Xuefan Gu

Determination of 7-ketocholesterol in plasma by LC-MS for rapid diagnosis of acid SMase-deficient Niemann-Pick disease.

Acid sphingomyelinase (ASMase)-deficient Niemann-Pick disease (NPD) is caused by mutations in the sphingomyelin phosphodiesterase 1 (SMPD1) gene, resulting in accumulation of sphingomyelin in the lysosomes and secondary changes in cholesterol metabolism. We hypothesized that the oxidation product of cholesterol, 7-ketocholesterol (7-KC), might increase in the plasma of patients with ASMase-deficient NPD. In this study, a rapid and nonderivatized method of measurement of plasma 7-KC by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. Plasma samples from healthy subjects, patients with ASMase-deficient NPD, nonaffected ASMase-deficient NPD heterozygotes, Niemann-Pick type C (NPC) disease, glycogen storage disorder type II (GSDII), Gaucher disease (GD), mucopolysaccharidosis type II (MPSII), Krabbe disease (KD), and metachromatic leukodystrophy (MLD) were tested retrospectively. Markedly elevated 7-KC was found in patients with ASMase-deficient NPD and NPC disease that showed significant differences from ASMase-deficient NPD heterozygotes; patients with GSDII, GD, MPSII, KD, and MLD; and normal controls. The analysis of plasma 7-KC by LC-MS/MS offers the first simple, quantitative, and highly sensitive method for detection of ASMase-deficient NPD and could be useful in the diagnosis of both ASMase-deficient NPD and NPC disease.

Diagnosis of Niemann-Pick disease type C with 7-ketocholesterol screening followed by NPC1/NPC2 gene mutation confirmation in Chinese patients

BackgroundIt has been reported that oxidation product of cholesterol, 7-ketocholesterol, increases in plasma of patients with NP-C. Previously, we established a rapid test to determine the plasma 7-ketocholesterol level and found it elevated significantly in patients with acid sphingomyelinase deficient NPD and NP-C disease.MethodsIndividuals randomly referred to our outpatient clinics in the past two years for hepatosplenomegaly or isolated splenomegaly, who have been excluded as acid sphingomyelinase deficient NPD or Gaucher disease, and individuals with newborn cholestasis, psychomotor regression/retardation, were screened for plasma 7-ketocholesterol level. Individuals with high 7-ketocholesterol level were then analyzed for NPC1 and NPC2 gene mutation to confirm the accuracy of NP-C diagnosis.ResultsBy screening the plasma 7-ketocholesterol of suspect individuals, 12 out of 302 (4%) had shown remarkable high levels compared with reference. All these twelve individuals were subsequently confirmed to be NP-C by DNA analysis of NPC1 and NPC2 genes, with the early infantile form (n = 7), the late infantile form (n = 1), the juvenile form (n = 1) and the adult form (n = 1). Furthermore, two NP-C patients without observable neuropsychiatric disability were picked up through this procedure. Only one patient had NP-C due to NPC2 gene mutations, with the rest due to NPC1 gene mutations. We found that in NP-C patients AST was usually mildly elevated and ALT was in a normal range when jaundice was not present. In total, 22 mutant alleles were identified in the NPC1 gene, including six novel small deletions/insertions, e.g., c.416_417insC, c.1030delT, c.1800delC, c.2230_2231delGT, c.2302_2303insG, and c.2795dupA; seven novel exonic point mutations, c.1502A>T (p.D501V), c.1553G>A (p.R518Q), c.1832A>G (p.D611G), c.2054T>C (p.I685T), c.2128C>T(p.Q710X), c.2177G>C (p.R726T), c.2366G>A (p.R789H), and one novel intronic mutation c.2912-3C>G. Small deletions/insertions constituted nearly half of the mutant alleles (10/22, 45%), indicating a unique mutation spectrum in this cohort of Chinese NP-C patients.ConclusionOur data confirm in a clinical setting that screening plasma 7-ketocholesterol is an efficient and practical diagnostic tool to identify NP-C patients from suspect individuals. Patients without neuropsychological involvement could also be identified by this method therefore allowing an opportunity for earlier treatment.

Mutational Spectrum of Phenylketonuria in the Chinese Han Population: A Novel Insight into the Geographic Distribution of the Common Mutations

The absence of a comprehensive analysis for phenylketonuria (PKU) mutations in the Chinese Han population has resulted in continued studies during the past 18 y to elucidate the mutational spectrum in patients from virtually all Chinese regions. Our study systematically investigated 13 exons and their surrounding introns of the phenylalanine hydroxylase (PAH) gene in 212 unrelated patients using PCR and direct sequencing. A total of 79 different mutations were identified in 405 of 424 mutant PAH alleles including 15 novel ones. Eight mutations, R243Q, Ex6–96A>G, IVS4 − 1G>A, R413P, Y356X, R111X, R241C, and V399V, with a relative frequency of 3% or more, accounted for two thirds of the identified ones. The data presented in this study indicates that the total pool of mutant PAH alleles in China consisted of a small number of common mutations and a very high number of rare mutations. Moreover, by merging the findings of previous studies to generate a more composite data set for the Chinese mainland, it is shown that there are no significant differences of the common mutations between southern and northern except for R413P statistically, raising questions about the previous hypothesis that great variations on mutation frequencies exist between above regions.

Identification of a distinct mutation spectrum in the SMPD1 gene of Chinese patients with acid sphingomyelinase-deficient Niemann-Pick disease

BackgroundClinical observations and molecular analysis of the SMPD1 gene in Chinese patients with acid sphingomyelinase deficiency Niemann-Pick disease (NPD) are scarce.MethodsA cohort of 27 Chinese patients diagnosed with acid sphingomyelinase deficiency, within the past five years, were collected and investigated for genotype, phenotype, and their correlations.ResultsThe majority of our patients (25/27) were under 18 years of age. From the cohort group, eight (30%) fulfilled characters of type A. Four other patients experienced neurologic involvement after two years of age, these were classified as intermediate type. The remaining fifteen presented without clear neurologic involvement and were regarded as type B. One patient, from the type B group, presented with the unusual symptom of a secondary amenorrhea. Three patients, one from the type B group and two from the intermediate group, presented with pronounced proteinuria, in the late stages of the disease, indicating possible kidney involvement in NPD. Twenty-four SMPD1 gene mutations had been identified; eighteen of these are novel ones. These included four exonic small deletions/duplications (c.4delC, c.147_150del4, c.842-849dup8, c.1307-1312dup6), one termination mutation (p.Glu248X), and thirteen exonic point mutations (p.Gly336Ser, p.Trp342Cys, p.Leu382Phe, p.Pro429Leu, p.Pro430Ser, p.Trp437Arg, p.Thr451Pro, p.His461Pro, p.Ala484Val, p.Ser486Arg, p.Tyr500His, p.Pro533Leu, p.Val559Leu). Notably, eight mutations had more than one occurrence with c.4delC and p.Glu248X accounting for ~30% of all alleles. Correlation analysis of genotype and phenotype indicated eight mutations, c.842-849dup8, p.Glu248X, p.Arg230Cys, p.Trp437Arg, p.His461Pro, p.Ala484Val p.Ser486Arg, and p.Pro533Leu,to be severe mutations. Five mutations, c.4delC, p.Leu382Phe, p.Pro429Leu, p.Pro430Ser and p.Val559Leu were projected to be mild mutations. Interestingly, three intermediate individuals carried combinations of a mild mutation, c.4delC, on one allele and a severe mutation on the other allele.ConclusionsThe Chinese population may have a comparably high incidence of sphingomyelinase-deficient Niemann-Pick disease type A. This study has identified some novel genotype and phenotype correlations in this rare and devastating disorder.

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

OBJECTIVE Maple 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. METHODS BCKDHA, 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. RESULTS Within 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. CONCLUSION We 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.

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.

Further delineation of the phenotype of truncating KMT2A mutations: The extended Wiedemann–Steiner syndrome

KMT2A mutations cause Wiedemann–Steiner syndrome (WDSTS), which is characterized by hypertrichosis cubiti, short stature, and distinct facial features in general. Here, we report two Chinese boys with novel nonsense KMT2A mutations. Most of their phenotypes are concordant with WDSTS. They, however, lack the key WDSTS feature—hypertrichosis cubiti. Additionally, their transverse palmar creases are absent. We further summarized the genotypes and phenotypes of the KMT2A mutation carriers. The consensus phenotypes include postnatal growth retardation, developmental delay, short stature, and intellectual disability. The common facial features include thick eyebrows, long eyelashes, downslanting, and narrow palpebral fissures, wide nasal bridge, and broad nasal tip. They have generalized hypertrichosis. A hairy back can be observed as frequently as hairy elbows in patients with KMT2A mutations. Absent palmar proximal transverse creases are only observed in these two Chinese boys. This might be due to the difference in ethnic background. Thus far, all mutations in KMT2A are located before the FYRC domain. They would truncate KMT2A mRNA transcripts. Haploinsufficiency of the histone methyltransferase activity would therefore influence transcriptional regulation.

21-hydroxylase deficiency-induced congenital adrenal hyperplasia in 230 Chinese patients: Genotype-phenotype correlation and identification of nine novel mutations.

Steroid 21-hydroxylase deficiency (21-OHD) caused by the CYP21A2 gene mutations accounts for more than 90% of congenital adrenal hyperplasia (CAH) cases. In this study, molecular defects of 230 patients with 21-OHD were investigated. Point mutations of CYP21A2 gene were analyzed by Sanger sequencing, and large gene deletions were detected by multiplex ligation-dependent probe amplification (MLPA). Nine micro-conversions and 18 spontaneous mutations accounted for 74.6% of alleles, while large gene deletions and large gene conversions accounted for 25.4% of alleles. The most frequent micro-conversion was c.292-13A/C>G (I2G) (35%), followed by p.I173N (14.3%), p.R357W (5.9%) and p.Q319* (4.6%). Nine novel mutations were identified in these patients, which were predicted to hamper the 21-hydroxylase protein function in varying degrees. Genotype and phenotype correlated well in 89.6% of our patients, but disparity in phenotypic appearance also appeared in a small portion of the patients. 16.1% of the patients carried homozygous genotypes while 83.9% of patients carried compound heterozygous mutations. We concluded that the frequency of CYP21A2 mutations in our study was slightly different from those reported for other ethnic groups. Micro-conversions were the main category of the mutation spectrum, while large deletions and large gene conversions could also cause 21-OHD. A large portion of different types of the compound heterozygous genotypes may partially contribute to the discordance in genotype-phenotype comparison. This study expanded the CYP21A2 mutation spectrum of Chinese patients and could be helpful in prenatal diagnosis and genetic counseling for 21-OHD patients.

Spectrum Analysis of Common Inherited Metabolic Diseases in Chinese Patients Screened and Diagnosed by Tandem Mass Spectrometry

Information concerning inherited metabolic diseases in China is scarce. We investigated the prevalence and age distributions of amino acid, organic acid, and fatty acid oxidation disorders in Chinese patients.

Mechanisms regulating superoxide generation in experimental models of phenylketonuria: An essential role of NADPH oxidase

This study was designed to investigate whether nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, a superoxide-producing enzyme, could be involved in phenylketonuria (PKU)-associated oxidative stress. A Pah(enu2)-BTBR PKU mouse model, and an in vitro cell culture model of PKU mimicking high phenylalanine insults in PKU, were employed for this study. The concentration of phenylalanine in mouse cerebral cortex was determined by liquid chromatography-tandem mass spectrometry. Superoxide production was displayed with dihydroethidium staining. NADPH oxidase expression level was measured by real-time RT-PCR, Western blotting and immunofluorescence. NADPH oxidase activity was measured by the colorimetric method. The phenylalanine concentrations in cerebral cortices of PKU mice were significantly higher than those in wild-type control mice. Similar results concerning superoxide production and NADPH oxidase protein expression and activity, were also found in this brain region. In addition, it was found that cerebral cortical neurons subjected to an in vitro high phenylalanine insult, displayed increased superoxide production accompanied by increases of NADPH oxidase protein expression and activity. Pretreatment with the inhibitor of this oxidase (diphenylene iodonium or apocynin) prevented this superoxide-increasing effect. Collectively, these findings provide evidence that NADPH oxidase might be a key enzyme involved in enhanced superoxide production in PKU and suggest that it may be a potential therapeutic target in neuroprotective strategies against phenylalanine-evoked oxidative brain injury in PKU.