Fluid-structure interaction study of the edge-to-edge repair technique on the mitral valve.

The effect of functional mitral regurgitation has been investigated in an anatomically sized, fluid-structure interaction mitral valve model, where simulated correction has been performed by applying: (1) edge-to-edge repair with annuloplasty and (2) edge-to-edge repair only. Initially defined in an open unstressed/corrected configuration, fluid-structure interaction simulations of diastole have been performed in a rigid ventricular volume. Comparison of the maximum principal stresses (during diastole) in the normal and repaired models has shown that the magnitude of stress in the repaired scenarios is ~200% greater. The combined edge-to-edge and annuloplasty procedure was found to spread the induced stresses across the free margin of the leaflets, whereas without annuloplasty a localised stress concentration in the region of the suture was observed. Fluid flow downstream of the corrected configurations was able to achieve the same magnitude as in the normal case, although the flow rate was impaired. The maximum flow rate was found to be reduced by 44-50% with the peak flow rate shifted from the end of the diastole in the normal case to the start in the repaired cases.

Absence of the vagus nerve in the stomach of Tbx1-/- mutant mice.

BACKGROUND: Tbx1 is a member of the Tbox family of binding domain transcription factors. TBX1 maps within the region of chromosome 22q11 deleted in humans with DiGeorge syndrome (DGS), a common genetic disorder characterized by numerous physical manifestations including craniofacial and cardiac anomalies. Mice with homozygous null mutations in Tbx1 phenocopy this disorder and have defects including abnormal cranial ganglia formation and cardiac neural crest cell migration. These defects prompted us to investigate whether extrinsic vagus nerve or intrinsic enteric nervous system abnormalities are prevalent in the gastrointestinal tract of Tbx1 mutant mice. METHODS: We used in situ hybridization for Ret, and immunohistochemical staining for neurofilament, HuC/D and ßIII-tubulin to study cranial ganglia, vagus nerve, and enteric nervous system development in Tbx1 mutant and control mice. KEY RESULTS: In Tbx1(-/-) embryos, cranial ganglia of the glossopharyngeal (IXth) and vagus (Xth) nerves were malformed and abnormally fused. In the gastrointestinal tract, the vagus nerves adjacent to the esophagus were severely hypoplastic and they did not extend beyond the gastro-esophageal junction nor project branches within the stomach wall, as was observed in Tbx1(+/+) mice. CONCLUSIONS & INFERENCES: Although cranial ganglia morphology appeared normal in Tbx1(+/-) mice, these animals had a spectrum of stomach vagus innervation defects ranging from mild to severe. In all Tbx1 genotypes, the intrinsic enteric nervous system developed normally. The deficit in vagal innervation of the stomach in mice mutant for a gene implicated in DGS raises the possibility that similar defects may underlie a number of as yet unidentified/unreported congenital disorders affecting gastrointestinal function.

Mitral valve dynamics in structural and fluid-structure interaction models.

Modelling and simulation of heart valves is a challenging biomechanical problem due to anatomical variability, pulsatile physiological pressure loads and 3D anisotropic material behaviour. Current valvular models based on the finite element method can be divided into: those that do model the interaction between the blood and the valve (fluid-structure interaction or 'wet' models) and those that do not (structural models or 'dry' models). Here an anatomically sized model of the mitral valve has been used to compare the difference between structural and fluid-structure interaction techniques in two separately simulated scenarios: valve closure and a cardiac cycle. Using fluid-structure interaction, the valve has been modelled separately in a straight tubular volume and in a U-shaped ventricular volume, in order to analyse the difference in the coupled fluid and structural dynamics between the two geometries. The results of the structural and fluid-structure interaction models have shown that the stress distribution in the closure simulation is similar in all the models, but the magnitude and closed configuration differ. In the cardiac cycle simulation significant differences in the valvular dynamics were found between the structural and fluid-structure interaction models due to difference in applied pressure loads. Comparison of the fluid domains of the fluid-structure interaction models have shown that the ventricular geometry generates slower fluid velocity with increased vorticity compared to the tubular geometry. In conclusion, structural heart valve models are suitable for simulation of static configurations (opened or closed valves), but in order to simulate full dynamic behaviour fluid-structure interaction models are required.

Molecular study of 33 families with Fraser syndrome new data and mutation review.

Fraser syndrome (FS) is an autosomal recessive malformation disorder characterized by cryptophthalmos, syndactyly, and abnormalities of the respiratory and urogenital tract. FS is considered to be the human equivalent of the murine blebbing mutants: in the mouse mutations at five loci cause a phenotype that is comparable to FS in humans, and thus far mutations in two syntenic human genes, FRAS1 and FREM2, have been identified to cause FS. Here we present the molecular analysis of 48 FS patients from 18 consanguineous and 15 nonconsanguineous families. Linkage analysis in consanguineous families indicated possible linkage to FRAS1 and FREM2 in 60% of the cases. Mutation analysis identified 11 new mutations in FRAS1 and one FREM2 mutation. Manifestations of these patients and previously reported cases with an FRAS1 mutation were compared to cases without detectable FRAS1 mutations to study genotype-phenotype correlations. Although our data suggest that patients with an FRAS1 mutation have more frequently skull ossification defects and low insertion of the umbilical cord, these differences are not statistically significant. Mutations were identified in only 43% of the cases suggesting that other genes syntenic to murine genes causing blebbing may be responsible for FS as well.

Mutations in SRD5B1 (AKR1D1), the gene encoding delta(4)-3-oxosteroid 5beta-reductase, in hepatitis and liver failure in infancy.

BACKGROUND: A substantial group of patients with cholestatic liver disease in infancy excrete, as the major urinary bile acids, the glycine and taurine conjugates of 7alpha-hydroxy-3-oxo-4-cholenoic acid and 7alpha,12alpha-dihydroxy-3-oxo-4-cholenoic acid. It has been proposed that some (but not all) of these have mutations in the gene encoding delta(4)-3-oxosteroid 5beta-reductase (SRD5B1; AKR1D1, OMIM 604741). AIMS: Our aim was to identify mutations in the SRD5B1 gene in patients in whom chenodeoxycholic acid and cholic acid were absent or present at low concentrations in plasma and urine, as these seemed strong candidates for genetic 5beta-reductase deficiency. PATIENTS AND SUBJECTS: We studied three patients with neonatal onset cholestatic liver disease and normal gamma-glutamyl transpeptidase in whom 3-oxo-delta(4) bile acids were the major bile acids in urine and plasma and saturated bile acids were at low concentration or undetectable. Any base changes detected in SRD5B1 were sought in the parents and siblings and in 50 ethnically matched control subjects. METHODS: DNA was extracted from blood and the nine exons of SRD5B1 were amplified and sequenced. Restriction enzymes were used to screen the DNA of parents, siblings, and controls. RESULTS: Mutations in the SRD5B1 gene were identified in all three children. Patient MS was homozygous for a missense mutation (662 C>T) causing a Pro198Leu amino acid substitution; patient BH was homozygous for a single base deletion (511 delT) causing a frame shift and a premature stop codon in exon 5; and patient RM was homozygous for a missense mutation (385 C>T) causing a Leu106Phe amino acid substitution. All had liver biopsies showing a giant cell hepatitis; in two, prominent extramedullary haemopoiesis was noted. MS was cured by treatment with chenodeoxycholic acid and cholic acid; BH showed initial improvement but then deteriorated and required liver transplantation; RM had advanced liver disease when treatment was started and also progressed to liver failure. CONCLUSIONS: Analysis of blood samples for SRD5B1 mutations can be used to diagnose genetic 5beta-reductase deficiency and distinguish these patients from those who have another cause of 3-oxo-delta(4) bile aciduria, for example, severe liver damage. Patients with genetic 5beta-reductase deficiency may respond well to treatment with chenodeoxycholic acid and cholic acid if liver disease is not too advanced.

A locus for asphyxiating thoracic dystrophy, ATD, maps to chromosome 15q13.

Asphyxiating thoracic dystrophy (ATD), or Jeune syndrome, is a multisystem autosomal recessive disorder associated with a characteristic skeletal dysplasia and variable renal, hepatic, pancreatic, and retinal abnormalities. We have performed a genome wide linkage search using autozygosity mapping in a cohort of four consanguineous families with ATD, three of which originate from Pakistan, and one from southern Italy. In these families, as well as in a fifth consanguineous family from France, we localised a novel ATD locus (ATD) to chromosome 15q13, with a maximum cumulative two point lod score at D15S1031 (Zmax=3.77 at theta=0.00). Five consanguineous families shared a 1.2 cM region of homozygosity between D15S165 and D15S1010. Investigation of a further four European kindreds, with no known parental consanguinity, showed evidence of marker homozygosity across a similar interval. Families with both mild and severe forms of ATD mapped to 15q13, but mutation analysis of two candidate genes, GREMLIN and FORMIN, did not show pathogenic mutations.

Expression and mutation analysis of BRUNOL3, a candidate gene for heart and thymus developmental defects associated with partial monosomy 10p.

Partial monosomy 10p is a rare chromosomal aberration. Patients often show symptoms of the DiGeorge/velocardiofacial syndrome spectrum. The phenotype is the result of haploinsufficiency of at least two regions on 10p, the HDR1 region associated with hypoparathyroidism, sensorineural deafness, and renal defects (HDR syndrome) and the more proximal region DGCR2 responsible for heart defects and thymus hypoplasia/aplasia. While GATA3 was identified as the disease causing gene for HDR syndrome, no genes have been identified thus far for the symptoms associated with DGCR2 haploinsufficiency. We constructed a deletion map of partial monosomy 10p patients and narrowed the critical region DGCR2 to about 300 kb. The genomic draft sequence of this region contains only one known gene, BRUNOL3 ( NAPOR, CUGBP2, ETR3). In situ hybridization of human embryos and fetuses revealed as well as in other tissues a strong expression of BRUNOL3 in thymus during different developmental stages. BRUNOL3 appears to be an important factor for thymus development and is therefore a candidate gene for the thymus hypoplasia/aplasia seen in partial monosomy 10p patients. We did not find BRUNOL3 mutations in 92 DiGeorge syndrome-like patients without chromosomal deletions and in 8 parents with congenital heart defect children.

Cloning of HOXD1 from unfertilised human oocytes and expression analyses during murine oogenesis and embryogenesis.

We describe the cloning of HOXD1 in human unfertilised oocytes and detailed expression analyses during mouse oogenesis and embryogenesis. The cDNA of 1991bp has an open reading frame of 987bp encoding a protein of 329 amino acids. A comparison of the amino acid sequence with the mouse homologue revealed an overall homology of 85.5% with 99% identity within the homeodomain. Expression was detected in unfertilised human oocytes and 2-, 4-, 8-cell and blastocyst stage embryos. Expression analyses in mature mouse ovaries, early embryos and isolated gut revealed expression in the oocytes of the primary and secondary ovarian follicles, and in embryonal mesodermal derivatives such as dermatomes, urogenital tubercle, tail bud, kidney, ovaries, testes and enteric mesoderm adjacent to the caecum where expression was up-regulated in vitro in response to increasing doses of retinoic acid. Our observations indicate a possible role for HOXD1/Hoxd1 in the ovarian oocytes and the establishment of mesodermal derivatives during embryogenesis.

Triallelic inheritance in Bardet-Biedl syndrome, a Mendelian recessive disorder.

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous disorder characterized by multiple clinical features that include pigmentary retinal dystrophy, polydactyly, obesity, developmental delay, and renal defects. BBS is considered an autosomal recessive disorder, and recent positional cloning efforts have identified two BBS genes (BBS2 and BBS6). We screened our cohort of 163 BBS families for mutations in both BBS2 and BBS6 and report the presence of three mutant alleles in affected individuals in four pedigrees. In addition, we detected unaffected individuals in two pedigrees who carry two BBS2 mutations but not a BBS6 mutation. We therefore propose that BBS may not be a single-gene recessive disease but a complex trait requiring three mutant alleles to manifest the phenotype. This triallelic model of disease transmission may be important in the study of both Mendelian and multifactorial disorders.

Replication and extension studies of inflammatory bowel disease susceptibility regions confirm linkage to chromosome 6p (IBD3).

Inflammatory bowel disease (IBD) is a chronic inflammatory disease of the intestine, commonly diagnosed as either ulcerative colitis (UC) or Crohn's disease (CD). Epidemiological studies have consistently shown that both genetic and environmental factors influence the pathogenesis of IBD. A number of genome scans have been conducted in cohorts of IBD families with affected sibling pairs (ASPs) to identify chromosomal regions that harbour IBD susceptibility genes. Several putative linked loci have been identified, including two loci on chromosomes 16 and 12, IBD1 and IBD2, which have subsequently been replicated by independent region-specific studies. We have conducted both a replication study on another linkage region, chromosome 6p (IBD3), and extension studies on two other regions, chromosomes 3p and 7q. Microsatellite markers across each region were genotyped in 284 IBD ASPs from 234 families. A nonparametric peak multipoint LOD score of 3.0 was observed near D6S291, replicating the previous linkage to chromosome 6p (IBD3). Nominal evidence of linkage was observed at both the 3p and 7q regions.

TBX1 is responsible for cardiovascular defects in velo-cardio-facial/DiGeorge syndrome.

Velo-cardio-facial syndrome (VCFS)/DiGeorge syndrome (DGS) is a human disorder characterized by a number of phenotypic features including cardiovascular defects. Most VCFS/DGS patients are hemizygous for a 1.5-3.0 Mb region of 22q11. To investigate the etiology of this disorder, we used a cre-loxP strategy to generate mice that are hemizygous for a 1.5 Mb deletion corresponding to that on 22q11. These mice exhibit significant perinatal lethality and have conotruncal and parathyroid defects. The conotruncal defects can be partially rescued by a human BAC containing the TBX1 gene. Mice heterozygous for a null mutation in Tbx1 develop conotruncal defects. These results together with the expression patterns of Tbx1 suggest a major role for this gene in the molecular etiology of VCFS/DGS.

Tbx1 haploinsufficieny in the DiGeorge syndrome region causes aortic arch defects in mice.

DiGeorge syndrome is characterized by cardiovascular, thymus and parathyroid defects and craniofacial anomalies, and is usually caused by a heterozygous deletion of chromosomal region 22q11.2 (del22q11) (ref. 1). A targeted, heterozygous deletion, named Df(16)1, encompassing around 1 megabase of the homologous region in mouse causes cardiovascular abnormalities characteristic of the human disease. Here we have used a combination of chromosome engineering and P1 artificial chromosome transgenesis to localize the haploinsufficient gene in the region, Tbx1. We show that Tbx1, a member of the T-box transcription factor family, is required for normal development of the pharyngeal arch arteries in a gene dosage-dependent manner. Deletion of one copy of Tbx1 affects the development of the fourth pharyngeal arch arteries, whereas homozygous mutation severely disrupts the pharyngeal arch artery system. Our data show that haploinsufficiency of Tbx1 is sufficient to generate at least one important component of the DiGeorge syndrome phenotype in mice, and demonstrate the suitability of the mouse for the genetic dissection of microdeletion syndromes.

Human HOX gene mutations.

HOX genes play a fundamental role in the development of the vertebrate central nervous system, axial skeleton, limbs, gut, urogenital tract and external genitalia, but it is only in the last 4 years that mutations in two of the 39 human HOX genes have been shown to cause congenital malformations; HOXD13, which is mutated in synpolydactyly, and HOXA13, which is mutated in Hand-Foot-Genital syndrome. Here we review the mutations already identified in these two genes, consider how these mutations may act, and discuss the possibility that further mutations remain to be discovered both in developmental disorders and in cancer.

The 22q11 deletion syndromes.

DiGeorge syndrome, velocardiofacial syndrome and various other malformations have been described in association with deletions and translocations involving human chromosome 22q11. Many of the structural malformations observed are also seen in animal models of neural crest disruption suggesting that the haplo-insufficiency resulting from the deletion somehow affects this group of cells or their interactions. Over the past few years it has been shown that the deletion predisposes to a range of psychotic conditions prompting the hypothesis that the deleted region may contain a predisposition locus for psychotic illness. The DiGeorge chromosomal region has been entirely sequenced and many of the genes mapping to the deletion interval have been studied in some detail. Despite these efforts, no gene has yet been proved to play a defined role in the pathogenesis of the syndrome. Current efforts are directed at the study of engineered chromosome mouse models which offer the potential to dissect at least some of the developmental pathways disrupted in this intriguing group of malformation syndromes.

Cortical dysgenesis in 2 patients with chromosome 22q11 deletion.

Two patients with chromosome 22q11 deletion and cortical dysgenesis (gyral abnormalities) are reported in this study. One had unilateral clubfoot in addition to multiple features suggestive of the Di George syndrome (DGS), and the other presented with leg asymmetry and seizures, with subsequent recognition of the velo-cardio-facial syndrome (VCFS). In each patient, gyral abnormalities were identified in the hemisphere contralateral to the limb abnormality. A wide range of central nervous system abnormalities have been reported in DGS and VCFS, including three prior reports of gyral abnormalities (lissencephaly, microgyria). The 2 patients reported herein strengthen the association between the 22q11 deletion spectrum and cortical dysgenesis, but the underlying pathogenetic mechanism (primary neural migration vs. vascular disruption) remains unclear.

Novel HOXA13 mutations and the phenotypic spectrum of hand-foot-genital syndrome.

Hand-foot-genital syndrome (HFGS) is a rare, dominantly inherited condition affecting the distal limbs and genitourinary tract. A nonsense mutation in the homeobox of HOXA13 has been identified in one affected family, making HFGS the second human syndrome shown to be caused by a HOX gene mutation. We have therefore examined HOXA13 in two new and four previously reported families with features of HFGS. In families 1, 2, and 3, nonsense mutations truncating the encoded protein N-terminal to or within the homeodomain produce typical limb and genitourinary abnormalities; in family 4, an expansion of an N-terminal polyalanine tract produces a similar phenotype; in family 5, a missense mutation, which alters an invariant domain, produces an exceptionally severe limb phenotype; and in family 6, in which limb abnormalities were atypical, no HOXA13 mutation could be detected. Mutations in HOXA13 can therefore cause more-severe limb abnormalities than previously suspected and may act by more than one mechanism.

Dual-probe fluorescence in situ hybridization assay for detecting deletions associated with VCFS/DiGeorge syndrome I and DiGeorge syndrome II loci.

Over 90% of patients with DiGeorge syndrome (DGS) or velocardiofacial syndrome (VCFS) have a microdeletion at 22q11.2. Given that these deletions are difficult to visualize at the light microscopic level, fluorescence in situ hybridization (FISH) has been instrumental in the diagnosis of this disorder. Deletions on the short arm of chromosome 10 are also associated with a DGS-like phenotype. Since deletions at 22q11.2 and at 10p13p14 result in similar findings, we have developed a dual-probe FISH assay for screening samples referred for DGS or VCFS in the clinical laboratory. This assay includes two test probes for the loci, DGSI at 22q11.2 and DGSII at 10p13p14, and centromeric probes for chromosomes 10 and 22. Of 412 patients tested, 54 were found to be deleted for the DGSI locus on chromosome 22 (13%), and a single patient was found deleted for the DGSII locus on chromosome 10 (0. 24%). The patient with the 10p deletion had facial features consistent with VCFS, plus sensorineural hearing loss, and renal anomalies. Cytogenetic analysis showed a large deletion of 10p [46, XX,del(10)(p12.2p14)] and FISH using a 10p telomere region-specific probe confirmed the interstitial nature of the deletion. Analysis for the DGSI and the DGSII loci suggests that the deletion of the DGSII locus on chromosome 10 may be 50 times less frequent than the deletion of DGSI on chromosome 22. The incidence of deletions at 22q11.2 has been estimated to be 1 in 4000 newborns; therefore, the deletion at 10p13p14 may be estimated to occur in 1 in 200,000 live births.

Frasier syndrome, part of the Denys Drash continuum or simply a WT1 gene associated disorder of intersex and nephropathy?

Dysfunction of the Wilms' Tumour gene (WT1), a transcription factor critical for normal development and function of the urogenital tract, can result in both tumourigenesis [corrected] and urogenital abnormalities. The association of WT1 gene mutations with most cases of Denys-Drash syndrome is well described. More recently WT1 mutations have also been described in a related condition, Frasier syndrome. We report a case where genetic analysis showed a WT1 mutation typically associated with Frasier syndrome: a 1228 + 5 guanine to adenine substitution at the 3' alternative splice donor site in intron 9. The case provides a focus for the discussion of recent evidence that Denys Drash and Frasier syndrome form two ends of a spectrum of disorders. In addition, it illustrates the increasing significance of genetic investigation within clinical practice for diagnostic, prognostic and therapeutic purposes and the importance of karyotype analysis in phenotypically normal girls with renal disease.