Allelic phenotype prediction of phenylketonuria based on the machine learning method.

BACKGROUND: Phenylketonuria (PKU) is caused by mutations in the phenylalanine hydroxylase (PAH) gene. Our study aimed to predict the phenotype using the allelic genotype. METHODS: A total of 1291 PKU patients with 623 various variants were used as the training dataset for predicting allelic phenotypes. We designed a common machine learning framework to predict allelic genotypes associated with the phenotype. RESULTS: We identified 235 different mutations and 623 various allelic genotypes. The features extracted from the structure of mutations and graph properties of the PKU network to predict the phenotype of PKU were named PPML (PKU phenotype predicted by machine learning). The phenotype of PKU was classified into three different categories: classical PKU (cPKU), mild PKU (mPKU) and mild hyperphenylalaninemia (MHP). Three hub nodes (c.728G>A for cPKU, c.721 for mPKU and c.158G>A for HPA) were used as each classification center, and 5 node attributes were extracted from the network graph for machine learning training features. The area under the ROC curve was AUC = 0.832 for cPKU, AUC = 0.678 for mPKU and AUC = 0.874 for MHP. This suggests that PPML is a powerful method to predict allelic phenotypes in PKU and can be used for genetic counseling of PKU families. CONCLUSIONS: The web version of PPML predicts PKU allele classification supported by applicable real cases and prediction results. It is an online database that can be used for PKU phenotype prediction http://www.bioinfogenetics.info/PPML/ .

[Newborn screening, clinical features and genetic analysis for Citrin deficiency in Henan province].

OBJECTIVE: To explore the prevalence, clinical features, genetic characteristics and prognosis of Citrin deficiency in Henan province of China. METHODS: A total of 986 565 neonates screened by tandem mass spectrometry at the Third Affiliated Hospital of Zhengzhou University from January 2013 to December 2021 were retrospectively analyzed. Analysis of SLC25A13 gene variants and parental verification were carried out for neonates suspected for Citrin deficiency by next-generation sequencing. The clinical, biochemical and genetic characteristics of Citrin deficiency patients were integrated to guide the diet treatment and follow up the growth and development. Paired-t test was used to compare the amino acid levels in the peripheral blood samples before and after the treatment. RESULTS: Nine cases of Citrin deficiency were diagnosed among the 986 565 neonates. Specific elevation of citrulline was observed in all of the 9 cases. Six variants were detected by genetic sequencing, among which c.852_855delTATG, c.615+5G>A, c.550C>T and IVS16ins3kb were known pathogenic variants, whilst c.1111_1112delAT and c.837T>A were unreported previously. The detection rate for c. 852_855delTATG was the highest (61.6%, 11/18), followed by IVS16ins3kb (16.7%, 3/18). The clinical symptoms of all patients were relieved after the treatment, and the blood amino acid profile and biochemical parameters were significantly improved by gradually falling within the normal range. By June 2022, all patients had shown a good prognosis. CONCLUSION: The prevalence of Citrin deficiency among neonates from Henan Province by tandem mass spectrometry is 1/109 618, and the carrier rate for the pathogenic variants of the SLC25A13 gene was 1/166. The c.852_855delTATG may be a hot spot variant among the patients. Discovery of the novel variants has enriched the mutational spectrum of the SLC25A13 gene. Above results have provided a basis for the early diagnosis, treatment, prognosis and genetic counseling for the affected families.

Identification of phenylketonuria patient genotypes using single-gene full-length sequencing.

BACKGROUND: Phenylketonuria (PKU) is a common, autosomal recessive inborn error of metabolism caused by PAH gene variants. After routine genetic analysis methods were applied, approximately 5% of PKU patients were still not diagnosed with a definite genotype. METHODS: In this study, for the first time, we identified PKU patients with unknown genotypes via single-gene full-length sequencing. RESULTS: The detection rate of PKU genotype increased from 94.6 to 99.4%, an increase of approximately 5%. The variants c.1199 + 502A > T and 1065 + 241C > A were found at a high frequency in Chinese PKU patients. CONCLUSION: Our study suggest that single-gene full-length sequencing is a rapid, efficient and cost-effective tool to improve the genotype detection rate of PKU patients. Moreover, we provides additional case data to support pathogenicity of deep intronic variants in PAH.

Elemental composition and microbial community differences between wastewater treatment plant effluent and local natural surface water: A Zhengzhou city study.

Wastewater treatment plant (WWTP) effluent is discharged into rivers as supplemental water, which may result in ecological risk. This study compares the element composition and microbial community of WWTP effluent and natural surface water (NSW) and reveals the potential ecological risk of WWTP effluent discharge. Twenty recently upgraded WWTPs and three relatively large reservoirs in Zhengzhou city, China, were selected. The contents of N, P, S, K, Ca, Mg, B, Si, Na, Fe, Mn, Co, Ni and Sn were significantly higher in WWTP effluent than in NSW, while those of Mo, V, Pb and Cd were significantly lower. There was no significant difference between WWTP effluent and NSW in terms of the element imbalance index (IMI) (representing the extent of imbalance of element proportions) relative to the class IV surface water quality standard (the control standard for most Chinese rivers). The macronutrient IMI relative to the Hoagland formula was significantly lower in WWTP effluent than in NSW, and WWTP effluent discharge could significantly lower this index in NSW; this may be an important cause of primary productivity explosion. The microbial diversity was significantly higher in WWTP effluent than in NSW. The predicted relative abundances of mobile genetic elements and oxidative-stress-tolerant phenotypes were significantly higher in WWTP effluent than in NSW, whereas the abundance of gram-negative phenotypes was significantly lower, and that of potential pathogenic phenotypes was slightly lower. The effluent from upgraded WWTPs exhibited a low risk of pathogen diffusion but a high risk of antibiotic resistance gene diffusion. The element composition and microbial community should be considered when evaluating the ecological risk of WWTP effluent discharge.

Dual CRISPR interference and activation for targeted reactivation of X-linked endogenous FOXP3 in human breast cancer cells.

BACKGROUND: Unlike autosomal tumor suppressors, X-linked tumor suppressors can be inactivated by a single hit due to X-chromosome inactivation (XCI). Here, we argue that targeted reactivation of the non-mutated allele from XCI offers a potential therapy for female breast cancers. METHODS: Towards this goal, we developed a dual CRISPR interference and activation (CRISPRi/a) approach for simultaneously silencing and reactivating multiple X-linked genes using two orthogonal, nuclease-deficient CRISPR/Cas9 (dCas9) proteins. RESULTS: Using Streptococcus pyogenes dCas9-KRAB for silencing XIST and Staphylococcus aureus dCas9-VPR for activating FOXP3, we achieved CRISPR activation of FOXP3 in various cell lines of human female breast cancers. In human breast cancer HCC202 cells, which express a synonymous heterozygous mutation in the coding region of FOXP3, simultaneous silencing of XIST from XCI led to enhanced and prolonged FOXP3 activation. Also, reactivation of endogenous FOXP3 in breast cancer cells by CRISPRi/a inhibited tumor growth in vitro and in vivo. We further optimized CRISPRa by fusing dCas9 to the demethylase TET1 and observed enhanced FOXP3 activation. Analysis of the conserved CpG-rich region of FOXP3 intron 1 confirmed that CRISPRi/a-mediated simultaneous FOXP3 activation and XIST silencing were accompanied by elevated H4 acetylation, including H4K5ac, H4K8ac, and H4K16ac, and H3K4me3 and lower DNA methylation. This indicates that CRISPRi/a targeting to XIST and FOXP3 loci alters their transcription and their nearby epigenetic modifications. CONCLUSIONS: The simultaneous activation and repression of the X-linked, endogenous FOXP3 and XIST from XCI offers a useful research tool and a potential therapeutic for female breast cancers.

Effect of wastewater treatment plant discharge on the bacterial community in a receiving river.

The effluent of wastewater treatment plants (WWTPs) is an important water resource for some rivers in regions with relatively low precipitation, which may pose ecological risks. Various pollutants and microorganisms are discharged into rivers, along with the WWTP effluent, but this process has not been thoroughly studied. The objective of this study was to evaluate the effect of WWTP effluent on the bacterial community in the sediment and water column of an urban river and to identify the relationship between the total and active bacterial communities. Five sites were sampled in the river, including the most upstream site of the river (Up-most), 200 m upstream of the WWTP (Up-200), at the point of effluent discharge of the WWTP (Eff-pl) and 50 m (Down-50) and 1000 m (Down-1000) downstream of the WWTP. Compared with the two upstream sites (Up-most and Up-200), the bacterial species composition of Eff-pl was significantly different (p < 0.05) in both the sediment and water columns, while the bacterial species composition at Down-1000 was significantly different (p < 0.05) in the sediment but not in the water. The relative abundance of Proteobacteria, Actinobacteriota and Verrucomicrobiota was significantly different (p < 0.05) at Eff-pl in both the sediment and water columns compared with that at the upstream sites. The shared bacterial species between the DNA and RNA 16 S rRNA analyses were only 45.5-62.2% and 43.2-52.3% for the sediment and water, respectively. Accordingly, WWTP effluent drainage significantly alters (p < 0.05) the bacterial composition in the receiving river but can be recovered in water within a short distance. However, in sediment, a longer recovery space is probably needed. Analyses of the combination of total and active bacterial compositions are recommended to evaluate the ecological consequences of WWTP effluent drainage on the bacterial composition.

[Analysis of GCH1 gene variant in a consanguineous Chinese pedigree affected with tetrahydrobiopterin deficiency].

OBJECTIVE: To explore the genetic basis for a child featuring tetrahydrobiopterin deficiency and global developmental delay. METHODS: Clinical and laboratory examinations were carried out for the child. Genomic DNA of the patient was subjected to high-throughput sequencing to identify genetic variants associated with hyperphenylalaninemia. Candidate variants were verified by Sanger sequencing. RESULTS: The result of blood tandem mass spectrometry showed that the Phenylalanine in the blood was 642.7 µmol/l, and the ratio of Phenylalanine/Tyrosine was 5.42. Analysis of urinary pterin: neopterin 0.09 mmol/mol Cr, biopterin 0.04 mmol/mol Cr, biopterin% 77%, which suggested tetrahydrobiopterin deficiency. The parents of the proband were first cousins. DNA sequencing revealed that the proband has harbored homozygous c.353A>T variants in exon 2 of the GCH1 gene, for which his great grandmother, grandfather, mother, uncle, father and elder brother were heterozygous carriers with normal phenotype and no clinical symptoms associated with dopa responsive dystonia. CONCLUSION: The homozygous c.353A>T variant of the GCH1 gene probably underlay the tetrahydrobiopterin deficiency in this pedigree of consanguineous marriage.

Aidi injection combined with chemotherapy in the treatment of cancer patients: a systematic review of systematic reviews and meta-analyses.

The objective of the study was to evaluate and summarize the evidence from systematic reviews and meta-analyses regarding the efficacy and safety of Aidi injection combined with chemotherapy in the treatment of cancer patients. PubMed, EMBASE, Web of Science, Cochrane Library, China National Knowledge Infrastructure, Chong qing VIP databases, and Wanfang databases were searched for systematic reviews/meta-analyses on the topic of Aidi treating cancer patients published from inception to 20 December 2020. Google Scholar and OpenGrey were searched for grey literature and International Prospective Register of Systematic Reviews for ongoing reviews. Two investigators independently selected eligible studies, extracted data, and assessed the methodological quality of included systematic reviews/meta-analyses using the measurement tool to assess systematic reviews 2 (AMSTAR-2) tool, and the strength of evidence was assessed with the grade of recommendation, assessment, development, and evaluation (GRADE) system. Twenty-seven systematic reviews/meta-analyses were identified in the study. The methodological quality of all 27 systematic reviews/meta-analyses were critically low when evaluated by AMSTAR-2, and the evidence quality of all outcomes rated as either low or very low based on the GRADE system. The available evidence is currently insufficient to support or refute the use of Aidi in the treatment of cancer patients, thus high-quality trials with large sample sizes are needed to explore its efficacy and safety in cancer patients.

Effect of influent salinity on the selection of macrophyte species in floating constructed wetlands.

Pilot-scale floating constructed wetlands (FCWs) under varying influent salinities were implemented, and the effects of influent salinity on pollutant removal efficiency (RE) and macrophyte species selection were identified. The results suggest that a salinity increase generally decreased pollutant REs, while some macrophytes, such as Iris pseudacorus, could effectively resist this decrease. The average coefficients of variation between macrophyte species in REs of chemical oxygen demand, ammonium nitrogen, nitrate nitrogen and total phosphorus increased from 28.6% at low salinity to 91.3% at high salinity, which suggests the greater importance of macrophyte selection under high salinity. With an increase in salinity, the rhizosphere bacterial community showed convergent evolution or convergence followed by slight divergent evolution between macrophyte species, while the importance of macrophyte parameter selection in characterizing pollutant REs decreased. Therefore, influent salinity is a key factor to consider in macrophyte selection and application, especially in FCWs without soil.

Enhanced organic matter decomposition in sediment by Tubifex tubifex and its pathway.

The role of Tubifex tubifex in organic matter (OM) decomposition in aquatic ecosystems has been widely studied, but considerable uncertainties exist in terms of the effect mechanism. The effect of T. tubifex on sediment OM decomposition in laboratory-scale microcosms was quantified, and possible pathways were identified. In the first 7 days of the decomposition of OM mixed in sediment, no significant effect of T. tubifex on organic matter loss (OML) was observed for both low- and high-OM treatments; meanwhile, from day 7-60, T. tubifex addition significantly improved OML from 55.0%-57.5% to 71.8%-77.7% in the low-OM treatments and from 55.5%-56.6% to 64.1%-68.7% in the high-OM treatments. The enhanced OML observed with T. tubifex was mainly due to the promoted decomposition of refractory organic components, e.g., cellulose, hemicellulose and lignin. The proportion of refractory components in the gut of T. tubifex was significantly lower than that in the sediments (p < 0.01), indicating a pathway corresponding to the ingestion and digestion of refractory components by T. tubifex. Although T. tubifex reduced the water dissolved oxygen (DO) by increasing the water chemical oxygen demand (COD), the oxygen supply was improved by T. tubifex, and this could be affected by the increase in the relative abundance of aerobic to anaerobic bacteria in the sediments. T. tubifex significantly increased the diversity of the bacterial and fungal communities in the sediments. Moreover, the community structure of bacteria and fungi was substantially different between gut and sediment. Therefore, multiple pathways of the effect of T. tubifex on OM decomposition were established, and the results have great significance for the artificial manipulation of OM circulation using T. tubifex and the restoration of damaged aquatic ecosystems.

[Association of related maternal factors with susceptibility to congenital hypothyroidism: a case-control study].

OBJECTIVE: To study the association of related maternal factors with the susceptibility to congenital hypothyroidism (CH) in neonates. METHODS: A case-control study was designed. The neonates who were diagnosed with CH in Neonatal Screening Center of Henan Province from January 1, 2016 to December 31, 2017 were enrolled as cases. Healthy neonates, matched for sex and age were enrolled as controls. A conditional logistic regression analysis and additive and multiplicative interaction analyses were used to identify the risk factors for susceptibility to CH. RESULTS: A total of 2 771 661 neonates were screened during this period, among whom 1 494 neonates were diagnosed with CH, with a crude incidence rate of 53.9/100 000. A total of 843 pairs of the cases and the controls completed the telephone survey and provided qualified data. The conditional logistic regression analysis showed that an older maternal age at delivery, a low educational level in mothers, living in the rural area, a family history of thyroid diseases, histories of exposure to formaldehyde during pregnancy, exposure to radiation during pregnancy, and medication during pregnancy, were risk factors for CH (P<0.05), while low maternal age at delivery and progesterone intake during pregnancy were protective factors against CH (P<0.05). CONCLUSIONS: An older maternal age at delivery, a low educational level in mothers, living in the rural area, a family history of thyroid diseases, and histories of exposure to formaldehyde during pregnancy, exposure to radiation during pregnancy and medication during pregnancy may increase the susceptibility to CH in neonates.

CRISPR/Cas9 for Human Genome Engineering and Disease Research.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system, a versatile RNA-guided DNA targeting platform, has been revolutionizing our ability to modify, manipulate, and visualize the human genome, which greatly advances both biological research and therapeutics development. Here, we review the current development of CRISPR/Cas9 technologies for gene editing, transcription regulation, genome imaging, and epigenetic modification. We discuss the broad application of this system to the study of functional genomics, especially genome-wide genetic screening, and to therapeutics development, including establishing disease models, correcting defective genetic mutations, and treating diseases.

Effect of cadmium contamination on the eutrophic secondary pollution of aquatic macrophytes by litter decomposition.

The objective of this study was to identify the effect of cadmium (Cd) contamination on the decomposition of aquatic macrophyte litter and its eutrophic secondary pollution. A laboratory experiment was conducted with three treatments: water Cd contamination (Cd-w), litter Cd contamination (Cd-l) and control (CK). The results showed that CK and Cd-w exhibited the typical decomposition dynamics of litter, i.e., early rapid decomposition followed by slow decomposition, while the litter biomass loss (BL) in Cd-l exhibited an approximately linear relationship with time over the 64-day experimental period. The BL in Cd-l was only 10.8% in the initial 4 days, while that in CK and Cd-w was 59.0% and 54.8%, respectively. Cd inhibited the fluctuation of the water chemical oxygen demand (COD) by reducing both the early increase and the subsequent decrease. The increases in water total nitrogen (TN) and total phosphorus (TP) were inhibited by Cd contamination throughout most of the decomposition period. The alterations of litter quality during the plant growth period and of the bacterial community during the litter decomposition period by Cd contamination could explain the variations in litter decomposition rate and its eutrophic secondary pollution during the early and late decomposition stages, respectively. The Cd inhibition of the eutrophic secondary pollution of aquatic macrophytes has great significance for the improved evaluation of Cd contamination.

[Clinical and genetic characteristics of primary carnitine deficiency identified by neonatal screening].

OBJECTIVE: To study the prevalence, clinical and genetic characteristics of primary carnitine deficiency (PCD). METHODS: From January 2013 to December 2017, 720 667 newborns and their mothers were tested for PCD by tandem mass spectrometry. Potential mutations of carnitine transporter gene SLC22A5 among suspected PCD patients were analyzed. Dietary guidance and L-carnitine supplementation were provided to the parents. Growth and intelligence development were surveyed during follow-up. RESULTS: In total 21 neonates and 6 mothers were diagnosed with PCD, which yielded an incidence of 1 in 34 317. Eighteen SLC22A5 mutations were detected, which included 4 novel mutations, namely c.1484T>C, c.394-1G>T, c.431T>C and c.265-266insGGCTCGCCACC. Eighteen patients were found to carry compound heterozygous mutations and 3 have carried homozygous SLC22A5 mutations. Three mothers carried compound heterozygous mutations and 2 carried homozygous mutations. Common mutations included c.1400C>G (42.3%), c.760C>T (11.5%) and c.51C>G (7.7%). During the 8-42 month follow-up, neonates with PCD showed no clinical symptoms but normal growth. Blood level of free carnitine was raised in all mothers after the treatment. CONCLUSION: The incidence of neonatal PCD in Henan is 1 in 34 317, with the most common mutation being c.1400C>G. Above finding has enriched the spectrum of SLC22A5 gene mutations.

Correction to: Spectrum of PAH gene variants among a population of Han Chinese patients with phenylketonuria from northern China.

Following publication of the original article [1], the authors reported an error in Table 3 on page 4. Variant No. 18 should be " p.Ser339Phe c.1016C>T " (as given in Number 117 of Additional file 2).

Spectrum of PAH gene variants among a population of Han Chinese patients with phenylketonuria from northern China.

BACKGROUND: Phenylketonuria (PKU), which primarily results from a deficiency of phenylalanine hydroxylase (PAH), is one of the most common inherited inborn errors of metabolism that impairs postnatal cognitive development. The incidence of various PAH variations differs by race and ethnicity. The aim of the present study was to characterize the PAH gene variants of a Han population from Northern China. METHODS: In total, 655 PKU patients and their families were recruited for this study; each proband was diagnosed both clinically and biochemically with phenylketonuria. Subjects were sequentially screened for single-base variants and exon deletions or duplications within PAH via direct Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). RESULTS: A spectrum of 174 distinct PAH variants was identified: 152 previously documented variants and 22 novel variants. While single-base variants were distributed throughout the 13 exons, they were particularly concentrated in exons 7 (33.3%), 11 (14.2%), 6 (13.2%), 12 (11.0%), 3 (10.4%), and 5 (4.4%). The predominant variant was p.Arg243Gln (17.7%), followed by Ex6-96A > G (8.3%), p.Val399 = (6.4%), p.Arg53His (4.7%), p.Tyr356* (4.7%), p.Arg241Cys (4.6%), p.Arg413Pro (4.6%), p.Arg111* (4.4%), and c.442-1G > A (3.4%). Notably, two patients were also identified as carrying de novo variants. CONCLUSION: The composition of PAH gene variants in this Han population from Northern China was distinct from those of other ethnic groups. As such, the construction of a PAH gene variant database for Northern China is necessary to lay a foundation for genetic-based diagnoses, prenatal diagnoses, and population screening.

Are the traditional large-scale drought indices suitable for shallow water wetlands? An example in the Everglades.

Numerous drought indices have been developed over the past several decades. However, few studies have focused on the suitability of indices for studies of ephemeral wetlands. The objective is to answer the following question: can the traditional large-scale drought indices characterize drought severity in shallow water wetlands such as the Everglades? The question was approached from two perspectives: the available water quantity and the response of wetland ecosystems to drought. The results showed the unsuitability of traditional large-scale drought indices for characterizing the actual available water quantity based on two findings. (1) Large spatial variations in precipitation (P), potential evapotranspiration (PE), water table depth (WTD) and the monthly water storage change (SC) were observed in the Everglades; notably, the spatial variation in SC, which reflects the monthly water balance, was 1.86 and 1.62 times larger than the temporal variation between seasons and between years, respectively. (2) The large-scale water balance measured based on the water storage variation had an average indicating efficiency (IE) of only 60.01% due to the redistribution of interior water. The spatial distribution of variations in the Normalized Different Vegetation Index (NDVI) in the 2011 dry season showed significantly positive, significantly negative and weak correlations with the minimum WTD in wet prairies, graminoid prairies and sawgrass wetlands, respectively. The significant and opposite correlations imply the unsuitability of the traditional large-scale drought indices in evaluating the effect of drought on shallow water wetlands.

A minimal nitrogen fixation gene cluster from Paenibacillus sp. WLY78 enables expression of active nitrogenase in Escherichia coli.

Most biological nitrogen fixation is catalyzed by molybdenum-dependent nitrogenase, an enzyme complex comprising two component proteins that contains three different metalloclusters. Diazotrophs contain a common core of nitrogen fixation nif genes that encode the structural subunits of the enzyme and components required to synthesize the metalloclusters. However, the complement of nif genes required to enable diazotrophic growth varies significantly amongst nitrogen fixing bacteria and archaea. In this study, we identified a minimal nif gene cluster consisting of nine nif genes in the genome of Paenibacillus sp. WLY78, a gram-positive, facultative anaerobe isolated from the rhizosphere of bamboo. We demonstrate that the nif genes in this organism are organized as an operon comprising nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV and that the nif cluster is under the control of a σ(70) (σ(A))-dependent promoter located upstream of nifB. To investigate genetic requirements for diazotrophy, we transferred the Paenibacillus nif cluster to Escherichia coli. The minimal nif gene cluster enables synthesis of catalytically active nitrogenase in this host, when expressed either from the native nifB promoter or from the T7 promoter. Deletion analysis indicates that in addition to the core nif genes, hesA plays an important role in nitrogen fixation and is responsive to the availability of molybdenum. Whereas nif transcription in Paenibacillus is regulated in response to nitrogen availability and by the external oxygen concentration, transcription from the nifB promoter is constitutive in E. coli, indicating that negative regulation of nif transcription is bypassed in the heterologous host. This study demonstrates the potential for engineering nitrogen fixation in a non-nitrogen fixing organism with a minimum set of nine nif genes.

[Genotype and phenotype correlation of phenylalanine hydroxylase deficiency among patients from Henan].

OBJECTIVE: To delineate the mutation spectrum of phenylalanine hydroxylase (PAH) gene among patients affected with phenylalanine hydroxylase deficiency (PAHD) in Henan Province of China, and to explore the correlation between the genotype and the phenotype. METHODS: A total of 155 affected children were recruited. Potential mutation of the PAH gene were analyzed by direct sequencing. The genotype-phenotype correlation was analyzed by matching the expected and observed phenotypes. RESULTS: Over 72 mutations and 108 genotypes have been identified. There were 7 homozygous mutations, including 1 case with EX6-96A>G/EX6-96A>G, 1 with R241C/R241C, 1 with R413P/R413P, and 4 with R243Q/R243Q. Among these, 6 patients have presented classic PKU phenotypes, except for a R241C/R241C genotype which has led to mild PKU. In 104 patients carrying compound PAH mutations, 52 were classic, 34 were mild and 39 had mild HPA. Patients who were heterozygous for EX6-96A>G/R241C, R243Q/A434D, EX6-96A>G/R413P and EX6-96A>G/ R241C were found with both the classic PKU and mild PKU phenotypes. Common mutations associated with mild HPA have included R53H, R243Q, V399V and H107R. The common mutations associated with mild PKU included R243Q, R241C, EX6-96A>G, and IVS4-1G>A. The prevalent mutations in classic PKU were R243Q, EX6-96A>G and V399V. The consistency between prediction of the biochemical genotype and observed phenotype was 77.78%, especially in classic PKU, the consistency was up to 82.14%. Significant correlations were disclosed between pretreatment levels of phenylalanine and AV sum (r=-0.6729, P < 0.01). CONCLUSION: The mutation spectrum of PAH gene in Henan seems to differ from that of other regions. Independent assortment of mutant alleles may result in a complex genotype-phenotype correlation, but the genotypes of PAHD patients have correlated with the phenotype.

Refactoring the nitrogen fixation gene cluster from Klebsiella oxytoca.

Bacterial genes associated with a single trait are often grouped in a contiguous unit of the genome known as a gene cluster. It is difficult to genetically manipulate many gene clusters because of complex, redundant, and integrated host regulation. We have developed a systematic approach to completely specify the genetics of a gene cluster by rebuilding it from the bottom up using only synthetic, well-characterized parts. This process removes all native regulation, including that which is undiscovered. First, all noncoding DNA, regulatory proteins, and nonessential genes are removed. The codons of essential genes are changed to create a DNA sequence as divergent as possible from the wild-type (WT) gene. Recoded genes are computationally scanned to eliminate internal regulation. They are organized into operons and placed under the control of synthetic parts (promoters, ribosome binding sites, and terminators) that are functionally separated by spacer parts. Finally, a controller consisting of genetic sensors and circuits regulates the conditions and dynamics of gene expression. We applied this approach to an agriculturally relevant gene cluster from Klebsiella oxytoca encoding the nitrogen fixation pathway for converting atmospheric N(2) to ammonia. The native gene cluster consists of 20 genes in seven operons and is encoded in 23.5 kb of DNA. We constructed a "refactored" gene cluster that shares little DNA sequence identity with WT and for which the function of every genetic part is defined. This work demonstrates the potential for synthetic biology tools to rewrite the genetics encoding complex biological functions to facilitate access, engineering, and transferability.