Variant-specific effects define the phenotypic spectrum of HNRNPH2-associated neurodevelopmental disorders in males

AbstractBain type of X-linked syndromic intellectual developmental disorder, caused by pathogenic missense variants in HRNRPH2, was initially described in six female individuals affected by moderate-to-severe neurodevelopmental delay. Although it was initially postulated that the condition would not be compatible with life in males, several affected male individuals harboring pathogenic variants in HNRNPH2 have since been documented. However, functional in-vitro analyses of identified variants have not been performed and, therefore, possible genotype–phenotype correlations remain elusive. Here, we present eight male individuals, including a pair of monozygotic twins, harboring pathogenic or likely pathogenic HNRNPH2 variants. Notably, we present the first individuals harboring nonsense or frameshift variants who, similarly to an individual harboring a de novo p.(Arg29Cys) variant within the first quasi-RNA-recognition motif (qRRM), displayed mild developmental delay, and developed mostly autistic features and/or psychiatric co-morbidities. Additionally, we present two individuals harboring a recurrent de novo p.(Arg114Trp), within the second qRRM, who had a severe neurodevelopmental delay with seizures. Functional characterization of the three most common HNRNPH2 missense variants revealed dysfunctional nucleocytoplasmic shuttling of proteins harboring the p.(Arg206Gln) and p.(Pro209Leu) variants, located within the nuclear localization signal, whereas proteins with p.(Arg114Trp) showed reduced interaction with members of the large assembly of splicing regulators (LASR). Moreover, RNA-sequencing of primary fibroblasts of the individual harboring the p.(Arg114Trp) revealed substantial alterations in the regulation of alternative splicing along with global transcriptome changes. Thus, we further expand the clinical and variant spectrum in HNRNPH2-associated disease in males and provide novel molecular insights suggesting the disorder to be a spliceopathy on the molecular level.

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Loss-of-function and missense variants in NSD2 cause decreased methylation activity and are associated with a distinct developmental phenotype

Genetics in Medicine ◽

10.1038/s41436-021-01158-1 ◽

2021 ◽

Author(s):

Paolo Zanoni ◽

Katharina Steindl ◽

Deepanwita Sengupta ◽

Pascal Joset ◽

Angela Bahr ◽

...

Keyword(s):

Loss Of Function ◽

Mild Phenotype ◽

Psychological Issues ◽

Missense Variants ◽

Mild Developmental Delay ◽

Pathogenic Variants ◽

In Silico Modeling ◽

And Function ◽

Methylation Activity

Abstract Purpose Despite a few recent reports of patients harboring truncating variants in NSD2, a gene considered critical for the Wolf–Hirschhorn syndrome (WHS) phenotype, the clinical spectrum associated with NSD2 pathogenic variants remains poorly understood. Methods We collected a comprehensive series of 18 unpublished patients carrying heterozygous missense, elongating, or truncating NSD2 variants; compared their clinical data to the typical WHS phenotype after pooling them with ten previously described patients; and assessed the underlying molecular mechanism by structural modeling and measuring methylation activity in vitro. Results The core NSD2-associated phenotype includes mostly mild developmental delay, prenatal-onset growth retardation, low body mass index, and characteristic facial features distinct from WHS. Patients carrying missense variants were significantly taller and had more frequent behavioral/psychological issues compared with those harboring truncating variants. Structural in silico modeling suggested interference with NSD2’s folding and function for all missense variants in known structures. In vitro testing showed reduced methylation activity and failure to reconstitute H3K36me2 in NSD2 knockout cells for most missense variants. Conclusion NSD2 loss-of-function variants lead to a distinct, rather mild phenotype partially overlapping with WHS. To avoid confusion for patients, NSD2 deficiency may be named Rauch–Steindl syndrome after the delineators of this phenotype.

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MED12-Related (Neuro)Developmental Disorders: A Question of Causality

Genes ◽

10.3390/genes12050663 ◽

2021 ◽

Vol 12 (5) ◽

pp. 663

Author(s):

Stijn van de Plassche ◽

Arjan PM de Brouwer

Keyword(s):

Developmental Disorders ◽

De Novo ◽

Expression Patterns ◽

Mediator Complex ◽

Gene Expression Patterns ◽

Facial Dysmorphism ◽

Regulation Of Transcription ◽

Feeding Difficulties ◽

Missense Variants ◽

Pathogenic Variants

MED12 is a member of the Mediator complex that is involved in the regulation of transcription. Missense variants in MED12 cause FG syndrome, Lujan-Fryns syndrome, and Ohdo syndrome, as well as non-syndromic intellectual disability (ID) in hemizygous males. Recently, female patients with de novo missense variants and de novo protein truncating variants in MED12 were described, resulting in a clinical spectrum centered around ID and Hardikar syndrome without ID. The missense variants are found throughout MED12, whether they are inherited in hemizygous males or de novo in females. They can result in syndromic or nonsyndromic ID. The de novo nonsense variants resulting in Hardikar syndrome that is characterized by facial clefting, pigmentary retinopathy, biliary anomalies, and intestinal malrotation, are found more N-terminally, whereas the more C-terminally positioned variants are de novo protein truncating variants that cause a severe, syndromic phenotype consisting of ID, facial dysmorphism, short stature, skeletal abnormalities, feeding difficulties, and variable other abnormalities. This broad range of distinct phenotypes calls for a method to distinguish between pathogenic and non-pathogenic variants in MED12. We propose an isogenic iNeuron model to establish the unique gene expression patterns that are associated with the specific MED12 variants. The discovery of these patterns would help in future diagnostics and determine the causality of the MED12 variants.

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Heterozygous Missense Pathogenic Variants Within the Second Spectrin Repeat of SPTBN2 Lead to Infantile-Onset Cerebellar Ataxia

Journal of Child Neurology ◽

10.1177/0883073819878917 ◽

2019 ◽

Vol 35 (2) ◽

pp. 106-110 ◽

Cited By ~ 1

Author(s):

Andrea Accogli ◽

Judith St-Onge ◽

Nassima Addour-Boudrahem ◽

Joël Lafond-Lapalme ◽

Alexandre Dionne Laporte ◽

...

Keyword(s):

Cognitive Impairment ◽

Cerebellar Ataxia ◽

De Novo ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Adult Onset ◽

Spinocerebellar Ataxias ◽

Spectrin Repeat ◽

Missense Variants ◽

Pathogenic Variants

The term spinocerebellar ataxia encompasses a heterogeneous group of neurodegenerative disorders due to pathogenic variants in more than 100 genes, underlying 2 major groups of ataxia: autosomal dominant cerebellar ataxias (ADCA, also known as spinocerebellar ataxias [SCAs]) due to heterozygous variants or polyglutamine triplet expansions leading to adult-onset ataxia, and autosomal recessive spinocerebellar ataxias (ARCAs, also known as SCARs) due to biallelic variants, usually resulting in more severe and earlier-onset cerebellar ataxia. Certain ataxia genes, including SPTBN2 which encodes β-III spectrin, are responsible for both SCA and SCAR, depending on whether the pathogenic variant occurs in a monoallelic or biallelic state, respectively. Accordingly, 2 major phenotypes have been linked to SPTBN2: pathogenic heterozygous in-frame deletions and missense variants result in an adult-onset, slowly progressive ADCA (SCA5) through a dominant negative effect, whereas biallelic loss-of-function variants cause SCAR14, an allelic disorder characterized by infantile-onset cerebellar ataxia and cognitive impairment. Of note, 2 heterozygous missense variants (c.1438C>T, p.R480 W; c.1309C>G, p.R437G), both lying in the second spectrin repeat of SPTBN2, have been linked to infantile-onset cerebellar ataxia, similar to SCAR14. Here, we report a novel de novo heterozygous pathogenic missense variant (c.1310G>A) in SPTBN2 in a child with infantile-onset cerebellar ataxia and mild cognitive impairment. This variant affects the same R437 residue of the second spectrin repeat but results in a different amino acid change (p.R437Q). We review previously reported cases and discuss possible pathomechanisms responsible for the early-onset cerebellar phenotype due to disease-causing variants in the second spectrin repeat.

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KVarPredDB: a database for predicting pathogenicity of missense sequence variants of keratin genes associated with genodermatoses

Human Genomics ◽

10.1186/s40246-020-00295-z ◽

2020 ◽

Vol 14 (1) ◽

Author(s):

Yuyi Ying ◽

Lu Lu ◽

Santasree Banerjee ◽

Lizhen Xu ◽

Qiang Zhao ◽

...

Keyword(s):

Amino Acids ◽

Web Application ◽

Functional Characterization ◽

Dominant Mode ◽

Heptad Repeat ◽

Wild Type ◽

Application Development ◽

Missense Variants ◽

Pathogenic Variants ◽

Development Framework

Abstract Background Germline variants of ten keratin genes (K1, K2, K5, K6A, K6B, K9, K10, K14, K16, and K17) have been reported for causing different types of genodermatoses with an autosomal dominant mode of inheritance. Among all the variants of these ten keratin genes, most of them are missense variants. Unlike pathogenic and likely pathogenic variants, understanding the clinical importance of novel missense variants or variants of uncertain significance (VUS) is the biggest challenge for clinicians or medical geneticists. Functional characterization is the only way to understand the clinical association of novel missense variants or VUS but it is time consuming, costly, and depends on the availability of patient’s samples. Existing databases report the pathogenic variants of the keratin genes, but never emphasize the systematic effects of these variants on keratin protein structure and genotype-phenotype correlation. Results To address this need, we developed a comprehensive database KVarPredDB, which contains information of all ten keratin genes associated with genodermatoses. We integrated and curated 400 reported pathogenic missense variants as well as 4629 missense VUS. KVarPredDB predicts the pathogenicity of novel missense variants as well as to understand the severity of disease phenotype, based on four criteria; firstly, the difference in physico-chemical properties between the wild type and substituted amino acids; secondly, the loss of inter/intra-chain interactions; thirdly, evolutionary conservation of the wild type amino acids and lastly, the effect of the substituted amino acids in the heptad repeat. Molecular docking simulations based on resolved crystal structures were adopted to predict stability changes and get the binding energy to compare the wild type protein with the mutated one. We use this basic information to determine the structural and functional impact of novel missense variants on the keratin coiled-coil heterodimer. KVarPredDB was built under the integrative web application development framework SSM (SpringBoot, Spring MVC, MyBatis) and implemented in Java, Bootstrap, React-mutation-mapper, MySQL, Tomcat. The website can be accessed through http://bioinfo.zju.edu.cn/KVarPredDB. The genomic variants and analysis results are freely available under the Creative Commons license. Conclusions KVarPredDB provides an intuitive and user-friendly interface with computational analytical investigation for each missense variant of the keratin genes associated with genodermatoses.

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Functional characterization of BRCC3 mutations in acute myeloid leukemia with t(8;21)(q22;q22.1)

Leukemia ◽

10.1038/s41375-019-0578-6 ◽

2019 ◽

Vol 34 (2) ◽

pp. 404-415 ◽

Cited By ~ 6

Author(s):

Tatjana Meyer ◽

Nikolaus Jahn ◽

Stefanie Lindner ◽

Linda Röhner ◽

Anna Dolnik ◽

...

Keyword(s):

Dna Damage ◽

Cell Lines ◽

De Novo ◽

Functional Characterization ◽

Interferon Response ◽

Excellent Outcome ◽

Recurrent Mutations ◽

Primary Mouse ◽

Inflammasome Activity

Abstract BRCA1/BRCA2-containing complex 3 (BRCC3) is a Lysine 63-specific deubiquitinating enzyme (DUB) involved in inflammasome activity, interferon signaling, and DNA damage repair. Recurrent mutations in BRCC3 have been reported in myelodysplastic syndromes (MDS) but not in de novo AML. In one of our recent studies, we found BRCC3 mutations selectively in 9/191 (4.7%) cases with t(8;21)(q22;q22.1) AML but not in 160 cases of inv(16)(p13.1q22) AML. Clinically, AML patients with BRCC3 mutations had an excellent outcome with an event-free survival of 100%. Inactivation of BRCC3 by CRISPR/Cas9 resulted in improved proliferation in t(8;21)(q22;q22.1) positive AML cell lines and together with expression of AML1-ETO induced unlimited self-renewal in mouse hematopoietic progenitor cells in vitro. Mutations in BRCC3 abrogated its deubiquitinating activity on IFNAR1 resulting in an impaired interferon response and led to diminished inflammasome activity. In addition, BRCC3 inactivation increased release of several cytokines including G-CSF which enhanced proliferation of AML cell lines with t(8;21)(q22;q22.1). Cell lines and primary mouse cells with inactivation of BRCC3 had a higher sensitivity to doxorubicin due to an impaired DNA damage response providing a possible explanation for the favorable outcome of BRCC3 mutated AML patients.

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Clear evidence of LAMA5 gene biallelic truncating variants causing infantile nephrotic syndrome

Kidney360 ◽

10.34067/kid.0004952021 ◽

2021 ◽

pp. 10.34067/KID.0004952021

Author(s):

Yukimasa Taniguchi ◽

China Nagano ◽

Kiyotoshi Sekiguchi ◽

Atsushi Tashiro ◽

Noriko Sugawara ◽

...

Keyword(s):

Nephrotic Syndrome ◽

Splice Site ◽

Japanese Patients ◽

Compound Heterozygous ◽

Missense Variants ◽

Targeted Next Generation Sequencing ◽

Pathogenic Variants ◽

Infantile Nephrotic Syndrome ◽

Splice Site Variant

Background: Pathogenic variants in single genes encoding podocyte-associated proteins have been implicated in about 30% of steroid resistant nephrotic syndrome (SRNS) patients in children. However, LAMA5 gene biallelic variants have been identified in only 7 patients so far, and most are missense variants of unknown significance. Furthermore, no functional analysis had been conducted for all but one of these variants. Here, we report three patients with LAMA5 gene biallelic truncating variants manifesting infantile nephrotic syndrome and one SRNS case with biallelic LAMA5 missense variants. Methods: We conducted comprehensive gene screening of Japanese patients with severe proteinuria. Using targeted next-generation sequencing, 62 podocyte-related genes were screened in 407 unrelated patients with proteinuria. For the newly discovered LAMA5 variants, we conducted in vitro heterotrimer formation assays. Results: Biallelic truncating variants in the LAMA5 gene (NM_005560) were detected in 3 patients from 2 families. All patients presented with proteinuria within 6 months of age. Patients 1 and 2 were siblings possessing a nonsense variant (c.9232C>T, p.(Arg3078*)) and a splice site variant (c.1282+1G>A) that led to exon 9 skipping and a frameshift. Patient 3 had a remarkable irregular contour of the glomerular basement membrane. She was subsequently found to have a nonsense variant (c.8185C>T, p.(Arg2720*)) and the same splice site variant in patients 1 and 2. By in vitro heterotrimer formation assays, both truncating variants produced smaller laminin α5 proteins that nevertheless formed trimers with laminin β1 and γ1 chains. Patient 4 showed SRNS at the age of eight and carried compound heterozygous missense variants (c.1493C>T, p.(Ala498Val) and c.8399G>A, p.(Arg2800His)). Conclusions: Our patients showed clear evidence of biallelic LAMA5 truncating variants causing infantile nephrotic syndrome. We also discerned the clinical and pathological characteristics observed in LAMA5-related nephropathy. LAMA5 variant screening should be performed in congenital/infantile nephrotic syndrome patients.

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KVarPredDB: A database for predicting pathogenicity of missense sequence variants of keratin genes associated with genodermatoses

10.21203/rs.3.rs-68420/v2 ◽

2020 ◽

Author(s):

Yuyi Ying ◽

Lu Lu ◽

Santasree Banerjee ◽

Lizhen Xu ◽

Qiang Zhao ◽

...

Keyword(s):

Amino Acids ◽

Web Application ◽

Functional Characterization ◽

Dominant Mode ◽

Heptad Repeat ◽

Wild Type ◽

Application Development ◽

Missense Variants ◽

Pathogenic Variants ◽

Development Framework

Abstract Background: Germline variants of ten keratin genes (K1, K2, K5, K6A, K6B, K9, K10, K14, K16 and K17) have been reported for causing different types of genodermatoses with an autosomal dominant mode of inheritance. Among all the variants of these ten keratin genes, most of them are missense variants. Unlike pathogenic and likely pathogenic variants, understanding the clinical importance of novel missense variants or variants of uncertain significance (VUS) is the biggest challenge for clinicians or medical geneticists. Functional characterization is the only way to understand the clinical association of novel missense variants or VUS but it is time-consuming, costly and depends on the availability of patient’s samples. Existing databases report the pathogenic variants of the keratin genes, but never emphasize the systematic effects of these variants on keratin protein structure and genotype-phenotype correlation.Results: To address this need, we developed a comprehensive database KVarPredDB, which contains information of all ten keratin genes associated with genodermatoses. We integrated and curated 400 reported pathogenic missense variants as well as 4629 missense VUS. KVarPredDB predicts the pathogenicity of novel missense variants as well as to understand the severity of disease phenotype, based on four criteria; firstly, difference in physico-chemical properties between the wild-type and substituted amino acids, secondly, the loss of inter/intra-chain interactions, thirdly, evolutionary conservation of the wild type amino acids and lastly, the effect of the substituted amino acids in the heptad repeat. Molecular docking simulations based on resolved crystal structures were adopted to predict stability changes and get the binding energy to compare the wild type protein with the mutated one. We use this basic information to determine the structural and functional impact of novel missense variants on the keratin coiled-coil heterodimer. KVarPredDB was built under the integrative web application development framework SSM (SpringBoot, Spring MVC, MyBatis) and implemented in Java, Bootstrap, React-mutation-mapper, MySQL, Tomcat. The website can be accessed through http://bioinfo.zju.edu.cn/KVarPredDB. The genomic variants and analysis results are freely available under the Creative Commons license.Conclusions: KVarPredDB provides an intuitive and user-friendly interface with computational analytical investigation for each missense variant of the keratin genes associated with genodermatoses.

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KVarPredDB: A Database for Predicting Pathogenicity of Missense Sequence Variants of Keratin Genes Associated With Genodermatoses

10.21203/rs.3.rs-68420/v1 ◽

2020 ◽

Author(s):

Santasree Banerjee ◽

Yuyi Ying ◽

Lu Lu ◽

Lizhen Xu ◽

Qiang Zhao ◽

...

Keyword(s):

Amino Acids ◽

Web Application ◽

Functional Characterization ◽

Dominant Mode ◽

Heptad Repeat ◽

Wild Type ◽

Application Development ◽

Missense Variants ◽

Pathogenic Variants ◽

Development Framework

Abstract Background: Germline variants of ten keratin genes (K1, K2, K5, K6A, K6B, K9, K10, K14, K16 and K17) have been reported for causing different types of genodermatoses with an autosomal dominant mode of inheritance. Among all the variants of these ten keratin genes, most of them are missense variants. Unlike pathogenic and likely pathogenic variants, understanding the clinical importance of novel missense variants or variants of uncertain significance (VUS) is the biggest challenge for clinicians or medical geneticists. Functional characterization is the only way to understand the clinical association of novel missense variants or VUS but it is time-consuming, costly and depends on the availability of patient’s samples. Existing databases report the pathogenic variants of the keratin genes, but never emphasize the systematic effects of these variants on keratin protein structure and genotype-phenotype correlation. Results: To address this need, we developed a comprehensive database KVarPredDB, which contains information of all ten keratin genes associated with genodermatoses. We integrated and curated 398 reported pathogenic missense variants as well as 4629 missense VUS. KVarPredDB predicts the pathogenicity of novel missense variants as well as to understand the severity of disease phenotype, based on four criteria; firstly, difference in physico-chemical properties between the wild-type and substituted amino acids, secondly, the loss of inter/intra-chain interactions, thirdly, evolutionary conservation of the wild type amino acids and lastly, the effect of the substituted mutation in the heptad repeat. Molecular docking simulations based on resolved crystal structures were adopted to predict stability changes and get the binding energy to compare the wild type protein with the mutated one. We use this basic information to determine the structural and functional impact of novel missense variants on the keratin coiled-coil heterodimer. KVarPredDB was implemented in Java, Bootstrap, React-mutation-mapper, common MySQL, Tomcat, and under the integrative web application development framework SSM (SpringBoot, Spring MVC, MyBatis). The web site can be accessed through http://bioinfo.zju.edu.cn/KVarPredDB. The genomic variants and analysis results are freely available under the Creative Commons license.Conclusions: KVarPredDB provides an intuitive and user-friendly interface with computational analytical investigation for each missense variant of the keratin genes associated with genodermatoses.

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Functional CDKN2A assay identifies frequent deleterious alleles misclassified as variants of uncertain significance

eLife ◽

10.7554/elife.71137 ◽

2022 ◽

Vol 11 ◽

Author(s):

Hirokazu Kimura ◽

Raymond M Paranal ◽

Neha Nanda ◽

Laura D Wood ◽

James R Eshleman ◽

...

Keyword(s):

Functional Characterization ◽

Ductal Adenocarcinoma ◽

Proliferation Assay ◽

Variants Of Uncertain Significance ◽

Pathogenic Variants ◽

Deleterious Alleles ◽

Increased Risk ◽

Uncertain Significance

Pathogenic germline CDKN2A variants are associated with an increased risk of pancreatic ductal adenocarcinoma (PDAC). CDKN2A variants of uncertain significance (VUSs) are reported in up to 4.3% of patients with PDAC and result in significant uncertainty for patients and their family members as an unknown fraction are functionally deleterious, and therefore, likely pathogenic. Functional characterization of CDKN2A VUSs is needed to reclassify variants and inform clinical management. 29 germline CDKN2A VUSs previously reported in patients with PDAC or in ClinVar were evaluated using a validated in vitro cell proliferation assay. 12 of the 29 CDKN2A VUSs were functionally deleterious (11 VUSs) or potentially functionally deleterious (1 VUS) and were reclassified as likely pathogenic variants. Thus, over 40% of CDKN2A VUSs identified in patients with PDAC are functionally deleterious and likely pathogenic. When incorporating VUSs found to be functionally deleterious, and reclassified as likely pathogenic, the prevalence of pathogenic/likely pathogenic CDKN2A in patients with PDAC reported in the published literature is increased to up to 4.1% of patients, depending on family history. Therefore, CDKN2A VUSs may play a significant, unappreciated role in risk of pancreatic cancer. These findings have significant implications for the counselling and care of patients and their relatives.

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Cell-based analysis of CAD variants identifies individuals likely to benefit from uridine therapy

Genetics in Medicine ◽

10.1038/s41436-020-0833-2 ◽

2020 ◽

Vol 22 (10) ◽

pp. 1598-1605 ◽

Cited By ~ 3

Author(s):

Francisco del Caño-Ochoa ◽

Bobby G. Ng ◽

Malak Abedalthagafi ◽

Mohammed Almannai ◽

Ronald D. Cohn ◽

...

Keyword(s):

Autosomal Recessive ◽

Clinical Presentation ◽

De Novo ◽

Clinical Phenotype ◽

Transient Transfection ◽

Missense Variants ◽

De Novo Biosynthesis ◽

Pathogenic Variants ◽

Large Size ◽

A Cell

Abstract Purpose Pathogenic autosomal recessive variants in CAD, encoding the multienzymatic protein initiating pyrimidine de novo biosynthesis, cause a severe inborn metabolic disorder treatable with a dietary supplement of uridine. This condition is difficult to diagnose given the large size of CAD with over 1000 missense variants and the nonspecific clinical presentation. We aimed to develop a reliable and discerning assay to assess the pathogenicity of CAD variants and to select affected individuals that might benefit from uridine therapy. Methods Using CRISPR/Cas9, we generated a human CAD-knockout cell line that requires uridine supplements for survival. Transient transfection of the knockout cells with recombinant CAD restores growth in absence of uridine. This system determines missense variants that inactivate CAD and do not rescue the growth phenotype. Results We identified 25 individuals with biallelic variants in CAD and a phenotype consistent with a CAD deficit. We used the CAD-knockout complementation assay to test a total of 34 variants, identifying 16 as deleterious for CAD activity. Combination of these pathogenic variants confirmed 11 subjects with a CAD deficit, for whom we describe the clinical phenotype. Conclusions We designed a cell-based assay to test the pathogenicity of CAD variants, identifying 11 CAD-deficient individuals who could benefit from uridine therapy.

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