Transcripts from a novel BMPR2 termination mutation escape nonsense mediated decay by downstream translation re-initiation: implications for treating pulmonary hypertension.

Bone morphogenetic protein receptor type 2 (BMPR2) gene mutations are a major risk factor for heritable pulmonary arterial hypertension (HPAH), an autosomal dominant fatal disease. We have previously shown that BMPR2 transcripts that contain premature termination codon (PTC) mutations are rapidly and nearly completely degraded through nonsense mediated decay (NMD). Here we report a unique PTC mutation (W13X) that did not behave in the predicted manner. We found that patient-derived cultured lymphocytes (CLs) contained readily detectable levels of the PTC-containing transcript. Further analysis suggested that this transcript escaped NMD by translational re-initiation at a downstream Kozak sequence, resulting in the omission of 173 amino acids. Treatment of CLs containing the PTC with an aminoglycoside decreased the truncated protein levels, with a reciprocal increase in full-length BMPR2 protein and, importantly, BMPR-II signaling. This is the first demonstration of aminoglycoside-mediated 'repair' of a BMPR2 mutation at the protein level in patient-derived cells and has obvious implications for treatment of HPAH where no disease-specific treatment options are available. Our data also suggest the need for a more thorough characterization of mutations prior to labeling them as haploinsufficient or dominant negative based simply on sequencing data.

Alterations in oestrogen metabolism: implications for higher penetrance of familial pulmonary arterial hypertension in females.

Mutations in bone morphogenetic protein receptor type 2 (BMPR2) cause familial pulmonary arterial hypertension (FPAH), but the penetrance is reduced and females are significantly overrepresented. In addition, gene expression data implicating the oestrogen-metabolising enzyme CYP1B1 suggests a detrimental role of oestrogens or oestrogen metabolites. We examined genetic and metabolic markers of altered oestrogen metabolism in subjects with a BMPR2 mutation. Genotypes for CYP1B1 Asn453Ser (N453S) were determined for 140 BMPR2 mutation carriers (86 females and 54 males). Nested from those subjects, a case-control study of urinary oestrogen metabolite levels (2-hydroxyoestrogen (2-OHE) and 16alpha-hydroxyoestrone (16alpha-OHE(1))) was conducted in females (five affected mutation carriers versus six unaffected mutation carriers). Among females, there was four-fold higher penetrance among subjects homozygous for the wild-type genotype (N/N) than those with N/S or S/S genotypes (p = 0.005). Consistent with this finding, the 2-OHE/16alpha-OHE(1) ratio was 2.3-fold lower in affected mutation carriers compared to unaffected mutation carriers (p = 0.006). Our findings suggest that variations in oestrogens and oestrogen metabolism modify FPAH risk. Further investigation of the role of oestrogens in this disease with profound sex bias may yield new insights and, perhaps, therapeutic interventions.

Impaired adrenocorticotropin-adrenal axis in combined pituitary hormone deficiency caused by a two-base pair deletion (301-302delAG) in the prophet of Pit-1 gene.

The Prophet of Pit-1 gene (PROP1) encodes a paired-like homeodomain protein, which is expressed early in pituitary gland development. When mutated, it is responsible for combined pituitary hormone deficiency (CPHD) in humans, as well as in Ames dwarf mice (df/df). Several independent mutations in the homeodomain of PROP1 have been identified as causative for the human CPHD phenotype, which has been characterized, thus far, as absence or low levels of GH, PRL, TSH, LH, and FSH. Here, we report 10 CPHD cases, 9 of which were born to consanguineous marriages occurring in a large family living in an isolated area in the Southeast of Brazil. All affected patients present complete absence of puberty and low GH, PRL, TSH, LH, and FSH associated with severe hypoplasia of the pituitary gland, as seen by MRI. All 3 exons of the PROP1 genes of these patients were sequenced. The 301-302delAG frameshift mutation was found in both alleles of each affected case. Surprisingly, we observed ACTH/cortisol insufficiency associated with the PROP1 phenotype. The patients' ages varied between 8 and 67 yr, and cortisol response impairment was identified in 5 of 6 of the older patients and in an 11-yr-old patient. Previous studies have not fully characterized patients at advanced ages, leading us to conclude that the phenotype of this PROP1 mutation includes late-onset adrenal insufficiency. We present an extensive clinical analysis of all of these patients. The presence of ACTH/cortisol deficiency in this family bearing the PROP1 301-302delAG mutation indicates the importance of a complete endocrine characterization and of life-long monitoring of PROP1 patients.

Growth disorders caused by genetic defects in the growth hormone pathway.

The growth hormone (GH) pathway is composed of a series of interdependent genes whose products are required for normal growth (Fig 1). The GH pathway genes include ligands (GH and insulin-like growth factor 1 [lGF-1]), transcription factors (prophet of pit 1, or prop 1 and pit 1), agonists and antagonists (growth hormone-releasing hormone [GHRH] and somatostatin), and receptors (GHRH receptor [GHRHR] and the GH receptor [GHR]). These genes are expressed in different organs and tissues, including the hypothalamus, pituitary, liver, and bone. Effective and regulated expression of the growth hormone pathway is essential for growth in stature as well as homeostasis of carbohydrate, protein, and fat metabolism.

The PROP1 2-base pair deletion is a common cause of combined pituitary hormone deficiency.

Combined pituitary hormone deficiency (CPHD) has an incidence of approximately 1 in 8000 births. Although the proportion of familial CPHD cases is unknown, about 10% have an affected first degree relative. We have recently reported three mutations in the PROP1 gene that cause CPHD in human subjects. We report here the frequency of one of these mutations, a 301-302delAG deletion in exon 2 of PROP1, in 10 independently ascertained CPHD kindreds and 21 sporadic cases of CPHD from 8 different countries. Our results show that 55% (11 of 20) of PROP1 alleles have the 301-302delAG deletion in familial CPHD cases. Interestingly, although only 12% (5 of 42) of the PROP1 alleles of our 21 sporadic cases were 301-302delAG, the frequency of this allele (in 20 of 21 of the sporadic subjects given TRH stimulation tests) was 50% (3 of 6) and 0% (0 of 34) in the CPHD cases with pituitary and hypothalamic defects, respectively. Using whole genome radiation hybrid analysis, we localized the PROP1 gene to the distal end of chromosome 5q and identified a tightly linked polymorphic marker, D5S408, which can be used in segregation studies. Analysis of this marker in affected subjects with the 301-302delAG deletion suggests that rather than being inherited from a common founder, the 301-302delAG may be a recurring mutation.

Clinical and molecular characterization of Brazilian patients with growth hormone gene deletions.

Genomic DNA from 23 patients with isolated growth hormone (GH) deficiency (12 males and 11 females: heights -4.9 +/- 1.4 SDS) was screened for GH gene deletions by restriction endonuclease analysis of polymerase chain reaction amplification products. Three unrelated patients had typical features of severe GH deficiency and deletions (6.7 kb in two and 7.6 kb in one) of the GH gene. The two patients with 6.7-kb deletions developed growth-attenuating anti-GH antibodies whereas the patient with the 7.6-kb deletion continued to grow with GH replacement therapy. Our finding that 3/23 (approximately 13%) Brazilian subjects had GH gene deletions agrees with previous studies of severe isolated GH deficiency subjects in other populations. Two of three subjects (67%) with deletions developed blocking antibodies despite administration of exogenous GH at low doses. Interestingly, only 1/10 of cases with affected relatives or parental consanguinity had GH-1 gene deletions.

Mutations in PROP1 cause familial combined pituitary hormone deficiency.

Combined pituitary hormone deficiency (CPHD) in man denotes impaired production of growth hormone (GH) and one or more of the other five anterior pituitary hormones. Mutations of the pituitary transcription factor gene POU1F1 (the human homologue of mouse Pit1) are responsible for deficiencies of GH, prolactin and thyroid stimulating hormone (TSH) in Snell and Jackson dwarf mice and in man, while the production of adrenocorticotrophic hormone (ACTH), luteinizing hormone (LH) and follicle stimulating hormone (FSH) is preserved. The Ames dwarf (df) mouse displays a similar phenotype, and appears to be epistatic to Snell and Jackson dwarfism. We have recently positionally cloned the putative Ames dwarf gene Prop1, which encodes a paired-like homeodomain protein that is expressed specifically in embryonic pituitary and is necessary for Pit1 expression. In this report, we have identified four CPHD families with homozygosity or compound heterozygosity for inactivating mutations of PROP1. These mutations in the human PROP1 gene result in a gene product with reduced DNA-binding and transcriptional activation ability in comparison to the product of the murine df mutation. In contrast to individuals with POU1F1 mutations, those with PROP1 mutations cannot produce LH and FSH at a sufficient level and do not enter puberty spontaneously. Our results identify a major cause of CPHD in humans and suggest a direct or indirect role for PROP1 in the ontogenesis of pituitary gonadotropes, as well as somatotropes, lactotropes and caudomedial thyrotropes.

Allelic variations in the human growth hormone-1 gene promoter of growth hormone-deficient patients and normal controls.

OBJECTIVE: Isolated growth hormone deficiency (IGHD) type IB is suggested to be more probably due to alterations in the genes directly involved in the hypothalamo-pituitary axis and/or in the specific transcriptional regulation (cis-trans coupling) of the hGH-1 gene than to alterations in the gene itself. In this study we analyzed the hGH-1 gene promoter region for structural alterations and allelic variations. METHODS: The hGH-1 gene promoter region was analyzed by PCR, cycle sequencing and direct-blotting electrophoresis in a total of 212 individuals including 113 patients with IGHD type IB, 21 unaffected family members and 78 normal controls. RESULTS: Twenty-two sequence variation sites were identified. Of these, 14% were located around the region of -1075bp, 77% between -550bp and the translational start site (+1bp) and 9% within the first intron. Only one variation site affected a characterized cis-acting element, namely that of NF-1. Importantly, all the variations found in patients were also observed in non-affected family members as well as in normal unrelated controls. CONCLUSIONS: These findings imply that it is not a single variation within the GH-1 gene promoter, and therefore in the cis-acting elements, which causes IGHD. However, we can not exclude the possibility that combinations of variations might perturb expression. Furthermore, these data illustrate the normal heterogeneity of the GH-1 gene promoter region, a fact that has to be borne in mind whenever transcriptional studies are performed.

A novel mechanism of aberrant pre-mRNA splicing in humans.

Eukaryotic pre-mRNA splicing is regulated by consensus sequences at the intron boundaries and branch site. Recently, Sirand-Pugnet et al. reported the importance of an additional intronic sequence, an (A/U)GGG repeat in chicken beta-tropomyosin that is a binding site for a protein required for spliceosome assembly. Interestingly, we have detected mutations in IVS3 of the human growth hormone (GH) gene that affect a putative, homologous consensus sequence and which also perturb splicing. In a series of dominant-negative GH mutations that cause exon skipping, we found two mutations that do not occur within the 5' and 3' splice sites, or branch consensus sites. The first mutation is a G-->A transition of the 28th base (+28G-->A) of and the second deletes 18 bp (del+28-45) of IVS3 of the human GH gene. These mutations segregated with autosomal dominant GH deficiency in both kindreds and no other allelic GH gene changes were detected. RT-PCR amplification of transcripts from expression vectors containing the +28G-->A or del+28-45 alleles yielded products showing a >10-fold preferred use of alternative splicing, similar to findings previously reported for IVS3 donor site mutations. Both mutations are located 28 bp downstream from the 5' splice site and examination of the sequences perturbed revealed an intronic XGGG repeat similar to the repeat found to regulate mRNA splicing in chicken beta-tropomyosin. Interestingly, the XGGG repeats involved in our mutations exhibit homologous spacing to those in a so-called 'winner' RNA sequence. Binding of A1 heterogeneous nuclear ribonucleoprotein (hnRNP) by 'winner' sequences in pre-mRNA transcripts is thought to play an important role in pre-mRNA packaging and transport as well as 5' splice site selection in pre-mRNAs that contain multiple 5' splice sites. Our findings suggest that (i) XGGG repeats may regulate alternative splicing in the human GH gene and (ii) mutations of these repeats cause GH deficiency by perturbing alternative splicing. Mutations of homologous intron sequences may underlie other human diseases.

Detection of growth hormone gene defects by dideoxy fingerprinting (ddF).

We carried out screening for mutations in the GH-1 gene in 29 sporadic Japanese subjects with severe Isolated Growth Hormone Deficiency (IGHD) by dideoxy fingerprinting (ddF). Three of 29 (approximately 10%) were heterozygous for each of the following GH-1 gene mutations including: 1) an G-->A transition in the third codon of the GH-1 signal peptide of exon 1 resulting in a Threonine to Alanine substitution, 2) a G-->A transition in the first base of the donor splice site of IVS 3 (+1G-->A) and 3) a G-->A transition in the 183rd codon of the GH-1 mature peptide of exon 5 resulting in an Arginine to Histidine substitution. One of three was heterozygous for both mutations of 1) and 2). The IVS 3 (+1G-->A) mutation has been previously reported in affected individuals from three unrelated families with IGHD type II (autosomal dominant form). This mutation destroys 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. Our findings indicate that 1) ddF screening of genomic DNAs provides a practical tool to detect GH gene mutations and 2) some sporadic cases of IGHD may be caused by GH gene alternations.

Genetic mapping of the human pituitary-specific transcriptional factor gene and its analysis in familial panhypopituitary dwarfism.

We have analyzed the human pituitary-specific transcription factor (Pit-1) gene using PCR amplification of DNA fragments that span intron III and contain portions of exons III and IV. A PCR restriction fragment length polymorphism (PCRFLP) was detected in intron III by RsaI digestion, which was used to assign the human Pit-1 locus to chromosome 3p by linkage analysis of the CEPH panel. Analysis of corresponding Pit-1 segments from six nonrelated probands with familial panhypopituitary dwarfism (FPD) did not reveal any alterations in size and co-segregation of Pit-1, or a tightly linked microsatellite marker (D3S1559), and FPD was excluded in all six kindreds. Our data (1) assign Pit-1 to human chromosome 3p by linkage, (2) provide a PCRFLP and identify a variety of tightly linked markers, for analysis of FPD, and (3) exclude Pit-1 defects as the basis of at least one form of FPD.

A recurring dominant negative mutation causes autosomal dominant growth hormone deficiency--a clinical research center study.

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 (+ 1G-->A) in IGHD II subjects from three nonrelated kindreds from Sweden, North America, and South Africa. This + 1G-->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 + 1G-->A alleles, and complementary DNAs from the resulting GH mRNAs were sequenced. The mutation was found 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 + 1G-->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 + 1G-->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) + 1G-->A IVS 3 mutations perturb GH mRNA splicing and cause IGHD II; and 2) these mutations can present as de novo GHD cases.

Phenotypic determinants of adenovirus E1A gene autoregulation: variable region between conserved coding domains 2 and 3.

Different serotypes and evolutionary variants of human adenoviruses exhibit distinctive patterns of positive and negative autoregulation of the viral E1A gene. An autoregulatory E1A promoter mutation of the adenovirus type 3 (Ad3) E1A gene renders Ad3hr15 incapable of growth in normally permissive cells. The promoter mutation is complemented in trans by E1A products of the heterologous helper adenovirus type 5 (Ad5). Second-site revertants of Ad3hr15 restore viability with high levels of E1A gene expression. The revertant E1A genotypes retain the mutant E1A promoter and have small in-frame deletions in the nonconserved region between the repression- and activation-associated conserved domains CR2 and CR3. Plasmid expression vectors were constructed as 12S and 13S cDNA forms of revertant E1A genes. These were used in cotransfection experiments with a reporter gene plasmid under transcriptional control of the mutant Ad3hr15 E1A promoter. The repression of the Ad3hr15 E1A promoter by helper Ad5 or revertant 12S E1A cDNA was consistently greater than that effected wild-type Ad3 12S cDNAs expression. Significantly greater levels of positive transactivation were observed in cotransfections with 13S cDNAs of Ad5 or with the 13S E1A cDNA of Ad3hr15 revertants, compared to the transactivation observed with the mutant-encoded wild-type Ad3 13S E1A cDNA. The Ad5 helper and dI-revertant phenotype of Ad3hr15 appear to be related to transactivation activities of coexpressed E1A genes. The nonconserved region which separates the conserved coding regions CR2 and CR3 of the type 3 E1A gene acts to attenuate E1A-mediated repression and transactivation of transcription.

Familial growth hormone deficiency: a model of dominant and recessive mutations affecting a monomeric protein.

Two families with familial isolated GH deficiency (IGHD) were studied, type II (autosomal dominant) and type I (autosomal recessive), whose GH1 genes exhibit cosegregation with IGHD. DNA sequencing of the GH1 genes of the first family (IGHD II) demonstrated heterozygosity for a T-->C transition in the sixth base of the donor splice site of intron III. The GH1 gene mutation in the second family (IGHD I) was found, in a previous study, to be a G-->C transversion altering the first base of the donor splice site of intron IV. Interestingly, analysis of the transcripts derived from the mutant IGHD II allele revealed that the sequences corresponding to exon III were absent due to an exon skip that causes the loss of amino acids 32-71 from the mature GH protein. In contrast, the IGHD I mutation activates a cryptic donor splice site 73 bases upstream of the normal exon IV donor splice site causing loss of amino acids 103-126 of exon IV followed by a reading frameshift and synthesis of 94 novel amino acids before chain termination 88 nucleotides downstream of the normal GH stop codon. It is hypothesized that, because of the loss of protein sequences derived from exons IV and V, the IGHD I mutation products are not transported to secretory granules and thus cannot perturb secretion of the normal monomeric GH protein. In contrast the T-->C IGHD II mutant allele product retains these sequences and is transported to secretory granules where it can interact with the normal allele product producing a dominant-negative effect at the protein level.

Heterogeneous growth hormone (GH) gene mutations in familial GH deficiency.

The GH1 genes of probands of two families with familial isolated GH deficiency (IGHD) were sequenced. Double stranded sequencing of the polymerase chain reaction (PCR) amplification products from genomic DNA of two affected cousins in a consanguineous Turkish family revealed a G-->A transition in the 20th codon of the GH1 signal peptide. This substitution converts a TGG (Trp) to a TAG (stop) codon and generates a new AluI recognition site. PCR amplification of the GH1 alleles of family members, followed by AluI digestion, revealed that the G-->A transition segregated with the IGHD phenotype. In a Saudi Arabian family, a G-->C transversion was found that alters the first base of the donor splice site of intron IV. This substitution should perturb mRNA splicing, resulting in an altered protein product which should be unstable or bioinactive. This transversion also destroys an HphI site, which was used to assay samples from relatives. Digestion of PCR amplification products with HphI demonstrated cosegregation of the G-->C transversion with IGHD. These results demonstrate that familial IGHD is a heterogeneous disease that perturbs different steps in the expression of the GH1 gene.

Functional diversity of E1A gene autoregulation among human adenoviruses.

Autoregulation of the adenovirus E1A gene involves its constitutive expression and positively and negatively regulated transcription. Dissection of this process will identify basal-level cis elements and autoregulatory targets of the E1A promoter and functional domains within the trans-acting E1A gene products. In this report, the DNA sequence of the human subgroup B adenovirus type 3 (Ad3) E1A gene is presented and compared with that of the E1A genes of similar and distantly related human adenoviruses. The cDNA forms of the Ad3 E1A gene, corresponding to two major early mRNA species, are cloned, sequenced, and subcloned into plasmid expression vectors. Cotransfections of cell cultures are performed with Ad5 or Ad3 E1A gene expression plasmids and a reporter gene under control of the Ad5 or Ad3 E1A promoter. The Ad5 and Ad3 E1A promoters are similarly repressed by either serotype's 12S cDNA gene products. The Ad3 E1A promoter responds much more strongly than the Ad5 E1A promoter to transactivation by 13S cDNA gene products. In contrast, the 13S cDNA gene of Ad5 has greater transactivation activity than that of Ad3. Experiments with missense mutations of the Ad5 E1A gene indicate that transactivation of the Ad5 E1A promoter is weak, just reversing or balancing negative autorepression. Single amino acid substitutions in the conserved, repressive functional domain 2 of the E1A gene modulate transactivating activity that is usually associated with the separate and distal conserved functional domain 3. These results suggest a strong structure-function relationship influenced by the variable sequences separating these conserved domains.