A rare missense PAX6 mutation causes atypical aniridia in a three-generation Chinese family

●AIM:To investigate the molecular diagnosis of a threegeneration Chinese family affected with aniridia,and further to identify clinically a PAX6 missense mutation in members with atypical aniridia.●METHODS:Eleven family members with and without atypical aniridia were recruited.All family members underwent comprehensive ophthalmic examinations.A combination of whole exome sequencing(WES)and direct Sanger sequencing were performed to uncover the causative mutation.●RESULTS:Among the 11 family members,8 were clinically diagnosed with congenital aniridia(atypical aniridia phenotype).A rare heterozygous mutation c.622C>T(p.Arg208Trp)in exon 8 of PAX6 was identified in all affected family members but not in the unaffected members or in healthy control subjects.●CONCLUSION:A rare missense mutation in the PAX6 gene is found in members of a three-generation Chinese family with congenital atypical aniridia.This result contributes to an increase in the phenotypic spectrum caused by PAX6 missense heterozygous variants and provides useful information for the clinical diagnosis of atypical aniridia,which may also contribute to genetic counselling and family planning.

Two missense STK11 gene variations impaired LKB1/adenosine monophosphate-activated protein kinase signaling in Peutz-Jeghers syndrome

BACKGROUND Peutz-Jeghers syndrome(PJS)is a rare hereditary neoplastic disorder mainly associated with serine/threonine kinase 11(STK11/LKB1)gene mutations.Preimplantation genetic testing can protect a patient’s offspring from mutated genes;however,some variations in this gene have been interpreted as variants of uncertain significance(VUS),which complicate reproductive decision-making in genetic counseling.AIM To identify the pathogenicity of two missense variants and provide clinical guidance.METHODS Whole exome gene sequencing and Sanger sequencing were performed on the peripheral blood of patients with PJS treated at the Reproductive and Genetic Hospital of Citic-Xiangya.Software was employed to predict the protein structure,conservation,and pathogenicity of the two missense variation sites in patients with PJS.Additionally,plasmids were constructed and transfected into HeLa cells to observe cell growth.The differences in signal pathway expression between the variant group and the wild-type group were compared using western blot and immunohistochemistry.Statistical analysis was performed using one-way analysis of variance.P<0.05 was considered statistically significant.RESULTS We identified two missense STK11 gene VUS[c.889A>G(p.Arg297Gly)and c.733C>T(p.Leu245Phe)]in 9 unrelated PJS families who were seeking reproductive assistance.The two missense VUS were located in the catalytic domain of serine/threonine kinase,which is a key structure of the liver kinase B1(LKB1)protein.In vitro experiments showed that the phosphorylation levels of adenosine monophosphate-activated protein kinase(AMPK)at Thr172 and LKB1 at Ser428 were significantly higher in transfected variation-type cells than in wild-type cells.In addition,the two missense STK11 variants promoted the proliferation of HeLa cells.Subsequent immunohistochemical analysis showed that phosphorylated-AMPK(Thr172)expression was significantly lower in gastric,colonic,and uterine polyps from PJS patients with missense variations than in non-PJS patients.Our findings indicate that these two missense STK11 variants are likely pathogenic and inactivate the STK11 gene,causing it to lose its function of regulating downstream phosphorylated-AMPK(Thr172),which may lead to the development of PJS.The identification of the pathogenic mutations in these two clinically characterized PJS patients has been helpful in guiding them toward the most appropriate mode of pregnancy assistance.CONCLUSION These two missense variants can be interpreted as likely pathogenic variants that mediated the onset of PJS in the two patients.These findings not only offer insights for clinical decision-making,but also serve as a foundation for further research and reanalysis of missense VUS in rare diseases.

Novel homozygous ADAMTS17 missense variant in Weill-Marchesani syndrome

AIM: To explore the phenotype and genotype of WeillMarchesani syndrome(WMS) in a Chinese family and review related literature.METHODS: Three WMS patients and other unaffected individuals in this family with a history of consanguineous marriage were included in this study. Medical history, comprehensive ophthalmic examinations, and systemic evaluation, as well as whole exome and Sanger sequencing of specific genomic regions, were performed. RESULTS: The three affected siblings presented with short stature, brachydactyly and ocular disorders, including very shallow anterior chamber, high myopia, microspherophakia lens subluxation with stretched zonules and glaucoma. Genetic analysis verified a homozygous missense mutation(c.2983C>T: p. Arg995Trp) in ADAMTS17,which was correlated with the diseases in this family, indicating an autosomal recessive inherited manner of WMS. This review aims to summarize the mutation sites of WMS genes, so as to prevent the disease and better guide clinical diagnosis and treatment.CONCLUSION: A novel homozygous missense variant of ADAMTS17 is identified in a WMS family with a history of consanguineous marriage. Our study expands the range of mutations associated with WMS and deepens our understanding of pathology in disease associated with ADAMTS17 variants.

Whole exome sequencing identifies an AMBN missense mutation causing severe autosomal-dominant amelogenesis imperfecta and dentin disorders

Tooth development is a complex process that involves precise and time-dependent orchestration of multiple genetic, molecular,and cellular interactions. Ameloblastin(AMBN, also named "amelin" or "sheathlin") is the second most abundant enamel matrix protein known to have a key role in amelogenesis. Amelogenesis imperfecta(AI [MIM: 104500]) refers to a genetically and phenotypically heterogeneous group of conditions characterized by inherited developmental enamel defects. The hereditary dentin disorders comprise a variety of autosomal-dominant genetic symptoms characterized by abnormal dentin structure affecting either the primary or both the primary and secondary teeth. The vital role of Ambn in amelogenesis has been confirmed experimentally using mouse models. Only two cases have been reported of mutations of AMBN associated with non-syndromic human AI. However, no AMBN missense mutations have been reported to be associated with both human AI and dentin disorders.We recruited one kindred with autosomal-dominant amelogenesis imperfecta(ADAI) and dentinogenesis imperfecta/dysplasia characterized by generalized severe enamel and dentin defects. Whole exome sequencing of the proband identified a novel heterozygous C-T point mutation at nucleotide position 1069 of the AMBN gene, causing a Pro to Ser mutation at the conserved amino acid position 357 of the protein. Exfoliated third molar teeth from the affected family members were found to have enamel and dentin of lower mineral density than control teeth, with thinner and easily fractured enamel, short and thick roots, and pulp obliteration. This study demonstrates, for the first time, that an AMBN missense mutation causes non-syndromic human AI and dentin disorders.

Achromatopsia caused by novel missense mutations in the CNGA3 gene

AIMTo identify the genetic defects in a Chinese family with achromatopsia.METHODSA 2.5-year-old boy, who displayed nystagmus, photophobia, and hyperopia since early infancy, was clinically evaluated. To further confirm and localize the causative mutations in this family, targeted region capture and next-generation sequencing of candidate genes, such as CNGA3, CNGB3, GNAT2, PDE6C, and PDE6H were performed using a custom-made capture array.RESULTSSlit-lamp examination showed no specific findings in the anterior segments. The optic discs and maculae were normal on fundoscopy. The unaffected family members reported no ocular complaints. Clinical signs and symptoms were consistent with a clinical impression of autosomal recessive achromatopsia. The results of sequence analysis revealed two novel missense mutations in CNGA3, c.633T&#x0003e;A (p.D211E) and c.1006G&#x0003e;T (p.V336F), with an autosomal recessive mode of inheritance.CONCLUSIONGenetic analysis of a Chinese family confirmed the clinical diagnosis of achromatopsia. Two novel mutations were identified in CNGA3, which extended the mutation spectrum of this disorder.

<i>In silico</i>analysis of influence of the missense mutation P629S on the molecular interaction and 3D properties of PIK3R5

PIK3R5 is the regulatory subunit of Phosphoinositide 3-kinase γ (PI3Kγ) that is responsible for phosphory-lating membrane lipids to activate the AKT pathway. PIK3R5 binds Gβγ and facilitates the interaction with p110γ catalytic subunit (PIK3CG) during PI3Kγ activation. The identification of PIK3R5 P629S mutation in AOA2 patients indicated a potential defect in the AKT pathway resulting from impaired PIK3R5 interaction with Gβγ and PIK3CG, defective AKT pathway can result in cerebellar cell death causing neurological symptoms. Our in silico macromolecular docking of the wild type and mutant PIK3R5 protein models with ligand revealed an energy requirement to maintain the mutant complexes compared to no energy required to maintain the wild type complexes, in addition, the mutant structures were loose compared to rigid wild type structures, such structural changes may impair the molecular function of the PIK3R5 and hence affect the AKT pathway.

Functional analysis of missense mutations in GLI2 and GLI3 involved in congenital heart disease

Objective:In this study,we aimed to explore the biological functions of 10 rare case-specific missense mutations in GLI2 and 4 in GLI3,which were previously screened in a cohort of 412 patients with congenital heart disease(CHD)and 213 normal controls from Shandong Province,China.Methods:A dual-luciferase reporter assay was used to assess the effects of these mutations in GLI2 and GLI3 on the activity of the sonic Hedgehog signaling pathway in HEK293T cells.Differences in protein levels between mutant and wild-type GLI2 and GLI3 were detected in HEK293T cells using Western blotting.Results:The dual-luciferase reporter assay showed that compared to the wild-type GLI2 protein,p.A1113V significantly increased activation of the sonic Hedgehog signaling pathway,whereas p.H78P and p.I1451S did not have a significant effect.The other mutations largely reduced the activation effect.Compared with the wild-type GLI3 protein,only p.A286V,among the four mutations,significantly reduced the activation effect on the SHH signaling pathway.Western blotting data showed reduced expression of GLI2 p.G716V,GLI2 p.K736N,GLI2 p.I1451S,and GLI3 p.A286V,whereas the remaining mutations had no significant effects.Conclusion:The mutations GLI2 c.2147G>T(p.G716V),GLI2 c.2208G>C(p.K736N),and GLI3 c.857C>T(p.A286V)involved in CHD affect the regulation of the sonic Hedgehog signaling pathway;thus,these rare missense mutations in GLI2 and GLI3 might increase the risk of CHD.

Abnormal function of EPHA2/p.R957P mutant in congenital cataract

AIM:To identify genetic defects in a Chinese family with congenital posterior polar cataracts and assess the pathogenicity.METHODS:A four-generation Chinese family affected with autosomal dominant congenital cataract was recruited.Nineteen individuals took part in this study including 5 affected and 14 unaffected individuals.Sanger sequencing targeted hot-spot regions of 27 congenital cataract-causing genes for variant discovery.The pathogenicity of the variant was evaluated by the guidelines of American College of Medical Genetics and InterVar software.Confocal microscopy was applied to detect the subcellular localization of fluorescence-labeled ephrin type-A receptor 2(EPHA2).Co-immunoprecipitation assay was implemented to estimate the interaction between EphA2 and other lens membrane proteins.The mRNA and protein expression were analyzed by reverse transcription-polymerase chain reaction(qRT-PCR)and Western blotting assay,respectively.The cell migration was analyzed by wound healing assay.Zebrafish model was generated by ectopic expression of human EPHA2/p.R957P mutant to demonstrate whether the mutant could cause lens opacity in vivo.RESULTS:A novel missense and pathogenic variant c.2870G>C was identified in the sterile alpha motif(SAM)domain of EPHA2.Functional studies demonstrated the variant’s impact:reduced EPHA2 protein expression,altered subcellular localization,and disrupted interactions with other lens membrane proteins.This mutant notably enhanced human lens epithelial cell migration,and induced a central cloudy region and roughness in zebrafish lenses with ectopic expression of human EPHA2/p.R957P mutant under differential interference contrast(DIC)optics.CONCLUSION:Novel pathogenic c.2870G>C variant of EPHA2 in a Chinese congenital cataract family contributes to disease pathogenesis.

Novel MIP gene mutation causes autosomal-dominant congenital cataract

●AIM:To identify disease-causative mutations in families with congenital cataract.●METHODS:Two Chinese families with autosomaldominant congenital cataract(ADCC)were recruited and underwent comprehensive eye examinations.Gene panel next-generation sequencing of common pathogenic genes of congenital cataract was performed in the proband of each family.Sanger sequencing was used to valid the candidate gene mutations and sequence the other family members for co-segregation analysis.The effect of sequence changes on protein structure and function was predicted through bioinformatics analysis.Major intrinsic protein(MIP)-wildtype and MIP-G29R plasmids were constructed and microinjected into zebrafish single-cell stage embryos.Zebrafish embryonic lens phenotypes were screened using confocal microscopy.●RESULTS:A novel heterozygous mutation(c.85G>A;p.G29R)in the MIP gene was identified in the proband of one family.A known heterozygous mutation(c.97C>T;p.R33C;rs864309693)in MIP was found in the proband of another family.In-silico prediction indicated that the novel mutation might affect the MIP protein function.Zebrafish embryonic lens was uniformly transparent in both wild-type PCS2+MIP and mutant PCS2+MIP.●CONCLUSION:Two missense mutations in the MIP gene in Chinese cataract families are identified,and one of which is novel.These findings expand the genetic spectrum of MIP mutations associated with cataracts.The functional studies suggest that the novel MIP mutation might not be a gain-of-function but a loss-of-function mutation.

Eight novel MUT loss-of-function missense mutations in Chinese patients with isolated methylmalonic academia

Background:Isolated methylmalonic acidemia is a rare autosomal recessive metabolic disorder mostly caused by mutations in the methylmalonyl coenzyme A mutase (MCM) gene (MUT).This study aimed to verify whether missense mutations in MUT in Chinese patients affect the stability and enzymatic activity of MCM.Methods:Eight Chinese patients were identified with novel mutations.Plasmids carrying the wild-type and mutated MUT cDNA were constructed and transfected into HEK293T cells for functional analyses.The expression and activity of MCM were determined by western blot and ultra-performance liquid chromatography,respectively.Results:All patients had high levels of blood propionylcarnitine and urinary methylmalonyl acid.By the end of the study,two patients were lost to follow-up,three died,and three survived with mental retardation.Compared to the wild-type protein,the expression levels of all missense mutations of in vitro MCM protein were decreased (P<0.05) except those for I597R,and the MCM activity of the mutations was reduced in a permissive assay.Conclusion:The missense mutations L140P,A141T,G161V,W309G,I505T,Q514K,I597R and G723D affected the stability and enzymatic activity of MCM,indicating that they had a disease-causing capacity.

A Novel Missense Mutation in Peripheral Myelin Protein-22 Causes Charcot-Marie-Tooth Disease

Background：Charcot-Marie-Tooth disease （CMT） is the most common inherited peripheral neuropathy.A great number of causative genes have been described in CMT,and among them,the heterozygous duplication of peripheral myelin protein-22 （PMP22） is the major cause.Although the missense mutation in PMP22 is rarely reported,it has been demonstrated to be associated with CMT.This study described a novel missense mutation of PMP22 in a Chinese family with CMT phenotype.Methods：Targeted next-generation sequencing （NGS） was used to screen the causative genes in a family featured with an autosomal dominant demyelinating form of CMT.The potential variants identified by targeted NGS were verified by Sanger sequencing and classified according to the American College of Medical Genetics and Genomics standards and guidelines.Further cell transfection studies were performed to characterize the function of the novel variant.Results：Using targeted NGS,a novel heterozygous missense variant in PMP22 （c.320G〉A,p.G107D） was identified.In vitro cell functional studies revealed that mutant PMP22 protein carrying p.G 107D mutation lost the ability to reach the plasma membrane,was mainly retained in the endoplasmic reticulum,and induced cell apoptosis.Conclusions：This study supported the notion that missense mutations in PMP22 give rise to a CMT phenotype,possibly through a toxic gain-of-function mechanism.

A novel DLL4 missense mutation in a Chinese patient with Adams-Oliver syndrome

To the Editor:Adams-Oliver syndrome (AOS,including 6 types) was initially reported in a three-generation family by Adams and Oliver in 1945,[1] with an estimated incidence of 1 in 225,000 live births.[2] Approximately 84% of AOS patients have terminal transverse limb defects,including amputations,syndactyly,brachydactyly,or oligodactyly.

A functional missense variant in ITIH3 affects protein expression and neurodevelopment and confers schizophrenia risk in the Han Chinese population

The Psychiatric Genomics Consortium(PGC)has recently identified 10 potential functional coding variants for schizophrenia.However,how these coding variants confer schizophrenia risk remains largely unknown.Here,we investigate the associations between eight potential functional coding variants identified by PGC and schizophrenia in a large Han Chinese sample(n=4022 cases and 9270 controls).Among the eight tested single nucelotide polymorphisms(SNPs),rs3617(a missense variant,p.K315 Q in the ITIH3 gene)showed genome-wide significant association with schizophrenia in the Han Chinese population(P=8.36×10-16),with the same risk allele as in PGC.Interestingly,rs3617 is located in a genomic region that is highly evolutionarily conserved,and its schizophrenia risk allele(C allele)was associated with lower ITIH3 mRNA and protein expression.Intriguingly,mouse neural stem cells stably overexpressing ITIH3 with different alleles of rs3617 exhibited significant differences in proliferation,migration,and differentiation,suggesting the impact of rs3617 on neurodevelopment.Subsequent transcriptome analysis found that the differentially expressed genes in neural stem cells stably overexpressing different alleles of rs3617 were significantly enriched in schizophrenia-related pathways,including cell adhesion,synapse assembly,MAPK and PI3 K-AKT pathways.Our study provides convergent lines of evidence suggesting that rs3617 in ITIH3 likely affects protein function and neurodevelopment and thereby confers risk of schizophrenia.

Rare inherited missense variants of POGZ associate with autism risk and disrupt neuronal development

Excess de novo likely gene-disruptive and missense variants within dozens of genes have been identified in autism spectrum disorder(ASD)and other neurodevelopmental disorders.However,many rare inherited missense variants of these high-risk genes have not been thoroughly evaluated.In this study,we analyzed the rare missense variant burden of POGZ in a large cohort of ASD patients from the Autism Clinical and Genetic Resources in China(ACGC)and further dissected the functional effect of diseaseassociated missense variants on neuronal development.Our results showed a significant burden of rare missense variants in ASD patients compared to the control population(P=4.6×10-5,OR=3.96),and missense variants in ASD patients showed more severe predicted functional outcomes than those in controls.Furthermore,by leveraging published large-scale sequencing data of neurodevelopmental disorders(NDDs)and sporadic case reports,we identified 8 de novo missense variants of POGZ in NDD patients.Functional analysis revealed that two inherited,but not de novo,missense variants influenced the cellular localization of POGZ and failed to rescue the defects in neurite and dendritic spine development caused by Pogz knockdown in cultured mouse primary cortical neurons.Significantly,L1CAM,an autism candidate risk gene,is differentially expressed in POGZ deficient cell lines.Reduced expression of L1cam was able to partially rescue the neurite length defects caused by Pogz knockdown.Our study showed the important roles of rare inherited missense variants of POGZ in ASD risk and neuronal development and identified the potential downstream targets of POGZ,which are important for further molecular mechanism studies.

A Novel Functional Missense Mutation p.T219A in Type Gaucher＇s Disease

Background： Gaucher＇s disease （GD） is an autosomal recessive disorder caused by a deficiency of acid β-glucosidase （glucocerebrosidase [GBA]） that results in the accumulation of glucocerebroside within macrophages. Many mutations have been reported to be associated with this disorder. This study aimed to discover more mutations and provide data for the genetic pattern of the gene, which will help the development of quick and accurate genetic diagnostic tools for this disease. Methods： Genomic DNA was obtained from peripheral blood leukocytes of the patient and Sanger sequencing is used to sequence GBA gene. Sequence alignments of mammalian β-GBA （GCase） and three-dimensional protein structure prediction of the mutation were made. A construct of this mutant and its compound heterozygous counterpart were used to measure GCase in vitro. Results： GCase is relatively conserved at p.T219A. This novel mutation differs from its wild-type in structure. Moreover, it also causes a reduction in GCase enzyme activity. Conclusion： This novel mutation （c.655A〉G, p.T219A） is a pathogenic missense mutation, which contributes to GD.

A computational approach to explore the functional missense mutations in the spindle check point protein Mad1

In this work, the most detrimental missense mutations of Madl protein that cause various types of cancer were identified computationally and the substrate binding efficiencies of those missense mutations were analyzed. Out of 13 missense mutations, I Mutant 2.0, SIFT and PolyPhen programs identified 3 variants that were less stable, deleterious and damaging respectively. Subsequently, modeling of these 3 variants was performed to understand the change in their conformations with respect to the native Madl by computing their root mean squared deviation （RMSD）. Furthermore, the native protein and the 3 mutants were docked with the binding partner Mad2 to explain the substrate binding efficiencies of those detrimental missense mutations. The docking studies identified that all the 3 mutants caused lower binding affinity for Mad2 than the native protein. Finally, normal mode analysis determined that the loss of binding affinity of these 3 mutants was caused by altered flexibility in the amino acids that bind to Mad2 compared with the native protein. Thus, the present study showed that majority of the substrate binding amino acids in those 3 mutants displayed loss of flexibility, which could be the theoretical explanation of decreased binding affinity between the mutant Madl and Mad2.

Functional and structural characterization of missense mutations in PAX6 gene

The PAX6 gene belongs to the Paired box （PAX） family of transcription factors that is tissue specific and required for the differentiation and proliferation of cells in embryonic development. PAX6 regulates the pattern formation in early developmental stages. This function of PAX6 protein enables the successful completion of neurogenesis and oculogenesis in most animals such as mice, Drosophila and some other model organisms including humans. A variation in the sequence of PAX6 gene may alter the function and structure of the protein. Such changes can produce adverse effects on functioning of the PAX6 protein which were clinically observed to occur in a broad range of ocular defects such as aniridia in humans. We employed in silico prediction methods such as SIFT, PolyPhen 2; I mutant 3.0, SNAP, SNPs＆GO, and PHD-SNP to screen the pathogenic missense mutation in PAX6 and DNA binding sites by BindN and BindN ＋. Furthermore, we employed KD4V server to examine the structural and functional modifications that occur in the PAX6 protein as a result of mutation. Based on the results obtained from the in silico prediction methods, we carried out modeling analysis for V53L, I56T, G64V, and I87R to visualize the impact of mutation in structural context.

Germline mutation analysis of hPMS2 gene in Chinese families with hereditary nonpolyposis colorectal cancer

AIM: To study the germline mutation of hPMS2 gene in 26 unrelated Chinese hereditary nonpolyposis colorectal cancer (HNPCC) probands and to fulfill the screening strategy for HNPCC in Chinese. METHODS: Genomic DNA was extracted from the peripheral blood. To avoid the interference of pseudogene in detection of the remaining 11 exons (exon 1-5, 9, 11-15), long-range polymerase chain reaction (PCR) was conducted to amplify the complete coding region of hPMS2 gene firstly. Then 1/8 of the PCR productswere used as template to amplify the individual exon respectively and DNA sequencing was done. Direct DNA sequencing of the conventional PCR products of exon 6, 7, 8 and 10 of hPMS2 gene was performed. The same analysis was made in 130 healthy persons without family histories of HNPCC to further investigate the pathological effects of the detected missense mutation. RESULTS: One HNPCC proband fulf illed Bethesda guidelines and was found to carry the germline mutation of hPMS2 gene, which has not been reported in Chinese HNPCC families. It was a missense mutation at c.1532C>T of exon 11. It was detected in three controls as well with an occurrence rate of 2.3% (3/130). Since it could not be found in the PMS2-single nucleotide polymorphism (SNP) database, this missense mutation is a new SNP unreported up to date. Meanwhile, 260 reported SNPs of hPMS2 gene were detected in the 26 HNPCC probands. The 2nd and 5th exons were probably the hot SNP regions of hPMS2 gene in Chinese HNPCC families involving 53.1% of all reported SNP. CONCLUSION: The germline mutation of hPMS2 gene may be rare in Chinese HNPCC families. The 2nd and 5th exons are hot SNP regions of hPMS2 gene.

Compound heterozygous mutations in CYP1B1 gene leads to severe primary congenital glaucoma phenotype

AIM: To identify the novel mutation alleles in the CYP1B1 gene of primary congenital glaucoma(PCG) patients at Shandong Province of China, and investigate their correlation with glaucomatous features.METHODS: The DNA from the peripheral blood of 13 congenital glaucoma patients and 50 ethnically matched healthy controls from the affiliated hospital of Qingdao University were extracted. The coding region of the CYP1B1 gene was amplified by PCR and direct DNA sequencing was performed. Disease causing-variants were analyzed by comparing the sequences and the structures of wild type and mutant CYP1B1 proteins by PyMOL software.RESULTS: Two missense mutations, including A330 F caused by c.988 G>T&c.989 C>T, and R390H caused by c.1169 G>A, were identified in one of the 13 PCG patients analyzed in our study. A330F mutation was observed to be novel in the Chinese Han population, which dramatically altered the protein structure of CYP1B1 gene, including the changes in the ligand-binding pocket. Furthermore, R390H mutation caused the changes in heme-protein binding site of this gene. In addition, the clinical phenotype displayed by PCG patient with these mutations was more pronounced than other PCG patients without these mutations. Multiple surgeries and combined drug treatment were not effective in reducing the elevated intraocular pressure in this patient.CONCLUSION: A novel A330F mutation is identified in the CYP1B1 gene of Chinese PCG patient. Moreover, in combination with other mutation R390H, this PCG patient shows significant difference in the CYP1B1 protein structure, which may specifically contribute to severe glaucomatous phenotype.

A novel mutation of LIM2 causes autosomal dominant membranous cataract in a Chinese family

AIM:To identify mutations in the genes of a fourgeneration Chinese family with congenital membranous cataracts and investigate the morphologic changes and possible functional damage underlying the role of the mutant gene.METHODS:Whole exome analysis of thirteen members of a four-generation pedigree affected with congenital membranous cataracts was performed;co-segregation analysis of identified variants was validated by Sanger sequencing.All members underwent detailed physical and complete eye examinations.The physical changes caused by the mutation were analyzed in silico through homology modeling.The lens fiber block from a patient was observed under a scanning electron microscope(SEM).Cell membrane proteins and cytoplasmic proteins from the human lenses donated by one patient with cataract in this family and from the dislocated lens resulted from the penetrating ocular trauma of a patient unrelated with this family were extracted,and the expression and localization of MP20 and Cx46 were detected by Western blot(WB)assay in these proteins.RESULTS:A novel LIM2 heterozygous mutation(c.388 C>T,p.R130 C)was identified with congenital membranous cataracts inherited by an autosomal dominant(AD)pattern.Nystagmus and amblyopia were observed in all patients of this family,and exotropia and long axial length were observed in most patients.A/B ultrasound scan and ultrasound biomicroscopy revealed obvious thin crystalline lenses from 1.7 to 2.7 mm in central thickness in all cataract eyes.The bioinformatic analysis showed that the mutation was deleterious to the physiological function of LIM2-encoded MP20.Furthermore,by SEM,ultrastructure of the cataract nucleus showed that lens fiber cells(LFCs)remained morphologic characteristics of immature fiber cells,including flap cell surface with straight edges and lacking normal ball-and-socket joint boundaries,which implied that the differentiation of LFCs might be inhibited.Accumulation of MP20 and Cx46 in the cytoplasm was observed in the cytoplasm of the LFCs in human cataract lens.CONCLUSION:We identify a novel heterozygous LIM2(c.388 C>T,p.R130 C)mutation inherited by an AD pattern.This LIM2 mutation causes the abnormal sub-localization of MP20 and Cx46 in LFCs resulting in membranous cataracts.