Mucopolysaccharidosis type IVA (MPS IVA) was described in 1929 by Luis Morquio from Uruguay and James Brailsford from England, and was later found as an autosomal recessive lysosomal storage disease. MPS IVA is caused by mutations in the gene encoding the enzyme, N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Reduced GALNS activity results in impaired catabolism of two glycosaminoglycans (GAGs), chondroitin-6-sulfate (C6S) and keratan sulfate (KS). Clinical presentations of MPS IVA reflect a spectrum of progression from a severe ”classical” phenotype to a mild “attenuated” phenotype. More than 180 different mutations have been identified in the GALNS gene, which likely explains the phenotypic heterogeneity of the disorder. Accumulation of C6S and KS manifests predominantly as short stature and skeletal dysplasia (dysostosis multiplex), including atlantoaxial instability and cervical cord compression. However, abnormalities in the visual, auditory, cardiovascular, and respiratory systems can also affect individuals with MPS IVA. Diagnosis is typically based on clinical examination, skeletal radiographs, urinary GAG, and enzymatic activity of GALNS in blood cells or fibroblasts. Deficiency of GALNS activity is a common assessment for the laboratory diagnosis of MPS IVA; however, with recently increased availability, gene sequencing for MPS IVA is often used to confirm enzyme results. As multiple clinical presentations are observed, diagnosis of MPS IVA may require multi-system considerations. This review provides a history of defining MPS IVA and how the understanding of the disease manifestations has changed over time. A summary of the accumulated knowledge is presented, including information from the International Morquio Registry. The classical phenotype is contrasted with attenuated cases, which are now being recognized and diagnosed more frequently. Laboratory based diagnoses of MPS IVA are also discussed.

Objectives Mucolipidoses II and III alpha/beta (ML II and ML III) are lysosomal disorders in which the essential mannose-6-phosphate recognition marker is not synthesized onto lysosomal hydrolases and other glycoproteins. The disorders are caused by mutations in GNPTAB, which encodes two of three subunits of the heterohexameric enzyme, N-acetylglucosamine-1-phosphotransferase. Clinical, biochemical, and molecular findings in 61 probands (63 patients) are presented in order to provide a broad perspective of these mucolipidoses. Methods GNPTAB was sequenced in all probands and/or parents. Activity of several lysosomal enzymes was measured in plasma, and GlcNac-1-phosphotransferase was assayed in leukocytes. Thirty-six patients were studied in detail, allowing extensive clinical data to be abstracted. Results ML II correlates with near total absence of phosphotransferase activity resulting from homozygosity or compound heterozygosity for frameshift or nonsense mutations. Craniofacial and orthopedic manifestations are evident at birth, skeletal findings become more obvious within the first year, and growth is severely impaired. Speech, ambulation, and cognitive function are impaired. ML III retains a low level of phosphotransferase activity due to at least one missense or splice site mutation. The phenotype is milder with minimal delays in milestones, the appearance of facial coarsening by early school age, and slowing of growth after age four years. Conclusions Fifty-one pathogenic changes in GNPTAB are presented, including 42 novel mutations. Ample clinical information improves criteria for delineation of ML II and ML III. Phenotype-genotype correlations suggested in more general terms in earlier reports on smaller groups of patients are specified and extended.

Mutations in the creatine transporter gene, SLC6A8 (MIM 30036), located in Xq28, have been found in families with X-linked mental retardation (XLMR) as well as in males with idiopathic mental retardation (MR). In order to estimate the frequency of such mutations in the MR population, a screening of 478 males with MR of unknown cause was undertaken. All 13 exons of SLC6A8 were sequenced using genomic DNA. Six novel potentially pathogenic mutations were identified that were not encountered in at least 588 male control chromosomes: two deletions (p.Asn336del, p.Ile347del) and a splice site alteration (c.1016+2C>T) are considered pathogenic based on the nature of the variant. A mutation (p.Arg391Trp) should be considered pathogenic owing to its localization in a highly conserved region. Two other missense variants (p.Lys4Arg, p.Gly26Arg) are not conserved but were not observed in over 300 male control chromosomes. Their pathogenicity is uncertain. A missense variant (p.Val182Met), was classified as a polymorphism based on a normal creatine/creatinine (Cr:Crn) ratio and cerebral creatine signal in proton magnetic resonance spectroscopy (H-MRS) in the patient. Furthermore, we found 14 novel intronic and neutral variants that were not encountered in at least 280 male control chromosomes and should be considered as unclassified variants. Our findings of a minimum of four pathogenic mutations and two potentially pathogenic mutations indicate that about 1% of males with MR of unknown etiology might have a SLC6A8 mutation. Thus, DNA sequence analysis and/or a Cr:Crn urine screen is warranted in any male with MR of unknown cause.

Optimal lysosome function requires maintenance of an acidic pH maintained by proton pumps in combination with a counterion transporter such as the Cl À /H þ exchanger, CLCN7 (ClC-7), encoded by CLCN7. The role of ClC-7 in maintaining lysosomal pH has been controversial. In this paper, we performed clinical and genetic evaluations of two children of different ethnicities. Both children had delayed myelination and development, organomegaly, and hypopigmentation, but neither had osteopetrosis. Whole-exome and-genome sequencing revealed a de novo c.2144A>G variant in CLCN7 in both affected children. This p.Tyr715Cys variant, located in the C-terminal domain of ClC-7, resulted in increased outward currents when it was heterologously expressed in Xenopus oocytes. Fibroblasts from probands displayed a lysosomal pH approximately 0.2 units lower than that of control cells, and treatment with chloroquine normalized the pH. Primary fibroblasts from both probands also exhibited markedly enlarged intracellular vacuoles; this finding was recapitulated by the overexpression of human p.Tyr715Cys CLCN7 in control fibroblasts, reflecting the dominant, gain-of-function nature of the variant. A mouse harboring the knock-in Clcn7 variant exhibited hypopigmentation, hepatomegaly resulting from abnormal storage, and enlarged vacuoles in cultured fibroblasts. Our results show that p.Tyr715Cys is a gain-of-function CLCN7 variant associated with developmental delay, organomegaly, and hypopigmentation resulting from lysosomal hyperacidity, abnormal storage, and enlarged intracellular vacuoles. Our data supports the hypothesis that the ClC-7 antiporter plays a critical role in maintaining lysosomal pH.

Polyamines (putrescine, spermidine, spermine) are ubiquitous, simple molecules that interact with a variety of other molecules in the cell, including nucleic acids, phospholipids and proteins. Various studies indicate that polyamines are essential for normal cell growth and differentiation. Furthermore, these molecules, especially spermine, have been shown to modulate ion channel activities of certain cells. Nonetheless, little is known about the specific cellular functions of these compounds, and extensive laboratory investigations have failed to identify a heritable condition in humans in which polyamine synthesis is perturbed. We report the first polyamine deficiency syndrome caused by a defect in spermine synthase (SMS). The defect results from a splice mutation, and is associated with the Snyder-Robinson syndrome (SRS, OMIM_309583), an X-linked mental retardation disorder. The affected males have mild-tomoderate mental retardation (MR), hypotonia, cerebellar circuitry dysfunction, facial asymmetry, thin habitus, osteoporosis, kyphoscoliosis, decreased activity of SMS, correspondingly low levels of intracellular spermine in lymphocytes and fibroblasts, and elevated spermidine/spermine ratios. The clinical features observed in SRS are consistent with cerebellar dysfunction and a defective functioning of red nucleus neurons, which, at least in rats, contain high levels of spermine. Additionally, the presence of MR reflects a role for spermine in cognitive function, possibly by spermine's ability to function as an 'intrinsic gateway' molecule for inward rectifier K þ channels.

A family with X-linked mental retardation characterized by severe mental retardation, speech and behavioral abnormalities, and seizures in affected male patients has been found to have a G1141C transversion in the creatine-transporter gene SLC6A8. This mutation results in a glycine being replaced by an arginine (G381R) and alternative splicing, since the G-->C transversion occurs at the -1 position of the 5' splice junction of intron 7. Two female relatives who are heterozygous for the SLC6A8 mutation also exhibit mild mental retardation with behavior and learning problems. Male patients with the mutation have highly elevated creatine in their urine and have decreased creatine uptake in fibroblasts, which reflects the deficiency in creatine transport. The ability to measure elevated creatine in urine makes it possible to diagnose SLC6A8 deficiency in male patients with mental retardation of unknown etiology.

Congenital disorders of glycosylation (CDG) arise from pathogenic mutations in over one hundred genes leading to impaired protein or lipid glycosylation. ALG1 encodes a β1,4 mannosyltransferase that catalyzes the addition of the first of nine mannose moieties to form a dolichol-lipid linked oligosaccharide intermediate (DLO) required for proper N-linked glycosylation. ALG1 mutations cause a rare autosomal recessive disorder termed ALG1-CDG. To date thirteen mutations in eighteen patients from fourteen families have been described with varying degrees of clinical severity. We identified and characterized thirty-nine previously unreported cases of ALG1-CDG from thirty-two families and add twenty-six new mutations. Pathogenicity of each mutation was confirmed based on its inability to rescue impaired growth or hypoglycosylation of a standard biomarker in an alg1-deficient yeast strain. Using this approach we could not establish a rank order comparison of biomarker glycosylation and patient phenotype, but we identified mutations with a lethal outcome in the first two years of life. The recently identified protein-linked xeno-tetrasaccharide biomarker, NeuAc-Gal-GlcNAc2, was seen in all twenty-seven patients tested. Our study triples the number of known patients and expands the molecular and clinical correlates of this disorder.

Background Primary deficiencies in mannosylation of N-glycans are seen in a majority of patients with congenital disorders of glycosylation (CDG). We report the discovery of a series of novel N-glycans in sera, plasma, and cultured skin fibroblasts from patients with CDG having deficient mannosylation. Method We used LC-MS/MS and MALDI-TOF-MS analysis to identify and quantify a novel N-linked tetrasccharide linked to the protein core, an N-tetrasaccharide (Neu5Acα2,6Galβ1,4-GlcNAcβ1,4GlcNAc) in plasma, serum glycoproteins, and a fibroblast lysate from patients with CDG caused by ALG1 (ALG1, chitobiosyldiphosphodolichol β-mannosyltransferase), PMM2 (phosphomannomutase 2), and MPI (mannose phosphate isomerase). Results Glycoproteins in sera, plasma, or cell lysate from ALG1-CDG, PMM2-CDG, and MPI-CDG patients had substantially more N-tetrasaccharide than unaffected controls. We observed a >80% decline in relative concentrations of the N-tetrasaccharide in MPI-CDG plasma after mannose therapy in 1 patient and in ALG1-CDG fibroblasts in vitro supplemented with mannose. Conclusions This novel N-tetrasaccharide could serve as a diagnostic marker of ALG1-, PMM2-, or MPI-CDG for screening of these 3 common CDG subtypes that comprise >70% of CDG type I patients. Its quantification by LC-MS/MS may be useful for monitoring therapeutic efficacy of mannose. The discovery of these small N-glycans also indicates the presence of an alternative pathway in N-glycosylation not recognized previously, but its biological significance remains to be studied.