In utero hematopoietic stem cell transplantation: a caprine model for prenatal therapy in inherited metabolic diseases.

OBJECTIVES: We explored the feasibility and efficacy of in utero hematopoietic stem cell transplantation in the caprine animal model system with the objectives of determining procedures for transplantation and establishing methods for detecting engraftment. METHODS: Male fetal liver hematopoietic stem cells were injected into female fetuses during the immunotolerant period, using either hysterotomy or ultrasound-guided injections. RESULTS: The rate of fetal death was much lower for the ultrasound-guided injections. Donor cells were observed in the peritoneal fluid of 4 fetuses 3 days after injection, but no donor cells were detected in tissues at longer time periods. CONCLUSIONS: Ultrasound-guided injection of hematopoietic stem cells into the abdomen of a developing fetus is safe and feasible. The parameters required for successful engraftment have not yet been identified.

Identification of a bovine beta-mannosidosis mutation and detection of two beta-mannosidase pseudogenes.

Beta-mannosidase deficiency results in beta-mannosidosis, a severe neurodegenerative lysosomal storage disease identified in cattle, goats, and humans. To more fully understand the molecular pathology of this disease, the mutation associated with bovine beta-mannosidosis was identified by sequence analysis of cDNA from an affected calf. A transition mutation of G to A at position 2574 of the cDNA coding sequence creates a premature stop codon near the 3' end of the protein coding region. To aid commercial breeders of Salers cattle, a PCR-based test was developed to detect the mutation for beta-mannosidosis carrier screening. Application of this test also revealed the presence of two beta-mannosidase pseudogenes. Portions of the pseudogenes were amplified with allele-specific primers and then sequenced. One pseudogene was highly homologous (>99% sequence identity) to the expressed cDNA sequence over the 1292 bp that were sequenced, while the other showed more divergence (83% sequence identity) in the 477 bp that were sequenced. Both are processed pseudogenes that are not expressed. The severity of the bovine beta-mannosidosis phenotype suggests that the 22 C-terminal amino acids of beta-mannosidase play an important role in the function of this enzyme.

Human beta-mannosidase cDNA characterization and first identification of a mutation associated with human beta-mannosidosis.

Human beta-mannosidosis is an autosomal recessive, lysosomal storage disease caused by a deficiency of the enzyme beta-mannosidase. Unlike the severe clinical manifestation of the disease in ruminants, in which it leads to neonatal death, the human disease phenotype is generally milder. In addition, the phenotypic manifestation among the reported cases of human beta-mannosidosis is variable, even among members of the same family. To understand the molecular basis of the human disease and the mechanisms for such clinical variability, we sequenced the entire coding region of the human beta-mannosidase gene using a combination of cDNA library screening, RT-PCR and 5' rapid amplification of cDNA ends (RACE). The composite cDNA is 3293 nt, consisting of an 87 nt 5'-untranslated region, 2640 nt coding region and 566 nt 3'-untranslated region. The gene was localized to human chromosome 4q22-25. Analysis of a multiple tissue northern blot demonstrated a single 3.7 kb transcript. Mutation analysis of a Czech gypsy family with two siblings differently affected with beta-mannosidosis demonstrated a homozygous A-->G transition 2 bp upstream of a splice acceptor site. The associated cryptic splice site activation and exon skipping caused by this mutation resulted in two abnormally spliced mutant mRNA species in both siblings.

Caprine beta-mannosidase: sequencing and characterization of the cDNA and identification of the molecular defect of caprine beta-mannosidosis.

The complete sequence of the caprine beta-mannosidase cDNA coding region has been determined, and a mutation that is associated with caprine beta-mannosidosis has been identified. Reverse transcriptase-polymerase chain reactions were performed using primers based on bovine and, later, goat cDNA sequences to produce an overlapping series of amplicons covering the entire coding region. The composite cDNA codes for an 879-amino-acid peptide that has four potential N-glycosylation sites. Comparison of the caprine and bovine cDNAs reveals that 96.3% of the nucleotides and 95.2% of the deduced amino acids are identical. A single-base deletion at position 1398 of the coding sequence was identified in the cDNA isolated from a goat affected with beta-mannosidosis. This deletion results in a shift in the reading frame and a premature termination of translation, yielding a deduced peptide of 481 amino acids. An assay, developed to determine the presence or absence of this mutation, confirmed that animals affected with beta-mannosidosis were homozygous for the mutation and that obligate carriers in a caprine beta-mannosidosis colony were heterozygous. This assay accurately distinguished between mutation carrier and noncarrier goats and was used for prenatal diagnosis using DNA collected from fetal fluids. The assay also confirmed chimerism in a goat with an atypically mild beta-mannosidosis phenotype. Thus, this application enables assessment of the efficacy of engraftment of hematopoietic stem cells after prenatal transfer from donor sources.

Cloning and sequence analysis of caprine N-acetylglucosamine 6-sulfatase cDNA.

Mucopolysaccharidosis IIID results from the deficiency of N-acetylglucosamine 6-sulfatase activity. A Nubian goat with this lysosomal storage disease has been identified. As a first step in developing this animal model for testing treatment methods, we cloned and sequenced the caprine N-acetylglucosamine 6-sulfatase cDNA coding region. Overall there is 88% nucleotide homology between the goat and human sequence and 94% homology of the deduced amino acid sequence. The human and two ruminant species differ by the presence of an imperfect trinucleotide (CCG) repeat in the ruminant signal sequence.

Molecular cloning and characterization of bovine beta-mannosidase.

Deficiency of lysosomal beta-mannosidase activity results in a severe neurodegenerative disease in goats and cattle and a relatively milder phenotype in humans. A cDNA coding for the entire beta-mannosidase protein is described. Mixed oligonucleotides derived from bovine beta-mannosidase peptide sequences were used to screen a bovine thyroid cDNA library. Clones covering about 80% of the C-terminal region were recovered. The missing 5'-region was obtained using the technique of 5'-rapid amplification of cDNA ends. The composite cDNA contains 3852 nucleotides, encoding 879 amino acids. The N-terminal methionine is followed by 16 amino acids displaying the characteristics of a typical signal peptide sequence. The deduced amino acid sequence is colinear with all peptide sequences determined by protein microsequencing. Northern blot analysis demonstrates a single 4.2-kilobase transcript in various tissues from both normal and affected goats and calves. The mRNA level is decreased in tissues of affected beta-mannosidosis animals. The gene encoding beta-mannosidase is localized to human chromosome 4 as shown by Southern analysis of rodent/human somatic cell hybrids. This is the first report of cloning of lysosomal beta-mannosidase.