A recurring dominant negative mutation causes ...

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type II (IGHD-II) is an autosomal dominant disorder that has been previously shown in some patients to be caused by heterogeneous. GH gene defects that affect ...
0021-972x/95/$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright 0 1995 by The Endocrine Society

Vol. 80, No. 12 Printed in U.S.A.

A Recurring Dominant Negative Mutation Causes Autosomal Dominant Growth Hormone DeficiencyA Clinical Research Center Study* JOY D. COGAN, BJORN RAMELI, MARKKU MELLISSA PRINCE, ROBERT M. BLIZZARD, BRAMMERT, AND LEIF GROOP

LEHTO, THOMY

JOHN J. L.

PHILLIPS RAVEL,

DE

III, MARGARETA

Department of Pediatrics, Vanderbilt University School of Medicine (J.D.C., J.A.P., M.P.), Nashville, Tennessee 37232-2578; the Department of Endocrinology, Lund University (B.R., M.L., B.G., L.G.), MAS S-21401 Malmo, Sweden; the Department of Pediatrics, University of Virginia (R.M.B.), Charlottesville, Virginia 22903; and the Department of Human Genetics, South African Institute for Medical Research and University of the Witwatersrand (T.J.L.R.), Johannesburg, South Africa ABSTRACT Familial isolated GH deficiency type II (IGHD-II) is an autosomal dominant disorder that has been previously shown in some patients to be caused by heterogeneous GH gene defects that affect GH messenger RNA (mRNA) splicing. We report here our finding of multiple G-A transitions of the first base of the donor splice site of IVS 3 (+ lG+A) in IGHD II subjects from three nonrelated kindreds from Sweden, North America, and South Africa. This + lG+A substitution creates an NlaIII site that was used to demonstrate that all affected individuals in all three families were heterozygous for the mutation. To determine the effect of this mutation on GH mRNA processing, HeLa cells were transfected with expression plasmids containing normal or mutant + lG+A alleles, and complementary DNAs from the resulting GH mRNAs were sequenced. The mutation was found

F

AMILIAL isolated GH deficiency type II (IGHD II) has an autosomal dominant mode of inheritance (l-3). One family with IGHD II has been reported in which a sixth base transition (+6T+C) of the intron 3 (IVS 3) donor splice site was found to cause aberrant GH messengerRNA (mRNA) splicing, resulting in deletion of exon 3 (2). This exon 3 skipping results in deletion of amino acids 32-71 in the mature GH protein. The truncated product corresponds to the 17.5-kilodalton isoform of the GH and may interact with the normal GH allele products at the protein level to prevent normal secretion of GH. Recently, a secondmutation causing IGHD II has been described that is an IVS 3 +lG-+C. This mutation has the same effect on splicing as that described above (3). We have recently discovered analogous defects in a number of unnrelated IGHD II kindreds. We report here our finding of multiple G-+A transitions of the first base of the Received April 12, 1995. Revision received June 22, 1995. Accepted June 26, 1995. Address all correspondence and requests for reprints to: Dr. Joy D. Cogan, Division of Genetics, Vanderbilt University School of Medicine, DD-2205 Medical Center North, Nashville, Tennessee 37232-2578. * This work was supported in part by NIH Grants DK-35592, HD28819, and RR-00095. The work in Sweden was supported by Medical Research Council Grant 10858. t Recipient of a scholarship from the Faculty of Medicine, University of Lund.

to destroy the GH IVS 3 donor splice site, causing skipping of exon 3 and loss of the codons for amino acids 32-71 of the mature GH peptide from the mutant GH mRNA. Our finding of exon 3 skipping in transcripts of the + lG+A mutant allele is identical to our previous report of a different sixth base transition (+6T+C) mutation of the IVS 3 donor splice site that also causes IGHD II. Microsatellite analysis of an affected subjects’ DNA from each of the three nonrelated kindreds indicates that the +lG+A mutation arose independently in each family. Finding that neither grandparent has the mutation in the first family suggests that it arose de novo in that family. Our data indicate that 1) +lG+A IVS 3 mutations perturb GH mRNA splicing and cause IGHD II; and 2) these mutations can present as de nouo GHD cases. (J CZin Endocrinol Metab 80: 3591-3595, 1995)

donor splice site of IVS 3 (+lG-+A) in affected individuals from three unrelated kindreds from Sweden, North America, and South Africa. In transient expression assays,this mutation also perturbs the GH IVS 3 donor splice site and causes skipping of exon 3 and loss of amino acids 32-71 of the mature GH product. This finding demonstrates that heterogeneous defects that perturb GH mRNA splicing can cause IGHD II. Interestingly, two findings indicate that the IVS 3 +lG*A mutation recurs. First, the mutation appears to have arisen de no~o in one of our three families. Second, microsatellite alleles closely linked to the IGHD II mutations differ. Theseobservations suggest that the CpG dinucleotide that constitutes the last and first basesof exon and IVS 3 may be prone to recurrent G-A mutations that are observed in other CpG dinucleotides. Our findings provide additional insight into to the defects and pathogenesis of familial IGHD II.

Subjects

and Methods

Subjects We studied DNAs from nine individuals affected with IGHD II and their three independent kindreds, whose pedigrees are shown in Fig. 1. The biochemical and physical characteristics of the subjects and their growth charts and responses to GH replacement are shown in Table 1. Family 1 includes two affected members, father and son, whereas

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COGAN Family

JCE & Me 1995 Vol80. No 12

ET AL.

3

Tanner classification for sexual development is IV, and he continues to grow. There is no evidence in this family of any tropic hormone deficiency other than GH. In family 3, the mother (patient 1) has proportionate dwarfism (119.4 cm; SD score, -7.1) and reports that her father was of a similar short stature. Her mother, three siblings, and both sets of grandparents are of normal height. Patient 1 has two affected daughters (see Fig. 1). The first daughter (patient 2) began, at 3.75 yr of age, GH replacement therapy (1 U/kg BW*week). An initial good response was followed by a period of little effect, possibly due to suboptimal compliance with therapy. At 4 yr, 11 months, her height is 89.8 cm (SD score, -3.6). The second daughter (patient 3) has not been fully investigated or started GH replacement therapy.

1. Pedigrees of IGHD II families, showing the +lG-+A splice site mutation in affected patients (G/A) and unaffected family members (G/G), as determined by digestion of the GH PCR products with A&III. 0, Unaffected females; 0, unaffected males. 0, IGHD II patients, females; n , IGHD II patients, male; q , GHD of uncertain etiology.

FIG.

both grandparents were of normal height (Fig. 1). Diagnosis of GHD was based on the clinical picture of short stature, growth retardation, and minimal GH response to arginine-insulin stimuli. No other hormone deficiencies were observed. The father received GH substitution at the age of 16 yr and grew 9 cm@ to reach a final length of 159 cm at the age of 18 yr. On examination, he had disproportionately short arms and legs and immature facial features. His affected son had immature features before the onset of GH treatment at the age of 3 yr. The height velocity of the son was 16 cm/yr the first year after treatment; at 8 yr, his height was plotted within - 1 SD. The onset of puberty and the closing of the epiphyses in the father’s case may explain the difference in the growth rate. Family 2 consists of four generations of affected individuals (Fig. 1) (4). Interestingly, each affected autosomal dominant individual in the first three generations married a spouse with growth deficiency of an undetermined etiology (Fig. 1). The only person with autosomal dominant GHD to receive GH was in the fourth generation. He and his mother received GH-releasing and arginine-insulin GH provocative tests. There was minimal GH released after either stimulus. The boy (patient 4 in Table 1) has now been treated since age 3.6 yr. At age 14.5 yr, his height was plotted on the 90th percentile of the growth chart. His TABLE

1. Clinical

characteristics

of IGHD Family la

Nationality Sex

GIT P& Poststimulus Somatomedin C (U/ml) IGF-I (ng/mL)’ T, (nmol/IJb TSH (mU/IJb Basal TRH GH response GH antibodies Ht velocity (cm&r) Pre-hGH On hGH (1st yr)

/ microsatellite

studies

DNAs were isolated from peripheral blood samples from all family members and controls. The samples were genotyped for a dinucleotide repeat polymorphism within the GH gene cluster, as described by Polymeropoulos et al. with some modifications (5). Twenty-five nanograms of genomic DNA were added to a 15-PL reaction mixture of 10 mmol/L Tris-HCI (pH 8.3),50 mmol/L KCl, 3 mmol/L MgCl, 0.1% Triton X-100, 200 pmol/L of each deoxy-NTP, 3 pmol reverse oligonucleotide primer, 3 pmol 5’-end-labeled forward oligonucleotide primer, and 0.5 U Tuq DNA polymerase (Promega Corp., Madison, WI). The forward and reverse primers corresponded to nucleotides 2572.5-25744 (5’-TCCAGCCTCGGAGACAGAAT-3’) and the complement of 25967-25947 (5’AGTCCTTTCTCCAGAGCAGGT-3’) of the GH gene cluster, respectively (6). The polymerase chain reaction @‘CR) reaction mixture was denatured for 5 min at 94 C, cycled 30 times (94 C, 1 min; 61 C, 1 min; and 72 C, 1 min), followed by a IO-mm extension at 72 C. The PCR products were separated on a 5% polyacrylamide gel and visualized by autoradiography (results not shown).

PCR amplification

of genomic

DNA and DNA sequencing

The GH, genes of an affected child from each family were PCR amplified (7). One microgram of genomic DNA was added to a lOO-PL reaction mixture of 10 mmol/L Tris-HCI (pH 8.3),50 mmol/L KCl, 1.5 mmol/L MgCI,, 0.1% Triton X-100,200 wol/L of each deoxy-NTP, 0.1 pmol/L of each primer, and 4 U Tuq DNA polymerase (Promega). The

II subjects 1

Family 2

1

Swedish

cm

Segregation

Male Adult (16jb

Male 3.25

Female Adult’

142.0 -6.0

75.0 -6.5

117.0 -6.0

20 - 293 ) 17.5

28

11.3

FIG. 4. Electrophoretic analysis of RT-PCR products derived from GH, mRNA extracted from HeLa cells transfected with normal (lane l), GGT-+C IGHD II (lane 2), and +lG+A IGHD II (lane 3) GH expression plasmids.

showed that each mutant GH, allele had a different microsatellite haplotype. In family 1, the microsatellite haplotype of the affected individual’s chromosome with the G to A transition was inherited from the paternal grandfather (data not shown). Sequencing

of GH, alleles

The sequencesof the GH, alleles of an affected child from each IGHD II family were determined by direct sequencing of PCR products (Fig. 2). The sequencing ladders represent the corresponding nucleotide sequencesfrom normal (Fig. 2, left) and patient (Fig. 2, right) DNAs. Note that the patient is heterozygous for a G+A transition in the first base of the donor splice site of IVS 3. Direct DNA sequencing of the GH, PCR products of one affected member from each family demonstrated that all were heterozygous for this G--+A transition (data not shown). Restriction

endonuclease

detection

The IVS 3 +lG+A mutation was found to generate an iWuII1 restriction endonuclease site that was used to demonstrate heterozygosity for the mutation in all affected family members (seeFig. 3 for results in family 2). Digestion of the

ET AL.

JCE & M . 1995 Vol80 . No 12

1690-bpPCR products from normal subjectswith Ma111generated three fragments of 1270,270, and 150bp. The presence of the IVS 3 +lG-+A transition caused cleavage of the 1270-bpfragment into 640-and 630-bp segments(Fig. 3). The presence of the 1270-bp fragment as well as the 640- and 630-bp fragments in the digested PCR products of affected family members (lanes 1,2,5, and 6) demonstrates that they are heterozygous for the IVS 3 +lG+A transition and confirm an autosomal dominant mode of inheritance. All of the unaffected family members are homozygous normal. Although the same results were found in families 1 and 3, in family 1 the mutation was not detected in either normal paternal grandparent (see Fig. 1). Analysis

of transcripts

of GH, alleles

To determine the effects of the G-A transition on GH mRNA splicing, expression plasmids containing the normal GH, gene, the +lG+A IGHD II mutant allele, or the previously reported +6T+C IGHD II mutant were transfected into HeLa cells, and the GH transcripts were analyzed by DNA sequencing of RT-PCR products (2). The normal GH, gene yielded normally spliced GH transcripts, whereas the +lG+A IGHD II allele, like the previously reported +6T+C, yielded transcripts whose exon 3 was skipped, causing loss of amino acids 32+71 (Fig. 4). Protein products of these mutant alleles are predicted to be 151 amino acids in length compared to the 191-amino acid length of the normal GH peptide. Discussion The IVS 3 + lG-+A transition was found on the samelocus with a different microsatellite marker in all three unrelated IGHD II families (data not shown). This finding suggeststhat each of the GH mutations may have arisen independently. Further evidence supporting this finding is that the normal paternal grandfather in family 1 has the microsatellite allele that was expected to be transmitted to his affected son, but does not have the IVS 3 +lG-+A transition. Regarding the recurrent nature of the mutation found in all three families, we noted that the G-+A transition occurs at a CpG dinucleotide that spansthe exon 3/IVS 3 boundary on the antisense strand. CpG dinucleotides are reported to have increased mutation rates due to spontaneous deamination of a methylated cytosine on the sense or antisense strand (11). For example, recently, a recurring G-+A transition of a CpG dinucleotide due to conversion of a C-+T on the antisense strand at codon 380 of the fibroblast growth factor receptor gene was found in 97% of 154 casesof achondroplasia, including 114 sporadic cases(12). To determine the mechanism by which the IVS 3 (+ lG+A) transition mutation causesIGHD II, expression plasmids containing the normal GH, gene or the mutant allele were transfected into HeLa cells. The resulting GH transcripts were analyzed by DNA sequencing of the RTPCR products. The products of the plasmid containing the +lG-+A mutation of the donor splice site of IVS 3 exhibited aberrant splicing, with skipping of exon 3 and loss of the codons for amino acids 32-71, which correspond to the 17.5-

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MUTATION

CAUSING

kilodalton isoform of GH (13). The associated deficiency of GH and the phenotype of GHD in individuals heterozygous for this mutation probably result from the same dominant negative mechanism previously reported for an IVS 3 +6T+C transition (2). Future studies to detect the frequency of these and other dominant negative mutations in IVS 3 are underway. In conclusion, our results demonstrate that GH, alleles with a +lG--+A transition in IVS 3 cause IGHD II by perturbing GH mRNA splicing. These mutations can apparently arise de nova, thereby causing some sporadic cases of GHD. Finally, the cause of these recurring G+A transitions may be the presence of a CpG dinucleotide at the exon 3/IVS 3 boundary. The C+T transitions expected on the antisense strand are proposed to give rise to the IVS 3 +lG+A mutations reported here to cause IGHD II. References 1.

Phillips growth

III JA, hormone

Cogan JD. 1994 Molecular

2. Cogan JD, Phillips

3.

deficiency.

J Clin

basis of familial human Endocrinol Metab. 7811-16.

III JA, Schenkman SS, Milner

RDG, Sakati N.

1994 Familial growth hormone deficiency: a model of dominant and recessive mutations affecting a monomeric protein. J Clin Endocrinol Metab. 79:1261-1265. Binder G, Ranke MB. 1995 Screening for growth hormone (GH) gene splice-site mutations in sporadic cases with severe isolated GH

IGHD-II deficiency using 80:1247-1252.

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transcript

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J Clin Endocrinol

RM, Foley TP, et al. 1985 Growth hormone and growth hormone: genetic studies in familial deficiency. Pediatr Res. 19:489-492. 5. Polymeropoulos MH, Rath DS, Xiao H, Merril CR. 1991 A simple sequence repeat polymorphism at the human growth hormone locus. Nucleic Acids Res. 19:689. 6. Chen EY, Liao YC, Smith DH, Barrera-Saldana HA, Gelinas RE, Seeburg PH. 1989 The human growth hormone locus: nucleotide sequence, biology, and evolution. Genomics. 4479-497. 7. Saiki RK, Gelfand DH, Stoffel S, et al. 1988 Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 239487-491. 8. Sanger F, Nicklen S, Coulson AR. 1977 DNA sequencing with chain-terminating inhibitors. Proc Nat1 Acad Sci USA. 74:5463-5467. 9. Kunkel TA. 1985 Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Nat1 Acad Sci USA. 82488-492. 10. Kunkel TA, Roberts JD, Zakour RA. 1987 Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 154367-382. 11. Cooper DN, Youssoufian H. 1988 The CpG dinucleotide and human genetic disease. Hum Genet. 78:151-155. 12. Bellus GA, Hefferon TW, Ortiz de Luna RI, et al. 1995 Achondroplasia is defined by recurrent G380R mutations of FGFR3. Am J Hum Genet. 56368-373. 13. Phillips III JA. 1995 Inherited defects in growth hormone synthesis and action. In Striver CR, Beaudet AL, Sly WS, Valle D, eds. The metabolic basis of inherited disease. New York: McGraw-Hill; 30233044. 4.

Rogol AD, Blizzard

Metab.

releasing hormone growth hormone

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