Xuefan Gu

Selective screening for inborn errors of metabolism on clinical patients using tandem mass spectrometry in China: A four-year report

SummaryWe have initiated clinical selective screening for inborn errors of metabolism in China by analysing amino acids and acylcarnitines in a dried blood filter-paper samples using tandem mass spectrometry. Samples from a total of 3070 children suspected of inborn errors of metabolism were collected through a study network which covered most provinces of China. The diagnoses were further confirmed through clinical symptoms, by gas chromatography–mass spectrometry and other biochemistry studies, and in a few cases by DNA analysis. In all, 212 cases were diagnosed (6.6%) including 92 (43.4%) with amino acids disorders (48 with phenylketonuria, 12 with ornithine carbamoyltransferase deficiency, 7 with tyrosinaemia type I, 9 with maple syrup urine disease, 5 with citrullinaemia type I, 8 with citrullinaemia type II, 2 with homocystinuria, and 1 with argininaemia); 107 (50.5%) with organic acid disorders (including 58 with methylmalonic acidaemia, 13 with propionic acidaemia, 6 with isovaleric acidaemia, 7 with glutaric acidaemia type I, 6 with 3-methylcrotonyl-CoA carboxylase deficiency, 2 with 3-hydroxy-3-methylglutaryl-CoA lyase deficiency, 10 with multiple carboxylase deficiency, and 5 with β-ketothiolase deficiency); and 13 (6.1%) with fatty acid oxidation disorders (including 1 with carnitine palmitoyltransferase deficiency type I, 1 with carnitine palmitoyltransferase deficiency type II, 1 with short-chain acyl-CoA dehydrogenase deficiency, 5 with medium-chain acyl-CoA dehydrogenase deficiency, 3 with very long-chain acyl-CoA dehydrogenase deficiency, and 2 with multiple acyl-CoA dehydrogenase deficiency). It is suggested that tandem mass spectrometry is useful for selective screening of clinically suspected patients. The majority of diseases (94%) in this study were amino acid disorders and organic acid disorders. Fatty acid oxidation disorders are relatively rare in the Chinese, but medium-chain acyl-CoA dehydrogenase deficiency should be further investigated.

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.

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.

Elevated Bile Acids in Newborns with Biliary Atresia (BA)

Biliary Atresia (BA), a result from inflammatory destruction of the intrahepatic and extrahepatic bile ducts, is a severe hepatobiliary disorder unique to infancy. Early diagnosis and Kasai operation greatly improve the outcome of BA patients, which encourages the development of early screening methods. Using HPLC coupled tandem mass spectrometry, we detected primary bile acids content in dried blood spots obtained from 8 BA infants, 17 neonatal jaundice and 292 comparison infants at 3–4 days of life. Taurocholate (TC) was significantly elevated in biliary atresia infants (0.98±0.62 µmol/L) compared to neonatal jaundice (0.47±0.30 µmol/L) and comparison infants (0.43±0.40 µmol/L), with p = 0.0231 and p = 0.0016 respectively. The area under receiver operating characteristic (ROC) curve for TC to discriminate BA and comparison infants was 0.82 (95% confidence interval: 0.72–0.92). A cutoff of 0.63 µmol/L produced a sensitivity of 79.1% and specificity of 62.5%. The concentrations of total bile acids were also raised significantly in BA compared to comparison infants (6.62±3.89 µmol/L vs 3.81±3.06 µmol/L, p = 0.0162), with the area under ROC curve of 0.75 (95% confidence interval: 0.61–0.89). No significant difference was found between the bile acids of neonatal jaundice and that of comparison infants. The early increase of bile acids indicates the presentation of BA in the immediate newborn period and the possibility of TC as newborn screening marker.

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.

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