multiple NF1-associated glomus tumors. We have observed mitotic recombination in 22% of molecularly characterized NF1-associated glomus tumors, suggesting that it is a not uncommon mechanism in the reduction to homozygosity of the NF1 germline mutation in these tumors. In tumor NF1-G12, we hypothesize that mitotic recombination also "unmasked" (reduced to homozygosity) a hypomorphic germline allele in a gene on chromosome arm 17q associated with chromosomal instability, resulting in the extreme polyploidy. © 2012 Wiley Periodicals, Inc. ispartof: Genes, Chromosomes & Cancer vol:51 issue:5 pages:429-437 ispartof: location:United States status: published

Neurofibromatosis type-1 (NF1), caused by heterozygous inactivation of the NF1 tumour suppressor gene, is associated with the development of benign and malignant peripheral nerve sheath tumours (MPNSTs). Although numerous germline NF1 mutations have been identified, relatively few somatic NF1 mutations have been described in neurofibromas. Here we have screened 109 cutaneous neurofibromas, excised from 46 unrelated NF1 patients, for somatic NF1 mutations. NF1 mutation screening (involving loss-of-heterozygosity (LOH) analysis, multiplex ligation-dependent probe amplification and DNA sequencing) identified 77 somatic NF1 point mutations, of which 53 were novel. LOH spanning the NF1 gene region was evident in 25 neurofibromas, but in contrast to previous data from MPNSTs, it was absent at the TP53, CDKN2A and RB1 gene loci. Analysis of DNA/RNA from neurofibroma-derived Schwann cell cultures revealed NF1 mutations in four tumours whose presence had been overlooked in the tumour DNA. Bioinformatics analysis suggested that four of seven novel somatic NF1 missense mutations (p.A330T, p.Q519P, p.A776T, p.S1463F) could be of functional/clinical significance. Functional analysis confirmed this prediction for p.S1463F, located within the GTPase-activating protein-related domain, as this mutation resulted in a 150-fold increase in activated GTP-bound Ras. Comparison of the relative frequencies of the different types of somatic NF1 mutation observed with those of their previously reported germline counterparts revealed significant (P=0.001) differences. Although non-identical somatic mutations involving either the same or adjacent nucleotides were identified in three pairs of tumours from the same patients (P

Neurofibromatosis type 1 (NF1) is a common disorder that arises secondary to mutations in the tumor suppressor gene NF1. Glomus tumors are small, benign but painful tumors that originate from the glomus body, a thermoregulatory shunt concentrated in the fingers and toes. We report 11 individuals with NF1 who harbored 20 glomus tumors of the fingers and 1 in the toe; 5 individuals had multiple glomus tumors. We hypothesized that biallelic inactivation of NF1 underlies the pathogenesis of these tumors. In 12 NF1-associated glomus tumors, we used cell culture and laser capture microdissection to isolate DNA. We also analyzed two sporadic (not NF1-associated) glomus tumors. Genetic analysis showed germ line and somatic NF1 mutations in seven tumors. RAS mitogen-activated protein kinase hyperactivation was observed in cultured NF1−/− glomus cells, reflecting a lack of inhibition of the pathway by functional neurofibromin, the protein product of NF1. No abnormalities in NF1 or RAS mitogen-activated protein kinase activation were found in sporadic glomus tumors. By comparative genomic hybridization, we observed amplification of the 3′-end of CRTAC1 and a deletion of the 5′-end of WASF1 in two NF1-associated glomus tumors. For the first time, we show that loss of neurofibromin function is crucial in the pathogenesis of glomus tumors in NF1. Glomus tumors of the fingers or toes should be considered as part of the tumor spectrum of NF1. [Cancer Res 2009;69(18):7393–401]

Background Keratinocytic epidermal nevus syndrome (KENS) is a complex disorder not only characterized by the presence of epidermal nevi but also by abnormalities in the internal organ systems. A small number of cases with KENS are molecularly characterized and reported in the literature with somatic activating RAS, FGFR3 and PIK3CA mutations. Case presentation In this study we present a patient with hyper- and hypopigmented regions, verrucous pigmented skin lesions and a paravertebral conglomerate tumour at the level of the cervical and thoracic spine. A large lipomatous dumbbell tumour caused atrophy of the spinal cord with progressive paraparesis. We identified a mosaic c.35G > A (p.Gly12Asp) KRAS mutation in the pigmented verrucous epidermal nevus tissue, the intraneural schwann cells and the lipoma. The c.35G > A (p.Gly12Asp) KRAS mutation was absent in the peripheral blood leukocytes. Conclusion We conclude that KENS, the intraneural Schwann cell proliferation and the lipoma in this individual were caused by a postzygotic and mosaic activating c.35G > A (p.Gly12Asp) KRAS mutation.

Loss-of-function mutations in the NF1 tumor suppressor gene underlie the familial cancer syndrome neurofibromatosis type I (NF1). The NF1 -encoded protein, neurofibromin, functions as a Ras-GTPase activating protein (RasGAP). Accordingly, deregulation of Ras is thought to contribute to NF1 development. However, the critical effector pathways involved in disease pathogenesis are still unknown. We show here that the mTOR pathway is tightly regulated by neurofibromin. mTOR is constitutively activated in both NF1 -deficient primary cells and human tumors in the absence of growth factors. This aberrant activation depends on Ras and PI3 kinase, and is mediated by the phosphorylation and inactivation of the TSC2 -encoded protein tuberin by AKT. Importantly, tumor cell lines derived from NF1 patients, and a genetically engineered cell system that requires Nf1 -deficiency for transformation, are highly sensitive to the mTOR inhibitor rapamycin. Furthermore, while we show that the activation of endogenous Ras leads to constitutive mTOR signaling in this disease state, we also demonstrate that in normal cells Ras is differentially required for mTOR signaling in response to various growth factors. Thus, these findings identify the NF1 tumor suppressor as an indispensable regulator of TSC2 and mTOR. Furthermore, our results also demonstrate that Ras plays a critical role in the activation of mTOR in both normal and tumorigenic settings. Finally, these data suggest that rapamycin, or its derivatives, may represent a viable therapy for NF1.

Neurofibromatosis type 1 (NF1) patients have an 8-13% lifetime risk of developing malignant peripheral nerve sheath tumors (MPNST) which have a very poor prognosis. In this study, cells from eight MPNSTs (six primary and two recurrences) of six clinically and genetically well-characterized NF1 patients were taken into culture. Tracing of loss of heterozygosity (LOH) of the NF1, p53, and p16 gene regions or of abnormal karyotypes enabled identification of tumor cells from five MPNSTs. In two other MPNST-derived cell cultures, LOH of the relevant regions in the original tumors could not be detected, indicating that the obtained cells were nonneoplastic cells. Cells from most MPNSTs grew only under standard culture conditions but not under conditions optimized for Schwann cells. These cells were S100-negative and did not exhibit spindle shape which is a characteristic of Schwann cells. Drastically increased proliferation rates were found for most of the MPNST cells in comparison to Schwann cells derived from benign neurofibromas. Our study demonstrates that genetic analysis is effective and essential for verification of MPNST tumor cells in culture. These verified MPNST cells are valuable for further investigations of the biology and pathogenesis of this malignancy as well as for in vitro pharmacologic studies essential for the development of new therapies.

The polycomb repressive complex 2 (PRC2) exerts oncogenic effects in many tumour types1. However, loss-of-function mutations in PRC2 components occur in a subset of haematopoietic malignancies, suggesting that this complex plays a dichotomous and poorly understood role in cancer2, 3. Here we provide genomic, cellular, and mouse modelling data demonstrating that the polycomb group gene SUZ12 functions as tumour suppressor in PNS tumours, high-grade gliomas and melanomas by cooperating with mutations in NF1. NF1 encodes a Ras GTPase-activating protein (RasGAP) and its loss drives cancer by activating Ras4. We show that SUZ12 loss potentiates the effects of NF1 mutations by amplifying Ras-driven transcription through effects on chromatin. Importantly, however, SUZ12 inactivation also triggers an epigenetic switch that sensitizes these cancers to bromodomain inhibitors. Collectively, these studies not only reveal an unexpected connection between the PRC2 complex, NF1 and Ras, but also identify a promising epigenetic-based therapeutic strategy that may be exploited for a variety of cancers.

Neurofibromatosis type 1 is a common autosomal dominant disorder caused by mutations of the NF1 gene on chromosome 17. In only 5%-10% of cases, a microdeletion including the NF1 gene is found. We analyzed a set of polymorphic dinucleotide-repeat markers flanking the microdeletion on chromosome 17 in a group of seven unrelated families with a de novo NF1 microdeletion. Six of seven microdeletions were of maternal origin. The breakpoints of the microdeletions of maternal origin were localized in flanking paralogous sequences, called "NF1-REPs." The single deletion of paternal origin was shorter, and no crossover occurred on the paternal chromosome 17 during transmission. Five of the six cases of maternal origin were informative, and all five showed a crossover, between the flanking markers, after maternal transmission. The observed crossovers flanking the NF1 region suggest that these NF1 microdeletions result from an unequal crossover in maternal meiosis I, mediated by a misalignment of the flanking NF1-REPs.

The cardiofaciocutaneous syndrome (CFC syndrome; OMIM 115 150) is a sporadic disorder with phenotypic similarities with Noonan syndrome (OMIM 163 950) and Costello syndrome (OMIM 218 040). It is characterised by congenital heart defects, facial dysmorphism, ectodermal abnormalities, failure to thrive, and developmental delay. Increased nuchal translucency has been reported previously in Noonan syndrome and Costello syndrome. Here we report prenatal findings in two patients with the CFC syndrome, both presenting increased nuchal translucency/cystic hygroma colli. The first case is a third pregnancy in a healthy non-consanguineous couple with an increased nuchal translucency of 4 mm at 12 weeks and after chorionic villus sampling, a normal male karyotype, 46,XY. An ultrasound at 23 weeks revealed a mild bilateral pyelectasia (7 mm), a polyhydramnios [amniotic fluid index (AFI) = 37 cm] with stomach filling and a mild cerebral ventriculomegaly (atria: bilateral 10.5 mm) and macrosomia (97th centile) with relatively short femur (5th centile). This remained stable throughout pregnancy. After pre-term pre-labour rupture of membranes, the boy was born at 34 weeks [weight: 3100 g (>95th centile)]. An echocardiography at the age of 6 months revealed a mild valvular pulmonary stenosis (PIG 40 mmHg). He was referred at 1 year of age with failure to thrive [weight: 6820 g (