Prognostic value of HPV circulating tumor DNA detection and quantification in locally advanced cervical cancer.

Background: Cervical cancers are mainly caused by an oncogenic HPV. For locally advanced stages, the standard treatment is radio-chemotherapy (RTCT) followed by brachytherapy. Nevertheless, the prognosis remains highly heterogeneous between patients. Objective: We investigated the prognostic value of HPV circulating tumor DNA (ctDNA) in locally advanced cervical cancers alongside that of Squamous Cell Carcinoma Antigen (SCC-A). Methods: This single-center retrospective study included patients treated in curative intent for an IB3 to IVA squamous cell cervical cancer. Quantification of HPV ctDNA in serum collected at diagnosis was performed using a multiplex digital PCR assay for the simultaneous detection of 8 HPV genotypes. Results: Among the 97 patients included, 76 patients (78.4%) were treated by RTCT, followed by brachytherapy for 57 patients (60%). HPV ctDNA was detected in 59/97 patients at diagnosis (60.8%). This detection was associated with lymph node invasion (p=0.04) but not with tumor stage. A high level of SCC-A at diagnosis was associated with tumor stage (p=0.008) and lymph node invasion (p=0.012). In univariate analysis, better disease-free survival (DFS) was associated with optimal RTCT regimen (p=0.002), exposure to brachytherapy (p=0.0001) and a low SCC-A at diagnosis (continuous analysis, p=0.002). Exploratory analysis revealed that 3/3 patients (100%) whose HPV ctDNA was still detectable at the end of treatment relapsed, while 6/22 patients (27.3%) whose HPV ctDNA was negative at the end of treatment relapsed. Conclusion: HPV ctDNA detection at diagnosis of locally advanced cervical squamous cell carcinomas is frequent and related to node invasion, but not to DFS. The prognostic value of HPV ctDNA detection after treatment warrants specific studies.

Upstream open reading frame-introducing variants in patients with primary familial brain calcification.

More than 50% of patients with primary familial brain calcification (PFBC), a rare neurological disorder, remain genetically unexplained. While some causative genes are yet to be identified, variants in non-coding regions of known genes may represent a source of missed diagnoses. We hypothesized that 5'-Untranslated Region (UTR) variants introducing an AUG codon may initiate mRNA translation and result in a loss of function in some of the PFBC genes. After reannotation of exome sequencing data of 113 unrelated PFBC probands, we identified two upstream AUG-introducing variants in the 5'UTR of PDGFB. One, NM_002608.4:c.-373C>G, segregated with PFBC in the family. It was predicted to create an upstream open reading frame (ORF). The other one, NM_002608.4:c.-318C>T, was found in a simplex case. It was predicted to result in an ORF overlapping the natural ORF with a frameshift. In a GFP reporter assay, both variants were associated with a dramatic decrease in GFP levels, and, after restoring the reading frame with the GFP sequence, the c.-318C>T variant was associated with a strong initiation of translation as measured by western blotting. Overall, we found upstream AUG-introducing variants in the 5'UTR of PDGFB in 2/113 (1.7%) undiagnosed PFBC cases. Such variants thus represent a source of putative pathogenic variants.

A postzygotic de novo NCDN mutation identified in a sporadic FTLD patient results in neurochondrin haploinsufficiency and altered FUS granule dynamics.

Frontotemporal dementia (FTD) is a heterogeneous clinical disorder characterized by progressive abnormalities in behavior, executive functions, personality, language and/or motricity. A neuropathological subtype of FTD, frontotemporal lobar degeneration (FTLD)-FET, is characterized by protein aggregates consisting of the RNA-binding protein fused in sarcoma (FUS). The cause of FTLD-FET is not well understood and there is a lack of genetic evidence to aid in the investigation of mechanisms of the disease. The goal of this study was to identify genetic variants contributing to FTLD-FET and to investigate their effects on FUS pathology. We performed whole-exome sequencing on a 50-year-old FTLD patient with ubiquitin and FUS-positive neuronal inclusions and unaffected parents, and identified a de novo postzygotic nonsense variant in the NCDN gene encoding Neurochondrin (NCDN), NM_014284.3:c.1206G > A, p.(Trp402*). The variant was associated with a ~ 31% reduction in full-length protein levels in the patient's brain, suggesting that this mutation leads to NCDN haploinsufficiency. We examined the effects of NCDN haploinsufficiency on FUS and found that depleting primary cortical neurons of NCDN causes a reduction in the total number of FUS-positive cytoplasmic granules. Moreover, we found that these granules were significantly larger and more highly enriched with FUS. We then examined the effects of a loss of FUS function on NCDN in neurons and found that depleting cells of FUS leads to a decrease in NCDN protein and mRNA levels. Our study identifies the NCDN protein as a likely contributor of FTLD-FET pathophysiology. Moreover, we provide evidence for a negative feedback loop of toxicity between NCDN and FUS, where loss of NCDN alters FUS cytoplasmic dynamics, which in turn has an impact on NCDN expression.

Description of Joint Alterations Observed in a Family Carrying p.Asn453Ser COMP Variant: Clinical Phenotypes, In Silico Prediction of Functional Impact on COMP Protein and Stability, and Review of the Literature.

The role of genetics in the development of osteoarthritis is well established but the molecular bases are not fully understood. Here, we describe a family carrying a germline mutation in COMP (Cartilage Oligomeric Matrix Protein) associated with three distinct phenotypes. The index case was enrolled for a familial form of idiopathic early-onset osteoarthritis. By screening potential causal genes for osteoarthritis, we identified a heterozygous missense mutation of COMP (c.1358C>T, p.Asn453Ser), absent from genome databases, located on a highly conserved residue and predicted to be deleterious. Molecular dynamics simulation suggests that the mutation destabilizes the overall COMP protein structure and consequently the calcium releases from neighboring calcium binding sites. This mutation was once reported in the literature as causal for severe multiple epiphyseal dysplasia (MED). However, no sign of dysplasia was present in the index case. The mutation was also identified in one of her brothers diagnosed with MED and secondary osteoarthritis, and in her sister affected by an atypical syndrome including peripheral inflammatory arthritis of unknown cause, without osteoarthritis nor dysplasia. This article suggests that this mutation of COMP is not only causal for idiopathic early-onset osteoarthritis or severe MED, but can also be associated to a broad phenotypic variability with always joint alterations.

Early-Onset Cerebral Amyloid Angiopathy and Alzheimer Disease Related to an APP Locus Triplication.

BACKGROUND AND OBJECTIVE: To report a triplication of the amyloid-ß precursor protein (APP) locus along with relative messenger RNA (mRNA) expression in a family with autosomal dominant early-onset cerebral amyloid angiopathy (CAA) and Alzheimer disease (AD). METHODS: Four copies of the APP gene were identified by quantitative multiplex PCR of short fluorescent fragments, fluorescent in situ hybridization (FISH), and array comparative genomic hybridization. APP mRNA levels were assessed using reverse-transcription-digital droplet PCR in the proband's whole blood and compared with 10 controls and 9 APP duplication carriers. RESULTS: Beginning at age 39 years, the proband developed severe episodic memory deficits with a CSF biomarker profile typical of AD and multiple lobar microbleeds in the posterior regions on brain MRI. His father had seizures and recurrent cerebral hemorrhage since the age of 37 years. His cerebral biopsy showed abundant perivascular amyloid deposits, leading to a diagnosis of CAA. In the proband, we identified 4 copies of a 506-kb region located on chromosome 21q21.3 and encompassing the whole APP gene without any other gene. FISH suggested that the genotype of the proband was 3 copies/1 copy corresponding to an APP locus triplication, which was consistent with the presence of 2 APP copies in the healthy mother and with the paternal medical history. Analysis of the APP mRNA level showed a 2-fold increase in the proband and a 1.8 fold increase in APP duplication carriers compared with controls. DISCUSSION: Increased copy number of APP is sufficient to cause AD and CAA, with likely earlier onset in case of triplication compared with duplication.

Haploinsufficiency of the Primary Familial Brain Calcification Gene SLC20A2 Mediated by Disruption of a Regulatory Element.

OBJECTIVE: Primary familial brain calcification (PFBC) is a rare cerebral microvascular calcifying disorder with diverse neuropsychiatric expression. Five genes were reported as PFBC causative when carrying pathogenic variants. Haploinsufficiency of SLC20A2, which encodes an inorganic phosphate importer, is a major cause of autosomal-dominant PFBC. However, PFBC remains genetically unexplained in a proportion of patients, suggesting the existence of additional genes or cryptic mutations. We analyzed exome sequencing data of 71 unrelated, genetically unexplained PFBC patients with the aim to detect copy number variations that may disrupt the expression of core PFBC-causing genes. METHODS: After the identification of a deletion upstream of SLC20A2, we assessed its consequences on gene function by reverse transcriptase droplet digital polymerase chain reaction (RT-ddPCR), an ex vivo inorganic phosphate uptake assay, and introduced the deletion of a putative SLC20A2 enhancer mapping to this region in human embryonic kidney 293 (HEK293) cells by clustered regularly interspaced short palindromic repeats (CRISPR) - CRISPR-associated protein 9 (Cas9). RESULTS: The 8p11.21 deletion, segregating with PFBC in a family, mapped 35 kb upstream of SLC20A2. The deletion carriers/normal controls ratio of relative SLC20A2 mRNA levels was 60.2% (P < 0.001). This was comparable with that of patients carrying an SLC20A2 premature stop codon (63.4%; P < 0.001). The proband exhibited a 39.3% decrease of inorganic phosphate uptake in blood (P = 0.015). In HEK293 cells, we observed a 39.8% decrease in relative SLC20A2 mRNA levels after normalization on DNA copy number (P < 0.001). DISCUSSION: We identified a deletion of an enhancer of SLC20A2 expression, with carriers showing haploinsufficiency in similar ranges to loss-of-function alleles, and we observed reduced mRNA levels after deleting this element in a cellular model. We propose a 3-step strategy to identify and easily assess the effect of such events. © 2020 International Parkinson and Movement Disorder Society.

Biallelic MYORG mutation carriers exhibit primary brain calcification with a distinct phenotype.

Primary familial brain calcification (PFBC) is a rare neurogenetic disorder with diverse neuropsychiatric expression. Mutations in four genes cause autosomal dominant PFBC: SLC20A2, XPR1, PDGFB and PDGFRB. Recently, biallelic mutations in the MYORG gene have been reported to cause PFBC with an autosomal recessive pattern of inheritance. We screened MYORG in 29 unrelated probands negatively screened for the autosomal dominant PFBC genes and identified 11 families with a biallelic rare or novel predicted damaging variant. We studied the clinical and radiological features of 16 patients of these 11 families and compared them to that of 102 autosomal dominant PFBC patients carrying a mutation in one of the four known autosomal dominant PFBC genes. We found that MYORG patients exhibited a high clinical penetrance with a median age of onset of 52 years (range: 21-62) with motor impairment at the forefront. In particular, dysarthria was the presenting sign in 11/16 patients. In contrast to patients with autosomal dominant PFBC, 12/15 (80%) symptomatic patients eventually presented at least four of the following five symptoms: dysarthria, cerebellar syndrome, gait disorder of any origin, akinetic-hypertonic syndrome and pyramidal signs. In addition to the most severe clinical pattern, MYORG patients exhibited the most severe pattern of calcifications as compared to the patients from the four autosomal dominant PFBC gene categories. Strikingly, 12/15 presented with brainstem calcifications in addition to extensive calcifications in other brain areas (lenticular nuclei, thalamus, cerebellar hemispheres, vermis, ±cortex). Among them, eight patients exhibited pontine calcifications, which were observed in none of the autosomal dominant PFBC patients and hence appeared to be highly specific. Finally, all patients exhibited cerebellar atrophy with diverse degrees of severity on CT scans. We confirmed the existence of cerebellar atrophy by performing MRI voxel-based morphometry analyses of MYORG patients with autosomal dominant PFBC mutation carriers as a comparison group. Of note, in three families, the father carried small pallido-dentate calcifications while carrying the mutation at the heterozygous state, suggesting a putative phenotypic expression in some heterozygous carriers. In conclusion, we confirm that MYORG is a novel major PFBC causative gene and that the phenotype associated with such mutations may be recognized based on pedigree, clinical and radiological features.

APP Mutations in Cerebral Amyloid Angiopathy with or without Cortical Calcifications: Report of Three Families and a Literature Review.

BACKGROUND: Specific APP mutations cause cerebral amyloid angiopathy (CAA) with or without Alzheimer's disease (AD). OBJECTIVE: We aimed at reporting APP mutations associated with CAA, describe the clinical, cerebrospinal fluid AD biomarkers, and neuroimaging features, and compare them with the data from the literature. METHODS: We performed a retrospective study in two French genetics laboratories by gathering all clinical and neuroimaging data from patients referred for a genetic diagnosis of CAA with an age of onset before 66 years and fulfilling the other Boston revised criteria. We studied the segregation of mutations in families and performed a comprehensive literature review of all cases reported with the same APP mutation. RESULTS: We screened APP in 61 unrelated French patients. Three mutations, located in the Aß coding region, were detected in five patients from three families: p.Ala692Gly (Flemish), p.Glu693Lys (Italian), and p.Asp694Asn (Iowa). Patients exhibited CAA and progressive cognitive impairment associated with cortical calcifications in the Iowa and Italian mutation carriers, but not the patient carrying the Flemish mutation. CONCLUSIONS: This is the first evidence of cortical calcification in patients with an APP mutation other than the Iowa mutation. We discuss the radiological, cerebrospinal fluid, and clinical phenotype of patients carrying these mutations in the literature.

Identification of partial SLC20A2 deletions in primary brain calcification using whole-exome sequencing.

Primary brain calcification (PBC) is a dominantly inherited calcifying disorder of the brain. SLC20A2 loss-of-function variants account for the majority of families. Only one genomic deletion encompassing SLC20A2 and six other genes has been reported. We performed whole-exome sequencing (WES) in 24 unrelated French patients with PBC, negatively screened for sequence variant in the known genes SLC20A2, PDGFB, PDGFRB and XPR1. We used the CANOES tool to detect copy number variations (CNVs). We detected two deletions of exon 2 of SLC20A2 in two unrelated patients, which segregated with PBC in one family. We then reanalyzed the same series using a QMPSF assay including one amplicon in each exon of SLC20A2 and detected two supplemental partial deletions in two patients: one deletion of exon 4 and one deletion of exons 4 and 5. These deletions were missed by the first screening step of CANOES but could finally be detected after readjustment of bioinformatic parameters and use of a genotyping step of CANOES. This study reports the first partial deletions of SLC20A2 and strengthens its position as the major PBC-causative gene. It is possible to detect short CNVs from WES data, although the sensitivity of such tools should be evaluated in comparison with other methods.

PDGFB partial deletion: a new, rare mechanism causing brain calcification with leukoencephalopathy.

Idiopathic basal ganglia calcification (IBGC) is a progressive cerebral disorder with diverse motor, cognitive, and psychiatric expression. It is inherited as an autosomal dominant trait. Three IBGC-causing genes have been identified in the past 2 years: SLC20A2, PDGFRB, and PDGFB. Biological and genetic evidence showed that loss of function of either SLC20A2 or the PDGFB/PDGFRB pathway was the mechanism underlying calcification in patients with a mutation. Recently, in a study focusing on SLC20A2, a large deletion at this locus was reported. No study has systematically searched for copy number variants (CNV) involving these three genes. We designed a quantitative PCR assay of multiple short fluorescent fragments (QMPSF) to detect CNVs involving one of these three genes in a single assay. Among the 27 unrelated patients from our IBGC case series with no mutation in SLC20A2, PDGFRB, and PDGFB, we identified in one patient a heterozygous partial deletion involving exons 2 to 5 of PDGFB. This patient exhibited both strio-pallido-dentate calcification and white matter hyperintensity of presumed vascular origin, associated with mood disorder, subtle cognitive decline, and gait disorder. We confirmed by RT-PCR experiments that the allele carrying the deletion was transcribed. The resulting cDNA lacks sequence for several critical functional domains of the protein. Intragenic deletion of PDGFB is a new and rare mechanism causing IBGC. CNVs involving the three IBGC-causing genes should be investigated in patients with no point mutation.

A de novo nonsense PDGFB mutation causing idiopathic basal ganglia calcification with laryngeal dystonia.

Idiopathic basal ganglia calcification (IBGC) is characterized by brain calcification and a wide variety of neurologic and psychiatric symptoms. In families with autosomal dominant inheritance, three causative genes have been identified: SLC20A2, PDGFRB, and, very recently, PDGFB. Whereas in clinical practice sporadic presentation of IBGC is frequent, well-documented reports of true sporadic occurrence are rare. We report the case of a 20-year-old woman who presented laryngeal dystonia revealing IBGC. Her healthy parents' CT scans were both normal. We identified in the proband a new nonsense mutation in exon 4 of PDGFB, c.439C>T (p.Gln147*), which was absent from the parents' DNA. This mutation may result in a loss-of-function of PDGF-B, which has been shown to cause IBGC in humans and to disrupt the blood-brain barrier in mice, resulting in brain calcification. The c.439C>T mutation is located between two previously reported nonsense mutations, c.433C>T (p.Gln145*) and c.445C>T (p.Arg149*), on a region that could be a hot spot for de novo mutations. We present the first full demonstration of the de novo occurrence of an IBGC-causative mutation in a sporadic case.

Phenotypic spectrum of probable and genetically-confirmed idiopathic basal ganglia calcification.

Idiopathic basal ganglia calcification is characterized by mineral deposits in the brain, an autosomal dominant pattern of inheritance in most cases and genetic heterogeneity. The first causal genes, SLC20A2 and PDGFRB, have recently been reported. Diagnosing idiopathic basal ganglia calcification necessitates the exclusion of other causes, including calcification related to normal ageing, for which no normative data exist. Our objectives were to diagnose accurately and then describe the clinical and radiological characteristics of idiopathic basal ganglia calcification. First, calcifications were evaluated using a visual rating scale on the computerized tomography scans of 600 consecutively hospitalized unselected controls. We determined an age-specific threshold in these control computerized tomography scans as the value of the 99th percentile of the total calcification score within three age categories: <40, 40-60, and >60 years. To study the phenotype of the disease, patients with basal ganglia calcification were recruited from several medical centres. Calcifications that rated below the age-specific threshold using the same scale were excluded, as were patients with differential diagnoses of idiopathic basal ganglia calcification, after an extensive aetiological assessment. Sanger sequencing of SLC20A2 and PDGFRB was performed. In total, 72 patients were diagnosed with idiopathic basal ganglia calcification, 25 of whom bore a mutation in either SLC20A2 (two families, four sporadic cases) or PDGFRB (one family, two sporadic cases). Five mutations were novel. Seventy-one per cent of the patients with idiopathic basal ganglia calcification were symptomatic (mean age of clinical onset: 39 ± 20 years; mean age at last evaluation: 55 ± 19 years). Among them, the most frequent signs were: cognitive impairment (58.8%), psychiatric symptoms (56.9%) and movement disorders (54.9%). Few clinical differences appeared between SLC20A2 and PDGFRB mutation carriers. Radiological analysis revealed that the total calcification scores correlated positively with age in controls and patients, but increased more rapidly with age in patients. The expected total calcification score was greater in SLC20A2 than PDGFRB mutation carriers, beyond the effect of the age alone. No patient with a PDGFRB mutation exhibited a cortical or a vermis calcification. The total calcification score was more severe in symptomatic versus asymptomatic individuals. We provide the first phenotypical description of a case series of patients with idiopathic basal ganglia calcification since the identification of the first causative genes. Clinical and radiological diversity is confirmed, whatever the genetic status. Quantification of calcification is correlated with the symptomatic status, but the location and the severity of the calcifications don't reflect the whole clinical diversity. Other biomarkers may be helpful in better predicting clinical expression.

Mutation of the PDGFRB gene as a cause of idiopathic basal ganglia calcification.

OBJECTIVES: To identify a new idiopathic basal ganglia calcification (IBGC)-causing gene. METHODS: In a 3-generation family with no SLC20A2 mutation, we performed whole exome sequencing in 2 affected first cousins, once removed. Nonsynonymous coding variants, splice acceptor and donor site variants, and frameshift coding indels (NS/SS/I) were filtered against dbSNP131, the HapMap Project, 1000 Genomes Project, and our in-house database including 72 exomes. RESULTS: Seventeen genes were affected by identical unknown NS/SS/I variations in the 2 patients. After screening the relatives, the p.Leu658Pro substitution within the PDGFRB gene remained the sole unknown mutation segregating with the disease in the family. This variation, which is predicted to be highly damaging, was present in 13 of 13 affected subjects and absent in 8 relatives without calcifications. Sequencing PDGFRB of 19 other unrelated IBGC cases allowed us to detect another potentially pathogenic substitution within PDGFRB, p.Arg987Trp, also predicted to be highly damaging. PDGFRB encodes a protein involved in angiogenesis and in the regulation of inorganic phosphate (Pi) transport in vascular smooth muscle cells via Pit-1, a Pi transporter encoded by SLC20A1. CONCLUSION: Mutations of PDGFRB further support the involvement of this biological pathway in IBGC pathophysiology.

Partial deletion of the MAPT gene: a novel mechanism of FTDP-17.

A heterozygous genomic deletion removing exons 6 to 9 of the microtubule associated protein tau (MAPT) gene, predicting to result into a truncated protein lacking the first microtubule binding domain, was detected in a patient with frontotemporal dementia (FTD). Cell culture experiments showed that the truncated tau isoforms had a dramatic decrease in the normal binding to microtubules but acquired the ability to bind microtubule associated protein-1B (MAP-1B). This indicates that this tauopathy likely results both from a loss of function mechanism and from a deleterious gain of function by which cytoplasmic deleted forms of tau sequester another MAP. Both mechanisms could contribute to impair microtubule dynamics.