Novel Germline PHD2 Variant in a Metastatic Pheochromocytoma and Chronic Myeloid Leukemia, but in the Absence of Polycythemia.

Background: Pheochromocytoma (Pheo) and paraganglioma (PGL) are rare tumors, mostly resulting from pathogenic variants of predisposing genes, with a genetic contribution that now stands at around 70%. Germline variants account for approximately 40%, while the remaining 30% is attributable to somatic variants. Objective: This study aimed to describe a new PHD2 (EGLN1) variant in a patient affected by metastatic Pheo and chronic myeloid leukemia (CML) without polycythemia and to emphasize the need to adopt a comprehensive next-generation sequencing (NGS) panel. Methods: Genetic analysis was carried out by NGS. This analysis was initially performed using a panel of genes known for tumor predisposition (EGLN1, EPAS1, FH, KIF1Bß, MAX, NF1, RET, SDHA, SDHAF2, SDHB, SDHC, SDHD, TMEM127, and VHL), followed initially by SNP-CGH array, to exclude the presence of the pathogenic Copy Number Variants (CNVs) and the loss of heterozygosity (LOH) and subsequently by whole exome sequencing (WES) comparative sequence analysis of the DNA extracted from tumor fragments and peripheral blood. Results: We found a novel germline PHD2 (EGLN1) gene variant, c.153G>A, p.W51*, in a patient affected by metastatic Pheo and chronic myeloid leukemia (CML) in the absence of polycythemia. Conclusions: According to the latest guidelines, it is mandatory to perform genetic analysis in all Pheo/PGL cases regardless of phenotype. In patients with metastatic disease and no evidence of polycythemia, we propose testing for PHD2 (EGLN1) gene variants. A possible correlation between PHD2 (EGLN1) pathogenic variants and CML clinical course should be considered.

Variants Disrupting CD40L Transmembrane Domain and Atypical X-Linked Hyper-IgM Syndrome: A Case Report With Leishmaniasis and Review of the Literature.

X-linked hyper-IgM (XHIGM) syndrome is caused by mutations of the CD40LG gene, encoding the CD40L protein. The clinical presentation is characterized by early-onset infections, with profound hypogammaglobulinemia and often elevated IgM, susceptibility to opportunistic infections, such as Pneumocystis jirovecii pneumonia, biliary tract disease due to Cryptosporidium parvum, and malignancy. We report a 41-year-old male presenting with recurrent leishmaniasis, hypogammaglobulinemia, and myopathy. Whole-exome sequencing (WES) identified a missense variant in the CD40LG gene (c.107T>A, p.M36K), involving the transmembrane domain of the protein and a missense variant in the carnitine palmitoyl-transferase II (CPT2; c.593C>G; p.S198C) gene, leading to the diagnosis of hypomorphic XHIGM and CPT2 deficiency stress-induced myopathy. A review of all the previously reported cases of XHIGM with variants in the transmembrane domain showcased that these patients could present with atypical clinical features. Variants in the transmembrane domain of CD40LG act as hypomorphic generating a protein with a lower surface expression. Unlike large deletions or extracellular domain variants, they do not abolish the interaction with CD40, therefore preserving some biological activity.

Pediatric High Grade Glioma Classification Criteria and Molecular Features of a Case Series.

Pediatric high-grade gliomas (pHGGs) encompass a heterogeneous group of tumors. Three main molecular types (H3.3 mutant, IDH mutant, and H3.3/IDH wild-type) and a number of subtypes have been identified. We provide an overview of pHGGs and present a mono-institutional series. We studied eleven non-related pHGG samples through a combined approach of routine diagnostic tools and a gene panel. TP53 and H3F3A were the most mutated genes (six patients each, 54%). The third most mutated gene was EGFR (three patients, 27%), followed by PDGFRA and PTEN (two patients each, 18%). Variants in the EZHIP, MSH2, IDH1, IDH2, TERT, HRAS, NF1, BRAF, ATRX, and PIK3CA genes were relatively infrequent (one patient each, 9%). In one case, gene panel analysis documented the presence of a pathogenic IDH2 variant (c.419G>A, p.Arg140Gln) never described in gliomas. More than one-third of patients carry a variant in a gene associated with tumor-predisposing syndromes. The absence of constitutional DNA did not allow us to identify their constitutional origin.

STOP Pain Project-Opioid Response in Pediatric Cancer Patients and Gene Polymorphisms of Cytokine Pathways.

Moderate to severe cancer pain treatment in children is based on the use of weak and strong opioids. Pharmacogenetics play a central role in developing personalized pain therapies, as well as avoiding treatment failure and/or intolerable adverse drug reactions. This observational study aimed to investigate the association between IL-6, IL-8, and TNFα genetic single nucleotide polymorphisms (SNPs) and response to opioid therapy in a cohort of pediatric cancer patients. Pain intensity before treatment (PIt0) significantly differed according to IL-6 rs1800797 SNP, with a higher PI for A/G and G/G individuals (p = 0.017), who required a higher dose of opioids (p = 0.047). Moreover, compared to G/G subjects, heterozygous or homozygous individuals for the A allele of IL-6 rs1800797 SNP had a lower risk of having a PIt0 > 4. Dose24h and Dosetot were both higher in G/G individuals for TNFα rs1800629 (p = 0.010 and p = 0.031, respectively), while risk of having a PIt0 > 4 and a ∆VAS > 2 was higher for G/G subjects for IL-6 rs1800795 SNP compared to carriers of the C allele. No statistically significant association between genotypes and safety outcomes was found. Thus, IL-6 and TNFα SNPs could be potential markers of baseline pain intensity and opioid dose requirements in pediatric cancer patients.

Expanding the phenotype of Wiedemann-Steiner syndrome: Craniovertebral junction anomalies.

Wiedemann-Steiner syndrome (WDSTS) is a rare autosomal dominant condition caused by heterozygous loss of function variants in the KMT2A (MLL) gene, encoding a lysine N-methyltransferase that mediates a histone methylation pattern specific for epigenetic transcriptional activation. WDSTS is characterized by a distinctive facial phenotype, hypertrichosis, short stature, developmental delay, intellectual disability, congenital malformations, and skeletal anomalies. Recently, a few patients have been reported having abnormal skeletal development of the cervical spine. Here we describe 11 such individuals, all with KMT2A de novo loss-of-function variants: 10 showed craniovertebral junction anomalies, while an 11th patient had a cervical abnormality in C7. By evaluating clinical and diagnostic imaging data we characterized these anomalies, which consist primarily of fused cervical vertebrae, C1 and C2 abnormalities, small foramen magnum and Chiari malformation type I. Craniovertebral anomalies in WDSTS patients have been largely disregarded so far, but the increasing number of reports suggests that they may be an intrinsic feature of this syndrome. Specific investigation strategies should be considered for early identification and prevention of craniovertebral junction complications in WDSTS patients.

Reverse Phenotyping after Whole-Exome Sequencing in Steroid-Resistant Nephrotic Syndrome.

BACKGROUND AND OBJECTIVES: Nephrotic syndrome is a typical presentation of genetic podocytopathies but occasionally other genetic nephropathies can present as clinically indistinguishable phenocopies. We hypothesized that extended genetic testing followed by reverse phenotyping would increase the diagnostic rate for these patients. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: All patients diagnosed with nephrotic syndrome and referred to our center between 2000 and 2018 were assessed in this retrospective study. When indicated, whole-exome sequencing and in silico filtering of 298 genes related to CKD were combined with subsequent reverse phenotyping in patients and families. Pathogenic variants were defined according to current guidelines of the American College of Medical Genetics. RESULTS: A total of 111 patients (64 steroid-resistant and 47 steroid-sensitive) were included in the study. Not a single pathogenic variant was detected in the steroid-sensitive group. Overall, 30% (19 out of 64) of steroid-resistant patients had pathogenic variants in podocytopathy genes, whereas a substantial number of variants were identified in other genes, not commonly associated with isolated nephrotic syndrome. Reverse phenotyping, on the basis of a personalized diagnostic workflow, permitted to identify previously unrecognized clinical signs of an unexpected underlying genetic nephropathy in a further 28% (18 out of 64) of patients. These patients showed similar multidrug resistance, but different long-term outcome, when compared with genetic podocytopathies. CONCLUSIONS: Reverse phenotyping increased the diagnostic accuracy in patients referred with the diagnosis of steroid-resistant nephrotic syndrome.

Bicuspid Aortic Valve: Role of Multiple Gene Variants in Influencing the Clinical Phenotype.

Background. Bicuspid aortic valve (BAV) is a common congenital heart defect with increased prevalence of aortic dilatation and dissection. BAV has an autosomal dominant pattern of inheritance with reduced penetrance and variable expressivity. BAV has been described as an isolated trait or associated with other clinical manifestations in syndromic conditions. Identification of a syndromic condition in a BAV patient is clinically relevant in order to personalize indication to aortic surgery. We aimed to point out how genetic diagnosis by next-generation sequencing (NGS) can improve management of a patient with complex BAV clinical picture. Methods and Results. We describe a 45-year-old Caucasian male with BAV, thoracic aortic root and ascending aorta dilatation, and connective features evocative but inconclusive for clinical diagnosis of Marfan syndrome (MFS). Targeted (91 genes) NGS was used. Proband genetic variants were investigated in first-degree relatives. Proband carried 5 rare variants in 4 genes: FBN1(p.Asn542Ser and p.Lys2460Arg), NOTCH1(p.Val1739Met), LTBP1(p.Arg1330Gln), and TGFBR3(p.Arg423Trp). The two FBN1 variants were inherited in cis by the mother, showing systemic features evocative of MFS, but with a milder phenotype than that observed in the proband. Careful clinical observation along with the presence of the FBN1 variants allowed diagnosis of MFS in the proband and in his mother. NOTCH1 variant was found in mother and brother, not exhibiting BAV, thus not definitely supporting/excluding association with BAV. Interestingly, the proband, his brother and father, all showing root dilatation, and his sister, with upper range aortic root dimension, were carriers of a TGFBR3 variant. LTBP1 might also modulate the vascular phenotype. Conclusions. Our results underline the usefulness of NGS together with family evaluation in diagnosis of patients with monogenic traits and overlapping clinical manifestations due to contribution of the same genes and/or presence of comorbidities determined by different genes.

SLMSuite: a suite of algorithms for segmenting genomic profiles.

BACKGROUND: The identification of copy number variants (CNVs) is essential to study human genetic variation and to understand the genetic basis of mendelian disorders and cancers. At present, genome-wide detection of CNVs can be achieved using microarray or second generation sequencing (SGS) data. Although these technologies are very different, the genomic profiles that they generate are mathematically very similar and consist of noisy signals in which a decrease or increase of consecutive data represent deletions or duplication of DNA. In this framework, the most important step of the analysis consists of segmenting genomic profiles for the identification of the boundaries of genomic regions with increased or decreased signal. RESULTS: Here we introduce SLMSuite, a collection of algorithms, based on shifting level models (SLM), to segment genomic profiles from array and SGS experiments. The SLM algorithms take as input the log-transformed genomic profiles from SGS or microarray experiments and output segmentation results. We apply our method to the analysis of synthetic genomic profiles and real whole genome sequencing data and we demonstrate that it outperforms the state of the art circular binary segmentation algorithm in terms of sensitivity, specificity and computational speed. CONCLUSION: The SLMSuite contains an R library with the segmentation methods and three wrappers that allow to use them in Python, Ruby and C++. SLMSuite is freely available at https://sourceforge.net/projects/slmsuite .

Next generation sequencing and functional analysis of patient urine renal progenitor-derived podocytes to unravel the diagnosis underlying refractory lupus nephritis.

Often the cause of refractory lupus nephritis (RLN) remains unclear. We performed next-generation sequencing for podocyte genes in an RLN patient and identified compound heterozygosity for APOL1 risk alleles G1 and G2 and a novel homozygous c.[1049C>T]+[1049C>T] NPHS1 gene variant of unknown significance. To test for causality renal progenitor cells isolated from urine of this patient were differentiated into podocytes in vitro. Podocytes revealed aberrant nephrin trafficking, cytoskeletal structure and lysosomal leakage, and increased detachment as compared with podocytes isolated from controls. Thus, lupus podocytopathy can be confirmed as a cause of RLN by functional genetics on patient-derived podocytes.

Molecular characterization of paediatric glioneuronal tumours with neuropil-like islands: a genome-wide copy number analysis.

Paediatric glioneuronal tumour with neuropil-like islands (GTNI) is a rare neoplasm of neuronal differentiation and diffusely infiltrating astroglial and oligodendrocyte-like components. The 2007 World Health Organization classification of central nervous system tumours considered it as a pattern variation of anaplastic astrocytoma. There are few data on paediatric GTNI probably both for their rarity and variable clinical aggressiveness. We studied by SNP/CGH array four tumour samples of GTNI from two males and two females (one new-born and three children aged from 4 to 8 years), in order to identify any possible common genomic alteration. All patients received chemo- and radiotherapy after their surgical treatment. No genomic instability nor recurrent alterations have been demonstrated in two of our GTNI cases. In the remaining two, we detected a mosaic trisomy 8 (15-20%) in one case, and an amplification at 5q14.1 involving DMGDH (partially), BHMT2 and BHMT genes, with the distal breakpoint falling at 23 Kbp from the 5'UTR of JMY, a p53 cofactor. Although the smallness of the sample impairs any clinical-histological correlation, GTNI appear different at the molecular level, with genomic imbalances playing a possible role in at least part of them. Our work gives an important contribution in knowledge and classification of this family of tumours.

Heterogeneous genetic alterations in sporadic nephrotic syndrome associate with resistance to immunosuppression.

In children, sporadic nephrotic syndrome can be related to a genetic cause, but to what extent genetic alterations associate with resistance to immunosuppression is unknown. In this study, we designed a custom array for next-generation sequencing analysis of 19 target genes, reported as possible causes of nephrotic syndrome, in a cohort of 31 children affected by sporadic steroid-resistant nephrotic syndrome and 38 patients who exhibited a similar but steroid-sensitive clinical phenotype. Patients who exhibited extrarenal symptoms, had a familial history of the disease or consanguinity, or had a congenital onset were excluded. We identified a genetic cause in 32.3% of the children with steroid-resistant disease but zero of 38 children with steroid-sensitive disease. Genetic alterations also associated with lack of response to immunosuppressive agents in children with steroid-resistant disease (0% of patients with alterations versus 57.9% of patients without alterations responded to immunosuppressive agents), whereas clinical features, age at onset, and pathologic findings were similar in steroid-resistant patients with and without alterations. These results suggest that heterogeneous genetic alterations in children with sporadic forms of nephrotic syndrome associate with resistance to steroids as well as immunosuppressive treatments. In these patients, a comprehensive screening using such an array may, thus, be useful for genetic counseling and may help clinical decision making in a fast and cost-efficient manner.

Genome-wide copy number analysis in pediatric glioblastoma multiforme.

Glioblastoma (GBM) is a very aggressive and lethal brain tumor with poor prognosis. Despite new treatment strategies, patients' median survival is still less than 1 year in most cases. Few studies have focused exclusively on this disease in children and most of our understanding of the disease process and its clinical outcome has come from studies on malignant gliomas in childhood, combining children with the diagnosis of GBM with other pediatric patients harboring high grade malignant tumors other than GBM. In this study we investigated, using array-CGH platforms, children (median age of 9 years) affected by GBM (WHO-grade IV). We identified recurrent Copy Number Alterations demonstrating that different chromosome regions are involved, in various combinations. These observations suggest a condition of strong genomic instability. Since cancer is an acquired disease and inherited factors play a significant role, we compared for the first time the constitutional Copy Number Variations with the Copy Number Alterations found in tumor biopsy. We speculate that genes included in the recurrent 9p21.3 and 16p13.3 deletions and 1q32.1-q44 duplication play a crucial role for tumorigenesis and/or progression. In particular we suggest that the A2BP1 gene (16p13.3) is one possible culprit of the disease. Given the rarity of the disease, the poor quality and quantity of bioptic material and the scarcity of data in the literature, our findings may better elucidate the genomic background of these tumors. The recognition of candidate genes underlying this disease could then improve treatment strategies for this devastating tumor.

Schwannomatosis associated with multiple meningiomas due to a familial SMARCB1 mutation.

Schwannomatosis (MIM 162091) is a condition predisposing to the development of central and peripheral schwannomas; most cases are sporadic without a clear family history but a few families with a clear autosomal dominant pattern of transmission have been described. Germline mutations in SMARCB1 are associated with schwannomatosis. We report a family with multiple schwannomas and meningiomas. A SMARCB1 germline mutation in exon 1 was identified. The mutation, c.92A>T (p.Glu31Val), occurs in a highly conserved amino acid in the SMARCB1 protein. In addition, in silico analysis demonstrated that the mutation disrupts the donor consensus sequence of exon 1. RNA studies verified the absence of mRNA transcribed by the mutant allele. This is the first report of a SMARCB1 germline mutation in a family with schwannomatosis characterized by the development of multiple meningiomas.

NF2 mutation screening by denaturing high-performance liquid chromatography and high-resolution melting analysis.

Neurofibromatosis type 2 (NF2) is an autosomal-dominant disorder caused by mutations in the NF2 gene and predisposing to the development of nervous system. Identification of germline mutations is essential to provide appropriate genetic counseling in NF2 patients, but it represents an extremely challenging task because the vast majority of mutations are unique and spread over the entire coding sequence. Moreover, about 30% of de novo patients are indeed mosaic, and direct sequencing can undetect mutated alleles present in a minority of cells. As most screening techniques do not meet the requirements for efficient NF2 testing, we have developed a semi-automated denaturing high-performance liquid chromatography (DHPLC) method for point mutation detection combined with a multiplex ligation-dependent probe amplification approach to screen for gene rearrangements. In addition, we have evaluated high-resolution melting analysis (HRMA) as an exon scanning procedure to identify point mutations in the NF2 gene. The results obtained in 92 NF2 patients expand the NF2 mutational spectrum and indicate DHPLC and HRMA as good systems to screen for point mutations in diseases with a heterogeneous spectrum of alterations.

Evidence of a four-hit mechanism involving SMARCB1 and NF2 in schwannomatosis-associated schwannomas.

Schwannomatosis is characterized by the onset of multiple intracranial, spinal, or peripheral schwannomas, without involvement of the vestibular nerve, which is instead pathognomonic of neurofibromatosis type 2 (NF2). Recently, a schwannomatosis family with a germline mutation of the SMARCB1 gene on chromosome 22 has been described. We report on the molecular analysis of the SMARCB1 and NF2 genes in a series of 21 patients with schwannomatosis and in eight schwannomatosis-associated tumors from four different patients. A novel germline SMARCB1 mutation was found in one patient; inactivating somatic mutations of NF2, associated with loss of heterozygosity (LOH) of 22q, were found in two schwannomas of this patient. This is the second report of a germline SMARCB1 mutation in patients affected by schwannomatosis and the first report of SMARCB1 mutations associated with somatic NF2 mutations in schwannomatosis-associated tumors. The latter observation suggests that a four-hit mechanism involving the SMARCB1 and NF2 genes may be implicated in schwannomatosis-related tumorigenesis.