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

Mycobacteria, which are highly successful pathogen, resist delivary to lysosomes and instead survive within a specialized vacuole, the mycobacterial phagosome. The bacteria survive intracellularly because they are able to actively recruit and retain TACO ( tryptophane aspartate-containing coat protein ) at the mycobacterial phagosome, where it prevents lysosomal delivary in a cholesterol-dependent manner. In this study, we investigated the difference of TACO expression is whether related to mutant in coro1a gene in patients with leprosy and normal volunteer. First, we screened for detection of a mutant in the leucine zipper motif within the exon 11, and then in the exon 9 to 10, and finally in the coiled-coil region. Interestingly, single base substitutions ( point mutation ) presents at assembly site of U1 snRNP, around of 5' splice site in the intron 9, there are a C to T and G to A transition are at 9 bp and 14 bp downstream of 5' splice site, respectively, and both of it. Among the 3 types of polymorphism, frequency of a G to A transition is markedly increased in patients of lepromatous type, which are new cases or relapsed. Both a C to T and G to A transitions are found in 1 case of tuberculoid type and 2 cases in lepromatoue type, but not found in control group. The silent mutation in leucine zipper motif within the exon 11 is located at codon at 454 ( CTG-->CTA), which is 1st leucine from C-terminal among four leucine zipper. In coiled-coil region, no mutation is found in genomic DNA of patients with leprosy. Further, we will do functional study about the identified point mutation and will screen any possible mutation in the region of promotor and WD repeat.
Bacteria ; Codon ; DNA ; Exons ; Healthy Volunteers ; Humans ; Introns ; Leprosy* ; Leucine ; Leucine Zippers ; Lysosomes ; Phagosomes ; Point Mutation* ; Ribonucleoprotein, U1 Small Nuclear ; RNA Splice Sites ; Tryptophan ; Vacuoles

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

OBJECTIVETo study the expression of plasma membrane Ca(2+)-ATPase isoform 2 (PMCA2) in spiral ganglion cell (SGC) from inner ear of newborn rats and further check PMCA2 splice variants at site A and C.

METHODSSpiral ganglion tissues isolated from cochlea of newborn rats (P3-P4) were cultured and identified in vitro. The cochlea of newborn rats (P3-P4) were isolated and cut into frozen sections. The expression of PMCA2 was detected by immunofluorescence analyses. The SGC cultured in 4 wells of the 6-well culture plate were collected and the total RNA was extracted by Trizol and reverse transcribed to cDNA. The site A and C splice variants of PMCA2 were respectively checked by nested PCR and common PCR.

RESULTSThe SGC grew well with good refraction and showed positive immunoreactivity for neuronal marker NF-200. Strong green fluorescence could be seen in cytomembrane, cytoplasm and neuritis, as well showing SGC immunoreactivity for PMCA2 antibody. In the cochlear sections, the spiral ganglion tissues were strongly stained by PMCA2. PMCA2z was present at splice site A, but PMCA2b and PMCA2c were present at splice site C.

CONCLUSIONSGC from newborn rats strongly expresses PMCA2 and different splice variants are present at PMCA2 splice site A and C.

Animals ; Female ; Male ; Plasma Membrane Calcium-Transporting ATPases ; metabolism ; Protein Isoforms ; metabolism ; RNA Splice Sites ; Rats ; Rats, Sprague-Dawley ; Spiral Ganglion ; cytology ; metabolism

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

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

OBJECTIVETo explore the expression of phospholipase C-gamma 1 (PLC-gamma1) alternative splicing variants in rats.

METHODSAccording to the sequence of human PLCG1 splicing variant, specific primers for rat PLC-gamma1 were designed and synthesized. The rat RNA was reverse transcribed into cDNA, which was amplified using the specific primers, and the PCR products were sequenced and analyzed using BLAST and bioinformatics methods. Totally 21 rat tissue samples were examined, including the heart, liver, lung, kidney, eyeball, and brain obtained in 3 different embryonic stages, 7 different early postnatal stages, and in adulthood.

RESULTSThe result did not show that rat PLC-gamma1 had the same splicing variant (PLC-gamma1a, NM_002660) as human does.

CONCLUSIONSThe same splicing variant of PLC-gamma1 detectable in human may not exist in rats, and the pre-mRNA may undergo splicing resulting predominantly in PLC-gamma1b mRNA. Very likely, the alternative splicing site of rat PLC-gamma1 is not identical to that of human.

Alternative Splicing ; Animals ; Base Sequence ; Molecular Sequence Data ; Phospholipase C gamma ; genetics ; RNA Precursors ; genetics ; RNA Splice Sites ; genetics ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA

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