Detection of somatic and germline pathogenic variants in adult cohort of drug-resistant focal epilepsies.

OBJECTIVE: This study investigates the prevalence of pathogenic variants in the mechanistic target of rapamycin (mTOR) pathway in surgical specimens of malformations of cortical development (MCDs) and cases with negative histology. The study also aims to evaluate the predictive value of genotype-histotype findings on the surgical outcome. METHODS: The study included patients with drug-resistant focal epilepsy who underwent epilepsy surgery. Cases were selected based on histopathological diagnosis, focusing on MCDs and negative findings. We included brain tissues both as formalin-fixed, paraffin-embedded (FFPE) or fresh frozen (FF) samples. Single-molecule molecular inversion probes (smMIPs) analysis was conducted, targeting the MTOR gene in FFPE samples and 10 genes within the mTOR pathway in FF samples. Correlations between genotype-histotype and surgical outcome were examined. RESULTS: We included 78 patients for whom we obtained 28 FFPE samples and 50 FF tissues. Seventeen pathogenic variants (22 %) were identified and validated, with 13 being somatic within the MTOR gene and 4 germlines (2 DEPDC5, 1 TSC1, 1 TSC2). Pathogenic variants in mTOR pathway genes were exclusively found in FCDII and TSC cases, with a significant association between FCD type IIb and MTOR genotype (P = 0.003). Patients carrying mutations had a slightly better surgical outcome than the overall cohort, however it results not significant. The FCDII diagnosed cases more frequently had normal neuropsychological test, a higher incidence of auras, fewer multiple seizure types, lower occurrence of seizures with awareness impairment, less ictal automatisms, fewer Stereo-EEG investigations, and a longer period long-life of seizure freedom before surgery. SIGNIFICANCE: This study confirms that somatic MTOR variants represent the primary genetic alteration detected in brain specimens from FCDII/TSC cases, while germline DEPDC5, TSC1/TSC2 variants are relatively rare. Systematic screening for these mutations in surgically treated patients' brain specimens can aid histopathological diagnoses and serve as a biomarker for positive surgical outcomes. Certain clinical features associated with pathogenic variants in mTOR pathway genes may suggest a genetic etiology in FCDII patients.

R106C TFG variant causes infantile neuroaxonal dystrophy "plus" syndrome.

TFG (tropomyosin-receptor kinase fused gene) encodes an essential protein in the regulation of vesicular trafficking between endoplasmic reticulum and Golgi apparatus. The homozygous variant c.316C > T within TFG has been previously associated with a complicated hereditary spastic paraplegia (HSP) phenotype in two unrelated Indian families. Here, we describe the first Italian family with two affected siblings harboring the same variant, who in childhood were classified as infantile neuroaxonal dystrophy (INAD) based on clinical and neuropathological findings. Twenty years after the first diagnosis, exome sequencing was instrumental to identify the genetic cause of this disorder and clinical follow-up of patients allowed us to reconstruct the natural history of this clinical entity. Investigations on patient's fibroblasts demonstrate the presence of altered mitochondrial network and inner membrane potential, associated with metabolic impairment. Our study highlights phenotypic heterogeneity characterizing individuals carrying the same pathogenic variant in TFG and provides an insight on tight connection linking mitochondrial efficiency and neuronal health to vesicular trafficking.

9q31.1q31.3 deletion in two patients with similar clinical features: a newly recognized microdeletion syndrome?

Interstitial deletions of the long arm of chromosome 9 are rare and most patients have been detected by conventional cytogenetic techniques. Disparities in size and localization are large and no consistent region of overlap has been delineated. We report two similar de novo deletions of 6.3 Mb involving the 9q31.1q31.3 region, identified in two monozygotic twins and one unrelated patient through array-CGH analysis. By cloning the deletion breakpoints, we could show that these deletions are not mediated by segmental duplications. The patients displayed a distinct clinical phenotype characterized by mild intellectual disability, short stature with high body mass index, thick hair, arched eyebrows, flat profile with broad chin and mild prognathism, broad, and slightly overhanging tip of the nose, short neck with cervical gibbus. The twin patients developed a metabolic syndrome (type 2 diabetes, hypercholesterolemia, vascular hypertension) during the third decade of life. Although long-term follow-up and collection of additional patients will be needed to obtain a better definition of the phenotype, our findings characterize a previously undescribed syndromic disorder associated with haploinsufficiency of the chromosome 9q31.1q31.3 region.

Prenatal genetic counseling referrals for advanced maternal age: still room for improvement.

OBJECTIVE: The objective of this study is to evaluate genetic risks already present before pregnancy in a cohort of pregnant women referred for prenatal genetic counseling exclusively for advanced maternal age (AMA). METHOD: We retrospectively reviewed the records of 1353 women referred over 1 year (2010) for pre-test genetic counseling with the only indication of AMA at three Italian Clinical Genetic Services. RESULTS: Of the 1353 women fulfilling the inclusion criteria of the study, 87 (6.4%) had cumulatively 94 genetic risk factors not previously identified (one risk factor in 80 patients and two risk factors in seven). Twenty-six risk factors (27.7%) concerned heterogeneous or multifactorial conditions and 68 (72.3%) Mendelian or chromosomal disorders and consanguinity.In nine out of these 87 women, the estimated risk for the offspring of a genetic disease or a significant structural anomaly was >5%. Additional testing according to the identified risks was performed in 36 of these 87 women/families. CONCLUSIONS: The proportion of cases with additional risk factors is smaller than reported in previous studies, but it remains substantial and confirms the need for strategies to increase awareness of the public and health professionals responsible for the care of women in childbearing age.

Mutations in MYH9 result in the May-Hegglin anomaly, and Fechtner and Sebastian syndromes. The May-Heggllin/Fechtner Syndrome Consortium.

The autosomal dominant, giant-platelet disorders, May-Hegglin anomaly (MHA; MIM 155100), Fechtner syndrome (FTNS; MIM 153640) and Sebastian syndrome (SBS), share the triad of thrombocytopenia, large platelets and characteristic leukocyte inclusions ('Döhle-like' bodies). MHA and SBS can be differentiated by subtle ultrastructural leukocyte inclusion features, whereas FTNS is distinguished by the additional Alport-like clinical features of sensorineural deafness, cataracts and nephritis. The similarities between these platelet disorders and our recent refinement of the MHA (ref. 6) and FTNS (ref. 7) disease loci to an overlapping region of 480 kb on chromosome 22 suggested that all three disorders are allelic. Among the identified candidate genes is the gene encoding nonmuscle myosin heavy chain 9 (MYH9; refs 8-10), which is expressed in platelets and upregulated during granulocyte differentiation. We identified six MYH9 mutations (one nonsense and five missense) in seven unrelated probands from MHA, SBS and FTNS families. On the basis of molecular modelling, the two mutations affecting the myosin head were predicted to impose electrostatic and conformational changes, whereas the truncating mutation deleted the unique carboxy-terminal tailpiece. The remaining missense mutations, all affecting highly conserved coiled-coil domain positions, imparted destabilizing electrostatic and polar changes. Thus, our results suggest that mutations in MYH9 result in three megakaryocyte/platelet/leukocyte syndromes and are important in the pathogenesis of sensorineural deafness, cataracts and nephritis.

Host response to EBV infection in X-linked lymphoproliferative disease results from mutations in an SH2-domain encoding gene.

X-linked lymphoproliferative syndrome (XLP or Duncan disease) is characterized by extreme sensitivity to Epstein-Barr virus (EBV), resulting in a complex phenotype manifested by severe or fatal infectious mononucleosis, acquired hypogammaglobulinemia and malignant lymphoma. We have identified a gene, SH2D1A, that is mutated in XLP patients and encodes a novel protein composed of a single SH2 domain. SH2D1A is expressed in many tissues involved in the immune system. The identification of SH2D1A will allow the determination of its mechanism of action as a possible regulator of the EBV-induced immune response.

MR Brain Screening in ADPKD Patients : To Screen or not to Screen?

BACKGROUND: Adult polycystic kidney disease (ADPKD) still represents a major cause of renal failure and intracranial aneurisms (IA) have a higher prevalence in ADPKD than in the general population. Current guidelines suggest performing brain MRI only in the subjects with a positive familiar history of IAs or subarachnoid hemorrhage (SAH). This is a retrospective case-control analysis to evaluate the usefulness of a MR screening program in ADPKD patients. METHODS: We retrospectively analyzed all ADPKD patients followed in our outpatient clinic between 2016 and 2019 who underwent a brain MRI screening. We evaluated the presence of IAs and others brain abnormalities and compared our results with a non-ADPKD population (n = 300). We performed univariate and multivariate regression analysis to evaluate if general and demographic features, laboratory findings, clinical parameters and genetic test results correlated with IAs or other brain abnormalities presence. RESULTS: Among the patients evaluated 17 out of 156 (13.6%) ADPKD patients had IAs, compared to 16 out of 300 (5.3%) non-ADPKD controls (p < 0.005). Considering ADPKD patients presenting IAs, 12 (70.6%) had no family history for IAs or SAH. Genetic analysis was available for 97 patients: in the sub-population with IAs, 13 (76.5%) presented a PKD1 mutation and none a PKD2 mutation. We found that arachnoid cysts (AC) (p < 0.001) and arterial anatomical variants (p < 0.04) were significantly more frequent in ADPKD patients. CONCLUSION: In our population ADPKD patients showed a higher prevalence of IAs, AC and arterial variants compared to non-ADPKD. Most of the IAs were found in patients presenting a PKD1 mutation. We found a significant number of alterations even in those patients without a family history of IAs or SAH. The practice of submitting only patients with familial IAs or kidney transplantation candidates to MRI scan should be re-evaluated.

Weekly combination of non-pegylated liposomal doxorubicin and taxane in first-line breast cancer: wALT trial (phase I-II).

BACKGROUND: Through different pharmacodynamic-kinetic interactions, weekly administration of proved efficacy agents can overcome resistance with lower toxicity and greater benefit. Based on this assumption, we designed a phase I-II trial with weekly non-pegylated liposomal anthracycline and taxane in first-line breast cancer patients. PATIENTS AND METHODS: We enrolled 56 previously untreated metastatic breast cancer patients; they were randomly assigned to receive paclitaxel (Taxol) (50 mg/mq) or docetaxel (Taxotere) (30 mg/mq) combined with non-pegylated liposomal anthracycline (25 mg/mq) on days 1, 8 and 15 every 4 weeks. The primary end points were the clinical benefit and treatment-related toxic effects assessment. Secondary end points were time-to-disease progression (TTP) and overall survival (OS). RESULTS: The overall clinical benefit was 87.04%. World Health Organization G3-4 toxic effects included neutropenia (45%), anemia (44%), complete alopecia (83%), severe onycholysis and neuropathy. The 24% of patients developed left ventricular ejection fraction reduction but none >10% with recover after treatment completion. The median absolute decrease from baseline was 1%. Median TTP was 11 months and median OS was 23 months. CONCLUSIONS: Combined weekly administration of taxane and non-pegylated liposomal anthracycline is well tolerated and clinical benefit data encourage phase III study design.

A family with autosomal dominant leukodystrophy linked to 5q23.2-q23.3 without lamin B1 mutations.

BACKGROUND AND PURPOSE: Duplications of lamin B1 (LMNB1) at 5q23 are implicated in adult-onset autosomal dominant leukodystrophy (ADLD) having been described in six families with diverse ethnic background but with a homogeneous phenotype. In a large Italian family, we recently identified a variant form of ADLD characterized clinically by absence of the autonomic dysfunction at onset described in ADLD and, on MRI, by milder cerebellar involvement with sparing of hemispheric white matter. Aim of this study was to investigate the genetic basis of this variant form of ADLD. METHODS: We carried out a genome-wide linkage analysis using microsatellite markers, and the genes in the candidate region were screened for point mutations. LMNB1 was also screened for deletions/duplications by real-time PCR, multiplex ligation-dependent probe amplification and Southern blot. RESULTS: We mapped the variant ADLD locus to 5q23.2-q23.3, a genomic region containing 11 genes including LMNB1. Neither gene copy-number defects nor point mutations in the LMNB1 gene were found. We also excluded point mutations in the coding exons of the other ten genes in the candidate region. However, expression of lamin B1 evaluated in lymphoblastoid cells was higher in patients than in healthy controls, and was similar to the lamin B1 expression levels found in a patient with LMNB1 duplication. CONCLUSIONS: This observation suggests that a mutation in an LMNB1 regulatory sequence underlies the variant ADLD phenotype. Thus, adult forms of ADLD linked to 5q23 appear to be more heterogeneous clinically and genetically than previously thought.

Detection of hepatic metastases from colorectal cancer: prospective evaluation of gray scale US versus SonoVue® low mechanical index real time-enhanced US as compared with multidetector-CT or Gd-BOPTA-MRI.

PURPOSE: To compare ultrasound (US), low-mechanical index contrast enhanced US (CEUS) and multidetector-CT (MDCT) for the detection of hepatic metastases from colorectal cancer. METHODS AND MATERIALS: From January to June 2006, 110 patients (65 males, 45 females; mean age 62 years; range 39-78) with suspected hepatic lesions from colorectal cancer were prospectively evaluated with US, CEUS and MDCT by two independent readers. Intraoperative ultrasonography (IOUS, n = 45) or a follow-up up for at least 6 months by using MDCT or Gd-BOPTA-enhanced MRI was considered the gold standard. McNemar test was employed. RESULTS: Reference standards revealed 430 metastases in 110 patients. On a patient-by-patients analysis, CEUS improved US sensitivity from 67.4-71.6% to 93.4-95.8% (p < 0.05). On a lesion-by-lesion analysis, CEUS improved the sensitivity of US from 60.9-64.9% to 85.3-92.8% (p < 0.001). The specificity increased from 50-60% to 76.7-83.3%. No significant differences in sensitivity or specificity between CEUS and MDCT were found. Contrast-enhanced US was significantly more sensitive than baseline US in the detection of metastases smaller than 1 cm (p < 0.001) with an increase in sensitivity from 29.1-35% to 63.3-76.6% no significant statistical difference was identified when compared with MDCT (sensitivity of 73.3-75.8%). CONCLUSIONS: CEUS is significantly more accurate than US and highly comparable with MDCT in the detection of liver metastases from colorectal cancer. Therefore, in the evaluation of patients with suspected hepatic metastases from colorectal tumour, US examination must be performed after contrast administration.

Mowat-Wilson syndrome: facial phenotype changing with age: study of 19 Italian patients and review of the literature.

Mowat-Wilson syndrome (MWS; OMIM #235730) is a genetic condition caused by heterozygous mutations or deletions of the ZEB2 gene, and characterized by typical face, moderate-to-severe mental retardation, epilepsy, Hirschsprung disease, and multiple congenital anomalies, including genital anomalies (particularly hypospadias in males), congenital heart defects, agenesis of the corpus callosum, and eye defects. Since the first delineation by Mowat et al. [Mowat et al. (1998); J Med Genet 35:617-623], approximately 179 patients with ZEB2 mutations, deletions or cytogenetic abnormalities have been reported primarily from Europe, Australia and the United States. Genetic defects include chromosome 2q21-q23 microdeletions (or different chromosome rearrangements) in few patients, and ZEB2 mutations in most. We report on clinical and genetic data from 19 Italian patients, diagnosed within the last 5 years, including six previously published, and compare them with patients already reported. The main purpose of this review is to underline a highly consistent phenotype and to highlight the phenotypic evolution occurring with age, particularly of the facial characteristics. The prevalence of MWS is likely to be underestimated. Knowledge of the phenotypic spectrum of MWS and of its changing phenotype with age can improve the detection rate of this condition.

Autosomal recessive hereditary spastic paraplegia with thin corpus callosum: a novel mutation in the SPG11 gene and further evidence for genetic heterogeneity.

BACKGROUND AND PURPOSE: Autosomal Recessive Hereditary Spastic Paraplegia with Thin Corpus Callosum (AR-HSPTCC) is a clinically and genetically heterogeneous complicated form of spastic paraplegia. Two AR-HSPTCC loci have been assigned to chromosome 15q13-15 (SPG11) and chromosome 8p12-p11.21 respectively. Mutations in the SPG11 gene, encoding the spatacsin protein, have been found in the majority of SPG11 families. In this study, involvement of the SPG11 or 8p12-p11.21 loci was investigated in five Italian families, of which four consanguineous. METHODS: Families were tested for linkage to the SPG11 or 8p12-p11.21 loci and the SPG11 gene was screened in all the affected individuals. RESULTS: Linkage was excluded in the four consanguineous families. In the only SPG11-linked family the same homozygous haplotype 4.2 cM across the SPG11 locus was shared by all the three affected siblings. A novel c.2608A>G mutation predicted to affect the splicing was found in exon 14 of the SPG11 gene. DISCUSSION: This collection of families contributes to highlight the intra and inter locus heterogeneity in AR-HSPTCC, already remarked in previous reports. In particular, it confirms heterogeneity amongst Italian families and reports a new mutation predicted to affect splicing in the spatacsin gene.

Refinement of the SPG9 locus on chromosome 10q23.3-24.2 and exclusion of candidate genes.

BACKGROUND AND PURPOSE: The hereditary spastic paraplegias (HSPs) are a heterogeneous group of neurodegenerative disorders, characterized by a progressive spasticity of the lower limbs. So far, 33 different loci (SPGs) have been mapped and the 15 genes responsible have been identified. We mapped a locus responsible for a form of spastic paraplegia, complicated by bilateral cataracts, gastroesophageal reflux with persisting vomiting and amyotrophy to chromosome 10q23.3-q24.2, in an Italian family. The critical region was in a 12 cm chromosomal interval between markers D10S564 and D10S603 (SPG9, MIM601162). In the same region, two other forms of HSP have been recently mapped: SPG27 and SPG33. In the latter case, the gene responsible has been identified. MATERIALS AND METHODS: To better characterize this region, we genotyped individuals from SPG9-linked families using additional markers and reduced the candidate region to a 4.8 Mb, excluding several genes by positional cloning. RESULTS: The refined SPG9 locus is positioned completely within SPG27 and does not include the SPG33 gene. DISCUSSION: Fifty-two transcripts are present in the refined critical region and 25 strong candidates have been excluded as disease causing genes by direct sequencing. Six of them were also excluded as responsible for SPG27.

INPP4B overexpression and c-KIT downregulation in human achalasia.

BACKGROUND: Achalasia is a rare motility disorder characterized by myenteric neuron and interstitial cells of Cajal (ICC) abnormalities leading to deranged/absent peristalsis and lack of relaxation of the lower esophageal sphincter. The mechanisms contributing to neuronal and ICC changes in achalasia are only partially understood. Our goal was to identify novel molecular features occurring in patients with primary achalasia. METHODS: Esophageal full-thickness biopsies from 42 (22 females; age range: 16-82 years) clinically, radiologically, and manometrically characterized patients with primary achalasia were examined and compared to those obtained from 10 subjects (controls) undergoing surgery for uncomplicated esophageal cancer (or upper stomach disorders). Tissue RNA extracted from biopsies of cases and controls was used for library preparation and sequencing. Data analysis was performed with the "edgeR" option of R-Bioconductor. Data were validated by real-time RT-PCR, western blotting and immunohistochemistry. KEY RESULTS: Quantitative transcriptome evaluation and cluster analysis revealed 111 differentially expressed genes, with a P ≤ 10-3 . Nine genes with a P ≤ 10-4 were further validated. CYR61, CTGF, c-KIT, DUSP5, EGR1 were downregulated, whereas AKAP6 and INPP4B were upregulated in patients vs controls. Compared to controls, immunohistochemical analysis revealed a clear increase in INPP4B, whereas c-KIT immunolabeling resulted downregulated. As INPP4B regulates Akt pathway, we used western blot to show that phospho-Akt was significantly reduced in achalasia patients vs controls. CONCLUSIONS & INFERENCES: The identification of altered gene expression, including INPP4B, a regulator of the Akt pathway, highlights novel signaling pathways involved in the neuronal and ICC changes underlying primary achalasia.

Weekly oxaliplatin, 5-fluorouracil and folinic acid (OXALF) as first-line chemotherapy for elderly patients with advanced gastric cancer: results of a phase II trial.

BACKGROUND: Elderly patients have been often excluded from or underrepresented in the study populations of combination chemotherapy trials. The primary end point of this study was to determine the response rate and the toxicity of the weekly oxaliplatin, 5-fluorouracil and folinic acid (OXALF) regimen in elderly patients with advanced gastric cancer. The secondary objective was to measure the time to disease progression and the survival time. METHODS: Chemotherapy-naive patients with advanced gastric cancer aged 70 or older were considered eligible for study entry. Patients received weekly oxaliplatin 40 mg/m2, fluorouracil 500 mg/m2 and folinic acid 250 mg/m2. All drugs were given intravenously on a day-1 schedule. RESULTS: A total of 42 elderly patients were enrolled. Median age was 73 years and all patients had metastatic disease. The response rate according to RECIST criteria was 45.2% (95% CIs: 30%-56%) with two complete responses, 17 partial responses, 13 stable diseases and 10 progressions, for an overall tumor rate control of 76.2% (32 patients). Toxicity was generally mild and only three patients discontinued treatment because of treatment related adverse events. The most common treatment-related grade 3/4 adverse events were fatigue (7.1%), diarrhoea (4.8%), mucositis (2.4%), neurotoxicity (2.4%) and neutropenia (4.8%). The median response duration was 5.3 months (95% CIs: 2.13 - 7.34), the median time to disease progression was 5.0 months (95% CIs: 3.75 - 6.25) and the median survival time was 9.0 months (95% CIs: 6.18 - 11.82). CONCLUSION: OXALF represents an active and well-tolerated treatment modality for elderly patients with locally advanced and metastatic gastric cancer.

RET mutations in exons 13 and 14 of FMTC patients.

RET is a receptor tyrosine kinase gene which is responsible for three different inherited cancer syndromes namely multiple endocrine neoplasia type 2A (MEN 2A), type 2B (MEN 2B) and familial medullary thyroid carcinoma (FMTC) as well as for Hirschsprung disease (HSCR), a congenital disorder affecting the intestinal motility. Germ-line mutations in the RET exons 10 and 11 were demonstrated in the majority of the MEN 2A and FMTC patients. On the other hand, one codon of RET exon 16 is preferentially changed in MEN 2B patients. Recently, a germ-line mutation in the exon 13 was described in one FMTC family as well as in four sporadic MTCs. In the present study, we observed the same exon 13 mutation in two FMTC families. In addition, we identified a previously unreported substitution of RET exon 14 in two unrelated FMTC families. Both mutations segregate with the disease in these four FMTC families and involve the tyrosine kinase domain of RET. Haplotype analysis using polymorphic markers tightly linked to the RET gene indicates that in each pedigree the mutation arose as an independent event.