Mannose 6-phosphate receptor-mediated uptake is defective in acid sphingomyelinase-deficient macrophages: implications for Niemann-Pick disease enzyme replacement therapy

Progressive accumulation of lipid-laden macrophages is a hallmark of the acid sphingomyelinase (ASM)-deficient forms of Niemann-Pick disease (i.e. Types A and B NPD). To investigate the mechanisms underlying enzyme replacement therapy for this disorder, we studied the uptake of recombinant, human ASM (rhASM) by alveolar macrophages from ASM knock-out (ASMKO) mice. The recombinant enzyme used for these studies was produced in Chinese hamster ovary cells and contained complex type, N-linked oligosaccharides. Binding of radiolabeled, rhASM to the ASMKO macrophages was enhanced as compared with normal macrophages, consistent with their larger size and increased surface area. However, internalization of the enzyme by the ASMKO cells was markedly reduced when compared with normal cells. Studies using receptor-specific ligands to inhibit enzyme uptake revealed that in normal cells rhASM was taken up by a combination of mannose and mannose 6-phosphate receptors (MR and M6PR, respectively), whereas in the ASMKO cells the M6PR had a minimal role in rhASM uptake. Expression of M6PR mRNA was normal in the ASMKO cells, although Western blotting revealed more receptors in these cells when compared with normal. We therefore hypothesized that lipid accumulation in ASMKO macrophages led to abnormalities in M6PR trafficking and/or degradation, resulting in reduced enzyme uptake. Consistent with this hypothesis, we also found that, when rhASM was modified to expose terminal mannose residues and target mannose receptors, the uptake of this modified enzyme form by ASMKO cells was approximately 10-fold greater when compared with the "complex" type rhASM. These findings have important implications for NPD enzyme replacement therapy, particularly in the lung.     

Alveolar lipoproteinosis in an acid sphingomyelinase-deficient mouse model of Niemann-Pick disease

Types A and B Niemann-Pick disease (NPD) are lipid storage disorders caused by the deficient activity of acid sphingomyelinase (ASM). In humans, NPD is associated with the dysfunction of numerous organs including the lung. Gene targeting of the ASM gene in transgenic mice produced an animal model with features typical of NPD, including pulmonary inflammation. To assess mechanisms by which ASM perturbed lung function, we studied lung morphology, surfactant content, and metabolism in ASM-deficient mice in vivo. Pulmonary inflammation, with increased cellular infiltrates and the accumulation of alveolar material, was associated with alterations in surfactant content. Saturated phosphatidylcholine (SatPC) content was increased twofold, and sphingomyelin content was increased 5.5-fold in lungs of the ASM knockout (ASMKO) mice. Additional sphingomyelin enhanced the sensitivity of surfactant inhibition by plasma proteins. Clearance of SatPC from the lungs of ASMKO mice was decreased. Catabolism of SatPC by alveolar macrophages from the ASMKO mouse was significantly decreased, likely accounting for decreased pulmonary SatPC in vivo. In summary, ASM is required for normal surfactant catabolism by alveolar macrophages in vivo. Alterations in surfactant composition, including increased sphingomyelin content, contributed to the abnormal surfactant function observed in the ASM-deficient mouse.     

Null alleles at the Huntington disease locus: implications for diagnostics and CAG repeat instability

PCR amplification of the CAG repeat in exon 1 of the IT15 gene is routinely undertaken to confirm a clinical diagnosis of Huntington disease (HD) and to provide predictive testing for at-risk relatives of affected individuals. Our studies have detected null alleles on the chromosome carrying the expanded repeat in three of 91 apparently unrelated HD families. Sequence analysis of these alleles has revealed the same mutation event, leading to the juxtaposition of uninterrupted CAG and CCG repeats. These data suggest that a mutation-prone region exists in the IT15 gene bounded by the CAG and CCG repeats and that caution should be exercised in designing primers that anneal to the region bounded by these repeats. Two of the HD families segregated null alleles with expanded uninterrupted CAG repeats at the lower end of the zone of reduced penetrance. The expanded repeats are meiotically unstable in these families, although this instability is within a small range of repeat lengths. The haplotypes of the disease-causing chromosomes in these two families differ, only one of which is similar to that reported previously as being specific for new HD mutations. Finally, no apparent mitotic instability of the uninterrupted CAG repeat was observed in the brain of one of the HD individuals.     

Compound heterozygosity at the sphingomyelin phosphodiesterase-1 (SMPD1) gene is associated with low HDL cholesterol

Type A and B forms of Niemann-Pick disease (NPD) are lipid storage disorders caused by deficient activity of the enzyme acid sphingomyelinase (aSMase) and the resulting accumulation of sphingomyelin in tissues. In the present study, we investigated two family members who had been diagnosed with Type B NPD and who had a severe decrease in plasma high density lipoprotein cholesterol (HDL-C). The proband (a 48-year-old male) had an HDL-C of 0.30 mmol/l (12 mg/dl) and his sister had values of 0.45 mmol/l (17 mg/dl) with severe premature coronary artery disease (CAD). Hypertriglyceridemia was found in both cases. aSMase activity measured in skin fibroblasts appeared markedly depressed. The SMPD1 gene, coding for aSMase, was sequenced in affected subjects and all family members. Compound heterozygosity (DeltaR608 and R441X) was identified in both affected patients. Carriers of the DeltaR608 mutation tended to have moderately to severe decreased HDL-C levels, whereas carriers of the R441X mutation, although present only in young subjects (<20 years of age) had normal HDL-C levels. To investigate the cause of the low HDL-C level in these patients, we studied apoA-I-mediated cellular cholesterol efflux in fibroblasts. Unlike patients with Tangier disease, cholesterol efflux was found to be normal under the experimental conditions used in the present study. On the other hand, we observed a significant increase in the free cholesterol:esterified cholesterol ratio in HDL fraction from these patients and a decrease in endogenous lecithin-cholesterol acyltransferase (LCAT) activity, as determined by the fractional esterification rate. Taken together, these results suggest that (1) compound heterozygosity at the SMPD1 gene causes a severe decrease in aSMase activity and in HDL-C and increases the risk of CAD, (2) this lipoprotein abnormality is not attributable to defective cellular cholesterol efflux, (3) abnormal HDL composition might cause a decrease in LCAT activity and a lack of HDL maturation.     

Metabolic heritability at birth: implications for chronic disease research

Recent genome-wide association studies of the adult human metabolome have identified genetic variants associated with relative levels of several acylcarnitines, which are important clinical correlates for chronic conditions such as type 2 diabetes and obesity. We have previously shown that these same metabolite levels are highly heritable at birth; however, no studies to our knowledge have examined genetic associations with these metabolites measured at birth. Here, we examine, in 743 newborns, 58 single nucleotide polymorphisms (SNPs) in 11 candidate genes previously associated with differing relative levels of short-chain acylcarnitines in adults. Six SNPs (rs2066938, rs3916, rs3794215, rs555404, rs558314, rs1799958) in the short-chain acyl-CoA dehydrogenase gene (ACADS) were associated with neonatal C4 levels. Most significant was the G allele of rs2066938, which was associated with significantly higher levels of C4 (P = 1.5 × 10^?29). This SNP explains 25 % of the variation in neonatal C4 levels, which is similar to the variation previously reported in adult C4 levels. There were also significant (P < 1 × 10^?4) associations between neonatal levels of C5-OH and SNPs in the solute carrier family 22 genes (SLC22A4 and SLC22A5) and the 3-methylcrotonyl-CoA carboxylase 1 gene (MCCC1). We have replicated, in newborns, SNP associations between metabolic traits and the ACADS and SLC22A4 genes observed in adults. This research has important implications not only for the identification of rare inborn errors of metabolism but also for personalized medicine and early detection of later life risks for chronic conditions.     

VEGF at the neurovascular interface: therapeutic implications for motor neuron disease

VEGF was discovered almost 25 years ago, and its angiogenic activity has been extensively studied ever since. Accumulating evidence indicates, however, that VEGF also has direct effects on neuronal cells. VEGF exerts neuroprotective effects on various cultured neurons of the central nervous system. In vivo, VEGF controls the correct migration of facial branchiomotor neurons in the developing hindbrain and stimulates the proliferation of neural stem cells in enriched environments and after cerebral ischemia. Transgenic mice expressing reduced levels of VEGF develop late-onset motor neuron degeneration, reminiscent of amyotrophic lateral sclerosis (ALS), whereas reduced levels of VEGF have been implicated in a polyglutamine-induced model of motor neuron degeneration. Recent data further reveal that intracerebroventricular delivery of recombinant VEGF protein delays disease onset and prolongs survival of ALS rats, whereas intramuscular administration of a VEGF-expressing lentiviral vector increases the life expectancy of ALS mice by as much as 30%. Deciphering the precise role of VEGF at the neurovascular interface promises to uncover new insights into the development and pathology of the nervous system, helpful to design novel strategies to treat (motor) neurodegenerative disorders.     

New complexities at the Qa-1 locus

Mouse strain and tissue distribution analyses indicate that the new antiserum A anti-A-Tla ^b recognizes the cell-surface product governed by the previously serologically undetectable Qa-I ^b allele. This cell-surface product has therefore been called Qa-1.2. Three levels of anti-Qa-1.2 cytotoxicity in the presence of complement have been observed: high, intermediate, and zero lysis. In general, high levels of lysis correlate with the presence of the Qa-1 b allele, while zero levels of lysis correlate with the presence of the Qa-1 ^aallele. The A.CA strain reacts with both anti-Qa-1.1 and anti-Qa-1.2 and may possess a third allele, Qa-1 ^d. Several strains including B6-H-2 ^k react in an intermediate fashion. Recombinant strain analyses indicate that this intermediate reaction may be due to modifying genes within the H-2D region.     

Short tandem-repeat polymorphism/alu haplotype variation at the PLAT locus: implications for modern human origins

Two dinucleotide short tandem-repeat polymorphisms (STRPs) and a polymorphic Alu element spanning a 22-kb region of the PLAT locus on chromosome 8p12-q11.2 were typed in 1,287-1,420 individuals originating from 30 geographically diverse human populations, as well as in 29 great apes. These data were analyzed as haplotypes consisting of each of the dinucleotide repeats and the flanking Alu insertion/deletion polymorphism. The global pattern of STRP/Alu haplotype variation and linkage disequilibrium (LD) is informative for the reconstruction of human evolutionary history. Sub-Saharan African populations have high levels of haplotype diversity within and between populations, relative to non-Africans, and have highly divergent patterns of LD. Non-African populations have both a subset of the haplotype diversity present in Africa and a distinct pattern of LD. The pattern of haplotype variation and LD observed at the PLAT locus suggests a recent common ancestry of non-African populations, from a small population originating in eastern Africa. These data indicate that, throughout much of modern human history, sub-Saharan Africa has maintained both a large effective population size and a high level of population substructure. Additionally, Papua New Guinean and Micronesian populations have rare haplotypes observed otherwise only in African populations, suggesting ancient gene flow from Africa into Papua New Guinea, as well as gene flow between Melanesian and Micronesian populations.     

Mice triallelic for the Ig heavy chain locus: implications for VHDJH recombination

V(H)DJ(H) recombination has been extensively studied in mice carrying an Ig heavy chain rearranged transgene. In most models, inhibition of endogenous Ig rearrangement occurs, consistently with the feedback model of IgH recombination. Nonetheless, an incomplete IgH allelic exclusion is a recurrent observation in these animals. Furthermore, transgene expression in ontogeny is likely to start before somatic recombination, thus limiting the use of Ig-transgenic mice to access the dynamics of V(H)DJ(H) recombination. As an alternative approach, we challenged the regulation of somatic recombination with the introduction of an extra IgH locus in germline configuration. This was achieved by reconstitution of RAG2(-/-) mice with fetal liver cells trisomic for chromosome 12 (Ts12). We found that all three alleles can recombine and that the ratio of Ig allotype-expressing B cells follows the allotypic ratio in trisomic cells. Although these cells are able to rearrange the three alleles, the levels of Ig phenotypic allelic exclusion are not altered when compared with euploid cells. Likewise, we find that most VDJ rearrangements of the silenced allele are unable to encode a functional mu-chain, indicating that the majority of these cells are also genetically excluded. These results provide additional support for the feedback model of allelic exclusion.     

Clinal variation for amino acid polymorphisms at the Pgm locus in Drosophila melanogaster

Clinal variation is common for enzymes in the glycolytic pathway for Drosophila melanogaster and is generally accepted as an adaptive response to different climates. Although the enzyme phosphoglucomutase (PGM) possesses several allozyme polymorphisms, it is unique in that it had been reported to show no clinal variation. Our recent DNA sequence investigation of Pgm found extensive cryptic amino acid polymorphism segregating with the allozyme alleles. In this study, we characterize the geographic variation of Pgm amino acid polymorphisms at the nucleotide level along a latitudinal cline in the eastern United States. A survey of 15 SNPs across the Pgm gene finds significant clinal differentiation for the allozyme polymorphisms as well as for many of the cryptic amino acid polymorphisms. A test of independence shows that pervasive linkage disequilibrium across this gene region can explain many of the amino acid clines. A single Pgm haplotype defined by two amino acid polymorphisms shows the strongest correlation with latitude and the steepest change in allele frequency across the cline. We propose that clinal selection at Pgm may in part explain the extensive amino acid polymorphism at this locus and is consistent with a multilocus response to selection in the glycolytic pathway.