Dual rare genetic diseases in five pediatric patients: insights from next-generation diagnostic methods.

BACKGROUND: Clinicians traditionally aim to identify a singular explanation for the clinical presentation of a patient; however, in some cases, the diagnosis may remain elusive or fail to comprehensively explain the clinical findings. In recent years, advancements in next-generation sequencing, including whole-exome sequencing, have led to the incidental identification of dual diagnoses in patients. Herein we present the cases of five pediatric patients diagnosed with dual rare genetic diseases. Their natural history and diagnostic process were explored, and lessons learned from utilizing next-generation diagnostic technologies have been reported. RESULTS: Five pediatric cases (3 boys, 2 girls) with dual diagnoses were reported. The age at diagnosis was from 3 months to 10 years. The main clinical presentations were psychomotor retardation and increased muscular tension, some accompanied with liver dysfunction, abnormal appearance, precocious puberty, dorsiflexion restriction and varus of both feet, etc. After whole-exome sequencing, nine diseases were confirmed in these patients: Angelman syndrome and Krabbe disease in case 1, Citrin deficiency and Kabuki syndrome in case 2, Homocysteinemia type 2 and Copy number variant in case 3, Isolated methylmalonic acidemia and Niemann-Pick disease type B in case 4, Isolated methylmalonic acidemia and 21-hydroxylase deficiency in case 5. Fifteen gene mutations and 2 CNVs were identified. Four novel mutations were observed, including c.15292de1A in KMT2D, c.159_164inv and c.1427G > A in SLC25A13, and c.591 C > G in MTHFR. CONCLUSIONS: Our findings underscore the importance of clinicians being vigilant about the significance of historical and physical examination. Comprehensive clinical experience is crucial for identifying atypical clinical features, particularly in cases involving dual rare genetic diseases.

Stable and ultralong room-temperature phosphorescent copolymers with excellent adhesion, resistance, and toughness.

Developing stable room-temperature phosphorescence (RTP) emission without being affected by moisture and mechanical force remains a great challenge for purely organic systems, due to their triplet states sensitive to the infinitesimal motion of phosphors and the oxygen quencher. We report a kind of highly robust phosphorescent systems, by doping a rigid phosphor into a copolymer (polyvinyl butyral resin) matrix with a balance of mutually exclusive features, including a rigidly hydrophilic hydrogen bond network and elastically hydrophobic constituent. Impressively, these RTP polymeric films have superior adhesive ability on various surfaces and showed reversible photoactivated RTP with lifetimes up to 5.82 seconds, which can be used as in situ modulated anticounterfeit labels. They can maintain a bright afterglow for over 25.0 seconds under various practical conditions, such as storage in refrigerators, soaking in natural water for a month, or even being subjected to strong collisions and impacts. These findings provide deep insights for developing stable ultralong RTP materials with desirable comprehensive performance.

Structure and dynamics of dynamic covalent cross-linked PEOs and PEO/LiPF6 electrolytes: a coarse-grained simulation study.

The incorporation of dynamic covalent bonds has been an attractive strategy to synthesize adaptive solid polymer electrolytes (SPEs). Here, we present molecular dynamics results concerning the relationship between ion transport and segmental dynamics for dynamic covalent cross-linked PEO-Li+ SPEs. To dissolve LiPF6 into PEO, a 1/r4-form approximation of ion-dipole interactions is employed as the solvation potential. Its parameters are estimated with the assistance of the Bayesian optimization algorithm and validated by comparing the resulting behaviors of PEO/LiPF6 with experimental observations. The dynamic associations of EO with Li+ and PF6- significantly reduce the segmental mobility of PEO, verifying the coupling of PEO segmental dynamics with ion transport. In order to reproduce the unique behaviors of associative covalent adaptive networks (CANs), the bond-exchange reaction is controlled by the collision probability and the user-defined activation energy (Ea ≥ 0) based on a hybrid of molecular dynamics and Monte Carlo methods. The dynamics of network topology, facilitated by the reshuffling of dynamic covalent bonds, is analyzed using graph theory. The network mesh size varies with time, which can be considered as one of the characteristics for associative CANs. The reshuffling of dynamic bonds releases the constraint from cross-linked structures, and enhances the long-range segmental mobility as well as the mobilities of Li+ and PF6-. By drawing comparisons with its conventionally cross-linked counterpart, the effect of dynamic-bond reshuffling on ion transport is studied for the dynamic covalent cross-linked PEO16-LiPF6 electrolyte in terms of self-diffusivities, cation transference number, and ionic conductivity.

Silica-based mesoporous ion-imprinted fluorescent sensors for the detection of Pb2+in aqueous environments.

In this work, an environment-friendly core-shell material based on CDs@SiO2as the core and mesoporous ion-imprinted layer as the shell was reported. As a highly sensitive and accurate fluorescent sensor for the detection of Pb2+in environmental water, the composition combined ion imprinting technology with quantum dots to selectively quench the fluorescence of CDs by metal coordination in the presence of Pb2+, and the visual change of gradually weakening blue color could be observed by the naked eye for visual detection. The mesoporous structure significantly improved the detection recognition rate of CDs@SiO2@MIIPs.The molecularly imprinted sensor presented a favorable linear relationship over a Pb2+concentration range from 10 nmol l-1to 100 nmol l-1and a detection limit of 2.16 nmol l-1for Pb2+. The imprinting factor of the CDs@SiO2@MIIPs was 5.13. The sensor has a fast detection rate, is highly selective in the identification of Pb2+, and can be reused up to 10 times. The applicability of the method was evaluated by the determination of Pb2+in spiked environmental water samples with satisfactory results.

Comparing amniotic fluid mass spectrometry assays and amniocyte gene analyses for the prenatal diagnosis of methylmalonic aciduria.

BACKGROUND: Methylmalonic aciduria (MMA), a rare inherited disorder, is the most common organic aciduria in China, and prenatal diagnosis has contributed to its prevention. However, the prenatal diagnosis of MMA using cultured amniocytes or chorionic villi to detect gene mutations is exclusively applicable to families with a definite genetic diagnosis. To evaluate the reliability of mass spectrometry assays for the prenatal diagnosis of MMA, we conducted a retrospective study of our 10 years' experience. MATERIALS AND METHODS: This retrospective compare study reviewed the medical records for maternal and fetuses data for 287 mothers with a family history of MMA from June 2010 to December 2020. Methylmalonate and propionylcarnitine in cell-free amniotic fluid were measured using a stable isotope dilution method (GC/MS) and MS/MS-based method (LC/MS/MS). Total homocysteine (tHcy) was measured by fluorescence polarization immunoassay. Depending on the presence of disease-causing gene mutations in probands, gene studies on amniocytes from 222 pregnant women were performed. RESULTS: For 222 fetuses of the families with definite genetic diagnosis, gene analyses were performed using cultured amniocytes. 52 fetuses were affected by MMA, whereas 170 were "unaffected". For GC/MS and LC/MS/MS, the specificity was 96.5% and 95.9%, sensitivity was 71.2% and 84.6%, respectively. The positive and negative predictive values were 86.0% and 91.6% and 86.3% and 95.3%, respectively. Propionylcarnitine/butyrylcarnitine ratio showed the highest accuracy and could thus serve as a sensitive indicator to identify those at a risk for MMA. When GC/MS and LC/MS/MS were performed in parallel, the specificity was 92.5% and sensitivity was 95.6%. When evaluating tHcy, the positive and negative predictive values were 95.0% and 96.1%, respectively. In 65 fetuses without family genetic diagnosis, 11 were finally confirmed to have MMA and 54 were "unaffected" by amniotic fluid biochemical assays. The 54 children showed normal urine organic acids and healthy development after birth. CONCLUSIONS: Amniotic fluid biochemical assays using GC/MS and LC/MS/MS in parallel increased the accuracy of prenatal diagnosis of MMA. Propionylcarnitine is a more reliable marker than methylmalonic acid in amniotic fluid. Further, tHcy is recommended for the prenatal diagnosis of combined MMA and homocysteinemia.

Discovery and Pharmacological Characterization of JNJ-64619178, a Novel Small-Molecule Inhibitor of PRMT5 with Potent Antitumor Activity.

The protein arginine methyltransferase 5 (PRMT5) methylates a variety of proteins involved in splicing, multiple signal transduction pathways, epigenetic control of gene expression, and mechanisms leading to protein expression required for cellular proliferation. Dysregulation of PRMT5 is associated with clinical features of several cancers, including lymphomas, lung cancer, and breast cancer. Here, we describe the characterization of JNJ-64619178, a novel, selective, and potent PRMT5 inhibitor, currently in clinical trials for patients with advanced solid tumors, non-Hodgkin's lymphoma, and lower-risk myelodysplastic syndrome. JNJ-64619178 demonstrated a prolonged inhibition of PRMT5 and potent antiproliferative activity in subsets of cancer cell lines derived from various histologies, including lung, breast, pancreatic, and hematological malignancies. In primary acute myelogenous leukemia samples, the presence of splicing factor mutations correlated with a higher ex vivo sensitivity to JNJ-64619178. Furthermore, the potent and unique mechanism of inhibition of JNJ-64619178, combined with highly optimized pharmacological properties, led to efficient tumor growth inhibition and regression in several xenograft models in vivo, with once-daily or intermittent oral-dosing schedules. An increase in splicing burden was observed upon JNJ-64619178 treatment. Overall, these observations support the continued clinical evaluation of JNJ-64619178 in patients with aberrant PRMT5 activity-driven tumors.

A Chemical Probe for the Methyl Transferase PRMT5 with a Novel Binding Mode.

Protein arginine methyltransferase 5 (PRMT5) is an enzyme that can symmetrically dimethylate arginine residues in histones and nonhistone proteins by using S-adenosyl methionine (SAM) as the methyl donating cofactor. We have designed a library of SAM analogues and discovered potent, cell-active, and selective spiro diamines as inhibitors of the enzymatic function of PRMT5. Crystallographic studies confirmed a very interesting binding mode, involving protein flexibility, where both the cofactor pocket and part of substrate binding site are occupied by these inhibitors.

Extremely Stretchable Vitrimers.

Vitrimers are covalent adaptable networks, having many interesting versatile abilities with unprecedented potentials. Here, the combination of a low-Tg polymer system with dioxaborolane metathesis is used to develop catalyst-free vitrimers that can be stretched to more than 8900× their original length at a moderate stretching rate (≈50 mm min-1 ). Superstretchable vitrimers are prepared from biodegradable xylitol-based polyol oligomers and cross-linked by dioxaborolane linkages. They are also found to be remarkable in terms of mechanical strength and other properties, such as malleability, self-healing ability, puncture resistance, and processing stability. Furthermore, the repeated rearrangements of dioxaborolane linkages and hydrogen bonds give rise to efficient energy dissipation with a maximum efficiency of 88%, allowing the superstretchable vitrimers to be promising for energy absorbing applications.

Microstructure and antibacterial efficacy of graphene oxide nanocomposite fibres.

Antibacterial polymer nanocomposite fibre meshes containing graphene oxide (GO) nanosheets were successfully prepared by pressurised gyration. The morphological and chemical composition of the resulting fibre meshes were determined using Scanning Electron Microscopy (SEM), Raman spectroscopy, Raman mapping and Fourier-Transform Infrared Spectroscopy (FT-IR). SEM showed the fibres to have an average diameter increasing from ~1-4 µm as the GO loading increased. FT-IR and Raman spectroscopy confirmed the inclusion of GO nanosheets on the fibre surface. The antibacterial potential of GO nanocomposite fibres were investigated using Escherichia coli K12. Average bacterial reduction ranged from 46 to 85 % with results favouring the strongest bioactivities of the nanocomposite containing 8 wt% of GO. Finally, bacterial toxicity of the nanocomposites was evaluated by reactive oxygen species (ROS) formation. A mechanism for the antibacterial behaviour of the nanocomposite fibres is presented. Stimulated Raman scattering imaging and spectra of the fibres post antibacterial studies showed flakes of GO distributed across the surface of the poly(methyl 2-methylpropenoate) (PMMA) fibres, which contribute to the high killing efficacy of the composites towards E. coli. GO nanosheets embedded in a polymer matrix have demonstrated the ability to retain their antibacterial properties, thus offering themselves as a promising antibacterial agent.

Preparation of 4'-Spirocyclobutyl Nucleoside Analogues as Novel and Versatile Adenosine Scaffolds.

Despite the large variety of modified nucleosides that have been reported, the preparation of constrained 4'-spirocyclic adenosine analogues has received very little attention. We discovered that the [2+2]-cycloaddition of dichloroketene on readily available 4'-exo-methylene furanose sugars efficiently results in the diastereoselective formation of novel 4'-spirocyclobutanones. The reaction mechanism was investigated via density functional theory (DFT) and found to proceed either via a non-synchronous or stepwise reaction sequence, controlled by the stereochemistry at the 3'-position of the sugar substrate. The obtained dichlorocyclobutanones were converted into nucleoside analogues, providing access to a novel class of chiral 4'-spirocyclobutyl adenosine mimetics in eight steps from commercially available sugars. Assessment of the biological activity of designed 4'-spirocyclic adenosine analogues identified potent inhibitors for protein methyltransferase target PRMT5.

Facile Fabrication of Porous Conductive Thermoplastic Polyurethane Nanocomposite Films via Solution Casting.

Porous conductive polymers are one of important materials, featuring lightweight, large specific surface area and high porosity. Non-solvent induced phase separation is widely employed to prepare porous polymer sheet materials. Through utilizing water vapor in ambient environment as the non-solvent, a facile approach was developed to produce porous conductive polymer nanocomposites using the conventional solution-casting method. Without using any non-solvent liquids, porous carbon nanofiber/thermoplastic polyurethane (CNF/TPU) nanocomposites were prepared directly by solution casting of their dimethylformamide (DMF) solutions under ambient conditions. The strength of the CNF framework played a key role in preventing the collapse of pores during DMF evaporation. The dependence of porous structures on CNF loading was studied by scanning electron microscopy and porosity measurement. The influence of CNF loading on the mechanical properties, electrical conductivity and piezoresistive behavior was explored.

Highly Stretchable Conductors Based on Expanded Graphite Macroconfined in Tubular Rubber.

Highly stretchable and durable conductors are significant to the development of wearable devices, robots, human-machine interfaces, and other artificial intelligence products. Although many respectable methods have been reported, it is still a challenge to fabricate stretchable conductors with a large elastic limit, high conductivity, and excellent reliability in rapid, effective, and economic ways. Herein, a facile method is offered to fabricate high-performance stretchable tubular conductors (TCs) based on a macroconfined structure of expanded graphite (EG) in rubber tubing by simply physical packing. The maximum original electrical conductivity of TCs reached a high value of 160.6 S/cm. Meanwhile, TCs showed more insensitive response of conductivity to increasing tensile strain compared to the TCs encapsulated with liquid metal or ionic liquid. The conductivity and effective stretchability of TCs can be adjusted by varying the packing density of EG. A low gauge factor below 3 was reached even under 400% stretching for TCs with a packing density of 1.233 g/cm3. The excellent resilience and good stability of conductivity of TCs during dynamic stretching-releasing cycles are attributed to the stable and rapid reconstruction of the percolation network of EG particles. The combination of high conductivity, tunable stretchability, and good reliability renders potential applications to TCs, such as highly stretchable interconnects or strain sensors, in human motion detection.

Facile and Scalable Synthesis Method for High-Quality Few-Layer Graphene through Solution-Based Exfoliation of Graphite.

Here we describe a facile and scalable method for preparing defect-free graphene sheets exfoliated from graphite using the positively charged polyelectrolyte precursor poly(p-phenylenevinylene) (PPV-pre) as a stabilizer in an aqueous solution. The graphene exfoliated by PPV-pre was apparently stabilized in the solution as a form of graphene/PPV-pre (denoted to GPPV-pre), which remains in a homogeneous dispersion over a year. The thickness values of 300 selected 76% GPPV-pre flakes ranged from 1 to 10 nm, corresponding to between one and a few layers of graphene in the lateral dimensions of 1 to 2 µm. Furthermore, this approach was expected to yield a marked decrease in the density of defects in the electronic conjugation of graphene compared to that of graphene oxide (GO) obtained by Hummers' method. The positively charged GPPV-pre was employed to fabricate a poly(ethylene terephthalate) (PET) electrode layer-by-layer with negatively charged GO, yielding (GPPV-pre/GO)n film electrode. The PPV-pre and GO in the (GPPV-pre/GO)n films were simultaneously converted using hydroiodic acid vapor to fully conjugated PPV and reduced graphene oxide (RGO), respectively. The electrical conductivity of (GPPV/RGO)23 multilayer films was 483 S/cm, about three times greater than that of the (PPV/RGO)23 multilayer films (166 S/cm) comprising RGO (prepared by Hummers method). Furthermore, the superior electrical properties of GPPV were made evident, when comparing the capacitive performances of two supercapacitor systems; (polyaniline PANi/RGO)30/(GPPV/RGO)23/PET (volumetric capacitance = 216 F/cm3; energy density = 19 mWh/cm3; maximum power density = 498 W/cm3) and (PANi/RGO)30/(PPV/RGO)23/PET (152 F/cm3; 9 mWh/cm3; 80 W/cm3).

Synthesis of Multiwalled Carbon Nanotube-Reinforced Polyborosiloxane Nanocomposites with Mechanically Adaptive and Self-Healing Capabilities for Flexible Conductors.

Intrinsic self-healing polyborosiloxane (PBS) and its multiwalled carbon nanotube (MWCNT)-reinforced nanocomposites were synthesized from hydroxyl terminated poly(dimethylsiloxane) (PDMS) and boric acid at room temperature. The formation of Si-O-B moiety in PBS was confirmed by Fourier transform infrared spectroscopy. PBS and its MWCNT-reinforced nanocomposites were found possessing water- or methanol-activated mechanically adaptive behaviors; the compressive modulus decreased substantially when exposed to water or methanol vapor and recovered their high value after the stimulus was removed. The compressive modulus was reduced by 76%, 86%, 90%, and 83% for neat PBS and its nanocomposites containing 3.0, 6.2, and 13.3 wt % MWCNTs, respectively, in water vapor, and the modulus reduction activated by methanol vapor was greater than by water vapor. MWCNTs at higher contents acted as a continuous electrical channel in PBS offering electrical conductivity, which was up to 1.21 S/cm for the nanocomposite containing 13.3 wt % MWCNTs. The MWCNT-reinforced PBS nanocomposites also showed excellent mechanically and electrically self-healing properties, moldability, and adhesion to PDMS elastomer substrate. These properties enabled a straightforward fabrication of self-repairing MWCNT/PBS electronic circuits on PDMS elastomer substrates.

[Psychomotor retardation and intermitent convulsions for 8 months in an infant].

This study reports a boy with psychomotor retardation and epilepsy due to maternal phenylketonuria (PKU). The boy was admitted at the age of 20 months because of psychomotor retardation and epilepsy. He had seizures from the age of 1 year. His development quotient was 43. He presented with microcephaly, normal skin and hair color. Brain MRI scan showed mild cerebral white matter demyelination, broadening bilateral lateral ventricle and foramen magnum stricture. Chromosome karyotype, urine organic acids, blood amino acids and acylcarnitines were normal. His mother had mental retardation from her childhood. She presented with learning difficulties and yellow hair. Her premarriage health examinations were normal. She married a healthy man at age of 26 years. When she visited us at 28 years old, PKU was found by markedly elevated blood phenylalanine (916.54 µmol/L vs normal range 20-120 µmol/L). On her phenylalanine hydroxylase (PAH) gene, a homozygous mutations c.611A>G (p.Y204C) was identified, which confirmed the diagnosis of PAH-deficient PKU. Her child carries a heterozygous mutation c.611A>G with normal blood phenylalanine. Her husband had no any mutation on PAH. It is concluded that family investigation is very important for the etiological diagnosis of the children with mental retardation and epilepsy. Carefully clinical and metabolic survey should be performed for the parents with mental problems to identify parental diseases-associated child brain damage, such as maternal PKU.

[Acute brainstem encephalitis and myelitis in a girl with isolated methylmalonic aciduria due to MUT gene defect].

Methylmalonyl CoA mutase deficiency due to MUT gene defect has been known as the main cause of isolated methylmalonic acidemia in Mainland China. This study reported a patient with isolated methylmalonic aciduria (MUT type) characterized as acute brainstem encephalitis and myelitis. The previously healthy girl presented with fever, lethargy and progressive weakness in her extremities at the age of 3 years and 2 months. Three day later, she had respiratory distress and consciousness. Cranial MRI revealed bilateral symmetrical lesion of pallidum, brain stem and spinal cord, indicating acute brainstem encephalitis and myelitis. Her blood propionylcarnitine (6.83 µmol/L vs normal range 1.0 to 5.0 µmol/L) and urinary methylmalonic acid (133.22 mmol/mol creatinine vs normal range 0.2 to 3.6 mmol/mol creatinine) increased significantly. Plasma total homocysteine was normal. On her MUT gene, a reported mutation (c.1630_1631GG>TA) and a novel mutation (c.1663C>T, p.A555T) were identified, which confirmed the diagnosis of methylmalonic aciduria (MUT type). After cobalamin injection, protein-restricted diet with the supplements of special formula and L-carnitine, progressive improvement has been observed. The clinical manifestation of patients with methylmalonic aciduria is complex. Metabolic study and gene analysis are keys for the diagnosis and treatment of the disorder.

[Seven patients of argininemia with spastic tetraplegia as the first and major symptom and prenatal diagnosis of two fetuses with high risk].

OBJECTIVE: Argininemia is a rare disorder of urea cycle defect. The clinical manifestations of this disorder are similar to those of cerebral palsy so that the diagnosis is usually much delayed. This study aimed to investigate the phenotypes and genotypes of seven Chinese patients suffering from argininemia. METHOD: Three boys and four girls with spastic tetraplegia were diagnosed as argininemia by blood aminoacids analysis and ARG1 gene study. Patients were given a protein-restricted diet, citrulline, sodium benzoate, and other treatment intervention. The mother of Patient 5 and 6 accepted genetic counseling and underwent prenatal diagnosis by amniocentesis. RESULT: Seven patients presented with progressive spastic tetraplegia and poor physical growth from the age of 1 month to 4 years. Argininemia was found at the age of 1 year and 10 months to 12 years. Five patients had mental retardations. Three had seizures. Their blood arginine elevated (86.66 to 349.83 µmol/L, normal controls 5 to 25 µmol/L). Liver dysfunction was found in six patients. Five patients had elevated blood ammonia levels. In four patients, cerebral atrophy was observed by cranial magnetic resonance imaging. Nine mutations in the ARG1 gene were identified from 7 patients. Only two mutations, c.703G > A in exon 7 and c.32T > C in exon 1 had been reported. c.34G > T, c.53G > A, c.67delG, c.232dupG, c.374C > T, c.539G > C and c.646-649delCTCA, were novel mutations of ARG1. A homozygous mutation c.703G > A was found in the amniocytes of Patient 5's mother, indicating that the fetus was affected by argininemia. Induced abortion was performed. c.53G > A from Patient 6 was not found in the amniocytes of her mother, indicating that the fetus was not affected by hepatocyte arginase deficiency. The result was confirmed by postnatal mutation analysis of cord blood and the normal blood arginine of the newborn. CONCLUSION: Argininemia is one of the few treatable causes of pediatric spastic paralysis. In this study, seven Chinese patients with spastic tetraplegia were detected by blood aminoacids analysis and confirmed by molecular analysis. Seven novel mutations on ARG1 gene were identified. Prenatal diagnosis of the fetus of a family was performed by amniocytes ARG1 gene analysis.

[A Chinese boy with methylmalonic aciduria cblB type and a novel mutation in the MMAB gene].

cblB defect is a rare type of methylmalonic aciduria. In this study, a Chinese boy was diagnosed with methylmalonic aciduria cblB type and a novel mutation in the MMAB gene. The clinical presentations, blood acylcarnitines profiles, urine organic acids and genetic features of the patient were reported. The boy presented with fever, feeding difficulty and lethargy at the age of 2 months. Seven days later, he had coma, cold limb, thrombocytopenia, metabolic acidosis and liver damage. His blood propionylcarnitine and urinary methylmalonic acid levels increased significantly, but the plasma total homocysteine level was in the normal range, which supported the diagnosis of isolated methylmalonic aciduria. Gene analysis was performed by direct sequencing. No mutation in the MUT gene was found. However, a reported mutation c.577G>A (p.E193K) and a novel mutation c.562G>A (p.V188M) in the MMAB gene were identified, which confirmed the diagnosis of methylmalonic aciduria cblB type. Progressive clinical and biochemical improvement has been observed after hydroxylcobalamin injection, protein-restricted diet with the supplements of special formula and L-carnitine. He is currently 3 years and 11 months old and has a normal development condition. The phenotypes of the patients with cblB defect are nonspecific. Metabolic analysis and MMAB gene analysis are keys for the diagnosis of the disorder.

[Acute encephalopathy induced by vaccination in an infant with methylmalonic aciduria cblA].

OBJECTIVE: We report the first case of acute encephalopathy induced by vaccination in an infant with methylmalonic aciduria cblA in China. METHOD: The clinical presentation, blood acylcarnitines analysis, urine organic acids analysis and gene studies of the patient were summarized. RESULT: The proband, a boy, was admitted at the age of 15 months because of recurrent vomiting, acidosis and development delay for 8 months. The previously healthy boy presented vomiting and coma just one hour after hepatitis B vaccination at the age of seven months. Moderate dehydration, electrolyte disturbance and metabolic acidosis had been found. Although his acute metabolic crisis had been corrected soon after intravenous transfusion, psychomotor retardation and recurrent vomiting had been observed. When he was 15 months old, vomiting and lethargy occurred again 3 hours after DTaP vaccination. He was weakened as the illness became worse and got coma with dyspnea 7 days later. He was hospitalized with the suspected diagnosis of viral encephalitis. Blood acylcarnitines analysis, urine organic acids analysis and gene study had been performed for the etiologic investigation.His blood propionylcarnitine (16.3 µmol/L vs. normal range 1.0-5.0 µmol/L) and propionylcarnitine/free carnitine ratio (0.27 vs. normal range 0.03 to 0.25) increased. Markedly elevated urinary methylmalonic acid (388.21 mmol/mol creatinine vs. normal range 0.2 to 3.6 mmol/mol creatinine) and normal plasma total homocysteine supported the diagnosis of isolated methylmalonic aciduria. Two mutations, c.650 T>A (p.L217X) and c.742 C>T (p.Q248X), were identified in his MMAA gene, confirmed the diagnosis of cblA. Each parent carried one of the two mutations. Progressive clinical and biochemical improvement has been observed after hydroxylcobalamin injection, protein-restricted diet with the supplements of special formula and L-carnitine. He is currently 2 years and 7 months old with normal development and general condition. CONCLUSION: A boy with cblA was firstly detected after the acute encephalopathy induced by vaccination in China. It is important to pay more attention to the patients with metabolic crisis or organ damage after vaccination. Metabolic studies are keys to the diagnosis of potential diseases and improve the outcome.

Site-specific protein modification using immobilized sortase in batch and continuous-flow systems.

Transpeptidation catalyzed by sortase A allows the preparation of proteins that are site-specifically and homogeneously modified with a wide variety of functional groups, such as fluorophores, PEG moieties, lipids, glycans, bio-orthogonal reactive groups and affinity handles. This protocol describes immobilization of sortase A on a solid support (Sepharose beads). Immobilization of sortase A simplifies downstream purification of a protein of interest after labeling of its N or C terminus. Smaller batch and larger-scale continuous-flow reactions require only a limited amount of enzyme. The immobilized enzyme can be reused for multiple cycles of protein modification reactions. The described protocol also works with a Ca(2+)-independent variant of sortase A with increased catalytic activity. This heptamutant variant of sortase A (7M) was generated by combining previously published mutations, and this immobilized enzyme can be used for the modification of calcium-senstive substrates or in instances in which low temperatures are needed. Preparation of immobilized sortase A takes 1-2 d. Batch reactions take 3-12 h and flow reactions proceed at 0.5 ml h(-1), depending on the geometry of the reactor used.