Biomedical subjects

Diana Baralle

Publications and source records attributed to Diana Baralle.

10 recordsLinked to original sources

PubMed2026

Proteomics identify disease-associated variants in patients with rare diseases undiagnosed after genome sequencing.

Despite the introduction of genome sequencing (GS) for rare disease diagnostics, a genetic cause is not identified in most patients. Here, we explored the potential of proteomics to improve the diagnostic yield in 424 patients with rare diseases from the 100,000 Genomes Project (100kGP) without a genetic diagnosis. Serum proteomic profiling was performed using the Olink Explore 1536 assay (N = 1463 proteins). For 13 patients without genetic diagnoses, detection of lower serum protein "outliers" (z-score < -2) led to confirmed genetic diagnoses by resolving variants of uncertain significance or prioritizing genes for targeted GS reanalysis. For 23 additional patients without genetic diagnoses (64% of findings), we identified candidate gene-disease links and variants through convergent evidence from lower protein outliers and variants ranked through the variant prioritization tool Exomiser. For example, we identified a candidate heterozygous missense variant [Genome Aggregation Database (gnomAD) minor allele frequency = 0.006%] in tyrosine kinase with immunoglobulin-like and epidermal growth factor homology domains 1 (TIE1) that was only present in a patient with lower TIE1 serum abundance (z-score = -5.12) and their father, both of whom were affected by the same monogenic cardiac disorder, but in no other individuals from the 100kGP. Missense (52.5%) and splice region (27.5%) variants accounted for most diagnostic or candidate variants prioritized. This proof-of-principle study demonstrated that serum proteomics can support rare disease diagnosis and identify disease-causing genes in patients undiagnosed after GS, although successful implementation will likely depend on tissue specificity of protein expression, detectability in blood, proteomic platform coverage, and sensitivity.

Humans↗

PubMed2026

RNA splicing evidence enables robust classification of BRCA1 exon 18 variants: Results from the ENIGMA consortium.

The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) research consortium conducted a comprehensive study to characterize spliceogenic variants in BRCA1 exon 18. The absence of systematic RNA-based assessment for these variants has led to inconsistent interpretation, limiting accurate classification and management of individuals and their families. The splicing profile of 166 variants was assessed using minigene assays; 32 were additionally analyzed in blood-derived RNA from 51 individuals and 18 in mouse embryonic stem cell (mESC)-based assays to evaluate homology-directed repair (HDR) capacity. mRNA assessment by RT-PCR in blood samples and minigene assays showed a significant positive correlation, with splicing analysis in mESCs displaying highly concordant results. The mESC-based HDR assay showed that the in-frame exon 18 skipping (Δ18) transcript encodes a non-functional protein lacking rescue activity. Linear regression analysis using mESC splicing and functional data indicated that ≥59% of full-length (FL) levels and <34% of Δ18 were associated with benign HDR activity. These thresholds differ from those recommended by the ClinGen ENIGMA BRCA1 and BRCA2 Variant Curation Expert Panel American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) specifications for applying BP7_strong(RNA): >30% functional transcripts or <70% non-functional transcripts. Incorporation of RNA splicing evidence into variant interpretation increased pathogenic (28.6%-31.7%) and benign (3.7%-24.4%) classifications while reducing likely pathogenic (19.5%-17.7%), uncertain (18.9%-8.5%), and likely benign (29.3%-17.7%) categories. Experimental mRNA profiling impacted the interpretation of 34% of variants and resolved uncertainty in approximately 10% of cases. Exon 18 skipping was less tolerated, indicating that the degree of splice perturbation required to impair BRCA1 function may depend on the nature of the resulting non-functional transcript.

Humans↗

PubMed2026

MAJIQ-CLIN: A novel tool to help identify Mendelian disease-causing variants from RNA-seq data.

PURPOSE: The current diagnostic rate for patients with suspected Mendelian genetic disorders is low, despite exome/genome sequencing being the standard of care. One reason for this low diagnostic rate is that traditional exome/genome sequencing analysis methods struggle to detect RNA splicing aberrations. Causative variants often involve splicing changes, with numerous splice-altering variants being responsible for known Mendelian disorders. Therefore, it is crucial to develop reliable tools to detect, quantify, prioritize, and visualize RNA splicing aberrations from patient RNA sequencing data. METHODS: We developed Modeling Alternative Junction Inclusion Quantification for Clinical Applications (MAJIQ-CLIN), a method to identify RNA splicing aberrations in patients' RNA sequencing data compared with a cohort of control samples. MAJIQ-CLIN can efficiently process large datasets, avoiding reprocessing when new data are added, while effectively detecting local splicing variations with deviations in a given patient, termed outlier local splicing variation, or unique to the patient, termed private local splicing variation. RESULTS: We performed a systematic evaluation of the accuracy of tools for detecting patients' RNA splicing aberrations from RNA sequence using synthetic data across several aberration types and transcript inclusion levels. Then, we used several real datasets to assess MAJIQ-CLINs ability to identify solved test cases and control for the effect of confounders such as batches. We showed that MAJIQ-CLIN compares favorably to existing tools in both accuracy and efficiency. We also used MAJIQ-CLIN to investigate several unsolved patient cases from the Undiagnosed Diseases Network. CONCLUSION: MAJIQ-CLIN offers an efficient, accurate, and user-friendly tool to aid in diagnosing Mendelian disease-causing variants from RNA sequence data.

Bioinformatics↗

PubMed2025

Utility of genome sequencing and group-enrichment to support splice variant interpretation in Marfan syndrome.

PURPOSE: To quantify the impact of noncanonical FBN1 splice site variants in undiagnosed Marfan syndrome (MFS), a connective tissue disorder associated with skeletal abnormalities and familial thoracic aortic aneurysm disease (FTAAD). METHODS: A systematic analysis of ultrarare FBN1 variants was performed using genome sequencing data from the 100,000 Genomes Project. Variants were annotated with SpliceAI and the significance of enrichment among individuals with FTAAD was assessed using Fisher's exact test. Experimental validation used RNA sequencing, reverse transcriptase polymerase chain reaction, minigene constructs, and replication analysis was with data from UK Biobank. RESULTS: Using aggregate data for 78,195 individuals, we identified 13,864 singleton single-nucleotide variants in FBN1 of which 21 were predicted to affect splicing (SpliceAI > 0.5). Incidence of candidate splice variants in individuals recruited with FTAAD (9/703) was significantly elevated compared with that seen in non-FTAAD participants (12/77,492; odds ratio = 84, P = 9.7 × 10-14). Additional analysis uncovered a further 14 families harboring 11 different FBN1 splice variants. A total of 20 candidate splice variants in 23 families were identified, of which 70% lay beyond the ±8 splice regions. RNA testing confirmed the predicted splice aberration in 16 of 20 and for 9 of 20, pseudoexonization was the likely splicing anomaly. CONCLUSION: Our findings indicate that noncanonical splice variants may account for approximately 3% of families with undiagnosed FTAAD, highlighting the importance of incorporating analysis of introns and confirmatory RNA testing into genetic testing for Marfan syndrome.

Humans↗

PubMed2006

NF1 mRNA biogenesis: effect of the genomic milieu in splicing regulation of the NF1 exon 37 region.

We have studied the splicing regulation of NF1 exons 36 and 37. We show that they not only require an intact exonic Splicing Enhancer (ESE) within exon 37, but also need the genomic region stretching from exons 31 to 38. Any nucleotide change in two exon 37 third codon positions disrupts the ESE. The extent of exons 36 and 37 skipping due to a mutated ESE depends on the genomic context. This is a unique example of what may be a more general phenomena involved in the tuning of pre-mRNA processing and gene expression modulation in the chromosomal setting.

Alternative Splicing↗

PubMed2006

PMS2 mutations in childhood cancer.

Until recently, the PMS2 DNA mismatch repair gene has only rarely been implicated as a cancer susceptibility locus. New studies have shown, however, that earlier analyses of this gene have had technical limitations and also that the genetic behavior of mutant PMS2 alleles is unusual, in that, unlike MLH1 or MSH2 mutations, PMS2 mutations show low heterozygote penetrance. As a result, a dominantly inherited cancer predisposition has not been a feature reported in families with PMS2 mutations. Such families have instead been ascertained through childhood-onset cancers in homozygotes or through apparently sporadic colorectal cancer in heterozygotes. We present further information on the phenotype associated with homozygous PMS2 deficiency in 13 patients from six families of Pakistani origin living in the United Kingdom. This syndrome is characterized by café-au-lait skin pigmentation and a characteristic tumor spectrum, including leukemias, lymphomas, cerebral malignancies (such as supratentorial primitive neuroectodermal tumors, astrocytomas, and glioblastomas), and colorectal neoplasia with an onset in early adult life. We present evidence for a founder effect in five families, all of which carried the same R802-->X mutation (i.e., arginine-802 to stop) in PMS2. This cancer syndrome can be mistaken for neurofibromatosis type 1, with important management implications including the risk of the disorder occurring in siblings and the likelihood of tumor development in affected individuals.

Adenosine Triphosphatases↗

PubMed2005

A centrosomal mechanism involving CDK5RAP2 and CENPJ controls brain size.

Autosomal recessive primary microcephaly is a potential model in which to research genes involved in human brain growth. We show that two forms of the disorder result from homozygous mutations in the genes CDK5RAP2 and CENPJ. We found neuroepithelial expression of the genes during prenatal neurogenesis and protein localization to the spindle poles of mitotic cells, suggesting that a centrosomal mechanism controls neuron number in the developing mammalian brain.

Animals↗

PubMed2004

hnRNP H binding at the 5' splice site correlates with the pathological effect of two intronic mutations in the NF-1 and TSHbeta genes.

We have recently reported a disease-causing substitution (+5G > C) at the donor site of NF-1 exon 3 that produces its skipping. We have now studied in detail the splicing mechanism involved in analyzing RNA-protein complexes at several 5' splice sites. Characteristic protein patterns were observed by pulldown and band-shift/super-shift analysis. Here, we show that hnRNP H binds specifically to the wild-type GGGgu donor sequence of the NF-1 exon 3. Depletion analyses shows that this protein restricts the accessibility of U1 small nuclear ribonucleoprotein (U1snRNA) to the donor site. In this context, the +5G > C mutation abolishes both U1snRNP base pairing and the 5' splice site (5'ss) function. However, exon recognition in the mutant can be rescued by disrupting the binding of hnRNP H, demonstrating that this protein enhances the effects of the +5G > C substitution. Significantly, a similar situation was found for a second disease-causing +5G > A substitution in the 5'ss of TSHbeta exon 2, which harbors a GGgu donor sequence. Thus, the reason why similar nucleotide substitutions can be either neutral or very disruptive of splicing function can be explained by the presence of specific binding signatures depending on local contexts.

Binding Sites↗

PubMed2004

Mutations of VMD2 splicing regulators cause nanophthalmos and autosomal dominant vitreoretinochoroidopathy (ADVIRC).

PURPOSE: To investigate the genetic basis of autosomal dominant vitreoretinochoroidopathy (ADVIRC), a rare, inherited retinal dystrophy that may be associated with defects of ocular development, including nanophthalmos. METHODS: A combination of linkage analysis and DNA sequencing in five families was used to identify disease-causing mutations in VMD2. The effect of these mutations on splicing was assessed using a minigene system. RESULTS: Three pathogenic sequence alterations in VMD2 were identified in five families with nanophthalmos associated with ADVIRC. All sequences showed simultaneous missense substitutions and exon skipping. CONCLUSIONS: VMD2 encodes bestrophin, a transmembrane protein located at the basolateral membrane of the RPE, that is also mutated in Best macular dystrophy. We support that each heterozygous affected individual produces three bestrophin isoforms consisting of the wild type and two abnormal forms: one containing a missense substitution and the other an in-frame deletion. The data showed that VMD2 mutations caused defects of ocular patterning, supporting the hypothesized role for the RPE, and specifically VMD2, in the normal growth and development of the eye.

Bestrophins↗

PubMed2003

Different mutations in the NF1 gene are associated with Neurofibromatosis-Noonan syndrome (NFNS).

The association of the Noonan phenotype with neurofibromatosis type 1 (NF1) was first noted by Allanson et al. [Am J Med Genet 1985;21:457-462.] and 30 further cases have subsequently been reported. It has been suggested that this phenotype is more common than previously appreciated, as Colley et al. [Clin Genet 1996;49:59-64.] examined 94 sequentially identified patients with NF1 from their genetic register and found Noonan features in 12. A 3-bp deletion of exon 17 of the NF1 neurofibromin gene was described in one family by Carey et al. [Proc Greenwood Genet Center 1997;17:52-53]. However, it remains unclear whether Neurofibromatosis-Noonan syndrome (NFNS) represents a form of NF1 (with mutations in the NF1 neurofibromin gene) or a separate syndrome. We have used a new, rapid sequence analysis technique-comparative sequence analysis (CSA)-to examine the NF1 gene in six patients with NFNS. None of the six patients had the previously identified mutation, nor did we observe other mutations within this exon. However, two other mutations were found: in exon 25, a 3-bp deletion 4312 del GAA, and in exon 23-2, a 2-bp insertion 4095 ins TG. The PTPN11 gene, now known to cause over 50% of Noonan syndrome was also examined in four cases of NFNS, and no mutations were found. These results show that NFNS can in some cases result from different mutations in the NF1 gene and therefore represents a variant form of NF1.

Adolescent↗