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The distal Xq28 region is very gene-rich, comprising a relatively large number of low-copy repeats (LCRs) predisposing to genomic rearrangements. The best-known rearrangement at this locus is the F8 intron 22 inversion, responsible for up to 45% of severe hemophilia A (HA) cases. An additional inversion of intron 1 of F8 has more recently been described, affecting 2%-5% of patients with severe HA. These "balanced" rearrangements are mediated by intrachromosomal homologous recombination between inversely oriented LCRs located in intron 1 or 22 and other extragenic copies positioned more telomerically outside the F8 gene. The successive innovations of semi-quantitative technologies like multiplex ligation-dependent probe amplification (MLPA) and array comparative genomic hybridization (array CGH) have rendered it possible to highlight a significant number of "unbalanced" rearrangements associated or not with these inversions. Some rearrangements are generated by the non-allelic homologous recombination (NAHR) pathway between directly oriented LCRs. Others are probably the result of unequal crossing-over or U-loop exchanges during female meiosis. This review sought to provide an overview of the mechanisms underlying rearrangements at the distal Xq28 locus and discuss their clinical impacts other than HA, such as risks of developing high inhibitor levels and spontaneous abortion, as well as other pathologies like cardiovascular disease or potentially X-linked intellectual disease.

Peu de temps après la découverte des différents gènes dit monogéniques (un gène = une maladie), la communauté médicale et scientifique s’est rapidement enthousiasmée pour le développement et la mise en place d’essais cliniques en thérapie génique dans l’espoir de corriger une activité génétique défaillante. Après le décès d’un patient atteint d’immunodéficience combiné sévère lié à l’X, largement rapporté dans la communauté scientifique, il fallut se rendre à l’évidence dans les années 2000 que l’on était encore loin d’une mise en place de traitements reconnus par thérapie génique. Cependant, on constate aujourd’hui un développement de nouveaux vecteurs dérivés de lentivirus et d’AVV, plus efficaces et plus sécurisés, qui permet l’aboutissement prometteur de plusieurs essais cliniques de traitements de diverses pathologies comme l’hémophilie, l’adrénoleucodystrophie, l’amaurose de Leber ou le cancer, [Gene therapy in 2017: overview and perspectives] Shortly after the discovery of the different so-called monogenic genes (one gene = one disease), the medical and scientific community became excited by the prospects of development and implementation of clinical trials in gene therapy aimed at correcting failing genetic activity. Following the death of a patient with severe combined X-linked immunodeficiency, widely reported in the scientific community, it was obvious that there was still much progress to be made in the 2000s for recognized gene therapies to be implemented. However, new vectors derived from lentiviruses and adenoviral vectors are currently in development, which promise to be more efficient and safer, with positive outcomes in several clinical trials on hemophilia, adrenoleukodystrophy, Leber's amaurosis, or cancer.

DNA structure alterations are the ultimate source of genetic variations. Without them, evolution would be impossible. While they are essential for DNA diversity, defects in DNA synthesis can lead to numerous genetic diseases. Due to increasingly innovative technologies, our knowledge of the human genome and genetic diseases has grown considerably over the last few years, allowing us to detect another class of variants affecting the chromosomal structure. DNA sequence can be altered in multiple ways: DNA sequence changes by substitution, deletion, or duplication of some nucleotides; chromosomal structure alterations by deletion, duplication, translocation, and inversion, ranging in size from kilobases to mega bases; changes in the cell's genome size. If the alteration is located within a gene and sufficiently deleterious, it can cause genetic disorders. Due to the F8 gene's high rate of new small mutations and its location at the tip of X chromosome, containing high repetitive sequences, a wide variety of genetic variants has been described as the cause of hemophilia A (HA). In addition to the F8 intron 22 repeat inversion, HA can also result from point mutations, other inversions, complex rearrangements, such as duplications or deletions, and transposon insertions causing phenotypes of variable severity characterized by complete or partial deficiency of circulating FVIII. This review aims to present the origins, mechanisms, and consequences of F8 alterations. A sound understanding of the multiple genetic mechanisms responsible for HA is essential to determine the appropriate strategy for molecular diagnosis and detected each type of genetic variant.

Roughly 40% of observed mutations responsible for hemophilia A (HA) are novel and present in either a single family or a limited number of unrelated families. During routine diagnostic analysis of 73 unrelated Belgian patients with mild HA, 4 out of 43 different mutations (p.Ser2030Asn, p.Arg2178Cys, p.Arg2178His, and p.Pro2311His) were detected in more than one family, representing 35% of total identified mutations. To discriminate between an independent recurrence or a founder effect, an analysis of intra- and -extragenic single nucleotide polymorphisms (SNPs) and short tandem repeats (STRs) flanking the F8 gene was conducted. SNP haplotype and microsatellite analysis revealed strong evidence that p.Ser2030Asn and p.Pro2311His mutations were probably associated with a founder effect. The two other mutations localized in an F8 cytosine-phosphate-guanine (CpG) site likely resulted from recurrent de novo events. This study suggests that missense mutations producing C-to-T or G-to-A substitutions in CpG dinucleotide can occur de novo with more repetition than other causal substitutions that do not affect the CpG site. Analysis of F8 database implied that CpG sites throughout the F8 gene are not all mutated with the same frequency. Causes are still unknown and remain to be identified.

The intron 22 inversion found in up to 50% of severe hemophilia A patients results from a recombination between three intron 22 homologous copies (int22h). This study evaluated the implication of these copies in the formation of extended duplications comprising exons 1-22 of the factor 8 (F8) gene and their association with hemophilia and mental retardation. Two hemophilic patients with moderate and severe phenotypes and a third nonhemophilic patient with developmental delay were studied. All exhibited a duplication of F8 gene exons 1-22 identified by multiplex ligation-dependent probe amplification along with abnormal patterns on Southern blotting and unexpected long-range PCR amplification. Breakpoint analysis using array comparative genomic hybridization was performed to delimit the extent of these rearrangements. These duplications were bounded on one side by the F8 intragenic int22h-1 repeat and on the other side by extragenic int22h-2 or int22h-3 copies. However, the simultaneous identification of a second duplication containing F8 gene exons 2-14 for the moderate patient and the classical intron 22 inversion for the severe patient are considered in this study as the genetic causal defects of hemophilia. This study shows that the well-known int22h copies are involved in extended duplications comprising F8 gene exons 1-22. These specific duplications are probably not responsible for hemophilia and intellectual disability, but should be carefully considered in genetic counseling, while continuing to investigate the causal mutation of hemophilia.European Journal of Human Genetics advance online publication, 9 January 2013; doi:10.1038/ejhg.2012.275.

Haemophilia A (HA) is caused by widespread mutations in the factor VIII gene. The high spontaneous mutation rate of this gene means that roughly 40% of HA mutations are private. This study aimed to describe the approaches used to confirm private disease-causing mutations in a cohort of Belgian HA patients. We studied 148 unrelated HA families for the presence of intron 22 and intron 1 inversion by Southern blotting and polymerase chain reaction (PCR). Multiplex ligation-dependent probe amplification (MLPA) assay was used to detect large genomic rearrangements. Detection of point mutations was performed by DNA sequencing. Predicting the causal impact of new non-synonymous changes was studied by two general strategies: (i) molecular approaches such as family cosegregation, evaluation of the implicated codon based on phylogenic separated species and absence of the mutation in the general Belgian population, and (ii) bioinformatics approaches to analyse the potential functional consequences of missense mutations. Among the 148 HA patients, in addition to common intron 22 and intron 1 inversions as well as large deletions or duplications, 67 different point mutations were identified, of which 42 had been reported in the HAMSTeRS database, and 25 were novel including 10 null variants for which RNA analyses confirmed the causal effect of mutations located in a splice site consensus and 15 missense mutations whose causality was demonstrated by molecular approaches and bioinformatics. This article reports several strategies to evaluate the deleterious consequences of unreported F8 substitutions in a large cohort of HA patients.

'History can change blood. And blood can change the course of history'. Haemophilia is an illustration of this, as this congenital hereditary coagulation disorder, passed through the majority of royal European families at the beginning of the 20th century by Queen Victoria of England and Empress of the Indies, had indisputable political consequences, which led to one of the most defining moments of contemporary history: the Bolshevik Revolution. Today, none of Queen Victoria's living descendents carry haemophilia. Because of this, the characterization of haemophilia (deficit of either factor VIII or XI) and the identification of the causal mutation are rendered impossible. In 1991, a tomb containing the remains of Czar Nicolas II's entire family was discovered. A second tomb was discovered in 2007, allowing Russian and American scientists to fill in this gap in medical history. Following a scientific approach combining current genetic experimentation tools and the development of biological information technology, researchers were able to identify each body, allowing them to obtain precious genetic material from the young Czar Alexis, who was stricken by the disease, which revealed a causal substitution in the splice acceptor site of exon 4 in the F9 gene. This mutation that is responsible for haemophilia B had traumatized European royal families throughout the 20th century!.

CTG triplet expansion was studied in lymphocytes and thyroid tissue in a patient with myotonic dystrophy (DM) and associated thyroid nodular disease. An approximately 7 fold larger amplification was found in abnormal thyroid tissue compared to lymphocytes, suggesting that anomalies in the putative DM kinase gene might contribute to thyroid dysfunction.