Intza Garin

Recessive mutations in the INS gene result in neonatal diabetes through reduced insulin biosynthesis

Heterozygous coding mutations in the INS gene that encodes preproinsulin were recently shown to be an important cause of permanent neonatal diabetes. These dominantly acting mutations prevent normal folding of proinsulin, which leads to beta-cell death through endoplasmic reticulum stress and apoptosis. We now report 10 different recessive INS mutations in 15 probands with neonatal diabetes. Functional studies showed that recessive mutations resulted in diabetes because of decreased insulin biosynthesis through distinct mechanisms, including gene deletion, lack of the translation initiation signal, and altered mRNA stability because of the disruption of a polyadenylation signal. A subset of recessive mutations caused abnormal INS transcription, including the deletion of the C1 and E1 cis regulatory elements, or three different single base-pair substitutions in a CC dinucleotide sequence located between E1 and A1 elements. In keeping with an earlier and more severe beta-cell defect, patients with recessive INS mutations had a lower birth weight (−3.2 SD score vs. −2.0 SD score) and were diagnosed earlier (median 1 week vs. 10 weeks) compared to those with dominant INS mutations. Mutations in the insulin gene can therefore result in neonatal diabetes as a result of two contrasting pathogenic mechanisms. Moreover, the recessively inherited mutations provide a genetic demonstration of the essential role of multiple sequence elements that regulate the biosynthesis of insulin in man.

Genome-Wide Allelic Methylation Analysis Reveals Disease-Specific Susceptibility to Multiple Methylation Defects in Imprinting Syndromes

Genomic imprinting is the parent‐of‐origin‐specific allelic transcriptional silencing observed in mammals, which is governed by DNA methylation established in the gametes and maintained throughout the development. The frequency and extent of epimutations associated with the nine reported imprinting syndromes varies because it is evident that aberrant preimplantation maintenance of imprinted differentially methylated regions (DMRs) may affect multiple loci. Using a custom Illumina GoldenGate array targeting 27 imprinted DMRs, we profiled allelic methylation in 65 imprinting defect patients. We identify multilocus hypomethylation in numerous Beckwith–Wiedemann syndrome, transient neonatal diabetes mellitus (TNDM), and pseudohypoparathyroidism 1B patients, and an individual with Silver–Russell syndrome. Our data reveal a broad range of epimutations exist in certain imprinting syndromes, with the exception of Prader–Willi syndrome and Angelman syndrome patients that are associated with solitary SNRPN‐DMR defects. A mutation analysis identified a 1 bp deletion in the ZFP57 gene in a TNDM patient with methylation defects at multiple maternal DMRs. In addition, we observe missense variants in ZFP57, NLRP2, and NLRP7 that are not consistent with maternal effect and aberrant establishment or methylation maintenance, and are likely benign. This work illustrates that further extensive molecular characterization of these rare patients is required to fully understand the mechanism underlying the etiology of imprint establishment and maintenance.

New mechanisms involved in paternal 20q disomy associated with pseudohypoparathyroidism

PURPOSE Type I pseudohypoparathyroidism (PHP-I) can be subclassified into Ia and Ib, depending on the presence or absence of Albrights hereditary osteodystrophys phenotype, diminished α-subunit of the stimulatory G protein (G(s)α) activity and multihormonal resistance. Whereas PHP-Ia is mainly associated with heterozygous inactivating mutations in G(s)α-coding exons of GNAS, PHP-Ib is caused by imprinting defects of GNAS. To date, just one patient with PHP and complete paternal uniparental disomy (UPD) has been described. We sought to identify the underlining molecular defect in twenty patients with parathyroid hormone resistance, hypocalcemia and hyperphosphatemia, and abnormal methylation pattern at GNAS locus. METHODS Microsatellite typing and comparative genome hybridization were performed for proband and parents. RESULTS We describe four patients with partial paternal UPD of chromosome 20 involving pat20qUPD in one case, from 20q13.13-qter in two cases, and pat20p heterodisomy plus interstitial 20q isodisomy in one patient. CONCLUSIONS These observations demonstrate that mitotic recombination of chromosome 20 can also give rise to UPD and PHP, a situation similar to other imprinting disorders, such as Beckwith-Wiedemann syndrome or neonatal diabetes.

Gsα activity is reduced in erythrocyte membranes of patients with psedohypoparathyroidism due to epigenetic alterations at the GNAS locus.

In pseudohypoparathyroidism (PHP), PTH resistance results from impairment of signal transduction of G protein–coupled receptors caused by a deficiency of the Gsα‐cAMP signaling cascade due to diminished Gsα activity in maternally imprinted tissues. In PHP‐Ia, inactivating mutations of the GNAS gene lead to haploinsufficiency in some tissues with biallelic expression, so in addition to PHP, Albrights hereditary osteodystrophy (AHO) is also present. In PHP‐Ib, caused by methylation defects at the GNAS locus, diminished Gsα activity was thought to be limited to maternally imprinted tissues, such as the renal proximal tubule and the thyroid, leading to a lack of AHO. Recently, we demonstrated methylation defects in patients with AHO signs, indicating a connection between epigenetic changes and AHO. Our objective was to determine Gsα activity in erythrocyte membranes in patients with epigenetic defects at the GNAS locus compared to normal controls and patients with inactivating GNAS mutations. Gsα activity and expression, mutation of the GNAS locus, and methylation status were studied in patients with PHP and mild signs of AHO (PHP‐Ia: 12; PHP‐Ib: 17, of which 8 had some features of AHO). Then, we statistically compared the Gsα activity of the different PHP subtypes. Patients with methylation defects at the GNAS locus show a significant decrease in erythrocyte Gsα activity compared to normal controls (PHP‐Ib versus controls, p < .001). This was significantly lower in patients with AHO signs (PHP‐Ib + mild‐AHO versus PHP‐Ib, p < .05). Our research shows that PHP‐Ia and PHP‐Ib classification is not only overlapped genetically, as reported, but also in terms of Gsα activity. Reduced expression of GNAS due to methylation defects could downregulate Gsα activity in other tissues beyond those described and could also be causative of AHO.

Detection of Hypomethylation Syndrome among Patients with Epigenetic Alterations at the GNAS Locus

CONTEXT Genomic imprinting is the modification of the genome so that genes from only one (rather than two) of the parental alleles are expressed. The mechanism underlying imprinting is epigenetic, occurring via changes in DNA methylation and histone modifications rather than through alterations in the DNA sequence. To date, nine different imprinting disorders have been clinically and genetically identified and a considerable research effort has been focused on determining the cause of the corresponding methylation defects. OBJECTIVE Our objective was to identify multilocus imprinting defects and characterize any mutations in trans-acting genes in patients with pseudohypoparathyroidism (PHP) caused by epigenetic alterations at GNAS locus. DESIGN We have investigated multilocus imprinting defects in 22 PHP patients with aberrant methylation at the GNAS locus not due to previously described deletions or to paternal uniparental disomy (UPD) of chromosome 20. RESULTS We found that, in contrast to what has been described in growth disorders, multilocus hypomethylation is an uncommon event in PHP patients. We were also unable to identify any genetic alteration causative of the epigenetic defects in the currently known methylation regulatory genes. CONCLUSION Our work suggests that a trans-acting gene regulating the establishment or maintenance of imprinting at GNAS locus, if it exists, should be specific to PHP cases caused by epigenetic defects at GNAS.

European guidance for the molecular diagnosis of pseudohypoparathyroidism not caused by point genetic variants at GNAS: an EQA study.

Pseudohypoparathyroidism is a rare endocrine disorder that can be caused by genetic (mainly maternally inherited inactivating point mutations, although intragenic and gross deletions have rarely been reported) or epigenetic alterations at GNAS locus. Clinical and molecular characterization of this disease is not that easy because of phenotypic, biochemical and molecular overlapping features between both subtypes of the disease. The European Consortium for the study of PHP (EuroPHP) designed the present work with the intention of generating the standards of diagnostic clinical molecular (epi)genetic testing in PHP patients. With this aim, DNA samples of eight independent PHP patients carrying GNAS genetic and/or epigenetic defects (three patients with GNAS deletions, two with 20q uniparental disomy and three with a methylation defect of unknown origin) without GNAS point mutations were anonymized and sent to the five participant laboratories for their routine genetic analysis (methylation-specific (MS)-MLPA, pyrosequencing and EpiTYPER) and interpretations. All laboratories were able to detect methylation defects and, after the data analysis, the Consortium compared the results to define technical advantages and disadvantages of different techniques. To conclude, we propose as first-level investigation in PHP patients copy number and methylation analysis by MS-MLPA. Then, in patients with partial methylation defect, the result should be confirmed by single CpG bisulphite-based methods (ie pyrosequencing), whereas in case of a complete methylation defect without detectable deletion, microsatellites or SNP genotyping should be performed to exclude uniparental disomy 20.

Multilocus methylation defects in imprinting disorders

Abstract Mammals inherit two complete sets of chromosomes, one from the father and one from the mother, and most autosomal genes are expressed from both maternal and paternal alleles. In imprinted genes, the expression of the allele is dependent upon its parental origin. Appropriate regulation of imprinted genes is important for normal development, with several genetic diseases associated with imprinting defects. A common process for controlling gene activity is methylation. The first steps for understanding the functions of DNA methylation and its regulation in mammalian development have led us to identify common (epi)genetic mechanisms involved in the eight human congenital imprinting disorders.

Genome-wide DNA methylation analysis of pseudohypoparathyroidism patients with GNAS imprinting defects

BackgroundPseudohypoparathyroidism (PHP) is caused by (epi)genetic defects in the imprinted GNAS cluster. Current classification of PHP patients is hampered by clinical and molecular diagnostic overlaps. The European Consortium for the study of PHP designed a genome-wide methylation study to improve molecular diagnosis.MethodsThe HumanMethylation 450K BeadChip was used to analyze genome-wide methylation in 24 PHP patients with parathyroid hormone resistance and 20 age- and gender-matched controls. Patients were previously diagnosed with GNAS-specific differentially methylated regions (DMRs) and include 6 patients with known STX16 deletion (PHPΔstx16) and 18 without deletion (PHPneg).ResultsThe array demonstrated that PHP patients do not show DNA methylation differences at the whole-genome level. Unsupervised clustering of GNAS-specific DMRs divides PHPΔstx16 versus PHPneg patients. Interestingly, in contrast to the notion that all PHP patients share methylation defects in the A/B DMR while only PHPΔstx16 patients have normal NESP, GNAS-AS1 and XL methylation, we found a novel DMR (named GNAS-AS2) in the GNAS-AS1 region that is significantly different in both PHPΔstx16 and PHPneg, as validated by Sequenom EpiTYPER in a larger PHP cohort. The analysis of 58 DMRs revealed that 8/18 PHPneg and 1/6 PHPΔstx16 patients have multi-locus methylation defects. Validation was performed for FANCC and SVOPL DMRs.ConclusionsThis is the first genome-wide methylation study for PHP patients that confirmed that GNAS is the most significant DMR, and the presence of STX16 deletion divides PHP patients in two groups. Moreover, a novel GNAS-AS2 DMR affects all PHP patients, and PHP patients seem sensitive to multi-locus methylation defects.

Brachydactyly E: isolated or as a feature of a syndrome

Brachydactyly (BD) refers to the shortening of the hands, feet or both. There are different types of BD; among them, type E (BDE) is a rare type that can present as an isolated feature or as part of more complex syndromes, such as: pseudohypopthyroidism (PHP), hypertension with BD or Bilginturan BD (HTNB), BD with mental retardation (BDMR) or BDE with short stature, PTHLH type. Each syndrome has characteristic patterns of skeletal involvement. However, brachydactyly is not a constant feature and shows a high degree of phenotypic variability. In addition, there are other syndromes that can be misdiagnosed as brachydactyly type E, some of which will also be discussed. The objective of this review is to describe some of the syndromes in which BDE is present, focusing on clinical, biochemical and genetic characteristics as features of differential diagnoses, with the aim of establishing an algorithm for their differential diagnosis. As in our experience many of these patients are recruited at Endocrinology and/or Pediatric Endocrinology Services due to their short stature, we have focused the algorithm in those steps that could mainly help these professionals.

Permanent Neonatal Diabetes Caused by Creation of an Ectopic Splice Site within the INS Gene

Background The aim of this study was to characterize the genetic etiology in a patient who presented with permanent neonatal diabetes at 2 months of age. Methodology/Principal Findings Regulatory elements and coding exons 2 and 3 of the INS gene were amplified and sequenced from genomic and complementary DNA samples. A novel heterozygous INS mutation within the terminal intron of the gene was identified in the proband and her affected father. This mutation introduces an ectopic splice site leading to the insertion of 29 nucleotides from the intronic sequence into the mature mRNA, which results in a longer and abnormal transcript. Conclusions/Significance This study highlights the importance of routinely sequencing the exon-intron boundaries and the need to carry out additional studies to confirm the pathogenicity of any identified intronic genetic variants.