Philippe Khau Van Kien

Mutations in myosin heavy chain 11 cause a syndrome associating thoracic aortic aneurysm/aortic dissection and patent ductus arteriosus

We have recently described two kindreds presenting thoracic aortic aneurysm and/or aortic dissection (TAAD) and patent ductus arteriosus (PDA) and mapped the disease locus to 16p12.2-p13.13 (ref. 3). We now demonstrate that the disease is caused by mutations in the MYH11 gene affecting the C-terminal coiled-coil region of the smooth muscle myosin heavy chain, a specific contractile protein of smooth muscle cells (SMC). All individuals bearing the heterozygous mutations, even if asymptomatic, showed marked aortic stiffness. Examination of pathological aortas showed large areas of medial degeneration with very low SMC content. Abnormal immunological recognition of SM-MHC and the colocalization of wild-type and mutant rod proteins in SMC, in conjunction with differences in their coimmunoprecipitation capacities, strongly suggest a dominant-negative effect. Human MYH11 gene mutations provide the first example of a direct change in a specific SMC protein leading to an inherited arterial disease.

Comparison of Clinical Presentations and Outcomes Between Patients With TGFBR2 and FBN1 Mutations in Marfan Syndrome and Related Disorders

Background— TGFBR2 mutations were recognized recently among patients with a Marfan-like phenotype. The associated clinical and prognostic spectra remain unclear. Methods and Results— Clinical features and outcomes of 71 patients with a TGFBR2 mutation (TGFBR2 group) were compared with 50 age- and sex-matched unaffected family members (control subjects) and 243 patients harboring FBN1 mutations (FBN1 group). Aortic dilatation was present in a similar proportion of patients in both the TGFBR2 and FBN1 groups (78% versus 79%, respectively) but was highly variable. The incidence and average age for thoracic aortic surgery (31% versus 27% and 35±16 versus 39±13 years, respectively) and aortic dissection (14% versus 10% and 38±12 versus 39±9 years) were also similar in the 2 groups. Mitral valve involvement (myxomatous, prolapse, mitral regurgitation) was less frequent in the TGFBR2 than in the FBN1 group (all P<0.05). Aortic dilatation, dissection, or sudden death was the index event leading to genetic diagnosis in 65% of families with TGFBR2 mutations, versus 32% with FBN1 mutations (P=0.002). The rate of death was greater in TGFBR2 families before diagnosis but similar once the disease had been recognized. Most pregnancies were uneventful (without death or aortic dissection) in both TGFBR2 and FBN1 families (38 of 39 versus 213 of 217; P=1). Seven patients (10%) with a TGFBR2 mutation fulfilled international criteria for Marfan syndrome, 3 of whom presented with features specific for Loeys-Dietz syndrome. Conclusions— Clinical outcomes appear similar between treated patients with TGFBR2 mutations and individuals with FBN1 mutations. Prognosis depends on clinical disease expression and treatment rather than simply the presence of a TGFBR2 gene mutation.

Mapping of Familial Thoracic Aortic Aneurysm/Dissection With Patent Ductus Arteriosus to 16p12.2–p13.13

Background—Three loci have been shown to be responsible for nonsyndromic familial thoracic aortic aneurysms (TAAs) and aortic dissections (ADs). We recently described a large family in which TAA/AD associates with patent ductus arteriosus (PDA) and provided genetic arguments for a unique pathophysiological entity. Methods and Results—Genome-wide scan was performed in 40 subjects belonging to 3 generations in this large pedigree. Using the 7 TAA/AD cases as affected, we observed positive 2-point LOD scores on adjacent markers at chromosome 16p, with a maximum LOD score value of 2.73 at &thgr;=0, a value that increased to 3.56 when 5 PDA cases were included. Multipoint linkage analysis yielded a maximum LOD score of 4.14 in the vicinity of marker D16S3103. Fine mapping allowed the observation of recombinant haplotypes that delimited a critical 20-cM interval at 16p12.2-p13.13. Automatic determination of aortic compliance with cine MRI showed that all subjects bearing the disease haplotype, even asymptomatic, displayed a very low level of aortic compliance and distensibility. Aortic stiffness was strongly associated with disease haplotype with a marked effect of age, indicating subclinical and early manifestation of the disease. Conclusions—Genetic analysis of this family identified a unique locus responsible for both TAA/AD and PDA at chromosome 16p12.2-p13.13 with aortic stiffness as an early hallmark of the disease. TAA/AD with PDA is a new monogenic entity among the genetically heterogeneous group of TAA/AD disease.

Cloacal exstrophy in an infant with 9q34.1-qter deletion resulting from a de novo unbalanced translocation between chromosome 9q and Yq

Cloacal exstrophy is a rare malformation, belonging to a spectrum of birth defects, which, in order of severity, includes phallic separation with epispadias, pubic diastasis, bladder exstrophy, and cloacal exstrophy. This malformation overlaps the OEIS complex (O = omphalocele, E = bladder exstrophy, I = imperforate anus, S = spinal defects). The etiology of cloacal exstrophy is unknown to date. It may result from either a single defect of early blastogenesis or a defect of mesodermal migration during the primitive streak period. We report an infant with cloacal exstrophy, exomphalos, right kidney agenesis, ambiguous external genitalia, and axial hypotonia. The karyotype showed a de novo unbalanced translocation between the long arm of chromosome 9 and the long arm of chromosome Y resulting in a 9q34.1‐qter deletion. Reviewing the literature, we did not find any observation of cloacal exstrophy associated with a structural chromosomal abnormality. The steroidogenic factor 1 (SF1) gene, included in the deleted region, was a good candidate gene but no pathogenic mutation was found by direct sequencing. We hypothesize that another gene, expressed early in embryogenesis and responsible for cloacal exstrophy, is present in the 9q34.1‐qter region.

Familial thoracic aortic aneurysm/dissection with patent ductus arteriosus: genetic arguments for a particular pathophysiological entity

Thoracic aortic aneurysm and aortic dissection (TAA and AD) are an important cause of sudden death. Familial cases could account for 20% of all cases. A genetic heterogeneity with two identified genes (FBN1 and COL3A1) and three loci (3p24–25 or MFS2/TAAD2, 5q13–q14 and 11q23.2–24) has been shown previously. Study of a single family composed of 179 members with an abnormally high occurrence of TAA/AD disease. A total of 40 subjects from three generations were investigated. In addition to five cases of stroke and three cases of sudden death, there were four cases of AD and four cases of TAA in adults. In all, 11 cases of patent ductus arteriosus (PDA) were observed, two of which were associated with TAA and one with AD. Segregation analysis showed that the distribution of these vascular abnormalities was more likely compatible with a single genetic defect with an autosomal dominant pattern of inheritance. There were no clinical signs of Marfan, Elhers–Danlos vascular type or Char syndromes. Genetic linkage analysis was performed for seven genes or loci implicated in familial TAA/AD disease (COL3A1, FBN1, 3p24–25 or MFS2/TAAD2, 5q13–q14 and 11q23.2–q24), Char syndrome (TFAP2B) or autosomal recessive PDA (12q24). Using different genetic models, linkage with these seven loci was excluded. Familial TAA/AD with PDA is likely to be a particular heritable vascular disorder, with an as yet undiscovered Mendelian genetic basis.

Homozygosity for a null allele of COL3A1 results in recessive Ehlers–Danlos syndrome

So far, mutations in the human COL3A1 gene have been associated with the predominantly inherited Ehlers–Danlos syndrome (EDS), vascular type. Genotype–phenotype correlation perspectives collapsed, as haploinsufficiency, which was long suggested to confer a milder or unrecognized phenotype, was reported in four patients with a phenotype similar to that of vascular EDS. Here, we study a case of recessive EDS in a young consanguineous girl of healthy parents. She fulfilled the vascular EDS criteria for laboratory testing. Total sequencing of COL3A1 cDNA identified a homozygous nucleotide duplication (c.479dupT) resulting in a premature termination codon (p.Lys161GlnfsX45). Studies in genomic DNA showed that this mutation was inherited from each parent. The expression analysis (RT-PCR, quantitative-PCR, immunohistochemistry, WB) showed strong mRNA decay and an absence of type III collagen in the proband. The expected COL3A1 haploinsufficiency in her healthy ascendants did not lead to the manifestations of vascular EDS. This case provides evidence of a stochastic effect of COL3A1 haploinsufficiency in humans, which could be explained by the relation between nonsense-mediated mRNA decay efficiency and the resulting dominant-negative effect depending on the position of the mutation and/or modifying factors. It opens up new perspectives for the understanding of COL3A1 genotype–phenotype correlations, which is required while considering targeted therapy.

Automatic determination of aortic compliance with cine-magnetic resonance imaging: An application of fuzzy logic theory

Lalande A, Khau van Kien P, Salvé N, et al. Automatic determination of aortic compliance with cine-magnetic resonance imaging: An application of fuzzy logic theory. Invest Radiol 2002;37:685–691. Rationale and Objectives. Aortic compliance is defined as the relative change in aortic cross-sectional area divided by the change in arterial pressure. Magnetic resonance imaging (MRI) is a useful imaging modality for the noninvasive evaluation of aortic compliance. However, manual tracing of the aortic contour is subject to important interobserver variations. To estimate the aortic compliance from cine-MRI, a method based on fuzzy logic theory was elaborated. Materials and Methods.Seven healthy volunteers and eight patients with Marfan syndrome were examined using an ECG gated cine-MRI sequence. The aorta was imaged in the transverse plane at the level of the pulmonary trunk. A method based on fuzzy logic was developed to automatically detect the aortic contour. Results. Through our robust automatic contouring method, the calculation of aortic cross-sectional areas allows an estimation of the aortic compliance. Conclusion. The aortic compliance can be obtained from a fuzzy logic based automatic contouring method, thereby avoiding the important interobserver variation often associated with manual tracing.

Pure intronic rearrangements leading to aberrant pseudoexon inclusion in dystrophinopathy: a new class of mutations?

We report on two unprecedented cases of pseudoexon (PE) activation in the DMD gene resulting from pure intronic double‐deletion events that possibly involve microhomology‐mediated mechanisms. Array comparative genomic hybridization analysis and direct genomic sequencing allowed us to elucidate the causes of the pathological PE inclusion detected in the RNA of the patients. In the first case (Duchenne phenotype), we showed that the inserted 387‐bp PE was originated from an inverted ∼57 kb genomic region of intron 44 flanked by two deleted ∼52 kb and ∼1 kb segments. In the second case (Becker phenotype), we identified in intron 56 two small deletions of 592 bp (del 1) and 29 bp (del 2) directly flanking a 166‐bp PE located in very close proximity (134 bp) to exon 57. The key role of del 1 in PE activation was established by using splicing reporter minigenes. However, the analysis of mutant constructs failed to identify cis elements that regulate the inclusion of the PE and suggested that other splicing regulatory factors may be involved such as RNA structure. Our study introduces a new class of mutations in the DMD gene and emphasizes the potential role of underdetected intronic rearrangements in human diseases. Hum Mutat 32:1–9, 2011.

Comprehensive oligonucleotide array-comparative genomic hybridization analysis: new insights into the molecular pathology of the DMD gene

We report on the effectiveness of a custom-designed oligonucleotide-based comparative genomic hybridization microarray (array-CGH) to interrogate copy number across the entire 2.2-Mb genomic region of the DMD gene and its applicability in diagnosis. The high-resolution array-CGH, we developed, successfully detected a series of 42 previously characterized large rearrangements of various size, localization and type (simple or complex deletions, duplications, triplications) and known intronic CNVs/Indels. Moreover, the technique succeeded in identifying a small duplication of only 191 bp in one patient previously negative for DMD mutation. Accurate intronic breakpoints localization by the technique enabled subsequent junction fragments identification by sequencing in 86% of cases (all deletion cases and 62.5% of duplication cases). Sequence examination of the junctions supports a role of microhomology-mediated processes in the occurrence of DMD large rearrangements. In addition, the precise knowledge of the sequence context at the breakpoints and analysis of the resulting consequences on maturation of pre-mRNA contribute to elucidating the cause of discrepancies in phenotype/genotype correlations in some patients. Thereby, the array-CGH proved to be a highly efficient and reliable diagnostic tool, and the new data it provides will have many potential implications in both, clinics and research.

De novo 15q21.1q21.2 deletion identified through FBN1 MLPA and refined by 244K array-CGH in a female teenager with incomplete Marfan syndrome.

Interstitial deletions involving the 15q21.1 band are very rare. Only 4 of these cases have been studied using molecular cytogenetic techniques in order to confirm the deletion of the whole FBN1 gene. The presence of clinical features of the Marfan syndrome (MFS) spectrum associated with mental retardation has been described in only 2/4 patients. Here we report on a 16-year-old female referred for suspicion of MFS (positive thumb and wrist sign, scoliosis, joint hyperlaxity, high-arched palate with dental crowding, dysmorphism, mitral insufficiency with dystrophic valve, striae). She had therefore 3 minor criteria according to the Ghent nosology. She also had speech disabilities but could follow normal school training. Direct sequencing of the FBN1, TGFBR1 and TGFBR2 genes was negative. MLPA revealed a genomic deletion of the whole FBN1 gene, confirmed by loss of heterozygosity of maternal alleles for several microsatellite markers surrounding the FBN1 gene. The deletion was confirmed by FISH using a FBN1 probe and was not found in the parents. Array-CGH permitted to define a 2.97 Mb deletion, which was the smallest 15q microdeletion including FBN1. Contrary to the other published observations, our proband does not exhibit mental retardation, but neuropsychological evaluations revealed an attention deficit as well as a deficit in information-processing speed. Haploinsufficiency of FBN1 is likely to contribute to the presence of MFS features. However, attenuated features could be explained because disturbances of TGF-beta signalling associated with FBN1 mutations do not exert full phenotypic effect through simple haploinsufficiency. Phenotypic variability in other patients with interstitial deletions including 15q21.1 band may reflect differences in deletion size and/or cys/trans modifying factors.