Growth Hormone Receptor (Ghr) 6ω Pseudoexon Activation: A Novel Cause Of Severe Growth Hormone Insensitivity (Ghi)

Abstract

Context: Severe forms of Growth Hormone Insensitivity (GHI) are characterized by extreme short stature, dysmorphism and metabolic anomalies.

Objective: Identification of the genetic cause of growth failure in 3 'classical' GHI subjects.

Design: A novel intronic GHR variant was identified, and in vitro splicing assays confirmed aberrant splicing. A 6Ω pseudoexon GHR vector and patient fibroblast analysis assessed the consequences of the novel pseudoexon inclusion and the impact on GHR function.

Results: We identified a novel homozygous intronic GHR variant (g.5:42700940T>G, c.618 + 836T> G), 44bp downstream of the previously recognized intronic 6Ψ GHR pseudoexon mutation in the index patient. Two siblings also harbored the novel intronic 6Ω pseudoexon GHR variant in compound heterozygosity with the known GHR c.181C>T (R43X) mutation. In vitro splicing analysis confirmed inclusion of a 151bp mutant 6Ω pseudoexon not identified in wild-type constructs. Inclusion of the 6Ω pseudoexon causes a frameshift resulting in a non-functional truncated GHR lacking the transmembrane and intracellular domains. The truncated 6Ω pseudoexon protein demonstrated extracellular accumulation and diminished activation of STAT5B signaling following growth hormone stimulation.

Conclusion: Novel GHR 6Ω pseudoexon inclusion results in loss of GHR function consistent with a severe GHI phenotype. This represents a novel mechanism of Laron syndrome and is the first deep intronic variant identified causing severe postnatal growth failure. The 2 kindreds originate from the same town in Campania, Southern Italy, implying common ancestry. Our findings highlight the importance of studying variation in deep intronic regions as a cause of monogenic disorders.

Keywords: GHR 6Ω pseudoexon; Short stature; growth hormone insensitivity; severe primary IGF-I deficiency.

Figure 1.

Clinical images and growth chart of patient 1. A and B, Clinical images of patient 1 (P1; index case) aged 1.7 years with classical Laron features of midfacial hypoplasia, depressed nasal bridge, and frontal bossing. C, Growth chart showing severe postnatal growth failure and response to recombinant human insulin-like growth factor 1 therapy.

Figure 2.

Height velocity charts of the patients demonstrating the benefits of recombinant human insulin-like growth factor 1 (IGF-1) therapy. A, Patient 1; B, patient 2; and C, patient 3.

Figure 3.

Growth chart of patient 2. Growth chart showing severe postnatal growth failure. Periods of recombinant human insulin-like growth factor 1 (IGF-1) therapy are indicated.

Figure 4.

Clinical images and growth chart of patient 3. A and B, Clinical images showing patient 3 (P3; younger sibling of P2) aged A, 3.5 months, and B, 5.4 years, displaying classical Laron features of midfacial hypoplasia, depressed nasal bridge, and frontal bossing. C, Growth chart showing severe postnatal growth failure. Periods of insulin-like growth factor 1 (IGF-1) therapy are indicated.

Figure 5.

Pedigrees and electropherograms for kindreds 1 and 2. Electropherograms and pedigrees showing the segregation of the c.618+836T > G growth hormone receptor (GHR) variant in affected families. A, Homozygous and heterozygous c.618+836T > G GHR variants in patient 1 (P1) and both parents, respectively. B, Patients 2 and 3 (P2 and 3) harbored compound heterozygous c.618+836T > G (inherited from the mother) and c.181C > T (R43X) (inherited from the father) GHR mutations.

Figure 6.

Effect of novel 6Ω growth hormone receptor (GHR) pseudoexon c.618+836T > G variant. A, The novel 6Ω GHR pseudoexon c.618+836T > G variant creates an AGGT splice donor site (red) downstream of the original GHR 6Ψ pseudoexon variant (c.618+792A > G) (green) that produces a CGGT splice donor site. The dormant intronic AGCC acceptor splice site involved in missplicing and inclusion of both pseudoexons is shown in purple. Dashed lines indicate interrupted intronic sequence. B, Schematic of the 6Ψ and novel 6Ω GHR pseudoexons inclusion events into the messenger RNA (mRNA). C, Schematic of the novel 6Ω GHR pseudoexon inclusion event and predicted GHR protein compared to wild-type (WT) sequence. The 6Ω pseudoexon inclusion is predicted to cause a frameshift and result in premature truncation of the GHR lacking both transmembrane (TM) and intracellular domains. D, Gel electrophoresis of complementary DNA splicing products following the splicing assay using an exon trap vector (MoBiTec-Exontrap cloning vector pET01). EV, empty vector, pET01 alone; WT-GHR, pET01 with 600 base pair (bp) of wild-type GHR intron 6 sequence inserted; patient 1, pET01 with 600 bp of patient 1 intron 6 sequence inserted (including the c.618+836T > G variant). GHR-6Ψ, pET01 with 600-bp sequence from a patient with the original GHR pseudoexon (6Ψ) c.618+792A > G variant. The spliced products were amplified by polymerase chain reaction and visualized on a 2% agarose gel. Lanes 1 and 2: A 250-bp band is seen in EV and WT sequence, as expected, representing the 2 exons of the exon trap vector and confirming normal splicing with WT sequence (lane 2). Lane 3: A 401-bp band is seen in the proband and sequencing revealed a 151-bp insert between the 2 exons of the exon trap vector (250 bp), confirming novel 6Ω pseudoexon inclusion. Lane 4: A 358-bp band is seen in the GHR 6Ψ patient sample and sequencing revealed a 108-bp insert between the 2 exons (250 bp) of the exon trap vector, confirming the original pseudoexon inclusion, as expected.

Figure 7.

Expression of wild-type (WT) and mutant transcripts in affected family members with the heterozygous c.618+836T > G growth hormone receptor (GHR) 6Ω variant. The GHR 6Ω pseudoexon also diminishes GH-dependent STAT5B activation and accumulates extracellularly. A, Complementary DNA (cDNA) was prepared from dermal fibroblasts derived from a healthy control (HC) and patients 2 (P2) and P3 and both parents. Schematic showing the locations of the primers (GHR cDNA exon 4F [forward] and GHR cDNA exon 8R [reverse] [blue arrows]) used to amplify the region encompassing WT GHR, the GHR 6Ω pseudoexon insertion. A “normal” 705–base pair (bp) polymerase chain reaction (PCR) product was seen in all samples. An additional larger (856-bp) PCR product was seen in P2, P3, and their mother, who were heterozygous for the c.618+836T > G GHR 6Ω variant, indicating the additional 151-bp 6Ω pseudoexon insertion. B, Schematic showing the locations of the primers (GHR cDNA pseudo F1 [forward] and GHR cDNA exon 8R [reverse] [blue arrows]). The forward primer at the junction of the 6Ω pseudoexon insertion means only sequences containing the GHR 6Ω pseudoexon insertion are amplified. The expected 387-bp PCR product is seen in patient 2, patient 3, and their mother, all of whom are heterozygous for the c.618+836T > G GHR variant. C, Whole-cell lysates from untreated or GH-stimulated (20 minutes) HEK293 cells transfected with pcDNA3.1 empty vector, WT GHR or 6Ω GHR mutant constructs. Representative immunoblots of 3 experiments are shown. D, Immunoblot analysis of conditioned media with anti-GH binding protein (BP) antibody from HEK293 cells transfected with the 6Ω GHR mutant construct showing extracellular accumulation of the truncated mutant 6Ω GHR protein. B actin, β actin; K2M, kindred 2 mother; K2F kindred 2 father.