Schwannomatosis (SWN) is a rare tumor predisposition syndrome caused by pathogenic variants in NF2, SMARCB1, or LZTR1. Mosaicism contributes to up to 30

Legius syndrome is an autosomal dominant disorder caused by variants in SPRED1. In this study, we generated four induced pluripotent stem cell (iPSC) lines derived from patients with Legius syndrome by reprogramming peripheral blood mononuclear cells. Using CRISPR/Cas9 or prime editing, the pathogenic variants were corrected to generate isogenic control lines. All patient and isogenic control lines exhibited a normal morphology and karyotype, expressed pluripotency markers, and possessed trilineage differentiation potential. This is the first established human iPSC model developed for Legius syndrome and is a valuable resource for investigating the molecular mechanisms underlying this condition.

Congenital smooth muscle hamartomas (CSMH) are benign hamartomatous proliferations (of the smooth muscles of the arrector pili muscles). They are most often present at birth and associated with many clinical and pathological signs common to Becker's nevus (BN). Pathogenic mosaic variants in the beta actin gene (ACTB) have been described in both entities. We present two children with CSMH in whom an identical and previously unreported likely pathogenic variant of ACTB was identified. This supports the hypothesis that Becker's nevus and CSMH are part of the same nosological spectrum; therefore, the term CSMH is more appropriate.

We would like to sincerely thank Dr [...].

PURPOSE:Patients with PTEN Hamartoma Tumor Syndrome (PHTS) have high hereditary cancer risks for breast, endometrial, and thyroid cancer. Patients develop multiple primary cancers, but these risks remain uncertain. We aimed to provide the second primary cancer risk. METHODS:This European cohort study assessed second primary cancer risks with Kaplan-Meier analyses using data from medical files, registries and/or patient questionnaires. RESULTS:Overall, 279 adult PHTS patients with (a history of) cancer were included (80% female). Among females, 106 (54%) developed a PHTS-related second primary cancer after a PHTS-related first primary cancer, whereas 10 (29%) males developed a PHTS-related second primary cancer after a PHTS-related first primary cancer. The 5- and 10-year PHTS-related second primary cancer risks were 24.5% (95% CI = 18.1-32.5) and 45.7% (95% CI = 36.9-55.4) in females and 14.5% (95% CI = 5.7-34.1) and 19.8% (95% CI = 8.6-41.9) in males, respectively. Furthermore, 5- and 10-year risks for a second primary breast cancer after a first primary breast cancer were 23.3% (95% CI = 14.9-35.2) and 45.6% (95% CI = 33.0-60.2) in females, respectively. CONCLUSION:This study demonstrated that PHTS patients have high second primary cancer risks, which is driven by breast cancer in females. Hence, identifying patients with PHTS before or at first primary cancer diagnosis is essential to enable potential early detection or prevention of a second primary cancer through surveillance or risk-reducing surgery.

PTEN hamartoma tumour syndrome (PHTS) patients have a high hereditary risk of cancer, especially breast (BC), endometrial (EC), and thyroid cancer (TC). However, the prognosis of PHTS-related cancers is unknown. This European cohort study included adult PHTS patients with data from medical files, registries, and/or questionnaires. Overall survival (OS) was assessed using Kaplan-Meier analyses and were compared with sporadic cancer and the general population using standardized mortality (SMR) and relative survival rates (RSR). Survival bias was addressed using left-truncation. Overall, 147 BC patients were included. The 10y-OS was 77% (95%CI = 66–90), decreasing with increasing stage from 90% (95%CI = 73–100) for stage 0 to 0% (95%CI = 0–0) for stage IV. BC relative survival was comparable to sporadic BC in the first two years (2y-RSR = 1.1; 95%CI = 1.1–1.1) and increasing thereafter (5y-RSR = 1.7; 95%CI = 1.6–1.7). For TC (N = 56) and EC (N = 35), 10y-OS was 87% (95%CI = 74–100) and 64% (95%CI = 38–100), respectively. Overall and cancer-specific mortality in female PHTS patients exceeded general population rates (SMR = 3.7; 95%CI = 2.6–5.0 and SMR = 2.7; 95%CI = 1.6–4.4). The prognosis of PHTS-related cancers was comparable to the general population. The higher overall mortality in PHTS patients is presumably related to their higher cancer incidence. These findings, and the high survival observed in early-stage cancer, emphasise the importance of recognising PHTS early to facilitate cancer surveillance.

Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic condition with a birth incidence of one in 2000 to one in 3000 [...]

A 6-year-old boy with multiple capillary malformations of the port-wine birthmark (PWB) type on the right leg since birth presented with a varicose vein and segmental overgrowth of the affected leg. Genetic testing on affected skin confirmed the presence of a somatic novel pathogenic HRAS 30 bp in-frame duplication/insertion in the switch II domain. This case illustrates the phenotypic overlap of different genotypes and shows that somatic HRAS pathogenic variants, especially in-frame duplications/insertions, must be added to the list of the underlying causes in capillary malformations.

Background Cutaneous epidermal nevi are genotypically diverse mosaic disorders. Pathogenic hotspot variants in HRAS, KRAS, and less frequently, NRAS and BRAF may cause isolated keratinocytic epidermal nevi and sebaceous nevi or several different syndromes when associated with extracutaneous anomalies. Therefore, some authors suggest the concept of mosaic RASopathies to group these different disorders.Methods In this paper, we describe three new cases of syndromic epidermal nevi caused by mosaic HRAS variants: one associating an extensive keratinocytic epidermal nevus with hypomastia, another with extensive mucosal involvement and a third combining a small sebaceous nevus with seizures and intellectual deficiency. Moreover, we performed extensive literature of all cases of syndromic epidermal nevi and related disorders with confirmed pathogenic postzygotic variants in HRAS, KRAS, NRAS or BRAF.Results Most patients presented with bone, ophthalmological or neurological anomalies. Rhabdomyosarcoma, urothelial cell carcinoma and pubertas praecox are also repeatedly reported. KRAS pathogenic variants are involved in 50% of the cases, especially in sebaceous nevi, oculoectodermal syndrome and encephalocraniocutaneous lipomatosis. They are frequently associated with eye and brain anomalies. Pathogenic variants in HRAS are rather present in syndromic keratinocytic epidermal nevi and phacomatosis pigmentokeratotica.Conclusion This review delineates genotype/phenotype correlations of syndromic epidermal nevi with somatic RAS and BRAF pathogenic variants and may help improve their follow-up.

Dear Editors, Neurofibromatosis type I (NF1) caused by pathogenic variants (PVs) in NF1, is a progressive condition characterized by multiple cafe-au-lait macules (CALMs), skinfold freckling, Lisch nodules, cutaneous, subcutaneous or plexiform neurofibromas, optic pathway glioma and distinctive osseous lesions, developing over time.1 According to the revised diagnostic criteria either at least two of the above mentioned clinical features or at least one clinical feature and the detection of a heterozygous NF1 PV are required in order to establish a diagnosis.2 Particularly in young children with multiple CALMs with/without skinfold freckling and no family history, the identification of an NF1 PV is frequently the only way to establish an early diagnosis and to distinguish NF1 from the clinically overlapping Legius syndrome, caused by SPRED1 PVs. The birth incidence of NF1 is 1/3,000, ∼50% being de novo cases.1 In a small subset of cases the PV arises postzygotically resulting in mosaic NF1 which is estimated to be 10–20 times rarer than constitutional NF1.3 In mosaics, severity and expression of the clinical phenotype depends on the affected tissues and the proportion of mutated cells. Early postzygotic PVs which involve tissues from different germ layers result in (mild) generalized NF1 and the PV is usually present also in the gametes (gono-somatic mosaicism).4 PVs occurring later in embryogenesis may result in purely somatic mosaicism presenting for example as neurofibromas and/or CALMs with a segmental distribution in NF1.3, 5 Because mosaicism frequently goes along with a milder phenotype,3, 5 some affected individuals may remain undiagnosed.6 This applies in particular to mosaicism for a PV which is restricted to gametes (gonadal or germline mosaicism) and, hence, does not lead to any clinical symptoms.4 Parental genetic mosaicism should always be taken into account when counselling parents of a child with “apparently” de novo NF1 as we illustrate with two families in which NF1 occurred in more than one child. Informed consent for publication was obtained for all individuals tested. Family 1: The index patient, a 2-year-old girl, presented with multiple CALMs and a facial plexiform neurofibroma. Genetic testing identified the NF1 PV NM_000267.3:c.1783_2001+205delins13, p.(Glu595Argfs*6). When her younger sister was clinically diagnosed with NF1 at the age of 2 years based on multiple CALMs, juvenile xanthogranulomas on the scalp and axillary freckling, the suspicion of mosaicism in one of the parents was raised. This was substantiated by identification of subtle bilateral inguinal freckling in the 37-year-old mother (Figure 1a, b). Using the sensitive method of locked nucleic acid analysis,7 we confirmed the presence of the NF1 PV in the daughter, at low percentages in blood (1.5%), left buccal swab (2.8%), urine (3.8%) and hair roots (2.9%) (Table 1) and, thus, gonado-somatic mosaicism in the mother. The mother developed at the age of 54 years an atypical neurofibroma in the left lower leg. Identification of the PV in this tumor also indicates Schwann cell involvement. Family 2: The female index patient presented with >15 CALMs at 7 months. NF1 was confirmed by identification of the recurrent NF1 PV NM_000267.3:c.910C>T, p.(Arg304Ter). Although NF1 clinical signs were absent in both parents, analysis of DNA from blood lymphocytes of both parents was requested. In neither of the two parents, Sanger sequencing revealed evidence for the presence of this NF1 PV. Due to the rare possibility of gonadal mosaicism, prenatal testing was offered to the parents on their inquiry in the next pregnancy, which unexpectedly detected the same NF1 PV in chorionic villi (Figure 2a). Gonadal mosaicism was retrospectively confirmed by Sanger sequencing detecting the PV in the sperm cells of the father (Figure 2b). The more sensitive digital droplet PCR revealed the mutated allele in 9% of sperm cells but not in blood, urine, buccal mucosa, and hair roots (Figure 2c, Table 1). Parents having a child with “apparently” de novo NF1 frequently raise worries of a recurrence risk in siblings. Case 1 illustrates the importance of a thorough dermatological and clinical examination of both parents followed by highly sensitive techniques to confirm low-level mosaicism in cases with suspected mosaicism in a parent. This strategy is superior to genetic testing of parental blood DNA for the PV with methods like Sanger sequencing, which due to limited sensitivity would not have detected the PV in the mosaic mother. Since freckling in the mother was restricted to one body part compatible with segmental NF1, case 1 as well as cases from the literature8 illustrate also the difficulties of predicting the transmission risk of the PV from the presentation of mosaicism in the patient. Only few cases of pure gonadal mosaicism have been reported in NF1.9-11 Nonetheless, as case 2 illustrates, parents should be made aware of this rare possibility to make an informed decision. Since the majority of point mutations are located on the paternal allele, analysis of sperm cells by Sanger sequencing, a method with limited sensitivity, may be informative as illustrated in case 2. Prenatal diagnosis should also be offered at the parents' request, as this is the only way to rule out the PV in subsequent pregnancies with certainty. We wish to thank the Austrian lay association NF Kinder (https://www.nfkinder.at/) for their continuous support of patients and their families with neurofibromatosis and of our work. None.

The SPRED family proteins act as negative regulators of the Ras-ERK pathway: the N-terminal EVH1 domain interacts with the Ras-GAP domain (GRD) of the NF1 protein, while the C-terminal Sprouty-related (SPR) domain promotes membrane localization of SPRED, thereby recruiting NF-1 to Ras. Loss-of-function mutations in the hSPRED1 cause Legius syndrome in an autosomal dominant manner. In this study, we investigated the effects of missense mutations in the SPR domain identified in patients with Legius syndrome. Among the 18 mutations we examined, six (C368S, M369L, V408E, P415A, P415L, and P422R) have defects in the palmitoylation of the SPRED1 protein, losing plasma membrane localization and forming cytoplasmic granular aggregates. To evaluate the in vivo effects of SPR mutations, knock-in (KI) mice with P415A and P415V substitutions or M417Afs∗4, a C-terminal 28 amino acid deletion, were generated. All these KI mice exhibited cranial malformations, a characteristic feature of Legius syndrome. However, both P415A and P415V mutants formed granular aggregates, whereas M417Afs∗4 showed a diffuse cytoplasmic distribution, and Spred1P415A and Spred1P415V mice, but not Spred1M417Afs∗4 mice, developed cerebellar ataxia and Purkinje cell loss with age. These data suggest that in addition to loss of palmitoylation, the C-terminal region is required for the granular aggregate formation and Purkinje cell loss. The autophagy inducer spermidine rescued the ataxia phenotypes and Purkinje cell loss in Spred1P415A mice. These results suggest that some, but not all, SPR mutations that lose lipid modification induce abnormal cytoplasmic aggregation, which could be a target for autophagic clearance, and potentially cause neurodegenerative diseases.

Neurofibromatosis type-1 is a genetic disorder caused by loss-of-function variants in the tumor-suppressor NF1. Approximately 4% to 11% of neurofibromatosis type-1 patients have a NF1 locus complete deletion resulting from nonallelic homologous recombination between low copy repeats. Codeleted genes probably account for the more severe phenotype observed in NF1-deleted patients. This genotype-phenotype correlation highlights the need for a detailed molecular description. A droplet digital PCR (ddPCR) set along the NF1 locus was designed to delimitate the three recurrent NF1 deletion breakpoints. The ddPCR was tested in 121 samples from nonrelated NF1-deleted patients. Classification based on ddPCR versus multiplex ligation-dependent probe amplification (MLPA) was compared. In addition, microsatellites were analyzed to identify parental origin of deletions. ddPCR identified 77 type-1 (64%), 20 type-2 (16%), 7 type-3 (6%), and 17 atypical deletions (14%). The results were comparable with MLPA, except for three atypical deletions misclassified as type-2 using MLPA, for which the SUZ12 gene was not deleted. A significant maternal bias (25 of 30) in the origin of deletions was identified. This study proposes a fast and efficient ddPCR quantification to allow fine NF1 deletion classification. It indicates that ddPCR can be implemented easily into routine diagnosis to complement the techniques dedicated to NF1 point variant identification. This new tool may help unravel the genetic basis conditioning phenotypic variability in NF1-deleted patients and offer tailored genetic counseling.

Encephalocraniocutaneous lipomatosis (ECCL) is a sporadic congenital condition characterised by ocular, cutaneous and central nervous system involvement. Mosaic activating variants inFGFR1andKRAShave been reported in several individuals with this syndrome. We report on a patient with neurofibromatosis type 1 (NF1) with a germline pathogenic variant in theNF1gene and an ECCL phenotype, suggesting ECCL to be part of a spectrum of malformations associated withNF1pathogenic variants. An anatomical hemispherectomy was performed for intractable epilepsy. Through genetic analysis of blood, cerebral tissue and giant cell lesions in both jaws, we identified the germlineNF1pathogenic variant in all samples and a second-hit pathogenicNF1variant in cerebral tissue and both giant cell lesions. BothNF1variants were located on different alleles resulting in somatic mosaicism for a biallelicNF1inactivation originating in early embryogenesis (second-hit mosaicism or Happle type 2 mosaicism). The biallelic deficit inNF1in the left hemicranium explains the severe localised, congenital abnormality in this patient. Identical first and second-hit variants in a giant cell lesion of both upper and lower jaws provide confirmatory evidence for an early embryonic second hit involving at least the neural crest. We suggest that the ECCL phenotype may be part of a spectrum of congenital problems associated with mosaicNF1nullisomy originating during early embryogenesis. The biallelicNF1inactivation during early embryogenesis mimics the severe activation of the RAS-MAPK pathway seen in ECCL caused by embryonic mosaic activatingFGFR1andKRASvariants in the cranial region. We propose that distinct mechanisms of mosaicism can cause the ECCL phenotype through convergence on the RAS-MAPK pathway.

Females with PTEN Hamartoma Tumor Syndrome (PHTS) have breast cancer risks up to 76%. This study assessed associations between breast cancer and lifestyle in European female adult PHTS patients. Data were collected via patient questionnaires (July 2020–March 2023) and genetic diagnoses from medical files. Associations between lifestyle and breast cancer were calculated using logistic regression corrected for age. Index patients with breast cancer before PHTS diagnosis (breast cancer index) were excluded for ascertainment bias correction. In total, 125 patients were included who completed the questionnaire at a mean age of 44 years (SD = 13). This included 21 breast cancer indexes (17%) and 39 females who developed breast cancer at 43 years (SD = 9). Breast cancer patients performed about 1.1 times less often 0–1 times/week physical activity than ≥2 times (ORtotal-adj = 0.9 (95%CI 0.3–2.6); consumed daily about 1.2–1.8 times more often ≥1 than 0–1 glasses of alcohol (ORtotal-adj = 1.2 (95%CI 0.4–4.0); ORnon-breastcancer-index-adj = 1.8 (95%CI 0.4–6.9); were about 1.04–1.3 times more often smokers than non-smokers (ORtotal-adj = 1.04 (95%CI 0.4–2.8); ORnon-breastcancer-index-adj = 1.3 (95%CI 0.4–4.2)); and overweight or obesity (72%) was about 1.02–1.3 times less common (ORtotal-adj = 0.98 (95%CI 0.4–2.6); ORnon-breastcancer-index-adj = 0.8 (95%CI 0.3–2.7)). Similar associations between lifestyle and breast cancer are suggested for PHTS and the general population. Despite not being statistically significant, results are clinically relevant and suggest that awareness of the effects of lifestyle on patients’ breast cancer risk is important.

Lynch syndrome (LS) and constitutional mismatch repair deficiency (CMMRD) are distinct cancer syndromes caused, respectively, by mono- and bi-allelic germline mismatch repair (MMR) variants. LS predisposes to mainly gastrointestinal and genitourinary cancers in adulthood. CMMRD predisposes to brain, haematological, and LS-spectrum cancers from childhood. Two suspected LS patients with first cancer diagnosis aged 27 or 38 years were found to be homozygous for an MMR (likely) pathogenic variant, MSH6 c.3226C>T (p.(Arg1076Cys)), or variant of uncertain significance (VUS), MLH1 c.306G>A (p.(Glu102=)). MLH1 c.306G>A was shown to cause leaky exon 3 skipping. The apparent genotype-phenotype conflict was resolved by detection of constitutional microsatellite instability in both patients, a hallmark feature of CMMRD. A hypomorphic effect of these and other variants found in additional late onset CMMRD cases, identified by literature review, likely explains a LS-like phenotype. CMMRD testing in carriers of compound heterozygous or homozygous MMR VUS may find similar cases and novel hypomorphic variants. Individualised management of mono- and bi-allelic carriers of hypomorphic MMR variants is needed until we better characterise the associated phenotypes.