Germline Mutation in KIF1Bß Gene Associated with Loss of Heterozygosity: Usefulness of Next-Generation Sequencing in the Genetic Screening of Patients with Pheochromocytoma.

The genetic approach of pheochromocytomas and paragangliomas has changed in the last two decades. Nowadays, we know that more than 40% of patients have a germline mutation in one of the susceptibility genes identified to date. Our aim is to underline how genetic diagnosis by next-generation sequencing (NGS) can improve the management of patients affected by pheochromocytomas and paragangliomas in our routine diagnostic screening. We reported a case presentation and next-generation sequencing analysis supported by in silico studies and evaluation of mitochondrial status in KIF1Bß tissue. A 46-year-old male affected by a left secreting pheochromocytoma underwent surgery in 2017. After surgery, the normetanephrine levels decreased very slowly and a suspected abdominal lymph node was detected. We found a novel germline KIF1Bß gene mutation, c.4052C > T, p. Pro1351Leu associated with tumor loss of heterozygosity, and resulted likely-pathogenetic by in silico studies. This mutation was also associated with an increased number of mitochondria through the electron microscopy compared with wild-type tissues as suggestive for mitochondria neoformation compensatory to the mitochondrial autophagic figures observed. Our results underline the usefulness of next-generation sequencing in the presence of multiple tumor predisposition genes and how, at the same time, its use may result challenging for the clinicians. To date, performing the genetic analysis according to the latest Consensus Statement is mandatory in patients affected by PHEO/PGL.

Defining the diagnostic effectiveness of genes for inclusion in panels: the experience of two decades of genetic testing for hypertrophic cardiomyopathy at a single center.

PURPOSE: Genetic testing in hypertrophic cardiomyopathy (HCM) has long relied on Sanger sequencing of sarcomeric genes. The advent of next-generation sequencing (NGS) has catalyzed routine testing of additional genes of dubious HCM-causing potential. We used 19 years of genetic testing results to define a reliable set of genes implicated in Mendelian HCM and assess the value of expanded NGS panels. METHODS: We dissected genetic testing results from 1,198 single-center HCM probands and devised a widely applicable score to identify which genes yield effective results in the diagnostic setting. RESULTS: Compared with early panels targeting only fully validated sarcomeric HCM genes, expanded NGS panels allow the prompt recognition of probands with HCM-mimicking diseases. Scoring by "diagnostic effectiveness" highlighted that PLN should also be routinely screened besides historically validated genes for HCM and its mimics. CONCLUSION: The additive value of expanded panels in HCM genetic testing lies in the systematic screening of genes associated with HCM mimics, requiring different patient management. Only variants in a limited set of genes are highly actionable and interpretable in the clinic, suggesting that larger panels offer limited additional sensitivity. A score estimating the relative effectiveness of a given gene's inclusion in diagnostic panels is proposed.

Mutation landscape in patients with myelofibrosis receiving ruxolitinib or hydroxyurea.

Refractoriness to ruxolitinib in patients with myelofibrosis (MF) was associated with clonal evolution; however, whether genetic instability is promoted by ruxolitinib remains unsettled. We evaluated the mutation landscape in 71 MF patients receiving ruxolitinib (n = 46) and hydroxyurea (n = 25) and correlated with response. A spleen volume response (SVR) was obtained in 57% and 12%, respectively. Highly heterogenous patterns of mutation acquisition/loss and/or changes of variant allele frequency (VAF) were observed in the 2 patient groups without remarkable differences. In patients receiving ruxolitinib, driver mutation type and high-molecular risk profile (HMR) at baseline did not impact on response rate, while HMR and sole ASXL1 mutations predicted for SVR loss at 3 years. In patients with SVR, a decrease of ≥ 20% of JAK2V617F VAF predicted for SVR duration. VAF increase of non-driver mutations and clonal progression at follow-up correlated with SVR loss and treatment discontinuation, and clonal progression also predicted for shorter survival. These data indicate that (i) ruxolitinib does not appreciably promote clonal evolution compared with hydroxyurea, (ii) VAF increase of pre-existing and/or (ii) acquisition of new mutations while on treatment correlated with higher rate of discontinuation and/or death, and (iv) reduction of JAK2V617F VAF associated with SVR duration.

Fundus phenotype in retinitis pigmentosa associated with EYS mutations.

PURPOSE: to report phenotypic and genotypic features in a group of autosomal recessive retinitis pigmentosa (arRP) patients associated with EYS mutations. METHODS: we retrospectively reviewed the clinical records and the molecular genetic data of arRP patients carrying mutations in the EYS gene. All the patients underwent a comprehensive opthalmological examination. Additional investigation included color fundus photography, fundus autofluorescence, Goldmann visual field, OCT scans and full-field standard electroretinography. RESULTS: we studied 10 RP patients (20 eyes) characterized by mutations in the EYS gene. Thirteen different sequence variants in the EYS gene were identified. In total, nine mutations found in our series had not previously been reported in the literature. All patients in our series complained of typical RP symptoms at the onset of the disease, namely night blindness and progressive constriction of the visual field. Visual acuity ranged from light perception to 20/20. Relevant findings reported in our series are Interdigitation-zone (IZ band) involvement, present even in the milder phenotypes and an estimated prevalence of 6.2% of arRP associated with EYS mutations. CONCLUSIONS: we reported the mutation spectrum of a group of EYS-related RP patients including nine novel mutations and the associated clinical phenotypes. Our series is the largest group of EYS-related arRP patients in the Italian population.

Validation of a method for noninvasive prenatal testing for fetal aneuploidies risk and considerations for its introduction in the Public Health System.

OBJECTIVE: The aim of this study was to validate noninvasive prenatal testing (NIPT) for fetal aneuploidies by whole-genome massively parallel sequencing (MPS). METHODS: MPS was performed on cell-free DNA (cfDNA) isolated from maternal plasma in two groups: a first set of 186 euploid samples and a second set of 195 samples enriched of aneuploid cases (n = 69); digital PCR for fetal fraction (FF) assessment was performed on 178/381 samples. Cases with <10 × 106 reads (n = 54) were excluded for downstream data analysis. Follow-up data (invasive testing results or neonatal information) were available for all samples. Performances in terms of specificity/sensitivity and Z-score distributions were evaluated. RESULTS: All positive samples for trisomy 21 (T21) (n = 43), trisomy 18 (T18) (n = 6) and trisomy 13 (T13) (n = 7) were correctly identified (sensitivity: 99.9%); 5 false positive results were reported: 3 for T21 (specificity = 98.9%) and 2 for T13 (specificity = 99.4%). Besides FF, total cfDNA concentration seems another important parameter for MPS, since it influences the number of reads. CONCLUSIONS: The overall test accuracy allowed us introducing NIPT for T21, T18 and T13 as a clinical service for pregnant women after 10 + 4 weeks of gestation. Sex chromosome aneuploidy assessment needs further validation due to the limited number of aneuploid cases in this study.

A Systematic Assessment of Accuracy in Detecting Somatic Mosaic Variants by Deep Amplicon Sequencing: Application to NF2 Gene.

The accurate detection of low-allelic variants is still challenging, particularly for the identification of somatic mosaicism, where matched control sample is not available. High throughput sequencing, by the simultaneous and independent analysis of thousands of different DNA fragments, might overcome many of the limits of traditional methods, greatly increasing the sensitivity. However, it is necessary to take into account the high number of false positives that may arise due to the lack of matched control samples. Here, we applied deep amplicon sequencing to the analysis of samples with known genotype and variant allele fraction (VAF) followed by a tailored statistical analysis. This method allowed to define a minimum value of VAF for detecting mosaic variants with high accuracy. Then, we exploited the estimated VAF to select candidate alterations in NF2 gene in 34 samples with unknown genotype (30 blood and 4 tumor DNAs), demonstrating the suitability of our method. The strategy we propose optimizes the use of deep amplicon sequencing for the identification of low abundance variants. Moreover, our method can be applied to different high throughput sequencing approaches to estimate the background noise and define the accuracy of the experimental design.

Clinical and genetic study of a family with a paternally inherited 15q11-q13 duplication.

Interstitial chromosome 15q11-q13 duplications are associated with developmental delay, behavioral problems and additional manifestations, including epilepsy. In most affected individuals the duplicated chromosome is maternally derived, whereas paternal inheritance is more often associated with a normal phenotype. Seizures have not been described in patients with paternal dup 15q11-q13. We describe a family with five individuals in three generations with a paternally-inherited 15q11-q13 duplication, four of whom exhibited abnormal phenotypic characteristics, including seizures. The 18-year-old female proband presented with moderate intellectual disability, obesity, and epilepsy. Her brother manifested learning disability and behavioral problems. They both inherited the 15q11-q13 dup from their father who had a normal phenotype. Their paternal uncle and grandfather also had the duplication and were reported to have had seizures. Array-CGH and MLPA analyses showed that the duplication included the TUBGCP5, CYFIP1, MKRN3, MAGEL2, NDN, SNRPN, UBE3A, ATP10A, GABRB3, GABRA5, GABRG3, and OCA2 genes. This report provides evidence for intrafamilial phenotypic variability of paternal dup 15q11-q13, ranging from normal to intellectual disability and seizures, and potentially expanding the phenotype of paternal 15q11-q13 interstitial duplications.

Growth hormone therapy-related hyperglycaemia in a boy with renal cystic hypodysplasia and a new mutation of the HNF1 beta gene.

We provide a molecular and pathophysiological characterization of an 11-year-old male patient, with a diagnosis of renal hypodysplasia, cysts and chronic renal failure. Although previously normoglycaemic and with a negative familial history for diabetes mellitus, he developed fasting hyperglycaemia within 12 months of the start of treatment with recombinant human growth hormone (rhGH). Direct sequencing of the HNF1 beta gene revealed a de novo heterozygous mutation in exon 2, c.535delC [Pro118LeuX7]+[=]. The appearance of fasting hyperglycaemia following rhGH treatment in children with renal cystic hypodysplasia suggests that investigation of the HNF1 beta gene is warranted, even when familial history is negative for diabetes. This is particularly important in regard to genetic counselling.