Validation of a next-generation sequencing (NGS) panel to improve the diagnosis of X-linked hypophosphataemia (XLH) and other genetic disorders of renal phosphate wasting.

BACKGROUND: Hypophosphataemic rickets (HR) comprise a clinically and genetically heterogeneous group of conditions, defined by renal-tubular phosphate wasting and consecutive loss of bone mineralisation. X-linked hypophosphataemia (XLH) is the most common form, caused by inactivating dominant mutations in PHEX, a gene encompassing 22 exons located at Xp22.1. XLH is treatable by anti-Fibroblast Growth Factor 23 antibody, while for other forms of HR such as therapy may not be indicated. Therefore, a genetic differentiation of HR is recommended. OBJECTIVE: To develop and validate a next-generation sequencing panel for HR with special focus on PHEX. DESIGN AND METHODS: We designed an AmpliSeq gene panel for the IonTorrent PGM next-generation platform for PHEX and ten other HR-related genes. For validation of PHEX sequencing 50 DNA-samples from XLH-patients, in whom 42 different mutations in PHEX and 1 structural variation have been proven before, were blinded, anonymised and investigated with the NGS panel. In addition, we analyzed one known homozygous DMP1 mutation and two samples of HR-patients, where no pathogenic PHEX mutation had been detected by conventional sequencing. RESULTS: The panel detected all 42 pathogenic missense/nonsense/splice-site/indel PHEX-mutations and in one the known homozygous DMP1 mutation. In the remaining two patients, we revealed a somatic mosaicism of a PHEX mutation in one; as well as two variations in DMP1 and a very rare compound heterozygous variation in ENPP1 in the second patient. CONCLUSIONS: This developed NGS panel is a reliable tool with high sensitivity and specificity for the diagnosis of XLH and related forms of HR.

EMQN best practice guidelines for molecular genetic testing and reporting of 21-hydroxylase deficiency.

Molecular genetic testing for congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD) is offered worldwide and is of importance for differential diagnosis, carrier detection and adequate genetic counseling, particularly for family planning. In 2008 the European Molecular Genetics Quality Network (EMQN) for the first time offered a European-wide external quality assessment scheme for CAH (due to 21-OH deficiency). The interest was great and over the last years at about 60 laboratories from Europe, USA and Australia regularly participated in that scheme. These best practice guidelines were drafted on the basis of the extensive knowledge and experience got from those annually organized CAH-schemes. In order to obtain the widest possible consultation with practicing laboratories the draft was therefore circulated twice by EMQN to all laboratories participating in the EQA-scheme for CAH genotyping and was updated by that input. The present guidelines address quality requirements for diagnostic molecular genetic laboratories, as well as criteria for CYP21A2 genotyping (including carrier-testing and prenatal diagnosis). A key aspect of that article is the use of appropriate methodologies (e.g., sequencing methods, MLPA (multiplex ligation dependent probe amplification), mutation specific assays) and respective limitations and analytical accuracy. Moreover, these guidelines focus on classification of variants, and the interpretation and standardization of the reporting of CYP21A2 genotyping results. In addition, the article provides a comprehensive list of common as well as so far unreported CYP21A2-variants.

A novel stop mutation (p.(Gln22*)) of DAX1 (NR0B1) results in late-onset X-linked adrenal hypoplasia congenita.

DAX1 (NR0B1) is an orphan nuclear receptor, which plays an important role in development and function of the adrenal glands and gonads. Mutations in DAX1 cause X-linked adrenal hypoplasia congenita (X-linked AHC), which is characterized by adrenal insufficiency (AI) and hypogonadotropic hypogonadism (HHG). Affected boys present with adrenal failure usually in childhood and, later in life, with delayed puberty. However, patients with a late-onset form of X-linked AHC have also been described in the past years. We report a male patient who presented with symptoms of an adrenal crisis at the age of 38 years and was later diagnosed with HHG. Family history was positive with several male relatives diagnosed with AI and compatible with the assumed X-chromosomal inheritance of the trait. Direct sequencing of DAX1 of the patient revealed a hemizygous cytosine-to-thymine substitution at nucleotide 64 in exon 1, which creates a novel nonsense mutation (p.(Gln22*)). In order to compare the clinical presentation of the patient to that of other patients with X-linked AHC, we searched the electronic database MEDLINE (PubMed) and found reports of nine other cases with delayed onset of X-linked AHC. In certain cases, genotype-phenotype correlation could be assumed. LEARNING POINTS: X-linked AHC is a rare disease characterized by primary AI and hypogonadotropic hypogonadism (HHG). The full-blown clinical picture is seen usually only in males with a typical onset in childhood.Patients with a late-onset form of X-linked AHC have also been described recently. Being aware of this late-onset form might help to reach an early diagnosis and prevent life-threatening adrenal crises.Adult men with primary AI of unknown etiology should be investigated for HHG. Detecting a DAX1 mutation may confirm the clinical diagnosis of late-onset X-linked AHC.In relatives of patients with genetically confirmed X-linked AHC, targeted mutation analysis may help to identify family members at risk and asymptomatic carriers, and discuss conscious family planning.

Frequency of AIP gene mutations in young patients with acromegaly: a registry-based study.

CONTEXT: Familial and sporadic GH-secreting pituitary adenomas are associated with mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene. Patients with an AIP mutation (AIPmut) tend to have more aggressive tumors occurring at a younger age. OBJECTIVE: The objective of the study was to investigate the frequency of AIPmut in patients diagnosed at 30 years of age or younger. DESIGN: The German Acromegaly Registry database (1795 patients in 58 centers) was screened for patients diagnosed with acromegaly at 30 years of age or younger (329 patients). Sixteen centers participated and 91 patients consented to AIPmut analysis. INTERVENTION: DNA was analyzed by direct sequencing and multiplex ligation dependent probe amplification Main outcome Measures: The number of patients with AIPmut was measured. RESULTS: Five patients had either a mutation (c.490C>T, c.844C>T, and c.911G>A, three males) or gross deletions of exons 1 and 2 of the AIP gene (n = 2, one female). The overall frequency of an AIPmut was 5.5%, and 2.3% or 2.4% in patients with an apparently sporadic adenoma or macroadenoma, respectively. By contrast, three of four patients (75%) with a positive family history were tested positive for an AIPmut. Except for a positive family history, there were no significant differences between patients with and without an AIPmut. CONCLUSIONS: The frequency of AIPmut in this registry-based cohort of young patients with acromegaly is lower than previously reported. Patients with a positive family history should be tested for an AIPmut, whereas young patients without an apparent family history should be screened, depending on the individual cost to benefit ratio.

The association of genetic markers for type 2 diabetes with prediabetic status - cross-sectional data of a diabetes prevention trial.

OBJECTIVE: To investigate the association of risk alleles for type 2 diabetes with prediabetes accounting for age, anthropometry, inflammatory markers and lifestyle habits. DESIGN: Cross-sectional study of 129 men and 157 women of medium-sized companies in northern Germany in the Delay of Impaired Glucose Tolerance by a Healthy Lifestyle Trial (DELIGHT). METHODS: Besides established risk factors, 41 single nucleotide polymorphisms (SNPs) that have previously been found to be associated with type 2 diabetes were analyzed. As a nonparametric test a random forest approach was used that allows processing of a large number of predictors. Variables with the highest impact were entered into a multivariate logistic regression model to estimate their association with prediabetes. RESULTS: Individuals with prediabetes were characterized by a slightly, but significantly higher number of type 2 diabetes risk alleles (42.5±4.1 vs. 41.3±4.1, p = 0.013). After adjustment for age and waist circumference 6 SNPs with the highest impact in the random forest analysis were associated with risk for prediabetes in a logistic regression model. At least 5 of these SNPs were positively related to prediabetic status (odds ratio for prediabetes 1.57 per allele (Cl 1.21-2.10, p = 0.001)). CONCLUSIONS: This explorative analysis of data of DELIGHT demonstrates that at least 6 out of 41 genetic variants characteristic of individuals with type 2 diabetes may also be associated with prediabetes. Accumulation of these risk alleles may markedly increase the risk for prediabetes. However, prospective studies are required to corroborate these findings and to demonstrate the predictive value of these genetic variants for the risk to develop prediabetes.

Characteristic genomic imbalances in pediatric pheochromocytoma.

Pheochromocytoma (PCC) in children is rare, genetically not well described, and often related to a poor prognosis. We detected genomic imbalances in all 14 tumors from children analyzed by comparative genomic hybridization. A combinatorial loss of chromatin from 3p and 11p was a common feature in 10 of 14 (72%) patients, which was a result of either a loss of a total chromosome 3 and a total chromosome 11 in 6 of 10 patients, or confined deletions of their p arms in 4 of 10 patients. All patients exhibiting a loss of 3p and 11p carried VHL mutations. The VHL mutations were constitutive in 9 cases and somatic and restricted to tumor DNA in the remaining tumor. On the other hand, VHL mutations were absent in 4 patients, 2 who had other familial syndromes (NF1, SDHD) and 2 with unknown etiology. Our data show that the pattern of imbalances in the tumor DNA of PCC patients strongly correlated with an underlying familial VHL mutation. Furthermore, we show that true sporadic PCC is rare in childhood. Thus, children with PCC should be checked for a related predisposing gene. This would also identify familial syndrome patients requiring long-term monitoring for other syndrome-related malignancies.

Coincidence of multiple endocrine neoplasia types 1 and 2: mutations in the RET protooncogene and MEN1 tumor suppressor gene in a family presenting with recurrent primary hyperparathyroidism.

CONTEXT: Primary hyperparathyroidism (HPT) presents as a part of inherited syndromes such as multiple endocrine neoplasia (MEN) types 1 and 2. In patients with MEN1, parathyroid hyperplasia or multiple adenomas occur in approximately 90-95%. MEN2A-related HPT is characterized by a mild hypercalcemia, which is mostly asymptomatic. OBJECTIVE: Here we present a family with coexistence of MEN1 gene mutation and RET mutation. RESULTS: Six family members carrying MEN1 gene mutation IVS5 + 1G>A only, one family member with RET mutation Y791F, and three family members with both MEN1 gene and RET mutation were studied. The key to diagnosis was recurrent HPT in a young male carrying RET mutation Y791F, a mutation not likely to give rise to recurrent HPT. CONCLUSION: MEN1 gene mutation and RET codon 791 mutation in the same patient did not affect the typical phenotype of MEN1 or MEN2, and also the course of diseases seems to be unchanged. The reason may be that both mutations, although contributing to tumor pathogenesis, do not interact and induce a worsening of the cancer syndromes.