Huiwen Zhang

Analysis of the IDS gene in 38 patients with Hunter syndrome: the c.879G>A (p.Gln293Gln) synonymous variation in a female create exonic splicing.

Background Hunter syndrome (mucopolysaccharidosis type II, MPS II) is a rare disease inherited in an X-linked autosomal recessive pattern. It is the prevailing form of the mucopolysaccharidoses in China. Here we investigated mutations of IDS (iduronate 2-sulfatase) gene in 38 unrelated Chinese patients, one of which is a female. Methods Peripheral leucocytes were collected from the patients and the IDS gene was amplified to looking for the variations. For a female patient, the X chromosome status was analyzed by androgen receptor X-inactivation assay and the mutation impact on RNA level was further performed by reverse transcription polymerase chain reaction. Results We discovered that point mutations constituted the major form while mutations in codon p.R468 defined the largest number of patients in our cohort. Consistent with data from other ethnic groups, exons 9 and 3 had comparatively more mutations, while exon 2 had quite a few mutations unique to Chinese patients. Of the 30 different mutations identified, only 9 were novel: one was a premature termination mutation, i.e., c.196C>T (p.Gln66X); three were missense mutations, i.e., c.200T>C (p.Leu67Pro), c.215T>C (p.Leu72Pro), c.389C>T (p.Thr130Ile); one was a small deletion, i.e., c.1104_1122del19 (p.Ser369ArgfsX16); and one was a deletion that spanned both exons 8 and 9 deletion leading to gross structural changes in the IDS gene. In addition, a synonymous mutation c.879G>A (p.Gln293Gln) was identified in a female Hunter disease patient, which resulted in loss of the original splicing site, activated a cryptic splicing site upstream, leading to a 28 bp deletion and a premature termination at p. Tyr285GlufsX47. Together with concurrent skewed X-inactivation this was believed to facilitate the development of Hunter disease in this girl. Conclusions In conclusion, the molecular analysis of IDS gene in Chinese patients confirmed the Hunter disease diagnosis and expanded the mutation and clinical spectrum of this devastating disorder.

Clinical, biochemical, and molecular analysis of combined methylmalonic acidemia and hyperhomocysteinemia (cblC type) in China

The most common inborn error of cobalamin (cbl) metabolism in China is the cblC type characterized by combined methylmalonic acidemia and hyperhomocysteinemia. The clinical presentation is relatively nonspecific, such as feeding difficulty, recurrent vomiting, hypotonia, lethargy, seizures, progressive developmental delay, and mental retardation, together with anemia and metabolic acidosis. More specific biochemical findings include high levels of propionylcarnitine (C3), free carnitine (C3/C0), and acetylcarnitine (C3/C2) measured by tandem mass spectrometry (MS/MS), elevation of methylmalonic acid (MMA) measured by gas chromatography–mass spectrometry (GC-MS), and increased total homocysteine with normal or decreased methionine. We report on 50 Chinese patients with combined methylmalonic acidemia and hyperhomocysteinemia. Forty-six belonged to the cblC complementation group. Mutation analysis of the MMACHC gene was performed to characterize the mutational spectrum of cblC deficiency, and 17 different mutations were found. Most were clustered in exons 3 and 4, accounting for 91.3% of all mutant alleles. Two mutations were novel, namely, c.315xa0C>G (p.Y105X) and c.470xa0G>C(p.W157S). In terms of genotype–phenotype correlation, the c.609xa0G>A mutation was associated with early-onset disease when homozygous. Unlike previous reports from other populations, c.609xa0G>A (p.W203X) was the most frequent cblC mutation detected in our study of Chinese patients, affecting 51 of 92 MMACHC alleles (55.4%). The high prevalence of this nonsense mutation could have potential therapeutic significance for Chinese cblC patients. Besides traditional approaches consisting of hydroxocobalamin injections, carnitine, betaine, and protein restriction, novel drugs that target premature termination codons may have a role in the future.

Analysis of gene mutations in Chinese patients with maple syrup urine disease

OBJECTIVEnMaple syrup urine disease (MSUD) is predominantly caused by mutations in the BCKDHA, BCKDHB and DBT genes, which encode for the E1α, E1β and E2 subunits of the branched-chain α-keto acid dehydrogenase complex, respectively. The aim of this study was to screen DNA samples from 16 Chinese MSUD patients and assess a potential correlation between genotype and phenotype.nnnMETHODSnBCKDHA, BCKDHB and DBT genes were analyzed by polymerase chain reaction (PCR) and direct sequencing. Segments bearing novel mutations were identified by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis.nnnRESULTSnWithin the variant alleles, 28 mutations (28/32, 87.5%), were detected in 15 patients, while one patient displayed no mutations. Mutations were comprised of 20 different: 6 BCKDHA gene mutations in 4 cases, 10 BCKDHB gene mutations in 8 cases and 4 DBT gene mutations in 3 cases. From these, 14 were novel, which included 3 mutations in the BCKDHA gene, 7 in the BCKDHB gene and 4 in the DBT gene. Only two patients with mutations in the BCKDHB and DBT genes were thiamine-responsive and presented a better clinical outcome.nnnCONCLUSIONnWe identified 20 different mutations within the BCKDHA, BCKDHB and DBT genes among 16 Chinese MSUD patients, including 14 novel mutations. The majority were non-responsive to thiamine, associating with a worse clinical outcome. Our data provide the basis for further genotype-phenotype correlation studies in these patients, which will be beneficial for early diagnosis and in directing the approach to clinical intervention.

Patients with progressive pseudorheumatoid dysplasia: from clinical diagnosis to molecular studies.

Progressive pseudorheumatoid dysplasia (PPD) is a rare inherited autosomal recessive disease for which no prevalent data have been reported in China. We aimed to identify PPD based on clinical manifestations and imaging analysis of the bony skeleton and then to investigate gene mutations of Wnt1-inducible signaling pathway protein 3 (WISP3) in Chinese patients with PPD. Seven patients (aged 9-49 years) from six unrelated Chinese families all presented with a waddling gait, progressive swelling and restricted joint movements, and all were diagnosed as having PPD according to clinical signs and symptoms, as well as radiographic imaging. The radiographic imaging revealed no erosive arthropathy, but showed platyspondyly, irregular or wedged/ovoid anterior end-plates of the vertebral bodies, coxa vara and widened epiphyses or metaphyses including the femoral head and the metacarpophalangeal and interphalangeal joints. Normal laboratory values were found for the erythrocyte sedimentation rate, C-reactive protein and rheumatoid factors in all patients. Molecular studies revealed that five patients carried c.624_625insA/c.729_735delGAGAAAA, c.624_625insA/c.866_867insA, c.866_867 insA/c.866_867insA, Q46X/C114W and C223G/C114W mutations, respectively. In conclusion, our findings suggest that in order to avoid misdiagnosis, physicians should carefully examine the entire skeleton, including the spine, in addition to the skeletal extremities. Mutation analysis of the WISP3 gene is useful for confirming the clinical and radiographic diagnosis of PPD.

Impaired Glucose Tolerance in a Mouse Model of Sidt2 Deficiency

Sidt2 was identified as a novel integral lysosomal membrane protein recently. We generated global Sidt2 knockout mice by gene targeting. These mice have a comparatively higher random and fasting glucose concentration. Intraperitoneal and oral glucose tolerance tests in Sidt2 knockout mice indicated glucose intolerance and decreased serum insulin level. Notably, the Sidt2−/− mice had hypertrophic islets compared with control mice. By Western blot and immunofluorescence, Sidt2−/− mouse islets were shown to have increased insulin protein, which actually contained more insulin secretory granules than their controls, demonstrated by electromicroscopy. Consistent with the in vivo study, isolated islet culture from the Sidt2−/− mice produced less insulin when stimulated by a high concentration of glucose or a depolarizing concentration of KCl. Under electromicroscope less empty vesicles and more mature ones in Sidt2−/− mice islets were observed, supporting impaired insulin secretory granule release. In conclusion, Sidt2 may play a critical role in the regulation of mouse insulin secretory granule secretion.

Maternal origin of a de novo microdeletion spanning the ERCC6 gene in a classic form of the Cockayne syndrome

The Cockayne syndrome is a rare autosomal recessive disease characterized by a general developmental delay, the unique face, and abnormal skin sensitivity to sunlight. It belongs to the family of disorders of the nucleotide excision repair system. Mutations of the ERCC6 and ERCC8 genes are the predominant cause of the Cockayne syndrome, whereby the ERCC6 gene mutation makes up approximately 70% of the cases. We report a Chinese case of a classic Cockayne syndrome, carrying the novel nonsense mutation c.1387C>T/Q463X in the ERCC6 gene in an apparently homozygous status. This mutation was found in a heterozygous status in this patients father, while the mother carried two wild-type ERCC6 alleles. A further molecular investigation of the family revealed that there was a de novo microdeletion including the ERCC6 gene of maternal origin in the proband. The determination of the deletion breakpoints by Illumina genome-wide DNA analysis beadchip showed that the deletion spanned 2.82 Mb in size. This case adds to the mutation spectrum of this DNA repair disorder.

A Large Deletion of PROP1 Gene in Patients with Combined Pituitary Hormone Deficiency from Two Unrelated Chinese Pedigrees

Background: Familial combined pituitary hormone deficiency (CPHD) appears to have a genetic cause, PROP1 gene mutations being the most common one. We investigated whether PROP1 plays a role in two Chinese familial cases of CPHD. Methods:PROP1 gene and adjacent sequences from genomic samples from two unrelated families were amplified to investigate molecular variations and define the extension of a potential deletion. A quantitative real-time polymerase chain reaction was conducted to analyze the copy number of PROP1 gene in the probands’ mothers. The relationship of the two distantly located families was further analyzed using microsatellite markers. Results: A segment of about 53.2 kilobases (kb) comprehending PROP1 and another gene encoding a hypothetical protein Q6ZTH3 was deleted in both pedigrees. The mother of one of the probands was hemizygous for this large deletion, which confirmed the assumption that the affected children inherited the deletion allele from their consanguineous parents. The difference of three microsatellites surrounding the absent segment indicated that the two pedigrees were genetically unrelated. Conclusion: We report the largest genomic deletion including PROP1 gene associated with CPHD. Q6ZTH3 is unlikely to exert an indispensable function during embryogenesis or organogenesis. The 7.7-kb segment upstream of the transcription of PROP1 probably harbors a fragile site that favors the occurrence of breakpoints.

Beta-galactosidase deficiencies and novel GLB1 mutations in three Chinese patients with Morquio B disease or GM1 gangliosidosis.

BackgroundThis paper aims to report GLB1 activities and mutation analysis of three patients from the mainland of China, one with Morquio B disease and two with GM1 gangliosidosis.MethodsGLB1 activity and GLB1 gene mutation were analyzed in the three patients who were clinically suspected of having Morquio B disease or GM1 gangliosidosis. Novel mutations were analyzed by aligning GLB1 homologs, 100 control chromosomes, and the PolyPhen-2 tool.ResultsThe enzymatic activity of GLB1 was found to be 5.03, 4.20, and 4.50 nmol/h/mg in the three patients, respectively. Patient 1 was a compound heterozygote for p.[Arg148Cys] and p.[Tyr485Cys] mutations in the GLB1 gene. Patient 2 was a compound heterozygote for p.[Tyr270Phe] and p.[Leu337Pro] mutations. Patient 3 was a homozygote for p.[Asp448Val] mutation. Three mutations (p.[Tyr485Cys], p.[Tyr270Phe] and p.[Leu337Pro]) were novel variants and were predicted to damage GLB1 function.ConclusionsThe enzymatic activity and related gene analysis of β-galactosidase should be performed in clinically suspected individuals to confirm diagnosis. The three novel mutations, p.[Tyr485Cys], p.[Tyr270Phe], and p.[Leu337Pro], are thought to be disease-causing mutations.

Diagnosis, treatment, follow-up and gene mutation analysis in four Chinese children with biotinidase deficiency

SummaryObjective: To report the clinical course and explore the gene mutation spectrum of four Chinese children with biotinidase deficiency. Methods: Four Chinese patients aged 4xa0months to 8xa0years were referred to this study. Tandem mass spectrometry, gas chromatography–mass spectrometry and the determination of biotinidase activities were performed for selective screening of biotinidase deficiency. Four patients with biotinidase deficiency were diagnosed, treated with biotin and followed. Results: (1) Four patients with biotinidase deficiency were diagnosed by characteristic metabolites, such as elevated blood levels of 3-hydroxyisovalerylcarnitine (6.22u2009±u20093.1xa0μmol/L), elevated 3-methylcrontonylglycine, methylcitrate and 3-hydroxypropionate in urine and very low biotinidase activities. (2) These patients have been treated with biotin for 1–8xa0years; two of them still have mental retardation, and two have irreversible hearing or vision disability. (3) In the four patients, six different mutations in the biotinidase gene were identified: c.98G:del7ins3, c.1369G>A (p. V457M), c.1384delA, c.1493_1494insT, c.1284C>A (p.Y428X) and c.1157G>A (p.W386X). The latter four mutations are novel variations. Seven out of eight mutations are located on exon 4 of the biotinidase gene. Conclusions: Early recognition of biotinidase deficiency is crucial to avoid permanent damage. Determination of biotinidase activity should be included in neonatal screening in China. Exon 4 may be a hot-spot for biotinidase gene mutations in Chinese patients. Four novel gene variations may be disease-causing mutations and should be confirmed by expression studies.

Recurring G12S Mutation of HRAS in a Chinese Child with Costello Syndrome with High Alkaline Phosphatase Level

Costello syndrome, Noonan syndrome, cardio-facio-cutaneous (CFC) syndrome, Leopard syndrome, neurofibromatosis type 1, along with neurofibromatosis type-1 like syndrome, represent a group of entities caused by mutations in genes encoding components of the Ras/MAPK signaling cascade. These clinically distinct syndromes have overlapping features, such as facial dysmorphism, heart defects, ectodermal abnormalities, and variable levels of delay in mental development. These genetic disorders also have interesting and fascinating molecular aspects. HRAS mutations have so far been identified only in patients with Costello syndrome. On the other hand, KRAS mutations have been identified in patients with CFC, Noonan, or Costello syndrome. PTPN11 mutations are encountered in about 50% of patients diagnosed with Noonan syndrome. No PTPN11 mutations have been identified in patients with Costello or CFC syndrome. SOS1 or RAF-1 mutations have been detected in another 40% of patients with Noonan syndrome (Tartaglia et al. 2007). Recently they were named Ras/MAPK syndromes (Aoki et al. 2008). Ras/MAPK syndromes are noteworthy because Ras genes (KRAS, HRAS, and NRAS), are well-known to be cancer-related. Aberrant somatic activation of Ras had been found in approximately 20–30% of all tumors. The vast majority of oncogenetic somatic mutations occur in codons 12, 13, or 61, where genomic mutations in the Ras/MAPK syndromes are also found. Ras signaling occurs in all cells and is activated by several stimuli, such as receptor tyrosine kinases, G-protein-coupled receptors, cytokine receptors, and extracellular matrix receptors, which would