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1.
Am J Hum Genet ; 111(9): 1953-1969, 2024 Sep 05.
Artículo en Inglés | MEDLINE | ID: mdl-39116879

RESUMEN

While it is widely thought that de novo mutations (DNMs) occur randomly, we previously showed that some DNMs are enriched because they are positively selected in the testes of aging men. These "selfish" mutations cause disorders with a shared presentation of features, including exclusive paternal origin, significant increase of the father's age, and high apparent germline mutation rate. To date, all known selfish mutations cluster within the components of the RTK-RAS-MAPK signaling pathway, a critical modulator of testicular homeostasis. Here, we demonstrate the selfish nature of the SMAD4 DNMs causing Myhre syndrome (MYHRS). By analyzing 16 informative trios, we show that MYHRS-causing DNMs originated on the paternally derived allele in all cases. We document a statistically significant epidemiological paternal age effect of 6.3 years excess for fathers of MYHRS probands. We developed an ultra-sensitive assay to quantify spontaneous MYHRS-causing SMAD4 variants in sperm and show that pathogenic variants at codon 500 are found at elevated level in sperm of most men and exhibit a strong positive correlation with donor's age, indicative of a high apparent germline mutation rate. Finally, we performed in vitro assays to validate the peculiar functional behavior of the clonally selected DNMs and explored the basis of the pathophysiology of the different SMAD4 sperm-enriched variants. Taken together, these data provide compelling evidence that SMAD4, a gene operating outside the canonical RAS-MAPK signaling pathway, is associated with selfish spermatogonial selection and raises the possibility that other genes/pathways are under positive selection in the aging human testis.


Asunto(s)
Mutación de Línea Germinal , Discapacidad Intelectual , Proteína Smad4 , Humanos , Masculino , Proteína Smad4/genética , Discapacidad Intelectual/genética , Contractura/genética , Adulto , Facies , Espermatozoides/metabolismo , Espermatozoides/patología , Criptorquidismo/genética , Trastornos del Crecimiento/genética , Deformidades Congénitas de la Mano/genética , Selección Genética , Alelos , Edad Paterna , Testículo/patología , Testículo/metabolismo
2.
Am J Hum Genet ; 111(3): 594-613, 2024 03 07.
Artículo en Inglés | MEDLINE | ID: mdl-38423010

RESUMEN

The endosomal sorting complex required for transport (ESCRT) machinery is essential for membrane remodeling and autophagy and it comprises three multi-subunit complexes (ESCRT I-III). We report nine individuals from six families presenting with a spectrum of neurodevelopmental/neurodegenerative features caused by bi-allelic variants in SNF8 (GenBank: NM_007241.4), encoding the ESCRT-II subunit SNF8. The phenotypic spectrum included four individuals with severe developmental and epileptic encephalopathy, massive reduction of white matter, hypo-/aplasia of the corpus callosum, neurodevelopmental arrest, and early death. A second cohort shows a milder phenotype with intellectual disability, childhood-onset optic atrophy, or ataxia. All mildly affected individuals shared the same hypomorphic variant, c.304G>A (p.Val102Ile). In patient-derived fibroblasts, bi-allelic SNF8 variants cause loss of ESCRT-II subunits. Snf8 loss of function in zebrafish results in global developmental delay and altered embryo morphology, impaired optic nerve development, and reduced forebrain size. In vivo experiments corroborated the pathogenicity of the tested SNF8 variants and their variable impact on embryo development, validating the observed clinical heterogeneity. Taken together, we conclude that loss of ESCRT-II due to bi-allelic SNF8 variants is associated with a spectrum of neurodevelopmental/neurodegenerative phenotypes mediated likely via impairment of the autophagic flux.


Asunto(s)
Epilepsia Generalizada , Atrofia Óptica , Animales , Humanos , Niño , Pez Cebra/genética , Atrofia Óptica/genética , Fenotipo , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética
3.
Am J Hum Genet ; 111(6): 1206-1221, 2024 06 06.
Artículo en Inglés | MEDLINE | ID: mdl-38772379

RESUMEN

Utilizing trio whole-exome sequencing and a gene matching approach, we identified a cohort of 18 male individuals from 17 families with hemizygous variants in KCND1, including two de novo missense variants, three maternally inherited protein-truncating variants, and 12 maternally inherited missense variants. Affected subjects present with a neurodevelopmental disorder characterized by diverse neurological abnormalities, mostly delays in different developmental domains, but also distinct neuropsychiatric signs and epilepsy. Heterozygous carrier mothers are clinically unaffected. KCND1 encodes the α-subunit of Kv4.1 voltage-gated potassium channels. All variant-associated amino acid substitutions affect either the cytoplasmic N- or C-terminus of the channel protein except for two occurring in transmembrane segments 1 and 4. Kv4.1 channels were functionally characterized in the absence and presence of auxiliary ß subunits. Variant-specific alterations of biophysical channel properties were diverse and varied in magnitude. Genetic data analysis in combination with our functional assessment shows that Kv4.1 channel dysfunction is involved in the pathogenesis of an X-linked neurodevelopmental disorder frequently associated with a variable neuropsychiatric clinical phenotype.


Asunto(s)
Trastornos del Neurodesarrollo , Adolescente , Adulto , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Epilepsia/genética , Secuenciación del Exoma , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Heterocigoto , Mutación Missense/genética , Trastornos del Neurodesarrollo/genética , Linaje , Fenotipo , Canales de Potasio Shal/genética
4.
Am J Hum Genet ; 110(11): 1938-1949, 2023 11 02.
Artículo en Inglés | MEDLINE | ID: mdl-37865086

RESUMEN

Fanconi anemia (FA) is a clinically variable and genetically heterogeneous cancer-predisposing disorder representing the most common bone marrow failure syndrome. It is caused by inactivating predominantly biallelic mutations involving >20 genes encoding proteins with roles in the FA/BRCA DNA repair pathway. Molecular diagnosis of FA is challenging due to the wide spectrum of the contributing gene mutations and structural rearrangements. The assessment of chromosomal fragility after exposure to DNA cross-linking agents is generally required to definitively confirm diagnosis. We assessed peripheral blood genome-wide DNA methylation (DNAm) profiles in 25 subjects with molecularly confirmed clinical diagnosis of FA (FANCA complementation group) using Illumina's Infinium EPIC array. We identified 82 differentially methylated CpG sites that allow to distinguish subjects with FA from healthy individuals and subjects with other genetic disorders, defining an FA-specific DNAm signature. The episignature was validated using a second cohort of subjects with FA involving different complementation groups, documenting broader genetic sensitivity and demonstrating its specificity using the EpiSign Knowledge Database. The episignature properly classified DNA samples obtained from bone marrow aspirates, demonstrating robustness. Using the selected probes, we trained a machine-learning model able to classify EPIC DNAm profiles in molecularly unsolved cases. Finally, we show that the generated episignature includes CpG sites that do not undergo functional selective pressure, allowing diagnosis of FA in individuals with reverted phenotype due to gene conversion. These findings provide a tool to accelerate diagnostic testing in FA and broaden the clinical utility of DNAm profiling in the diagnostic setting.


Asunto(s)
Anemia de Fanconi , Humanos , Anemia de Fanconi/diagnóstico , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Metilación de ADN/genética , Proteínas/genética , ADN/metabolismo
5.
Am J Hum Genet ; 110(5): 774-789, 2023 05 04.
Artículo en Inglés | MEDLINE | ID: mdl-37054711

RESUMEN

The Integrator complex is a multi-subunit protein complex that regulates the processing of nascent RNAs transcribed by RNA polymerase II (RNAPII), including small nuclear RNAs, enhancer RNAs, telomeric RNAs, viral RNAs, and protein-coding mRNAs. Integrator subunit 11 (INTS11) is the catalytic subunit that cleaves nascent RNAs, but, to date, mutations in this subunit have not been linked to human disease. Here, we describe 15 individuals from 10 unrelated families with bi-allelic variants in INTS11 who present with global developmental and language delay, intellectual disability, impaired motor development, and brain atrophy. Consistent with human observations, we find that the fly ortholog of INTS11, dIntS11, is essential and expressed in the central nervous systems in a subset of neurons and most glia in larval and adult stages. Using Drosophila as a model, we investigated the effect of seven variants. We found that two (p.Arg17Leu and p.His414Tyr) fail to rescue the lethality of null mutants, indicating that they are strong loss-of-function variants. Furthermore, we found that five variants (p.Gly55Ser, p.Leu138Phe, p.Lys396Glu, p.Val517Met, and p.Ile553Glu) rescue lethality but cause a shortened lifespan and bang sensitivity and affect locomotor activity, indicating that they are partial loss-of-function variants. Altogether, our results provide compelling evidence that integrity of the Integrator RNA endonuclease is critical for brain development.


Asunto(s)
Proteínas de Drosophila , Enfermedades del Sistema Nervioso , Adulto , Animales , Humanos , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Mutación/genética , ARN Mensajero
6.
Hum Mol Genet ; 32(3): 473-488, 2023 01 13.
Artículo en Inglés | MEDLINE | ID: mdl-36018820

RESUMEN

Kinesins are motor proteins involved in microtubule (MT)-mediated intracellular transport. They contribute to key cellular processes, including intracellular trafficking, organelle dynamics and cell division. Pathogenic variants in kinesin-encoding genes underlie several human diseases characterized by an extremely variable clinical phenotype, ranging from isolated neurodevelopmental/neurodegenerative disorders to syndromic phenotypes belonging to a family of conditions collectively termed as 'ciliopathies.' Among kinesins, kinesin-1 is the most abundant MT motor for transport of cargoes towards the plus end of MTs. Three kinesin-1 heavy chain isoforms exist in mammals. Different from KIF5A and KIF5C, which are specifically expressed in neurons and established to cause neurological diseases when mutated, KIF5B is an ubiquitous protein. Three de novo missense KIF5B variants were recently described in four subjects with a syndromic skeletal disorder characterized by kyphomelic dysplasia, hypotonia and DD/ID. Here, we report three dominantly acting KIF5B variants (p.Asn255del, p.Leu498Pro and p.Leu537Pro) resulting in a clinically wide phenotypic spectrum, ranging from dilated cardiomyopathy with adult-onset ophthalmoplegia and progressive skeletal myopathy to a neurodevelopmental condition characterized by severe hypotonia with or without seizures. In vitro and in vivo analyses provide evidence that the identified disease-associated KIF5B variants disrupt lysosomal, autophagosome and mitochondrial organization, and impact cilium biogenesis. All variants, and one of the previously reported missense changes, were shown to affect multiple developmental processes in zebrafish. These findings document pleiotropic consequences of aberrant KIF5B function on development and cell homeostasis, and expand the phenotypic spectrum resulting from altered kinesin-mediated processes.


Asunto(s)
Cinesinas , Animales , Humanos , Cinesinas/genética , Cinesinas/metabolismo , Mamíferos/metabolismo , Hipotonía Muscular , Neuronas/metabolismo , Fenotipo , Pez Cebra/genética , Pez Cebra/metabolismo
7.
J Pathol ; 263(2): 166-177, 2024 06.
Artículo en Inglés | MEDLINE | ID: mdl-38629245

RESUMEN

Infantile fibrosarcomas (IFS) and congenital mesoblastic nephroma (CMN) are rare myofibroblastic tumors of infancy and early childhood commonly harboring the ETV6::NTRK3 gene fusion. IFS/CMN are considered as tumors with an 'intermediate prognosis' as they are locally aggressive, but rarely metastasize, and generally have a favorable outcome. A fraction of IFS/CMN-related neoplasms are negative for the ETV6::NTRK3 gene rearrangement and are characterized by other chimeric proteins promoting MAPK signaling upregulation. In a large proportion of these tumors, which are classified as IFS-like mesenchymal neoplasms, the contributing molecular events remain to be identified. Here, we report three distinct rearrangements involving RAF1 among eight ETV6::NTRK3 gene fusion-negative tumors with an original histological diagnosis of IFS/CMN. The three fusion proteins retain the entire catalytic domain of the kinase. Two chimeric products, GOLGA4::RAF1 and LRRFIP2::RAF1, had previously been reported as driver events in different cancers, whereas the third, CLIP1::RAF1, represents a novel fusion protein. We demonstrate that CLIP1::RAF1 acts as a bona fide oncoprotein promoting cell proliferation and migration through constitutive upregulation of MAPK signaling. We show that the CLIP1::RAF1 hyperactive behavior does not require RAS activation and is mediated by constitutive 14-3-3 protein-independent dimerization of the chimeric protein. As previously reported for the ETV6::NTRK3 fusion protein, CLIP1::RAF1 similarly upregulates PI3K-AKT signaling. Our findings document that RAF1 gene rearrangements represent a recurrent event in ETV6::NTRK3-negative IFS/CMN and provide a rationale for the use of inhibitors directed to suppress MAPK and PI3K-AKT signaling in these cancers. © 2024 The Pathological Society of Great Britain and Ireland.


Asunto(s)
Fibrosarcoma , Nefroma Mesoblástico , Proteínas de Fusión Oncogénica , Proteínas Proto-Oncogénicas c-raf , Humanos , Fibrosarcoma/genética , Fibrosarcoma/patología , Proteínas Proto-Oncogénicas c-raf/genética , Lactante , Proteínas de Fusión Oncogénica/genética , Nefroma Mesoblástico/genética , Nefroma Mesoblástico/patología , Femenino , Masculino , Neoplasias Renales/genética , Neoplasias Renales/patología , Fusión Génica , Transducción de Señal/genética , Proteínas Proto-Oncogénicas c-ets/genética , Proliferación Celular , Reordenamiento Génico , Proteína ETS de Variante de Translocación 6 , Receptor trkC
8.
Hum Mol Genet ; 31(4): 561-575, 2022 02 21.
Artículo en Inglés | MEDLINE | ID: mdl-34508588

RESUMEN

Germline-activating mutations in HRAS cause Costello syndrome (CS), a cancer prone multisystem disorder characterized by reduced postnatal growth. In CS, poor weight gain and growth are not caused by low caloric intake. Here, we show that constitutive plasma membrane translocation and activation of the GLUT4 glucose transporter, via reactive oxygen species-dependent AMP-activated protein kinase α and p38 hyperactivation, occurs in primary fibroblasts of CS patients, resulting in accelerated glycolysis and increased fatty acid synthesis and storage as lipid droplets. An accelerated autophagic flux was also identified as contributing to the increased energetic expenditure in CS. Concomitant inhibition of p38 and PI3K signaling by wortmannin was able to rescue both the dysregulated glucose intake and accelerated autophagic flux. Our findings provide a mechanistic link between upregulated HRAS function, defective growth and increased resting energetic expenditure in CS, and document that targeting p38 and PI3K signaling is able to revert this metabolic dysfunction.


Asunto(s)
Síndrome de Costello , Síndrome de Costello/genética , Síndrome de Costello/metabolismo , Fibroblastos/metabolismo , Humanos , Oxidación-Reducción , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Transducción de Señal/genética
9.
Hum Mol Genet ; 31(16): 2766-2778, 2022 08 23.
Artículo en Inglés | MEDLINE | ID: mdl-35348676

RESUMEN

We previously molecularly and clinically characterized Mazzanti syndrome, a RASopathy related to Noonan syndrome that is mostly caused by a single recurrent missense variant (c.4A > G, p.Ser2Gly) in SHOC2, which encodes a leucine-rich repeat-containing protein facilitating signal flow through the RAS-mitogen-associated protein kinase (MAPK) pathway. We also documented that the pathogenic p.Ser2Gly substitution causes upregulation of MAPK signaling and constitutive targeting of SHOC2 to the plasma membrane due to the introduction of an N-myristoylation recognition motif. The almost invariant occurrence of the pathogenic c.4A > G missense change in SHOC2 is mirrored by a relatively homogeneous clinical phenotype of Mazzanti syndrome. Here, we provide new data on the clinical spectrum and molecular diversity of this disorder and functionally characterize new pathogenic variants. The clinical phenotype of six unrelated individuals carrying novel disease-causing SHOC2 variants is delineated, and public and newly collected clinical data are utilized to profile the disorder. In silico, in vitro and in vivo characterization of the newly identified variants provides evidence that the consequences of these missense changes on SHOC2 functional behavior differ from what had been observed for the canonical p.Ser2Gly change but converge toward an enhanced activation of the RAS-MAPK pathway. Our findings expand the molecular spectrum of pathogenic SHOC2 variants, provide a more accurate picture of the phenotypic expression associated with variants in this gene and definitively establish a gain-of-function behavior as the mechanism of disease.


Asunto(s)
Anomalías Múltiples , Péptidos y Proteínas de Señalización Intracelular , Síndrome del Cabello Anágeno Suelto , Anomalías Múltiples/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Síndrome del Cabello Anágeno Suelto/genética , Fenotipo , Proteínas ras/genética , Proteínas ras/metabolismo
10.
Am J Hum Genet ; 108(1): 100-114, 2021 01 07.
Artículo en Inglés | MEDLINE | ID: mdl-33352116

RESUMEN

Chiari I malformation (CM1), the displacement of the cerebellum through the foramen magnum into the spinal canal, is one of the most common pediatric neurological conditions. Individuals with CM1 can present with neurological symptoms, including severe headaches and sensory or motor deficits, often as a consequence of brainstem compression or syringomyelia (SM). We conducted whole-exome sequencing (WES) on 668 CM1 probands and 232 family members and performed gene-burden and de novo enrichment analyses. A significant enrichment of rare and de novo non-synonymous variants in chromodomain (CHD) genes was observed among individuals with CM1 (combined p = 2.4 × 10-10), including 3 de novo loss-of-function variants in CHD8 (LOF enrichment p = 1.9 × 10-10) and a significant burden of rare transmitted variants in CHD3 (p = 1.8 × 10-6). Overall, individuals with CM1 were found to have significantly increased head circumference (p = 2.6 × 10-9), with many harboring CHD rare variants having macrocephaly. Finally, haploinsufficiency for chd8 in zebrafish led to macrocephaly and posterior hindbrain displacement reminiscent of CM1. These results implicate chromodomain genes and excessive brain growth in CM1 pathogenesis.


Asunto(s)
Malformación de Arnold-Chiari/genética , Proteínas de Unión al ADN/genética , Polimorfismo de Nucleótido Simple/genética , Adulto , Animales , Malformación de Arnold-Chiari/patología , Encéfalo/patología , Estudios de Casos y Controles , Femenino , Haploinsuficiencia/genética , Humanos , Imagen por Resonancia Magnética/métodos , Masculino , Siringomielia/genética , Secuenciación del Exoma/métodos , Pez Cebra/genética
11.
Am J Hum Genet ; 108(11): 2112-2129, 2021 11 04.
Artículo en Inglés | MEDLINE | ID: mdl-34626534

RESUMEN

Upregulated signal flow through RAS and the mitogen-associated protein kinase (MAPK) cascade is the unifying mechanistic theme of the RASopathies, a family of disorders affecting development and growth. Pathogenic variants in more than 20 genes have been causally linked to RASopathies, the majority having a dominant role in promoting enhanced signaling. Here, we report that SPRED2 loss of function is causally linked to a recessive phenotype evocative of Noonan syndrome. Homozygosity for three different variants-c.187C>T (p.Arg63∗), c.299T>C (p.Leu100Pro), and c.1142_1143delTT (p.Leu381Hisfs∗95)-were identified in four subjects from three families. All variants severely affected protein stability, causing accelerated degradation, and variably perturbed SPRED2 functional behavior. When overexpressed in cells, all variants were unable to negatively modulate EGF-promoted RAF1, MEK, and ERK phosphorylation, and time-course experiments in primary fibroblasts (p.Leu100Pro and p.Leu381Hisfs∗95) documented an increased and prolonged activation of the MAPK cascade in response to EGF stimulation. Morpholino-mediated knockdown of spred2a and spred2b in zebrafish induced defects in convergence and extension cell movements indicating upregulated RAS-MAPK signaling, which were rescued by expressing wild-type SPRED2 but not the SPRED2Leu381Hisfs∗95 protein. The clinical phenotype of the four affected individuals included developmental delay, intellectual disability, cardiac defects, short stature, skeletal anomalies, and a typical facial gestalt as major features, without the occurrence of the distinctive skin signs characterizing Legius syndrome. These features, in part, characterize the phenotype of Spred2-/- mice. Our findings identify the second recessive form of Noonan syndrome and document pleiotropic consequences of SPRED2 loss of function in development.


Asunto(s)
Mutación con Pérdida de Función , Síndrome de Noonan/genética , Fenotipo , Proteínas Represoras/genética , Alelos , Animales , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Noqueados , Pez Cebra
12.
Am J Hum Genet ; 108(1): 115-133, 2021 01 07.
Artículo en Inglés | MEDLINE | ID: mdl-33308444

RESUMEN

Signal peptide-CUB-EGF domain-containing protein 3 (SCUBE3) is a member of a small family of multifunctional cell surface-anchored glycoproteins functioning as co-receptors for a variety of growth factors. Here we report that bi-allelic inactivating variants in SCUBE3 have pleiotropic consequences on development and cause a previously unrecognized syndromic disorder. Eighteen affected individuals from nine unrelated families showed a consistent phenotype characterized by reduced growth, skeletal features, distinctive craniofacial appearance, and dental anomalies. In vitro functional validation studies demonstrated a variable impact of disease-causing variants on transcript processing, protein secretion and function, and their dysregulating effect on bone morphogenetic protein (BMP) signaling. We show that SCUBE3 acts as a BMP2/BMP4 co-receptor, recruits the BMP receptor complexes into raft microdomains, and positively modulates signaling possibly by augmenting the specific interactions between BMPs and BMP type I receptors. Scube3-/- mice showed craniofacial and dental defects, reduced body size, and defective endochondral bone growth due to impaired BMP-mediated chondrogenesis and osteogenesis, recapitulating the human disorder. Our findings identify a human disease caused by defective function of a member of the SCUBE family, and link SCUBE3 to processes controlling growth, morphogenesis, and bone and teeth development through modulation of BMP signaling.


Asunto(s)
Huesos/metabolismo , Proteínas de Unión al Calcio/metabolismo , Discapacidades del Desarrollo/metabolismo , Osteogénesis/fisiología , Transducción de Señal/fisiología , Animales , Proteína Morfogenética Ósea 2/metabolismo , Proteína Morfogenética Ósea 4/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Línea Celular , Línea Celular Tumoral , Femenino , Regulación del Desarrollo de la Expresión Génica/fisiología , Células HEK293 , Células Hep G2 , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Células MCF-7 , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL
13.
Mol Med ; 30(1): 47, 2024 Apr 09.
Artículo en Inglés | MEDLINE | ID: mdl-38594640

RESUMEN

BACKGROUND: RASopathies are genetic syndromes affecting development and having variable cancer predisposition. These disorders are clinically related and are caused by germline mutations affecting key players and regulators of the RAS-MAPK signaling pathway generally leading to an upregulated ERK activity. Gain-of-function (GOF) mutations in PTPN11, encoding SHP2, a cytosolic protein tyrosine phosphatase positively controlling RAS function, underlie approximately 50% of Noonan syndromes (NS), the most common RASopathy. A different class of these activating mutations occurs as somatic events in childhood leukemias. METHOD: Here, we evaluated the application of a FRET-based zebrafish ERK reporter, Teen, and used quantitative FRET protocols to monitor non-physiological RASopathy-associated changes in ERK activation. In a multi-level experimental workflow, we tested the suitability of the Teen reporter to detect pan-embryo ERK activity correlates of morphometric alterations driven by the NS-causing Shp2D61G allele. RESULTS: Spectral unmixing- and acceptor photobleaching (AB)-FRET analyses captured pathological ERK activity preceding the manifestation of quantifiable body axes defects, a morphological pillar used to test the strength of SHP2 GoF mutations. Last, the work shows that by multi-modal FRET analysis, we can quantitatively trace back the modulation of ERK phosphorylation obtained by low-dose MEK inhibitor treatment to early development, before the onset of morphological defects. CONCLUSION: This work proves the usefulness of FRET imaging protocols on both live and fixed Teen ERK reporter fish to readily monitor and quantify pharmacologically- and genetically-induced ERK activity modulations in early embryos, representing a useful tool in pre-clinical applications targeting RAS-MAPK signaling.


Asunto(s)
Síndrome de Noonan , Pez Cebra , Animales , Humanos , Adolescente , Pez Cebra/genética , Pez Cebra/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Síndrome de Noonan/genética , Mutación , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo
14.
Genet Med ; : 101283, 2024 Sep 28.
Artículo en Inglés | MEDLINE | ID: mdl-39355979

RESUMEN

BACKGROUND: ARID1A/ARID1B haploinsufficiency leads to Coffin-Siris syndrome, duplications of ARID1A lead to a distinct clinical syndrome, whilst ARID1B duplications have not yet been linked to a phenotype. METHODS: We collected patients with duplications encompassing ARID1A and ARID1B duplications. RESULTS: 16 ARID1A and 13 ARID1B duplication cases were included with duplication sizes ranging from 0.1-1.2 Mb(1-44 genes) for ARID1A and 0.9-10.3 Mb(2-101 genes) for ARID1B. Both groups shared features, with ARID1A patients having more severe intellectual disability, growth delay and congenital anomalies. DNA methylation analysis showed that ARID1A patients had a specific methylation pattern in blood, which differed from controls and from patients with ARID1A or ARID1B loss-of-function variants. ARID1B patients appeared to have a distinct methylation pattern, similar to ARID1A duplication patients, but further research is needed to validate these results. Five cases with duplications including ARID1A or ARID1B initially annotated as duplications of uncertain significance were evaluated using PhenoScore and DNA methylation re-analysis, resulting in the reclassification of two ARID1A and two ARID1B duplications as pathogenic. CONCLUSION: Our findings reveal that ARID1B duplications manifest a clinical phenotype and ARID1A duplications have a distinct episignature that overlaps with that of ARID1B duplications, providing further evidence for a distinct and emerging BAFopathy caused by whole gene duplication rather than haploinsufficiency.

15.
Genet Med ; 26(11): 101241, 2024 Aug 10.
Artículo en Inglés | MEDLINE | ID: mdl-39140257

RESUMEN

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.

16.
Mov Disord ; 39(7): 1225-1231, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-38685873

RESUMEN

BACKGROUND: The MRPS36 gene encodes a recently identified component of the 2-oxoglutarate dehydrogenase complex (OGDHC), a key enzyme of the Krebs cycle catalyzing the oxidative decarboxylation of 2-oxoglutarate to succinyl-CoA. Defective OGDHC activity causes a clinically variable metabolic disorder characterized by global developmental delay, severe neurological impairment, liver failure, and early-onset lactic acidosis. METHODS: We investigated the molecular cause underlying Leigh syndrome with bilateral striatal necrosis in two siblings through exome sequencing. Functional studies included measurement of the OGDHC enzymatic activity and MRPS36 mRNA levels in fibroblasts, assessment of protein stability in transfected cells, and structural analysis. A literature review was performed to define the etiological and phenotypic spectrum of OGDHC deficiency. RESULTS: In the two affected brothers, exome sequencing identified a homozygous nonsense variant (c.283G>T, p.Glu95*) of MRPS36. The variant did not affect transcript processing and stability, nor protein levels, but resulted in a shorter protein lacking nine residues that contribute to the structural and functional organization of the OGDHC complex. OGDHC enzymatic activity was significantly reduced. The review of previously reported cases of OGDHC deficiency supports the association of this enzymatic defect with Leigh phenotypic spectrum and early-onset movement disorder. Slightly elevated plasma levels of glutamate and glutamine were observed in our and literature patients with OGDHC defect. CONCLUSIONS: Our findings point to MRPS36 as a new disease gene implicated in Leigh syndrome. The slight elevation of plasma levels of glutamate and glutamine observed in patients with OGDHC deficiency represents a candidate metabolic signature of this neurometabolic disorder. © 2024 International Parkinson and Movement Disorder Society.


Asunto(s)
Complejo Cetoglutarato Deshidrogenasa , Enfermedad de Leigh , Enfermedad de Leigh/genética , Humanos , Masculino , Complejo Cetoglutarato Deshidrogenasa/genética , Complejo Cetoglutarato Deshidrogenasa/deficiencia , Proteínas Mitocondriales/genética , Preescolar , Lactante
17.
Am J Med Genet A ; 194(8): e63627, 2024 08.
Artículo en Inglés | MEDLINE | ID: mdl-38613168

RESUMEN

Casitas B-lineage lymphoma (CBL) encodes an adaptor protein with E3-ligase activity negatively controlling intracellular signaling downstream of receptor tyrosine kinases. Somatic CBL mutations play a driver role in a variety of cancers, particularly myeloid malignancies, whereas germline defects in the same gene underlie a RASopathy having clinical overlap with Noonan syndrome (NS) and predisposing to juvenile myelomonocytic leukemia and vasculitis. Other features of the disorder include cardiac defects, postnatal growth delay, cryptorchidism, facial dysmorphisms, and predisposition to develop autoimmune disorders. Here we report a novel CBL variant (c.1202G>T; p.Cys401Phe) occurring de novo in a subject with café-au-lait macules, feeding difficulties, mild dysmorphic features, psychomotor delay, autism spectrum disorder, thrombocytopenia, hepatosplenomegaly, and recurrent hypertransaminasemia. The identified variant affects an evolutionarily conserved residue located in the RING finger domain, a known mutational hot spot of both germline and somatic mutations. Functional studies documented enhanced EGF-induced ERK phosphorylation in transiently transfected COS1 cells. The present findings further support the association of pathogenic CBL variants with immunological and hematological manifestations in the context of a presentation with only minor findings reminiscent of NS or a clinically related RASopathy.


Asunto(s)
Mutación de Línea Germinal , Proteínas Proto-Oncogénicas c-cbl , Humanos , Proteínas Proto-Oncogénicas c-cbl/genética , Mutación de Línea Germinal/genética , Masculino , Síndrome de Noonan/genética , Síndrome de Noonan/patología , Trastorno del Espectro Autista/genética , Trastorno del Espectro Autista/patología , Trastorno del Espectro Autista/inmunología , Trastorno del Espectro Autista/sangre , Predisposición Genética a la Enfermedad , Preescolar , Niño , Animales , Fenotipo , Células COS , Trombocitopenia/genética , Trombocitopenia/patología
18.
Am J Med Genet A ; 194(11): e63810, 2024 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-38958480

RESUMEN

Noonan syndrome (NS) is an autosomal dominant condition characterized by facial dysmorphism, congenital heart disease, development delay, growth retardation and lymphatic disease. It is caused by germline pathogenic variants in genes encoding proteins in the Ras/mitogen-activated protein kinase signaling pathway. Nerve enlargement is not generally considered as a feature of NS, although some cases have been reported. High-resolution nerve ultrasound enables detailed anatomical assessment of peripheral nerves and can show enlarged nerves. This retrospective cohort study aims to describe the sonographic findings of patients with NS performed during a 1-year time period. Data on the degree of enlargement, the relation to increasing age, pain in extremities, genotype on the gene level and clinical features were collected. Twenty-nine of 93 patients visiting the NS Center of Expertise of the Radboud University Medical Center Nijmegen underwent high-resolution ultrasound. In 24 patients (83%) nerve enlargement was found. Most of them experienced pain. We observed a weak correlation with increasing age and the degree of nerve enlargement but no association with pain, genotype at the gene level or clinical features. This study shows that patients with NS have a high predisposition for sonographic nerve enlargement and that the majority experience pain.


Asunto(s)
Síndrome de Noonan , Humanos , Síndrome de Noonan/genética , Síndrome de Noonan/patología , Masculino , Femenino , Niño , Adolescente , Preescolar , Adulto , Estudios Retrospectivos , Ultrasonografía , Lactante , Adulto Joven , Nervios Periféricos/patología , Nervios Periféricos/diagnóstico por imagen , Genotipo
19.
Int J Mol Sci ; 25(15)2024 Jul 30.
Artículo en Inglés | MEDLINE | ID: mdl-39125883

RESUMEN

Bardet-Biedl syndrome (BBS) is a rare recessive multisystem disorder characterized by retinitis pigmentosa, obesity, postaxial polydactyly, cognitive deficits, and genitourinary defects. BBS is clinically variable and genetically heterogeneous, with 26 genes identified to contribute to the disorder when mutated, the majority encoding proteins playing role in primary cilium biogenesis, intraflagellar transport, and ciliary trafficking. Here, we report on an 18-year-old boy with features including severe photophobia and central vision loss since childhood, hexadactyly of the right foot and a supernumerary nipple, which were suggestive of BBS. Genetic analyses using targeted resequencing and exome sequencing failed to provide a conclusive genetic diagnosis. Whole-genome sequencing (WGS) allowed us to identify compound heterozygosity for a missense variant and a large intragenic deletion encompassing exon 12 in BBS9 as underlying the condition. We assessed the functional impact of the identified variants and demonstrated that they impair BBS9 function, with significant consequences for primary cilium formation and morphology. Overall, this study further highlights the usefulness of WGS in the diagnostic workflow of rare diseases to reach a definitive diagnosis. This report also remarks on a requirement for functional validation analyses to more effectively classify variants that are identified in the frame of the diagnostic workflow.


Asunto(s)
Síndrome de Bardet-Biedl , Secuenciación Completa del Genoma , Síndrome de Bardet-Biedl/genética , Síndrome de Bardet-Biedl/diagnóstico , Humanos , Masculino , Adolescente , Cilios/patología , Cilios/genética , Proteínas del Citoesqueleto
20.
Int J Mol Sci ; 25(17)2024 Sep 05.
Artículo en Inglés | MEDLINE | ID: mdl-39273584

RESUMEN

Multiple acyl-CoA dehydrogenase deficiency (MADD) is a rare inborn error of metabolism affecting fatty acid and amino acid oxidation with an incidence of 1 in 200,000 live births. MADD has three clinical phenotypes: severe neonatal-onset with or without congenital anomalies, and a milder late-onset form. Clinical diagnosis is supported by urinary organic acid and blood acylcarnitine analysis using tandem mass spectrometry in newborn screening programs. MADD is an autosomal recessive trait caused by biallelic mutations in the ETFA, ETFB, and ETFDH genes encoding the alpha and beta subunits of the electron transfer flavoprotein (ETF) and ETF-coenzyme Q oxidoreductase enzymes. Despite significant advancements in sequencing techniques, many patients remain undiagnosed, impacting their access to clinical care and genetic counseling. In this report, we achieved a definitive molecular diagnosis in a newborn by combining whole-genome sequencing (WGS) with RNA sequencing (RNA-seq). Whole-exome sequencing and next-generation gene panels fail to detect variants, possibly affecting splicing, in deep intronic regions. Here, we report a unique deep intronic mutation in intron 1 of the ETFDH gene, c.35-959A>G, in a patient with early-onset lethal MADD, resulting in pseudo-exon inclusion. The identified variant is the third mutation reported in this region, highlighting ETFDH intron 1 vulnerability. It cannot be excluded that these intronic sequence features may be more common in other genes than is currently believed. This study highlights the importance of incorporating RNA analysis into genome-wide testing to reveal the functional consequences of intronic mutations.


Asunto(s)
Flavoproteínas Transportadoras de Electrones , Intrones , Proteínas Hierro-Azufre , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH , Humanos , Deficiencia Múltiple de Acil Coenzima A Deshidrogenasa/genética , Flavoproteínas Transportadoras de Electrones/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Proteínas Hierro-Azufre/genética , Intrones/genética , Recién Nacido , Mutación , Masculino , Femenino , Secuenciación Completa del Genoma
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