Diagnosis of patients with mucopolysaccharidosis type II via RNA sequencing.

BACKGROUND: Mucopolysaccharidosis type II (MPS II) is an X-linked recessive lysosomal storage disorder caused by various variants in the IDS gene. It is known that genomic recombinants between IDS and its homologous pseudogene IDSP1 account for a small number of patients, for whom genetic diagnosis usually relies on restriction enzyme digestion at specific loci. Nevertheless, such approach cannot reveal the impact of rearrangements on IDS transcription, which is crucial for the interpretation of the pathogenicity of rearrangement variants. METHODS: RNA sequencing (RNA-seq) was explored to analyze transcriptional alterations in four male MPS II patients who were negative for Sanger sequencing of the IDS gene. Reverse transcription-polymerase chain reaction and TA clone sequencing were used to validate RNA-seq analysis results. The IDS-IDSP1 recombinant was determined by sequencing the indicated loci in genome. RESULTS: Differential expression analysis showed the expression levels of IDS gene in patients were largely reduced compared to the healthy individuals. Differential splicing analysis revealed skipping of exons 8 and 9 of IDS, without any splice-junction defects at the genomic level. In addition, two types of fusion transcripts, IDS_EOLA1 and IDS_EOLA1-DT_EOLA1 were identified by gene fusion analysis. Sequencing of the known rearrangement alleles showed these four patients have the same type of IDS-IDSP1 recombinant. CONCLUSION: We establish an RNA-seq workflow to analyze transcriptional characteristics of IDS gene from multiple perspectives. Our study validates the diagnostic value of RNA-seq in MPS II, including the discovery of transcriptional alterations and the potential to suggest genome-level rearrangements in IDS.

Efficient nitrite accumulation and elemental sulfur recovery in partial sulfide autotrophic denitrification system: Insights of seeding sludge, S/N ratio and flocculation strategy.

Partial sulfide autotrophic denitrification (PSAD) has been proposed as a promising process to achieve elemental sulfur recovery and nitrite accumulation, which is required for anaerobic ammonium oxidation reaction. This study investigated the effect of seeding sludge on the start-up performance of PSAD process, with different sludge taken from the oxidation zone (S-o) of wastewater treatment plants, partial denitrification reactor (S-PD), and anoxic/oxic reactor (S-A/O). The results showed that the PSAD process could be achieved rapidly in three systems on day 22, 29 and 26, respectively. In particular, the S-O system completed the start-up in the shortest time of 22 d, with NO3--N and S2- removal efficiency of 85.3% and 99.3%, respectively. Selected the S-O system to operate long term, the nitrite (NO2--N) and biological elemental sulfur (S0) accumulation efficiencies were systematically investigated under different S/N ratios (in a range of 0.71-1.2). The maximum NO2--N and S0 accumulation efficiencies were 85.2% and 73.5%, respectively, at the S/N ratio of 1.1. In addition, the separation and recovery of S0 in effluent was achieved by employing polyaluminum chloride (PAC) as a flocculant. Using 2D Gaussian function as quadratic model for the maximizing of S0 flocculant efficiency (SFR), an optimal condition of PAC dosage 7.92 mL/L and pH 5.14 was obtained, and the SFR reached 94.1%, under such conditions. The findings offered useful information to facilitate the application of the PSAD process.

[Partial Nitritation and Anaerobic Ammonia Oxidation Synergistic Denitrification to Remove Nitrogen and Carbon from Domestic Sewage].

A sequencing batch reactor-anaerobic sequencing batch reactor(SBR-ASBR) process was used to treat domestic sewage. In the SBR, the effects of the anoxic/aerobic time ratio and temperature on the realization of partial nitritation(PN) were investigated. In the ASBR, the effects of different COD/NO2--N(C/N) ratios on the removal of nitrogen and carbon using anaerobic ammonia oxidation(ANAMMOX) and denitrification were studied. The results illustrated that:① After three single cycles and on the 22nd day, the NO2--N accumulation rate(NiAR) was 98.06%, and the nitrate nitrogen generation rate(SNiPR, calculated as N/VSS) was 0.28g·(g·d)-1, and simultaneous nitrification and denitrification removal the TN and COD were 12.29 and 110.36mg·L-1, respectively(temperature=25℃, anoxic/aerobic time ratio=30 min:30 min). ② At an anoxic/aerobic time ratio of 30 min:30 min, the filamentous sludge bulked, the sludge activity decreased, and sludge settleability was poor at 15℃. Furthermore, the conversion rate of NH4+-N to NO2--N was 86.83%, indicating that the effluent NH4+-N concentration was too low to provide suitable matrix concentrations for ANAMMOX at 30℃. The effluent concentrations of NH4+-N and NO2--N were 31.58 mg·L-1 and 35.04mg·L-1, respectively, matching the ratio of the ANAMMOX substrate at 25℃. ③ The SBR-ASBR combined process showed good denitrification performance; the effluent TN, NH4+-N, and COD concentrations were stable at 13.13, 4.83, and 69.96mg·L-1, respectively, and the removal rates were 83.10%, 93.64%, and 75.11%, respectively. When the influent C/N of the ASBR was 2.5, 2.0, and 1.5, respectively, anaerobic ammonia oxidation and denitrification showed the best performance with respect to nitrogen and carbon removal with a C/N of 2.0. The effluent NH4+-N, NO2--N, NO3--N, and COD were 0.09, 0.25, 1.04, and 32.73 mg·L-1, respectively.

A genomic copy number variant analysis implicates the MBD5 and HNRNPU genes in Chinese children with infantile spasms and expands the clinical spectrum of 2q23.1 deletion.

BACKGROUND: Infantile spasms (IS) is a specific type of epileptic encephalopathy associated with severe developmental disabilities. Genetic factors are strongly implicated in IS, however, the exact genetic defects remain unknown in the majority of cases. Rare mutations in a single gene or in copy number variants (CNVs) have been implicated in IS of children in Western countries. The objective of this study was to dissect the role of copy number variations in Chinese children with infantile spasms. METHODS: We used the Agilent Human Genome CGH microarray 180 K for genome-wide detection of CNVs. Real-time qPCR was used to validate the CNVs. We performed genomic and medical annotations for individual CNVs to determine the pathogenicity of CNVs related to IS. RESULTS: We report herein the first genome-wide CNV analysis in children with IS, detecting a total of 14 CNVs in a cohort of 47 Chinese children with IS. Four CNVs (4/47 = 8.5%) (1q21.1 gain; 1q44, 2q31.1, and 17p13 loss) are considered to be pathogenic. The CNV loss at 17p13.3 contains PAFAH1B1 (LIS1), a causative gene for lissencephaly. Although the CNVs at 1q21.1, 1q44, and 2q23.1 have been previously implicated in a wide spectrum of clinical features including autism spectrum disorders (ASD) and generalized seizure, our study is the first report identifying them in individuals with a primary diagnosis of IS. The CNV loss in the 1q44 region contains HNRNPU, a strong candidate gene recently suggested in IS by the whole exome sequencing of children with IS. The CNV loss at 2q23.1 includes MBD5, a methyl-DNA binding protein that is a causative gene of ASD and a candidate gene for epileptic encephalopathy. We also report a distinct clinical presentation of IS, microcephaly, intellectual disability, and absent hallux in a case with the 2q23.1 deletion. CONCLUSION: Our findings strongly support the role of CNVs in infantile spasms and expand the clinical spectrum associate with 2q23.1 deletion. In particular, our study implicates the HNRNPU and MBD5 genes in Chinese children with IS. Our study also supports that the molecular mechanisms of infantile spasms appear conserved among different ethnic backgrounds.