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PubMed2005

Novel TRPM6 mutations in 21 families with primary hypomagnesemia and secondary hypocalcemia.

Primary hypomagnesemia with secondary hypocalcemia is a rare autosomal recessive disorder characterized by profound hypomagnesemia associated with hypocalcemia. Pathophysiology is related to impaired intestinal absorption of magnesium accompanied by renal magnesium wasting as a result of a reabsorption defect in the distal convoluted tubule. Recently, mutations in the TRPM6 gene coding for TRPM6, a member of the transient receptor potential (TRP) family of cation channels, were identified as the underlying genetic defect. Here, the results of a TRPM6 mutational analysis of 21 families with 28 affected individuals are presented. In this large patient cohort, a retrospective clinical evaluation based on a standardized questionnaire was also performed. Genotype analysis revealed TRPM6 mutations in 37 of 42 expected mutant alleles. Sixteen new TRPM6 mutations were identified, including stop mutations, frame-shift mutations, splice-site mutations, and deletions of exons. Electrophysiologic analysis of mutated ion channels after heterologous expression in Xenopus oocytes proved complete loss of function of TRPM6. Clinical evaluation revealed a homogeneous clinical picture at manifestation with onset in early infancy with generalized cerebral convulsions. Initial laboratory evaluation yielded extremely low serum magnesium levels, low serum calcium levels, and inadequately low parathyroid hormone levels. Treatment usually consisted of acute intravenous magnesium supplementation leading to relief of clinical symptoms and normocalcemia, followed by lifelong oral magnesium supplementation. Serum magnesium levels remained in the subnormal range despite adequate therapy. This is best explained by a disturbed magnesium conservation in the distal convoluted tubule, which emerged in all patients upon magnesium supplementation. Delay of diagnosis resulted in permanent neurologic damage in three patients.

DNA Mutational Analysis↗

PubMed2006

Rapid one-step carrier detection assay of mucolipidosis IV mutations in the Ashkenazi Jewish population.

Two mutations in the MCOLN1 mucolipidosis IV (ML IV) gene represent approximately 95% of the mutations in Ashkenazi-Jewish patients with ML IV. The mutations, a splice site mutation (IVS3-2A>G) and an approximately 6.4-kb deletion (511del6434), account for 72% and 23% of ML IV alleles in this population, respectively. An automated high-throughput assay was developed using the 5'-nuclease (TaqMan) method for the simultaneous detection of both mutations in a single reaction. Three fluorescent probes specifically detected wild-type, IVS3-2A>G, and 511del6434 alleles in each reaction real-time. Data collected were automatically analyzed, and genotype results were uploaded into a laboratory information management system. The assay was validated using genomic controls, demonstrating high robustness and accuracy. Carrier screening of 10,527 samples revealed 77 heterozygote carriers of IVS3-2A>G, 25 heterozygote carriers of 511del6434, and two compound heterozygote of both mutant alleles. The frequency of mutated alleles was 0.73% for IVS3-2A>G and 0.24% for 511del6434. The combined carrier frequency was 1:103 with predicted disease incidence of 1:42,436 individuals in this population, slightly lower than previously described frequencies. This automated high-throughput assay is labor saving, because two mutations can be detected in a single reaction. The method has potential for use in other assays requiring simultaneous detection of two mutations.

Alleles↗

PubMed1999

[Auditory brainstem responses in group A xeroderma pigmentosum].

Xeroderma pigmentosum has been known to result from disturbance in the repair of injured DNA caused by the ultra violet light. According to cell fusion studies, this disease is classified into 8 groups. Among these groups, group A (A-XP) shows the most severe type of neurological disturbance. Slowly progressive diffuse impairment both in the central and the peripheral nervous systems has been reported. Although hearing loss occurs in all the patients, few papers have described the chronological changes of this disability. This study aimed to clarify the development of audiological abnormalities in A-XP patients. We recorded auditory brainstem evoked responses (ABRs) in 20 Japanese children with A-XP. All patients had homozygous intron 3 splicing mutations of xeroderma pigmentosum group A complementing gene, the most common type mutations in Japan. ABR threshold, peak latency of 5th wave and the peak interval latency between the 1st and 5th waves (I-V interpeak latency) were measured, and were compared with those obtained from age-matched controls. ABRs were well detected in all patients examined under 4 years old. The I-V interpeak latencies became shorter with age as in the controls. In 3 ears, the 5th wave was recorded without 1st wave. In 4 ears which no ABRs were detected with 90 dBHL stimulation, the only 5th wave was detected. No waves were obtained from the patients aged over 10 years. The current study revealed that ABR disturbance in A-XP patients became obvious after 4 years of age. In addition, no ABR was found to be identified after 10 years of age. During 4 to 10 years of age, some patients showed the elevation of the threshold of the 5th wave. In A-XP patients, the peripheral nerve was hypothesized to be affected earlier than the central pathway involved in ABR.

Adolescent↗

PubMed1999

Molecular analysis of hemophilia B in Poland: 12 novel mutations of the factor IX gene.

We examined the molecular basis of factor IX deficiency in 53 unrelated Polish patients with hemophilia B. Heteroduplex analysis and direct sequencing of polymerase chain reaction (PCR) products were applied to identify the gene defect. Forty-three different point mutations were detected in the factor IX gene of 47 patients. There were 29 missense mutations, 9 nonsense mutations, 4 splice site mutations and 1 point mutation in the promoter region. Twelve mutations were novel. The results of this study emphasize a very high degree of heterogeneity of hemophilia B.

Adolescent↗

PubMedDate not supplied

[Presenilins in the genesis of Alzheimer s disease].

INTRODUCTION: With their discovery in 1995, presenilins were put forward as molecules of unknown function but central to the aetiology of Alzheimer s disease. DEVELOPMENT: The fact that point and splice mutations lead to an increase in the amount of Ab peptide produced by the cells, provided further support for the amyloid theory about the origin of the disease and, on the other hand, placed these molecules in the central part of this theory. Different groups showed that these proteins had also roles in several cellular pathways, among those, the notch pathway is one of the most important, as it is with the interaction among presenilin 1 and b catenin. Recently, several works have suggested that presenilins are molecules involved in the processing of APP by being an integral part of the protein complex that process the precursor to produce the amyloid peptide. CONCLUSION: In this paper, I will give an overview of some of the most recent work developed towards the identification of presenilin function and its involvement in Alzheimer s disease.

Alzheimer Disease↗

PubMedDate not supplied

[Role of uric acid in hypertension and in the progression of chronic renal disease].

In humans, uric acid (UA) is the main urinary metabolite of purines, whereas in other species (avians) it is the chief nitrogen compound excreted in the urine. The absence of uricase, due to nonsense or splice mutations occurring during the evolution of primates, results in blood urate levels much higher in humans than in other mammalians. This could have favorable implications, including protection from oxidative damage, as well as possibly allowing better blood pressure (BP) control in settings of low dietary Na+ intake. UA is a stimulus of the renin-angiotensin system (RAS). Moreover, blood UA levels are a major marker of proximal tubular function and overall volume status. On the other hand, hyperuricemia is a co-factor in Na+ -sensitive arterial hypertension, a marker and perhaps itself responsible for microvascular and systemic damage through RAS stimulation, inhibitory effects on endothelial cells, and the proliferation of perivascular smooth muscle cells. Recent studies in rats rendered hyperuricemic through administration of oxonic acid demonstrate the induction of hypertension and severe microvascular damage when associated with subtotal nephrectomy or chronic cyclosporine treatment. The opportunity to pharmacologically manipulate blood UA levels through inhibitors of synthesis (allopurinol), uricosuric agents (benziodarone), or recombinant urate oxidase (rasburicase) provides a relevant therapeutic potential in UA metabolism disorders, tumor lysis syndrome, and possibly in essential hypertension, as well as chronic progressive renal disease.

Animals↗

PubMed1996

Aggressive osteoblastoma with focal malignant transformation and development of pulmonary metastases. A case report with a review of literature.

We report the case of a 57-year-old woman with an unusually fast-growing and destructive osteoblastic tumor affecting the left humeral head. On histopathologic examination, most of the initial tumor revealed the characteristic morphologic features of a benign-appearing aggressive osteoblastoma. Based upon the presence of a few small scattered areas composed of atypical osteoblasts in abundant lace-like osteoid showing vascular permeation, the definitive diagnosis was that of an osteoblastoma with focal malignant transformation to well-differentiated osteosarcoma. Molecular biologic analysis revealed a splice mutation at the exon 5 donor site of the p53 gene, clearly indicating a malignant potential of the tumor. The proximal third of the humerus was resected en bloc and replaced by an uncemented modular endoprosthesis. Five months after surgery, an extensive local soft tissue recurrence occurred. Eight months postoperatively, a further massive recurrent tumor had developed an multiple pulmonary metastases became evident. Chemotherapy caused a marked decrease in the size of the soft tissue recurrences and the lung metastases showed no further increase of their number and size. Osteoblastomas with conversion to osteosarcoma should be considered a separate clinicopathologic tumor entity to be distinguished from genuine osteosarcoma. All cases of malignantly transformed conventional and aggressive osteoblastomas reported to date have shown a conversion to low- or high-grade osteosarcomas only in recurrent tumors. The present case supports the concept that osteoblastomas may primarily undergo early malignant transformation. Osteoblastomas with conversion to osteosarcoma require an aggressive surgical approach followed by chemotherapy in the hope of prolonging life expectancy or obtaining a cure.

Bone Neoplasms↗

PubMed2004

Mutations of VMD2 splicing regulators cause nanophthalmos and autosomal dominant vitreoretinochoroidopathy (ADVIRC).

PURPOSE: To investigate the genetic basis of autosomal dominant vitreoretinochoroidopathy (ADVIRC), a rare, inherited retinal dystrophy that may be associated with defects of ocular development, including nanophthalmos. METHODS: A combination of linkage analysis and DNA sequencing in five families was used to identify disease-causing mutations in VMD2. The effect of these mutations on splicing was assessed using a minigene system. RESULTS: Three pathogenic sequence alterations in VMD2 were identified in five families with nanophthalmos associated with ADVIRC. All sequences showed simultaneous missense substitutions and exon skipping. CONCLUSIONS: VMD2 encodes bestrophin, a transmembrane protein located at the basolateral membrane of the RPE, that is also mutated in Best macular dystrophy. We support that each heterozygous affected individual produces three bestrophin isoforms consisting of the wild type and two abnormal forms: one containing a missense substitution and the other an in-frame deletion. The data showed that VMD2 mutations caused defects of ocular patterning, supporting the hypothesized role for the RPE, and specifically VMD2, in the normal growth and development of the eye.

Bestrophins↗

PubMed1991

Inactivation of the p53 gene expression by a splice donor site mutation in a human T-cell leukemia cell line.

An abnormally sized 3.5 kb p53 transcript was detected in the KE-37R human leukemic T-cell line in which no p53 protein could be detected by immunoprecipitation. S1 nuclease protection experiments and sequencing analysis indicated conservation of the entire intron 4 (755 bp) in the 3.5 kb transcript and the presence of a G to A substitution in the last exonic nucleotide of the splice donor site. These data support the notion that p53 gene inactivation by point mutations in splice junctions also exists in hemopoietic neoplasia.

Animals↗

PubMed2002

Krit1 missense mutations lead to splicing errors in cerebral cavernous malformation.

At least 40% of families affected with cerebral cavernous malformation have a mutation in Krit1. We previously identified two point mutations in Krit1 leading to changes in amino acids (D137G and Q210E) in two different families. Further RNA analysis reveals that both point mutations actually activate cryptic splice-donor sites, causing aberrant splicing and leading to a frameshift and protein truncation. To date, no simple missense mutations have been detected in Krit1.

Alleles↗

PubMed1993

Abnormal mRNA splicing resulting from three different mutations in the CFTR gene.

Three different putative splicing mutations in the CFTR gene have been studied by analysing mRNA extracted from nasal epithelial cells harvested from patients with cystic fibrosis. Six patients were analysed, all of whom had classical symptoms of cystic fibrosis (CF). Two patients carried the 621 + 1G-->T mutation, 3 patients carried the 1717 - 1G-->A mutation and 1 patient carried the 1898 + 1G-->A mutation. All patients carried the delta F508 mutation on the other chromosome. Ten non-CF control subjects were also studied. The 621 + 1G-->T mutation resulted in activation of an alternative splice site within exon 4 in one patient and activation of this site or skipping of exon 4 in the other patient. The 1717 - 1G-->A mutation resulted in skipping of exon 11 in all 3 patients studied and the 1898 + 1G-->T mutation resulted in skipping of exon 12. These experiments demonstrate that these mutations do result in aberrant splicing of CFTR mRNA as predicted from the changes in genomic sequence.

Base Sequence↗

PubMed2005

Analysis of mRNA in hemophilia A patients with undetectable mutations reveals normal splicing in the factor VIII gene.

BACKGROUND: haemophilia A (HA) is characterized by partial or total deficiency of factor VIII (FVIII) protein activity. It is caused by a broad spectrum of mutations in the FVIII gene. Despite tremendous improvements in mutation screening methods, in about 2% of HA patients no DNA change could be found, even after sequencing the whole coding part of the FVIII gene including the flanking splice sites, as well as the promotor and the 3' UTR regions. OBJECTIVES, PATIENTS AND METHODS: In the present study we performed a detailed RNA analysis of three groups of patients. The first included control patients with known splicing defects, the second included two patients with already identified nucleotide changes close to splicing sites, that could potentially alter the normal splicing process, and a third group of 11 unrelated patients whose genomic DNA have already been screened for mutations by DHPLC and direct sequencing with no mutation being identified. RESULTS: Both candidate splice site mutations were shown to result in either skipping or alternative splicing of at least one exon, therefore these DNA changes must be considered as causal for the patients' HA phenotype. In contrast, no abnormalities on the RNA level were observed in any of 11 unrelated patients without mutations in the FVIII gene. CONCLUSIONS: These findings exclude mutations that could be located deep in the introns and affecting either normal splicing or lead to mechanisms causing some unknown rearrangements of the FVIII gene. In fact, our results point to the presence of still unknown factor(s) causing HA, which might be either allelic or in the close proximity of the FVIII gene or non-allelic associated with other genetic loci that are involved in the processing of the FVIII protein.

DNA Mutational Analysis↗

PubMed1990

Nucleotide sequence analysis of human beta-globin gene by the quantification method: mutations in 3'-splice junction sequence and beta-thalassemia.

The nucleotide sequence at the intron-exon junction in the human beta-globin gene was analyzed by the quantification method (categorical discriminant analysis) proposed previously. Using the sample score of a 16-nucleotide sequence at a 3'-splice junction, we studied to what extent such a sequence contains the 3'-splice signal. To examine the applicability of our method, we further studied several mutants of beta-thalassemia, where nucleotide changes exist at 3'-splice junction sequences of the first and second introns. Other mutants involve point mutations which generate new 3'-splice signals within the first intron. Experimental results on the abnormal splicing in those mutants could be explained in terms of the sample scores of 16-nucleotide sequences and their locations relative to the branch point.

Base Sequence↗

PubMed1994

Severe homozygous protein C deficiency: identification of a splice site missense mutation (184, Q-->H) in exon 7 of the protein C gene.

Single strand conformation polymorphism (SSCP) analysis of exon 7 of the protein C gene has identified a novel splice site missense mutation (184, Q-->H), in a newborn child with purpura fulminans and undetectable protein C levels. The mutations, seen in the homozygous state in the child and in the heterozygous state in her mother, was characterized and found to be a G to C nucleotide substitution at the -1 position of the donor splice site of intron 7 of the protein C gene, which changes histidine 184 for glutamine (184, Q-->H). According to analysis of the normal and mutated sequences, this mutation should also abolish the function of the donor splice site of intron 7 of the protein C gene. Since such a mutation is compatible with the absence of gene product in plasma and since DNA sequencing of all protein C gene exons in this patient did not reveal any other mutation, we postulate that mutation 184, Q-->H results in the absence of protein C gene product in plasma, which could be the cause of the severe phenotype observed in this patient.

Base Sequence↗

PubMed2006

A splice-supporting intronic mutation in the last bp position of a cryptic exon within intron 6 of the CYBB gene induces its incorporation into the mRNA causing chronic granulomatous disease (CGD).

Chronic granulomatous disease (CGD) is caused by a defect in both the host's defenses and its regulation of inflammation normally provided by phagocytes and other leukocytes. As in the case described here, it is not uncommon that CGD patients are diagnosed late, only after organ-damaging manifestations have already progressed. In this patient, we found that CGD arose due to a splice-supporting mutation in the last position of a cryptic exon towards the middle of intron 6 of the CYBB (gp91-phox) gene. The mutation led to the insertion of 56 bp into most of the CYBB mRNA of leukocytes causing a frame shift and a premature stop codon. The normal cryptic exon was also found to be mildly active in some tissues other than leukocytes in healthy donors, to be conserved in many primates, and to a lesser degree in other mammals. Some sequence similarity suggests that the cryptic exon may have originated from a mammalian interspersed repetitive (MIR) element. Taken together, we clarify an unusual disease-causing mutation, indicate its evolutionary background and emphasize the importance of a timely diagnosis of CGD.

Base Sequence↗

PubMed2001

Activation of multiple cryptic donor splice sites by the common congenital afibrinogenemia mutation, FGA IVS4 + 1 G-->T.

Our recent studies on the molecular basis of the autosomal recessive disorder congenital afibrinogenemia showed that the most common mutation is a donor splice mutation in FGA intron 4, IVS4 + 1 G-->T, accounting for approximately half of disease alleles. The effect of this mutation on messenger RNA (mRNA) splicing, however, remained unproven. COS-7 cells transfected with a normal plasmid construct produced 100% mRNA molecules with correct splicing, whereas cells transfected with a mutant construct produced multiple aberrant mRNAs, due to utilization of cryptic donor splice sites situated in exon 4 and intron 4. One particular site situated 4 base pairs (bp) downstream of the normal site was used in 85% of transcripts causing afibrinogenemia by a 4-bp insertion-frameshift, leading to premature alpha-chain truncation. Our results confirm the utility of transfecting COS-7 cells to study mRNA splice-site mutations and demonstrate that the common FGA IVS4 variant is a null mutation leading to afibrinogenemia.

Afibrinogenemia↗

PubMed1998

Isolation and characterization of EMS induced splicing defective point mutations within the intron of the nrdB gene of bacteriophage T4.

The nrdB gene of bacteriophage T4 codes for the small subunit of ribonucleotide reductase and contains a 598-base-pair self-splicing intron which is closely related to other group I introns of T4 and eukaryotes. The screening, isolation, and mapping of 31 nrdB intron mutations were conducted by the strategic usage of the white halo phenotype exhibited by T4 mutants defective in dyhydrofolate reductase or thymidylate synthase. These intron mutations cluster towards the ends, mainly the 3' end, and show a defect in self-splicing. These mutations map in regions of conserved structural elements, thus supporting secondary structure predictions. A distinct pattern of clustering is observed with the highest number of mutations mapping within three of the smaller regions (A, C, and D) of the nrdB intron and no mutations mapping in the largest (B) region. The highest density of mutations mapped in the smallest region (C) of the intron, containing only 96 bases, thus showing a distinct pattern of clustering within the catalytic core.

Bacteriophage T4↗

PubMed1998

Defective RNA splicing resulting from a mutation in the cyclic guanosine monophosphate-phosphodiesterase beta-subunit gene.

PURPOSE: The authors have previously reported a 3' splice site mutation in intron 2 of the rod cyclic guanosine monophosphate-phosphodiesterase (cGMP) beta-subunit (beta-PDE) gene in a patient with compound heterozygous autosomal recessive retinitis pigmentosa. The purpose of this study is to determine whether this mutation interferes with RNA splicing, what products it generates, and whether the resultant mRNA is able to support the synthesis of the native protein. METHODS: Two expression constructs were prepared by subcloning genomic DNA fragments (one from the control subject DNA and the other from the patient's DNA) to the pCIS2 expression vector. Recombinant plasmid DNA was introduced into 293 human embryonic kidney cells using the calcium phosphate-mediated transfection procedure. Northern blot hybridization, reverse transcription-polymerase chain reaction, and sequencing were used for RNA analysis. RESULTS: Four major products were present in the RNA pool isolated from cells transfected with the expression construct containing the splice site mutation. One of the transcripts resulted from the activation of a cryptic 3' splice site located in exon 3, 12 nucleotides downstream of the mutated site. The second fragment was longer than the correctly spliced mRNA by approximately 1 kb and contained unspliced intron 2. Two other high molecular weight products corresponded to intermediate lariats. CONCLUSIONS: An acceptor splice site mutation in intron 2 of the beta-PDE gene leads to the accumulation of pre-mRNA and intermediate lariats and a 12-nucleotide shorter beta-PDE transcript produced by the use of a cryptic splice site located in exon 3. In the normal beta-PDE mRNA, these 12 nucleotides code for ValPheLeuLys. These amino acids are highly conserved in the putative noncatalytic cGMP-binding domain I of beta-PDE from several species and, probably, are important for the correct folding and function of the protein.

3',5'-Cyclic-GMP Phosphodiesterases↗