Clinical practice guideline on the management of vestibular schwannoma.

INTRODUCTION: Vestibular schwannoma (VS) is the most common tumour of the cerebellopontine angle. The greater accessibility to radiological tests has increased its diagnosis. Taking into account the characteristics of the tumour, the symptoms and the age of the patient, three therapeutic strategies have been proposed: observation, surgery or radiotherapy. Choosing the most appropriate for each patient is a frequent source of controversy. MATERIAL AND METHODS: This paper includes an exhaustive literature review of issues related to VS that can serve as a clinical guide in the management of patients with these lesions. The presentation has been oriented in the form of questions that the clinician usually asks himself and the answers have been written and/or reviewed by a panel of national and international experts consulted by the Otology Commission of the SEORL-CCC. RESULTS: A list has been compiled containing the 13 most controversial thematic blocks on the management of VS in the form of 50 questions, and answers to all of them have been sought through a systematic literature review (articles published on PubMed and Cochrane Library between 1992 and 2023 related to each thematic area). Thirty-three experts, led by the Otology Committee of SEORL-CCC, have analyzed and discussed all the answers. In Annex 1, 14 additional questions divided into 4 thematic areas can be found. CONCLUSIONS: This clinical practice guideline on the management of VS offers agreed answers to the most common questions that are asked about this tumour. The absence of sufficient prospective studies means that the levels of evidence on the subject are generally medium or low. This fact increases the interest of this type of clinical practice guidelines prepared by experts.

Lymphatic Malformations in Parkes Weber's Syndrome: Retrospective Review of 16 Cases in a Vascular Anomalies Center.

INTRODUCTION: Parkes Weber's syndrome (PWS) is a rare genetic disorder characterized by overgrowth and vascular malformations, primarily affecting the extremities. While PWS is known to be associated with arteriovenous and capillary malformations, the potential involvement of lymphatic malformations (LMs) has not been previously reported. The objective of this study is to investigate the presence of lymphatic anomalies in PWS patients and their role in the development of limb asymmetry. MATERIALS AND METHODS: This is a retrospective study of patients diagnosed with PWS in a Vascular Anomalies Center from 1994 to 2020. Clinical data were obtained from medical records including diagnostic imaging, lymphoscintigraphy, and genetic testing. The Institutional Review Board and Ethics Committee have approved this study. RESULTS: A total of 16 patients aged 18 interquartile range 14.7 years diagnosed with PWS were included (50% female). Six of the 16 patients with PWS had clinical and imaging data suggestive of LM (37.5%) and 3 of them had genetic variants in RASA1 (2/3) or KRAS (1/3). Limb asymmetry was greater in patients with isolated PWS (2.6 ± 0.8 cm) than in the PWS-lymphatic anomalies population (2 ± 0.7 cm), although not significant (p = 0.247). One in 6 patients with PWS-LM required amputation (16.6%) versus 1 in 10 in isolated PWS (10%). CONCLUSION: Lymphatic anomalies may be present in a significant number of patients with PWS and could have a role in limb asymmetry and outcomes. It is paramount to investigate their existence and distinguish them from true overgrowth.

Undergrowth Of First Toe In PiK3CA-Related Overgrowth Spectrum (PROS).

BACKGROUND: PIK3CA-related overgrowth syndrome (PROS) include a heterogeneous group of disorders characterized by segmental overgrowth secondary to somatic mosaic activating variants in PIK3CA. Segmental undergrowth is more uncommon and has been less studied but pathogenic variants in PIK3CA have also been found. With this in mind, we have noticed a group of patients with PROS that present an undergrowth component associated with their focal overgrowth. METHODS: Retrospective review of patients with PROS presenting overgrowth of the lower limb and undergrowth of the ipsilateral first toe was performed. RESULTS: Six patients were included, 4 female and 2 male with a median age of 16.8 years. All patients presented a PROS phenotype with overgrowth of the lower limb and undergrowth of ipsilateral first toe. A PIK3CA pathogenic variant was confirmed in all patients. Patients underwent multiple treatments, currently all are receiving alpelisib with a mean duration of 15.8 months (1-39) and partial response in lipomatosis and vascular anomalies but no response in overgrowth and undergrowth so far. CONCLUSIONS: Pathogenic variants in the same gene can create different phenotypes depending on the time and place of the mutation. There is little information regarding opposing phenotpyes in the same patient with PROS. The presence of undergrowth in our series might be explained by genetic, embryogenic, maternal, or placental factors but needs to be further investigated.

Thoracic venous malformation: a particular form of a visceral variant.

A teenage boy was admitted due to a thoracic mass with previous respiratory infections. The CT scan showed phleboliths in a cystic lesion with large draining channels. He also presented a mild thrombocytosis, elevated fibrinogen and D-dimer. Arteriogram revealed no abnormal arterial supply but venography proved venous draining channels as the major components of the lesion. The most important venous pedicle was embolised. However, 6 months later, CT scan showed no reduction in lesion size. Surgical resection was performed. Anatomopathological study described a venous malformation (VM) with a lymphatic component, and genetic testing found a typical mutation in PIK3CA and genetic variant in MAP3K3 This case reports a very rare pattern of thoracic vascular tumour. The authors aim to highlight the importance of genetic studies of VM with atypical presentation in order to achieve a definitive diagnosis.

Phenotypic and molecular characterization of five patients with PIK3CA-related overgrowth spectrum (PROS).

Somatic and germline PI3K-AKT-mTOR pathway pathogenic variants are involved in several segmental overgrowth phenotypes such as the PIK3CA-related overgrowth spectrum (PROS), Proteus syndrome, and PTEN hamartoma tumor syndrome. In this study, we describe five patients with PROS. We identified by high-throughput sequencing four different somatic PIK3CA pathogenic variants in five individuals. The Glu726Lys variant, which was previously reported in megalencephaly-capillary malformation-polymicrogyria (MCAP) syndrome, was identified in two patients with unclassified PROS. The Cys420Arg substitution, which was previously reported in CLOVES, was found in a patient with fibroadipose hyperplasia. Additionally, relatively rare pathogenic variants, His1047Tyr and Tyr1021Cys, were detected in two patients with MCAP. Therefore, we suggest performing deep sequencing of PIK3CA in all patients with suspected PROS, instead of targeted polymerase chain reaction for hotspot pathogenic variants.

KRAS mutation identified in a patient with melorheostosis and extended lymphangiomatosis treated with sirolimus and trametinib.

Detection of KRAS mutation in skin biopsy in a patient with melorheostosis, lymphantiomatosis and vascular stenosis. She was successfully treated with trametinib.

Familial hypercholesterolemia: A single-nucleotide variant (SNV) in mosaic at the low density lipoprotein receptor (LDLR).

BACKGROUND AND AIMS: Familial hypercholesterolemia is most frequently caused by genetic variants in the LDLR gene. Most of LDLR pathogenic variants are missense, followed by splicing and deletion/insertions variants. Mosaicism is a genetic condition in which an individual shows more than one clone of cells with different genotypes. The objective of this article was the molecular characterization of a patient with hypercholesterolemia. METHODS AND RESULTS: Genetic analysis of DNA from peripheral blood and saliva was performed by NGS, Sanger sequencing and pyrosequencing technologies. NGS analysis detected the pathogenic variant LDLR:c.1951G > T:p.(Asp651Tyr) in 9%-12% of reads. The presence of the variant was confirmed by pyrosequencing analysis. The variant found was functional characterized using an in vitro model (CHO-ldlA7 cells). Activity and expression of cell surface LDLR were measured by flow cytometry. Colocalization LDLR-Dil-LDL was detected by immunofluorescence. The LDLR activity showed 80% uptake, 50% binding and 53% expression of cell surface LDLR regarding wild type. CONCLUSIONS: Herein, we report the first case of a mosaic single nucleotide variant affecting the LDLR gene in a patient with familial hypercholesterolemia. As it has been described for other pathologies, mosaicism could be underestimated in FH and its detection will improve with the introduction of NGS technologies in the diagnostic routine.

Biomarkers in Vestibular Schwannoma-Associated Hearing Loss.

Vestibular schwannomas (VSs) are benign tumors composed of differentiated neoplastic Schwann cells. They can be classified into two groups: sporadic VS and those associated with neurofibromatosis type 2 (NF2). VSs usually grow slowly, initially causing unilateral sensorineural hearing loss (HL) and tinnitus. These tumors cause HL both due to compression of the auditory nerve or the labyrinthine artery and due to the secretion of different substances potentially toxic to the inner ear or the cochlear nerve. As more and more patients are diagnosed and need to be managed, we are more than ever in need of searching for biomarkers associated with these tumors. Owing to an unknown toxic substance generated by the tumor, HL in VS may be linked to a high protein amount of perilymph. Previous studies have identified perilymph proteins correlated with tumor-associated HL, including µ-Crystallin (CRYM), low density lipoprotein receptor-related protein 2 (LRP2), immunoglobulin (Ig) γ-4 chain C region, Ig κ-chain C region, complement C3, and immunoglobulin heavy constant γ 3. Besides, the presence of specific subtypes of heat shock protein 70 has been suggested to be associated with preservation of residual hearing. It has been recently demonstrated that chemokine receptor-4 (CXCR4) is overexpressed in sporadic VS as well as in NF2 tumors and that hearing disability and CXCR4 expression may be correlated. Further, the genetic profile of VS and its relationship with poor hearing has also been studied, including DNA methylation, deregulated genes, growth factors, and NF2 gene mutations. The knowledge of biomarkers associated with VS would be of significant value to maximize outcomes of hearing preservation in these patients.

Constitutional mosaicism in RASA1-related capillary malformation-arteriovenous malformation.

Capillary malformation-arteriovenous malformation (CM-AVM) is caused by germline RASA1 and EPHB4 alterations. RASA1 intralesional second hits have also been reported. Here we report RASA1 constitutional mosaicism, defined here as the presence of a mosaic variant in all cell types of an individual, in two patients with CM-AVM. High-throughput sequencing was used to search for RASA1 pathogenic variants in blood samples from two unrelated patients with CM-AVM. An affected tissue sample from one of the patients was also analyzed. Both patients showed different nonsense RASA1 variants in mosaic, ranging from 7% to 21.5%, in blood samples and in the corresponding affected tissue sample from one of the patients. In conclusion, we report for the first time the presence of RASA1 constitutional mosaicism in CM-AVM. Constitutional mosaicism has implications for accurate molecular diagnosis and recurrence risk and helps to explain the great phenotypic variability in CM-AVM.

Somatic activating mutations in PIK3CA cause generalized lymphatic anomaly.

Generalized lymphatic anomaly (GLA) is a vascular disorder characterized by diffuse or multifocal lymphatic malformations (LMs). The etiology of GLA is poorly understood. We identified four distinct somatic PIK3CA variants (Glu542Lys, Gln546Lys, His1047Arg, and His1047Leu) in tissue samples from five out of nine patients with GLA. These same PIK3CA variants occur in PIK3CA-related overgrowth spectrum and cause hyperactivation of the PI3K-AKT-mTOR pathway. We found that the mTOR inhibitor, rapamycin, prevented lymphatic hyperplasia and dysfunction in mice that expressed an active form of PIK3CA (His1047Arg) in their lymphatics. We also found that rapamycin reduced pain in patients with GLA. In conclusion, we report that somatic activating PIK3CA mutations can cause GLA, and we provide preclinical and clinical evidence to support the use of rapamycin for the treatment of this disabling and deadly disease.

Variants in members of the cadherin-catenin complex, CDH1 and CTNND1, cause blepharocheilodontic syndrome.

Blepharocheilodontic syndrome (BCDS) consists of lagophthalmia, ectropion of the lower eyelids, distichiasis, euryblepharon, cleft lip/palate and dental anomalies and has autosomal dominant inheritance with variable expression. We identified heterozygous variants in two genes of the cadherin-catenin complex, CDH1, encoding E-cadherin, and CTNND1, encoding p120 catenin delta1 in 15 of 17 BCDS index patients, as was recently described in a different publication. CDH1 plays an essential role in epithelial cell adherence; CTNND1 binds to CDH1 and controls the stability of the complex. Functional experiments in zebrafish and human cells showed that the CDH1 variants impair the cell adhesion function of the cadherin-catenin complex in a dominant-negative manner. Variants in CDH1 have been linked to familial hereditary diffuse gastric cancer and invasive lobular breast cancer; however, no cases of gastric or breast cancer have been reported in our BCDS cases. Functional experiments reported here indicated the BCDS variants comprise a distinct class of CDH1 variants. Altogether, we identified the genetic cause of BCDS enabling DNA diagnostics and counseling, in addition we describe a novel class of dominant negative CDH1 variants.

In-frame Variants in FLNA Proximal Rod 1 Domain Associate With a Predominant Cardiac Valvular Phenotype.

INTRODUCTION AND OBJECTIVES: X-linked cardiac valvular dysplasia is a rare form of male-specific congenital heart defect mainly characterized by myxomatous degeneration of the atrioventricular valves with variable hemodynamic consequences. It is caused by genetic defects in FLNA-encoded filamin A, a widely expressed actin-binding protein that regulates cytoskeleton organization. Filamin A loss of function has also been associated with often concurring neurologic and connective tissue manifestations, with mutations in the first half of the Rod 1 domain apparently expressing the full cardiac phenotype. We contribute to previous genotype-phenotype correlations with a multidisciplinary approach in a newly-described family. METHODS: Cardiologic, dysmorphologic, and genetic evaluation of available members were complemented with transcriptional and X-chromosome inactivation studies. RESULTS: A novel FLNA mutation c.1066-3C>G cosegregated with a male-expressed, apparently isolated, cardiac phenotype with no skewed X-inactivation pattern in female carriers. This variant was shown to result in an in-frame deletion of 8 amino acid residues near the N-terminal region of the protein. CONCLUSIONS: A nonimprinted, partial loss of function of filamin A proximal Rod 1 domain seems to be the pathogenetic mechanism of cardiac valvular dysplasia, with some cases occasionally expressing associated extracardiac manifestations.

Phenotypic Variation in Patients with Homozygous c.1678G>T Mutation in EVC Gene: Report of Two Mexican Families with Ellis-van Creveld Syndrome.

BACKGROUND Ellis-van Creveld syndrome is an autosomal recessive chondro-ectodermal dysplasia characterized by disproportionate short stature, limb shortening, narrow chest, postaxial polydactyly and dysplastic nails and teeth. In addition, 60% of cases present congenital heart defects. Ellis-van Creveld syndrome is predominantly caused by mutations in the EVC or EVC2 (4p16) genes, with only a few cases caused by mutations in WDR35.  CASE REPORT Here, we report on two Mexican families with patients diagnosed with Ellis-van Creveld syndrome. Family 1 includes four patients: three females of 15, 18, and 23 years of age and a 7-year old male. Family 2 has only one affected newborn male. All patients exhibited multiple features including hypodontia, dysplastic teeth, extra frenula, mild short stature, distal limb shortening, postaxial polydactyly of hands and feet, nail dystrophy, and knee joint abnormalities. Only two patients had an atrial septal defect. In all cases, molecular analysis by Sanger sequencing identified the same homozygous mutation in exon 12 of EVC, c.1678G>T, which leads to a premature stop codon.  CONCLUSIONS The mutation c.1678G>T has been previously reported in another Mexican patient and it appears to be a recurrent mutation in Mexico which could represent a founder mutation. The large number of patients in this case allows the clinical variability and spectrum of manifestations present in individuals with Ellis-van Creveld syndrome even if they carry the same homozygous mutation in a same family.

Hypoinsulinaemic, hypoketotic hypoglycaemia due to mosaic genetic activation of PI3-kinase.

OBJECTIVE: Genetic activation of the insulin signal-transducing kinase AKT2 causes syndromic hypoketotic hypoglycaemia without elevated insulin. Mosaic activating mutations in class 1A phospatidylinositol-3-kinase (PI3K), upstream from AKT2 in insulin signalling, are known to cause segmental overgrowth, but the metabolic consequences have not been systematically reported. We assess the metabolic phenotype of 22 patients with mosaic activating mutations affecting PI3K, thereby providing new insight into the metabolic function of this complex node in insulin signal transduction. METHODS: Three patients with megalencephaly, diffuse asymmetric overgrowth, hypoketotic, hypoinsulinaemic hypoglycaemia and no AKT2 mutation underwent further genetic, clinical and metabolic investigation. Signalling in dermal fibroblasts from one patient and efficacy of the mTOR inhibitor Sirolimus on pathway activation were examined. Finally, the metabolic profile of a cohort of 19 further patients with mosaic activating mutations in PI3K was assessed. RESULTS: In the first three patients, mosaic mutations in PIK3CA (p.Gly118Asp or p.Glu726Lys) or PIK3R2 (p.Gly373Arg) were found. In different tissue samples available from one patient, the PIK3CA p.Glu726Lys mutation was present at burdens from 24% to 42%, with the highest level in the liver. Dermal fibroblasts showed increased basal AKT phosphorylation which was potently suppressed by Sirolimus. Nineteen further patients with mosaic mutations in PIK3CA had neither clinical nor biochemical evidence of hypoglycaemia. CONCLUSIONS: Mosaic mutations activating class 1A PI3K cause severe non-ketotic hypoglycaemia in a subset of patients, with the metabolic phenotype presumably related to the extent of mosaicism within the liver. mTOR or PI3K inhibitors offer the prospect for future therapy.

Array CGH Analysis of Paired Blood and Tumor Samples from Patients with Sporadic Wilms Tumor.

Wilms tumor (WT), the most common cancer of the kidney in infants and children, has a complex etiology that is still poorly understood. Identification of genomic copy number variants (CNV) in tumor genomes provides a better understanding of cancer development which may be useful for diagnosis and therapeutic targets. In paired blood and tumor DNA samples from 14 patients with sporadic WT, analyzed by aCGH, 22% of chromosome abnormalities were novel. All constitutional alterations identified in blood were segmental (in 28.6% of patients) and were also present in the paired tumor samples. Two segmental gains (2p21 and 20q13.3) and one loss (19q13.31) present in blood had not been previously described in WT. We also describe, for the first time, a small, constitutive partial gain of 3p22.1 comprising 2 exons of CTNNB1, a gene associated to WT. Among somatic alterations, novel structural chromosomal abnormalities were found, like gain of 19p13.3 and 20p12.3, and losses of 2p16.1-p15, 4q32.5-q35.1, 4q35.2-q28.1 and 19p13.3. Candidate genes included in these regions might be constitutively (SIX3, SALL4) or somatically (NEK1, PIAS4, BMP2) operational in the development and progression of WT. To our knowledge this is the first report of CNV in paired blood and tumor samples in sporadic WT.

Impact of NGS in the medical sciences: Genetic syndromes with an increased risk of developing cancer as an example of the use of new technologies.

The increased speed and decreasing cost of sequencing, along with an understanding of the clinical relevance of emerging information for patient management, has led to an explosion of potential applications in healthcare. Currently, SNP arrays and Next-Generation Sequencing (NGS) technologies are relatively new techniques used to scan genomes for gains and losses, losses of heterozygosity (LOH), SNPs, and indel variants as well as to perform complete sequencing of a panel of candidate genes, the entire exome (whole exome sequencing) or even the whole genome. As a result, these new high-throughput technologies have facilitated progress in the understanding and diagnosis of genetic syndromes and cancers, two disorders traditionally considered to be separate diseases but that can share causal genetic alterations in a group of developmental disorders associated with congenital malformations and cancer risk. The purpose of this work is to review these syndromes as an example of a group of disorders that has been included in a panel of genes for NGS analysis. We also highlight the relationship between development and cancer and underline the connections between these syndromes.

Gene expression analysis of aberrant signaling pathways in meningiomas.

Examining aberrant pathway alterations is one method for understanding the abnormal signals that are involved in tumorigenesis and tumor progression. In the present study, expression arrays were performed on tumor-related genes in meningiomas. The GE Array Q Series HS-006 was used to determine the expression levels of 96 genes that corresponded to six primary biological regulatory pathways in a series of 42 meningiomas, including 32 grade I, four recurrent grade I and six grade II tumors, in addition to three normal tissue controls. Results showed that 25 genes that were primarily associated with apoptosis and angiogenesis functions were downregulated and 13 genes frequently involving DNA damage repair functions were upregulated. In addition to the inactivation of the neurofibromin gene, NF2, which is considered to be an early step in tumorigenesis, variations of other biological regulatory pathways may play a significant role in the development of meningioma.

Expression analysis of tumor-related genes involved in critical regulatory pathways in schwannomas.

PURPOSE: Gene expression array analysis is providing key data on the potential candidate genes and biological pathways involved in schwannoma origin and development. In this way we performed expression array studies on tumor-related genes in schwannomas. METHODS: The GE Array Q Series HS-006 (SuperArray, Bethesda, MD, USA) was used to determine the expression levels of 96 genes corresponding to 6 primary biological regulatory pathways in a series of 23 schwannomas. RESULTS: We identified 15 genes down-regulated, primarily corresponding to signal transduction functions, and 26 genes up-regulated, most frequently involving cell adhesion functions. CONCLUSIONS: In addition to the NF2 inactivation (considered as an early step), variations of other biological regulatory pathways might play a key role in schwannoma.