Marina Schlicke

Novel heterozygous IGF1R mutation in two brothers with developing impaired glucose tolerance.

Abstract Infants born small for gestational age (SGA) are at risk to develop metabolic complications. Insulin-like growth factor 1 (IGF-1) resistance due to IGF-1 receptor (IGF1R) mutations is a rare genetic condition that causes proportionate growth retardation. The contribution of an impaired IGF1R function to the development of comorbidities such as disturbed glucose homeostasis is not well understood. Genetic analysis and detailed auxological, endocrine and psychological investigations in two male SGA siblings were performed. The two patients and their father bear a novel heterozygous mutation (p.Cys1248Tyr) in the IGF1R gene. Both brothers displayed very similar growth pattern before and during recombinant human growth hormone treatment, whereas oral glucose tolerance tests showed variable manifestations of progressive impaired glucose tolerance. The father had already developed type 2 diabetes mellitus. Growth retardation in our patients is likely caused by the IGF1R mutation that might predispose to disturbances of carbohydrate homeostasis. Therefore, a close metabolic monitoring of affected patients is indicated, particularly if growth hormone therapy is commenced.

Genetics of Human Stature: Insight from Single Gene Disorders

Mutations of numerous genes encoding proteins that affect multiple pathways responsible for regulation of cell proliferation can cause growth disturbances in humans. Genes such as HESX1, PROP1, PIT1/POU1F1 and GLI2 have been shown to cause pituitary hormone deficiency. In addition, heterozygous mutations or gene deletions in the growth hormone-insulin-like growth factor (GH-IGF) axis such as the GH, GH-releasing hormone receptor, GH receptor, STAT5b, IGF-I, IGF-I receptor and the acid labile subunit have also been observed in children with growth failure and short stature. More recently, mutations of genes encoding regulators of cell proliferation and division, i.e., the pericentrin gene, have also resulted in severe growth disturbances.

Directed evolution of an error-prone T7 DNA polymerase that attenuates viral replication.

Experimental evidence exists that RNA viruses replicate with extremely high mutation rates that result in significant genetic diversity. The diverse nature of viral populations allows rapid adaptation to dynamic environments, and evolution of resistances to vaccines as well as antiviral substances. For DNA viruses that replicate at much greater fidelities, as yet, neither diverse structures in the population nor their responses to increased mutation rates have been sufficiently described. By using the example of DNA bacteriophage T7, we describe the identification of virus‐specific DNA polymerase variants with decreased replication fidelities, and their impact on the efficiency of the viral infection cycle.

Alu-Mediated Recombination Defect in IGF1R: Haploinsufficiency in a Patient with Short Stature

Background: The insulin-like growth factor (IGF) receptor (IGF1R) is essential for normal development and growth. IGF1R mutations cause IGF-1 resistance resulting in intrauterine and postnatal growth failure. The phenotypic spectrum related to IGF1R mutations remains to be fully understood. Methods: Auxological and endocrinological data of a patient identified previously were assessed. The patients fibroblasts were studied to characterize the IGF1R deletion, mRNA fate, protein expression and signalling capabilities. Results: The boy, who carries a heterozygous IGF1R exon 6 deletion caused by Alu element-mediated recombination and a heterozygous SHOX variant (p.Met240Ile), was born appropriate for gestational age but developed proportionate short stature postnatally. IGF-1 levels were low-normal. None of the stigmata associated with SHOX deficiency or sporadically observed in IGF1R mutation carriers were present. Nonsense-mediated mRNA decay led to a substantial decline of IGF1R dosage and IGF-1-dependent receptor autophosphorylation but not impaired downstream signalling. Conclusion: We present the first detailed report of an intragenic IGF1R deletion identified in a patient who, apart from short stature, deviates from all established markers that qualify a growth-retarded child for IGF1R analysis. Although such children will usually escape routine clinical mutation screenings, they can contribute to the understanding of factors and mechanisms that cooperate with the IGF1R.

Combined pituitary hormone deficiency due to gross deletions in the POU1F1 (PIT-1 ) and PROP1 genes

Pituitary development depends on a complex cascade of interacting transcription factors and signaling molecules. Lesions in this cascade lead to isolated or combined pituitary hormone deficiency (CPHD). The aim of this study was to identify copy number variants (CNVs) in genes known to cause CPHD and to determine their structure. We analyzed 70 CPHD patients from 64 families. Deletions were found in three Turkish families and one family from northern Iraq. In one family we identified a 4.96 kb deletion that comprises the first two exons of POU1F1. In three families a homozygous 15.9 kb deletion including complete PROP1 was discovered. Breakpoints map within highly homologous AluY sequences. Haplotype analysis revealed a shared haplotype of 350 kb among PROP1 deletion carriers. For the first time we were able to assign the boundaries of a previously reported PROP1 deletion. This gross deletion shows strong evidence to originate from a common ancestor in patients with Kurdish descent. No CNVs within LHX3, LHX4, HESX1, GH1 and GHRHR were found. Our data prove multiplex ligation-dependent probe amplification to be a valuable tool for the detection of CNVs as cause of pituitary insufficiencies and should be considered as an analytical method particularly in Kurdish patients.

Genetic analyses of bone morphogenetic protein 2, 4 and 7 in congenital combined pituitary hormone deficiency.

BackgroundThe complex process of development of the pituitary gland is regulated by a number of signalling molecules and transcription factors. Mutations in these factors have been identified in rare cases of congenital hypopituitarism but for most subjects with combined pituitary hormone deficiency (CPHD) genetic causes are unknown. Bone morphogenetic proteins (BMPs) affect induction and growth of the pituitary primordium and thus represent plausible candidates for mutational screening of patients with CPHD.MethodsWe sequenced BMP2, 4 and 7 in 19 subjects with CPHD. For validation purposes, novel genetic variants were genotyped in 1046 healthy subjects. Additionally, potential functional relevance for most promising variants has been assessed by phylogenetic analyses and prediction of effects on protein structure.ResultsSequencing revealed two novel variants and confirmed 30 previously known polymorphisms and mutations in BMP2, 4 and 7. Although phylogenetic analyses indicated that these variants map within strongly conserved gene regions, there was no direct support for their impact on protein structure when applying predictive bioinformatics tools.ConclusionsA mutation in the BMP4 coding region resulting in an amino acid exchange (p.Arg300Pro) appeared most interesting among the identified variants. Further functional analyses are required to ultimately map the relevance of these novel variants in CPHD.

Copy number variations in "classical" obesity candidate genes are not frequently associated with severe early-onset obesity in children

Abstract Background: Obesity is genetically heterogeneous and highly heritable, although polymorphisms explain the phenotype in only a small proportion of obese children. We investigated the presence of copy number variations (CNVs) in “classical” genes known to be associated with (monogenic) early-onset obesity in children. Methods: In 194 obese Caucasian children selected for early-onset and severe obesity from our obesity cohort we screened for deletions and/or duplications by multiplex ligation-dependent probe amplification reaction (MLPA). As we found one MLPA probe to interfere with a polymorphism in SIM1 we investigated its association with obesity and other phenotypic traits in our extended cohort of 2305 children. Results: In the selected subset of most severely obese children, we did not find CNV with MLPA in POMC, LEP, LEPR, MC4R, MC3R or MC2R genes. However, one SIM1 probe located at exon 9 gave signals suggestive for SIM1 insufficiency in 52 patients. Polymerase chain reaction (PCR) analysis identified this as a false positive result due to interference with single nucleotide polymorphism (SNP) rs3734354/rs3734355. We, therefore, investigated for associations of this polymorphism with obesity and metabolic traits in our extended cohort. We found rs3734354/rs3734355 to be associated with body mass index-standard deviation score (BMI-SDS) (p = 0.003), but not with parameters of insulin metabolism, blood pressure or food intake. Conclusions: In our modest sample of severely obese children, we were unable to find CNVs in well-established monogenic obesity genes. Nevertheless, we found an association of rs3734354 in SIM1 with obesity of early-onset type in children, although not with obesity-related traits.

Heterozygous Mutation within a Kinase-Conserved Motif of the Insulin-Like Growth Factor I Receptor Causes Intrauterine and Postnatal Growth Retardation

Context: Micro-RNAs (miRNAs) have been recently involved in the modulation of several biological activities including cancer. Many human tumors show deregulated expression of miRNAs targeting oncogenes and/or tumor suppressors, thus identifying miRNAs as new molecular targets for cancer therapy. Objectives: Nuclear factor (NF)B is strongly activated in human anaplastic thyroid carcinomas (ATCs). Because the regulation of miRNA expression is under control of RNA polymerase II-dependent transcription factors, we stably inactivated NFB in the ATC-derived FRO cell line and analyzed its miRNA profile in comparison with the parental counterpart by using a miRNA chip microarray. Results: The analysis revealed that a number of miRNAs were differentially expressed in the two cell lines. Among others, the miR-146a showed a strong down-regulation that was confirmed by quantitative real time RT-PCR. The expression of miR-146a was almost undetectable in mouse embryonic fibroblasts isolated from the RelA knockout mice and was restored after reexpression of RelA, thus indicating that miR-146a transcription was controlled by NFB. The inhibition of miR-146a expression in FRO cells decreased their oncogenic potential and increased the susceptibility to chemotherapeutic drug-induced apoptosis. No difference was found in the growth rate between untransfected and miR-146a-null FRO cells. Importantly, the miR-146a resulted in overexpression of human ATC specimens compared with the normal thyroid tissue. Conclusions: Our results show that NFB contributes to anaplastic thyroid cancer up-regulating the expression of miR-146a. Heterozygous Mutation within a Kinase-Conserved Motif of the Insulin-Like Growth Factor I Receptor Causes Intrauterine and Postnatal Growth Retardation Tassilo Kruis, Jurgen Klammt, Assimina Galli-Tsinopoulou, Tillmann Wallborn, Marina Schlicke, Eva Muller, Jurgen Kratzsch, Antje Korner, Rasha Odeh, Wieland Kiess, and Roland Pfaffle (J Clin Endocrinol Metab, 10.1210/jc.2009-1433) ABSTRACT Background: IGF-I receptor (IGFIR) plays an essential role in human intrauterine and postnatal development. Few heterozygous mutations in IGF1R leading to IGF-I resistance and intrauterine and postnatal growth retardation have been described to date. Objective: The clinical and functional relevance of a novel heterozygous IGF1R mutation identified in a girl with short stature and six relatives was evaluated. Patients: Affected individuals showed birth lengths between 1.40 and 1.82 SD score (SDS) and birth weights between 1.84 and 2.19 SDS. Postnatal growth retardation ranged between 1.51 and 3.93 height SDS. Additional phenotypic findings were variable including microcephaly, clinodactyly, delayed menarche, and diabetes mellitus type 2. Genetic analyses were initiated due to elevated IGF-I levels of the girl. Results: Denaturing HPLC screening and direct DNA sequencing revealed a heterozygous G3464C IGF1R mutation in exon 19 located within a phylogenetically conserved motif of the kinase domain. The resultant mutation of glycine 1125 to alanine (G1125A) did not affect IGF1R protein expression in transiently transfected COS-7 cells and Igf1R deficient mouse fibroblasts (R ) but abrogated IGF-I-induced receptor autophosphorylation and phosphorylation of downstream kinases protein kinase B/Akt and MAPK/ERK. Cotransfection of wild-type and mutant IGF1R resulted in reduced autophosphorylation of 36 10% of wild-type levels, suggesting a partial dominant-negative effect. Mol Endocrinol, February 2010, 24(2):468–470 mend.endojournals.org 469Background: IGF-I receptor (IGFIR) plays an essential role in human intrauterine and postnatal development. Few heterozygous mutations in IGF1R leading to IGF-I resistance and intrauterine and postnatal growth retardation have been described to date. Objective: The clinical and functional relevance of a novel heterozygous IGF1R mutation identified in a girl with short stature and six relatives was evaluated. Patients: Affected individuals showed birth lengths between 1.40 and 1.82 SD score (SDS) and birth weights between 1.84 and 2.19 SDS. Postnatal growth retardation ranged between 1.51 and 3.93 height SDS. Additional phenotypic findings were variable including microcephaly, clinodactyly, delayed menarche, and diabetes mellitus type 2. Genetic analyses were initiated due to elevated IGF-I levels of the girl. Results: Denaturing HPLC screening and direct DNA sequencing revealed a heterozygous G3464C IGF1R mutation in exon 19 located within a phylogenetically conserved motif of the kinase domain. The resultant mutation of glycine 1125 to alanine (G1125A) did not affect IGF1R protein expression in transiently transfected COS-7 cells and Igf1R deficient mouse fibroblasts (R ) but abrogated IGF-I-induced receptor autophosphorylation and phosphorylation of downstream kinases protein kinase B/Akt and MAPK/ERK. Cotransfection of wild-type and mutant IGF1R resulted in reduced autophosphorylation of 36 10% of wild-type levels, suggesting a partial dominant-negative effect. Mol Endocrinol, February 2010, 24(2):468–470 mend.endojournals.org 469

A Heterozygous Mutation of the Insulin-Like Growth Factor-I Receptor Causes Retention of the Nascent Protein in the Endoplasmic Reticulum and Results in Intrauterine and Postnatal Growth Retardation

Background: Mutations in the IGF-I receptor (IGF1R) gene can be responsible for intrauterine and postnatal growth disorders. Objective: Here we report on a novel mutation in the IGF1R gene in a female patient. The aim of our study was to analyze the functional impact of this mutation. Patient: At birth, the girl’s length was 47 cm [ 1.82 SD score (SDS)], and her weight was 2250 g ( 2.26 SDS). Clinical examination revealed microcephaly and retarded cognitive development. She showed no postnatal catch-up growth but had relatively high IGF-I levels ( 1.83 to 2.17 SDS). Results: Denaturing HPLC screening and direct DNA sequencing disclosed a heterozygous missense mutation resulting in an amino acid exchange from valine to glutamic acid at position 599 (V599E-IGF1R). Using various cell systems, we found that the V599E-IGF1R mutant was not tyrosine phosphorylated and had an impaired downstream signaling in the presence of IGF-I. Flow cytometry and live cell confocal laser scanning microscopy revealed a lack of cell surface expression due to an extensive retention of V599E-IGF1R proteins within the endoplasmic reticulum. Conclusion: The V599E-IGF1R mutation interferes with the receptor’s trafficking path, thereby abrogating proreceptor processing and plasma membrane localization. Diminished cell surface receptor density solely expressed from the patient’s wild-type allele is supposed to lead to insufficient IGF-I signaling. We hypothesize that this mechanism results in intrauterine and postnatal growth retardation of the affected patient. The reported retention of the nascent IGF1R in the endoplasmic reticulum presents a novel mechanism of IGF-I resistance. Modulatory Effect of Raloxifene and Estrogen on the Metabolic Action of Growth Hormone in Hypopituitary Women Vita Birzniece, Udo Meinhardt, James Gibney, Gudmundur Johannsson, Robert C. Baxter, Markus J. Seibel, and Ken K. Y. Ho (J Clin Endocrinol Metab, published March 5, 2010, 10.1210/jc.2009-2743)