[Analysis of DPYS gene variants in a child with dihydropyrimidase deficiency].
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To explore the genetic basis for a child with dihydropyrimidase (DHP) deficiency.High-throughput sequencing was carried out for the child. Suspected variants were verified by using Sanger sequencing.The proband was found to carry compound heterozygous variants of the DPYS gene, namely c.1468C>T (a missense variant) and c.1339-1363del (a frameshifting variant).The compound heterozygous variants of the DPYS gene probably underlie the DHP in this child. Above result has enabled genetic counseling and prenatal diagnosis for his parents.Keywords:
Sanger sequencing
Proband
Compound heterozygosity
Summary Background Hereditary leukonychia is a rare nail dystrophy characterized by distinctive whitening of the nail plate. Mutations in the PLCD1 gene have been identified as a major causative factor in hereditary leukonychia (HL). However, few reports have analyzed the relationship between genotype and phenotype, especially in Chinese HL patients. Our study aims to explore the typical clinical features of hereditary leukonychia cases in Chinese Han pedigree and the correlations with PLCD1 gene mutation. Patients and methods In this study, two Chinese patients presented with leukonychia and koilonychia. Whole‐exome sequencing (WES) was performed to screen for the mutations in PLCD1 gene and other candidate genes for hereditary leukonychia. Parents with PLCD1 mutation were selected for Sanger sequencing. Results A novel heterozygote missense mutation in exon 9 of PLCD1 gene was identified in the proband and his mother. Whole‐exome sequencing revealed both, the proband (III.5) and his mother (II.4) carrying c.1451A>G mutation, while other family members had a normal sequence of the PLCD1 gene. Conclusion For the first time, a hereditary leukonychia case with PLCD1 mutation has been described in Chinese Han pedigree. This finding suggests the PLCD1 mutation maybe involved in hereditary leukonychia.
Sanger sequencing
Proband
Compound heterozygosity
Genotype-phenotype distinction
Mutation Testing
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To detect potential mutation in a pedigree affected with autosomal recessive non-syndromic deafness.Mutation analysis was carried out by next generation sequencing, and suspected mutations were verified by Sanger sequencing.A heterozygous c.235delC mutation of the GJB2 gene, together with compound heterozygous mutations of the OTOF gene [c.1194T>A (p.D398E) and c.2180A>G (p.N727S)] were detected in the proband. The sister of the proband (also had hearing loss) has carried a heterozygous c.235delC mutation in the GJB2 gene, in addition with a heterozygous c.2180A>G(p.N727S) mutation of the OTOF gene. By Sanger sequencing, a heterozygous IVS1+2T>A mutation was further detected in the non-coding region of the GJB2 gene in both sisters.The compound heterozygous c.235delC and IVS1+2T>A mutations of the GJB2 gene probably account for the hearing loss in the two sisters, among which IVS1+2T>A is considered as a novel pathogenic mutation of the GJB2 gene.
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Compound heterozygosity
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To analyze the genetic cause of a family with autosomal recessive neuronal ceroid lipofuscinoses (NCL).The proband was screened for mutations within the coding region of the candidate genes through high-throughput targeted sequencing. Potential causative mutations were verified by PCR and Sanger sequencing in the proband and his parents. RT-PCR and TA clone sequencing were performed to investigate whether the mRNAs were abnormally spliced.The sequencing results revealed compound heterozygous mutations of CLN6:c.486+2T>C and c.486+4A>T, which were respectively inherited from his parents. RT-PCR and TA cloning sequencing suggested that the mRNAs were abnormally spliced in two forms due to both mutations.The compound heterozygous mutations of CLN6:c.486+2T>C and c.486+4A>T are possibly the genetic causes of the NCL family. Detection of the novel mutation has extended mutation spectrum of CLN6.
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Neuronal ceroid lipofuscinosis
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Coding region
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To explore the genetic etiology of two patients with Perrault syndrome (PRLTS) in a family.Whole exome sequencing (WES) was carried out to screen potential variants within genomic DNA extracted from the proband. Suspected variants were validated by clinical data and results of Sanger sequencing.WES has identified two heterozygous variants of TWNK gene, namely c.1172G>A (p.Arg391His) and c.1844G>C (p.Gly615Ala). Sanger sequencing confirmed that the c.1172G>A (p.Arg391His), a known pathogenic variant, was derived from her father, while the c.1844G>C (p.Gly615Ala), a novel variant, was derived from her mother. Her brother, who was similarly affected, has carried the same compound heterozygous variants.The compound heterozygous variants c.1172G>A (p.Arg391His) and c.1844G>C (p.Gly615Ala) of the TWNK gene probably underlie PRLTS in the sib pair. The above results have facilitated genetic counseling and prenatal diagnosis for the affected family.
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genomic DNA
Exome
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Sanger sequencing
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Penetrance
Mutation Testing
Compound heterozygosity
Genetic Analysis
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Alport syndrome (AS) is an inherited progressive nephropathy caused by mutations in one or two of the type IV collagen novel chains (α3, α4 and α5), which are encoded by COL4A3, COL4A4 and COL4A5, respectively. To date, three genetic forms of AS have been reported, including X-linked AS, autosomal recessive AS, and autosomal dominant AS, and ~80% of patients have X-linked AS caused by mutations in COL4A5. In the present study, four novel and one previously reported COL4A5 mutations were identified using targeted next-generation sequencing in Chinese patients suspected of having AS. The results were confirmed by Sanger sequencing, which revealed two novel missense mutations resulting in the substitution of various glycine residues in a collagenous domain containing Gly-X-Y triplet sequence repeats [c.4198G>C, p.(Gly1400Arg) and c.3428G>T, p.(Gly1143Val)], a previously reported missense mutation [c.3071G>A, p.(Gly1024Glu)], a splice site mutation (c.2146+2T>A) and one frameshift mutation [c.1810delC (p.Thr605Ilefs*13)]. After analyzing the affected family members, it was shown that the identified mutations were associated with severe clinical phenotypes. These results broaden the known spectrum of mutations of the COL4A5 gene associated with AS and may have implications for genetic diagnosis, therapy and genetic counseling of affected families.
Alport syndrome
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To detect mutations of SLC25A13 gene in 20 families affected with citrin deficiency and provide prenatal diagnosis for them.The 20 probands and their parents were subjected to high-frequency mutation screening combined with Sanger sequencing. After confirming the genotype of each pedigree, genetic counseling and prenatal diagnosis were performed for their subsequent pregnancies.Biallelic pathogenic mutations of the SLC25A13 gene were identified in all probands. These included three deletions (c.851del4, c.1092_1095delT, and c.495delA), two splice-site mutations (IVS6+5G to A and IVS11+1G to A), two nonsense mutations (c.775C to T (p.Q259X) and c.72T to A (p.Y24X)), one duplication mutation (c.1638_1660dup), one insertion (IVSl6ins3kb), and one missense mutation (c.1775A to C (p.Q592P)). Among 24 fetuses undergoing prenatal diagnosis, 8 had normal genotypes, 11 were mutation carriers, while 5 harbored biallelic mutations. Those with wild type alleles or heterozygous SLC25A13 mutations were delivered. Two fetuses harboring homozygous c.851del4 mutations were also delivered. Three fetuses harboring biallelic mutations were terminated.Analysis of SLC25A13 gene mutations in families affected by citrin deficiency can provide evidence for molecular diagnosis and facilitate genetic counseling and prenatal diagnosis for the subsequent pregnancy, which can effectively reduce the risk of birth of further affected children.
Sanger sequencing
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Nonsense mutation
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Abstract Background Holocarboxylase synthetase (HLCS) deficiency is a rare inborn disorder of biotin metabolism, which results in defects in several biotin-dependent carboxylases and presents with metabolic ketoacidosis and skin lesions. Case presentation In this paper, we report a Chinese Han pedigree with HLCS deficiency diagnosed by using next-generation sequencing and validated with Sanger sequencing of the HLCS and BTD genes. The Chinese proband carries the common missense mutation c.1522C > T (p.Arg508Trp) in exon 9 of the HLCS gene, which generates an increased K m value for biotin. A novel frameshift mutation c.1006_1007delGA (p.Glu336Thrfs*15) in exon 6 of the HLCS gene is predicted to be deleterious through PROVEAN and MutationTaster. A novel heterozygous mutation, c.638_642delAACAC (p.His213Profs*4), in the BTD gene is also identified. Conclusions The Chinese proband carries the reported Arg508Trp variant, the novel 2-bp frameshift mutation c.1006_1007delGA (p.Glu336Thrfs*15), which expands the mutational spectrum of the HLCS gene, and the novel heterozygous mutation c.638_642delAACAC (p.His213Profs*4), which expands the mutational spectrum of the BTD gene. Furthermore, reversible hearing damage is rarely reported in patients with HLCS deficiency, which deserves further discussion.
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Sanger sequencing
Compound heterozygosity
INDEL Mutation
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To explore the genetic basis for a child with dihydropyrimidase (DHP) deficiency.High-throughput sequencing was carried out for the child. Suspected variants were verified by using Sanger sequencing.The proband was found to carry compound heterozygous variants of the DPYS gene, namely c.1468C>T (a missense variant) and c.1339-1363del (a frameshifting variant).The compound heterozygous variants of the DPYS gene probably underlie the DHP in this child. Above result has enabled genetic counseling and prenatal diagnosis for his parents.
Sanger sequencing
Proband
Compound heterozygosity
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To carry out mutation analysis for a pedigree affected with maple syrup urine disease (MSUD).Clinical data of the proband was collected. Potential mutations of the BCKDHA and BCKDHB genes were analyzed by PCR and Sanger sequencing. Prenatal diagnosis was provided to a high-risk fetus at 12th gestational week through chorionic villus sampling.Two heterozygous mutations c.284G>C (p.Gly95Ala) and c.853C>T (p.Arg285*) of the BCKDHB gene were identified in the proband, which were inherited from his mother and father, respectively. Among these, c.853C>T (p.Arg285*) was known to be pathogenic, while c.284G>C (p.Gly95Ala) was a novel mutation. Prenatal diagnosis showed that the fetus has inherited the c.284G>C (p.Gly95Ala) mutation from its mother but no mutation from its father. After birth, the infant appeared to be healthy.The compound heterozygous mutations c.284G>C (p.Gly95Ala) and c.853C>T (p.Arg285*) probably underlie the pathogenesis of MUSD in the proband. Mutation analysis can facilitate prenatal diagnosis and genetic counseling for the affected families.
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Compound heterozygosity
Sanger sequencing
Chorionic villus sampling
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