Recessive osteogenesis imperfecta caused by LEPRE1 mutations: clinical documentation and identification of the splice form responsible for prolyl 3-hydroxylation

Abstract: Background: Recessive forms of osteogenesis imperfecta (OI) may be caused by mutations in LEPRE1, encoding prolyl 3-hydroxylase-1 (P3H1) or in CRTAP, encoding cartilage associated protein. These proteins constitute together with cyclophilin B (CyPB) the prolyl 3-hydroxylation complex that hydroxylates the Pro986 residue in both the type I and type II collagen alpha 1-chains. Methods: We screened LEPRE1, CRTAP and PPIB (encoding CyPB) in a European/Middle Eastern cohort of 20 lethal/severe OI patients without a type I collagen mutation. Results: Four novel homozygous and compound heterozygous mutations were identified in LEPRE1 in four probands. Two probands survived the neonatal period, including one patient who is the eldest reported patient (17(7/12) years) so far with P3H1 deficiency. At birth, clinical and radiologic features were hardly distinguishable from those in patients with autosomal dominant (AD) severe/lethal OI. Follow-up data reveal that the longer lived patients develop a severe osteochondrodysplasia that overlaps with, but has some distinctive features from, AD OI. A new splice site mutation was identified in two of the four probands, affecting only one of three LEPRE1 mRNA splice forms, detected in this study. The affected splice form encodes a 736 amino acid (AA) protein with a "KDEL'' endoplasmic reticulum retention signal. While western blotting and immunocytochemical analysis of fibroblast cultures revealed absence of this P3H1 protein, mass spectrometry and SDS-urea-PAGE data showed severe reduction of alpha 1(I) Pro986 3-hydroxylation and overmodification of type I (pro) collagen chains in skin fibroblasts of the patients. Conclusion: These findings suggest that the 3-hydroxylation function of P3H1 is restricted to the 736AA splice form

Long-term acquired everolimus resistance in pancreatic neuroendocrine tumours can be overcome with novel PI3K-AKT-mTOR inhibitors

Background:The mTOR-inhibitor everolimus improves progression-free survival in advanced pancreatic neuroendocrine tumours (PNETs). However, adaptive resistance to mTOR inhibition is described.Methods:QGP-1 and BON-1, two human PNET cell lines, were cultured with increasing concentrations of everolimus up to 22 weeks to reach a dose of 1 μM everolimus, respectively, 1000-fold and 250-fold initial IC 50. Using total DNA content as a measure of cell number, growth inhibitory dose-response curves of everolimus were determined at the end of resistance induction and over time after everolimus withdrawal. Response to ATP-competitive mTOR inhibitors OSI-027 and AZD2014, and PI3K-mTOR inhibitor NVP-BEZ235 was studied. Gene expression of 10 PI3K-Akt-mTOR pathway-related genes was evaluated using quantitative real-time PCR (RT-qPCR).Results:Long-term everolimus-treated BON-1/R and QGP-1/R showed a significant reduction in everolimus sensitivity. During a drug holiday, gradual return of everolimus sensitivity in BON-1/R and QGP-1/R led to complete reversal of resistance after 10-12 weeks. Treatment with AZD2014, OSI-027 and NVP-BEZ235 had an inhibitory effect on cell proliferation in both sensitive and resistant cell lines. Gene expression in BON-1/R revealed downregulation of MTOR, RICTOR, RAPTOR, AKT and HIF1A, whereas 4EBP1 was upregulated. In QGP-1/R, a downregulation of HIF1A and an upregulation of ERK2 were observed.Conclusions:Long-term everolimus resistance was induced in two human PNET cell lines. Novel PI3K-AKT-mTOR pathway-targeting drugs can overcome everolimus resistance. Differential gene expression profiles suggest different mechanisms of everolimus resistance in BON-1 and QGP-1

Loss-of-function mutations in the X-linked biglycan gene cause a severe syndromic form of thoracic aortic aneurysms and dissections.

Thoracic aortic aneurysm and dissection (TAAD) is typically inherited in an autosomal dominant manner, but rare X-linked families have been described. So far, the only known X-linked gene is FLNA, which is associated with the periventricular nodular heterotopia type of Ehlers-Danlos syndrome. However, mutations in this gene explain only a small number of X-linked TAAD families. We performed targeted resequencing of 368 candidate genes in a cohort of 11 molecularly unexplained Marfan probands. Subsequently, Sanger sequencing of BGN in 360 male and 155 female molecularly unexplained TAAD probands was performed. We found five individuals with loss-of-function mutations in BGN encoding the small leucine-rich proteoglycan biglycan. The clinical phenotype is characterized by early-onset aortic aneurysm and dissection. Other recurrent findings include hypertelorism, pectus deformity, joint hypermobility, contractures, and mild skeletal dysplasia. Fluorescent staining revealed an increase in TGF-β signaling, evidenced by an increase in nuclear pSMAD2 in the aortic wall. Our results are in line with those of prior reports demonstrating that Bgn-deficient male BALB/cA mice die from aortic rupture. In conclusion, BGN gene defects in humans cause an X-linked syndromic form of severe TAAD that is associated with preservation of elastic fibers and increased TGF-β signaling.Genet Med 19 4, 386-395

Genotype-Phenotype Correlation in NF1: Evidence for a More Severe Phenotype Associated with Missense Mutations Affecting NF1 Codons 844–848

Neurofibromatosis type 1 (NF1), a common genetic disorder with a birth incidence of 1:2,000–3,000, is characterized by a highly variable clinical presentation. To date, only two clinically relevant intragenic genotype-phenotype correlations have been reported for NF1 missense mutations affecting p.Arg1809 and a single amino acid deletion p.Met922del. Both variants predispose to a distinct mild NF1 phenotype with neither externally visible cutaneous/plexiform neurofibromas nor other tumors. Here, we report 162 individuals (129 unrelated probands and 33 affected relatives) heterozygous for a constitutional missense mutation affecting one of five neighboring NF1 codons—Leu844, Cys845, Ala846, Leu847, and Gly848—located in the cysteine-serine-rich domain (CSRD). Collectively, these recurrent missense mutations affect ∼0.8% of unrelated NF1 mutation-positive probands in the University of Alabama at Birmingham (UAB) cohort. Major superficial plexiform neurofibromas and symptomatic spinal neurofibromas were more prevalent in these individuals compared with classic NF1-affected cohorts (both p < 0.0001). Nearly half of the individuals had symptomatic or asymptomatic optic pathway gliomas and/or skeletal abnormalities. Additionally, variants in this region seem to confer a high predisposition to develop malignancies compared with the general NF1-affected population (p = 0.0061). Our results demonstrate that these NF1 missense mutations, although located outside the GAP-related domain, may be an important risk factor for a severe presentation. A genotype-phenotype correlation at the NF1 region 844–848 exists and will be valuable in the management and genetic counseling of a significant number of individuals

Hereditary angioedema in 2 sisters due to paternal gonadal mosaicism

We report the occurrence of type I hereditary angioedema in 2 sisters with unaffected parents caused by paternal gonadal mosaicism. Gonadal mosaicism is a rather uncommon genetic phenomenon, but nevertheless, correct diagnosis is important because it may affect further family planning

Novel microdeletions on chromosome 14q32.2 suggest a potential role for non-coding RNAs in Kagami-Ogata syndrome

In approximately 20% of individuals with Kagami-Ogata syndrome (KOS14, MIM 608149), characterized by a bell-shaped thorax with coat-hanger configuration of the ribs, joint contractures, abdominal wall defects and polyhydramnios during the pregnancy, the syndrome is caused by a maternal deletion of the imprinted gene cluster in chromosome 14q32.2. Most deletions reported so far included one or both of the differentially methylated regions (DMRs) – DLK1/MEG3 IG-DMR and MEG3-DMR. We present two unrelated families with two affected siblings each, presenting with classical KOS14 due to maternally inherited microdeletions. Interestingly, all four patients have lived through to adulthood, even though mortality rates for patients with KOS14 due to a microdeletion are relatively high. In the first family, none of the DMRs is included in the deletion and the methylation status is identical to that of controls. Deletions that do not encompass the DMRs in this region are thus sufficient to elicit the full KOS14 phenotype. In the second family, a partially overlapping deletion including both DMRs and MEG3 was detected. In summary, we show that patients with KOS14 can live into adulthood, that causal deletions do not have to include the DMRs and that consequently a normal methylation pattern does not exclude KOS14