Structure-specific nucleases: role in Okazaki fragment maturation.

Proper function of structure-specific nucleases is key for faithful Okazaki fragment maturation (OFM) process completion. Deregulation of such nucleases leads to aberrant OFM and causes a spectrum of mutations, some of which may confer survival outcomes under specific stresses and serve as attractive targets for therapeutic intervention in human cancers.

New SNCA mutation and structures of α-synuclein filaments from juvenile-onset synucleinopathy.

A 21-nucleotide duplication in one allele of SNCA was identified in a previously described disease with abundant α-synuclein inclusions that we now call juvenile-onset synucleinopathy (JOS). This mutation translates into the insertion of MAAAEKT after residue 22 of α-synuclein, resulting in a protein of 147 amino acids. Both wild-type and mutant proteins were present in sarkosyl-insoluble material that was extracted from frontal cortex of the individual with JOS and examined by electron cryo-microscopy. The structures of JOS filaments, comprising either a single protofilament, or a pair of protofilaments, revealed a new α-synuclein fold that differs from the folds of Lewy body diseases and multiple system atrophy (MSA). The JOS fold consists of a compact core, the sequence of which (residues 36-100 of wild-type α-synuclein) is unaffected by the mutation, and two disconnected density islands (A and B) of mixed sequences. There is a non-proteinaceous cofactor bound between the core and island A. The JOS fold resembles the common substructure of MSA Type I and Type II dimeric filaments, with its core segment approximating the C-terminal body of MSA protofilaments B and its islands mimicking the N-terminal arm of MSA protofilaments A. The partial similarity of JOS and MSA folds extends to the locations of their cofactor-binding sites. In vitro assembly of recombinant wild-type α-synuclein, its insertion mutant and their mixture yielded structures that were distinct from those of JOS filaments. Our findings provide insight into a possible mechanism of JOS fibrillation in which mutant α-synuclein of 147 amino acids forms a nucleus with the JOS fold, around which wild-type and mutant proteins assemble during elongation.

Characterization of a large UNC13D gene duplication in a patient with familial hemophagocytic lymphohistiocytosis type 3.

Familial hemophagocytic lymphohistiocytosis (FHL) type 3 is a life-threatening immune dysregulation syndrome caused by mutations in the UNC13D gene, encoding the munc13-4 protein, which is important for function of cytotoxic lymphocytes. FHL3 accounts for 30-40% of FHL cases, and more than 100 mutations in the UNC13D gene have been described to date. We describe the first case of FHL3 carrying an intragenic duplication of UNC13D, apparently mediated by recombination of Alu elements. NK cell degranulation and munc13-4 protein expression assays are useful for early identification of such mutations, which may be missed by analysis of genomic DNA alone.

FLCN intragenic deletions in Chinese familial primary spontaneous pneumothorax.

Primary spontaneous pneumothorax (PSP) is a significant clinical problem, affecting tens of thousands patients annually. Germline mutations in the FLCN gene have been implicated in etiology of familial PSP (FPSP). Most of the currently identified FLCN mutations are small indels or point mutations that detected by Sanger sequencing. The aim of this study was to determine large FLCN deletions in PSP families that having no FLCN sequence-mutations. Multiplex ligation-dependent probe amplification (MLPA) assays and breakpoint analyses were used to detect and characterize the deletions. Three heterozygous FLCN intragenic deletions were identified in nine unrelated Chinese families including the exons 1-3 deletion in two families, the exons 9-14 deletion in five families and the exon 14 deletion in two families. All deletion breakpoints are located in Alu repeats. A 5.5 Mb disease haplotype shared in the five families with exons 9-14 deletion may date the appearance of this deletion back to approximately 16 generations ago. Evidences for founder effects of the other two deletions were also observed. This report documents the first identification of founder mutations in FLCN, as well as expands mutation spectrum of the gene. Our findings strengthen the view that MLPA analysis for intragenic deletions/duplications, as an important genetic testing complementary to DNA sequencing, should be used for clinical molecular diagnosis in FPSP.

Identification of a Novel Homozygous Mutation in PRDM12 Gene in a Patient with Hereditary Sensory and Autonomic Neuropathy Type VIII.

Hereditary sensory autonomic neuropathy type VIII (HSAN-VIII) is a rare genetic disease that occurs due to mutations in the PRDM12 gene. Here, we describe a novel homozygous mutation c.826_840dupTGCAACCGCCGCTTC (p.Cys276_Phe280dup) on exon 5 in the PRDM12 gene identified by WES and confirmed using Sanger sequencing method.

Molecular genetic features and clinical manifestations in Chinese familial cerebral cavernous malformation: from a novel KRIT1/CCM1 mutation (c.1119dupT) to an overall view.

Cerebral cavernous malformations (CCMs) are common vascular anomaly diseases in the central nervous system associated with seizures, cerebral microbleeds, or asymptomatic mostly. CCMs can be classified as sporadic or familial, with familial cerebral cavernous malformations (fCCMs) being the autosomal dominant manner with incomplete penetrance. Germline mutations of KRIT1, CCM2, and PDCD10 are associated with the pathogenesis of fCCMs. Till now, little is known about the fCCMs mutation spectrum in the Han Chinese population. In this study, we enrolled a large, aggregated family, 11/26 of the family members were diagnosed with CCMs by pathological or neuroradiological examination, with a high percentage (5/9) of focal spinal cord involvement. Genomic DNA sequencing verified a novel duplication mutation (c.1119dupT, p.L374Sfs*9) in exon 9 of the Krev interaction trapped 1 (KRIT1) gene. The mutation causes a frameshift and is predicted to generate a truncated KRIT1/CCM1 protein of 381 amino acids. All our findings confirm that c.1119dupT mutation of KRIT1 is associated with fCCMs, which enriched the CCM genes' mutational spectrum in the Chinese population and will be beneficial for deep insight into the pathogenesis of Chinese fCCMs. Additionally, with a retrospective study, we analyzed the molecular genetic features of Chinese fCCMs, most of the Chinese fCCMs variants are in the KRIT1 gene, and all these variants result in the functional deletion or insufficiency of the C-terminal FERM domain of the KRIT1 protein.

Identification of a novel three-nucleotide duplication in ECM1 in Chinese siblings affected with lipoid proteinosis.

Lipoid proteinosis (LP) is a rare autosomal recessive disorder caused by pathological mutations in the glycoprotein extracellular matrix protein 1 gene (ECM1). In this study, we examined two sibling patients who were suspected of LP in a consanguineous Chinese family for clinical manifestations and sequenced the all coding exonic regions of ECM1 in the proband. Both siblings were detected a homozygous three-nucleotide duplication, c.506_508dupCTG in the exon 6 of ECM1. This mutation introduces an alanine addition between two highly conserved amino acids (Pro169 and Gly170), designated as p.169_170insA, within one of the two tandem repeat domains which are functional important for protein-protein interactions. Their parents were unaffected and heterozygous for this mutation. This mutation wasn't found in one hundred normal Chinese individuals screened and wasn't previously reported elsewhere, excluding it as a common neutral polymorphism. These evidences supported this duplication as the causative mutation of LP. Our finding expanded the spectrum of disease-causing mutations in LP and provides further evidence for the importance of ECM1 gene in the development of this rare genodermatosis.

A Novel Duplication Mutation in the Myelin Protein Zero Gene Causing Mild, Nonprogressive Demyelinating Neuropathy.

Mutations in the myelin protein zero (MPZ) gene can cause a variety of clinical and electrophysiological forms of genetic neuropathies including Charcot-Marie-Tooth (CMT) type 1B disease which is characterized by demyelinating features. We present a father and daughter with neuropathy carrying a novel 31 base pair duplication mutation in the 5' untranslated region of the MPZ gene, c.-23_8dup31. Genetic analysis and protein modeling indicated that this is a frameshift mutation resulting in premature truncation of the encoded protein. The daughter underwent repeat neurological examination and electromyography testing over an 11-year time span demonstrating no clinical or electrophysiological change. Our study expands the clinical and genetic spectrum of mutations that can cause CMT type 1B disease and demonstrates the value of sequence analysis of noncoding portions of a gene that are not intronic.

Structural and functional annotation of PR/SET Domain (PRDM) protein family: In-silico study elaborating role of PRDM12 mutation in congenital insensitivity to pain.

Congenital insensitivity to pain (CIP), classified as a type of hereditary sensory and autonomic neuropathies, is a rare disease in which the affected individuals fail to perceive sensation of pain. One of the PR/SET Domain Proteins, PRDM12, has been identified in recent past as a candidate gene for congenital insensitivity to pain. In the present study, we performed whole exome sequencing in a Pakistani family with CIP phenotype to ascertain the causative mutation. We identified a previously described alanine repeat duplication in PRDM12 (Ala353_Ala359dup) in this family. After this, we performed structural annotations for PR/SET Domain (PRDM) containing protein family to prognosticate the potential hypothetical structure of PRDM proteins with physical and chemical parameters. Out of nineteen members of this family, four members (PRDM5, PRDM8, PRDM12 and PRDM13) were specially focused because of their role in neurological disorders. Predictions about structure and interactions of these proteins revealed novel interacting molecules and pathways. Detailed in silico analysis of PRDM12 was performed to elaborate importance of its domain structure in interaction with other proteins and its role in pain insensitivity phenotype. These results have substantially enhanced our understanding regarding the etiology of congenital pain insensitivity and would stimulate further research on therapy and prevention.

Therapy-related acute promyelocytic leukemia with FMS-like tyrosine kinase 3-internal tandem duplication mutation in solitary bone plasmacytoma: A case report.

BACKGROUND: Therapy-related acute promyelocytic leukemia (t-APL) is a rare complication observed in solitary bone plasmacytoma (SBP), and SBP after radiotherapy evolving to APL harboring the FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutation has never been reported. Here, we present the first case reported until now. CASE SUMMARY: We describe a 64-year-old woman who presented with lumbar pain and was initially diagnosed with SBP. However, after one year of radiotherapy treatment, this patient experienced a long-standing bone-marrow-suppressive period and finally developed APL harboring the FLT3-ITD mutation, as confirmed by analyses of clinical features, bone marrow morphology, flow cytometry, cytogenetic examination, and molecular biology. On admission, the patient had disseminated intravascular coagulation and intracranial hemorrhage, and the peripheral blood and bone marrow smear displayed abundant abnormal promyelocytes. Unfortunately, she died when the definite diagnosis was made. CONCLUSION: The patient with t-APL harboring FLT3-ITD mutation evolving from SBP after radiotherapy had not been reported and had poor clinical outcomes. FLT3-ITD mutation in t-APL may be a potential pathogenesis of leukemogenesis. We should consider the potential risk of secondary neoplasms in SBP patients after radiotherapy.

Prognosis and outcome of patients with acute myeloid leukemia based on FLT3-ITD mutation with or without additional abnormal cytogenetics.

The FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) gene mutation is present in ~20% of patients with de novo acute myeloid leukemia (AML). Patients with an FLT3-ITD mutation have a poor prognosis. However, the prognostic function of FLT3-ITD combined with other cytogenetic abnormalities are not clear. In the present study, a retrospective analysis of 103 newly diagnosed patients with AML was performed. The results revealed that the overall survival (OS) and recurrence-free survival (RFS) times were significantly longer in patients with an FLT3-ITD mutation combined with other favorable risk genes, compared with in those patients with a single FLT3-ITD mutation (P=0.0361 and P=0.0426). Sorafenib combined with chemotherapy significantly improved the overall response rate (ORR) when compared with mono-chemotherapy (P=0.039), but no significant differences were observed in the OS and RFS. In conclusion, favorable-risk cytogenetics may improve the clinical outcomes of patients with FLT3-ITD-mutated AML, but adverse-risk cytogenetics may not further worsen the prognosis. Sorafenib combined with chemotherapy may increase the ORR but would not result in a longer OS and RFS.

A novel D90_K91insN mutation in exon 4 of the SOD1 gene caused familial amyotrophic lateral sclerosis in a Chinese pedigree.

We reported a novel heterozygous duplication mutation (c.272_274dupACA, D90_K91insN) in exon 4 of the SOD1 gene in a Chinese pedigree. This pedigree demonstrates an autosomal dominant pattern of inheritance, with potentially reduced penetrance. The clinical phenotype was rather uniform with a distal lower extremity onset, predominant involvement of lower motor neurons (LMNs), and a relatively short survival time (mean 2.6 years) compared with other mutations in the loop V structure of SOD1. We also detected that the average SOD1 activity in D90_K91insN mutation carriers is 68.5% of wild-type controls. In conclusion, we identified the first non-frameshift duplication mutation in loop V of the human SOD1 in the Chinese population, suggesting the importance of the loop V structure in the pathogenicity of FALS.

Gene mutation analysis of a family with familial generalized lentiginosis / 中华皮肤科杂志

Objective:To analyze clinical and genetic characteristics of a family with familial generalized lentiginosis, and to identify the causative gene mutation.Methods:Clinical characteristics and inherited pattern were analyzed in a family with familial generalized lentiginosis. Peripheral blood samples were obtained from the proband, his affected father and healthy mother, and genomic DNA was extracted. PCR was performed to amplify all exons and their flanking sequences of the SASH1 gene, followed by DNA sequencing. The proband′s mother and 100 unrelated healthy controls served as controls to determine the mutation site. Previous literature and gene mutation databases were searched to rule out the possibility that the SASH1 gene mutations were single nucleotide polymorphisms, and to determine whether it was a known mutation.Results:A 4-generation family consisting of 17 members was investigated, and there were 9 patients in the family, including 7 males and 2 females. Patients existed in each generation, and the disease was inherited in an autosomal dominant manner in this family. Gene sequencing revealed a heterozygous duplication mutation c.49_54dupCCCGAG in exon 1 of the SASH1 gene in the proband and his father. This mutation was not found in his mother or healthy controls, and had not been reported in previous literature or gene mutation databases.Conclusion:The heterozygous duplication mutation c.49_54dupCCCGAG in the SASH1 gene is a pathogenic mutation for the clinical manifestations of familial generalized lentiginosis in this family.

Bayesian inference for duplication-mutation with complementarity network models.

We observe an undirected graph G without multiple edges and self-loops, which is to represent a protein-protein interaction (PPI) network. We assume that G evolved under the duplication-mutation with complementarity (DMC) model from a seed graph, G0, and we also observe the binary forest Γ that represents the duplication history of G. A posterior density for the DMC model parameters is established, and we outline a sampling strategy by which one can perform Bayesian inference; that sampling strategy employs a particle marginal Metropolis-Hastings (PMMH) algorithm. We test our methodology on numerical examples to demonstrate a high accuracy and precision in the inference of the DMC model's mutation and homodimerization parameters.

Slowly progressing lower motor neuron disease caused by a novel duplication mutation in exon 1 of the SOD1 gene.

Familial amyotrophic lateral sclerosis accounts for about 5% of all cases of the neurodegenerative disorder amyotrophic lateral sclerosis. Genetic mutations in Cu/Zn superoxide dismutase (SOD1) have been associated with one kind of familial amyotrophic lateral sclerosis (ALS1). We identified a novel duplication mutation in exon 1 of the SOD1 gene in a Japanese family whose members had lower motor neuron diseases. The patients showed slow disease progression, with the onset of lower limb muscle weakness and exertional dyspnea. Some patients had mild motor and sensory neuropathy and/or bladder dysfunction, which is further evidence that SOD1 mutation results in a predominantly lower motor neuron phenotype.