Typical NF2 and LTZR1 mutations are retained in an immortalized human schwann cell model of schwannomatosis.

Human SCs play a primary role in SWN, a rare genetic disorder in which patients develop multiple schwannomas. So that, their isolation and immortalization could represent an irreplaceable tool to investigate the disease etiopathology. Although few clones of tumoural SCs have been obtained, unfortunately they present genetic, morphological and biological characteristics that do not fully represent the original cells. Herein we isolated, characterized and immortalized primary SCs from human schwannomas. Our immortalized human SCs present typical NF2 and LTZR1 genetic mutations of SWN and retain original phenotype characteristics, representing a valuable tool for further genetic, functional and biomolecular in vitro studies.

Heterozygosity for loss-of-function variants in LZTR1 is associated with isolated multiple café-au-lait macules.

PURPOSE: Pathogenic LZTR1 variants cause schwannomatosis and dominant/recessive Noonan syndrome (NS). We aim to establish an association between heterozygous loss-of-function LZTR1 alleles and isolated multiple café-au-lait macules (CaLMs). METHODS: A total of 849 unrelated participants with multiple CaLMs, lacking pathogenic/likely pathogenic NF1 and SPRED1 variants, underwent RASopathy gene panel sequencing. Data on 125 individuals with heterozygous LZTR1 variants were collected for characterizing their clinical features and the associated molecular spectrum. In vitro functional assessment was performed on a representative panel of missense variants and small in-frame deletions. RESULTS: Analysis revealed heterozygous LZTR1 variants in 6.0% (51/849) of participants, exceeding the general population prevalence. LZTR1-related CaLMs varied in number, displayed sharp or irregular borders, and were generally isolated but occasionally associated with features recurring in RASopathies. In 2 families, CaLMs and schwannomas co-occurred. The molecular spectrum mainly consisted of truncating variants, indicating loss-of-function. These variants substantially overlapped with those occurring in schwannomatosis and recessive NS. Functional characterization showed accelerated protein degradation or mislocalization, and failure to downregulate mitogen-activated protein kinase signaling. CONCLUSION: Our findings expand the phenotypic variability associated with LZTR1 variants, which, in addition to conferring susceptibility to schwannomatosis and causing dominant and recessive NS, occur in individuals with isolated multiple CaLMs.

Loss-of-function variants in ERF are associated with a Noonan syndrome-like phenotype with or without craniosynostosis.

Pathogenic, largely truncating variants in the ETS2 repressor factor (ERF) gene, encoding a transcriptional regulator negatively controlling RAS-MAPK signaling, have been associated with syndromic craniosynostosis involving various cranial sutures and Chitayat syndrome, an ultrarare condition with respiratory distress, skeletal anomalies, and facial dysmorphism. Recently, a single patient with craniosynostosis and a phenotype resembling Noonan syndrome (NS), the most common disorder among the RASopathies, was reported to carry a de novo loss-of-function variant in ERF. Here, we clinically profile 26 individuals from 15 unrelated families carrying different germline heterozygous variants in ERF and showing a phenotype reminiscent of NS. The majority of subjects presented with a variable degree of global developmental and/or language delay. Their shared facial features included absolute/relative macrocephaly, high forehead, hypertelorism, palpebral ptosis, wide nasal bridge, and low-set/posteriorly angulated ears. Stature was below the 3rd centile in two-third of the individuals, while no subject showed typical NS cardiac involvement. Notably, craniosynostosis was documented only in three unrelated individuals, while a dolichocephalic aspect of the skull in absence of any other evidence supporting a premature closing of sutures was observed in other 10 subjects. Unilateral Wilms tumor was diagnosed in one individual. Most cases were familial, indicating an overall low impact on fitness. Variants were nonsense and frameshift changes, supporting ERF haploinsufficiency. These findings provide evidence that heterozygous loss-of-function variants in ERF cause a "RASopathy" resembling NS with or without craniosynostosis, and allow a first dissection of the molecular circuits contributing to MAPK signaling pleiotropy.

A Misdiagnosed Familiar Brooke-Spiegler Syndrome: Case Report and Review of the Literature.

Aim of the report: Brooke-Spiegler syndrome (BSS) is a rare autosomal dominant disease characterized by the growth of cylindromas, spiradenomas, trichoepitheliomas, or their combination. These neoplasms usually begin in the second decade and progressively increase in number and size over the years. Diagnosis necessitates consideration of family history, clinical examination, histological findings, and genetic analysis. The aim of this paper is to explore the clinical overlap between Brooke-Spiegler syndrome (BSS) and neurofibromatosis type 1 (NF1). We aim to highlight the challenges associated with their differential diagnosis and emphasize the lack of standardized diagnostic criteria and treatment approaches. Case presentation: Hereby, we introduce the case of a 28-year-old male referred for suspicion of neurofibromatosis type 1 (NF1) who initially declined the recommended surgical excision for a scalp mass. After four years, he returned with larger masses of the scalp, and underwent excision of multiple masses, revealing cylindromas, spiradenomas, and spiradenocylindromas. Family history reported similar tumors in his father, who was also diagnosed with NF1 for the presence of multiple subcutaneous lesions on the scalp. Clinical overlap led to a genetic consultation, but testing for CYLD mutations yielded no significant variations. Despite this, the strong family history and consistent findings led to a revised diagnosis of Brooke-Spiegler syndrome, correcting the initial misdiagnosis of NF1 syndrome. Conclusions: Thanks to the evolving landscape of BSS research over the past two decades, its molecular underpinnings, clinical presentation, and histopathological features are now clearer. However, a thorough family history assessment is mandatory when BSS is suspected. It is our belief that a multidisciplinary approach and cooperation between specialists are essential when dealing with BSS. By sharing this case, we hope to underscore the importance of considering BSS as a differential diagnosis, especially in cases with atypical presentations or overlapping features with other syndromes like NF1.

Ambra1 deficiency impairs mitophagy in skeletal muscle.

BACKGROUND: Maintaining healthy mitochondria is mandatory for muscle viability and function. An essential surveillance mechanism targeting defective and harmful mitochondria to degradation is the selective form of autophagy called mitophagy. Ambra1 is a multifaceted protein with well-known autophagic and mitophagic functions. However, the study of its role in adult tissues has been extremely limited due to the embryonic lethality caused by full-body Ambra1 deficiency. METHODS: To establish the role of Ambra1 as a positive regulator of mitophagy, we exploited in vivo overexpression of a mitochondria-targeted form of Ambra1 in skeletal muscle. To dissect the consequence of Ambra1 inactivation in skeletal muscle, we generated muscle-specific Ambra1 knockout (Ambra1fl/fl :Mlc1f-Cre) mice. Mitochondria-enriched fractions were obtained from muscles of fed and starved animals to investigate the dynamics of the mitophagic flux. RESULTS: Our data show that Ambra1 has a critical role in the mitophagic flux of adult murine skeletal muscle and that its genetic inactivation leads to mitochondria alterations and myofibre remodelling. Ambra1 overexpression in wild-type muscles is sufficient to enhance mitochondria clearance through the autophagy-lysosome system. Consistently with this, Ambra1-deficient muscles display an abnormal accumulation of the mitochondrial marker TOMM20 by +76% (n = 6-7; P < 0.05), a higher presence of myofibres with swollen mitochondria by +173% (n = 4; P < 0.05), and an alteration in the maintenance of the mitochondrial membrane potential and a 34% reduction in the mitochondrial respiratory complex I activity (n = 4; P < 0.05). Lack of Ambra1 in skeletal muscle leads to impaired mitophagic flux, without affecting the bulk autophagic process. This is due to a significantly decreased recruitment of DRP1 (n = 6-7 mice; P < 0.01) and Parkin (n = 6-7 mice; P < 0.05) to the mitochondrial compartment, when compared with controls. Ambra1-deficient muscles also show a marked dysregulation of the endolysosome compartment, as the incidence of myofibres with lysosomal accumulation is 20 times higher than wild-type muscles (n = 4; P < 0.05). Histologically, Ambra1-deficient muscles of both 3- and 6-month-old animals display a significant decrease of myofibre cross-sectional area and a 52% reduction in oxidative fibres (n = 6-7; P < 0.05), thus highlighting a role for Ambra1 in the proper structure and activity of skeletal muscle. CONCLUSIONS: Our study indicates that Ambra1 is critical for skeletal muscle mitophagy and for the proper maintenance of functional mitochondria.

Choroidal Abnormalities in Pediatric NF1: A Cohort Natural History Study.

The purpose of this study was to assess the long-term natural history of choroidal abnormalities (CAs) in a large pediatric neurofibromatosis type 1 (NF1) population, quantifying their progression in number and dimensions. Pediatric patients (<16 years old) affected by NF1 with a minimum follow-up of 3 years with at least one CA in one eye were consecutively recruited. Near-infrared (NIR) imaging was performed to identify CAs, which were quantified in number and size. The CAs area and perimeter were normalized for the optic disc dimensions to avoid possible bias related to the growing process of the eye. Ninety-nine eyes of 53 patients were evaluated. The CAs number, area and perimeter significantly increased during follow-up (p < 0.0001 for each parameter). The patient age at baseline was inversely correlated with the CAs number over time (coefficient = −0.1313, p = 0.0068), while no correlation was found between the patient age and CAs progression in size. In conclusion, we provide evidence that, in NF1 pediatric patients, CAs change over time, increasing both in number and dimensions, independently from the physiological growth of the eye. While the increase of the CAs number occurs particularly at an earlier age, the increase in the CAs dimensions is a slow process that remains constant during childhood.

Epilepsy in NF1: Epidemiologic, Genetic, and Clinical Features. A Monocentric Retrospective Study in a Cohort of 784 Patients.

An increased lifetime risk of epilepsy has been reported in neurofibromatosis type 1 (NF1) patients, ranging between 4% and 14%. To further analyze the correlation between NF1 and epilepsy, we retrospectively reviewed the epidemiologic, clinical, radiological, and molecular data of 784 unselected patients diagnosed with NF1 and referred to the neurofibromatosis outpatient clinics at the University Hospital of Padua. A crude prevalence of epilepsy of 4.7% was observed. In about 70% of cases, seizures arose in the context of neuroradiological findings, with the main predisposing factors being cerebral vasculopathies and hydrocephalus. In the absence of structural abnormalities, the prevalence of epilepsy was found to be 1.27%, which is approximately equal to the total prevalence in the general population. NF1 patients with seizures exhibit a higher incidence of intellectual disability and/or developmental delay, as well as of isolated learning disabilities. The comparison of causative NF1 mutations between the two groups did not reveal a specific genotype-phenotype correlation. Our data refine the current knowledge on epileptological manifestations in NF1 patients, arguing against the hypothesis that specific mechanisms, inherent to neurofibromin cellular function, might determine an increased risk of epilepsy in this condition.

Integrated CGH/WES Analyses Advance Understanding of Aggressive Neuroblastoma Evolution: A Case Study.

Neuroblastoma (NB) is the most common extra-cranial malignancy in preschool children. To portray the genetic landscape of an overly aggressive NB leading to a rapid clinical progression of the disease, tumor DNA collected pre- and post-treatment has been analyzed. Array comparative genomic hybridization (aCGH), whole-exome sequencing (WES), and pharmacogenetics approaches, respectively, have identified relevant copy number alterations (CNAs), single nucleotide variants (SNVs), and polymorphisms (SNPs) that were then combined into an integrated analysis. Spontaneously formed 3D tumoroids obtained from the recurrent mass have also been characterized. The results prove the power of combining CNAs, SNVs, and SNPs analyses to assess clonal evolution during the disease progression by evidencing multiple clones at disease onset and dynamic genomic alterations during therapy administration. The proposed molecular and cytogenetic integrated analysis empowers the disease follow-up and the prediction of tumor recurrence.

Hybrid Minigene Assay: An Efficient Tool to Characterize mRNA Splicing Profiles of NF1 Variants.

Neurofibromatosis type 1 (NF1) is caused by heterozygous loss of function mutations in the NF1 gene. Although patients are diagnosed according to clinical criteria and few genotype-phenotype correlations are known, molecular analysis remains important. NF1 displays allelic heterogeneity, with a high proportion of variants affecting splicing, including deep intronic alleles and changes outside the canonical splice sites, making validation problematic. Next Generation Sequencing (NGS) technologies integrated with multiplex ligation-dependent probe amplification (MLPA) have largely overcome RNA-based techniques but do not detect splicing defects. A rapid minigene-based system was set up to test the effects of NF1 variants on splicing. We investigated 29 intronic and exonic NF1 variants identified in patients during the diagnostic process. The minigene assay showed the coexistence of multiple mechanisms of splicing alterations for seven variants. A leaky effect on splicing was documented in one de novo substitution detected in a sporadic patient with a specific phenotype without neurofibromas. Our splicing assay proved to be a reliable and fast method to validate novel NF1 variants potentially affecting splicing and to detect hypomorphic effects that might have phenotypic consequences, avoiding the requirement of patient's RNA.

Wilms tumor in patients with osteopathia striata with cranial sclerosis.

Germline pathogenic variants in AMER1 cause osteopathia striata with cranial sclerosis (OSCS: OMIM 300373), an X-linked sclerosing bone disorder. Female heterozygotes exhibit metaphyseal striations in long bones, macrocephaly, cleft palate, and, occasionally, learning disability. Male hemizygotes typically manifest the condition as fetal or neonatal death. Somatically acquired variants in AMER1 are found in neoplastic tissue in 15-30% of patients with Wilms tumor; however, to date, only one individual with OSCS has been reported with a Wilms tumor. Here we present four cases of Wilms tumor in unrelated individuals with OSCS, including the single previously published case. We also report the first case of bilateral Wilms tumor in a patient with OSCS. Tumor tissue analysis showed no clear pattern of histological subtypes. In Beckwith-Wiedemann syndrome, which has a known predisposition to Wilms tumor development, clinical protocols have been developed for tumor surveillance. In the absence of further evidence, we propose a similar protocol for patients with OSCS to be instituted as an initial precautionary approach to tumor surveillance. Further evidence is needed to refine this protocol and to evaluate the possibility of development of other neoplasms later in life, in patients with OSCS.

RETINAL VASCULAR ABNORMALITIES RELATED TO NEUROFIBROMATOSIS TYPE 1: Natural History and Classification by Optical Coherence Tomography Angiography in 473 Patients.

PURPOSE: To analyze and classify neurofibromatosis Type 1 (NF1)-related retinal vascular abnormalities (RVAs), their natural history and correlation with disease severity, in a large cohort of patients. METHODS: This was an observational longitudinal study with prospective enrollment. Four hundred and seventy-three patients affected by NF1 and 150 age-matched healthy subjects were consecutively enrolled. Retinal vascular abnormalities were detected by means of near-infrared reflectance and studied by optical coherence tomography angiography. The superficial vascular plexus and the deep vascular complex (DVC) were quantitatively and qualitatively analyzed. RESULTS: We identified RVAs in 82 of 473 (17%) NF1 patients, but in none of the 150 healthy subjects. A comparison revealed that NF1 patients with RVAs showed a higher number of NF1 diagnostic criteria (4.3 ± 1.5 vs. 3.9 ±1.5, respectively; P = 0.02) than patients without RVAs. Three different RVA types were identified on optical coherence tomography angiography: macrovascular angiomatosis of the sole superficial vascular plexus; macrovascular angiomatosis of the superficial vascular plexus combined with microvascular angiomatosis of the deep vascular complex; and combined macrovascular angiomatosis of both superficial vascular plexus and deep vascular complex. The prospective analysis of optical coherence tomography angiography images showed no significant longitudinal evolution of RVAs (mean follow-up: 3.7 ± 2.8 years). A single patient developed a de novo single RVA, and two RVAs showed detectable changes during follow-up. CONCLUSION: In NF1 patients, RVAs are a characteristic sign that correlates with a more severe systemic disease expression, usually remaining stable during time. Optical coherence tomography angiography allows for the identification of different RVAs subtypes.

Breast cancer in neurofibromatosis 1: survival and risk of contralateral breast cancer in a five country cohort study.

PURPOSE: Neurofibromatosis 1 (NF1) is an autosomal dominant condition caused by pathogenic variants of the NF1 gene. A markedly increased risk of breast cancer is associated with NF1. We have determined the breast cancer survival and risk of contralateral breast cancer in NF1. METHODS: We included 142 women with NF1 and breast cancer from five cohorts in Europe and 335 women without NF1 screened for other familial breast cancers. Risk of contralateral breast cancer and death were assessed by Kaplan-Meier analysis with delayed entry. RESULTS: One hundred forty-two women with NF1 were diagnosed for breast cancer at a median age of 46.9 years (range 27.0-84.3 years) and then followed up for 1235 person-years (mean = 8.70 years). Twelve women had contralateral breast cancer with a rate of 10.5 per 1000 years. Cumulative risk for contralateral breast cancer was 26.5% in 20 years. Five and 10-year all-cause survival was 64.9% (95% confidence interval [CI] = 54.8-76.8) and 49.8% (95%CI = 39.3-63.0). Breast cancer-specific 10-year survival was 64.2% (95% CI = 53.5-77.0%) compared with 91.2% (95% CI = 87.3-95.2%) in the non-NF1 age-matched population at increased risk of breast cancer. CONCLUSION: Women with NF1 have a substantial contralateral breast cancer incidence and poor survival. Early start of breast cancer screening may be a way to improve the survival.

Correction: Breast cancer in neurofibromatosis 1: survival and risk of contralateral breast cancer in a five country cohort study.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Optic Pathway Glioma in Type 1 Neurofibromatosis: Review of Its Pathogenesis, Diagnostic Assessment, and Treatment Recommendations.

Type 1 neurofibromatosis (NF1) is a dominantly inherited condition predisposing to tumor development. Optic pathway glioma (OPG) is the most frequent central nervous system tumor in children with NF1, affecting approximately 15-20% of patients. The lack of well-established prognostic markers and the wide clinical variability with respect to tumor progression and visual outcome make the clinical management of these tumors challenging, with significant differences among distinct centers. We reviewed published articles on OPG diagnostic protocol, follow-up and treatment in NF1. Cohorts of NF1 children with OPG reported in the literature and patients prospectively collected in our center were analyzed with regard to clinical data, tumor anatomical site, diagnostic workflow, treatment and outcome. In addition, we discussed the recent findings on the pathophysiology of OPG development in NF1. This review provides a comprehensive overview about the clinical management of NF1-associated OPG, focusing on the most recent advances from preclinical studies with genetically engineered models and the ongoing clinical trials.

Vanillic Acid Restores Coenzyme Q Biosynthesis and ATP Production in Human Cells Lacking COQ6.

Coenzyme Q (CoQ), a redox-active lipid, is comprised of a quinone group and a polyisoprenoid tail. It is an electron carrier in the mitochondrial respiratory chain, a cofactor of other mitochondrial dehydrogenases, and an essential antioxidant. CoQ requires a large set of enzymes for its biosynthesis; mutations in genes encoding these proteins cause primary CoQ deficiency, a clinically and genetically heterogeneous group of diseases. Patients with CoQ deficiency often respond to oral CoQ10 supplementation. Treatment is however problematic because of the low bioavailability of CoQ10 and the poor tissue delivery. In recent years, bypass therapy using analogues of the precursor of the aromatic ring of CoQ has been proposed as a promising alternative. We have previously shown using a yeast model that vanillic acid (VA) can bypass mutations of COQ6, a monooxygenase required for the hydroxylation of the C5 carbon of the ring. In this work, we have generated a human cell line lacking functional COQ6 using CRISPR/Cas9 technology. We show that these cells cannot synthesize CoQ and display severe ATP deficiency. Treatment with VA can recover CoQ biosynthesis and ATP production. Moreover, these cells display increased ROS production, which is only partially corrected by exogenous CoQ, while VA restores ROS to normal levels. Furthermore, we show that these cells accumulate 3-decaprenyl-1,4-benzoquinone, suggesting that in mammals, the decarboxylation and C1 hydroxylation reactions occur before or independently of the C5 hydroxylation. Finally, we show that COQ6 isoform c (transcript NM_182480) does not encode an active enzyme. VA can be produced in the liver by the oxidation of vanillin, a nontoxic compound commonly used as a food additive, and crosses the blood-brain barrier. These characteristics make it a promising compound for the treatment of patients with CoQ deficiency due to COQ6 mutations.

The Arg1038Gly missense variant in the NF1 gene causes a mild phenotype without neurofibromas.

BACKGROUND: Neurofibromatosis type 1 (NF1) is an autosomal dominant condition caused by inactivating mutations of the NF1 gene. The wide allelic heterogeneity of this condition, with more than 3,000 pathogenic variants reported so far, is paralleled by its high clinical variability, which is observed even within the same family. The definition of genotype-phenotype correlations has been hampered by the complexity of the NF1 gene and, although a few exceptions have been recognized, the clinical course remains unpredictable in most patients. METHODS: Sequencing of NF1 in patients with cafè-au-lait spots identified the c.3112A>G variant. RNA analysis and a minigene assay were employed to investigate splicing. RESULTS: Here we report a novel genotype-phenotype correlation in NF1: the identification of the missense variant NM_000267.3:c.3112A>G p.(Arg1038Gly) in seven individuals from two unrelated families with a mild phenotype. All the patients manifest cafè-au-lait spots without neurofibromas or other NF1-associated complications, and Noonan syndrome features in most cases. The missense variant was not previously reported in available databases, segregates with the phenotype and involves a highly conserved residue. Both a minigene assay and patient's RNA analysis excluded an effect on splicing. CONCLUSION: Our data support the correlation of the p.Arg1038Gly missense substitution with the cutaneous phenotype without neurofibromas or other complications. This finding may have relevant implications for patients and genetic counseling, but also to get insights into the function of neurofibromin.

Usefulness and Limitations of Comprehensive Characterization of mRNA Splicing Profiles in the Definition of the Clinical Relevance of BRCA1/2 Variants of Uncertain Significance.

Highly penetrant variants of BRCA1/2 genes are involved in hereditary predisposition to breast and ovarian cancer. The detection of pathogenic BRCA variants has a considerable clinical impact, allowing appropriate cancer-risk management. However, a major drawback is represented by the identification of variants of uncertain significance (VUS). Many VUS potentially affect mRNA splicing, making transcript analysis an essential step for the definition of their pathogenicity. Here, we characterize the impact on splicing of ten BRCA1/2 variants. Aberrant splicing patterns were demonstrated for eight variants whose alternative transcripts were fully characterized. Different events were observed, including exon skipping, intron retention, and usage of de novo and cryptic splice sites. Transcripts with premature stop codons or in-frame loss of functionally important residues were generated. Partial/complete splicing effect and quantitative contribution of different isoforms were assessed, leading to variant classification according to Evidence-based Network for the Interpretation of Mutant Alleles (ENIGMA) consortium guidelines. Two variants could be classified as pathogenic and two as likely benign, while due to a partial splicing effect, six variants remained of uncertain significance. The association with an undefined tumor risk justifies caution in recommending aggressive risk-reduction treatments, but prevents the possibility of receiving personalized therapies with potential beneficial effect. This indicates the need for applying additional approaches for the analysis of variants resistant to classification by gene transcript analyses.

Correlation of peripapillary retinal nerve fibre layer thickness with visual acuity in paediatric patients affected by optic pathway glioma.

PURPOSE: To evaluate peripapillary retinal nerve fibre layer (RNFL) thickness, measured by spectral-domain optical coherence tomography (SD-OCT), as a surrogate of visual function in a population of paediatric patients affected by optic pathway glioma (OPG) associated with neurofibromatosis type 1 (NF1). METHODS: A total of 38 paediatric patients (66 eyes) affected by MRI-proven OPG were included. Each patient underwent complete ophthalmological examination, including age-appropriate visual acuity (VA) assessment and RNFL analysis by SD-OCT. Visual acuity was classified as normal or pathologic using age-based normative data. Visual acuity was correlated to mean RNFL thickness of the whole peripapillary area and of each single analyzed sector (nasal, superior, temporal, inferior). RESULTS: Visual acuity was normal in 43 (65%) and pathologic in 23 (35%) eyes. Mean parapapillary RNFL thickness of each analyzed sector was significantly lower in eyes with abnormal VA (p < 0.05). The best balanced cut-off value of global RNFL thickness allowing to discriminate between eyes with normal and pathologic VA was 76.25 µm (91%, 76%, 67% and 94% of sensitivity, specificity, positive and negative predicting value, respectively). Considering best balanced cut-off values of other analyzed RNFL sectors, the superior (p = 0.0029) and the inferior (p = 0.0024) sectors reached the higher sensitivity (87% and 87%, respectively) and specificity (81% and 79%, respectively). CONCLUSION: Retinal nerve fibre layer thickness is directly related to VA in children affected by NF1-related OPG, and should be considered as a potential surrogate marker of VA. Retinal nerve fibre layer thickness cut-off values can be used in paediatric patients to discriminate false-positive results obtained by VA measurement.

Recessive Spondylocarpotarsal Synostosis Syndrome Due to Compound Heterozygosity for Variants in MYH3.

Spondylocarpotarsal synostosis syndrome (SCTS) is characterized by intervertebral fusions and fusion of the carpal and tarsal bones. Biallelic mutations in FLNB cause this condition in some families, whereas monoallelic variants in MYH3, encoding embryonic heavy chain myosin 3, have been implicated in dominantly inherited forms of the disorder. Here, five individuals without FLNB mutations from three families were hypothesized to be affected by recessive SCTS on account of sibling recurrence of the phenotype. Initial whole-exome sequencing (WES) showed that all five were heterozygous for one of two independent splice-site variants in MYH3. Despite evidence indicating that three of the five individuals shared two allelic haplotypes encompassing MYH3, no second variant could be located in the WES datasets. Subsequent genome sequencing of these three individuals demonstrated a variant altering a 5' UTR splice donor site (rs557849165 in MYH3) not represented by exome-capture platforms. When the cohort was expanded to 16 SCTS-affected individuals without FLNB mutations, nine had truncating mutations transmitted by unaffected parents, and six inherited the rs557849165 variant in trans, an observation at odds with the population allele frequency for this variant. The rs557849165 variant disrupts splicing in the 5' UTR but is still permissive of MYH3 translational initiation, albeit with reduced efficiency. Although some MYH3 variants cause dominant SCTS, these data indicate that others (notably truncating variants) do not, except in the context of compound heterozygosity for a second hypomorphic allele. These observations make genetic diagnosis challenging in the context of simplex presentations of the disorder.

Parent-of-origin effect of hypomorphic pathogenic variants and somatic mosaicism impact on phenotypic expression of retinoblastoma.

Retinoblastoma is the most common eye cancer in children. Numerous families have been described displaying reduced penetrance and expressivity. An extensive molecular characterization of seven families led us to characterize the two main mechanisms impacting on phenotypic expression, as follows: (i) mosaicism of amorphic pathogenic variants; and (ii) parent-of-origin-effect of hypomorphic pathogenic variants. Somatic mosaicism for RB1 splicing variants (c.1960+5G>C and c.2106+2T>C), leading to a complete loss of function was demonstrated by high-depth NGS in two families. In both cases, the healthy carrier parent (one with retinoma) showed a variant frequency lower than that expected for a heterozygous individual, indicating a 56-60% mosaicism level. Previous evidences of a ~3-fold excess of RB1 maternal canonical transcript led us to hypothesize that this differential allelic expression could influence phenotypic outcome in families at risk for RB onset. Accordingly, in five families, we identified a higher tumor risk associated with paternally inherited hypomorphic pathogenic variants, namely a deletion resulting in the loss of 37 amino acids at the N-terminus (c.608-16_608del), an exonic substitution with a "leaky" splicing effect (c.1331A>G), a partially deleterious substitution (c.1981C>T) and a truncating C-terminal variant (c.2663+2T>C). The identification of these mechanisms changes the genetic/prenatal counseling and the clinical management of families, indicating a higher recurrence risk when the hypomorphic pathogenic variant is inherited from the father, and suggesting the need for second tumor surveillance in unaffected carriers at risk of developing adult-onset cancer such as osteosarcoma or leiomyosarcoma.