Eduardo Fernández-Rebollo

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.

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.

Coexistence of two different pseudohypoparathyroidism subtypes (Ia and Ib) in the same kindred with independent Gsα coding mutations and GNAS imprinting defects.

Background Pseudohypoparathyroidism (PHP) defines a rare group of disorders whose common feature is resistance to the parathyroid hormone. Patients with PHP-Ia display additional hormone resistance, Albright hereditary osteodystrophy (AHO) and reduced Gsα activity in easily accessible cells. This form of PHP is associated with heterozygous inactivating mutations in Gsα-coding exons of GNAS, an imprinted gene locus on chromosome 20q13.3. Patients with PHP-Ib typically have isolated parathyroid hormone resistance, lack AHO features and demonstrate normal erythrocyte Gsα activity. Instead of coding Gsα mutations, patients with PHP-Ib display imprinting defects of GNAS, caused, at least in some cases, by genetic mutations within or nearby this gene. Patients Two unrelated PHP families, each of which includes at least one patient with a Gsα coding mutation and another with GNAS loss of imprinting, are reported here. Results One of the patients with GNAS imprinting defects has paternal uniparental isodisomy of chromosome 20q, explaining the observed imprinting abnormalities. The identified Gsα coding mutations include a tetranucleotide deletion in exon 7, which is frequently found in PHP-Ia, and a novel single nucleotide change at the acceptor splice junction of intron 11. Conclusions These molecular data reveal an interesting mixture, in the same family, of both genetic and epigenetic mutations of the same gene.

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.

Exclusion of the GNAS locus in PHP-Ib patients with broad GNAS methylation changes: evidence for an autosomal recessive form of PHP-Ib?

Most patients with autosomal dominant pseudohypoparathyroidism type Ib (AD‐PHP‐Ib) carry maternally inherited microdeletions upstream of GNAS that are associated with loss of methylation restricted to GNAS exon A/B. Only few AD‐PHP‐Ib patients carry microdeletions within GNAS that are associated with loss of all maternal methylation imprints. These epigenetic changes are often indistinguishable from those observed in patients affected by an apparently sporadic PHP‐Ib form that has not yet been defined genetically. We have now investigated six female patients affected by PHP‐Ib (four unrelated and two sisters) with complete or almost complete loss of GNAS methylation, whose healthy children (11 in total) showed no epigenetic changes at this locus. Analysis of several microsatellite markers throughout the 20q13 region made it unlikely that PHP‐Ib is caused in these patients by large deletions involving GNAS or by paternal uniparental isodisomy or heterodisomy of chromosome 20 (patUPD20). Microsatellite and single‐nucleotide variation (SNV) data revealed that the two affected sisters share their maternally inherited GNAS alleles with unaffected relatives that lack evidence for abnormal GNAS methylation, thus excluding linkage to this locus. Consistent with these findings, healthy children of two unrelated sporadic PHP‐Ib patients had inherited different maternal GNAS alleles, also arguing against linkage to this locus. Based on our data, it appears plausible that some forms of PHP‐Ib are caused by homozygous or compound heterozygous mutation(s) in an unknown gene involved in establishing or maintaining GNAS methylation.

Loss of XLαs (extra-large αs) imprinting results in early postnatal hypoglycemia and lethality in a mouse model of pseudohypoparathyroidism Ib.

Maternal deletion of the NESP55 differentially methylated region (DMR) (delNESP55/ASdel3-4m, delNASm) from the GNAS locus in humans causes autosomal dominant pseudohypoparathyroidism type Ib (AD-PHP-IbdelNASm), a disorder of proximal tubular parathyroid hormone (PTH) resistance associated with loss of maternal GNAS methylation imprints. Mice carrying a similar, maternally inherited deletion of the Nesp55 DMR (ΔNesp55m) replicate these Gnas epigenetic abnormalities and show evidence for PTH resistance, yet these mice demonstrate 100% mortality during the early postnatal period. We investigated whether the loss of extralarge αs (XLαs) imprinting and the resultant biallelic expression of XLαs are responsible for the early postnatal lethality in ΔNesp55m mice. First, we found that ΔNesp55m mice are hypoglycemic and have reduced stomach-to-body weight ratio. We then generated mice having the same epigenetic abnormalities as the ΔNesp55m mice but with normalized XLαs expression due to the paternal disruption of the exon giving rise to this Gnas product. These mice (ΔNesp55m/Gnasxlm+/p−) showed nearly 100% survival up to postnatal day 10, and a substantial number of them lived to adulthood. The hypoglycemia and reduced stomach-to-body weight ratio observed in 2-d-old ΔNesp55m mice were rescued in the ΔNesp55m/Gnasxlm+/p− mice. Surviving double-mutant animals had significantly reduced Gαs mRNA levels and showed hypocalcemia, hyperphosphatemia, and elevated PTH levels, thus providing a viable model of human AD-PHP-Ib. Our findings show that the hypoglycemia and early postnatal lethality caused by the maternal deletion of the Nesp55 DMR result from biallelic XLαs expression. The double-mutant mice will help elucidate the pathophysiological mechanisms underlying AD-PHP-Ib.

The majority of cases of neonatal diabetes in Spain can be explained by known genetic abnormalities

Background  Neonatal diabetes is a rare disease characterized by hyperglycaemia within the first 3 months of life and requiring insulin treatment; it can either be transient (TNDM) or permanent (PNDM). Alterations at band 6q24 and heterozygous activating mutations in KCNJ11, the gene encoding the pore‐forming subunit of the KATP channel, can cause neonatal diabetes.

Haploinsufficiency at GCK gene is not a frequent event in MODY2 patients.

Objective  The aim of this study was to characterize glucokinase (GCK) alterations in maturity‐onset diabetes of the young 2 (MODY2)‐suspected patients and to investigate their clinical characteristics in relation to the parental origin of the mutation.

New mutation type in pseudohypoparathyroidism type Ia

Context  The GNAS gene encodes the α‐subunit of the stimulatory G proteins, which play a crucial role in intracellular signal transduction of peptide and neurotransmitter receptors. Heterozygous inactivating maternally inherited mutations of GNAS (including translation initiation mutations, amino acid substitutions, nonsense mutations, splice site mutations and small insertions or deletions) lead to a phenotype in which Albright hereditary osteodystrophy is associated with pseudohypoparathyroidism type Ia.