Canine GM2-Gangliosidosis Sandhoff Disease Associated with a 3-Base Pair Deletion in the HEXB Gene.

BACKGROUND: GM2-gangliosidosis is a fatal neurodegenerative lysosomal storage disease (LSD) caused by deficiency of either ß-hexosaminidase A (Hex-A) and ß-hexosaminidase B (Hex-B) together, or the GM2 activator protein. Clinical signs can be variable and are not pathognomonic for the specific, causal deficiency. OBJECTIVES: To characterize the phenotype and genotype of GM2-gangliosidosis disease in an affected dog. ANIMALS: One affected Shiba Inu and a clinically healthy dog. METHODS: Clinical and neurologic evaluation, brain magnetic resonance imaging (MRI), assays of lysosomal enzyme activities, and sequencing of all coding regions of HEXA, HEXB, and GM2A genes. RESULTS: A 14-month-old, female Shiba Inu presented with clinical signs resembling GM2-gangliosidosis in humans and GM1-gangliosidosis in the Shiba Inu. Magnetic resonance imaging (MRI) of the dog's brain indicated neurodegenerative disease, and evaluation of cerebrospinal fluid (CSF) identified storage granules in leukocytes. Lysosomal enzyme assays of plasma and leukocytes showed deficiencies of Hex-A and Hex-B activities in both tissues. Genetic analysis identified a homozygous, 3-base pair deletion in the HEXB gene (c.618-620delCCT). CONCLUSIONS AND CLINICAL IMPORTANCE: Clinical, biochemical, and molecular features are characterized in a Shiba Inu with GM2-gangliosidosis. The deletion of 3 adjacent base pairs in HEXB predicts the loss of a leucine residue at amino acid position 207 (p.Leu207del) supporting the hypothesis that GM2-gangliosidosis seen in this dog is the Sandhoff type. Because GM1-gangliosidosis also exists in this breed with almost identical clinical signs, genetic testing for both GM1- and GM2-gangliosidosis should be considered to make a definitive diagnosis.

Alkaline phosphatase: placental and tissue-nonspecific isoenzymes hydrolyze phosphoethanolamine, inorganic pyrophosphate, and pyridoxal 5'-phosphate. Substrate accumulation in carriers of hypophosphatasia corrects during pregnancy.

Hypophosphatasia features selective deficiency of activity of the tissue-nonspecific (liver/bone/kidney) alkaline phosphatase (ALP) isoenzyme (TNSALP); placental and intestinal ALP isoenzyme (PALP and IALP, respectively) activity is not reduced. Three phosphocompounds (phosphoethanolamine [PEA], inorganic pyrophosphate [PPi], and pyridoxal 5'-phosphate [PLP]) accumulate endogenously and appear, therefore, to be natural substrates for TNSALP. Carriers for hypophosphatasia may have decreased serum ALP activity and elevated substrate levels. To test whether human PALP and TNSALP are physiologically active toward the same substrates, we studied PEA, PPi, and PLP levels during and after pregnancy in three women who are carriers for hypophosphatasia. Hypophosphatasemia corrected during the third trimester because of PALP in maternal blood. Blood or urine concentrations of PEA, PPi, and PLP diminished substantially during that time. After childbirth, maternal circulating levels of PALP decreased, and PEA, PPi, and PLP levels abruptly increased. In serum, unremarkable concentrations of IALP and low levels of TNSALP did not change during the study period. We conclude that PALP, like TNSALP, is physiologically active toward PEA, PPi, and PLP in humans. We speculate from molecular/crystallographic information, indicating significant similarity of structure of the substrate-binding site of ALPs throughout nature, that all ALP isoenzymes recognize these same three phosphocompound substrates.

Optimized transduction of canine paediatric CD34(+) cells using an MSCV-based bicistronic vector.

We have used a murine MSCV-based bicistronic retroviral vector, containing the common gamma chain (gammac) and enhanced green fluorescent protein (EGFP) cDNAs, to optimize retroviral transduction of canine cells, including an adherent canine thymus fibroblast cell line, Cf2Th, as well as normal canine CD34(+) bone marrow (BM) cells. Both canine cell types were shown to express Ram-1 (the amphotropic retroviral receptor) mRNA. Supernatants containing infectious viruses were produced using both stable (PA317) and transient (Phoenix cells) amphotropic virus producer cell lines. Centrifugation (spinfection) combined with the addition of polybrene produced the highest transduction efficiencies, infecting approximately 75% of Cf2Th cells. An average of 11% of highly enriched canine CD34(+) cells could be transduced in a protocol that utilized spinfection and plates coated with the fibronectin fragment CH-296 (Retronectin). Indirect assays showed the vector-encoded canine gammac cDNA produced a gammac protein that was expressed on the cell surface of transduced cells. This strategy may result in the transduction of sufficient numbers of CD34(+) BM cells to make the treatment of canine X-linked severe combined immunodeficiency and other canine genetic diseases feasible.

Aberrant properties of alkaline phosphatase in patient fibroblasts correlate with clinical expressivity in severe forms of hypophosphatasia.

The markedly variable clinical expressivity of hypophosphatasia was explored by examining biochemical properties of alkaline phosphatase (ALP) in fibroblasts cultured from 16 patients with severe autosomal recessive forms of this metabolic bone disease. Outcome ranged from death in utero to survival into childhood. Mean ALP activity in patients was 4.3% of controls. Gel filtration analysis indicated a mixture of dimeric and tetrameric ALP in both subject groups. Control cells produced levels of bone ALP cross-reacting material that correlated strongly with ALP activity. Patient bone ALP cross-reacting material levels averaged 41% of the control mean with a wide range of individual values that did not correlate with ALP activity. Control ALP activity was stable in 3% SDS and during electrodialysis. Patient ALP activity was generally unstable under both conditions but with a considerable range of individual values. Fibroblast ALP from every patient exhibited some aberrancy in physicochemical and immunoreactive properties. These data strongly correlated (r = 0.95) with clinical severity. There appeared to be specific associations of tissue nonspecific (bone/liver/kidney isoenzyme) ALP (TNSALP) gene mutations with aberrant enzyme properties and disease severity. We conclude that a spectrum of aberrant biochemical properties of the TNSALP enzyme, caused by different combinations of TNSALP gene missense mutations, reflects the variable clinical expressivity of hypophosphatasia.

Hypophosphatasia: levels of bone alkaline phosphatase immunoreactivity in serum reflect disease severity.

Hypophosphatasia is a rare metabolic bone disease characterized biochemically by deficient enzymatic activity of the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP). All isoforms of TNSALP (e.g. bone and liver TNSALP), apparently differing only by posttranslational modifications, are affected. To explore the biochemical basis and extremely variable severity of hypophosphatasia, we used 2-site immunoradiometric assays that quantify in serum either 1) bone TNSALP (iBALP) alone, or 2) both bone and liver TNSALP (iTNSALP). Sera from 33 patients in 26 kindreds reflecting all 6 clinical types of the disorder were studied. In every patient, except the two with pseudohypophosphatasia, serum iBALP and iTNSALP levels were decreased compared to age-appropriate control ranges. The magnitude of the decrease in iBALP and iTNSALP levels correlated with clinical severity. The mean ratio of iBALP or iTNSALP level to total ALP activity was unremarkable for the mild childhood, adult, and odonto forms of hypophosphatasia. For the severe perinatal and infantile forms, the mean ratios were low. If our finding of reduced iBALP levels in the circulation reflects a similar abnormality in the skeleton, the pathogenesis of hypophosphatasia could involve decreased amounts of extracellular TNSALP in bone and cartilage. How TNSALP gene mutations affect the enzyme and cause skeletal disease requires further investigation.

A cDNA encoding canine muscle-type phosphofructokinase.

The canine muscle-type-phosphofructokinase-encoding gene (M-PFK) was sequenced by using a combination of cDNA cloning and RT-PCR amplification. The canine M-PFK sequence shares 88 and 90% identity with rabbit and human M-PFK, respectively. The canine ORF was determined to be 6-bp longer than either human or rabbit M-PFK due to a 6-bp insertion at the end of exon 13.

Canine X-linked severe combined immunodeficiency. A model for investigating the requirement for the common gamma chain (gamma c) in human lymphocyte development and function.

Our laboratory has identified and characterized an X-linked severe combined immunodeficiency (XSCID) in dogs that is due to mutations in the common gamma (gamma c) subunit of the interleukin-2 (IL2), IL4, IL7, IL9, and IL15 receptors. Canine XSCID, unlike genetically engineered gamma c-deficient mice, has a clinical and immunologic phenotype virtually identical to human XSCID. It appears that species-specific differences exist in the role of the gamma c and its associated cytokines in mice compared to their role in humans and dogs, suggesting gamma c-deficient dogs may be a more relevant model for studying the role of the gamma c in humans. We are utilizing this model for a variety of studies to address: 1. Fundamental questions concerning the role of the gamma c in cytokine regulation and lymphocyte development. 2. The pathogenesis of XSCID. 3. Strategies for improving bone marrow transplantation outcome. 4. Development and evaluation of strategies for gene therapy. 5. Human hematopoietic stem cell development.

Mild autosomal dominant hypophosphatasia: in utero presentation in two families.

We describe four pregnancies in two families in which mild hypophosphatasia, apparently transmitted as an autosomal dominant trait, manifested in utero as severe long bone bowing. Postnatally, there was spontaneous improvement of the skeletal defects. Recognition of this presentation for hypophosphatasia by family investigation and assessment of the fetal skeleton for degree of ossification and chest size using ultrasonography is important. The prognosis for this condition is considerably better than for more severe forms of hypophosphatasia and for many other disorders that cause skeletal defects with long bone bowing in utero.

Canine X-linked severe combined immunodeficiency.

Canine X-linked severe combined immunodeficiency (XSCID) is due to mutations in the common gamma (gamma c) subunit of the IL-2, IL-4, IL-7, IL-9 and IL-15 receptors. The most striking clinical feature is a failure to thrive or 'stunted' growth. Recurrent or chronic infections begin at the time of decline of maternal antibody, usually between six and eight weeks of age. Affected dogs rarely survive past three to four months of age. The major pathologic feature of canine XSCID is a small, dysplastic thymus. Grossly identifiable lymph nodes, tonsils, and Peyer's patches are absent in XSCID dogs. During the neonatal period, XSCID dogs have few, if any, peripheral T cells and increased number of peripheral B cells. Some XSCID dogs do develop phenotypically mature, nonfunctional T cells with age, however, the absolute number of peripheral T cells remain significantly decreased compared to age-matched normal dogs. An interesting finding is that as soon as T cells begin to appear in XSCID dogs they rapidly switch from a CD45RA+ (naive) phenotype to a CD45RA- (activated or memory phenotype). One of the characteristic findings in XSCID dogs is an absent or markedly depressed blastogenic response of T cells in response to stimulation through the T cell receptor and when the necessary second messengers for cellular proliferation are directly provided that by-pass signals delivered through ligand-receptor interaction. The proliferative defect is due to the inability of T cells to express a functional IL-2 receptor. Canine XSCID B cells do not proliferate following stimulation with T cell-dependent B cell mitogens, however, they proliferate normally in response to T cell-independent B cell mitogens. Canine XSCID B cells are capable of producing IgM but are incapable of class-switching to IgG antibody production following immunization with the T cell-dependent neoantigen, bacteriophage phiX174. The number of thymocytes in the XSCID thymus is approximately 0.3% of the thymocytes present in the thymus of age-matched normal dogs. The proportion of CD4-CD8- thymocytes in XSCID dogs is increased 3.5-fold and the CD4+CD8+ population is decreased 2.3-fold. These findings demonstrate that (1) a functional gamma c is required for normal B and T cell function, (2) early T cell development is highly dependent upon a functional gamma c, and (3) B cell development can occur through a gamma c-independent pathway.

Bone marrow transplantation for canine X-linked severe combined immunodeficiency.

Canine X-linked severe combined immunodeficiency (XSCID) is due to mutations in the common gamma chain which is a subunit of the receptors of IL-2, IL-4, IL-7, IL-9 and IL-15. Bone marrow transplantation (BMT) of human XSCID patients without pretransplant conditioning (cytoablation) results in engraftment of donor T-cells and reconstitution of T-cell function but engraftment of few, if any, donor B cells with resultant poor reconstitution of humoral immune function. In this study, we show that XSCID dogs can be transplanted with allogeneic bone marrow cells resulting in engraftment of both donor B and T cells and reconstitution of full systemic immune function including normal humoral immune function without the need for cytoablation.

A single nucleotide insertion in the canine interleukin-2 receptor gamma chain results in X-linked severe combined immunodeficiency disease.

The immunologic and genetic analysis of a 14-week-old-male cardigan Welsh corgi puppy that presented with failure to thrive, diarrhea, and intermittent vomiting are described. The lack of palpable lymph nodes, the premature death of a male sibling, and similar clinical signs in a male cousin suggested that a primary immunodeficiency disease might be responsible for his poor clinical condition. Quantitation of serum immunoglobulins revealed low concentrations of IgG and undetectable IgA, yet normal concentrations of IgM. A complete blood cell count showed a slight anemia and lymphopenia. Although the peripheral blood contained a normal percentage of T cells, with an increased CD4:CD8 ratio, they were unable to proliferate in response to phytohemagglutinin (PHA) and/or interleukin 2 (IL-2). Furthermore, following PHA activation, the peripheral blood lymphocytes (PBL) demonstrated a nearly complete lack of IL-2 binding. All of these laboratory findings were identical with our previous findings from dogs with X-linked severe combined immunodeficiency (XSCID) that is due to a mutation in their IL-2 receptor gamma (IL-2R gamma) chain. Examination of the corgi's IL-2R gamma cDNA revealed an insertion of a cytosine following nucleotide 582, resulting in a premature stop codon prior to the transmembrane domain. The insertion also created an EcoO109 restriction enzyme site that enabled us to detect the mutation in the patient's genomic DNA. This new mutation in the IL-2R gamma chain discovered in a cardigan Welsh corgi puppy results in XSCID with similar immunologic abnormalities as observed in dogs with the same disease resulting from a different IL-2R gamma chain mutation.

Sry-negative XX sex reversal in a family of Norwegian Elkhounds.

Two related female Norwegian Elkhounds were evaluated at 6 and 8 months of age for enlarged clitori. Both had a 78 XX karyotype. Histology of their internal reproductive tracts demonstrated 1 to be an XX true hermaphrodite with bilateral ovotestes and the other to be an XX male with bilateral aspermatogenic testes. Polymerase chain reaction-based tests of genomic DNA showed that both dogs lacked Sry, the testis-determining gene. Pedigree analysis was consistent with an autosomal recessive mode of inheritance, as has been reported in the American Cocker Spaniel and the German Shorthaired Pointer. This is the 1st reported case of familial Sry-negative XX sex reversal in the Norwegian Elkhound. A summary of 34 previously unreported cases of dogs with masculinized external genitalia and a normal 78 XX karyotype seen from 1980 to 1997 is given.

SLC3A1 and SLC7A9 mutations in autosomal recessive or dominant canine cystinuria: a new classification system.

BACKGROUND: Cystinuria, one of the first recognized inborn errors of metabolism, has been reported in many dog breeds. HYPOTHESIS/OBJECTIVES: To determine urinary cystine concentrations, inheritance, and mutations in the SLC3A1 and SLC7A9 genes associated with cystinuria in 3 breeds. ANIMALS: Mixed and purebred Labrador Retrievers (n = 6), Australian Cattle Dogs (6), Miniature Pinschers (4), and 1 mixed breed dog with cystine urolithiasis, relatives and control dogs. METHODS: Urinary cystinuria and aminoaciduria was assessed and exons of the SLC3A1 and SLC7A9 genes were sequenced from genomic DNA. RESULTS: In each breed, male and female dogs, independent of neuter status, were found to form calculi. A frameshift mutation in SLC3A1 (c.350delG) resulting in a premature stop codon was identified in autosomal-recessive (AR) cystinuria in Labrador Retrievers and mixed breed dogs. A 6 bp deletion (c.1095_1100del) removing 2 threonines in SLC3A1 was found in autosomal-dominant (AD) cystinuria with a more severe phenotype in homozygous than in heterozygous Australian Cattle Dogs. A missense mutation in SLC7A9 (c.964G>A) was discovered in AD cystinuria in Miniature Pinschers with only heterozygous affected dogs observed to date. Breed-specific DNA tests were developed, but the prevalence of each mutation remains unknown. CONCLUSIONS AND CLINICAL IMPORTANCE: These studies describe the first AD inheritance and the first putative SLC7A9 mutation to cause cystinuria in dogs and expand our understanding of this phenotypically and genetically heterogeneous disease, leading to a new classification system for canine cystinuria and better therapeutic management and genetic control in these breeds.

Missense mutations of the tissue-nonspecific alkaline phosphatase gene in hypophosphatasia.

Hypophosphatasia is an inborn error of metabolism that is characterized clinically by defective bone mineralization and biochemically by deficient activity of the tissue-nonspecific isoenzyme of alkaline phosphatase (TNSALP) in serum and in tissues. Clinical severity is extremely variable, ranging from death in utero to pathologic fractures first presenting in adulthood. Severe forms of the disease are inherited in an autosomal recessive fashion; the modes of transmission of mild forms are uncertain. Deficiency of TNSALP activity in this condition suggests that mutations in the TNSALP "candidate" gene are the primary defects. This hypothesis was supported in 1988 by the demonstration, in one inbred infant, that an identical missense mutation in both alleles of the gene encoding TNSALP caused lethal hypophosphatasia. Here we summarize the work leading to that discovery and discuss the recent identification of additional missense mutations in the TNSALP gene associated with the entire clinical spectrum of hypophosphatasia.

Infantile hypophosphatasia: successful prenatal assessment by testing for tissue-non-specific alkaline phosphatase isoenzyme gene mutations.

We successfully assessed a fetus at risk for lethal infantile hypophosphatasia using amniocyte DNA and allele-specific oligonucleotide (ASO) probes for two missense mutations in the tissue-non-specific alkaline phosphatase isoenzyme (TNSALP) gene. The nucleotide changes had been discovered in a sister who died at 8 months of age from this inborn error of metabolism. The mother was known to carry the 747 (cDNA) G-->A transition, whereas her husband and 5-year-old daughter, who were also healthy, carried the 1309 A-->T transversion. Amniocytes, obtained at 16 weeks' gestation, provided genomic DNA for polymerase chain reaction (PCR) amplification of the appropriate TNSALP gene exons. ASO hybridization revealed absence of the 747A mutation and presence of the 1309T base changes in the fetus, indicating a carrier for hypophosphatasia. At 8 months of age, the offspring was in excellent health and without any radiological evidence of skeletal disease. His serum ALP activity and plasma pyridoxal 5'-phosphate level were decreased and increased, respectively, at levels consistent with the prenatal assessment. The ASO studies were confirmed postnatally using peripheral blood leukocyte DNA. This is the first application of direct mutational analysis to assess a fetus at risk for hypophosphatasia.

Identification of a retrovirus-like repetitive element in human DNA.

We describe a 5- to 6-kilobase-pair repetitive family in human DNA. One member of this family is linked to the beta-globin gene cluster and is close to the 3' breakpoints of three different naturally occurring deletions involving this gene cluster. Sequence analysis indicates that this element includes terminal direct repeats of 415 base pairs that exhibit the features of long terminal repeats (LTRs) of retroviruses. A potential histidine tRNA primer binding site occurs just 3' to the 5' direct repeat. This retrovirus-like element interrupts a member of the Kpn I family of repeated DNA and is bracketed by a 5-base-pair directly repeated sequence. When attempts are made to clone the element in bacteriophage, homologous recombination between the LTR-like sequences is very frequently observed. Copy number estimates by two methods indicate that the element is repeated 800-1000 times in the human genome. We term this Homo sapiens family of retrovirus-like elements having a histidine tRNA primer binding site the hsRTVL-H family.

A retrovirus-like element occurs between the 3' breakpoints of two large deletions in the human beta-globin gene cluster.

We have identified a novel repetitive family in human DNA. One member of this family is found downstream of the beta-globin gene cluster between the 3' breakpoints of the deletions associated with Chinese G gamma + (A gamma delta beta)O thalassemia and HPFH-2. This repetitive element is approximately 6 kbp in length and includes terminal direct repeats of 415 bp. Numerous DNA sequence features of the direct repeats (LTRs) and their flanking regions indicate that the element is a retrovirus-like structure. The most striking of these features is the presence of a histidine tRNA binding site just 3' to the 5' LTR. Accordingly the element is named RTVL-H (retrovirus-like element-histidine). The LTRs of the RTVL-H element are not strongly homologous to the LTRs of any previously described mammalian retrovirus or retrovirus-like element. Copy number estimates suggest that there are approximately 1000 RTVL-H elements in the human genome. The element found 3' (greater than 60 kbp) to the beta-globin gene appears to be a stable part of the normal genome. This retrovirus-like element is brought close to the fetal gamma-globin locus by the Chinese thalassemia deletion but is deleted in HPFH-1 and HPFH-2.