Clinical Characteristics and Novel ZEB2 Gene Mutation Analysis of Three Chinese Patients with Mowat-Wilson Syndrome.

Purpose: Mowat-Wilson syndrome (MWS) is an autosomal dominant disease caused by a pathogenic variant of the ZEB2 gene. The main clinical manifestations include special facial features, Hirschsprung disease (HSCR), global developmental delay and other congenital malformations. Here, we summarize the clinical characteristics and genetic mutation analysis of three Chinese patients with MWS. Patients and Methods: The clinical characteristics of the patients were monitored and the treatment effect was followed up. DNA was extracted from peripheral blood and analyzed by sequencing. Whole exome sequencing was then performed. Results: Three novel ZEB2 gene mutations were identified in 3 patients (c.1147_1150dupGAAC, p.Q384Rfs*7, c.1137_1146del TAGTATGTCT, p.S380Nfs *13 and c.2718delT, p.A907Lfs*23). They all had special facial features, intellectual disability, developmental delay, microcephaly, structural brain abnormalities and other symptoms. After long-term regular rehabilitation treatment, the development quotient of each functional area of the patient was slightly improved. Conclusion: Our study expanded the mutation spectrum of ZEB2 and enriched our understanding of the clinical features of MWS. It also shows that long-term standardized treatment is of great significance for the prognosis of patients.

A Chinese Boy with Mowat-Wilson Syndrome Caused by a 10 bp Deletion in the ZEB2 Gene.

PURPOSE: Mowat-Wilson syndrome (MWS) is a rare complex malformation syndrome which is characterized by typical facial dysmorphism, moderate to severe intellectual disability, global developmental delay, and multiple congenital anomalies. Here, we summarize the clinical characteristics and gene mutation analysis of a Chinese boy with MWS. PATIENTS AND METHODS: The clinical features of the patient were monitored. DNA extracted from peripheral blood was subjected to sequencing analysis. Then, the whole-exome sequencing was performed. RESULTS: A novel deletion mutation (c.1137_1146del TAGTATGTCT) was identified in exon 8 of the ZEB2 gene. The deletion mutation was predicted to produce a truncated protein (p.S380Nfs*13), resulting in haploinsufficiency. The patient presented with short stature, microcephaly, congenital heart defects, cryptorchidism, corpus callosum agenesis, global developmental delay, and intellectual disability. Furthermore, he demonstrated bilateral sensorineural hearing loss. This manifestation is less common in MWS. It is first reported in Chinese patients with MWS. Clinical follow-up showed that the facial features of MWS developed with time. The facial features of the patient were not obvious except for the uplifted ear lobes at the age of 3 months. At the age of 22 months, the facial characteristics of the patient included ocular hypertelorism, frontal bossing, rounded nasal tip, sparse eyebrows, prominent chin, widely spaced teeth, and uplifted ear lobes with a central depression. CONCLUSION: A novel deletion mutation of the ZEB2 gene was identified. This work contributes to expanding the mutation spectra of MWS. Our results may reflect the variability of the phenotype in MWS.

Bainbridge-ropers syndrome caused by loss-of-function variants in ASXL3: Clinical abnormalities, medical imaging features, and gene variation in infancy of case report.

BACKGROUND: Bainbridge-Ropers syndrome (BRPS) is a recently described developmental disorder caused by de novo truncating mutations in the Additional sex combs-like 3 (ASXL3) gene. Only four cases have been reported in China and are limited to the analysis of its clinical abnormalities, medical imaging features and gene variation. The aim of this study was to investigate the clinical phenotype, imaging manifestations and genetic characteristics of BPRS syndrome caused by ASXL3 gene mutation. Clinical data, medical imaging data and gene test results of BRPS in infant patients were retrospectively analyzed, and related literature was summarized. CASE PRESENTATION: At the age of 8 months, brain MRI showed that the subarachnoid space of the forehead was widened, part of the sulci was deepened, and the corpus callosum was thin. The development quotient (DQ) was determined using the 0~6-year-old pediatric examination table of neuropsychological development at 6 months and 8 months. The DQ of both tests was less than 69. Whole-exome sequencing revealed a heterozygous frameshift mutation c.3493_3494deTG in exon 12 of the ASXL3 gene, resulting in the amino acid change p. (Cys1165Ter). No variation was present at this site in her parents. Sanger sequencing of family members validated this analysis, suggesting a de novo mutation. The de novo ASXL3 mutations generated stop codons and were predicted, in silico, to generate a truncated ASXL3. CONCLUSIONS: The main clinical features of the patient included psychomotor development retardation, difficulty in feeding, hypotonia, and special facial features. MRI features showed that brain development lagged behind that of normal children. Genetic testing is helpful in the early diagnosis of BRPS.

The first study of successful pregnancies in Chinese patients with Phenylketonuria.

BACKGROUND: Since the inception of newborn screening programs in China in the 1990s, pregnancy among patients with inherited, metabolic disorders has become more common. This study explores the management and outcomes of planned, full-term pregnancies in patients with phenylketonuria (PKU). METHOD: Married patients with PKU from 2012 to 2017 were enrolled to receive prenatal counseling and regular health assessments. Study-related assessments included the timing of Phe-restricted diets, maternal weight gain, gestational age, pregnancy complications, and blood Phe concentrations (both pre-conception and during pregnancy), obstetrical data, and offspring outcomes(e.g. anthropomorphic measurements and developmental quotients [DQs]). RESULTS: A total of six offspring were successfully delivered. The mean ± SD (range) age of the mother at delivery was 26.3 ± 4.7 (range: 21.1-32.5) years. The mean duration of Phe control before pregnancy was 5.5 ± 1.3(range: 3.1-6.5) months. During pregnancy, the proportion of blood Phe concentrations within the clinically-recommended target range (120-360 µmol/L) ranged from 63.2-83.5%. Low birth weight (< 2500 g) offspring occurred in two women who experienced suboptimal metabolic control. In addition, offspring DQ was related to the proportion of blood Phe levels per trimester that were within the recommended range (r = 0.886, p = 0.016). CONCLUSION: This is the first report of women in China with PKU who successfully gave birth to clinically healthy babies. Infant outcomes were related to maternal blood Phe management prior to and during pregnancy. In maternal PKU patients with poor compliance to dietary treatment, sapropterin dihydrochloride (6R-BH4) may be an option to improve the management of blood Phe levels.

Clinical presentation, molecular analysis and follow-up of patients with mut methylmalonic acidemia in Shandong province, China.

BACKGROUND: The mut methylmalonic acidemia (MMA) caused by the deficiency of methylmalonyl-CoA mutase (MCM) activity, which results from defects in the MUT gene. The aim of this study was to summarize the clinical and biochemical data, spectrum of mutations, treatment regime and follow-up of patients with mut MMA from Jan 2013 to Dec 2017 in Shandong province, China. METHODS: Twenty patients were diagnosed with isolated mut MMA by elevated C3, C3/C2, and urine methylmalonic acid levels without hyperhomocysteinemia. The MUT gene was amplified and sequenced. Most patients received treatment with specific medical nutrition and oral l-carnitine after diagnosis. Metabolic parameters, clinical presentation and mental development were followed up. RESULTS: Among 20 patients with mut MMA, 14 had clinical presentations, and 12 presented in the neonatal period. Three patients died of metabolic crises triggered by infection. Twenty-three different mutations were detected, and four mutations (c.613G > A, c.446A > G, c.920-923delTCTT and c.1359delT) were novel. Most patients received timely treatment and had favorable metabolic responses, with reductions in C3, C3/C2 and urine MMA. We obtained 16 records of DQ/IQ assessments. Six patients exhibited normal development, but ten patients suffered from neurological symptoms of varying degrees and had low DQ/IQ scores. CONCLUSION: Our study contributes toward expanding the knowledge of the genetic basis of mut MMA. The c.914T > C was the most frequent mutation, and four novel mutations were detected. Patients diagnosed by newborn screening and treated at the presymptomatic stage may have better outcomes. However, these limited data do not allow any definitive statements on possible genotype-phenotype correlations that can influence the outcomes of mut MMA. Nonetheless, it is necessary for high-risk families to have early prenatal diagnoses.

Two novel mutations in the BCKDHB gene that cause maple syrup urine disease.

BACKGROUND: Maple syrup urine disease (MSUD) is a rare metabolic disorder of autosomal recessive inheritance caused by decreased activity of branched-chain α-ketoacid dehydrogenase complex (BCKD). Mutations in the three genes (BCKDHA, BCKDHB and DBT) are associated with MSUD. Here, we describe the presenting symptoms, clinical course and gene mutation analysis of a Chinese boy with MSUD. METHODS: Plasma amino acid analysis was performed by tandem mass spectrometry and the levels of organic acids in urine were measured with gas chromatography-mass spectrometry. The BCKDHB gene was sequenced by Sanger method. Furthermore, the significance of the novel mutations was predicted by Polyphen and Mutationtaster. After diagnosis, the patient was fed with protein-restricted diet to reduce intake of BCAA and was treated with l-carnitine. Metabolic parameters, clinical presentation and mental development were followed up. RESULTS: The patient was diagnosed as MSUD. Two novel BCKDHB mutations (c.523 T > C and c.478-25_552del100) were identified. In silico analysis predicted that the two mutations were "disease causing". The boy tolerated the treatment well and had symptomatic improvement. He presented with mild hypotonia and had nearly normal DQ scores at the age of 10 months. The two novel mutations resulted in the clinical manifestations of MSUD. Our results may reflect the heterogeneity of the pathogenic variants found in patients with MSUD.

Correlation study of cough variant asthma and mycoplasma pneumonia infection in children.

Cough variant asthma (CVA), as one of bronchitis diseases, features with repeated cough. In clinics, CAV does not show significant abnormal signs, therefore antibiotic therapy can hardly achieve satisfactory treatment effect. With the development of scientific technologies, the correlation between mycoplasma pneumoniae infection and CVA has become a hot research topic in clinics. In clinics, mycoplasma pneumoniae is extensively regarded as major cause for CVA, with complex pathogenic mechanism. The symptoms of CVA is characterized by chronic non abnormal inflammation, normally accompanied with bronchospasm and intestinal infection. Clinical practices show that about 6% of CVA children mainly show continuous cough till midnight during onset period, which is easily to misdiagnosed as bronchitis. Mycoplasma pneumoniae infection is a common disease threatening Children's health in China. With the annual increase of incidence of infantile pneumonia in China, mycoplasma pneumoniae infection has become a major reason leading to the death of child in China. More such mycoplasma pneumoniae infection is a sporadic disease spreading in a small range, and can onset in four seasons, making great impact on living quality and health of children. On this basis, this paper analyzes the correlation between Children's CVA and mycoplasma pneumoniae infection, in the hope of providing valuable reference for clinical treatment.

Household financial burden of phenylketonuria and its impact on treatment in China: a cross-sectional study.

BACKGROUND: Phenylketonuria (PKU) is a rare inborn disease, which, untreated, leading to severe neurobehavioral dysfunction. Considering its complexity, the management of PKU may bring a formidable economic burden to parents and caregivers. It is still unknown what the out-of-pocket expenses are for a patient with PKU in China. This paper explores the household financial burden of classical PKU and its impact on Chinese families in a quantitative manner for the first time. METHODS: A non-interventional and observational study was conducted at the China-Japan Friendship Hospital, one of the national centers for inherited metabolic disorders in China. The medical and non-medical household financial burdens were consolidated into a questionnaire to evaluate the out-of-pocket costs (OOPCs) of PKU treatment and follow-up. FINDINGS: The total OOPCs were USD$3766.1 (0y), USD$3795.2 (1-2 ys), USD$4657.7 (3-4 ys), USD$5979.9 (5-8 ys), and USD$5588.7 (9 ys and older) for PKU patients of different age groups. The median economic burden of classical PKU was 75.0 % of total annual family income (range 1.0-779.1 %), and 94.4 % of the families exceeding the threshold considered as catastrophic expenditure. There was a negative correlation between the financial burden and the proportion of time when Phe concentrations were in the desired target range (120-250 µmol/L) in 0-4-ys group (r = -0.474, p = 0.026). CONCLUSIONS: The management of PKU is associated with a severe financial burden on patients' families, which may lead to insufficient treatment or variation of blood Phe concentration. The current reimbursement policies are as yet inadequate. A national reimbursement system targeting treatment practices for PKU patients and other rare diseases across China is imperative.

Clinical presentation, gene analysis and outcomes in young patients with early-treated combined methylmalonic acidemia and homocysteinemia (cblC type) in Shandong province, China.

OBJECTIVES: To estimate the incidence of MMA on newborn screening in Shandong province from May 2011 to May 2014 and summarize the clinical presentation, biochemical features, mutation analysis, and treatment regime of early-treated patients with cblC disease. METHODS: Between May 2011 and May 2014, 35,291 newborns were screened for MMA in Jinan maternal and Child Care Hospital, Shandong province. The levels of C3, C3/C2, methionine and tHcy were measured. Most patients received treatment with intramuscular hydroxocobalamin after diagnosis. Metabolic parameters, clinical presentation and mental development were followed up. RESULTS: Nine patients were identified among 35,291 by newborn screening, giving an estimated incidence of 1:3920 live births for MMA, and all were classified as cblC disease. Among them, five patients received treatment with intramuscular hydroxocobalamin and two patients did not receive any treatment. One patient died of metabolic crises triggered by infection at the age of 38 days. Seven different mutations (c.609G>A, c.455_457delCCC, c.394C>T, c.445_446insA, c.658_660delAAG, c.452A>G and IVS1+1G>A) were detected. The mutations (c.455_457delCCC and IVS1+1G>A) are novel. Five patients who received treatment had favorable metabolic response, with both reduction of urine MMA and tHcy and increase of methionine. We obtained 7 records of DQ assessment. The five patients who received treatment presented with developmental delay and obvious neurological manifestations. In two patients who did not receive any treatment, case 8 presented with severe mental retardation and developmental delay, while case 9 had nearly normal DQ values at the age of 1(1/12)years. CONCLUSION: Our study characterized variable phenotypes of neurodevelopment in early-treated cblC patients diagnosed on newborn screening. The long-term outcomes of cblC disease are unsatisfactory in spite of conventional treatment and improvement of biochemical abnormalities. Although the number of patients is too small, the information provided in this work is of value in highlighting possible genotype-phenotype correlation that influences outcomes in cblC disease by future studies.

A De novo PAX8 mutation in a Chinese child with congenital thyroid dysgenesis.

BACKGROUND: Thyroid dysgenesis (TD) is the most frequent cause of congenital hypothyroidism (CH), but its pathogenesis remains unclear. As a thyroid transcription factor, paired box transcription factor 8 (PAX8) is essential for thyroid organogenesis and development. AIM: To screen PAX8 mutations and characterize the features of these mutations in Chinese TD patients. MATERIALS AND METHODS: Blood samples were collected from 63 TD patients in Shandong Province, China, and genomic DNA was extracted from peripheral blood leukocytes. Exon 3~4 of PAX8 were analyzed by PCR and direct sequencing. RESULTS: Direct sequencing of PAX8 revealed a heterozygous missense mutation (c.155G/C, P.Arg52Pro) in one child with agenesis. Genetic screening of the child's family revealed that the clinically unaffected parents do not carry the mutation, suggesting that the identified sequence change is a de novo mutation. CONCLUSION: We report a heterozygous missense de novo mutation in PAX8 in one out of 63 unrelated Chinese TD patients, showing that the PAX8 mutation rate is very low in TD patients in China. However, de novo mutation and epigenetic mechanisms need to be considered in the future study.

Diagnosis, treatment and follow-up of patients with tetrahydrobiopterin deficiency in Shandong province, China.

OBJECTIVES: To summarize the clinical and biochemical data, mutation analysis, treatment, outcome and the follow-up data of patients with BH4 deficiency from 2004 to 2012 in Shandong province, China. METHODS: We analyzed the clinical, biochemical and treatment data of 40 patients with BH4 deficiency. Urinary neopterin and biopterin were analyzed. Further BH4 loading tests were performed in suspected patients with abnormal urinary pterin profiles. The patients with BH4 deficiency were treated with BH4 and neurotransmitter after diagnosis. Blood phenylalanine level, clinical symptoms and mental development were followed up. RESULTS: 40 cases with BH4 deficiency were identified and all classified as PTPS deficiency between 2004 and 2012 in Shandong province, China. They were diagnosed at the age of 20d - 41m and most patients received treatment with BH4, l-dopa and 5-HTP after diagnosis. Seven different mutations (P87S, K91R, T106M, D96N, N52S, S21R, and L127F) were detected in 11 patients. But outcome assessments were not always available. We obtained 19 records of DQ/IQ assessment. In 9 patients (7 early and 2 late diagnosed) no development delay is observed, while in 10 patients (8 early and 2 late diagnosed) development was delayed. CONCLUSIONS: Our study emphasized that screening for BH4 deficiency should be carried out in all patients with HPA in order to minimize misdiagnosis. Although the outcomes of BH4 deficiency are highly variable, early diagnosis and treatment is essential for good outcomes.

A three-plasmid system for construction of armed oncolytic adenovirus.

There is growing interest in the use of oncolytic virus as a tool in cancer gene therapy. However, construction of oncolytic adenovirus (Ad) is not an easy task due to lack of convenient, robust methods. A three-plasmid system was introduced for construction of armed oncolytic Ad. Besides the pShuttle-CMV and pAdEasy-1, a third plasmid (pTE-ME1), harboring the E1 region of Ad5, was generated and included in this system. In pTE-ME1, the promoter of E1A was deleted and replaced with a multiple-cloning site (MCS). A therapeutic gene and tissue-specific promoter (TSP) could be inserted routinely into the MCS of pShuttle-CMV and pTE-ME1, respectively. The modified E1 region could then be excised from pTE-ME1 and integrated into the therapeutic gene-containing pShuttle-CMV to form the final shuttle plasmid. This shuttle plasmid was recombined with pAdEasy-1 in Escherichia coli strain BJ5183 to generate Ad plasmid. Finally, the oncolytic Ad could be rescued in Ad plasmid-transfected packaging cells. The GFP gene and the promoter of telomerase reverse transcriptase (TERTp) were chosen as the transgene and TSP, respectively, to test this system. Two oncolytic Ads, Ad-GFP-TPE and Ad-GFP-D19K, were generated successfully. Their oncolytic and replicating abilities were investigated in TERT-positive tumor cells. The results suggest that the three-plasmid system was practicable and could be used to construct other transcriptionally regulated oncolytic Ads carrying a therapeutic gene.

A recombinant adenovirus prime-virus-like particle boost regimen elicits effective and specific immunities against norovirus in mice.

Prime-boost vaccination using recombinant viral vectors and proteins has emerged as a highly effective strategy for protecting against viral pathogens. However, the ability of such regimens to provide immunity against norovirus (NV), an important cause of acute epidemic gastroenteritis worldwide, has never been assessed. In this study, we analyzed NV-specific humoral, mucosal, and cellular immune responses following intranasal immunization with the recombinant adenovirus expressing the NV GGII4 capsid protein (rAd) prime-NV virus-like particle (VLP) boost, VLP prime-rAd boost, or repeated NV VLP regimens. Our results show that mice primed with rAd and boosted with VLP had stronger humoral, mucosal, and interferon-gamma responses than those immunized with VLP prime-rAd boost or VLP alone. These results demonstrate that adenovirus prime-VLP boost vaccination is an effective strategy for induction of immune responses against NV and is a promising strategy to improve current VLP-based NV vaccine development.

Intranasal administration of a recombinant adenovirus expressing the norovirus capsid protein stimulates specific humoral, mucosal, and cellular immune responses in mice.

Norovirus (NV) is a major cause of acute, epidemic nonbacterial gastroenteritis in individuals of all ages. The immunological mechanism of NV infection and the approaches used to prevent infection remain to be elucidated. In this study, the specific immune responses of BALB/c mice were assessed following intranasal immunization with a recombinant adenovirus vector expressing the genogroup II4 (GGII/4) norovirus capsid protein. Analysis of IgM, IgG, and IgA antibodies specific for the recombinant virus-like particles (VLPs) of NV demonstrated that a high level of humoral immunity developed following immunization. Mucosal immune responses were also detectable in stool, intestinal homogenates, lung homogenates, and lung lavage samples. Specific cellular immune responses were observed in NV VLPs-restimulated splenocytes by ELISPOT and Th1/Th2 cytokine cytometric array (CBA). Serum IgG subclass analysis showed that a balanced Th1- and Th2-like cellular immune response was induced in BALB/c mice following immunization with recombinant adenovirus. These findings demonstrate that the intranasal immunization of a recombinant adenovirus expressing the NV capsid protein is an efficient strategy to stimulate systemic, mucosal, and cellular Th1/Th2 immune responses in mice, and could serve as a novel approach for designing NV vaccines.

[Improved replication of enteric adenovirus type 41 in Hep2 cell line expressing E1B55K].

Adenovirus type 40 and 41 (Ad40, Ad41), which belong to human adenovirus subgroup F, are called fastidious adenoviruses due to their property of poor growth in cultured cell lines in vitro The effect of expression of exogenous E1B55K in Hep2 on Ad41 replication in this cell line was investigated. E1B55K gene was amplified by PCR with DNA extracted from Ad41-positive feces supernatant as template. Eukaryotic expression plasmid (pcDNA3) carrying E1B55K was constructed, purified, and transferred into Hep2 cell. Expression of E1B55K in G418-resistant clones was assayed by RT-PCR, and one clone named as Hep2-E1B4#4 could produce more Ad41 progenies when compared with other clones by the method of inducing complete cytopathic effect (CPE) in 293 cells. Infection of equivalent Ad41 caused more significant cytopathic effect (CPE) in Hep2-E1B#4 than that in the control cells of Hep2 or Hep2-DNA3, also suggesting enhanced viral replication in Hep2-E1B#4. The titer of Ad41 was further determined by method of immunocytochemical staining, and semi-quantity PCR was employed to compare the copy number of Ad41 genome DNA. The results showed that the yield of Ad41 in Hep2-E1B#4 was more than 9 times of that in control cells when equal amount of seed viruses were incubated, and the copy number of Ad41 genome increased 4 times in the raw extract from the infected Hep2-E1B#4 when compared with that from control cells. In conclusion, E1B55K gene transfer improved the ability of Hep2 in packaging Ad41, and the Hep2-E1B#4 cell line, which expressed E1B55K constitutively, would be helpful in isolation, cultivation and amplification of Ad41.