Increasing knowledge in IGF1R defects: lessons from 35 new patients.

BACKGROUND: The type 1 insulin-like growth factor receptor (IGF1R) is a keystone of fetal growth regulation by mediating the effects of IGF-I and IGF-II. Recently, a cohort of patients carrying an IGF1R defect was described, from which a clinical score was established for diagnosis. We assessed this score in a large cohort of patients with identified IGF1R defects, as no external validation was available. Furthermore, we aimed to develop a functional test to allow the classification of variants of unknown significance (VUS) in vitro. METHODS: DNA was tested for either deletions or single nucleotide variant (SNV) and the phosphorylation of downstream pathways studied after stimulation with IGF-I by western blot analysis of fibroblast of nine patients. RESULTS: We detected 21 IGF1R defects in 35 patients, including 8 deletions and 10 heterozygous, 1 homozygous and 1 compound-heterozygous SNVs. The main clinical characteristics of these patients were being born small for gestational age (90.9%), short stature (88.2%) and microcephaly (74.1%). Feeding difficulties and varying degrees of developmental delay were highly prevalent (54.5%). There were no differences in phenotypes between patients with deletions and SNVs of IGF1R. Functional studies showed that the SNVs tested were associated with decreased AKT phosphorylation. CONCLUSION: We report eight new pathogenic variants of IGF1R and an original case with a homozygous SNV. We found the recently proposed clinical score to be accurate for the diagnosis of IGF1R defects with a sensitivity of 95.2%. We developed an efficient functional test to assess the pathogenicity of SNVs, which is useful, especially for VUS.

Quantification of the methylation at the GNAS locus identifies subtypes of sporadic pseudohypoparathyroidism type Ib.

BACKGROUND: Pseudohypoparathyroidism type Ib (PHP-Ib) is due to epigenetic changes at the imprinted GNAS locus, including loss of methylation at the A/B differentially methylated region (DMR) and sometimes at the XL and AS DMRs and gain of methylation at the NESP DMR. OBJECTIVE: To investigate if quantitative measurement of the methylation at the GNAS DMRs identifies subtypes of PHP-Ib. DESIGN AND METHODS: In 19 patients with PHP-Ib and 7 controls, methylation was characterised at the four GNAS DMRs through combined bisulfite restriction analysis and quantified through cytosine specific real-time PCR in blood lymphocyte DNA. RESULTS: A principal component analysis using the per cent of methylation at seven cytosines of the GNAS locus provided three clusters of subjects (controls n=7, autosomal dominant PHP-Ib with loss of methylation restricted to the A/B DMR n=3, and sporadic PHP-Ib with broad GNAS methylation changes n=16) that matched perfectly the combined bisulfite restriction analysis classification. Furthermore, three sub-clusters of patients with sporadic PHP-Ib, that displayed different patterns of methylation, were identified: incomplete changes at all DMRs compatible with somatic mosaicism (n=5), profound epigenetic changes at all DMRs (n=8), and unmodified methylation at XL in contrast with the other DMRs (n=3). Interestingly, parathyroid hormone concentration at the time of diagnosis correlated with the per cent of methylation at the A/B DMR. CONCLUSION: Quantitative assessment of the methylation in blood lymphocyte DNA is of clinical relevance, allows the diagnosis of PHP-Ib, and identifies subtypes of PHP-Ib. These epigenetic findings suggest mosaicism at least in some patients.

Presenting features and molecular genetics of primary hyperparathyroidism in the paediatric population.

AIM: To describe the presenting features and molecular genetics of primary hyperparathyroidism (PHPT) in the paediatric population. METHODS: Retrospective study of 63 children diagnosed with primary PHPT from 1998 to 2018. RESULTS: Compared to older children, infants were often asymptomatic (54% vs 15%, P = 0.002) with a milder form of PHPT. When symptomatic, children and adolescents mostly presented with non-specific complaints such as asthenia, depression, weight loss, vomiting or abdominal pain. A genetic cause of PHPT was identified in about half of this cohort (52%). The infancy period was almost exclusively associated with mutation in genes involved in the calcium-sensing receptor (CaSR) signalling pathway (i.e. CaSR and AP2S1 genes, 'CaSR group'; 94% of infants with mutations) whereas childhood and adolescence were associated with mutation in genes involved in parathyroid cell proliferation (i.e. MEN1, CDC73, CDKN1B and RET genes, 'cell proliferation group'; 69% of children and adolescents with mutations). Although serum calcium levels did not differ between the two groups (P = 0.785), serum PTH levels and the urinary calcium/creatinine ratio were significantly higher in 'cell proliferation group' patients compared to those in the 'CaSR group' (P = 0.001 and 0.028, respectively). CONCLUSION: Although far less common than in adults, PHPT can develop in children and is associated with significant morbidity. Consequently, this diagnosis should be considered in children with non-specific complaints and lead to monitoring of mineral homeostasis parameters. A genetic cause of PHPT can be identified in about half of these patients.

Should 45,X/46,XY boys with no or mild anomaly of external genitalia be investigated and followed up?

OBJECTIVE: Few studies of patients with a 45,X/46,XY mosaicism have considered those with normal male phenotype. The purpose of this study was to evaluate the clinical outcome of 45,X/46,XY boys born with normal or minor abnormalities of external genitalia, notably in terms of growth and pubertal development. METHODS: Retrospective longitudinal study of 40 patients followed between 1982 and 2017 in France. RESULTS: Twenty patients had a prenatal diagnosis, whereas 20 patients had a postnatal diagnosis, mainly for short stature. Most patients had stunted growth, with abnormal growth spurt during puberty and a mean adult height of 158 ± 7.6 cm, i.e. -2.3 DS with correction for target height. Seventy percent of patients presented Turner-like syndrome features including cardiac (6/23 patients investigated) and renal malformations (3/19 patients investigated). Twenty-two patients had minor abnormalities of external genitalia. One patient developed a testicular embryonic carcinoma, suggesting evidence of partial gonadal dysgenesis. Moreover, puberty occurred spontaneously in 93% of patients but 71% (n = 5) of those evaluated at the end of puberty presented signs of declined Sertoli cell function (low inhibin B levels and increased FSH levels). CONCLUSION: This study emphasizes the need to identify and follow-up 45,X/46,XY patients born with normal male phenotype until adulthood, as they present similar prognosis than those born with severe genital anomalies. Currently, most patients are diagnosed in adulthood with azoospermia, consistent with our observations of decreased testicular function at the end of puberty. Early management of these patients may lead to fertility preservation strategies.

Chromosome 14q32.2 Imprinted Region Disruption as an Alternative Molecular Diagnosis of Silver-Russell Syndrome.

Context: Silver-Russell syndrome (SRS) (mainly secondary to 11p15 molecular disruption) and Temple syndrome (TS) (secondary to 14q32.2 molecular disruption) are imprinting disorders with phenotypic (prenatal and postnatal growth retardation, early feeding difficulties) and molecular overlap. Objective: To describe the clinical overlap between SRS and TS and extensively study the molecular aspects of TS. Patients: We retrospectively collected data on 28 patients with disruption of the 14q32.2 imprinted region, identified in our center, and performed extensive molecular analysis. Results: Seventeen (60.7%) patients showed loss of methylation of the MEG3/DLK1 intergenic differentially methylated region by epimutation. Eight (28.6%) patients had maternal uniparental disomy of chromosome 14 and three (10.7%) had a paternal deletion in 14q32.2. Most patients (72.7%) had a Netchine-Harbison SRS clinical scoring system ≥4/6, and consistent with a clinical diagnosis of SRS. The mean age at puberty onset was 7.2 years in girls and 9.6 years in boys; 37.5% had premature pubarche. The body mass index of all patients increased before pubarche and/or the onset of puberty. Multilocus analysis identified multiple methylation defects in 58.8% of patients. We identified four potentially damaging genetic variants in genes encoding proteins involved in the establishment or maintenance of DNA methylation. Conclusions: Most patients with 14q32.2 disruption fulfill the criteria for a clinical diagnosis of SRS. These clinical data suggest similar management of patients with TS and SRS, with special attention to their young age at the onset of puberty and early increase of body mass index.

GH deficiency with central precocious puberty: a new rare disorder associated with a developmental defect of the hypothalamic-pituitary area.

CONTEXT: GH deficiency (GHD) associated with central precocious puberty (CPP) has been widely reported in cases of arachnoid cyst, septo-optic dysplasia, brain tumors, or after cerebral radiation therapy. However, idiopathic GHD associated with CPP has been reported in only one isolated case. OBJECTIVE: To evaluate the occurrence and clinical features of the association of nonacquired GHD and CPP. DESIGN AND SETTING: This was a retrospective multicenter study. PATIENTS AND METHODS: The study population was identified through a French nationwide multicenter network (about 3000 patients). We reviewed the medical records of all subjects diagnosed with nonacquired GHD and CPP, with or without developmental abnormalities of the hypothalamic-pituitary axis on cerebral magnetic resonance imaging (MRI), and without any known associated anomaly. RESULTS: We identified four patients with either isolated GHD (n=1) or multiple anterior pituitary hormone deficiencies (n=3). Clinical signs of CPP occurred at 6.4 +/- 2.3 years in boys and 7.5 +/- 0.5 years in girls, and GnRH analog therapy was started at 4.2 +/- 1.6 years after the initiation of recombinant human GH treatment. Cerebral MRI demonstrated ectopic neurohypophysis associated with anterior pituitary hypoplasia in three out of the four patients. The morphology and position of the anterior pituitary and neurohypophysis were normal in one patient who displayed a persistence of the craniopharyngeal canal. CONCLUSIONS: CPP is very rare in patients with nonacquired GHD and is mostly associated with developmental defects in the hypothalamic-pituitary area. Whether molecular mechanisms governing development and activation of the hypothalamic-pituitary axis share dependent factors remains to be explored.

NNT mutations: a cause of primary adrenal insufficiency, oxidative stress and extra-adrenal defects.

OBJECTIVE: Nicotinamide nucleotide transhydrogenase (NNT), one of the several genes recently discovered in familial glucocorticoid deficiencies (FGD), is involved in reactive oxygen species detoxification, suggesting that extra-adrenal manifestations may occur, due to the sensitivity to oxidative stress of other organs rich in mitochondria. Here, we sought to identify NNT mutations in a large cohort of patients with primary congenital adrenal insufficiency without molecular etiology and evaluate the degree of adrenal insufficiency and onset of extra-adrenal damages. METHODS: Sanger or massive parallel sequencing of NNT and patient monitoring. RESULTS: Homozygous or compound heterozygous NNT mutations occurred frequently (26%, 13 unrelated families, 18 patients) in our cohort. Seven new mutations were identified: p.Met337Val, p.Ala863Glu, c.3G>A (p.Met1?), p.Arg129*, p.Arg379*, p.Val665Profs*29 and p.Ala704Serfs*19. The most frequent mutation, p.Arg129*, was found recurrently in patients from Algeria. Most patients were diagnosed belatedly (8-18 months) after presenting severe hypoglycemia; others experiencing stress conditions were diagnosed earlier. Five patients also had mineralocorticoid deficiency at onset. One patient had congenital hypothyroidism and two cryptorchidism. In follow-up, we noticed gonadotropic and genitalia impairments (precocious puberty, testicular inclusions, interstitial Leydig cell adenoma, azoospermia), hypothyroidism and hypertrophic cardiomyopathy. Intrafamilial phenotype heterogeneity was also observed. CONCLUSIONS: NNT should be sequenced, not only in FGD, but also in all primary adrenal insufficiencies for which the most frequent etiologies have been ruled out. As NNT is involved in oxidative stress, careful follow-up is needed to evaluate mineralocorticoid biosynthesis extent, and gonadal, heart and thyroid function.