Lluı̈sa Vilageliu

Unusual expression of Gaucher's disease: cardiovascular calcifications in three sibs homozygous for the D409H mutation.

Three sisters suffering from an unusual form of Gauchers disease are described. These patients had cardiovascular abnormalities consisting of calcification of the ascending aorta and of the aortic and mitral valves. Neurological findings included ophthalmoplegia and saccadic eye movements in two patients, and tonic-clonic seizures in the third. The three patients died, two of them after having undergone aortic valve replacement. Tissue was obtained from one of the sibs and fibroblast and liver beta-glucocerebrosidase activity was reduced to 4% and 11% of mean normal values. Genotype analysis indicated that the patient was homozygous for the D409H mutation. It is tempting to relate the phenotype of severe cardiac involvement to the D409H/D409H genotype, although further cases will be needed before this association can be confirmed.

Identification of 14 novel GLB1 mutations, including five deletions, in 19 patients with GM1 gangliosidosis from South America

GM1 gangliosidosis is a lysosomal storage disorder caused by the absence or reduction of lysosomal β‐galactosidase activity because of mutations in the GLB1 gene. Three major clinical forms have been established: type I (infantile), type II (late infantile/juvenile) and type III (adult). A mutational analysis was performed in 19 patients with GM1 gangliosidosis from South America, mainly from Argentina. Two of them were of Gypsy origin. Main clinical findings of the patients are presented. All 38 mutant alleles were identified: of the 22 different mutations found, 14 mutations are described here for the first time. Among the novel mutations, five deletions were found. Four of them are relatively small (c.435_440delTCT, c.845_846delC, c.1131_1145del15 and c.1706_1707delC), while the other one is a deletion of 1529 nucleotides that includes exon 5 and is caused by an unequal crossover between intronic Alu sequences. All the described patients with GM1 gangliosidosis were affected by the infantile form, except for four unrelated patients classified as type II, III, and II/III (two cases). The two type II/III patients bore the previously described p.R201H mutation, while the adult patient bore the new p.L155R. The juvenile patient bore two novel mutations: p.S434L and p.G554E. The two Gypsy patients are homozygous for the p.R59H mutation as are all Gypsy patients previously genotyped.

Two new mild homozygous mutations in Gaucher disease patients: Clinical signs and biochemical analyses

Gaucher disease (GD) is a lysosomal storage disorder resulting from impaired activity of lysosomal beta-glucocerebrosidase. More than 60 mutations have been described in the GBA gene. They have been classified as lethal, severe, and mild on the basis of the corresponding phenotype. The fact that most GD patients are compound heterozygous and that most type 1 patients bear the N370S allele, which by itself causes a mild phenotype, make it difficult to correlate the clinical signs with the mutations. Besides N370S, about 10 mild mutations have been described, but only one undoubtedly classified as mild was found at homozygosity. Here we report 2 novel mutations, I402T and V375L, at homozygosity in 2 adult Italian type 1 GD patients. Some properties of the I402T fibroblast enzyme have been compared to those of the enzyme from cells of several N370S/N370S patients. Analysis of the catalytic properties and heat stability as well as the response to phosphatidylserine and sphingolipid activator protein indicate a marked similarity between the 2 enzymes. The finding of another, unrelated patient bearing the I402T mutation (in this case as a compound heterozygote with mutation N370S) suggests that this allele might be quite frequent in the area of Sicily from where both patients originated. In conclusion, the phenotypic expression in the 2 homozygous patients presented here and the biochemical data for one of them allowed the classification of these mutations as mild thus extending the group of mild mutations found at homozygosity.

Expression and characterization of 14 GLB1 mutant alleles found in GM1-gangliosidosis and Morquio B patients

GM1-gangliosidosis and Morquio B disease are lysosomal storage disorders caused by β-galactosidase deficiency attributable to mutations in the GLB1 gene. On reaching the endosomal-lysosomal compartment, the β-galactosidase protein associates with the protective protein/cathepsin A (PPCA) and neuraminidase proteins to form the lysosomal multienzyme complex (LMC). The correct interaction of these proteins in the complex is essential for their activity. More than 100 mutations have been described in GM1-gangliosidosis and Morquio B patients, but few have been further characterized. We expressed 12 mutations suspected to be pathogenic, one known polymorphic change (p.S532G), and a variant described as either a pathogenic or a polymorphic change (p.R521C). Ten of them had not been expressed before. The expression analysis confirmed the pathogenicity of the 12 mutations, whereas the relatively high activity of p.S532G is consistent with its definition as a polymorphism. The results for p.R521C suggest that this change is a low-penetrant disease-causing allele. Furthermore, the effect of these β-galactosidase changes on the LMC was also studied by coimmunoprecipitations and Western blotting. The alteration of neuraminidase and PPCA patterns in several of the Western blotting analyses performed on patient protein extracts indicated that the LMC is affected in at least some GM1-gangliosidosis and Morquio B patients.

Autosomal recessive retinitis pigmentosa in Spain: evaluation of four genes and two loci involved in the disease

Autosomal recessive retinitis pigmentosa (ARRP) is a genetically heterogeneous form of retinal degeneration. The genes for the β‐subunit of rod phosphodiesterase (PDEB), rhodopsin (RHO), peripherin/RDS (RDS) and the rod outer segment membrane protein 1 (ROM1), as well as loci at 6p and 1q, have previously been reported as the cause of ARRP. In order to determine whether they are responsible for the disease in Spanish pedigrees, linkage and homozygosity studies using markers at these loci were carried out on 47 Spanish ARRP families. SSCP analysis was performed to search for mutations in the genes cosegregating with the disease in particular pedigrees. Three homozygous mutations in the PDEB gene were found, thus accounting for 6% of the cases. No other disease‐causing mutation was observed in the other genes analysed, nor was significant evidence found for the involvement of the loci at 6p or 1q. On the basis of these data, it is unlikely that these genes and loci account for a considerable proportion of ARRP cases.

Novel homozygous mutation in the alpha subunit of the rod cGMP gated channel (CNGA1) in two Spanish sibs affected with autosomal recessive retinitis pigmentosa

Retinitis pigmentosa (RP, MIM 268000) comprises a clinically and genetically heterogeneous group of disorders leading to progressive dysfunction of the rod photoreceptors of the retina. Patients suffer early night blindness followed by loss of peripheral vision associated with pigment accumulation in the outer retina and attenuation of the retinal vessels. The pathophysiology of RP involves apoptosis of rod cells. Although macular vision is preserved in the initial stages of the disease, visual field defects gradually increase and cone affectation follows. RP can be inherited as an autosomal dominant, autosomal recessive, digenic, or X linked trait. Several RP genes and loci have been described to date (RetNet web site: http://utsph.sph.uth.tmc.edu/www/Wtsph/RetNet/home.htm). Altogether, the genes known to cause the autosomal recessive forms explain a small proportion of cases, while the great majority remain unexplained, which illustrates the extreme genetic heterogeneity of this condition. As expected, many of the ARRP genes are rod specific, some are only expressed in the retinal pigment epithelium, and none is cone specific. Cyclic nucleotide gated channels (CNCG) are a group of non-selective ion channels present in different tissues. The rod cGMP gated cation channel is located in the outer segment of the plasma membrane and is involved in the last step of the phototransduction cascade. In spite of a similar function, rod and cone cells express different CNCG genes.1 Rod channels are predicted to form heterotetramers of two alpha and beta subunit dimers. Each contains a core structural unit of six membrane spanning segments, a pore region, and a cGMP binding domain. The alpha peptide ( CNGA1 , MIM 123825, GDB 127557) forms a functional channel by itself and is considered the main functional subunit, while the beta counterpart ( CNGB1, MIM 600724, GDB 434397) modulates the activity of the channel and is unable to promote ion transfer by …

Unsuccessful chimeraplast strategy for the correction of a mutation causing Gaucher disease.

A number of gene therapy approaches have been developed for the treatment of genetic diseases, most of them based on the use of viral vectors. However, in general, they have not been successful and some complications, such as immune reactions induced by adenoviral vectors or random integration of retroviral vectors, have been reported frequently. To overcome these limitations, novel strategies have recently emerged. One of them is chimeraplasty, based on the correction of single-base mutations by mismatch repair mechanisms using chimeric RNA/DNA oligonucleotides, named chimeraplasts. Several papers have reported the use of this method to correct a number of pathological mutations underlying different diseases. In Gaucher disease, the most prevalent spingoliposis, mutation c.1448C->T (L444P), is one of the most common mutations in many populations. We have attempted to correct mutation c.1448C->T in fibroblasts from a Gaucher disease patient using a chimeraplast approach. Although we have shown that the chimeraplast reaches the fibroblast nucleus by colocalization with nuclear structures, no genomic correction was detected. To evaluate whether fibroblast and hepatocyte extracts are able to effect correction in vitro, we followed a cell-free extract assay using Escherichia coli cells. Our results show a very low efficiency (if any) of chimeraplast correction. A growing number of negative results for chimeraplast experiments have recently been reported. This promising technique has turned out to be inconsistent and impossible to replicate in most laboratories and many of the first successful results are now being questioned. Our negative data are consistent with this criticism.

Recurrence of the D409H mutation in Spanish Gaucher disease patients: description of a new homozygous patient and haplotype analysis.

Gaucher disease results, in most patients, from mutations in the gene encoding glucocerebrosidase. Mutation D409H is the third most frequent in Spanish patients, accounting for 5.7% of all mutated alleles. This allele is associated mainly with the neurological forms of the disease. Recently, homozygosity for the D409H mutation has been associated with a particular phenotype, including specific cardiovascular symptoms. Here we report a second Spanish patient bearing the D409H/D409H genotype with a very early manifestation of the disease. The patient started enzyme replacement therapy at 3 months of age. A common origin for the Spanish D409H alleles was ruled out by haplotype analysis using an internal polymorphism of the glucocerebrosidase gene and two external microsatellite markers.

Screening of CD96 and ASXL1 in 11 patients with Opitz C or Bohring-Opitz syndromes.

Opitz C trigonocephaly (or Opitz C syndrome, OTCS) and Bohring‐Opitz syndrome (BOS or C‐like syndrome) are two rare genetic disorders with phenotypic overlap. The genetic causes of these diseases are not understood. However, two genes have been associated with OTCS or BOS with dominantly inherited de novo mutations. Whereas CD96 has been related to OTCS (one case) and to BOS (one case), ASXL1 has been related to BOS only (several cases). In this study we analyze CD96 and ASXL1 in a group of 11 affected individuals, including 2 sibs, 10 of them were diagnosed with OTCS, and one had a BOS phenotype. Exome sequences were available on six patients with OTCS and three parent pairs. Thus, we could analyze the CD96 and ASXL1 sequences in these patients bioinformatically. Sanger sequencing of all exons of CD96 and ASXL1 was carried out in the remaining patients. Detailed scrutiny of the sequences and assessment of variants allowed us to exclude putative pathogenic and private mutations in all but one of the patients. In this patient (with BOS) we identified a de novo mutation in ASXL1 (c.2100dupT). By nature and location within the gene, this mutation resembles those previously described in other BOS patients and we conclude that it may be responsible for the condition. Our results indicate that in 10 of 11, the disease (OTCS or BOS) cannot be explained by small changes in CD96 or ASXL1. However, the cohort is too small to make generalizations about the genetic etiology of these diseases.

Activity and High-Order Effective Connectivity Alterations in Sanfilippo C Patient-Specific Neuronal Networks

Summary Induced pluripotent stem cell (iPSC) technology has been successfully used to recapitulate phenotypic traits of several human diseases in vitro. Patient-specific iPSC-based disease models are also expected to reveal early functional phenotypes, although this remains to be proved. Here, we generated iPSC lines from two patients with Sanfilippo type C syndrome, a lysosomal storage disorder with inheritable progressive neurodegeneration. Mature neurons obtained from patient-specific iPSC lines recapitulated the main known phenotypes of the disease, not present in genetically corrected patient-specific iPSC-derived cultures. Moreover, neuronal networks organized in vitro from mature patient-derived neurons showed early defects in neuronal activity, network-wide degradation, and altered effective connectivity. Our findings establish the importance of iPSC-based technology to identify early functional phenotypes, which can in turn shed light on the pathological mechanisms occurring in Sanfilippo syndrome. This technology also has the potential to provide valuable readouts to screen compounds, which can prevent the onset of neurodegeneration.