Functional and structural basis of the nuclear localization signal in the ZIC3 zinc finger domain

Disruptions in ZIC3 cause heterotaxy, a congenital anomaly of the left-right axis. ZIC3 encodes a nuclear protein with a zinc finger (ZF) domain that contains five tandem C2H2 ZF motifs. Missense mutations in the first ZF motif (ZF1) result in defective nuclear localization, which may underlie the pathogenesis of heterotaxy. Here we revealed the structural and functional basis of the nuclear localization signal (NLS) of ZIC3 and investigated its relationship to the defect caused by ZF1 mutation. The ZIC3 NLS was located in the ZF2 and ZF3 regions, rather than ZF1. Several basic residues interspersed throughout these regions were responsible for the nuclear localization, but R320, K337 and R350 were particularly important. NMR structure analysis revealed that ZF1-4 had a similar structure to GLI ZF, and the basic side chains of the NLS clustered together in two regions on the protein surface, similar to classical bipartite NLSs. Among the residues for the ZF1 mutations, C253 and H286 were positioned for the metal chelation, whereas W255 was positioned in the hydrophobic core formed by ZF1 and ZF2. Tryptophan 255 was a highly conserved inter-finger connector and formed part of a structural motif (tandem CXW-C-H-H) that is shared with GLI, Glis and some fungal ZF proteins. Furthermore, we found that knockdown of Karyopherin alpha1/alpha6 impaired ZIC3 nuclear localization, and physical interactions between the NLS and the nuclear import adapter proteins were disturbed by mutations in the NLS but not by W255G. These results indicate that ZIC3 is imported into the cell nucleus by the Karyopherin (Importin) system and that the impaired nuclear localization by the ZF1 mutation is not due to a direct influence on the NLS.     

Genetic variants in ZIC1, ZIC2, and ZIC3 are not major risk factors for neural tube defects in humans

Neural tube defects (NTD) are congenital malformations arising from incomplete neural tube closure during early embryogenesis. Most NTD in humans show complex inheritance patterns, with both genetic and environmental factors involved in the etiology of this malformation. More than 120 mouse models for human NTD exist. NTD have been observed in mice deficient for the Zic family genes, Zic1, Zic2, and Zic3. We performed mutation analysis in the human orthologs of these genes using DNA material from a large panel of NTD patients. In ZIC2 we identified a deletion of one codon that encodes an alanine residue located in the amino terminal alanine stretch of the protein. The deletion was present in one patient, but not in 364 controls. That may suggest a role-albeit small-of this variant in the etiology of NTD in humans. Transmission disequilibrium testing of a frequent polymorphism in the ZIC2 gene (1059C > T, H353H) in parent-spina bifida aperta child triads showed no association with NTD. One silent polymorphism (858G > A, V286V) of unknown significance was identified in ZIC3. Neither mutations nor polymorphisms were found in the coding region or flanking sequences of ZIC1. Our data indicate that ZIC1, ZIC2, and ZIC3 are not major risk factors for NTD in humans.     

鋅指转录因子ZIC3在内脏异位中的研究现状及进展

内脏异位是由于左右非对称性发育异常所致,常与胸腹腔器官的异常偏侧化有关。心脏经常受累,且心脏受累的严重程度通常决定其预后效果。内脏异位患者有特征性的心血管畸形、内脏器官的异常排列以及中线结构发育畸形。在内脏异位患者中第一个被发现有突变的基因是编码锌指转录因子的ZIC3。很多研究证实,ZIC3突变可导致X连锁内脏异位,而且在孤立性先心病中也发现了ZIC3的突变。至今,在内脏异位患者中发现有13个ZIC3突变,其中包括无义突变、错义突变、沉默突变、移码突变以及易位突变等。然而,ZIC3基因在内脏异位,特别是伴复杂先心病中的致病机理仍不是很清楚。本文就ZIC3结构、作用、突变以及其在内脏异位伴先心病中的研究现状及存在的问题做一综述。     

神经管畸形患者锌指蛋白ZIC2基因编码区罕见突变的分子遗传分析

目的筛选ZIC2外显子区域基因突变，分析突变对神经管畸形（NTDs）的影响。方法采用病例对照研究方法，收集来自山西省和江苏省苏州市经孕期B超或病理学解剖证实为NTDs的胎儿为NTDs组，以常规体检排除出生缺陷和重大疾病的胎儿和健康成年人为对照组。采用酚-氯仿法抽提基因组DNA，通过PCR扩增ZIC2的3个外显子及邻近的部分内含子序列，扩增产物采用ABI Prism Bigdye system进行测序，对于测序发现的基因突变进行反向测序验证。结果NTDs组163例，对照组576例。在NTDs组中发现一个同义突变c．1140G〉A，未发现错义突变或重复缺失。同义突变c．1140G〉A位于第2外显子，为1例妊娠期37周诊断为枕部脑膜脑膨出的女性胎儿。KEGG数据库分析ZIC2结构发现该同义突变位于ZIC2的一个C2H2锌指结构中。在对照组中未检测到相同突变。结论ZIC2是一个高度保守基因，其编码区不含有与NTDs相关的SNP和罕见突变。     

Genetic and Functional Analyses of ZIC3 Variants in Congenital Heart Disease

Mutations in zinc‐finger in cerebellum 3 (ZIC3) result in heterotaxy or isolated congenital heart disease (CHD). The majority of reported mutations cluster in zinc‐finger domains. We previously demonstrated that many of these lead to aberrant ZIC3 subcellular trafficking. A relative paucity of N‐ and C‐terminal mutations has, however, prevented similar analyses in these regions. Notably, an N‐terminal polyalanine expansion was recently identified in a patient with VACTERL, suggesting a potentially distinct function for this domain. Here we report ZIC3 sequencing results from 440 unrelated patients with heterotaxy and CHD, the largest cohort yet examined. Variants were identified in 5.2% of sporadic male cases. This rate exceeds previous estimates of 1% and has important clinical implications for genetic testing and risk‐based counseling. Eight of 11 were novel, including 5 N‐terminal variants. Subsequent functional analyses included four additional reported but untested variants. Aberrant cytoplasmic localization and decreased luciferase transactivation were observed for all zinc‐finger variants, but not for downstream or in‐frame upstream variants, including both analyzed polyalanine expansions. Collectively, these results expand the ZIC3 mutational spectrum, support a higher than expected prevalence in sporadic cases, and suggest alternative functions for terminal mutations, highlighting a need for further study of these domains.     

The molecular basis of UDP-galactose-4-epimerase (GALE) deficiency galactosemia in Korean patients.

PURPOSE: UDP-galactose-4-epimerase (GALE) deficiency galactosemia is an autosomal recessive disorder and the prevalence of the disease varies among ethnic groups. We aimed to investigate molecular characteristics of the Korean patients with attenuated GALE activity and elevated galactose-1-phosphate levels in blood. METHODS: In order to characterize the molecular defects underlying GALE deficiency, the GALE gene of 7 patients showing severe activity decreases was sequenced. PCR-RFLP was performed to confirm the presence of the mutations identified by sequencing. RESULTS: Nine mutations were identified: 8 missense mutations (p.A25V, p.R40C, p.D69E, p.E165K, p.R169W, p.R239W, p.G302D, and p.R335H) and one nonsense mutation (p.W336X). Except for p.R335H, all of these mutations are novel. Six patients were compound heterozygotes (p.D69E/p.G302D, p.R40C/p.R169W, p.D69E/p.E165K, p.R239W/p.R335H, p.A25V/p.R169W, and p.G302D/p.R335H) and the remaining patient had only one mutation (p.W336X/not detected). Thirty patients with moderately reduced GALE activity were also tested by PCR-RFLP for the presence of the above mutation, and mutations were detected in 17 of these 30 patients. The frequency of p.G302D (9/30), p.R239W (6/30) and p.R169W (5/30) in our Korean patients with GALE deficiency galactosemia was relatively high. CONCLUSIONS: We detected 9 mutations of the GALE gene in Korean galactosemia patients, and confirmed allelic heterogeneity in this disease.     

骨巨细胞瘤H3.3免疫组织化学阴性病例的H3F3A测序分析

目的探讨骨巨细胞瘤(giant cell tumor of bone,GCTB)H3.3免疫组织化学阴性病例存在的H3F3A基因突变类型。方法收集2017年1月至2019年1月就诊于北京积水潭医院的181例GCTB,行光镜观察并结合临床影像学明确诊断。EnVision二步法免疫组织化学H3.3染色检测H3F3A G34W突变蛋白的表达,对免疫组织化学结果阴性的病例采用Sanger测序法检测H3F3A基因的突变类型。结果免疫组织化学H3.3以细胞核阳性反应为阳性标准,181例GCTB:阳性164例,阴性17例,阳性率90.61%。对免疫组织化学结果阴性的17例采用DNA Sanger测序法检测H3F3A基因突变情况。测序结果显示8例存在突变,分别为:3例G34L(glycine 34 to leucine,3/181,1.66%),3例G34V(glycine 34 to valine,3/181,1.66%),2例G34R(glycine 34 to arginine,2/181,1.10%),其余9例均为野生型(glycine 34,9/181,4.97%)。经测序分析证实免疫组织化学H3.3阴性的病例中并无G34W突变。H3.3免疫组织化学结合测序分析,诊断GCTB总体阳性率可达95.03%。结论H3.3免疫组织化学可检测存在H3F3A G34W突变的GCTB,对其他罕见突变类型及野生型不具诊断价值。     

Identification of 12 novel mutations and two new polymorphisms in the arylsulfatase A gene: Haplotype and genotype–phenotype correlation studies in spanish metachromatic leukodystrophy patients

Arylsulfatase A (ARSA) deficiency is the main cause of metachromatic leukodystrophy (MLD), a lysosomal disorder with no specific treatment. In view of the importance of genetic counseling, analyses of mutations and polymorphisms, including the ARSA pseudodeficiency allele, were carried out in 18 unrelated Spanish MLD patients. A systematic search allowed us to identify 100% of the alleles involving 17 different mutations, 12 of which are novel: G32S, L68P, R84W, P94A, G99V, P136S, W193X, H227Y, R288H, G308D, T327I, and IVS6‐12C→G. Two new polymorphisms, 2033C>T and 2059C>T, were identified in intron 6 which, in combination with two polymorphisms previously described (2161C>G and 2213C>G), gave rise to four different haplotypes in the control population. In addition, we also studied polymorphism 842G>T. Linkage disequilibrium was detected between mutations IVS2+1G→A, D255H, and T327I and specific haplotypes, suggesting a unique origin for these mutations. Moreover, mutation T327I was always associated with the T allele of the new rare variant A210A (893C>T). The distribution of mutation D255H (frequency 19.4%) among patients with different MLD clinical presentation revealed a clear genotype–phenotype correlation paralleling that reported for mutation IVS2+1G→A (frequency 25%). Among the novel mutations, only P136S and R288H occurred on a background of the ARSA pseudodeficiency allele. Screening 182 normal chromosomes identified a frequency of 8.8% of this allele; moreover, we identified two unrelated subjects with the polyA‐ mutation in the absence of the N350S mutation, and this infrequent haplotype reinforced the heterogeneity of conditions with ARSA deficiency. Hum Mutat 14:240–248, 1999. © 1999 Wiley‐Liss, Inc.     

Targeted Resequencing of 29 Candidate Genes and Mouse Expression Studies Implicate ZIC3 and FOXF1 in Human VATER/VACTERL Association

The VATER/VACTERL association describes the combination of congenital anomalies including vertebral defects, anorectal malformations, cardiac defects, tracheoesophageal fistula with or without esophageal atresia, renal malformations, and limb defects. As mutations in ciliary genes were observed in diseases related to VATER/VACTERL, we performed targeted resequencing of 25 ciliary candidate genes as well as disease‐associated genes (FOXF1, HOXD13, PTEN, ZIC3) in 123 patients with VATER/VACTERL or VATER/VACTERL‐like phenotype. We detected no biallelic mutation in any of the 25 ciliary candidate genes; however, identified an identical, probably disease‐causing ZIC3 missense mutation (p.Gly17Cys) in four patients and a FOXF1 de novo mutation (p.Gly220Cys) in a further patient. In situ hybridization analyses in mouse embryos between E9.5 and E14.5 revealed Zic3 expression in limb and prevertebral structures, and Foxf1 expression in esophageal, tracheal, vertebral, anal, and genital tubercle tissues, hence VATER/VACTERL organ systems. These data provide strong evidence that mutations in ZIC3 or FOXF1 contribute to VATER/VACTERL.     

Reciprocal effect of Waardenburg syndrome mutations on DNA binding by the Pax-3 paired domain and homeodomain

The Pax-3 protein contains two DNA-binding domains, a paired domain and a homeodomain. Mutations in Pax-3 cause Waardenburg syndrome (WS) in humans and the mouse Splotch (Sp) phenotype. In the Sp-delayed mouse, a mutation in the Pax-3 paired domain (G9R) abrogates the DNA-binding activity of both the paired domain and the homeodomain, suggesting that they may functionally interact. To investigate this possibility further, we have analyzed the DNA-binding properties of additional point mutants in the Pax-3 paired domain and homeodomain that occur in WS patients (F12L, N14H, G15S, P17L, R23L, G48A, S51F and G66D in the paired domain, V47F and R53G in the homeodomain), the Pax-1 un mutation (G15A) and a substitution associated with Peters' anomaly in the PAX-6 gene (R23G). Within the paired domain, seven of 10 mutations were found to abrogate DNA-binding by the paired domain. Remarkably, these seven mutations also affected DNA binding by the homeodomain, causing either a complete loss (P17L and G66D), a reduction (R23G, R23L, G15S and G15A) or an increase in DNA-binding activity (N14H). In addition, the effect of paired domain mutations occurred at the level of monomer formation by the homeodomain, while the dimerization potential of this domain seemed unaffected in mutants where it could be analyzed. Furthermore, while both homeodomain mutations were found to abolish DNA binding by this domain, the R53G mutation also abrogated DNA binding by the paired domain. The important observation that independent mutations in either domain can affect DNA binding by the other in the intact Pax- 3 protein strongly suggests that the two domains are not functionally independent but bind DNA through cooperative interactions. Modeling the deleterlous mutations on the three-dimensional structure of the paired domain of Drosophila Prd shows that these mutations cluster at the DNA interface, thus suggesting that a series of DNA contacts are essential for DNA binding by both the paired domain and the homeodomain of Pax-3.      

GATA3 abnormalities and the phenotypic spectrum of HDR syndrome

We report on GATA3 analysis and the phenotypic spectrum in nine Japanese families with the HDR syndrome (hypoparathyroidism, sensorineural deafness, and renal dysplasia) (MIM 146255). Fluorescence in situ hybridisation and microsatellite analyses showed heterozygous gross deletions including GATA3 in four families. Sequence analysis showed heterozygous novel mutations in three families: a missense mutation within the first zinc finger domain at exon 4 (T823A, W275R), an unusual mutation at exon 4 (900insAA plus 901insCCT or C901AACCCT) resulting in a premature stop at codon 357 with loss of the second zinc finger domain, and a nonsense mutation at exon 6 (C1099T, R367X). No GATA3 abnormalities were identified in the remaining two families. The triad of HDR syndrome was variably manifested by patients with GATA3 abnormalities. The results suggest that HDR syndrome is primarily caused by GATA3 haploinsufficiency and is associated with a wide phenotypic spectrum.


Keywords: GATA3; HDR syndrome; phenotypic spectrum; mutation analysis     

Molecular and biochemical characterization of Tunisian patients with glycogen storage disease type III

Glycogen storage disease type III (GSD III) is an autosomal recessive inborn error of metabolism caused by mutations in the glycogen debranching enzyme amylo-1,6-glucosidase gene, which is located on chromosome 1p21.2. GSD III is characterized by the storage of structurally abnormal glycogen, termed limit dextrin, in both skeletal and cardiac muscle and/or liver, with great variability in resultant organ dysfunction. The spectrum of AGL gene mutations in GSD III patients depends on ethnic group. The most prevalent mutations have been reported in the North African Jewish population and in an isolate such as the Faroe Islands. Here, we present the molecular and biochemical analyses of 22 Tunisian GSD III patients. Molecular analysis revealed three novel mutations: nonsense (Tyr1148X) and two deletions (3033_3036del AATT and 3216_3217del GA) and five known mutations: three nonsense (R864X, W1327X and W255X), a missense (R524H) and an acceptor splice-site mutation (IVS32-12A>G). Each mutation is associated to a specific haplotype. This is the first report of screening for mutations of AGL gene in the Tunisian population.     

Evaluation of germline BMP4 mutation as a cause of colorectal cancer

Transforming growth factor-β (TGF-β) signalling plays a key role in colorectal cancer (CRC). Bone morphogenetic protein-4 (BMP4) is a member of the TGF-β family of signal transduction molecules. To examine if germline mutation in BMP4 causes CRC we analysed 504 genetically enriched CRC cases (by virtue of early-onset disease, family history of CRC) for mutations in the coding sequence of BMP4. We identified three pathogenic mutations, p.R286X (g.8330C>T), p.W325C (g.8449G>T) and p.C373S (g.8592G>C), amongst the CRC cases which were not observed in 524 healthy controls. p.R286X localizes to the N-terminal of the TGF-β1 prodomain truncating the protein prior to the active domain. p.W325C and p.C373S mutations are predicted from protein homology modelling with BMP2 to impact deleteriously on BMP4 function. Segregation of p.C373S with adenoma and hyperplastic polyp in first-degree relatives of the case suggests germline mutations may confer a juvenile polyposis-type phenotype. These findings suggest mutation of BMP4is a cause of CRC and the value of protein-based modelling in the elucidation of rare disease-causing variants.