The molecular defect underlying canine fucosidosis.

Fucosidosis is a lysosomal storage disease which affects humans and English springer spaniel dogs. The disease is recessively inherited in both species and results from a deficiency of the enzyme alpha-L-fucosidase. We have recently cloned and sequenced the canine fucosidase gene (EMBL sequence admission number X92448 (cDNA) and X92671-X92678 (individual exonic data)). The gene spans 12 kb and consists of eight exons. SSCP based mutation analysis of affected animals was carried out on the coding region of this gene both with exonic primers, and intronic primer pairs for each exon. A 14 base pair deletion of the cDNA was identified at the 3'' end of exon 1 in fucosidosis affected animals. Surprisingly, PCR based genomic cloning of DNA from these animals showed an identical deletion in this DNA, ending at the start of intron 1. This change causes a frameshift and, in consequence, 25 novel codons are transcribed in exon 2 before the first of two adjacent premature stop codons is encountered.     

Reproductive failure and parental chromosome abnormalities

The effect of ascertaining on estimates of the frequency ofparental chromosome abnormalities in couples with a previoushistory of pregnancy wastage was investigated by comparing threesamples which differ in the ascertainment modality but not inthe cytogenetical approach. The incidence of chromosome abnormalitieswas higher in the sample of 441 couples selected essentiallyon clinical criteria (6%) than in the two samples (659 and 479couples) selected retrospectively from the files of two cytogeneticlaboratories (4.6 and 3.2%). The comparison of these resultswith similar data, based on large samples reported in the literature,indicated that sample size may be relevant in producing thewide ranges of variation of the frequency of chromosomal abnormalities.Using our data and those from three other samples of >300couples a reasonable estimate of the overall incidence of chromosomalabnormalities in couples with a previous history of fetal wastageis {small tilde}5%.     

Intrafamilial variability in fucosidosis

Two families with five patients affected with fucosidosis are described. Within each family, both type I and type II fucosidosis are present. This suggests that environmental factors or "modifying genes" different from the fucosidase structural gene contribute to the phenotype of a fucosidosis patient.     

Copy number abnormalities in sporadic canine colorectal cancers

Human colorectal cancer (CRC) is one of the better-understood systems for studying the genetics of cancer initiation and progression. To develop a cross-species comparison strategy for identifying CRC causative gene or genomic alterations, we performed array comparative genomic hybridization (aCGH) to investigate copy number abnormalities (CNAs), one of the most prominent lesion types reported for human CRCs, in 10 spontaneously occurring canine CRCs. The results revealed for the first time a strong degree of genetic homology between sporadic canine and human CRCs. First, we saw that between 5% and 22% of the canine genome was amplified/deleted in these tumors, and that, reminiscent of human CRCs, the total altered sequences directly correlated to the tumor''s progression stage, origin, and likely microsatellite instability status. Second, when mapping the identified CNAs onto syntenic regions of the human genome, we noted that the canine orthologs of genes participating in known human CRC pathways were recurrently disrupted, indicating that these pathways might be altered in the canine CRCs as well. Last, we observed a significant overlapping of CNAs between human and canine tumors, and tumors from the two species were clustered according to the tumor subtypes but not the species. Significantly, compared with the shared CNAs, we found that species-specific (especially human-specific) CNAs localize to evolutionarily unstable regions that harbor more segmental duplications and interspecies genomic rearrangement breakpoints. These findings indicate that CNAs recurrent between human and dog CRCs may have a higher probability of being cancer-causative, compared with CNAs found in one species only.Cancer is a disease of the genome, and genomic instability is a hallmark of cancer (Hanahan and Weinberg 2000). As cancer progresses, more extensive genomic instability develops, and more abnormal changes accumulate on the genome (e.g., copy number abnormalities or CNAs, translocations, and inversions) (Albertson et al. 2003). While some of these aberrations disrupt normal cellular processes and indeed contribute to cancer development and progression, others emerge simply as passenger alterations of cancer genomic instability and play no role in disease etiology. Clearly, finding genomic abnormalities is important, but identifying those that are cancer-causative is even more meaningful.A central aim of cancer research has been to identify causative (or driver) alterations. This has become both increasingly challenging and urgent in recent years with the launch of high-throughput cancer genome projects, such as the Cancer Genome Atlas (http://cancergenome.nih.gov/), the International Cancer Genome Consortium (http://www.icgc.org/), and others (e.g., Greenman et al. 2007; Jones et al. 2008). Researchers have been tackling this challenge by improving experimental conditions (e.g., high-resolution microarray to refine the boundaries of amplicons to narrow down the “driver” genes; see Haverty et al. 2008) and by developing more sophisticated statistical models and functional analysis strategies to systematically (Aebersold et al. 2009) identify significant abnormalities (e.g., Beroukhim et al. 2007; Greenman et al. 2007; Jones et al. 2008).We are developing a cross-species comparison strategy that differs fundamentally from the current published approaches described above (which study humans only). We hypothesize that causative alteration candidates can be distinguished from consequent candidates by examining orthologous genes or genomic loci with tumors from multiple species having the same type of cancer. Provided these species share similar molecular and genetic pathways of cancer development and progression, abnormalities that are recurrent among different species will be deemed causative, whereas those that are found in only one species and are located in evolutionarily unstable sites will be considered consequent (bystanders or passengers) (Fig. 1). In our study, evolutionarily unstable sites are defined as regions enriched with interspecies genomic rearrangement breakpoints (ISGRBPs) (Pevzner and Tesler 2003; Zhao et al. 2004) and segmental duplications (SDs) (Bailey et al. 2002, 2004; Turner et al. 2007; Kidd et al. 2008).Open in a separate windowFigure 1.(A) Cross-species comparison for causative (or driver) aberration identification. Once we demonstrate that the same types of cancer from the human and the dog share similar molecular and genetic pathways of cancer development and progression, we will consider abnormalities recurrent between the two species as driver candidates (solid gray area), and those found in only one species and falling in evolutionarily unstable sites (EIN sites) as passenger candidates (small squared areas). Those in the parallel-lined areas need further studies. (B) The advantage of the human–dog comparison strategy over the human-only strategy for cancer driver gene identification. The cross-species comparison strategy can make use of the difference in the genomic location of orthologous genes between the human and the dog, a result of evolutionary genomic rearrangements that occurred since the two species diverged more than 75 million years ago. This shows that two genes, which are nearby in the human genome but distant in the dog genome, are both disrupted in the human cancer (boxed with broken lines). However, in the dog cancer, only one gene is disrupted, which will be considered as driver, and the other is intact (boxed with unbroken lines), which will be deemed as passenger.Our hypothesis rests on the same rationale that cancer researchers have been using for years: abnormalities recurrent among different cases are more likely to be causative, compared with nonrecurrent events. The difference is we are searching for events that are recurrent not only among different cases within the same species, but also among different species. Compared with single-species approaches, this multispecies comparison strategy can better distinguish causative events from passenger alterations of cancer genomic instability, by taking account of the difference in the genomic location of orthologous genes and loci caused by interspecies genomic rearrangements that occurred during evolution (Fig. 1). For instance, chromosome region 18q is often found to be deleted in human colorectal cancer (CRC). Except for SMAD4, which is clearly demonstrated to be a CRC driver gene (Kinzler and Vogelstein 1996), the roles of many 18q genes in CRC development and progression remain unclear, and it is possible that some of these genes are deleted in human CRC simply because they are near SMAD4. We have found that interspecies genomic rearrangements have dispatched many such genes far away from SMAD4 via translocations or inversions in the dog genome. Studying dog CRCs could shed light on whether these genes are drivers or passengers, provided we can first show that dog and human CRCs share similar molecular and genetic pathways of cancer development and progression.To test this cross-species strategy, we have been conducting a comparative study between sporadic human and canine CRCs. Human CRC is one of the better-understood systems for studying the genetics of cancer initiation and progression. The proposed stepwise model of human colorectal carcinogenesis (Kinzler and Vogelstein 1996; Rajagopalan et al. 2003) highlights the key role of genomic instability, which occurs in the form of either chromosomal instability (CIN) (Lengauer et al. 1997) or microsatellite instability (MSI) (Toft and Arends 1998). MSI, which is characterized by a high level of single- or oligo-base mutations, due to defective DNA mismatch repair (MMR) (Grady 2004), and recognized by indel mutations in microsatellite loci (Boland et al. 1998), occurs in ∼13% of human sporadic CRCs. CIN, characterized by CNAs of relatively narrow genomic regions ranging from single loci to entire chromosomes, occurs in 87% of human sporadic CRCs. Although the role that CIN plays in causing cancer is unclear, the occurrence of CNAs could result in inactivation of tumor suppressors (e.g., APC, SMAD4, and TP53), as well as overactivation of oncogenes, such as KRAS. This could, in turn, disrupt key signaling pathways of Wnt, TGF-beta, p53, and others that play fundamental roles in CRC development and progression (Kinzler and Vogelstein 1996; Gallahan and Callahan 1997; Rajagopalan et al. 2003; Grady 2004; Sancho et al. 2004; Kaiser et al. 2007).Because of its importance in human CRCs, we investigated genomic instability in 10 spontaneously occurring dog CRCs and reported the results here. This study yielded, for the first time, several pieces of evidence in support of dog CRCs possibly following similar molecular pathways of cancer development and progression as human CRCs. First, via array comparative genome hybridization (aCGH) analyses, we found that reminiscent of human CRCs, between 5% to 22% of the canine genome was either amplified or deleted in these canine tumors, and that the total amount of altered genomic sequences directly correlated to the tumor''s progression stage, origin, and likely MSI status. Second, mapping of the identified dog CNAs to the better-annotated human genome revealed that the canine orthologs of many genes involved in known human CRC development and progression pathways were disrupted, indicating that these pathways might also be altered in these canine CRCs. Third, we observed a significant overlapping of CNAs between human and canine tumors, and tumors from the two species were clustered according to the tumor subtypes, but not according to the species. Besides these genetic similarities between the two species, our analyses revealed that the CNAs found in only one species (especially the human) localize to evolutionarily unstable genomic regions that harbor more SDs and ISGRBPs compared with CNAs found in both species. This indicates that shared CNAs might be more significant in CRC etiology compared with species-specific (especially human-specific) CNAs.     

Spectrum of mutations in fucosidosis

Fucosidosis is a lysosomal storage disorder characterised by progressive psychomotor deterioration, angiokeratoma and growth retardation. It is due to deficient alpha-l-fucosidase activity leading to accumulation of fucose-containing glycolipids and glycoproteins in various tissues. Fucosidosis is extremely rare with less than 100 patients reported worldwide, although the disease occurs at a higher rate in Italy, in the Hispanic-American population of New Mexico and Colorado, and in Cuba. We present here a review study of the mutational spectrum of fucosidosis. Exon by exon mutation analysis of FUCA1, the structural gene of alpha-l-fucosidase, has identified the mutation(s) in nearly all fucosidosis patients investigated. The spectrum of the 22 mutations detected to date includes four missense mutations, 17 nonsense mutations consisting of seven stop codon mutations, six small deletions, two large deletions, one duplication, one small insertion and one splice site mutation. All these mutations lead to nearly absent enzymatic activity and severely reduced cross-reacting immunomaterial. The observed clinical variability is, therefore, not due to the nature of the fucosidosis mutation, but to secondary unknown factors.     

Reproductive tract abnormalities in rats treated neonatally with DES

Neonatal female Sprague-Dawley rats were given daily injections of 3 μg diethylstilbestro (DES) for 5 days beginning within 24 hours of birth. Some of these rats were ovariectomized between the 15th and 18th postnatal days, and on the 60th postnatal day were given daily injections of 3 μg estradiol for 1, 3, or 5 weeks. Intact rats were sacrificed at 60, 95, or 130 days. The morphology of the upper vagina, cervix, and lower uterus was examined by light and scanning electron microscopy. Two abnormalities resulting from neonatal DES exposure were found. The first was squamous metaplasia observed in the uteri of rats given DES and later exposed to exogenous or endogenous estrogen. Metaplasia was seen in both the luminal and glandular epithelium. The longer the rats were exposed to exogenous or endogenous estrogen, the more extensive was the metaplasia. The second was a gross morphologic abnormality seen in all rats given DES regardless of any later treatment. That part of the cervix that protrudes into the cranial limit of the vagina was absent and, thus, a vaginal fornix was nonexistent. Previous investigations have emphasized abnormalities of the lower reproductive tract. The present study indicates that the upper reproductive tract also must be considered in investigations of the effects of hormones administered to neonates.     

Prenatal diagnosis of fucosidosis

A pregnancy from a family at risk for fucosidosis was monitored. Determinations of fucosidase and mannosidase were performed on the serum and white blood cells of several members of the family, on amniotic fluid and amniotic fluid cells of the fetus at several passages, and on fibroblast cell lines from index cases. The fetus was diagnosed as being free from the disease.
This conclusion was confirmed after birth by fucosidase determination in plasma and white cells from cord blood, and in the placenta. Fluctuations in fucosidase activity were observed in extracts from cultured amniotic cells at various passages. The possible causes of this variability are discussed.     

A 5'' splice site mutation in fucosidosis.

Fucosidosis is a rare, autosomal recessive, lysosomal storage disease, resulting from a deficiency of the enzyme alpha-fucosidase (EC 3.2.1.51). It is characterised clinically by progressive mental and motor deterioration, growth retardation, coarse facies, and often recurrent infections, but the course of the disease is variable. The gene encoding lysosomal alpha-fucosidase has been mapped to the short arm of chromosome 1 at position 1p34.1-36.1 and has been called FUCA1. Two mutations causing disease have been described previously, a C-->T change in exon 8 giving rise to a premature, in frame TAA stop codon, and a deletion of at least two exons from the 3' end of the gene. In this paper we present evidence that a homozygous G-->A transition in the first position of the 5' splice site of intron 5 of FUCA1 is the disease causing mutation in a 9 year old child of distantly related parents. A new banding pattern was detected in the patient by Southern blotting of genomic DNA using TaqI restriction and a cDNA FUCA1 probe. The patient was homozygous for this pattern. Three sibs with alpha-fucosidase activity below the normal reference range and both parents were heterozygous. This pattern was not detected in 26 other fucosidosis patients and has not been found in any controls. The mutation was localised by a combination of restriction mapping using different cDNA probes, single stranded conformational polymorphism analysis of exons and flanking regions amplified by the polymerase chain reaction, and by direct sequencing of the amplified sequence. A view of the nature of the mutation, its cosegregation with the disease mutation and its absence in controls, it is probable that the 5' splice site mutation causes fucosidosis in this child.     

Mutation analysis of a Japanese patient with fucosidosis

Fucosidosis is a rare autosomal recessive disorder resulting from a deficiency of α-L-fucosidase. Recently, various mutations have been reported in this disease, but it is difficult to elucidate the phenotype from the genetic mutations. We report a patient with chronic infantile type fucosidosis, with a compound heterozygote of a nonsense mutation (W148X, Trp at codon 148 to stop codon) and a large deletion, including all exons. This is the first report of a large deletion demonstrated in fucosidosis. It is interesting that this patient has a relatively mild clinical course despite the absence of the mRNA. This case also indicates the difficulty in determining the phenotype from the genotype in fucosidosis. Received: February 19, 1999 / Accepted: April 16, 1999     

Alpha-L-fucosidase in cultured bone marrow fibroblasts from fucosidosis patients.

Bone marrow fibroblasts were cultured from two patients with fucosidosis type 2, six control subjects, and three patients with other lysosomal disorders. Optimal conditions for measuring alpha-L-fucosidase activity in lysates of these cells with the fluorogenic substrate 4-methylumbelliferyl-alpha-L-fucoside were established. The pH profile of normal bone marrow fibroblasts showed three peaks and a shoulder of enzymatic activity, with maximum activity at pH 4.75. In cells derived from fucosidosis patients two peaks of apparent alpha-L-fucosidase activity were obtained; the pH optimum was 4.5. alpha-L-Fucosidase activity (mean +/- SD) in the fucosidosis and control bone marrow fibroblasts was 2.5 and 312.4 +/- 10.9 nmoles 4-methylumbelliferone per milligram protein per hour, respectively. A reduction in the apparent specific enzymatic activity in the fucosidosis cells was observed by using increasing concentrations of cellular protein in the assay system. Mixing experiments between normal and fucosidosis cells gave the expected activities. These findings indicate that cultured bone marrow fibroblasts can be used for the diagnosis and study of fucosidosis.     

Biochemical studies on an unusual case of fucosidosis

Biochemical studies have been performed on the spleen and liver of a patient suspected of having fucosidosis. The specific, complete deficiency of 4-methylumbelliferyl (4-MU)-α-L-fucosidase activity coupled with the massive liver accumulation of polar glycolipids (comparable to those found in a known case of fucosidosis), confirms the diagnosis of fucosidosis. The patient is unusual in that his liver retains 25–50 % of normal α-L-fucosidase activity for the p-nitrophenyl (PNP) substrate. The patient's spleen showed complete deficiency of α-L-fucosidase activity for both 4-MU- and PNP-α-L-fucopyranoside. The patient's crude liver α-L-fucosidase was not significantly different in thermostability when compared to the crude enzyme from a normal liver.
After partial purification on agarose-ɛ-aminocaproyl-fucosamine and sepharose-concanavalin A, the patient's liver α-L-fucosidase was found to possess both PNP and 4-MU activities. This suggests that components in the patient's crude liver supernatant may inhibit 4-MU-α-L-fucosidase activity. This was demonstrated further when the patient's liver supernatant was found to inhibit significantly normal liver 4-MU-α-L-fucosidase activity. Kinetic and immunochemical characterization indicated that the patient's α-fucosidase was similar, if not identical, to the normal liver enzyme. Apparent Km's for 4-MU-α-L-fucopyranoside were determined to be 0.1 mM and 0.12 mM for the patient and normal liver enzyme, respectively. The IgG fraction of antiserum prepared against purified normal liver α-L-fucosidase immunoprecipitated 81 % of the patient's α-L-fucosidase, suggesting similarity of antigenic determinants.     

Two patients with an unusual form of type II fucosidosis

Two brothers with an unusual form of fucosidosis are presented, providing further evidence for the clinical heterogeneity of the disease. The patients have several characteristics of type II, but from the point of view of progression rate and survival, they resemble more type III. However, the characteristic skin lesions of type 111 are not seen. In contrast to all other subtypes, a dry, thin skin is observed. The authors tend to classify these patients as a "slow motion" type II variant. Both in leukocyte lysates and plasma, severe α-L-fucosidase deficiency was established. In the parents, intermediate α-L-fucosidase activities were observed in leukocytes, but normal values in plasma, indicating that plasma is not suitable for carrier detection in this family. It is felt that no conclusions regarding clinical subtypes of fucosidosis can be drawn without careful characterization of the mutant enzyme in the primarily affected tissues.     

Immunologic abnormalities in canine juvenile polyarteritis syndrome: a naturally occurring animal model of Kawasaki disease.

This study describes the immunologic abnormalities during the acute phase of juvenile polyarteritis syndrome (JPS), a multisystem necrotizing vasculitis of young dogs with a predilection for the coronary arteries. JPS has striking clinical, laboratory, and pathologic similarities to Kawasaki disease (KD), the most common cause of acquired heart disease in children in the United States. The immunologic abnormalities include an increase in serum IgA, an increase in the percentage of peripheral B cells and a decrease in the percentage of total peripheral T cells, a marked suppression of the blastogenic response to mitogenic stimulation, an inability to generate immunoglobulin-secreting plasma cells following polyclonal activation, the presence of antineutrophil cytoplasmic antibodies, and evidence of monocyte/macrophage activation. These immunoregulatory abnormalities are similar to those observed in children during the acute phase of KD. This unique, naturally occurring animal model of necrotizing vasculitis may prove useful for investigating novel therapeutic interventions in the treatment of necrotizing vasculitis and may yield insight into the immunopathology and etiology of KD.     

Mutation identification and characterization of a Taiwanese patient with fucosidosis

Fucosidosis is a rare lysosomal storage disease caused by a defect of the α-l-fucosidase (FUCA1) gene. Worldwide 26 mutations underlying the disease have been reported. By direct DNA sequencing of exons and flanking introns, homozygous Y126X mutation and Q281R polymorphism were found in a Taiwanese patient with fucosidosis. Upon expressing in COS-7 cells, 97.4% of α-l-fucosidase activity compared with that of the wild-type construct was observed in the cDNA containing Q281R polymorphism. Western blot analysis revealed a 58-kDa precursor and 56-kDa mature forms for cells transfected with wild-type and Q281R enzymes. Using the fluorogenic substrate, the Michaelis constants and maximal velocities of both enzymes were very similar. While no appreciable enzyme activity (0.0%) was observed with Y126X mutation, no apparent decrease in FUCA1 mRNA level was seen with Y126X mutation. The expressed truncated Y126X protein was unstable and largely degraded. The delineation of the molecular defect could serve to complement future prenatal diagnosis for this family when necessary. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Shuan-Pei Lin and Jui-Hung Chang have contributed equally to this work.     

Six additional mutations in fucosidosis: three nonsense mutations and three frameshift mutations

The rare lysosomal storage disease, fucosidosis results froman almost complete deficiency of a-L-fucosldase (EC 3.2.1.51 [EC] ).We have identified six new potential disease causing mutationsdetected by PCR amplification and sequencing of all 8 exonsof the a-L-fucosidase gene FUCA1. (1) A C to T mutation (Q77X)in exon 1 of two Jewish - Italian siblings. This mutation waspresent in one allele and was found also In the mother who wasof Italian origin. (2) A C to A mutation (W382X) In exon 6 Inan Italian patient. This mutation was found In one allele andobliterates a unique Hphl site. (3) A C to A mutation (Y211X)in exon 3 in a Belgian patient. This mutation obliterates aunique Rsal site and was present in both alleles. (4) A homozygoussingle base (C) deletion In exon 2 In an Italian patient. Thisdeletion results In a frameshift mutation (P141fs) and obliteratesa unique Eael site. (5) A homozygous single base (C) deletionIn exon 5 In a Portuguese patient, which also results in a frameshiftmutation (S265fs). (6) A single base (A) deletion In exon 3In a Canadian - Indian patient, which also results In a frameshiftmutation (S216fs). The S216fs mutation was found In only oneallele; the mutation in the other allele is not yet known.     

α-L-fucosidase in cultured bone marrow fibroblasts from fucosidosis patients

Bone marrow fibroblasts were cultured from two patients with fucosidosis type 2, six control subjects, and three patients with other lysosomal disorders. Optimal conditions for measuring α-L-fucosidase activity in lysates of these cells with the fluorogenic substrate 4-methylumbelliferyl-α-L-fucoside were established. The pH profile of normal bone marrow fibroblasts showed three peaks and a shoulder of enzymatic activity, with maximum activity at pH 4.75. In cells derived from fucosidosis patients two peaks of apparent α-L-fucosidase activity were obtained; the pH optimum was 4.5. α-L-Fucosidase activity (mean ± SD) in the fucosidosis and control bone marrow fibroblasts was 2.5 and 312.4 ± 10.9 nmoles 4-methylumbelliferone per milligram protein per hour, respectively. A reduction in the apparent specific enzymatic activity in the fucosidosis cells was observed by using increasing concentrations of cellular protein in the assay system. Mixing experiments between normal and fucosidosis cells gave the expected activities. These findings indicate that cultured bone marrow fibroblasts can be used for the diagnosis and study of fucosidosis.     

Cultured fucosidosis fibroblasts: A simple technique demonstrating storage of tritiated-fucose labeled material

Incorporation of tritiated fucose into the acid-soluble and acid-insoluble fractions of cultured fucosidosis and control fibroblast monolayers was determined over a 10-day period. Fucosidosis cells incorporated significantly more label into the acid-soluble, but not the acid-insoluble fraction, than control cells. The ratio of acid-soluble to acid-insoluble radioactivity increased linearly from 1 to 10 days for the fucosidosis cells, indicating the storage of low molecular weight partial degradation products.