Clustered de novo start-loss variants in GLUL result in a developmental and epileptic encephalopathy via stabilization of glutamine synthetase.

Glutamine synthetase (GS), encoded by GLUL, catalyzes the conversion of glutamate to glutamine. GS is pivotal for the generation of the neurotransmitters glutamate and gamma-aminobutyric acid and is the primary mechanism of ammonia detoxification in the brain. GS levels are regulated post-translationally by an N-terminal degron that enables the ubiquitin-mediated degradation of GS in a glutamine-induced manner. GS deficiency in humans is known to lead to neurological defects and death in infancy, yet how dysregulation of the degron-mediated control of GS levels might affect neurodevelopment is unknown. We ascertained nine individuals with severe developmental delay, seizures, and white matter abnormalities but normal plasma and cerebrospinal fluid biochemistry with de novo variants in GLUL. Seven out of nine were start-loss variants and two out of nine disrupted 5' UTR splicing resulting in splice exclusion of the initiation codon. Using transfection-based expression systems and mass spectrometry, these variants were shown to lead to translation initiation of GS from methionine 18, downstream of the N-terminal degron motif, resulting in a protein that is stable and enzymatically competent but insensitive to negative feedback by glutamine. Analysis of human single-cell transcriptomes demonstrated that GLUL is widely expressed in neuro- and glial-progenitor cells and mature astrocytes but not in post-mitotic neurons. One individual with a start-loss GLUL variant demonstrated periventricular nodular heterotopia, a neuronal migration disorder, yet overexpression of stabilized GS in mice using in utero electroporation demonstrated no migratory deficits. These findings underline the importance of tight regulation of glutamine metabolism during neurodevelopment in humans.

Menke-Hennekam syndrome; delineation of domain-specific subtypes with distinct clinical and DNA methylation profiles.

CREB-binding protein (CBP, encoded by CREBBP) and its paralog E1A-associated protein (p300, encoded by EP300) are involved in histone acetylation and transcriptional regulation. Variants that produce a null allele or disrupt the catalytic domain of either protein cause Rubinstein-Taybi syndrome (RSTS), while pathogenic missense and in-frame indel variants in parts of exons 30 and 31 cause phenotypes recently described as Menke-Hennekam syndrome (MKHK). To distinguish MKHK subtypes and define their characteristics, molecular and extended clinical data on 82 individuals (54 unpublished) with variants affecting CBP (n = 71) or p300 (n = 11) (NP_004371.2 residues 1,705-1,875 and NP_001420.2 residues 1,668-1,833, respectively) were summarized. Additionally, genome-wide DNA methylation profiles were assessed in DNA extracted from whole peripheral blood from 54 individuals. Most variants clustered closely around the zinc-binding residues of two zinc-finger domains (ZZ and TAZ2) and within the first α helix of the fourth intrinsically disordered linker (ID4) of CBP/p300. Domain-specific methylation profiles were discerned for the ZZ domain in CBP/p300 (found in nine out of 10 tested individuals) and TAZ2 domain in CBP (in 14 out of 20), while a domain-specific diagnostic episignature was refined for the ID4 domain in CBP/p300 (in 21 out of 21). Phenotypes including intellectual disability of varying degree and distinct physical features were defined for each of the regions. These findings demonstrate existence of at least three MKHK subtypes, which are domain specific (MKHK-ZZ, MKHK-TAZ2, and MKHK-ID4) rather than gene specific (CREBBP/EP300). DNA methylation episignatures enable stratification of molecular pathophysiologic entities within a gene or across a family of paralogous genes.

Integrated in silico and experimental assessment of disease relevance of PCDH19 missense variants.

PCDH19 is a nonclustered protocadherin molecule involved in axon bundling, synapse function, and transcriptional coregulation. Pathogenic variants in PCDH19 cause infantile-onset epilepsy known as PCDH19-clustering epilepsy or PCDH19-CE. Recent advances in DNA-sequencing technologies have led to a significant increase in the number of reported PCDH19-CE variants, many of uncertain significance. We aimed to determine the best approaches for assessing the disease relevance of missense variants in PCDH19. The application of the American College of Medical Genetics and Association for Molecular Pathology (ACMG-AMP) guidelines was only 50% accurate. Using a training set of 322 known benign or pathogenic missense variants, we identified MutPred2, MutationAssessor, and GPP as the best performing in silico tools. We generated a protein structural model of the extracellular domain and assessed 24 missense variants. We also assessed 24 variants using an in vitro reporter assay. A combination of these tools was 93% accurate in assessing known pathogenic and benign PCDH19 variants. We increased the accuracy of the ACMG-AMP classification of 45 PCDH19 variants from 50% to 94%, using these tools. In summary, we have developed a robust toolbox for the assessment of PCDH19 variant pathogenicity to improve the accuracy of PCDH19-CE variant classification.

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.

Deletion of Exon 1 in AMER1 in Osteopathia Striata with Cranial Sclerosis.

Osteopathia striata with cranial sclerosis (OSCS) is an X-linked dominant condition characterised by metaphyseal striations, macrocephaly, cleft palate, and developmental delay in affected females. Males have a more severe phenotype with multi-organ malformations, and rarely survive. To date, only frameshift and nonsense variants in exon 2, the single coding exon of AMER1, or whole gene deletions have been reported to cause OSCS. In this study, we describe two families with phenotypic features typical of OSCS. Exome sequencing and multiplex ligation-dependent probe amplification (MLPA) did not identify pathogenic variants in AMER1. Therefore, genome sequencing was employed which identified two deletions containing the non-coding exon 1 of AMER1 in the families. These families highlight the importance of considering variants or deletions of upstream non-coding exons in conditions such as OSCS, noting that often such exons are not captured on probe or enrichment-based platforms because of their high G/C content.

Biallelic loss-of-function variants in TBC1D2B cause a neurodevelopmental disorder with seizures and gingival overgrowth.

The family of Tre2-Bub2-Cdc16 (TBC)-domain containing GTPase activating proteins (RABGAPs) is not only known as key regulatorof RAB GTPase activity but also has GAP-independent functions. Rab GTPases are implicated in membrane trafficking pathways, such as vesicular trafficking. We report biallelic loss-of-function variants in TBC1D2B, encoding a member of the TBC/RABGAP family with yet unknown function, as the underlying cause of cognitive impairment, seizures, and/or gingival overgrowth in three individuals from unrelated families. TBC1D2B messenger RNA amount was drastically reduced, and the protein was absent in fibroblasts of two patients. In immunofluorescence analysis, ectopically expressed TBC1D2B colocalized with vesicles positive for RAB5, a small GTPase orchestrating early endocytic vesicle trafficking. In two independent TBC1D2B CRISPR/Cas9 knockout HeLa cell lines that serve as cellular model of TBC1D2B deficiency, epidermal growth factor internalization was significantly reduced compared with the parental HeLa cell line suggesting a role of TBC1D2B in early endocytosis. Serum deprivation of TBC1D2B-deficient HeLa cell lines caused a decrease in cell viability and an increase in apoptosis. Our data reveal that loss of TBC1D2B causes a neurodevelopmental disorder with gingival overgrowth, possibly by deficits in vesicle trafficking and/or cell survival.

The X-linked filaminopathies: Synergistic insights from clinical and molecular analysis.

The X-linked filaminopathies represent a diverse group of clinical conditions, all caused by variants in the gene FLNA. FLNA encodes the widely expressed actin binding protein, filamin A that has multiple roles during embryonic development including cell migration, mechanical sensing, and cell signaling. In this review, we discuss the 10 distinct X-linked filaminopathy conditions that between them affect almost all organ systems, including the brain, skeleton, heart, and skin, highlighting the critical role of this protein in human development. We review each of the phenotypes and discuss their pathogenesis, where known. Assigning pathogenicity to variants in FLNA can prove difficult, especially for missense variants and small indels, in-part because of the X-linked nature of the phenotypes, the overlap of phenotypic features between conditions, and poor understanding of the function of certain protein domains. We outline here approaches to characterize phenotypes, highlight hotspot regions within FLNA commonly mutated in these conditions, and approaches to resolving some variants of uncertain significance.

Expanding the molecular and clinical phenotypes of FUT8-CDG.

Pathogenic variants in the Golgi localised alpha 1,6 fucosyltransferase, FUT8, cause a rare inherited metabolic disorder known as FUT8-CDG. To date, only three affected individuals have been reported presenting with a constellation of symptoms including intrauterine growth restriction, severe delays in growth and development, other neurological impairments, significantly shortened limbs, respiratory complications, and shortened lifespan. Here, we report an additional four unrelated affected individuals homozygous for novel pathogenic variants in FUT8. Analysis of serum N-glycans revealed a complete lack of core fucosylation, an important diagnostic biomarker of FUT8-CDG. Our data expands both the molecular and clinical phenotypes of FUT8-CDG and highlights the importance of identifying a reliable biomarker for confirming potentially pathogenic variants.

Biallelic variants in EFEMP1 in a man with a pronounced connective tissue phenotype.

Connective tissue disorders are a spectrum of diseases that affect the integrity of tissues including skin, vasculature, and joints. They are often caused by variants that disrupt genes encoding components of extracellular matrix (ECM). The fibulin glycoproteins are ECM proteins important for integrity of tissues including dermis, retina, fascia, and vasculature. The fibulin family consists of seven members (fibulins-1 to -7) and is defined by a fibulin-type domain at the C-terminus. The family is associated with human diseases, for instance a variant in FBLN1, encoding fibulin-1, is associated with synpolydactyly, while one in EFEMP1, encoding fibulin-3, causes Doyne honeycomb degeneration of the retina. Loss-of-function of fibulins-4 and -5 causes cutis laxa, while variants in fibulins-5 and -6 are associated with age-related macular degeneration. Of note, EFEMP1 is not currently associated with any connective tissue disorder. Here we show biallelic loss-of-function variants in EFEMP1 in an individual with multiple and recurrent abdominal and thoracic herniae, myopia, hypermobile joints, scoliosis, and thin translucent skin. Fibroblasts from this individual express significantly lower EFEMP1 transcript than age-matched control cells. A skin biopsy, visualised using light microscopy, showed normal structure and abundance of elastic fibres. The phenotype of this individual is remarkably similar to the Efemp1 knockout mouse model that displays multiple herniae with premature aging and scoliosis. We conclude that loss of EFEMP1 function in this individual is the cause of a connective tissue disorder with a novel combination of phenotypic features, and can perhaps explain similar, previously reported cases in the literature.

Germline mutations and somatic inactivation of TRIM28 in Wilms tumour.

Wilms tumour is a childhood tumour that arises as a consequence of somatic and rare germline mutations, the characterisation of which has refined our understanding of nephrogenesis and carcinogenesis. Here we report that germline loss of function mutations in TRIM28 predispose children to Wilms tumour. Loss of function of this transcriptional co-repressor, which has a role in nephrogenesis, has not previously been associated with cancer. Inactivation of TRIM28, either germline or somatic, occurred through inactivating mutations, loss of heterozygosity or epigenetic silencing. TRIM28-mutated tumours had a monomorphic epithelial histology that is uncommon for Wilms tumour. Critically, these tumours were negative for TRIM28 immunohistochemical staining whereas the epithelial component in normal tissue and other Wilms tumours stained positively. These data, together with a characteristic gene expression profile, suggest that inactivation of TRIM28 provides the molecular basis for defining a previously described subtype of Wilms tumour, that has early age of onset and excellent prognosis.