Prevalence of inherited metabolic disorders among newborns in Zhuzhou, a southern city in China.

Background and aims: Defective enzymes, cofactors, or transporters of metabolic pathways cause inherited metabolic disorders (IMDs), a group of genetic disorders. Several IMDs have serious consequences for the affected neonates. Newborn screening for IMDs is conducted by measuring specific metabolites between 3 and 7 days of life. Herein, we analyzed the incidence, spectrum, and genetic characteristics of IMDs in newborns in the Zhuzhou area. Methods: Tandem mass spectrometry was conducted on 90,829 newborns who were admitted to the Women and Children Healthcare Hospital of Zhuzhou and requested for screening for IMDs. These newborns were subsequently subjected to next-generation sequencing and further validated using Sanger sequencing. Results: 30 IMDs cases were found in 90,829 cases of newborns screened for IMDs, and the overall incidence was 1/3,027. The incidence of amino acid, organic acid, fatty acid oxidation and urea cycle disorders were 1/8,257, 1/18,165, 1/7,569, and 1/45,414, respectively. Additionally, 9 cases of maternal IMDs were found in our study, and unreported gene mutations of 3 cases IMDs were identified. Conclusion: Our data indicated that IMDs are never uncommon in zhuzhou, meanwhile, we also found that primary carnitine deficiency was the only disorder of fatty acid oxidation in Zhuzhou, and the incidence (1/7,569) was higher than the national level, organic acid metabolic diseases are mostly inherited. Therefore, our study has clarified the disease spectrum and genetic backgrounds, contributing to the treatment and prenatal genetic counseling of these disorders in this region.

Characterization of a rare mosaic X-ring chromosome in a patient with Turner syndrome.

BACKGROUND: Ring chromosomes can be formed by terminal breaks of two arms of a chromosome and their rejoining, leading to a loss of genetic material. They may also be formed by telomere-telomere fusions with no deletion, resulting in the formation of a complete ring. Mosaic X-ring chromosomes are extremely rare and have highly variable phenotypes. Here, we report a case with a mosaic X-ring chromosome in a patient with Turner syndrome, and we illustrate the unreported complicated mechanism using chromosome analysis and fluorescence in situ hybridization (FISH). CASE PRESENTATION: A 10-year-old girl of short stature presenting Turner syndrome was admitted to our hospital. The patient's clinical characteristics were subsequently documented. Genetic analysis showed a karyotype of mostly 45,X[140]/46,X,r(X)[60]. The X ring chromosome was cytogenetically characterized as 45,X/46,X,r(X)(p22.32q21.1), with a length of approximately 74 Mb. CONCLUSIONS: Taken together, we report a rare case with a mosaic X ring chromosome in Turner syndrome and we believe this case expands our collective knowledge of mosaic structural chromosomal disorders and provides new insight into clinical management and genetic counseling for Turner syndrome.

A Chinese Patient with Spastic Paraplegia Type 4 with a De Novo Mutation in the SPAST Gene.

BACKGROUND: Spastic paraplegia type 4 (SPG4) is the most common type of hereditary spastic paraplegia (HSP) caused by mutations in the SPAST gene. Case Presentation. We report the case of a 27-year-old pregnant Chinese woman with HSP in whom we identified a missense mutation in the SPAST gene (c.1496G>A, p.Arg499His) and a nonsense mutation in the NEFH gene (c.289G>T, p.Glu97 ∗ ) via whole-exome sequencing; this finding corroborated that of Sanger sequencing. The patient exhibited the pure SPG4 phenotype with onset during childhood. The SPAST mutation was absent in the parents and paternal relatives. However, the NEFH mutation was identified in five people with no clinical phenotype. Based on theoretical conjecture and the family gene segregation information, we concluded that the SPAST mutation, but not the NEFH mutation, accounted for the proband's phenotype. Eventually, the woman gave birth to a healthy baby girl with the NEFH mutation. CONCLUSION: In this report, we identified a missense mutation in the SPAST gene (p.Arg499His) in a 27-year-old pregnant Chinese woman with HSP. We believe that this study expands the knowledge about the clinical parameters and mutation spectrum of SPG4.

[Analysis of NRXN1 gene deletion in an autistic patient].

OBJECTIVE: To explore the genetic basis for a patient with autism. METHODS: High-throughput sequencing was carried out to detect copy number variations in the patient. RESULTS: DNA sequencing found that the patient has carried a 0.11 Mb deletion in distal 2p16.3 spanning from genomic position 50 820 001 to 50 922 000, which resulted removal of exon 6 and part of intron 7 of the NRXN1 gene. The same deletion was not found his parents and brother. CONCLUSION: Partial deletion of the NRXN1 gene may underlie the disease in this patient.

A dominant negative FGFR1 mutation identified in a Kallmann syndrome patient.

Kallmann syndrome (KS) is characterized by isolated hypogonadotropic hypogonadism (IHH) with anosmia. Fibroblast growth factor receptor 1 (FGFR1) is one of KS-associated genes, accounts for approximately 10% of total patients. FGFR1 mutations have also been identified in more severe craniosynostosis syndromes, and a subset of craniosynostosis syndromes-associated FGFR1 mutations show dominant negative effect. In this study, we identified a novel FGFR1 mutation (c.867G>A; p.W289X) in a KS patient. The p.W289X mutation leads premature termination, producing a truncated FGFR1 without the transmembrane and intracellular domains. Indeed, the W289X FGFR1 was secreted into culture medium. Further, W289X FGFR1 interfered with the function of wild type receptor to induce ERK1/2 phosphorylation. We therefore identified a dominant negative FGFR1 mutation in the KS patient, and this mutant FGFR1 may be used to decipher the physiological function of FGFR1.

Functional analysis of a nonstop mutation in MITF gene identified in a patient with Waardenburg syndrome type 2.

Waardenburg syndrome (WS) is an autosomal dominant inherited neurogenic disorder with the combination of various degrees of sensorineural deafness and pigmentary abnormalities affecting the skin, hair and eye. The four subtypes of WS were defined on the basis of the presence or absence of additional symptoms. Mutation of human microphthalmia-associated transcription factor (MITF) gene gives rise to WS2. Here, we identified a novel WS-associated mutation at the stop codon of MITF (p.X420Y) in a Chinese WS2 patient. This mutation resulted in an extension of extra 33 amino-acid residues in MITF. The mutant MITF appeared in both the nucleus and the cytoplasm, whereas the wild-type MITF was localized in the nucleus exclusively. The mutation led to a reduction in the transcriptional activities, whereas the DNA-binding activity was not altered. We show that the foremost mechanism was haploinsufficiency for the mild phenotypes of WS2 induced in X420Y MITF.

[Analysis of nuclear localization and signal function of MITF protein predisposing to Warrdenburg syndrome].

OBJECTIVE: To study the role of dysfunction of nuclear localization signals (NLS) of MITF protein in the pathogenesis of Waardenburg syndrome. METHODS: Eukaryotic expression plasmid pCMV-MITF-Flag was used as a template to generate mutant plasmid pCMV-MITF△NLS-Flag by molecular cloning technique in order to design the mutagenic primers. The UACC903 cells were transfected transiently with MITF and MITF△NLS plasmids, and the luciferase activity assays were performed to determine their impact on the transcriptional activities of target gene tyrosinase (TYR). The oligonucleotide 5'-GAACGAAGAAGAAGATTT-3' was subcloned into pEGFP-N1 to generate recombinant eukaryotic expression plasmid pEGFP-N1-MITF-NLS. The NIH3T3 cells were transfected separately with MITF, MITF△NLS, pEGFP-N1 and pEGFP-N1-NLS plasmids, and their subcellular distribution was observed by immunoflorescence assays. RESULTS: Expression plasmids for the mutant MITF△NLS with loss of core NLS sequence and pEGFP-N1-NLS coupled with MITF△NLS were successfully generated. Compared with the wild-type MITF, MITF△NLS was not able to transactivate the transcriptional activities of promoter TYR and did not affect the normal function of MITF. MITF△NLS was only localized in the cytoplasm and pEGFP-N1 was found in both the cytoplasm and nucleus, whereas pEGFP-N1-NLS was mainly located in the nucleus. CONCLUSION: This study has confirmed the localization function of NLS sequence 213ERRRRF218 within the MITF protein. Mutant MITF with loss of NLS has failed to transactivate the transcriptional activities of target gene TYR, which can result in melanocyte defects and cause WS.

LEF-1 Regulates Tyrosinase Gene Transcription In Vitro.

TYR, DCT and MITF are three important genes involved in maintaining the mature phenotype and producing melanin; they therefore participate in neural crest cell development into melanocytes. Previous studies have revealed that the Wnt signaling factor lymphoid enhancer-binding factor (LEF-1) can enhance DCT and MITF gene expression. However, whether LEF-1 also affects TYR gene expression remains unclear. In the present study, we found that LEF-1 regulated TYR transcription in vitro. LEF-1 overexpression increased TYR gene promoter activity, whereas LEF-1 knockdown by RNA interference significantly decreased TYR expression. Moreover, the core GTTTGAT sequence (-56 to -50) within the TYR promoter is essential for the effect of LEF-1 on TYR expression, and chromatin immunoprecipitation (ChIP) assay indicated that endogenous LEF-1 interacts with the TYR promoter. In addition, we observed a synergistic transactivation of the TYR promoter by LEF-1 and MITF. These data suggest that Wnt signaling plays an important role in regulating melanocyte development and differentiation.

[Construction and analysis of recombinant eukaryotic expression plasmids for SOX10, the causative gene of Warrdenburg syndrome].

OBJECTIVE: To study the exogenous expression and subcellular localization of wild type (WT) and mutant SOX10 proteins in vitro through generation of expression plasmids in order to reveal the pathogenesis of Waardenburg syndrome (WS). METHODS: The plasmids pECE-SOX10 and pCMV-Flag were ligated after they were subjected to double enzyme digestion using molecular cloning technique to generate recombinant eukaryotic expression plasmid pCMV-SOX10-Flag, which was as a template to generate expression plasmids for novel mutations G37fs, G38fs and E248fs of the SOX10 gene. The constructs were verified by direct sequencing. NIH3T3 cells were transiently transfected with the expression plasmids of wide type SOX10, G37fs, G38fs and E248fs, respectively. The exogenous expression of WT SOX10 protein and mutant G37fs, G38fs and E248fs proteins were analyzed using Western blot assay, while their subcellular distribution were observed with an immunofluorescence assay. RESULTS: The DNA sequences of expression plasmids for SOX10 and its mutant G37fs, G38fs and E248f were all correct. Both WT and mutant SOX10 proteins were detected at the expected site. WT SOX10 and E248fs proteins have only localized in the nucleus, whereas G37fs and G38fs proteins showed aberrant localization in both cytoplasm and nucleus. CONCLUSION: Recombinant eukaryotic expression plasmids for the SOX10 gene and its mutants were successfully constructed. Preliminary analysis showed that the mutations have affected the subcellular distribution of WT SOX10 proteins, which has laid a basis for further study of the molecular mechanism of WS caused by SOX10 gene mutations.

O-GlcNAcylation of BMAL1 regulates circadian rhythms in NIH3T3 fibroblasts.

Various physiological processes and behaviors show a circadian rhythm of approximately 24 h, which is crucial in coordinating internal metabolic processes and environmental signals. Post-translational modifications play an important role in regulating circadian core proteins. In this study, we demonstrated that BMAL1 was modified with an O-linked ß-N-acetylglucosamine (O-GlcNAc), which stabilized BMAL1 and enhanced its transcriptional activity. Conversely, inhibition of O-GlcNAcylation resulted in inhibition of circadian rhythms of clock gene expression. Because O-GlcNAcylation is sensitive to the glucose level, such a modification may provide a new mechanism connecting metabolism with circadian rhythms.

Functional analysis of MITF gene mutations associated with Waardenburg syndrome type 2.

MITF mutations results in an abnormal melanocyte development and lead to Waardenburg syndrome type 2 (WS2). Here, we analyzed the in vitro activities of two recently identified WS2-associated MITF mutations (p.R217I and p.T192fsX18). The R217I MITF retained partial activity, normal DNA-binding ability and nuclear distribution, whereas the T192fsX18 MITF failed to activate TYR promoter and showed aberrant subcellular localization which may be caused by deletion of nuclear localization signal (NLS) at aa 213-218 (ERRRRF). These results suggest that haploinsufficiency may be the underlying mechanism for the mild phenotypes of WS2 caused by these two mutations.

Functional analysis of Waardenburg syndrome-associated PAX3 and SOX10 mutations: report of a dominant-negative SOX10 mutation in Waardenburg syndrome type II.

Waardenburg syndrome (WS) is an auditory-pigmentary disorder resulting from melanocyte defects, with varying combinations of sensorineural hearing loss and abnormal pigmentation of the hair, skin, and inner ear. WS is classified into four subtypes (WS1-WS4) based on additional symptoms. PAX3 and SOX10 are two transcription factors that can activate the expression of microphthalmia-associated transcription factor (MITF), a critical transcription factor for melanocyte development. Mutations of PAX3 are associated with WS1 and WS3, while mutations of SOX10 cause WS2 and WS4. Recently, we identified some novel WS-associated mutations in PAX3 and SOX10 in a cohort of Chinese WS patients. Here, we further identified an E248fsX30 SOX10 mutation in a family of WS2. We analyzed the subcellular distribution, expression and in vitro activity of two PAX3 mutations (p.H80D, p.H186fsX5) and four SOX10 mutations (p.E248fsX30, p.G37fsX58, p.G38fsX69 and p.R43X). Except H80D PAX3, which retained partial activity, the other mutants were unable to activate MITF promoter. The H80D PAX3 and E248fsX30 SOX10 were localized in the nucleus as wild type (WT) proteins, whereas the other mutant proteins were distributed in both cytoplasm and nucleus. Furthermore, E248fsX30 SOX10 protein retained the DNA-binding activity and showed dominant-negative effect on WT SOX10. However, E248fsX30 SOX10 protein seems to decay faster than the WT one, which may underlie the mild WS2 phenotype caused by this mutation.

Disease-causing mutation in PKR2 receptor reveals a critical role of positive charges in the second intracellular loop for G-protein coupling and receptor trafficking.

Prokineticins are a pair of signal factors involved in many physiological processes by binding to two closely related G-protein-coupled receptors, PKR1 and PKR2. Recently, mutations in prokineticin 2 (PK2) and PKR2 are found to be associated with Kallmann syndrome and/or idiopathic hypogonadotropic hypogonadism, disorders characterized by delayed puberty and infertility. However, little is known how PKRs interact and activate G-proteins to elicit signal transduction. In the present study, we took advantage of one disease-associated mutation (R164Q) located in the second intracellular (IL2) loop of PKR2, to investigate the role of IL2 loop in the cell signaling, G-protein binding and receptor trafficking. R164Q mutant PKR2 showed normal cell surface expression and ligand binding capacity. However, the PKR2 signaling was abolished by R164Q mutation. We demonstrated that R164Q mutation disrupted the interaction of IL2 loop to the Gα(q), Gα(i), and Gα(16)-proteins. A positive-charged amino acid at this position is required for proper function, and the signaling efficacy and potency depend on the net amount of positive charges. We also demonstrated that the interactive partner of Arg-164 may localize in the C-terminal five residues of Gα(q)-protein. A series of mutation analysis indicated that the basic amino acids at the C terminus of IL2 loop may function cooperatively in GPCRs. Furthermore, R164Q mutation also results in minimal ligand-induced endocytosis of PKR2. As many GPCRs share structural homology in the C terminus of IL2 loop, our findings may have general application in understanding structure and function of GPCRs.