Novel loss-of-function variants expand ABCC9-related intellectual disability and myopathy syndrome.

Loss-of-function mutation of ABCC9, the gene encoding the SUR2 subunit of ATP sensitive-potassium (KATP) channels, was recently associated with autosomal recessive ABCC9-related intellectual disability and myopathy syndrome (AIMS). Here we identify nine additional subjects, from seven unrelated families, harbouring different homozygous loss-of-function variants in ABCC9 and presenting with a conserved range of clinical features. All variants are predicted to result in severe truncations or in-frame deletions within SUR2, leading to the generation of non-functional SUR2-dependent KATP channels. Affected individuals show psychomotor delay and intellectual disability of variable severity, microcephaly, corpus callosum and white matter abnormalities, seizures, spasticity, short stature, muscle fatigability and weakness. Heterozygous parents do not show any conserved clinical pathology but report multiple incidences of intra-uterine fetal death, which were also observed in an eighth family included in this study. In vivo studies of abcc9 loss-of-function in zebrafish revealed an exacerbated motor response to pentylenetetrazole, a pro-convulsive drug, consistent with impaired neurodevelopment associated with an increased seizure susceptibility. Our findings define an ABCC9 loss-of-function-related phenotype, expanding the genotypic and phenotypic spectrum of AIMS and reveal novel human pathologies arising from KATP channel dysfunction.

Functional analyses of rare germline BRCA1 variants by transcriptional activation and homologous recombination repair assays.

BACKGROUND: Damaging alterations in the BRCA1 gene have been extensively described as one of the main causes of hereditary breast and ovarian cancer (HBOC). BRCA1 alterations can lead to impaired homologous recombination repair (HRR) of double-stranded DNA breaks, a process which involves the RING, BRCT and coiled-coil domains of the BRCA1 protein. In addition, the BRCA1 protein is involved in transcriptional activation (TA) of several genes through its C-terminal BRCT domain. METHODS: In this study, we have investigated the effect on HRR and TA of 11 rare BRCA1 missense variants classified as variants of uncertain clinical significance (VUS), located within or in close proximity to the BRCT domain, with the aim of generating additional knowledge to guide the correct classification of these variants. The variants were selected from our previous study "BRCA1 Norway", which is a collection of all BRCA1 variants detected at the four medical genetic departments in Norway. RESULTS: All variants, except one, showed a significantly reduced HRR activity compared to the wild type (WT) protein. Two of the variants (p.Ala1708Val and p.Trp1718Ser) also exhibited low TA activity similar to the pathogenic controls. The variant p.Trp1718Ser could be reclassified to likely pathogenic. However, for ten of the variants, the total strength of pathogenic evidence was not sufficient for reclassification according to the CanVIG-UK BRCA1/BRCA2 gene-specific guidelines for variant interpretation. CONCLUSIONS: When including the newly achieved functional evidence with other available information, one VUS was reclassified to likely pathogenic. Eight of the investigated variants affected only one of the assessed activities of BRCA1, highlighting the importance of comparing results obtained from several functional assays to better understand the consequences of BRCA1 variants on protein function. This is especially important for multifunctional proteins such as BRCA1.

Functional Analyses of Rare Germline Missense BRCA1 Variants Located within and outside Protein Domains with Known Functions.

The BRCA1 protein is implicated in numerous important cellular processes to prevent genomic instability and tumorigenesis, and pathogenic germline variants predispose carriers to hereditary breast and ovarian cancer (HBOC). Most functional studies of missense variants in BRCA1 focus on variants located within the Really Interesting New Gene (RING), coiled-coil and BRCA1 C-terminal (BRCT) domains, and several missense variants in these regions have been shown to be pathogenic. However, the majority of these studies focus on domain specific assays, and have been performed using isolated protein domains and not the full-length BRCA1 protein. Furthermore, it has been suggested that BRCA1 missense variants located outside domains with known function are of no functional importance, and could be classified as (likely) benign. However, very little is known about the role of the regions outside the well-established domains of BRCA1, and only a few functional studies of missense variants located within these regions have been published. In this study, we have, therefore, functionally evaluated the effect of 14 rare BRCA1 missense variants considered to be of uncertain clinical significance, of which 13 are located outside the well-established domains and one within the RING domain. In order to investigate the hypothesis stating that most BRCA1 variants located outside the known protein domains are benign and of no functional importance, multiple protein assays including protein expression and stability, subcellular localisation and protein interactions have been performed, utilising the full-length protein to better mimic the native state of the protein. Two variants located outside the known domains (p.Met297Val and p.Asp1152Asn) and one variant within the RING domain (p.Leu52Phe) were found to make the BRCA1 protein more prone to proteasome-mediated degradation. In addition, two variants (p.Leu1439Phe and p.Gly890Arg) also located outside known domains were found to have reduced protein stability compared to the wild type protein. These findings indicate that variants located outside the RING, BRCT and coiled-coiled domains could also affect the BRCA1 protein function. For the nine remaining variants, no significant effects on BRCA1 protein functions were observed. Based on this, a reclassification of seven variants from VUS to likely benign could be suggested.

Epidemiology and natural history in 101 subjects with FKRP-related limb-girdle muscular dystrophy R9. The Norwegian LGMDR9 cohort study (2020).

We aimed to investigate the epidemiology and natural history of FKRP-related limb-girdle muscular dystrophy R9 (LGMDR9) in Norway. We identified 153 genetically confirmed subjects making the overall prevalence 2.84/100,000, the highest reported figure worldwide. Of the 153 subjects, 134 (88 %) were homozygous for FKRP c.826C>A giving a carrier frequency for this variant of 1/101 in Norway. Clinical questionnaires and patient notes from 101 subjects, including 88 c.826C>A homozygotes, were reviewed, and 43/101 subjects examined clinically. Age of onset in c.826C>A homozygotes demonstrated a bimodal distribution. Female subjects showed an increased cumulative probability of wheelchair dependency and need for ventilatory support. Across the cohort, the need for ventilatory support preceded wheelchair dependency in one third of the cases, usually due to sleep apnea. In c.826C>A homozygotes, occurrence of cardiomyopathy correlated positively with male gender but not with age or disease stage. This study highlights novel gender differences in both loss of ambulation, need for ventilatory support and the development of cardiomyopathy. Our results confirm the need for vigilance in order to detect respiratory insufficiency and cardiac involvement, but indicate that these events affect males and females differently.

Novel mutations in the HADHB gene causing a mild phenotype of mitochondrial trifunctional protein (MTP) deficiency.

Mitochondrial trifunctional protein (MTP) deficiency is an ultrarare hereditary recessive disorder causing a broad spectrum of phenotypes with lethal infantile cardiomyopathy at the most severe end. Attenuated forms with polyneuropathy have been reported combined with myoglobinuria or rhabdomyolysis as key features. We here report three young adults (two siblings) in which three variants in the HADHB-gene were identified. All three cases had a similar mild phenotype with axonal neuropathy and frequent intermittent weakness episodes but without myoglobinuria. Special dietary precautions were recommended to minimize complications especially during infections and other catabolic states. MTP deficiency is therefore an important differential diagnosis in patients with milder fluctuating neuromuscular symptoms. Take­home message: Axonal neuropathy and recurrent muscular weakness without concomitant rhabdomyolysis may be due to MTP deficiency.

BRCA1 Norway: comparison of classification for BRCA1 germline variants detected in families with suspected hereditary breast and ovarian cancer between different laboratories.

Pathogenic germline variants in Breast cancer susceptibility gene 1 (BRCA1) predispose carriers to hereditary breast and ovarian cancer (HBOC). Through genetic testing of patients with suspected HBOC an increasing number of novel BRCA1 variants are discovered. This creates a growing need to determine the clinical significance of these variants through correct classification (class 1-5) according to established guidelines. Here we present a joint collection of all BRCA1 variants of class 2-5 detected in the four diagnostic genetic laboratories in Norway. The overall objective of the study was to generate an overview of all BRCA1 variants in Norway and unveil potential discrepancies in variant interpretation between the hospitals, serving as a quality control at the national level. For a subset of variants, we also assessed the change in classification over a ten-year period with increasing information available. In total, 463 unique BRCA1 variants were detected. Of the 126 variants found in more than one hospital, 70% were interpreted identically, while 30% were not. The differences in interpretation were mainly by one class (class 2/3 or 4/5), except for one larger discrepancy (class 3/5) which could affect the clinical management of patients. After a series of digital meetings between the participating laboratories to disclose the cause of disagreement for all conflicting variants, the discrepancy rate was reduced to 10%. This illustrates that variant interpretation needs to be updated regularly, and that data sharing and improved national inter-laboratory collaboration greatly improves the variant classification and hence increases the accuracy of cancer risk assessment.

Kennedy disease in two sisters with biallelic CAG expansions of the androgen receptor gene.

We present a retrospective 21-year follow-up of two sisters with X-linked biallelic CAG expansions in the androgen receptor (AR) gene causing Kennedy disease. Two sisters inherited CAG expansions from their mother who was a carrier and their father who had Kennedy disease. Genetic testing revealed alleles comprising 43/45, and 43/43 CAG repeats in the younger and older sister, respectively. They were referred to a neurologist for further evaluation. Both reported similar symptoms with chronic backache, pain and cramps in upper- and lower extremities, and fasciculations in their faces and extremities. Neurological examination demonstrated postural hand tremor in both and EMG revealed chronic neurogenic changes. Reevaluation of the patients at ages 74 and 83 showed slight progression of clinical manifestations. As opposed to male patients, these two females showed minimal disease progression and have maintained normal level of function into old age.

Detection of Quebec Polyomavirus DNA in Samples from Different Patient Groups.

Polyomaviruses infect many species, including humans. So far, 15 polyomaviruses have been described in humans, but it remains to be established whether all of these are genuine human polyomaviruses. The most recent polyomavirus to be detected in a person is Quebec polyomavirus (QPyV), which was identified in a metagenomic analysis of a stool sample from an 85-year-old hospitalized man. We used PCR to investigate the presence of QPyV DNA in urine samples from systemic lupus erythematosus (SLE) patients (67 patients; 135 samples), multiple sclerosis patients (n = 35), HIV-positive patients (n = 66) and pregnant women (n = 65). Moreover, cerebrospinal fluid from patients with suspected neurological diseases (n = 63), nasopharyngeal aspirates from patients (n = 80) with respiratory symptoms and plasma samples from HIV-positive patients (n = 65) were examined. QPyV DNA was found in urine from 11 (16.4%), 10 (15.4%) and 5 (14.3%) SLE patients, pregnant women, and multiple sclerosis patients, respectively. No QPyV DNA could be detected in the other samples. Alignment with the only available QPyV sequence in the GenBank revealed amino acid substitutions in the HI-loop of capsid protein VP1 in 6/28 of the isolates. Our results show that QPyV viruria can occur, but whether it may cause clinical symptoms in the patients remains to be determined.

HPyV6 and HPyV7 in urine from immunocompromised patients.

BACKGROUND: Human polyomavirus 6 (HPyV6) and HPyV7 are two of the novel polyomaviruses that were originally detected in non-diseased skin. Serological studies have shown that these viruses are ubiquitous in the healthy adult population with seroprevalence up to 88% for HPyV6 and 72% for HPyV7. Both viruses are associated with pruritic skin eruption in immunocompromised patients, but a role with other diseases in immunoincompetent patients or malignancies has not been established. METHODS: PCR was used to determine the presence of HPyV6 and HPyV7 DNA in urine samples from systemic lupus erythematosus (n = 73), multiple sclerosis (n = 50), psoriasis vulgaris (n = 15), arthritic psoriasis (n = 15) and HIV-positive patients (n = 66). In addition, urine from pregnant women (n = 47) and healthy blood donors (n = 20) was investigated. RESULTS: HPyV6 DNA was detected in 21 (28.8%) of the urine specimens from SLE patients, in 6 (9.1%) of the urine samples from the HIV-positive cohort, and in 19 (40.4%) samples from pregnant women. HPyV7 DNA was only found in 6 (8.2%) of the urine specimens from SLE patients and in 4 (8.5%) samples from pregnant women. No HPyV6 and HPyV7 viruria was detected in the urine samples from the other patients. CONCLUSIONS: HPyV6, and to a lesser extend HPyV7, viruria seems to be common in SLE and HIV-positive patients, and pregnant women. Whether these viruses are of clinical relevance in these patients is not known.

Identifying sequence variants contributing to hereditary breast and ovarian cancer in BRCA1 and BRCA2 negative breast and ovarian cancer patients.

Families with breast and ovarian cancer are often tested for disease associated sequence variants in BRCA1 and BRCA2. Pathogenic sequence variants (PVs) in these two genes are known to increase breast and ovarian cancer risks in females. However, in most families no PVs are detected in these two genes. Currently, several studies have identified other genes involved in hereditary breast and ovarian cancer (HBOC). To identify genetic risk factors for breast and ovarian cancer in a Norwegian HBOC cohort, 101 breast and/or ovarian cancer patients negative for PVs and variants of unknown clinical significance (VUS) in BRCA1/2 were screened for PVs in 94 genes using next-generation sequencing. Sixteen genes were closely scrutinized. Nine different deleterious germline PVs/likely pathogenic variants (LPVs) were identified in seven genes in 12 patients: three in ATM, and one in CHEK2, ERCC5, FANCM, RAD51C, TP53 and WRN. Additionally, 32 different VUSs were identified and these require further characterization. For carriers of PV/LPV in many of these genes, there are no national clinical management programs in Norway. The diversity of genetic risk factors possibly involved in cancer development show the necessity for more knowledge to improve the clinical follow-up of this genetically diverse patient group.

ABCC9-related Intellectual disability Myopathy Syndrome is a KATP channelopathy with loss-of-function mutations in ABCC9.

Mutations in genes encoding KATP channel subunits have been reported for pancreatic disorders and Cantú syndrome. Here, we report a syndrome in six patients from two families with a consistent phenotype of mild intellectual disability, similar facies, myopathy, and cerebral white matter hyperintensities, with cardiac systolic dysfunction present in the two oldest patients. Patients are homozygous for a splice-site mutation in ABCC9 (c.1320 + 1 G > A), which encodes the sulfonylurea receptor 2 (SUR2) subunit of KATP channels. This mutation results in an in-frame deletion of exon 8, which results in non-functional KATP channels in recombinant assays. SUR2 loss-of-function causes fatigability and cardiac dysfunction in mice, and reduced activity, cardiac dysfunction and ventricular enlargement in zebrafish. We term this channelopathy resulting from loss-of-function of SUR2-containing KATP channels ABCC9-related Intellectual disability Myopathy Syndrome (AIMS). The phenotype differs from Cantú syndrome, which is caused by gain-of-function ABCC9 mutations, reflecting the opposing consequences of KATP loss- versus gain-of-function.

Fetal sex determination in twin pregnancies using non-invasive prenatal testing.

Non-invasive prenatal testing (NIPT) is accurate for fetal sex determination in singleton pregnancies, but its accuracy is not well established in twin pregnancies. Here, we present an accurate sex prediction model to discriminate fetal sex in both dichorionic diamniotic (DCDA) and monochorionic diamniotic/monochorionic monoamniotic (MCDA/MCMA) twin pregnancies. A retrospective analysis was performed using a total of 198 twin pregnancies with documented sex. The prediction was based on a multinomial logistic regression using the normalized frequency of X and Y chromosomes, and fetal fraction estimation. A second-step regression analysis was applied when one or both twins were predicted to be male. The model determines fetal sex with 100% sensitivity and specificity when both twins are female, and with 98% sensitivity and 95% specificity when a male is present. Since sex determination can be clinically important, implementing fetal sex determination in twins will improve overall twin pregnancies management.

Trans-activation-based risk assessment of BRCA1 BRCT variants with unknown clinical significance.

BACKGROUND: Deleterious variants in the tumour suppressor BRCA1 are known to cause hereditary breast and ovarian cancer syndrome (HBOC). Missense variants in BRCA1 pose a challenge in clinical care, as their effect on protein functionality often remains unknown. Many of the pathogenic missense variants found in BRCA1 are located in the BRCA1 C-terminal (BRCT) domains, domains that are known to be vital for key functions such as homologous recombination repair, protein-protein interactions and trans-activation (TA). We investigated the TA activity of 12 BRCA1 variants of unknown clinical significance (VUSs) located in the BRCT domains to aid in the classification of these variants. RESULTS: Twelve BRCA1 VUSs were investigated using a modified version of the dual luciferase TA activity assay (TA assay) that yielded increased sensitivity and sample throughput. Variants were classified according to American College of Medical Genetics and Genomics (ACMG) criteria using TA assay results and available data. In combining our TA-assay results and available data, in accordance with the ACMG guidelines for variant classification, we proposed the following variant classifications: c.5100A>G, c.5326C>T, c.5348T>C and c.5477A>T as likely benign (class 2) variants. c.5075A>C, c.5116G>A and c.5513T>G were likely pathogenic (class 4), whereas c.5096G>A likely represents a likely pathogenic variant with moderate penetrance. Variants c.5123C>T, c.5125G>A, c.5131A>C and c.5504G>A remained classified as VUSs (class 3). CONCLUSIONS: The modified TA assay provides efficient risk assessment of rare missense variants found in the BRCA1 BRCT-domains. We also report that increased post-transfection incubation time yielded a significant increase in TA assay sensitivity.

A Role of Sp1 Binding Motifs in Basal and Large T-Antigen-Induced Promoter Activities of Human Polyomavirus HPyV9 and Its Variant UF-1.

Human polyomavirus 9 (HPyV9) was originally detected in the serum of a renal transplant patient. Seroepidemiological studies showed that ~20-50% of the human population have antibodies against this virus. HPyV9 has not yet been associated with any disease and little is known about the route of infection, transmission, host cell tropism, and genomic variability in circulating strains. Recently, the HPyV9 variant UF-1 with an eight base-pair deletion, a thirteen base-pair insertion and with point mutations, creating three putative Sp1 binding sites in the late promoter was isolated from an AIDS patient. Transient transfection studies with a luciferase reporter plasmid driven by HPyV9 or UF1 promoter demonstrated that UF1 early and late promoters were stronger than HPyV9 promoters in most cell lines, and that the UF1 late promoter was more potently activated by HPyV9 large T-antigen (LTAg). Mutation of two Sp1 motifs strongly reduced trans-activation of the late UF1 promoter by HPyV9 LTAg in HeLa cells. In conclusion, the mutations in the UF1 late promoter seem to strengthen its activity and its response to stimulation by HPyV9 LTAg in certain cells. It remains to be investigated whether these promoter changes have an influence on virus replication and affect the possible pathogenic properties of the virus.

Characterization of BRCA1 and BRCA2 variants found in a Norwegian breast or ovarian cancer cohort.

Germline mutations in BRCA1 and BRCA2 cause hereditary breast and ovarian cancer. Molecular screening of these two genes in patients with a family history of breast or ovarian cancer has revealed pathogenic variants as well as genetic variants of unknown significance (VUS). These VUS may cause a challenge in the genetic counseling process regarding clinical management of the patient and the family. We investigated 32 variants previously detected in 33 samples from patients with a family history of breast or ovarian cancer. cDNA was analyzed for alternative transcripts and selected missense variants located in the BRCT domains of BRCA1 were assessed for their trans-activation ability. Although an extensive cDNA analysis was done, only three of the 32 variants appeared to affect the splice-process (BRCA1 c.213-5T>A, BRCA1 c.5434C>G and BRCA2 c.68-7T>A). In addition, two variants located in the BRCT domains of BRCA1 (c.5075A>C p.Asp1692Ala and c.5513T>G p.Val1838Gly) were shown to abolish the BRCT domain trans-activation ability, whereas BRCA1 c.5125G>A p.Gly1709Arg exhibited equal trans-activation capability as the WT domain. These functional studies may offer further insights into the pathogenicity of certain identified variants; however, this assay is only applicable for a subset of missense variants.

A heterozygous 21-bp deletion in CAPN3 causes dominantly inherited limb girdle muscular dystrophy.

Limb girdle muscular dystrophy type 2A is the most common limb girdle muscular dystrophy form worldwide. Although strict recessive inheritance is assumed, patients carrying a single mutation in the calpain 3 gene (CAPN3) are reported. Such findings are commonly attributed to incomplete mutation screening. In this investigation, we report 37 individuals (age range: 21-85 years, 21 females and 16 males) from 10 families in whom only one mutation in CAPN3 could be identified; a 21-bp, in-frame deletion (c.643_663del21). This mutation co-segregated with evidence of muscle disease and autosomal dominant transmission in several generations. Evidence of muscle disease was indicated by muscle pain, muscle weakness and wasting, significant fat replacement of muscles on imaging, myopathic changes on muscle biopsy and loss of calpain 3 protein on western blotting. Thirty-one of 34 patients had elevated creatine kinase or myoglobin. Muscle weakness was generally milder than observed in limb girdle muscular dystrophy type 2A, but affected the same muscle groups (proximal leg, lumbar paraspinal and medial gastrocnemius muscles). In some cases, the weakness was severely disabling. The 21-bp deletion did not affect mRNA maturation. Calpain 3 expression in muscle, assessed by western blot, was below 15% of normal levels in the nine mutation carriers in whom this could be tested. Haplotype analysis in four families from three different countries suggests that the 21-bp deletion is a founder mutation. This study provides strong evidence that heterozygosity for the c.643_663del21 deletion in CAPN3 results in a dominantly inherited muscle disease. The normal expression of mutated mRNA and the severe loss of calpain 3 on western blotting, suggest a dominant negative effect with a loss-of-function mechanism affecting the calpain 3 homodimer. This renders patients deficient in calpain 3 as in limb girdle muscular dystrophy type 2A, albeit in a milder form in most cases. Based on findings in 10 families, our study indicates that a dominantly inherited pattern of calpainopathy exists, and should be considered in the diagnostic work-up and genetic counselling of patients with calpainopathy and single-allele aberrations in CAPN3.

Characterization of the non-coding control region of polyomavirus KI isolated from nasopharyngeal samples from patients with respiratory symptoms or infection and from blood from healthy blood donors in Norway.

Seroepidemiological studies showed that the human polyomavirus KI (KIPyV) is common in the human population, with age-specific seroprevalence ranging from 40-90 %. Genome epidemiological analyses demonstrated that KIPyV DNA is predominantly found in respiratory tract samples of immunocompromised individuals and children suffering from respiratory diseases, but viral sequences have also been detected in brain, tonsil, lymphoid tissue studies, plasma, blood and faeces. Little is known about the sequence variation in the non-coding control region of KIPyV variants residing in different sites of the human body and whether specific strains dominate in certain parts of the world. In this study, we sequenced the non-coding control region (NCCR) of naturally occurring KIPyV variants in nasopharyngeal samples from patients with respiratory symptoms or infection and in blood from healthy donors in Norway. In total 86 sequences were obtained, 44 of which were identical to the original isolated Stockholm 60 variant. The remaining NCCRs contained one or several mutations, none of them previously reported. The same mutations were detected in NCCRs amplified from blood and nasopharyngeal samples. Some patients had different variants in their specimens. Transient transfection studies in HEK293 cells with a luciferase reporter plasmid demonstrated that some single mutations had a significant effect on the relative early and late promoter strength compared with the Stockholm 60 promoter. The effect of the NCCR mutations on viral replication and possible virulence properties remains to be established.

Early and late promoters of BK polyomavirus, Merkel cell polyomavirus, Trichodysplasia spinulosa-associated polyomavirus and human polyomavirus 12 are among the strongest of all known human polyomaviruses in 10 different cell lines.

Recently, 11 new human polyomaviruses (HPyVs) have been isolated and named KI, WU, Merkel cell polyomavirus (MCPyV), HPyV6, -7, -9, -10 and -12, Trichodysplasia spinulosa-associated polyomavirus (TSPyV), STLPyV and NJPyV-2013. Little is known about cell tropism of the novel HPyVs, and cell cultures allowing virus propagation are lacking. Because viral tropism partially depends on the interaction of cellular transcription factors with the viral promoter, we monitored the promoter activity of all known HPyVs. Therefore, we compared the relative early and late promoter activity of the BK polyomavirus (BKPyV) (WW strain) with the corresponding activities of the other HPyVs in 10 different cell lines derived from brain, colon, kidney, liver, lung, the oral cavity and skin. Our results show that the BKPyV, MCPyV, TSPyV and HPyV12 early promoters displayed the strongest activity in most cell lines tested, while the remaining HPyV had relative low early promoter activity. HPyV12 showed the highest late promoter activity of all HPyVs in most cell lines, but also the BKPyV, MCPyV and TSPyV late promoters belonged to the stronger ones among HPyVs. The HPyVs with weak early promoter activity had in general also weak late promoter activity, except for HPyV10 whose late promoter was relatively strong in six of the 10 cell lines. A 20 bp deletion in the promoter of an HPyV12 variant significantly affected both early and late promoter activity in most cell lines. In conclusion, our findings suggest which cell lines may be suitable for virus propagation and may give an indication of the cell tropism of the HPyVs.

Agrin mutations lead to a congenital myasthenic syndrome with distal muscle weakness and atrophy.

Congenital myasthenic syndromes are a clinically and genetically heterogeneous group of rare diseases resulting from impaired neuromuscular transmission. Their clinical hallmark is fatigable muscle weakness associated with a decremental muscle response to repetitive nerve stimulation and frequently related to postsynaptic defects. Distal myopathies form another clinically and genetically heterogeneous group of primary muscle disorders where weakness and atrophy are restricted to distal muscles, at least initially. In both congenital myasthenic syndromes and distal myopathies, a significant number of patients remain genetically undiagnosed. Here, we report five patients from three unrelated families with a strikingly homogenous clinical entity combining congenital myasthenia with distal muscle weakness and atrophy reminiscent of a distal myopathy. MRI and neurophysiological studies were compatible with mild myopathy restricted to distal limb muscles, but decrement (up to 72%) in response to 3 Hz repetitive nerve stimulation pointed towards a neuromuscular transmission defect. Post-exercise increment (up to 285%) was observed in the distal limb muscles in all cases suggesting presynaptic congenital myasthenic syndrome. Immunofluorescence and ultrastructural analyses of muscle end-plate regions showed synaptic remodelling with denervation-reinnervation events. We performed whole-exome sequencing in two kinships and Sanger sequencing in one isolated case and identified five new recessive mutations in the gene encoding agrin. This synaptic proteoglycan with critical function at the neuromuscular junction was previously found mutated in more typical forms of congenital myasthenic syndrome. In our patients, we found two missense mutations residing in the N-terminal agrin domain, which reduced acetylcholine receptors clustering activity of agrin in vitro. Our findings expand the spectrum of congenital myasthenic syndromes due to agrin mutations and show an unexpected correlation between the mutated gene and the associated phenotype. This provides a good rationale for examining patients with apparent distal myopathy for a neuromuscular transmission disorder and agrin mutations.

Are human polyomaviruses co-factors for cancers induced by other oncoviruses?

Presently, 12 human polyomaviruses are known: BK polyomavirus (BKPyV), JCPyV, KIPyV, WUPyV, Merkel cell polyomavirus (MCPyV), HPyV6, HPyV7, Trichodysplasia spinulosa-associated polyomavirus, HPyV9, HPyV10, STLPyV and HPyV12. In addition, the non-human primate polyomavirus simian virus 40 (SV40) seems to circulate in the human population. MCPyV was first described in 2008 and is now accepted to be an etiological factor in about 80% of the rare but aggressive skin cancer Merkel cell carcinoma. SV40, BKPyV and JCPyV or part of their genomes can transform cells, including human cells, and induce tumours in animal models. Moreover, DNA and RNA sequences and proteins of these three viruses have been discovered in tumour tissue. Despite these observations, their role in cancer remains controversial. So far, an association between cancer and the other human polyomaviruses is lacking. Because human polyomavirus DNA has been found in a broad spectrum of cell types, simultaneous dwelling with other oncogenic viruses is possible. Co-infecting human polyomaviruses may therefore act as a co-factor in the development of cancer, including those induced by other oncoviruses. Reviewing studies that report co-infection with human polyomaviruses and other tumour viruses in cancer tissue fail to detect a clear link between co-infection and cancer. Directions for future studies to elaborate on a possible auxiliary role of human polyomaviruses in cancer are suggested, and the mechanisms by which human polyomaviruses may synergize with other viruses in oncogenic transformation are discussed.