Analysis of clinical features and GALNS gene mutation in a patient with mucopolysaccharidosis type IV A / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To detect potential mutation of galactosamine-6-sulfate (GALNS) gene in a Chinese girl affected with mucopolysaccharidosis type IV A (Morquio A syndrome).</p><p><b>METHODS</b>The patient was diagnosed by assaying the activities of mucopolysaccharidosis-related enzymes in leukocytes. Potential mutation in the GALNS gene was detected with PCR and Sanger sequencing.</p><p><b>RESULTS</b>The patient was characterized by short stature, skeletal deformities, normal intelligence, and auditory dysfunction. The activities of GALNS enzymes were low. A compound heterozygous missense mutation, c.1094G>T (p.Gly365Val)/c.938C>T (p.Thr313Met), was detected in the GALNS gene. The mutations were respectively inherited from her father and mother. Among them, the c.1094G>T (p.Gly365Val) mutation was not reported previously.</p><p><b>CONCLUSION</b>The mutations c.1094G>T (p.Gly365Val)/c.938C>T (p.Thr313Met) probably underlie the pathogenesis of the disease in our patient.</p>

Analysis of GALNS gene mutation in thirty-eight Chinese patients with mucopolysaccharidosis type IVA / 中华儿科杂志

<p><b>OBJECTIVE</b>Mucopolysaccharidosis (MPS) type IVA (MPS IVA) is an autosomal recessive lysosomal storage disease caused by deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS) needed to degrade glycosaminoglycanes (GAGs), accumulation of GAGs in the tissue resulting in disorder of function. So far, the small number of articles about clinical study of Chinese MPS IVA were published and only one paper about gene mutation analysis was published. This study aimed to investigate the mutation spectrum and characteristic of GALNS gene in Chinese patients with MPS IVA who were diagnosed in our hospital.</p><p><b>METHOD</b>Thirty-eight patients from 36 families (male 17, female 21) were diagnosed as MPS IVA by GALNS activity determination [(0.85 ± 1.33) nmol/(17 h·mg)] and clinical symptoms during 2006-2012. The average age of diagnosis was (5.7 ± 3.6) years. Mutation analysis of GALNS gene performed performed by PCR-direct DNA sequencing for 38 patients. PCR-restriction fragment length polymorphism analysis was used for validating novel mutation, and also to assess amino acid conservation for novel missense variants in five different species. PolyPhen-2 tool was used to predict the possible impact of missense mutations on the structure and function of the human GALNS protein, etc. Analysis of GALNS activity and gene mutation in amniotic fluid were performed to provide the prenatal diagnosis for some families with MPS type IVA.</p><p><b>RESULT</b>(1) Thirty-eight kinds of mutation in GALNS gene were identified in 38 patients of them, 71% were missense mutations. p. M318R was a hot-spot mutation (21%) tested. Five kinds of mutation i.e., p. P163H, p.G168L, p. A324E, p. L366P and p. F452L were only found in Chinese patients with MPS IVA. Eighteen kinds of novel mutation were detected including p. E315K, p.G304D, p.R251Q, p.Y240C, p.G161E, p.N32D, p.L390P, p. D60E, p. P420S, W403C/T404S, p.L454P, for p.W405X, p. M1I, c.409_ c.420del12, c.1176_1178del3, c.1046delG, c.1188delG and IVS9-2A>C. (2) The polymorphism of novel missense variants were ruled out by the PCR-restriction fragment length polymorphism analysis and no related mutations were found in 50 normal controls. A splice site mutation IVS9-2A>C had been validated by reverse transcription PCR direct sequencing. The amino acid of mutant position of 10 kinds of missense variants are highly conserved and only p. L454 is moderately conserved position. These missense variants were predicted to cause damage to the structure and function of human GALNS protein possibly according to the PolyPhen-2 tool, so these novel missense variants may be disease-causing mutations. (3) Prenatal diagnosis was provided for 7 families and three fetuses were diagnosed as MPS IVA.</p><p><b>CONCLUSION</b>The GALNS gene mutation spectrum in Chinese patients with MPS IVA is really different from that in other countries, five kinds of mutation were only found in Chinese patients with MPS IVA. The reports of hot-spot mutation in Chinese patients were also different, and should be analyzed by more data of gene mutation analysis and epidemiological study.</p>

GLB1 gene mutation and clinical characteristics of a patient with mucopolysaccharidosis type IVB / 中华儿科杂志

<p><b>OBJECTIVE</b>To report the results of clinical characteristics, enzyme activity determination and mutation analysis of GLB1 gene in a Chinese patient with mucopolysaccharidosis (MPS) type IVB (Morquio B disease).</p><p><b>METHOD</b>A 14-year-old Chinese boy with MPS type IVB was firstly diagnosed by blood leucocytes galactosamine-6-sulfate sulfatase (GALNS) and β-galactosidase (GLB1) determination, who was characterized by short stature, multiplex skeletal abnormalities, difficulty in walking. PCR-sequencing analysis was applied to detect the mutations in GLB1 of the patient.</p><p><b>RESULT</b>The patient was characterized by dwarfism, pectus carinatum, kyphosis, normal intelligence, and no neurologic damage of spasms, linguistic capacity and so on. The patient had normal GALNS enzyme activity and very low GLB1 enzyme activity [5.03 nmol/(h·mg) vs. normal value 118 - 413 nmol/(h·mg) ] in leukocytes. A compound heterozygous missense mutations c.442C > T(p.R148C)/c.1454A > G(p.Y485C) in GLB1 gene were detected in this patient. The mutation p.Y485C is a novel variant. With the method of gene analysis of new variant, the mutation p.Y485C was considered to be a pathogenic mutation.</p><p><b>CONCLUSION</b>The MPS IVB patient showed severe multiple skeletal deformities, normal intelligence, no neurologic damage and very low GLB1 enzyme activity, who carries compound heterozygous mutations p.R148C/p.Y485C. The mutation p.Y485C in GLB1 gene may be a novel pathologic mutation of MPS type IVB.</p>

Rapid prenatal genetic diagnosis of a fetus with a high risk for Morquio A syndrome / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To provide rapid and accurate prenatal genetic diagnosis for a fetus with high risk of Morquio A syndrome.</p><p><b>METHODS</b>Based on ascertained etiology of the proband and genotypes of the parents, particular mutations of the GALNS gene were screened at 10th gestational week with amplification refractory mutation system (ARMS), denaturing high performance liquid chromatography (DHPLC), and direct DNA sequencing.</p><p><b>RESULTS</b>DHPLC screening has identified abnormal double peaks in the PCR products of exons 1 and 10, whilst only a single peak was detected in normal controls. Amplification of ARMS specific primers derived a specific product for the fetus's gene, whilst no similar product was detected in normal controls. Sequencing of PCR products confirmed that exons 1 and 10 of the GALNS gene from the fetus contained a heterozygous paternal c.106-111 del (p.L36-L37 del) deletion and a heterozygous maternal c.1097 T>C (p.L366P) missense mutation, which resulted in a compound heterozygote status.</p><p><b>CONCLUSION</b>The fetus was diagnosed with Morquio A syndrome and a genotype similar to the proband. Termination of the pregnancy was recommended. Combined ARMS, DHPLC and DNA sequencing are effective for rapid and accurate prenatal diagnosis for fetus with a high risk for Morquio A syndrome. Such methods are particularly suitable for early diagnosis when pathogenesis is clear. Furthermore, combined ARMS and DHPLC are suitable for rapid processing of large numbers of samples for the identification of new mutations.</p>

Identification of a novel mutation of GALNS gene from a Chinese pedigree with mucopolysaccharidosis type IV A / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study the molecular genetic mechanism of mucopolysaccharidosis type IV A(MPS IV A), and reveal the relationship between the genotype and phenotype, and provide a basis for prenatal gene diagnosis in the future.</p><p><b>METHODS</b>A preliminary diagnosis was made by qualitative detection of urinary glycosaminoglycans of the suspected MPS IV A proband. Then, mutation detection was performed on the proband and her family members with PCR and direct sequencing of the PCR products. After a novel c.1567T to G mutation was detected, Xsp I restriction enzyme digestion and amplification refractory mutation system (ARMS) fast specific identification were established to analyze the sequences of exon 14 in GALNS gene, including 110 randomly selected healthy controls, the proband and other pedigree members. At the same time, bioinformatic approaches for protein secondary, tertiary structure prediction were applied to identify the novel pathologic mutation.</p><p><b>RESULTS</b>The proband's urine GAGs test was a weak positive(± ), and a c.1567T to G heterozygous termination codon mutation in exon 14 and a c.374C to T heterozygous missense mutation in exon 4 were found. The proband was compound heterozygous of the two mutations, so was her younger sister. Her mother was a carrier with only a c.1567T to G heterozygous mutation in exon 14. Her father had a heterozygous mutation of c.374C to T in exon 4. After Xsp I restriction enzyme digestion, healthy controls had three bands including 28 bp, 120 bp and 399 bp, while the proband and her mother had four bands consisting of 28 bp, 120 bp, 148 bp and 399 bp. For amplification by ARMS specific primers, it was negative for the controls, while it was positive for the proband and the carrier. The results of protein secondary and tertiary structure prediction showed that the c.1567T to G mutation located in the stop codon, resulted in stop codon (TAG) changing to glutamic acid (GAG), with the peptide chain extending 92 amino acid residues, and secondary and tertiary protein structure change, which were not found in the controls. The result of enzyme assay showed that the activity of GALNS enzyme in the affected child was 8.3 nmol/17h/mg pr, which was obviously lower than the normal value (the normal range is 41.9-92.1 nmol/17h/mg pr).</p><p><b>CONCLUSION</b>These results illustrate that the c.1567 T to G is a novel pathologic mutation, which is the main cause of the disease in this family.</p>

Enzymatic and hemolytic activities of Candida dubliniensis strains

Candida dubliniensis is an opportunistic yeast that has been recovered from several body sites in many populations; it is most often recovered from the oral cavities of human immunodeficiency virus-infected patients. Although extensive studies on epidemiology and phylogeny of C. dubliniensis have been performed, little is known about virulence factors such as exoenzymatic and hemolytic activities. In this study we compared proteinase, hyaluronidase, chondroitin sulphatase and hemolytic activities in 18 C. dubliniensis and 30 C. albicans strains isolated from AIDS patients. C. albicans isolates produced higher amounts of proteinase than C. dubliniensis (p < 0.05). All the tested C. dubliniensis strains expressed hyaluronidase and chondroitin sulphatase activities, but none of them were significantly different from those observed with C. albicans (p > 0.05). Hemolytic activity was affected by CaCl2; when this component was absent, we did not notice any significant difference between C. albicans and C. dubliniensis hemolytic activities. On the contrary, when we added 2.5 g percent CaCl2, the hemolytic activity was reduced on C. dubliniensis and stimulated on C. albicans tested strains (p < 0.05).

C. dubliniensis é uma levedura oportunista que, embora já tenha sido isolada de vários sítios anatômicos é, com maior frequência, encontrada na boca de pacientes infectados pelo HIV. Embora tenham sido realizados numerosos estudos sobre a epidemiologia e filogenia, seus fatores de virulência como atividade exoenzimática e atividade hemolítica, são, ainda, pouco conhecidos. Neste estudo comparou-se a atividade in vitro de proteinase, hialuronidase, condroitin sulfatase e atividade hemolítica de 18 cultivos de C. dubliniensis com 30 cultivos de C. albicans, todos isolados de pacientes com SIDA. Foi evidenciada maior atividade de proteinase em C. albicans em relação a C. dubliniensis (p < 0,05). Todos os isolados de C. dubliniensis evidenciaram atividade de hialuronidase e condroitin-sulfatase de forma similar ao observado com C. albicans (p > 0,05). Constatou-se que a atividade hemolítica foi influenciada pelo CaCl2; em sua ausência não foram observadas diferenças na atividade hemolítica das duas espécies; todavia, ao se agregar 2,5 por cento de CaCl2, a atividade hemolítica de C. dubliniensis foi reduzida enquanto a de C. albicans, estimulada (p < 0,05).

Production and characterization of monoclonal antibodies to shark cartilage proteoglycan

1. Two proteoglycans, PG1 and PG2, have been isolated from shark cartilage. Both are highly polydisperse and large (molecular mass: 1-10 x 10**6 Daltons) and contain chondroitin sulfate and keratan sulfate side chains, but PG2 is somewhat smaller tham PG1 and contains less keratan sulfate. 2. Monoclonal antibodies were raised against PG1. Many antibodies were obtained and one of them, MST1, was subcloned and furter characterized. This monoclonal antibody reacts with PG1 and PG2 from shark cartilage and also with aggrecan from bovine trachea cartilage. Chondroitinase AC-treated proteglycans react MST1, indicating that the antibody does not reconize chondroitin sulfate. MST1 also recognizes aggrecan from human cartilage and a proteoglycan from bovine brain (neurocan) but it does reconize proteoglycans from rat Walker tumor, fetal calf muscle and decorin from human myoma. 3. Using MST1 we were able to demonstrate that both PG1 aggregate with hyaluronic acid

Enzimas histolíticas produzidas por leveduras do gênero Candida / Histolytic enzymes produced by yeasts of the genus Candida

Verificou-se a produçäo de hialuronidase, condroitin sulfatase, lecitinase e gelatinase, por amostras de leveduras do gênero Candida, isoladas da cavidade bucal. Todas as amostras de C. albicans, produziram hialuronidase e condroitin sulfatase, mas näo lecitinase e gelatinase. Das 17 amostras de C. parapsilosis testadas, apenas 2 produziram lecitinase e 4 produziram gelatinase. C. tropicalis e C. guillermondi, näo produziram qualquer das enzimas estudadas