The growth-promoting effects of IGF-I is mediated through the IGF-I receptor (IGF1R), a widely expressed cell-surface tyrosine kinase receptor. IGF1R copy number variants (CNV) can cause pre- and ...postnatal growth restriction or overgrowth.
Whole exome sequence (WES), chromosomal microarray, and targeted IGF1R gene analyses were performed on 3 unrelated children who share features of small for gestational age, short stature, and elevated serum IGF-I, but otherwise had clinical heterogeneity. Fluorescence-activated cell sorting (FACS) analysis of cell-surface IGF1R was performed on live primary cells derived from the patients.
Two novel IGF1R CNV and a heterozygous IGF1R nonsense variant were identified in the 3 patients. One CNV (4.492 Mb) was successfully called from WES, utilizing eXome-Hidden Markov Model (XHMM) analysis. FACS analysis of cell-surface IGF1R on live primary cells derived from the patients demonstrated a ∼50% reduction in IGF1R availability associated with the haploinsufficiency state.
In addition to conventional methods, IGF1R CNV can be identified from WES data. FACS analysis of live primary cells is a promising method for efficiently evaluating and screening for IGF1R haploinsufficiency. Further investigations are necessary to delineate how comparable IGF1R availability leads to the wide spectrum of clinical phenotypes and variable responsiveness to rhGH therapy.
The majority of insulin-like growth factor (IGF)-I and IGF-II circulate in the serum as a complex with the insulin-like growth factor binding protein (IGFBP)-3 or IGFBP-5, and an acid-labile subunit ...(ALS). The function of ALS is to prolong the half-life of the IGF-I-IGFBP-3/IGFBP-5 binary complexes. Fourteen different mutations of the human IGFALS gene have been identified in 17 patients, suggesting that ALS deficiency may be prevalent in a subset of patients with extraordinarily low serum levels of IGF-I and IGFBP-3 that remain abnormally low upon growth hormone stimulation. Postnatal growth was clearly affected. Commonly, the height standard deviation score before puberty was between -2 and -3, and approximately 1.4 SD shorter than the midparental height SDS. Pubertal delay was found in 50% of the patients. Circulating IGF-II, IGFBP-1, -2 and -3 levels were reduced, with the greatest reduction observed for IGFBP-3. Insulin insensitivity was a common finding, and some patients presented low bone mineral density. Human ALS deficiency represents a unique condition in which the lack of ALS proteins results in the disruption of the entire IGF circulating system. Despite a profound circulating IGF-I deficiency, there is only a mild impact on postnatal growth. The preserved expression of locally produced IGF-I might be responsible for the preservation of linear growth near normal limits.
Laron syndrome (LS) is a genetic disorder caused by mutations in the growth hormone receptor (GHR) gene. The most frequent GHR mutation is E180splice (rs121909360), which was initially found in an ...inbred population of Spanish descent in Ecuador and subsequently in Israel, Brazil, Chile, and the United States. The aim of the present study is to determine if the E180splice mutation arose from a common origin. We studied 22 patients with LS from Ecuador, Israel (of Moroccan origin), Brazil, Chile, and the United States (of Mexican origin) who were homozygous for the E180splice mutation and compared them to control individuals for markers surrounding the GHR, intragenic polymorphisms, and Y-chromosome STR. An identical haplotype was found in all but one of the subjects carrying the E180splice mutation: D5S665: 150/150; D5S2082: 192/192; D5S2087: 246/246; rs6179 G/G; and rs6180 C/C. One patient differed from the others only at D5S2082 (168/192). This haplotype is rare (~1%) in control individuals and confirmed that the E180splice-associated haplotype was not derived from independent origins but represented recombination from a common ancestor. The analysis of paternal lineage markers showed that 50% belong to haplogroup R1b (found in Portugal and Spain) and 40% to haplogroups J and E (typical in the Middle East and in Eastern European Jews). The germline E180Splice mutation appears to have originated from a single common ancestor. The presence of Y-chromosome markers associated with Sephardic populations in persons harboring the E180splice mutation provides genetic evidence in support of the historical tracking of the exodus of this specific population.
Abstract
Context:
Autosomal-recessive mutations in the growth hormone receptor (GHR) are the most common causes for primary growth hormone insensitivity (GHI) syndrome with classical GHI ...phenotypically characterized by severe short stature and marked insulin-like growth factor (IGF)-I deficiency. We report three families with dominant-negative heterozygous mutations in the intracellular domain of the GHR causing a nonclassical GHI phenotype.
Objective:
To determine if the identified GHR heterozygous variants exert potential dominant-negative effects and are the cause for the GHI phenotype in our patients.
Results:
All three mutations (c.964dupG, c.920_921insTCTCAAAGATTACA, and c.945+2T>C) are predicted to result in frameshift and early protein termination. In vitro functional analysis of variants c.964dupG and c.920_921insTCTCAAAGATTACA (c.920_921ins14) suggests that these variants are expressed as truncated proteins and, when coexpressed with wild-type GHR, mimicking the heterozygous state in our patients, exert dominant-negative effects. Additionally, we provide evidence that a combination therapy of recombinant human growth hormone (rhGH) and rhIGF-I improved linear growth to within normal range for one of our previously reported patients with a characterized, dominant-negative GHR (c.899dupC) mutation.
Conclusion:
Dominant-negative GHR mutations are causal of the mild GHI with substantial growth failure observed in our patients. Heterozygous defects in the intracellular domain of GHR should, therefore, be considered in cases of idiopathic short stature and IGF-I deficiency. Combination therapy of rhGH and rhIGF-I improved growth in one of our patients.
In patients with mild GHI, heterozygous, truncating GHR mutations exerted dominant-negative effects. A combined therapy of rhGH plus rhIGF-I is an effective treatment option.
Human genetic defects in the growth hormone (GH)-IGF-I axis affecting the IGF system present with growth failure as their principal clinical feature. This is usually associated with GH insensitivity ...(GHI) presenting in childhood as severe or mild short stature. Dysmorphic features and metabolic abnormalities may also be present. The field of GHI due to mutations affecting GH action has evolved rapidly since the first description of the extreme phenotype related to homozygous GH receptor (GHR) mutations in 1966. A continuum of genetic, phenotypic, and biochemical abnormalities can be defined associated with clinically relevant defects in linear growth. The mechanisms of the GH-IGF-I axis in the regulation of normal human growth is discussed followed by descriptions of mutations in GHR, STAT5B, IGF-I, IGFALS, IGF1R, and GH1 defects causing bio-inactive GH or anti-GH antibodies. These GH-IGF-I axis defects are associated with a range of clinical, and hormonal characteristics. An up-dated approach to the clinical assessment of the patient with GHI focusing on investigation of the GH-IGF-I axis and relevant molecular studies contributing to the identification of causative genetic defects is also discussed.
IGF-binding protein (IGFBP)-related protein 1 (IGFBP-rP1) has been shown to bind both IGFs and insulin, albeit with low affinity, and to inhibit insulin signaling. We hypothesized that IGFBP-rP1 is ...associated with insulin resistance and components of the IGF system in humans. To this aim, a cross-sectional study was conducted in 113 nondiabetic and 43 type 2 diabetic men. Insulin sensitivity (insulin sensitivity index S(i) from intravenous glucose tolerance tests in nondiabetic subjects, or the rate constant for disappearance of glucose K(ITT) from insulin tolerance tests in type 2 diabetic subjects), circulating IGFBP-rP1 (from enzyme-linked immunosorbent assay), adiponectin (from radioimmunoassay), C-reactive protein (CRP; from immunoturbidimetry), soluble tumor necrosis factor receptor 2 (sTNFR2; from enzyme-amplified sensitivity immunoassay), and IGF system parameters (IGF-I, free IGF-I, and IGFBP-1 from immunoradiometric assay) were assessed in all subjects. Among nondiabetic men, those in the highest quartile for circulating IGFBP-rP1 exhibited decreased S(i) and adiponectin (both P < 0.01) as well as increased CRP and sTNFR2 (both P < 0.05). Circulating IGFBP-rP1 was also found to be increased in previously undiagnosed type 2 diabetic patients (P = 0.01) but not in known type 2 diabetic patients receiving pharmacological therapy. Although no changes in IGF system components were evident by IGFBP-rP1 quartiles in nondiabetic subjects, independent positive associations of IGFBP-rP1 with circulating fasting IGFBP-1 were evident after adjustment for insulin resistance parameters in both nondiabetic and type 2 diabetic subjects, with IGFBP-rP1 explaining 2 and 11% of IGFBP-1 variance, respectively. In additional multivariate analyses, S(i), sTNFR2, and age stood as independent predictive variables of IGFBP-rP1 (together explaining 18% of its variance) in nondiabetic subjects, and BMI became the only independent predictive variable of IGFBP-rP1 (explaining 26% of its variance) in type 2 diabetic men. These findings show for the first time that circulating IGFBP-rP1 is increased with insulin resistance, and they also suggest novel interactions between IGFBP-rP1 and the IGF system in humans.
Context: Primary GH insensitivity (GHI) or Laron syndrome, caused by mutations of the GH receptor (GHR) gene, has a clinical phenotype of postnatal growth failure associated with normal elevated ...serum concentrations of GH and low serum levels of IGF-I.
Objective: We investigated the clinical and biochemical implications of molecular defects in the GHR gene in an Austrian family with two daughters who were GHI.
Patients: Patient 1 height, −4.8 sd score (SDS) and patient 2 (height, −5.0 SDS) had elevated circulating levels of GH, low-normal levels of GH-binding protein, and abnormally low IGF-I (−5.0 SDS and −2.6 SDS, respectively) and IGF-binding protein-3 (−2.6 SDS and −2.0 SDS, respectively).
Results: Both patients carry novel compound, missense, heterozygous GHR mutations, C94S and H150Q. In vitro reconstitution experiments demonstrated that whereas each of the mutants could be stably expressed, GHR(C94S) lost its affinity for GH and could neither activate signal transducer and activator of transcription (STAT)-5b nor drive STAT5b-dependent gene transcription in response to GH (1–100 ng/ml). GHR(H150Q) showed normal affinity for GH but impaired capacity for signal transduction. The compound heterozygote and C94S heterozygote, but not the H150Q heterozygote, showed significant deficiency in activating GH-induced gene expression, corroborating diminished GH-induced STAT5b activation in fibroblasts carrying GHR(C94S) as either a compound heterozygote (in the patients) or a simple heterozygote (in one parent).
Conclusions: Each of the compound heterozygous mutations contributed additively to the pathological condition seen in the patients, and the more detrimental of the two mutations, C94S, may cause (partial) primary GHI, even in a heterozygous state.
Patients with aggrecan (ACAN) deficiency present with dominantly inherited short stature, often with advanced skeletal maturation and premature growth cessation. There is a paucity of information on ...the effects of growth-promoting interventions.
The aim of this study was to evaluate the efficacy and safety of recombinant human growth hormone (rhGH) therapy on linear growth in children with ACAN deficiency.
Open-label, single-arm, prospective study at Cincinnati Children's Hospital Medical Center. Ten treatment-naïve patients were recruited. Inclusion criteria were a confirmed heterozygous mutation in ACAN, age ≥2 years, prepubertal, bone age (BA) ≥chronological age (CA), and normal insulin-like growth factor I concentration. Treatment was with rhGH (50 µg/kg/day) over 1 year. Main outcomes measured were height velocity (HV) and change in (Δ) height SD score (HtSDS).
Ten patients (6 females) were enrolled with median CA of 5.6 years (range 2.4-9.7). Baseline median HtSDS was -2.5 (range -4.3 to -1.1). Median baseline BA was 6.9 years (range 2.5-10.0), with median BA/CA of 1.2 (range 0.9-1.5). Median pretreatment HV was 5.2 cm/year (range 3.8-7.1), increased to 8.3 cm/year (range 7.3-11.2) after 1 year of therapy (P = .004). Median ΔHtSDS after 1 year was +0.62 (range +0.35 to +1.39) (P = .002). Skeletal maturation did not advance inappropriately (median ΔBA/CA -0.1, P = .09). No adverse events related to rhGH were observed.
Treatment with rhGH improved linear growth in a cohort of patients with short stature due to ACAN deficiency.
A predicted alanine to proline substitution in Stat5b that results in profound short stature, growth hormone insensitivity, and immunodeficiency represents the first natural mutation of this ...transcription factor in a human. To understand the mechanisms responsible for these pathophysiological abnormalities, we have studied the biochemical and biophysical properties of the mutant Stat5b molecule. In a cellular reconstitution model growth hormone robustly stimulated tyrosine phosphorylation and transcriptional activity of wild-type Stat5b while Stat5bA630P was minimally modified and did not promote reporter gene expression. Steady state levels of Stat5bWT were ∼3-fold higher than Stat5bA630P in cell extracts prepared with nonionic detergents. Although initial rates of biosynthesis of both proteins were similar, pulse-chase experiments established that the apparent half-life of newly synthesized soluble Stat5bA630P was <15% of Stat5bWT (3.5 h versus >24 h). Stat5bA630P accumulated in cells primarily in cytoplasmic inclusion bodies. Structural analysis of the isolated SH2 domain containing the A630P mutation showed that it resembled the wild-type SH2 segment but that it exhibited reduced thermodynamic stability and slower folding kinetics, displayed an increased hydrophobic surface, and was prone to aggregation in solution. Our results are compatible with a model in which Stat5bA630P is an inactive transcription factor by virtue of its aberrant folding and diminished solubility triggered by a misfolded SH2 domain. The potential for aggregation and formation of cytoplasmic inclusions raises the possibility that Stat5bA630P could produce additional defects through inhibition of proteasome function.
Genotype-phenotype associations for common diseases are often compounded by pleiotropy and metabolic state. Here, we devised a pooled human organoid-panel of steatohepatitis to investigate the impact ...of metabolic status on genotype-phenotype association. En masse population-based phenotypic analysis under insulin insensitive conditions predicted key non-alcoholic steatohepatitis (NASH)-genetic factors including the glucokinase regulatory protein (GCKR)-rs1260326:C>T. Analysis of NASH clinical cohorts revealed that GCKR-rs1260326-T allele elevates disease severity only under diabetic state but protects from fibrosis under non-diabetic states. Transcriptomic, metabolomic, and pharmacological analyses indicate significant mitochondrial dysfunction incurred by GCKR-rs1260326, which was not reversed with metformin. Uncoupling oxidative mechanisms mitigated mitochondrial dysfunction and permitted adaptation to increased fatty acid supply while protecting against oxidant stress, forming a basis for future therapeutic approaches for diabetic NASH. Thus, “in-a-dish” genotype-phenotype association strategies disentangle the opposing roles of metabolic-associated gene variant functions and offer a rich mechanistic, diagnostic, and therapeutic inference toolbox toward precision hepatology.
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•En masse liver organoid analysis informs NASH genotype-phenotype associations•Gene editing in organoids delineates GCKR-rs1260326 impact on glycolysis and lipogenesis•GCKR variant has opposing impacts on NASH severity with or without diabetes•Mitochondrial dysfunction is associated with GCKR variant-defined diabetic NASH
Human organoid modeling combined with genotype-phenotype association studies disentangles the unique opposing roles of a steatohepatitis-susceptible gene variant.
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