Incidental finding of unreported large duplication in F8 gene during prenatal analysis: Which management for genetic counselling?

Detection of incidental finding and variant of unknown significance (VUS) during prenatal diagnosis has particularly increased with the emergence of genetic tests such chromosomal microarray analysis (CMA). Many factors and clear guidelines need to be applied in the interpretation of the potential clinical consequences of unreported complex copy number variations (deletions/duplications). From a clinical case where an unreported and not completely intragenic duplication in F8 gene has been identified in a 12-week-old fetus without haemophilia A history documented in the family, we will examine and study the difficulties of interpretation and the challenges that the detection of such variant has on genetic counselling.

Review of molecular mechanisms at distal Xq28 leading to balanced or unbalanced genomic rearrangements and their phenotypic impacts on hemophilia.

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.

Principles of genetic variations and molecular diseases: applications in hemophilia A.

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.

Tandem inversion duplication within F8 Intron 1 associated with mild haemophilia A.

In approximately 90% of mild haemophilia A (HA) patients, a missense mutation can be identified using complete gene sequencing. In this study, multiplex ligation-dependent probe amplification analysis was performed as a second step in 10 French-speaking Belgian with mild HA presenting no detectable causal mutation by complete sequencing of the factor VIII (FVIII) (F8) gene's 26 exons and its 1.2 kb of contiguous promoter sequence. This gene dosage technique enabled the detection of exon 1 duplications of F8 in three apparently unrelated subjects. Using array-comparative genomic hybridization, breakpoint analysis delimited the duplication extent to 210 kb in the F8 intron 1 and VBP1 gene intragenic position. We postulated that the rearrangement responsible for this duplication, never before reported, could be attributed to a symmetrical tandem inversion duplication, resulting in a large 233 kb rearrangement of F8 intron 1. This rearranged intron should lead to the production of a small number of normal mRNA transcripts in relation to the mild HA phenotype. Our analysis of the entire F8 mRNA from index case 1, particularly the segment containing exons 1-9, revealed normal amplification and sequencing. Reduced plasma FVIII antigen levels caused by cross-reacting material is associated with a quantitative deficiency of plasma FVIII. Male patients were unresponsive to desmopressin (1-deamino-8-D-arginine vasopressin). All patients displayed identical F8 haplotypes, despite not being related, which suggests a possible founder effect caused by a 210 kb duplication involving F8 exon 1.

Overrepresentation of missense mutations in mild hemophilia A patients from Belgium: founder effect or independent occurrence?

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.

Prevalence and spectrum of SDHx mutations in pheochromocytoma and paraganglioma in patients from Belgium: an update.

Since the early 2000s, the prevalence and spectrum of mutations in genes encoding subunits of succinate dehydrogenase (SDHx) were reported in large cohorts of patients with pheochromocytoma (PC) and paraganglioma (PGL) from most Western countries. Unfortunately, in Belgium, no equivalent work was performed thus far. Therefore, the aim of the work was to look for mutations in SDHx genes and genotype-phenotype correlations in patients with PC and/or PGL from Belgium. Screening of the coding parts of SDHx genes and deletion search were performed in all patients with PC and/or PGL referred to the -Cliniques Universitaires Saint-Luc from 05/2003 to 05/2011. Genetic screening was performed in 59 unrelated head and neck (hn)PGLs (8 fami-lial) and 53 PCs (7 extra-adrenal; 3 metastatic). In hnPGLs, 10 different SDHD mutations (3 substitutions, 5 deletions, 2 splice site mutations) were detected in 16 patients, including 7 familial cases and 9 apparently sporadic cases. In the same subset, we found 8 different SDHB mutations (5 substitutions, 1 splice site mutation, 1 deletion, 1 duplication) in 10 patients with sporadic hnPGL without evidence of malignancy. No SDHx mutation was detected in patients harboring PCs and no SDHC mutation whatsoever. In conclusion, in our multicentric database of PC-PGLs from Belgium, (i) the prevalence of SDHx mutations was high in hnPGLs (44% in the whole subset, 37% of apparently sporadic cases); (ii) in sporadic cases, the prevalence of SDHB mutations was high (20%), similar to that of SDHD (18%); and (iii) no SDHx mutation was found in a subset of mostly adrenal, benign PCs.

Computational and molecular approaches for predicting unreported causal missense mutations in Belgian patients with haemophilia A.

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.

The 'royal disease'--haemophilia A or B? A haematological mystery is finally solved.

'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!

Identification of de novo deletion in the factor VIII gene by MLPA technique in two girls with isolated factor VIII deficiency.

Blood coagulation evaluation performed preoperatively in two unrelated girls with isolated prolongation of the activated partial thromboplastin time (APTT) and no family history of bleeding disorder revealed a mild factor VIII deficiency. Quantitative and qualitative defect of von Willebrand factor was not present. Genetic analysis of the F8 gene identified no mutations. In contrast, quantitative gene screening using multiplex ligation dependent probe amplification (MLPA) revealed a large heterozygous deletion of the F8 gene in both patients consistent with a carrier status of sporadic severe haemophilia A. This report illustrates that MLPA technique represents an efficient method to screen for large F8 gene deletions in sporadic undiagnosed carriers of haemophilia A.

Novel germline mutations in the APC gene and their phenotypic spectrum in familial adenomatous polyposis kindreds.

Among 23 germline mutations identified in the APC screening of 45 familial adenomatous polyposis (FAP) patients, we have found 10 different novel frameshift mutations in 11 apparently unrelated patients. In two cases, an additional missense mutation was detected. One previously described as a causative germline mutation (S2621C), associated with a 1-bp insertion (4684insA) on the opposite allele, did not segregate with the FAP phenotype in the family and was therefore considered as being non-pathogenic. The other (Z1625H) was located 2 codons before a 1-bp deletion (4897delC). Both mutations were transmitted together from an FAP father to his affected son. The FAP phenotype of these 10 novel truncating mutations was clinically documented within their kindreds. Important variability was observed in the phenotype. Interestingly, we noted that a mutation (487insT) localized at the boundary of the 5' attenuated APC phenotype region in two unrelated families resulted in classical polyposis. A clear-cut genotype-phenotype correlation could be drawn in only two instances. In one family, a 4684insA mutation led to a mild polyposis associated with early inherited osteomas and, in the family bearing the double mutation (Z1625H + 4897delC), the phenotype was obviously a 3' attenuated type. Our data illustrate the wide genetic and phenotypic heterogeneity of this condition between and within the families, making the establishment of correlations complex and any prediction in this disease difficult, although targeting the mutation site may be helpful in some specific cases.

Isolated polycystic liver disease as a distinct genetic disease, unlinked to polycystic kidney disease 1 and polycystic kidney disease 2.

Polycystic liver disease (PLD) is proven to occur either sporadically or in association with autosomal dominant polycystic kidney disease (ADPKD), whereas the existence of an isolated (i.e., without any kidney cyst) familial form is disputed. We describe a family with definitely isolated PLD transmitted through three generations and exclude the linkage of the disease to the genetic markers of PKD1 and PKD2, the two main loci responsible for ADPKD. These findings strongly support the existence of PLD as a genetic disease distinct from the known forms of ADPKD.

The genetic background of familial adenomatous polyposis. Linkage analysis, the APC gene identification and mutation screening.

Familial adenomatous polyposis (FAP) is an autosomal, dominantly inherited disease that predisposes to colorectal cancer and is characterized by the presence of hundreds to thousands of adenomas covering the colon and rectum. Mapping of the FAP locus to 5q21-q22 by linkage studies in families ultimately allowed the identification of the APC (Adenomatous Polyposis Coli) gene itself. The APC gene comprises 15 exons with a 9 kilobase RNA transcript and a 312 kilodalton final protein product. This discovery transformed the diagnosis of FAP and offered direct identification of defective gene carriers by mutation screening. Currently used techniques have been successful in detecting mutations in 15 to 67 percent of patients. To date, at least 136 different mutations have been described in 301 unrelated FAP patients, most of which (98%) are translation terminating mutations leading to a truncated final protein product. Promising applications or development of novel procedures, like the protein truncation test (PTT), are under way for the remaining FAP patients. With the exception of the description of a critical boundary in exon 9 for the presence or absence of CHRPE, there are no clear genotype-phenotype relationships, but mutations located in the 5' half of exon 15 seem to lead to a more severe phenotype. Very little is know about the APC protein product function. The APC protein could be involved in cell-to-cell signalling and/or cell adhesion functions. The APC gene is a tumour suppressor gene involved in early stages of sporadic colorectal carcinogenesis. Further understanding of the APC gene function may define a rational approach for early detection, prevention strategies, assessment of prognosis and treatment of colorectal cancer. In this regard, animal models of FAP, like the MIN (Multiple Intestinal Neoplasia) mouse or the APC 1638 mouse, are promising and powerful tools.

High CTG repeat number in nodular thyroid tissue from a myotonic dystrophy patient.

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.

Cloning of DNA segments of phage 2C, which allows autonomous plasmid replication in Bacillus subtilis.

The chromosome of Bacillus subtilis phage 2C is a 100-MDa double-stranded DNA molecule, containing hydroxymethyluracil in place of thymine and carrying redundant ends each encompassing 10% of the genome. 2C DNA was cleaved with EcoRI and HindIII, and cloned in the shuttle plasmids pSC 540 and pCP 115, both containing segments originating from B. subtilis and Escherichia coli plasmids. These chimaerical plasmids, carrying the chloramphenicol resistance gene, were unable to replicate in B. subtilis; this ability was restored, however, after the insertion of viral DNA segments. Physical maps of the recombinant plasmids were made; a large deletion of the E. coli-derived segment of pSC 540 was observed (which paralleled a loss of replication in this host), whereas addition of 2C DNA segments in pCP 115 was not accompanied by deletion (replication in E. coli was conserved in this case). Cloned viral segments mapped mostly, but not exclusively, within the redundant ends of 2C DNA. It is suggested that the thirteen recombinant clones carried the replication origin region of phage 2C DNA, and that these sequences originated within or close to the redundant extremities of the viral chromosome.