Molecular Genetics Sanfilippo A

The gene encoding HNS is situated on chromosome 17q25.3, contains eight exons and extends over 11 kb of DNA. It produces a mRNA transcript of ~3.1 kb, which encodes a protein of 502 amino acids with a predicted molecular mass of 54.67 kDa.[7,8] The reaction catalyzed by HNS is the removal of the N-linked sulfate group from the nonreducing terminal glucosamine residues of heparan sulfate. Recombinant HNS produced in Chinese hamster ovary (CHO) cells exists in two forms, a major species of 62-64 kDa (precursor) and a minor species of 56-58 kDa

(mature). Both forms have the same size and N-terminus as the comparable forms isolated from liver.[9] The native molecular mass of HNS as determined by fast-performance liquid chromatography (FPLC) is 115 kDa so it exists as a dimer inside lysosomes.[10] Heparan-N-sulfatase has five potential N-glycosylation sites and all are used, with three high mannose or complex oligosaccharide chains and two with tri- and/or tetracentenary complex-like and biantennary chains.[11] At least one mannose group will be phosphorylated because mannose-6-phosphate groups on all soluble lysosomal enzymes are used to transport the enzymes from the trans Golgi to the lysosome via mannose-6-phosphate receptors. Heparan-N-sulfatase is a member of a highly conserved group of sulfatases that undergo a common and unique posttranslational modification necessary for catalytic function. This process involves the conversion of an active site cysteine residue to formylglycine and is catalyzed by the C(alpha)-formyl-glycine generating enzyme (FGE). The gene for FGE (SUMF1) was recently cloned and shown to be defective in multiple sulfatase deficiency patients, in whom the activity of all sulfatases is reduced.[12'13]

Sanfilippo B

The gene encoding NAGLU is situated on chromosome 17q21.1, extends approximately 8.2 kb, and contains six exons. The cDNA is 2.7 kb long and encodes a protein of 743 amino acids that has a predicted molecular mass of 82 kDa.[14'15] Alpha-N-acetylglucosaminidase acts as an exoglycosidase on the nonreducing end of alpha-N-acetyl-glucosaminide residues of heparan sulfate. Two forms of NAGLU have been purified from human placenta with apparent molecular weights of 77 and 80 kDa, representing the mature and precursor forms, respectively.1-14-1 There are seven potential glycosylation sites and it has been predicted that the site at residue 272 is occupied and carries a phosphorylated carbohydrate side chain necessary for lysosomal targeting.

Mutation Analysis

Following the characterization of the HNS and NAGLU genes, many mutations have been identified in Sanfilippo A and B patients. The initial step is to perform polymerase chain reaction (PCR) amplification on the coding exons and intron/exon boundaries and numerous sets of primers have been published. Initially single-stranded conformation polymorphism (SSCP) analysis was used to screen the PCR products for possible mutations. Nowadays, all of the PCR fragments are purified and sequenced directly, either by radioactive methods or by fluorescent sequencing technology. Many different mutations have now been identified and both disorders show extensive genetic heterogeneity.

Sanfilippo A

To date, 70 mutations have been identified in Sanfilippo A patients. These consist of 50 missense, 4 nonsense, 8 deletions, 7 insertions and 1 splice site mutation. Some mutations have been found in more than one patient whereas others are unique to individual families. Some mutations have been found at high frequencies in particular populations. The R245H, R74C, 1079delC, and S66W were the most frequent mutations in the Dutch (56.7%),[16] Polish (56%),[17] Spanish (36.5%),[18] and Italian (33%)[19] populations, respectively. Several non-disease-causing polymorphisms have been identified including R456H, which has a high frequency of 55% in the normal Australian population.1-20-1 A study of four polymorphic markers in the Spanish Sanfilippo A patients with the 1079delC mutation showed that they had a conserved haplotype, suggesting a common origin for this mutation.[18]

Sanfilippo B

To date, 99 mutations have been identified in Sanfilippo B patients. These consist of 60 missense, 8 nonsense, 16 deletions, 12 insertions and 2 splice site mutations. Again some mutations have been found in more than one patient whereas others are unique to individual families. In contrast to Sanfilippo A, common mutations have not been identified in patients of different national origins. However, in a recent study of Greek Sanfilippo B patients, three mutations were found at a high frequency, with mutations Y140C, H414R, and R626X accounting for almost 70% of the mutant alleles.[21]

Genotype/Phenotype Correlation

The relationship between the severity of a monogenic disorder and the mutations is called the genotype/ phenotype correlation. If the mutation causes the production of a truncated protein either by a nonsense mutation or by a deletion or insertion (frameshift), this is predicted to be severe and the protein nonfunctional. Patients homozygous for these types of mutations are predicted to be severely affected and this is true in both Sanfilippo A and B. The effect of missense mutations on the protein is more difficult to predict because some will allow the production of functional enzyme albeit at lower levels and others will not. It depends on the function of a particular amino acid within the protein as some will have a more critical role than others. This can only be investigated by expression studies where the mutation is generated in a normal copy of the gene, transfected into mammalian cells, such as CHO, and the resulting enzyme assayed for activity. All missense mutations should be investigated in this way, firstly to confirm their pathogenicity and secondly to determine which ones produce residual activity. Without performing such a procedure it is possible that a rare polymorphism could be reported as a mutation. By carrying out further studies such as immunoprecipi-tation of mutant enzyme and cellular localization by confocal microscopy, more information can be obtained about the effect of the mutation on the processing and transport of the enzyme. This will increase our understanding of the effect of the mutation on enzyme function and further our knowledge of the disease process. The majority of missense mutations reported in Sanfilippo A and B have been found in severely affected patients either in homozygous form or in combination with a truncating mutation so it can be deduced that they have a serious effect on enzyme function. Only a small number of attenuated, late onset patients have been described in Sanfilippo A and B, and, as predicted, they all have at least one missense mutation, which will be responsible for the residual enzyme activity. The effect of polymorphisms on enzyme activity still requires investigation in Sanfi-lippo A and B.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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