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PubMed2026

Dynamic Protein Structure Paradox: An Integrative Framework for Endpoint-Conditioned Evidentiary Sufficiency in Structure-to-Function Claims.

Accurate coordinates for a represented protein state do not, by themselves, establish activity or any other condition-specific function. This article defines the Dynamic Protein Structure Paradox (DPSP) as the apparent conflict between structural accuracy and functional underdetermination and develops it as an integrative evidentiary assessment framework rather than a new theory or paradigm. The underlying problem has been longstanding, since structural genomics, function annotation, allostery, and disorder research each established that fold does not determine function and that function does not determine fold. DPSP consolidates those results into one endpoint-conditioned rule. Once a measurable endpoint is defined, it assesses four coupled dimensions: relevant-state completeness, context completeness, ensemble or kinetic dependence, and chemical dependence. A rubric rates each dimension as adequate, uncertain, or missing, and a materiality test determines which gaps influence the stated decision. The outcome is one of three mutually exclusive modes of utilization: geometry-led, conditional, or function-measured. The deliverable is a concise evidence statement delineating what the structure supports, which decisive variable remains unmeasured, and what corroboration is necessary. DPSP complements, rather than replaces, existing structural, ensemble, and computational approaches. The framework remains unvalidated, its thresholds are provisional, and the studies necessary to confirm or refute it are specified.

Proteins↗

PubMed2026

Rapidly evolving aphid gall effector proteins exhibit saposin-like folds.

Many insects manipulate plants by injecting effector proteins. In one extreme example of this molecular "hijacking," Hormaphis cornu aphids inject bicycle proteins into Hamamelis virginiana, contributing to the development of novel organs called galls. Bicycle proteins share no amino acid sequence similarity with proteins of known function. Here, we report the crystal structures of two divergent bicycle proteins. Both proteins contain saposin-like folds: one with multiple disulfide bonds exhibits a swapped domain topology; the other has no disulfide bonds and possesses two distinct, tandem domains. To explore the structural evolution of bicycle proteins, we attempted to predict bicycle protein structures with Alphafold2 (AF2) and other deep learning programs. While AF2 did not recover the two experimental structures using existing databases, it succeeded when provided with multiple sequence alignments (MSAs) of protein sequences from newly sequenced closely related species. Using this approach, we generated 2,400 high-confidence bicycle protein predictions from seven aphid species. While all aphid bicycle proteins contain predicted saposin-like folds, they display a vast diversity of structural and physicochemical properties. While this diversity thwarts prediction of conserved functions encoded in structure, it suggests that bicycle proteins have evolved to target diverse plant processes and/or to evade plant immune surveillance. Our extension of AF2 with custom MSAs of proteins from closely related species provides a generalizable, powerful approach for predicting structures of rapidly evolving protein families.

Animals↗

PubMed2025

The functional study of novel KLHL3 missense mutations associated with pseudohypoaldosteronism type II.

BACKGROUND: Pseudohypoaldosteronism type II (PHA II) is an inherited tubulopathy, clinically defined by three hallmark features, including secondary hypertension, hyperchloremic metabolic acidosis, and persistent hyperkalemia occurring despite maintained glomerular filtration function. Herein, we aim to investigate the association of kelch like family member 3 (KLHL3) gene mutations with PHA II. METHODS: Compound heterozygous KLHL3 mutations were identified through whole-exome sequencing and Sanger validation. AlphaFold-based structural modeling, site-directed mutagenesis of Flag-tagged plasmids, and co-immunoprecipitation (Co-IP)/immunoblotting in vivo were combined to analyze mutant protein interactions and ubiquitination effects. RESULTS: A Chinese patient was identified with two previously unreported KLHL3 variants (c.131G > A [p.R44Q] and c.744 C > G [p.Y248*]), exhibiting a biochemical triad of asymptomatic hyperkalemia, mild metabolic acidosis, and borderline hypertension. Administration of thiazide diuretics effectively normalized the patient’s hyperkalemia and hypertension. A p.R44Q missense mutation predicted as variants of uncertain significance (VOUS) by American College of Medical Genetics and Genomics (ACMG) guidelines, and a p.Y248* nonsense mutation predicted as variants of likely pathogenic. Functional study revealed that the two KLHL3 mutations impair its ubiquitination of with-no-lysine kinase 1 (WNK1) and with-no-lysine kinase 4 (WNK4), and further increase phosphorylation of both SPAK (sterile20/sporulation-specific protein-1 related proline/alanine-rich kinase)/OSR1 (oxidative stress response kinase-1) and Na-Cl-cotransporter (NCC). CONCLUSIONS: Our study characterized two previously unreported KLHL3 mutations, followed by comprehensive in vitro functional analyses to elucidate their pathophysiological contributions at the molecular level.

Humans↗

PubMed2026

A novel PKHD1 missense variant disrupting splicing in a fetus with Caroli disease.

BACKGROUND: Caroli disease (CD) is a rare inherited disorder characterized by dilatation of intrahepatic bile ducts, and prenatal diagnosis of this disease is extremely rare. PKHD1 is the only known causative gene, yet the pathogenicity of most missense variants remains unclear. METHODS: Exome sequencing (ES) was performed on a fetus with clinical features of CD. Candidate variants were validated by Sanger sequencing in the family. The impact of the novel missense variant on pre-mRNA splicing was assessed using minigene assays, and structural modeling of the PKHD1 protein was conducted with AlphaFold 3. RESULTS: At 23 weeks of gestation, the fetus showed hepatic cysts on ultrasound and a "central dot" sign on MRI, suggesting a diagnosis of CD. The fetus also exhibited features of autosomal recessive polycystic kidney disease and oligohydramnios. ES identified and Sanger sequencing confirmed three PKHD1 variants: a paternal nonsense variant c.5323C>T; p.(Arg1775*), and two maternal missense variants c.6682G>C; p.(Glu2228Gln) and c.8012G>T; p.(Arg2671Leu). The variant c.6682G>C is novel and minigene assays demonstrated that it caused exon 40 skipping, leading to an in‑frame deletion (c.6491_6682del; p.(Gly2164_Arg2227del)). Structural modeling predicts that this deletion lies within a large β‑barrel domain and may compromise its structural stability. Conclusion We characterize a novel missense variant that causes aberrant splicing of PKHD1 in CD. This finding underscores the necessity of functional analysis for evaluating the pathogenicity of missense variants, especially those at the last nucleotide of an exon. Our study expands the mutation spectrum of PKHD1 and provides insights into genotype‑phenotype correlations.

Humans↗

PubMed2026

[Genetic analysis of a fetus with Short-rib thoracic dysplasia 8 with or without polydactyly due to variants of DYNC2I1 gene].

OBJECTIVE: To investigate the clinical characteristics of a fetus with Short-rib thoracic dysplasia 8 with or without polydactyly (SRTD8) due to variants of DYNC2I1 gene. METHODS: A fetus identified to have short ribs, short long bones, and narrow thorax at 26+1 weeks of gestation at the Women and Children's Hospital of Ningbo University in September 2024 was selected as study subject. The fetus underwent termination of pregnancy at 35+5 weeks of gestation. Clinical data of the fetus were retrospectively collected. Whole exome sequencing (WES) was carried out on fetal tissue, and candidate variants were validated by Sanger sequencing. Difference between the wild type and variant DYNC2I1 proteins was analyzed using AlphaFold v3.0.1 and PyMOL v2.5.6 software. Pathogenicity of the variant was rated based on guidelines from the American College of Medical Genetics and Genomics (ACMG). Using keywords such as "DYNC2I1 gene", previous literature on patients due to biallelic DYNC2I1 gene variants were retrieved from the PubMed databases, CNKI, and Wanfang Data Knowledge Service Platform, and the genetic variant and clinical phenotypes of patients were analyzed. The literature retrieval time was set from the establishment of database to December 31, 2025. This study was approved by the Medical Ethics Committee of the hospital (Ethics No.: 2023-094). RESULTS: Prenatal ultrasound revealed that the fetus had short ribs, short long bones, and narrow thorax at 26+1 gestational weeks. WES and Sanger sequencing revealed that the fetus has harbored compound heterozygous variants of the DYNC2I1 gene, namely c.265_268 (p.Gln89GlyfsTer15) in exon 3 and c.1777C>T (p.Arg593Trp) in exon 14, which were inherited from his father and mother, respectively. Prediction of the DYNC2I1 protein structure suggested that the c.265_268del variant has formed a premature termination codon, which may significantly alter the protein's secondary structure. The c.1777C>T variant may disrupt the electrostatic interaction between Arg593 and Asp729. Based on the ACMG guidelines, the c.265_268del (p.Gln89GlyfsTer15) variant was predicted to be likely pathogenic (PM2_Supporting +PVS1), whilst the c.1777C>T(p.Arg593Trp) variant was rated as uncertain significance (PM2_Supporting+PM3+PP4). Literature search has identified five articles related to biallelic DYNC2I1 variants involving a total of 11 fetuses/patients. Together with the fetus from this study, typical phenotypes included short ribs (6 cases), narrow thorax (6 cases), short limb bones (6 cases), and hand polydactyly (6 cases), and foot polydactyly (5 cases), albeit with significant clinical heterogeneity. A total of 12 genetic variants were identified, among which c.44delC was the most common (16.7%, 4/24), followed by c.1777C>T, c.2246C>T, and c.2305G>A (each accounting for 12.5%). No mutational hotspot was identified. CONCLUSION: The c.265_268del (p.Gln89GlyfsTer15) and c.1777C>T (p.Arg593Trp) compound heterozygous variants of the DYNC2I1 gene probably underlay the pathogenesis of SRTD8 in this fetus. This study has enriched the mutational spectrum of the DYNC2I1 gene and facilitated etiological diagnosis and treatment of DYNC2I1-related diseases.

Humans↗

PubMed2025

Whole-genome sequencing, as a powerful diagnostic tool in hearing loss, reveals novel variants in PTPRQ missed by whole-exome sequencing.

BACKGROUND/OBJECTIVES: Hearing loss (HL) is one of the most common congenital disorders, affecting 1-2 in 1,000 newborns. Modern genetic diagnostics using large gene panels and/or whole exome analysis (WES) can identify disease-causing mutations in 25-50 % of patients, with higher solve rates in individuals with earlier onset. RESULTS: Here, we used whole-genome sequencing (WGS) to reanalyze 14 index patients/families who remained without genetic diagnosis by WES. We were able to identify the genetic cause of HL in 6 families (43 %). Two families were diagnosed with DFNB84A caused by compound heterozygous recessive mutations in PTPRQ. Three of the four underlying variants, including a structural variant, a deep intronic variant, and a splice variant, escaped detection by WES. Minigene assays confirmed the pathogenicity of the intronic and the splice variants. In addition, we used protein 3D structure prediction and rigid ligand docking to study the pathogenicity of variants that escape nonsense-mediated decay. CONCLUSION: In our study, we present four novel variants in PTPRQ, three of which were detected only by WGS. To our knowledge, we report here the first pathogenic deep intronic PTPRQ variant causing HL. Our results suggest that the mutational spectrum of PTPRQ is not well covered by standard WES and that PTPRQ-associated hearing loss may be more frequent than previously thought. WGS provides an additional layer of information in the diagnostics of HL.

Humans↗

PubMed2026

[Genetic analysis of a male with Multiple morphological abnormalities of sperm flagella combined with sperm head abnormalities due to compound heterozygous variants of DNAH1 gene and a literature review].

OBJECTIVE: To explore the clinical phenotype and genetic etiology of a male with Multiple morphological abnormalities of sperm flagella (MMAF) combined with sperm head abnormalities due to compound heterozygous variants of DNAH1 gene, with an aim to provide guidance for assisted reproductive technology in his family. METHODS: A man with MMAF combined with sperm head abnormalities who visited Women and Children's Hospital of Ningbo University in October 2024 was selected as study subject. Clinical data of the patient's family were retrospectively collected. Peripheral blood samples were collected from the patient and his spouse, and G-banding karyotyping and whole exome sequencing (WES) were carried out. Candidate variants were validated by Sanger sequencing. Conservation of the DNAH1 protein was queried on the UCSC website. The difference between wild type and variant DNAH1 proteins were analyzed using AlphaFold v3.0.1 and PyMOL v2.5.6. The pathogenicity of variant was rated based on the guidelines from American College of Medical Genetics and Genomics (ACMG). Previous literature was searched using keywords "DNAH1 gene" and "multiple morphological abnormalities of the sperm flagella" on CNKI, Wanfang Data Knowledge Service Platform, and PubMed database to identify cases of MMAF attributed to biallelic DNAH1 gene variants. The retrieval period was set from the establishment of the databases to December 31, 2025. The genotypes and clinical phenotypes of patients with biallelic DNAH1 mutations were analyzed. This study was approved by the Medical Ethics Committee of the hospital (Ethics No.: EC2023-094). RESULTS: The 30-year-old patient and his 30-year-old wife had infertility for 2 years. Semen analysis revealed no motile sperm and a 99.0% abnormal morphology rate. Typical MMAF was observed with phase-contrast microscopy. Sperm morphology analysis revealed abnormalities of the head, neck, and tail with an approximate ratio of 9:5:1. The patient's karyotype was 46,XY, and his wife's karyotype was 45,X[4]/47,XXX[1]/46,XX[84]. WES and Sanger sequencing revealed that the patient harbored compound heterozygous variants of the DNAH1 gene, namely c.1435_1444+3del and c.12204_12206del (p.Asn4069del), but their origin remained unidentified. UCSC genome browser query results showed that the amino acid residue at position 4 069 of the DNAH1 protein is highly conserved across various species. Protein structure prediction reveals that, in the wild-type DNAH1 protein, the Asparagine at position 4 069 (Asn4069) can form hydrogen bonds with the Leucine on the main chain at position 4 086 (Leu4086) and the Serine on the side chain at position 4 087 (Ser4087). The c.12204_12206del variant, resulting in deletion of Asn4069, disrupts these hydrogen bonds and does not generate any compensatory interactions. Based on the ACMG guidelines, the c.1435_1444+3del variant was predicted to be likely pathogenic (PM2_Supporting+PVS1), and the c.12204_12206del(p.Asn4069del) variant was rated as likely pathogenic (PM2_Supporting+PM4+PM3+PP4). The couple had elected for in vitro fertilization using donor sperm. During this cycle, 12 oocytes were retrieved, 10 oocytes were successfully fertilized, 1 embryo and 6 blastocysts were obtained. Following the first transfer of a frozen-thawed blastocyst, implantation of an empty gestational sac occurred, which led to a miscarriage. After the second transfer of a high-quality blastocyst, the embryo split into twins following implantation, and the spouse had selected fetal reduction. The gestational age was 33+3 weeks on June 1, 2026. Literature review identified three studies reporting biallelic mutations of the DNAH1 gene in association with MMAF combined with sperm head abnormalities. Together with the patient from this study, a total of 20 patients were included in the analysis. The rate of sperm flagellar abnormalities in these patients was above 80.0%, while the rate of sperm head abnormalities has ranged from 12.0% to 100.0%. In four patients, the genetic basis was unknown. In the remaining 16 patients, 35 mutations were detected, with c.8626-1G>A being the most common (22.9%, 8/35). CONCLUSION: This patient showed MMAF with frequent sperm head defects. Compound heterozygous variants of the DNAH1 gene probably underlay these abnormalities, which in turn has led to his primary infertility. This study revealed the phenotypic variability of MMAF and broadened the mutational spectrum of the DNAH1 gene.

Humans↗

PubMed2025

Newly Developed Structure-Based Methods Do Not Outperform Standard Sequence-Based Methods for Large-Scale Phylogenomics.

Recent developments in protein structure prediction have allowed the use of this previously limited source of information at genome-wide scales. It has been proposed that the use of structural information may offer advantages over sequences in phylogenetic reconstruction, due to their slower rate of evolution and direct correlation to function. Here, we examined how recently developed methods for structure-based homology search and tree reconstruction compare with current state-of-the-art sequence-based methods in reconstructing genome-wide collections of gene phylogenies (i.e. phylomes). While structure-based methods can be useful in specific scenarios, we found that their current performance does not justify using the newly developed structure-based methods as a default choice in large-scale phylogenetic studies. On the one hand, the best performing sequence-based tree reconstruction methods still outperform structure-based methods for this task. On the other hand, structure-based homology detection methods provide larger lists of candidate homologs, as previously reported. However, this comes at the expense of missing hits identified by sequence-based methods, as well as providing sets of homolog candidates with higher fractions of false positives. These insights help to guide the use of structural data in comparative genomics and highlight the need to continue improving structure-based approaches. Our pipeline is fully reproducible and has been implemented in a Snakemake workflow. This will facilitate a continuous assessment of future improvements of structure-based tools in the AlphaFold era.

Phylogeny↗