DPH1 syndrome: two novel variants and structural and functional analyses of seven missense variants identified in syndromic patients

Abstract # Authors contributed equally to this work. ~ Currently at Genomics England Ltd, London, United Kingdom Next generation sequencing is transforming our understanding of human genetic variation and is becoming a routine part of human genetic analysis. The identification of millions of new variants, which are mainly rare and assessing their implications for human health presents new challenges to researchers and clinicians. We have analyzed missense variants in the ITGB2A and ITGB3 genes obtained from whole exome and whole genome sequencing (WES & WGS) data from 5 databases: The Human Genome Mutation Database, the 1000 Genomes project, the UK10K Whole Exome Sequencing project, the UK10K Whole Genome Sequencing project, and The National Heart, Lung and Blood Institute Exome Sequencing Project. Together, these encompass variants of the platelet αIIbβ3 integrin receptor from ~32,000 alleles derived from 16,108 individuals. We identified 111 missense variants that have previously been associated with Glanzmann thrombasthenia (GT), 20 variants associated with alloimmune thrombocytopenia, and 5 variants associated with aniso/macrothrombocytopenia. None of the GT variants were found in the last four databases, indicating that they have minor allele frequencies (MAF) less than ~0.01%, attesting to both their rarity and the likelihood that they entered the population within the last ~2,500 years. We also identified 114 novel missense variants in ITGB2A affecting ~11% of the amino acids and 68 novel missense variants in ITGB3 affecting ~9% of the amino acids. 96% of the novel variants had MAF <0.1%, indicating their rarity. Based on sequence conservation, MAF, and/or location of the substituted residue on a complete model of αIIbβ3 that suggested a possible effect on protein folding, we selected three novel variants (αIIb P943A and P176H, and β3 C547G) that affect amino acids previously associated with GT for expression in HEK 293 cells. Both αIIb P176H and β3 C547G severely affected αIIbβ3 expression, whereas αIIb P943A had only a partial effect on expression and no effect on DTT-induced fibrinogen binding. We were not surprised that the latter variant did not have a severe effect on expression or function because it has an MAF (0.46%) that is much higher than the MAFs of the other GT-causing variants. To estimate the percentage of the 114 novel identified variants that are likely to be deleterious we used 3 different algorithms, CADD, Polyphen 2-HDVI, and SIFT. The algorithms showed moderate concordance in their rankings of the likelihood that a variant is deleterious. To compare their predictive powers, we performed receiver operating characteristic (ROC) analysis based on their ability to discriminate confirmed GT missense variants (positive controls) from alloantigens (negative controls); the area under the curve (AUC) values were 0.91, 0.88, and 0.90, respectively. At cutoff values that achieved greater than 95% sensitivity for each algorithm: 1) the specificity values were 75%, 65%, and 60%, and 2) the percentages of novel αIIb+β3 missense variants predicted to be deleterious were 43%, 56%, and 58%. Polyphen 2-HDVI and SIFT identified αIIb P176H and β3 C547G as highly likely to be deleterious and αIIb P943A as much less likely to be deleterious, whereas CADD did not differentiate them in the same way. We conclude that ~1.1% of individuals in the populations studied carry at least one missense variant in αIIb or β3 and that 0.6% carry a variant that might be deleterious and therefore may result in a hemorrhagic GT-like phenotype. The rarity of almost all of the novel missense variants identified indicates that they entered the population recently. Despite having detailed knowledge of the structure and function of αIIbβ3, it is difficult to predict with certainty the impact of any single missense variant. This will pose serious challenges as more individuals undergo WES and WGS; we anticipate that linkage to health record data, as will happen for the UK 100,000 Genomes project, will aid clinical interpretation. Finally, “hypomorphic” gene variants that produce only a partial decrease in expression, such as αIIb P943A, may contribute to the wide variation in αIIbβ3 surface expression observed in the healthy population. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Genetic Analysis of 28 Chinese Families With Tyrosinase-Positive Oculocutaneous Albinism

Frontiers in Genetics ◽

10.3389/fgene.2021.715437 ◽

2021 ◽

Vol 12 ◽

Author(s):

Linya Ma ◽

Jianjian Zhu ◽

Jing Wang ◽

Yazhou Huang ◽

Jibo Zhang ◽

...

Keyword(s):

Autosomal Recessive ◽

Oculocutaneous Albinism ◽

Type Ii ◽

Related Disorder ◽

Chinese Families ◽

Missense Variants ◽

Clinical Presentations ◽

Splicing Variants ◽

Novel Variants ◽

Generation Sequencing

BackgroundTyrosinase-positive oculocutaneous albinism (OCA, type II, OCA2) is an autosomal recessive genetic disease in which the biosynthesis of melanin decreases in the skin, hair, and eyes. OCA2 disease is caused by mutations in OCA2 gene. The gene product plays a role in regulating the pH of melanosomes. Up to now, hundreds of OCA2 mutations have been reported and novel variants are still being discovered.MethodsIn this study, we reviewed the records of OCA2 patients who had conducted albinism genetic testing, and then analyzed the clinical and genetic information of 28 OCA2 patients who had been genetically diagnosed by using Sanger sequencing and next-generation sequencing.ResultsIn this study, we reported 31 variants screened from 28 Chinese OCA2 families, and characterized the detailed molecular and clinical presentations. There were 12 novel variants among all detected variants, including 3 missense variants (p.G393V, p.T482A, and p.R720P), 4 frameshift variants (p.R53Gfs∗49, p.N279Kfs∗17, p.I469Lfs∗4, p.I655Nfs∗12), 2 splicing variants (c.1637-2A > G, c.1951 + 1G > C), 2 stopgain variants (p.L278X, p.W652X) and 1 insertion variants (p.P315LinsT). One potential cluster of missense variants was implicated indicating the important roles of the underlying domains in OCA2 pathogenesis.ConclusionOur results were beneficial for diagnosis and precision clinical management for OCA2-related disorder, and this study expanded the mutation spectrum of oculocutaneous albinism.

Download Full-text

New pathogenic variants and insights into pathogenic mechanisms in GRK1-related Oguchi disease

10.1101/2020.02.20.936880 ◽

2020 ◽

Author(s):

James A. Poulter ◽

Molly S. C. Gravett ◽

Rachel L. Taylor ◽

Kaoru Fujinami ◽

Julie De Zaeytijd ◽

...

Keyword(s):

Protein Function ◽

Kinase Domain ◽

Missense Variants ◽

Pathogenic Variants ◽

Oguchi Disease ◽

Whole Exome ◽

Novel Variants ◽

Variation Database ◽

Biallelic Mutations ◽

The Impact

AbstractPurposeBiallelic mutations in G-Protein coupled receptor kinase 1 (GRK1) cause Oguchi disease, a rare subtype of congenital stationary night blindness (CSNB). The purpose of this study was to identify pathogenic GRK1 variants and use in-depth bioinformatic analyses to evaluate how their impact on protein structure could lead to pathogenicity.MethodsPatients’ genomic DNA was sequenced by whole genome, whole exome or focused exome sequencing. Pathogenic variants, published and novel, were compared to nondisease associated missense variants. The impact of GRK1 missense variants at the protein level were then predicted using a series of computational tools.ResultsWe identified eleven previously unpublished cases with biallelic pathogenic GRK1 variants, including seven novel variants, and reviewed all GRK1 pathogenic variants. Further structure-based scoring revealed a hotspot for missense variants in the kinase domain. Additionally, to aid future clinical interpretation, we identified the bioinformatics tools best able to differentiate pathogenic from non-pathogenic variants.ConclusionWe identified new GRK1 pathogenic variants in Oguchi disease patients and investigated how disease-causing variants may impede protein function, giving new insights into the mechanisms of pathogenicity. All pathogenic GRK1 variants described to date have been collated into a Leiden Open Variation Database (http://dna2.leeds.ac.uk/GRK1_LOVD/genes/GRK1).

Download Full-text

L-Type Calcium Channel: Predicting Pathogenic/Likely Pathogenic Status for Variants of Uncertain Clinical Significance

Membranes ◽

10.3390/membranes11080599 ◽

2021 ◽

Vol 11 (8) ◽

pp. 599

Author(s):

Svetlana I. Tarnovskaya ◽

Anna A. Kostareva ◽

Boris S. Zhorov

Keyword(s):

Calcium Channels ◽

Clinical Significance ◽

Heart Defects ◽

Clinical Manifestations ◽

Specific Protein ◽

Bioinformatics Tool ◽

Missense Variants ◽

Conduction Disorders ◽

Pathogenic Variants ◽

Novel Variants

(1) Background: Defects in gene CACNA1C, which encodes the pore-forming subunit of the human Cav1.2 channel (hCav1.2), are associated with cardiac disorders such as atrial fibrillation, long QT syndrome, conduction disorders, cardiomyopathies, and congenital heart defects. Clinical manifestations are known only for 12% of CACNA1C missense variants, which are listed in public databases. Bioinformatics approaches can be used to predict the pathogenic/likely pathogenic status for variants of uncertain clinical significance. Choosing a bioinformatics tool and pathogenicity threshold that are optimal for specific protein families increases the reliability of such predictions. (2) Methods and Results: We used databases ClinVar, Humsavar, gnomAD, and Ensembl to compose a dataset of pathogenic/likely pathogenic and benign variants of hCav1.2 and its 20 paralogues: voltage-gated sodium and calcium channels. We further tested the performance of sixteen in silico tools in predicting pathogenic variants. ClinPred demonstrated the best performance, followed by REVEL and MCap. In the subset of 309 uncharacterized variants of hCav1.2, ClinPred predicted the pathogenicity for 188 variants. Among these, 36 variants were also categorized as pathogenic/likely pathogenic in at least one paralogue of hCav1.2. (3) Conclusions: The bioinformatics tool ClinPred and the paralogue annotation method consensually predicted the pathogenic/likely pathogenic status for 36 uncharacterized variants of hCav1.2. An analogous approach can be used to classify missense variants of other calcium channels and novel variants of hCav1.2.

Download Full-text

αIIbβ3 variants defined by next-generation sequencing: Predicting variants likely to cause Glanzmann thrombasthenia

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1422238112 ◽

2015 ◽

Vol 112 (15) ◽

pp. E1898-E1907 ◽

Cited By ~ 28

Author(s):

Lorena Buitrago ◽

Augusto Rendon ◽

Yupu Liang ◽

Ilenia Simeoni ◽

Ana Negri ◽

...

Keyword(s):

Amino Acids ◽

Next Generation Sequencing ◽

Hek293 Cells ◽

Next Generation ◽

Glanzmann Thrombasthenia ◽

Missense Variants ◽

Sorting Intolerant From Tolerant ◽

Fibrinogen Binding ◽

Novel Variants ◽

Generation Sequencing

Next-generation sequencing is transforming our understanding of human genetic variation but assessing the functional impact of novel variants presents challenges. We analyzed missense variants in the integrin αIIbβ3 receptor subunit genes ITGA2B and ITGB3 identified by whole-exome or -genome sequencing in the ThromboGenomics project, comprising ∼32,000 alleles from 16,108 individuals. We analyzed the results in comparison with 111 missense variants in these genes previously reported as being associated with Glanzmann thrombasthenia (GT), 20 associated with alloimmune thrombocytopenia, and 5 associated with aniso/macrothrombocytopenia. We identified 114 novel missense variants in ITGA2B (affecting ∼11% of the amino acids) and 68 novel missense variants in ITGB3 (affecting ∼9% of the amino acids). Of the variants, 96% had minor allele frequencies (MAF) < 0.1%, indicating their rarity. Based on sequence conservation, MAF, and location on a complete model of αIIbβ3, we selected three novel variants that affect amino acids previously associated with GT for expression in HEK293 cells. αIIb P176H and β3 C547G severely reduced αIIbβ3 expression, whereas αIIb P943A partially reduced αIIbβ3 expression and had no effect on fibrinogen binding. We used receiver operating characteristic curves of combined annotation-dependent depletion, Polyphen 2-HDIV, and sorting intolerant from tolerant to estimate the percentage of novel variants likely to be deleterious. At optimal cut-off values, which had 69–98% sensitivity in detecting GT mutations, between 27% and 71% of the novel αIIb or β3 missense variants were predicted to be deleterious. Our data have implications for understanding the evolutionary pressure on αIIbβ3 and highlight the challenges in predicting the clinical significance of novel missense variants.

Download Full-text

Hepatic lipase (LIPC) sequencing in individuals with extremely high and low high-density lipoprotein cholesterol levels

PLoS ONE ◽

10.1371/journal.pone.0243919 ◽

2020 ◽

Vol 15 (12) ◽

pp. e0243919

Author(s):

Dilek Pirim ◽

Clareann H. Bunker ◽

John E. Hokanson ◽

Richard F. Hamman ◽

F. Yesim Demirci ◽

...

Keyword(s):

Hepatic Lipase ◽

Plasma Lipid ◽

Density Lipoprotein ◽

Cholesterol Levels ◽

Novel Variants ◽

Low Hdl ◽

Functional Features ◽

Regulatory Potential ◽

Functional Analyses ◽

Coding Variants

Common variants in the hepatic lipase (LIPC) gene have been shown to be associated with plasma lipid levels; however, the distribution and functional features of rare and regulatory LIPC variants contributing to the extreme lipid phenotypes are not well known. This study was aimed to catalogue LIPC variants by resequencing the entire LIPC gene in 95 non-Hispanic Whites (NHWs) and 95 African blacks (ABs) with extreme HDL-C levels followed by in silico functional analyses. A total of 412 variants, including 43 novel variants were identified; 56 were unique to NHWs and 234 were unique to ABs. Seventy-eight variants in NHWs and 89 variants in ABs were present either in high HDL-C group or low HDL-C group. Two non-synonymous variants (p.S289F, p.T405M), found in NHWs with high HDL-C group were predicted to have damaging effect on LIPC protein by SIFT, MT2 and PP2. We also found several non-coding variants that possibly reside in the circRNA and lncRNA binding sites and may have regulatory potential, as identified in rSNPbase and RegulomeDB databases. Our results shed light on the regulatory nature of rare and non-coding LIPC variants as well as suggest their important contributions in affecting the extreme HDL-C phenotypes.

Download Full-text

Functional and Structural Consequences of NineCYP21A2Mutations Ranging from Very Mild to Severe Effects

International Journal of Endocrinology ◽

10.1155/2016/4209670 ◽

2016 ◽

Vol 2016 ◽

pp. 1-10 ◽

Cited By ~ 5

Author(s):

Débora de Paula Michelatto ◽

Leif Karlsson ◽

Ana Letícia Gori Lusa ◽

Camila D’Almeida Mgnani Silva ◽

Linus Joakim Östberg ◽

...

Keyword(s):

Enzyme Activities ◽

Maximum Velocity ◽

The Novel ◽

Wild Type ◽

Phenotype Correlation ◽

Novel Variants ◽

Order Of Magnitude ◽

Classic Cah ◽

17 Hydroxyprogesterone ◽

Functional Analyses

We present the functional and structural effects of seven novel (p.Leu12Met, p.Arg16Cys, p.Ser101Asn, p.Ser202Gly, p.Pro267Leu, p.Gln389_Ala391del, and p.Thr450Met) and two previously reported but not studied (p.Ser113Phe and p.Thr450Pro)CYP21A2mutations. Functional analyses were complemented within silicoprediction of mutation pathogenicity based on the recently crystallized human CYP21A2 structure. Mutated proteins were transiently expressed in COS-1 cells and enzyme activities towards 17-hydroxyprogesterone and progesterone were determined. Residual enzyme activities between 43% and 97% were obtained for p.Arg16Cys, p.Ser101Asn, p.Ser202Gly, p.Pro267Leu, and p.Thr450Met, similar to the activities of the well-known nonclassic mutations p.Pro453Ser and p.Pro482Ser, whereas the p.Leu12Met variant showed an activity of 100%. Conversely, the novel p.Ser113Phe, p.Gln389_Ala391del, and p.Thr450Pro mutations drastically reduced the enzyme function below 4%. TheKmvalues for all novel variants were in the same order of magnitude as for the wild-type protein except for p.The450Met. The maximum velocity was decreased for all mutants except for p.Leu12Met. We conclude that p.Leu12Met is a normal variant; the mutations p.Arg16Cys, p.Ser101Asn, p.Ser202Gly, p.Pro267Leu, and p.Thr450Met could be associated with very mild nonclassic CAH, and the mutations p.Ser113Phe, p.Gln389_Ala391del, and p.Thr450Pro are associated with classic CAH. The obtained residual activities indicated a good genotype-phenotype correlation.

Download Full-text

Genotype-phenotype correlations and novel molecular insights into the DHX30-associated neurodevelopmental disorders

10.1101/2020.09.24.20196097 ◽

2020 ◽

Author(s):

Ilaria Mannucci ◽

Nan Cher Yeo ◽

Hannes Huber ◽

Jaclyn Murry ◽

Jeff Abramson ◽

...

Keyword(s):

De Novo ◽

Neurodevelopmental Disorder ◽

Missense Variant ◽

Stress Granule ◽

Helicase Activity ◽

Speech Impairment ◽

Granule Formation ◽

Missense Variants ◽

Functional Analyses ◽

Global Translation

Background We aimed to define the clinical and mutational spectrum, and to provide novel molecular insights into DHX30-associated neurodevelopmental disorder. Methods Clinical and genetic data from affected individuals were collected through family support group, GeneMatcher and our network of collaborators. Novel missense variants were investigated by in-vitro and in-vivo assays. These analyses included investigation of stress granule formation, global translation, ATPase and helicase activity, as well as the effect of selected variants on embryonal development in Zebrafish. Results We identified altogether 25 previously unreported individuals. All 19 individuals harboring heterozygous missense variants within helicase core motifs (HCMs) have global developmental delay, intellectual disability, severe speech impairment and gait abnormalities. These variants impair the ATPase and helicase activity of DHX30 and global translation, trigger stress granule formation, and cause developmental defects in a zebrafish model. Notably, 4 individuals harboring heterozygous variants resulting either in haploinsufficiency or truncated proteins presented a milder clinical course, similar to an individual bearing a de novo mosaic missense variant within HCM. Late-onset severe ataxia was observed in an individual with a de novo missense variant within the ratchet-like domain, and early-onset lethal epileptic encephalopathy in an individual with a homozygous missense variant within the helicase core region but not within a HCM. We report ten novel variants, two of which are recurrent, and provide evidence of gonadal mosaicism in one family. Functional analyses confirmed pathogenicity of all missense variants, and suggest the existence of clinically distinct subtypes that correlate with their location and nature. Moreover, we established here DHX30 as an ATP-dependent RNA helicase. Conclusions Our study highlights the usefulness of social media in order to define novel Mendelian disorders, and exemplifies how functional analyses accompanied by clinical and genetic findings can define clinically distinct subtypes for ultra-rare disorders. Such approaches require close interdisciplinary collaboration between families/legal representatives of the affected, clinicians, molecular genetics diagnostic laboratories and research laboratories.

Download Full-text

Phenotype and Genotype Study of Chinese POMT2-Related α-Dystroglycanopathy

Frontiers in Genetics ◽

10.3389/fgene.2021.692479 ◽

2021 ◽

Vol 12 ◽

Author(s):

Xiao-Yu Chen ◽

Dan-Yu Song ◽

Li Jiang ◽

Dan-Dan Tan ◽

Yi-Dan Liu ◽

...

Keyword(s):

Muscular Dystrophy ◽

Congenital Muscular Dystrophy ◽

Genetic Characteristics ◽

Apparent Lack ◽

Missense Variants ◽

Novel Variants ◽

Bioinformatics Software ◽

Uncertain Significance ◽

Genetic Features

ObjectiveAlpha-dystroglycanopathy (α-DGP) is a subtype of muscular dystrophy caused by defects in the posttranslational glycosylation of α-dystroglycan (α-DG). Our study aimed to summarize the clinical and genetic features of POMT2-related α-DGP in a cohort of patients in China.MethodsPedigrees, clinical data, and laboratory tests of patients diagnosed with POMT2-related α-DGP were analyzed retrospectively. The pathogenicity of variants in POMT2 were predicted by bioinformatics software. The variants with uncertain significance were verified by further analysis.ResultsThe 11 patients, comprising eight males and three females, were from nine non-consanguineous families. They exhibited different degrees of muscle weakness, ambulation, and intellectual impairment. Among them, three had a muscle-eye-brain disease (MEB)-like phenotype, five presented congenital muscular dystrophy with intellectual disability (CMD-ID), and three presented limb-girdle muscular dystrophy (LGMD). Overall, nine novel variants of POMT2, including two non-sense, one frameshift and six missense variants, were identified. The pathogenicity of two missense variants, c.1891G > C and c.874G > C, was uncertain based on bioinformatics software prediction. In vitro minigene analysis showed that c.1891G > C affects the splicing of POMT2. Immunofluorescence staining with the IIH6C4 antibody of muscle biopsy from the patient carrying the c.874G > C variant showed an apparent lack of expression.ConclusionThis study summarizes the clinical and genetic characteristics of a cohort of POMT2-related α-DGP patients in China for the first time, expanding the mutational spectrum of the disease. Further study of the pathogenicity of some missense variants based on enzyme activity detection is needed.

Download Full-text