Comprehensive Transcriptomic and Proteomic Profiling of CILD40 with Novel Compound Heterozygous Mutations of DNAH9

A Novel Homozygous Mutation in ARL2BP Causes Multiple Morphological Abnormalities of the Flagella and Primary Ciliary Dyskinesia

Primary ciliary dyskinesia (PCD) and multiple morphological abnormalities of the sperm flagella (MMAF) frequently co‐occur in male infertility. However, the genetic basis of this syndromic presentation remains unclear. Using whole‐exome sequencing, we identified a novel homozygous ARL2BP splice‐site mutation (c.294‐2A>G) in a 23‐year‐old infertile male from a consanguineous family who presented with syndromic PCD and MMAF. This variant causes aberrant pre‐mRNA splicing and triggers nonsense‐mediated mRNA decay, resulting in the complete absence of ARL2BP protein expression. Transmission electron microscopy revealed extensive disorganization of flagellar axonemes with consistent central pair (CP) microtubule depletion and disorganization of peripheral doublets. Immunofluorescence confirmed a severe deficiency of the CP protein SPAG6 in the sperm flagella. Notably, the patient presented without retinal symptoms. Given that ARL2BP‐related retinitis pigmentosa generally emerges during the third decade, long‐term ophthalmological follow‐up is essential to detect delayed‐onset retinal degeneration. In conclusion, these findings confirm ARL2BP as a causative gene for both PCD and MMAF, expanding the genotypic and phenotypic spectrum of ciliopathies.

Biallelic RSPH4A loss-of-function variants cause primary ciliary dyskinesia in a Chinese patient

Background Primary ciliary dyskinesia (PCD) is a rare autosomal recessive disorder characterized by defective motile cilia function, affecting approximately one in 7,500 to one in 10,000 live births. Pathogenic variants in radial spoke head genes, including RSPH4A, cause PCD with distinctive central-microtubular-pair defects. However, the functional consequences of novel RSPH4A variants remain poorly characterized, limiting genetic counseling and prenatal diagnostic capabilities. This study aims to reveal the genetic etiology of PCD in an affected family and the clinical significance of the two novel RSPH4A variants identified in this PCD-affected pedigree. Methods We recruited a five-member Chinese family including an 11-year-old female PCD proband presenting with chronic bronchiectasis and recurrent respiratory infections. Comprehensive clinical evaluations, whole exome sequencing (WES), and Sanger sequencing were performed to identify genetic variants. Bioinformatics analyses including protein sequence alignment and structural modeling were conducted. Experimental validation employed site-directed mutagenesis, quantitative real-time PCR, and Western blotting in HEK293T cells to characterize variant effects on mRNA stability and protein expression. Results WES identified compound heterozygous RSPH4A variants in the proband: RSPH4A (NM_001010892.3): c.2T>C (p.Met1Thr) inherited from the mother and RSPH4A (NM_001010892.3): c.854delA (p.Lys286Serfs*22) inherited from the father, showing strict co-segregation with the disease phenotype. The c.2T>C variant disrupted the translation initiation codon, while c.854delA introduced a premature termination codon within the radial spoke head domain. Cross-species analysis demonstrated high conservation of the affected region across 15 vertebrate species. Structural modeling predicted complete loss of the radial spoke head domain in the truncated protein. Functional studies revealed that c.2T>C severely impaired protein translation despite intact mRNA levels, whereas c.854delA triggered nonsense-mediated mRNA decay and produced unstable truncated protein. Both variants resulted in substantial reduction in functional RSPH4A protein expression. Conclusion This study identifies novel loss-of-function RSPH4A variants causing PCD through distinct molecular mechanisms, expanding the mutational spectrum of radial spoke head protein-related ciliopathies. These findings offer compelling proof in favor of a molecular diagnosis of PCD in this family and enable carrier screening for at-risk relatives. The experimental validation strategy establishes a framework for interpreting variants of uncertain significance in PCD genes, facilitating accurate prenatal diagnosis and preimplantation genetic testing to reduce the recurrence risk and birth defect incidence in PCD. Furthermore, understanding the precise functional consequences of RSPH4A variants informs genotype-phenotype correlations and may guide future development of targeted therapeutic interventions for this debilitating respiratory disorder.

PIK3CA in-frame deletion-activated PI3K/AKT signaling pathway drives macrodactyly.

Cross-species functional analysis of a de novo DCLK1 variant associated with a neurodevelopmental disorder

The findings implicate DCLK1 in a previously unrecognized progressive neurodevelopmental disorder and demonstrate the power of integrative cross-species functional genomics in resolving ultra-rare disease variants.

Biallelic Truncating Variant in LRGUK Is Associated With Severe Multiple Morphological Abnormalities of the Sperm Flagella and Sperm Nuclear Defects in Humans.

Multiple morphological abnormalities of the flagella (MMAF) is a severe form of male infertility characterized by immotile spermatozoa with absent, short, coiled, or irregular flagella. Despite advances in whole-exome sequencing, many cases remain genetically unexplained. Here, we identify a homozygous truncating variant in LRGUK (c.1063C>T; p.Arg355Ter) in an infertile man presenting with a typical MMAF phenotype. The variant, identified by whole-exome sequencing in a cohort of 168 MMAF patients and confirmed by Sanger sequencing, is predicted to result in loss of the C-terminal guanylate kinase-like domain. Immunofluorescence showed absence of LRGUK protein in patient spermatozoa, supporting a loss-of-function effect. Semen analysis revealed impaired motility and complete teratozoospermia. Morphological and ultrastructural analyzes demonstrated severe defects affecting both sperm head and flagellum, including disorganized axonemal architecture and central pair abnormalities. Nuclear analyzes showed increased nuclear size, defective chromatin compaction, and elevated DNA fragmentation. Immunostaining further indicated alterations of central apparatus-associated proteins, supporting a role for LRGUK in C1b projection organization. These findings identify LRGUK as a novel gene involved in male infertility, essential for spermatid morphogenesis and flagellum assembly.

TTC14 dysfunction contributing to microcephaly and lissencephaly spectrum features through protein mislocalization and impaired RNA processing

Tetratricopeptide Repeat Domain 14 plays a crucial role in RNA metabolism during neurodevelopment, and that the p.His30Arg variant impairs its function, possibly leading to a neurodevelopmental disorder within the lissencephaly spectrum.