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>A (p.D211E) and c.1006G>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.

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.

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.

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.

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 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.

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.

In silico analysis of a disease-causing mutation in PCDH15 gene in a consanguineous Pakistani family with Usher phenotype

AIM： To map Usher phenotype in a consanguineous Pakistani family and identify disease-associated mutation in a causative gene to establish phenotype-genotype correlation.· METHODS： A consanguineous Pakistani family in which Usher phenotype was segregating as an autosomal recessive trait was ascertained. On the basis of results of clinical investigations of affected members of this family disease was diagnosed as Usher syndrome（USH）. To identify the locus responsible for the Usher phenotype in this family, genomic DNA from blood sample of each individual was genotyped using microsatellite Short Tandem Repeat（STR） markers for the known Usher syndrome loci. Then direct sequencing was performed to find out disease associated mutations in the candidate gene.· RESULTS： By genetic linkage analysis, the USH phenotype of this family was mapped to PCDH15 locus on chromosome 10q21.1. Three different point mutations in exon 11 of PCDH15 were identified and one of them,c.1304AC was found to be segregating with the disease phenotype in Pakistani family with Usher phenotype.This, c.1304 A C transversion mutation predicts an amino-acid substitution of aspartic acid with an alanine at residue number 435（p.D435A） of its protein product.Moreover, in silico analysis revealed conservation of aspartic acid at position 435 and predicated this change as pathogenic.·CONCLUSION：Theidentificationofc.1304ACpathogenic mutation in PCDH15 gene and its association with Usher syndrome in a consanguineous Pakistani family is thefirst example of a missense mutation of PCDH15 causing USH1 phenotype. In previous reports, it was hypothesized that severe mutations such as truncated protein of PCDH15 led to the Usher I phenotype and that missense variants are mainly responsible for non-syndromic hearing impairment.

Novel mutations of the Alstr?m syndrome 1 gene in an infant with dilated cardiomyopathy:A case report

BACKGROUND Alstr?m syndrome(AS)is a rare autosomal recessive disease that is generally induced by mutations of the Alstr?m syndrome 1(ALMS1)gene.We report a case of AS,extend the spectrum of ALMS1 mutations and highlight the biological role of ALMS1 to explore the relationship between dilated cardiomyopathy(DCM)and mutations in ALMS1.CASE SUMMARY We present the case of an infant with AS mainly manifesting with DCM that was caused by a novel mutation of the ALMS1 gene.Whole-exome sequencing revealed a simultaneous large deletion and point mutation in ALMS1,leading to frameshift and missense mutations,respectively,rather than nonsense or frameshift mutations,which have been reported previously.Upon optimized anti-remodeling therapy,biohumoral exams and arrhythmic burden of the infant were alleviated at follow-up after 6 mo.CONCLUSION We identified novel mutations of ALMS1 and extended the spectrum of ALMS1 mutations in an infant with AS.

Circulating tumor DNA genomic profiling reveals the complicated olaparib-resistance mechanism in prostate cancer salvage therapy: A case report

BACKGROUND The poly(ADP-ribose)polymerase(PARP)inhibitor olaparib has displayed superior clinical effect in metastatic castration-resistant prostate cancer(mCRPC)patients with the homologous recombination repair(HRR)genes mutations.However,when a patient’s tumor tissue volume is insufficient for genomic profiling of HRR gene mutations,circulating tumor DNA(ctDNA)may be useful in helping to determine and monitor the efficacy of olaparib,as well as in abiraterone-combination treatment,and for understanding any resistance mechanism related to such mutations.CASE SUMMARY A 61-year-old man who was diagnosed with metastatic prostate adenocarcinoma was initially hormone sensitivity,showing high Gleason score(5+5=10)and absolute positive rate(14/14 biopsied specimens).Following failure of several standard therapies,the patient progressed to mCRPC.Surprisingly,the patient showed good response to olaparib-abiraterone-prednisone combination treatment(an androgen-deprivation therapy,provided as the‘final choice’in China).Serum total prostate-specific antigen(TPSA)level reduced and symptoms remitted for 4 months.However,thereafter,serum TPSA levels began slowly increasing,indicating development of olaparib resistance.Subsequent comprehensive genomic profiling of ctDNA, screening 508 cancer-related genes by next-generation sequencing,identified 10 somatic variants as well as 3 copy number alterations. Two identified reversemissense mutations in partner and localizer of BRCA2 (PALB2) may have recovered the readingframe, restoring function of the primary germline PALB2 mutation and causing resistance to thePARP inhibitor olaparib.CONCLUSIONReverse mutations in PALB2, discovered via genomic profiling of ctDNA, may represent apotential resistance mechanism against olaparib in mCRPC.

USH2A mutation and specific driver mutation subtypes are associated with clinical efficacy of immune checkpoint inhibitors in lung cancer

This study aimed to identify subtypes of genomic variants associated with the efficacy of immune checkpoint inhibitors(ICIs)by conducting systematic literature search in electronic databases up to May 31,2021.The main outcomes including overall survival(OS),progression-free survival(PFS),objective response rate(ORR),and durable clinical benefit(DCB)were correlated with tumor genomic features.A total of 1546 lung cancer patients with available genomic variation data were included from 14 studies.The Kirsten rat sarcoma viral oncogene homolog G12C(KRAS^(G12C))mutation combined with tumor protein P53(TP53)mutation revealed the promising efficacy of ICI therapy in these patients.Furthermore,patients with epidermal growth factor receptor(EGFR)classical activating mutations(including EGFRL858Rand EGFRΔ19)exhibited worse outcomes to ICIs in OS(adjusted hazard ratio(HR),1.40;95%confidence interval(CI),1.01-1.95;P=0.0411)and PFS(adjusted HR,1.98;95%CI,1.49-2.63;P<0.0001),while classical activating mutations with EGFR^(T790)Mshowed no difference compared to classical activating mutations without EGFR^(T790)Min OS(adjusted HR,0.96;95%CI,0.48-1.94;P=0.9157)or PFS(adjusted HR,0.72;95%CI,0.39-1.35;P=0.3050).Of note,for patients harboring the Usher syndrome type-2A(USH2A)missense mutation,correspondingly better outcomes were observed in OS(adjusted HR,0.52;95%CI,0.32-0.82;P=0.0077),PFS(adjusted HR,0.51;95%CI,0.38-0.69;P<0.0001),DCB(adjusted odds ratio(OR),4.74;95%CI,2.75-8.17;P<0.0001),and ORR(adjusted OR,3.45;95%CI,1.88-6.33;P<0.0001).Our findings indicated that,USH2A missense mutations and the KRAS^(G12C)mutation combined with TP53 mutation were associated with better efficacy and survival outcomes,but EGFR classical mutations irrespective of combination with EGFR^(T790)Mshowed the opposite role in the ICI therapy among lung cancer patients.Our findings might guide the selection of precise targets for effective immunotherapy in the clinic.

Clinical features and MUT gene mutation spectrum in Chinese patients with isolated methylmalonic acidemia:identification of ten novel allelic variants

Background: This study aims to study MUT gene mutation spectrum in Chinese patients with isolated methylmalonic academia (MMA) and their clinical features for the potential genotype-phenotype correlation. Methods: Forty-three patients were diagnosed with isolated MMA by elevated blood propionylcarnitine, propionylcarnitine to acetylcarnitine ratio, and urine methylmalonate without hyperhomocysteinemia. The MUT gene was amplifi ed by polymerase chain reaction and directly sequenced. Those patients with at least one variant allele were included. The novel missense mutations were assessed by bioinformatic analysis and screened against alleles sequenced from 50 control participants. Results: Among the 43 patients, 38 had typical clinical presentations, and the majority (30/38) experienced early-onset MMA. Eight patients died and seven were lost to follow-up. Twenty patients had poor outcomes and eight showed normal development. The 43 identified MUT gene mutations had at least one variant allele, whereas 35 had two mutant alleles. Of the 33 mutations reported before, eight recurrent mutations were identified in 32 patients, and c.729_730insTT (p.D244Lfs*39) was the most common (12/78) in the mutant alleles. Of the 10 novel mutations, six were missense mutations and four were premature termination codon mutations. The six novel missense mutations seemed to be pathogenic. Conclusions: A total of 10 novelMUT mutations were detected in the Chinese population. c.729_730insTT (p.D244Lfs*39) was the most frequent mutation. A genotype-phenotype correlation could not be found, but the genotypic characterization indicated the need of genetic counseling for MMA patients and early prenatal diagnoses for high-risk families.

A novel point mutation in CD18 causing leukocyte adhesion deficiency in a Chinese patient

Background Leukocyte adhesion deficiency type 1 （LAD-l） is a rare, autosomal recessive inherited immunodeficiency disease characterized by recurrent severe bacterial infection, impaired pus formation, poor wound healing, associated with the mutation in the CD18 gene responsible for the ability of the leucocytes to migrate from the blood stream towards the site of inflammation. Correct and early diagnosis of LAD-1 is vital to the success of treatment and prevention of aggressive infections. The purpose of this study was to collect the clinical findings of the disease and to identify the genetic entity. Methods CD18 expression in the peripheral blood leukocytes from the patient, his parents and normal control was measured with flow cytometry. The entire coding regions of the CD18 gene were screened with direct sequencing genomic DNA. Results CD18 expression level on this patient＇s leukocyte surface was significantly decreased, with normal level in control group, his father and mother. Gene analysis revealed that this patient had a homozygous c.899A〉T missense mutation in exon 8 of CD18 gene, causing the substitution of Asp to Val at the 300 amino acid. His parents were both heterozygous carriers while no such mutation was found in 50 normal controls. Conclusion This study disclosed a novel point mutation Asp 300 Val located in a highly conserved region （HCR） of CD18 and confirmed the heterogeneity of the mutations causing LAD-1, indicating it was quite beneficial to establish correct and early diagnosis in children with severe LAD-1.