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  • Author: Alexander A L Jorge x
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Edoarda V A Albuquerque, Renata C Scalco and Alexander A L Jorge

Tall stature is defined as a height of more than 2 standard deviations (s.d.) above average for same sex and age. Tall individuals are usually referred to endocrinologists so that hormonal disorders leading to abnormal growth are excluded. However, the majority of these patients have familial tall stature or constitutional advance of growth (generally associated with obesity), both of which are diagnoses of exclusion. It is necessary to have familiarity with a large number of rarer overgrowth syndromes, especially because some of them may have severe complications such as aortic aneurysm, thromboembolism and tumor predisposition and demand-specific follow-up approaches. Additionally, endocrine disorders associated with tall stature have specific treatments and for this reason their recognition is mandatory. With this review, we intend to provide an up-to-date summary of the genetic conditions associated with overgrowth to emphasize a practical diagnostic approach of patients with tall stature and to discuss the limitations of current growth interruption treatment options.

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Patricia N Pugliese-Pires, Carlos A Tonelli, Jose M Dora, Paulo C A Silva, Mauro Czepielewski, Genoir Simoni, Ivo J P Arnhold and Alexander A L Jorge

Background

GH insensitivity (GHI) syndrome caused by STAT5B mutations was recently reported, and it is characterized by extreme short stature and immune dysfunction. Treatment with recombinant human IGF1 (rhIGF1) is approved for patients with GHI, but the growth response to this therapy in patients with STAT5B mutations has not been reported.

Objectives

To report the clinical features, molecular findings, and the short-term growth response to rhIGF1 therapy in patients with STAT5B mutation.

Subjects and methods

Hormonal and immunological evaluations were performed in two male siblings with GHI associated with atopic eczema, interstitial lung disease, and thrombocytopenic purpura. STAT5B genes were directly sequenced. The younger sibling was treated with rhIGF1 at a dose of 110 μg/kg BID.

Results

Both siblings had laboratory findings compatible with GHI associated with hyperprolactinemia. Lymphopenia and reduced number of natural killer cells without immunoglobulin abnormalities were observed. STAT5B sequence revealed a homozygous frameshift mutation (p.L142fsX161) in both siblings. The younger sibling (9.9 years of age) was treated with rhIGF1 at appropriate dosage, and he did not present any significant change in his growth velocity (from 2.3 to 3.0 cm/year after 1.5 years of therapy). The presence of a chronic illness could possibly be responsible for the poor result of rhIGF1 treatment. Further studies in patients with STAT5B defects are necessary to define the response to rhIGF1 treatment in this disorder.

Conclusion

GHI associated with immune dysfunction, especially interstitial lung disease, and hyperprolactinemia is strongly suggestive of a mutation in STAT5B in both sexes.

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Luciana R Montenegro, Andrea C Leal, Debora C Coutinho, Helena P L Valassi, Mirian Y Nishi, Ivo J P Arnhold, Berenice B Mendonca and Alexander A L Jorge

Background

Hypomethylation of the paternal imprinting center region 1 (ICR1) is the most frequent molecular cause of Silver–Russell syndrome (SRS). Clinical evidence suggests that patients with this epimutation have mild IGF1 insensitivity.

Objective

To assess in vitro IGF1 action in fibroblast culture from a patient with SRS and IGF1 insensitivity.

Methods

Fibroblast cultures from one patient with SRS due to ICR1 demethylation and controls were established. The SRS patient has severe growth failure, elevated IGF1 level, and poor growth rate during human recombinant GH treatment. IGF1 action was assessed by cell proliferation, AKT, and p42/44-MAPK phosphorylation. Gene expression was determined by real-time PCR.

Results

Despite normal IGF1R sequence and expression, fibroblast proliferation induced by IGF1 was 50% lower in SRS fibroblasts in comparison with controls. IGF1 and insulin promoted a p42/44-MAPK activation in SRS fibroblasts 40 and 36%, respectively, lower than that in control fibroblasts. On the other hand, p42/44-MAPK activation induced by EGF stimulation was only slightly reduced (75% in SRS fibroblasts in comparison with control), suggesting a general impairment in MAPK pathway with a greater impairment of the stimulation induced by insulin and IGF1 than by EGF. A PCR array analysis disclosed a defect in MAPK pathway characterized by an increase in DUSP4 and MEF2C gene expressions in patient fibroblasts.

Conclusion

A post-receptor IGF1 insensitivity was characterized in one patient with SRS and ICR1 hypomethylation. Although based on one unique severely affected patient, these results raise an intriguing mechanism to explain the postnatal growth impairment observed in SRS patients that needs confirmation in larger cohorts.

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Renata C Scalco, Vivian Hwa, Horacio M Domené, Héctor G Jasper, Alicia Belgorosky, Roxana Marino, Alberto M Pereira, Carlos A Tonelli, Jan M Wit, Ron G Rosenfeld and Alexander A L Jorge

Context and objective

GH insensitivity with immune dysfunction caused by STAT5B mutations is an autosomal recessive condition. Heterozygous mutations in other genes involved in growth regulation were previously associated with a mild height reduction. Our objective was to assess for the first time the phenotype of heterozygous STAT5B mutations.

Methods

We genotyped and performed clinical and laboratory evaluations in 52 relatives of two previously described Brazilian brothers with homozygous STAT5B c.424_427del mutation (21 heterozygous). Additionally, we obtained height data and genotype from 1104 adult control individuals from the same region in Brazil and identified five additional families harboring the same mutation (18 individuals, 11 heterozygous). Furthermore, we gathered the available height data from first-degree relatives of patients with homozygous STAT5B mutations (17 individuals from seven families). Data from heterozygous individuals and non-carriers were compared.

Results

Individuals carrying heterozygous STAT5B c.424_427del mutation were 0.6 SDS shorter than their non-carrier relatives (P=0.009). Heterozygous subjects also had significantly lower SDS for serum concentrations of IGF1 (P=0.028) and IGFBP3 (P=0.02) than their non-carrier relatives. The 17 heterozygous first-degree relatives of patients carrying homozygous STAT5B mutations had an average height SDS of −1.4±0.8 when compared with population-matched controls (P< 0.001).

Conclusions

STAT5B mutations in the heterozygous state have a significant negative impact on height (∼3.9 cm). This effect is milder than the effect seen in the homozygous state, with height usually within the normal range. Our results support the hypothesis that heterozygosity of rare pathogenic variants contributes to normal height heritability.

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Ana P M Canton, Sílvia S Costa, Tatiane C Rodrigues, Debora R Bertola, Alexsandra C Malaquias, Fernanda A Correa, Ivo J P Arnhold, Carla Rosenberg and Alexander A L Jorge

Background

The etiology of prenatal-onset short stature with postnatal persistence is heterogeneous. Submicroscopic chromosomal imbalances, known as copy number variants (CNVs), may play a role in growth disorders.

Objective

To analyze the CNVs present in a group of patients born small for gestational age (SGA) without a known cause.

Patients and methods

A total of 51 patients with prenatal and postnatal growth retardation associated with dysmorphic features and/or developmental delay, but without criteria for the diagnosis of known syndromes, were selected. Array-based comparative genomic hybridization was performed using DNA obtained from all patients. The pathogenicity of CNVs was assessed by considering the following criteria: inheritance; gene content; overlap with genomic coordinates for a known genomic imbalance syndrome; and overlap with CNVs previously identified in other patients with prenatal-onset short stature.

Results

In 17 of the 51 patients, 18 CNVs were identified. None of these imbalances has been reported in healthy individuals. Nine CNVs, found in eight patients (16%), were categorized as pathogenic or probably pathogenic. Deletions found in three patients overlapped with known microdeletion syndromes (4q, 10q26, and 22q11.2). These imbalances are de novo, gene rich and affect several candidate genes or genomic regions that may be involved in the mechanisms of growth regulation.

Conclusion

Pathogenic CNVs in the selected patients born SGA were common (at least 16%), showing that rare CNVs are probably among the genetic causes of short stature in SGA patients and revealing genomic regions possibly implicated in this condition.

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Patricia N Pugliese-Pires, Jean-Philippe Fortin, Thais Arthur, Ana Claudia Latronico, Berenice B Mendonca, Sandra Mara F Villares, Ivo J P Arnhold, Alan S Kopin and Alexander A L Jorge

Background

A limited number of mutations in the GH secretagogue receptor gene (GHSR) have been described in patients with short stature.

Objective

To analyze GHSR in idiopathic short stature (ISS) children including a subgroup of constitutional delay of growth and puberty (CDGP) patients.

Subjects and methods

The GHSR coding region was directly sequenced in 96 independent patients with ISS, 31 of them with CDGP, in 150 adults, and in 197 children with normal stature. The pharmacological consequences of GHSR non-synonymous variations were established using in vitro cell-based assays.

Results

Five different heterozygous point variations in GHSR were identified (c.−6 G>C, c.251G>T (p.Ser84Ile), c.505G>A (p.Ala169Thr), c.545 T>C (p.Val182Ala), and c.1072G>A (p.Ala358Thr)), all in patients with CDGP. Neither these allelic variants nor any other mutations were found in 694 alleles from controls. Functional studies revealed that two of these variations (p.Ser84Ile and p.Val182Ala) result in a decrease in basal activity that was in part explained by a reduction in cell surface expression. The p.Ser84Ile mutation was also associated with a defect in ghrelin potency. These mutations were identified in two female patients with CDGP (at the age of 13 years, their height SDS were −2.4 and −2.3). Both patients had normal progression of puberty and reached normal adult height (height SDS of −0.7 and −1.4) without treatment.

Conclusion

This is the first report of GHSR mutations in patients with CDGP. Our data raise the intriguing possibility that abnormalities in ghrelin receptor function may influence the phenotype of individuals with CDGP.

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Rafael A Carvalho, Betsaida Urtremari, Alexander A L Jorge, Lucas S Santana, Elisangela P S Quedas, Tomoko Sekiya, Viviane C Longuini, Fabio L M Montenegro, Antonio M Lerario, Sergio P A Toledo, Stephen J Marx, Rodrigo A Toledo and Delmar M Lourenço Jr

Background

Loss-of-function germline MEN1 gene mutations account for 75–95% of patients with multiple endocrine neoplasia type 1 (MEN1). It has been postulated that mutations in non-coding regions of MEN1 might occur in some of the remaining patients; however, this hypothesis has not yet been fully investigated.

Objective

To sequence for the entire MEN1 including promoter, exons and introns in a large MEN1 cohort and determine the mutation profile.

Methods and patients

A target next-generation sequencing (tNGS) assay comprising 7.2 kb of the full MEN1 was developed to investigate germline mutations in 76 unrelated MEN1 probands (49 familial, 27 sporadic). tNGS results were validated by Sanger sequencing (SS), and multiplex ligation-dependent probe amplification (MLPA) assay was applied when no mutations were identifiable by both tNGS and SS.

Results

Germline MEN1 variants were verified in coding region and splicing sites of 57/76 patients (74%) by both tNGS and SS (100% reproducibility). Thirty-eight different pathogenic or likely pathogenic variants were identified, including 13 new and six recurrent variants. Three large deletions were detected by MLPA only. No mutation was detected in 16 patients. In untranslated, regulatory or in deep intronic MEN1 regions of the 76 MEN1 cases, no point or short indel pathogenic variants were found in untranslated, although 33 benign/likely benign and three new VUS variants were detected.

Conclusions

Our study documents that point or short indel mutations in non-coding regions of MEN1 are very rare events. Also, tNGS proved to be a highly effective technology for routine genetic MEN1 testing.

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João L O Madeira, Alexander A L Jorge, Regina M Martin, Luciana R Montenegro, Marcela M Franca, Everlayny F Costalonga, Fernanda A Correa, Aline P Otto, Ivo J P Arnhold, Helayne S Freitas, Ubiratan F Machado, Berenice B Mendonca and Luciani R Carvalho

Context

Mutations in the GH1 promoter are a rare cause of isolated growth hormone deficiency (IGHD).

Objective

To identify the molecular aetiology of a family with IGHD.

Design

DNA sequencing, electromobility shift (EMSA) and luciferase reporter assays.

Setting

University Hospital.

Patients

Three siblings (2M) born to consanguineous parents presented with IGHD with normal pituitary on MRI.

Methods

The GH1 proximal promoter, locus control region, five exons and four introns as well as GHRHR gene were sequenced in genomic DNA by Sanger method. DNA–protein interaction was evaluated by EMSA in nuclear extracts of GH3 pituitary cells. Dual-luciferase reporter assays were performed in cells transiently transfected with plasmids containing four different combinations of GH1 allelic variants (AV).

Results

The patients harboured two homozygous variants (c.-185T>C and c.-223C>T) in the GH1 promoter within a highly conserved region and predicted binding sites for POU1F1/SP1 and SP1 respectively. The parents and brother were carriers and these variants were absent in 100 controls. EMSA demonstrated absent binding of GH3 nuclear extract to the c.-223C>T variant and normal binding of both POU1F1 protein and GH3 nuclear extract to the c.-185T>C variant. In contrast to GH1 promoter with AV only at c.-185, the GH1 promoter containing the AV only at c.-223 and at both positions drove significantly less expression of luciferase compared with the promoter containing either positions wild type in luciferase reporter assays.

Conclusion

To our knowledge, c.-223C>T is the first homozygous point mutation in the GH1 promoter that leads to short stature due to IGHD.