No abstract

Hemophilia B (HB) associates with pathogenic F9-variants. The literature showed that hemizygous deletions encompassing F9 and vicinal genes may express extra-phenotypes suggesting new causal relationships. Aim: Analyze the molecular basis of syndromic cases of HB, obesity (OB), severe global developmental delay (SGDD) and generalized hypotonia (GH). Whole F9-deletions were detected in 3 hemizygous probands with HB. Dense SNP-array and case-specific STS walking strategies allowed amplification and characterization of the deletion breakpoints. Bioinformatic/statistical analyses included data mining in HPO (Human Phenotype Ontology), OMIM, STRING (protein-protein interaction networks) databases and estimation of null-hypothesis-based Expected-values. Patients (cases#3-case#1) showed complete F9 deletions involving 0.16-4.34-Mb and 1-17 additional genes on Xq26.3-q27.2. Bioinformatic/statistical approaches revealed highly significant STRING-associations (P[?]0.00115) between case#1/#2 common deleted genes (SOX3, FGF13, CXorf66) and those HPO associated with OB (20/343), GH (36/923) and SGDD (10/119). Only case#2 showed two extra-phenotypic abnormalities, anal atresia and pituitary hypothyroidism, and one extra-deleted gene, MAGEC2. Our bioinformatic/statistical approaches confirmed a previous involvement of SOX3 in OB suggesting additional roles in GH and SGDD, similar to FGF13 and CXorf66 (experimentally linked to POMGNT1, HPO-connected with all 3 phenotypes). Our findings highlight the importance to characterize X-chromosome deletion syndromes to unveil functional associations of the involved genomic regions. Syndromic cases of hemophilia B and morbid obesity due to contiguous gene deletions on Xq26.3-q27.2: unsuspected phenotype-genotype associations by bioinformatics and extensive clinical data mining

Here, we report on the identification of a highly polymorphic tetranucleotide repeat (named DXpS) mapping to within a CpG island on the Xp. This island is 191 bp upstream from the start of the repeat element, and contains sites for the HhaI, HpaII and BstUI methyl-sensitive restriction enzymes ( Figure 1). We developed the DXpS and the HUMARA markers into a biplex methylation-based quantitative fluorescent PCR assay. For DXpS we observed twelve alleles with negligible stuttering (Figure 2). DXpS exhibited a heterozygosity rate of 87% (n = 60), matching that of HUMARA. The combined informativeness of the biplex assay was 98%. Random and nonrandom X-inactivation patterns inferred with DXpS in phenotypically normal females and haemophiliac females carrying a defective F8 gene were highly concordant (r 2 = 0.96) with HUMARA patterns (Figures 3, 4 and 5).

Our work depicts a familial Duchenne muscular dystrophy case with a complex structural variant (cxSV) and a manifesting pregnant woman. Were our aims to provide molecular diagnosis and hypothesize mechanisms underlying the origin of the cxSV. We implemented a multi-technique approach including MLPA, STRs-segregation, AR-assay, SNP-array, WGS and a bioinformatic algorithm for identification of double strand breaks (DSB) stimulator motifs. We established the carrier status of the prenatal sample and explained its mother s symptomatology by skewed X-chromosome inactivation. Furthermore, an ancestral familial ex38-43 duplication plus a de novo ex45-54 deletion was revealed in the proband, who carried the cxSV in a recombinant maternal X-chromosome. Characterization of cxSV s breakpoints junction and its surrounding sequences allowed us to identify DSB stimulator motifs. The replication-dependent "Fork Stalling and Template Switching" mechanism was predicted to be the most likely scenario for the duplication s origin. Whilst, the de novo deletion could arise from a germline event of inter-chromosome non-allelic recombination involving the "Non-Homologous End Joining" mechanism. The multi-technique strategy enabled precise diagnosis, accurate genetic assessment and widen the understanding of the molecular mechanisms involved in SVs' generation. Finally, the further comprehension of the occurrence of DMD variants, favors the development of new gene therapy strategies.

Here, we report on the identification of a highly polymorphic tetranucleotide repeat (named DXpS) mapping to within a CpG island on the Xp. This island is 191 bp upstream from the start of the repeat element, and contains sites for the HhaI, HpaII and BstUI methyl-sensitive restriction enzymes ( Figure 1). We developed the DXpS and the HUMARA markers into a biplex methylation-based quantitative fluorescent PCR assay. For DXpS we observed twelve alleles with negligible stuttering (Figure 2). DXpS exhibited a heterozygosity rate of 87% (n = 60), matching that of HUMARA. The combined informativeness of the biplex assay was 98%. Random and nonrandom X-inactivation patterns inferred with DXpS in phenotypically normal females and haemophiliac females carrying a defective F8 gene were highly concordant (r 2 = 0.96) with HUMARA patterns (Figures 3, 4 and 5).