Perrault syndrome is caused by recessive mutations in CLPP, encoding a mitochondrial ATP-dependent chambered protease.

Perrault syndrome is a genetically and clinically heterogeneous autosomal-recessive condition characterized by sensorineural hearing loss and ovarian failure. By a combination of linkage analysis, homozygosity mapping, and exome sequencing in three families, we identified mutations in CLPP as the likely cause of this phenotype. In each family, affected individuals were homozygous for a different pathogenic CLPP allele: c.433A>C (p.Thr145Pro), c.440G>C (p.Cys147Ser), or an experimentally demonstrated splice-donor-site mutation, c.270+4A>G. CLPP, a component of a mitochondrial ATP-dependent proteolytic complex, is a highly conserved endopeptidase encoded by CLPP and forms an element of the evolutionarily ancient mitochondrial unfolded-protein response (UPR(mt)) stress signaling pathway. Crystal-structure modeling suggests that both substitutions would alter the structure of the CLPP barrel chamber that captures unfolded proteins and exposes them to proteolysis. Together with the previous identification of mutations in HARS2, encoding mitochondrial histidyl-tRNA synthetase, mutations in CLPP expose dysfunction of mitochondrial protein homeostasis as a cause of Perrault syndrome.

Cognition in children with neurofibromatosis type 1: data from a population-based study.

AIM: This study aimed to investigate the core cognitive deficits in children with neurofibromatosis type 1 (NF1). METHOD: The study recruited 49 children with NF1 (25 males, 24 females; mean age 11y 9mo [SD 3y 2mo]), 19 healthy siblings of children with NF1 (sibling comparisons; mean age 12y 7mo [SD 2y 7mo], 9 males, 10 females) and 29 healthy children from the community (community comparisons; mean age 11y [SD 2y 7mo], 12 males, 17 females). Participants completed a battery of cognitive tests including tests of intelligence, academic achievement, attention, visuoperceptual functioning, visual learning, executive functioning, and non-verbal working memory tests. RESULTS: Our study, using a population-based sample, confirmed previous findings from studies using variable sampling methods. Children with NF1 had significantly lower Full-scale IQs (p=0.04) and lower academic achievement (p=0.026-0.005) than their siblings. Compared with their siblings, they also had significantly poorer visuospatial processing (p=0.007), visual associate learning (p=0.014), non-verbal working memory (p=0.023), and executive function (p<0.001). Data from the community comparisons were not included because they were subject to significant selection bias. INTERPRETATION: Population-based frequencies for cognitive deficits in children with NF1 are similar to the frequencies in non-population based samples. This study highlights the heterogeneous nature of cognitive problems in children with NF1 and the need for monitoring and support at school.

Behaviour in children with neurofibromatosis type 1: cognition, executive function, attention, emotion, and social competence.

AIM: This systematic review aimed to pull together the findings from research into behavioural systems and attention in children with neurofibromatosis type 1 (NF1) and to identify areas that need further study. METHOD: Relevant papers were identified through searches of electronic databases (MEDLINE, PsycINFO, EMBASE) and manual searches through reference lists. In total, 5746 articles were identified and 57 met the inclusion criteria. The data were synthesized using the narrative approach, as the studies varied considerably in terms of participants and measures. RESULTS: The results of the review showed that intelligence, academic skills, visuospatial skills, social competence, and attention are impaired in children with NF1. Evidence of deficits in memory, motor functioning, language, and executive functions was less clear. INTERPRETATION: Research has made marked progress in outlining the behavioural phenotype of NF1. However, although the general areas of impairment are becoming better known, the exact nature of the impairment is still not understood in many areas of behaviour. Care needs to be taken with the way in which behavioural constructs are defined and measured, and the variability of problems in NF1 is a particular challenge. Nevertheless, research is steadily moving towards comprehensive understanding of behaviour in children with NF1.

Autism and other psychiatric comorbidity in neurofibromatosis type 1: evidence from a population-based study.

AIM: To investigate psychopathology in children with neurofibromatosis type 1 (NF1), particularly the prevalence of autism spectrum disorder (ASD) and attention-deficit-hyperactivity disorder (ADHD) symptomatology, using a population-based sampling approach. METHOD: Standard questionnaire screen reports were analysed for ASD (Social Responsiveness Scale, SRS), ADHD (Conners' Parent Rating Scale- Revised, CPRS-R), and other psychiatric morbidity (Strengths and Difficulties Questionnaire, SDQ) from parents and teachers of children aged from 4 to 16 years (112 females, 95 males) on the UK North West Regional Genetic Service register for NF1. RESULTS: Parental response rate was 52.7% (109/207 children; 59 females, 50 males, mean age 9 y 11 mo, SD 3 y 3 mo). The SRS showed that in 29.4% (32/109) of children, autism was in the severe, clinical range (T-score>75) and in 26.6% (29/109) in the mild to moderate range (T-score 60-75). CPRS-R scores showed that in 53.8% (57/106) of children autism was in the clinical ADHD range (ADHD index T-score>65). Based on their scores on the SDQ total difficulties scale, 41.5% (44/106) of children were in the abnormal range and 14.2% (15/106) were in the borderline range. Twenty-five per cent (26/104) of children met criteria for both clinical autism and ADHD. INTERPRETATION: This representative population-based sample of children with NF1 indicates a high prevalence of ASD symptoms associated with NF1 as well as substantial co-occurrence with ADHD symptoms. The findings clarify the psychopathology of NF1 and show the disorder as a potentially important single-gene cause for autism symptoms.

Report of two brothers with short stature, microcephaly, mental retardation, and retinoschisis-A new mental retardation syndrome?

Involvement of genes on the X-chromosome as a cause of mental retardation has been recognized for a long time. X-linked phenotypes of mental retardation have been divided into non-syndromic and syndromic based on associated manifestations. At present, more than 140 syndromic X-linked mental retardation (XLMR) conditions have been reported and a causative gene mutation has been identified in almost half of these. Here, we report on two brothers with short stature, microcephaly, severe mental retardation, and retinoschisis. Results of karyotype analysis, fragile-X and neuroimaging studies were normal. Fundus examination showed bilateral retinoschisis at variable stages in both sibs. X-linked retinoschisis is a retinal dystrophy caused by mutations in the RS1 gene at Xp22.1, which lead to splitting of the neural retina and reduced visual acuity in affected men. However, as yet there have been no reports of mental retardation in X-linked retinoschisis although genetic loci for XLMR and short stature have been mapped to Xp22.1. Sequencing and microarray analysis failed to find any alteration of RS1 gene or copy number alteration in the region. In addition, genotype analysis of Xp22.1 provided evidence against linkage to this region. The associated findings of retinoschisis and mental retardation in two brothers suggest a new mental retardation syndrome likely to be an X linked trait.

Newly recognized recessive syndrome characterized by dysmorphic features, hypogonadotropic hypogonadism, severe microcephaly, and sensorineural hearing loss maps to 3p21.3.

We present a newly recognized, likely autosomal recessive, pleiotropic disorder seen in four individuals (three siblings and their nephew) from a consanguineous family of Pakistani origin. The condition is characterized by hypogonadotropic hypogonadism, severe microcephaly, sensorineural deafness, moderate learning disability, and distinctive facial dysmorphic features. Autozygosity mapping using SNP array genotyping defined a single, large autozygous region of 13.1 Mb on chromosome 3p21 common to the affected individuals. The critical region contains 227 genes and initial sequence analysis of a functional candidate gene has not identified causative mutations.

Fibulin 5 forms a compact dimer in physiological solutions.

Fibulin 5 is a 52-kDa calcium-binding epidermal growth factor (cbEGF)-rich extracellular matrix protein that is essential for the formation of elastic tissues. Missense mutations in fibulin 5 cause the elastin disorder cutis laxa and have been associated with age-related macular degeneration, a leading cause of blindness. We investigated the structure, hydrodynamics, and oligomerization of fibulin 5 using small angle x-ray scattering, EM, light scattering, circular dichroism, and sedimentation. Compact structures for the monomer were determined by small angle x-ray scattering and EM, and are supported by close agreement between the theoretical sedimentation of the structures and the experimental sedimentation of the monomer in solution. EM showed that monomers associate around a central cavity to form a dimer. Light scattering and equilibrium sedimentation demonstrated that the equilibrium between the monomer and the dimer is dependent upon NaCl and Ca2+ concentrations and that the dimer is dominant under physiological conditions. The dimerization of fragments containing just the cbEGF domains suggests that intermolecular interactions between cbEGFs cause dimerization of fibulin 5. It is possible that fibulin 5 functions as a dimer during elastinogenesis or that dimerization may provide a method for limiting interactions with binding partners such as tropoelastin.

Mutation-specific effects of polymorphism H558R in SCN5A-related sick sinus syndrome.

INTRODUCTION: Mutations in SCN5A, the gene encoding alpha subunit of cardiac type sodium channel, Na(v)1.5, lead to familial sick sinus syndrome (SSS). Although several molecular mechanisms for this genetic condition have been explored, the underlying mechanisms for the variable genotype-phenotype relationships have not been well addressed. One of the important contributors to such relationships is the genetic background such as single-nucleotide polymorphisms. METHODS AND RESULTS: To clarify the effects of a common polymorphism in SCN5A gene, H558R, on SCN5A-related SSS phenotype, we investigated the electrophysiological properties of all of the 13 known SSS-related hNa(v)1.5 mutant channels on both H558 and R558 background. Electrophysiological properties of hNa(v)1.5 mutant channels were investigated by the whole-cell patch clamp technique in HEK293 cells. When peak currents were affected by the mutation, cell surface biotinylation was performed to quantify the fraction of correctly cell membrane-targeted mutant channels. Loss-of-function defect of D1275N in SCN5A was rescued by R558 through enhancing cell surface targeting and improving steady-state activation of the mutant channels. In contrast, the defects of mutants E161K, P1298L, and R1632H were aggravated in the R558 background, mainly due to the reduced steady-state availability of mutant channels. The electrophysiological properties of the remaining SSS-related hNa(v)1.5 mutants including the missense mutants (L212P, T220I, DelF1617, T187I, R878C, G1408R), and the truncated mutants (W1421X, K1578fs/52, R1623X) were not significantly affected by H558R. CONCLUSION: We conclude that polymorphism H558R has mutation-specific effects on SCN5A-related SSS. Our data highlight the importance of common genetic variants in modulating phenotypes of genetic diseases.

Wild-type and missense mutants of retinoschisin co-assemble resulting in either intracellular retention or incorrect assembly of the functionally active octamer.

The X-linked disease retinoschisis is caused by mutations in the RS1 gene encoding retinoschisin, most commonly missense mutations leading to a lack of secretion of functional protein. One potential approach to treat this disease would be the introduction of the wild-type protein by gene therapy in affected individuals. Retinoschisin normally forms homo-octamers, so co-expression of the wild-type protein with the mutant could result in their co-assembly. In the present study, we show that retinoschisin assembles into an octamer before transport from the endoplasmic reticulum and that co-assembly of wild-type and mutant protein can occur when they are co-expressed in the same cell. This co-assembly results in the retention of some, but not all, expressed wild-type retinoschisin. Moreover, when the wild-type protein is expressed with a missense mutant that is normally secreted, co-assembly occurs resulting in the secretion of a heterogeneous mixture of oligomers. Missense mutations of retinoschisin which cause intracellular retention also lead to an unfolded protein response. However, this is not sufficient to decrease cell viability suggesting that the pathology of the disease is not likely to be linked to programmed cell death.

An overview of Notch3 function in vascular smooth muscle cells.

Proteins of the Notch family are cell surface receptors that transduce signals between neighbouring cells. The Notch signalling pathway is highly evolutionarily conserved and critical for cell fate determination during embryonic development, including many aspects of vascular development. The interaction of Notch receptors with ligands leads to cleavage of the Notch intracellular domain (NICD) which then translocates to the nucleus and activates the transcription factor CBF1/JBP-Jkappa, regulating downstream gene expression. To date four Notch receptors have been found in mammals. Of these, Notch3 is predominantly expressed in adult arterial smooth muscle cells in human. NOTCH3 gene mutations cause the autosomal dominant condition, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoecephelopathy (CADASIL), an inherited early stroke syndrome leading to dementia due to systemic vascular degeneration. This suggests that Notch3 plays a critical role in maintaining the phenotypic stability of vascular smooth muscle cells (VSMCs). Recent publications indicate that Notch3 is involved in vascular injury and is a determinant of VSMC survival, but its exact function is unknown. The molecular mechanisms underlying CADASIL pathology are therefore intriguing. Investigation of CADASIL mutant Notch3 shows that the majority of mutations do not change CBF1/JBP-Jkappa mediated classic Notch activation, so the pathological consequences of NOTCH3 mutations in CADASIL patients can not be simply explained by loss- or gain-of-function in the classic Notch signalling pathway. This suggests that a novel Notch3-mediated signalling pathway may be present in VSMCs, or cross-regulation of Notch3 to other signalling pathway(s) may play a critical role on VSMCs survival. Alternatively, the mutant Notch3 may gain a novel or toxic function in VSMCs. This review will focus on recent findings of Notch3 in vascular development and in regulating the VSMC behaviour and phenotype, and will use findings on investigating the molecular pathology of the single gene disorder CADASIL to understand the function of Notch3 in VSMCs.

Circadian regulation of retinoschisin in the chick retina.

PURPOSE: To investigate the circadian regulation and acute illumination effects on the expression and secretion of retinoschisin from vertebrate retinas. METHODS: Retinas were studied on the second day of constant darkness (DD) after several days of entrainment to 12-hour light/12-hour dark (LD) cycles in ovo or in vitro. Quantitative real-time PCR and Western immunoblotting were used to examine the mRNA and protein expressions of retinoschisin at different circadian time points. Pharmacologic treatments in whole retina and dissociated retinal cell cultures were used to investigate the cellular mechanisms underlying the circadian regulation of retinoschisin content and secretion. Different illumination conditions were given to examine changes in retinoschisin content in association with acute light/dark adaptation. RESULTS: The mRNA level, protein expression, and secretion of retinoschisin were under circadian control, all of which were higher at night and lower during the day. The Ras, MAP kinase Erk, CaMKII pathway served as part of the circadian output regulating the rhythmicity of retinoschisin. Blockage of L-type VGCCs dampened the retinoschisin rhythm, but inhibition of L-type VGCCs did not completely abolish the secretion of retinoschisin. The protein expression of retinoschisin also responded to acute illumination changes. CONCLUSIONS: The mRNA and protein expression, as well as retinoschisin secretion, are under circadian control. L-type VGCCs play a role in the circadian regulation of retinoschisin, but the molecular mechanism underlying retinoschisin secretion does not depend on L-type VGCCs. Protein expression of retinoschisin in response to acute illumination changes depends on previous light exposure experience.

Notch3 activation modulates cell growth behaviour and cross-talk to Wnt/TCF signalling pathway.

Notch3 is one of the four Notch receptors identified in mammal and expressed mainly in the arterial smooth muscle cells of human adult. Signalling via Notch3 is thought to be important in maintaining the phenotypic stability of the cells, but the nature of the signalling and its regulation to other signalling pathways are largely unknown. To understand further of the cellular function of Notch3 signalling, we generated cell lines stably expressing a constitutively active form of human Notch3 comprising of its soluble intracellular domain (N3IC). The N3IC expressing cells showed accelerated proliferation, decreased migration, increased cell surface N-cadherin, and growth in a colonised fashion that was reversible by N-cadherin blockade. N3IC expressing cells were also protected significantly against staurosporine-induced apoptosis and exhibited lower caspase 3/7 activity, accompanied by up-regulation of pAKT compared to control cells. We also found a complex cross-talk between Notch3 signalling and the Wnt pathway. N3IC stimulated Wnt-independent T-cell factor (TCF, the target transcription factor in the Wnt pathway) activation which was associated with increased Tyr-142 phosphorylation of beta-catenin. In contrast N3IC suppressed TCF activation in response to LiCl, which mimics the Wnt-dependent TCF activation mechanism. We conclude that Notch3 promotes cell growth and survival by activating PI3-kinase/AKT pathway; N-cadherin participates in the change of cell growth caused by Notch3 activation; and Notch3 signalling has dual-effects on the Wnt/TCF pathway suggesting a buffering role that Notch3 signalling may play in balancing these two important signalling pathways in regulating cell function.

X-linked retinoschisis: an update.

X-linked retinoschisis is the leading cause of macular degeneration in males and leads to splitting within the inner retinal layers leading to visual deterioration. Many missense and protein truncating mutations have now been identified in the causative retinoschisis gene (RS1) which encodes a 224 amino acid secretory retinal protein, retinoschisin. Retinoschisin octamerisation is implicated in cell-cell interactions and cell adhesion perhaps by interacting with beta2 laminin. Mutations cause loss of retinoschisin function by one of the three mechanisms: by interfering with protein secretion, by preventing its octamerisation or by reducing function in the secreted octamerised protein. The development of retinoschisis mouse models have provided a model system that closely resembles the human disease. Recent reports of RS1 gene transfer to these models and the sustained restoration of some retinal function and morphology suggest gene replacement may be a possible future therapy for patients.

Allelic variation of the FRMD7 gene in congenital idiopathic nystagmus.

OBJECTIVES: To perform a genotype-phenotype correlation study in an X-linked congenital idiopathic nystagmus pedigree (pedigree 1) and to assess the allelic variance of the FRMD7 gene in congenital idiopathic nystagmus. METHODS: Subjects from pedigree 1 underwent detailed clinical examination including nystagmology. Screening of FRMD7 was undertaken in pedigree 1 and in 37 other congenital idiopathic nystagmus probands and controls. Direct sequencing confirmed sequence changes. X-inactivation studies were performed in pedigree 1. RESULTS: The nystagmus phenotype was extremely variable in pedigree 1. We identified 2 FRMD7 mutations. However, 80% of X-linked families and 96% of simplex cases showed no mutations. X-inactivation studies demonstrated no clear causal link between skewing and variable penetrance. CONCLUSIONS: We confirm profound phenotypic variation in X-linked congenital idiopathic nystagmus pedigrees. We demonstrate that other congenital nystagmus genes exist besides FRMD7. We show that the role of X inactivation in variable penetrance is unclear in congenital idiopathic nystagmus. Clinical Relevance We demonstrate that phenotypic variation of nystagmus occurs in families with FRMD7 mutations. While FRMD7 mutations may be found in some cases of X-linked congenital idiopathic nystagmus, the diagnostic yield is low. X-inactivation assays are unhelpful as a test for carrier status for this disease.

Reduced secretion of fibulin 5 in age-related macular degeneration and cutis laxa.

Age-related macular degeneration (ARMD) is the leading cause of irreversible visual loss in the Western world, affecting approximately 25 million people worldwide. The pathogenesis is complex and missense mutations in FBLN5 have been reported in association with ARMD. We have investigated the role of fibulin 5 in ARMD by completing the first European study of the gene FBLN5 in ARMD (using 2 European cohorts of 805 ARMD patients and 279 controls) and by determining the functional effects of the missense mutations on fibulin 5 expression. We also correlated the FBLN5 genotype with the ARMD phenotype. We found two novel sequence changes in ARMD patients that were absent in controls and expressed these and the other nine reported FBLN5 mutations associated with ARMD and two associated with the autosomal recessive disease cutis laxa. Fibulin 5 secretion was significantly reduced (P<0.001) for four ARMD (p.G412E, p.G267S, p.I169 T, and p.Q124P) and two cutis laxa (p.S227P, p.C217R) mutations. These results suggest that some missense mutations associated with ARMD lead to decreased fibulin 5 secretion with a possible corresponding reduction in elastinogenesis. This study confirms the previous work identifying an association between FBLN5 mutations and ARMD and for the first time suggests a functional mechanism by which these mutations can lead to ARMD. It further demonstrates that FBLN5 mutations can be associated with different phenotypes of ARMD (not limited to the previously described cuticular drusen type). Such knowledge may ultimately lead to the development of novel therapies for this common disease.

Inherited eye disease: cause and late effect.

Molecular genetics has provided relatively few insights into late-onset eye disorders, but epidemiological data indicate that genetic factors are important in some late-onset eye disorders that cause major health burdens. Much clinical genetic research is based on the belief that developmental and late-onset disorders are not necessarily the result of defects in different genes, but are often caused by different mutations in the same collection of genes. Thus, mutations that either abolish or radically change gene function might cause early-onset disorders, whereas more-subtle changes in gene expression might underlie late-onset diseases. We present arguments and examples that indicate that this principle might be a fruitful guide to investigating the causes of late-onset eye disorders.

Mutations in the CACNA1F and NYX genes in British CSNBX families.

X-linked congenital stationary night blindness (CSNBX) is a genetically and phenotypically heterogeneous non-progressive disorder, characterised by impaired night vision but grossly normal retinal appearance. Other more variable features include reduction in visual acuity, myopia, nystagmus and strabismus. Genetic mapping studies by other groups, and our own studies of British patients, identified key recombination events indicating the presence of at least 2 disease genes on Xp11. Two causative genes (CACNA1F and NYX) for CSNBX have now been identified through positional cloning strategies. In this report, we present the results of comprehensive mutation screening in 14 CSNBX families, three with mutations in the CACNA1F gene and 10 with mutations in the NYX gene. In one family we failed to identify the mutation after testing RP2, RPGR, NYX and CACNA1F. NYX gene mutations are a more frequent cause of CSNBX, although there is evidence for founder mutations. Our report of patient population mutation screening for both CSNBX genes, and our exclusion of RP2 and RGPR, indicates that mutations in CACNA1F and NYX are likely to account for all CSNBX.

Refinement of the NHS locus on chromosome Xp22.13 and analysis of five candidate genes.

Nance-Horan syndrome (NHS) is an X-linked condition characterised by congenital cataracts, dental abnormalities, dysmorphic features, and mental retardation in some cases. Previous studies have mapped the disease gene to a 2 cM interval on Xp22.2 between DXS43 and DXS999. We report additional linkage data resulting from the analysis of eleven independent NHS families. A maximum lod score of 9.94 (theta=0.00) was obtained at the RS1 locus and a recombination with locus DXS1195 on the telomeric side was observed in two families, thus refining the location of the gene to an interval of around 1 Mb on Xp22.13. Direct sequencing or SSCP analysis of the coding exons of five genes (SCML1, SCML2, STK9, RS1 and PPEF1), considered as candidate genes on the basis of their location in the critical interval, failed to detect any mutation in 12 unrelated NHS patients, thus making it highly unlikely that these genes are implicated in NHS.

Multiple loss-of-function mechanisms contribute to SCN5A-related familial sick sinus syndrome.

BACKGROUND: To identify molecular mechanisms underlying SCN5A-related sick sinus syndrome (SSS), a rare type of SSS, in parallel experiments we elucidated the electrophysiological properties and the cell surface localization of thirteen human Na(v)1.5 (hNa(v)1.5) mutant channels previously linked to this disease. METHODOLOGY/PRINCIPAL FINDINGS: Mutant hNa(v)1.5 channels expressed by HEK293 cells and Xenopus oocytes were investigated by whole-cell patch clamp and two-microelectrode voltage clamp, respectively. HEK293 cell surface biotinylation experiments quantified the fraction of correctly targeted channel proteins. Our data suggested three distinct mutant channel subtypes: Group 1 mutants (L212P, P1298L, DelF1617, R1632H) gave peak current densities and cell surface targeting indistinguishable from wild-type hNa(v)1.5. Loss-of-function of these mutants resulted from altered channel kinetics, including a negative shift of steady-state inactivation and a reduced voltage dependency of open-state inactivation. Group 2 mutants (E161K, T220I, D1275N) gave significantly reduced whole-cell currents due to impaired cell surface localization (D1275N), altered channel properties at unchanged cell surface localization (T220I), or a combination of both (E161K). Group 3 mutant channels were non-functional, due to an almost complete lack of protein at the plasma membrane (T187I, W1421X, K1578fs/52, R1623X) or a probable gating/permeation defect with normal surface localisation (R878C, G1408R). CONCLUSIONS/SIGNIFICANCE: This study indicates that multiple molecular mechanisms, including gating abnormalities, trafficking defects, or a combination of both, are responsible for SCN5A-related familial SSS.

Structural effects of fibulin 5 missense mutations associated with age-related macular degeneration and cutis laxa.

PURPOSE: AMD has a complex etiology with environmental and genetic risk factors. Ten fibulin 5 sequence variants have been associated with AMD and two other fibulin 5 mutations cause autosomal-recessive cutis laxa. Fibulin 5 is a 52-kDa calcium-binding epidermal growth factor (cbEGF)-rich extracellular matrix protein that is essential for the formation of elastic tissues. Biophysical techniques were used to detect structural changes in the fibulin 5 mutants and to determine whether changes are predictive of pathogenicity. METHODS: Native PAGE, nonreduced SDS-PAGE, size-exclusion column multiangle laser light scattering, sedimentation velocity, and circular dichroism (CD) were used to investigate the mobility, hydrodynamic radii, folding, and oligomeric states of the fibulin 5 mutants in the absence and presence of Ca(2+). RESULTS: CD showed that all mutants are folded, although perturbations to secondary structure contents were detected. Both cutis laxa mutants increased dimerization. Most other mutants slightly increased self-association in the absence of Ca(2+) but this was also demonstrated by G202R, a polymorphism detected in a control individual. The AMD-associated mutant G412E showed lower-than-expected mobility during native-PAGE, the largest hydrodynamic radius for the monomer form and the highest levels of aggregation in both the absence and presence of Ca(2+). CONCLUSIONS: The results identified structural differences for the disease-causing cutis laxa mutants and for one AMD variant (G412E), suggesting that this may also be pathogenic. Although the other AMD-associated mutants showed no gross structural differences, they cannot be excluded as pathogenic by differences outside the scope of this study-for example, disruption of heterointeractions.