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PubMed2026

Clinical and functional characterization of a novel homozygous non-canonical splice mutation (c.1910-15_1910-11delinsTTACA) in CEP290 causing Joubert syndrome.

BACKGROUND: Joubert syndrome (JS) is a rare, predominantly autosomal recessive neurodevelopmental disorder characterized by hypotonia, motor delay, intellectual disability, oculomotor apraxia, and the hallmark "molar tooth sign" on axial view of MRI. JS is genetically heterogeneous, with pathogenic variants identified in more than 40 genes involved in primary cilia function. Among these, CEP290 is one of the most frequently mutated genes. RESULTS: In this study, we investigated two children-an 11-year-old boy (the proband) and his 5-year-old sister-both presenting with a similar phenotype consistent with JS. The parents, who self-identified as Chechen, reported distant consanguinity. The family also included a healthy 13-year-old daughter. The proband had previously been evaluated by a neurologist and underwent whole-genome sequencing (WGS); however, no causative variants were identified initially. After phenotype reassessment by a clinical geneticist, we performed a reanalysis of the raw WGS data and identified a novel homozygous intronic variant of uncertain significance (VUS), c.1910-15_1910-11delinsTTACA in CEP290 (NM_025114.4). Sanger sequencing confirmed that both the proband and his affected sister were homozygous for this variant, which they inherited from their heterozygous parents. Their healthy sister did not carry the variant. mRNA-sequencing and targeted cDNA sequencing (read depth ~ 100,000x) demonstrated that this intronic variant causes completely aberrant splicing of CEP290 pre-mRNA. Predominantly this variant causes the skipping of exon 20 in the main CEP290 transcript. Alternatively, the variant results in partial inclusion of intron 19 into the mRNA, elongation of exon 20 by 58 nucleotides, and a homozygous substitution chr12:88114573 (ACTGTGTA> TTACAGTA). No canonical mRNA isoform was detected when the variant was homozygous. Both the predicted severe truncation and the likely degradation of aberrant transcripts through nonsense-mediated decay (NMD) would correspond to complete loss of CEP290 function. Following the reclassification of this VUS to likely pathogenic, the family was able to pursue in vitro fertilization (IVF) with preimplantation genetic testing for monogenic disorders (PGT-M). CONCLUSION: Our study highlights the critical importance of proper phenotyping prior to referral for WES/WGS as well as of combining NGS with functional mRNA studies to achieve a molecular diagnosis for patients with predicted splice-site mutations in JS-associated genes. It also emphasizes the need for functional reassessment of VUS when genomic data are expected to guide reproductive decision-making within affected families.

Humans↗
PubMed1998

Germline mutations in the vhl gene in patients presenting with phaeochromocytomas.

It has been shown that an appreciable percentage of patients presenting with primary, apparently sporadic phaeochromocytomas may in fact have von-Hippel-Lindau (VHL) disease. In order to investigate this, we retrospectively screened 68 patients, who had been operated on for phaeochromocytomas, for the presence of germline mutations in the vhl gene. DNA was isolated from peripheral-blood leukocytes and used to screen the entire coding sequence and the intron-exon boundaries of the vhl gene for mutations, using a PCR-based SSCP strategy. When an abnormal pattern was found in the SSCP analysis, sequence analysis was carried out. We found SSC variants in the vhl gene in 8 of the 68 patients. Of 6 patients, 2 turned out to be related (an uncle and his nephew), and they carried the same mis-sense mutation: R64P. In 4 other patients, mis-sense mutations, P25L, L63P, G144Q and I147T, were also identified. None of these mutations has been described, and 3 of them (P25L, L63P and R64P) are located closer to the N terminus of the vhl protein than any reported vhl mutation. In the remaining 2 cases, the mutations were localized not in the coding sequence but in the intronic sequence (but not within splice-sites), adjacent to the exon, so they were probably not related to the disease. Our results show that a relatively high proportion (6/68, or 8.8%), though not as high as the 20% reported earlier, of patients with apparently sporadic phaeochromocytomas may carry germline mutations in the vhl gene.

Adrenal Gland Neoplasms↗
PubMed2000

Fabry disease: twenty-two novel mutations in the alpha-galactosidase A gene and genotype/phenotype correlations in severely and mildly affected hemizygotes and heterozygotes.

BACKGROUND: Fabry disease, an inborn error of glycosphingolipid catabolism, results from mutations in the X-chromosomal gene encoding the lysosomal exoglycosidase, alpha-galactosidase A (alpha-Gal A; EC 3.2.1.22). The nature of the molecular lesions in the alpha-Gal A gene in 36 unrelated families was determined in order to provide precise heterozygote detection, prenatal diagnosis, and to define genotype/phenotype correlations. METHODS: Genomic DNA was isolated from affected males and/or carrier females from 36 unrelated families with Fabry disease. The entire alpha-Gal A coding region and flanking intronic sequences were analyzed by PCR amplification and solid-phase or cycle sequencing. Markers closely linked to the alpha-Gal A gene were analyzed to determine if probands with the same mutations were related. RESULTS: Twenty-two novel mutations were identified including 10 missense (P40L, W95S, S148N, C172R, M187V, N224S, W226R, A230T, D266H, N320Y), three nonsense (Y134X, C142X, W204X in two families), three splice-site defects (IVS2(+1), IVS3(+1), IVS4(+1)) and six small deletions or insertions (26delA in two families, 672ins37, 774delAC, 833insA, 1139delC, 1188insT). Of the remaining 12 families (33.3%), each had a previously identified mutation, eight of which occurred at CpG dinucleotides including R112C (two families), R112H, R227Q, R227X (three families), and R301Q. Haplotype analysis of the mutant alleles that occurred in two or three presumably unrelated families revealed that the families with the rare novel alleles (W204X and 26delA) were probably related, whereas those with mutations involving CpG dinucleotides (R112C and R227X) were not, the latter being consistent with their origins as independent mutational events. Genotype/phenotype correlations revealed that certain mutations previously found in mild variant patients also were found in classic patients. In addition, the genotypes and spectrum of phenotypic severity were determined in five heterozygotes with no family history. CONCLUSIONS: These results illustrate the molecular heterogeneity of the lesions causing Fabry disease and emphasize the fact that CpG dinucleotides constitute important hot spots for mutation in the alpha-Gal A gene. These studies also permit precise heterozygote detection and prenatal diagnosis in these families, and delineate phenotype-genotype correlations in this disease.

Amino Acid Sequence↗
PubMed1997

Autosomal glycogenosis of liver and muscle due to phosphorylase kinase deficiency is caused by mutations in the phosphorylase kinase beta subunit (PHKB).

Glycogen storage disease due to phosphorylase kinase deficiency occurs in several variants that differ in mode of inheritance and tissue-specificity. This heterogeneity is suspected to be largely due to mutations affecting different subunits and isoforms of phosphorylase kinase. The gene of the ubiquitously expressed beta subunit, PHKB, was a candidate for involvement in autosomally transmitted phosphorylase kinase deficiency of liver and muscle. To identify such mutations, the complete PHKB coding sequence was amplified by RT-PCR of RNA isolated from blood samples of patients and analyzed by direct sequencing of PCR products. The characterization of mutations was complemented by PCR of genomic DNA. In one female and four male patients, we identified five independent nonsense mutations (Y418ter; R428ter; Y974H+E975ter; Q656ter in two cases), one single-base insertion in codon N421, one splice-site mutation affecting exon 31, and a large deletion involving the loss of exon 8. Although these severe translation-disrupting mutations occur in constitutively expressed sequences of the only known beta subunit gene of phosphorylase kinase, PHKB, they are associated with a surprisingly mild clinical phenotype, affecting virtually only the liver, and relatively high residual enzyme activity of approximately 10%.

Adolescent↗
PubMed2002

Application of polymerase chain reaction with oligoligation assay to determine genotype in individuals presenting with congenital adrenal hyperplasia.

BACKGROUND: Current methods for the molecular diagnosis of the 21-hydroxylase deficiency variant of congenital adrenal hyperplasia use cumbersome combinations of Southern blotting and polymerase chain reaction (PCR). The aim of the present study was to develop a practical genetic test for the unambiguous diagnosis of this condition, and to use this procedure to determine the range of mutations in Scottish patients. In addition, we wished to obtain further information to that currently available in the literature regarding the correlation of genotype with phenotype in any identified carriers. METHODS: We studied five Scottish probands and their family members. To try to obviate the need for Southern blotting, we investigated a technique that uses the oligoligation chain reaction after gene-specific PCR. RESULTS: We found a spectrum of mutations in the ten unrelated mutant alleles studied. These consisted of at least three different 30-kb deletions, two intron 2 splice-site mutations and single occurrences of the 1172N, V281L and R356W substitutions. CONCLUSIONS: The genotype-phenotype correlations agreed with those previously described. In addition, our results suggest that there is no predominant Scottish genotype.

Adrenal Hyperplasia, Congenital↗
PubMed1997

Phosphorylase-kinase-deficient liver glycogenosis with an unusual biochemical phenotype in blood cells associated with a missense mutation in the beta subunit gene (PHKB).

We have identified mutations in the phosphorylase kinase (Phk) beta subunit gene in a male patient with liver glycogenosis caused by Phk deficiency. The patient's DNA has been analyzed for mutations in the genes encoding the alpha L, beta, and gamma TL subunits of Phk, all of which can be responsible for liver glycogenosis, by a strategy primarily based on reverse transcription/polymerase chain reaction of blood RNA and complemented by analysis of genomic DNA. His alpha L and gamma TL coding sequences are normal, whereas he is compound-heterozygous for two mutations in the beta subunit gene, PHKB. The first is a splice-site mutation (IVS4 [-2A-->G]) causing the reading-frame-disrupting deletion of exon 5 in the mRNA from this allele. The second is an Ala117Pro missense mutation, also in exon 5. This is the first missense mutation identified in PHKB, as opposed to nine translation-terminating mutations described to date. It offers an explanation for the unique biochemical phenotype of this patient. In his leukocytes, low Phk activity is measured when tested with the endogenous liver isoform of phosphorylase as the protein substrate, but normal activity is observed when tested with muscle phosphorylase added in vitro. In contrast, Phk activity in his erythrocytes is low with both substrates. The missense mutation may selectively impair the interaction of Phk with one isoform of its substrate protein and may destabilize the enzyme in a cell-type-specific way. This phenotype shares some aspects with X-linked liver glycogenosis subtype 2 (XLG2), a variant of liver Phk deficiency arising from missense mutations in the alpha L subunit gene (PHKA2), but differs from XLG2 in other respects. The present case demonstrates that mutations in Phk genes other than PHKA2 can also be associated with untypically high activity in certain blood cell types. Moreover, it emphasizes that missense mutations in Phk may cause unusual patterns of tissue involvement that would not be predicted a priori from the tissue specificity of expression of the mutated gene sequences.

Blood Cells↗
PubMed2001

High prevalence of glucokinase mutations in Italian children with MODY. Influence on glucose tolerance, first-phase insulin response, insulin sensitivity and BMI.

AIMS/HYPOTHESIS: The aim of this study was to assess the prevalence of glucokinase gene mutations in Italian children with MODY and to investigate genotype/phenotype correlations of the mutants. METHODS: Screening for sequence variants in the glucokinase gene was performed by denaturing gradient gel electrophoresis and direct sequencing in 132 children with maturity onset diabetes of the young (MODY) and in 9 children with chronic fasting hyperglycaemia but without laboratory evidence for Type I (insulin-dependent) diabetes mellitus and with normoglycaemic parents ("non-classical" MODY). RESULTS: Altogether 54 mutations were identified in the MODY group (54/132 or 41%) and 3 among the "non-classical" MODY individuals (3/9 or 33%). Paternity testing indicated that the latter mutations have arisen de novo. Mean fasting plasma glucose concentrations of the children with the mutant glucokinase was in the expected impaired fasting glucose range. In contrast, results of the oral glucose tolerance test showed a wide range from normal glucose tolerance (Group 1: 2-h OGTT = 6.7 +/- 1.1 mmol/l; 11 patients) to diabetes (Group 2: 2-h OGTT = 11.5 +/- 0.5 mmol/l; 9 patients), with the remaining in the impaired glucose tolerance range. Disruptive mutations (i.e. nonsense, frameshifts, splice-site) were equally represented in Groups 1 and 2 and were not clearly associated with an impaired first-phase insulin response. Surprisingly, 5 out of 11 children (or 45%) in Group 1 were found to be overweight but no children in Group 2 were overweight. Sensitivity index (SI), calculated by a recently described method, was found to be significantly lower in Group 2 than in Group 1 (SI Group 2 = 0.0013 +/- 0.0009 ml Kg(-1) min(-1)/muU/ml; SI Group 1 = 0.0068 +/- 0.0048, p < 0.0035). CONCLUSION/INTERPRETATION: Mutations in glucokinase are the first cause of MODY among Italian children selected through a low threshold limit of fasting plasma glucose (i. e. > 5.5 mmol). The lack of correlation between the molecular severity of glucokinase mutations, insulin secretion at intravenous glucose tolerance test and differences in glucose tolerance suggests that factors outside the beta cell are also involved in determining post-load glucose concentrations in these subjects. Our results seem to indicate that the differences observed in the 2-h responses at the OGTT among children with MODY 2 could be related to individual differences in insulin sensitivity.

Amino Acid Substitution↗
PubMed2003

Mutational analysis of 206 families with cavernous malformations.

OBJECT: A gene contributing to the autosomal-dominant cerebral cavernous malformation (CCM) phenotype, KRIT1 (an acronym for Krev Interaction Trapped 1), has been identified through linkage analysis and mutation screening. The authors collected blood samples from 68 patients with familial CCM and 138 patients with apparently sporadic CCM as well as from their families, in an effort to characterize the prevalence and spectrum of disease-causing sequence variants in the KRIT1 gene. METHODS: The authors used single-strand conformational polymorphism analysis to identify genomic variants in KRIT1, which were sequenced to determine the specific mutation. Among 43 Hispanic-American kindreds who immigrated to the southwestern US from northern Mexico, 31 share an identical founder mutation. This Q455X mutation is found in 18 (86%) of 21 persons with a positive family history and in 13 (59%) of 22 persons with apparently sporadic CCM. This mutation was not found among 13 persons with CCM who were recruited from Mexico. These findings establish the key role of a recent founder mutation in Hispanic persons with CCM who live in the US. Although nearly all Hispanic families in the US in which there are multiple CCM cases linked to the CCM1 locus, only 13 of 25 non-Hispanic CCM-carrying families have displayed evidence of linkage to the CCM1 locus. Among these 13 families, the authors identified eight independent mutations in nine kindreds. They identified four additional mutations among 22 familial CCM kindreds with no linkage information, bringing the total number of independent mutations to 12. Inherited KRIT1 mutations were not detected among 103 non-Hispanic persons in whom a family history of CCM was rigorously excluded. CONCLUSIONS: All mutations were nonsense mutations, frame-shift mutations predicting premature termination, or splice-site mutations located throughout the KRIT1 gene, suggesting that these are genetic loss-of-function mutations. These genetic findings, in conjunction with the clinical phenotype, are consistent with a two-hit model for the occurrence of CCM.

Codon↗
PubMed2003

RNA processing defects of the helicase gene RECQL4 in a compound heterozygous Rothmund-Thomson patient.

Rothmund-Thomson syndrome (RTS) (OMIM 268400) is an autosomal recessive genodermatosis associated with genomic instability and increased risk of mesenchymal cancers. Mutations in the RECQL4 gene, encoding a protein of the family of Werner (WRN) and Bloom (BLM) helicases, have been identified in a subset of RTS patients. Apart from congenital poikiloderma, the clinical presentation of RTS is widely variable, raising the question of the possible existence of a second locus. Results herein reported on a sporadic Caucasian patient emphasize the concept that mutation analyses at both DNA and RNA level complement the genetic defect suggested by clinical and cytogenetic signs. The patient presented with typical congenital poikiloderma and bone defects and exhibited significant genomic instability in the peripheral blood karyotype. By RECQL4 DNA mutation analysis, he was found to carry a 1473delT (mut 5) on one allele and an AG to AC change at the 3'-splice site of exon 13 (a variant of mut 4) on the second allele. RT-PCR analysis of RECQL4 cDNA encompassing the entire helicase domain showed diffuse splicing defects indicating that the loss of a single 3'-splice signal motif disregulates the correct splice-site selection and affects the overall RNA processing. The presence of an unstable minisatellite which ends at 3'-splice site of IVS12 may enhance the mutation at this site. This genomic feature together with a number of short introns in the RECQL4 gene may account for the common missplicing of RECQL4 mRNA. While it is possible that defects of RECQL4 mRNA processing might account for part of the clinical variability observed for this syndrome, only a thorough analysis at both genomic and RNA level may allow a genotype-phenotype correlation in RTS patients, restricting the search of a second RTS locus to the specific patients.

Adenosine Triphosphatases↗
PubMed2001

[Splice site mutations and atherosclerosis: mechanisms and prediction models].

Nucleotide variants in genes of the lipid metabolism influence the risk of premature atherosclerosis. Ten percent of all single nucleotide substitutions in these genes involve splice sites. The effects of these changes on mRNA splicing and phenotypic severity, however, are not inherently obvious from the nucleotide sequence. This review presents various genes of lipid metabolism with splicing mutations known to influence the risk of premature atherosclerosis. Mechanisms of pre-mRNA splicing are illustrated and different models for prediction of the effect of nucleotide substitutions on splice-site function are presented. The role of information theory-based models is emphasized along with its role for prediction of splice-site function and phenotypic severity of atherosclerosis.

Age Factors↗
PubMed1992

Heterozygosity for the IVS-I-5 (G-->C) mutation with a G-->A change at codon 18 (Val-->Met; Hb Baden) in cis and a T-->G mutation at codon 126 (Val-->Gly; Hb Dhonburi) in trans resulting in a thalassemia intermedia.

We have analyzed the hemoglobins of a young German patient with beta-thalassemia intermedia and of his immediate family and included in these studies an evaluation of possible nucleotide changes in the beta-globin genes through sequencing of amplified DNA. One chromosome of the propositus and one of his father's carried the GTG-->GGG mutation at codon 126 leading to the synthesis of Hb Dhonburi or alpha 2 beta (2)126(H4)Val-->Gly; this variant is slightly unstable and is associated with mild thalassemic features. His second chromosome and one of his mother's had the common IVS-I-5 (G-->C) mutation that leads to a rather severe beta(+)-thalassemia and the GTG-->ATG mutation at codon 18, resulting in the replacement of a valine residue by a methionine residue. This newly discovered beta-chain variant, named Hb Baden, was present for only 2-3% in both the patient and his mother. This low amount results from a decreased splicing of RNA at the donor splice-site of the first intron that is nearly completely deactivated by the IVS-I-5 (G-->C) thalassemic mutation. The chromosome with the codon 18 (GTG-->ATG) and the IVS-I-5 (G-->C) mutations has thus far been found only in this German family; analysis of 51 chromosomes from patients with the IVS-I-5 (G-->C) mutation living in different countries failed to detect the codon 18 (GTG-->ATG) change.

Amino Acid Sequence↗
PubMed2005

Splicing for alternative structures of Cav1.2 Ca2+ channels in cardiac and smooth muscles.

An estimate of up to 60% of genes are subjected to alternative splicing, and 15% of human genetic diseases are associated with mutation of the splice sites [Krawczak M, Reiss J, and Cooper DN. The mutational spectrum of single base-pair substitutions in mRNA splice junctions of human genes: causes and consequences. Hum Genet 1992; 90: 41-54; Cooper TA, and Mattox W. The regulation of splice-site selection, and its role in human disease. Am J Hum Genet 1997; 61: 259-66; Modrek B and Lee CJ. Alternative splicing in the human, mouse and rat genomes is associated with an increased frequency of exon creation and/or loss. Nat Genet 2003; 34: 177-80; Modrek B, Resch A, Grasso C, and Lee C. Genome-wide detection of alternative splicing in expressed sequences of human genes. Nucleic Acids Res 2001; 29: 2850-9; Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, et al. Initial sequencing and analysis of the human genome. Nature 2001; 409: 860-921] . The molecular diversity of alternatively spliced transcripts provides templates for a myriad of protein structures that are potentially crucial to sustaining the complexity of human physiology. The extensive alternative splicing of the alpha(1)1.2-subunit of the L-type Ca(v)1.2 channel, producing splice variants with distinct electrophysiological and pharmacological properties, would impact directly on the function of the cardiovascular system. Cell-selective expression of Ca(v)1.2 channels containing a specific alternatively spliced exon increases the functional variations for specific cellular activities in response to changing physiological signals. However, the regulation or control of the alpha(1)1.2-subunit alternative splicing machinery is unknown, and the role of numerous splice variants expressed in a cell is a mystery. A systematic and concerted effort is required to determine all the possible combinations of alternatively spliced exons in alpha(1)1.2-subunits in smooth and cardiac muscles. This will provide useful information to monitor changes on the usage of the entire suite of alternatively spliced exons to help relate altered Ca(v)1.2 channel function to physiology and disease.

Alternative Splicing↗
PubMed2005

Epidermolysis bullosa simplex associated with pyloric atresia is a novel clinical subtype caused by mutations in the plectin gene (PLEC1).

Epidermolysis bullosa (EB) is an inherited mechano-bullous disorder of the skin, and is divided into three major categories: EB simplex (EBS), dystrophic EB, and junctional EB (JEB). Mutations in the plectin gene (PLEC1) cause EBS associated with muscular dystrophy, whereas JEB associated with pyloric atresia (PA) results from mutations in the alpha6 and beta4 integrin genes. In this study, we examined three EB patients associated with PA from two distinct families. Electron microscopy detected blister formation within the basal keratinocytes leading to the diagnosis of EBS. Surprisingly, immunohistochemical studies using monoclonal antibodies to a range of basement membrane proteins showed that the expression of plectin was absent or markedly attenuated. Sequence analysis demonstrated four novel PLEC1 mutations. One proband was a compound heterozygote for a nonsense mutation of Q305X and a splice-site mutation of 1344G-->A. An exon-trapping experiment suggested that the splice-site mutation induced aberrant splicing of the gene. The second proband harbored a heterozygous maternal nonsense mutation, Q2538X and homozygous nonsense mutations R1189X. Analysis of the intragenic polymorphisms of PLEC1 suggested that R1189X mutations were due to paternal segmental uniparental isodisomy. These results indicate that PLEC1 is a possible causative gene in this clinical subtype, EBS associated with PA. Furthermore, two patients out of our three cases died in infancy. In terms of clinical prognosis, this novel subtype is the lethal variant in the EBS category.

Epidermolysis Bullosa Simplex↗
PubMed2000

Null alleles of the COL5A1 gene of type V collagen are a cause of the classical forms of Ehlers-Danlos syndrome (types I and II).

Ehlers-Danlos syndrome (EDS) types I and II, which comprise the classical variety, are well characterized from the clinical perspective, but it has been difficult to identify the molecular basis of the disorder in the majority of affected individuals. Several explanations for this failure to detect mutations have been proposed, including genetic heterogeneity, failure of allele expression, and technical difficulties. Genetic heterogeneity has been confirmed as an explanation for such failure, since causative mutations have been identified in the COL5A1, COL5A2, and tenascin X genes and since they have been inferred in the COL1A2 gene. Nonetheless, in the majority of families with autosomal dominant inheritance of EDS, there appears to be linkage to loci that contain the COL5A1 or COL5A2 genes. To determine whether allele-product instability could explain failure to identify some mutations, we analyzed polymorphic variants in the COL5A1 gene in 16 individuals, and we examined mRNA for the expression of both alleles and for alterations in splicing. We found a splice-site mutation in a single individual, and we determined that, in six individuals, the mRNA from one COL5A1 allele either was not expressed or was very unstable. We identified small insertions or deletions in five of these cell strains, but we could not identify the mutation in the sixth individual. Thus, although as many as one-half of the mutations that give rise to EDS types I and II are likely to lie in the COL5A1 gene, a significant portion of them result in very low levels of mRNA from the mutant allele, as a consequence of nonsense-mediated mRNA decay.

Adolescent↗
PubMed2003

Mutations in the ZNF41 gene are associated with cognitive deficits: identification of a new candidate for X-linked mental retardation.

Nonsyndromic X-linked mental retardation (MRX) is defined by an X-linked inheritance pattern of low IQ, problems with adaptive behavior, and the absence of additional specific clinical features. The 13 MRX genes identified to date account for less than one-fifth of all MRX, suggesting that numerous gene defects cause the disorder in other families. In a female patient with severe nonsyndromic mental retardation and a de novo balanced translocation t(X;7)(p11.3;q11.21), we have cloned the DNA fragment that contains the X-chromosomal and the autosomal breakpoint. In silico sequence analysis provided no indication of a causative role for the chromosome 7 breakpoint in mental retardation (MR), whereas, on the X chromosome, a zinc-finger gene, ZNF41, was found to be disrupted. Expression studies indicated that ZNF41 transcripts are absent in the patient cell line, suggesting that the mental disorder in this patient results from loss of functional ZNF41. Moreover, screening of a panel of patients with MRX led to the identification of two other ZNF41 mutations that were not found in healthy control individuals. A proline-to-leucine amino acid exchange is present in affected members of one family with MRX. A second family carries an intronic splice-site mutation that results in loss of specific ZNF41 splice variants. Wild-type ZNF41 contains a highly conserved transcriptional repressor domain that is linked to mechanisms of chromatin remodeling, a process that is defective in various other forms of MR. Our results suggest that ZNF41 is critical for cognitive development; further studies aim to elucidate the specific mechanisms by which ZNF41 alterations lead to MR.

Base Sequence↗
PubMed1995

Screening for growth hormone (GH) gene splice-site mutations in sporadic cases with severe isolated GH deficiency using ectopic transcript analysis.

We screened 10 children with sporadic severe isolated GH deficiency (IGHD) for GH-1 gene splice site mutations using ectopic transcript analysis. None had a history of birth trauma, congenital defects, thyroid disorders, or PRL deficiency. The mean age of these patients at diagnosis was 3.5 yr; the mean height at diagnosis was -4.0 SD score. GH-1 gene deletion was excluded in all cases. Ribonucleic acid (RNA) from lymphocytes was reverse transcribed and amplified by nested polymerase chain reaction, using two primer pairs with annealing sites within exons 2 and 5 of the GH-1 gene. The main polymerase chain reaction fragment obtained was 460 basepairs and proved to be the amplification of the GH-1 transcript. We also found three shorter fragments which were alternatively spliced GH-1 transcripts, including a variant devoid of the first 45 basepairs of exon 3, a second lacking the whole exon 3, and a third one, not previously described, lacking both exon 3 and exon 4. We found the same pattern of alternative splicing in RNA from GH-producing pituitary tumor tissue, which served as a positive control. In 1 of 10 patients, a pathologically shortened main fragment lacking exon 3 was detected. As proved by sequencing genomic DNA, this was the result of a heterozygous splice site mutation, with transversion from G to C of the first base of the donor splice site of intron III generating a new DdeI recognition site. The other allele had no mutation. DdeI digestion enabled us to rule out the defect in the parents' DNA. Thus, the mutation was de novo. As the patient with the mutation displayed the most severe and earliest growth retardation in the study group and had virtually no GH in serum, it must be assumed that the heterozygous genetic defect resulted in a dominant negative effect. The reason for this is still unclear. Recently, within a family that exhibited the autosomal dominant phenotype of IGHD (IGHD-II), a heterozygous point mutation was located 5 bases down-stream from that we describe here. A similar effect on splicing was observed. In conclusion, analysis of ectopic GH-1 transcripts enabled us to detect 1) a new alternatively spliced GH-1 messenger RNA variant lacking exons 3 and 4, and 2) 1 of 10 sporadic cases of severe idiopathic IGHD due to a heterozygous de novo splice site mutation in the GH-1 gene that changes G to C in the first base of intron III.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗
PubMed2004

Computational comparative analyses of alternative splicing regulation using full-length cDNA of various eukaryotes.

We previously reported a computational approach to infer alternative splicing patterns from Mus musculus full-length cDNA clones and microarray data. Although we predicted a large number of unreported splice variants, the general mechanisms regulating alternative splicing were yet unknown. In the present study, we compared alternative exons and constitutive exons in terms of splice-site strength and frequency of potential regulatory sequences. These regulatory features were further compared among five different species: Homo sapiens, M. musculus, Arabidopsis thaliana, Oryza sativa, and Drosophila melanogaster. Solid statistical validations of our comparative analyses indicated that alternative exons have (1) weaker splice sites and (2) more potential regulatory sequences than constitutive exons. Based on our observations, we propose a combinatorial model of alternative splicing mechanisms, which suggests that alternative exons contain weak splice sites regulated alternatively by potential regulatory sequences on the exons.

Alternative Splicing↗
PubMed1988

Identification of a mutation that causes exon skipping during collagen pre-mRNA splicing in an Ehlers-Danlos syndrome variant.

Recent biochemical studies have shown that the fibroblasts from a patient with Ehlers-Danlos Syndrome Type VIIB produce nearly equal amounts of normal and shortened pro-alpha 2(I) collagen chains (Wirtz, M.K., Glanville, R. W., Steinmann, B., Rao, V. H., and Hollister, D. (1987) J. Biol. Chem. 262, 16376-16385). Compositional and sequencing studies of the abnormal pro-alpha 2(I) chain identified an interstitial deletion of 18 residues corresponding to the N-telopeptide of the collagen molecule. Since this region is encoded by a 54-base pair exon, number 6, the protein defect could have been caused by gene deletion, abnormal pre-mRNA splicing, or both. Here, in order to elucidate the molecular nature of this mutation we have analyzed the sequences of pro-alpha 2(I) collagen cDNA and genomic clones obtained from RNA and DNA of the patient's fibroblasts. Using oligomer-specific cloning we identified a cDNA that contains a 54-base pair deletion corresponding precisely to the sequence of exon 6. Identification of the normal gene was based on the finding of an identical sequence polymorphism in a normal cDNA and in the genomic clone derived from one of the two collagen alleles. The other gene, instead, displayed a base substitution (T to C) in the obligatory GT dinucleotide of the 5' splice-site sequence of intron 6. Analysis of nearly 100 base pairs immediately 5' to exons 5, 6, and 7, and 3' to exons 5 and 7 did not reveal any additional change. Therefore, the data strongly suggest that the observed GT-to-GC transition at the splice donor site of intron 6 generates an abnormally spliced mRNA in which the sequence of exon 5 is joined to the sequence of exon 7. Since skipping of exon 6 does not interfere with the coding frame of the mRNA, the resulting shortened polypeptide, albeit utilized in the assembly of a procollagen trimer, ultimately causes the Ehlers-Danlos Syndrome Type VII phenotype.

Alleles↗