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Department of Pediatrics, Vanderbilt University School of Medicine (C.T.M., M.A.P., J.A.P.), Nashville, Tennessee 37232-2578; and Department of Endocrinology, University Children’s Hospital, Inselspital (P.E.M.), Bern, Switzerland
Address all correspondence and requests for reprints to: Dr. John A. Phillips III, Division of Genetics, Vanderbilt University School of Medicine, DD-2205 Medical Center North, Nashville, Tennessee 37232-2578. E-mail: chanda.moseley{at}mcmail.vanderbilt.edu
Abstract
Familial isolated GH deficiency type II (IGHD II) is caused, in some cases, by heterogeneous IVS3 mutations that affect GH mRNA splicing. We report here our finding an A
G transition of the fifth base of exon 3 (E3+ 5 AG) in affected individuals from an IGHD II family. This mutation disrupts a (GAA)n exon splice enhancer (ESE) motif immediately following the weak IVS2 3' splice site. The mutation also destroys an MboII site used to demonstrate heterozygosity in all affected family members. To determine the effect of ESE mutations on GH mRNA processing, GH3 cells were transfected with expression constructs containing the normal ESE, +5 AG, or other ESE mutations, and cDNAs derived from the resulting GH mRNAs were sequenced. All ESE mutations studied reduced activation of the IVS2 3' splice site and caused either partial E3 skipping, due to activation of an E3+ 45 cryptic 3' splice site, or complete E3 skipping. Partial or complete E3 skipping led to loss of the codons for amino acids 32–46 or 32–71, respectively, of the mature GH protein. Our data indicate that the E3+ 5 AG mutation causes IGHD II because it perturbs an ESE required for GH splicing.
GH DEFICIENCY (GHD) is a disorder that has been estimated to occur in 1 of 4,000 to 1 of 10,000 births, with 5–30% of patients having affected relatives (1, 2, 3). An autosomal dominant form of GHD (IGHD II) is caused by mutations in intron 3 (IVS3) of the GH1 gene. One such mutation is a sixth base transition (+6 TC) of the IVS3 5' splice site that causes aberrant GH mRNA splicing, resulting in deletion of exon 3 (E3) (4, 5) and loss of amino acids 32–71 from the mature 22-kDa GH protein, which produces a truncated product corresponding to the 17.5-kDa isoform of GH (6). IVS3 mutations +1 GC (5, 7), +1 GA (5, 8, 9), and +5 GA (9, 10) have also been reported to cause IGHD II and have the same effects on splicing as those described above. Additional IVS3 mutations +28 GA (5, 11) and del +28–45 (11) have been found to cause IGHD II. Analyses of these mutations led to the localization of an intron splice enhancer within IVS3 of the GH gene (12).
We report here our finding an AG transition of the fifth base (+5 AG) in E3 in affected individuals from a family with IGHD II and the implication of this finding. This mutation is located in an exon splice enhancer (ESE) motif (13) that immediately follows the weak 3' splice site of IVS2. In transient expression assays the natural E3+ 5 AG mutation and a variety of additional ESE point mutations all cause reduced activation of the IVS2 3' splice site, resulting in aberrant GH mRNA splicing. This aberrant splicing causes deletion of the first 15 nucleotides of E3 and loss of amino acids 32–46 of the GH product as a result of the activation of a cryptic 3' splice site located at base 45 of E3 (E3+ 45). The truncated product corresponds to the 20-kDa isoform of GH (6). Aberrant splicing also results in the complete skipping of E3 and loss of amino acids 32–71 of the mature GH product as described above in the case of the IVS3 mutations. These data indicate that the +5 AG mutation perturbs an ESE in E3 and causes IGHD II. Our findings show that aberrant GH pre-mRNA splicing is caused by point mutations of an ESE, demonstrating the importance of normal ESE function for the production of the mature 22-kDa GH protein.
Subjects and Methods
Subjects
We studied DNAs from members of a Swiss IGHD II family whose pedigree and clinical characteristics (14, 15) are shown in Fig. 1
and Table 1, respectively (written informed consent was obtained from all family members). There is no known history of consanguinity. IGF-I and IGF-binding protein-3 were measured in family members IV1 and IV2 and ranged from -3.2 to -2.8 SD score and from -2.8 to -2.4 SD score, respectively.
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The GH1 genes of an affected child from the IGHDII family (Fig. 1
, arrow) were PCR amplified using forward and reverse oligonucleotide primers corresponding to nucleotides 4556–4575 (5'-CCAGCAATGCTCAGGGAAAG-3') and the complement of 7255–7226 (5'-TGTCCCACCGGTTGGGCATGGCAGGTAGCC-3') of the GH gene cluster, respectively (16). PCR mixtures were denatured for 2 min at 92 C and cycled 35 times (92 C for 1 min; 63 C for 45 sec; 68 C for 3 min). The resulting PCR products containing GH1 sequences (2700 bp) were cleaned by filtration with a Microcon-50 microconcentrator (Amicon, Danvers, MA) and used as templates for direct sequencing. Sequencing was performed by the dideoxy method (17) using the Thermo Sequenase Cycle Sequencing Kit (Amersham Pharmacia Biotech, Piscataway, NJ), and the results are shown in Fig. 2.
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Aliquots of the 2700-bp GH1 PCR amplification products were digested with MboII (New England Biolabs, Inc., Beverly, MA) at 37 C for 2.5 h. The fragments were separated on a 6% polyacrylamide gel and visualized by ethidium bromide staining (Fig. 3).
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Human GH expression vectors containing mutations were constructed using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) and mutant oligonucleotide primers (see Table 2). After confirming that the desired mutation was present, fragments containing GH1 mutations were excised from the mutant vectors by digestion with AvrII and BglII (New England Biolabs, Inc.) and ligated to the corresponding AvrII/BglII sites of the GH1 expression vector pXGH5 (Nichols Institute Diagnostics, San Juan Capistrano, CA). DNA transfections were carried out in rat somatotroph (GH3) cells using Lipofectamine 2000 (Life Technologies, Inc., Gaithersburg, MD). Cells were grown in 35-mm tissue culture dishes to 40–60% confluence and transfected with 4 µg wild-type or mutant pXGH5 constructs and 12 µl Lipofectamine 2000/dish. Total RNA was purified from cells 48 h posttransfection using TRIzol reagent (according to the manufacturer’s directions; Life Technologies, Inc.), then treated with deoxyribonuclease I (amplification grade, Life Technologies, Inc.).
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First-strand GH1 cDNA was synthesized from total RNA using the SuperScript Preamplification System (Life Technologies, Inc.) and the GH1-specific reverse primer 5'-ACAAGGCTGGTGGGCACTGGAGT-3', corresponding to the complement of 6775–6753 of the GH gene cluster. The first-strand cDNA products were cleaned by filtration with a Microcon 50 microconcentrator (Amicon), then used as templates for 25-µl PCRs for cycle curves and quantitative PCR analysis (18). 5'-End-labeled GH1-specific forward primer (5'-CGTCTGCACCAGCTGGCCTTT-3') and reverse primer (5'-CCACAGCTGCCCTCCACAGA-3') corresponding to 5563–5583 and the complement of 6686–6667 of the GH gene cluster, respectively, were used to amplify GH1 cDNAs. To determine the optimal cycle number at which cDNAs of different lengths were amplified exponentially, GH1 cDNAs from two independent transfections with wild-type pXGH5 constructs were amplified in PCR mixtures denatured for 10 min at 95 C and cycled 22–32 times (94 C for 45 sec, 72 C for 30 sec, 72 C for 2 min). Cycle curves were plotted, and it was determined that the linear range of PCR amplification was 23–25 cycles for all products. Thus, 25 µl quantitative PCRs using the cDNA products from transfections with wild-type or mutant pXGH5 constructs were performed for 23 or 24 cycles following the above-mentioned cycling conditions. The RT-PCR products were separated on 5% nondenaturing polyacrylamide gels that were dried following electrophoresis and exposed to a phosphorimager screen for 24 h. The relative amounts of full-length, cryptic, and E3-skipped cDNA amplicons were quantitated with a PhosphorImager and ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA).
Results
Sequencing of GH1 alleles
The sequences of the GH1 alleles of an affected child from our IGHD II family were determined by direct sequencing of PCR products (Fig. 2). The sequencing ladders represent the corresponding nucleotide sequences derived from normal (Fig. 2, left) and patient (Fig. 2, right) DNAs. Note that the patient is heterozygous for an AG transition at the fifth base of E3 (E3+ 5 AG). Also note that this base is part of a GAAGAA ESE motif that immediately follows the 3' splice site of IVS2 (Fig. 2B).
Restriction endonuclease detection
The E3+ 5 AG mutation was found to destroy an MboII restriction endonuclease site that was used to demonstrate heterozygosity for the mutation in all affected family members (Fig. 3). Digestion of the 2700-bp PCR products from normal subjects with MboII generated eight fragments: the 1322-, 371-, 363-, and 266-bp fragments shown in Fig. 3 and the 211-, 115-, 45-, and 7-bp fragments (data not shown). The presence of the E3+ 5 AG transition destroyed an MboII site and, as a result, yielded an abnormal 1588-bp fragment instead of the normal 1322- and 266-bp fragments (Fig. 3). The presence of the 1588-bp as well as the 1322- and 266-bp fragments in the digested PCR products of affected family members (Fig. 3, lanes 2 and 5–8) demonstrates that they are heterozygous for the E3+ 5 AG transition, in agreement with an autosomal dominant mode of inheritance. All of the unaffected family members are homozygous normal (Fig. 3, lanes 1, 3, and 9).
Analysis of transcripts of GH1 alleles
To determine the effects of the AG transition on GH mRNA splicing, expression constructs containing the normal GH1 gene (E3+ 5A) or the E3+ 5 AG mutant allele were transfected into GH3 cells, and the GH transcripts were analyzed by DNA sequencing of quantitative RT-PCR products (18). The normal GH1 gene yielded normally spliced GH transcripts (Fig. 4, lane 1), whereas the +5 AG allele yielded an increased proportion of transcripts whose E3 was either partially or completely skipped, causing the loss of amino acids 32–46 or 32–71, respectively (Fig. 4, lane 2, 368 and 293 bp, respectively). To determine the effects of other ESE base changes on GH mRNA splicing, expression constructs containing point mutations at the second, third, fourth, or sixth position in the ESE motif were transfected into GH3 cells, and the quantitative RT-PCR products were analyzed. Each mutant GH1 allele yielded an increased percentage of transcripts with partial or complete skipping of E3 (Fig. 4, lanes 3–8).
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The E3+ 5 AG transition was only found in affected individuals in our IGHD II family. All family members with IGHD II were determined to be heterozygous for the mutation, in agreement with an autosomal dominant mode of inheritance. The E3+ 5 AG mutation was not detected in 49 CEPH controls (98 chromosomes) or in 26 samples (52 chromosomes) from other IGHD subjects.
The E3+ 5 AG transition is a missense mutation that encodes a glutamate (GAA) to glycine (GGA) substitution at codon 33 (E33G). It is possible that this amino acid substitution may affect the secondary structure of the GH protein due to a change from the bulky, acidic, polar side-chain in glutamate to the smaller, nonpolar side-chain in glycine. However, it is unlikely that this substitution would affect binding to GH receptor, because amino acid 33 does not appear to be a part of the GH receptor-binding site (19).
Interestingly, the E3+ 5 AG transition is located within a GAAGAA ESE motif (13) immediately following the IVS2 3' splice site. Pre-mRNA splicing enhancer elements are capable of activating weak splice sites in nearby introns (20). It is not surprising, therefore, to discover this ESE, since the IVS2 3' splice site was known to be weak compared with other downstream acceptor splice sites (21). We hypothesize that under normal conditions, this ESE promotes activation of the IVS2 3' splice site and, in turn, production of the normal 22-kDa GH protein.
To determine the mechanism by which the E3+ 5 AG transition in the ESE motif causes IGHD II, expression constructs containing the normal GH1 gene or the mutant allele were transfected into GH3 cells. The resulting GH transcripts were analyzed by DNA sequencing of RT-PCR products. Some transcripts of constructs containing the +5 AG ESE mutation exhibited aberrant splicing, with activation of an E3+ 45 cryptic 3' splice site, resulting in deletion of the first 15 nucleotides of E3 and loss of amino acids 32–46, corresponding to the 20-kDa isoform of GH (6). Other mutant transcripts arose by aberrant splicing that resulted in skipping of E3 and loss of the codons for amino acids 32–71, which correspond to the 17.5-kDa isoform of GH (6). The deficiency of GH and the GHD phenotype of individuals heterozygous for the E3+ 5 A G mutation suggest the dominant negative mechanism reported previously for an IVS3 +6 TC transition (4).
Analysis of RT-PCR products showed that approximately 62% of mutant transcripts exhibiting aberrant splicing lack E3 completely. It is unclear why, in the presence the E3+ 5 A G mutation that disrupts an ESE motif, E3 skipping is favored over use of the E3+ 45 cryptic splice site, which is the next available 3' splice site following the weak IVS2 3' splice site. It is possible that other elements, such as silencers, act, in addition to the E3 ESE we have detected, to prevent utilization of the E3+ 45 cryptic site in favor of activation of the IVS3 3' splice site, which results in large amounts of 17.5-kDa isoforms.
To determine the effects of other ESE base changes on GH1 pre-mRNA splicing, expression constructs containing single base ESE mutations were transfected into GH3 cells, and the resulting GH transcripts were analyzed by DNA sequencing of the RT-PCR products. Importantly, all mutant alleles, including two that have no effect on ESE purine content or the encoded amino acid (GAAGAAGAGGAA and GAAGAAGAAGAG), exhibited aberrant splicing similar to that of the +5AG transition. Thus, altering any base within this ESE motif disrupts the function of the ESE, resulting in aberrant splicing.
In conclusion, our data show that GH1 alleles with a +5 AG transition in E3 cause IGHD II by perturbing GH pre-mRNA splicing due to the disruption of an ESE. It has been estimated that 15% of point mutations resulting in human genetic diseases cause splicing defects (22), and many of these mutations affect a sequence that resembles an ESE (23). Thus, we provide here an example of an ESE mutation that causes a human genetic disease by deranging alternative splicing (23, 24, 25, 26). Our finding that translationally silent point mutations (GAAGAAGAGGAA or GAAGAAGAAGAG) can affect splicing by perturbing ESEs shows that analogous changes in other genes may also derange splicing. Thus, single base substitutions that cause disease should be more carefully evaluated by studying their potential effects on pre-mRNA splicing.
Footnotes
This work was supported in part by NIH Grant DK-35592. The work in Switzerland was supported by the Swiss National Science Foundation (Grant 32-53714.98).
Abbreviations: E3, Exon 3; ESE, exon splice enhancer; GHD, GH deficiency; IGHD II, isolated GH deficiency type II.
Received February 26, 2001.
Accepted November 3, 2001.
References
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, Unaffected females; 
, unaffected males; •, IGHD II patients, female; 
, IGHD II patients, male; 
, probable GHD. The arrow indicates a patient found, by DNA sequencing, to have the +5 A
