Genotype-Phenotype Comparison in POGZ-Related Neurodevelopmental Disorders by Using Clinical Scoring

POGZ-related disorders (also known as White-Sutton syndrome) encompass a wide range of neurocognitive abnormalities and other accompanying anomalies. Disease severity varies widely among POGZ patients and studies investigating genotype-phenotype association are scarce. Therefore, our aim was to collect data on previously unreported POGZ patients and perform a large-scale phenotype-genotype comparison from published data. Overall, 117 POGZ patients′ genotype and phenotype data were included in the analysis, including 12 novel patients. A severity scoring system was developed for the comparison. Mild and severe phenotypes were compared with the types and location of the variants and the predicted presence or absence of nonsense-mediated RNA decay (NMD). Missense variants were more often associated with mild phenotypes (p = 0.0421) and truncating variants predicted to escape NMD presented with more severe phenotypes (p < 0.0001). Within this group, variants in the prolin-rich region of the POGZ protein were associated with the most severe phenotypes (p = 0.0004). Our study suggests that gain-of-function or dominant negative effect through escaping NMD and the location of the variants in the prolin-rich domain of the protein may play an important role in the severity of manifestations of POGZ–associated neurodevelopmental disorders.

An ensemble of toxic channel types underlies the severity of the de novo variant G375R of the human BK channel

De novo mutations play a prominent role in neurodevelopmental diseases including autism, schizophrenia, and intellectual disability. Many de novo mutations are dominant and so severe that the afflicted individuals do not reproduce, so the mutations are not passed into the general population. For multimeric proteins, such severity may result from a dominant-negative effect where mutant subunits assemble with WT to produce channels with adverse properties. Here we study the de novo variant G375R heterozygous with the WT allele for the large conductance voltage- and Ca2+-activated potassium (BK) channel, Slo1. This variant has been reported to produce devastating neurodevelopmental disorders in three unrelated children. If mutant and WT subunits assemble randomly to form tetrameric BK channels, then ~6% of the assembled channels would be wild type (WT), ~88% would be heteromeric incorporating from 1-3 mutant subunits per channel, and ~6% would be homomeric mutant channels consisting of four mutant subunits. To test this hypothesis, we analyzed the biophysical properties of single BK channels in the ensemble of channels expressed following a 1:1 injection of mutant and WT cRNA into oocytes. We found ~3% were WT channels, ~85% were heteromeric channels, and ~12% were homomeric mutant channels. All of the heteromeric channels as well as the homomeric mutant channels displayed toxic properties, indicating a dominant negative effect of the mutant subunits. The toxic channels were open at inappropriate negative voltages, even in the absence of Ca2+, which would lead to altered cellular function and decreased neuronal excitability.

The MADS-box gene FveSEP3 plays essential roles in flower organogenesis and fruit development in woodland strawberry

Flower and fruit development are two key steps for plant reproduction. The ABCE model for flower development has been well established in model plant species; however, the functions of ABCE genes in fruit crops are less understood. In this work, we identified an EMS mutant named R27 in woodland strawberry (Fragaria vesca), showing the conversion of petals, stamens, and carpels to sepaloid organs in a semidominant inheritance fashion. Mapping by sequencing revealed that the class E gene homolog FveSEP3 (FvH4_4g23530) possessed the causative mutation in R27 due to a G to E amino acid change in the conserved MADS domain. Additional fvesep3CR mutants generated by CRISPR/Cas9 displayed similar phenotypes to fvesep3-R27. Overexpressing wild-type or mutated FveSEP3 in Arabidopsis suggested that the mutation in R27 might cause a dominant-negative effect. Further analyses indicated that FveSEP3 physically interacted with each of the ABCE proteins in strawberry. Moreover, both R27 and fvesep3CR mutants exhibited parthenocarpic fruit growth and delayed fruit ripening. Transcriptome analysis revealed that both common and specific differentially expressed genes were identified in young fruit at 6–7 days post anthesis (DPA) of fvesep3 and pollinated wild type when compared to unpollinated wild type, especially those in the auxin pathway, a key hormone regulating fruit set in strawberry. Together, we provided compelling evidence that FveSEP3 plays predominant E functions compared to other E gene homologs in flower development and that FveSEP3 represses fruit growth in the absence of pollination and promotes fruit ripening in strawberry.

Genetic Diversity of Genes Controlling Unilateral Incompatibility in Japanese Cultivars of Chinese Cabbage

In recent years, unilateral incompatibility (UI), which is an incompatibility system for recognizing and rejecting foreign pollen that operates in one direction, has been shown to be closely related to self-incompatibility (SI) in Brassica rapa. The stigma- and pollen-side recognition factors (SUI1 and PUI1, respectively) of this UI are similar to those of SI (stigma-side SRK and pollen-side SP11), indicating that SUI1 and PUI1 interact with each other and cause pollen-pistil incompatibility only when a specific genotype is pollinated. To clarify the genetic diversity of SUI1 and PUI1 in Japanese B. rapa, here we investigated the UI phenotype and the SUI1/PUI1 sequences in Japanese commercial varieties of Chinese cabbage. The present study showed that multiple copies of nonfunctional PUI1 were located within and in the vicinity of the UI locus region, and that the functional SUI1 was highly conserved in Chinese cabbage. In addition, we found a novel nonfunctional SUI1 allele with a dominant negative effect on the functional SUI1 allele in the heterozygote.

A non-transmembrane channel formed by Ca2+-bound calsequestrin-2

Although it is well known that ion channels conduct ions across biomembranes, whether ions are conducted by some non-membrane proteins is not known because of the lack of a detection method. Calsequestrin-2 (CSQ2) is a sarcoplasmic reticulum (SR) Ca2+-binding protein suppling Ca2+ for the ryanodine receptor Ca2+ release during the excitation–contraction coupling in cardiomyocytes. CSQ2 mutations, even in some heterozygous occasions, causes catecholaminergic polymorphic ventricular tachycardia (CPVT2), suggesting that CSQ2 may function beyond a Ca2+ buffer. Here, we identify a non-transmembrane channel in Ca2+-enriched CSQ2 dimers, which facilitates fast Ca2+ mobilization. Using crystallography, we solved the high-resolution structure of Ca2+-bound CSQ2 and discovered that the negatively charged residues at the dimer interface encompassed a tubular channel-like structure, dubbed “tunnel,” in which ∼15 Ca2+ ions aligned across the ∼5 nm tunnel path. To determine the potential tunnel conductance, we developed a graphene-based nanoelectronic technology to connect a CSQ2 dimer into a nanocircuit. In the Tyrode solution containing 1 mM Ca2+, a CSQ2 dimer exhibited a conductance one order of magnitude higher than the background level. This conductance was Ca2+ dependent, and was largely suppressed by the single-residue mutation D309N at the bottleneck region of the tunnel path, indicating that the tunnel conducted Ca2+ fluxes. When the D309N mutant CSQ2 was expressed in wild-type rat cardiomyocytes by adenoviral vectors, isoproterenol treatment induced chaotic Ca2+ waves, delayed after-depolarizations and trigged activities resembling those occurring in CPVT2 models. This dominant negative effect of CSQ2 mutation agreed well with our structural observation that CSQ2 tunnels were interconnected to form a tunnel network. Taken together, these results revealed that CSQ2 builds a nano-highway network for energy-efficient Ca2+ mobilization in the SR. Factors that block the Ca2+ highway may lead to arrhythmogenesis.

TP53 Allelic Status in Myeloid Malignancies

Introduction TP53 is a tumor suppressor gene involved in regulating cell division and apoptosis in response to DNA damage. In hematologic malignancies, TP53 alterations are present in both myeloid and lymphoid malignancies. TP53 alterations including both sequence level mutations and deletions occur in 8-10% of de novo acute myeloid leukemia (AML), and are significantly enriched in patients with therapy-related myeloid neoplasms with a frequency of 25-40%. TP53 alterations are associated with complex karyotype, resistance to traditional cytotoxic chemotherapy and dismal outcome, and are well established poor prognostic markers for both AML and myelodysplastic syndrome (MDS). While both biallelic and monoallelic TP53 alterations are seen, biallelic TP53 alteration is more frequent in both AML and MDS. In the instances of monoallelic TP53 alterations, the remaining wild-type allele can be inhibited by the dominant negative effect of the mutant p53. The clinical implication of TP53 allelic status remains controversial. Biallelic TP53 alterations and/or high TP53 mutation variant allele frequency (VAF) are associated with older patients, complex karyotype, few co-occurring mutations and poor outcome in both AML and MDS. Other studies demonstrate that biallelic vs. monoallelic TP53 alterations or high vs. low TP53 VAF have similar prognosis in myeloid malignancies. Current European LeukemiaNet (ELN) recommends testing TP53 deletion using karyotype analysis and TP53 mutations by molecular testing. Methods: We performed a retrospective review of patients with myeloid malignancies, myeloid next generation sequencing (NGS) panel and cytogenetic tests performed at ARUP Laboratories. We identified 18 patients with myeloid malignancies and TP53 mutations. We used a combination of karyotype analysis, FISH, chromosomal microarray (CMA) and NGS to determine the TP53 allelic status. Results: 18 patients diagnosed with myeloid malignancies and TP53 mutations were identified. Among them, 6 were diagnosed with AML, 10 had MDS, one with CMML and one had post essential thrombocythemia myelofibrosis. Age range is from 31 to 78 with a median age 65.5 years. 23 TP53 mutations were identified among 18 patients. The majority (78%) of TP53 mutations are located in the DNA binding domain. Co-occurring mutations are uncommon in patients with TP53 mutations. 11 out of 18 patients did not have co-occurring mutations in other myeloid malignancy related genes. Three cases had 1, three cases had 2, and one case had 3 co-occurring mutations at the time of diagnosis. Karyotype analysis and FISH were performed on all 18 patients. CMA was performed on 9 patients. 17p abnormalities, defined by the deletion or copy-neutral loss of heterozygosity (CN-LOH) of 17p, were seen in 9 out of 18 cases. The 17p abnormalities in 8 out of these 9 cases were visible by karyotype. Case 11 had a CN-LOH identified by CMA which was cryptic by karyotype. Typical complex karyotype was seen in 16 out of 18 cases. Biallelic TP53 alterations are defined by either the presence of a TP53 mutation with a 17p abnormality (deletion or CN-LOH), or two TP53 mutations with similar VAF, or one TP53 mutation with VAF &gt;50%. Biallelic TP53 alterations were seen more frequently compared with monoallelic TP53 alterations (14 vs. 4 patients) and enriched in older patients (Figure 1). All 6 AML patients had biallelic TP53 alterations. Outcome data is available in 10 patients including 8 patients with biallelic and 2 patients with monoallelic TP53 alterations. Patients with biallelic TP53 alterations have dismal outcome (Figure 2). Patient 7 represents an atypical patient with TP53 alteration in our study cohort. She was diagnosed with MDS at the age of 31. She had monoallelic TP53 mutation with a relatively low VAF and normal karyotype through her disease course. Her MDS never progressed and she remained in remission after transplant for more than four years and still doing well. Conclusion: In this study, we evaluated the TP53 allelic status in 18 patients with myeloid malignancies. Biallelic TP53 alterations are more frequent than monoallelic TP53 alterations, and are associated with older patients, and fewer co-occurring mutations. Biallelic TP53 alterations are associated with typical complex karyotype and dismal outcome. Our study supports the importance to differentiate between biallelic and monoallelic TP53 alterations. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

A novel I551F variant of Na+/HCO3- cotransporter NBCe1-A shows reduced cell surface expression, resulting in diminished transport activity

Homozygous mutations in SLC4A4, encoding the electrogenic Na+/HCO3- cotransporter NBCe1, cause proximal renal tubular acidosis (pRTA) associated with extrarenal symptoms. Although 17 mutated sites in SLC4A4 have thus far been identified among pRTA patients, physiological significance of other nonsynonymous single nucleotide variants (SNVs) remains largely undetermined. Here, we investigated the functional properties of SNVs in NBCe1. From NCBI dbSNP database, we identified 13 SNVs that have not previously been characterized in highly conserved, transmembrane domains of NBCe1-A. Immunocytochemical analysis revealed that I551F variant was present predominantly in the cytoplasm in HEK293 cells, whereas all other SNVs did not show as dramatic a change in subcellular distribution. Western blot analysis in HEK293 cells demonstrated that the I551F variant showed impaired glycosylation and a 69 % reduction in cell surface levels. To determine the role of I551 in more detail, we examined the significance of various artificial mutants both in non-polarized HEK293 cells and polarized MDCK cells, which indicated that only I551F substitution resulted in cytoplasmic retention. Moreover, functional analysis using Xenopus oocytes demonstrated that the I551F variant had a significantly reduced activity corresponding to 39 % of that of wild-type, whereas any other SNVs and artificial I551 mutants did not show significant changes in activity. Finally, immunofluorescence study in HEK293 cells indicated that the I551F variant retains wild-type NBCe1-A in the cytoplasm. These data demonstrate that I551F-NBCe1-A shows impaired transport activity predominantly through cytoplasmic retention, and suggest that the variant can have a dominant-negative effect by forming complexes with wild-type NBCe1-A.

Distinct Morphological and Behavioural Alterations in ENU-Induced Heterozygous Trpc7K810Stop Mutant Mice

Trpc7 (transient receptor potential cation channel, subfamily C, member 7; 862 amino acids) knockout mice are described showing no clear phenotypic alterations, therefore, the functional relevance of the gene remains unclear. A complementary approach for the functional analysis of a given gene is the examination of individuals harbouring a mutant allele of the gene. In the phenotype-driven Munich ENU mouse mutagenesis project, a high number of phenotypic parameters was used for establishing novel mouse models on the genetic background of C3H inbred mice. The phenotypically dominant mutant line SMA002 was established and further examined. Analysis of the causative mutation as well as the phenotypic characterization of the mutant line were carried out. The causative mutation was detected in the gene Trpc7 which leads to the production of a truncated protein due to the novel stop codon at amino acid position 810 thereby affecting the highly conserved cytoplasmic C terminus of the protein. Trpc7 heterozygous mutant mice of both sexes were viable and fertile, but showed distinct morphological and behavioural alterations which is in contrast to the published phenotype of Trpc7 knockout mice. Thus, the Trpc7K810Stop mutation leads to a dominant negative effect of the mutant protein.

Dominant negative effects of SCN5A missense variants

Introduction: Up to 30% of patients with Brugada Syndrome (BrS) carry loss-of-function (LoF) variants in the cardiac sodium channel gene SCN5A. Recent studies have suggested that the SCN5A protein product NaV1.5 can form dimers and exert dominant negative effects. Methods: We identified 35 LoF variants (<10% peak current compared to wild type (WT)) and 15 partial LoF variants (10-50% peak current compared to WT) that we assessed for dominant negative behavior. SCN5A variants were studied in HEK293T cells alone or in heterozygous co-expression with WT SCN5A using automated patch clamp. To assess clinical risk, we compared the prevalence of dominant negative vs. putative haploinsufficient (frameshift/splice site) variants in a BrS case consortium and the gnomAD population database. Results: In heterozygous expression with WT, 32/35 LoF variants and 6/15 partial LoF showed reduction to <75% of WT-alone peak INa, demonstrating a dominant negative effect. Carriers of dominant negative LoF missense variants had an enriched disease burden compared to putative haploinsufficient variant carriers (2.7-fold enrichment in BrS cases, p=0.019). Conclusions: Most SCN5A missense LoF variants exert a dominant negative effect. Cohort analyses reveal that this class of variant confers an especially high burden of BrS.

RAD50 Loss of Function Variants in the Zinc Hook Domain Associated with Higher Risk of Familial Esophageal Squamous Cell Carcinoma

Unbiased whole-exome sequencing approaches in familial esophageal squamous cell carcinoma (ESCC) initially prioritized RAD50 as a candidate cancer predisposition gene. The combined study with 3289 Henan individuals from Northern China identified two pathogenic RAD50 protein truncation variants, p.Q672X and a recurrent p.K722fs variant at the zinc hook domain significantly conferring increased familial ESCC risk. Effects of ~10-fold higher familial ESCC risk were observed, when compared to East Asians from the gnomAD database. Functional characterization suggested that the RAD50Q672X mutation contributes a dominant-negative effect in DNA repair of double-stranded breaks. Overexpression of the RAD50Q672X and RAD50L1264F missense mutation also sensitized cell death upon replication stress stimuli induced by formaldehyde treatment and the CHK1 inhibitor, AZD7762. Our study suggested the novel insight of the potential for synthetic lethal therapeutic options for RAD50Q672X and the East-Asian-specific RAD50L1264F variants and CHK1 inhibitors. Our study also suggested the association of RAD50 LOF variants in the zinc hook domain with a higher risk of familial ESCC in Chinese.