This case highlights the diagnostic value of comprehensive genomic analysis and subsequent RNA sequencing for identifying and analyzing deep intronic variants in genetically unexplained cases of XLH and underscores the importance of re-evaluating patients with a strong clinical diagnosis but previously negative genetic results.
Abstract
X-linked hypophosphatemia (XLH) is a rare, genetically determined disorder of phosphate metabolism, most commonly caused by mutations in the PHEX gene. These mutations lead to overexpression of the phosphaturic hormone FGF23, resulting in renal phosphate wasting and impaired bone mineralization. In up to 16% of clinically diagnosed cases, no causative variant can be identified using standard sequencing approaches. We report on a female patient with a clearly defined clinical XLH phenotype, in whom no causative mutation had been detected over several years despite extensive genetic testing. The aim was to identify a previously undetected genetic cause using extended DNA and RNA methods. After unremarkable short-read whole exome sequencing (WES), short-read whole genome sequencing (WGS) was performed. For confirmation of splice effect, RNA was extracted from peripheral blood, amplified via RT-PCR, and analyzed using Nanopore long-read sequencing. A novel deep intronic variant in the PHEX gene (c.2070 + 601C>T) was identified and confirmed as de novo. The variant caused two aberrant transcripts with pseudoexon inclusions, each leading to a premature stop codon. This aberrant splicing supports the pathogenicity of the variant in the context of a loss-of-function mechanism. Following molecular diagnosis, the patient was successfully initiated on Burosumab therapy, resulting in clinical improvement. This case highlights the diagnostic value of comprehensive genomic analysis and subsequent RNA sequencing for identifying and analyzing deep intronic variants in genetically unexplained cases of XLH. The findings expand the known PHEX mutation spectrum and emphasize the importance of re-evaluating patients with a strong clinical diagnosis but previously negative genetic results. In the future, such technologies may play a crucial role in improving diagnostics for rare monogenic diseases.
Abstract Background Non-obstructive azoospermia (NOA) is a severe form of male infertility characterized by impaired spermatogenesis. Although chromosomal abnormalities and Y-chromosome microdeletions are known causes, many cases remain idiopathic, suggesting that other genetic factors may be involved. RAD54 like (RAD54L) is a key component of the homologous recombination pathway required for DNA double-strand break repair and meiotic progression. Objective To identify the potential genetic cause of NOA using whole-genome sequencing (WGS) in affected individuals from consanguineous families. Materials and Methods For performing this experimental study, 10 NOA men who were referred to Infertility Center of the Yazd Reproductive Sciences Institute, Yazd, Iran underwent clinical, hormonal, and genetic evaluations. Genomic DNA was extracted from peripheral blood and was subjected to WGS. Variants were filtered and prioritized based on allele frequency, predicted pathogenicity, inheritance pattern, and gene expression data. In silico splicing analyses were performed using SpliceAI and Human Splicing Finder. Results WGS identified an ultrarare homozygous splice-site variant, c.1375+1G > T, in the RAD54L gene. The variant affects a canonical donor splice site and is predicted to cause loss of normal splicing, likely resulting in exon 12 skipping, frameshift, and premature termination codon formation. RAD54L shows the highest expression in testicular tissue and plays a critical role in homologous recombination and meiotic DNA repair. The patient exhibited hormonal findings consistent with primary testicular failure. Conclusion This study proposes RAD54L as a potential novel candidate gene linked to NOA and underscores the efficacy of WGS in identifying rare genetic etiologies of idiopathic male infertility. Further functional and clinical studies are needed to validate this association.
Razieh Ebrahimi Askari, A. Malcher, Sara Sadeghzadeh et al.· International Journal of Rep...· 0 citations
X-linked hypophosphatemic rickets (XLH) is the most common inherited cause of renal phosphate wasting. It is typically caused by pathogenic variants in the phosphate-regulating endopeptidase homolog, X-linked (PHEX gene), which lead to increased levels of fibroblast growth factor 23 (FGF23). However, noncanonical intronic variants represent a significant diagnostic challenge, as they may not be detected by conventional genetic studies and require interpretation supported by bioinformatic tools. We present the case of a pediatric patient with postnatal short stature and progressive genu varum. Biochemical studies revealed hypophosphatemia with reduced tubular reabsorption of phosphate, while calcium, parathyroid hormone (PTH), and vitamin D levels were within normal ranges. Radiological findings were consistent with rickets. Initial clinical exome sequencing was negative. Subsequently, a targeted gene panel identified a previously unreported intronic variant in the PHEX gene (c.1768+4dup; chrX:22,219,105 T>TA). In silico analysis using SpliceAI demonstrated a high probability of donor splice site loss (DS_DL = 0.80), while Pangolin predicted a splice-disrupting effect (score = 0.72); collectively, these findings support a deleterious effect on mRNA splicing. This case highlights the importance of integrating clinical, biochemical, genetic, and computational data for the interpretation of noncanonical intronic variants, expanding the mutational spectrum described for PHEX in XLH.
Carlos F. Panqueba Arias, Ingry K. Rojas Rodriguez, Rossi I. Quero et al.· Cureus· 0 citations
It is demonstrated that cryptic pathogenic APC variants beyond the detection limits of routine genetic testing can be resolved through WGS combined with transcript‐level validation, highlighting the importance of transcript‐aware variant interpretation and supports the integration of genome‐wide sequencing and RNA‐based analyses into the diagnostic evaluation of genetically unexplained FAP.
Sung-Ryung Choi, Min Jung Kim, Ja-Lok Ku et al.· Human Mutation· 0 citations
A male infant with a severe FINCA-like phenotype is reported, including early-onset hemolytic anemia, pulmonary involvement, neurodevelopmental impairment, growth failure, recurrent infections, and fatal progression at 8.5 months.
A. Rozhkova, Anton A. Esibov, Aleksandra Borkovskaia et al.· International Journal of Mol...· 0 citations
CHARGE syndrome is a rare congenital disorder primarily attributed to heterozygous pathogenic variants of the CHD7 gene. Most pathogenic CHD7 variants are loss-of-function (LoF) variants, whereas the interpretation of missense variants remains challenging in the absence of functional evidence for their pathogenicity. We report a female infant presenting with clinical features characteristic of CHARGE syndrome. Targeted sequencing identified a heterozygous CHD7 variant (NM_017780.4:c.5273A>G), initially annotated as a missense substitution p.Asp1758Gly. This variant has been previously reported and registered with conflicting pathogenicity classifications; however, its transcript-level consequences remain unclear. Long-PCR-based RNA sequencing of total RNA from peripheral blood mononuclear cells revealed two aberrant splicing patterns associated with the variant: a predominant transcript carrying a 28-bp deletion due to cryptic donor splice-site activation, and a minor transcript with partial intron 24 retention. Both transcripts were predicted to result in premature termination codons. These findings demonstrate that c.5273A>G functions as a LoF variant through dual aberrant splicing rather than a simple missense substitution. This case underscores the importance of RNA-level splicing analysis for the accurate interpretation and classification of CHD7 missense variants.
Takashi Okuno, Tatsuto Shimizu, Aiko Igarashi et al.· American Journal of Medical...· 0 citations
The co-occurrence of variants in AKAP4 and RNF220 may suggest an oligogenic etiology of NOA and contribute to the phenotypic variability associated with AKAP4 variants.
Razieh Ebrahimi Askari, A. Malcher, Fateme Sefid et al.· International Journal of Mol...· 0 citations
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