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PubMed2026

TTC19 and FMNL2 gene variants in a pediatric case of mitochondrial disorder with renal tubular acidosis.

Mitochondrial complex III deficiency caused by pathogenic variants in TTC19 is a heterogeneous disorder typically presenting with progressive neurological involvement in late childhood. Early-onset of disease with predominant renal manifestations are uncommon and may complicate diagnosis. We report a child presenting with developmental delay, failure to thrive, lactic acidosis, and distal renal tubular acidosis (dRTA), raising suspicion of an underlying mitochondrial disorder. Whole exome sequencing (WES) analysis identified a homozygous intron-exon boundary deletion of 31 bp (c.463-19_474del) in TTC19 predicted to disrupt splicing, with functional evidence demonstrating aberrant transcript formation, reduced gene expression, and mitochondrial dysfunction in patient-derived fibroblasts. Based on the biochemical findings, re-analysis of exome data revealed a novel homozygous canonical splice-site variant (c.783-1G>A) in FMNL2. The splicing assay showed the skipping of exon 9, and reduced expression in the fibroblasts. This case expands the clinical spectrum of TTC19-related mitochondrial complex III deficiency with early-onset renal tubular acidosis. While TTC19 is the most plausible primary disease-causing gene, the functional disruption of FMNL2 suggests a potential contributory role or association with the renal phenotype. Hence, these findings highlight the importance of genomic re-analysis along with functional studies in resolving complex multisystem disorders.

Female↗

PubMed1989

RNA structure, not sequence, determines the 5' splice-site specificity of a group I intron.

The group I self-splicing introns act at exon-intron junctions without recognizing a particular sequence. In order to understand splice-site selection, we have developed an assay system based on the Tetrahymena ribozyme to allow the study of numerous 5'-splice-site variants. Cleavage at the correct site requires formation of the correct secondary structure and occurs most efficiently within a 3-base-pair window centered on base pair 5 from the bottom of the P1 stem. Within this window the ribozyme recognizes and cleaves at a "wobble" base pair; the base pair above the cleavage site also influences splicing efficiency. The recognition of RNA structure rather than sequence explains the ability of these transposable introns to splice out of a variety of sequence contexts.

Animals↗

PubMed2026

[Genetic and functional characterization of a novel KIT splicing variant in a Chinese three-generation pedigree with piebaldism].

OBJECTIVES: To investigate the genetic etiology of a three-generation pedigree affected with piebaldism. METHODS: Next-generation sequencing and Sanger sequencing were employed to detect and verify gene variants. Bioinformatics tools were used to predict the effects of candidate variants on splicing and protein function. RT-PCR and Sanger sequencing were further performed to validate the impact of the variant on RNA splicing, and homology modeling was applied to predict its effect on the three-dimensional structure of the KIT protein. The pathogenicity of the variant was then classified according to the guidelines of the American College of Medical Genetics and Genomics (ACMG) and the UK Association for Clinical Genomic Science (ACGS). RESULTS: A heterozygous insertion variant near the splice site, c.1990+8_1990+9insTGCACCATTGGAGGTAAA, was identified in the KIT gene in the proband and was found to co-segregate with the phenotype within the family. RT-PCR and cDNA sequencing revealed that this variant led to aberrant splicing during transcription, resulting in a 21 bp in-frame insertion in the mRNA, which encodes an extra 7 amino acids within the tyrosine kinase domain and may thus affect protein function. In silico predictions, together with the experimental findings, supported classification of this variant as likely pathogenic according to relevant variant interpretation guidelines. CONCLUSIONS: The heterozygous splice-site insertion variant KIT:c.1990+8_1990+9insTGCACCATTGGAGGTAAA is the genetic cause of piebaldism in this pedigree.

Genetics diagnosis↗

PubMed1987

Genes of nuclear encoded mitochondrial proteins: evidence for a variant of the 3' splice-site consensus sequence.

The introns of animal nuclear genes and of viral genes encoding protein possess at their 3' splice-site the consensus sequence (CT)11NTCAG (Mount, S.M. (1982) Nucl. Acids Res. 10, 459-472; Green, M.R. (1986) Ann. Rev. Genet. 20, 671-708). However, the total 39 introns of the 5 imported mitochondrial proteins of higher eucaryotes whose gene structure has been determined to date show a predominance of 44% for base T at position -4. Apparently, a variant consensus sequence, i.e. (CT)11TTCAG, characterizes the genes of nuclear encoded mitochondrial proteins.

5-Aminolevulinate Synthetase↗

PubMed2024

A germline PDGFRB splice site variant associated with infantile myofibromatosis and resistance to imatinib.

PURPOSE: Infantile myofibromatosis is characterized by the development of myofibroblastic tumors in young children. In most cases, the disease is caused by somatic gain-of-function variants in platelet-derived growth factor (PDGF) receptor beta (PDGFRB). Here, we reported a novel germline intronic PDGFRB variant, c.2905-8G>A, in 6 unrelated infants with multifocal myofibromatosis and their relatives. METHODS: We performed constitutional and tumor DNA and RNA sequencing to identify novel variants, which were subsequently characterized in cellular assays. RESULTS: All patients had multiple skin nodules, 4 had bone lesions, and 2 had aggressive disease with bowel obstruction. The c.2905-8G>A substitution creates an alternative acceptor splice site in intron 21, inserting 2 codons in the PDGFRB transcript. Functional studies revealed that the splice change induced a partial loss of function, contrasting with previously described variants. In 4 tumor samples, we identified a second somatic hit at position Asp850 in PDGFRB exon 18, triggering constitutive receptor activation and resistance to imatinib. In addition to vinblastine and methotrexate, 2 patients received imatinib without objective response. One of them switched to dasatinib with concomitant improvement. CONCLUSION: This splice-site PDGFRB variant favors the development of myofibroma, featuring an acquired oncogenic variant in the same gene and resistance to targeted therapy.

Humans↗

PubMed1992

Albumin Rugby Park: a truncated albumin variant caused by a G-->C splice-site mutation in intron 13.

Three members of a family were found to be heterozygous for a fast albumin variant (Albumin Rugby Park) that made up only 8% of total serum albumin. Isoelectric focussing indicated an increased negative charge on the C-terminal CNBr peptide and C-terminal sequence analysis of the native protein showed an aberrant sequence of -Ser-Phe. Sequence analysis of PCR-amplified DNA indicated a G-->C mutation at position 1 of the 13th intron and this was confirmed by restriction digestion. The replacement of the obligate GT sequence by CT at the exon/intron boundary prevents splicing of the 13th intron and translation continues for 21 nucleotides until a stop codon is reached. The new protein lacks the 14 amino acids coded for in the 14th exon (GKKLVAASQAALGH), but these are replaced by 7 new residues (LLQFSSF), giving a truncated albumin of 578 residues.

Albumins↗

PubMed1991

Identification of a splice-site mutation in the human growth hormone-variant gene.

The human growth-hormone-variant (hGH-V) gene normally expresses two alternatively spliced forms of mRNA--hGH-V and hGH-V2--in the placenta. hGH-V2 mRNA differs from hGH-V rDNA by the retention of intron 4 and represents approximately 15% of transcripts at term. In a survey of hGH-V gene expression in 20 placentas of gestational age 8-40 wk, we detected a single placenta that contained, in addition to the two normal hGH-V mRNA species, a set of two slightly larger hGH-V mRNAs. Sequence analysis of the elongated hGH-V mRNA demonstrated retention of the first 12 bases of intron 2, resulting from both a base substitution at the intron 2 splice-donor dinucleotide (GT----AT) and activation of a cryptic splice-donor site 12 bases downstream. Survey of a total of 60 additional chromosomes failed to reveal additional incidence of this mutation. The mutation, which we have designated hGH-Vintron 2, pos 1 (G----A), represents both an initial example of a nondeletional mutation within the hGH-V gene and corresponding structural alteration in the encoded hGH-V hormone.

Base Sequence↗

PubMed1995

Homologous nuclear-encoded mitochondrial and cytosolic isoproteins. A review of structure, biosynthesis and genes.

Mitochondrial and cytosolic proteins may be expected to differ in specific traits due to their different intracellular location. However, the identification of these differences between mitochondrial and cytosolic proteins is complicated by the heterogeneity of the two protein groups. These difficulties have been overcome by comparing traits of homologous genes, which are derived from a common ancestor gene, and their gene products. An earlier report [Hartmann, C., Christen, P. & Jaussi, R. (1991) Nature 352, 762-763] describing a positive net charge difference between the mature parts of nuclear-encoded mitochondrial proteins and their homologous cytosolic isoproteins, could be corroborated by extending the data collection. New data were gathered from computer databases and published studies. The average isoelectric points of the mitochondrial and cytosolic isoproteins are 7.5 and 6.5, respectively. Depending on the type of protein, the observed difference results from differences in the number of basic and/or acidic amino acid residues in the isoproteins. Probably both the conditions required for mitochondrial protein import and the local conditions within the organelle furthered the evolution of basic protein structures. The contribution of the mitochondrial targeting peptide to the positive charge of precursors of nuclear-encoded mitochondrial proteins is largest when the value of the isoelectric point of the mature protein is small. This mutual dependence of the charge of the targeting peptide and the mature protein part supports the notion that positive charge is essential for mitochondrial protein import. Several traits other than electric charge, i.e. codon usage, chromosome location, structural organization or regulation of the genes, do not show specific differences between the sets of the heterotopic isoproteins. There is no preference of gene location for any of the gene sets; only rarely are the genes for a mitochondrial and a cytosolic isoprotein located on the same chromosome. A variant of the 3' splice-site consensus exists in genes of nuclear-encoded mitochondrial proteins. This is most likely a consequence of the evolution of the genes in separate lineages before endosymbiosis led to the formation of mitochondria. Some of the original mRNA group II intron self-splicing functions of the endosymbiont seem to persist in part of the cytosolic splicing machinery and apparently require a specific consensus sequence [Juretic, N., Jaussi, R., Mattes, U. & Christen, P. (1987) Nucleic Acids Res.15, 10083-10086].

Animals↗

PubMed2026

Dual Aberrant Splicing Caused by an Apparently Missense CHD7 Variant, c.5273A>G (p.Asp1758Gly), in CHARGE Syndrome.

CHARGE syndrome is a rare congenital disorder primarily attributed to heterozygous pathogenic variants of the CHD7 gene. Most pathogenic CHD7 variants are loss-of-function (LoF) variants, whereas the interpretation of missense variants remains challenging in the absence of functional evidence for their pathogenicity. We report a female infant presenting with clinical features characteristic of CHARGE syndrome. Targeted sequencing identified a heterozygous CHD7 variant (NM_017780.4:c.5273A>G), initially annotated as a missense substitution p.Asp1758Gly. This variant has been previously reported and registered with conflicting pathogenicity classifications; however, its transcript-level consequences remain unclear. Long-PCR-based RNA sequencing of total RNA from peripheral blood mononuclear cells revealed two aberrant splicing patterns associated with the variant: a predominant transcript carrying a 28-bp deletion due to cryptic donor splice-site activation, and a minor transcript with partial intron 24 retention. Both transcripts were predicted to result in premature termination codons. These findings demonstrate that c.5273A>G functions as a LoF variant through dual aberrant splicing rather than a simple missense substitution. This case underscores the importance of RNA-level splicing analysis for the accurate interpretation and classification of CHD7 missense variants.

CHD7↗

PubMed1990

A difference in the splicing patterns of the closely related normal and variant human growth hormone gene transcripts is determined by a minimal sequence divergence between two potential splice-acceptor sites.

The human growth hormone (hGH-N) and the closely related human growth hormone-variant (hGH-V) genes differ in their patterns of splice-site selection. In 9% of hGH-N transcripts exon 2 is spliced to an alternative acceptor site located 45 bases within exon 3. mRNA spliced in this manner encodes a 20-kDa hGH-N isoform instead of the normal 22-kDa hGH-N. The hGH-V transcript fails to utilize this alternative splicing pathway. A region of the hGH-N and hGH-V genes critical to this difference in splice-site selection has been identified by reciprocal exchange of corresponding genomic fragments and has been defined in detail by a series of reciprocal site-specific exchanges. Three base differences located between the two potential splice-acceptor sites are both necessary and sufficient in defining the respective splicing patterns. One of these bases may serve as a lariat branch point critical for the alternative acceptor site activity while the remaining two bases appear to modulate the frequency with which this site is selected.

Animals↗

PubMed1995

Molecular genetics of the glycophorin gene family, the antigens for MNSs blood groups: multiple gene rearrangements and modulation of splice site usage result in extensive diversification.

The purpose of the review is to describe a system of human erythrocyte membrane glycoproteins exhibiting extensive diversity. Glycophorins A and B (GPA and GPB) are the antigens of the MNSs blood groups; thus individuals bearing variant glycophorins can be readily identified by serological typing. Examination of the wide array of variants of these antigens showed that they include many forms, possibly made evident by lack of constraints due to the apparent dispensability of the parent molecules. This article reviews the molecular genetics of 25 variants of the glycophorin gene family, whose common denominator is that they arise from unequal gene recombinations or gene conversions coupled to splice-site mutations. Most rearrangements occurred within a 2-kb region mainly within GPA and GPB of the gene family and only rarely within the third member, GPE. The key feature is the shuffling of sequences within two specific exons (one of which is silent), homologous in the two parent genes. This has resulted in expression of a mosaic of sequences within this region, leading to polymorphism. The common pattern of recombinations coupled to pre-mRNA splicing was the predominant mechanism of the origin of glycophorin diversity. Thus far this mechanism appears to be unique among human gene families. It could have occurred by chance rearrangements among closely linked genes and been driven by a biological advantage, not as yet identified. This remains to be established. Nevertheless, gene rearrangements observed here are akin to those reported for the major histocompatibility complex (MHC). In the glycophorin family the small size of the region within which gene interactions have occurred and the participation of essentially only two alleles makes this relatively simpler system more focused and easier to dissect and describe molecularly.

Chromosome Mapping↗

PubMed1990

Two mutations of the factor IX gene including a donor splice consensus deletion and a point mutation in a Dutch patient with severe hemophilia B.

The abnormal factor IX gene of a patient with severe hemophilia B (hemophilia B Ursem) was selected for study. All of the coding and their flanking regions and parts of the 5'- and 3'-untranslated regions of the factor IX gene were amplified from the patient's genomic DNA by using the polymerase chain reaction (PCR). By analyzing the nucleotide sequence of the PCR products we have identified two mutations in the patient's factor IX gene, viz. a tetranucleotide deletion (GAGT, nt 6492 to 6495) or (TGAG, nt 6491 to 6494) in the 5'-donor splice site consensus at the exon 2-intron B boundary, and a point mutation at nucleotide 31103 in the catalytic domain (exon 8) of factor IXa, which changes the codon for valine 328 (GTT) to one for isoleucine (ATT). PCR-amplified exon 8 from 45 normal males and 55 normal females had the codon for valine-328. We propose that the deletion within the donor splice-site consensus is the cause of the disease in this individual, whereas the substitution of valine-328 by isoleucine may be a neutral variant which is, at least, very rare in the normal population. In a family study the DNA sequence of the patient's mother shows both the G to A transition in exon 8 and the 5'-donor splice consensus deletion in intron B in one allele.

Amino Acid Sequence↗

PubMed2026

A Novel Homozygous Mutation in ARL2BP Causes Multiple Morphological Abnormalities of the Flagella and Primary Ciliary Dyskinesia.

Primary ciliary dyskinesia (PCD) and multiple morphological abnormalities of the sperm flagella (MMAF) frequently co-occur in male infertility. However, the genetic basis of this syndromic presentation remains unclear. Using whole-exome sequencing, we identified a novel homozygous ARL2BP splice-site mutation (c.294-2A>G) in a 23-year-old infertile male from a consanguineous family who presented with syndromic PCD and MMAF. This variant causes aberrant pre-mRNA splicing and triggers nonsense-mediated mRNA decay, resulting in the complete absence of ARL2BP protein expression. Transmission electron microscopy revealed extensive disorganization of flagellar axonemes with consistent central pair (CP) microtubule depletion and disorganization of peripheral doublets. Immunofluorescence confirmed a severe deficiency of the CP protein SPAG6 in the sperm flagella. Notably, the patient presented without retinal symptoms. Given that ARL2BP-related retinitis pigmentosa generally emerges during the third decade, long-term ophthalmological follow-up is essential to detect delayed-onset retinal degeneration. In conclusion, these findings confirm ARL2BP as a causative gene for both PCD and MMAF, expanding the genotypic and phenotypic spectrum of ciliopathies.

Humans↗

PubMed2026

Phenotypic and transcriptomic characterization of biallelic RNU2-2 developmental and epileptic encephalopathy.

OBJECTIVE: A significant proportion of individuals with suspected genetic developmental and epileptic encephalopathies (DEEs) remain unsolved following whole genome sequencing (WGS). Here we describe biallelic RNU2-2 variants causing a recently reported, severe, recessive DEE. METHODS: We screened individuals who have received WGS analyses at the Genomic Medicine Centre Karolinska for Rare Diseases for biallelic RNU2-2 variants. Deep phenotyping was performed through reviewing entire medical histories and phenotypic traits were transcribed to their corresponding Human Phenotype Ontology (HPO) term. HPO terms were used to generate pairwise phenotypic similarity scores and assess for significantly shared phenotype enrichment in the RNU2-2 sub-cohort. RNA sequencing analyses were performed in fibroblast and blood tissues to compare splicing events between RNU2-2 individuals and two independent control groups. RESULTS: We identified 14 individuals from nine families with 12 ultra-rare biallelic RNU2-2 variants clustering in the conserved 5' domains. Genotype data from 13 of 14 individuals has been reported previously as part of a larger cohort. All individuals presented with a highly concordant, severe DEE, characterized by severe to profound intellectual disability, inability to walk or communicate, hyperkinesia, and refractory seizures. Infantile spasms and tonic seizures were the predominant seizure types and a Lennox-Gastaut syndrome-like phenotype was common. These individuals had a significantly similar phenotypic signature when compared with 703 individuals with complex pediatric epilepsies (two-sided Monte Carlo permutation test, p = .005). RNA sequencing analyses showed aberrant splicing, with the most pronounced effects in fibroblast tissues in mutually exclusive exon and alternate 3' splice-site events, which were not detectable in blood. SIGNIFICANCE: We present deep phenotyping data and transcriptomic analyses that provide support for rare, 5' clustering biallelic RNU2-2 variants causing this novel, severe DEE. We propose an RNA sequencing methodology on fibroblast tissue for future validation of RNU2-2 variants.

autosomal recessive disease↗

PubMed1993

Interaction of polypyrimidine tract binding protein with the encephalomyocarditis virus mRNA internal ribosomal entry site.

Translation of encephalomyocarditis virus (EMCV) mRNA occurs in a cap-independent manner, requiring instead a cis-acting element termed the internal ribosomal entry site (IRES). Binding of a 57-kDa ribosome-associated protein (p57) to the EMCV IRES has been found to correlate with cap-independent translation. p57 has recently been reported to be very similar, if not identical, to the polypyrimidine tract binding protein (pPTB), a spliceosome-associated factor possibly involved in U2 snRNP/pre-mRNA complex formation of 3'-splice-site recognition. The interaction between purified pPTB and the EMCV IRES was characterized in this study using nitrocellulose filter binding and UV cross-linking assays. pPTB bound the EMCV IRES with high affinity (Kd = 40 nM at 25 degrees C, pH 5.5, 80 mM ionic strength). pPTB also bound strongly to RNA fragments containing either the 5'-end, 3'-end, or an internal stem-loop of the IRES. The binding properties of 16 RNA variants derived from the IRES revealed however that purified pPTB bound with less specificity than pPTB in a mixture of cytoplasmic HeLa cell polypeptides. The addition of HeLa extract to purified pPTB increased the binding specificity, suggesting that factors within the extract alter the binding specificity of pPTB. The binding of pPTB to the full-length IRES and three IRES-derived fragments was studied in detail. Complex formation was optimal at low pH and was driven entirely by entropy. As many as four ion pairs are formed upon binding, with electrostatic interactions accounting for approximately 35% of the total free energy of complex formation.

Base Sequence↗

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↗

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↗

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↗