Compound heterozygous CNGA3 mutations (R436W, L633P) in a 
Japanese patient with congenital achromatopsia

Congenital achromatopsia is a stationary retinal disorder with autosomal recessive inheritance that is characterized by loss of color discrimination, low visual acuity, photophobia, and nystagmus. This disorder has been shown to be associated with CNGA3, CNGB3, and GNAT2 mutations, and the frequency of mutations in the CNGA3 gene (encoding α subunit of the cone-specific cGMP-gated cation channel) was 23–33% in European populations. The aim of this study was to test the hypothesis that CNGA3 mutations are also responsible for congenital achromatopsia in Japanese patients. DNA from venous blood samples from a total of 14 patients from 13 Japanese pedigrees was prepared. Mutation screening of the CNGA3 gene was performed using direct sequencing and PCR-single-strand conformation polymorphism analysis. Compound heterozygous missense mutations (p.R436W and p.L633P, the latter of which was novel) were identified in one patient only, a 22-year-old female. Neither of these two mutations was found in 150 Japanese control individuals. The patient's parents and sister carried one of these mutations each but were not affected. No mutations in the CNGB3 or GNAT2 genes were identified in the patient. Clinically, best-corrected visual acuity was 0.1 in both eyes. No specific findings were obtained in funduscopy. Optical coherence topography revealed a normal foveal thickness but a 20% decrease in parafoveal thickness. Ganzfeld full-field electroretinograms (ERGs) showed normal responses in rod and mixed rod-plus-cone ERGs but no response in cone or 30-Hz flicker ERGs. Spectral sensitivity on a white background revealed a curve with only one peak at around 500 nm, which fits the absorption spectrum of human rhodopsin. L633, conserved among vertebrate orthologs of human CNGA3, is a hydrophobic residue forming part of the carboxy-terminal leucine zipper (CLZ) domain, which is functionally important in the mediation of intracellular interactions. To our knowledge, this is the first report of a Japanese complete achromat with CNGA3 mutations, and of any patient with a missense mutation within the CLZ domain. The outcome suggests low frequency (7%, 1/14) of CNGA3 mutations in Japanese patients.

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Molecular study of eight Japanese cases of glucose-6-phosphate dehydrogenase deficiency by nonradioisotopic single-strand conformation polymorphism analysis [see comments]

Blood ◽

10.1182/blood.v83.11.3363.3363 ◽

1994 ◽

Vol 83 (11) ◽

pp. 3363-3368 ◽

Cited By ~ 29

Author(s):

A Hirono ◽

S Miwa ◽

H Fujii ◽

F Ishida ◽

K Yamada ◽

...

Keyword(s):

Direct Sequencing ◽

Japanese Patients ◽

Single Strand Conformation Polymorphism ◽

Single Strand ◽

Sscp Analysis ◽

Polymorphism Analysis ◽

Missense Mutations ◽

G6pd Gene ◽

Glucose 6 Phosphate Dehydrogenase ◽

Conformation Polymorphism

Abstract Using a newly developed nonradioisotopic method of polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis combined with the direct sequencing using the fluorescence-labeled terminator, we identified seven missense mutations, 527 A-->G, 1003 G-- >A, 1159 C--eT, 1160 G-->A, 1229 G-->A, 1246 G-->A, and 1361 G-->A, in eight Japanese patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Except for the 527 A-->G, each mutation has been reported to cause variants G6PD Chatham, G6PD Guadalajara, G6PD Beverly Hills, G6PD “Japan”, G6PD Tokyo, and G6PD Andalus, respectively. In addition, a single base deletion in intron 5 was found in the patients with G6PD Guadalajara or G6PD Andalus. The variant with unique 527 A-->G was characterized and designated as G6PD Shinshu. We also characterized G6PD “Japan” and found that the variant had the striking resemblance with G6PD Riverside, bearing a missense mutation in the same codon, but causing a different amino acid substitution. Our modified PCR-SSCP analysis using minigel and ethidium bromide staining could detect six of the eight diverse mutations in the G6PD gene. Because it is easy and requires no special apparatus, this modified method will be useful for screening mutations in the G6PD gene.

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Molecular study of eight Japanese cases of glucose-6-phosphate dehydrogenase deficiency by nonradioisotopic single-strand conformation polymorphism analysis [see comments]

Blood ◽

10.1182/blood.v83.11.3363.bloodjournal83113363 ◽

1994 ◽

Vol 83 (11) ◽

pp. 3363-3368

Author(s):

A Hirono ◽

S Miwa ◽

H Fujii ◽

F Ishida ◽

K Yamada ◽

...

Keyword(s):

Direct Sequencing ◽

Japanese Patients ◽

Single Strand Conformation Polymorphism ◽

Single Strand ◽

Sscp Analysis ◽

Polymorphism Analysis ◽

Missense Mutations ◽

G6pd Gene ◽

Glucose 6 Phosphate Dehydrogenase ◽

Conformation Polymorphism

Using a newly developed nonradioisotopic method of polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis combined with the direct sequencing using the fluorescence-labeled terminator, we identified seven missense mutations, 527 A-->G, 1003 G-- >A, 1159 C--eT, 1160 G-->A, 1229 G-->A, 1246 G-->A, and 1361 G-->A, in eight Japanese patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. Except for the 527 A-->G, each mutation has been reported to cause variants G6PD Chatham, G6PD Guadalajara, G6PD Beverly Hills, G6PD “Japan”, G6PD Tokyo, and G6PD Andalus, respectively. In addition, a single base deletion in intron 5 was found in the patients with G6PD Guadalajara or G6PD Andalus. The variant with unique 527 A-->G was characterized and designated as G6PD Shinshu. We also characterized G6PD “Japan” and found that the variant had the striking resemblance with G6PD Riverside, bearing a missense mutation in the same codon, but causing a different amino acid substitution. Our modified PCR-SSCP analysis using minigel and ethidium bromide staining could detect six of the eight diverse mutations in the G6PD gene. Because it is easy and requires no special apparatus, this modified method will be useful for screening mutations in the G6PD gene.

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A Family with Gitelman Syndrome with Asymptomatic Phenotypes while Carrying Reported SLC12A3 Mutations

Case Reports in Nephrology and Dialysis ◽

10.1159/000507845 ◽

2020 ◽

Vol 10 (2) ◽

pp. 71-78

Author(s):

Moena Ishikawa ◽

Yumi Tada ◽

Hiromu Tanaka ◽

Wataru Morii ◽

Masako Inaba ◽

...

Keyword(s):

Venous Blood ◽

Genetic Diagnosis ◽

Autosomal Recessive Disorder ◽

Gitelman Syndrome ◽

Compound Heterozygous ◽

Missense Mutations ◽

Maternal Grandmother ◽

Phenotypic Effect ◽

Normal Limits ◽

Old Japanese

Gitelman syndrome (GS) is an autosomal recessive disorder characterized by alkalosis, hypokalemia, and hypomagnesemia. Although hundreds of genetic variants associated with GS have been reported, many of them are categorized as of uncertain significance in ClinVar. Here, we describe a pediatric GS patient from a three-generation family whose mother and maternal grandmother were asymptomatic. The proband was a 16-year-old Japanese girl with muscle weakness and continuous hypokalemic metabolic alkalosis. The patient, her mother, and her maternal grandmother were compound heterozygous for, and each expressing a different combination of, previously reported SLC12A3variants in GS patients. The mother and the maternal grandmother had no symptoms related to GS, and blood gas tests showed that the blood potassium levels and venous pH were within normal limits; however, the venous blood HCO3- levels were slightly elevated. The phenotypic effect of missense mutations is difficult to evaluate, and accumulation of genotypic data with accurate phenotyping, including those of “healthy” and “asymptomatic” individuals in various ethnic populations, will improve the genetic diagnosis of GS.

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Precise Carrier Diagnosis in Families with Haemophilia A: Use of Conformation Sensitive Gel Electrophoresis for Mutation Screening and Polymorphism Analysis

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615052 ◽

1998 ◽

Vol 79 (04) ◽

pp. 723-726 ◽

Cited By ~ 62

Author(s):

I. J. Williams ◽

A. Abuzenadah ◽

P. R. Winship ◽

F. E. Preston ◽

G. Dolan ◽

...

Keyword(s):

Factor Viii ◽

Gel Electrophoresis ◽

Direct Sequencing ◽

Mutation Screening ◽

Gene Mutations ◽

Carrier Status ◽

Polymorphism Analysis ◽

Female Carrier ◽

Haemophilia A ◽

Factor Viii Gene

SummaryCausative mutations in the factor VIII gene of seven unrelated patients with severe haemophilia A were identified using the mutation screening procedure conformation sensitive gel electrophoresis (1) and characterised by direct sequencing. Female family members of all patients had requested either carrier status determination or prenatal diagnosis. However, lack of the factor VIII gene inversion, a prior family history or informative polymorphisms prevented diagnosis in these families. Identification of a mutation in each family enabled female carrier status to be determined in all cases. Six mutations were previously unreported. One Afro-Caribbean patient had two sequence changes; A670 2G and A6769G. The latter, resulting in Met2238Val and previously reported as a FVIII mutation, was shown to be polymorphic with a 42% heterozygosity rate in an Afro-Caribbean population. Conformation sensitive gel electrophoresis was found to be technically simple and efficient at locating previously unknown FVIII gene mutations.

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Phenotypic and molecular characterisation of type 3 von Willebrand disease in a cohort of Indian patients

Thrombosis and Haemostasis ◽

10.1160/th12-10-0737 ◽

2013 ◽

Vol 109 (04) ◽

pp. 652-660 ◽

Cited By ~ 9

Author(s):

Ulrich Budde ◽

Rifat Jan ◽

Florian Oyen ◽

Meganathan Kannan ◽

Reinhard Schneppenheim ◽

...

Keyword(s):

Direct Sequencing ◽

Mutation Screening ◽

Strand Break ◽

Von Willebrand Disease ◽

Missense Mutations ◽

Large Deletions ◽

High Propensity ◽

Type 3 ◽

Von Willebrand ◽

Willebrand Factor

SummarySevere type 3 VWD (VWD3) is characterised by complete absence or presence of trace amounts of non-functional von Willebrand factor (VWF). The study was designed to evaluate the VWF mutations in VWD3 patients and characterise the breakpoints of two identified homozygous novel large deletions. Patients were diagnosed by conventional tests and VWF multimer analysis. Mutation screening was performed in 19 VWD3 patients by direct sequencing of VWF including flanking intronic sequence and multiplex ligation-dependent probe amplification (MLPA) analysis. Breakpoint characterisation of two identified novel large deletions was done using walking primers and long spanning PCR. A total of 21 different mutations including 15 (71.4%) novel ones were identified in 17 (89.5%) patients. Of these mutations, five (23.8%) were nonsense (p.R1659*, p.R1779*, p.R1853*, p.Q2470*, p.Q2520*), one was a putative splice site (p.M814I) and seven (33.3%) were deletions (p.L254fs*48, p.C849fs*60, p.L1871fs*6, p.E2720fs*24) including three novel large deletions of exon 14–15, 80,830bp (−41510_657+7928A*del) and 2,231bp [1534–2072T_c.1692G*del(p.512fs*terminus)] respectively. A patient carried gene conversion comprising of pseudogene harbouring mutations. The missense mutations (p.G19R, p.K355R, p.D437Y, p.C633R, p.M771V, p.G2044D, p.C2491R) appear to play a major role and were identified in seven (36.8%) patients. In conclusion, a high frequency of novel mutations suggests the high propensity of VWF for new mutations. Missense and deletion mutations found to be a common cause of VWD3 in cohort of Indian VWD3 patients. Breakpoints characterisation of two large deletions reveals the double strand break and non-homologous recombination as deletions mechanism.

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Changes in Resurgent Sodium Current Contribute to the Hyperexcitability of Muscles in Patients with Paramyotonia Congenita

Biomedicines ◽

10.3390/biomedicines9010051 ◽

2021 ◽

Vol 9 (1) ◽

pp. 51

Author(s):

Chiung-Wei Huang ◽

Hsing-Jung Lai ◽

Pi-Chen Lin ◽

Ming-Jen Lee

Keyword(s):

Sodium Current ◽

Mutation Screening ◽

Chinese Hamster ◽

Muscle Stiffness ◽

Missense Mutations ◽

Slow Inactivation ◽

Fast Inactivation ◽

Paramyotonia Congenita ◽

Α Subunit ◽

Resurgent Sodium Current

Paramyotonia congenita (PMC) is a rare hereditary skeletal muscle disorder. The major symptom, muscle stiffness, is frequently induced by cold exposure and repetitive exercise. Mutations in human SCN4A gene, which encodes the α-subunit of Nav1.4 channel, are responsible for PMC. Mutation screening of SCN4A gene from two PMC families identified two missense mutations, p.T1313M and p.R1448H. To elucidate the electrophysiological abnormalities caused by the mutations, the p.T1313M, p.R1448H, and wild-type (WT) SCN4A genes were transient expressed on Chinese hamster ovary (CHO-K1) cells. The detailed study on the gating defects of the mutant channels using the whole-cell patch clamping technique was performed. The mutant Nav1.4 channels impaired the basic gating properties with increasing sustained and window currents during membrane depolarization and facilitated the genesis of resurgent currents during repolarization. The mutations caused a hyperpolarization shift in the fast inactivation and slightly enhanced the slow inactivation with an increase in half-maximal inactivation voltage. No differences were found in the decay kinetics of the tail current between mutant and WT channels. In addition to generating the larger resurgent sodium current, the time to peak in the mutant channels was longer than that in the WT channels. In conclusion, our results demonstrated that the mutations p.T1313M and p.R1448H in Nav1.4 channels can enhance fast inactivation, slow inactivation, and resurgent current, revealing that subtle changes in gating processes can influence the clinical phenotype.

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Mutation Screening of DUOX2 Gene in Children with Congenital Hypothyroidism

10.21203/rs.2.22276/v1 ◽

2020 ◽

Author(s):

Xiao-Yu Chen ◽

Yong Liu ◽

Jian-Hua Liu ◽

Xiaosong Qin

Keyword(s):

Congenital Hypothyroidism ◽

Mutation Screening ◽

Venous Blood ◽

Chinese Patients ◽

Dimensional Structure ◽

Compound Heterozygous Mutation ◽

Heterozygous Mutation ◽

Compound Heterozygous ◽

Gene Variants ◽

The Relationship

Abstract Background: Congenital hypothyroidism(CH) is generally known as the most common neonatal endocrine disorder. However, the mutational spectrum of DUOX2 gene and the relationship between genotype and phenotype have not been fully established among Chinese CH patients. Therefore, The aim of this study was to screen DUOX2 mutations in Chinese patients with CH and to research the relationship between DUOX2 genotype and clinical phenotype.Methods: Eighty-six patients with CH were recruited from northeastern region of China. Peripheral venous blood samples were collected, genomic DNA was extracted , PCR and next-generation sequencing (NGS) were used to analyze all exons of DUOX2. Detailed medical data were collected, and the relationship between DUOX2 genotype and the clinical phenotype was preliminarily investigated.Results: NGS analysis of DUOX2 gene identified a total of 20 different gene variants in 26 patients(30.2%), which was consistent with the gene variants in Asian populations, among these variants 16 were known to be pathogenic or likely to be pathogenic, and four were suspected to be of uncertain significance. Three mutations (p.K530X, p.L1343F and p.R1110Q) were highly recurrent in our patient cohort. By using protein homology modeling method, the analysis of its three-dimensional structure suggested that the mutations p.336_337del and p.T1107fs caused the change of the protein.Conclusions: In our study, p.336_337del and p.T1107fs were found to be novel variants and p.K530X with the highest prevalence. Children with DUOX2 single allele heterozygous mutation or compound heterozygous mutation exhibited different morphological developments of the thyroid.

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A Novel Mutation Glyl672→Arg in Type 2A and a Homozygous Mutation in Type 2B von Willebrand Disease

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650564 ◽

1996 ◽

Vol 76 (02) ◽

pp. 253-257 ◽

Cited By ~ 14

Author(s):

Takeshi Hagiwara ◽

Hiroshi Inaba ◽

Shinichi Yoshida ◽

Keiko Nagaizumi ◽

Morio Arai ◽

...

Keyword(s):

Missense Mutation ◽

Novel Mutation ◽

Point Mutations ◽

Japanese Patients ◽

Von Willebrand Disease ◽

Homozygous Mutation ◽

Missense Mutations ◽

Reaction Products ◽

Type 3 ◽

Von Willebrand

SummaryGenetic materials from 16 unrelated Japanese patients with von Willebrand disease (vWD) were analyzed for mutations. Exon 28 of the von Willebrand factor (vWF) gene, where point mutations have been found most frequent, was screened by various restriction-enzyme analyses. Six patients were observed to have abnormal restriction patterns. By sequence analyses of the polymerase chain-reaction products, we identified a homozygous R1308C missense mutation in a patient with type 2B vWD; R1597W, R1597Q, G1609R and G1672R missense mutations in five patients with type 2A; and a G1659ter nonsense mutation in a patient with type 3 vWD. The G1672R was a novel missense mutation of the carboxyl-terminal end of the A2 domain. In addition, we detected an A/C polymorphism at nucleotide 4915 with HaeIII. There was no particular linkage disequilibrium of the A/C polymorphism, either with the G/A polymorphism at nucleotide 4391 detected with Hphl or with the C/T at 4891 detected with BstEll.

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Anti-Myelin Oligodendrocyte Glycoprotein Encephalomyelitis and Extensive Longitudinal Transverse Myelitis Associated with Compound Heterozygous NLRP3 Missense Mutations in a Young Child

Journal of Pediatric Neurology ◽

10.1055/s-0040-1721434 ◽

2020 ◽

Author(s):

Deirdre O'Sullivan ◽

Michael Moore ◽

Susan Byrne ◽

Andreas O. Reiff ◽

Susanna Felsenstein

Keyword(s):

Young Child ◽

Genetic Basis ◽

Transverse Myelitis ◽

Acute Disseminated Encephalomyelitis ◽

Myelin Oligodendrocyte Glycoprotein ◽

Compound Heterozygous ◽

Missense Mutations ◽

Childhood Onset

AbstractAcute disseminated encephalomyelitis in association with extensive longitudinal transverse myelitis is reported in a young child with positive anti-myelin oligodendrocyte glycoprotein (MOG) antibody with heterozygous NLRP3 missense mutations; p.(Arg488Lys) and p.(Ser159Ile). This case may well present an exceptional coincidence, but may describe a yet unrecognized feature of the spectrum of childhood onset cryopyrinopathies that contribute to the understanding of the genetic basis for anti-MOG antibody positive encephalomyelitis. Based on this observation, a larger scale study investigating the role of NLRP3 and other inflammasomes in this entity would provide important pathophysiological insights and potentially novel avenues for treatment.

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Mutation screening of the thyroid peroxidase gene in a cohort of 55 Portuguese patients with congenital hypothyroidism

Acta Endocrinologica ◽

10.1530/eje.1.01826 ◽

2005 ◽

Vol 152 (2) ◽

pp. 193-198 ◽

Cited By ~ 35

Author(s):

Carina Rodrigues ◽

Paula Jorge ◽

José Pires Soares ◽

Isaura Santos ◽

Regina Salomão ◽

...

Keyword(s):

Congenital Hypothyroidism ◽

Mutation Screening ◽

Gene Mutations ◽

Human Thyroid ◽

Thyroid Peroxidase ◽

Molecular Testing ◽

Missense Mutations ◽

Human Thyroid Peroxidase ◽

Iodide Organification Defect ◽

Organification Defect

Objective: Defects in the human thyroid peroxidase (TPO) gene are reported to be one of the causes of congenital hypothyroidism (CH) due to a total iodide organification defect. The aim of the present study was to determine the nature and frequency of TPO gene mutations in patients with CH, characterised by elevated TSH levels and orthotopic thyroid gland, identified in the Portuguese National Neonatal Screening Programme. Subjects and methods: The sample comprised 55 patients, from 53 unrelated families, with follow-up in the endocrinology clinics of the treatment centres of Porto and Lisbon. Mutation screening in the TPO gene (exons 1–17) was performed by single-strand conformational analysis followed by sequencing of fragments with abnormal migration patterns. Results: Eight different mutations were detected in 13 patients (seven homozygotes and six compound heterozygotes). Novel mutations included three missense mutations, namely 391T > C (S131P), 1274A > G (N425S) and 2512T > A (C838S), as well as the predictable splice mutation 2748G > A (Q916Q/spl?). The undocumented polymorphism 180-47A > C was also detected. Conclusion: The results are in accordance with previous observations confirming the genetic heterogeneity of TPO defects. The proportion of patients in which the aetiology was determined justifies the implementation of this molecular testing in our CH patients with dyshormonogenesis.

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