Translational Diagnostics: An In-House Pipeline to Validate Genetic Variants in Children with Undiagnosed and Rare Diseases.

Diagnosis is essential for the management and treatment of patients with rare diseases. In a group of patients, the genetic study identifies variants of uncertain significance or inconsistent with the phenotype; therefore, it is urgent to develop novel strategies to reach the definitive diagnosis. Herein, we develop the in-house Translational Diagnostics Program (TDP) to validate genetic variants as part of the diagnostic process with the close collaboration of physicians, clinical scientists, and research scientists. The first 7 of 33 consecutive patients for whom exome-based tests were not diagnostic were investigated. The TDP pipeline includes four steps: (i) phenotype assessment, (ii) literature review and prediction of in silico pathogenicity, (iii) experimental functional studies, and (iv) diagnostic decision-making. Re-evaluation of the phenotype and re-analysis of the exome allowed the diagnosis in one patient. In the remaining patients, the studies included either cDNA cloning or PCR-amplified genomic DNA, or the use of patients' fibroblasts. A comparative computational analysis of confocal microscopy images and studies related to the protein function was performed. In five of these six patients, evidence of pathogenicity of the genetic variant was found, which was validated by physicians. The current research demonstrates the feasibility of the TDP to support and resolve intramural medical problems when the clinical significance of the patient variant is unknown or inconsistent with the phenotype.

Optimised molecular genetic diagnostics of Fanconi anaemia by whole exome sequencing and functional studies.

PURPOSE: Patients with Fanconi anaemia (FA), a rare DNA repair genetic disease, exhibit chromosome fragility, bone marrow failure, malformations and cancer susceptibility. FA molecular diagnosis is challenging since FA is caused by point mutations and large deletions in 22 genes following three heritability patterns. To optimise FA patients' characterisation, we developed a simplified but effective methodology based on whole exome sequencing (WES) and functional studies. METHODS: 68 patients with FA were analysed by commercial WES services. Copy number variations were evaluated by sequencing data analysis with RStudio. To test FANCA missense variants, wt FANCA cDNA was cloned and variants were introduced by site-directed mutagenesis. Vectors were then tested for their ability to complement DNA repair defects of a FANCA-KO human cell line generated by TALEN technologies. RESULTS: We identified 93.3% of mutated alleles including large deletions. We determined the pathogenicity of three FANCA missense variants and demonstrated that two FANCA variants reported in mutations databases as 'affecting functions' are SNPs. Deep analysis of sequencing data revealed patients' true mutations, highlighting the importance of functional analysis. In one patient, no pathogenic variant could be identified in any of the 22 known FA genes, and in seven patients, only one deleterious variant could be identified (three patients each with FANCA and FANCD2 and one patient with FANCE mutations) CONCLUSION: WES and proper bioinformatics analysis are sufficient to effectively characterise patients with FA regardless of the rarity of their complementation group, type of mutations, mosaic condition and DNA source.

Detectable clonal mosaicism in blood as a biomarker of cancer risk in Fanconi anemia.

Detectable clonal mosaicism for large chromosomal events has been associated with aging and an increased risk of hematological and some solid cancers. We hypothesized that genetic cancer predisposition disorders, such as Fanconi anemia (FA), could manifest a high rate of chromosomal mosaic events (CMEs) in peripheral blood, which could be used as early biomarkers of cancer risk. We studied the prevalence of CMEs by single-nucleotide polymorphism (SNP) array in 130 FA patients' blood DNA and their impact on cancer risk. We detected 51 CMEs (4.4-159 Mb in size) in 16 out of 130 patients (12.3%), of which 9 had multiple CMEs. The most frequent events were gains at 3q (n = 6) and 1q (n = 5), both previously associated with leukemia, as well as rearrangements with breakpoint clustering within the major histocompatibility complex locus (P = 7.3 × 10-9). Compared with 15 743 age-matched population controls, FA patients had a 126 to 140 times higher risk of detectable CMEs in blood (P < 2.2 × 10-16). Prevalent and incident hematologic and solid cancers were more common in CME carriers (odds ratio [OR] = 11.6, 95% confidence interval [CI] = 3.4-39.3, P = 2.8 × 10-5), leading to poorer prognosis. The age-adjusted hazard risk (HR) of having cancer was almost 5 times higher in FA individuals with CMEs than in those without CMEs. Regarding survival, the HR of dying was 4 times higher in FA individuals having CMEs (HR = 4.0, 95% CI = 2.0-7.9, P = 5.7 × 10-5). Therefore, our data suggest that molecular karyotyping with SNP arrays in easy-to-obtain blood samples could be used for better monitoring of bone marrow clonal events, cancer risk, and overall survival of FA patients.

Ancient Haplotypes at the 15q24.2 Microdeletion Region Are Linked to Brain Expression of MAN2C1 and Children's Intelligence.

The chromosome bands 15q24.1-15q24.3 contain a complex region with numerous segmental duplications that predispose to regional microduplications and microdeletions, both of which have been linked to intellectual disability, speech delay and autistic features. The region may also harbour common inversion polymorphisms whose functional and phenotypic manifestations are unknown. Using single nucleotide polymorphism (SNP) data, we detected four large contiguous haplotype-genotypes at 15q24 with Mendelian inheritance in 2,562 trios, African origin, high population stratification and reduced recombination rates. Although the haplotype-genotypes have been most likely generated by decreased or absent recombination among them, we could not confirm that they were the product of inversion polymorphisms in the region. One of the blocks was composed of three haplotype-genotypes (N1a, N1b and N2), which significantly correlated with intelligence quotient (IQ) in 2,735 children of European ancestry from three independent population cohorts. Homozygosity for N2 was associated with lower verbal IQ (2.4-point loss, p-value = 0.01), while homozygosity for N1b was associated with 3.2-point loss in non-verbal IQ (p-value = 0.0006). The three alleles strongly correlated with expression levels of MAN2C1 and SNUPN in blood and brain. Homozygosity for N2 correlated with over-expression of MAN2C1 over many brain areas but the occipital cortex where N1b homozygous highly under-expressed. Our population-based analyses suggest that MAN2C1 may contribute to the verbal difficulties observed in microduplications and to the intellectual disability of microdeletion syndromes, whose characteristic dosage increment and removal may affect different brain areas.