Deficiency of TET3 leads to a genome-wide DNA hypermethylation episignature in human whole blood (vol 6, 92, 2021)

Genetics of disease, diagnosis and treatmen

From Harmony to Dissonance : An organoid-based study of chromosomal instability and aneuploidy in colorectal cancer progression

The development of colorectal cancer (CRC) is associated with the accumulation of genetic mutations that provide cells with malignant properties. Cancer cells carry defects in genome surveillance mechanisms that lead to genetic instability, providing increased chance to generate the mutations needed for transformation. One of the main manifestations of genetic instability is aneuploidy, a karyotype that is not a multiple of the haploid set. Elevated chromosome missegregation during mitosis underlie the accumulation of aneuploidy in CRC, and this reduced mitotic fidelity gives rise to a specific from of genetic instability known as chromosomal instability (CIN). This thesis encompasses a series of studies that aim to understand the causes and consequences of CIN in CRC development. In chapter 2 we provide an in-depth discussion concerning current literature on aneuploidy and CIN in cancer biology. We discuss how aneuploidy and CIN are fundamentally different traits and how each differently influence cancer biology. Therefore, we emphasize the need to study CIN and aneuploidy individually and to that end CIN should be based on the measurement of chromosome missegregation itself. We finally provide a theoretical framework to hypothesize how aggravating CIN might be a therapeutically exploitable option in treating CIN tumors. Chapter 3 describes the first study that employs the CRISPR/Cas9 technique in combination with organoid cultures. We combine both techniques to develop an artificial CRC progression model by introducing the most commonly mutated genes in CRC that are associated with the adenoma-to-carcinoma sequence. Studying the resulting set of genetically engineered tumor progression organoids confirmed that mutation of all four genes is needed for full malignant transformation. Based on time-lapse imaging of the progression set, we furthermore find that loss of p53 induces CIN in colon organoids. We continue to use the set of progression organoids in chapter 4 to decipher the role of p53 in preventing chromosome segregation errors. We find that loss of p53 renders cells dependent on p38 activity to prevent a specific form of mitotic error that we term ‘bulky anaphase bridges’ (BABs). BABs are associated with DNA-damage and are dependent on active DNA-damage signaling in G2. Finally, we show that p53 deficient, CRC patient derived organoids depend on active p38 to prevent excessive CIN as well. This study therefore reveals previously unanticipated roles for p53 and p38 in preventing genetic instability. Furthermore, the interaction between p53 and p38 might provide therapeutic opportunities. The study described in chapter 5 is the first to assess chromosome segregation errors over the course of tumor progression. Assessment of CIN in organoids derived from benign, pre-malignant and malignant tissues from mice and men revealed that CIN is associated with malignant transformation. CIN levels do not increase from primary tumor to metastasis and do not correlate with aneuploidy scores. In chapter 6 we address the question whether each chromosome has an equal chance to missegregate. We find that a more peripheral location in the interphase nucleus correlates with increased missegregation proportions, revealing a previously unanticipated relation between interphase nuclear organization and mitotic behavior

Deep intronic TIMMDC1 variant delays diagnosis of rapidly progressive complex I deficiency

Complex I deficiency is the most common pediatric mitochondrial disease. It can cause a wide range of clinical disorders, including Leigh syndrome. TIMMDC1 encodes an assembly protein of complex I and has been recently associated with early onset mitochondrial disease in three unrelated families. In all three families the same homozygous deep intronic variant was identified leading to inclusion of a new exon resulting in a frameshift and premature stop codon (c.596 + 2146A > G, p.Gly199_Thr200ins5*). Herein, we describe two brothers of Dutch descent, presenting in infancy with hypotonia and respiratory insufficiency and a rapidly progressive and fatal disease course. Laboratory findings and metabolic investigations revealed no specific abnormalities, notably no raised plasma lactate. MRI showed transient lesions in the basal ganglia of brother 1. A muscle biopsy demonstrated complex I deficiency in brother 2. Exome sequencing yielded a novel heterozygous TIMMDC1 variant: c.385C > T, p.(Arg129*). Targeted sequencing revealed the previously published deep intronic variant c.596 + 2146A > G, p.(Gly199_Thr200ins5*) on the second allele which is not detected by exome sequencing. In summary, we present the fourth family with TIMMDC1-related disease, with a novel nonsense variant. This report illustrates the importance of considering mitochondrial disease even when laboratory findings are normal, and the added value of targeted sequencing of introns

Deficiency of TET3 leads to a genome-wide DNA hypermethylation episignature in human whole blood

TET3 encodes an essential dioxygenase involved in epigenetic regulation through DNA demethylation. TET3 deficiency, or Beck-Fahrner syndrome (BEFAHRS; MIM: 618798), is a recently described neurodevelopmental disorder of the DNA demethylation machinery with a nonspecific phenotype resembling other chromatin-modifying disorders, but inconsistent variant types and inheritance patterns pose diagnostic challenges. Given TET3's direct role in regulating 5-methylcytosine and recent identification of syndrome-specific DNA methylation profiles, we analyzed genome-wide DNA methylation in whole blood of TET3-deficient individuals and identified an episignature that distinguishes affected and unaffected individuals and those with mono-allelic and bi-allelic pathogenic variants. Validation and testing of the episignature correctly categorized known TET3 variants and determined pathogenicity of variants of uncertain significance. Clinical utility was demonstrated when the episignature alone identified an affected individual from over 1000 undiagnosed cases and was confirmed upon distinguishing TET3-deficient individuals from those with 46 other disorders. The TET3-deficient signature - and the signature resulting from activating mutations in DNMT1 which normally opposes TET3 - are characterized by hypermethylation, which for BEFAHRS involves CpG sites that may be biologically relevant. This work expands the role of epi-phenotyping in molecular diagnosis and reveals genome-wide DNA methylation profiling as a quantitative, functional readout for characterization of this new biochemical category of disease.Genetics of disease, diagnosis and treatmen

Correction to: De novo variants in SNAP25 cause an early-onset developmental and epileptic encephalopathy.

Unfortunately, in the first sentence in the abstract, a spelling mistake was introduced during the production process after our proofreading. The wording was changed from “aims” to “aimsed.” Heather M. McLaughlin was not listed among the authors. The original article has been corrected

De novo variants in <em>SNAP25</em> cause an early-onset developmental and epileptic encephalopathy.

Purpose: This study aimsed to provide a comprehensive description of the phenotypic and genotypic spectrum of SNAP25 developmental and epileptic encephalopathy (SNAP25-DEE) by reviewing newly identified and previously reported individuals. Methods: Individuals harboring heterozygous missense or loss-of-function variants in SNAP25 were assembled through collaboration with international colleagues, matchmaking platforms, and literature review. For each individual, detailed phenotyping, classification, and structural modeling of the identified variant were performed. Results: The cohort comprises 23 individuals with pathogenic or likely pathogenic de novo variants in SNAP25. Intellectual disability and early-onset epilepsy were identified as the core symptoms of SNAP25-DEE, with recurrent findings of movement disorders, cerebral visual impairment, and brain atrophy. Structural modeling for all variants predicted possible functional defects concerning SNAP25 or impaired interaction with other components of the SNARE complex. Conclusion: We provide a comprehensive description of SNAP25-DEE with intellectual disability and early-onset epilepsy mostly occurring before the age of two years. These core symptoms and additional recurrent phenotypes show an overlap to genes encoding other components or associated proteins of the SNARE complex such as STX1B, STXBP1, or VAMP2. Thus, these findings advance the concept of a group of neurodevelopmental disorders that may be termed “SNAREopathies.”

Delineation of a KDM2B-related neurodevelopmental disorder and its associated DNA methylation signature.

PURPOSE: Pathogenic variants in genes involved in the epigenetic machinery are an emerging cause of neurodevelopment disorders (NDDs). Lysine-demethylase 2B (KDM2B) encodes an epigenetic regulator and mouse models suggest an important role during development. We set out to determine whether KDM2B variants are associated with NDD. METHODS: Through international collaborations, we collected data on individuals with heterozygous KDM2B variants. We applied methylation arrays on peripheral blood DNA samples to determine a KDM2B associated epigenetic signature. RESULTS: We recruited a total of 27 individuals with heterozygous variants in KDM2B. We present evidence, including a shared epigenetic signature, to support a pathogenic classification of 15 KDM2B variants and identify the CxxC domain as a mutational hotspot. Both loss-of-function and CxxC-domain missense variants present with a specific subepisignature. Moreover, the KDM2B episignature was identified in the context of a dual molecular diagnosis in multiple individuals. Our efforts resulted in a cohort of 21 individuals with heterozygous (likely) pathogenic variants. Individuals in this cohort present with developmental delay and/or intellectual disability; autism; attention deficit disorder/attention deficit hyperactivity disorder; congenital organ anomalies mainly of the heart, eyes, and urogenital system; and subtle facial dysmorphism. CONCLUSION: Pathogenic heterozygous variants in KDM2B are associated with NDD and a specific epigenetic signature detectable in peripheral blood

Loss-of-function variants in SRRM2 cause a neurodevelopmental disorder

PURPOSE: SRRM2 encodes the SRm300 protein, a splicing factor of the SR-related protein family characterized by its serine- and arginine-enriched domains. It promotes interactions between messenger RNA and the spliceosome catalytic machinery. This gene, predicted to be highly intolerant to loss of function (LoF) and very conserved through evolution, has not been previously reported in constitutive human disease. METHODS: Among the 1000 probands studied with developmental delay and intellectual disability in our database, we found 2 patients with de novo LoF variants in SRRM2. Additional families were identified through GeneMatcher. RESULTS: Here, we report on 22 patients with LoF variants in SRRM2 and provide a description of the phenotype. Molecular analysis identified 12 frameshift variants, 8 nonsense variants, and 2 microdeletions of 66 kb and 270 kb. The patients presented with a mild developmental delay, predominant speech delay, autistic or attention-deficit/hyperactivity disorder features, overfriendliness, generalized hypotonia, overweight, and dysmorphic facial features. Intellectual disability was variable and mild when present. CONCLUSION: We established SRRM2 as a gene responsible for a rare neurodevelopmental disease

Pathogenic <em>SPTBN1</em> variants cause an autosomal dominant neurodevelopmental syndrome.

SPTBN1 encodes βII-spectrin, the ubiquitously expressed β-spectrin that forms micrometer-scale networks associated with plasma membranes. Mice deficient in neuronal βII-spectrin have defects in cortical organization, developmental delay and behavioral deficiencies. These phenotypes, while less severe, are observed in haploinsufficient animals, suggesting that individuals carrying heterozygous SPTBN1 variants may also show measurable compromise of neural development and function. Here we identify heterozygous SPTBN1 variants in 29 individuals with developmental, language and motor delays; mild to severe intellectual disability; autistic features; seizures; behavioral and movement abnormalities; hypotonia; and variable dysmorphic facial features. We show that these SPTBN1 variants lead to effects that affect βII-spectrin stability, disrupt binding to key molecular partners, and disturb cytoskeleton organization and dynamics. Our studies define SPTBN1 variants as the genetic basis of a neurodevelopmental syndrome, expand the set of spectrinopathies affecting the brain and underscore the critical role of βII-spectrin in the central nervous system