FOXG1 variants can be associated with milder phenotypes than congenital Rett syndrome with unassisted walking and language development.

Since 2008, FOXG1 haploinsufficiency has been linked to a severe neurodevelopmental phenotype resembling Rett syndrome but with earlier onset. Most patients are unable to sit, walk, or speak. For years, FOXG1 sequencing was only prescribed in such severe cases, limiting insight into the full clinical spectrum associated with this gene. Next-generation sequencing (NGS) now enables unbiased diagnostics. Through the European Reference Network for Rare Malformation Syndromes, Intellectual and Other Neurodevelopmental Disorders, we gathered data from patients with heterozygous FOXG1 variants presenting a mild phenotype, defined as able to speak and walk independently. We also reviewed data from three previously reported patients meeting our criteria. We identified five new patients with pathogenic FOXG1 missense variants, primarily in the forkhead domain, showing varying nonspecific intellectual disability and developmental delay. These features are not typical of congenital Rett syndrome and were rarely associated with microcephaly and epilepsy. Our findings are consistent with a previous genotype-phenotype analysis by Mitter et al. suggesting the delineation of five different FOXG1 genotype groups. Milder phenotypes were associated with missense variants in the forkhead domain. This information may facilitate prognostic assessments in children carrying a FOXG1 variant and improve the interpretation of new variants identified with genomic sequencing.

Case report: PIK3CA somatic mutation leading to Klippel Trenaunay Syndrome and multiple tumors.

We report a case of Klippel Trenaunay Syndrome that was monitored both clinically and molecularly over a period of 9 years. A somatic mosaic mutation of PIK3CA (p(E545G)) was identified using both cfDNA NGS liquid biopsy and tissue biopsy. At the age of 56, due to intervening clonal mutations in PIK3CA background, she developed a squamous cell carcinoma in the right affected leg which was treated surgically. Nine years later, lung bilateral adenocarcinoma arose on PIK3CA mutated tissues supported by different clonal mutations. One year later, the patient died from metastases led by a new FGFR3 clone unresponsive to standard-of-care, immunotherapy-based. Our results highlight the presence of a molecular hallmark underlying neoplastic transformation that occurs upon an angiodysplastic process and support the view that PIK3CA mutated tissues must be treated as precancerous lesions. Importantly, they remark the effectiveness of combining cfDNA NGS liquid and tissue biopsies to monitor disease evolution as well as to identify aggressive clones targetable by tailored therapy, which is more efficient than conventional protocols.

Elucidating the clinical and molecular spectrum of SMARCC2-associated NDD in a cohort of 65 affected individuals.

PURPOSE: Coffin-Siris and Nicolaides-Baraitser syndromes are recognizable neurodevelopmental disorders caused by germline variants in BAF complex subunits. The SMARCC2 BAFopathy was recently reported. Herein, we present clinical and molecular data on a large cohort. METHODS: Clinical symptoms for 41 novel and 24 previously published affected individuals were analyzed using the Human Phenotype Ontology. For genotype-phenotype correlations, molecular data were standardized and grouped into non-truncating and likely gene-disrupting (LGD) variants. Missense variant protein expression and BAF-subunit interactions were examined using 3D protein modeling, co-immunoprecipitation, and proximity-ligation assays. RESULTS: Neurodevelopmental delay with intellectual disability, muscular hypotonia, and behavioral disorders were the major manifestations. Clinical hallmarks of BAFopathies were rare. Clinical presentation differed significantly, with LGD variants being predominantly inherited and associated with mildly reduced or normal cognitive development, whereas non-truncating variants were mostly de novo and presented with severe developmental delay. These distinct manifestations and non-truncating variant clustering in functional domains suggest different pathomechanisms. In vitro testing showed decreased protein expression for N-terminal missense variants similar to LGD. CONCLUSION: This study improved SMARCC2 variant classification and identified discernible SMARCC2-associated phenotypes for LGD and non-truncating variants, which were distinct from other BAFopathies. The pathomechanism of most non-truncating variants has yet to be investigated.

SPTBN5, Encoding the ßV-Spectrin Protein, Leads to a Syndrome of Intellectual Disability, Developmental Delay, and Seizures.

Whole exome sequencing has provided significant opportunities to discover novel candidate genes for intellectual disability and autism spectrum disorders. Variants in the spectrin genes SPTAN1, SPTBN1, SPTBN2, and SPTBN4 have been associated with neurological disorders; however, SPTBN5 gene-variants have not been associated with any human disorder. This is the first report that associates SPTBN5 gene variants (ENSG00000137877: c.266A>C; p.His89Pro, c.9784G>A; p.Glu3262Lys, c.933C>G; p.Tyr311Ter, and c.8809A>T; p.Asn2937Tyr) causing neurodevelopmental phenotypes in four different families. The SPTBN5-associated clinical traits in our patients include intellectual disability (mild to severe), aggressive tendencies, accompanied by variable features such as craniofacial and physical dysmorphisms, autistic behavior, and gastroesophageal reflux. We also provide a review of the existing literature related to other spectrin genes, which highlights clinical features partially overlapping with SPTBN5.

Nosological and Theranostic Approach to Vascular Malformation through cfDNA NGS Liquid Biopsy.

Several different nosological classifications have been used over time for vascular malformations (VMs) since clinical and pathological signs are largely overlapping. In a large proportion of cases, VMs are generated by somatic mosaicism in key genes, belonging to a few different molecular pathways. Therefore, molecular characterization may help in the understanding of the biological mechanisms related to the development of pathology. Tissue biopsy is not routinely included in the diagnostic path because of the need for fresh tissue specimens and the risk of bleeding. Bypassing the need for bioptic samples, we took advantage of the possibility of isolating cell-free DNA likely released by the affected tissues, to molecularly characterize 53 patients by cfDNA-NGS liquid biopsy. We found a good match between the identified variant and the clinical presentation. PIK3CA variants were found in 67% of Klippel Trenaunay Syndrome individuals; KRAS variants in 60% of arteriovenous malformations; MET was mutated in 75% of lymphovenous malformations. Our results demonstrate the power of cfDNA-NGS liquid biopsy in VMs clinical classification, diagnosis, and treatment. Indeed, tailored repurposing of pre-existing cancer drugs, such as PIK3CA, KRAS, and MET inhibitors, can be envisaged as adjuvant treatment, in addition to surgery and/or endovascular treatment, in the above-defined VMs categories, respectively.

Digenic Alport Syndrome.

Digenic Alport syndrome refers to the inheritance of pathogenic variants in COL4A5 plus COL4A3 or COL4A4 or in COL4A3 plus COL4A4 Where digenic Alport syndrome includes a pathogenic COL4A5 variant, the consequences depend on the sex of the affected individual, COL4A5 variant "severity," and the nature of the COL4A3 or COL4A4 change. A man with a pathogenic COL4A5 variant has all his collagen IV α3α4α5-heterotrimers affected, and an additional COL4A3 or COL4A4 variant may not worsen disease. A woman with a pathogenic COL4A5 variant has on average 50% of her heterotrimers affected, which is increased to 75% with a further COL4A3 or COL4A4 variant and associated with a higher risk of proteinuria. In digenic Alport syndrome with pathogenic COL4A3 and COL4A4 variants, 75% of the heterotrimers are affected. The COL4A3 and COL4A4 genes occur head-to-head on chromosome 2, and inheritance is autosomal dominant when both variants affect the same chromosome (in cis) or recessive when they affect different chromosomes (in trans). This form of digenic disease results in increased proteinuria and a median age of kidney failure intermediate between autosomal dominant and autosomal recessive Alport syndrome. Previous guidelines have suggested that all pathogenic or likely pathogenic digenic variants should be identified and reported. Affected family members should be identified, treated, and discouraged from kidney donation. Inheritance within a family is easier to predict if the two variants are considered independently and if COL4A3 and COL4A4 variants are known to be inherited on the same or different chromosomes.

Monoallelic KIF1A-related disorders: a multicenter cross sectional study and systematic literature review.

BACKGROUND: Monoallelic variants in the KIF1A gene are associated with a large set of clinical phenotypes including neurodevelopmental and neurodegenerative disorders, underpinned by a broad spectrum of central and peripheral nervous system involvement. METHODS: In a multicenter study conducted in patients presenting spastic gait or complex neurodevelopmental disorders, we analyzed the clinical, genetic and neuroradiological features of 28 index cases harboring heterozygous variants in KIF1A. We conducted a literature systematic review with the aim to comparing our findings with previously reported KIF1A-related phenotypes. RESULTS: Among 28 patients, we identified nine novel monoallelic variants, and one a copy number variation encompassing KIF1A. Mutations arose de novo in most patients and were prevalently located in the motor domain. Most patients presented features of a continuum ataxia-spasticity spectrum with only five cases showing a prevalently pure spastic phenotype and six presenting congenital ataxias. Seventeen mutations occurred in the motor domain of the Kinesin-1A protein, but location of mutation did not correlate with neurological and imaging presentations. When tested in 15 patients, muscle biopsy showed oxidative metabolism alterations (6 cases), impaired respiratory chain complexes II + III activity (3/6) and low CoQ10 levels (6/9). Ubiquinol supplementation (1gr/die) was used in 6 patients with subjective benefit. CONCLUSIONS: This study broadened our clinical, genetic, and neuroimaging knowledge of KIF1A-related disorders. Although highly heterogeneous, it seems that manifestations of ataxia-spasticity spectrum disorders seem to occur in most patients. Some patients also present secondary impairment of oxidative metabolism; in this subset, ubiquinol supplementation therapy might be appropriate.

New Candidates for Autism/Intellectual Disability Identified by Whole-Exome Sequencing.

Intellectual disability (ID) is characterized by impairments in the cognitive processes and in the tasks of daily life. It encompasses a clinically and genetically heterogeneous group of neurodevelopmental disorders often associated with autism spectrum disorder (ASD). Social and communication abilities are strongly compromised in ASD. The prevalence of ID/ASD is 1-3%, and approximately 30% of the patients remain without a molecular diagnosis. Considering the extreme genetic locus heterogeneity, next-generation sequencing approaches have provided powerful tools for candidate gene identification. Molecular diagnosis is crucial to improve outcome, prevent complications, and hopefully start a therapeutic approach. Here, we performed parent-offspring trio whole-exome sequencing (WES) in a cohort of 60 mostly syndromic ID/ASD patients and we detected 8 pathogenic variants in genes already known to be associated with ID/ASD (SYNGAP1, SMAD6, PACS1, SHANK3, KMT2A, KCNQ2, ACTB, and POGZ). We found four de novo disruptive variants of four novel candidate ASD/ID genes: MBP, PCDHA1, PCDH15, PDPR. We additionally selected via bioinformatic tools many variants in unknown genes that alone or in combination can contribute to the phenotype. In conclusion, our data confirm the efficacy of WES in detecting pathogenic variants of known and novel ID/ASD genes.

13q Deletion Syndrome Involving RB1: Characterization of a New Minimal Critical Region for Psychomotor Delay.

Retinoblastoma (RB) is an ocular tumor of the pediatric age caused by biallelic inactivation of the RB1 gene (13q14). About 10% of cases are due to gross-sized molecular deletions. The deletions can involve the surrounding genes delineating a contiguous gene syndrome characterized by RB, developmental anomalies, and peculiar facial dysmorphisms. Overlapping deletions previously found by traditional and/or molecular cytogenetic analysis allowed to define some critical regions for intellectual disability (ID) and multiple congenital anomalies, with key candidate genes. In the present study, using array-CGH, we characterized seven new patients with interstitial 13q deletion involving RB1. Among these cases, three patients with medium or large 13q deletions did not present psychomotor delay. This allowed defining a minimal critical region for ID that excludes the previously suggested candidate genes (HTR2A, NUFIP1, PCDH8, and PCDH17). The region contains 36 genes including NBEA, which emerged as the candidate gene associated with developmental delay. In addition, MAB21L1, DCLK1, EXOSC8, and SPART haploinsufficiency might contribute to the observed impaired neurodevelopmental phenotype. In conclusion, this study adds important novelties to the 13q deletion syndrome, although further studies are needed to better characterize the contribution of different genes and to understand how the haploinsufficiency of this region can determine ID.

Corrigendum: Exome Sequencing in BRCA1-2 Candidate Familias: The Contribution of Other Cancer Susceptibility Genes.

[This corrects the article DOI: 10.3389/fonc.2021.649435.].

Exome Sequencing in 200 Intellectual Disability/Autistic Patients: New Candidates and Atypical Presentations.

Intellectual disability (ID) and autism spectrum disorder (ASD) belong to neurodevelopmental disorders and occur in ~1% of the general population. Due to disease heterogeneity, identifying the etiology of ID and ASD remains challenging. Exome sequencing (ES) offers the opportunity to rapidly identify variants associated with these two entities that often co-exist. Here, we performed ES in a cohort of 200 patients: 84 with isolated ID and 116 with ID and ASD. We identified 41 pathogenic variants with a detection rate of 22% (43/200): 39% in ID patients (33/84) and 9% in ID/ASD patients (10/116). Most of the causative genes are genes responsible for well-established genetic syndromes that have not been recognized for atypical phenotypic presentations. Two genes emerged as new candidates: CACNA2D1 and GPR14. In conclusion, this study reinforces the importance of ES in the diagnosis of ID/ASD and underlines that "reverse phenotyping" is fundamental to enlarge the phenotypic spectra associated with specific genes.

Clinical, molecular and glycophenotype insights in SLC39A8-CDG.

BACKGROUND: SLC39A8, a gene located on chromosome 4q24, encodes for the manganese (Mn) transporter ZIP8 and its detrimental variants cause a type 2 congenital disorder of glycosylation (CDG). The common SLC39A8 missense variant A391T is associated with increased risk for multiple neurological and systemic disorders and with decreased serum Mn. Patients with SLC39A8-CDG present with different clinical and neuroradiological features linked to variable transferrin glycosylation profile. Galactose and Mn supplementation therapy results in the biochemical and clinical amelioration of treated patients. RESULTS: Here, we report clinical manifestations, neuroradiological features and glycophenotypes associated with novel SLC39A8 variants (c.1048G > A; p.Gly350Arg and c.131C > G; p.Ser44Trp) in two siblings of the same Italian family. Furthermore, we describe a third patient with overlapping clinical features harbouring the homozygous missense variant A391T. The clinical phenotype of the three patients was characterized by severe developmental disability, dystonic postural pattern and dyskinesia with a more severe progression of the disease in the two affected siblings. Neuroimaging showed a Leigh syndrome-like pattern involving the basal ganglia, thalami and white matter. In the two siblings, atrophic cerebral and cerebellum changes consistent with SLC39A8-CDG were detected as well. Serum transferrin isoelectric focusing (IEF) yielded variable results with slight increase of trisialotransferrin isoforms or even normal pattern. MALDI-MS showed the presence of hypogalactosylated transferrin N-glycans, spontaneously decreasing during the disease course, only in one affected sibling. Total serum N-glycome depicted a distinct pattern for the three patients, with increased levels of undergalactosylated and undersialylated precursors of fully sialylated biantennary glycans, including the monosialo-monogalacto-biantennary species A2G1S1. CONCLUSIONS: Clinical, MRI and glycosylation features of patients are consistent with SLC39A8-CDG. We document two novel variants associated with Leigh syndrome-like disease presentation of SLC39A8-CDG. We show, for the first time, a severe neurological phenotype overlapping with that described for SLC39A8-CDG in association with the homozygous A391T missense variant. We observed a spontaneous amelioration of transferrin N-glycome, highlighting the efficacy of MS-based serum glycomics as auxiliary tool for the diagnosis and clinical management of therapy response in patients with SLC39A8-CDG. Further studies are needed to analyse more in depth the influence of SLC39A8 variants, including the common missense variant, on the expression and function of ZIP8 protein, and their impact on clinical, biochemical and neuroradiological features.

Exome sequencing in BRCA1-2 candidate familias: the contribution of other cancer susceptibility genes

Hereditary Breast and Ovarian Cancer (HBOC) syndrome is a condition in which the risk of breast and ovarian cancer is higher than in the general population. The prevalent pathogenesis is attributable to inactivating variants of the BRCA1-2 highly penetrant genes, however, other cancer susceptibility genes may also be involved. By Exome Sequencing (WES) we analyzed a series of 200 individuals selected for genetic testing in BRCA1-2 genes according to the updated National Comprehensive Cancer Network (NCCN) guidelines. Analysis by MLPA was performed to detect large BRCA1-2 deletions/duplications. Focusing on BRCA1-2 genes, data analysis identified 11 cases with pathogenic variants (4 in BRCA1 and 7 in BRCA1-2) and 12 with uncertain variants (7 in BRCA1 and 5 in BRCA2). Only one case was found with a large BRCA1 deletion. Whole exome analysis allowed to characterize pathogenic variants in 21 additional genes: 10 genes more traditionally associated to breast and ovarian cancer (ATM, BRIP1, CDH1, PALB2, PTEN, RAD51C, and TP53) (5% diagnostic yield) and 11 in candidate cancer susceptibility genes (DPYD, ERBB3, ERCC2, MUTYH, NQO2, NTHL1, PARK2, RAD54L, and RNASEL). In conclusion, this study allowed a personalized risk assessment and clinical surveillance in an increased number of HBOC families and to broaden the spectrum of causative variants also to candidate non-canonical genes.

Shorter androgen receptor polyQ alleles protect against life-threatening COVID-19 disease in European males.

BACKGROUND: While SARS-CoV-2 similarly infects men and women, COVID-19 outcome is less favorable in men. Variability in COVID-19 severity may be explained by differences in the host genome. METHODS: We compared poly-amino acids variability from WES data in severely affected COVID-19 patients versus SARS-CoV-2 PCR-positive oligo-asymptomatic subjects. FINDINGS: Shorter polyQ alleles (≤22) in the androgen receptor (AR) conferred protection against severe outcome in COVID-19 in the first tested cohort (both males and females) of 638 Italian subjects. The association between long polyQ alleles (≥23) and severe clinical outcome (p = 0.024) was also validated in an independent cohort of Spanish men <60 years of age (p = 0.014). Testosterone was higher in subjects with AR long-polyQ, possibly indicating receptor resistance (p = 0.042 Mann-Whitney U test). Inappropriately low serum testosterone level among carriers of the long-polyQ alleles (p = 0.0004 Mann-Whitney U test) predicted the need for intensive care in COVID-19 infected men. In agreement with the known anti-inflammatory action of testosterone, patients with long-polyQ and age ≥60 years had increased levels of CRP (p = 0.018, not accounting for multiple testing). INTERPRETATION: We identify the first genetic polymorphism that appears to predispose some men to develop more severe disease. Failure of the endocrine feedback to overcome AR signaling defects by increasing testosterone levels during the infection leads to the polyQ tract becoming dominant to serum testosterone levels for the clinical outcome. These results may contribute to designing reliable clinical and public health measures and provide a rationale to test testosterone as adjuvant therapy in men with COVID-19 expressing long AR polyQ repeats. FUNDING: MIUR project "Dipartimenti di Eccellenza 2018-2020" to Department of Medical Biotechnologies University of Siena, Italy (Italian D.L. n.18 March 17, 2020) and "Bando Ricerca COVID-19 Toscana" project to Azienda Ospedaliero-Universitaria Senese. Private donors for COVID-19 research and charity funds from Intesa San Paolo.