OBJECTIVE: To identify the pathogenic variants from a patient with suspected congenital contractural arachnodactyly, and to explore the possible molecular genetic pathogenesis, so as to provide evidence for clinical diagnosis. METHODS: Whole exome sequencing was performed for the patient. The splicing site variation of candidate pathogenic genes was verified by Sanger sequencing, and the new transcript sequence was determined by RT-PCR and TA-cloning sequencing. RESULTS: The patient carried a heterozygous c.533-1G>C variant of FBN2 gene, which was not reported. The sequencing of mRNA showed that the variant leaded to the disappearance of the canonical splice acceptor site of FBN2 gene and the activation of a cryptic splice acceptor site at c.533-71, resulting in the insertion of 70 bp sequence in the new transcript. It was speculated that the polypeptide encoded by the new transcript changed from valine (Val) to serine (Ser) at amino acid 179, and prematurely terminated after 26 aminoacids. According to the guidelines of American College of Medical Genetics and Genomics, the variant of FBN2 gene c. 533-1G>C was determined as pathogenic (PVS1+PM2+PP3 ). CONCLUSION A novel splicing variant of FBN2 gene (c.533-1G>C) was identified, which can lead to congenital contractural arachnodactyly.
Arachnodactyly/genetics* ; Contracture/genetics* ; Fibrillin-2/genetics* ; Humans ; Mutation ; RNA Splice Sites ; Whole Exome Sequencing

Adolescent ; Androgen-Insensitivity Syndrome/genetics* ; Cytosine ; Female ; Guanine ; Humans ; Male ; Mutation ; RNA Splice Sites/genetics* ; Receptors, Androgen/genetics*

OBJECTIVETo detect potential mutation of EXT1 gene in a pedigree affected with multiple osteochondroma and explore its pathogenic mechanism.

METHODSThe coding regions and their flanking sequences of the EXT1/EXT2 genes were subjected to PCR amplification and Sanger sequencing. Suspected mutations were verified by excluding possible single nucleotide polymorphisms and bioinformatics analysis. Transcripts of the EXT1 gene in the proband were analyzed by TA clone-sequencing, with its abundance compared with that of healthy controls.

RESULTSDNA sequencing has identified in the proband a novel heterozygous point mutation (c.1164+1G to A) at the 5'splice sites of intron 3 of the EXT1 gene. The same mutation was not found in the healthy controls. Bioinformatics analysis indicated that the mutation is highly conserved and can lead to skipping of exon 3 or aberrant splicing. TA clone-sequencing indicated that the numbers of transcripts with skipping of exon 3 has significantly increased in the proband (< 0.05) compared with the controls.

CONCLUSIONThe c.1164+1G to A mutation has resulted in skipping of exon 3 in a proportion of EXT1 gene transcripts. As the result, the number of transcripts with tumor suppressing function is relatively reduced and has ultimately led to the tumors.

Adult ; Base Sequence ; Child ; Exostoses, Multiple Hereditary ; genetics ; Female ; Humans ; Male ; Molecular Sequence Data ; N-Acetylglucosaminyltransferases ; genetics ; Point Mutation ; RNA Splice Sites ; RNA Splicing

Vitamin D hydroxylation-deficient rickets type 1A (VDDR1A) is an autosomal recessively-inherited disorder caused by mutations in CYP27B1 encoding the 1α-hydroxylase enzyme. We report on a female patient with VDDR1A who presented with hypocalcemic seizure at the age of 13 months. The typical clinical and biochemical features of VDDR1A were found, such as hypocalcemia, increased alkaline phosphatase, secondary hyperparathyroidism and normal 25-hydroxyvitamin D3 (25(OH)D₃). Radiographic images of the wrist showed metaphyseal widening with cupping and fraying of the ulna and distal radius, suggesting rickets. A mutation analysis of the CYP27B1 gene identified a homozygous mutation of c.589+1G>A in the splice donor site in intron 3, which was known to be pathogenic. Since that time, the patient has been under calcitriol and calcium treatment, with normal growth and development. During the follow-up period, she did not develop genu valgum, scoliosis, or nephrocalcinosis.
25-Hydroxyvitamin D3 1-alpha-Hydroxylase* ; Alkaline Phosphatase ; Calcifediol ; Calcitriol ; Calcium ; Female ; Follow-Up Studies ; Genu Valgum ; Growth and Development ; Humans ; Hyperparathyroidism, Secondary ; Hypocalcemia ; Introns ; Nephrocalcinosis ; Radius ; Rickets* ; RNA Splice Sites ; Scoliosis ; Seizures ; Ulna ; Vitamin D* ; Vitamins* ; Wrist

X-linked hypophosphatemia (XLH) is the most common form of familial hypophosphatemic rickets and it is caused by loss-of-function mutations in the PHEX gene. Recently, a wide variety of PHEX gene defects in XLH have been revealed; these include missense mutations, nonsense mutations, splice site mutations, insertions, and deletions. Recently, we encountered a 2-year-9-month-old female with sporadic hypophosphatemic rickets. She underwent osteotomy, dental abscess was evident, and there was severe bowing of the legs. A low serum phosphorus level in combination with elevated serum alkaline phosphatase activity and normal serum calcium is suggestive of hypophosphatemic rickets. PHEX gene analysis revealed a splice acceptor site mutation, c.934-1G>T (IVS8-1G>T), at the intron8 and exon9 junction. To the best of our knowledge, this mutation is novel and has not been reported. The results of this study expand and improve our understanding of the clinical and molecular characteristics and the global pool of patients with sporadic hypophosphatemic rickets.
Abscess ; Alkaline Phosphatase ; Calcium ; Codon, Nonsense ; Familial Hypophosphatemic Rickets ; Female ; Humans ; Leg ; Mutation, Missense ; Osteotomy ; Phosphorus ; Rickets, Hypophosphatemic* ; RNA Splice Sites

Fanconi anemia (FA) is a rare genetic disorder affecting multiple body systems. Genetic testing, including prenatal testing, is a prerequisite for the diagnosis of many clinical conditions. However, genetic testing is complicated for FA because there are often many genes that are associated with its development, and large deletions, duplications, or sequence variations are frequently found in some of these genes. This study describes successful genetic testing for molecular diagnosis, and subsequent prenatal diagnosis, of FA in a patient and his family in Korea. We analyzed all exons and flanking regions of the FANCA, FANCC, and FANCG genes for mutation identification and subsequent prenatal diagnosis. Multiplex ligation-dependent probe amplification analysis was performed to detect large deletions or duplications in the FANCA gene. Molecular analysis revealed two mutations in the FANCA gene: a frameshift mutation c.2546delC and a novel splice-site mutation c.3627-1G>A. The FANCA mutations were separately inherited from each parent, c.2546delC was derived from the father, whereas c.3627-1G>A originated from the mother. The amniotic fluid cells were c.3627-1G>A heterozygotes, suggesting that the fetus was unaffected. This is the first report of genetic testing that was successfully applied to molecular diagnosis of a patient and subsequent prenatal diagnosis of FA in a family in Korea.
Base Sequence ; Child, Preschool ; Exons ; Fanconi Anemia/*diagnosis/genetics ; Fanconi Anemia Complementation Group A Protein/genetics ; Fanconi Anemia Complementation Group C Protein/genetics ; Fanconi Anemia Complementation Group G Protein/genetics ; Female ; Frameshift Mutation ; Genetic Testing ; Heterozygote ; Humans ; Karyotyping ; Male ; Pregnancy ; Prenatal Diagnosis ; RNA Splice Sites ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA

This study was aimed to analyze the β-globin gene mutations in a patient with β-thalassemia minor. Genomic DNA was extracted from peripheral blood cells of the patient. The full-length DNA sequence coding for β-globin was amplified by polymerase chain reaction, and the gene mutation was determined by DNA sequencing. The results indicated that a heterogeneous A→G mutation was found at position 129 in intron 1 of the β-thalassemia minor patient. It is concluded that the IVS-I-129(A→G) mutation is a splicing site mutation leading to a splicing error in immature messenger RNA and a protein translation error for the β-globin gene. Thus, the IVS-I-129(A→G) is a novel mutation.
Adult ; Base Sequence ; DNA Mutational Analysis ; Female ; Humans ; Introns ; Point Mutation ; Protein Biosynthesis ; RNA Splice Sites ; beta-Globins ; genetics ; beta-Thalassemia ; genetics

Spliceosomal RNAs are a family of small nuclear RNAs (snRNAs) that are essential for pre-mRNA splicing. All vertebrate spliceosomal snRNAs are extensively pseudouridylated after transcription. Pseudouridines in spliceosomal snRNAs are generally clustered in regions that are functionally important during splicing. Many of these modified nucleotides are conserved across species lines. Recent studies have demonstrated that spliceosomal snRNA pseudouridylation is catalyzed by two different mechanisms: an RNA-dependent mechanism and an RNA-independent mechanism. The functions of the pseudouridines in spliceosomal snRNAs (U2 snRNA in particular) have also been extensively studied. Experimental data indicate that virtually all pseudouridines in U2 snRNA are functionally important. Besides the currently known pseudouridines (constitutive modifications), recent work has also indicated that pseudouridylation can be induced at novel positions under stress conditions, thus strongly suggesting that pseudouridylation is also a regulatory modification.
Animals ; Base Sequence ; Molecular Sequence Data ; Nucleic Acid Conformation ; Nucleotides ; metabolism ; Oocytes ; cytology ; metabolism ; Pseudouridine ; metabolism ; RNA Precursors ; metabolism ; RNA Splice Sites ; RNA Splicing ; RNA, Messenger ; genetics ; metabolism ; RNA, Small Nuclear ; genetics ; metabolism ; Ribonucleoproteins, Small Nuclear ; genetics ; metabolism ; Saccharomyces cerevisiae ; genetics ; metabolism ; Saccharomyces cerevisiae Proteins ; genetics ; metabolism ; Spliceosomes ; genetics ; metabolism ; Uridine ; analogs & derivatives ; metabolism ; Xenopus ; genetics ; metabolism

BACKGROUND: Neurofibromatosis type 2 (NF2) is an autosomal dominant syndrome caused by the NF2 tumor suppressor gene. However, the NF2 mutation characteristics in Korean patients are not sufficiently understood. In this study, we conducted a comprehensive mutational analysis in 7 Korean NF2 patients by performing direct sequencing and gene-dosage assessment. METHODS: We analyzed all exons and flanking regions of NF2 by direct sequencing and screened the deletions or duplications involving NF2 by multiplex ligation-dependent probe amplification. RESULTS: Four novel NF2 mutations, including 2 splice-site mutations (c.364-1G>A and c.886-3C>G), 1 frameshift mutation (c.524delA), and 1 missense mutation (c.397T>C; p.Cys133Arg), were identified in our patients. No large deletion or duplication was identified in our series. Subsequently, we identified an abnormal splicing product by using reverse transcription-PCR and direct sequencing in 2 patients with a novel splice-site mutation. The missense mutation c.397T>C was predicted to have harmful effects on protein function. CONCLUSIONS: The detection rate of NF2 mutations in Korean patients (57%) is similar to those in other populations. Our results provided a greater insight into the mutational spectrum of the NF2 gene in Korean subjects.
3' Flanking Region/genetics ; 5' Flanking Region/genetics ; Adult ; Aged ; Amino Acid Sequence ; Asian Continental Ancestry Group/*genetics ; Child, Preschool ; Exons ; Female ; Frameshift Mutation ; *Genes, Neurofibromatosis 2 ; Humans ; Male ; Middle Aged ; Molecular Sequence Data ; *Mutation ; Mutation, Missense ; Neurofibromatosis 2/diagnosis/*genetics ; RNA Splice Sites ; Republic of Korea ; Sequence Analysis, DNA ; Young Adult

OBJECTIVETo investigate the existence of alternatively spliced variants of constitutive androstane receptor (CAR) in liver of mouse.

METHODSThe nucleotide from liver of mouse was purified and the CAR cDNA was amplified by PCR. The fragments of CAR cDNA were cloned to T vector and sequence analysis was performed.

RESULTVarious spliced variants of CAR in liver mouse were confirmed by DNA sequencing.

CONCLUSIONThere are alternatively spliced variants in CAR, which are located in the ligand binding sequence of CAR.

Alternative Splicing ; Amino Acid Sequence ; Animals ; DNA, Complementary ; genetics ; Liver ; metabolism ; Male ; Mice ; Molecular Sequence Data ; RNA Splice Sites ; Receptors, Cytoplasmic and Nuclear ; genetics